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1.
Abstract

Thirteen monoclonal antibodies (MAbs) were prepared against the extracellular product (ECP) of Mycobacterium sp., strain TB267, isolated from chevron snakehead Channa striata, and five MAbs were prepared against the reference strain Mycobacterium chelonei. The reactivity of the MAbs was examined against seven different strains of mycobacteria by an enzyme-linked immunosorbent assay (ELISA), and Western blot analysis was used to establish the molecular weight of antigens recognized by the MAbs. Western blot analysis proved essential for the selection of the MAbs because some that did not react by ELISA were found to be positive in this assay. All MAbs recognized a 65-kilodalton (kDa) protein present in ECPs, whole cell sonicates, and lysate preparations of the mycobacteria examined, and the epitopes recognized by the MAbs were located on molecules susceptible to protease activity. The 65-kDa protein, one of the major protein constituents of the mycobacterial preparations, was found in periplasmic spaces and cell walls of the bacteria via immunogold staining with MAbs.  相似文献   

2.
Antibody production in rainbow trout to extracellular antigens was investigated. The following antigen preparations and immunisation regimes were used: native extracellular products (ECP) in Freund's complete adjuvant (FCA), intraperitoneally (i.p.) with and without booster; formalinized ECP in FCA, i.p. with and without booster; washed, formalinized A. salmonicida cells in FCA, i.p., with booster; native ECP in saline, i.m., four weekly injections at two different doses, 45 micrograms and 6 micrograms each injection (after the protocol of Shieh, 1985). Using crossed normal rainbow trout serum, i.p., single injection (after the protocol of Sakai, 1985). Using crossed immunoelectrophoresis all antisera contained precipitating antibodies to three to five ECP components except that from fish immunised i.m. with 6 micrograms protein where antibodies were undetectable. In no case were specific antibodies to ECP protease or haemolysin detected. In a rabbit immunised with formalinized ECP in FCA under a similar regime to the rainbow trout, antibodies to at least 15 ECP components, including protease and haemolysin, were detected. The assumption of a specific immune response to the protease, at least in respect of antibody production, in recent reports of protection afforded by vaccines composed of either crude ECP or partially purified protease (Shieh, 1985) or partially purified protease inactivated by normal serum (Sakai, 1985) is not supported by the present findings.  相似文献   

3.
Abstract

In this study, we investigated the characteristics of inhibitor(s) of infectious pancreatic necrosis virus (IPNV) found in the serum of normal rainbow trout Oncorhynchus mykiss (RTS). The molecular size, stability to pH and temperature, and ontogeny of the inhibitor in trout were studied, and the effect of cations (Ca2+ and Mg2+) on the activity of the inhibitor was tested. The strongest inhibition of virus was obtained at approximately 150 kDa as measured by ultracentrifugation, sieve gel chromatography, and ultrafiltration. The inhibition decreased significantly when RTS was dialyzed or filtered in the absence of divalent cations, but replacement of at least one cation restored activity. Activity was stable at temperatures ranging from 30°C to 50°C, but 55°C completely destroyed the inhibitory capacity of RTS. The inhibitory activity of RTS was not reduced between pH 4 and 10 but was diminished below pH 4 and above pH 10; such activity was not abrogated by proteases. Additionally, pretreatment of RTS with the polysaccharide mannan significantly reduced inhibition. Thus, the serum inhibitor(s) had many characteristics of a lectin. To determine the ontogeny of inhibition, serum samples were taken from normal rainbow trout, beginning at 2 weeks posthatch; consistent inhibition was not obtained until the rainbow trout had reached the age of 23 weeks posthatch.  相似文献   

4.
Abstract

Laboratory exposures to the infectious stages (triactinomyxons) of Myxobolus cerebralis demonstrated a range of susceptibility to whirling disease among four species of inland salmonids. Replicate groups of each species were exposed to two concentrations of triactinomyxons, a low dose (100–200 per fish) and a high dose (1,000–2,000 per fish). Exposed fish were evaluated for clinical signs, for severity of microscopic lesions at 35 d, 2 and 5 months, and for spore concentrations in the head cartilage at 5 months. A standard strain of rainbow trout Oncorhynchus mykiss matched for age served as a susceptible species control. Rainbow trout, westslope cutthroat trout O. clarki lewisi, Yellowstone cutthroat trout O. clarki bouvieri, and bull trout Salvelinus confluentus were susceptible to M. cerebralis infections. Clinical signs, including radical swimming (“whirling”) and black tails, were observed at 7 weeks postexposure among rainbow and cutthroat trout challenged at 3 weeks of age. Clinical signs were rare among bull trout exposed at an age of 4 weeks and absent among rainbow and cutthroat trout exposed at 3 months posthatch. Most rainbow, cutthroat, and bull trout were found to be infected when examined at 5 months postexposure. The most severe microscopic lesions among infected fish at 5 months postexposure were found among rainbow trout. Cutthroat trout had less severe lesions, bull trout had mild infections, and no evidence of infection was found among Arctic grayling Thymallus arcticus. Mean spore concentrations among infected fish correlated with the severity of microscopic lesion scores. Rainbow trout had mean concentrations of spores in head cartilage reaching 106, whereas more resistant species such as bull trout had 104 spores; no spores were found among Arctic grayling at 5 months postexposure.  相似文献   

5.
Abstract

A lectin that agglutinates rabbit red blood cells (RBCs) and human type B RBCs was isolated from ova of rainbow trout Oncorhynchus mykiss. Hemagglutination of rabbit RBCs was inhibited completely by 10 mM L-rhamnose but not by certain other sugars, 100 mM EDTA, or 100 mM 2-mercaptoethanol. Partial purification of this hemagglutinating material was achieved by affinity chromatography of an H2O-dialyzed yolk homogenate on rhamnose-linked Sepharose. A polyacrylamide gel electrophoresis (SDS-PAGE) performed on this sample revealed two polypeptides with approximate molecular masses of 19 kilodaltons (kDa) and 30 kDa. By fast-phase liquid chromatography, proteins with a molecular mass of less than 20 kDa were separated from other elements of the affinity-purified hemagglutinating material. These proteins were found to lack hemagglutinating activity. When a western blot with rabbit antilectin antiserum was performed against yolk extract, rainbow trout serum, or yolk from larvae, a 30-kDa polypeptide was detected within all three samples. If the rainbow trout serum and egg lectins are the same molecule, then the biological function of the rainbow trout egg lectin may include host defense or perhaps a basic, homeostatic mechanism such as glycoprotein transport  相似文献   

6.
SUMMARY The BACTEC radiometric culture method for detection of Mycobacterium paratuberculosis was evaluated on faeces from cattle on a farm in quarantine for Johne's disease. A multiplex polymerase chain reaction (PCR) based on the IS900 sequence specific for M paratuberculosis and a genus specific 16S rRNA region was developed and used to test cultures showing evidence of mycobacterial growth in the BACTEC liquid radiometric culture medium. Using the BACTEC - PCR combination, confirmation of M paratuberculosis from faeces and tissue of clinically affected animals was achieved within 2 to 4 weeks and 1 week, respectively, a substantial improvement on traditional culture and identification methods. The PCR provided rapid exclusion of M paratuberculosis when other Mycobacterium spp were grown. The radiometric culture medium proved to be very sensitive for culturing Mycobacterium spp.  相似文献   

7.
Differences in susceptibility to the myxozoan parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD), between four strains of rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) were evaluated. Fish were exposed to water enzootic for the parasite in the field for 5 days and were subsequently transferred to the laboratory. Relative parasite load was determined after 2, 3 and 4 weeks post-exposure (wpe) by quantitative real-time PCR (qPCR) of kidney samples and number of parasite stages was determined in immunohistochemical stained sections of kidney, liver and spleen tissues. According to qPCR results, the highest amount of parasite DNA per equal amount of host tissue at all time points was measured in brown trout. Two of the rainbow trout strains showed lower relative parasite load than all other groups at the beginning of the experiment, but the parasite multiplied faster in these strains resulting in an equal level of relative parasite load for all rainbow trout strains at 4 wpe. A weak negative correlation of fish size and parasite load was detected. Only in samples of a few fish, single stages of T. bryosalmonae were found in sections stained by immunohistochemistry impeding quantitative evaluation of parasite numbers by this method. The results indicate a differential resistance to T. bryosalmonae between the rainbow trout strains investigated and between rainbow trout and brown trout.  相似文献   

8.
The incidence of bovine tuberculosis (bTB) is increasingly giving rise to large economic losses in the agricultural industry. The current methods used for detection and control of bTB (skin test and interferon-gamma) lack desired sensitivity and specificity. Therefore, the development of a rapid and reliable bTB serological based assay is urgently required. An antibody assay using combinations of strain-specific mycobacterial antigens could resolve both specificity and sensitivity issues. We analyzed the ability of a series of selected mycobacterial antigens to outline a humoral immune response in a rabbit model experimentally challenged with different mycobacterium. Antibodies specific for three antigens, MTB40, ESAT6 and CFP10, were present in serum 2 weeks post-challenge (early indicator), while two other antigens, Rv3870 and Rv1580c, could be detected from 8 to 11 weeks post-challenge. These selected mycobacterial antigens did not exhibit any cross-reactivity with avian PPD and only a very low positivity with bovine PPD. This data suggests that this panel of strain-specific mycobacterial antigens could be used for identification of Mycobacteriumbovis infection in serum samples. The combinatorial application of these antigens could form part of a serum field test which may assist the future diagnosis of TB.  相似文献   

9.
Abstract

This study examined whether supplemental amino acid-chelated trace elements were effective in improving disease resistance in rainbow trout Oncorhynchus mykiss. Four semipurified diets containing trace elements from either sulfates or an amino acid chelate source were fed to the fish for 18 weeks. Subsequently, fish weighing about 20–40 g were challenged with β-hemolytic Streptococcus sp. The cumulative mortality was less in the chelate-supplemented groups but not significantly different from that of the sulfate groups. Antibody titer was significantly higher in the chelate-fed fish. Lysozyme activity and total immunoglobulin content were slightly more enhanced in the chelate-fed than in the sulfate-fed groups. Despite the increased antibody production, no distinct improvement in disease resistance against Streptococcus sp. was achieved by supplementing rainbow trout diets with amino acid-chelated trace elements. Further confirmatory studies focusing on the amounts of the trace elements used are needed to shed light on the influence of amino acid chelates on disease resistance.  相似文献   

10.
SUMMARY Experimental transmission of epizootic haematopoietic necrosis virus (EHNV) to adult redfin perch Perca fluviatilis and juvenile rainbow trout Oncorhynchus mykiss was undertaken at different water temperatures using intraperitoneal (IP) and bath inoculation. Redfin perch were highly susceptible to EHNV by both routes of infection. Bath inoculation with as few as 0.08 TCID50. mL-1 was lethal. The incubation period in redfin perch was about 11 days at a water temperature of 19–21°C but was longer at colder temperatures and disease did not occur at temperatures below 12°C. The longest incubation period recorded in redfin perch was 28 days. Rainbow trout were not susceptible to infection by bath inoculation but the disease was reproduced after IP inoculation with 105.6 TCID50 at water temperatures ranging from 8–21°C. The incubation period was 3–10 days at 19–21°C, but was up to 32 days at 8–10°C. Persistent infection with EHNV was detected by virus isolation in a clinically unaffected rainbow trout after 63 days. The implications of these findings in the understanding of the epidemiology of EHNV infection are discussed.  相似文献   

11.
Abstract

Two sizes of fingerling Snake River cutthroat trout Oncorhynchus clarkii behnkei and Colorado River rainbow trout O. mykiss were raised at hatcheries testing negative for Myxobolus cerebralis and stocked into the Dolores and Cache la Poudre rivers from 1999 to 2001. Populations were resampled over a 2-year period to determine which species and size combination had the highest growth and survival rates. Fish were tested for M. cerebralis via polymerase chain reaction and pepsin?trypsin digest analyses. Growth and survival rates between the species and size groups were not significantly different in either river. In the Dolores River, annual survival for both species and sizes of fish combined ranged from 0.063 to 0.12. In the Cache la Poudre River, survival for both sizes of rainbow trout was 0.004; survival for cutthroat trout ranged from 0.182 to 0.53. Larger fish had higher growth rates than smaller fish, and cutthroat trout had higher rates than similar sizes of rainbow trout. In both rivers, a higher percentage of the rainbow trout sample was infected than in the cutthroat trout sample. Rainbow trout also had a higher mean number of spores per head than cutthroat trout, and small rainbow trout had higher spore counts than large rainbow trout. Survival rates for cutthroat trout in the Cache la Poudre River were the highest of any of the groups, suggesting a difference that is biologically significant. Raising fingerlings to sizes greater than 100 mm can improve poststocking survival. If rainbow trout are stocked into contaminated waters, raising fingerlings to a larger size does not appear to improve growth or survival rates. Stocking rainbow trout in the spring could maximize growth rates but will expose fish to greater triactinomyxon densities, resulting in higher intensities of infection.  相似文献   

12.
Abstract

Spontaneous outbreaks of bacterial gill disease (BGD) occurred in 17-44-g rainbow trout Oncorhynchus mykiss after they were placed in a water recirculation system. In each of five groups stocked from September 1991 through July 1992, BGD occurred within 6–8 d after stocking. In each instance, BGD was followed by a secondary amoeba infestation. The spontaneous BGD outbreaks did not occur among previously stocked groups that had recovered from earlier BGD disease outbreaks. Examination of gill tissue by Gram stain and indirect fluorescent antibody technique showed increased numbers of filamentous bacteria associated with BGD after the rainbow trout were stocked into the system. Bacterial numbers decreased after a 1-h treatment with chloramine-T at concentrations of 9–15 mg/L but increased within 2 d after treatment. Although the chloramine-T treatments controlled mortality related to BGD, the amoeba infestation persisted. Histological examination of gills showed some focal hyperplasia before the rainbow trout were placed into the recirculation system, but hyperplasia became more extensive and lamellar fusion and mild telangiectasis developed within a week after placement in the system. The density at which the fingerling rainbow trout were stocked and the suspended solids present in tank water may have contributed to the BGD outbreaks. Exposure of juvenile rainbow trout to tank water from the recirculation system before they were placed into the system did not afford them protection against BGD after stocking.  相似文献   

13.
This study aimed to assess systemic and mucosal immune responses of Atlantic salmon (Salmo salar) exposed to two protein-hapten antigens – dinitrophenol (DNP) and fluorescein isothiocyanate (FITC) each conjugated with keyhole limpet haemocyanin (KLH) – administered using different delivery strategies. Fish were exposed to the antigens through different routes, and were given a booster 4 weeks post initial exposure. Both systemic and mucosal antibody responses were measured for a period of 12 weeks using an enzyme-linked immunosorbent assay (ELISA). Only fish exposed to both antigens via intraperitoneal (IP) injection showed increased systemic antibody response starting 6 weeks post immunization. No treatment was able to produce a mucosal antibody response; however there was an increase in antibody levels in the tissue supernatant from skin explants obtained 12 weeks post immunization from fish injected with FITC. Western blots probed with serum and culture supernatant from skin explants showed a specific response against the antigens. In conclusion, IP injection of hapten-antigen in Atlantic salmon was the best delivery route for inducing an antibody response against these antigens in this species. Even though IP injection did not induce an increase in antibody levels in the skin mucus, there was an increased systemic antibody response and an apparent increase of antibody production in mucosal tissues as demonstrated by the increased level of specific antibody levels in supernatants from the tissue explants.  相似文献   

14.
Abstract

Triploid (heat-shocked) and diploid groups of rainbow trout Oncorhynchus mykiss, brook trout Salvelinus fontinalis, coho salmon Oncorhynchus kisutch, and reciprocal hybrids were produced, monitored for early life stage survival, and evaluated for susceptibility to infectious hematopoietic necrosis virus (IHNV). The female rainbow trout × male brook trout triploid hybrids had significantly greater (P < 0.01) survival than the diploid hybrids of this cross. The heat-shocked hybrid group of the female rainbow trout × male coho salmon also exhibited significantly greater survival to the eyed egg stage of development than the untreated group of this hybrid. Studies of the susceptibility of treatment groups to a 1990 IHNV isolate from the Hagerman Valley were conducted by using a standardized immersion exposure procedure at one or two different mean body weights. The diploid brook trout and coho salmon and two triploid hybrids (female rainbow trout × male brook trout or male coho salmon) were significantly less (P < 0.05) susceptible to IHNV than the pure-species diploid and triploid rainbow trout groups.  相似文献   

15.
The phagocytosis of Vibrio anguillarum by peritoneal macrophages from normal rainbow trout was enhanced by antibody and complement. Treatment of either macrophages or the bacteria by antibody also enhanced opsonisation. Five weeks after immunisation with V anguillarum, the phagocytic activity of macrophages from rainbow trout was increased significantly compared with the activity of those from unvaccinated fish. Although agglutinin titres did not increase until three weeks after immunisation, seven out of 10 fish challenged one week after immunisation survived, indicating that immunised fish had developed resistance to vibrio infection before significant levels of antibody or phagocytic activity became detectable.  相似文献   

16.
Abstract

Cell lines from white sturgeon Acipenser transmontanus were derived from peripheral blood cells, heart, and spleen. Incubated with infectious hematopoietic necrosis virus (IHNV) for 8 d at l5°C, these cell lines produced 0.7–53.2 plaque-forming units (PFU)/cell. Waterborne exposure of larval white sturgeons (60 d posthatch) to 106 PFU/mL of IHNV resulted in 10% mortality 5–6 d postinfection, with virus concentrations consistently greater than 105 PFU/g. A replicate group of larval white sturgeons that were sampled at different times post-IHNV exposure had no detectable virus at 24 h, but 72% of the fish had IHNV concentrations of 102-106 PFU/g when they were examined 2–9 d postinfection. Juvenile white sturgeons (mean weight, 35 g) immersed in or injected with IHNV exhibited no mortality, and virus was only detected immediately postexposure in just 25% of the fish tested. Juvenile white sturgeons fed either virus-free rainbow trout Oncorhynchus mykiss or dead IHNV-infected rainbow trout had no viable virus in their feces. Juvenile white sturgeons fed or exposed to IHNV failed to transmit the virus to cohabiting rainbow trout fry. These results suggest that IHNV can replicate in larval white sturgeons but presumably not in juveniles or adults. Virus neutralization activity was detected in serum from adult white sturgeons (4–6 years old) cultured with rainbow trout exposed to IHNV but not in white sturgeons kept in a pathogen-free environment and fed a manufactured diet. White sturgeon serum with IHNV-neutralizing activity was used to passively immunize rainbow trout, and it provided significant (P < 0.01) protection against IHNV challenge.  相似文献   

17.
Neutrophil activity in rainbow trout (Oncorhynchus mykiss) is increased upon antigenic stimulation with the Yersinia ruckeri O-antigen bacterin. The characteristics of neutrophil attachment to glass and nitroblue tetrazolium (NBT) staining were used to determine the effectiveness of immunization programs with fingerling rainbow trout. Fish immunized by intraperitoneal injection with doses of 100, 10, or 1 μg of the bacterin showed the highest responses in that order in numbers of glass adherent, NBT-positive neutrophils. Studies on the kinetics of the occurrence of numbers of glass-adherent, NBT-positive staining cells from the fish injected with the 10 μg dose showed the numbers of positive cells were largest on Day 2 after injection. The specific immune response was confirmed by demonstrating the presence of plaque-forming cells by the passive hemolytic plaque assay and the rise in humoral antibody titers by passive hemagglutination 12 days after injection. The effects of immunization in trout could be detected earlier by using the neutrophil glass adherence and NBT reduction assays than by using assays based on observations of the specific immune response.  相似文献   

18.
In this study, effect of ellagic acid on some haematological, immunological and antioxidant parameters in the blood and various tissues of rainbow trout (Oncorhynchus mykiss) were examined. Four groups of rainbow trout were fed experimental diets containing either no ellagic acid (control) or supplemented with ellagic acid at 50 mg/kg diet (EA‐50), 100 mg/kg diet (EA‐100) or 150 mg/kg diet (EA‐150) for 21 days. Samples of the blood and tissue (liver, kidney and spleen) were collected at the end of the experiment and analysed for their haematological profile (the red blood cell count, the haemoglobin concentration and the haematocrit level), immune response (the white blood cell count, the oxidative radical production (NBT activity), the total plasma protein and total immunoglobulin level) and oxidant/antioxidant status (the malondialdehyde level, the superoxide dismutase, catalase and glutathione peroxidase activity as well as the reduced glutathione concentration). The findings of this study demonstrated that ellagic acid had a positive effect on the haematological parameters, the immune response and the antioxidant enzyme activities of the fish.  相似文献   

19.
Abstract

Juvenile rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta acclimated to freshwater or salinities of 9.0‰ or less were exposed to Yersinia ruckeri, the bacterial pathogen that causes enteric redmouth disease (ERM). Both species of fish were kept in the same recirculating systems after bacterial exposure. Rainbow trout mortality was significantly (P < 0.05) different in each salinity: 96.5% in freshwater, 89.5% in water of 1.1‰ salinity, 81.3% in 3.0‰ salinity, and 75.0% in 9.0‰ salinity (model SE = 1.0). All deaths occurred between 3 and 12 d after exposure to Y. ruckeri. Only 2.3% of brown trout in all salinities died, and differences among treatments were not significant. For both fish species, Y. ruckeri was isolated from liver, spleen, and trunk kidney of fish dying during this experiment, and lesions of rainbow trout were consistent with ERM. Yersinia ruckeri was not isolated from brown trout surviving for 21 d after bacterial exposure but was isolated from 3 of 24 surviving rainbow trout; a polymerase chain reaction assay detected the DNA of Y. ruckeri in 3 additional rainbow trout survivors. Neither the lesions of fish with ERM nor the percentage of surviving fish subclinically infected with Y. ruckeri was affected by salinity. Bacterial growth in vitro was not affected by low (≤9.0‰) salinity; however, bacterial adhesion to polystyrene was significantly reduced as salinity increased. Although mortality caused by Y. ruckeri was significantly lower for rainbow trout in water with slightly increased salinity, none of the salinities tested was effective in preventing serious losses caused by this pathogen in recirculating systems.  相似文献   

20.
Abstract

Blood chemistry analytes are determined for fish from either serum or plasma samples. Serum and plasma are similar in that they both represent the fluid component of blood; however, plasma contains clotting factors that are not present in serum. This study was conducted to determine whether the type of sample—plasma or serum—had an effect on measured blood analytes in rainbow trout Oncorhynchus mykiss, channel catfish Ictalurus punctatus, hybrid tilapia Oreochromis spp., and hybrid striped bass (striped bass Morone saxatilis × white bass M. chrysops). Paired plasma and serum samples were analyzed for the following standard biochemical analytes: total protein, albumin, globulin, creatinine, total bilirubin, alkaline phosphatase, aspartate aminotransferase, sodium, potassium, chloride, calcium magnesium, phosphorus, glucose, and cholesterol. For all four taxa, values for potassium were lower in the serum and magnesium and phosphorus values were higher in the serum. Glucose values were lower in the serum from rainbow trout, hybrid striped bass, and channel catfish; whereas cholesterol values were higher in the serum of rainbow trout, channel catfish, and hybrid tilapias. The differences observed between serum and plasma were distinct from changes occurring with hemolysis and, therefore, do not represent release of erythrocyte constituents. The differences most likely represent metabolic utilization of blood constituents while the blood was clotting. This work indicates that plasma should be used preferentially to serum for biochemical analysis because analyte levels determined from serum may not accurately reflect those found in circulating blood.  相似文献   

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