Terry S. McDowell

Effect of water temperature on the development, release and survival of the triactinomyxon stage of Myxobolus cerebralis in its oligochaete host.

The development of the triactinomyxon stage of Myxobolus cerebralis and release of mature spores from Tubifex tubifex were shown to be temperature dependent. In the present work, the effect of temperature over a range of 5-30 degrees C on the development and release of the triactinomyxon stages of M. cerebralis was studied. Infected T. tubifex stopped releasing triactinomyxon spores 4 days after transfer from 15 degrees C to 25 degrees C or 30 degrees C. Transmission electron microscopic examinations of the tubificids held at 25 degrees C and 30 degrees C for 3 days showed that all developmental stages degenerated and transformed to electron-dense clusters between the gut epithelial cells of T. tubifex. In contrast, tubificid worms held at 5 degrees C and 10 degrees C examined at the same time were heavily infected with many early developmental stages of triactinomyxon. At 15 degrees C, the optimal temperature for development, maturing and mature stages of the parasite were evident. Infected T. tubifex transferred from 15 degrees C to 20 degrees C stopped producing triactinomyxon spores after 15 days. However, 15 days at 20 degrees C was not sufficient to destroy all developmental stages of the parasite. When the tubificid worms were returned to 15 degrees C, the one-cell stages and the binucleate-cell stages resumed normal growth. It was also demonstrated that T. tubifex cured of infection by holding at 30 degrees C for 3 weeks and shifted to 15 degrees C could be re-infected with M. cerebralis spores. The waterborne triactinomyxon spores of M. cerebralis did not appear to be as short-lived as previously reported. More than 60% of experimentally produced waterborne triactinomyxon spores survived and maintained their infectivity for rainbow trout for 15 days at water temperatures up to 15 degrees C. In natural aquatic systems, the triactinomyxon spores may survive and keep their infectivity for periods even longer than 15 days.

Susceptibility of Selected Inland Salmonids to Experimentally Induced Infections with Myxobolus cerebralis, the Causative Agent of Whirling Disease

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 ...

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.

Two Cell Lines from White Sturgeon

Abstract Cell lines were established from spleen and heart tissues of a juvenile white sturgeon Acipenser transmontanus and designated WSS-2 and WSH-1, respectively. Isoenzyme analyses of both lines confirmed their host of origin. Both lines were hypotetraploid and possessed micro- and macrochromosomes. The modal chromosome number for the WSS-2 line was 219; the WSH-1 line had a bimodal distribution with modes of 237 and 243. The effects of temperature on growth and susceptibility to selected viral pathogens were examined for the WSS-2 line. Cell replication occurred at 15–30°C, optimum temperature was 25°C. Cell survival was observed after 11 d at 10°C but not at 35°C. The WSS-2line was refractory to infections with channel catfish virus (CCV) and infectious pancreatic necrosis virus (IPNV) but susceptible to infectious hematopoietic necrosis virus (IHNV) and white sturgeon iridovirus (WSIV). The need for cell lines from the host of origin in studies of viral pathogens of fish was demonstrated by the abi...

Susceptibility of Koi Carp, Common Carp, Goldfish, and Goldfish × Common Carp Hybrids to Cyprinid Herpesvirus-2 and Herpesvirus-3

Abstract Hybrid goldfish (male goldfish Carassius auratus × female common carp Cyprinus carpio) were completely resistant to mortality following experimental infections with cyprinid herpesvirus-2 (CyHV-2) and moderately resistant to cyprinid herpesvirus-3 (CyHV-3). In contrast, examination of the parental species demonstrated that goldfish were highly susceptible to CyHV-2 infections and that both common carp and koi (a variant of common carp) were highly susceptible to CyHV-3. Approximately 50% of the hybrids examined at 25 d after intraperitoneal injection with either CyHV-2 or CyHV-3 possessed viral genomic DNA, as detected by polymerase chain reaction assays. These initial trials suggest that hybrid goldfish have a natural resistance to mortality following experimental challenge with CyHV-2 and CyHV-3. However, additional studies that examine virus replication, including the potential for the development of a carrier state in hybrids, are needed. The production of hybrids for the food fish market may...

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 p...

Susceptibility of Three Species of Anadromous Salmonids to Experimentally Induced Infections with Myxobolus cerebralis, the Causative Agent of Whirling Disease

Abstract The susceptibility of three species of anadromous salmonids to whirling disease was examined after their experimental exposures to the infectious stages of Myxobolus cerebralis. Chinook salmon Oncorhynchus tshawytscha exposed as alevins were very susceptible to infection; the appearance of clinical signs, prevalence of infection, severity of microscopic lesions, and spore counts at 130 d postexposure are similar to those of age-matched rainbow trout O. mykiss exposed at the same dose. In contrast, coho salmon O. kisutch demonstrated no clinical signs of infection and had a lower prevalence of infection and spore numbers than did the exposed rainbow trout. A comparison of two strains of steelhead (anadromous rainbow trout), one from an enzootic site (San Lorenzo River) where M. cerebralis has been present for the past 35 years and a second from a site where the parasite is not found (Dry Creek), showed them both to be highly susceptible to experimental infections with M. cerebralis. These controll...

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.

Genetic Relationships among Herpes-Like Viruses Isolated from Sturgeon

Abstract We report the identification of partial DNA polymerase gene sequences of seven herpes-like viruses found in sturgeon Acipenser spp. from North America and Europe. Phenetic comparisons using nucleic acid and deduced protein alignments divided the sturgeon herpes-like viruses into three genogroups. The first genogroup includes the previously described Acipenserid herpesvirus 1 (AciHV-1) and two new isolates from farmed white sturgeon A. transmontanus from California and wild white sturgeon from Idaho. The second genogroup contains the previously described Acipenserid herpesvirus 2 (AciHV-2) and a new isolate from wild white sturgeon found in Oregon. The third genogroup includes two viruses with identical amino acid sequences found in farmed white sturgeon from Italy and Canada. We propose to name the third sturgeon herpes-like genogroup Acipenserid herpesvirus 3 (AciHV-3). The phylogram used for comparing the relationships of these viruses shows strong bootstrap support (82%) for two separate clade...

Detection of a Nonoccluded Baculovirus in the Freshwater Crayfish Cherax quadricarinatus in North America

Abstract A nonoccluded baculovirus was detected during a routine health examination of the tropical freshwater blue crayfish Cherax quadricarinatus reared in a farm in California, USA. The crayfish were progeny of captive-reared adults imported from Australia. There were no external or internal signs of disease in infected crayfish, but histological examination revealed eosinophilic to amphophilic intranuclear inclusions within the tubular epithelial cells of the hepatopancreas. The infected cells occurred throughout the hepatopancreas but never exceeded 10% of the cells constituting the tubule epithelium. Electron microscopy revealed numerous loosely enveloped, rod-shaped baculoviruses. The enveloped virions had a length of 292 ± 15 nm and a diameter of 102 ± 7 nm (mean ± SD). The cylindrical nucleocapsids often possessed squared ends and were 216 ± 13 nm in length by 47 ± 3 nm in width. There was no evidence of occlusion body formation similar to that known for certain other crustacean baculoviruses. Th...