Farm size, seining practices, and salt use: risk factors for Aeromonas hydrophila outbreaks in farm-raised catfish, Alabama, USA.

In freshwater aquaculture systems, Aeromonas hydrophila is usually considered to be an opportunistic pathogen most often associated with secondary bacterial infections. Since 2009, the U.S. catfish industry, especially in West Alabama, has been affected by mortality from a strain of A. hydrophila that has been acting as a primary pathogen. Tens of millions of pounds of catfish production have been lost as a consequence of this disease. This study used data from two whole-population farmer surveys to examine farm-level risk factors for two A. hydrophila outbreaks in foodsize Alabama catfish, one in 2009 (surveyed in 2010), and one in 2011 (surveyed in 2012). The response to the 2010 survey was 85% and the response to the 2012 survey was 82%. Univariate analyses were used to examine biologically plausible variables (farm size, pond stocking density, seine exposure, use of salt (NaCl) in ponds), and used categorical disease outcome and dependent variables. Farm size was included in bivariate analyses with the other variables, because it was a potential confounding variable. For both study years, the odds of an A. hydrophila outbreak were significantly greater for farms larger than the mean size (2009: mean=132 acres (53.4 hectares), odds ratio (OR)=8.2; 95% confidence interval (CI)=3.3-20.6, p<0.001; 2011: mean=116 acres (46.9 hectares), OR=5.3, CI=1.7-17.0, p=0.009). Compared with 42% of control farms, every case farm was seined by a commercial or processing plant seining crew in 2009. The bivariate analysis of the 2011 variable "average number of times each pond was seined per year" indicated that regardless of farm size, farms with ponds that were seined more than twice per year had a significantly greater odds of an A. hydrophila outbreak (OR=4.1, CI=1.2-14.4, p=0.02). For 2009, the results of the bivariate analyses of chloride concentrations indicated that farms that had chloride concentrations >135 ppm had a significantly lower odds of experiencing A. hydrophila outbreaks (OR=0.2, CI=0.05-0.6, p-value=0.004). To achieve economies of scale, catfish farmers raise fish on large farms at higher stocking densities, but this practice may result in increased susceptibility to disease outbreaks. Producers should prioritize implementing biosecurity measures such as improved seining practices and other management practices to protect fish grown at high population densities. Further work will determine what the detailed seining protocols should include, and whether the use of salt, and at what concentrations, reduces the risk of A. hydrophila outbreaks.

Use of vaccination against enteric septicemia of catfish and columnaris disease by the U.S. catfish industry.

Vaccination is an effective strategy used for the protection of food animals against infectious diseases. A 2010 U.S. Department of Agriculture questionnaire examined U.S. catfish industry use (in 2009) of two commercial vaccines that provide protection against enteric septicemia of catfish (ESC) and columnaris disease, catfish producers' opinions regarding the percentage of vaccinated fish they expect to be protected, and producers' general expectations regarding survival of vaccinated fish compared with unvaccinated fish. During 2009, 9.7% of the total fingerling operations used one or both vaccines; 12.3% of the total industry fry production was vaccinated against ESC, and 17.0% was vaccinated against columnaris disease. Of the producers who grew food-sized catfish to harvest, 6.7% used vaccinated catfish. The farms that did not use vaccinated fish for grow out had a mean size of 63.4 water surface hectares (156.6 water surface acres). The operations that used vaccinated fish were larger (mean size = 206.6 water surface hectares, or 510.6 water surface acres). The producers that stocked ESC-vaccinated fish for grow out represented 19.0% of the total water surface area of food fish production; producers that stocked columnaris-vaccinated fish represented 16.6% of the total area. Of the producers that stocked ESC-vaccinated catfish, 41.9% thought that survival was better in vaccinated fish than in unvaccinated fish; of the producers that stocked columnaris-vaccinated catfish, 46.2% thought that vaccinated fish displayed better survival. However, 37.5% of producers that used the ESC vaccine and 39.7% of producers that used the columnaris vaccine did not know whether vaccination improved survival rates. When all producers were asked about their expectations regarding the percentage of vaccinated fish that would be protected from disease, 52.4% responded that they expected 100% of their fish to be protected. More producer information about reasonable expectations regarding vaccine efficacy, the conditions under which immunosuppression and vaccine failure can occur, and assessment of vaccine performance may result in increased use of vaccination as a tool for the catfish industry.

Identification of 'Candidatus Piscichlamydia salmonis' in Arctic charr Salvelinus alpinus during a survey of charr production facilities in North America.

Arctic charr Salvelinus alpinus production facilities, nonproduction water sources and effluents in the United States and Canada were sampled to determine if chlamydiae associated with epitheliocystis were present in water and were associated with inclusions of epitheliocystis in gill tissue. Gills from 607 fish from 13 sites were processed for histopathologic examination and DNA extraction. Water was collected from 21 locations for DNA testing. Eighteen fish from one location had inclusions of epitheliocystis with proliferative and inflammatory gill lesions. Inclusions were stained using the Gimenez technique and, at the ultrastructural level, consisted of intracytoplasmic membrane-bound vacuoles containing reticulate and intermediate bodies in a fibrillar matrix. PCR using Order Chlamydiales-specific primers performed on DNA extracts from 12 of 13 infected fish yielded amplicons that were identical to (GQ302988) or differed at one base from (GQ302987) the 16S ribosomal RNA gene signature sequence of 'Candidatus Piscichlamydia salmonis', which is the chlamydia that was previously identified in epitheliocystis inclusions of farmed Atlantic salmon. In situ hybridization using a approximately 1.5 kb riboprobe corresponding to the 'Candidatus Piscichlamydia salmonis' 16S rRNA genetic sequence (AY462244) confirmed its presence within Arctic charr gill inclusions. DNA isolated from water samples was tested by Chlamydiales-specific PCR and yielded 54 partial 16S rRNA genetic sequences spanning the signature region; however, no 16S rRNA genetic sequences associated with epitheliocystis were identified. This is the first report of 'Candidatus Piscichlamydia salmonis' associated with epitheliocystis in Arctic charr, the first identification of 'Candidatus Piscichlamydia salmonis' from a freshwater production location, and the first reported occurrence in North America.

Survival of vaccinated, feed-trained largemouth bass fry (Micropterus salmoides floridanus) during natural exposure to Flavobacterium columnare.

Vaccinated, feed-trained largemouth bass fry (Micropterus salmoides floridanus) were cohabited with sham-vaccinated fish. Fish were exposed, under natural conditions, to Flavobacterium columnare, a ubiquitous bacterium associated with columnaris disease. During every time interval, the probability that a vaccinated fish would survive past time, t, was greater than for sham-vaccinated fish and survivor functions were significantly different (p-value <0.001). Overall, vaccinated fish had a 43% lower risk of death during the field trial. Overall incidence was 1.7 times greater for the sham-vaccinated (1.4%/d) as compared to the vaccinated fish (0.8%/d). Vaccination with AQUAVAC-COL (Intervet/Schering-Plough Animal Health) significantly reduced the risk of death from columnaris disease in feed-trained largemouth bass fry.

Characterization of a Neochlamydia-like bacterium associated with epitheliocystis in cultured Arctic charr Salvelinus alpinus.

Infections of branchial epithelium by intracellular gram-negative bacteria, termed epitheliocystis, have limited culture of Arctic charr Salvelinus alpinus. To characterize a bacterium associated with epitheliocystis in cultured charr, gills were sampled for histopathologic examination, conventional and immunoelectron microscopy, in situ hybridization, 16S ribosomal DNA (rDNA) amplification, sequence analysis and phylogenetic inference. Sampling was conducted at the Freshwater Institute (Shepherdstown, West Virginia, USA) during outbreaks of epitheliocystis in April and May 2002. Granular, basophilic, cytoplasmic inclusions in charr gill were shown to stain with Macchiavello, Lendrum's phloxine-tartrazine and Gimenez histochemical techniques. Ultrastructurally, inclusions were membrane-bound and contained round to elongate reticulate bodies that were immunoreactive to an antibody against chlamydial lipopolysaccharide, suggesting the presence of similar epitopes. DNA extracted from gills supported amplification of the most polymorphic and phylogenetically relevant region of the 16S rRNA gene, which had 97 to 100% identity with several uncultured clinical Neochlamydia spp. (order Chlamydiales) Clones WB13 (AY225593.1) and WB258 (AY225594.1). Sequence-specific riboprobes localized to inclusions during in situ hybridization experiments. Taxonomic affiliation was inferred by distance- and parsimony-based phylogenetic analyses of the 16S sequence, which branched with Neochlamydia hartmannellae in the order Chlamydiales with high confidence. This is the first molecular characterization of a chlamydia associated with epitheliocystis in Arctic charr and the fourth Neochlamydia spp. sequence to be associated with epitheliocystis. Presence of a clinical neochlamydial sequence, first identified from a cat, in Arctic charr suggests a possible mammalian and piscine host range for some environmental chlamydiae.

Improved husbandry to control an outbreak of rainbow trout fry syndrome caused by infection with Flavobacterium psychrophilum.

CASE DESCRIPTION: A cohort of 35,200, 13-week-old, female rainbow trout at a fish farm was evaluated because of a 2-week history of anorexia and lethargy and a mortality rate of approximately 100 fish/d. CLINICAL FINDINGS: Affected fish were lethargic and thin and had disequilibrium, bilateral exophthalmia, pale red gills and kidneys, red-tinged coelomic fluid, and pale brown livers. Some fish were differentially pigmented bilaterally. The presumptive diagnosis was bacterial or viral septicemia. The definitive diagnosis was rainbow trout fry syndrome caused by infection with Flavobacterium psychrophilum. TREATMENT AND OUTCOME: A strategy for controlling the outbreak based on reducing pathogen numbers in affected tanks and reducing pathogen spread among tanks was developed. The option of treating with antimicrobial-medicated feed was discussed with the farmer, but was declined. After changes were made, mortality rate declined quickly, with no more deaths within 10 days after the initial farm visit. CLINICAL RELEVANCE: Bacterial coldwater disease is the most common manifestation of infection with F psychrophilum in fingerling and adult rainbow trout. However, the organism can also cause rainbow trout fry syndrome. This condition should be included on a list of differential diagnoses for septicemia in hatchery-reared rainbow trout fry.

Susceptibility of Arctic Char to Experimental Challenge with Infectious Hematopoietic Necrosis Virus (IHNV) and Infectious Pancreatic Necrosis Virus (IPNV).

Arctic char Salvelinus alpinus were exposed to the fish pathogens infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) to estimate susceptibility of the species to infection. Fish were immersion challenged at 46, 74, and 95 d posthatch for 5 h in viral concentration of 103 or 105 PFU(plaque-forming units)/mL and were monitored for 21 d postchallenge. Fish that died were assayed individually for virus-associated mortality, and survivors were assayed individually to estimate prevalence of virus carrier status. Arctic char succumbed to IHNV infection with population losses as high as 41%, and prevalence of IHNV carriers in surviving fish was 24% or less with virus titers in carrier fish ranging from 2.5 × 102 PFU/g to greater than 1.3 × 106 PFU/g. Infections with IPNV resulted in losses of 20% or less, and prevalence of IPNV carriers in surviving fish was as high as 82% with virus titers in carrier fish ranging from 2.5 × 102 PFU/g to greater than 5.0 × 106 PFU/g. Logistic regression models were developed for the mortality and infection data for each virus. For IHNV, the odds of dying or becoming infected were significantly greater for the fish from the 74-d age-group than for the fish from the 46-d or 95-d age-groups. Fish exposed to 105 PFU of IHNV/mL had significantly greater odds of dying or becoming infected than fish exposed to 103 PFU/mL. For IPNV, the odds of dying were significantly greater for the 74-d age-group compared with the other two age-groups. The 74-d fish were also significantly more likely to become infected than the 95-d-old fish. Fish exposed to 105 PFU of IPNV/mL had significantly greater odds of dying or becoming infected than fish exposed to 103 PFU of IPNV /mL.