Progress 10/01/03 to 09/30/08
Outputs
OUTPUTS: The P.I. has retired for some time and there is nothing to report. PARTICIPANTS: The P.I. has retired for some time and there is nothing to report. TARGET AUDIENCES: The P.I. has retired for some time and there is nothing to report. PROJECT MODIFICATIONS: The P.I. has retired for some time and there is nothing to report.
Impacts
The P.I. has retired for some time and there is nothing to report.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
A study was conducted at two power plants located in Alabama to determine the health status and pathogen prevalence in threadfin shad, gizzard shad, and freshwater drum impinged at the water intake of an electric generating plant. In comparison to reference fish, impinged gizzard shad had diminished weight at equivalent lengths (~10%) and higher prevalence of F. columnare infections (78% in impinged vs 18% in the reference). The number of external protozoan parasites was also significantly different with a 19.2 higher prevalence occurring in the impinged fish. Impinged freshwater drum, also had significantly higher prevalence of pathogenic bacteria compared to reference fish (difference of 25.9%) as well as in external protozoan parasites (a difference of 36.2% higher in the impinged fish). No differences in pathogens or in length-weight frequencies were observed in the threadfin shad. An additional observation that noted from the gizzard shad was the prevalence of
melanomic tumors. Gizzard shad were examined from the Black Warrior River at Plant Gorgas (10 cases out of 600 fish observed). Tumors were raised and pigmented, varying in size as weell as location on the body. These tumors were cutaneous spindle cell melanomas that have only been previously reported in Oklahoma. No etiology has been determined. Current methods of detecting channel catfish virus (CCV) are lethal and require relatively high titer of active virus which is only present during an outbreak of susceptible fish. The objective of this study is to use polymerase chain reaction (PCR) to non-lethally screen brood fish for CCV. Barbel samples were taken from channel catfish brood stock during spawning. DNA was extracted from frozen barbel samples, and PCR primers specific for CCV were used to run the PCR following standard protocols. Tissue samples were collected from 137 individual brood fish in 2006 and eighty-five of those fish were PIT tagged for future identification and
re-sampling in 2007. Twenty-five fish were found positive for CCV in 2006. Tissue samples were collected from 150 brood fish in 2007, and 80 samples have been screened. Four of those 80 individuals were found positive for CCV. Utilizing this method may allow us to monitor the "carrier" status of brood fish over several spawning periods and could be beneficial in maintaining virus-free brood stocks. Two novel bacteriophages that infect E. ictaluri were isolated. The phages are double-stranded DNA viruses with approximate genome sizes of 39 and 45 kb, respectively. The latent period for phage infection was 70 min, with an estimated burst size of 1200. All E. ictaluri strains tested were susceptible to phage infection with variable plaquing efficiencies. These phages have not been observed to produce plaques on any other bacterial species. The specificity and lytic activity of these bacteriophages for E. ictaluri strains makes them attractive candidates as diagnostic agents and for
bacteriophage therapy of ESC.
Impacts
Fish diseases are important in both wild fish populations and aquaculture. Improved diagnostic methods, including methods for detecting pathogens in subclinical infections, will enable better management of affected fish populations. Knowledge of factors affecting the spread, prevalence, and pathogenesis of fish diseases is important for control of the disease and will enable managers to make appropriate provisions in population management.
Publications
- Arias, C.R., O. Olivares-Fuster, K. Hayden, C.A. Shoemaker, J.M. Grizzle, and P.H. Klesius. 2007. First report of Yersinia ruckeri biotype 2 in the USA. Journal of Aquatic Animal Health 19:35-40.
- Oliveres-Fuster, O., J. L. Baker, J. S. Terhune, C. A. Shoemaker, P. H. Klesius, and C. R. Arias. 2007. Host-specific association between Flavobacterium columnare genomovars and fish species. Journal of Systematic and Applied Microbiology 30: 624-633.
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Progress 01/01/06 to 12/31/06
Outputs
The adhesion of bacteria to gills and skin is an essential step in pathogenesis of columnaris disease, caused by Flavobacterium columnare, and differences in bacterial adhesiveness could affect virulence. To test this hypothesis, virulence and adhesion of F. columnare were determined in simultaneous experiments with newly hatched zebrafish in multiwell plates. Virulence of three isolates of F. columnare was determined by adding bacteria to the water and observing the fish for four days. In the adhesion assay, each well received the same F. columnare suspensions used for the virulence assay. After a 1-h exposure to the bacteria, adhesion of the bacteria to fish in the adhesion assay corresponded to the virulence of the isolates. These results indicated that simultaneous measurement of adhesion and virulence provided better comparison of these variables than when the experiments were done at different times. Detection of largemouth bass virus (LMBV) was compared for two
types of samples: gills and a pool of visceral organs. Based on the results from both the viscera and gill, 23 of the 70 largemouth bass tested were infected with LMBV. Of the 23 LMBV-positive fish, gills were positive for LMBV in nine fish and were the only positive sample for five of these fish. Testing for LMBV by using only gill samples resulted in a detection sensitivity of 39%, compared to 78% for viscera. This level of sensitivity for gill samples was not satisfactory for detection of LMBV in a fish populations. The health of fish impinged on the cooling-water intake screens at Plant Barry, a coal-fired steam generating plant located on the Mobile River, Mobile County, AL, was compared with the health condition of reference fish collected from the river population. Fish species sampled were threadfin shad, blue catfish, channel catfish, and freshwater drum with sampling occurring in the spring and fall of 2005. Impinged threadfin shad, blue catfish, and freshwater drum weighed
significantly less (7-30%) during the spring, and impinged threadfin shad and freshwater drum weighed significantly less (12-32%) during the fall when compared with reference fish of equivalent lengths. Additionally, lesions were significantly more common for all species of impinged fish than for reference fish during the spring (14-73%) and for species except threadfin shad during the fall (25-64%). Lesions observed included, necrotic gills, eroded fins and fin bases, ulcers, skin depigmentation, and hemorrhaging. Liver, trunk kidney, and the periphery of lesions on gills and skin were sampled for bacteria. Flavobacterium columnare was detected in a significantly greater percentage of impinged fish than in reference fish during the spring (13-61%) and fall (16-74%). The protozoan parasite, Ichthyobodo necator, was also observed at a significantly higher prevalence during the spring (50%) and fall (30%) in impinged freshwater drum. These data suggest that water intake structures may
be selecting diseased fish from the population and could provide a method for detecting diseases present in a low proportion of the population.
Impacts
Fish diseases, such as columnaris disease and largemouth bass virus disease, are important in both wild fish populations and aquaculture. Improved diagnostic methods, including methods for detecting pathogens in subclinical infections, will enable better management of affected fish populations. Knowledge of factors affecting the spread and pathogenesis of fish diseases is important for control of the disease and will enable managers to include the effects of diseases in population management.
Publications
- Beck, B.H., R.S. Bakal, C.J. Brunner, and J.M. Grizzle. 2006. Virus distribution and signs of disease after immersion exposure to largemouth bass virus. Journal of Aquatic Animal Health 18:176-183.
- Maceina, M.J., and J.M. Grizzle. 2006. The relation of largemouth bass virus to largemouth bass population metrics in five Alabama reservoirs. Transactions of the American Fisheries Society 135:545-555.
- Schramm, H.L., Jr., A.R. Walters, J.M. Grizzle, B.H. Beck, L.A. Hanson, and S.B. Rees. 2006. Effects of live-well conditions on mortality and largemouth bass virus prevalence in largemouth bass caught during summer tournaments. North American Journal of Fisheries Management 26:812-825.
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Progress 01/01/05 to 12/31/05
Outputs
To improve the cell-culture methods to detect largemouth bass virus (LMBV), we tested inoculation method, adsorption time, incubation temperature, and various cell lines to determine the conditions that would provide the most sensitive cell culture assay for LMBV. The optimal inoculation procedure tested was to remove the culture medium from the culture well before the addition of the inoculum, and the optimal adsorption procedure tested was to allow the virus to adsorb for 40 min while the plates were on an orbital shaker. Following inoculation, incubation at 30C resulted in a higher number of viral plaques than incubation at 25C or 32C. Four cell lines (BF-2, FHM, EPC, and CCO) inoculated with LMBV had similar susceptibility to infection. Similar percentages of LMBV positive samples were detected in BF-2 and FHM cell cultures inoculated with homogenized organ samples from largemouth bass; however, the use of two cell lines increased the number of infected samples
discovered. A blind passage also increased the number of positive samples detected in cell culture. Subcultivation to confirm virus positive samples was useful for reducing false-positive results. Largemouth bass virus was also studied in a laboratory challenge experiment. Largemouth bass were immersed for 1 h in water containing LMBV and then fish were necropsied daily for 27 d. Cell culture was used to detect LMBV, and the following organs were tested: gill, liver, spleen, trunk kidney, head kidney, swim bladder, heart, brain, gonad, intestinal cecum, and intestine. Of the 225 fish exposed to LMBV, 2 dead and 5 moribund fish were collected during the 27 d after immersion. All of the moribund fish tested positive for LMBV, and 31% of fish with no clinical signs of disease were infected with LMBV. Largemouth bass virus was detected systemically at the end of the 1-h exposure period and for up to 24 d postexposure. The gill, swim bladder, and trunk kidney were the organs most
frequently infected with LMBV and had the highest titers of LMBV. Fish collected during days 0 through 8 postexposure had a greater number of LMBV infected organs than fish collected on days 9 through 27.
Impacts
Largemouth bass virus disease is a recently discovered problem in wild fish. Improved diagnostic methods, including methods for detecting this virus, will enable better management of affected fish populations. Knowledge of factors affecting the spread of this pathogen will be important for control of the disease and will enable managers to include the effects of LMBV disease in population management.
Publications
- Grizzle, J.M. 2005. The Southeastern Cooperative Fish Disease Project: 40 years of service to the southeastern United States. Proceedings of the Annual Conference Southeastern Association of Fish and Wildlife Agencies 58(2004):187-195
- Manning, B. B., Terhune, J. S., Li, M. H., Robinson, E. H. and Wise, D. J. 2005. Exposure to feed-borne mycotoxins T-2 toxin or ochratoxin A causes increased mortality of channel catfish challenged with Edwardsiella ictaluri. Journal of Aquatic Animal Health 17:147-152.
- McClenahan, S.D., Beck, B.H. and Grizzle, J.M. 2005. Evaluation of cell culture methods for detection of largemouth bass virus. Journal of Aquatic Animal Health 17:365-372
- McClenahan, S.D., Grizzle, J.M. and Schneider, J.E., Jr. 2005. Evaluation of unpurified cell culture supernatant as template for the polymerase chain reaction (PCR) with largemouth bass virus. Journal of Aquatic Animal Health 17:191-196.
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Progress 01/01/04 to 12/31/04
Outputs
Three types of assays for adhesiveness of Flavobacterium columnare were developed and then compared with a previously developed, plastic-plate assay. For the gill assay, gills were dissected from channel catfish, bluegill, and common carp. There was a significant difference among F. columnare isolates for gills from bluegill (8 isolates) and common carp (3 isolates). Only two isolates were tested with normal channel catfish gills; however, there was a significant increase in the number of bacteria adherent to gill of channel catfish with proliferative gill disease or with Aeromonas infection. For the assay with larval zebrafish, there were significant differences in adhesiveness of 11 isolates of F. columnare, and the CFU/mg of fish varied over a 150-fold range. For the assay with cultured cells, 7 isolates of F. columnare were tested for adhesiveness in hypotonic conditions (well water), and there was no significant difference among isolates. Four isolates were
tested in saline, and one isolated had a significantly reduced adhesiveness. The assay with cultured cells was not satisfactory because the high concentration of sodium chloride in saline reduces the adhesiveness of F. columnare. The use of fresh water during the incubation of cells with F. columnare resulted in swelling of the cultured cells because of the hypotonic conditions. The variation in adhesiveness among the F. columnare isolates tested was small for the cultured-cell and gill assays. The plastic plate assay and the larval fish assay appear most promising for future studies. The polymerase chain reaction (PCR) has been used to identify largemouth bass virus (LMBV) in cell culture, and this method has included extraction of the sample DNA. To simplify this procedure, we centrifuged the fathead minnow or bluegill fry cell culture fluid to remove cellular debris and then used the supernatant directly in the PCR without DNA extraction. For supernatants from cell cultures
inoculated with 1:100 dilutions of homogenized swim bladder or pooled spleen and trunk kidney, only 70.0% of the LMBV positive samples (based on all available evidence) tested positive with this new PCR method. After a 1:500 dilution of these cell culture supernatants, 85% of the failed samples became PCR positive. In contrast, 97.2% of LMBV positive samples were PCR positive when undiluted supernatants from subcultivations (cells inoculated with supernatant from cell cultures showing cytopathic effect [CPE]) were used directly in the PCR. Unpurified cell culture fluid supernatant from blind passage samples (cells inoculated with supernatant from CPE negative cultures) was PCR positive for 93.8% of the samples that were eventually shown to be LMBV positive. The use of cell culture supernatant directly in the PCR reduced the cost and time required to identify LMBV isolated in cell culture. Extraction of DNA from cell cultures to obtain template for PCR identification of LMBV was
required for only 2% of the LMBV positive samples.
Impacts
Results of these experiments will enable fish farmers to reduce losses caused by disease. An improved understanding of fish diseases will result in better management of wild fish populations.
Publications
- Terhune, J.S., Grizzle, J.M., Hayden, K., McClenahan, S.D., Lamprecht, S.D. and White, M.G. 2004. First report of koi herpesvirus in wild common carp in the Western Hemisphere. Fish Health Newsletter 32(3):8-9.
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Progress 01/01/03 to 12/31/03
Outputs
Research was initiated in the following areas: evaluation of a vaccine for columnaris disease, development of methods to evaluate virulence of columnaris isolates, improved detection of largemouth bass virus (LMBV), and studies of LMBV pathogenesis.
Impacts
Results of these experiments will enable fish farmers to reduce losses caused by disease. An improved understanding of fish diseases will result in better management of wild fish populations.
Publications
- Grizzle, J.M. and Brunner, C.J. 2003. Review of largemouth bass virus. Fisheries 28(11):10-14.
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