Production of Ceratonova shasta Myxospores from Salmon Carcasses: Carcass Removal Is Not a Viable Management Option.

Severe infection by the endemic myxozoan parasite, Ceratonova (synonym, Ceratomyxa) shasta, has been associated with declines in and impaired recovery efforts of populations of fall-run Chinook Salmon Oncorhynchus tshawytscha in the Klamath River, California. The parasite has a complex life cycle involving a polychaete worm host as well as a salmon host. Myxospore transmission of this parasite, from salmon to polychaete, is a life cycle step during which there is a potential for applied disease management. A 3-year data set on prevalence, intensity, and spore characteristics of C. shasta myxospores was obtained from adult Chinook Salmon carcasses surveyed in the main stem of the Klamath River and three of its tributaries, Bogus Creek and the Shasta and Trinity rivers. Annual prevalence of myxospore detection in salmon intestines ranged from 22% to 52%, and spore concentration values per intestinal scraping ranged from 3.94 × 10(2) to 1.47 × 10(7) spores. A prevalence of 7.3% of all carcasses examined produced >5.0 × 10(5) spores, and these carcasses with "high" spore counts accounted for 76-95% of the total spores in a given spawning season. Molecular analysis of visually negative carcasses showed that 45-87% of these samples had parasite DNA, indicating they contained either low spore numbers or presporogonic stages of the parasite. Myxospores were rarely found in carcasses of freshly spawned adults but were common in decomposed carcasses of both sexes. The date of collection or age (based indirectly on FL) did not influence detection. The longer prespawn residence time for spring-run Chinook Salmon compared with that for fall-run Chinook Salmon in the Trinity River was associated with higher spore loads. The dye exclusion method for assessing spore viability in fresh smears indicated an inverse relationship in spore integrity and initial spore concentration. A carcass-removal pilot project in Bogus Creek for 6 weeks in the fall of 2008 (907 carcasses removed) and 2009 (1,799 carcasses removed) failed to measurably influence the DNA quantity of C. shasta in targeted waters. Combined with the high numbers of carcasses that contributed myxospores, we therefore deemed that this labor-intensive approach is not a viable management option to reduce the infectivity of C. shasta in Chinook Salmon in the Klamath River. Received January 23, 2015; accepted September 28, 2015.

Development and validation of a quantitative PCR to detect Parvicapsula minibicornis and comparison to histologically ranked infection of juvenile Chinook salmon, Oncorhynchus tshawytscha (Walbaum), from the Klamath River, USA.

Parvicapsula minibicornis is a myxosporean parasite that is associated with disease in Pacific salmon during their freshwater life history phase. This study reports the development of a quantitative (real-time) polymerase chain reaction (QPCR) to detect P. minibicornis DNA. The QPCR assay targets the 18S ribosomal subunit gene. A plasmid DNA control was developed to calibrate cycle threshold (C(T)) score to plasmid molecular equivalent (PME) units, a measure of gene copy number. Assay validation revealed that the QPCR was sensitive and able to detect 50 ag of plasmid DNA, which was equivalent to 12.5 PME. The QPCR assay could detect single P. minibicornis actinospores well above assay sensitivity, indicating a single spore contains at least 100 times the 18S DNA copies required for detection. The QPCR assay was repeatable and highly specific; no detectable amplification was observed using DNA from related myxozoan parasites. The method was validated using kidney tissues from 218 juvenile Chinook salmon sampled during the emigration period of March to July 2005 from the Klamath River. The QPCR assay was compared with histological examination. The QPCR assay detected P. minibicornis infection in 88.1% of the fish sampled, while histological examination detected infection in 71.1% of the fish sampled. Good concordance was found between the methods as 80% of the samples were in agreement. The majority of the disconcordant fish were positive by QPCR, with low levels of P. minibicornis DNA, but negative by histology. The majority of the fish rated histologically as having subclinical or clinical infections had high QPCR levels. The results of this study demonstrate that QPCR is a sensitive quantitative tool for evaluating P. minibicornis infection in fish health monitoring studies.

Longevity of Ceratomyxa shasta and Parvicapsula minibicornis actinospore infectivity in the Klamath River.

Infectious Ceratomyxa shasta and Parvicapsula minibicornis actinospores were present in Klamath River samples collected in April, May, and June 2005. Juvenile Chinook salmon Oncorhynchus tshawytscha exposed to river water maintained at the ambient Klamath River temperature for 0, 4, 24, 72, and 168 h (7 d) developed asymptomatic infections from both parasites. Elevated water temperature (18 degrees C) in June may have reduced actinospore viability, as both C. shasta and P. minibicornis infection markedly declined in fish exposed for over 72 h. As judged by the prevalence of infection for both parasites, the number of infectious actinospores tended to increase or remain steady through the spring. Ceratomyxa shasta infections were characterized by the presence of a few trophozoites within granulomatous foci in mesentery adipose tissue and were consistently observed outside of the intestine. Similarly, low numbers of P. minibicornis were observed in kidney glomeruli and tubules but were not associated with inflammation. Parvicapsula minibicornis DNA was consistently detected by quantitative real-time polymerase chain reaction in filtered water samples collected each month and from each time posttransfer. These data and the high prevalence of infection observed in the exposed fish indicate that P. minibicornis actinospores were at a relatively high concentration in the river during the spring. In contrast, C. shasta DNA was only detected in half of the water sample sets and its detection did not correspond well to C. shasta infectivity. An approximately threefold increase in river flow from the April to the May water collection was not associated with a decline in either the detection of actinospores (particularly for P. minibicornis) or the prevalence of infection for both parasites. Actinospores of these myxosporean parasites have the potential to infect salmonids for at least 7 d after release from the alternate polychaete host.

Genome-wide screen for atopy susceptibility alleles in the Hutterites.

A genome-wide screen for loci influencing positive skin prick tests (SPT) to airborne allergens was conducted in the Hutterites, a founder population of European ancestry. Positive SPT to 14 standardized allergens was measured in 370 subjects in our primary sample and 324 subjects in a replication sample. Evidence for linkage to positive SPT was assessed using the transmission disequilibrium test (TDT) with 337 autosomal markers (average spacing 9.13 cM, SD = 7.8 cM). Three loci showed the strongest overall evidence of linkage to atopy, with at least one allele-specific and a locus-specific p< 1 x 10(-4). This study provides evidence for at least three atopy-susceptibility loci in the Hutterites on chromosomes 1, 6 and 16.

The effect of gamma irradiation on a xenograft tendon bioprosthesis.

Radiation sterilisation of xenograft prostheses has been shown to cause damage to the material. Such damage has been imputed to affect the mechanical and biological properties and may contribute to long-term failure. This study has examined the effect of 25 kGy (2.5 Mrad) of gamma radiation on the mechanical and physicochemical properties and the biological function of a xenograft tendon bioprosthesis derived from glutaraldehyde cross-linked kangaroo tail tendon. The ultimate tensile stress of nonirradiated glutaraldehyde cross-linked tendon was greater than that of fresh frozen tendon, but irradiated cross-linked tendon did not differ. Irradiation did not alter the response to prolonged collagenase exposure. There was a decrease in overall apparent cross-link density (as determined by thermal denaturation temperature). After 12 months implantation, there was a slightly more active cellular response around irradiated tendon and the very peripheral fibres were infiltrated, but the mechanical properties of the retrieved implants were the same for irradiated and nonirradiated material. Gamma irradiation would appear to be a satisfactory method of sterilisation for glutaraldehyde cross-linked tendon materials. However, as damage to the cross-linked structure was detected in this study, it may not be appropriate in other applications.