Salmonellae in Fish Feces Analyzed by In Situ Hybridization and Quantitative Polymerase Chain Reaction

Abstract

The potential of fish to transfer salmonellae from heterogeneous aquatic biofilms into feces was assessed in controlled aquarium studies with Suckermouth Catfish Hypostomus plecostomus and with biofilms inoculated with salmonellae. Neither the presence of catfish nor inoculation with salmonellae had detectable effects on the abundance of the microbial community. Densities of the microbial community were about 10⁵ cells/mL in the water during a 1-week period, whereas densities of the microbial community increased 10-fold (10⁶ to 10⁷ cells/mg) in catfish feces during the same period. Salmonellae were detected by both quantitative polymerase chain reaction (qPCR) and situ hybridization in water samples immediately after inoculation, in numbers of about 10⁴ cells/mL, representing up to 20% of the cells of the microbial community. Numbers decreased by three orders of magnitude within the first 3 d of the study, which represented only 0.01% of the community, and became undetectable after day 5. In catfish feces, numbers of Salmonella initially increased to up to 6% of the cells of the community but then declined. These results suggest that Salmonella are not biomagnified during gut passage, and thus, fish only provide a means for the translocation of this pathogen.

Received November 25, 2012; accepted March 25, 2013     

Experimental Induction of Proliferative Gill Disease in Specific-Pathogen-Free Channel Catfish

Abstract

Specific-pathogen-free channel catfish Ictalurus punctatus were exposed to sediment and mud from a pond containing channel catfish with proliferative gill disease. In one experiment, fish were to exposed to mud and sediment for 2 months in water maintained at 19°C. Fish were necropsied weekly, and certain tissues were examined histologically and ultrastructurally. Four trials were conducted with sediment samples from different epizootics of proliferative gill disease. In a second experiment, fish were exposed to sediment for 7 d in water maintained at 16, 19, or 26°C; the fish were then moved to clean water held at 16, 19, or 26°C. Fish were necropsied before transfer to clean water and weekly thereafter for 2 months. Channel catfish held at 19°C developed proliferative gill disease within 2 d of exposure to sediment. Primary cells of a uninucleate myxosporean parasite were present in the gills at the base of lamellae. These developed into plasmodia with numerous secondary cells, and some primary cells disintegrated, releasing their internal secondary cells. Similar development was observed in internal organs 1 week after appearance of the parasite in gills. Complete sporogony did not occur over the 2 months of this study. Plasmodia became necrotic and were not detected after 60 d. In fish exposed to sediment for 7 d at 16, 19, and 21°C, similar organisms were detected, but clinical disease occurred only at 19 and 26°C. Proliferative gill disease may be attributed to extrasporogonic stages of a myxosporean resembling Sphaerospora spp.     

Pesticide/herbicide pollutants in the Kafue River and a preliminary investigation into their biological effect through catalase levels in fish

The study determined the types of pesticide/herbicide pollutants in water, sediment and fish from the Kafue River. A preliminary investigation of the oxidative stress from these pesticides/herbicides was also assessed by measurement of catalase activity. Water, sediment and fish samples were collected upstream, midstream and downstream the Kafue river in Chingola, Kitwe, Kafue National Park and Kafue Town. Water, sediment and fish muscle were sampled and analysed for pesticides using Gas chromatography. For catalase activity fish liver samples only were examined. The pesticides/herbicides detected in all samples collectively included: Heptachlor, pp'-DDE, Cypermethrin, Chlordane, Toxaphene, Terbufos, Kelthane, Endosulfan, Dieldrin, pp'-DDD, pp'-DDT, Atrazine, Disulfoton, d-trans-Allethrin and Endrin. On the other hand, catalase activity was detected in all fish liver samples from all sites. Its levels increased significantly from Chingola upstream to sites downstream with highest being in Kafue town. This study therefore, demonstrates that there is widespread contamination of the Kafue River with pesticides/ herbicides. It also demonstrates that organochlorides are found throughout the river especially in fish samples. The spectrum of pesticides/herbicides was much wider in fish probably due to bioaccumulation. It was also observed that fish are subjected to oxidative stress as determined by catalase levels. The stress is more pronounced downstream where the catalase levels were significantly higher than Chingola. The observation that more pesticide varieties are also found downstream may suggest a likely causative effect of the pesticides on oxidative stress although this needs further investigation. This study further tentatively highlights the potential dangers of these agro-related substances to dependants of the Kafue River water body and the need to carry out risk assessments and thereafter institute corrective measures to help reduce contamination and adverse effects.     

Vibrio anguillarum: prevalence of typical and atypical strains in marine recipients with special reference to carbohydrate pollution

Primary isolates of Vibrio anguillarum-like organisms could be separated into typical V. anguillarum (VA) and atypical V. anguillarum (AVA) by biochemical tests. The prevalence of the fish pathogenic V. anguillarum was highly influenced by carbohydrate pollution as compared to the AVA. Water an sediment counts of VA generally increased at the polluted sites during April-May and persisted at a level of approx. 100/ml water and 1,000/g sediment until October-November. A further increase in VA counts could be registered locally at the time when the sugar beet processing season started (September-October). At the control site VA counts increased during June-July to a level of 10/ml persisting until August, while the only increase in sediment counts occurred in September (100/g). The maximum counts in water and sediment were at the control site 10/ml and 100/g and the polluted sites 100,000/ml and 50,000/g, respectively.     

Essential oil of Aloysia citriodora Paláu and citral: sedative and anesthetic efficacy and safety in Rhamdia quelen and Ctenopharyngodon idella

ObjectiveTo verify the efficacy of citral in inducing sedation and anesthesia in silver catfish (Rhamdia quelen) and grass carp (Ctenopharyngodon idella) and to assess the safety of essential oil (EO) of Aloysia citriodora and citral in inducing and maintaining anesthesia in silver catfish.Study designClinical study, randomized, parallel, multi-arm with control group in target species.AnimalsA total of 96 juvenile and 72 adult silver catfish and 80 juvenile grass carp were used.MethodsSilver catfish and grass carp were exposed to different concentrations of citral, 15–675 and 15–600 μL L^–1, respectively, during the maximum period of 30 minutes to verify sedation and anesthesia induction and recovery times. In addition, for anesthetic induction, silver catfish were exposed to the EO of A. citriodora and citral at 225 μL L^–1 for 3.5 minutes. Then, fish were transferred to an anesthesia maintenance solution at 50 μL L^–1 for 10 minutes to assess hematologic and biochemical variables at 60 minutes, 2 and 6 days after treatment.ResultsCitral only induced sedation from 15, 25 and 40 μLL^–1 in both species. Anesthesia without mortality was induced in silver catfish at 50–600 μL L^–1 and grass carp at 75–450 μL L^–1. At 675 and 600 μL L^–1, mortality was recorded in silver catfish and grass carp, respectively. The EO of A. citriodora and citral were safe in inducing and maintaining anesthesia in silver catfish, with mean corpuscular hemoglobin concentration being the only variable that varied in relation to time and treatments.Conclusions and clinical relevanceCitral was effective in inducing sedation and anesthesia in both species. In addition, A. citriodora EO and citral were safe in inducing and maintaining anesthesia in silver catfish. Both agents are promising substances for the development of new drugs for fish.     

Effects of selenium supplementation in cattle on aquatic ecosystems in northern California.

The potential impact on aquatic ecosystems of supplementing the diets of beef cattle with selenium (Se) was studied on 4 northern California ranches. All study sites included an area of concentrated use by cattle that had diets supplemented with Se. In each case, a stream flowed through the site and provided a control sampling area upstream and a treated sampling area downstream. Specimens of water, sediment, algae, aquatic plants, aquatic invertebrates, and fish were analyzed fluorometrically for total Se content. Significant differences in Se concentration were not found between specimens from upstream control areas and those from downstream areas subjected to use by Se-treated cattle. Evidence was not found that Se supplementation in cattle at maximal permitted concentrations caused Se accumulation in associated aquatic ecosystems.     

Massive Lernaea cyprinacea Infestations Damaging the Gills of Channel Catfish Polycultured with Bighead Carp

Abstract

During June and July 1998, at least three Arkansas fish farms polyculturing bighead carp Hypophthalmichthys nobilis with channel catfish Ictalurus punctatus suffered major losses of catfish associated with massive infestations by the crustacean parasite Lernaea cyprinacea. The channel catfish had few adult Lernaea attached to their skin, but there were 8–50 Lernaea copepodids on the surface of each catfish gill filament. The copepodids were found grazing on the gill tissue, and their feeding activity was associated with gill damage including epithelial hyperplasia, telangiectasis, and hemorrhage. Bighead carp in the same ponds were reported to have had numerous adult Lernaea on their skin but did not die during the epizootic. It is possible that the filter-feeding apparatus of the carp captured the copepodids, thus preventing heavy infestation of the gill filaments. Lernaea copepodids have not been implicated previously in fish losses resulting from parasite damage to the gills. The loss of catfish in these cases is likely to be due to their polyculture with the bighead carp, a species that provides an excellent host for adult Lernaea.     

Massive mortality of common carp (Cyprinus carpio carpio) in the St. Lawrence River in 2001: diagnostic investigation and experimental induction of lymphocytic encephalitis

A massive fish kill affecting exclusively common carp (Cyprinus carpio carpio) in the St. Lawrence River, Québec, Canada, during the summer of 2001 was investigated by use of laboratory diagnostic methods and by an attempt to experimentally induce the disease. The ultimate causes of mortality were opportunistic bacterial infections with Aeromonas hydrophila and Flavobacterium sp. secondary to immunosuppression induced by physiologic (i.e., spawning) and environmental (i.e., high temperatures and low water levels) stressors, and possibly enhanced by an infection causing lymphocytic encephalitis observed in 9 of 18 (50%) fish examined. Experimental induction of disease was attempted in captured wild carp by administration of crude and filtered (particulate <0.22 microm) inocula prepared from a homogenate of tissues from carp affected by the natural outbreak. Although significant clinical disease or mortality was not induced by experimental challenge, lymphocytic encephalitis similar to the one observed in naturally affected carp was induced in four of seven (57%) fish administered crude inoculum and four of seven (57%) fish administered filtered inoculum. None of the control fish inoculated with sterile phosphate-buffered saline (n = 6) were affected by encephalitis. The cause of the encephalitis observed in carp from the natural outbreak and in experimentally inoculated fish could not be determined by use of virus isolation and transmission electron microscopy.     

Modulative influence of lysozyme dimer on defence mechanisms in the carp (Cyprinus carpio) and European sheatfish (Silurus glanis) after suppression induced by herbicide Roundup

Immunomodulation is a commonly used method of prophylaxis in humans and animals. Lysozyme dimer (KLP-602) was used at a dose of 50 ug/kg b.w. in order to correct the immunosuppression caused by the action of herbicide glyphosate (Roundup- Monsanto), which was used in a single bath for 10 minutes in a concentration of 100 mg/l of water. The investigations were carried out on 2 species of fish: the carp (Cyprinus carpio L.) and european catfish (Silurus glanis L.). Herbicide glyphosate caused a decrease in metabolic and phagocytic activity (RBA and PKA) and in proliferative response stimulated by Con A and LPS in carp and european catfish. The immunosuppression sustained for about 2 weeks. The results obtained indicate the possibility of correction of immunosuppression applying lysozyme dimmer (KLP-602) after use of which, the level of the studied indexes increased.     

Metacercarial distribution of Centrocestus formosanus among fish hosts in the Guadalupe River drainage of Texas

We examined the gills of wild fish collected from central Texas for Centrocestus formosanus metacercariae to determine whether this temperature-restricted parasite had invaded the thermally dynamic Guadalupe River via an introduced population in its thermally stable tributary, the Comal River. We collected fish from three sites in the Guadalupe River near its confluence with the Comal River (upstream, at, and downstream) and one site in the Comal River. Centrocestus formosanus infected 14 of the 25 species examined (56.0%) and 171 of the individual fish (27.1%). Several of the infected fish represent new host records for the parasite, and two are listed as species of special concern by the state of Texas. Mean metacercarial intensities varied from 8 to 616 among species, and the highest recorded intensity was greater than 800 in two Guadalupe roundnose minnow Dionda nigrotaeniata. Among the 24 species examined from the Guadalupe River, 11 (45.8%) were infected with C. formosanus. Thorough surveys at the study sites yielded no living specimens of the first obligate intermediate snail host (red-rim melania Melanoides tuberculatus), which must be present to perpetuate the parasite. Thus, the infections were probably due to drifting cercariae that had been shed into the water column upstream of the study area in the Comal River. We therefore investigated spatial patterns in cercarial acquisition using caged fish to determine whether drifting cercariae were present in the water column at the study sites. Of 57 uninfected blacktail shiners Cyprinella venusta exposed to Guadalupe River water downstream from and at the confluence, 52 (91.2%) became infected with C. Formosanus metacercariae at a mean rate of 4 metacercariae/d. This finding extends the known geographic range of this invasive exotic parasite and is the first report of the life cycle being advanced in the fish assemblage of a thermally variable temperate stream in the USA.     

Influence of methisoprinol on the replication of rhabdoviruses isolated from carp (Cyprinus carpio) and catfish (Ictalurus melas): in vitro study

Rhabdoviruses constitute one of the most pathogenic viruses isolated from rainbow trout and carp culture. Several viruses were also isolated from other species of fish. These viruses are mostly associated with epizootics and heavy losses. Spring viraemia of carp virus (SVCV) and pike fry rhabdovirus (PFRV) have been the most extensively studied, due to their significant economic impact. Significant progress has been made towards controlling the major bacterial fish diseases using vaccines, but this approach has not yet been successful in preventing viral diseases in fish culture. However, for an effective therapeutic approach, specific drugs should be developed to selectively inhibit virus replication and/or stimulate antiviral protection. In this investigation we examined the in vitro influence of methisoprinol on the SVCV and virus isolated from catfish (Ictalurus melas) replication by measuring their RNA synthesis. The viruses were propagated in EPC cells and cell cultures containing methisoprinol were followed by infection with SVCV or catfish rhabdovirus suspension containing 10(7) TCID50/ml. Methisoprinol (Polfa, Poland) at concentrations of 0, 100, 200, 300, 400 and 500 microg/ml of medium (Glasgow MEM) was used in this study. The results of this study show the strong inhibition of incorporation (cpm) of [3H]-uridine into SVCV and catfish rhabdovirus RNA in cell culture exposed to methisoprinol at various concentrations. The highest percent of inhibition of viral RNA at 72 h after infection with two rhabdoviruses were observed in doses of 400 and 500 microg/ml of methisoprinol in medium. The results of this in vitro study showed that methisoprinol inhibits the rhabdoviruses isolated from carp and catfish.     

Effects of variable periods of food deprivation on the development of enteric septicemia in channel catfish

Enteric septicemia of catfish (ESC), caused by the bacterium Edwardsiella ictaluri, is the most significant bacterial disease affecting channel catfish Ictalurus punctatus. Withholding feed during outbreaks of ESC is a widely accepted industry practice used to control losses from the disease. Scientific evidence concerning the validity of the practice is contradictory. Two studies were conducted to further evaluate the survival of channel catfish fingerlings following variable periods of feed deprivation before and after exposure to E. ictaluri in controlled aquarium experiments. In the first study, feed was withheld for varying time periods before bacterial challenge. After bacterial challenge, feed was either withheld or fish were fed daily. The second study utilized fish fed daily or fish deprived of feed 7 d before bacterial challenge. Daily feeding was resumed 4, 48, and 96 h after fish were exposed to E. ictaluri. In both experiments, the prechallenge feed treatments did not affect mortality. In contrast, withholding feed after bacterial challenge reduced mortalities by 52% in experiment 1 and by 45% in experiment 2. The highest mortality was observed when fish were fed immediately after immersion exposure and the lowest when fish were completely denied feed or fed daily starting 96 h after challenge. This reduction in mortality occurred when the concentration of E. ictaluri in aquarium water was negligible. These data suggest that when E. ictaluri is present in the water, feeding fish increases mortality by enhancing oral exposure to the pathogen.     

Nutrition of aquaculture species.

Dietary requirements for amino acids and fatty acids have been reported for channel catfish (Ictalurus punctatus), salmonids (Oncorhynchus spp.), common carp (Cyprinus carpio), tilapias (Oreochromis spp.), and eel (Anguilla japonicus). Most of the vitamin and mineral requirements are available for channel catfish and salmonids, and some are available for common carp, tilapia, eel, and other finfish and crustaceans. From this available information, cost-effective feeds can be formulated for the major commercial aquaculture species. Major differences in nutrient requirements between fish and mammals or birds are as follows: fish have a lower digestible energy:protein ratio (8 to 10 kcal of DE/g of CP for fish vs 15 to 20 kcal of DE/g of CP for livestock); fish require n-3 fatty acids and land animals require n-6; fish can absorb minerals from the water, which negates the need for some minerals in the diet; and fish have limited ability to synthesize vitamin C and must depend on a dietary source. Areas for further research include 1) refinement of nutrient requirements of the major culture species considering effects of fish size, temperature, and management; 2) nutrient requirements of crustaceans; 3) effects of nutrition on fish health and product quality; and 4) feeding technology.     

Salmonella serotypes isolated from the aquatic environment (Wabash River, Indiana, 1973-1976).

During 1973-1976, 833 isolations of salmonellae were made from the aquatic biosphere of the Wabash River, Lafayette, Ind. Of the isolants, 643 were definitively serotyped. Salmonella typhimurium represented 34.4% of the cultures. Salmonella eimsbuettel previously had not been recognized in Indiana. A total of 35 serotypes and 1 untypable group C1 was obtained from the aquatic samples, ie, river water, river bottom sediment, fish, and mussels. Most of the isolants were presumed to be of human origin, because many samplings were made in close proximity to sewage treatment plant outfalls. During the survey period, epidemic salmonellosis had not been reported for the Lafayette-West Lafayette metropolitan area.     

Establishment of a time-resolved fluoroimmunoassay for measuring plasma insulin-like growth factor I (IGF-I) in fish: effect of fasting on plasma concentrations and tissue mRNA expression of IGF-I and growth hormone (GH) in channel catfish (Ictalurus punctatus)

A time-resolved fluoroimmunoassay (TR-FIA) was established and validated that allows for the determination of plasma concentrations of insulin-like growth factor I (IGF-I) in three domestically cultured fishes: channel catfish (Ictalurus punctatus), hybrid striped bass (Morone chrysopsxM. saxatilis), and rainbow trout (Oncorhynchus mykiss). Sensitivity of the assay was 0.20 ng/ml. Intra- and inter-assay coefficients of variation (CV) were <7 and <12%, respectively. Serial dilutions of plasma from each species were parallel to the standard curve. Recovery of IGF-I from spiked plasma samples was >90% for all three species of fishes. The IGF-I TR-FIA was biologically validated via its use to determine the effect of fasting on circulating IGF-I levels in channel catfish. Fasting-induced changes in plasma growth hormone (GH), hepatic IGF-I mRNA expression, and pituitary GH mRNA expression were also determined. Fasted channel catfish lost 5.6 and 15.6% body mass after 2 and 4 weeks of fasting, respectively. Plasma IGF-I concentrations were depressed (P<0.05) relative to fed controls following 2 and 4 weeks of fasting. Plasma GH concentrations were not different (P>0.05) in fasted fish after 2 weeks, but significantly increased (P<0.05) by 4 weeks of fasting. Hepatic IGF-I mRNA expression after 2 and 4 weeks of fasting was reduced (P<0.05) relative to fed controls. Pituitary GH mRNA expression was similar (P>0.05) between 2-week-fasted catfish and fed controls, but was increased (P<0.05) in 4-week-fasted catfish. The IGF-I TR-FIA was sensitive, accurate, and precise for all three species of fishes, and provided a low-cost, and non-radioisotopic method for quantifying plasma IGF-I levels in fed and fasted channel catfish.     

Environmental pollutants and diseases of sexual development in humans and wildlife in South Africa: harbingers of impact on overall health?

This study deals with disorders of sexual development in humans, wildlife and animals in an urban nature reserve (RNR) and a currently DDT-sprayed malarial area. High levels of oestrogenic chemical residues in water, sediment and tissue; skewed sex ratios; reduced biodiversity; gonadal malformations in sharptooth catfish and freshwater snails; intersex in catfish; and impaired spermatogenesis in catfish and striped mouse are of serious concern in the RNR. Persistent eggshell thinning in African darter eggs, intersex in male Mozambican tilapia, follicular atresia in females and impaired spermatogenesis in males following laboratory exposure of parent fish to environmentally relevant DDT and DDE concentrations, and abnormalities in freshwater snails were found in the DDT-sprayed area. Human studies related to DDT exposure indicated impaired semen quality, a weak association with sperm chromatin defects and higher risks for external urogenital birth defects in those who were born to mothers whose houses were sprayed and those who were homemakers (stay at home mother) instead of being employed. These findings indicate that diseases of sexual development occurred in both human and wildlife populations exposed to environmental endocrine disruptor chemicals in South Africa. The chemical mixtures, possibly related to disorders of sexual differentiation (DSD), were very different between the two. However, DSD occurred concurrently in the malarial area, possibly indicating that humans and wildlife shared exposures. Moreover, it emphasizes the importance of suspecting disease in the other when disease is found in either human or wildlife populations.     

Metacercarial Distribution of Centrocestus formosanus among Fish Hosts in the Guadalupe River Drainage of Texas

Abstract

We examined the gills of wild fish collected from central Texas for Centrocestus formosanus metacercariae to determine whether this temperature-restricted parasite had invaded the thermally dynamic Guadalupe River via an introduced population in its thermally stable tributary, the Comal River. We collected fish from three sites in the Guadalupe River near its confluence with the Comal River (upstream, at, and downstream) and one site in the Comal River. Centrocestus formosanus infected 14 of the 25 species examined (56.0%) and 171 of the individual fish (27.1%). Several of the infected fish represent new host records for the parasite, and two are listed as species of special concern by the state of Texas. Mean metacercarial intensities varied from 8 to 616 among species, and the highest recorded intensity was greater than 800 in two Guadalupe roundnose minnow Dionda nigrotaeniata. Among the 24 species examined from the Guadalupe River, 11 (45.8%) were infected with C. formosanus. Thorough surveys at the study sites yielded no living specimens of the first obligate intermediate snail host (red-rim melania Melanoides tuberculatus), which must be present to perpetuate the parasite. Thus, the infections were probably due to drifting cercariae that had been shed into the water column upstream of the study area in the Comal River. We therefore investigated spatial patterns in cercarial acquisition using caged fish to determine whether drifting cercariae were present in the water column at the study sites. Of 57 uninfected blacktail shiners Cyprinella venusta exposed to Guadalupe River water downstream from and at the confluence, 52 (91.2%) became infected with C. formosanus metacercariae at a mean rate of 4 metacercariae/d. This finding extends the known geographic range of this invasive exotic parasite and is the first report of the life cycle being advanced in the fish assemblage of a thermally variable temperate stream in the USA.

Received October 18, 2010; accepted April 7, 2011     

Gonadosomatic index and testis morphology of common carp (Cyprinus carpio) in rivers contaminated with estrogenic chemicals

To study the effect of estrogenic chemicals on fish, the gonadosomatic index (GSI = [testis weight/body weight] x 100) and testis histology of mature common carp (Cyprinus carpio) from 2 contaminated sites (Ishizu and Wada rivers, Osaka) and a control site were examined between June 1998 and March 2001. The concentration of nonylphenol, bisphenol A and 17beta-estradiol in the Ishizu river was 3-4 times higher than in the Wada river. In the pre-breeding and breeding seasons, there were no significant differences in body weight among carp from the 3 sites, the body weight of Ishizu river carp being significantly lower (p<0.05) than that of Wada river fish only in the post-breeding season. The GSI and testis weight in fish from the Ishizu river were significantly lower (p<0.05) than in control fish during all phases of gonadal cycle and lower than in Wada river fish in the pre-breeding and post-breeding season. No histological abnormalities were found in the testes of the males examined. Histological observation of the testes revealed a delay in the onset of spermatogenesis in fish from the Ishizu river compared with those from the other sites. These results clearly imply that the estrogenic chemicals in the Ishizu river adversely affect the testis development of the fish.     

Distribution of Edwardsiella ictaluri in California

Abstract

Wild and domestic populations of channel catfish Ictalurus punctatus were examined to determine the distribution of the disease called enteric septicemia of catfish (ESC) in California. The causative agent of ESC, Edwardsiella ictaluri, was isolated from five separate sites in California. Two of these isolations were from rectal swabs of asymptomatic fish, confirming that a carrier state may exist. Normal-appearing fish with serum antibody titer to E. ictaluri were commonly found in domestic channel catfish populations, suggesting that many fish become infected but recover. Wild channel catfish with antibody to E. ictaluri were also found in major reservoirs and water distribution canals. Edwardsiella ictaluri appears to be widely distributed within California.     

Detection of botulinum type E toxin in channel catfish with visceral toxicosis syndrome using catfish bioassay and endopep mass spectrometry.

Visceral toxicosis of catfish (VTC) is a syndrome characterized by sudden mortality in apparently healthy market- and brooder-sized catfish (Ictalurus punctatus). This paper reports the design of a catfish neutralization assay to detect botulinum in catfish with VTC and verification by Endopep mass spectrometry (Endopep-MS). Sera from 6 affected catfish were incubated with botulinum antitoxin serotypes A, B, C, D, E, or F. For each serum sample, 3 experimental fingerlings were injected intracoelomically with each serotype-serum mixture and placed separately in an aquarium. Three fish were injected with VTC-affected serum only, and 3 fish were injected with unaffected serum only and also placed in separate aquaria. Signs of morbidity and mortality were seen in fish injected with sera combined with serotype A, B, C, or D, as well as in positive controls. No morbidity or mortality was seen in fish injected with sera combined with antitoxin serotypes E or F or negative control serum. Sera from affected and unaffected catfish were sent to Centers for Disease Control and Prevention for detection and differentiation of botulinum neurotoxin. Aliquots of 0.5 ml of sera were incubated with magnetic beads coated with antibodies to botulinum, and the beads were subjected to the Endopep-MS reaction. Sera from affected catfish tested positive for botulinum E. Sera from 34 unaffected catfish tested negative for botulinum. Although there was not enough botulinum present in affected samples to obtain exact quantification, the estimated quantity of botulinum E in these sera samples was between 0.01 and 0.5 mouse LD50/ml.