Comparative Susceptibility of Deschutes River,Oregon, Tubifex tubifex Populations to Myxobolus cerebralis

Abstract

Dams along the Deschutes River (DR) in central Oregon have blocked fish migration for over 40 years. Reestablishment of anadromous fish runs above the dams as part of a fish passage plan may introduce fish pathogens, such as Myxobolus cerebralis, the myxozoan parasite that causes salmonid whirling disease. This parasite is carried by adult salmon that stray into the DR system during their return to enzootic areas of the upper Columbia River basin, and it is now known to be established in at least one lower DR tributary. The life cycle of M. cerebralis involves two obligate hosts: a salmonid and the oligochaete worm Tubifex tubifex. To determine the likelihood of parasite establishment above the DR dams, we conducted benthic sediment surveys between 1999 and 2007 and found that T. tubifex had a patchy distribution and low relative abundance. Mitochondrial 16S ribosomal DNA gene analysis indicated that two lineages of T. tubifex (III and VI) were present both above and below the dams. Laboratory susceptibility studies to characterize differences in infection prevalence and parasite production between nine T. tubifex populations revealed that production varied considerably among exposed groups and was proportional to the number of lineage III worms present. Our results suggest that M. cerebralis could become established above the dams if infected fish are allowed passage into the upper DR system, but not all areas of the DR basin can be classified as having the same likelihood for parasite establishment, and the potential impact will be location dependent.

Received July 2, 2010; accepted October 24, 2010     

Potential for dissemination of the nonnative salmonid parasite Myxobolus cerebralis in Alaska

Myxobolus cerebralis, the myxozoan parasite responsible for whirling disease in salmonids, was first introduced into the United States in 1958 and has since spread across the country, causing severe declines in wild trout populations in the intermountain western United States. The recent detection of the parasite in Alaska is further evidence of the species' capability to invade and colonize new habitat. This study qualitatively assesses the risk of further spread and establishment of M. cerebralis in Alaska. We examine four potential routes of dissemination: human movement of fish, natural dispersal by salmonid predators and straying salmon, recreational activities, and commercial seafood processing. Potential for establishment was evaluated by examining water temperatures, spatial and temporal overlap of hosts, and the distribution and genetic composition of the oligochaete host, Tubifex tubifex. The most likely pathway of M. cerebralis transport in Alaska is human movement of fish by stocking. The extent of M. cerebralis infection in Alaskan salmonid populations is unknown, but if the parasite becomes dispersed, conditions are appropriate for establishment and propagation of the parasite life cycle in areas of south-central Alaska. The probability of further establishment is greatest in Ship Creek, where the abundance of susceptible T. tubifex, the presence of susceptible rainbow trout Oncorhynchus mykiss, and the proximity of this system to the known area of infection make conditions particularly suitable for spread of the parasite.     

Predicted Redistribution of Ceratomyxa shasta Genotypes with Salmonid Passage in the Deschutes River,Oregon

Abstract

A series of dams on the Deschutes River, Oregon, act as migration barriers that segregate the river system into upper and lower basins. Proposed fish passage between basins would reunite populations of native potamodromous fish and allow anadromous fish of Deschutes River origin access to the upper basin. We assessed the potential redistribution of host-species-specific genotypes (O, I, II, III) of the myxozoan parasite Ceratomyxa shasta that could occur with fish passage and examined the influence of nonnative fish on genotype composition. To determine the present distribution of the parasite genotypes, we exposed eight salmonid species—three native and five stocked for sport fishing—in present and predicted anadromous salmonid habitats. We monitored fish for infection by C. shasta and sequenced a section of the parasite ribosomal DNA gene from fish and water samples to determine parasite genotype. Genotype O was present in both upper and lower basins and detected only in steelhead Oncorhynchus mykiss. Genotype I was spatially limited to the lower basin, isolated predominately from Chinook salmon O. tshawytscha, and lethal for this species only. Genotype II was detected in both basins and in multiple species, but only as a minor component of the infection. Genotype III was also present in both basins, had a wide host range, and caused mortality in native steelhead and multiple nonnative species. Atlantic salmon Salmo salar and kokanee O. nerka were the least susceptible to infection by any genotype of C. shasta. Our findings confirmed the host-specific patterns of C. shasta infections and indicated that passage of Chinook salmon would probably spread genotype I into the upper Deschutes River basin, but with little risk to native salmonid populations.

Received April 20, 2012; accepted July 19, 2012     

Effects of freezing, drying, ultraviolet irradiation, chlorine, and quaternary ammonium treatments on the infectivity of myxospores of Myxobolus cerebralis for Tubifex tubifex

The effects of freezing, drying, ultraviolet irradiation (UV), chlorine, and a quaternary ammonium compound on the infectivity of the myxospore stage of Myxobolus cerebralis (the causative agent of whirling disease) for Tubifex tubifex were examined in a series of laboratory trials. Freezing at either -20 degrees C or -80 degrees C for a period of 7 d or 2 months eliminated infectivity as assessed by the absence of production of the actinospore stage (triactinomyxons [TAMs]) from T. tubifex cultures inoculated with treated myxospores over a 4-5-month period. Myxospores retained infectivity when held in well water at 5 degrees C or 22 degrees C for 7 d and when held at 4 degrees C or 10 degrees C d for 2 months. In contrast, no TAMs were produced from T. tubifex cultures inoculated with myxospores held at 20 degrees C for 2 months. Drying of myxospores eliminated any evidence of infectivity for T. tubifex. Doses of UV from 40 to 480 mJ/cm2 were all effective for inactivating myxospores of M. cerebralis, although a few TAMs were detected in one replicate T. tubifex culture at 240 mJ/cm2 and in one replicate culture at 480 mJ/cm2. Treatments of myxospores with chlorine bleach at active concentrations of at least 500 mg/L for 15 min largely inactivated myxospore infectivity for T. tubifex. Likewise, there was no evidence of TAMs produced by T. tubifex inoculated with myxospores treated with alkyl dimethyl benzyl ammonium chloride (ADBAC) at 1,500 mg/L for 10 min. Treatments of myxospores with 1,000-mg/L ADBAC for 10 min reduced TAM production in T. tubifex cultures sevenfold relative to that in cultures inoculated with an equal number of untreated myxospores. These results indicate that myxospores of M. cerebralis demonstrate a selective rather than broad resistance to selected physical and chemical treatments, and this selective resistance is consistent with conditions that myxospores are likely to experience in nature.     

Distribution of Myxobolus cerebralis in Salmonid Fishes in Montana

Abstract

Over an approximately 2-year period, 20,974 fish (trout and other salmonid species) from 230 separate waters (creeks, rivers, lakes, reservoirs, ponds, hatcheries, and irrigation ditches) within 21 of the 22 major drainages in Montana were examined for Myxobolus cerebralis. Nine of the major river drainages have waters containing infected fish: Beaverhead, Big Hole, Blackfoot, Clark Fork above the Bitterroot River, Flathead below the south fork of the Flathead River, Jefferson, Madison, Missouri above the Marias River, and Yellowstone above the Bighorn River. The Beaverhead, Clark Fork above the Bitterroot River, Jefferson, Madison, and Missouri above the Marias River have the greatest number of waters containing fish infected with M. cerebralis. Comparisons of infection levels (number of pooled samples that contain fish infected with M. cerebralis) between species among these drainages show significantly lower levels of infection in brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss in the Missouri above the Marias River drainage and significantly higher levels of infection of rainbow trout in the Jefferson. Comparisons of differences in infection levels between drainages among species show that, in the Beaverhead, Clark Fork above the Bitterroot River, and Madison, infection levels in brown trout are significantly higher than in rainbow trout. This is partially attributed to losses of juvenile rainbow trout because of M. cerebralis infection, leading to biased samples. Histopathologic studies showed lesions were consistently less severe in brown trout than other species and occurred in a different location (gill arches versus ventral calvarium). In six of the nine affected drainages (Beaverhead, Blackfoot, Clark Fork above the Bitterroot River, Flathead below the South Fork, Jefferson, and Madison), infected fish were found at or near the time that intensive sampling was initiated in the spring of 1995. In the three remaining affected drainages (Missouri above the Marias River, Yellowstone above the Bighorn River, and the Big Hole), infected fish were not identified until at least 15 months after the initiation of widespread testing. This indicates that in the first six drainages listed above, the infection was well established prior to 1995 but spread into the last three drainages in the ensuing months. Methods of transmission and the sources of infection are unknown, although the absence of infected fish in state, private, and federal hatcheries in Montana indicates hatchery fish from these sources are not likely to be responsible.     

Evaluation of five diagnostic methods for the detection and quantification of Myxobolus cerebralis.

Diagnostic methods were used to identify and quantify Myxobolus cerebralis, a myxozoan parasite of salmonid fish. In this study, 7-week-old, pathogen-free rainbow trout (Oncorhynchus mykiss) were experimentally infected with M. cerebralis and at 7 months postinfection were evaluated with 5 diagnostic assays: 1) pepsin-trypsin digest (PTD) to detect and enumerate spores found in cranial cartilage, 2) 2 different histopathology grading scales that provide a numerical score for severity of microscopic lesions in the head, 3) a conventional single-round polymerase chain reaction (PCR), 4) a nested PCR assay, and 5) a newly developed quantitative real-time TaqMan PCR. There were no significant differences (P > 0.05) among the 5 diagnostic assays in distinguishing between experimentally infected and uninfected control fish. The 2 histopathology grading scales were highly correlated (P < 0.001) for assessment of microscopic lesion severity. Quantification of parasite levels in cranial tissues using PTD and real-time TaqMan PCR was significantly correlated r = 0.540 (P < 0.001). Lastly, 104 copies of the 18S rDNA gene are present in the M. cerebralis genome, a feature that makes this gene an excellent target for PCR-based diagnostic assays. Also, 2 copies of the insulin growth factor-I gene are found in the rainbow trout genome, whose detection can serve both as an internal quality control for amplifiable DNA and as a basis to quantify pathogen genome equivalents present in quantitative PCR assays.     

Health status of salmonids in river systems in Natal. I. Collection of fish and parasitological examination

A detailed microbiological and parasitological survey of salmonids and indigenous fish in the upper reaches of selected river systems in Natal was undertaken. Fish were collected from the rivers by electro-fishing, from dams by gill netting and from hatcheries along the rivers. A total of 678 fish from 26 different sites along 8 river systems were collected. No parasites could be detected on fish from four of the rivers tested. Parasites were, however, detected on fish from large production sites in Natal and in the rivers feeding these sites. Parasites which were detected were Ichthyopthirius multifiliis, various Trichodina spp., Apiosoma sp. and Gyrodactylus sp. The heads of all fish estimated to be less than one year of age were collected and examined for the spores of Myxosoma cerebralis. All of the samples were found to be free of this parasite.     

The effects of Myxobolus cerebralis on the physiological performance of whirling disease resistant and susceptible strains of rainbow trout

The development of rainbow trout Oncorhynchus mykiss strains that are resistant to whirling disease has shown promise as a management tool for populations in areas where Myxobolus cerebralis is present. However, the physiological effects of the disease on characteristics necessary for fish survival in natural river conditions have not been tested in many of these strains. Five rainbow trout strains were evaluated for their swimming ability and growth characteristics in relation to M. cerebralis exposure: the resistant German rainbow trout (GR) strain (Hofer strain), the susceptible Colorado River rainbow trout (CRR) strain, and three intermediate (hybrid) strains (F1 = GR x CRR; F2 = F1 x F1; B2 = backcross of F1 x CRR). Three broad response patterns among strain and exposure were evident in our study. First, exposure metrics, growth performance, and swimming ability differed among strains. Second, exposure to the parasite did not necessarily produce differences in growth or swimming ability. Exposure to M. cerebralis did not affect batch weight for any strain, and critical swimming velocity did not differ between exposed and unexposed families. Third, although exposure did not necessarily affect growth or swimming ability, individuals that exhibited clinical deformities did show reduced growth and swimming performance; fish with clinical deformities were significantly smaller and had lower critical swimming velocities than exposed fish without clinical deformities. Research and management have focused on GR x CRR hybrid strains; however, given the performance of the GR strain in our study, it should not be discounted as a potential broodstock. Additional field trials comparing the GR and F1 strains should be conducted before wholesale adoption of the GR strain to reestablish rainbow trout populations in Colorado.     

Detection of early stages of Myxobolus cerebralis in fin clips from rainbow trout (Orynchus mykiss).

A nested polymerase chain reaction (PCR) assay was used to detect early stages of Myxobolus cerebralis in caudal and adipose fin samples from rainbow trout (RT). To determine sensitivity, groups of 10 RT were exposed to 2,000 M. cerebralis triactinomyxons/fish for 1 hour at 15 degrees C and subsequently moved to clean recirculating water. Fish were held for 2 and 6 hours and 1, 2, 3, 5, 7, 10, 30, and 60 days before sampling by nonlethal fin biopsy. Nested PCR performed on fin clips showed that M. cerebralis DNA was detected in caudal fin tissue in 100% of fish up to 5 days postexposure. At days 7 and 10 postexposure, 80% of fish were positive, and at 60 days postexposure, 60% of fish were positive using this technique. Conversely, testing on adipose fin clips proved less sensitive, as positive fish dropped from 80% at day 7 to below 20% at day 10 postinfection. Since detection of M. cerebralis infection using caudal fin samples coupled with nested PCR is an effective method for detection of early parasite stages, use of this technique provides for accurate, nonlethal testing.     

Disease Susceptibility of Hatchery Snake River Spring–Summer Chinook Salmon with Different Juvenile Migration Histories in the Columbia River

Abstract

Various methods have been developed to mitigate the effects of dams on juvenile Pacific salmon Oncorhynchus spp. migrating to the Pacific Ocean through the Columbia River basin. In this study, we examined the health of hatchery Snake River spring and summer Chinook salmon relative to two mitigating strategies: dam bypass and transportation (e.g., barging). The health of out-migrants was assessed in terms of the difference in the incidence of mortality among fish, categorically grouped into no-bypass, bypass, and transportation life histories, in response to challenge with the marine pathogen Listonella anguillarum during seawater holding. These three life histories were defined as follows: (1) fish that were not detected at any of the juvenile bypass systems above Bonneville Dam were classified as having a no-bypass life history; (2) fish that were detected at one or more juvenile bypass systems above Bonneville Dam were classified as having a bypass life history; and (3) fish that were barged were classified as having the transportation life history. Barged fish were found to be less susceptible to L. anguillarum than in-river fish—whether bypassed or not—which suggests that transportation may help mitigate the adverse health effects of the hydropower system of the Columbia River basin on Snake River spring–summer Chinook salmon. The findings of this study are not necessarily transferable to other out-migrant stocks in the Columbia River basin, given that only one evolutionarily significant unit, that is, Snake River spring–summer Chinook salmon, was used in this study.     

Use of site occupancy models to estimate prevalence of Myxobolus cerebralis infection in trout

Empirical estimates of pathogen prevalence in samples of fish may underestimate true prevalence because available detection techniques are incapable of perfect detection. Trout of several species were collected from enzootic (Myxobolus cerebralis, causative agent in whirling disease) habitats, and individual fish were examined for presence of the parasite two or six times by one of four methods: pepsin-trypsin digest (brown trout Salmo trutta), plankton centrifuge (brown trout), polymerase chain reaction (rainbow trout Oncorhynchus mykiss), or histopathology (brook trout Salvelinus fontinalis). The presence-absence data were modeled for prevalence of infection (psi) and probability of detection (p) of the parasite via occupancy models that accounted for imperfect detection of the organism. Based on estimates from the most-supported model for comparison, two myxospore concentration methods underestimated prevalence by about 12% for whole-head results and 34% for the expected value of half-head analysis. Polymerase chain reaction and histopathology gave virtually the same prevalence estimates for whole-head results as the best models but underestimated prevalence by about 6% and 12%, respectively, for the expected value of half-head analysis. The probability of detecting the parasite in a single survey of a fish head, conditional on the parasite's presence, was 0.66 for myxospore concentration methods, 0.81 for histopathology, and 1.0 (left halves) or 0.89 (right halves) for polymerase chain reaction. The occupancy models used in this study may be extended to large-scale monitoring of M. cerebralis to estimate expansion or contraction of the parasite's range over time.     

Detection of Myxobolus cerebralis in Rainbow Trout and Oligochaete Tissues by Using a Nonradioactive in Situ Hybridization (ISH) Protocol

Abstract

A nonradioactive in situ hybridization (ISH) protocol was developed to detect Myxobolus cerebralis, the causative organism of whirling disease, in its primary host, rainbow trout Oncorhynchus mykiss, and in its alternate oligochaete host, Tubifex tubifex. A cocktail of three oligonucleotide primers (derived from the small subunit ribosomal DNA sequence) directed at target sequences of the parasite DNA was tailed at the 3′ end with digoxigenin-labeled deoxyuridine triphosphate (DIG-dUTP). Labeled probes were hybridized to parasite DNA present in deparaffinized tissue sections from infected trout and oligochaetes. The bound probes were visualized after modifications of existing ISH protocols. By using the new ISH procedure, the parasite was found in target tissues of subclinically and clinically infected fish and tubificid oligochaetes after exposures of these hosts to triactinomyxons and mature spores, respectively. The probe did not bind with salmonid tissues infected with two other myxosporean parasites, Ceratomyxa shasta or the PKX organism, or to a Myxobolus sp. infecting the cartilage of plain sculpin Myoxocephalus jaok. These initial results indicate that ISH is an effective and specific test for detecting Myxobolus cerebralis in its fish and oligochaete hosts.     

Evaluation of nested polymerase chain reaction, pepsin/trypsin digest, and histology for the detection of Myxobolus cerebralis in salmonid fishes of Indiana and Michigan.

The objectives of this study were to survey fish from state hatcheries in Indiana and Michigan and to compare the nested polymerase chain reaction (PCR) test with pepsin/trypsin digest (PTD) and histopathology for the diagnosis of whirling disease (WD). One group of 40 and 9 groups of 60 fish heads, for a total of 580 samples, were submitted from hatcheries in Indiana and Michigan. These samples were examined for myxozoan spores using histopathology, PTD, and PCR tests. The heads were hemisectioned, and one half was fixed in 10% neutral-buffered formalin for histopathologic examination. The other half was processed for PTD. Some of the sediment was examined for the presence of myxozoan spores, and the rest was prepared for the nested PCR. Histologic examinations did not reveal Myxobolus cerebralis in any of the 580 samples. One hundred serial step sections, taken at 5-microm intervals, were evaluated for samples with positive spore identification by PTD. Histologic examination of these sections failed to reveal any myxozoan parasites. Myxozoan spores were observed in 16.9% (98/580) of samples in sediment after PTD. Spores morphologically similar to those of M. cerebralis were observed in 1.0% of PTD samples (n = 6). The nested PCR indicated that M. cerebralis spores were present in 0.5% of samples (n = 3). All 3 nested PCR-positive samples came from the same hatchery, however, spores of M. cerebralis were seen in 1 sample, spores of other myxozoan species were seen in the second sample, and spores were not seen in the third sample. When comparing the PTD to the nested PCR test, the PTD diagnosed 1 true positive, 5 false positives, 2 false negatives, and 572 true negatives, for a sensitivity of 33% and a specificity of 99.1%. Screening for M. cerebrallis infection in this study indicated a low prevalence of the disease. Histopathology was a very insensitive indicator of WD. The PCR test was highly specific and was used to differentiate spores of M. cerebralis from similar spores of other species.     

Myxobolus cerebralis infection in rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) exposed under natural stream conditions.

From early April into mid-June 1977, sequential groups of juvenile rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) were each exposed for 10 days to the parasite Myxobolus cerebralis by immersion in a stream inhabited by infected wild trout. Following incubation in a M. cerebralis-free facility, trout were subsequently killed, and heads and gill arches were examined by routine histologic methods. A grading scale to quantify lesion severity was developed and applied. Percentage infected, lesion severity scores, effects of water temperature and flow rates on percentage infected and lesion severity scores, and resulting pathology were determined for each species at each exposure period. The percentage of rainbow trout infected with M. cerebralis was significantly higher than the percentage of brown trout infected for each exposure period. The percentages of rainbow trout infected in exposure periods later in the calendar year were significantly higher than those in earlier periods. The percentages of brown trout infected were not significantly different among exposure periods. Overall average lesion severity scores were significantly higher in rainbow than in brown trout. Lesion severity scores in rainbow trout increased over time (a positive correlation with exposure period). Lesion severity scores were not significantly different for brown trout among exposure periods. A significant correlation existed between water temperature and percentage of rainbow trout infected; a significant correlation also existed between water temperature and lesion severity scores in rainbow trout. Similar correlations did not exist for percentage of brown trout infected or accompanying lesion severity scores. In rainbow trout, ventral calvarium was the most common site of M. cerebralis replication, followed by gill arches. In brown trout, lesions were virtually confined to gill arches. Early lesions consisted of foci of cartilage necrosis with small numbers of M. cerebralis developmental stages. More advanced lesions consisted of multifocal areas of cartilage necrosis with numerous M. cerebralis developmental stages and/or mature myxospores bordered and/or infiltrated by mono- and multinuclear leukocytes. Lesions in brown trout were smaller and had fewer associated leukocytes and M. cerebralis developmental stages and/or mature myxospores. Higher infection rates, lesion severity scores, and differences in lesion location in rainbow versus brown trout explain in part why numbers of rainbow but not brown trout have fallen in western rivers inhabited with M. cerebralis-infected trout.     

Detection of Myxobolus (Myxosoma) cerebralis in Salmonid Fishes in Oregon

Abstract

Myxobolus (Myxosoma) cerebralis, the etiological agent of whirling disease, was detected in salmonid fish populations in northeastern Oregon. This is the first record of M. cerebralis in the Pacific Northwest of the USA. During an epizootiological survey for the parasite, two methods for spore detection were compared, and an efficient procedure for determining M. cerebralis infection in adult fish was developed. The enzyme digest method was more efficient than the plankton centrifuge procedure for examination of numerous individual lots of fish processed during the survey. Sampling only the area around the otoliths was at least as effective as sampling entire heads for detection of spores in infected fish.     

Distribution of Tubifex tubifex Lineages and Myxobolus cerebralis Infection in the Tailwater of the San Juan River,New Mexico

Abstract

We chose a hypolimnetic-release tailwater of the San Juan River, New Mexico, to characterize the etiology of whirling disease, a parasitic infection of salmonids. We sampled a 2-km reach of the tailwater in August and December 2001 and June 2002 to characterize environmental factors influencing the distribution and density of Tubifex tubifex lineages and Myxobolus cerebralis infection rates. Shortly after the scouring flow, organic matter in sediments and T. tubifex densities increased within deep habitats. In contrast, no differences were observed in T. tubifex densities and organic matter collected from shallow habitats throughout the three sampling dates. Within this study area, we found three sympatric lineages of T. tubifex (lineages I, III, and VI). Lineage VI dominated riffle reaches, whereas lineages I, III, and VI were observed in pool habitats. Myxobolus cerebralis infection rates were higher in T. tubifex collected in pool habitats (3.01%) than in those collected in riffle habitats (0.51%). Only lineage III exhibited infection with M. cerebralis. We suggest that the habitat and genotype of T. tubifex are important in characterizing prevalence of disease within the San Juan River tailwater. Scouring flow may have a beneficial effect on disease severity in salmonid hosts by reducing organic loading and hence T. tubifex abundance in deep habitats.     

Development of Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) in bryozoan hosts (as examined by light microscopy) and quantitation of infective dose to rainbow trout (Oncorhynchus mykiss)

The myxozoan parasite Tetracapsuloides bryosalmonae is the causative agent of proliferative kidney disease (PKD), a highly damaging disease of cultured salmonid fish. Within this study, phylactolaemate bryozoans were collected from a river known to be endemic for PKD and subsequently cultured in the laboratory. Sequential developmental stages of T. bryosalmonae were studied by light microscopy within the living bryozoan colonies, allowing the identification of stages attached to host peritoneum, consistent with previous molecular evidence of cryptic stages. Infection resulted in the production of large numbers of spores, which were released from the bryozoans. Experimental exposure of rainbow trout (Oncorhynchus mykiss) to medium in which infected bryozoans were cultured resulted in clinical PKD. Rainbow trout were exposed to known numbers of T. bryosalmonae spores collected by micromanipulation, which had been released from mature spore sacs within colonies of the bryozoan Fredericella sultana. Exposure to one spore was sufficient to lead to development of PKD. These findings indicate that small numbers of bryozoans are capable of releasing sufficient spores to infect large numbers of fish, having implications for future control methods for PKD in salmonid farming.     

Transformation of Theileria parva derived from African buffalo (Syncerus caffer) by tick passage in cattle and its use in infection and treatment immunization.

A sporozoite stabilate (St. 199) of Theileria parva was obtained by feeding nymphal Rhipicephalus appendiculatus on an African buffalo (Syncerus caffer) and was used to immunize cattle by the infection and treatment method. Nymphal ticks were applied to one of the steers 90 days later and it was shown that the resultant adult tick had become infected. Using tick/cattle passage, two passage lines of T. parva were established. By the fifth tick/cattle passage, the parasite stocks had changed their behaviour to that of T. parva derived from cattle as the parasite produced relatively high schizont parasitosis and piroplasm parasitaemia in cattle, and had become highly infective to ticks. At various passage levels the parasite populations were characterized by behaviour and by monoclonal antibodies against T. parva schizonts using infected cell culture isolates from cattle during acute infections. The monoclonal antibody profile showed little evidence of antigen change of the parasite during passage through cattle, which was confirmed in a two-way cross-immunity experiment using sporozoite stabilate derived from ticks obtained from the buffalo and fourth passage in cattle. The implication of these results, particularly in relationship to immunization of cattle against T. parva derived from buffalo, is discussed.     

Comparison of hatchery and field performance between a whirling-disease-resistant strain and the Ten Sleep strain of rainbow trout

A whirling-disease-resistant strain of rainbow trout Oncorhynchus mykiss (GRHL strain) derived from a backcross of an F1 hybrid of two strains (German strain x Harrison Lake strain) with German strain females, was compared with the Ten Sleep (TS) strain of rainbow trout. The GRHL strain had consistently superior growth and feed conversion in two consecutive hatchery trials. Hatching and mortality rates were similar between strains. Both strains were stocked into two Utah reservoirs (Hyrum, Porcupine), and a third, Causey Reservoir, was monitored as a control for seasonal variation in prevalence of Myxobolus cerebralis. A total of 1,323 salmonids captured by gill net in spring and fall sampling between 2006 and 2008 were tested for M. cerebralis via pepsin-trypsin digest methods. Only eight of these (< 1% per species) had clinical signs consistent with whirling disease. In both reservoirs, GRHL survived better than the TS and had higher growth rates. The prevalence of M. cerebralis was significantly lower for GRHL (18.1%) than TS (50.0%) in Porcupine Reservoir. In Hyrum Reservoir the trend was similar, but prevalence was lower and did not significantly differ between GRHL (9.6%) and TS (23.1%). For infected fish, no significant differences were observed between strains in myxospore counts in either Hyrum (GRHL = 911-28,244 spores/fish [spf], TS = 1,822-155,800 spf) or Porcupine (GRHL = 333-426,667spf, TS = 333-230,511 spf) reservoirs. Unmarked rainbow trout in both reservoirs had significantly higher myxospore counts than stocked fish of either strain. There were significant differences in M. cerebralis prevalence and myxospore loads among other naturally reproducing salmonids in the reservoirs. The trend in susceptibility was cutthroat trout Oncorhynchus clarkii > kokanee Oncorhynchus nerka > brown trout Salmo trutta. The GRHL performed well in both hatchery and field settings and is recommended for stocking programs.     

Significance of two different ranking methods for studying fish fauna: a case study on the fish fauna from upper and middle portions from Mekong River

Studies on fish fauna should not only focus on fish composition and the comparison of fish composition among the study region and adjacent regions, but should also explore the origin and uniqueness of different taxa as well as the substitution of genera and species. In this study, the value of floristic presence method was modified and renamed the value of fish fauna presence (VFFP) method. The specific steps of the VFFP method and the traditional fish fauna analysis (TFFA) method were refined and standardized. Then, the VFFP and TFFA methods were applied to study the fish fauna of the upper and middle portions of the Mekong River basin. The results indicate that the TFFA method reflects the families (subfamilies) and genera that constitute the main body of fish in the studied river basin. The results of the VFFP method show which families (subfamilies) and genera are representative for the basin. Therefore, combining the TFFA and VFFP methods to analyze the composition of fish fauna can reflect the characteristics of fish fauna from different perspectives. The case study shows that the fish fauna of the Mekong River is a part of the fish fauna of Southeast Asia. Although it shows some similarities to the composition of South Asian fish fauna, it does not belong to the South Asian fish fauna as a whole, and is essentially different from the East Asian fish fauna. This study provides an objective, quantitative, and verifiable method for studying fish fauna.