Putative immune response of rainbow trout, Salmo gairdneri, to Diplostomum spathaceum infections

The immune response of rainbow trout to infection with the cercariae of the digenean parasite Diplostomum spathaceum was investigated. Rainbow trout infected with cercariae at weekly intervals, or injected with a suspension of dead cercariae, did not produce a specific humoral response against D. spathaceum cercariae, as tested using immunoperoxidase, agglutination and cytotoxicity assays. However, a significant difference occurred in the rate of infection of rainbow trout given weekly exposure to cercariae of D. spathaceum in winter and summer. Rainbow trout injected with a suspension of dead cercariae acquired significantly fewer metacercariae than controls when exposed to a challenge infection. This suggested a specific immune response by the host and is the first example of a reduction in the infection rate of rainbow trout immunized against a digenean parasite, when exposed to a challenge infection.     

Studies on the infectivity of Diplostomum spathaceum in rainbow trout (Oncorhynchus mykiss)

The infectivity of Diplostomum spathaceum (Digenea: Trematoda) cercariae to rainbow trout and the efficacy of the diplostomule migration to the lens following different routes of administration was examined. The optimum age of infectivity for cercariae was between 0-5 h after liberation from the snail and for intraperitoneally injected diplostomules, 5 h post-transformation in vitro through fish skin. After exposure of the entire fish body or head to cercariae, metacercariae first appeared in the lens at 5 h and their numbers gradually increased until 22 h. Following exposure of the tail region of rainbow trout to cercariae, metacercariae first appeared in the lens at 14 h. Significantly more metacercariae established in the lens of fish following exposure of the fish head compared with the tail region; 40% of penetrating cercariae reached the lens of fish following exposure of the head or entire body, 20% of cercariae or diplostomules injected either intraperitoneally, intramuscularly or intracardially reached the lens while only 5% of cercariae established as metacercariae following exposure of the tail region.     

Metacercariae of Diplostomum spathaceum in the eyes of fishes from Yellowstone Lake, Wyoming

Fish were collected from Yellowstone Lake, Wyoming (USA). Metacercariae of Diplostomum spathaceum was found in the lens of 11 of 12 longnose suckers (Catostomus catostomus). The mean number of metacercariae per sucker was 59 and the average age of the fish was 11.6 yr. There was no correlation between age and intensity of parasites (r = 0.24). Of 10 cutthroat trout (Salmo clarki) examined, there were metacercariae present in six. The metacercariae were found outside of the lens tissue in the trout; they occurred in the vitreous humor and the retina. These may be a different species from those found in the suckers.     

Serum agglutinating titres against Renibacterium salmoninarum the causative agent of bacterial kidney disease, in rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L.

Serum agglutinating titres against Renibacterium salmoninarum were determined from clinically infected, sub-clinically infected and non-infected rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L. Titres ≥16 occurred in clinically infected rainbow trout, but declined after a few months when disease signs were absent. Non-infected rainbow trout had titres ≤ 16. No correlation between the agglutinating titre and the level of infection was found in farmed Atlantic salmon. Variable titres were found in wild salmon broodstock although there was no evidence of BKD during 4 years of testing.     

Biochemical genetic variation in populations of golden trout, Salmo aguabonita: evidence of the threatened Little Kern River golden trout, S.a. whitei.

Eight wild populations of the High Sierra golden trout, Salmo aguabonita, and one domestic stock of rainbow trout, Salmo gairdneri, were examined for biochemical-genetic variation in eight protein systems. Variation within the eight systems was determined by at least 10 loci in both golden and rainbow trout and all the alleles identified in rainbow trout were observed as electro-phoretically identical phenotypes in golden trout. Variation was observed at an average of 51 percent of the loci in the golden trout samples and for five of the 10 loci in the rainbow trout. Average heterozygosity ranged from 12.6 to 13.9 percent for seven of the golden trout populations with one showing a low value of 5.4 percent. A comparable estimate of 12.1 percent was found for the rainbow stock. On the basis of genetic variation and allele frequencies at three loci, the eight golden trout populations were divided into two distinct groups. Three populations sampled from the Little Kern River basin tended to be genetically distinct from two additional Little Kern River basin populations and from three geographically distinct populations sampled from the eastern Kern River area. The former three populations were hypothesized to be of a recent rainbow-golden hybrid origin. Trout in the other two Little Kern River basin populations, sampled in head-waters of a stream tributary to the Little Kern River, were considered to be the threatened Little Kern golden trout, S. a. whitei Evermann, due to their high degree of genetic similarity to the geographically distinct subspecies S. a. aguabonita sampled from the eastern Kern River area. The finding of substantial genetic variation in the wild golden trout populations indicates that this threatened species is not at present genetically impoverished and thus does not appear to be in immediate danger of extinction through lack of adaptive capability.     

Cardiac pathology associated with the infection of Oncorhynchus mykiss Walbaum with Apatemon gracilis Rud. 1819

The chronological development of gross pathological and histopathological changes associated with the infection of rainbow trout, Oncorhynchus mykiss Walbaum, with metacercariae of Apatemon gracilis Rudolphi was investigated.     

Rhabdovirus infection induces public and private T cell responses in teleost fish.

Many viruses induce a strong T cell response that contributes to the elimination of infected cells presenting viral peptides by MHC molecules. The structure and expression of genes encoding molecules homologous to mammalian alphabeta TCRs have been recently characterized in rainbow trout and in several teleost species, but the alphabeta T cell response against pathogens has not been directly demonstrated. To study the modifications of the T cell repertoire during an acute viral infection in rainbow trout, we adapted the immunoscope methodology, which consists of spectratyping the complementarity-determining region 3 length of the TCRbeta chain. We showed that the naive T cell repertoire is polyclonal and highly diverse in the naive rainbow trout. Using viral hemorrhagic septicemia virus (VHSV), which provokes an acute infection in rainbow trout, we identified skewed complementarity-determining region 3 size profiles for several VbetaJbeta combinations, corresponding to T cell clonal expansions during primary and secondary response to VHSV. Both public and private T cell expansions were shown by immunoscope analysis of spleen cells from several infected individuals of a rainbow trout clone sharing the same genetic background. The public response to VHSV consisted of expansion of Vbeta4Jbeta1 T cell, which appeared early during the primary response and was strongly boosted during the secondary response.     

Detection of Yersinia ruckeri in rainbow trout blood by use of the polymerase chain reaction

We evaluated a polymerase chain reaction (PCR) method for detecting Yersinia ruckeri, the bacterial pathogen causing enteric redmouth disease (ERM), in blood of rainbow trout Oncorhynchus mykiss. Identification of the PCR product was confirmed by Southern blot hybridization with a 32P-labeled oligonucleotide probe matching a sequence within the small subunit ribosomal RNA gene of Y. ruckeri. Following a 1 h immersion of rainbow trout in water with 4.5 x 10(6) colony-forming units of Y. ruckeri l(-1), the PCR was positive for all blood samples from 1 h (first sample) to 5 d and was negative from 9 to 30 d (last sample). Fish in this experiment did not show signs of disease, probably because they had been vaccinated against Y. ruckeri. To test this method with naturally infected fish, 42 rainbow trout from hatcheries were examined. Four of these fish had clinical signs of ERM and were infected with Y. ruckeri based on bacteriological culture. The PCR method detected Y. ruckeri in blood, intestine, liver, and trunk kidney from the 4 fish with ERM and from 5 additional rainbow trout that were bacteriologically negative for Y. ruckeri. Three of 5 rainbow trout from streams receiving effluent from hatcheries were positive for Y. ruckeri when tested with PCR, although there was no growth of Y. ruckeri on culture plates inoculated with the same samples. Samples were successfully stored for 1 wk in lysis buffer at 25 degrees C. This study demonstrated that a non-lethal blood sample can be used with PCR to detect Y. ruckeri.     

Prevalence of Zoonotic Trematode Metacercariae in Freshwater Fish from Gangwon-do,Korea

The infection status of zoonotic trematode metacercariae was investigated in a total of 2,293 freshwater fish collected from 11 rivers or streams in 9 administrative regions of Gangwon-do, Korea for 5 years (2009-2013). All fish were collected by netting methods and examined using the artificial digestion methods. Clonorchis sinensis metacercariae were detected in 4 fish species, i.e., Pungtungia herzi, Squalidus japonicus coreanus, Acheilognathus rhombeus, and Ladislabia taczanowskii, from only Hantangang in Cheorwon-gun. Metagonimus spp. metacercariae were found in 1,154 (50.3%) fish and their average number per infected fish was 55.8. Among the positive fish species, especially Tribolodon hakonensis from Namdaecheon in Yangyang-gun and Plecoglossus altivelis from Osipcheon in Samcheok-si were most heavily infected. Centrocestus armatus metacercariae were detected in 611 (26.7%) fish and the average metacercarial burden per infected fish was 1,032. Two chub species, Zacco platypus and Zacco temminckii were highly and heavily infected with C. armatus metacercariae in almost all regions surveyed. Echinostoma spp. metacercariae were also found in 24 fish from a few localities, but their numbers per fish infected were very low. From the above results, it is confirmed that the metacercariae of intestinal flukes, especially Metagonimus spp. and C. armatus, were heavily infected, while C. sinensis metacercariae were rarely found in fish from Gangwon-do, Korea.     

Survey of Zoonotic Trematode Metacercariae in Fish from Water systems of Geum-gang (River) in Republic of Korea

The infection status of zoonotic trematode metacercariae (ZTM) was surveyed in freshwater fishes from the water systems of Geum-gang (River) in the Republic of Korea (Korea). A total of 1,161 freshwater fishes from 6 local sites of Geum-gang were examined with the artificial digestion method for 4 years (2012–2015). Clonorchis sinensis metacercariae were detected in 122 (37.2%) out of 328 fishes in the positive fish species from 4 surveyed areas, and their mean intensity was 43 per fish infected. Metagonimus spp. metacercariae were found in 432 (51.7%) out of 835 fishes in the positive fish species from all 6 surveyed areas, and their mean intensity was 30 per fish infected. Centrocestus armatus metacercariae were detected in 285 (75.0%) out of 380 fishes in the positive fish species from 6 surveyed areas, and their mean intensity was 2,100 per fish infected. Echinostoma spp. metacercariae were found in 56 (19.7%) out of 284 fishes in the positive fish species from 5 surveyed areas, and their mean intensity was 10 per fish infected. Clinostomum complanatum metacercariae were detected in 98 (57.3%) out of 171 fishes in the positive fish species from only 2 surveyed areas, and their mean intensity was 11 per fish infected. Conclusively, the endemicity of ZTM is not so high in fishes from water systems of Geum-gang in Korea although it is more or less different by fish species, surveyed areas and ZTM species.     

Occurrence of Flavobacterium psychrophilum in fish-farming environments

Occurrence of Flavobacterium psychrophilum in fish farms and fish-farming environments was studied using agar plate cultivation, the immunoflourescence antibody technique (IFAT) and nested PCR. Characteristics of 64 F. psychrophilum isolates from rainbow trout Oncorhynchus mykiss, fish farm rearing water, ovarian fluid and wild fish were serotyped, ribotyped and compared biochemically. Virulence of F. psychrophilum isolates from different sources was compared by injection into rainbow trout. Additionally, the number of F. psychrophilum cells shed by naturally infected rainbow trout was determined. F. psychrophilum was detected and isolated from skin mucus, skin lesions and internal organs of diseased rainbow trout and from fish without clinical disease. The pathogen was also present in wild perch Perca fluviatilis, roach Rutilus rutilus, and ovarian fluids of farmed rainbow trout brood fish. Isolates were biochemically homogenous, excluding the capability to degrade elastin. Five different agglutination patterns with different antisera against F. psychrophilum were found among the isolates studied. Although several different ribopatterns were found (ClaI: 12 ribopatterns and HaeIII: 9 ribopatterns), ribotype A was the most dominant. Farmed rainbow trout brood fish carried a broad-spectrum of serologically and genetically different F. psychrophilum in ovarian fluids. Virulence of the tested isolates in rainbow trout varied and naturally infected rainbow trout shed 10(4) to 10(8) cells fish(-1) h(-1) of F. psychrophilum into the surrounding water.     

Distribution of the flagellate Hexamita salmonis Moore, 1922 and the microsporidian Loma salmonae Putz, Hoffman and Dunbar, 1965 in brown trout, Salmo trutta L., and rainbow trout, Salmo gairdneri Richardson, in the River Itchen (U.K.) and three of its fish farms

The occurrence of Hexamita salmonis Moore, 1922 and Loma salmonae Putz, Hoffman and Dunbar, 1965 was investigated at 10 sites on the R. Itchen (five for brown trout only, three for rainbow trout only, and two for both brown trout and rainbow trout) and at three of its nine fish farms (two for rainbow trout, one for brown trout). Hexamita salmonis was recorded in brown trout from three river sites and the farm, and in rainbow trout from both farms and four river sites. Prevalence of Hexamita salmonis in farmed rainbow trout was higher than in farmed brown trout and was consistent with the former species being more susceptible to infection. H. salmonis was at significantly higher prevalence in rainbow trout from farm no. 5 than farm no. 2 for three size classes of fish. In wild brown trout and feral rainbow trout, the highest prevalences of H. salmonis were recorded at sites in the vicinity of farm no. 2. This distribution was consistent with an area of naturally high infection levels, and with infected fish unintentionally released from farm no. 2 serving as a source of infection, the infection subsequently becoming established in the river fish. Loma salmonae was recorded in wild brown trout and in rainbow trout from both farms. This appears to be the first recording of this parasite from British salmonids and also the first recording of the parasite from brown trout. The distribution of the parasite (particularly the prevalence being higher at farm no. 2 than farm no. 5) was consistent with it being introduced into the R. Itchen via rainbow trout from farm no. 2 (and probably no. 3) much of whose stock derived from imported Californian 'Shasta' rainbow trout.     

Comparative susceptibility of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta to Myxobolus cerebralis, the cause of salmonid whirling disease.

The susceptibility of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta to Myxobolus cerebralis, the cause of salmonid whirling disease, was assessed following dosed exposures to the infectious stages (triactinomyxons). Parallel groups of age-matched brown trout and rainbow trout were exposed to 10, 100, 1000 or 10,000 triactinomyxons per fish for 2 h and then placed in aquaria receiving single pass 15 degrees C well water. Severity of infection was evaluated by presence of clinical signs (whirling and/or black tail), prevalence of infection, severity of microscopic lesions, and spore counts 5 mo after exposure. Clinical signs of whirling disease, including a darkened caudal region (black tail) and radical tail chasing swimming (whirling), occurred first among rainbow trout at the highest dose at 6 to 7 wk post exposure. Black tail and whirling occurred among rainbow trout receiving 1000 and 100 triactinomyxons per fish at 8 to 9 wk post exposure. Only 1 of 20 fish had a black tail among rainbow trout receiving 10 triactinomyxons per fish, although 30% of the fish were infected at 5 mo post exposure. Black tails were observed in brown trout at 1000 and 10,000 triactinomyxons per fish beginning at 11 and 7 wk post exposure, respectively. There was no evidence of the tail chasing swimming (whirling) in any group of brown trout. The prevalence of infection, spore numbers, and severity of microscopic lesions due to M. cerebralis among brown trout were less at each exposure dose when compared to rainbow trout. Infections were found among rainbow trout at all doses of exposure but only among brown trout exposed to doses of 100 triactinomyxons per fish or greater. Risk of infection analyses showed that rainbow trout were more apt to be infected at each exposure dose than brown trout. Spore counts reached 1.7 x 10(6) per head among rainbow trout at the highest dose of exposure compared to 1.7 x 10(4) at the same exposure dose among brown trout. Spore numbers increased with dose of exposure in rainbow trout but not in brown trout. As microscopic lesion scores increased from mild to moderate, spore numbers increased in rainbow trout but not brown trout. The mechanisms by which brown trout resist infections with M. cerebralis were not determined. Cellular immune functions, including those of eosinophilic granular leukocytes that were more prominent in brown trout than rainbow trout, may be involved.     

The population dynamics of eyeflukes Diplostomum spathaceum and Tylodelphys clavata (Digenea: Diplostomatidae) in rainbow and brown trout in Rutland Water: 1974–1978

The development of populations of eyeflukes Diplostomum spathaceum and Tylodelphys clavata in trout introduced into Rutland Water is related to the availability of infected Limnaea pereger, the first intermediate host, and the differential resistance to eyeflukes in Salmo gairdneri and S. trutta. The numbers of snail and fish intermediate hosts were monitored from impoundment of the Gwash in 1974 to completion of the reservoir in 1978. High infection rates in coarse fish in the feeder streams in 1974 are related to high infection rates in Limnaea pereger. Large eyefluke populations in introduced rainbow trout but not in brown trout confirm their differential resistance. Differences in levels of infection in seine-netted and rod-caught rainbow trout suggest that heavily infected fish could not respond to the fishermens' lures. Reduced numbers of eyeflukes in rainbow trout from 1979 onwards are related to the vast increase in volume of the reservoir in 1977 and decrease in abundance of Limnaea pereger.     

Detection of rainbow trout antibodies against viral haemorrhagic septicaemia virus (VHSV) by neutralisation test is highly dependent on the virus isolate used

Three serological tests, enzyme linked immunosorbent assay (ELISA), 50% plaque neutralisation test (50%PNT) and Western blotting (WB), were used to detect antibodies against viral haemorrhagic septicaemia virus (VHSV) in 50 rainbow trout broodstock from a rainbow trout farm endemically infected with VHS but with no clinical signs of infection. When the sera were examined by 50%PNT using the VHSV reference isolate DK-F1 or the heat attenuated DK-F25 mutant strain, no neutralizing antibodies were found. In contrast, when one of the virus isolates from the farm (homologous virus) was used in the 50%PNT, 90% of the fish were found to be positive. By examining a panel of different VHSV isolates in 50%PNT, it was demonstrated that the virus isolate used as test antigen could significantly affect the sensitivity and titre determination in 50%PNT for detection of rainbow trout antibodies against VHSV. When the sera were examined for the presence of VHSV antibodies by ELISA or WB, 61% were found to be positive. When conducting WB analysis, the viral glycoprotein was the protein most frequently recognized, followed by the viral nucleoprotein.     

The role of alpha 2-macroglobulin in furunculosis: a comparison of rainbow trout and brook trout.

1. The molecular basis for the high survival rate of rainbow trout, Oncorhynchus mykiss, infected with furunculosis was investigated. 2. Alpha 2-macroglobulin (alpha 2M), a major serum protease inhibitor, was partially purified from rainbow trout and brook trout, Salvelinus fontinalis, sera; the latter species shows marked disease susceptibility. 3. It is shown that a 10-fold species-based difference in alpha 2M inhibitory activity exists against a furunculosis associated bacterial protease. 4. A possible basis for the observed disparity is discussed. 5. Results suggest that the high mol. wt form of teleost (trout) albumin is a dimer composed of two 85,000 subunits.     

Staphylococcal coagglutination, a rapid method of identifying infectious hematopoietic necrosis virus.

A staphylococcal coagglutination test was developed for the rapid detection of infectious hematopoietic necrosis virus (IHNV) in cell cultures and infected fish. The test could be completed in 15 min but required a minimum IHNV titer of 10(6) PFU/ml to obtain a positive reaction. All IHNV isolates, representing the five electropherotypes taken from a wide variety of species and different geographic ranges, caused coagglutination of Staphylococcus aureus cells sensitized with rabbit polyclonal serum against the Round Butte IHNV isolate. The coagglutination reaction was blocked by preincubation of IHNV with homologous antiserum, and IHNV did not cause coagglutination of S. aureus cells sensitized with normal rabbit serum. In specificity tests, cells sensitized with rabbit anti-IHNV serum or normal serum did not coagglutinate in the presence of infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, cell culture medium components, or media from cultures of cell lines of salmonid and nonsalmonid origin. Most importantly, the coagglutination test was able to detect and identify IHNV directly from experimentally infected rainbow trout fry, the organs of naturally infected adult kokanee salmon and winter steelhead trout, and ovarian fluids of the winter steelhead trout. The coagglutination test is very suitable for field use, since it is inexpensive, simple to interpret, sensitive, and rapid and requires no specialized equipment.     

Staphylococcal coagglutination, a rapid method of identifying infectious hematopoietic necrosis virus.

A staphylococcal coagglutination test was developed for the rapid detection of infectious hematopoietic necrosis virus (IHNV) in cell cultures and infected fish. The test could be completed in 15 min but required a minimum IHNV titer of 10(6) PFU/ml to obtain a positive reaction. All IHNV isolates, representing the five electropherotypes taken from a wide variety of species and different geographic ranges, caused coagglutination of Staphylococcus aureus cells sensitized with rabbit polyclonal serum against the Round Butte IHNV isolate. The coagglutination reaction was blocked by preincubation of IHNV with homologous antiserum, and IHNV did not cause coagglutination of S. aureus cells sensitized with normal rabbit serum. In specificity tests, cells sensitized with rabbit anti-IHNV serum or normal serum did not coagglutinate in the presence of infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, cell culture medium components, or media from cultures of cell lines of salmonid and nonsalmonid origin. Most importantly, the coagglutination test was able to detect and identify IHNV directly from experimentally infected rainbow trout fry, the organs of naturally infected adult kokanee salmon and winter steelhead trout, and ovarian fluids of the winter steelhead trout. The coagglutination test is very suitable for field use, since it is inexpensive, simple to interpret, sensitive, and rapid and requires no specialized equipment.     

Sequence analysis of OmNramp alpha and quantitative expression of Nramp homologues in different trout strains after infection with Myxobolus cerebralis

Salmonid whirling disease caused by the metazoan parasite Myxobolus cerebralis is an ongoing problem in wild and farmed rainbow trout Oncorhynchus mykiss populations. Rainbow trout from different strains vary in susceptibility to the parasite. Identification of underlying mechanisms could be a starting point for improved control of the disease. We conducted infection trials using 2 rainbow trout strains and brown trout Salmo trutta fario, a species not susceptible to the parasite, to investigate host immune response and resistance mechanisms. We compared expression levels of 2 natural resistance-associated macrophage proteins (Nramp alpha and beta) after infection with M. cerebralis. Total RNA was extracted from skin, muscle, kidney, head and spinal column, and gene expression was quantified by real-time PCR. Significant decreases in expression of both genes were observed at different time points in the infected susceptible rainbow trout compared to the non-infected group. Furthermore, the OmNramp alpha (O. mykiss natural resistance-associated macrophage protein alpha) sequences in 2 resistant and 1 non-resistant rainbow trout strain were analysed and compared for sequence aberrations.