Whole body net ion fluxes, plasma electrolyte concentrations and haematology during a Loma salmonae infection in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum)

Loma salmonae infections of salmonids culminate in the development of branchial xenomas and subsequent focal hyperplasia of the lamellar or filament epithelium following xenoma rupture and spore release. The effects of this acute branchial disruption upon net ionic flux rates and plasma electrolyte concentrations were determined in juvenile rainbow trout given an experimental oral exposure to L. salmonae. Mean numbers of branchial xenomas peaked at week 5 post-exposure (PE), which coincided with a reduction in the specific growth rate, although there were no significant differences in mass, length or condition of Loma-exposed fish compared with unexposed controls. Following exposure, negative net whole body Na(+) and K(+) fluxes decreased, whereas net Cl(-) fluxes remained unchanged compared with non-exposed control fish. At week 3 PE during the initial branchial xenoma formation stage, there was a significant negative whole body net K(+) flux in Loma-exposed trout compared with other points during the exposure and subsequent infection. Additionally, Loma-exposed fish had marginally elevated plasma Na(+) and Cl(-) concentrations, whilst K(+) levels remained unchanged, compared with control fish. Although there was a progressive decrease in leucocrit, haematocrit remained unchanged over the course of the Loma exposure and subsequent infection. These results suggest that ionic compensation can occur at the gills during the development of xenomas during exposure to L. salmonae and the resultant infection, therefore allowing defence of plasma electrolyte concentrations, unlike the acute ionic disturbances seen with some other parasitic diseases.     

Interaction of water temperature and challenge model on xenoma development rates for Loma salmonae (Microspora) in rainbow trout, Oncorhynchus mykiss (Walbaum)

This study evaluated the regulatory effects of water temperature on the development of branchial xenomas caused by Loma salmonae using a high-dose per os-challenge model compared with a low-dose cohabitation-challenge model. Approximately 275 juvenile rainbow trout (RBT), Oncorhynchus mykiss, were randomly distributed to six tanks with two tanks each maintained at 11, 15 and 19 degrees C. Fish in one tank from each temperature setting were exposed per os to macerated L. salmonae-infected gill material and fish in the other tank from each temperature setting were exposed to L. salmonae using the cohabitation-challenge model. Fish were monitored for the development of branchial xenomas beginning at day 21 post-exposure. Survival analyses were used to evaluate the effect of water temperature and challenge model on the number of days until the first visible branchial xenoma was detected. The survivor curves for the per os-challenge model revealed that there was at least one significant difference, whereas the cohabitation challenge did not reveal any significant differences amongst the temperature settings. The proportional hazards model revealed a significant interaction between the challenge model used and water temperature. This indicated that the effect of water temperature was different depending on challenge model. Additionally, from the mean xenoma intensities, on average, the per os-challenged fish showed higher xenoma intensity compared with the cohabitation-challenged fish. Overall, the impact of water temperature on disease pathogenesis was greater when the RBT were per os challenged compared with using the cohabitation model.     

The transmission potential of Loma salmonae (Microspora) in the rainbow trout, Oncorhynchus mykiss (Walbaum), is dependent upon the method and timing of exposure

The ability of a parasite to transmit from one fish to another is important in the dissemination of disease. Groups of 25 naive rainbow trout (RBT), Oncorhynchus mykiss (Walbaum), were exposed to Loma salmonae by feeding on the viscera (gills, hearts and spleens) from L. salmonae -infected donor RBT (DRBT) or by cohabitation with infected DRBT. Exposure occurred 3, 7, 11 and 15 weeks after the DRBT were infected. All naive RBT were examined 7 weeks post-exposure (PE) to the DRBT. Naive RBT, exposed to DRBT at week 3 PE, by feeding on viscera or by cohabitation, failed to develop visible branchial xenomas. Cohabiting naive RBT with DRBT, at week 7 PE and week 11 PE, resulted in the development of branchial xenomas. Xenomas failed to develop in naive RBT exposed via cohabitation to week 15 PE DRBT. Naive RBT, exposed by feeding on the viscera of DRBT at week 7 PE, week 11 PE and week 15 PE, developed branchial xenomas. The transmission potential of viscera from L. salmonae -infected DRBT at week 15 and week 20 PE was also examined. Naive RBT, fed with viscera free of visible branchial xenomas, from DRBT at week 15 PE and week 20 PE, developed branchial xenomas by week 7 PE. A polymerase chain reaction (PCR) was used to detect L. salmonae DNA from the water and sediments of a tank of L. salmonae -infected RBT at week 7 PE. The method and timing of exposure of naive fish to L. salmonae -infected fish are important in disease transmission and may be useful in predicting and preventing disease outbreaks in aquaculture.     

Influence of feeding ratio and size on susceptibility to microsporidial gill disease caused by Loma salmonae in rainbow trout, Oncorhynchus mykiss (Walbaum)

Two trials were designed to quantify the effect of feeding ratio and fish size on the cohabitation transmission of Loma salmonae, the causative agent of microsporidial gill disease (MGD) in salmonids, Oncorhynchus spp. To evaluate the effect of feeding ratio on disease onset, groups of 45 rainbow trout, O. mykiss (Walbaum) (RBT), were fed daily at 1% (low), 2% (medium) or 4% (high) of the fish biomass in the tank. There were three tanks at each feeding level: two tanks were exposed to the pathogen and one was a control. For the second objective, 300 RBT were separated into seven tanks so that the weight classifications were small (17-23 g), medium (32-38 g) and large (57-63 g). Each size class was done in duplicate with one control tank containing medium-sized fish. Separately for each trial, on day 0 post-exposure (PE) five highly infectious RBT were added to each tank (not including the control tanks) to begin the cohabitation exposure period. Beginning on day 21 PE and continuing biweekly until days 70 and 77 PE for the feeding and fish size trials, respectively, each fish was evaluated for visible branchial xenomas to determine disease onset time. Using survival analysis, the survival curves for the low, medium and high feedings were not significantly different from each other. However, there were significant differences amongst the small, medium and large weight classes in the size trial. The median numbers of days to the development of branchial xenomas was 31, 38 and 42 for small, medium and large size fish, respectively. On any given day, a medium or large sized fish had a hazard ratio for developing branchial xenomas of 0.66 and 0.63, respectively, compared with a small fish. In addition to host species and host strain differences, fish size is now considered a host risk factor for the development of MGD.     

Differences in Ichthyophonus prevalence and infection severity between upper Yukon River and Tanana River chinook salmon, Oncorhynchus tshawytscha (Walbaum), stocks

Two genetically distinct populations of chinook salmon, Oncorhynchus tshawytscha (Walbaum), were simultaneously sampled at the confluence of the Yukon and Tanana rivers in 2003. Upper Yukon-Canadian fish had significantly higher infection prevalence as well as more severe infections (higher parasite density in heart tissue) than the lower Yukon-Tanana River fish. Both populations had migrated the same distance from the mouth of the Yukon River at the time of sampling but had significantly different distances remaining to swim before reaching their respective spawning grounds. Multiple working hypotheses are proposed to explain the differences between the two stocks: (1) the two genetically distinct populations have different inherent resistance to infection, (2) genetically influenced differences in feeding behaviour resulted in temporal and/or spatial differences in exposure, (3) physiological differences resulting from different degrees of sexual maturity influenced the course of disease, and (4) the most severely infected Tanana River fish either died en route or fatigued and were unable to complete their migration to the Tanana River, thus leaving a population of apparently healthier fish.     

Efficacy of manual removal and ivermectin gavage for control of Salmincola californiensis (Wilson) infestation of chinook salmon, Oncorhynchus tshawytscha (Walbaum), captive broodstocks

Captive broodstocks of spring chinook salmon, Oncorhynchus tshawytscha , were initiated from collections of naturally produced parr from the Lemhi River, a tributary of the Salmon River, ID, USA. These fish were subsequently demonstrated to be infested with the copepod parasite Salmincola californiensis . The initial prevalence of visible adult parasites for 4 years of observations made shortly after collection varied from 19.7 to 71.6%. Both the prevalence and intensity of the infestation increased in the freshwater culture of these fish. Manual removal was initiated as a means of control and practiced at monthly intervals. The number of Salmincola removed decreased in the ensuing 5 months, but the prevalence was not greatly affected. Ivermectin (22,23 dihydroavermectin), was diluted with saline and delivered by gavage at the rate of 0.20 mg kg⁻¹ body weight when the groups were being handled for the manual removal of parasites. Either two or three ivermectin treatments were given to four broodstocks of chinook salmon depending on the severity of the infestation and on the extent of gill pathology. The combination of manual removal and ivermectin gavage eliminated live Salmincola and resolved all associated necrosis of the gill tissues. There was no trend to indicate that individual chinook salmon possessed a natural resistance to reinfestation.     

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.