ORAL SESSION ABSTRACTS
| SESSION
1. BACTERIOLOGY I |
[TOP] |
S1-1
Intrinsic Resistance of Edwardsiella ictaluri to Cationic Antimicrobial Peptides is Mediated by UDP-Glc Dehydrogenase, an Enzyme Required for the O-polysaccharide Synthesis
Santander* and R Curtiss III
| Arizona State University, Biodesign Institute, Center for Infectious Diseases and Vaccinology, Tempe, AZ, USA jsantanderm@asu.edu |
The genus Edwardsiella comprises a genetically distinct taxon related to other members of the Enterobacteriaceae. It consists of bacteria differing strongly in their biochemical and physiological features, natural habitats, and pathogenic properties. Intrinsic resistance to cyclic cationic antimicrobial peptides (colistin and polymyxin B) is a particular property of the genus Edwardsiella. Here we evaluated the role of the O-polysaccharide (O-PS) in the resistance to cyclic cationic antimicrobial peptides in Edwardsiella ictaluri (Hawke, 1981), the causative agent of fatal systemic infections in channel catfish (Ictalurus punctatus). The putative genes (wibT, galF, gne, and ugd) related to the synthesis of O-PS were systematically inframe deleted by suicide vector technology. Individual deletions of wibT, gne and ugd abolished synthesis of the O-PS, causing auto-agglutination and rough colonies. The wibT and gne mutants conserve their resistance to cyclic antimicrobial peptides. However, deletion of ugd, the gene that encodes the UDP-Glc dehydrogenase enzyme, causes sensitivity to cyclic cationic antimicrobial peptides, without revertants. LPS profile analysis indicates that deletion of ugd affects the lipopolysaccharide (LPS) core. The outer membrane protein profile showed up regulation of a single protein in wibT, gne and ugd mutants. These results indicate that the UDP-Glc dehydrogenase enzyme, and the structure of the LPS core, are essential for the Edwardsiella resistance mechanism to cyclic antimicrobial peptides.
S1-2
Evaluation of the Edwardsiella ictaluri Type IV Secretion System Role in Virulence
AA Graff1, ML Rogge1 and RL Thune*1,2
| 1 |
Department of Pathobiological Sciences, LSU School of Veterinary Medicine, Skip Bertman Drive, Baton Rouge, LA 70803 USA graff.allison@gmail.com, mrogge1@lsu.edu |
| 2 |
Department of Veterinary Science, Louisiana State University,Baton Rouge, LA USA thune@vetmed.lsu.edu |
Type IV secretion systems (T4SS) have roles in the secretion of both DNA and proteins for the purposes of conjugation and virulence, respectively. Edwardsiella ictaluri carries a pathogenicity island encoding genes with homology to T4SSs of other pathogens. In addition to the T4SS, E. ictaluri encodes type III and type VI secretion systems, both of which are important for pathogenesis in the channel catfish (Ictalurus punctatus). To determine if the T4SS is also involved in E. ictaluri pathogenesis, a mutation was constructed in the virD4-like gene, which is putatively involved in coupling secretion substrates to the secretion apparatus. Such a mutation should knock out function of the entire type IV system. Subsequent phenotypic characterization of the mutant included evaluation of invasion and replication in channel catfish cells, as well as in vivo colonization and virulence. No defect in invasion or intracellular replication was observed ex vivo, and the mutant strain colonized the head kidney, persisted, and was virulent. These results demonstrate that virulence of E. ictaluri is not dependent on the T4SS-encoded virD4, but this does not preclude the T4SS from having a role in virulence that is not detectable by the assays conducted. Further characterization is needed to determine if the T4SS components are functional, under what environmental conditions they are expressed, and how they may be involved in virulence.
S1-3
The Edwardsiella ictaluri Type III and Type
VI Secretion Systems Are Co-Regulated and Are Both Required for Virulence in
the Channel Catfish
ML
Rogge*1 and RL Thune1,2
| 1 |
Departmentof
Pathobiological Sciences, LSU School of Veterinary Medicine, Baton Rouge,
LA,USA mrogge1@lsu.edu
|
| 2 |
Departmentof Veterinary Science,
Louisiana State University, Baton Rouge, LA, USA thune@vetmed.lsu.edu
|
Type III and type VI secretion systems (T3SSs and
T6SSs) are found in many pathogenic bacteria and mediate the translocation of
virulence proteins from the bacterial cytoplasm to the cytoplasm of the host
cell. The Edwardsiella ictaluri T3SS
is required for intracellular replication and virulence in channel catfish (Ictalurus punctatus). Previous work
demonstrated that secretion of a T6SS protein, EvpC, is dependent on the T3SS-encoded
regulatory proteins EsrB and EsrC, suggesting the coordinated regulation of two
secretion systems by these regulatory proteins. To evaluate the T6SS role in
virulence, a mutation was made in EvpC, a putative T6SS translocon protein.
Knockout of the translocon protein should prohibit the T6SS from translocating
effector proteins. The mutant strain was evaluated for its ability to replicate
within channel catfish head kidney-derived macrophages (HKDM), colonize head
kidney tissue in vivo, and cause mortality
in channel catfish. The T6SS mutant maintained the ability to replicate within
HKDM, and was able to successfully colonize the head kidney. Following
immersion challenge, however, mortality of catfish exposed to the T6SS mutant
was significantly less than with the wild-type, indicating an important
virulence function for the T6SS. Further research is required to identify the
proteins secreted by the T6SS and determine what their function is in
virulence. Additionally, the role of the T3SS-encoded EsrB and EsrC in the
regulation of the T6SS and the integrated role of the T3SS and T6SS in the
pathogenesis of E. ictaluri requires
further study.
S1-4
Evaluation of the Role of Ammonia and Urea
Transporters on the Pathogenesis of Edwardsiella
ictaluri
J Beekman and RThune*
| Department of Pathobiological Sciences, School of
Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803 USA
thune@vetmed.lsu.edu
|
Edwardsiella ictaluri is a facultative
intracellular pathogen that survives and replicates within channel catfish macrophages. We previously reported that the E. ictaluri genome contains a urease
operon expressing a functional urease that converts urea to ammonia in vitro, and which plays a role in
survival of E. ictaluri within cultured
head kidney derived macrophages (HKDM). Taken together, this data suggests that urease contributes to survival
within the macrophage by producing ammonia to neutralize the acidic endosome. Interestingly, the E. ictaluri genome contains two genes in tandem just downstream of
the urease operon, one encoding a urea transporter and the second a truncated,
but potentially functional, ammonia transporter. The objective of this study was to evaluate the role
these transporters play in the movement of the urease substrate, urea, and the
urease product, ammonia, across the E.
ictaluri membrane. Strains of E. ictaluri containing deletion
mutations in either the urea or the ammonia transporter were found to be
attenuated by 48% and 36%, respectively, compared to wild type in a live
catfish challenge. There was no
difference in survival, however, between the mutants and the wild type within
cultured HKDM. RT-PCR of RNA derived
from E. ictaluri grown in BHI
detected low levels of transporter mRNA in the WT but not the mutants, as
measured by qPCR. The transporter
proteins, however, were undetectable by Western blot analysis of E. ictaluri whole-cell lysates. The live challenge results suggest a
role for the urea and ammonia transporters in the pathogenesis of E. ictaluri, but further phenotypic
analysis suggests that the effects are subtle and difficult to measure. Experiments are underway to detect the
transporter proteins in isolated bacterial membranes and to directly measure pH
changes in HKDM infected with either WT or transporter mutants using
fluorescent microscopy and ratio-metric imaging
S1-5
High Total Ammonia
Nitrogen Concentrations Increase Survival of Channel Catfish Experimentally
Infected with Flavobacterium columnare
AJ
Mitchell* and B Farmer
| Harry K. Dupree Stuttgart
National Aquaculture Research Center, United States Department of
Agriculture/Agricultural Research Stuttgart, AR USA drew.mitchell@ars.usda.gov
|
Although it is generally accepted that elevated
ammonia levels in the water increase mortalities in Flavobacterium columnare-infected fish, recent observations at our
laboratory have indicated otherwise. Two similar studies were set up to determine the effect of a single
high-level treatment of total ammonia nitrogen (TAN: 15 mg/L; un-ionized
ammonia: 0.4 mg/L) on the survival of channel catfish experimentally infected
with F. columnare (strain LV-359-01
at ~1x108 bacteria/mL of water). In both studies, channel catfish were stocked at 50 g/L in
10 L of water with four water exchanges/d and were given four replicates of one
of the following treatments: no ammonia or bacterial challenge (Treatment 1),
ammonia only (Treatment 2), bacterial challenge only (Treatment 3), or both
ammonia and bacterial challenge (Treatment 4). As trial and trial x treatment
interaction effects between the two studies were not significantly different,
survival data from the two studies were combined in the final determination of
differences among treatment means. There was no significant
difference between the survival of fish that received Treatment 1 (95.2±1.2%)
versus Treatment 2 (95.6±1.0%). These two treatments were significantly different from Treatments 3
(8.5±4.5%) and 4 (41.8±12.7%), and fish that received Treatment 4 had a
significantly higher survival rate than those that received Treatment 3. At the TAN levels tested in this study,
ammonia itself did not negatively influence survival of the fish but did either
limit the survival of F. columnare or
otherwise interfere with the infection process.
S1-6
Ultrastructure of
Skin Lesions on Channel Catfish, Ictalurus
punctatus Exposed to Flavobacterium
columnare– Insights on Skin Discoloration and Epidermal Damage
A
McElwain*1, O Olivares-Fuster, CR Arias2 and SA Bullard1
| 1 |
Departmentof Fisheries and
Allied Aquacultures (FAA), Aquatic Parasitology Laboratory (APL), 203 Swingle
Hall, Auburn University (AU), Auburn, AL azm0034@auburn.edu
|
| 2 |
FAA,Aquatic Microbiology
Laboratory, crarias@auburn.edu
|
Flavobacterium
columnare,
a Gram-negative rod-shaped bacterium, is the causative agent of “columnaris
disease” and a serious pathogen of freshwater fishes in aquaculture. A columnaris infection is typically
characterized by the presence of an opaque, dorso-lateral band referred to as
“saddleback” lesion. Although this
is a well-known sign of infection, little detailed information is available
regarding how this lesion develops in channel catfish, Ictalurus punctatus. In an effort to better understand columnaris pathogenesis, we used the
JCM-21327 strain of F. columnare in
an immersion challenge of specific pathogen free channel catfish and compared
skin samples from infected and control fish using scanning electron microscopy
(SEM). SEM samples were fixed in
10% neutral buffered formalin for 48 hrs, rinsed in distilled water, dehydrated
in a graded ethanol series using an automated tissue processor, immersed in
hexamethyldisilazane, and mounted on metal stubs before being sputter coated
with gold-palladium. In our
samples, saddleback lesions generally developed dorso-laterally, between the
posterior margin of the opercle and the adipose fin. A ragged epidermal margin consisting of wispy tissue
presumed to be epidermal remnants comprised the boundary between infected and
uninfected skin. Infected skin
appeared to be devoid of epidermis, and rod-shaped bacteria were present on the
exposed dermis. Bacterial cells on
the dermis were 0.20–0.40 mm ´ 8–10 mm and loosely arranged. The foregoing represents a step towards
understanding the development of skin lesions during a columnaris
infection. These results support
the notion that saddleback discoloration results from epidermal obliteration in
infected catfish.
| SESSION
2. IMMUNOLOGY I: Vaccines |
[TOP] |
S2-1
Expression Profiles
of Cobia (Rachycentron canadum)
Spleen and Head Kidney Following Vaccination Against Vibrio carchariae, Proteus
vulgaris and Streptococcus sp.
J J
Feng*1, Z Guo1, Y Su1,
L Xu1, J Wang1, Y Hou1,2 and L Mao1,2
| 1 |
Department of Fish Disease, South China Sea Fisheries Research Institute, CAFS. Guangzhou,
Gangdong, China janej_feng@msn.com, guozhixun1@163.com,
vetsyl@163.com, lwxu@163.com, wjy104@163.com
|
| 2 |
Department of Veterinary Science, South China Agriculture University, Guangdong Province,
Guangzhou, 510642, P R China houyifan920@126.com, maolina2003@163.com
|
To investigate the response of cobia to vaccination and pathogen
exposure, a full-length cDNA library was constructed from head kidney and
spleen mRNA collected following vaccination against Vibrio carchariae, Proteus
vulgaris and Streptococcus sp.
After sequencing 6012 readable sequences were obtained of which 854 clones
(14.20%) contained cell/organism defence genes including immunoglobulin, MHC
class I and II, interferon consensus sequence binding protein, B-cell
receptor-associated protein, early B-cell factor, 10 complement components,
heat shock protein 70 and 90, antimicrobial peptides hepcidin type 1 and 2, and
CC chemokine (macrophage inflammatory protein-1 beta-like chemokine,
MIP-1beta). These results indicate that this cDNA library contains many
important genes involved in the immune response, making it an important
resource for studying the response of cobia to vaccination or pathogen
exposure. At the same time, the characteristic of Ig k and µ chain, MHC II α and β were
primarily studied.
S2-2
Expression Attenuated Francisella asiatica iglC Mutant Induces Protective Immunity to
Francisellosis in Tilapia
E Soto*1, J Wiles1, PH
Elzer1, 2, KR Macaluso1 and JP Hawke1
| 1 |
Departmentof Pathobiological
Sciences, LSU-School of Veterinary Medicine, Dr., Baton Rouge, LA, USA esoto1@lsu.edu, jwiles@vetmed.lsu.edu, Kmacaluso@mail.vetmed.lsu.edu jhawke1@lsu.edu
|
| 2 |
LSUAgCenter, Department of
Veterinary Science, Baton Rouge, Louisiana,, USA pelzer@agctr.lsu.edu
|
Francisella
asiatica is a Gram negative, facultative intracellular
bacteria that causes fish francisellosis. Fish francisellosis is a severe
sub-acute to chronic granulomatous disease with high mortalities and high
infectivity rates in cultured and wild fish. To date, there is no approved
vaccine for this widespread emergent disease. The goal of this study was to
characterize the efficacy of a defined F. asiatica mutant (ΔiglC) as a live
attenuated vaccine against subsequent immersion challenge with the wild-type
(WT) organism. In previous work, the ΔiglC was found to be
attenuated upon intraperitoneal injection and immersion challenges. In vitro, the ΔiglC exhibited reduced growth in tilapia head kidney derived
macrophages, and was significantly attenuated (p<0.001) as demonstrated by
cytopathogenic and apoptosis assays. In this study, the ΔiglC was tested to
determine its ability to protect tilapia against challenge with high doses
(lethal dose 80) of WT bacteria. Naive tilapia vaccinated by immersion with a
suspension of the ΔiglC and subsequently challenged with WT F. asiatica were
protected (90% mean percent survival) from the lethal challenges. F.
asiatica-specific antibodies produced in response to immunization with the ΔiglC were
subsequently found to protect naive tilapia against high-dose F. asiatica challenge
in passive immunization experiments. Significant protection (p<0.05) was
obtained when fish were passively immunized and challenged with 104 and 105 CFU/fish of WT F. asiatica; but not when challenged
with 106 CFU/fish. This is the first report of a defined live
attenuated strain providing protection against F. asiatica in fish.
S2-3
An Experimental Vaccine Against Aeromonas
hydrophila Can Induce Protection in Rainbow Trout
SE LaPatra*1, KP Plant2,
WD Shewmaker1, RA Burkhart1 and AW Morton1
| 1 |
Clear
Springs Foods, Inc., Research Division, Buhl, ID, USA scottl@clearsprings.com
|
| 2 |
Hagerman Fish Culture Experiment Station, University
of Idaho, Hagerman, ID, USA kplant@uidaho.edu
|
A candidate vaccine against Aeromonas hydrophila in rainbow trout (Oncorhynchus mykiss) was developed using a bacterial lysate. To
test the strength of protection, A.
hydrophila challenge models were compared using injection into both the
peritoneal cavity (IP) and the dorsal sinus (DS) with selected doses of live
bacteria washed in saline or left untreated. Unlike the IP route, injection
into the DS with either saline washed or unwashed cells resulted in consistent
cumulative mortality and a clear dose response pattern that could be used to
establish a standard challenge having an LD50 of approximately 3 x107 colony forming units per fish. Survivors of the challenge suffered significantly
lower mortality upon re-challenge than naïve fish, suggesting a high level of
acquired resistance was elicited by infection. Passive immunization using serum
from hyper-immunized fish also resulted in significantly reduced mortality
indicating protection can be transferred and that some portion of resistance
may be antibody mediated. Vaccination of groups of rainbow trout with A. hydrophila lysate resulted in
significant protection against a high challenge dose but only when injected
along with Freunds complete adjuvant. At a low challenge dose, mortality in all
groups was low, but the bacterial lysate alone appeared to offer some
protection.
S2-4
Confirmation of Safety
and Efficacy for a Live Attenuated Flavobacterium
psychrophilum Vaccine
TJ Johnson*1, N Parvez1,
DR Call2, BR LaFrentz3, T Miura4 and KD Cain1
| 1 |
Departmentof Fish and Wildlife Resources,
University of Idaho, Moscow, USA
john3002@vandals.uidaho.edu, nparvez@uidaho.edu, kcain@uidaho.edu
|
| 2 |
DepartmentofVeterinary Microbiology and
Pathology, Washington State University, Pullman, WA USA drcall@vetmed.wsu.edu |
| 3 |
USDA-ARSAAHRU, Auburn, AL USA benjamin.lafrentz@ars.usda.gov
|
| 4 |
Departmentof Microbiology, Molecular Biology,
and Biochemistry, University of Idaho, Moscow, ID USA tmiura@uidaho.edu
|
Bacterial coldwater
disease (CWD) caused by Flavobacterium
psychrophilum causes severe economic losses in both private (food fish) and
public aquaculture facilities. A
live attenuated F. psychrophilum strain has recently developed and shows promise as a CWD vaccine. This strain, 259-93B.17 (B.17) was
further evaluated for safety and efficacy. The B.17 strain was passed 5X in rainbow trout (Oncorhynchus mykiss) via immersion and
injection at approximately 10X the reported commercialization dose. No mortality was observed during the
trials and fish did not develop clinical signs of disease, indicating that
there is no risk of reversion to virulence. The B.17 strain was re-isolated in the immersion group at
the 4th and 5th passage and in the injection group at the
3rd, 4th, and 5th passage showing that it was
able to survive in fish for a period of time. Evaluation of the efficacy of the B.17 strain was expanded
from earlier studies to include feed delivery as an additional mass vaccination
strategy. Immunization groups
included injection, immersion (with and without adipose fin clip), and oral
delivery of B.17 on feed in fish oil. Rainbow trout were immunized at 0.7 g and boosted at 4 and 6 weeks post
initial vaccination before challenging fish at 8 weeks. Serum samples were collected before the
first booster vaccination and at the time of challenge to determine the
antibody (Ab) response. Results
showed the highest protection and Ab titers (1707) in fish injection immunized
with a relative percent survival (RPS) of 91%. Fish immunized by immersion or
feed delivery had RPS values between 20% and 23%.
S2-5
Development,
Characterization and Early Evaluation of New Modified Live Vaccines Against
Columnaris Disease
CR
Arias* and O Olivares-Fuster
| Departmentof Fisheries and Allied Aquacultures, Auburn University, AL USA
ariascr@auburn.edu |
Columnaris disease, caused by Flavobacterium columnare, has been described as a
globally-distributed, acute to chronic, bacterial infection of freshwater and
brackish water fish. Many commercially important species are affected by
columnaris disease, including channel catfish (Ictalurus punctatus Rafinesque), common carp (Cyprinus carpio Linnaeus), rainbow trout (Oncorhynchus mykiss Walbaum) and other salmonids, Japanese eel (Anguilla japonica Temminck &
Schlegel), tilapia (Oreochromis spp.)
and a variety of ornamental species such as goldfish (Carassius auratus Linnaeus). In aquaculture settings, columnaris
outbreaks are often related to environmental stress associated with high
stocking densities, increased feeding rates, elevated organic loads and high
temperatures. Given the ubiquity of F.
columnare in aquatic environments, it is unrealistic to consider
eradication as an option, but prevention through vaccination has the potential
to lower the impact of columnaris disease. This study aimed at developing new
avirulent mutants from highly virulent strains of F. columnare, which could be used as modified live vaccines against
columnaris disease. Thirteen rifampin-resistant mutants from F. columnare genomovar II were obtained
and characterized. Mutants were confirmed to be stable, and avirulent for
channel catfish. Genetic and phenotypic differences between parent and mutant
strains were identified and found to be mutant-specific. Vaccination
experiments of channel catfish showed that all tested mutants offered
significant protection against columnaris disease with relative percent
survivals between 26% to 50%. Two of the new mutants showed greater protection
than the commercial vaccine, AQUAVAC-COLÓ.
S2-6
Initial Evaluation of
an Edwardsiella ictaluri Type III
Secretion System Mutant as a Vaccine Candidate Against Enteric Septicemia of
Catfish
ML
Rogge*1 and RL Thune1,2
| 1 |
Departmentof Pathobiological
Sciences, LSU School of Veterinary Medicine, Skip Bertman Drive, Baton Rouge, LA 70803 USA mrogge1@lsu.edu
|
| 2 |
DepartmentofVeterinary
Science, Louisiana State University, 111 Dalrymple Building, Baton Rouge, LA
70803 USA thune@vetmed.lsu.ed |
Although Edwardsiella
ictaluri encodes a type III secretion system (T3SS) that is required for
virulence in channel catfish (Ictalurus
punctatus) and for intracellular replication within channel catfish cells,
the mechanisms for these capabilities are not yet known. In this study, a
putative T3SS regulatory gene, esrC,
was mutated and analyzed for its ability to replicate in channel catfish head
kidney-derived macrophages (HKDM), persist in channel catfish tissues, and
provide protection against wild-type E.
ictaluri challenge. The esrC mutant strain replicated as well as the wild type strain in HKDM, and while
colonization of the channel catfish head kidney was similar to that of the
wild-type, there were significantly fewer mutant bacteria present in the head
kidney three days post-infection than for the wild-type, with mutant
persistence extending to six days post-infection. In spite of intra-macrophage
replication and in vivo persistence
for six days, the esrC mutant caused
no mortality in channel catfish compared to 64% mortality by the wild-type.
Subsequent challenge of the esrC mutant
survivors with wild-type E. ictaluri 28 days after the initial challenge resulted in a relative percent survival of
81%. The results of these data demonstrate the potential of an esrC mutant as a vaccine candidate for
enteric septicemia of catfish.
| SESSION
3. PARASITOLOGY I: Myxozoan Workshop 1 |
[TOP] |
S3-1
An Overview of
Myxozoan Parasites Found in Smallmouth Bass (Micropterus dolomieu) from Three Tributary Rivers of the Chesapeake
Bay, USA
HL
Ellery*, VS Blazer, LR Iwanowicz and DD Iwanowicz
| United States Geological Survey, National Fish Health Research
Laboratory, Kearneysville, WV, USA hellery@usgs.gov, vblazer@usgs.gov,
liwanowicz@usgs.gov, diwanowicz@usgs.gov |
The health of
aquatic organisms is commonly used as a biological indicator of environmental pollution. Over the past eight years, the
Shenandoah and Potomac Rivers have experienced serious kills/skin lesions of
adult fishes, indicating that the health of these river systems is in some way
being compromised. The
Susquehanna River in Pennsylvania has also dealt with the mortality of young of
the year bass. Many theories on
the cause of these fish kills have included low dissolved oxygen, fluctuations
in pH, contaminants, agricultural runoff, bacteria and parasites. The occurrence of some helminth
parasites in wild fishes is normal, but a high intensity in tissues may cause
debilitation and contribute to mortality. Smallmouth bass (Micropterus dolomieu) were
collected from selected sites and a full health examination was conducted using
chemical, bacterial, histopathological and parasite analyses. The existence and abundance of parasite
infestation was reviewed with histological techniques, morphological
identification, and DNA sequencing. One type of parasite that was often identified in the smallmouth bass
was myxozoans parasites. Myxozoan
parasites were observed in macrophage aggregates, kidney tubules, gills, skin,
and to a lesser extent in the bile duct of the liver. As with many types of parasites, low intensity infections of
myxozoans may have minimal effect on the health of a fish, but when infections
become extreme, mortality can occur. Histological results indicate heavy infections of the myxozoan in the
gills and bile ducts may have had an impact on the health of the adult bass,
while a third species may contribute to lesions and mortality of the
young. The use of DNA sequencing
has revealed that at least one of the myxozoans is possibly a new species. Seasonal and site comparisons have been
made in an effort to better understand the dynamic between the fish kills and
myxozoan parasites.
S3-2
Sphaeromyxa sp. (Myxosporea: Sphaeromyxidae), a Parasite of Lined Seahorses, Hippocampus erectus, from the Gulf of Mexico
B F
Sears*1, P Anderson2 and E C Greiner3
| 1 |
Department of
Integrative Biology, University of South Florida, Tampa, USA bsears@mail.usf.edu |
| 2 |
The Florida Aquarium, Inc., Tampa, Florida, USA panderson@flaquarium.org |
| 3 |
College of
Veterinary Medicine, University of Florida, Gainesville, FL USA greinere@vetmed.ufl.edu
|
A Sphaeromyxa species is
described from the bile ducts of aquaria-maintained lined seahorses (Hippocampus erectus) from the Gulf of
Mexico. Spores are linear, 17-18
μm long and 5-6 μm wide, with flattened tips; polar capsules measure
4 x 3 μm. The morphmetrics appear distinct from previously described
species of Sphaeromyxa. Necropsies of
healthy H. erectus following
euthanasia revealed liver pathology and myxozoan parasites in the bile ducts of
11 of the 40 animals sampled (27.5%). The presence of Sphaeromyxa sp. in an aquaculture setting should prompt keepers to
carefully quarantine new animals and exclude annelid fauna, a potential
intermediate host of myxozoans, from aquaria. A manuscript describing this
parasite, and proposing to erect a new species for it, has been submitted for
publication.
S3-3
Henneguya
zschokkei s.l. (Myxozoa) in
Whitefish (Coregonus lavaretus) in SW
Finland, and Possible Interaction between Wild and Farmed Fish
H-P
Fagerholm*1, B Szostakowska2 and T Wiklund3
| 1 |
Laboratory of Aquatic Pathobiology,
Department of biosciences, Åbo Akademi University, Åbo/Turku, Finland hafagerh@abo.fi, twiklund@abo.fi |
| 2 |
Medical University of Gdańsk, Department of Tropical
Parasitology, Interfaculty Institute of Maritime and Tropical Medicine, Gdynia,
Poland bszost@gumed.edu |
The myxozoans seriously threatening whitefish farming in Finland has
been studied. Our preliminary results suggests the presence of two genetically
isolated parasite types, one from brackish water, and one from freshwater
sites. The parasites form cysts in the muscles the fish host. Both appear
different from forms previously described, and are noted here as Henneguya zschockei sensu lato. Based on
knowledge of the life-history of the parasite (where the invertebrate annelid
recently has been isolated by Saarikoski et al., 2010) it is of considerable
importance to find control strategies in specific cases. In a project aimed at
establishing the identity, extent of infection, and strategies for preventing
the infection of Henneguya zschokkei sensu
lato in farmed whitefish Coregonus
lavaretus, both fresh and brackish water fish farms in Finland were studied
from 2008 - 2010. The prevalence of the infection ranged from 0% to 55%. In one
case, the origin of the infection most probably stemmed from a specific fresh
water area from which the fish had been brought for further growth in a
brackish water site. By defining the likely origin of the infection, control of
the infection can be considered. Development of control methods is important because of the recent
increase in farming of whitefish. Infected fish cannot be used for human
consumption because of hygienic and aesthetic reasons. The infection results in
heavy economic losses for the farmers and the fish industry.
S3-4
Application of a
Real-time PCR Assay for the Detection of Henneguya
ictaluri in Channel Catfish Ponds
MJ
Griffin*1, LM Pote2, AC Camus3, MJ Mauel1,
TE Greenway1 and DJ Wise1
| 1 |
Thad Cochran National
Warmwater Aquaculture Center, Mississippi State University, Stoneville, MS USA |
| 2 |
Department of Basic
Sciences, College of Veterinary Medicine, Mississippi State University,
Mississippi State, MS USA |
| 3 |
Department of Pathology,
College of Veterinary Medicine, University of Georgia, Athens, Georgia USA |
Proliferative gill
disease (PGD) in channel catfish (Ictalurus
punctatus) is caused by the myxozoan
parasite Henneguya ictaluri. Prolonged exposure of channel catfish
to the actinospore stage of the parasite results in extensive gill damage,
leading to reduced production and significant mortality in commercial
operations. A H. ictaluri-specific real-time PCR (QPCR) assay was developed to
determine parasite levels in commercial channel catfish ponds and evaluate the
risk of losing fish when ponds are initially stocked or when ponds are
restocked following PGD epizootics. Water samples collected on 2 separate days
with a minimum of 1 week between sampling, not only determine
the approximate concentrations of actinospores in the pond but also lends
insight into whether these parasite levels are increasing, decreasing or
staying the same. Increases in mean
actinospore concentrations correlated with increased mortality in sentinel fish
(r = 0.63; p = 0.003). Direct
detection and quantification of the organism in pond water is more rapid than
current protocols for evaluating the PGD status of a catfish pond, which rely
on the use of sentinel fish. This
work identified actinospore levels that correlate to both high and low risk of
losing fish to PGD. This assay
also provides a method of determining the overall prevalence of H. ictaluri throughout the industry,
significantly reducing the labor associated with sentinel fish trials. Current research regarding the
year-round prevalence of Henneguya
ictaluri in catfish ponds will also be discussed.
S3-5
Predicting Salmon
Mortality from Ceratomyxa shasta by
Measuring Parasite Densities in Water Samples
SL Hallett*, RA Ray, RA Holt, SD Atkinson and
JL Bartholomew
| Department of
Microbiology, Nash Hall 220, Oregon State University, Corvallis, OR USA
halletts@science.oregonstate.edu
|
The
myxozoan parasite, Ceratomyxa shasta, is responsible for losses among both wild and
hatchery-reared juvenile salmonids. It is a significant pathogen in the Klamath River, where it has been
identified as a key factor limiting salmon recovery. Monitoring studies involving sentinel fish exposures,
polychaete sampling, and water analysis have increased our understanding of the
temporal and spatial distribution of the parasite and have moved us closer to
reaching management goals of reducing mortality to below 40% in native stocks. Water sampling in particular has proven
to be a simple, high-resolution tool to determine and map total parasite
density (number of spores per liter) in river water. We have now used combined water sampling and fish exposures
to address the following question: how does parasite density relate to
population effects in each salmon species? In other words, can we identify threshold dose levels? We exposed different salmon species in
cages at five lower Klamath River index sites in the spring, summer, and fall
from 2005 through 2009. In
parallel, we collected triplicate 1-L water samples on the first and final days
of the three-day exposures. Fish
were then held at the Salmon Disease Laboratory (OSU) for up to 90 days, and
mortality due to C. shasta and mean
day to death were recorded. Water samples were filtered; the captured DNA,
extracted; and C. shasta, quantified
by TaqMan qPCR. We are now
correlating the two data sets to discover patterns of fish mortality, parasite
density, parasite genotype, water temperature, and flow.
S3-6
Evaluation of the
Risk of Myxobolus cerebralis Introduction as a Result of
Fish Passage
CM
Zielinski, SL Hallett and JL Bartholomew*
| Department
of Microbiology, Oregon State University, Corvallis, OR Halletts@science.oregonstate.edu, bartholj@science.oregonstate.edu
|
Dams along the Deschutes
River (DR) in central Oregon, USA, have blocked fish migration for over 40
years. Re-establishment of anadromous fish runs
above the dams, as part of a fish passage plan, may introduce fish
pathogens such as Myxobolus cerebralis. This myxozoan parasite, which causes
salmonid whirling disease, is carried into the DR basin by adult salmon that
stray during their return to enzootic areas of the upper Columbia River basin,
and we have confirmed that it has become established in at least one lower DR
tributary. To determine the likelihood of parasite
establishment above the dams, we conducted benthic sediment surveys between 1998
and 2007. We found that T.
tubifex, an oligochaete worm that is one of two obligate hosts for M. cerebralis, had a patchy distribution
and low relative abundance. Mitochondrial 16S rDNA gene analysis indicated that
two lineages of T. tubifex, III and VI, were present both above and
below the dams. Laboratory
susceptibility studies, designed to characterize differences in infection
prevalence and parasite production between nine T. tubifex populations,
revealed that production varied considerably among exposed groups and was
proportional to the number of lineage III worms present. Our results suggest that M. cerebralis could become established
above the dams if infected fish are allowed passage. Not all areas of the Deschutes River basin have the same likelihood
for parasite establishment, however; thus the potential impact will be
location-dependent.
S3-7
Myxobolus
cerebralis in Fishes in Reservoirs of Kamchatka
TV Gavruseva*
| Fish and Shellfish Disease Laboratory, Kamchatka
Research Institute of Fishery and Oceanography (KamchatNIRO), 18 Naberezhnaya
Street, Petropavlovsk-Kamchatski, 683000 Russia gavrt2004@mail.ru
|
The myxosporidian Myxobolus (syn. Myxosoma) cerebralis, the agent of whirling
disease, developing within the cartilage of farmed salmonids including trout,
can cause skeletal deformities, whirling behaviour and mortality. There is
little known about the occurrence of this parasite in wild fish in the
Kamchatka region of Russia. Previously M.
cerebralis was found in reared fingerlings of Oncorhynchus keta and Oncorhynchus
gorbuscha (Ushkovskii hatchery, Kamchatka) (Bogdanova, 1970). Then for many
years (1989 to 2006) this pathogenic agent was not recorded in the reservoirs
of Kamchatka, nor in salmonids in the Far East (Boutorina, 2008). However, in
2000 to 2010, histological and histochemical examinations of 2,750 wild and
cultured young salmonids (O. keta, Oncorhynchus kisutch, Oncorhyncus nerka, Oncorhynchus tschawytscha)
and trout (Salvelinus malma) from
Kamchatka during revealed M. cerebralis in wild fingerlings of sockeye salmon, with pathological signs only from Lake
Nachikinskoe (western sea coast of Kamchatka) in August 2006. In August 2009,
spores of M. cerebralis were determined in the dead nine-spined stickleback Pungitius pungitius from Lake
Nalychevskoe (eastern sea coast of Kamchatka). Pungitius pungitius is a typical hydrobiont of Kamchatka rivers and
lakes. Histological examination of sockeye salmon showed that spores and
developing stages of the parasite caused local necrosis and lysis of the
chondrocytes. There was marked formation of epithelial granuloma around the
affected area of a tissue. The cartilaginous tissue of a spinal column of
nine-spined stickleback has been completely replaced with mature spores of the
parasite without granuloma formation. We suggest that P. pungitius is natural
carrier and source of M. cerebralis for salmonid fish.
| SESSION
4. FISH HEALTH SURVEYS |
[TOP] |
S4-1
The National Wild
Fish Health Survey: Selected Findings and Limitations
SL
Mumford*
Olympia Fish Health Center,
US Fish and Wildlife Service, Olympia, WA 98506 USA Sonia_mumford@fws
Since 1996, the USFWS with the help of partnering
organizations has been collecting wild fish and testing for specific fish
pathogens. The pathogens selected
have been important in cultured fishes, and additional pathogens are added as
needed. The nine USFWS fish health
centers use standardized methods for assays, which makes comparisons between
species, areas, and watersheds possible. The major goal of the National Wild Fish Health Survey (NWFHS) is to
determine the geographical distribution of certain pathogens in wild fish. With a better understanding of pathogen
distribution, managers can better assess the risks and benefits of stocking and
fish transport activities. Data
collected from the National Wild Fish Health Survey has been or is currently
being used for: management decisions such as broodstock selection and egg
distribution for restoration programs, assessing interactions between wild and
hatchery fishes, monitoring dam removal projects (ie Elwha, and White Salmon
River), and surveillance for exotic pathogens (ie Spring Viremia or Carp Virus
(SVCV)) along with other potentially devastating viruses (ie Viral Hemorrhagic
Septicemia Virus). Other useful
information from the NWFHS includes unexpected findings such detection of
pathogens in species not previously thought to be susceptible (ie SVCV in
Bluegill and Largemouth bass). While much information can be gained from the NWFHS,
there are also limitations. The
data provides a snapshot in time at a specific location in the species of fish
that are collected. Since only
limited funds are available for collection of fish, selection of sampling sites
have largely been opportunistic, and fish numbers are not always ideal. Through the support of our partners,
there are some sites that provide statistically significant data, while other
sites can only supply supportive data. Due to sampling biases (difficulty in collecting clinically ill wild
fish), differences in number of fish collected, and numbers of sampling
locations over time, one should not assume the data shown reflects prevalence
data or an emergence of a given pathogen.
S4-2
Demonstration of New
Web Interface for the National Wild Fish Health Survey
KK
Peters*1 and JL Bradley2
| 1 |
US Fish
and Wildlife Service, Bozeman Fish Health Center, Bozeman,
MT, USA ken_peters@fws.gov |
| 2 |
US Fish
and Wildlife Service, Division of Fisheries and Aquatic Resource Conservation,
Idaho Fish Health Center, Orofino, ID USA
|
Beginning in 1996, the U. S. Fish and Wildlife
Service (Service) has been working with aquatic resource partners, under
auspices of the National Wild Fish Health Survey (Survey), to better understand
the occurrence of important fish pathogens in free-ranging fish. During this time, nearly 220,000 fish
have been sampled from some 4,600 distinct sample sites, representing 2,560
bodies of water. Samples are
tested using standardized protocols and procedures for the presence or absence
of fish pathogens. Data generated
by these tests are managed in regional databases by the Service’s network of
nine fish health laboratories. Additionally, spatial information including state, county, hydrologic
unit code, latitude and longitude, and name of water body are recorded for each
sample site. Records from regional
laboratories are periodically uploaded to a centralized national database
residing on a dedicated server in Denver, Colorado. In June 2010, the Service released a new, public, web-based
interface for searching and displaying information from the national
database. The interface is
designed to display search options, tabular search results, and an interactive
map simultaneously in a web-browser. Users can download case reports and create custom maps of search
results. Search results may also
be downloaded for use in spreadsheet applications (CSV) or “earth-browsers”
such as Google Earth® (KML). The web interface is accessed by navigating to the Survey homepage at
[http://www.fws.gov/wildfishsurvey] and then clicking on the “National Wild
Fish Health Survey Database” button, followed by “Start the Database” button.
S4-3
Results of Fish
Health Assessments of Glass Eels, Anguilla
rostrata, from Canadian Maritime Rivers from 2006-2010
D Groman*1, R Threader2, D Wadowska3,
T Maynard2 and L Blimke4
| 1 |
Aquatic Diagnostic Services, Atlantic Veterinary College,
University of Prince Edward Island,
Charlottetown, Canada. groman@upei.ca
|
| 2 |
Ontario Power Generation, Renfrew, ON Canada. liisa.blimke@opg.com ron.threader@opg.com
|
| 3 |
Electron
Microscopy Laboratory, Atlantic Veterinary College, University of Prince Edward
Island Charlottetown, Canada wadowska@groupwise.upei.ca
|
| 4 |
Kleinschimidt
Associates, Essex, CT 06426 USA. Tracy.Maynard@kleinschmidtusa.com
|
This paper reports on the results of fish health
assessments for newly captured glass eels (Anguilla
rostrata) from Nova Scotia and New Brunswick in Atlantic Canada, which were
part of a regional re-stocking program for Lake Ontario from 2006-2010. Glass eel populations were segregated
by watershed or river of capture, held in quarantine and subsequently assessed
for selected virology, bacteriology and parasitology. Results of virus isolation and molecular assays of pooled
samples from all years were negative for all viruses of interest. Bacteriological culture of the 2009-10
pooled samples revealed one lot, held on surface water prior to sampling, was
harboring Yersinia ruckeri. Gross parasitological examination
revealed sporadic regions of integumental hyperplasia, that were histologically
determined to be trophozoite stages of the ciliate Ichthyophthirius multifiliis. Histopathological examination of individuals from all lots revealed
necrotizing hepatitis and associated intranuclear microsporidian,
morphologically consistent with a Nucleospora
sp. One lot of older elvers
from the estuary of the St. Mary’s River in Guysbourgh County, Nova Scotia, was
found to be harboring larval and pre-adult nematodes in association with the
wall and lumen of the gas bladder. These nematodes were morphologically consistent with Anquillicoloides crassus. In addition, this lot was infected with an
as yet identified Myxosporidian infection of the urethra and urinary
bladder. The significance of these
results will be discussed.
S4-4
Cutthroat Trout Virus Surveys in New Mexico and Arizona
TD Lewis*,
EM Rodarte, and DD Hampton
| Dexter
Fish Health Center, USFWS Region 2, P. O. Box 219, Dexter NM 88230 USA
teresa_lewis@fws.gov,
marlene_rodarte@fws.gov, dave_hampton@fws.gov
|
Although cutthroat trout virus (CTV) has been
characterized as a benign virus in common trout species, there is a lack of
knowledge relating to the susceptibility and transmission potential of this
virus in listed trout species, specifically Apache, Gila, and Rio Grande
cutthroat trout in the southwest United States. Enhanced CTV surveillance has been ongoing in federal
hatcheries rearing trout in New Mexico and Arizona since 2008. Tissue culture diagnostics conducted
throughout 2009 did not identify any viral cytopathic effect (CPE). In 2010, ovarian fluid from trout at
federal and state hatcheries in Arizona and New Mexico developed CPE
characteristic of CTV and was confirmed positive for the virus using
RT-PCR. An experiment using
archived Apache trout ovarian fluid that was negative by cell culture tested
CTV positive using RT-PCR. Since
that time, the USFWS and partners have been conducting wild fish health surveys
of native trout and surrogates (brown trout, hybrid cutthroat trout, others) to
establish a baseline for CTV in the southwest. Cell culture and RT-PCR methods have been used in tandem for
this baseline. While
diagnosis of CTV continues to rely upon isolation in cell culture there is
mounting evidence that viral cDNA may be amplified in samples that do not evidence
CPE in culture. Despite the
apparent avirulent nature of CTV, concerns linger over its potential impacts in
ways that do not clearly result in morbidity or mortality (i.e., effects on
reproduction, stress responses, immunocompetence), especially in threatened or
endangered species.
Epidemics and Control
Strategies for Diseases of Farmed Salmonids: A Parameter Study
ART Jonkers1,2,
KJ Sharkey3, M Thrush4, JF Turnbull5 and K
Morgan*6
| 1 |
Institute for
Geophysics, University of Münster, Corrensstrasse 24, Münster 48149, Germany |
| 2 |
Department of Earth
& Ocean Sciences, University of Liverpool, 4 Brownlow Street, Liverpool,
L69 3GP, UK
|
| 3 |
Department of
Mathematical Sciences, University of Liverpool, Liverpool, L69 7ZL, UK
|
| 4 |
Centre for
Environment Fisheries and Aquaculture Science (Cefas), Barrack Road, Weymouth
DT4 8UB, UK
|
| 5 |
Institute of
Aquaculture, University of Stirling, Stirling, Stirlingshire, FK9 4LA, UK
|
| 6 |
Department of
Veterinary Clinical Science, University of Liverpool, Leahurst, Neston, CH64
7TE, UK
|
Simulated contact networks can help us understand properties of
epidemics including dynamics and risk, as well as their sensitivity to changes
in likelihood of transmission delays, and mitigation measures. When such
simulations closely reflect observed reality, inferences may be drawn regarding
the relative importance of specific parameters that have direct implications
for biosecurity and disease control policy decisions. Here we present a
large-scale simulation study of the English and Welsh fish farming and
fisheries industry, comprising 2,090 sites as recorded in 2004. We explore this
network’s resilience for different conditions and mitigation strategies. A survey of stream flow speeds demonstrated that transmission via
rivers takes less than two days on average. Directed river and transport
contacts in this network causes the risk of outward transmissions to be
geographically more confined than that of incoming transmissions. Balanced analysis of variance shows
that detection delay is the most important outbreak-limiting factor, followed
by laboratory capacity and culling delay. Furthermore, a reactive policy with
some additional proactive dangerous-contact tracing, performs better than
either strategy on its own. Finally, we distinguish fish farm sites from
fisheries in terms of specific biosecurity weaknesses, and derive some general,
quantified policy guidelines to mitigate future epidemics on this network.
| SESSION
5. DIAGNOSTIC TESTING |
[TOP] |
S5-1
Development of a U.S. National Fish Health Laboratory
Network
AE Goodwin*1, JB Rolland2 and KR Snekvik3
| 1 |
University of Arkansas at Pine Bluff, Aquaculture/Fisheries
Center, Pine Bluff, AR USA agoodwin@uaex.edu |
| 2 |
USDA/APHIS/VS/ASEP,
Riverdale, MD USA Jill.B.Rolland@aphis.usda.gov |
| 3 |
Washington Animal Disease Diagnostic Laboratory, College of
Veterinary Medicine, Washington State University, Pullman, WA, USA ksnek@vetmed.wsu.edu
|
An important part of the new U.S.
National Aquatic Animal Health Plan (NAAHP) is the development of a national
network of fish health laboratories that will work together to protect the health of wild
and cultured fish and shellfish, provide quality inspections in support of
interstate and international trade, and meet challenges associated with
implementation of the NAAHP. Development of the network has been a joint project undertaken by the
U.S. Animal Health Association, the American Association of Veterinary
Laboratory Diagnosticians, and the three federal co-competent authorities for
fish health: the U.S. Department of Agriculture's Animal and Plant Health
Inspection Service, the National Oceanic and Atmospheric Administration,
and the U.S. Fish and Wildlife Service. Implementation of the network is directed by a steering committee
composed of these three federal agencies, aquatic animal diagnostic
laboratories, and the aquaculture industry. The network is founded on the following principles: 1)
membership in the network may include all qualified state, federal, academic,
extension, and private laboratories; 2) laboratories will use a single
standardized protocol; 3) there will be quality assurance systems in place; 4)
there will be a reporting system in place; 5) results from the laboratories
will be recognized by the federal authorities as appropriate for export
inspections; and 6) labs will only remain eligible for participation in the
network if they meet training and proficiency requirements. The network will begin with a trial
period that will include only a protocol for the detection of VHSV IVb in cell
culture with an RT-PCR confirmatory test.
S5-2
ICHV-2 (the Other Catfish Herpesvirus): Detection by qPCR
AE Goodwin1*, E Marecaux1 and LA Hanson2
| 1 |
University of Arkansas at Pine Bluff, Aquaculture/Fisheries
Center, Pine Bluff, AR USA agoodwin@uaex.edu
|
| 2 |
College of Veterinary Medicine, Mississippi State, MS USA
hanson@cvm.msstate.edu
|
Channel catfish herpesvirus (Ictalurid herpesvirus-1 or IcHV-1) is a
common and cosmopolitan pathogen that causes sporadic mortality in
young-of-the-year channel catfish during hot summer months in the Southeast
USA. Less well know is Ictalurid herpesvirus-2 (IcHV-2), a
pathogen of cultured black bullhead (Ameiurus
melas) that produces high mortality in experimental exposures to channel
catfish (Ictalurus punctatus). During acute infections, the virus
grows readily in cell cultures but produces a cytopathic effect (CPE) similar
to that of IcHV-1 and the channel catfish reovirus. We have developed a quantitative PCR assay that can be used
to detect IcHV-2 in fish tissues and cell-culture supernatants. The assay does not amplify other fish
herpesviruses tested, or host DNA. It is quantitative over a range of eight logs, and the limit of
detection is less than 10 copies per reaction. In replicate assays carried out on different days, the
coefficient of variability was 10%. Our assay detected acute IcHV-2 infections best in the spleen and
kidney. We have used it to screen
a large collection of U.S. catfish herpesvirus isolates, and have confirmed
them all to be IcHV-1. This
assay should be useful for the diagnosis of IcHV-2 disease, the identification
of syncytial CPEs in cell cultures, and the detection of latent infections in
carrier fish.
S5-3
Use of Diagnostic
Assays to Screen Rainbow Trout (Oncorhynchus
mykiss) Broodstock for Flavobacterium
psychrophilum
A
Long*1, DR Call2 and KD Cain1
| 1 |
Department of Fish and
Wildlife Resources, University of Idaho, Moscow, ID USA
long8524@vandals.uidaho.edu; kcain@uidaho.edu |
| 2 |
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA USA
drcall@vetmed.wsu.edu
|
Flavobacterium
psychrophilum,
the causative agent of Bacterial Coldwater Disease (BCWD), affects several
economically important species of fish in the Pacific Northwest. F.
psychrophilum has been shown to survive both on the exterior of eggs and in
fluid from within the egg indicating it may be vertically transmitted. To further evaluate this, our lab has
developed an ELISA and membrane filtration fluorescent antibody test (MF-FAT)
using a monoclonal antibody specific to F.
psychrophilum. Evaluation of
these diagnostic assays as tools for managing BCWD is underway. Sixty rainbow trout broodstock were
screened for F. psychrophilum using
various diagnostic assays including ELISA and MF-FAT. Five families showing F.
psychrophilum infection levels ranging from below detection limits to 106 CFU ml-1 were selected for further experiments. Eyed eggs were shipped to UI and fry
are currently being reared on pathogen-free water and sampled for F. psychrophilum on a weekly basis. After five weeks of sampling, disinfected
egg and fry homogenates from each family have tested positive by nested PCR at
least once. To determine if
progeny from infected broodstock have a greater risk of developing BCWD, fish
will be exposed to chronic gas supersaturation, a stressor that has been linked
to disease outbreaks in hatcheries, and monitored for BCWD outbreaks. We will also test if progeny from
heavily infected broodstock are more susceptible to F. psychrophilum than progeny from uninfected broodstock when
directly challenged with the bacterium. Results will provide insight on the use of these assays to set threshold
levels for screening broodstock.
S5-4
Development of Immunochromatography Assay Kit for Ranavirus Determination
RK Young1, A Bayley2,
KD Thompson3, A Adams3, T Aoki1 and TS Jung*1
| 1 |
Aquatic
Biotechnology Center of WCU project, College of Veterinary Medicine, Gyeongsang
National University, Jinju, 660-701, Korea yl0808@lycos.co.kr; aoki@kaiyodai.ac.jp; jungts@gnu.ac.kr
|
| 2 |
Cefas, Weymouth laboratory,
Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB, UK amanda.bayley@cefas.co.uk
|
| 3 |
Institute of Aquaculture, University of
Stirling, Stirling FK9 4LA, Scotland, UK k.d.thompson@stir.ac.uk;
alexandra.adams@stir.ac.uk
|
Ranaviruses are serious pathogens of
fish, amphibians and reptiles and cause disease problems worldwide. In the
present study, we isolated ranavirus from both diseased tadpoles and frogs (Rana plancyi chosenica) and identified
the virus involved as Korean
ranavirus-1 (KRV-1). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to characterise the virus and the
results were compared to the profile obtained with frog virus-3 (FV-3). Several bands were observed between 35 to 49
kDa for isolates of both FV-3 and KRV-1 and these were identified as the major
capsid proteins (MCPs) of the virus by MALDI-TOF MASS analysis. The antigenic
proteins of KRV-1 were identified using sera from frogs infected with KRV-1.
Protein A-HRP was used to recognize the frog antibodies in the Western blot
analysis with antigen bands observed at 34 and 93 kDa. These bands were
identified as MCP and neurofilament triplet H1-like protein (NF-H1) by
MALDI-TOF/TOF. Monoclonal antibodies (MAbs) were produced against ranavirus
KRV-1 for the development of an immunochromatography
assay kit. The antigenic proteins recognised by these MAbs were also identified
by MALDI-TOF/TOF
and found to be MCP and NF-H1. The sensitivity
of the kit was similar to that of PCR, with a detection limit of 10 TCID50ml-1.
S5-5
Development of Two
Novel Quantitative Polymerase Chain Reaction (qPCR) Assays to Measure
Alphaherpesviral Load in Bottlenose Dolphins (Tursiops truncatus)
HTD
Maness*1, S Venn-Watson2, LL Archer3, R Rivera4,
ED Jensen5 and HH Nollens6
| 1 |
Aquatic Animal Health
Program and the Department of Small Animal Clinical Sciences, College of
Veterinary Medicine, University of Florida, Gainesville, FL USA
htdaniel@ufl.edu
|
| 2 |
National Marine Mammal
Foundation, San Diego, CA USA stephanie@epitracker.com
|
| 3 |
Department of Small Animal Clinical
Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL USA archerl@ufl.edu
|
| 4 |
Hubbs-SeaWorld Research
Institute, San Diego, CA USA rrivera@hswri.org
|
| 5 |
U.S. Navy Marine Mammal
Program, Space and Naval Warfare Systems Center Pacific, San Diego, CA USA
eric.d.jensen@navy.mil
|
| 6 |
SeaWorld San Diego, San
Diego, CA USA hendrik.nollens@seaworld.com
|
During viral surveillance of a collection of
bottlenose dolphins (Tursiops truncatus),
two distinct alphaherpesviruses, tentatively named Delphinid herpesvirus 2 (DeHV-2) and Delphinid herpesvirus 8 (DeHV-8), were detected using conventional
polymerase chain reaction (PCR) in the white blood cell (WBC) fraction of a
22-year-old female with a history of persistent lymphocytosis. Alphaherpesviruses
are generally considered to persist in the sensory nerve ganglion, thus, the
presence of herpesvirus in circulating blood cells was presumed to be
indicative of infection with active virus replication. Real-time quantitative
PCR (qPCR) assays targeting the DNA-dependent DNA polymerase gene were
developed to measure circulating DeHV-2 and DeHV-8 loads among healthy dolphins
and a series of samples from the case dolphin. Both qPCR assays were found to
be more sensitive (detection limit: 10 virus copies/sample), and faster, than
conventional PCR. Circulating viral load of DeHV-2 in the case dolphin (N = 16)
ranged from 0 to 1.3 virons per ng of DNA during a 30 month period. A
cross-sectional survey of the WBC fraction among clinically healthy bottlenose
dolphins (N = 55) revealed a slightly higher prevalence for DeHV-2 (N =2) than
for DeHV-8 (N = 0). We conclude that the qPCR assays are capable of detecting
DeHV-2 and DeHV-8 in bottlenose dolphin blood. Further studies are needed for a
clearer understanding of the clinical relevance of these two viruses in dolphin
populations.
| SESSION
6. GLOBAL EMERGENCY RESPONSE AND PREPAREDNESS: Emerging Aquatic Issues |
[TOP] |
S6-1
Sinking or
Swimming in the OIE Aquatic Animal Health Code
MD Remmenga* and LL Gustafson
| National Surveillance Unit,
USDA, APHIS, VS, Centers for Epidemiology & Animal Health, Fort Collins CO,
USA marta.d.remmenga@aphis.usda.gov, marta.d.remmenga@aphis.usda.gov |
The OIE Aquatic Animal Health Code provides flexible guidelines
for designing surveillance systems to demonstrate disease freedom. This flexibility does introduce a level
of complexity that can lead to confusion when developing a new system. Because there are multiple paths to
achieving disease freedom under the OIE guidelines, it may not be immediately
evident whether a facility or State surveillance program that claims to follow
the guidelines is truly adequate. Unfortunately, prescribed systems (e.g., AFS Blue Book) may not be
sufficient in all situations, and can lead to under- or over-sampling depending
on the risk of disease in the sampled population. Understanding the role of three different types of evidence
for demonstrating disease freedom can remove some confusion and frustration
with the OIE guidelines. First,
sampling and testing can be used to establish the current disease status of the
population. Second, appropriately documented historical evidence can reduce the
sampling required. Finally,
knowledge about the risk of disease introduction and about how the bio-security
measures reduce risk informs how frequently retesting is required. The flexibility
in the OIE guidelines provides an opportunity to create sustainable
surveillance systems that are also cost effective. Example scenarios will be
provided.
S6-2
Perspectives on Global Preparedness for Aquatic Animal
Diseases
KH
Hartman*
| USDA,
Animal and Plant Health Inspection Service, Veterinary Services, Ruskin, FL USA kathleen.h.hartman@aphis.usda.gov
|
Aquatic
animal health authorities, state and federal, as well as aquaculture facilities
must be prepared to efficiently and effectively respond to outbreaks of aquatic
animal diseases. Being prepared encompasses specific threat
identification, response plans, identification of resources, training resources
for responders, and conducting drills and exercises to test the procedures and
the plan. Implementing the
incident command system (ICS) is an important way to functionally organize the
response to a disaster. ICS
implementation in association with specific disaster types and required operations
remains a continuing challenge, and will be discussed.
S6-3
Developing,
Implementing, Auditing and Certifying Veterinary Biosecurity Programs
AD
Scarfe*1, D Palić2 and CI Walster3
| 1 |
American Veterinary Medical Association, Schaumburg,
IL USA. dscarfe@avma.org
|
| 2 |
College of Veterinary Medicine, Iowa State
University, Ames, IA USA. dulep@iastate.edu
|
| 3 |
The Island Veterinary Associates, Staffordshire,
UK. chris.walster@onlinevets.co.uk
|
One outcome of a 2009 International Aquaculture Biosecurity
Conference in Norway was a standardized approach developed by international
experts, for applying core elements necessary for practical and effective
biosecurity programs. When
correctly applied to epidemiological units (individual tanks or ponds, whole
farms, compartments, zones or watersheds, or geopolitical regions) they are
viable for any type of aquaculture operation and for any infectious and
contagious disease of concern, and may meet legislative or regulatory
requirements, including OIE standards. In order for biosecurity programs to be functional, effective and
justifiable they need to involve and integrate several formal processes
including: hazard and risk analysis (hazard identification and prioritization,
risk assessment/evaluation, risk management/mitigation and risk communication);
analysis and remediation of critical control points (including evaluation and
mitigation plans for correcting practices where disease could enter or leave
the epidemiological unit); applied epidemiological practices (including disease
diagnostics, surveillance, monitoring and determination of disease status or
freedom); emergency preparedness (contingency protocols for disease control and
eradication); auditing of procedures and records; and certification (providing
assurance of disease freedom). Optimally, for biosecurity programs to be effective, useful and
justifiable to all stakeholders (i.e, producers, industries, service providers
and government agencies) programs need to include: (1) documented disease
prevention, control and eradication practices; (2) a sound science basis; (3)
easily understood practical approaches; (4) economical viability; and, (5)
strong incentives to encourage compliance – whether voluntarily initiated
at farm or industry level, or mandated through government legislation and
regulations.
S6-4
Emerging Diseases of Important Mollusks
R Smolowitz*
|
Aquatic Diagnostic Laboratory, Roger Williams
University, Bristol, RI USA, rsmolowitz@rwu.edu
|
The types of mollusks
cultured for food worldwide include various types of bivalves (oysters, clams,
mussels, etc.) as well as gastropods (abalone). The effects of disease on the aquaculture of this important
food source can be devastating and not only destroy the availability of this
food animal, but also cause significant local and region economic impacts. Many emerging diseases in molluscan
animals are a result of transference of the disease agent to another
susceptible host or transferring the infected host to a different environment
resulting in significant disease. Perkinus marinus has caused
significant mortality of both cultured and wild eastern oysters (Crassostrea virginica) along the entire
eastern and gulf coastlines of the U.S. since the 1940’s. Genetic evidence suggests that disease
occurred when the Pacific oysters containing the agent (but no disease) were
transferred to the east coast of the U.S. Perkinus
olseni, a close relative of P.
marinus, is the cause of significant disease in several types of molluscan
animals in the Asia-Pacific area. It is listed by O.I.E. as a disease of concern and trade of infected
animals between countries is not recommended. Recently, examination of a group of Tridacna sp., giant clams, which are imported routinely to the U.S.
as part of the aquarium trade, showed infection and resulting disease caused by P. olseni. Other examples of emerging diseases include Herpes virus
infections of Pacific oysters in Europe and ganglioneuritis of abalone in
Australia.
S6-5
Pet Industry Efforts
to Control Chytridiomycosis in Amphibians
N
Saint-Erne*
| PetSmart,
Inc., Phoenix, Arizona USA nsainterne@ssg.petsmart.com
|
Chytridiomycosis (Batrachochytrium dendrobatidis) is a fungal infection of amphibians
that has been spreading across the globe and is linked to the decimation of
wild frog populations. The fungus
was first identified in southern Africa as a stable endemic infection years
before any positive specimen was found outside Africa. The global transportation of frogs and
other amphibians for scientific research and the pet trade brings with it the
risk of spreading this fungal infection. African dwarf frogs (Hymenochirus
curtipes) are popular aquarium pets, but they can be carriers of this
devastating fungal infection. These frogs sold in pet stores can be treated for
the fungus with a variety of topical antifungal medications (Betadine,
Benzalkonium chloride), as well as through heating the water to a temperature
that kills the fungus but is safe for the frogs. Preventive treatments using one of these
methods will reduce the risk of spreading this serious fungal infection, and
reduce the losses associated with the fungus in aquatic frogs kept as pets. The lesions associated with chytridiomycosis include hyperplasia
of the epidermis and hyperkeratosis of the stratum corneum. Typical clinical
signs in frogs infected with chytridiomycosis include lethargy, inappetence,
skin discoloration, presence of excessive sloughed skin, and sitting
unprotected during the day with their hind legs held loosely to the body. Frogs
become moribund in the terminal stages with loss of their righting reflex, and
death usually occurs a few days after the onset of clinical signs. Cause of
death is associated with electrolyte loss through the damaged epidermis.
S6-6
Aquarium and
Aquaculture Facility Emergency Preparedness and Recovery
BR
Whitaker*
| National
Aquarium - Baltimore, Baltimore, Maryland USA bwhitaker@aqua.org
|
Disasters in aquarium and aquaculture facilities can
be avoided or minimized by careful planning and risk management. A disaster can
be defined as any tragic event with great loss stemming from events such as
earthquakes, floods, catastrophic accidents, fires, or explosions
[http://en.wikipedia.org/wiki/Disaster]. Emergency situations are likely to
occur within the lifespan of a zoo, aquarium, aquaculture facility, research
laboratory or other aquatic animal facility. Direct damage to the facility or
disruption of supporting services can lead to a loss of animal life and
production. It can also cause injury or even death to staff attempting to
control or mitigate the impacts of the emergency. Preparing for such an event,
referred to as “emergency preparedness”, will minimize the risk of loss or
injury to both animals and humans. A solid plan is easier to prepare in
advance, provides staff with knowledge and confidence to manage a situation
when it occurs, and will result in avoiding or minimizing the disaster and
facilitating recovery. Following every event is the opportunity to improve your
emergency preparedness plan. Once life returns to normal, take the time to
review the event, the actions taken during the event, and the final outcome of
the emergency. This is your opportunity to learn and make improvements to your
plan that will change the outcome of future events.
| SESSION
7. PARASITOLOGY II: Myxozoan Workshop 2 |
[TOP] |
S7-1
Spatial, Temporal and Host Factors Structure the Ceratomyxa shasta (Myxozoa) Population in the Klamath River Basin
SD
Atkinson*1,2 and JL Bartholomew2
| 1 |
School of Chemistry and Molecular Biosciences, The
University of Queensland, Australia stephen@2am.com.au
|
| 2 |
Department of Microbiology, Oregon State University,
Corvallis, Oregon, USA
|
The myxozoan parasite Ceratomyxa shasta is a virulent pathogen of salmonid fish in the
Klamath River, Oregon/California, USA. We previously defined four principal
genotypes of the parasite (O, I, II, III) based on a trinucleotide repeat (ATC)0-3 in Internal Transcribed Spacer region 1 sequences. Genotypes occur in sympatry
and show marked host preference: I in Chinook salmon (Oncorhynchus tschawytscha) and II in non-native rainbow trout (O. mykiss). In the present study, we
sequenced the parasite from river water samples collected in May, June and
September at three localities below, above and between the Klamath’s five dams.
We also sampled adult and juvenile coho salmon (O. kisutch), steelhead trout (O.
mykiss, anadromous form) and native redband rainbow trout (O. mykiss, freshwater form) and
additional Chinook salmon and non-native rainbow trout. We found that the C. shasta population was highly
structured spatially, temporally and with respect to fish host species.
Genotype O was present in water throughout the basin but detected almost
exclusively in steelhead and native rainbow trout. Genotype I was in water only
below the dams and detected only in Chinook salmon. Genotype II was detected in
coho salmon below the dams, and in non-native rainbow trout exposed both above
and below the dams. The same genotypes were detected in adult and juvenile fish
of the same species. These findings have major implications for the design of
effective surveillance and control programs for this economically and
ecologically important fish parasite.
S7-2
Host-specific
Virulence and Transmission of Ceratomyxa
shasta Genotypes I and II
CN
Hurst* and JL Bartholomew
| Department
of Microbiology, Oregon State University, Corvallis, OR USA, hurstch@onid.orst.edu,
bartholj@science.oregonstate.edu
|
The myxozoan Ceratomyxa
shasta is endemic to the Klamath River (KR) basin in the Pacific Northwest
region of the United States. The parasite is dependent upon both a polychaete (Manayunkia speciosa) and a salmonid to
complete its life cycle and is
established throughout the main-stem KR. Chinook salmon (Oncorhynchus tshawytscha) were extirpated from upper KR basin with
the construction of Copco dam in 1917, and the severe effects of the parasite
on Chinook in the lower KR raises questions about the outcome of reintroducing
these fish into the upper basin. Recent studies demonstrate that C. shasta is comprised of at least four
genotypes with different salmonid host specificities. To support these
findings, we seeded populations of
polychaetes with parasite genotypes I and II and exposed three species of
salmonids present in the KR basin; Chinook and coho (Oncorhynchus kisutch) salmon and rainbow trout (Oncorhynchus mykiss). Genetic analyses
of laboratory exposed fish support the hypothesis of host-specific virulence.
We found that genotype I caused mortality in Chinook, while genotype II was
responsible for rainbow trout mortality. To determine if host-specific
transmission occurs, Chinook and rainbow trout were exposed to genotypes I and
II in the field. We analyzed blood samples taken within the first two weeks
post exposure and results demonstrate that genotypes I and II are transmitted
to both fish species, but the genotype associated with mortality is dominant.
S7-3
The Dynamics of Mixed
Myxozoan Infections and Potential Mechanisms of Within-Host Interactions
AS Holzer*1,2, R Wootten3 and C
Sommerville3
| 1 |
Institute
for Specific Prophylaxis and Tropical Medicine, Medical University Vienna,
Kinderspitalgasse 15, 1090 Vienna, Austria
|
| 2 |
Cavanilles Institute of Biodiversity and Evolutionary Biology, University of
Valencia, P.O. Box 22085, 46071 Valencia, Spain astrid.holzer@uv.es
|
| 3 |
Institute
of Aquaculture, University of Stirling, Stirling FK9 4LA, UK cs3@stir.ac.uk
rw3@stir.ac.uk
|
Different
myxozoan parasite species frequently co-occur in the same fish host, however,
nothing is known of the infection dynamics of mixed infections in relation to
the age and life stage of fish. The current study investigated the spatial and
temporal patterns of invasion, migration and expansion of mixed infections of
myxozoans in Atlantic cod, Gadus morhua, and
in the brown trout, Salmo trutta,
using PCR assays and single- or double-label in situ hybridisation protocols, which were developed specifically
for each myxozoan. These techniques allowed for the detection, differentiation
and localisation of different myxozoan developmental stages in fish during host
development. In G. morhua, despite concurrent initial infection
and continuing exposure of fish to infective stages of the 2 renal myxozoans Gadimyxa atlantica and Zschokkella hildae, proliferation and
sporogony of the two species was clearly separated in both time and space. A
similar condition was found in S. trutta,
where microhabitat separation in the kidney was less obvious but where the
development of Chloromyxum schurovi seems to be strongly influenced by the invasion of Tetracapsuloides bryosalmonae. The results strongly indicate that
within-host competition, not previously observed in this parasite group, takes
place between myxozoan species. The blood stream was identified as the first
and possibly most important site of encounter and interaction, especially in
species where this site is essential for parasite proliferation prior to host
organ invasion. The potential mechanisms of interaction are discussed in
relation to the literature on other parasite groups occurring in the blood and
to ongoing in vitro studies.
S7-4
Histopathological
Features of the Infection by the Myxosporean Zschokkella hildae Auerbach, 1910 in the
Kidney of Atlantic cod (Gadus morhua Linnaeus,
1758).
B
Gorgoglione* and C Sommerville
| Institute
of Aquaculture, University of Stirling, Stirling, Scotland, UK
b.gorgoglione@abdn.ac.uk
|
Zschokkella hildae is a common myxozoan
parasite of Atlantic cod, which acts as the vertebrate host in the two-host
myxosporean life cycle. Samples were obtained from 200 cultured Atlantic cod
aged 1+ from a fish farm on the west coast of Scotland. Z. hildae was identified from spore morphology in fresh squashes
and confirmed by PCR using species-specific 18S rDNA. Localisation and
distribution were confirmed using ISH. Kidney tissues were examined using
standard histological methods. Sporogonic plasmodia of Z. hildae formed large clusters attached to the epithelial lining
of the collecting ducts and ureters and commonly occluded the lumen. Rarely,
sporogonic plasmodia were seen in interstitial tissue. Pre-sporogonic stages
were seen in blood, in interstitial tissue and in the tubular epithelium,
suggesting the parasite route into the ureters. Pathological features included
granulocyte infiltrations, hyperplasia of supporting-connective tissue,
proliferation of interstitial tissue and occasional glomerular oedema. A marked
feature of the infection was the rodlet cell invasion of the parasitised tissues,
which was always associated with plasmodia. Discharging rodlet cells attached
directly to large plasmodia. Our observations provide evidence for the
potential pathogenicity of Z. hildae in Atlantic cod.
S7-5
Chinook Salmon Host
Response to Ceratomyxa shasta (Myxozoa)
S J
Bjork*1, Y Zhang2, JO Sunyer2 and J L
Bartholomew1
| 1 |
Department of Microbiology,
Oregon State University, Corvallis, OR, USA bjorksa@onid.orst.edu, bartholj@science.oregonstate.edu
|
| 2 |
Department of Pathobiology, School of Veterinary Medicine,
University of Pennsylvania, Philadelphia, PA, USA yonganzh@vet.upenn.edu, sunyer@vet.upenn.edu
|
Stocks
of Chinook salmon (Oncorhynchus
tshawytscha) have varying levels of resistance to the myxozoan parasite, Ceratomyxa shasta. In this study, the
progress of C. shasta infection and
host responses of highly susceptible and resistant Chinook salmon stocks were
compared. There were no differences in the invasion of the gills indicating
that resistance does not occur at the site of entry. At a sub-lethal parasite
dose, resistant fish eliminated the parasite from the blood 2 weeks after
infection, limiting the numbers that reach the intestine. On day 12 p.i.
infection intensity scores in the intestine were higher in susceptible than
resistant fish but inflammation in resistant fish was more severe. The
expression of IL-10 and IFNγ in the blood and IL-6, IL-8, IL-10 and
IFNγ in the intestine was up-regulated in both strains 12 days after
infection. IL-10 and IFNγ
increased in the blood of resistant fish and IFNγ expression increased in
the intestine on day 25, whereas all of the susceptible fish died by this time.
On day 90, resistant fish had resolved the inflammation and cytokine expression
was similar to the control group. Thus, it appears that the inflammatory
response of the susceptible strain was delayed and incapable of containing or
eliminating C. shasta but resistant
fish: 1) limited the number of parasites that reached the intestine through
clearance from the blood; 2) elicited a rapid and effective inflammatory
response in the intestine that contained and eliminated C. shasta and 3) resolved the inflammation and can recover from
infection.
| SESSION
8. BACTERIOLOGY II |
[TOP] |
S8-1
Virulence Properties
of Moritella viscosa Extracellular
Products
B Bjornsdottir*, T
Gudmundsdottir and BK Gudmundsdottir
Institute for Experimental
Pathology, University of Iceland, Keldur v/Vesturlandsveg, Iceland
bryndisb@hi.is, tota@hi.is, bjarngud@hi.is
|
The pathogenic bacterium Moritella viscosa causes winter ulcer disease in fish reared in
seawater at low temperatures. The
disease causes significant financial losses in aquaculture, mainly in salmon
farming, and thus vaccines are being developed. It has been shown that extracellular products (ECP) from M. viscosa cause disease symptoms, even
leading to death in Atlantic salmon (Salmo
salar L.); however, little
is known about the virulence of M.
viscosa. The aim of the
present study was to evaluate the virulence properties of M. viscosa ECPs. We
obtained ECPs of 22 M. viscosa strains isolated from different
geographical regions and host species, and evaluated their lethality to salmon,
and their cytotoxic and hemolytic activities, enzymatic activities, and
siderophore levels. The ECPs of
strains isolated from salmon showed lower cytotoxic and hemolytic activities
than did ECPs of strains isolated from other species of fish. No correlation was found between
lethality to salmon and cytotoxic or hemolytic activities. Two strains isolated from salmon in
Canada were found to be non-virulent, and their respective ECPs were
non-lethal. Variable levels of ECP
protein concentration, different enzymatic activities, and siderophore levels
could not explain the differences of in
vivo and in vitro virulence (that is to say, the lethality to salmon and
cytotoxic/hemolytic activities). The results indicate that cytotoxicity
assaying of cell-culture models may not be an optimal method for determining
the virulence of M. viscosa strains,
and that the major extracellular virulence factors of M. viscosa are still unidentified.
S8-2
Detection
of Yersinia ruckeri in Juvenile
Cultured Atlantic cod (Gadus morhua)
in a Marine Nursery Located in Atlantic Canada.
NY O’Brien1, D Groman*2, J.
Giles2 and DS Whelan1
| 1 |
Newfoundland
and Labrador Department of Fisheries and Aquaculture, Aquatic Animal Health Division, St. John’s Canada. nicoleobrien@gov.nl.ca, darylswhelan@gov.nl.ca
|
| 2 |
Aquatic
Diagnostic Services, Atlantic Veterinary College , University of Prince Edward
Island,
Charlottetown Canada. groman@upei.ca, jgiles@upei.ca
|
This case study describes a Yersinia ruckeri infection of juvenile
Atlantic cod (Gadus morhua),
averaging 10 g in weight. In December of 2009, 30,000 Atlantic cod maintained
in a marine nursery in Atlantic Canada were reported as experiencing elevated
mortality and morbidity (~5% cumulative mortality). Clinical signs observed
during an on-site veterinary visit included erythema at the base of the
pectoral fins and spiraling within the tanks. Samples were obtained for
diagnostic analysis, which included histopathology, bacteriology and virology. Samples were negative for Betanodavirus by both tissue
culture using E-11 cells and RT-PCR analyses. The bacterial pathogen, Yersinia ruckeri, (Type-01) was isolated
from the population. The
confirmatory identification methods for the isolate included, biochemical
reactions, serum agglutination, API-20E system and by MALDI-TOF Mass
Spectrometry. The isolate was
found to be sensitive by the Kirby Bauer Method to the following antibiotics:
Enrofloxacin, Aquaflor, Trimeth/sulfa and Oxytetracycline. Histopathology examinations revealed
that affected fish were suffering from a severe septicemia, with granulomatous/
histiocytic infiltrates present in many of the affected tissue lesions. The fish were treated with an in-feed
antibiotic. After treatment,
mortality and morbidity remained unchanged. The population was subsequently
culled and a random sample of the population was obtained to screen for
concurrent pathogens/diseases and the prevalence of Yersinia ruckeri was determined.
S8-3
Characterization of
Isolates of Group B Streptococci from Diseased Farmed and Wild Marine Fish from
the United States Gulf of Mexico Coast
E Soto, R Wang, JE
Wiles, W Baumgartner, and JP Hawke*
| Department of Pathobiological Sciences, LSU
School of Veterinary Medicine, Baton Rouge, LA USA. esoto1@lsu.edu, rwang@tigers.lsu.edu,
wbaumg1@tigers.lsu.edu, jwiles@vetmed.lsu.edu,
jhawke1@lsu.edu
|
Group
B streptococci have been described as disease causing agents from various
species of marine fish over a wide geographic and host range. Group B Streptococcus sp. have been described as
causative agents of fish kills of wild marine and euryhaline species in coastal
estuaries of the northern United States Gulf of Mexico coast since the early
1970’s. Isolates of group B streptococci have been obtained from cultured
hybrid striped bass (Morone saxatilis x
M. chrysops) and cocahoe minnow (Fundulus
grandis) from coastal Louisiana fish farms since the late 1980’s. In this
study, we identify these United States Gulf of Mexico coast isolates as Streptococcus agalactiae group B type Ib
and describe their biochemical
phenotype, antibiotic susceptibility, and phylogenetic relationship to
other Group B streptococci from fish cultured in Thailand, Ecuador, and
Honduras. Genetic analysis was performed using partial sequence comparison of
the 16S rRNA gene, sipA which encodes a surface immunogenic protein, cspA which encodes a cell
surface-associated protein, and secY which encodes components of a general protein-secretion pathway. Phylogenies inferred from secY and cspA gene sequence comparisons were more discriminative than those
inferred from 16S rRNA and sipA gene
sequences. In agreement with the molecular findings, biochemical and
antimicrobial resistance analysis demonstrated similar profiles for the
isolates recovered from the Gulf of Mexico coast when compared to those from
other locations. Laboratory challenge methods for inducing streptococcosis in
cocahoe minnows were performed using strain LADL-97-151. The lethal dose 50
value, 14 days post-challenge, was 10-9.43 (2 CFU/fish) by
intraperitoneal injection.
S8-4
A Wide Analysis of Fish Bacterial
Pathogen Streptococcus parauberis: Approach
to Complementary Genomics and Proteomics
SW
Nho*1, SB Park1, HB Jang1, IS Cha1,
MA Ha1, YR Kim1, J Hikima1, M Othani1,
T Aoki1, 2 and TS Jung1
| 1 |
Aquatic Biotechnology Center of WCU project,
College of Veterinary medicine, Gyoungsang National University, Jinju, Korea. shyguy0126@gmail.com
|
| 2 |
Laboratory
of Genome Science Course of Applied Marine Biosciences Graduate School of
Marine Science and Technology, Tokyo, Japan.
|
Streptococcus
parauberis is a causative agent of streptococcosis in different farmed fishes, and is also known to cause cellulitis of soft
tissues in humans. Streptococcosis caused by S. parauberis has a drastic
impact on Olive flounder (Paralichthys olivaceus) farming, the major
aquaculture industry in Korea, causing severe economic losses. This study was
performed to identify the antigenic proteins of an S. parauberis isolate
by employing immunoproteomic techniques using a specific flounder anti-S. parauberis sera generated by
experimental S. parauberis challenge.
Sixty-two antigenic proteins from approximately 200 spots, observed on
2-DE profiles of S. parauberis were
reacted with the flounder anti-sera. A part of antigenic proteins were
identified by mass spectrometry (MS) and peptide mass fingerprinting analyses
(http://www.matrixscience.com/) as follows: molecular chaperone, GAPDH,
enolase, IMPDH, OCT, FBA, and arginine deiminase. These
proteins are associated with invasion, adhesion and escape from defense
mechanisms to establish infection within the host environment. Simultaneously, to
investigate the phylogeny, metabolism, drug-resistance and virulence of S. parauberis, whole genome shotgun
sequencing was conducted using GS-FLX. The S. parauberis genome possesses a single chromosome
of approx. 2.1 Mbp containing 2,074 predicted ORFs. Using blast search
analyses (http://www.ncbi.nlm.nih.gov/BLAST/), 1,469 of these genes were
identified with S. uberis 0140J strain with high query coverage. To identify additional
antigenic proteins, the MS data were re-analyzed with database of amino acid
sequences deduced from the annotated gene sequences. The proteomics and
genomics alliance produces almost complete and accurate gene catalogues for
microbial genomes, a comprehensiveness which is essential for efficient systems
biology.
S8-5
Ecological and
Epidemiological Aspects of Bacterial Kidney Disease in Marine Phase Chinook
Salmon
LD
Rhodes*1, CA Rice1, CM Greene1, DJ Teel2,
M Trudel3, SL Nance4, T Zubkowski3, E Smith1
and P Moran1
| 1 |
NOAA Fisheries, Northwest
Fisheries Science Center, Seattle, WA 98112 USA
|
| 2 |
NOAA Fisheries, Northwest
Fisheries Science Center, Manchester Research, Laboratory, Port Orchard,
Washington, 98366 USA
|
| 3 |
Department of Fisheries and Oceans, Pacific Biological
Station, Nanaimo, BC, Canada
|
| 4 |
Aquatic Farms, Ltd., Kaneohe, HI USA
|
Bacterial kidney disease (BKD) is an endemic disease among
Pacific Northwest populations of salmonids. Infections can occur in freshwater or seawater, but there
are few investigations of infection during the marine phase. A study of neritic (nearshore) habitat
utilization by juvenile Chinook salmon (Oncorhynchus tshawytscha) throughout Puget Sound
has identified factors associated with infection likelihood as well as factors
that do not appear to contribute to infection. For example, the density of Chinook salmon measured in each
tow positively correlated with infection prevalence across a range of spatial
scales, suggesting a potential role for density in infection transmission. Surprisingly, the density of river
lamprey (Lampetra ayresis) per tow
was a significant risk factor for infection. When we examined the stomach contents and kidneys of river
lamprey, intact cells of the etiologic agent, Renibacterium salmoninarum,
were identified, indicating river lamprey could be a transmission vector during
emergence to seawater. Studies of
the offshore marine phase have also yielded unanticipated observations. Fish with levels of bacterial DNA and
protein typically associated with morbidity were collected in offshore
sampling, demonstrating that substantial infection burdens can be sustained in
free-ranging fish. These types of
studies complement freshwater studies by providing information about the
epidemiology and ecology of BKD during a poorly characterized part of the
salmon life history.
S8-6
Possible Common Etiological Agent for Strawberry Disease and Red Mark
Syndrome in Rainbow Trout.
M Metselaar*1, KD Thompson1, RML Gratacap1,
MJL Kik2, SE LaPatra3, SJ Lloyd4, DR Call4, PD Smith5 and A Adams1
| 1 |
Aquatic Vaccine Unit, Institute of
Aquaculture, University of Stirling, UK Matthijs.metselaar@stir.ac.uk
|
| 2 |
Department
of Pathobiology, Pathology, University of Utrecht, Utrecht, Netherlands
|
| 3 |
Clear
Springs Foods, Inc., Buhl, Idaho 83316, USA.
|
| 4 |
Department of Veterinary Microbiology
and Pathology, Washington State University, Pullman, WA, USA
|
| 5 |
Intervet Schering-Plough
Animal Health, Aquaculture Centre, Essex, UK
|
Strawberry Disease (SD), in the USA appears to
affect mostly rainbow trout (Oncorhynchus mykiss) and has recently been
associated a Rickettsia-like organism
(RLO) with SD, based on analysis of 16S rDNA sequences. A disease with similar
clinical signs to SD, referred to as Red Mark Syndrome (RMS), has been reported
in the UK. The research presented here indicates the presence of a common intracellular
bacterium in both SD- and RMS-affected fish. Using two different polyclonal
antisera raised against Piscirickettsia
salmonis and a number of different anti-P. salmonis monoclonal
antibodies (MAbs), a positive reaction was observed in skin lesions, liver,
kidney and spleen of RMS-affected fish sampled from several locations within
the UK, using immunohistochemistry (IHC). The same panel of anti-P. salmonis antibodies showed similar staining in the tissue of SD-affected fish by IHC. In
addition, polymerase chain reaction (PCR) performed (using RLO-specific
primers) on RMS-affected fish from the UK, indicated that these samples were
positive for the RLO 16S rRNA sequence. These findings suggest that the same
aetiological agent may in fact be responsible for RMS in the UK and SD in the
USA. Manageable
| SESSION
9. HUSBANDRY AND NUTRITION |
[TOP] |
S9-1
Isotonic Egg-washing Reduces
Bacteria and Viruses on Salmon Eggs in Aquaculture
M Kohara*1, S Ogawa2,
H Kasai3 and M Yoshimizu3
| 1 |
Graduate School of Fisheries Sciences,
Hokkaido University, Hakodate, Hokkaido 041-8611, Japan
kohara-masakazu@pref.nagano.jp
|
| 2 |
Nagano Prefectural Fisheries
Experimental Station, Akashina, Azumino, Nagano, 399-7102, Japan
ogawa-shigeru@pref.nagano.jp
|
| 3 |
Faculty of Fisheries Sciences, Hokkaido
University, Hakodate, Hokkaido 041-8611, Japan hisae@fish.hokudai.ac.jp,
yosimizu@fish.hokudai.ac.jp
|
Egg-washing
with isotonic solution has been used to improve the artificial fertilization in
fresh-water salmon in aquaculture. Usually, unfertilized eggs are rinsed two or
three times with isotonic solution before fertilization. We investigated the
effect of isotonic egg washing using rainbow trout eggs to reduce colonization
with Flavobacterium psychrophilum, Aeromonas.
salmonicida, infectious hematopoietic necrosis virus
(IHNV) and Oncorhynchus masou virus
(OMV) quantitatively. Newly spawned
eggs were divided into three treatment groups that were challenged with varied
concentrations of F. psychrophilum:
(1) immersion-challenged unfertilized eggs followed by water hardening; (2) immersion-challenged fertilized eggs
followed by water hardening; and (3) immersion-challenged eggs that were rinsed
with isotonic solution. Eggs were cultured on modified Cytophaga Agar. F. psychrophilum was isolated from the contents of eggs
at a high prevalence; however, no growth was observed at lower challenge
concentrations of F. psychrophilum. Interestingly, A. salmonicida and Renibacterium.
salmoninarum were also isolated from egg contents with a CBB medium when
bacterial numbers were high. Although, F. psychrophilum and R. salmoninarum were cultured from the egg contents, A. salmonicida was not cultured at seven
days after immersion-challenge. The number of live bacterial cel1s or virus
infectivity titers which were eliminated by egg-washing, was 103~5 CFU /ml or 104 TCID50/ml. The ratio of intra-ovum
infection with F. psychrophilum was
clearly less in rinsed eggs than in non-treated eggs, and result of detection
from showered eggs was negative. Results suggest that egg-washing with isotonic
solution is effective in reducing certain bacteria and viruses on the egg
surface, and may help to decrease the risk of egg infection.
S9-2
Bioexclusion in
Site-Level Biosecurity Plans
GA
Karreman*
| Syndel
Laboratories Ltd., 958 Chatsworth Road, Qualicum Beach, BC, V9K 1V5 Canada,
gracek@wchemical.com
|
Biosecurity can be defined as a system of processes
(i.e., inputs, movements and other activities), each with a set of procedures
that taken together minimize the risk of introduction and spread of infectious
organisms within or between aquatic animal populations. Biosecurity measures at
the site level include bioexclusion (prevention of pathogens from entry),
within-site infectious disease control (management of pathogens within a
facility) and biocontainment (prevention of pathogens from release). This talk
will focus on bioexclusion. When designing a biosecurity plan, an on-site
analysis provides the scenario for pathogen introduction into the site. The
analysis requires a working understanding of the production biology of the
species, the physical layout of the facility and the process flow for
operations. It also requires a working knowledge of the epidemiology of the
pathogen(s) to be excluded. A risk assessment estimates the risks of
introducing pathogenic organisms or disease into an aquatic animal site and
provides recommendations for mitigation of those risks. Specific points of
pathogen introduction can then be identified by systematically evaluating every
step in the process flow for potential introduction of the pathogen into
the site.
S9-3
Effect of Common Aquaculture Chemicals Against Selected Fish Pathogenic
Bacteria
SA Smith*
| Aquatic
Medicine Laboratory, Department of Biomedical Sciences and Pathobiology,
Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic
Institute and State University, Blacksburg, VA USA stsmith7@vt.edu
|
Bacterial pathogens are one of the most important
disease-causing agents in the aquaculture industry. As most pathogenic bacterial species are water-borne,
disinfection of aquaculture systems and equipment is essential for both disease
control and for enhanced biosecurity. This study examined a variety of
commercially available compounds for their ability to reduce or eliminate
specific bacterial species from the water. The compounds evaluated included
Clorox®, ethanol, Roccal®, Lysol®, iodine, formalin, Chloramine-T®,
glutaraldehyde and Virkon S®. Each
compound was tested at various concentrations over a 60-minute time interval
against Mycobacterium marinum, Vibrio
anguillarum, V. ordalii, Edwardsiella ictaluri, E. tarda, Aeromonas hydrophila,
A. salmonicida subsp. salmonicida, A. salmonicida subsp. achromogenes, Flavobacterium columnare, and F.
psychrophilum. In addition, the three Aeromonas species were tested at four different temperatures (22°, 15°, 10°, and 5° Celsius). For M. marinum, ethanol and Lysol® were the
most effective disinfectants, each reducing or eliminating the number of
detectable bacteria within one minute of contact time. Sodium hypochlorite was
moderately effective, but required a minimum contact time of 10 minutes to
reduce bacterial counts. For the Vibrio spp., Chloramine-T®, Clorox®, ethanol, iodine, Lysol®, Roccal®, and Virkon S®
eliminated all growth of both species of bacteria within one minute of contact
time. All species of Edwardsiella and Aeromonas were killed within one minute of contact time by all
compounds except Chloramine-T® and formalin. In addition, all three Aeromonas species were killed within one
minute of exposure by the effective compounds regardless of temperature.
S9-4
The Importance of Hygiene for Live Feed Management: Lessons Learned
from New Zealand Groper (Polyprion
oxygeneios) Juveniles
S. Anderson*1, I. Salinas1, L.
Tubbs2, J.S. Lumsden2, B. Diggles3, V. Webb1,
Y. Gublin4, S. Pether4, G. Irvine4 and S.
Walker4
| 1 |
National
Institute of Water and Atmospheric Research (NIWA) Ltd. Private Bag 14901,
Wellington 6241, New Zealand. sa.anderson@niwa.co.nz, i.salinas@niwa.co.nz,
v.webb@niwa.co.nz
|
| 2 |
Fish
Pathology Laboratory, Department of Pathobiology, Ontario Veterinary College,
Guelph, Ontario, Canada N1G 2W1. ltubbs@uoguelph.ca, jsl@uoguelph.ca
|
| |
DigsFish
Services Pty Ltd. 32 Bowsprit Cres. Banksia Beach, Bribie Island, Queensland
4507, Australia. ben@digsfish.com
|
| 3 |
National
Institute of Water and Atmospheric Research (NIWA) Ltd, PO Box 147, Ruakaka
0151, New Zealand. y.gublin@niwa.co.nz, s.pether@niwa.co.nz,
g.irvine@niwa.co.nz, s.walker@niwa.co.nz
|
New Zealand hāpuku or groper (Polyprion oxygeneios) is currently
undergoing development for aquaculture. This involves the production of groper
eggs, larvae, and juveniles. A critical period in groper larvae and juvenile
rearing is when the yolk sac is re-adsorbed and the larvae are transitioned
onto live feed. For groper, this begins with live rotifers at 9-11dph, followed
by transition over a 3-8 day period to live brine shrimp (Artemia salina) at
18-22dph. The present study investigates the possible contribution of Artemia to groper mortality events over
2 production seasons, and describes histopathological changes. In 2008, the
introduction of Artemia resulted in
inappetance and a period of elevated mortality; dead and moribund fish
presented with distended abdomens. Histology revealed a necrotising enteritis
in these fish, with empty Artemia cysts present in the intestine. In 2009, a similar increase in mortality
occurred when groper juveniles were fed an Artemia diet. In addition to a similar enteritis, a focally extensive hepatitis
containing intra-lesional bacteria was observed indicating progression to a
systemic bacteremia. In this instance, mortality rates increased rapidly with
100% mortality 6 days after the initial symptoms appeared. Microbiological
analysis indicated the presence of Vibrio-like
bacteria, later identified as Vibrio
ichthyoenteri. This bacterium was also isolated from live Artemia samples, and it is likely to be
the pathogen source. It appears that poor Artemia feed quality contributed to juvenile groper mortalities during 2008 and 2009,
underlining the critical importance of maintaining hygienic practices for live
feed production.
| SESSION
10. PARASITOLOGY III: Myxozoan Workshop 3 |
[TOP] |
S10
PARASITOLOGY III : MYXOZOAN WORKSHOP 3
(DISCUSSION)
| SESSION
11. SHRIMP AND LOBSTER DISEASE AND IMMUNOLOGY |
[TOP] |
S11-1
Molecular Immunology
of Kuruma Shrimp, Marsupenaeus japonicus
I Hirono*1, T Koyama1,
A Kaizu1,
A Shitara1,
MBB Maningas2,
FF Fagutao1,
H Yamada1,
K Komiya1,
T Aoki1 and H Kondo1
| 1 |
Laboratory of Genome
Science, Tokyo University of Marine Science and Technology Tokyo, Japan
hirono@kaiyodai.ac.jp
|
| 2 |
Department of Biological Sciences, University of Santo
Tomas, Espana, Philippines marybethmaningas@yahoo.com
|
Because of
the importance of penaeid shrimp in world aquaculture, there is much interest
in understanding their immune systems to improve their resistance to pathogenic
microorganisms. Unequivocal evidence points to the efficiency of RNA
interference (RNAi) in studying gene function and their involvement in mounting
antiviral responses in eukaryotes. The potency of double stranded RNA (dsRNA)
to knock-down gene expression (gene silencing) has been successfully
demonstrated in studying gene function in shrimp. From these studies, a shrimp
pathogen has been found to be engaged in the innate (non-sequence specific) and
RNAi-related (sequence specific) antiviral responses. Recent studies have shown
that it is indeed possible to block viral disease progression by injecting
shrimp with dsRNA specific to viral genes. Most of the studies conducted that
used RNAi technology, however, focused on the shrimp pathogens and few have
been done on the host itself. Here, some of our recent research results of
shrimp immune-responses against microbial pathogens after in vivo silencing of defense-related genes including clotting
protein, TGase, penaeidin-like protein lysozyme and prophenoloxidase is
presented. Genomic information of penaeid shrimp is essential for understanding
further their immune defense mechanisms. We found several different homologue
genes of white spot syndrome virus (WSSV), a virulent pathogen known to
significantly affect the health of shrimp. The expression of some of these
genes were upregulated in shrimp after WSSV infection.
S11-2
Characterization of Nitric
Oxide Synthase Gene, MjNOS, in Kuruma Shrimp Marsupenaeus
japonicus and its mRNA Variants
M Inada*1, S Okugawa1, J Nishi1, M
Yoshimine1, R Sudhakaran2, T Kono3, T
Mekata4, T Yoshida2, M
Sakai2 and T Itami2
| 1 |
Graduate School of Agriculture, University of Miyazaki, Miyazaki, Japan agg902u@student.miyazaki-u.ac.jp
|
| 2 |
Faculty of Agriculture, University of Miyazaki,
Miyazaki, Japan itamit@cc.miyazaki-u.ac.jp
|
| 3 |
Interdisciplinary
Research Organization, University of Miyazaki, Miyazaki, Japan
|
| 4 |
National Research Institute
of Aquaculture, Fisheries Research Agency, Tsuiura, Kamiura, Saiki, Oita Pref., Japan mekata@affrc.go.jp
|
Nitric oxide (NO) signaling is involved in many
physiological processes in vertebrates and invertebrates. In mammals, three types
of NO synthase (NOS) were reported, namely neuronal NOS (nNOS), endothelial NOS
(eNOS) and inducible NOS (iNOS). In crustaceans, NOS plays a significant role
in the regulation of nervous system and innate immunity. Here, we describe the
entire cDNA sequence (4,616 bp) and the open reading frame (1,187 amino acids)
of the kuruma shrimp Marsupenaeus japonicus NOS (MjNOS). Highly conserved amino acid sequences in heme and
tetrahydrobiopterin were observed in the oxygenase domain. FMN, FAD and NADPH
were found in the reductase domain. When heat-killed Vibrio penaeicida cells
were injected into the kuruma shrimp, MjNOS was expressed in the brain, gill, heart, lymphoid organ, intestine and thoracic
ganglion. MjNOS expression in the
gill reached its peak at 12 hours and decreased to its normal level
24 hours after V. penaeicida injection. In MjNOS mRNA, three variants were detected. Partial deletion of oxygenase
domain sequence was found in variant 1 , whereas complete deletion of reductase
domain with or without novel 104 bp insertion were found in variant 2 and
variant 3. The total NO concentration in hemocytes was determined using the Griess
reaction and high concentration was found at 12 hours after injection of heat-killed V. penaeicida cells.
Molecular Cloning and Characterization of the Dicer2 Gene in Kuruma Shrimp Marsupenaeus japonicus
J Nishi*1, S Okugawa1, M Inada1,
M Yoshimine1, T Yoshida2, Y Suzuki3, T Kono4,
M Sakai2, T Mekata5 and T
Itami2
| 1 |
Graduate School of
Agriculture, University of Miyazaki, Miyazaki Japan agg905u@student.miyazaki-u.ac.jp
|
| 2 |
Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan itamit@cc.miyazaki-u.ac.jp
|
| 3 |
Faculty of Engineering,
University of Miyazaki, Miyazaki Japan suzuki@civil.miyazaki-u.ac.jp
|
| 4 |
Interdisciplinary Research
Organization, University of Miyazaki, Miyazaki, Japan
|
| 5 |
National Research Institute of Aquaculture, Fisheries Research Agency, Saiki,
Japan mekata@affrc.go.jp
|
RNA silencing is a common antiviral defense mechanism in many eukaryotes from plants to animals. The generation of microRNA or small interfering RNA is dependent on the RNase ? enzyme Dicer gene. The Dicer gene plays an important role in the RNA silencing, and it has been reported that there are two types of Dicer genes in insects. However, only one type of Dicer gene was reported in kuruma shrimp (Marsupenaeus japonicus). We report here on the characterization of a second Dicer gene (MjDicer2) in kuruma shrimp. This is the first report of the presence of two types of Dicer genes in a crustacean. The full length of MjDicer2 cDNA was 4967 bp, including a 5' untranslated region (UTR) of 119 bp, a 3' UTR of 321 bp, and an open reading frame of 4,527 bp encoding a polypeptide of 1,509 amino acids. Analysis of the deduced amino acid sequence indicated that the mature peptide contained all six recognized functional domains, and showed the highest similarity to the black cottonwood (Populus trichocarpa) Dicer-like protein sequence with a similarity of 28.7%. Quantitative RT–PCR analyses showed a high expression level of MjDicer2 in the gill, lymphoid organ, and heart. MjDicer2 is most highly expressed in hemocytes 2 h after stimulation with white spot syndrome virus (WSSV). This study was supported by the Research and Development Program for New Bio-industry Initiatives and the Japan Society for the Promotion of Science.
S11-4
Characterization of Tumor Necrosis Factor
Ligand Superfamily Gene in Kuruma Shrimp, Marsupenaeus
japonicus
S Okugawa*1, R Sudhakaran2,
M Inada1, T Mekata3, J Nishi 1, M Yoshimine1,
T Yoshida2, Y Suzuki4, T Kono5, M Sakai2 and T
Itami2
| 1 |
Graduate School of
Agriculture, University of Miyazaki, Miyazaki Japan agg905u@student.miyazaki-u.ac.jp
|
| 2 |
Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan itamit@cc.miyazaki-u.ac.jp
|
| 3 |
National Research Institute of
Aquaculture, Fisheries Research Agency, Oita Japan mekata@affrc.go.jp
|
| 4 |
Faculty of Engineering,
University of Miyazaki, Miyazaki Japan suzuki@civil.miyazaki-u.ac.jp
|
| 5 |
Interdisciplinary Research
Organization, University of Miyazaki, Miyazaki, Japan tkono@cc.miyazaki-u.ac.jp
|
A tumor necrosis factor (TNF) gene has
been isolated and characterized in kuruma shrimp, Marsupenaeus japonicus, providing the
first conclusive evidence for the existence of the TNF ligand in shrimp. The kuruma
shrimp TNF (MjTNF) cDNA was composed
of 1868 bp with a 262 bp 50-untranslated region (UTR) and
a 220 bp 30-UTR, which was translated into a protein of 462 amino acid residues
that included a predicted transmembrane domain of 23
amino acid residues (Trp20-Val42) and the TNF family signature
(Pro321-Leu448). Homology analysis of MjTNF showed 30.7% and 26.7% identities
with fruit fly (Drosophila
melanogaster) Eiger and human (Homo
sapiens) ectodysplasin A, respectively. The MjTNF gene was constitutively expressed in
unstimulated organs of shrimp such as the muscle, stomach, brain and gill.
In lymphoid organ cells, an enhanced expression of the MjTNF gene was observed following stimulation with
peptidoglycan and polycytidylic acid. A high expression level of MjTNF was observed in vivo 2 h and 4 h after stimulation with
lipopolysaccharide and Vibrio penaeicida,
respectively. These observations suggest that MjTNF plays a role in the innate immune
defense in kuruma shrimp. The discovery of shrimp
TNF will allow a more complete and concrete understanding of shrimp
inflammatory responses. This study was supported by Research and Development Program for New
Bio-industry Initiatives and Japan society for the promotion of
science.
S11-5
Reciprocal Effects
between the Lethal Spiny Lobster Virus PaV1 and the Florida Lobster Fishery
DC
Behringer*1, JA Moss2, JD Shields2 and MJ
Butler IV3
| 1 |
University of Florida,
School of Forest Resources and Conservation, Fisheries and Aquatic Sciences,
Gainesville, Florida USA behringer@ufl.edu
|
| 2 |
Virginia Institute of Marine
Science, Gloucester Point, VA USA jeff@vims.edu, jamoss@vims.edu
|
| 3 |
Old Dominion University,
Department of Biological Sciences, Norfolk, Virginia USA mbutler@odu.edu
|
The Caribbean spiny lobster (Panulirus argus) is one of the most
economically valuable fisheries in the Florida Keys and tops this list for most
of the Caribbean. In 2001, the
fishery in Florida experienced a major decline in landings of ~30% from which
it has not recovered. This decline
was coincident with discovery of a lethal viral pathogen, PaV1, found infecting
juvenile lobsters in the Florida Keys. P. argus has a complex life
history and is exploited throughout its range – two factors that have
made it difficult to determine the cause of the decline. Here we describe the first assessment of
PaV1 within the fished segment of the population. We used PCR analysis to measure PaV1 prevalence from
lobsters caught in commercial traps throughout the Florida Keys. We also tested the effect of diseased
lobsters within traps on trap attractiveness, and on the transmission of PaV1
to other trapped, but healthy, lobsters. We found a mean prevalence of 11% in
the fished population with PCR+ lobsters as large as 95 mm carapace length (76
mm is legal). We also found that
traps harboring an infected lobster caught significantly fewer lobsters than
traps containing healthy lobsters. Furthermore, healthy lobsters confined in traps with diseased lobsters
became infected with PaV1 more frequently than those confined with other
healthy lobsters. This study demonstrates the indirect and subtle effects that
pathogens can have on fishery function through altered animal behavior and the
unintended consequences of fishery practices on pathogen epidemiology.
S11-6
Population Effects of
Bitter Crab Disease on Snow and Tanner Crabs From Alaska and Newfoundland.
JF Morado*1, MSM Siddeek2, DR
Mullowney3, EG Dawe3, CA Shavey1, PC Jensen1,
VC Lowe1 and RJ Cawthorn4
| 1 |
NOAA, National Marine
Fisheries Service, Alaska Fisheries Science Center, Seattle, WA USA frank.morado@noaa.gov,
christie.sahvey@noaa.gov, pam.jensen@noaa.gov, vanessa.lowe@noaa.gov
|
| 2 |
Alaska Department of Fish and Game, Division of
Commercial Fisheries, Juneau, AK shareef.siddeek@alaska.gov
|
| 3 |
Science
Branch, Fisheries and Oceans Canada, Northwest Atlantic Fisheries
Centre, St John's, NL, Canada darrell.mullowney@dfo-mpo.gc.ca,
earl.dawe@dfo-mpo.gc.ca
|
| 4 |
University of Prince Edward
Island, Atlantic Veterinary College, AVC Lobster Science Centre, Charlottetown,
PEI, Canada C1A 4P3 cawthorn@upei.ca
|
Bitter Crab Disease (BCD) is a fatal disease of
North Pacific snow (Chionoecetes opilio)
and Tanner (C. bairdi) crabs, and
North Atlantic snow crabs. Both crab species support major commercial fisheries
and the disease is caused by a parasitic dinoflagellate of the genus Hematodinium. The method employed in
disease monitoring varies and is often dictated by logistical considerations.
Regardless of the monitoring method, data from snow and Tanner crab populations
are now being sufficiently accumulated for various types of analyses and
inclusion into population models. As the Bering Sea Tanner crab stock
approaches an overfished level and a rebuilding plan is considered, it was of
interest to determine what role, if any, BCD may have on the rebuilding plan.
Simulation modeling indicated that BCD would have a delayed, but minimal
rebuilding effect on the western stock. However, to compare these potential
results with an area of known high prevalence, a similar simulation was
performed on the Tanner crab population from Stephens Passage, southeast
Alaska. In that instance, under a conservative exploitation rate, rebuilding of
the Stephens Passage stock was not possible under short or medium terms. In
coastal Newfoundland, BCD is highly prevalent across all pot-sampled size ranges,
but most prevalent in small or juvenile crabs. The data indicate that two
epizootics have occurred in the study area, but more interestingly, BCD appears
to be a good predictor of recruitment strength for subsequent years.
The significance of these findings on the respective crab stocks will
be discussed further.
| SESSION
12. ANTIMICROBIAL USE IN AQUACULTURE: Resistance, Residues and Research |
[TOP] |
S12-1
Current Veterinary Approaches
to Judicious Antimicrobial Use
AD
Scarfe*
| American Veterinary Medical
Association, Schaumburg, IL, USA dscarfe@avma.org
|
Since 1863, the American Veterinary Medical
Association (AVMA) has fulfilled its mission and objective to improve animal
and human health by seeking input on emerging issues from its members (today
numbering ~83,000). Input is channeled through numerous councils, committees,
task forces and other entities and utilized to formulate AVMA Executive Board
approved opinion (positions and policies) or guidance on numerous issues,
including the appropriate use of veterinary drugs. In 1998, to address concern for emerging resistance
of pathogens to drugs (particularly antibiotics) during the 1990s, with the
assistance of a Steering Committee on Antimicrobial Resistance, the AVMA
approved 15 “Principles for the Judicious Use of Antimicrobials.” These have served as the foundation of
numerous subsequent guidance documents and policies involving antimicrobial use
in all species – including aquatic animals. Many guidance documents have
been developed and distributed in collaboration with other veterinary
organizations and government agencies. AVMA policies are also used to guide the
profession’s response to legislation and regulations, and in many cases they
are also utilized by others. Concerns for resistance to antimicrobials approved
for veterinary use increased during the last decade, particularly with respect
to the potential implications in human medicine and the use of FDA approved
non-therapeutic (growth promotion) antibiotics in livestock. The debates that
incurred have, and will continue to influence legislation, regulations,
policies, public opinion and guidance (including those at international levels,
e.g. Codex Alimentarius, WHO, FAO and OIE) that affect the judicious use of
antimicrobials in all animals – including aquatic species.
S12-2
Aquaculture: Its Contribution
to the Aquatic Resistome?
DW
Verner –Jeffreys* and C Baker-Austin
| Cefas Weymouth laboratory,
Weymouth, Dorset, UK david.verner-jeffreys@cefas.ac.uk
|
The continued and accelerating development of
antimicrobial resistance (AMR) is one of the most serious threats to animal,
plant and human health we are presently facing. The lack of development of new antibiotics, the persistence,
even in the absence of selection, of antimicrobial resistance in several bacterial
pathogens means that our ability to treat bacterial infections in farmed
animals and man is increasingly threatened. There is strong scientific evidence that much of the
resistance in bacterial pathogens is encoded by ‘mobile’ genetic elements capable
of efficient cross genus transfer of resistance genes sometimes in large
genetically linked clusters. Together with the resistance genes, come a whole host of genes of
unknown function which may confer an entirely different selectable phenotype. The reservoir of genes which transfer
under the selective pressure of antibiotic usage is largely unknown. A large
body of scientific evidence shows that bacteria harboring these mobile AMR
genes are widely disseminated, and often at high prevalence, in all
environments studied to date (e.g. soil, mining waste, livestock production
sites, water treatment effluent, marine and freshwater water column and
sediment samples). The potential threat that these environmental ‘reservoirs’ of mobile AMR genes pose
to human and animal (including fish) health has not yet been ascertained, but is likely to be considerable. For this presentation we
will particularly focus on the aquatic environment and discuss the extent to
which aquaculture practices are affected by, and may contribute to, the aquatic
‘resistome’.
S12-3
Efforts by the U.S.
Food and Drug Administration’s Center for Veterinary Medicine to Address
Antimicrobial Resistance in Aquaculture
J
Matysczak*
| US Food and Drug
Administration, Rockville, MD, USA. Jennifer.matysczak@fda.hhs.gov
|
Antimicrobial drugs have
been widely used in human and veterinary medicine for more than 50 years with
tremendous benefits to both human and animal health. However, a number of scientific reports have raised concerns
over the use of medically important antimicrobial drugs in food-producing
animals. The FDA has recommended phasing in measures that would limit the use
of medically important antimicrobial drugs in food-producing animals to uses
that are considered necessary for assuring animal health and that include
veterinary oversight or consultation. This presentation will
provide a brief introduction to how the FDA considers antimicrobial resistance
in its review of a new animal drug application for food-producing animals, its
efforts to encourage judicious use of antimicrobials in aquaculture
domestically and in collaboration with international governments and
organizations, and the recent draft guidance published for comment.
S12-4
Resistant or Susceptible May
Not Always be Your Answer
RA Miller*
| Center for Veterinary Medicine, U.S. Food and Drug
Administration, Rockville, MD USA
|
In recent years, new
terminology has emerged to more accurately describe and interpret in vitro antimicrobial susceptibility
test results [minimal inhibitory concentrations (MICs) and zone diameters
(ZDs)]. Epidemiologic cut-off
values [also called wild-type cutoffs] separate populations on the basis of MIC
and/or ZD distributions, and are used to predict the presence of resistance mechanisms
in a given bacterial isolate. These are not to be confused with the more robust clinical breakpoint
categories, susceptible, intermediate, and resistant, which have been validated
in the animal and are defined by MIC and/or ZD values. Technically, it is incorrect for
clinicians to describe any aquatic isolate as susceptible or resistant,
with one exception; if oxytetracycline and/or oxolinic acid is tested against an isolate of Aeromonas salmonicida (see CLSI Supplement M42/M49-S1 for
details). To define an isolate as susceptible would mean the clinician has
validated the isolate’s in vitro test
result and correlated it previously with a positive clinical outcome in the
patient (population). Instead,
this isolate should be categorized as wild-type (i.e., there are not likely to be resistance mechanisms present) or nonwild-type (the isolate likely
possesses acquired and/or
mutational resistance mechanisms). Recent survey data has shown fish diagnostic laboratories use a wide
range of ZDs to define their lab-specific clinical breakpoints. However, a more harmonized approach to
how in vitro susceptibility data is
generated, reported, and interpreted could lessen some of the confusion
encountered when interpreting data from within a country, much less across
countries, and simultaneously maintain prudent antimicrobial use principles.
S12-5
Development of
Antimicrobial Susceptibility Testing Methods for Flavobacterium columnare and F.
psychrophilum.
CM
Gieseker*1and TD Mayer1
| US
Food and Drug Administration, Center for Veterinary Medicine, Office of
Research, 8401 Muirkirk Road, Laurel, MD 20708 USA charles.gieseker@fda.hhs.gov
|
The goal of this study was to develop reliable broth
microdilution methods to test the drug susceptibility of the fish pathogens Flavobacterium columnare and F. psychrophilum. Broth microdilution methods that use
dilute 1:5 Mueller-Hinton broth (4g/L) were developed and/or confirmed, then
used to test F. columnare (n=23,
28°C) and F. psychrophilum (n=23,
18°C) to determine the appropriate drug concentration ranges to use for
standardized testing. The quality
control bacteria Escherichia coli,
ATCC25922 and Aeromonas salmonicida,
ATCC 33658 were also tested along with the Flavobacteria at both temperatures. Custom
frozen broth microdilution panels were used to test the susceptibility of these
isolates to ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine,
gentamicin, oxolinic acid, oxytetracycline, sulfamethoxazole/trimethoprim, and
sulfadimethoxine/ormetoprim. To
validate these broth microdilution methods, F.
columnare (n=24, 28°C) and F.
psychrophilum (n=24, 18°C) isolates were tested simultaneously by agar
dilution and broth microdilution for their susceptibility to florfenicol and
oxytetracycline. The minimum
inhibitory concentration (MIC) results from broth microdilution were similar to
agar dilution. An intra-laboratory
trial was conducted with 4 technicians separately testing the susceptibility of F. columnare (n=6), F. psychrophilum (n=6) and both QC
bacteria to ampillicin, florfenicol, oxytetracycline, and
sulfadimethoxine/ormetoprim using our broth microdilution methods. The technicians agreed >90%
within one MIC dilution for both QC bacteria at both test temperatures and for
most of the Flavobacteria isolates. These results demonstrate that these broth microdilution methods are
robust and can be used to standardize antimicrobial susceptibility testing for F. columnare and F. psychrophilum.
S12-6
Analysis of Bacterial
Antibiotic Resistant Genes in Aquaculture
JY Wang*, YJ Geng, RX Wang and J Feng
| South
China Sea Fisheries Institute, Chinese Academy of Fishery Sciences, Guangzhou,
China wjy104@163.com
|
Fifty-four
bacterial strains derived from aquaculture sources in different regions were
tested for the drug sensitivity of 10 common antibiotics determined by
Kirby-Bauer disk diffusion according to NCCLS. With specific primers designed
according to the reported sequences in GenBank, polymerase chain reaction (PCR)
assay were used to amplify the antibiotic resistant genes and study the mechanisms
of drug resistance. Forty-two of the 54 gram-negative bacteria were identified
as resistant bacteria (77.8%), and 37 strains (68.5%) had multi-resistances for
resistance to more than 3 antibiotics. Resistant genes on plasmids were detected using PCR methods. Nine strains
were tested including trimethoprim-sulfamethoxazole resistance gene sul2, 3
strains possessed chloramphenicol resistant gene cat2, 4 strains had
chloramphenicol resistant gene cat3, only 1 strain was amplified to have chloramphenicol
resistant gene cat4, and 3 strains were detected with kanamycin resistant gene
aadB. Detection ratios of resistant genes in plasmid were 50%, 27%, 36%, 9%, 60%
respectively.
| SESSION
13. WILDLIFE, ORNAMENTAL AND AQUARIUM MEDICINE |
[TOP] |
S13-1
Apicomplexan Parasites
in the Brain of an Immature Indo-Pacific Green Sea Turtle
M Flint*1,2,
JC Patterson-Kane3, CJ Limpus4 and PC Mills2
| 1 |
Aquatic Animal Health, College of Veterinary Medicine, University of
Florida, Gainesville FL USA. Mark.Flint@MyFWC.com
|
| 2 |
Veterinary-Marine Animal Research, Teaching and Investigation
(Vet-MARTI) unit, School of Veterinary Science, The University of
Queensland, St. Lucia, Queensland Australia. p.mills@uq.edu.au
|
| 3 |
School of
Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, United Kingdom. j.patterson-kane@vet.gla.ac.uk
|
| 4 |
Queensland Department of Environment, Resources and Management,
Brisbane, Queensland Australia. col.limpus@derm.qld.gov.au
|
Apicomplexan protozoan
parasites of interest to aquatic animal caregivers and indigenous people
include Eimeria, Neospora, Isospora and Toxoplasma spp. Toxoplasma gondii has been responsible for isolated to mass
mortality events in several marine mammal species. However, it has not been
shown to cause a true infectious state in wild reptiles. In October 2007, a
79.5 cm curved carapace length, large immature male green turtle (Chelonia mydas) stranded in Moreton Bay,
Australia. Initial clinical examination revealed it to have a buoyancy disorder
(“floater syndrome”), be emaciated and lethargic, and display neurologic
deficits (right-sided head tilt). After 12 days of palliative care at SeaWorld, Australia the turtle died.
At necropsy the turtle was emaciated, a fishing hook was embedded in the
gastric mucosa, and 10-15 1-mm
diameter cream, nodular lesions, were noted
in the small intestinal serosa. Tissue specimens were fixed in 10% neutral
buffered formalin, processed routinely for histologic examination and stained
with hematoxylin and eosin. The brain contained multiple protozoal cysts
intermingled with inflammatory changes; the latter associated with spirorchiid
trematode eggs. There was
meningeal fibroplasia with evidence of infiltration by mixed leukocytes.
Diagnosis based on histopathology was cerebral toxoplasmosis, however
immunolabelling for Toxplasma gondii was inconclusive and negative for Neospora spp. Molecular analysis of the formalinized sample was unsuccessful.
Ultrastructurally, the protozoal elements showed features consistent with being
Apicomplexans. Very little is known about such parasites in marine turtles and
any possible clinical effects. Detailed investigation including conducting
complete gross and histological necropsy examinations inclusive of the brain is
warranted.
S13-2
The Importance of
Emerging Exotic Viral Pathogens of Ornamental Fish in Australia
AE Rimmer*, JA Becker, A Tweedie and RJ Whittington
| Farm Animal and Veterinary
Public Heath, Faculty of Veterinary Science, University of Sydney, Camden,
Australia anneke.rimmer@sydney.edu.au
|
The movement of ornamental fish through exporting
and importing practices provides a transmission pathway for the introduction
and establishment of exotic viral pathogens. The viral agents cyprinid herpesvirus-2 (CyHV-2) and megalocytivirus (gourami iridovirus) represent significant exotic pathogens
in Australia, having been implicated as the cause of severe disease and
mortality in intensively raised fish and ornamental fish. The disease threat to the aquaculture
industry, and to the native fish of Australia, has driven the development of
molecular diagnostic tests capable of detecting these ornamental fish viruses. Optimized
PCR assays for these pathogenic viruses were employed in a survey of ornamental
fish from Australian pet shops. Twelve of 37 goldfish (Carassius
auratus) tested positive for CyHV-2, while 8 of 23 dwarf gouramis (Colisa lalia) and 2 of 7 blue/gold
gouramis (Trichogaster trichopterus)
tested positive for megalocytivirus. The murray cod (Maccullochella
peelii peelii) is also susceptible to megalocytivirus. An iconic species of high conservation
value in Australia, the murray cod is a member of the Family Pericichthydae,
several members of which are listed as vulnerable or endangered in Australia,
including trout cod (Maccullochella
macquariensis), golden perch (Macquaria
ambigua ambigua), and Macquarie perch (Macquaria
australasica). To further study
the impact of megalocytivirus on Australian fish, an experimental challenge
model was developed in murray cod. The outcomes from this research have important implications for both
quarantine policy and management of ornamental fish in Australia.
S13-3
Correlations of
Swimming Patterns with Vertebral Deformities in Captive Sandtiger Sharks, Carcharias taurus
EE
Tate* 1, 2, PA Anderson1 and IK Berzins3
| 1 |
The
Florida Aquarium Center for Conservation, Tampa, Florida USA
panderson@flaquarium.org
|
| 2 |
The University of Tampa,
Tampa, FL USA etate07@gmail.com
|
| 3 |
John G. Shedd Aquarium,
Chicago, IL USA Iberzins@sheddaquarium.org
|
Swimming behavior is hypothesized to contribute to
the development of vertebral deformities in captive sandtiger sharks (Carcharias taurus). Public aquaria across the United
States were surveyed and asked to record video of individual C. taurus swimming from the top and from
the side of the exhibits for ten minutes per view. Fifty-three individuals from
11 institutions were recorded, and the records sent for ethological observation
and analysis. Thirteen sharks had vertebral deformities (via gross appearance,
radiography, and/or veterinary diagnosis), the remaining 40 were healthy. Data was
recorded and analyzed using JWatcher software. Behaviors measured included
duration and time percent swimming in a direction, gliding percentage, and tail
beat duration. Combined, C. taurus spent
a median of 98.9% of time swimming and 0.62% of time gliding; furthermore,
healthy sharks spent a median of 0.67% of time gliding versus a median 0% for afflicted sharks, suggesting an
increased swim-to-glide ratio. All sharks swam asymmetrically (in a circular
pattern, clockwise or counter-clockwise) at a median of 99.7% of the time.
Specimens with vertebral deformities had tail beat durations of 3.36 ± 0.22 s
vs 2.72 ± 0.10 s for healthy sharks. Stereotyped swimming patterns in accordance with an increase in swim-to-glide
ratios support the hypothesis that swimming patterns, especially in correlation
with smaller tanks and exhibits, may be causative of, or at least may
exacerbate, vertebral deformity in C.
taurus. Behavioral enrichment programs and implementation of larger and
non-circular tank designs are recommended to decrease the likelihood of the
deformity occurring in captive specimens.
S13-4
Prospecting for
Biomarkers of Health in Whale Sharks, Rhincodon
typus, Using Metabolomic Methods
ADM
Dove*1, J Leisen2, M Zhou2, K Lim-Hing2,
DH Webb1, L Gelbaum2, J Byrne3, M Viant3,
F Fernandez2 and J Kubanek2
| 1 |
Georgia Aquarium Research Center, Georgia Aquarium,
Atlanta, GA USA adove@georgiaaquarium.org
|
| 2 |
Georgia Institute of Technology, Atlanta GA
Julia.kubanek@biology.gatech.edu
|
| 3 |
School of Biosciences, University of Birmingham, Birmingham UK m.viant@bham.ac.uk
|
Whale sharks are plankton-eating sharks
that live in tropical oceans throughout the world; they are the largest of all
fishes. Despite their size, or
perhaps because of it, relatively little is known about their physiology or
biochemistry. The long-term
management of a population of whale sharks in an aquarium in Atlanta has
provided opportunities to redress this situation. Terminal illness in two of six animals allowed for
comparison of serum chemistry between healthy and moribund animals, with the
goal of identifying promising biomarker compounds to serve as indicators of
health in this species. Serum was
analyzed by complimentary metabolomic methods that could identify and sometimes
quantify unknown metabolites in serum samples. Several 1D and 2D 1H nuclear magnetic resonance
experiments were followed by analyses using DART mass spectrometry that
confirmed putative identifications and also provided extensive lists of
candidate compounds based on mass/charge ratios of individual metabolites. Serum of whale sharks included a
diverse array of metabolites, among which osmolytes (particularly TMAO), amino
acids and hydroxy acids were the most frequently encountered. Multivariate analyses were used to show
metabolic trajectories of the moribund animals. Homarine, or n-methyl picolinic acid, was a prominent driver
of these trajectories; direct quantification by NMR confirmed that it had a
significantly lower concentration in moribund whale sharks compared to healthy
animals and is therefore a promising biomarker. This is the first application of metabolomics to sharks and
the first report of homarine in an elasmobranch, but its function remains
unknown.
S13-5
Megalocytiviruses of
Ornamental Fish and Implications for Aquaculture Species
J
Go*1, M Lancaster2, K Deece1, O Dhungyel1 and RJ Whittington1
| 1 |
Faculty of Veterinary
Science, University of Sydney Australia jeffey.go@sydney.edu.au
|
| 2 |
Victorian Institute of
Animal Science, Attwood, Australia
|
Research was undertaken to determine whether viruses present in ornamental fish are linked to emerging viral diseases in aquaculture. The present study was prompted by a disease outbreak in February 2003 in Australian native Murray cod (Maccullochella peeli peeli) resulting in losses of up to 90% in two groups of fingerlings at a Victorian aquaculture facility. Histology revealed pathology consistent with Megalocytivirus infection, including those found in ornamental fish, and distinct from Epizootic Haematopoietic Necrosis Virus – the only systemic piscine iridovirid known to occur naturally in Australia. Using a molecular epidemiological approach, DNA was extracted from samples collected during the disease outbreak, and PCR primers were designed to amplify DNA from the major capsid protein (MCP), ATPase and RNA polymerase genes as well as CY15 and IRB6 amplicons. Sequencing results from PCR products indicated that there was extremely close homology between the Murray cod iridovirus (MCIV) isolate and viruses in the genus Megalocytivirus, particularly infectious spleen and kidney necrosis virus (ISKNV) and dwarf gourami iridovirus (DGIV) sequences in Genbank. Screening of routine gourami mortalities from pet shops in Sydney, using PCR primers designed against the MCP gene, revealed 22% were positive for Megalocytivirus DNA. When organ filtrates of PCR positive samples were injected intraperitoneally into Murray cod juveniles, 29 of 30 died within 21 days, and cohabitation of juvenile Murray cod with live gouramis resulted in transmission of Megalocytivirus through water and death of 11 of 30 Murray cod. Sequencing of this Megalocytivirus isolate derived from dwarf gouramis revealed 99.95% similarity, over 4527 nucleotides, between this isolate and MCIV, suggesting a likely origin of MCIV through the trade in ornamental fish.
S13-6
Correlations of
Capture, Transport, and Nutrition with Vertebral Deformities in Captive
Sandtiger Sharks (Carcharias taurus)
PA Anderson*1, DR Huber2, DE
Noaker2 and IK Berzins3
| 1 |
The Florida Aquarium Center for Conservation, Tampa,
Florida 33602 USA panderson@flaquarium.org
|
| 2 |
The University of Tampa, Tampa, FL USA dhuber@ut.edu dnoaker@ut.edu
|
| 3 |
John G. Shedd Aquarium, Chicago, IL USA Iberzins@sheddaquarium.org
|
Captive sandtiger sharks (Carcharias taurus) commonly develop vertebral deformities. U.S. public aquaria participated in a
three-stage epidemiological study of resident sharks: 1) a history and husbandry survey, 2) hematology, clinical
chemistry, and radiography requested during health exams, and 3) a necropsy
protocol upon expiration or euthanasia. Eighteen aquaria submitted data and/or samples from 73 specimens,
including 19 affected sharks (35.2%). Smaller sharks caught off the Rhode Island coast and/or by pound net
demonstrated a higher prevalence of deformity than larger sharks caught from
other areas or with hook and line. Affected sharks had lower leukocrits, disputing an infectious etiology,
but suggesting a chronic stress response to the condition. Affected sharks were also deficient in
zinc, potassium, and vitamins C and E relative to healthy sharks. Zinc and vitamin C play critical roles
in proper cartilage/collagen development and maintenance. Flexural stiffness of
intact vertebral columns and compressive stiffness, yield strength, and yield
strain of individual vertebrae from healthy animals were greater than those
from deformed animals; attributable to deficits in both the quantity and
distribution of mineral within the vertebrae of deformed specimens.
Additionally, compressive stiffness and ultimate
strength of vertebrae from healthy specimens were still lower than those of
other species tested, suggesting either a species-specific predisposition for
vertebral deformity, or abnormalities in tissue modeling due to nutrient
deficiencies from captive diets. Hence, aquarists should consider choice of collector and size at
capture, and should supplement diets to provide nutrients required for proper
development and maintenance of cartilaginous tissue.
| SESSION
14. MYCOSES |
[TOP] |
S14-1
Ulcerative Mycoses
Caused by Aphanomyces invadans and Ochroconis sp. in Wild and Cultured
Marine Fish Species.
JP Hawke*, W Baumgartner, and E Soto
| Department
of Pathobiological Sciences, LSU School of Veterinary Medicine, Baton Rouge,
LA, USA. jhawke1@lsu.edu, wbaumg1@tigers.lsu.edu, esoto1@lsu.edu.
|
Ulcerative
mycotic diseases are described from wild populations of red drum Sciaenops ocellatus and black drum Pogonius cromis and cultured cobia Rachycentron canadum. This is the first
report of an invasive fungal infection in cobia. The diseases were diagnosed
from specimens voluntarily submitted to the Louisiana Aquatic Diagnostic
Laboratory at the School of Veterinary Medicine, Louisiana State University.
Adult red drum and black drum were collected from the inshore marsh habitat in
Louisiana and the juvenile cobia (10-15 cm) were from a recirculating culture
system. Specimens presented with
sharply demarcated, circular to irregular, ulcerative lesions in the skin and
penetrating into the underlying muscle tissue. Oomycete and dematiaceous hyphomycete fungi were isolated by
aseptic necropsy on peptone yeast glucose (PYG) agar from infected muscle
tissue. The Aphanomyces colonies grew
slowly on the agar medium at 25°C and produced white velvety colonies that grew
above and below the agar surface. The hyphomycete fungus Ochroconis sp. also grew slowly at 25°C and produced brown
pigmented colonies on the surface of PYG agar. Histopathology of Aphanomyces infected fish revealed
non-septate hyphae penetrating deep into the muscle surrounded by granulomatous
inflammation and multinucleated giant cells. Histopathology of Ochroconis infections revealed
melanized, septate, fungal hyphae surrounded by epithelioid granulomatous
inflammation in skin, muscle, intestine, and kidney tissues. Identification of the pathogens was
accomplished using a combination of morphological characters and molecular
analysis by amplification of the internal transcribed spacer (ITS) region
located between the 18S and 28S rRNA genes and sequencing.
S14-2
Exophialiosis in
Bonito (Sarda chiliensis) from a
Public Aquarium
K
Hirokawa*1, M Murray2, A Reed1 and ES Weber
III1
| 1 |
Aquatic Animal Health
Laboratory, University of California at Davis, Davis, CA, USA
khirokawa@gmail.com, aigialos@yahoo.com, epweber@undavis.edu
|
| 2 |
Monterey Bay Aquarium,
Monterey, CA USA MMurray@mbayaq.org
|
Two wild-caught pacific bonito (Sarda chiliensis) from Monterey bay died during the quarantine
without prior clinical signs. The gross necropsy findings included skin
ulceration, enlarged spleen, swollen kidney with black nodules, granulomas in
the spleen, and black fungal hyphae were identified in renal impression smears
using light microscopy. Tissue samples were submitted for histopathologic
examination to the University of California, Davis. The results of
histopathologic examination revealed black septate fungus in the kidney and
skin. The kidney and skin cultures grew exophiala species, and Vibrio alginolyticus and Photobacteruim damsela from kidney,
skin, and blood. Exophiala samples were sent to a laboratory in Texas for
speciation. Previously, exophiala species have been cultured from marine and
fresh water fish including striped jack, Atlantic salmon, sandlance, smooth
dogfish, and leafy sea dragon in captive and cultured animals. In the current
case, while the origin of infection is not yet established for the bonito, the
previously reported cases all involve captive or cultured fish. Thus far, no
wild animal cases of exophialiosis have been reported, and nosocomial infection
is suspected in the current case. As far as we are aware, this is the first
case of exophialiosis reported in bonito. Exophiala are ubiquitous in soil and
water, and is a significant pathogen which can cause systemic disease,
resulting in significant mortality and morbidity in some fish. It has also
caused severe disease in valuable aquarium fish such as sea dragons. The
prevention and control of systemic mycotic disease is critical in maintaining
healthy aquarium animal collections.
S14-3
Effects of
Environmental Temperature on the Kinetics of Ichthyophoniasis in Juvenile
Pacific Herring (Clupea pallasii)
JL
Gregg*1, JJ Vollenweider2, CA Grady1, RA
Heintz2, and PK Hershberger1
| 1 |
U.S. Geological Survey,
Western Fisheries Research Center, Marrowstone Marine Field Station, Nordland,
WA USA jgregg@usgs.gov
|
| 2 |
NOAA National Marine
Fisheries Service, Alaska Fisheries Science Center, Auke Bay Laboratories,
Juneau, AK USA
|
Ichthyophonus, a widely distributed
parasite of the Atlantic and Pacific Oceans, significantly impacts commercially
important fish stocks through acute epizootics (fish kills), chronic persistent
mortality, reduced fitness, and reduced product quality. Primarily considered a disease of older
adult cohorts, Ichthyophonus may also
play a role in depressing survival of juvenile fish and thus limiting their
recruitment to fisheries. Here we
present the results of experiments conducted to examine the effect that Ichthyophonus has on juvenile Pacific
herring (Clupea pallasii) undergoing
a simulated over-winter fast. These experiments were carried out at three temperatures and have given
us new insights into the effects that environmental temperature has on disease
processes and mortality. Temperature had no effect in herring with established disease; however,
temperature did affect mortality in newly infected fish. Both lowering and raising the
temperature tended to reduce mortality. Warmer temperatures likely improve host immune function, while cooler
temperatures slow parasitic growth. When the exposure dose was reduced, we detected an inverse relationship
between infection prevalence and temperature. These data indicate that the environmental temperature
at the time of parasite exposure may determine the prevalence of disease in
wild fish populations over ensuing months and years.
S14-4
Molecular Detection
of Ichthyophonus in Walleye Pollock and the Search for
Planktonic Reservoirs Using Quantitative PCR
VL Lowe*1,2, CS Friedman1 and
JF Morado2
| 1 |
School of Aquatic and
Fishery Sciences, University of Washington, Seattle, WA USA carolynf@u.washington.edu
|
| 2 |
NOAA Fisheries, National
Oceanic and Atmospheric Administration, Alaska Fisheries Science Center,
Seattle, WA USA Vanessa.Lowe@noaa.gov, Frank.Morado@noaa.gov
|
Ichthyophoniasis is a disease of economic
significance in both wild and cultured fisheries and is caused by the protist, Ichthyophonus.
The parasite has low host specificity and is acquired through feeding on
infected prey. Zooplankton may act as potential reservoirs for Ichthyophonus and aid in transmission to
fish hosts. An Ichthyophonus-like
parasite was identified in walleye pollock, Theragra chalcogramma, an
ecologically and commercially important North Pacific fish. Intense parasite
infections can adversely affect product quality by altering cooked fillet
texture, causing increased fillet screening by the industry. To estimate Ichthyophonus prevalence in walleye
pollock and identify infected prey items, a probe-based quantitative PCR (qPCR)
assay has been developed based on the 18S rDNA gene of Ichthyophonus. Paired samples for qPCR and histology were collected
from the Bering Sea in 2006 and 2007 to compare detection methods. Age-0
pollock (<11cm) were examined by histology and plankton samples collected in
2007 were screened using qPCR. Parasite prevalence in age-0 fish was estimated
at 2%, and increased with fish size to 28% in adult pollock. Suspect Ichthyophonus has been detected in mixed plankton samples, but
these results should be approached with caution without confirmation by other
methods. On-going studies are
focused on parasite detection in sorted plankton taxa found in the diet of
age-0 pollock. Validation of this assay delivers an additional tool for Ichthyophonus research and may aid in
the characterization of alternative infection pathways.
| SESSION
15. PARASITOLOGY IV: Fish 1 |
[TOP] |
S15-1
A New Microsporidian from Common Stingray Dasyatis
pastinaca in the Mediterranean Sea
A Diamant*1,,
M Goren2, MB Yokeş3, BS Galil4, Y Klopman2, D Huchon2, A Szitenberg2 and SÜ
Karhan5
| 1 |
National Center for Mariculture, Israel Oceanographic and Limnological Research, Eilat, Israel diamant@agri.huji.ac.il |
| 2 |
Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel. |
| 3 |
Haliç University, Faculty of Arts & Sciences, Department of Molecular Biology & Genetics, Okmeydani, 34384 Istanbul, Turkey. |
| 4 |
National Institute of Oceanography, Israel Oceanographic and Limnological Research Haifa, Israel |
| 5 |
Division of Hydrobiology, Department of Biology, Faculty of Science, Istanbul University, Istanbul, Turkey. |
A new microsporidian infecting the Mediterranean
common stingray Dasyatis pastinaca was found in Iskenderun Bay, Turkey.
Fish samples were taken by bottom trawl during summer 2009. The parasite
invaded the stingray disc muscles, producing spindle-shaped subcutaneous
swellings and tumor-like masses measuring up to 11 cm in length. The masses
contained a yellowish-white substance which contained degraded host tissue and
parasites developmental stages. The ripe spore contained a single nucleated
sporoplasm and had a large posterior vacuole. Prevalence was 21% in a sample of
143 host individuals examined. While normal appearing, uninfected individuals
reached 3.8 kg, all infected stingrays were within the weight class of 300 -
800 g. There is only a single previous report of a microsporidian in a
cartilaginous fish: and infection in captive sharks in a public aquarium. The
present study reports on the first documentation from a wild elasmobranch
population. Analyses of the 16S rRNA gene indicated that this microsporidian
belongs to the Pleistophoridae. It clustered with species of the genera Heterosporis Schubert, 1969 and Ovipleistophora Pekkarinen, Lom & Nilsen, 2002. However, the morphology, development and
host differed distinctly from all currently known species, and thus the new
genus Dasyatispora is erected, with the new species named D.
levantinae gen. et sp. nov.
S15-2
Diversity of
Microsporidia Among Marine Parasitic Copepods (Caligidae) in the Northeast
Pacific Ocean
SRM Jones*, G Prosperi-Porta and E Kim
| Department of Fisheries and
Oceans, Pacific Biological Station, Nanaimo, British Columbia Canada. simon.jones@dfo-mpo.gc.ca
|
Desmozoon
lepeophtherii is a microsporidian parasite of the salmon louse (Lepeophtheirus salmonis) infesting farmed Atlantic salmon in
Scotland and Norway. Microsporidia
with similarities to D. lepeophtheirii were observed in salmon lice from Puget Sound, Washington. The prevalence of microsporidia in
salmon lice was further documented by examining specimens from Puget Sound (PS)
and British Columbia (BC), Canada, and those belonging to five other Lepeophtheirus species from BC. Analyses of the small subunit ribosomal
gene amplified from 157 copepods revealed three distinct microsporidian
sequences that differed in host association, prevalence and geographic
distribution. Type A was found
only in L. salmonis with a prevalence
of 15% in PS and 4% in BC. The genetic distance of Type A from that of D. lepeophtherii was 0.004. Type B occurred in L. salmonis (43% prevalence in PS, 30% in BC), L. cuneifer (50% prevalence) and L. parviventris (30% prevalence). The genetic distance between Types A and B was 0.525. Type C was obtained from a single
specimen of L. hospitalis and
differed from Types A and B with genetic distances of 0.507 and 0.287,
respectively. Type A clustered
with strong support among the enterocytozoonids whereas Types B and C were weakly
supported among microsporidia of marine origin. Microsporidia associated with sequence types A and B
displayed distinct patterns of development and tissue distribution. Thus a geographic variant of D. lepeophtherii and two novel
microsporidia occur among caligid copepods in the northeast Pacific Ocean.
S15-3
Effect of Essential
Oils on the Viability of Caligus
rogercresseyi Using a Novel in vitro Assay Based on the Inhibition of Lice Frontal Filament Formation.
J Troncoso*
and J González
| EWOS Innovation, Colaco Research Center, Av. Benavente 550 piso 10A,
Puerto Montt, Chile jose.troncoso@ewos.com
|
Three in vitro assays were performed to assess a product based on a mixture of essential oils
obtained from cinnamon, eucalyptus and oregano, against the sea lice Caligus rogercresseyi. Two assays were
conducted to determine the LD50 of the oils against copepodids and adult lice,
respectively. The first trial was performed using 10 different doses of the
essential oil mix ranging from 0.175 to 175 μL/L. In the second assay,
seven different doses of the essential oil mix were tested, ranging from 0.175
to 52.5 μL/L. A third assay was performed using a novel method to evaluate
the degree of inhibition of the formation of the frontal filament of
copepodids. In this last assay, four different concentrations of the essential
oil mix were tested: 0, 0.175, 6.869 and 13.563 μL/L. The essential oils
induced mortality of copepodids and adults of Caligus rogercresseyi. The
calculated LD50 against copepodids of C. rogercresseyi was 21.6 ppm
(18.5-25.8, 95% credible interval) at 48 h of exposure. Similarly, the
calculated LD50 against adults of C. rogercresseyi was 22.1 ppm
(18.4-27.4, 95% credible interval) at 48 h of exposure. The method to test the
inhibition of the formation of the frontal filament was used as a measure of
copepodid viability. The dose of essential oils needed to inhibit the formation
of the frontal filament in 80% was significantly lower than the LD50 required
to kill copepodids (0.175 ppm vs 21.6 ppm). The method to determine the
capacity of essential oils to inhibit the formation of the frontal filament of
copepodids of C. rogercresseyi proved
to be a sensitive test to evaluate potential anti-sea lice compounds
S15-4
Conceptual Framework
for the Use of Parasites of Fishes as Bioindicators of Acute and Chronic
Environmental Perturbation after the 2010 Deepwater
Horizon Oil Spill (DHOS) in the Gulf of Mexico
SA
Bullard*1 and GW Benz2
| 1 |
Department of Fisheries and
Allied Aquacultures, Aquatic Parasitology Laboratory, Auburn University,
Auburn, AL 36849, USA ash.bullard@auburn.edu
|
| 2 |
Department of Biology,
Middle Tennessee State University, Murfreesboro, TN, USA gbenz@mtsu.edu
|
Fish parasites comprise a large portion of marine
biodiversity but have been virtually ignored as Gulf of Mexico (GoMex)
bioindicators. Shifts in parasite diversity, prevalence, and intensity
resulting from the DHOS could indicate spill-related changes to water quality,
abundances and immunological health of free-living organisms, or the GoMex food
web. Ectoparasites with direct life cycles (no intermediate host or food-web
mediated transmission) may be sentinels for acute spill effects, as they are
typically small, have high surface area to volume ratios, and remain immersed
in seawater. Endoparasites with indirect life cycles (intermediate host[s]
required) involving food-web mediated transmission may be sentinels for
detecting chronic spill effects, as they reside in a host where they are less
vulnerable to toxins and have larvae requiring predator/prey transmission.
Environmental perturbations affecting food-web structure (chronic effect)
typically impact endoparasites more than ectoparasites. A ratio of the numbers
of ectoparasite species to endoparasite species per fish species per geographic
locality has been informative regarding oil pollution events: a relatively
smaller ratio signals acute environmental effects (i.e., ectoparasites die;
endoparasites survive), a relatively larger ratio signals chronic environmental
effects (i.e., ectoparasites recolonize hosts as water normalizes;
endoparasites are extirpated as food-web links are broken by loss of
intermediate hosts). Our recently funded RAPID Response study (National Science
Foundation grant DEB-1048523) focuses on these matters in hope of gaining
critical new insights about parasites as bioindicators on a regional, basin
scale while providing insights on the environmental “ripple effects” of the
DHOS. Supported by NSF grant DEB-1048523.
S15-5
Molecular Identification, Ultrastructure and
Histopathological Studies of Microsporidian Parasite of Transparent and Albino Puntius tetrazona
O Chang, K Li, F Wang, C Liu and S Wu
| Pearl
River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou,
Guangdong, China changouqin@163.com
|
Specimens of the transparent and albino Puntius tetrazona, obtained by
intraspecies hybridization at the Pearl River Fisheries Research
Institute, were discovered to have numerous cystic masses in their muscle
tissue. After pressure was applied
to the muscle tissue, many sporocysts and spores were observed. We set out to identify these spores
using ultrastructural, histopathological, and molecular methods. Under a transmission electron
microscope, the mature spores were uninucleate, ovoid or pyriform, and
approximately 7-8 μm in length and 3-4 μm in width. They possessed a polar tube surrounding
the posterior vacuole, which occupied about half of the spore’s total
volume. Under a scanning electron
microscope, sporocysts with a thick, dense wall were observed in the muscle
tissue, and many mature ovoid spores were present within the sporocysts. To
further characterize the parasite, we prepared histopathological sections from the
muscle of infected fish, and stained them with hematoxylin and eosin. The spores and sporocysts were in
different developmental stages, all of which developed within a wall-covered
structure called the sporophorocyst. Together, these ultrastructural and
histopathological features suggested that the spores represent a microsporidian Pleistophora parasite, which was
confirmed by molecular methods. Furthermore, when healthy transparent and albino Puntius tetrazona were fed and injected with spores isolated from
infected muscle, the ultrastructural and histopathological features described
above were observed. Our results
suggest that the microsporidian parasite was Pleistophora, and that the transparent and albino Puntius tetrazona would be a good model
organism for Pleistophora infection.
| SESSION
16. VIROLOGY I |
[TOP] |
S16-1
Isolation and Identification
of a Novel Reovirus by Using a Newly Established Cell Line Derived from Kidney
of Channel Catfish, Ictalurus punctatus, Rafinnesque
LB
Zeng*, J Xu, XL Li, XS Luo, Y Xiao, and Y Zhou
| Yangtze
River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Jingzhou
City, Hubei China zenglingbing@gmail.com
|
A cell line, designated CCK, derived
from the kidney of Channel catfish, Ictalurus
punctatus, Rafinnesque, has been newly established and
characterized. A novel reovirus has been isolated in CCK cells from
channel catfish fingerlings suffered with severe hemorrhage, and has been
identified as channel catfish reovirus (CCRV). In an artificial infection test
in fish, the typical naturally occurring symptoms of channel catfish hemorrhage
could be reproduced. The isolated virus could cause typical cytopathic effect
in CCK cells. Electron microscopy observation of ultra-thin section samples of
CCRV infected CCK cells revealed that the virus replicated in the cytoplasm,
was present in crystalline arrays, and had a non-enveloped double capsid with a
diameter of 60-70nm. Frozen-thawed, 56oC 1h, chloroform and ether
had no significant effects on CCRV titre, 65oC 1h could
significantly inactivate the viral infectivity. The CCRV genome SDS-PAGE
analysis and nuclease sensitivity test showed that the virus genome was the
same as that of viruses in Aquareovirudae, and consisted of 11 segments of dsRNA assigned into three
classes L1, L2, L3; M1, M2, M3 and S1, S2, S3, S4, S5 with a range of size from
0.9 to 4.4bp. The cloning and sequencing of the CCRV S4 segment indicated the
nucleic acid number of CCRV S4 was 909bp, which was exactly the same in length
as that of GCRV S4 (AF403396) and GSRV S4 (AF403407) segments. The BLAST of
CCRV S4 sequence in NCBI GenBank showed that it had a 99% and 90% similarity in
sequence to the GCRV S4 and GSRV S4 segments, respectively.
S16-2
Re-activation of Koi Herpesvirus (KHV) in
Persistent Infected Fish Used for Virus Detection After Non-lethal Sampling
SM Bergmann*
| Friedrich-Loeffler-Institut
(FLI), German Reference Laboratory for KHVD, Institute of Infectology, Federal
Research Institute for Animal Health, Greifswald-Insel Riems, Germany
sven.bergmann@fli.bund.de
|
KHV
disease (KHVD) has spread world-wide by transfer of infected but non-diseased
carp (Cyprinus carpio). Due to
limited sensitivity of CCB or KF-1 cells, molecular tools represent the “gold
standard” for KHV detection. As a
herpesviral characteristic, fish become “healthy” persistent carriers after
infection. In persistent phase, virus load can decrease often to 5 genomic
particles measured by real-time PCR. The reaction in fish after KHV infection
depends on water temperatures, virus concentration and virulence as well as on
immune status of the fish. While concentrations such as 105 TCID50 / ml at 23°C may lead to a severe KHVD outbreak, concentrations of 102 TCID50 / ml at 12°C cause often a persistent asymptomatic infection
(Meyer 2007). Virus eclipse represents a severe diagnostical problem. Routinely
used detection methods are in an unfavourable position because of
false-negative results. To overcome this and to provide a loophole, KHV
infected carp which had shown KHVD for 2 - 3 weeks p.i., were kept under the
same conditions (20°C, re-circulating system) further on. These carp, without
any sign of KHVD, were controlled weekly for KHV presence by non-lethal
sampling. In a basin, fish were netted to simulate transportation. Daily, gill
swabs and dropping samples were taken and investigated by molecular methods
(Bergmann et al. 2010). In daily taken swabs an individually different increase
of the virus load was detected between days 1 and 3 after netting. In
droppings, KHV was only detected at day 3 after netting. Generally, virus
concentration decreased dramatically in swabs from day 4 after netting. Even
this stress did not provoke KHVD. In conclusion, it was shown that non-lethal
sampling methods after stress induction are a useful tool for diagnosis of KHV
in persistent infected fish without killing those carp.
S16-3
Detection of KHV in Healthy Non-carp Carrier Fish in Polish Inland
Waters by PCR.
J
Kempter, M Kielpinski, R Panicz* and J Sadowski
| Department
of Aquaculture, West Pomeranian University of Technology, Szczecin, Poland remigiusz.panicz@zut.edu.pl
|
Wild-
as well as cultured fishes were examined for the presence of the
Koi-Herpes-Virus (KHV) genome. The epizootic map of the Odra River drainage was
compiled based on the results covering 17 fish-rearing facilities and 49
open-water sites. In total, 525
fish specimens were studied. A potential for transmitting the virus to common
carp by: vimba, grass carp, and silver carp was also tested. The transmission
experiment was based on co-habitation of specific pathogen free (SPF) carp with
vimba, tench, and grass carp. The studies were carried out in three
experimental treatments, differing in temperature regimes, each in two
repetitions. Obtained results clearly showed that virus transmission in tench
and vimba aquariums were present in all temperatures (18, 23, 27 °C). The second set of experiments focused
on testing inter-specific hybrids (Cyprinus carpio × Carasius auratus, Cyprinus
Carpio × Carasius carasius) for their resistance to KHV infection and for their chances to develop
clinical signs of the disease. The fish farms, diagnosed herewith as KHV
positive, should not rear carp for a minimum of two seasons. In the meantime, they may culture other
fish species after performing disinfection procedures. It should be emphasized
that a full rearing potential of an aquaculture enterprise, following a disease
outbreak, is very costly and it cannot be successful without public money.
Leaving such fish farm on its own, without public support and with a
KHV-positive label would result in the farm bankruptcy. The data
gathered within the present project were compiled to create the epizootic map
of the Odra River drainage, which is intended to provide the relevant
information to fish farmers and users of open waters about the current
KHV-infection state of this drainage.
S16-4
Analysis of
Attenuation in a Gene 50 Deleted Channel Catfish Virus (Ictalurid herpesvirus
1) Construct Suggests That More is Involved than Lack of Gene 50 Expression.
Larry
A. Hanson*1, Nathalie Vanderheijden2, Joseph A. Martial2,
Dusan Kunec1 and Lorelei Ford1
| 1 |
Department of Basic
Sciences, College of Veterinary Medicine, Mississippi State University,
Mississippi State, MS, USA hanson@cvm.msstate.edu
|
| 2 |
GIGA-Research - Unité de
Biologie Moléculaire et Génie Génétique, Université de Liège, Sart Tilman,
Belgium
|
We had previously established a recombinant channel
catfish virus (CCVdl50) that was highly attenuated and has potential for use as
a CCV vaccine (Vanderheijden et al. US patent 6,322,793). This
virus was established by replacing the gene 50, which encodes an excreted glycoprotein,
with Escherichia coli lac Z gene. In this study we evaluated the cause of the attenuation
observed in CCVdl50. Because gene 50 is close upstream to the dUTPase gene, we
generated CCV recombinants deleted in just the 5’ region of gene 50 using infectious
BAC technology. These recombinants did not express the excreted glycoprotein
but were only moderately attenuated, suggesting that combined effects may have
attenuated CCVdl50. To further evaluate this phenomenon we generated a series
of CCVdl50 revertants by co-transfecting the gene 50 encoding region with
CCVdl50 genomic DNA into CCO cells. The resulting revertants expressed the gene 50 glycoprotein and regained
some virulence. However they were
still highly to moderately attenuated compared to the wild type. The revertants demonstrated some
variation in the size of the gene 50 and we hypothesize that the repetitive
nature of gene 50 sequence caused sequence variability in the revertants,
confounding this analysis. The
combined data suggest that the attenuated nature of CCVdl50 is caused by
interrupting gene 50 expression and another uncharacterized factor.
S16-5
A Fatal Reovirus is
Highly Prevalent in Blue Crab, Callinectes
sapidus, in Aquaculture and Soft Shell Crab Production Facilities
HA
Bowers1, L Carrion2, GA Messick3, O Zmora4,
A Hanif1, R Jagus1 and EJ Schott*1
| 1 |
Institute of Marine and Environmental Technology,
University of Maryland Center for Environmental Science, Baltimore, MD USA schott@umces.edu
|
| 2 |
Coveside Crabs, Inc., Dundalk, Maryland USA ldcupuc@verizon.net
|
| 3 |
Center for Coastal Environmental Health &
Biomolecular Research at Charleston, USDOC/NOAA/NOS/NCCOS, Oxford, Maryland
USA gretchen.messick@noaa.gov
|
| 4 |
Department
of Marine Biology, University of Maryland Baltimore County, Baltimore, MD USA zmorao@umbc.edu
|
The blue crab, Callinectes
sapidus, supports a $150 million/year fishery in Atlantic and Gulf states
of the USA. Fishing pressure has
necessitated harvest restrictions in many states. Fishermen can increase the
value their catch by setting aside pre-molt animals (“peelers”) for production
of high value soft-shell crabs. However, crabs in soft-shell production facilities typically suffer
mortalities of 25-50%. We
suspected that virus infections were responsible for some mortalities. By
enriching double stranded RNA from crabs, we uncovered a reo-like viral RNA
genome pattern in over 50% of the dead and dying animals but in fewer than 5%
of healthy peelers. Virus passage
was accomplished by injection of crab homogenate, and was always fatal. Electron microscopic analysis of
virus-injected crabs shows reo-like virions within hemocytes. Analyses of dying peelers from other
soft-shell systems in MD, GA and FL revealed a >60% prevalence of
reovirus. We cloned part of the
reovirus genome, and designed and validated a sensitive Rt-PCR to detect
it. Using these molecular tools,
we have screened wild blue crabs collected by a NOAA/NCCOS project to assess
Chesapeake Bay health. The
prevalence of reovirus was <4%, though a single sample showed a prevalence
of 66%. The effects of this virus on blue crab populations in the wild may be
underappreciated. We are continuing to monitor and conduct transmission
experiments to better understand the impact of this virus on wild crabs and to
understand why the prevalence in captive crabs is so high.
S16-6
Metagenomic Studies
Reveal Novel Viruses in Shrimp
TFF
Ng*1, S Alavandi2 and M Breitbart1
| 1 |
University of South Florida,
Saint Petersburg, FL, USA terryfeifan@gmail.com
|
| 2 |
Central Institute of Brackishwater Aquaculture,
Chennai, India
|
The emergence of viral pathogens is a serious threat
to aquaculture and fisheries worldwide. However, our response to viral
pathogens has been largely reactive – a new pathogen is usually not discovered
until it has already reached epidemic proportions. Current diagnostic methods
such as PCR, immunological assays, and pan-viral microarrays are limited in
their ability to identify novel viruses. Viral metagenomics, which involves
viral particle purification and shotgun sequencing, has proven to be useful for
describing novel viruses. Using viral metagenomics, this study investigated
the virus diversity in hepatopancreas, heart and hemolymph from asymptomatic
wild shrimp (Farfantepenaeus duorarum)
collected from Tarpon Springs, Florida. Metagenomic sequencing of DNA and RNA
viruses purified from these shrimp led to the discovery of a novel circovirus
and two novel nodaviruses. The novel shrimp circovirus had a single-stranded
DNA genome of 1955 nucleotides, and shared less than 50% amino acid identity to
any known viruses in the Genbank database. All previously described
circoviruses infect birds or pigs, making this shrimp circovirus the first
circovirus described in an invertebrate. The shrimp circovirus represented a
phylogenetic branch distinct from the known avian and porcine circoviruses. Two novel nodaviruses, which shared
less than 60% amino acid identity to known shrimp nodaviruses and likely
represent a novel virus genus, were also identified in the shrimp. Future
research needs to examine the prevalence and ecological impact of these
viruses; however, this study has demonstrated the potential of viral
metagenomics to characterize novel viruses in aquatic species, and its
application for viral disease prevention and monitoring.
| SESSION
17. DIAGNOSTIC CHALLENGES |
[TOP] |
Special Session
DIAGNOSTIC CHALLENGES
| SESSION
18. IMMUNOLOGY II |
[TOP] |
S18-1
Construction and
Screening of a Library of Attenuated Mutants in Edwardsiella ictaluri, Identification of Genes Involved in
Bacterial Invasion and Potential as Vaccines
S Menanteau-Ledouble* and ML Lawrence
| Department
of Basic Sciences, College of Veterinary Medicine, Mississippi State
University, MS, USA smenanteau@cvm.msstate.edu, lawrence@cvm.msstate.edu
|
Attachment and invasion of host cells are very
important factors in the virulence of a number of pathogenic bacteria.
Previously, we investigated these mechanisms in the enterobacterial catfish pathogen Edwardsiella ictaluri by constructing
a random MarTxT7 library of E. ictaluri mutants
harboring the bioluminescence plasmid pAKLux. This library was then screened in
a series of three nested challenges to identify mutants that showed decreased
ability to colonize catfish epithelium. We identified 18 colonization-deficient
mutants that were then confirmed to be attenuated in another challenge. Gene mutations were identified by
single primer PCR and sequencing and included a dipeptide transport system, an
outer-membrane lipoprotein, as well as a putative adhesin. Four of these mutants were used in a
vaccination trial using 14 day old catfish fry followed 4 weeks later by
experimental exposure to wild-type E.
ictaluri. Results suggested
that these mutants are attenuated and provide a limited level of protection
against ESC. This study allowed not only for the identification of several
genes involved in skin colonization but also yielded several promising
candidates for the development of vaccines against this challenging pathogen
S18-2
Efficacy of a Parenteral and Oral DNA Vaccine in Fish for Aquatic
Mycobacteriosis
SA Smith *
| Aquatic
Medicine Laboratory, Department of Biomedical Sciences and Pathobiology,
Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic
Institute and State University, Blacksburg, Virginia USA stsmith7@vt.edu
|
Aquatic mycobacteriosis is a chronic progressive
disease that leads to systemic infections and death. It is an economically important bacterial disease of
virtually all species of freshwater, brackish and marine fish, and is most
commonly caused by the intracellular pathogens, Mycobacterium marinum, M. fortuitum, or M. chelonae. While the
immune response against aquatic Mycobacterium spp. has been characterized, an effective vaccine against mycobacteriosis
has not been developed. Because of
the potential to induce a protective immune response against intracellular
pathogens, a DNA vaccine was chosen as an appropriate construct for aquatic
mycobacteriosis. The vaccine was assembled by cloning the M. marinum 85A gene in the pcDNA 3.1 eukaryotic expression vector.
Hybrid striped bass (Morone saxatilis x M. chrysops), a documented susceptible species, were used as a model
to test the vaccine. Intramuscular
or intraperitoneal vaccinations of the pCMV-85A construct were administered on
days 0 and 14, along with appropriate positive and negative control
vaccinations. Blood samples were obtained every two weeks from the fish to
evaluate the humoral immune response by ELISA. On day 70, the fish were
challenged with live M. marinum. Morbidity and mortality observations,
along with routine histological examination and splenic bacterial counts were
used to determine the relative degree of protection among the different
vaccination groups. Experimental groups administered the DNA vaccine
demonstrated a reduced severity of pathology and a slower progression of
disease. Based on these results, the vaccine was compounded with chitosan or
polyactide-co-glycolide (PLG) nanoparticles and orally administered to separate
groups of hybrid striped bass. Blood samples were again obtained every two
weeks from the fish to evaluate the humoral immune response by ELISA. Only the
fish administered the PLG-DNA vaccine orally had an increased humoral immune
response similar to fish administered the DNA vaccine intramuscularly.
S18-3
The Identification and
Characterization of Interleukin-17 Family Genes and Receptors in Fish
H Korenaga*1, T Kono2, H Kuse1, H
Takayama1 and M Sakai1
| 1 |
Faculty of Agriculture, University of Miyazaki, 1-1
Gakuenkibanadai-nishi, Miyazaki, Japan hiro102wes@gmail.com
|
| 2 |
Interdisciplinary Research Organization, University
of Miyazaki, 1-1 Gakuenkibanadai-nishi, Miyazaki, Japan tkono@cc.miyazaki-u.ac.jp
|
Interleukin-17 (IL-17) is a
new cytokine family with six members (A-F) identified in human. The IL-17
family has been classified as pro-inflammatory cytokines because of its ability
to induce the expression of many mediators of inflammation properties. The
uniqueness of IL-17 family as a separate cytokine family was proved by the
isolation of a receptor, IL-17R, which binds IL-17 family and is required for
its signaling. Five additional receptors (IL-17RA-E) have been identified in
human. In fish, there are few reports on the identification for IL-17 family
ligands (L) / receptors (R). In this study, we describe a detailed
identification, cloning and genomic characterization of five IL-17 family
ligands from medaka (Oryzias latipes)
and genomic characterization of IL-17 family receptors from Fugu, zebrafish, stickleback, and medaka
genome database. The screening of medaka genome database revealed the existence
of five scaffolds containing six IL-17 ligand genes (AF-1, 2, 3, C, D and N).
Among these genes, IL-17N identified the particular gene in teleost from
analyzing the available genome database. Phylogenetic analysis using
neighbor-joining method showed that medaka IL-17 family ligands clustered with
other teleost IL-17 counterparts. Expression analysis by RT-PCR revealed
expression of IL-17 family ligand genes in normal tissues and LPS-stimulated
intestine. The expression patterns of IL-17 ligand family genes were different
with other teleosts. In the LPS-stimulated intestine, the expression of IL-17
family genes was significantly increased. Moreover, the existence of IL-17
family receptors (RA, RC, RD and RE) was isolated from fish genome database.
S18-4
Pathogen Recognition
Proteins in Rainbow Trout Plasma (Onchorhynchus mykiss)
S Russell*, K Young, M Edwards, A Peterson, A Reid
and JS Lumsden.
| Fish Pathology Laboratory, Department of
Pathobiology, University of Guelph, Guelph, ON, Canada sprussel@uoguelph.ca
|
Soluble innate immune
proteins are essential components of defense and play a critical role
recognizing and eliminating infectious
agents. Using binding assays, 2D
PAGE and mass spectrometry we identified two rainbow trout plasma proteins,
ladderlectin (RTLL) and intelectin (RTInt), which bound chitin and various
Gram-negative bacteria that cause disease in trout. Similar studies found RTLL,
but not RTInt, bound to purified viral hemorrhagic septecimia virus. RTLL is a C-type plasma lectin while
RTInt is an X-type lectin with homology to carp, human, and murine intelectins.
Both lectins show mannose specific, calcium dependent binding to target
surfaces. To quantify both proteins, rabbit polyclonal antibodies were produced
for use in enzyme immunoassays. Neither lectin was induced in trout experimentally
infected with Aeromonas salmonicida or injected with either A. salmonicida lipopolysaccharide (LPS) or a commercial A.
salmonicida vaccine. However,
in tissues of clinically diseased rainbow trout, both proteins were localized
by immunohistochemistry in intimate association with bacteria, fungi and
protozoa and within inflammatory leukocytes. These studies demonstrate that
both proteins are not detectably induced during an acute phase response but
they provide further evidence that RTLL and RTInt have microbial binding
abilities similar to other soluble pattern recognition receptors in higher
vertebrates.
S18-5
Antibody Response in
Ranched Southern Bluefin Tuna (Thunnus
maccoyii) Naturally Infected with the Blood Fluke Cardicola forsteri
NT
Kirchhoff*, MJ Leef, V Valdenegro, C Hayward, and BF Nowak
| National
Centre for Marine Conservation and Resource Sustainability, University of
Tasmania, Launceston, Australia nicolek1@amc.edu.au,
mleef@amc.edu.au vav@amc.edu.au, Hayward.Craig@saugov.sa.gov.au,
bnowak@amc.edu.au
|
The acquired immunity to a natural infection of
blood fluke, Cardicola forsteri, was
investigated in ranched southern bluefin tuna (Thunnus maccoyii) (SBT) in Port Lincoln, South Australia. Cardicola forsteri is
a aporocotylid blood fluke, which infects southern bluefin tuna. Prevalence of infection increases from
5-10% when SBT are introduced into the ranching pontoons, to 100% after three
months of ranching. This
presentation will characterize the speed of antibody generation, antibody
activity at various stages of blood fluke infection, and the specificity of
antibody response. Approximately 400 ranched SBT were used for the investigation,
representing ranching durations spanning from 0 (wild) to 21 weeks post
transfer into ranching pontoons. Antibody activity was quantified in serum through an ELISA using adult C. forsteri as an antigen source. The specificity of the antibody
response was examined against the other common parasites of ranched SBT: Caligus chiastos, Hexostoma thynni, Pseudocycnus
appendiculatus, and Euryphorus
brachypterus. Histology of the
heart and gills was used to characterize the presence of Cardicola forsteri eggs, number of eggs per square centimeter
within the heart, and presence or absence within the gills. Antibody activity was then correlated
against the different stages of infection. Speed of antibody response to infection was also calculated
in relation to culture duration and stage of infection. This study is the first
to detail the egg stage of Cardicola
forsteri infection within ranched SBT. Results indicate 100% prevalence of eggs within the heart
after 3 months ranching and an increase in specific antibody activity with
culture duration.
S18-6
The Zebrafish Model – A Flow
Cytometry-based Investigation of Mechanisms of Endocytosis
C
Hohn*, S-R Lee, LM Pinchuk, and L Petrie-Hanson
| Department
of Basic Sciences, College of Veterinary Medicine, Mississippi State, MS USA.
hohn@cvm.msstate.edu
|
Zebrafish
are utilized as models for human and fish diseases. Mutant zebrafish in
particular are being utilized to investigate mechanisms of host-pathogen
interaction. We established a zebrafish model for the infectious disease
Enteric Septicemia of Catfish caused by the intracellular pathogen Edwardsiella ictaluri. Rag1-/- mutant
zebrafish, known to lack an acquired immune system, are useful to investigate
the innate immune functions like endocytosis during infection. With
an average length of 3 – 5 cm and blood yield of 10 μl per
zebrafish, commercially available endocytosis test kits are
non-practical for use in these fish. We describe a new procedure utilizing flow-cytometry
to overcome this limitation. Pooled zebrafish kidney hematopoietic tissues were
used as a source of phagocytic cells for flow cytometry based endocytic assays.
FITC-Dextran, Lucifer Yellow and FITC-Edwardsiella
ictaluri were used to evaluate selective and non-selective mechanisms of
uptake in zebrafish phagocytes. We show for the first time that zebrafish
phagocytes utilize macropinocytosis and Ca2+ dependant endocytosis
mechanisms. Furthermore, 2 hour in
situ exposure of kidney cells to live E. ictaluri altered subsequent fluid phase uptake mechanisms in
phagocytic cells. We conclude that flow cytometry is a quick, reproducible, and
objective method to evaluate the endocytic capacity of zebrafish renal
phagocytes. The proposed method is easy to implement and will enable
researchers to study the effects of pathogens, environmental toxins, and stress
on fish immune health. It also provides an immunological method that makes
zebrafish a more attractive model system for addressing immunology related questions.
| SESSION
19. BACTERIOLOGY III |
[TOP] |
S19-1
Detection and
Quantification of Renibacterium
salmoninarum in Fluid Samples by Solid Phase Cytometry
CL
McKibben1, DG Elliott*1, and J-L Drocourt2
| 1 |
U.S.
Geological Survey, Western Fisheries Research Center, Street, Seattle, USA
cmckibben@usgs.gov, dgelliott@usgs.gov
|
| 2 |
Chemunex
SA, 3, Allée de la Seine, Ivry sur Seine, France jl.drocourt@aeschemunex.com
|
Many methods commonly used for detection of
waterborne pathogens or indicator organisms (e.g., bacteria, protozoa,
zooplankton and phytoplankton) have limitations in speed, sensitivity, and
accuracy. A new technique, solid phase laser scanning cytometry (SPC), has been
developed for rapid detection, identification and quantification of low numbers
of organisms in filterable samples. Organisms are first captured by membrane
filtration and labeled with fluorescent dye. Fluorescent probes for organism
identification (via immunofluorescence or fluorescence in situ hybridization)
or assessing viability are often used. The SPC instrument scans the entire
surface of the filter in less than 5 minutes, records and analyzes each
fluorescent event, and discriminates target cells from background and
auto-fluorescence. The position of each event is graphically displayed on a
computer screen, and a motorized microscope stage allows the operator to
visually validate each result. We are testing SPC for detection of the salmonid
pathogen Renibacterium salmoninarum in water samples and in ovarian fluid of spawning female fish. For optimization
of R. salmoninarum detection by SPC,
we tested several commercial polyclonal and monoclonal primary antibodies
(unlabeled and fluorochrome-labeled) and polyclonal secondary antibodies
(labeled), as well as procedures to reduce background fluorescence and to
increase and prolong specific fluorescence signals. We optimized SPC pre-scan
instrument settings and post-scan discriminant settings for R. salmoninarum detection, and are
testing its specificity, sensitivity, and repeatability according to standard
procedures.
S19-2
Aeromonas
salmonicida subsp. achromogenes and the Effect of the Autoinducer Synthase AsaI on
Bacterial Virulence
J Schwenteit, T Gudmundsdottir, B Bjornsdottir and BK Gudmundsdottir*
| Institute for Experimental
Pathology, University of Iceland, Keldur v/Vesturlandsveg, 112 Reykjavik,
Iceland IW650@gmx.de; tota@hi.is, bryndisb@hi.is, bjarngud@hi.is
|
Quorum sensing (QS) is a cell–cell
communication system that enables bacteria to synchronize gene expression with
population density. LuxIR-type QS via N-acyl-homoserinelactones
(AHLs) autoinducers are used by many Gram-negative proteobacteria for intraspecies QS. LuxI is the autoinducer synthase and LuxR is an AHL-dependent transcriptional
regulator. The Gram-negative fish pathogenic bacterium Aeromonas salmonicida possesses the LuxIR-type quorum sensing (QS) system, termed
AsaIR, and secretes only one type of N-acyl-homoserinelactone
(AHL), N-butanoyl-L-homoserine
lactone (C4-HSL). This study was performed to investigate the role of QS in Aer.
salmonicida subsp. achromogenes virulence and pigment
production. A knock out mutant of
AsaI, constructed by allelic exchange, did not produce a detectable QS signal
and its virulence in fish was significantly impaired, as LD50 of the
AsaI-deficient mutant was 20 fold higher than that of the isogenic wt strain
and the mean day to death of the mutant was significantly prolonged. Furthermore, the expression of two
virulence factors, a toxic peptidase, AsaP1, and a repeat in toxin, RTX, and a
brown pigment were reduced in the mutant. AsaP1 production was inhibited by
synthetic QS inhibitors (N-(propylsulfanylacetyl)- L-homoserine lactone; N-(pentylsulfanylacetyl)- L-homoserine lactone; and N-(heptylsulfanylacetyl)-
L-homoserine lactone) at concentrations that did not affect bacterial
growth. The observation that the Aer.
salmonicida subsp. achromogenes LuxIR-type QS system regulates
virulence and the ability of synthetic QSI to inhibit an important virulence
factor without affecting bacterial growth, makes Aer. salmonicida subsp. achromogenes an interesting target organism for the development of novel
antibacterial therapeutics.
S19-3
Weissella sp. Outbreaks in Brazilian Rainbow Trout (Onchorynchus
mykiss) Farms: Aetiologic
Agent, Genotyping, Transmission Routes and Antibiotic Resistance Profile
HCP Figueiredo*1,2,
CAG Leal2 , FAA Costa2 and GA Carvalho- Castro2
| 1 |
General Coordinator of Aquatic Animal Health,
Ministry of Fisheries and Aquaculture, Brasília, DF, Brazil.
|
| 2 |
AQUAVET, Laboratory of Aquatic Animal Diseases, Department of
Preventive Veterinary Medicine, Federal University of Minas Gerais, Belo
Horizonte, MG, Brazil.
|
Since 2006, rainbow trout (Onchorynchus
mykiss) farms in Brazil have
been suffering with outbreaks of a hemorrhagic septicemic disease. A similar
illness was described in China as the first case of Weissella sp.
infection in fish in 2009. In the summers of 2009 and 2010, seven outbreaks in
Brazilian rainbow trout farms were studied to address important aspects of
disease. The clinical signs observed were anorexia, lethargy, exophthalmia,
ascites, and hemorrhages in the mouth, tongue and eyes. A total of 107
bacterial isolates from 44 fish were obtained. Biochemical tests and 16S rRNA
sequences of Brazilian strains showed similarity (100%) with sequences of Weissella sp. isolated in China. The genetics of the strains were characterized by pulsed-field
gel electrophoresis (PFGE), using the restriction enzyme smaI, and results
indicated that the strains were clone related. The bacteria were isolated from
the intestinal tract of diseased rainbow trout but not from healthy ones. In
order to fulfill the Koch´s postulate and address the potential infectious
routes of bacteria, rainbow trout were experimentally infected by
intraperitoneal injection, immersion, and cohabitation between diseased and
healthy fish with no direct contact. The disease was successfully reproduced
and bacteria could be transmitted through the water. The antibiotic resistance
profile for five antibiotics were determined by disc diffusion test
(CLSI,2006). Based on normalized resistance interpretation (NRI) analysis, all
isolates were resistant to sulfonamide and susceptible to florfenicol, and a
few strains had reduced susceptibility to erythromycin, oxytetracycline and
norfloxacyn. This is the first description of multiple cases of Weissella sp. infection in rainbow trout farms including PFGE genotyping, antibiotic
resistance profile, and transmission routes for disease.
S19-4
Analysis of the Serological Variability of Vibrio tapetis, Causative Agent of BRD
in clams
S Balboa, A Bastardo, A
Doce, JL Barja, AE Toranzo, JL Romalde*
| Departamento de
Microbiología y Parasitología. CIBUS-Facultad de Biología. Universidad de
Santiago de Compostela. Spain
|
The etiological agent of
brown ring disease (BRD) was identified more than ten years ago as a new member
of the genus Vibrio designated as Vibrio tapetis. First studies indicated
that strains of this pathogen constituted a homogenous group on the basis of
their phenotypic and genotypic characteristics. However, as new strains were
isolated from different hosts, including fish, some variability within the
bacterial species was demonstrated, allowing for the recognition of three main
genetic subgroups, correlated to host type. A total of 28 strain of V.
tapetis with different geographical and host origin were employed to this
study, including isolates from Manila clam (Ruditapes
philippinarum), carpet-shell clam (R.
decussatus), Pullet carpet-shell clam (Venerupis
pullastra), cockle (Cerastoderma
edule), halibut (Hippoglossus
hippoglossus), corking wrasse (Symphodus
melops), corb (Umbrina cirrosa)
and wedge sole (Dicologoglossa cuneata).
The study of these isolates was done using the following serological
techniques; slide agglutination and dot-blot assays, as well as analysis of
LPS, total and outer membrane proteins and extracellular proteins. Three
antisera, obtained against the O-antigen of representative strains of the three
groups previously described for this bacterial species, were used for the
analysis. Most strains could be
divided into three major groups on the basis of both slide agglutination and
dot-blot assays, which coincided with the genetic groups previously described.
In addition, the wedge sole, corb and corking wrasse isolates showed some
cross-reactivity, being arranged in two new groups. LPS and protein analyses confirmed
these results.
S19-5
Mycobacterial Infections in Resident Chesapeake
Bay Striped Bass Morone saxatilis
CB
Stine*1,2, AM Baya1,3, L Hungerford3, MA
Matsche 4, L Pieper 4, K Rosemary 4, C
Driscoll 4 and AS Kane1,5,6
| 1 |
University of Maryland, Department of Veterinary Medicine,
College Park, MD USA
|
| 2 |
United States Food and Drug
Administration, Center for Veterinary Medicine, Laurel, MD USA
cynthia.stine@fda.hhs.gov (current address)
|
| 3 |
Maryland Department of
Agriculture, College Park, MD USA ambaya@umd.edu, Laura.Hungerford@fda.hhs.gov
|
| 4 |
Maryland Department of
Natural Resources, Sarbanes Cooperative Oxford Laboratory Oxford, MD USA
MMatsche@dnr.state.md.us, LPieper@dnr.state.md.us, KRosemary@dnr.state.md.us,
CDriscoll@dnr.state.md.us
|
| 5 |
University of Maryland,
Department of Epidemiology and Preventative Medicine, Baltimore, MD USA
|
| 6 |
Emerging Pathogens
Institute, University of Florida, Gainesville, FL USA (current address)
kane@ufl.edu
|
Striped bass, aged 0+ to 3+, were collected from the
Upper Bay and the Choptank, Nanticoke, Pocomoke and Potomac rivers in 2003-2005
to investigate the risk factors associated with mycobacterial
colonization. Three multivariate
models of mycobacterial colonization were fit in order to account for putative
variations in migratory behavior at different ages. As 0+ striped bass remain within their natal river, location
of capture other than the Pocomoke River, presence of grossly observable
parasites and presence of bacteria other than mycobacteria were associated with
prevalence of mycobacterial infections. As 1+ fish remain close to their natal river, only location of capture
other than the Pocomoke River was associated with prevalence of colonization. 2+ and 3+ striped bass generally
migrate within the Chesapeake Bay and presence of acanthacephalans in the
intestine and presence of moderate to severe trichodinid infections on the skin
and gills were associated with prevalence of mycobacterial colonization. The existence of multiple risk factors,
and the variability introduced into the models by age-dependant life history
factors, indicate mycobacterial colonizations in Chesapeake Bay striped bass
are a result of a series of complex processes.
S19-6
Genetic Diversity of Mycobacterium chelonae in Aquatic
Environments as Determined by PFGE
S
Sanchez*1, C Still1, J Lane1, J Saliki1,
A Camus2, B Bossart3, T Clauss3, T Mullican3 and A Dove3
| 1 |
Department of Infectious
Diseases and Athens Veterinary Diagnostic Laboratory, College of Veterinary
Medicine, The University of Georgia, Athens, GA USA ssanchez@uga.edu
|
| 2 |
Department of Pathology,
College of Veterinary Medicine, The University of Georgia, Athens, GA USA.
|
| 3 |
The Georgia Aquarium,
Atlanta GA USA.
|
Mycobacteria are aerobic, mycolic acid containing
bacteria that are characteristically acid-fast. All members of this group fall
within one genus Mycobacterium, which
includes important pathogens for both fish and mammals. The most common of
these opportunistic pathogens is M.
chelonae. In spite of the importance of this bacterium, very little is
known about its genetic diversity in animal and aquatic environments. The
purpose of the present study was to study the genetic relatedness of isolates
obtained from enclosed aquatic environments, fish, mammals and biofilms.
Fifty-five M. chelonae isolates were
grown in Muller-Hinton broth media to a 4.0 MacFarland standard. Pulsed-field
gel electrophoresis was performed using restriction enzyme XbaI and DraI. The
generated Tiff images were uploaded to Bionumerics (version 4.6, Applied Maths,
Kortrijk, Belgium), using the Dice coefficient to analyze the similarity of
banding patterns, and the unweighted pair group method using arithmetic
averages (UPGMA) for cluster analysis. The data generated by both enzymes
showed that M. chelonae isolates
cluster by source, such as water, fish or mammal species they were isolated
from, and differed from each other in 1 to 2 bands. This is expected as they
are genetically very homogenous. Nevertheless, some isolates identified as M. chelonae phenotypically, and by DNA
sequencing, had PFGE patterns that differed in 5-10 bands. This seems to
indicate that some M. chelonae may be
adapted to a particular host or niche. Furthermore, there may be some isolates
that current diagnostic methodology fails to properly identify as rapidly
growing Mycobacterium at the species
level.
| SESSION
20. PARASITOLOGY V: Shellfish |
[TOP] |
S20-1
The Frequency and
Distribution of Genetic Strains of Hematodinium,
a Parasitic Dinoflagellate of the Blue Crab (Callinectes sapidus) along the Delmarva Peninsula, VA
KM
Pagenkopp*1, JD Shields1, J Xiao1, TL Miller2,
HJ Small1, AR Place3, and KS Reece1
| 1 |
The
Virginia Institute of Marine Science, The College of William and Mary, 1208
Greate Road, Gloucester Point, VA, USA pagenkopp@vims.edu, jeff@vims.edu, jxiao@vims.edu, hamish@vims.edu, kreece@vims.edu
|
| 2 |
Biodiversity Program,
Queensland Museum, PO Box 3300, South Brisbane, QLD 4101. Australia terrence.miller@qm.qld.gov.au
|
| 3 |
Institute of Marine and
Environmental Technology, University of Maryland Center for Environmental
Research, 701 East Pratt Street, Baltimore, MD, USA place@umbi.umd.edu
|
The dinoflagellate Hematodinium sp. is a significant pathogen of blue crabs on the
Eastern seaboard of the USA. We designed 15 microsatellite markers (9
polymorphic and 6 monomorphic) from an enriched microsatellite library
constructed from a non-clonal culture of Hematodinium from a blue crab (Callinectes sapidus).
Using the microsatellites, we genotyped Hematodinium strains in blue crabs, and alternate hosts, from six sites along the Delmarva
Peninsula. Multiple genetic
strains of Hematodinium were found,
some of which were seen throughout the entire region, while others were only
found in the southern portion of the Delmarva Peninsula. The reasons for this
distribution are still being investigated, but possibilities include a source
of Hematodinium from other geographic
regions, or varying environmental tolerances among different strains.
Additionally, simultaneous infections were identified, indicating that multiple
genetic strains occur in blue crabs; up to four different strains of Hematodinium were found in an individual
crab. Strains of the parasite were compared to those in alternate hosts, as
well as to those from blue crabs from Georgia and Florida.
S20-2
Discovery and
Characterization of New Perkinsus Species
KS
Reece*1, GP Scott1, CF Dungan2, PM
da Silva3, C Adams1,4 and EM Burreson1
| 1 |
Virginia
Institute of Marine Science, College of William & Mary, Gloucester Point,
VA 23062, USA kreece@vims.edu
|
| 2 |
Maryland Department of
Natural Resources, Cooperative Oxford Laboratory, Oxford, MD 21654, USA
|
| 3 |
Universidade Federal de
Sergipe, Centro de Ciências Biológicas e da Saúde, Departamento de Engenharia
Agronômica. Av. Marechal Rondon,
s/n Jardim Rosa Elze, 49100-000 Sao Cristovao, SE, Brasil
|
| 4 |
Swarthmore College, 500
College Avenue, Swarthmore, PA 19081, USA
|
For decades since the first description of Perkinsus marinus in 1950, there were
only a few recognized species in the genus Perkinsus. However, modern molecular detection and
characterization techniques have facilitated the identification and description
of five new species within the past 10 years. In addition, P.
atlanticus was synonymized with P.
olseni, and P. andrewsi with P. chesapeaki. We have used PCR, in situ
hybridization (ISH), DNA sequence-based phylogenetic analyses, and field and
laboratory studies to examine the host and geographic distributions of Perkinsus species worldwide. Potential molluscan host tissues from
six continents were screened for parasite DNAs using PCR primers designed
initially to target members of the genus Perkinsus based on approximately 50 different internal transcribed spacer (ITS) region
sequences from three Perkinsus species. These primers successfully
amplify the DNA of all currently described Perkinsus species. The PCR primers along with an ISH probe targeting the large subunit
ribosomal RNA gene of Perkinsus species
have been key tools for identifying and characterizing many of these
species. We found a potential new Perkinsus species in Australian abalone,
whose ITS-region sequences group with those from clams being imported into
Florida from Vietnam for the aquarium trade. We are currently screening Vietnamese molluscs for this parasite.
Most recently we have discovered a new strain or species of Perkinsus in Saccostrea culcullata oysters from India, which based on genetic
data is closely related to P. beihaiensis described from southern Chinese oysters and a Perkinsus sp. recently found in Brazilian Crassostrea gasar oysters.
S20-3
Haplosporidiosis in
Bivalve Molluscs: New Perspectives on Haplosporidium
costale, Agent of SSO Disease in Crassostrea
virginica
NA
Stokes* and RB Carnegie
| Department of Environmental
and Aquatic Animal Health, Virginia Institute of Marine Science, P.O. Box 1346,
Gloucester Point, VA 23062, USA stokes@vims.edu, carnegie@vims.edu
|
The protistan parasite Haplosporidium costale infects the eastern oyster, Crassostrea virginica, and is the agent
of SSO (seaside organism) disease. The parasite has been reported from
higher salinity waters (>25psu) on the Atlantic coast of the USA from
Virginia to Maine, causing peak mortalities in May-June. In the absence of
sporulation, this parasite is indistinguishable from the notorious pathogen Haplosporidium nelsoni, and
differentiating plasmodial infections by H.
costale from the more common infections by H. nelsoni has long been problematic. Recent analyses by our
laboratory and others have shown that the distribution, and in some cases also
the impact, of H. costale is greater
than previously appreciated. Detection of H.
costale by morphological, and/or DNA approaches, has revealed its presence
in oysters from a broad geographical range, including Atlantic Canada,
California, China and Australia; furthermore, infections have been detected in
three host species, C. virginica, Crassostrea gigas and in Saccostrea glomerata. Infections have
been detected in nearly every month of the year, not only in spring as
previously known, and unexpectedly high prevalences (reaching 44%) have been
observed in Virginia embayments that previously have not been considered H. costale-enzootic. There have been
enough moderate to heavy H. costale infections to indicate that up to 10% of oysters are experiencing serious SSO
disease and mortality. Species-specific in
situ hybridizations have confirmed that the observed plasmodia are H. costale and not H. nelsoni. A contrasting size-specific infection profile has
emerged, with more H. costale infections in larger oysters and more H.
nelsoni infections in smaller oysters. We hypothesize that this may reflect
the difference between acute (H. nelsoni)
and chronic (H. costale) infection
and disease.
S20-4
Oyster Parasites Common to
Lake Worth Lagoon, Florida
SE Laramore*1 and J Scarpa2
| 1 |
Harbor Branch Oceanographic Institute at Florida
Atlantic University, 5600 US 1 North, Fort Pierce, FL 34946 USA slaramo1@hboi.fau.edu
|
| 2 |
Harbor Branch Oceanographic
Institute at Florida Atlantic University, 5600
US 1 North, Fort Pierce, FL 34946 USA jscarpa1@hboi.fau.edu
|
Three eastern oyster (Crassostrea virginica)
reefs in the Lake Worth Lagoon, a 21-mile long estuary in Palm Beach County,
Florida, were monitored for two years (March 2008-February 2010). Sites were
natural reefs located at John D. MacArthur Beach State Recreation Park at the
northern end of the lagoon and at Ibis Isle in the central lagoon. The third site was a created reef at
the Snook Islands Natural Area in the central lagoon. Oysters (n=5-6) were
collected monthly from each site to determine prevalence and intensity of Dermo
(Perkinsus marinus) using Ray’s fluid thioglycollate medium and other
parasites using standard H&E histological techniques. Water temperature was
similar between sites, but salinity was on average 5 ppt higher and more stable
at the northern site (MacArthur). Dermo prevalence was similar at all three
sites (50-100% spring/summer/fall) and lowest at all sites during the winter,
as well as during low-salinity events at the central sites. Dermo intensity
ranged from 0-4.5 Mackin scale with an average annual intensity range of
0.85-1.2; MacArthur had the highest and Snook Island the lowest. MSX (Haplosporidium
nelsoni) was not found at any site. Other parasites detected histologically
included the gregarine protozoan Nematopsis sp., turbellarians, the
trematode Bucephalus, the cestode Tylocephalum, and
ciliates. Symbiotic parasites (cnidarian Eutima and pea crab Pinnotheres)
were also present. Annual, seasonal, and site variation prevalence was noted
for different parasites. The diversity of parasites found in oysters is
indicative of a healthy ecosystem that supports a diverse assortment of other
species.
S20-5
Potential Bivalve Reservoirs
of Bonamia sp. in Florida’s Indian River Lagoon
SE Laramore*1 and AL Lave2
| 1 |
Harbor Branch Oceanographic Institute at Florida
Atlantic University, 5600
US 1 North, Fort Pierce, FL 34946 slaramo1@hboi.fau.edu
|
| 2 |
Jacksonville University, 2800
University Blvd. North, Jacksonville, FL 32211 alave@jacksonville.edu
|
In 2007, suminoe oysters (Crassostrea ariakensis)
and eastern oysters (Crassostrea virginica) exposed to Indian River
Lagoon, FL waters were found to be PCR positive for Bonamia sp.,
although infection was histologically confirmed only in C. ariakensis. Both species were also infected with Dermo (Perkinsus marinus). In the
summer of 2010 we began to look for bivalve reservoirs of Bonamia sp. in
the Indian River Lagoon. Ten to twenty representatives of various bivalve
species were collected from eight locations within the southern portion of the
Indian River Lagoon (St. Lucie Co. and Martin Co.) and tested for the presence
of the Bonamia sp., P. marinus, and Haplosporidium
nelsoni (causative agent of MSX) using polymerase chain reaction (PCR). Bivalves
collected and tested varied by location, but included various oyster species (C.
virginica, Ostreola equestris, Isognomon alatus, I. bicolor),
mussels (Brachidontes exustus, B. modiolus, Geukensia granossima, Ischadium
recurvum), arks (Arca imbricata, A. zebra), and clams (Mercenaria mercenaria, M. campechiensis, Chione elevata). PCR positive results have been obtained for Bonamia sp.
in C. virginica, I. alatus, I. bicolor, and G. granossima and
for P. marinus in C. virginica, I. alatus, and G. granossima. Analysis (PCR and histology) is
ongoing; however the presence of Bonamia may indicate a wider geographic
range for Bonamia than previously reported in the US.
S20-6
Evolutionary Ecology
of Crassostrea virginica and Its
Parasites
RB
Carnegie*1 and EM Burreson2
| Department of Environmental
and Aquatic Animal Health, Virginia Institute of Marine Science, P.O. Box 1346,
Gloucester Point, VA 23062 USA carnegie@vims.edu,
gene@vims.edu
|
The oyster Crassostrea virginica has long been plagued by the parasites Perkinsus marinus and Haplosporidium nelsoni. In the 1950s in the Chesapeake Bay,
USA, H. nelsoni joined an established C. virginica-P. marinus system. The goal of our study was to determine how the
arrival of H. nelsoni perturbed this
system. We proceeded along two tracks: First, we analyzed data from
contemporary samples of wild and sentinel oysters and placed this data in the
context of a sixty-year span; second, we re-evaluated archival histological
materials to assess changes in parasite presentation and disease. Data analyses have revealed decreasing H. nelsoni infection in wild oysters
despite increasing infection pressure on naïve sentinels, suggesting that wild
oysters have become more resistant to H.
nelsoni. The oysters have also
become more resistant to disease caused by P.
marinus, though this parasite continues to result in substantial
mortality. Since the mid-1980s,
disease caused by P. marinus has seen
a dramatic intensification, which has been attributed to a greater abundance of P. marinus since the drought years of
that decade. Retrospective
histopathology, however, revealed a fundamental change in the parasite
presentation since 1986: relatively large and scarce P.
marinus cells that divide by schizogony have been replaced by relatively
small and abundant cells that divide in binary fashion. We hypothesize that this change relates to the
observed disease intensification and an increase in parasite virulence and,
furthermore, that it is a response by P.
marinus to the introduction of H.
nelsoni.
| SESSION
21. TREATMENTS |
[TOP] |
S21-1
Comparison of in-vitro and in-vivo Efficacy of Florfenicol for Treatment of Francisella asiatica Infection in
Tilapia
E Soto*1, RG Endris2 and JP Hawke1
| 1 |
Louisiana State
University, Department of Pathobiological Sciences, School of Veterinary
Medicine, Baton Rouge, LA USA1 esoto1@lsu.edu, jhawke1@lsu.edu
|
| 2 |
Intervet/Schering-Plough Animal Health, Roseland, New Jersey USA
richard.endris@sp.intervet.com
|
Francisellosis caused
by the emergent fish pathogen Francisella
asiatica is an important bacterial disease in warm water aquaculture. The
lack of effective treatments led us to investigate the efficacy of florfenicol
to F. asiatica in vitro and to
potentially treat piscine francisellosis in
vivo. The minimal inhibitory concentration (MIC) of florfenicol for F. asiatica, as determined by the
broth-dilution method, was 2 µg/ml. In tilapia head kidney derived macrophages
(THKDM) assays, addition of florfenicol to the medium at 10 µg/ml resulted in
uptake of the drug and significantly reduced bacterial loads. Moreover,
cytopathogenicity assays done in infected THKDM also demonstrated drug efficacy
as concentrations of lactate dehydrogenase (LDH) released from infected THKDM
were significantly lower in macrophages treated with florfenicol (p<0.001)
than controls. In medicated feed
trials, treated fish were fed 15mg/kg of florfenicol for 10 days, either at 1,
3, or 6 days post-challenge. Immersion challenges resulted in 70% mortality in
non-treated fish. Fish fed
medicated feed 1 and 3 days post infection showed significantly higher survival
rates (p<0.001). Bacterial concentrations in the spleen of surviving fish
were evaluated by enumerating colony forming units (CFU) on cystine heart agar
(CHA) plates 30 days post-challenge. Significantly higher numbers (p<0.001)
of bacteria were recovered from non-treated fish than from fish fed medicated
feed 1 and 3 days post-infection. There were no significant differences in
bacterial burden between fish treated 6 days post-infection and untreated
controls. If administered during early infection,
florfenicol may effectively treat piscine francisellosis, including via
intracellular penetration and bacterial clearance.
The Administration of a Bronopol-Based
Chemical Treatment to Control Ichthyophthirius
multifiliis (Ciliophora) Infections
SM
Picón-Camacho*, JE Bron and AP Shinn
| Aquatic Parasitology Research Laboratory, Institute
of Aquaculture, University of Stirling, UK sara.picon@stir.ac.uk, jeb1@stir.ac.uk, aps1@stir.ac.uk
|
Whitespot infections caused
by the ciliate protozoan Ichthyophthirius
multifiliis cause serious economic losses, annually and globally, for
commercial and ornamental freshwater aquaculture. Few effective
chemotherapeutants are available for its control in commercial-scale
aquaculture. The present study explores the usage of bronopol, commercialized
by Novartis Ltd. as PycezeTM, as
a biocide for the treatment of I.
multifiliis through a series of in vitro and in vivo trials. In vitro exposure of tomonts to bronopol (0, 20, 50 and 100 mg L-1)
killed 0%, 76.2%, 97.2% and 100% after 30 min. Long exposures to 1 mg L-1 bronopol (up to 48 h) were successful in reducing the numbers of theronts
surviving after 12 h (29.16%) and 24 h (0% survival). In the in vivo trial, the efficacy of
continuous low doses of bronopol was explored in reducing the number of
trophonts establishing on juvenile O. mykiss. The effect of bronopol on
the time of infection was investigated by starting the administration of 2 mg L-1 24 h prior to the infection, and for 72 h afterwards. The effect of bronopol on
maturing trophonts and tomonts was assessed through the delivery of 1, 2 and 5
mg L-1 at 10-36 d post-infection. The trial showed that 2 mg L-1 administered prior to infection reduced the number of theronts surviving
by 35-48%. The 2 and 5 mg L-1 bronopol in the second cycle of
infection reduced the number of trophonts by 46-83%, while doses during the
third cycle gave further reductions of 83-97%.
S21-3
Efficacy of Florfenicol,
Copper Sulfate and Potassium Permanganate in Controlling a Natural Infection of Aeromonas hydrophila and Flavobacterium columnare in Sunshine
Bass, Morone chrysops Female ´ Morone saxatilis Male
AM
Darwish*
| Harry
K. Dupree-Stuttgart National Aquaculture Research Center, Agricultural Research
Service, United States Department of Agriculture, Stuttgart, AR, USA
Ahmed.Darwish@ars.usda.gov
|
Sunshine bass (Morone chrysops female ´ Morone saxatilis male) naturally infected with Aeromonas hydrophila and Flavobacterium columnare were randomly assigned to six
treatments: 1) two treatments of waterborne exposures to copper sulfate (CuSO4),
at 2.1 and at 4.2 mg/L (approximately one and two percent the water alkalinity)
for 24 h, 2) two treatments of waterborne exposures to potassium permanganate
(KMnO4) at 2 mg/L above the KMnO4 demand (PPD) for 24 h
and at 10 mg/L above the PPD for 1 h, 3) a treatment of florfenicol-medicated
feed at 15 mg/kg of fish body weight (BW) for 10 d, and 4) non-treated fish as
control. The fish were observed
for 32 d. The control fish had 71%
survival compared to, 88 and 79% survival for the CuSO4 treatments
at 2.1 and at 4.2 mg/L, respectively, 72 and 77% survival for the KMnO4 treatments
at 2 mg/L for 24 h and at 10mg/L for 1h, respectively, and 90% survival for the
fish treated with florfenicol-medicated feed at 15 mg/kg of fish BW for 10
d. The final survival of fish fed
florfenicol-medicated feed (90%) was significantly higher (P<0.1) than the control fish (71%). According to logrank comparison the survival curves of the
florfenicol-medicated treatment and the CuSO4 treatment at 2.1 mg/L
for 24 h were significantly different from the control fish; the rate of death
was significantly higher in the control fish. This is the first controlled study to demonstrate the
beneficial effect of florfenicol and CuSO4 against a mixed infection
of A. hydrophila and F. columnare.
S21-4
Evaluation of Novel
Oral Therapeutics Against Monogenean Parasites
LA
Tubbs*1,2, B Wang3 and M Hamann3
| 1 |
National
Institute of Water and Atmospheric Research Ltd, Viaduct Precinct, Auckland
Central, New Zealand
|
| 2 |
Fish
Pathology Laboratory, Department of Pathobiology, Ontario Veterinary College,
University of Guelph, Guelph, ON, Canada (Present
address) ltubbs@uoguelph.ca
|
| 3 |
Department of Pharmacognosy,
School of Pharmacy, University of Mississippi, MS 38677, USA
mthamann@olemiss.edu
|
A fundamental aspect of drug discovery strategies is
the ability to characterise the relationship between the pharmacodynamics (PD),
pharmacokinetics (PK), efficacy and toxicity of potential therapeutics. The
present study evaluated several marine compounds as potential oral therapeutics
against a monogenean parasite of kingfish (Seriola
lalandi) using in vitro techniques and experimental dosing. A panel of 35 compounds were initially
screened by immersing detached parasites in equimolar concentrations (1μM,
10μM and 100μM) of each compound; efficacy was compared to an
established anthelmintic (praziquantel (PZQ)). The most effective compounds
were advanced to a secondary screen using a modified Ussing chamber, where
parasites were attached to a piece of excised kingfish skin then exposed to
each compound via a supportive media, providing an in vitro assay which highly effectively models the oral dosing of
an infected fish. Four compounds with efficacy comparable to PZQ were selected
in this manner for in vivo assessment
of toxicity and PK. Toxicity was determined using a dose-escalation approach to
determine the maximum tolerated oral dose within the range 1 to 500 mg.kg-1 body weight. The relative PK of the least toxic, most effective, compound was
determined in the skin, muscle and plasma of kingfish using LC-MS using two
empirically derived, repetitive dosing regimens: 3 days with a 12h dosing
interval and 5 days with a 24 h interval. The plasma concentration of this
compound was between 10 and 100 fold lower than the concentration in muscle and
skin, indicating rapid clearance. These results demonstrate useful mechanisms
for predicting the outcome of potential therapeutics.
S21-5
Using Vaccines in
Aquaculture
H
Mitchell*
| Aqua Life Veterinary
Services, Aquatic Life Sciences (Western & Syndel), 12015 115th Ave NE, Kirkland, WA 98034 USA hughm@aqualifesci.com
|
Vaccines are relatively old medical technologies pre-dating germ
theory and have been credited with no less than revolutionizing human and
animal medicine. Their commercial
use in aquaculture is less than 40 years old, however their application in many
fish species still disproportionately languishes behind that of terrestrial
animals. Not unlike terrestrial production medicine, the current use and
understanding of fish vaccines can be controversial. As recognized in
agriculture medicine: “The
veterinary practitioner is often asked for advice about the use of vaccines for
the control of infectious diseases of food producing animals. There is often
more controversy and uncertainty about the efficacy of these vaccines than
about almost any other topic in livestock production”. How can the
terrestrial experience be used to foster more development, use and
effectiveness in fish production? Part of the answer lies in understanding exactly what a vaccine is: “…when
veterinarians recommend use of vaccine, the unspoken, implied expectation is
that the product will end disease losses. Avoidance of this misunderstanding requires precise and accurate
communication of reasonable expectations.” The history of fish vaccinology with current status in
aquaculture will be briefly reviewed. The various reasons for vaccinating will be discussed, including variables
involved in defining and assessing vaccine efficacy. The approach to using vaccines within a fish production
environment will be outlined in the context of an integrated fish health
management program where the other essential interacting components
include: genetics; risk factor
reduction; biosecurity and pathogen load reduction; and early diagnosis and
treatment.
| SESSION
22. PARASITOLOGY VI:
Fish 2 |
[TOP] |
S22-1
Health and Reproductive Consequences for Sciaenops
ocellatus (Perciformes: Sciaenidae) Infected with the Ovarian Parasite Philometra
floridensis (Nematoda: Philometridae)
MD Bakenhaster*1, S Lowerre-Barbierrie1,
Y Kiryu1, S Walters1, and EJ Fajer-Avila2
| 1 |
Florida Fish and Wildlife
Conservation Commission, Fish and Wildlife Research Institute, St. Petersburg, FL USA Micah.Bakenhaster@myfwc.com, Susan.Lowerre-Barbierrie@myfwc.com, Yasu.Kiryu@myfwc.com, Sara.Walters@myfwc.com
|
| 2 |
Centro de Investigación en
Alimentación y Desarrollo, A.C., Unidad Mazatlán en Acuicultura y Manejo
Ambiental, C.P. Mazatlán Sinaloa, México efajer@ciad.mx
|
The parasitic nematode Philometra floridensis is known to infect the ovary of the economically important fish Sciaenops
ocellatus, but its life history, the factors influencing host
susceptibility, and potential pathogenic effects are unknown. Here we
report new information on these topics based on evaluations of reproductively
mature wild hosts, both infected and uninfected, collected from the Gulf of
Mexico off Tampa Bay, Florida. To check for ontogenetic differences in
host susceptibility to the nematode, we evaluated the length and age of fish
vis-à-vis nematode prevalence. About 46% of female fish, and no male fish, were infected
with P. floridensis, with prevalence and intensity tending to be higher
in larger specimens. To evaluate health and reproductive consequences of
infection, we looked for parasite effects on histological conditions, condition
factor (K), batch fecundity estimates (BF), and gonadosomatic
index (GSI). We observed substantial pathological changes in
ovarian tissue associated with female nematodes but only in regions close to
worms. Both K and BF were higher for
infected fish at a given size. Together, these results suggest
that P. floridensis has a negative impact on its host’s reproduction at
the cellular level but not at the organismal level. Several factors, including
the higher prevalence of infection in larger and older hosts, may have
prevented our detection of some health impacts; however, at the intensities
observed in this study, infection did not appreciably reduce reproductive
fitness.
S22-2
Unknown Clinostomum sp. Associated with
Pathology in Channel Catfish (Ictalurus
punctatus) in Mississippi (USA)
CM
Doffitt*1, L Khoo2, LMPote1 and DT King3.
| 1 |
Department of Basic
Sciences, College of Veterinary Medicine (CVM), Mississippi State University,
Mississippi State, MS USA lpote@cvm.msstate.edu,
doffitt@cvm.msstate.edu
|
| 2 |
Department of Population and
Preventative Medicine, Thad Cochran Warmwater Aquaculture Center, (CVM/MSU),
Stoneville, MS USA lhk2@msstate.edu
|
| 3 |
USDA/Wildlife
Services/National Wildlife Research Center, Mississippi State, MS USA tommy.king@aphis.usda.gov
|
As the commercial catfish industry has expanded in
the southern United States, there has been an increase in piscivorous bird
populations in this region. Many
of these birds are infected with digeneans that utilize fish as intermediate
hosts. Clinostomum marginatum and C. complanatum have been reported in channel catfish; however,
these infections do not usually cause morbidity or mortality. Recently a clinostomoid-like trematode
was identified that was associated with morbidity and high parasite loads. A challenge study was done to determine
the pathology of this parasite and confirm its genomic identification. Cercariae were isolated from snails, Planorbella trivolvis, collected from
commercial catfish ponds. Parasite-free channel catfish fingerlings were exposed to 200
cercariae/fish for 2 hours. The
fish were sampled at 7, 14, 21, 28, 40, 80, and 120 days post-infection (dpi).
Metacercariae were observed at 14 dpi in the musculature, ocular cavity, and
oral cavity. By 120 dpi, the
metacercariae were well-developed with most internal structures evident. At all sampling points, pathology was
limited to the metacercarial site; however, the infected catfish showed reduced
growth rates and a relatively high parasite load (up to 24 metacercariae/fish).
Histological analysis of the metacercariae showed little host immunological
response or histopathology. Sequence analysis of cercariae and metacercariae revealed a 99% homology
with a Clinostomum sp. The high parasite infection combined
with a lack of mortality and severe pathology associated with this trematode is
unusual. More research is needed
to identify this species and its avian host. Research supported by The Berryman
Institute.
S22-3
DNA Barcoding
Platyhelminths that Parasitize Aquatic Animals
SA Locke*1, JD McLaughlin2 and DJ Marcogliese1
| 1 |
St.
Lawrence Centre, Environment Canada, Montreal Canada sean.locke@ec.gc.ca
|
| 2 |
Department of Biology, Concordia University, Montreal Canada
|
The ability to distinguish
species of pathogens can play an important role in monitoring and controlling
disease in aquaculture and wildlife management. DNA barcoding is a method of
distinguishing species using sequence from a standardized region of the
cytochrome oxidase I gene in the mitochondrion. In a recent survey of 20 fish
species in the St. Lawrence River near Montreal, DNA barcodes from ~1000 parasites collected revealed much higher
diversity and, in most cases, higher host specificity than expected in
metacercariae in the Diplostomoidea (Platyhelminthes: Digenea) (Locke et al, 2010, Int J Parasitol 40, 333-343; Locke et al, 2010, Mol Ecol 19,
2813-2827). Here we present additional results from ongoing sampling that is
expanded in terms of both geographic coverage and host taxa, including
amphibians, mammals, birds and additional fish species from across North
America. The two main patterns observed in the St. Lawrence River –
widespread host specificity and high diversity among diplostomoids – are
not contradicted by the results of the expanded sampling program. DNA barcodes
have also been obtained from other trematode taxa and from cestodes. The
performance, applications and limitations of DNA barcoding are discussed in the
context of wildlife parasitology.
S22-4
Life Histories of the
Trematodes Found in Double-crested Cormorant Populations in the Mississippi
Delta and the Potential Impact of these Parasites on Commercial and Wild Fish
Species Found in this Region
MM
O’Hear*1, LM Pote1, DT King2, CM Doffitt1,
C Panuska1 and LE Miranda3
| 1 |
Department of Basic
Sciences, College of Veterinary Medicine, Mississippi State University,
Mississippi State, MS 39762 mohear@cvm.msstate.edu,
lpote@cvm.msstate.edu
|
| 2 |
United States Department of
Agriculture/Wildlife Services/ National Wildlife Research Center, Mississippi
State, MS 39762 tommy.king@aphis.usda.gov
|
| 3 |
Department of Wildlife,
Fisheries and Aquaculture, Mississippi State University, Mississippi State, MS
39762 smiranda@cfr.msstate.edu
|
The population of double-crested cormorants (Phalacrocorax auritus) has steadily
increased over the last few decades in the Mississippi Delta. This piscivorous bird serves as a
definitive host for numerous trematodes, many of which have immature stages
that infect numerous fish species. The goal of this study was to identify and determine the impact of these
trematodes on both commercial and wild fish populations in the Mississippi
Delta. All adult trematodes were
enumerated and identified from cormorants (n=35) collected in close proximity
to commercial catfish ponds in the Mississippi Delta from January 2003 to March
2004. Trematodes found in the cormorants were Pseudopsilostoma varium, Drepanocephalus spathans, Austrodiplostomum
ostrowskiae, Hysteromorpha triloba and Ascotyle longus. Three of
these A. ostrowskiae, H. triloba and
A. longus are reported to have
fish as an intermediate host while the life cycles of the other two are
unknown. In addition, fish were collected from an ox-bow lake located near
commercial catfish operations that had a large resident cormorant population.
Fish were examined externally and internally for the presence of parasites. A
large percentage of these fish were infected with Posthodiplostomum minimum, which was found in multiple organs in 7
fish species. The only larval trematode stage recovered from this fish
population was P. minimum. Research
is underway to identify the hosts for P. minimum in this lake and identify the
life stages of P. varium and D. spathans using molecular sequence
comparisons. Research was
partially supported by the Berryman Institute.
S22-5
Infection Dynamics of
Ecto- and Endoparasites of Channel Catfish (Ictalurus
punctatus), Blue Catfish (Ictalurus
furcatus), and C×B Hybrid Catfish (Female Ictalurus punctatus × Male Ictalurus furcatus) in Earthen Pond Aquaculture
TN
Truong*1 and SA Bullard2
| 1 |
Department of Fisheries and
Allied Aquacultures (FAA), Aquatic Parasitology Laboratory (APL), Auburn
University, Auburn, AL USA, tnt0004@auburn.edu
|
| 2 |
FAA, APL, sab0019@auburn.edu
|
It is reported that C×B hybrid catfish (HC)
exhibit better growth, feed conversion ratios, tolerance to low dissolved
oxygen, and dress-out percentages, but few controlled, in-pond studies have
quantified parasite prevalence and biodiversity in HC compared with channel
(CC) and blue (BC) catfishes. This twelve-month study is underway to reveal the
ecological and taxonomic diversity of parasites infecting HC. Fingerlings of
HC, CC, and BC were communally stocked at equal density (750
individuals/species/pond) in three 0.1 acre earthen ponds at the EW
Shell Fisheries Center (Auburn, AL) in January 2010 and sampled
monthly (10 specimens per catfish species per pond) for the presence of ecto-
and endoparasites using stereomicroscopy and subsequent histology of brain,
eye, gill, skin, fins, kidney, swim bladder, heart, liver, spleen, intestine,
and stomach. Six major taxa of parasitic metazoans were found: Monogenea
(HC– 104 infected of 106 examined [98.1%]; CC– 63 of 63 [100%];
BC– 54 of 56 [96.4%]), Cestoda (HC– 31 of 106 [29.2%]; CC– 24
of 63 [38.1%]; BC– 15 of 56 [26.8%]), Copepoda (HC– 9 of 106
[8.5%]; CC– 15 of 63 [23.8%]; BC– 15 of 56 [26.8%]), Nematoda
(HC– 1 of 106 [0.9%]; CC– 1 of 63 [1.6%]; BC– 0%), Unionidae
(HC– 5 of 106 [4.7%]; CC– 0%; BC– 0%), and Myxozoa (HC–
22 of 106 [20.8%]; CC– 50 of 63 [79.4%]; BC– 3 of 56 [5.4%]). These
preliminary results show no evidence that HC is refractory to initial infection
by each major metazoan parasite taxon that infects its parental species.
S22-6
Observation and Description of
Five New Species of Cestode Parasites in Fishes from Ogba River, Benin City,
Edo State, Nigeria.
EE Obano*
| Department
of Fisheries, Faculty of Agriculture, University of Benin, Benin City, Edo
State, Nigeria. efeobano2002@yahoo.com
|
The entire
length of Ogba River is about 200 km, with many settlements and villages, whose
inhabitants are mainly farmers and fisher folks that depend on the river for
their domestic, farming and fishing needs. The study area was the bridge
segment of the Ogba River, which is about 10m in width with steep sandy slopes,
bordered by a large multipurpose agricultural farm complex, a forest reserve, a
zoological garden and an open drainage channel fed by the Benin City Master
drainage system. A
survey of the helminth fish parasites was carried out at Ogba River, Benin
City, Edo State. The following
helminth parasites were recovered: Caryophyllaeides sp, Crescentovitus sp, Bialovarium sp, Lytocestus sp, Monobothrium sp, Stocksia sp, Cucullanus sp, Procamallanus
laeviconchus, and Spinitectus sp. Cestodes accounted for 78% of
the infection, while the nematodes accounted for 22%. The main location of
parasite occurrence in fish was the intestine. Sex did not influence the degree of helminth parasite
infection in fishes. Small sized
and younger fishes were mostly infected. Five of the parasites (Stocksia sp, Monobothrium sp. Bialovarium sp, Crescentovitus sp and Caryophyllaeides sp) were new parasites recorded in the fishes of Ogba River.
| SESSION
23. VIROLOGY II: VHS |
[TOP] |
S23-1
Results of 2009 Surveillance Efforts for Viral
Hemorrhagic Septicemia Virus in Lake Ontario and Lake Superior
ER Cornwell*1,
GE Eckerlin2, RG Getchell1, GH Groocock1,
TH Thompson3, WN Batts3, G Kurath3, JR Winton3,
PR Bowser1, MB Bain4, and JW Casey1.
| 1 |
Aquatic
Animal Health Program, Department of Microbiology and Immunology, College of
Veterinary Medicine, Cornell University, Ithaca, NY USA erc58@cornell.edu, rgg4@cornell.edu,
ghg3@cornell.edu, prb4@cornell.edu, and jwc3@cornell.edu
|
| 2 |
Department
of Environmental and Forest Biology, SUNY College of Environmental Science and
Forestry, Syracuse, NY USA geeckerl@syr.edu
|
| 3 |
USGS
Western Fisheries Research Center, Seattle, WA 98115 USA tthompson@usgs.gov, bbatts@usgs.gov,
gkurath@usgs.gov, jwinton@usgs.gov
|
| 4 |
Department
of Natural Resources, Cornell University, Ithaca, NY USA Mark.Bain@Cornell.edu
|
Viral Hemorrhagic Septicemia Virus (VHSV) was first
isolated in the Great Lakes from an archived muskellunge (Esox masquinongy) collected from Lake St. Clair in 2003. In 2005, the first mass mortality event
in the Great Lakes attributable to VHSV occurred in freshwater drum (Aplodinotus grunniens) in Lake
Ontario. Throughout 2005, 2006,
and 2007, additional mortality events occurred throughout Lake Ontario, Huron,
Michigan, and Erie, as well as smaller inland lakes in the Great Lakes
basin. To date, no mortalities
attributable to VHSV have been reported in Lake Superior. We conducted a surveillance effort of
asymptomatic fish in Lake Ontario and Lake Superior in 2009 to determine
whether VHSV was still present in Lake Ontario, and if there was evidence for
its presence in Lake Superior. All
fish were first screened using a qRT-PCR assay, and presumed positive tissues
are currently being confirmed using traditional cell culture techniques. Using qRT-PCR, we detected VHSV in 382
out of 2,010 fish in Lake Ontario, with an apparent prevalence of 27% in round
goby (Neogobius malenostomus) and 8%
in yellow perch (Perca flavescens). In Lake Superior, we collected 847 fish
at 7 sites and detected VHSV at 4 sites. Our results were independently confirmed by re-isolation of RNA and
nested RT-PCR amplification of a different region from original tissues at a
separate laboratory. This is the
earliest evidence for the presence of VHSV in Lake Superior. Our results confirm
that VHSV remains in Lake Ontario and suggest that it is present in Lake
Superior.
S23-2
Generation of a Mutant Viral Hemorrhagic Septicemia Virus of the Great Lakes Strain Which is Attenuated in Yellow Perch (Perca flavescens)
A Ammayappan1, TM Thompson2, G Kurath2 and VN Vakharia*1
| 1 |
Department of Marine Biotechnology, University of Maryland Baltimore County, Baltimore, MD USA vakharia@umbc.edu
|
| 2 |
U.S. Geological Survey,
Western Fisheries Research Center, Seattle, WA USA gkurath@usgs.gov
|
Viral hemorrhagic septicemia virus (VHSV), belonging
to the genus Novirhabdovirus in the
family of Rhabdoviridae, causes a
highly contagious disease of fresh and saltwater fish worldwide. Recently, a
novel genotype of VHSV, designated IVb, has invaded the Great Lakes causing
large-scale epidemics in wild fish. An efficient reverse genetics system was
developed to generate a recombinant VHSV of genotype IVb from cloned cDNA. The
recombinant VHSV (rVHSV) was comparable to the parental wild type strain both in vitro and in vivo, causing high mortality in yellow perch (Perca flavescens). A modified
recombinant VHSV was generated in which the NV gene was substituted with an
enhanced green fluorescent protein gene (rVHSV-?NV-EGFP), and another
recombinant was made by inserting the EGFP gene into the full-length viral
clone between the P and M genes (rVHSV-EGFP). The in vitro replication kinetics of rVHSV-EGFP was similar to rVHSV;
however, the rVHSV-?NV-EGFP grew 2 logs lower. In yellow perch challenges,
wtVHSV and rVHSV induced 82% to 100% cumulative percent mortality (CPM),
respectively, whereas rVHSV-EGFP produced 62% CPM and rVHSV-?NV-EGFP caused
only 15% CPM. No reversion of mutation was detected in the recovered viruses
and the recombinant viruses stably maintained the foreign gene after several
passages. These results indicate that the NV gene of VHSV is not essential for
viral replication in vitro and in vivo, but it plays an important role
in viral replication efficiency, pathogenicity, and attenuation.
S23-3
Development of Tools
to Forecast the Potential for Viral Hemorrhagic Septicemia Epizootics in Wild
Fish Populations
PK Hershberger*1,
JL Gregg1, MK Purcell2, CA Grady1, JC Woodson2 and JR Winton2
| 1 |
U.S. Geological Survey,
Western Fisheries Research Center (WFRC), Marrowstone Marine Field Station,
Nordland, WA USA phershberger@usgs.gov
|
| 2 |
U.S. Geological Survey,
WFRC, Seattle, WA USA
|
Traditional disease surveillance efforts in wild
fishes are based on periodic infection and disease surveys combined with
mobilization of epidemiological investigations in response to observed fish
kills. This approach, although
well-suited for identifying pathogens of concern and providing a list of
probable causes for the observed epizootics, is likely to overlook acute
disease epizootics that occur non-synchronously with scheduled surveillances or
those that occur among highly migratory species that move outside the surveyed
area. Further, the traditional
approach provides no capacity for mitigating or preventing the impacts of
diseases to the affected populations. We contend that by understanding basic disease mechanisms unique to the
host-pathogen relationships, we can develop population-screening tools that are
capable of forecasting the potential for future disease epizootics. These tools can then be employed to
design adaptive disease management strategies intended to prevent or mitigate
population-level impacts of these diseases. Using epizootiological and immunological principles unique
to viral hemorrhagic septicemia (VHS) in Pacific herring, we describe the
development of several tools (an in vitro fin explant assay, a passive immunization
assay, and an Enzyme-Linked-Immunosorbent Assay (ELISA) to detect herring
antibodies against VHSV) that may be capable of forecasting the potential for
future VHS epizootics in wild herring populations.
S23-4
Experimental Susceptibility of Sea Lamprey and Tiger Muskellunge to
VHSV IVb
RG
Getchell*1, GH Groocock1, ER Cornwell1, LL
Coffee2, GA Wooster1, and PR Bowser1
| 1 |
Department of Microbiology and Immunology, College of Veterinary Medicine,
Cornell University, Ithaca, NY, USA rgg4@cornell.edu,
ghg3@cornell.edu, erc58@cornell.edu, gaw5@cornell.edu, prb4@cornell.edu
|
| 2 |
Department of Biomedical Sciences, College of Veterinary Medicine, Cornell
University, Ithaca, NY, USA llc77@cornell.edu
|
During 2009 the experimental susceptibility of sea
lamprey Petromyzon marinus and tiger
muskellunge Esox masquinongy X Esox lucius to the Great Lakes strain of
viral hemorrhagic septicemia virus (VHSV IVb) was tested by intraperitoneal
(IP) injection of 106 pfu of the MI03 isolate. Prior to challenge, 30 wild lamprey
from Cayuga Lake were assayed for VHSV using cell culture and a quantitative
RT-PCR (qRT-PCR) method. No
clinical signs of VHSV were observed in the fish collected. Five-fish pools of
liver, heart, spleen, anterior and posterior kidney were assayed for VHSV by
cell culture, and all pools were negative. Sixty lamprey from Cayuga Lake were subsequently collected
for the susceptibility experiment, which was carried out at 10 ºC. Nine of thirty lamprey challenged were
positive by cell culture and 26 of 30 positive by qRT-PCR. No mortalities occurred during the
28-day trial. The mean quantity of
VHSV measured by qRT-PCR was not significantly different at days 1, 3, 7, 14,
or 28. None of the 30 media-injected
controls tested positive by qRT-PCR or cell culture. The tiger muskellunge were
obtained from the NYSDEC’s South Otselic Hatchery and held at 15 ºC for at
least 2 weeks. The tiger
muskellunge were temperature shocked by dropping the tank water to 10 ºC the
day of the viral challenge. None
of the 25 media-injected controls tested positive by qRT-PCR or cell
culture. All 55 fish injected IP
with VHSV tested positive by qRT-PCR and cell culture. There was 85% mortality during the
14-day trial.
S23-5
Detection of VHSV IVb Within
the Gonads of Great Lakes Fish Using In
Situ Hybridization
LJ
Al-Hussinee*and JS Lumsden
| Fish Pathology
Laboratory, Department of Pathobiology, Ontario Veterinary College, University
of Guelph, Guelph, ON, Netherlands lalhussi@uoguelph.ca
|
Among several species of the Great lakes, freshwater drum (Aplodinotus grunniens) and muskellunge (Esox masquinongy) have been impacted by viral hemorrhagic septicaemia virus genotype IVb
(VHSV IVb) in the spring of 2005 and 2006. The viral agent belongs to the genus Novirhabdovirus, and globally this
agent causes high economic impact to aquaculture. Fish with VHSV IVb from
Ontario waters were characterized by moderate to marked necrosis of the
myocardium and other tissues and by a vasculitis. The pattern of lesions varied
with the species affected to some degree. Immunohistochemistry (IHC), using a
rabbit polyclonal antibody to purified VHSV IVb, identified viral antigen in
tissues with lesions but also in the oocytes and testicular tissues of several
fish species. In the current
research, we employed in situ hybridization (ISH) to examine the ovaries from naturally infected freshwater drum and muskellunge, as
well as experimentally infected rainbow trout (Oncorhynchus mykiss) and fathead minnows (Pimphales
promelas). Although the ovaries and testes of fish infected with VHSV IVb had no
significant histological changes, viral RNA was present to varying degrees
within these tissues. The abundance of viral RNA was moderate to marked within
oocytes of muskellunge and freshwater drum with lesser amounts in oocytes of
rainbow trout and fathead minnow. Viral RNA was mainly situated inside yolk
granule and/or
surrounding yolk vacuoles of oocytes, and around primary and secondary
spermatocytes. As determined by ISH, VHSV IVb may reside with oocytes and
spermatozoa; however, survival of virus within gametes needs to be determined.
This method is also another useful technique to study the pathogenicity of VHSV
infection.
S23-6
Humoral Immune Response of Muskellunge, Esox masquinongy, to Viral Hemorrhagic
Septicemia Virus
EV Millard*1 and M Faisal1, 2
| 1 |
Department of Pathobiology and
Diagnostic Investigation, College of Veterinary Medicine, S-112 Plant Biology
Building, Michigan State University, East Lansing, MI USA millarde@cvm.msu.edu
|
| 2 |
Department of Fisheries and
Wildlife, S-110 Plant Biology Building, Michigan State University, East
Lansing, MI USA faisal@cvm.msu.edu
|
Viral Hemorrhagic Septicemia Virus
(VHSV) genotype IVb emerged in the Laurentian Great Lakes causing widespread
fish kills. There are mounting
concerns about the effects this pathogen may have on the health of resident
fish populations. Our previous
studies have demonstrated the high susceptibility of the esocid muskellunge, Esox masquinongy, to VHSV-IVb, though
additional studies have provided evidence that this species can survive
infection if the dose of exposure is relatively low. Therefore this study was designed to determine if
circulating anti-VHSV antibodies are present in muskellunge survivors. Serum samples were collected from two
groups of muskellunge; adults from Lake St. Clair (Lake Erie watershed,
Michigan, USA) over four years, and juveniles that were experimentally exposed
to VHSV. Initially, sera were
analyzed for the presence of antibodies by the complement-dependent 50% plaque
neutralization test. Our findings
indicated that Lake St. Clair muskellunge mount a strong antibody response that
ranged from <40 to 40,960, and that high titers were detectable even in
years when the virus itself was not isolated from the lake. In the case of experimentally infected
muskellunge, antibodies could be measured as early as 5 weeks post infection
and titers ranged from <40 to 20,480. High titers were still detectable at 16 weeks. The samples were then assessed using a recently developed
competitive enzyme-linked immunosorbent assay (cELISA), using VHSV recombinant
proteins as the antigen. This
approach provides a comparison between the two assays and gives an accurate
account of the dynamics of anti-VHSV antibodies in a susceptible species.
S23-7
Viral Hemorrhagic
Septicemia (VHS) Surveillance on Wisconsin Fish Farms
MJ
Kebus*
| Wisconsin Department of
Agriculture, Trade & Consumer Protection, Division of Animal Health,
Madison WI USA Myron.kebus@datcp.state.wi.us
|
This study reflects work conducted between the
Wisconsin Department of Agriculture, Trade and Consumer Protection (DATCP) and the United States Department of
Agriculture, Animal and Plant Health Inspection Service (APHIS), Veterinary
Services under a Cooperative Agreement Award. In Wisconsin the DATCP is responsible for health requirements of imported
fish, farm-raised fish and all fish stocked into waters of the state. Wisconsin
has an abundance of trained veterinarians who are available to fish farmers for
VHS sampling and other disease work including observational surveillance. The
DATCP has collaborated with the University of Wisconsin-Madison, School of
Veterinary Medicine, to develop an online Fish Health Medicine Certificate
Program with accompanying hands-on training that has trained over 230 private
practice, state, and federal veterinarians from the U.S. in fish farm
veterinary medicine, including VHS sample collection. All fish being stocked into state waters are required to
have a fish health certificate prior to stocking. This requirement has been in place prior to the diagnosis of
VHS in the Great Lakes. Since the APHIS Amended VHS Federal Order was put in
place in the fall of 2006, the volume of fish health certificates has increased
from 100 to 500 annually. This
increase resulted in part due to the requirements of the Amended Federal Order
and in part due to the response by the VHS-affected states to add additional
fish health certificate requirements beyond those required by APHIS for import
into their states. As a result of
the increased volume of fish health certificates, there is now a desire from
Wisconsin fish farmers, natural resource agencies and DATCP to develop
electronic fish health certificate and an electronic database to help analyze
and manage the fish health certificates in Wisconsin.
| SESSION
24. VIROLOGY III: IHN/ENV |
[TOP] |
S24-1
Biology and Genetic Characterization of
the Fish and Amphibian Herpesviruses (Family: Alloherpesviridae)
TB Waltzek*1, GO Kelley2, ME Alfaro3, T
Kurobe1, AJ Davison4 and RP Hedrick1
| 1 |
Department of Medicine and Epidemiology, School of Veterinary Medicine,
University of California, Davis, CA, USA tbwaltzek@ucdavis.edu
|
| 2 |
California Department of Fish and Game, Fish Health Laboratory, Rancho
Cordova, CA, USA gokelley@dfg.ca.gov
|
| 3 |
Department of Ecology and Evolutionary Biology, University of California,
Los Angeles, CA, USA michaelalfaro@ucla.edu
|
| 4 |
MRC Virology Unit, Institute of Virology,
University of Glasgow, Glasgow UK a.davison@mrcvu.gla.ac.uk
|
Phylogenetic relationships among herpesviruses (HVs) of mammals,
birds, and reptiles have been studied extensively, whereas those among other
HVs are relatively unexplored. We have reconstructed the phylogenetic
relationships among 13 fish and amphibian HVs using maximum likelihood and
Bayesian analyses of amino acid sequences predicted from parts of the DNA
polymerase and terminase genes. The relationships among 6 of these viruses were
confirmed using the partial DNA polymerase data plus the complete sequences of
the terminase, helicase, and triplex protein genes. The position of these
viruses among all other sequenced HVs was also investigated using the complete
terminase gene. The results established the monophyly of the fish and amphibian
HVs (Alloherpesviridae) separate from the HVs of mammals, birds, and
reptiles (Herpesviridae) and the single recognized HV of bivalve
mollusks (Malacoherpesviridae) in the order Herpesvirales. Two
major clades in the family Alloherpesviridae were recognized: one
consisting of viruses from cyprinid and anguillid hosts and the other of viruses
from ictalurid, salmonid, acipenserid, and ranid hosts. A comparison of virus
and host phylogenies suggested that closely related HVs in this family may have
coevolved with their hosts, whereas significant codiversification was not
apparent for the more distantly related viruses.
S24-2
Genogroup-Specificity
of DNA Vaccines Against Infectious Hematopoietic Necrosis Virus (IHNV) in
Rainbow Trout (Oncorhynchus mykiss)
Ma. M D Peñaranda*1, 2, 3, S LaPatra4, and G Kurath1,2
| 1 |
Graduate Program
in Pathobiology, Department of Global Health, University of Washington, Seattle
WA USA mmdpena@uw.edu
|
| 2 |
Western
Fisheries Research Center, Biological Resources Discipline, USGS, Seattle, WA
USA gkurath@usgs.gov
|
| 3 |
Present
affiliation: Southeast Asian Fisheries Development Center – Aquaculture
Department, Tigbauan, Iloilo, Philippines
|
| 4 |
Clear Springs
Foods, Inc., Research Division, Buhl, Idaho USA scottl@clearsprings.com
|
Infectious hematopoietic necrosis virus (IHNV) is a
fish rhabdovirus that causes significant mortality in salmonid species. IHNV
has three major genogroups, designated M, U, and L. Host-specific virulence of
M and U genogroup IHNV has been established, both in the field and in
experimental challenges, with M isolates being highly virulent in rainbow trout
(Oncorhynchus mykiss) and U isolates
being more virulent in sockeye salmon (Oncorhynchus
nerka). In the present study, the efficacy of DNA vaccines containing
either M (pM) or U (pU) virus glycoprotein gene was investigated during intra-
and cross-genogroup challenges in rainbow trout. Correlates of protection was
also investigated by looking at the expression of the interferon-stimulated
gene Mx-1, and the production of neutralizing antibodies (NAbs) following pM or
pU DNA vaccination. At 7 days post-vaccination (early antiviral response), both
pM and pU were highly protective against either M or U challenge. At 28 days
post-vaccination (late antiviral response), the homologous pM vaccine was
significantly more protective than pU against M IHNV challenge, while both pM
and pU induced comparably high protection against U IHNV challenge. Mx-1 gene
expression, measured as an indicator of the host innate immune response, was
significantly higher in pM than pU vaccine when injected in rainbow trout at
equal vaccine dose. Neutralizing antibodies produced in response to the two
vaccines did not show a clear correlation with protection, suggesting that
there are other adaptive immune response mechanisms that are important in
providing late protection against IHNV in rainbow trout.
S24-3
Vector Potential of the Salmon Louse (Lepeophtheirus salmonis) in the
Transmission of Infectious Haematopoietic Necrosis Virus (IHNV)
E Jakob*1,2, DE Barker1and KA Garver2
| 1 |
Fisheries & Aquaculture
Department, Vancouver Island University, Nanaimo, BC Canada Eva.Jakob@viu.ca,
Duane.Barker@viu.ca
|
| 2 |
Fisheries & Oceans
Canada, Pacific Biological Station, Nanaimo, BC Canada
Kyle.Garver@dfo-mpo.gc.ca
|
Previous studies have isolated viral and bacterial
aquatic pathogens from various piscine ectoparasites, including leeches and
parasitic crustaceans, implicating them as possible carriers and/or reservoirs
of aquatic pathogens. However, the role of ectoparasites in the transmission of
aquatic diseases remains unclear. To this end, we investigated the potential of
the salmon louse, Lepeophtheirus
salmonis, to harbour the aquatic rhabdovirus IHNV and transmit it to naïve
Atlantic salmon (Salmo
salar) under laboratory conditions. One hour waterborne immersion trials of lice with
105 PFU/ml IHNV
revealed that lice can acquire and then harbour infectious virus up to 24 hours
post exposure. Furthermore, studies investigating the parasitism of the salmon
louse on IHNV infected Atlantic salmon demonstrated that lice are able to
acquire high titers of IHNV. Moreover, in a preliminary trial it was
investigated whether lice are able to transfer the virus to naïve fish (a)
after being exposed to IHNV through waterborne exposure and (b) after feeding
on IHNV infected fish. These studies provide the first evidence to support a
direct role of L. salmonis in the
transmission of IHNV to Atlantic salmon.
24-4
Estimation of Viral Parameters Associated with
Waterborne Transmission of Infectious Hematopoietic Necrosis Virus (IHNV) in
Atlantic Salmon.
KA Garver*1, AM Grant1,
and J Richard1
| Fisheries & Oceans Canada, Pacific Biological Station, Nanaimo, B.C.
Canada Kyle.Garver@dfo-mpo.gc.ca,
Amelia.Mahony@dfo-mpo.gc.ca, Jon.Richard@dfo-mpo.gc.ca
|
Multiple
epizootics of Infectious Hematopoietic Necrosis Virus (IHNV) have occurred in
farmed Atlantic salmon in the marine waters of British Columbia. During an outbreak occurring from
2001-2003, IHNV spread to 36 farm sites resulting in the loss of over 12
million fish due to either mortality or culling. To better understand the transmission of IHNV in marine
salmon sites, we empirically determined the minimum infectious dose and
shedding rates for IHNV in Atlantic salmon as well as the stability of virus in
seawater. Waterborne challenges
ranging in viral dose from 10-104 PFU/ml demonstrated that the
minimum dose required to induce mortality in Atlantic salmon post-smolts was 10
PFU/ml. Ultrafiltration of
seawater collected from flow-through virus challenge tanks (104 PFU/ml, 1h, 10oC) revealed detectable levels of shed virus beginning
at 7 days post-challenge. Peak shedding was observed at day 16
and was estimated to be 2 x 105 PFU fish-1 hour-1.
Virus stability studies indicate that IHNV decay rates in raw seawater is rapid
(~105 PFU/day) yet dependent on temperature and local microbial
flora. This information is being
incorporated in a water circulation model to provide more accurate geospatial
predictions of risk for IHNV in farmed Atlantic salmon.
S24-5
Molecular
Characterization and Infection Kinetics of Erythrocytic Necrosis Virus (ENV) in
Pacific Herring
EJ
Emmenegger*1, JA Glenn1, WN Batts1, CG Grady2,
JL Gregg2, SE Roon2, JR Winton1, and PK
Hershberger2
| 1 |
USGS
Western Fisheries Research Center, Seattle, WA USA eemmenegger@usgs.gov,
jglenn@usgs.gov, bbatts@usgs.gov, jwinton@usgs.gov
|
| 2 |
USGS Marrowstone Marine Field
Station, Nordland, WA USA cgrady@usgs.gov,
jgregg@usgs.gov, sroon@usgs.gov, phershberger@usgs.gov
|
Viral erythrocytic necrosis (VEN) periodically
causes epizootics in populations of herring and some salmonids in coastal
marine waters of the Atlantic and Pacific Oceans. VEN is classically diagnosed by microscopic examination of
Giemsa-stained blood films for the presence of inclusion bodies within the
cytoplasm of affected erythrocytes. Little is known about VEN disease
progression or the causative agent, erythrocytic necrosis virus (ENV), because
it fails to show cytopathic effect in any known cell line. ENV is presumed to
be an iridovirus based on electron micrographs, however viral identity has not
been confirmed. We are attempting multiple strategies to molecularly
characterize ENV and have initiated a study to understand the kinetics of
viremia during an ENV infection. We followed the course of the disease after
specific pathogen-free Pacific herring (Clupea
pallasii) were exposed to the putative iridovirus and we demonstrated that
the prevalence of erythrocytic inclusion bodies increased from 0% at 0-4 days
post-exposure, to 94% after 28 days. The kinetics of viremia throughout this
period will be quantified by enumerating virions in paired blood and
spleen/kidney samples using transmission electron microscopy. A better understanding of the
relationship between viral load and cytoplasmic inclusion bodies will
facilitate collection of blood samples containing the highest concentration of
virus, which will aid in the molecular identification of ENV and ultimately in
the development of a diagnostic PCR assay.
| SESSION
25. IMMUNOLOGY III |
[TOP] |
S25-1
Recombination
Activation Gene 1 (rag1) Mutant
Zebrafish for Immunology Research
L Petrie-Hanson, C Hohn* and L Hanson
| Department
of Basic Sciences, College of Veterinary Medicine, Mississippi State
University, Mississippi State, MS USA hohn@cvm.msstate.edu
|
We confirmed the presence of the 1 gene point
mutation rag1t26683 in our rag1-/- mutant population.
Mutants survived caudal fin sampling by continuous bath treatment in 42 mg/L
aquarium salt and 0.04 g/L Jungle Fungus eliminator during recovery. Rag1+/-, rag1-/- and rag1+/+ adults were differentiated by visible implant elastomer (VIE) tagging. The
dorsal fin base was the optimal site and optimal colors were red, pink, orange
and yellow. Peripheral blood differentials indicated that mutants had decreased
lymphocyte populations (34.7%) compared to wild types (70.5%), and increased
granulocyte populations (52.7%) compared to wild types (17.6%).
Monocyte/macrophage populations were similar between mutants and wild-types,
12.6% and 11.3%, respectively. FACS Calibur flow cytometric analyses
demonstrated that 7% and 43% of gated cells from mutants were lymphocytes and
myeloid cells, respectively, while wild types had 26% and 21% lymphocytes and
myeloid cells, respectively. Reverse transcriptase polymerase chain reaction
(RT-PCR) assays demonstrated mutant zebrafish kidney hematopoietic tissue
expressed mRNA encoding Non-specific Cytotoxic cell receptor protein-1
(NCCRP-1) and Natural Killer cell (NK) lysin but lacked T cell receptor (TCR)
and immunoglobulin (Ig) VH1 transcript expression, while wild type zebrafish
kidney hematopoietic tissue expressed NCCRP-1, NK lysin, TCR and Ig VH1
transcript expression.
S25-2
Adhesion Dynamics of Flavobacterium columnare to Channel
Catfish (Ictalurus punctatus) and
Zebrafish (Danio rerio) after
Immersion Challenge
O
Olivares-Fuster, SA Bullard, A McElwain, and CR Arias*
| Department
of Fisheries and Allied Aquacultures, Auburn University, AL USA
ariascr@auburn.edu
|
Adhesion of Flavobacterium
columnare to skin and gill of channel catfish (Ictalurus punctactus) and gill of zebrafish (Danio rerio) was evaluated by immersion challenge followed by
bacterial plate count and confirmatory observations of gill-adhered bacterial
cells using scanning electron microscopy. Two strains of F. columnare (ARS-1 and BGFS-27 from genomovar I and II, respectively) were compared. At 0.5
h post-challenge, both strains adhered to gill of channel catfish at comparable
levels (106 colony forming units (CFU)/g) but significant
differences in adhesion were found later in the time course. Channel catfish was able to effectively
reduce ARS-1 cells on gill; whereas, BGFS-27 persisted in gill beyond the first
24 h post-challenge. No significant difference was found between both strains
when adhered to skin, but adhered cell numbers were lower (103 CFU/g) than those found in gill and were not detectable at 6 h post-challenge.
Adhesion of BGFS-27 cells to gill of zebrafish also occurred at high numbers
(>106 CFU/g) while only <102 CFU/g of ARS-1 cells
were detected in this fish. The results of the present study show that
particular genomovars of F. columnare exhibit different levels of specificity to their fish hosts.
S25-3
Effects of the Anesthesia
Agents Benozocaine and Tricaine Methanesulfonate
(MS-222) on Immune Parameters of Tadpoles of the Cuban Tree Frog (Osteopilus septentrionalis)
B F Sears*, P Snyder, and J R Rohr
| Department of Integrative Biology, University of South
Florida, Tampa, FL USA bsears@usf.edu, psnyder@usf.edu,
jrohr@usf.edu
|
Benzocaine and tricaine methanesulfonate (MS-222) are
widely used in the anesthetization of poikilothermic vertebrates. While the
physiological effects of anesthesia via these agents have been characterized in
fish and adult amphibians, little is known about the physiological consequences
of anesthesia in larval amphibians. Because anesthesia is frequently employed
when studying the behavior of larval amphibians, including their avoidance of
parasites, we tested whether anesthesia reduces immunocompetence by reducing
the number of circulating white blood cells (WBC). Tadpoles of the Cuban tree
frog (Osteopilus septentrionalis) were exposed to artificial spring water (ASW; control), MS-222, or benzocaine.
Animals were sacrificed at intervals post-anesthesia, and blood smear slides
were prepared for microscopical examination of circulating WBC. Benzocaine did
not result in significantly different numbers of circulating WBC when compared
to the artificial spring water control. In contrast, MS-222 did significantly
reduce levels of circulating thrombocytes. We are currently conducting
experiments to examine changes in resistance to experimental trematode
infections after anesthesia.
S25-4
Antiviral Activities of an Intracellular
Viral RNA Sensor, MDA5 in Japanese Flounder (Paralichthys olivaceus)
M
Ohtani*1, J Hikima1, TS Jung1 and T Aoki1,
2
| 1 |
Aquatic
Biotechnology Center, College of Veterinary Medicine, Gyeongsang National
University,
Gyeongnam, South Korea makky34@gmail.com, j.hikima@gmail.com, and
jungts@gnu.ac.kr
|
| 2 |
Laboratory
of Genome Science, Tokyo University of Marine Science and Biotechnology, Minato, Tokyo, Japan
aoki@kaiyodai.ac.jp
|
Infected viral RNAs are
sensed with pattern recognition receptors including toll-like receptors (TLRs)
and retinoic acid inducible gene-like receptors (RLRs). Melanoma
differentiation-associated gene 5 (MDA5), a member of RLRs, recognizes the
viral RNA in cytoplasm and enhances the antiviral response in the host cells.
MDA5 gene in Japanese flounder (Paralichthys olivaceus) was
cloned. The MDA5 transcripts in the kidney infected
with viral hemorrhagic septicemia virus (VHSV) showed a significant increase in
the expression levels as well as the MDA5 expression in the kidney
leukocytes stimulated with poly I:C in
vitro. To reveal the antiviral activity of the flounder MDA5, five expression constructs
containing pcDNA4-MDA5 (full-length), pcDNA4-MDA5ΔRD (regulatory domain deleted), pcDNA4-MDA5ΔCARD (CARD deleted), pcDNA4-MDA5ΔHeli (RNA
helicase region deleted) and pcDNA4-Empty (as
control) were transfected into the flounder natural embryo (HINAE) cell line. After 48 hours transfection,
the transfected cells were infected with VHSV or infectious pancreatic
necrosis virus (IPNV) at 15?C. The cytopathic effect appearance of the virus-infected HINAE cells
transfected with pcDNA4-MDA5, pcDNA4-MDA5ΔRD and pcDNA4-MDA5ΔHeli was delayed in contrast to
the pcDNA4-Empty or -MDA5ΔCARD. To confirm the antiviral
responses, the expression level of interferon inducible genes (Mx and ISG15) in
HINAE cells transfected with the expression vectors were quantified by Q-PCR. The
expressions of Mx and ISG15 mRNAs were induced by pcDNA4-MDA5, pcDNA4-MDA5ΔRD and pcDNA4-MDA5ΔHeli in the presence of poly
I:C. These results suggested that the flounder MDA5 induced the inhibition of viral replication through the induction
of interferon inducible gene expressions.
S25-5
Lymphocyte Deficient
Zebrafish Demonstrate Specific Protection Following Bacterial Re-exposure
C Hohn and L Petrie-Hanson*
| Department
of Basic Sciences, College of Veterinary Medicine, Mississippi State
University, Mississippi State, MS USA, lora@cvm.msstate.edu
|
Recombination activation gene 1 deficient (rag1-/-) mutant zebrafish lack functional B and T
lymphocytes, and therefore are entirely dependant on their innate immunity for
protection against pathogens. The innate immune system is thought to lack
adaptive characteristics that provide enhanced protection to a second exposure
of the same pathogen. In the present study we evaluated the ability of rag1-/- mutant zebrafish
to mount such a protective response to the intracellular bacterium Edwardsiella ictaluri. Following
secondary challenge with a lethal dose of homologous bacteria 4 and 8 weeks
after a primary vaccination dose, both wild-type and lymphocyte deficient rag1-/- mutant zebrafish
demonstrated equivalent levels of protection. Heterologous bacterial exposures
did not provide protection in either wild-type or rag1-/- mutant zebrafish. Adoptive leukocyte
transfers from previously exposed mutants conferred protective immunity in naïve mutants when exposed to homologous bacteria. Our findings show that a component
of the innate immune system mounted a response that provided long-term specific protection.
Further, adoptive cell transfers demonstrated that kidney interstitial
leukocytes from lymphocyte deficient zebrafish transferred this protective
immunity. This
is the first report of any lymphocyte-deficient vertebrate mounting a
protective secondary immune response to a bacterial pathogen, and suggests that
innate immune cells can mediate adaptive immunity.
S25-6
Generation of a Monoclonal
Antibody Specific for ORF68 of Koi Herpesvirus
T Aoki*1, I Hirono1, H Kondo1, T Takano1,
S Unajak1, T Taki2, J Hikima3 and TS Jung3
| 1 |
Laboratory of
Genome Science, Tokyo University of Marine Science and Technology, Tokyo, Japan, aoki@kaiyodai.ac.jp, hirono@kaiyodai.ac.jp, h-kondo@kaiyodai.ac.jp, takano5320@hotmail.com, and fscissmn@ku.ac.th
|
| 2 |
Department of
Microbiology, Faculty of Hygienic Technology, School of Allied Health Sciences,
Kitasato University, Kanagawa, Japan, tatabox@kitasato-u.ac.jp
|
| 3 |
Aquatic
Biotechnology Center, College of Veterinary Medicine, Gyeongsang National
University, Jinju, Gyeongnam, South Korea, j.hikima@gmail.com, and jungts@gmail.com
|
Outbreak of Koi herpesvirus [KHV or Cyprinid
herpesvirus 3 (CyHV-3)] disease in carp is still a serious problem worldwide.
KHV is related closely to other two cyprinid herpesviruses, including pox
herpesvirus [CHV or Cyprinid herpesvirus 1 (CyHV-1)], haematopoietic necrosis
herpesvirus of goldfish [Cyprinid herpesvirus 2 (CyHV-2)], and KHV. It is said
that these viral species have common antigens, therefore, identifying specific
antigens from KHV is important to possibly control the disease caused by this
virus. In a previous study, two major KHV antigenic proteins, encoded by open
reading frame (ORF) 62 and ORF68 were identified by immunoscreening from the
genomic phage library using KHV-specific rabbit polyclonal antibody. To
generate the monoclonal antibody (MAb) against ORF62 or ORF68 proteins, BALC/c
mice were immunized
with the recombinant antigenic proteins of ORF62 or ORF68. The spleen cells
from immunized mice were fused with Sp2/o myeloma cells using polyethylene
glycol. After the screening by enzyme-linked immunoassay (ELISA) and Western
blot analysis, we obtained one MAb against ORF68 (7C6) but no MAb against
ORF62. In Western blot analysis, the anti-ORF68- MAb demonstrated specific
reaction with KHV-infected KF-1 cells lysate but not with CHV- or
CyHV-2-infected KF-1 cells lysate. Similar results were shown in fluorescent antibody (FA) test using infected KF-1 cells and immunohistochemical (IHC) investigation
conducted by haematoxylin and eosin (H&E) stain for infected liver sections or FITC detection for infected spleen
sections. These suggested that the anti-ORF68-MAb (7C6) is capable of specifically
detecting the KHV ORF68 protein.
| SESSION
26. TOXICOLOGY AND ENVIRONMENTAL STRESS |
[TOP] |
S26-1
Histopathologic
Effects of 17 Beta-estradiol on the Gonads of Larval Xenopus laevis
JC
Wolf *1, I Lutz2, W Kloas2, TA Springer3,
HO Krueger3, LR Holden4 and AJ Hosmer5
| 1 |
Experimental Pathology
Laboratories, Inc., Sterling, VA USA jwolf@epl-inc.com
|
| 2 |
Inland
Fisheries, Leibniz-Institute of Freshwater Ecology and Inland Fisheries,
Berlin, Germany werner.kloas@igb-berlin.de ilka.lutz@igb-berlin.de
|
| 3 |
Wildlife International Ltd.,
8598 Commerce Drive, Easton, MD, 21601 USA tspringe@wildlifeinternational.com hkrueger@wildlifeinternational.com
|
| 4 |
Sielken & Associates
Consulting, Inc., Bryan, TX USA larry@sielkenassociates.com
|
| 5 |
Syngenta Crop Protection,
Inc., Greensboro, NC USA. alan.hosmer@syngenta.com
|
Traditionally, histopathology has been used in a
limited fashion to investigate reproductive effects of xenoestrogens in
anurans. The purpose of this study
was to fully characterize the histopathologic effects of 17 beta-estradiol (E2)
on the gonads of larval Xenopus laevis exposed under tightly controlled laboratory conditions. This study was performed as three
separate experiments at two separate facilities, and involved more than two
thousand control and E2-exposed frogs. Microscopic findings were recorded according to standardized criteria
and terminology that were ultimately refined by the process. Throughout all experiments, the most
consistent effects of E2 exposure were increased proportions of phenotypic
females, and increased prevalences (as compared to untreated controls) of mixed
sex, dilated testicular tubules, dividing gonocytes in the testis, and internal
melanophores in the testis. The
diagnosis of dilated testicular tubules was an especially robust E2 indicator. These experiments demonstrated the
benefits of a detailed histopathologic examination for detecting
endocrine-active effects.
S26-2
Mortalities, Skin
Lesions and Intersex in Smallmouth Bass: Possible Association with Contaminants
of Emerging Concern
VS
Blazer*1, LR Iwanowicz1, DD Iwanowicz1 and H
Ellery1,2
| 1 |
National Fish Health
Research Laboratory, U.S. Geological Survey, Kearneysville, WV USA vblazer@usgs.gov; liwanowicz@usgs.gov; diwanowicz@usgs.gov
|
| 2 |
Cooperative Fish and
Wildlife Unit, West Virginia University, Morgantown, WV USA hellery@usgs.gov
|
Since 2002, major fish kills of centrarchids have
occurred in the Potomac, James and Susquehanna river drainages. The mortalities have varied in location
and severity and are generally accompanied by skin lesions. In the Potomac and
James rivers it is primarily the mature, adult smallmouth bass and redbreast
sunfish that are affected in the spring. In certain areas population effects
have been reported. Surveys over the past six to eight years have indicated
that a variety of bacterial and viral pathogens are present, heavy parasite loads
are observed and other lesions such as papillomas, gill lamellar fusion,
oxidative damage and a high prevalence of intersex or testicular oocytes are
noted. No one pathogen is consistently isolated and the variety of lesions
suggest the fish are immunosuppressed, and hence susceptible to a number of
potential pathogens, including Aeromonas
salmonicida, motile Aeromonads, Flavobacterium
columnare and largemouth bass virus. Heavy infestations of trematode
metacercariae and a number of myxozoan parasites are observed. The coincidental
finding of high prevalence and the severity of testicular oocytes at all areas
at which skin lesions and mortalities occur suggests that contaminants of
emerging concern may be involved, particularly estrogenic chemicals. Many of
these have been shown to influence disease resistance and be reproductive
endocrine-modulators. Sources of these chemicals (natural and synthetic
hormones, pesticides, herbicides, personal care products, and pharmaceuticals)
include human wastewater treatment plant effluent, stormwater runoff and
agriculture. Ongoing studies including chemical analyses of water and fish
tissue, and landscape analyses, will be discussed.
S26-3
Investigating Fish Kills in Young-of-Year Smallmouth Bass
in the Susquehanna River Drainage.
L
Iwanowicz*1, V A Blazer1, C Starliper1, H
Ellery and K Crawford2
| 1 |
US
Geological Survey, Leetown Science Center, Fish
Health Branch, 11649 Leetown Rd. Kearneysville WV, 25430 USA Luke_Iwanowicz@usgs.gov
|
| 2 |
US
Geological Survey, Columbia Environmental Research Center, 4200 New Haven Road, Columbia, MO 65201-8709,
USA
|
Smallmouth bass mortality throughout the Potomac and
Susquehanna River drainages has attracted the concern of the public and
resource management agencies during recent years. In the Susquehanna River
drainage, SMB mortality in the Susquehanna is primarily observed in
young-of-year (YOY) bass. Flavobactrerium
columnare has been isolated from bass during mortality events, but
histological evidence suggests that this pathogen may not be the singular or
primary cause of mortality. Chronic granulomatous inflammation in multiple
tissues was consistently identified in YOY bass examined in 2007.
Interestingly, within these areas of inflammation a putative rickettsia-like organism
has been noted. Largemouth bass virus has been isolated from moribund bass and
large myxozoan cysts have been observed in the skeletal muscle of these fish.
Extensive bacterial and viral culture efforts in addition to culture
independent methods are in progress to identify the assortment of microbes
associated with these mortality events. Here results of our findings from 2007-2010 will be reported.
S26-4
Disappearing Golden Shiner Phenomenon- Is
Hydrogen Sulfide the Culprit?
AM
Kelly* and S Kumaran
University
of Arkansas at Pine Bluff, Fish Health Services, Lonoke, AR 72086, USA
akelly@uaex.edu, skumaran@uaex.edu
|
Throughout the year, when water temperatures are
between 70 and 750C, golden shiner (Notemigonus crysoleucas) stocks
seem to disappear from ponds that have been in production for two or more
years. Investigations into the
possible causes for this phenomenon have lead to the conclusion that hydrogen
sulfide may play a role. Since the toxic form of hydrogen sulfide is more
abundant at lower pH values, a study conducted in ponds with fluctuating pH and
temperatures was conducted. Twenty-five ponds on a commercial fish farm were
outfitted with a cage containing 12 sentinel fish. In these 25 ponds, monitors were used to record daily
fluctuations in pH and temperature. Cages were checked twice a week and the number of fish in the cages and
their health status were recorded. At the end of the study, the number of live fish remaining was also recorded. Hydrogen sulfide concentrations were
taken in ponds before and after disturbing the mud bottoms. Ponds were categorized into low
<0.005-0.01 mg/L, medium 0.01-<0.75 mg/L and high >0.075 mg/L hydrogen
sulfide producing ponds. Fish in the low hydrogen sulfide ponds had
significantly higher survival rates (61.8%) versus those in the medium (17.7%)
and high (3.3%) hydrogen sulfide ponds. Future studies include determining
median lethal levels for hydrogen sulfide to golden shiners at various pH
values. Studies on the hydrogen sulfate and hydrogen sulfide concentration in
ponds as well as the 24 hour hydrogen sulfide cycle will be presented.
[TOP]
Contents
- Scientific Sessions
- Plenary Abstracts - Poster Abstracts - Special
Session Abstracts - Author
Index