Protection against atypical Aeromonas salmonicida infection in carp (Cyprinus carpio L.) by oral administration of humus extract

Humic substances are formed during the decomposition of organic matter in humus, and are found in many natural environments in which organic materials and microorganisms have been present. In the present study, oral administration of humus extract to common carp (Cyprinus carpio L.) induced effective protection against experimental atypical Aeromonas salmonicida infection. Mortality of fish and development of skin lesions such as hemorrhages and ulcers were significantly suppressed in carp treated with 10%, 5% or 1% humus extract adsorbed on dry feeding pellets. The median surviving days was also greater in fish treated with 10% or 5% humus extract than in untreated fish. Atypical A. salmonicida was isolated from ulcerative lesions of part of dead fish, but Aeromonas hydrophila and Flavobacterium sp. were also isolated from these fish, verifying bacterial population changes during the progression of skin lesions. These results clearly show that treatment of fish with humus extract is effective in preventing A. salmonicida disease.     

Virulence, cytotoxic and inflammatory activities of Vibrio anguillarum and Aeromonas salmonicida isolated from cultivated salmonid fish in Sweden

Extracellular products in culture filtrates of Aeromonas salmonicida subsp. achromogenes and Vibrio anguillarum isolated from infected fish have been shown to possess skin inflammatory factor. The extracellular products from Vibrio anguillarum were cytotoxic in HeLa and CHO cells. In addition to the skin lesions, the culture filtrates of V. anguillarum caused necrotic reaction on the rabbit skin. Five of 6 strains of V. anguillarum were lethal to mice after intraperitoneal administration of 3×10⁷ CFU. Only 1 strain of 5 A. salmonicida subsp. achromogenes produced extracellular products which elicited cytotoxic effects in the CHO cells. None of the A. salmonicida subsp. achromogenes strains were lethal to mice. The cytotoxins were inactivated when heated at 65°C for 30 min. The results indicate that the thermolabile exotoxins are non-enterotoxic since they failed to stimulate fluid accumulation in the rabbit ileal loop and did not cause elongation of the CHO cells. The rounding off of CHO cells, as well as of HeLa cells indicate that the exotoxins may play an important role in fish diseases.     

Development of a PCR protocol for the detection of Aeromonas salmonicida in fish by amplification of the fstA (ferric siderophore receptor) gene

The aims of the study were to evaluate a new PCR protocol designed to detect Aeromonas salmonicida in fish tissues and to develop a non-destructive method for the diagnosis of furunculosis. A set of primers (Fer3, Fer4), flanking a fragment of the fstA gene (coding for the ferric-siderophore receptor) was designed, showing to be sensitive and specific. When compared to PCR methods previously reported, the new protocol recognized all the 69 A. salmonicida strains evaluated, with no cross-reactions with the other bacterial species analysed. Sensitivity assays were performed in fish tissues seeded with serial dilutions of pure cultures of A. salmonicida and mixed cultures of this bacterium with Vibrio anguillarum and Aeromonas hydrophila. Detection limits obtained were of 60 and 450 bacterial cells 100 mg(-1) of tissue, respectively. Mucus and blood were evaluated in order to develop a non-destructive tool to detect the pathogen. The detection limits in seeded mucus and blood samples were 2.5 x 10(2) and 1 x 10(5) bacterial cells mL(-1), respectively. When the method was used to detect A. salmonicida in asymptomatic wild salmon, four samples of mucus and six of blood were positive, corresponding to 6 out of the 31 fish examined, whereas only one of the samples resulted positive by culture methods. It is concluded that the PCR protocol evaluated is fast, specific and sensitive to detect A. salmonicida in infected and asymptomatic fish, and will be helpful for the control of the disease through the prompt detection of carriers within fish populations.     

Clinical and physiologic effects of sodium chloride baths in goldfish (Carassius auratus)

Sodium chloride (salt; NaCl) has been used for freshwater fish to decrease stress and manage a variety of disease conditions. Recommendations for dose and duration vary greatly. The purpose of this study was to determine the potential adverse clinical and physiologic side effects of different concentrations of saltwater baths on goldfish. Eleven goldfish (Carassius auratus) were used in a cross-over study to assess the effects of three different salt concentrations (5, 10, and 20 g/L) on plasma biochemistries and clinical response. Baseline plasma chemistries were obtained and analyzed immediately prior to placing the goldfish into the saltwater bath and after the fish was removed. A 2-wk washout period was used in-between each treatment. Significant differences were found in fish in the sodium (10 g/L, P = 0.007; 20 g/L, P = 0.01), chloride (10 g/L, P = 0.006; 20 g/L, P = 0.001), and alanine aminotransferase (10 g/L, P = 0.002; 20 g/L, P = 0.004) after their exposure to 10 and 20 g/L saltwater. Glucose levels were found to differ significantly after exposure to all three NaCl concentrations (5 g/L, P = 0.0009; 10 g/L, P = 0.0001; 20 g/L, P = 0.0005). Clinically, 5 g/L and 10 g/L saltwater baths were well tolerated by the fish for the duration of the intended 12-hr treatments, with only one goldfish being removed during the 10 g/L bath at 7 hr for listlessness. The average time goldfish spent in the 20 g/L salt bath was 43 min, with six (54%) of the fish remaining in the 20 g/L salt bath for the intended 60-min treatment period. The remaining 5 (46%) goldfish were removed because they became listless or dyspneic. All of the fish recovered from the treatments without complication. The results of this study suggest that goldfish tolerate saltwater baths but that physiologic disturbances can occur at the higher doses.     

Protection against atypical Aeromonas salmonicida infection in common carp, Cyprinus carpio L., by oral administration of a mixed microbial culture of Lactobacillus paracasei, Pichia membranifaciens and Saccharomyces cereviciae

A microbial culture was prepared by co-cultivation of Lactobacillus paracasei, Pichia membranifaciens and Saccharomyces cereviciae for 48 hr at 30°C in rice bran extract medium, supplemented with dextrose. Oral administration of the resulting non-viable heat-inactivated microbial culture to common carp, Cyprinus carpio L., delivered in feed for four weeks, induced effective protection against experimental atypical Aeromonas salmonicida infection which causes "ulcer disease". After challenge of the carp by immersion, fish mortality and development of skin lesions such as hemorrhages and ulcers were significantly suppressed in carp treated with mixed microbial culture adsorbed on dry pellets relative to carp treated with medium or without extract. Atypical A. salmonicida was re-isolated from ulcerative lesions in parts of dead and surviving fish, but Aeromonas hydrophila and Flavobacterium sp. were also isolated from these fish, verifying microbial population changes during the progression of skin lesions. Among interleukin-1β (IL-1β), tumor necrosis factor-α, as well as CXC-α and CXC-β chemokines, gene expression of IL-1β was up regulated in the spleen and head kidney three weeks after administration of the mixed microbial culture. These results clearly show that this mixed microbial culture, delivered in feed, is effective in preventing A. salmonicida disease in carp.     

Polymerase chain reaction amplification of repetitive intergenic consensus and repetitive extragenic palindromic sequences for molecular typing of Pseudomonas anguilliseptica and Aeromonas salmonicida

The aim of this study was to evaluate the use of two molecular techniques, repetitive extragenic palindromic polymerase chain reaction (REP-PCR) and repetitive intergenic consensus PCR (ERIC-PCR), as epidemiological tools with which to discriminate among genetically distinct strains within two bacterial fish pathogens, Pseudomonas anguilliseptica and Aeromonas salmonicida. A total of 30 A. salmonicida and 52 P. anguilliseptica were analyzed. For P. anguilliseptica, three different major fingerprints were obtained with both techniques, which defined three genomic groups: one was composed of strains isolated from eels Anguilla spp., the second of strains from turbot Scophthalmus maximus and blackspot seabream (also known as red seabream) Pagellus bogaraveo, and the third of strains from other fish species, such as gilthead seabream (also known as gilthead bream) Sparus auratus, sea bass Dicentrarchus labrax (also known as European bass Morone labrax), and salmonids. In the case ofA. salmonicida, promising results were obtained with both techniques for subspecies differentiation. Thus, two genomic profiles were obtained by ERIC-PCR. The first profile consisted of A. salmonicida subsp. salmonicida strains isolated from the different hosts. The second profile was composed of two A. salmonicida subsp. masoucida and one A. salmonicida subsp. achromogenes. Using REP-PCR, three genotypes were obtained within this pathogen that were related to the diverse subspecies analyzed. In summary, both methodologies are useful for typing distinct strains associated with different host species and therefore are helpful in epidemiological studies of P. anguilliseptica. In contrast, in the case of A. salmonicida, more studies are needed to determine their utility in discriminating the subspecies salmonicida from the other two subspecies.     

Modulation of juvenile brook trout (Salvelinus fontinalis) cellular immune system after Aeromonas salmonicida challenge

In fish, the first line of defence against infectious microorganisms is based on non-specific cellular immune mechanisms (innate immunity). In this study, we measured the non-specific immune parameters (natural cytotoxic cells (NCC) activity, lymphoproliferation, percentage of phagocytosis and phagocytic activity) in brook trout (Salvelinus fontinalis) infected by a virulent strain of Aeromonas salmonicida. Eight days post-infection, the mortality of infected fish reached 70%. A transient immunostimulation of the NCC activity was noticed 24h post-infection, but there was no significant difference at 48 h. Then, infection of brook trout with A. salmonicida induced a biphasic immune response. At 24h post-infection, lymphoproliferation was drastically depressed but returned to control level at 96 h. A slight increase in the percentage of phagocytosis and the phagocytic activity was noticed throughout the experiment. Conversely the cell mortality was significantly higher in infected fish compared to control. The modulation of immunological parameters might reveal important clues on how innate immunity might protect fish from bacterial infections.     

Contribution of food deprivation to the immune response in rainbow trout (Oncorhynchus mykiss) vaccinated against Cryptobia salmositica and Aeromonas salmonicida

The aims of the present study were to determine (a) the effectiveness of an attenuated live Cryptobia salmositica vaccine; (b) the effects of food deprivation on the immune response and its duration in rainbow trout (Oncorhynchus mykiss) immunised with a live C. salmositica vaccine or with a killed Aeromonas salmonicida vaccine. The fish were divided into three groups (I, II and III; 14 fish per group), those in Groups I and II were under food deprivation (0.40% of body weight), while Group III fish were fed to satiety. The study showed that the attenuated strain of C. salmositica did not cause anaemia and disease, and the fish were protected from clinical disease when they were challenged with virulent parasites. Parasitaemia in all fish vaccinated and challenged with virulent C. salmositica fluctuated and was relatively low; however, fish in Group III had higher parasitaemia than those in Groups I and II between weeks 8 and 14. The numbers of activated neutrophils increased [nitroblue tetrazolium (NBT) assay] after immunisation with both Cryptobia and Aeromonas vaccines and they remained high throughout the experiment. Antibody production (ELISA values) increased after vaccination and were slightly higher in Group III. ELISA titres against A. salmonicida increased after vaccination and decreased after 5 weeks. The titres increased again after the vaccinated fish were given booster, and they were higher than those in the first vaccinated fish.     

Identification of a cyprinid fish, the tench Tinca tinca L., as a carrier of the bacterium Aeromonas salmonicida, causative agent of furunculosis in salmonids.

A typical, pigment-producing strain of Aeromonas salmonicida (A. sal.), the causative agent of furunculosis in salmonid fish species, was isolated from a cyprinid species, the tench Tinca tinca L. with papilloma-like skin alterations. Histopathology of the papilloma-like skin alterations in tench revealed "round holes", distinctly lined by thick layers of epithelial cells, but no bacteria. The organism was isolated from skin, gills and fins, but not internal organs. The isolate proved highly virulent for both juvenile tench and brown trout Salmo trutta L. in experimental infection, but it did not reproduce the clinical picture. The causative role of A. sal. for the surface lesions remains questionable. However, there is a perceived risk of the organism's transmission between tench and other susceptible species of fish, especially farmed trout.     

The effects of gamma irradiation on the lymphoid organs of rainbow trout and subsequent susceptibility to fish pathogens

Fish were irradiated with 60Co gamma rays at doses ranging from 10 to 50 Gy. Lethal doses were determined in fishes of different ages. For a given dose, fry and fingerlings were more susceptible than subadults. Whatever the irradiation dose was, the fish displayed a sharp decrease in blood leucocyte count. At the lowest doses, this acute leucopaenia was reversible. The cellular damage in the lymphoid organs was particularly obvious in the thymus. The depletion of lymphoid cells from immunocompetent organs decreased (viral hemorrhagic septicaemia, VHS) or increased (Y. ruckeri, A salmonicida) the susceptibility of trout to pathogens. The suppressive effect of radiation was age dependent. Irradiation appeared to be a reliable technique to detect asymptomatic carrier fish.     

Immunostimulants added to injected Aeromonas salmonicida bacterin enhance the defense mechanisms and protection in rainbow trout (Oncorhynchus mykiss).

Immunostimulants were given to rainbow trout for assaying effects on modulating non-specific defense mechanisms, specific immune response, and protection levels against pathogen challenge with Aeromonas salmonicida. Three drugs, levamisole (an approved veterinary drug in the USA), a quaternary ammonium compound (QAC), and a short-chain polypeptide (ISK) were found to affect the non-specific defense mechanism activities, which were measured by changes in circulatory neutrophil and phagocytic activity levels, and the specific immune response factors, which were measured by numbers of plaque-forming cells, and circulatory antibody levels. When given alone, the immunostimulants elevated the non-specific factors. When injected in combination with an A. salmonicida O-antigen bacterin, the non-specific factors were further elevated, and the specific response was raised over samples taken from fish given the bacterin without the immunostimulants. Challenge tests with the virulent pathogen, A. salmonicida, showed a 5-6 day delay in the onset of mortalities in the fish given the immunostimulants alone, and a 12-14 day delay when immunostimulants given were combined with the bacterin. In the groups given the QAC or ISK with the bacterin, there was a 20% and 40% survival rate, respectively.     

Blood changes in carp (Cyprinus carpio) induced by ulcerative Aeromonas salmonicida infections

Only recently Aeromonas salmonicida has been recognized as a significant bacterial pathogen in ulcerative disease of cyprinid fish. Our attempts to formulate a vaccine based on bacterial surface antigens were unsuccessful in conferring reliable protection against lethal challenge. This lead us to study pathological changes in the humoral defense system during ulcerative A. salmonicida infection in carp. High numbers of opportunist pathogens such as A. hydrophila and Pseudomonas sp. were frequently recovered from the internal organs of moribund fish, in addition to A. salmonicida. These findings together with leucopenia in moribund fish suggest that pathogenesis is characterized by a state of immune suppression. In addition, fish which had sustained a sublethal infection were not protected against a subsequent lethal challenge. However, fish previously injected with a concentrated and inactivated culture supernatant showed protection. Differential blood cell counts did not differ between experimental and control groups during sublethal infection in contrast to serum proteins. Furthermore infected non-immune carp showed a progressive decrease of immunoglobulin and total serum protein levels before the day of peak mortality whereas protected carp maintained the immunoglobulin concentration despite a decrease in protein. Our observations suggest the involvement of multiple pathogenic events, affecting different parts of the humoral defense system during ulcerative A. salmonicida infection. The immunosuppressive effects can be minimized by prior vaccination with culture supernatant.     

Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens

A multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of the five major fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida, Flavobacterium columnare, Renibacterium salmoninarum, and Yersinia ruckeri. Each of the five pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The detection limits of the multiplex PCR was in the range of 2, 1, 1, 3, and 1CFU for A. hydrophila, A. salmonicida, F. columnare, R. salmoninarum, and Y. ruckeri, respectively. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria. The multiplex PCR assay was useful for the detection of the bacteria in naturally infected fish. This assay is a sensitive and specific and reproducible diagnostic tool for the simultaneous detection of five pathogenic bacteria that cause disease in fish. Therefore, it could be a useful alternative to the conventional culture based method.     

Evaluation of emamectin benzoate for the control of experimentally induced infestations of Argulus sp. in goldfish and koi carp

The effect of 0.2% emamectin benzoate (SLICE; Intervet/ Schering-Plough Animal Health, Roseland, New Jersey) administered in top-dressed, pelleted commercial fish feed was evaluated for control of freshwater Argulus sp. in goldfish Carassius auratus and koi carp, a variant of common carp Cyprinus carpio, in freshwater aquaria at 24-25 degrees C. Sixteen individually housed goldfish were each exposed to 37 Argulus. The number of fish lice attached to each fish at the start of the experiment was not determined; however, the total number of motile fish lice in each aquarium (on fish and in the water) was determined at the start and end of each experiment. Eight goldfish were fed the control diet (0 microg x kg fish biomass(-1) x d(-1)) and eight were fed the medicated diet (50 microg x kg fish biomass(-1) x d(-1)) for seven consecutive days. After treatment, fish louse infestation in controls was 20.5 +/- 1.5 (mean +/- SE) lice per fish. No Argulus were found on fish in the treated group. In a separate experiment, 10 individually housed koi were each exposed to 128 Argulus. Five koi were fed the control diet and five were fed a low-dose medicated diet (5 microg x kg fish biomass(-1) x d(-1)) for 7 d. After treatment, fish louse infestation among the controls was 14.6 +/- 3.8 lice per koi. No Argulus were found on koi in the treated group. Hence, a 7-d regimen of oral emamectin benzoate controlled experimental infestation of Argulus when administered to goldfish at 50 microg x kg fish biomass(-1) x d(-1) and to koi at 5 microg x kg fish biomass(-1) x d(-1).     

Polycystic kidney disease in goldfish (Carassius auratus) from Hamilton Harbour, Lake Ontario, Canada

Severe kidney enlargement was observed in 6.3% (5 of 80) of goldfish collected from a heavily polluted industrial basin. Externally the fish had generalized swelling and abdominal distension. The kidneys contained numerous large, clear, fluid-filled cysts (polycystic) that ranged in size from microscopic to 4 cm in diameter. Affected kidneys had a wide range of histological changes-including the presence of large multiple cysts that caused severe distension and compression of normal renal tissue, multifocal granulomas, and signs of early, proliferative glomerulonephritis. The histology of affected kidneys is compared with other goldfish from Hamilton Harbour, and with goldfish collected from an alternate site (a population apparently free of polycystic kidney enlargements). This afflication is rare in feral fish populations, and its occurrence in a deteriorated environment such as Hamilton Harbour may provide further evidence of a link between fish health and environmental quality.     

Evaluation of Dairy–Yeast Prebiotic Supplementation in the Diet of Juvenile Goldfish in the Presence or Absence of Phytoplankton and Zooplankton

Abstract

Prebiotics recently have been shown to increase immune responses and disease resistance in certain fish species; therefore, the current study was conducted to evaluate the commercially available dairy–yeast prebiotic, GroBiotic-A, for use with juvenile goldfish Carassius auratus. The study consisted of two 10-week feeding trials in which juvenile goldfish were fed practical diets that were either unsupplemented or supplemented with the dairy–yeast prebiotic at 2% by dry weight. Juvenile fish were sorted by size and stocked into 12 units within each of two culture systems: one indoor system supplied with recirculated well water and one system located outdoors with a continuous flow of pond water to provide a source of phytoplankton and zooplankton. Both diets were fed to fish in six units within each system at the same fixed percentage of body weight twice daily. Culture system (i.e., presence or absence of phytoplankton and zooplankton) was the primary factor influencing (P < 0.0001) percent weight gain, feed efficiency, and survival of goldfish during the feeding trials. No dietary effect was detected, although there was a significant (P < 0.05) interaction between culture system and diet, with supplementation of the dairy–yeast prebiotic tending to improve weight gain and feed efficiency of fish in the presence of phytoplankton/zooplankton. During a controlled disease challenge with an intraperitoneally administered dose of Aeromonas hydrophila that was equivalent to a predetermined LD50 (dose lethal to 50% of test fish), average survival values ranged between 67% and 83% for fish that previously had access to phytoplankton/zooplankton compared with 17–33% for fish that had no access to phytoplankton/zooplankton. The dairy–yeast prebiotic, however, did not enhance resistance of goldfish to the bacterial pathogen and did not greatly alter microbiota of the anterior or posterior gastrointestinal tract based on denaturing gradient gel electrophoresis analysis. In conclusion, the dairy–yeast prebiotic did not improve feed efficiency in goldfish or resistance to a bacterial pathogen as previously observed in golden shiners Notemigonus crysoleucas and hybrid bass (white bass Morone chrysops × striped bass M. saxatilis).     

Simulating the effect of eliminating routine bacterial screening on the negative predictive value of the Ontario fish hatchery disease monitoring program.

We determined the impact of eliminating routine screening for Aeromonas salmonicida and Yersinia ruckeri on the efficacy of the Ontario Ministry of Natural Resources (OMNR) fish disease monitoring program, using Monte Carlo simulation. Because the main purpose of the program is to prevent transferring infected fish among OMNR hatcheries, or to wild fish populations through stocking waterways, the hatchery-level negative predictive value (HNPV) was used as an indicator of monitoring efficacy. The present program (which includes both routine screening of asymptomatic hatchery fish, and diagnostic testing of hatchery mortalities and clinically diseased fish) was confirmed to have a high median HNPV (0.999) for both study pathogens. Simulations suggested that the median probabilities that a hatchery would be pathogen-free if only diagnostic testing were continued (i.e. if no asymptomatic lots were screened), and all diseased lots tested negative for A. salmonicida and Y. ruckeri would be 0.994 for both pathogens (with <5% probability that HNPV would be less than 0.953 and 0.957, respectively) - indicating acceptable monitoring efficacy. However, limitations of the theoretical monitoring model must be considered.     

Development of a minimum-anesthetic-concentration depression model to study the effects of various analgesics in goldfish (Carassius auratus)

Teleost fish demonstrate the neurophysiologic capacity to experience pain and analgesia. A common model for assessing analgesic effect is the reduction of minimum anesthetic concentration (MAC). The present study adapted the model of MAC depression to evaluate the analgesic effects of morphine, butorphanol, medetomidine, and ketoprofen in goldfish (Carassius auratus). MAC was determined by an up-down method of sequential population sampling, anesthetizing fish with tricaine methanesulfonate (MS-222) in concentration increments of 10 parts per million (ppm), and using intramuscular needle insertion as a supramaximal noxious stimulus. Baseline MAC was determined in triplicate at the beginning (MACi) and conclusion (MACe) of the experiment (approximately 60 days). For drug trials, MAC was redetermined 1 hr after administration of morphine (10, 20, 40 mg/kg i.m.), butorphanol (0.1, 0.2, 0.4 mg/kg i.m.), medetomidine (0.01, 0.015, 0.025 mg/kg i.m.), ketoprofen (0.5, 1.0, 2.0 mg/kg i.m.), or saline control. Each drug/dose was tested in random order with a > 6-day washout period. MACi and MACf were 163 and 182 ppm, respectively, and were significantly different from each other (P = 0.02). All doses of morphine and ketoprofen decreased MAC below MACi. The highest dose of medetomidine decreased MAC below MACi. The lowest dose of butorphanol decreased MAC below MACi, but higher doses increased MAC above MACf. The authors conclude that MAC determination in fish using MS-222 was feasible and reproducible in the short term. The fact that MAC increased over time and/or exposure may limit the usefulness of MS-222 in MAC depression studies. Morphine and ketoprofen decrease anesthetic needs in goldfish and may provide analgesia.     

Administration of recombinant parasite beta-tubulin to goldfish (Carassius auratus L.) confers partial protection against challenge infection with Trypanosoma danilewskyi Laveran and Mesnil, 1904

The infection of carp and other cyprinid fish with Trypanosoma danilewskyi was reported to cause significant morbidity and mortality in aquaculture. Tubulin is a component of parasite excretory/secretory (ES) products recognized by antibodies present in the serum of recovered hosts. To assess the role of parasite tubulin in the induction of a protective immune response in the goldfish, recombinant T. danilewskyi beta-tubulin was produced in Escherichia coli and used to immunize goldfish against challenge with live parasites. Affinity purified rabbit anti-recombinant tubulin IgG bound to both surface and internal structures of trypanosomes, and when added to parasite cultures caused a dose-dependent inhibition of their growth in vitro. Immunization of goldfish i.p. with either 40 microg or 80 microg of endotoxin-free beta-tubulin+Freund's complete adjuvant (FCA) caused a significant decrease in parasitemia during the establishment phase of the infection (days 3 and 7) and increased the time required to reach the maximal mean number of parasites compared to non-immunized sham-injected control fish. The serum from immune fish contained antibodies that recognized trypanosomes as determined by confocal immunofluorescence microscopy and specific antibodies that recognized recombinant tubulin as measured by ELISA. Thus, the immunization of goldfish with recombinant parasite beta-tubulin conferred partial antibody-mediated protection against a challenge infection with live trypanosomes. This is a first report that parasite tubulin is immunogenic in poikilothermic vertebrates.