Invasion and initial replication of ultraviolet irradiated waterborne infective stages of Myxobolus cerebralis results in immunity to whirling disease in rainbow trout.

Myxobolus cerebralis is a microscopic metazoan parasite (Phylum Myxozoa: Myxosporea) associated with salmonid whirling disease. There are currently no vaccines to minimise the serious negative economical and ecological impacts of whirling disease among populations of salmonid fish worldwide. UV irradiation has been shown to effectively inactivate the waterborne infective stages or triactinomyxons of M. cerbralis in experimental and hatchery settings but the mechanisms by which the parasite is compromised are unknown. Treatments of triactinomyxons with UV irradiation at doses from 10 to 80 mJ/cm(2) either prevented (20-80 mJ/cm(2)) or significantly inhibited (10 mJ/cm(2)) completion of the parasite life cycle in experimentally exposed juvenile rainbow trout (Oncorhynchus mykiss). However, even the highest doses of UV irradiation examined (80 mJ/cm(2)) did not prevent key steps in the initiation of parasite infection, including attachment and penetration of the epidermis of juvenile rainbow trout as demonstrated by scanning electron and light microscopy. Furthermore, replication of UV-treated parasites within the first 24h following invasion of the caudal fin was suggested by the detection of concentrations of parasite DNA by quantitative PCR comparable to that among fish exposed to an equal concentration of untreated triactinomyxons. Subsequent development of parasites treated with an 80 mJ/cm(2) dose of UV irradiation however, was impaired as demonstrated by the decline and then lack of detection of parasite DNA; a trend beginning at 10 days and continuing thereafter until the end of the study at 46 days post parasite exposure. Treatments of triactinomyxons with a lower dose of UV irradiation (20 mJ/cm(2)) resulted in a more prolonged survival with parasite DNA detected, although at very low concentrations, in fish up to 49 days post parasite exposure. The successful invasion but only short-term survival of parasites treated with UV in rainbow trout resulted in a protective response to challenges with fully infective triactinomyxons. Prior treatments of juvenile rainbow trout with UV-treated triactinomyxons (10 and 20 mJ/cm(2)) resulted in a reduced prevalence of infection and significantly lower concentrations of cranial myxospores (two direct measures of the severity of whirling disease) compared with trout receiving no prior treatments when assessed 5 months post parasite exposure to fully infective triactinomyxons.

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.

Effects of temperature on infectivity and of commercial freezing on survival of the North American strain of viral hemorrhagic septicemia virus (VHSV).

Temperature affected the growth of the North American strain of viral hemorrhagic septicemia virus (VHSV) in experimentally infected cell cultures and in Pacific sardine Sardinops sagax. In addition, commercial freezing significantly reduced the infectivity of VHSV in tissues of experimentally infected sardine. Isolates of VHSV representing the geographic range of North American VHSV replicated in the EPC (Epithelioma papulosum cyprini) cell line at 10, 15 and 20 degrees C, but the more northern isolates from British Columbia, Canada, demonstrated significantly reduced growth at 20 degrees C compared to VHSV from more southern locations (p <0.001). An injection challenge of Pacific sardine with VHSV from California resulted in 66.7% mortality at a seawater temperature of 13 degrees C compared to 6.7% at 20 degrees C. Commercial blast-freezing of sardine experimentally infected with VHSV reduced median concentrations of virus in the kidney and spleen from 5.25 x 10(6) to 5.5 x 10(3) pfu (plaque-forming units) g(-1). Decreased growth of the California isolate of VHSV at higher temperatures following experimental infection of the sardine and reduced virus survival following commercial freezing of infected sardine are factors that would lessen the risk of transmission of VHSV through frozen baitfishes.

Evaluation of quantitative real-time PCR for rapid assessments of the exposure of sentinel fish to Myxobolus cerebralis.

Pathogen-free rainbow trout (Oncorhynchus mykiss) aged 735 degree days were experimentally exposed to a low dose of infectious Myxobolus cerebralis (20 triactinomyxons fish(-1)). Three time periods were chosen for sampling that included 10 days (d), 67 d, and 5 months (mo) post exposure. Five diagnostic assays were used: (1) conventional single-round polymerase chain reaction (PCR), (2) nested PCR, (3) real-time TaqMan PCR, (4) pepsin-trypsin digest, and (5) histopathology. M. cerebralis was detected among individual rainbow trout by all of the PCR diagnostic tests employed at each of the three sampling time points. This result demonstrates that any of these three diagnostic approaches are capable of detecting the parasite from infected fish tissues under the conditions tested. Real-time PCR provided good biological evidence that parasite replication increases temporally as shown by quantification values that were significantly different (P<0.0001) at 10 d as compared to 67 d and 5 mo postexposure. Although sampling at 10 d by real-time PCR may be too early to accurately predict quantities of the parasite that will be present at 5 mo, it does forecast the proportions of fish that are likely to be infected at 67 d and 5 mo postparasite exposure. Real-time PCR could potentially be used as a quantitative diagnostic PCR tool to predict parasite load and outcome of M. cerebralis infection.

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.

Susceptibility of two strains of rainbow trout (one with suspected resistance to whirling disease) to Myxobolus cerebralis infection.

The susceptibility of 2 strains of rainbow trout Oncorhynchus mykiss, 1 from North America (TL) and 1 from Germany (GR), to Myxobolus cerebralis (the cause of salmonid whirling disease) was assessed following exposure to the infectious stages (triactinomyxons). Two laboratory experiments were conducted with age-matched rainbow trout of each strain. At the beginning of the study, the 2 trout strains were aged ca. 570 degree-days in Expt 1, and ca. 999 degree-days in Expt 2. In both experiments, replicate groups of each trout strain were exposed to 10, 100, 1000 or 10000 triactinomyxons (TAMs) fish(-1) for 2 h. The fish were then held in aquaria receiving 15 degrees C well-water. Severity of infection was evaluated 5 mo after exposure by presence of clinical signs (whirling and/or black tail), prevalence of infection, severity of microscopic lesions, and spore counts. Clinical signs of whirling disease were evident only in the younger fish exposed in Expt 1: These occurred first among TL rainbow trout at the highest dose at 6 to 7 wk post exposure and then 2 wk later in fish at the 1000 TAMs dose. Black tail was also observed among GR rainbow trout at the 10000 TAMs dose only, but in fewer fish. The prevalence of infection, spore numbers, and severity of microscopic lesions due to M. cerebralis among GR rainbow trout were less at all doses compared to TL rainbow trout. Risk of infection analyses showed that TL rainbow trout were more prone to infection at the lower doses than GR trout. Mean spore counts were consistently (10- to 100-fold) less in GR than TL trout at doses of 1000 TAMs or lower. Microscopic lesions increased with increasing dose in both strains of rainbow trout. The mechanisms underlying the greater resistance of the GR strain to M. cerebralis infections are unknown, but are under investigation as part of a long-term project to determine the basis for resistance and susceptibility of salmonid fishes to whirling disease.