UNASSIGNED: Little is known about the proteomic changes at the portals of entry in rainbow trout after infection with the myxozoan parasites, Myxobolus cerebralis, and Tetracapsuloides bryosalmonae. Whirling disease (WD) is a severe disease of salmonids, caused by the myxosporean M. cerebralis, while, proliferative kidney disease (PKD) is caused by T. bryosalmonae, which instead belongs to the class Malacosporea. Climate change is providing more suitable conditions for myxozoan parasites lifecycle, posing a high risk to salmonid aquaculture and contributing to the decline of wild trout populations in North America and Europe. Therefore, the aim of this study was to provide the first proteomic profiles of the host in the search for evasion strategies during single and coinfection with M. cerebralis and T. bryosalmonae.
UNASSIGNED: One group of fish was initially infected with M. cerebralis and another group with T. bryosalmonae. After 30 days, half of the fish in each group were co-infected with the other parasite. Using a quantitative proteomic approach, we investigated proteomic changes in the caudal fins and gills of rainbow trout before and after co-infection.
UNASSIGNED: In the caudal fins, 16 proteins were differentially regulated post exposure to M. cerebralis, whereas 27 proteins were differentially modulated in the gills of the infected rainbow trout post exposure to T. bryosalmonae. After co-infection, 4 proteins involved in parasite recognition and the regulation of host immune responses were differentially modulated between the groups in the caudal fin. In the gills, 11 proteins involved in parasite recognition and host immunity, including 4 myxozoan proteins predicted to be virulence factors, were differentially modulated.
UNASSIGNED: The results of this study increase our knowledge on rainbow trout co-infections by myxozoan parasites and rainbow trout immune responses against myxozoans at the portals of entry, supporting a better understanding of these host-parasite interactions.

Tetracapsuloides bryosalmonae is a malacosporean endoparasite that infects a wide range of salmonids and causes proliferative kidney disease (PKD). Brown trout serves as a carrier host whereas rainbow trout represents a dead-end host. We thus asked if the parasite adapts to the different hosts by changing molecular mechanisms. We used fluorescent activated cell sorting (FACS) to isolate parasites from the kidney of brown trout and rainbow trout following experimental infection with T. bryosalmonae. The sorted parasite cells were then subjected to RNA sequencing. By this approach, we identified 1120 parasite transcripts that were expressed differentially in parasites derived from brown trout and rainbow trout. We found elevated levels of transcripts related to cytoskeleton organisation, cell polarity, peptidyl-serine phosphorylation in parasites sorted from brown trout. In contrast, transcripts related to translation, ribonucleoprotein complex biogenesis and subunit organisation, non-membrane bounded organelle assembly, regulation of protein catabolic process and protein refolding were upregulated in rainbow trout-derived parasites. These findings show distinct molecular adaptations of parasites, which may underlie their distinct outcomes in the two hosts. Moreover, the identification of these differentially expressed transcripts may enable the identification of novel drug targets that may be exploited as treatment against T. bryosalmonae. We here also describe for the first time how FACS based isolation of T. bryosalmonae cells from infected kidney of fish fosters research and allows to define differentially expressed parasite transcripts in carrier and dead-end fish hosts.

Heterogeneity in immunity occurs across numerous disease systems with individuals from the same population having diverse disease outcomes. Proliferative kidney disease (PKD) caused by Tetracapsuloides bryosalmonae, is a persistent parasitic disease negatively impacting both wild and farmed salmonids. Little is known of how PKD is spread or maintained within wild susceptible populations. We investigated an aspect of fish disease that has been largely overlooked, that is, the role of the host phenotypic heterogeneity in disease outcome. We examined how host susceptibility to T. bryosalmonae infection, and the disease PKD, varied across different infection life-history stages and how it differs between naïve, re-infected and persistently infected hosts. We investigated the response to parasite exposure in host phenotypes with (a) different ages and (b) heterogeneous infection life histories. Among (a) the age phenotypes were young-of-the-year (YOY) fish and juvenile 1+ fish (fish older than one) and, for (b) juvenile 1+ infection survivors were either re-exposed or not re- exposed to the parasite and response phenotypes were assigned post-hoc dependant on infection status. In fish not re-exposed this included fish that cleared infection (CI) or had a persistent infection (PI). In fish re-exposed these included fish that were re-infected (RI), or re-exposed and uninfected (RCI). We assessed both parasite-centric (infection prevalence, parasite burden, malacospore transmission) and host-centric parameters (growth rates, disease severity, infection tolerance and the immune response). In (a), YOY fish, parasite success and disease severity were greater and differences in the immune response occurred, demonstrating an ontogenetic decline of susceptibility in older fish. In (b), in PI and RI fish, parasite success and disease severity were comparable. However, expression of several adaptive immunity markers was greater in RI fish, indicating concomitant immunity, as re-exposure did not intensify infection. We demonstrate the relevance of heterogeneity in infection life history on disease outcome and describe several distinctive features of immune ontogeny and protective immunity in this model not previously reported. The relevance of such themes on a population level requires greater research in many aquatic disease systems to generate clearer framework for understanding the spread and maintenance of aquatic pathogens.

Diagnostic accuracy of pathogen detection depends upon the selection of suitable tests. Problems can arise when the selected diagnostic test gives false-positive or false-negative results, which can affect control measures, with consequences for the population health. The aim of this study was to compare sensitivity of different diagnostic methods IHC, PCR and qPCR detecting Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish and as a consequence differences in disease prevalence. We analysed tissue from 388 salmonid specimens sampled from a recirculating system and rivers in the Czech Republic. Overall prevalence of T. bryosalmonae was extremely high at 92.0%, based on positive results of at least one of the above-mentioned screening methods. IHC resulted in a much lower detection rate (30.2%) than both PCR methods (qPCR32: 65.4%, PCR: 81.9%). While qPCR32 produced a good match with IHC (60.8%), all other methods differed significantly (p < .001) in the proportion of samples determined positive. Both PCR methods showed similar sensitivity, though specificity (i.e., the proportion of non-diseased fish classified correctly) differed significantly (p < .05). Sample preservation method significantly (p < .05) influenced the results of PCR, with a much lower DNA yield extracted from paraffin-embedded samples. Use of different methods that differ in diagnostic sensitivity and specificity resulted in random and systematic diagnosis errors, illustrating the importance of interpreting the results of each method carefully.

Tetracapsuloides bryosalmonae, a myxozoan endoparasite, causes proliferative kidney disease in salmonids. The life cycle of T. bryosalmonae occurs between invertebrate bryozoan and vertebrate fish hosts. T. bryosalmonae develops in the body cavity of colonial bryozoan and spores are released from mature spore sacs into the water likely through the vestibular pore and infect fish by attaching to their gills. However, very little is known about the transcriptome of this important parasite, which hampers studies into the molecular mechanisms of host-parasite interactions and understanding the parasite biology. In order to circumvent this limitation, we performed de novo transcriptome assembly on the sacs of T. bryosalmonae, collected from infected bryozoan Fredericella sultana. A total of 111.5 million filtered paired-end reads was obtained and assembled into 25,908 contigs corresponding to putative transcripts that were functionally annotated. More than 50% of the assembled transcripts (13,071 contigs) had a significant hit in NCBI non-redundant database. Based on Gene ontology annotation, the most highly scored categories of molecular function of the contigs were related to binding and catalytic activities in T. bryosalmonae. This study provides a global overview of the T. bryosalmonae transcriptome that will be a valuable resource for identifying virulence factors, gene discovery, genome annotation, and vaccine development applications. This data is accessible via NCBI BioProject (PRJNA680464).

Proliferative kidney disease (PKD) is an emerging disease of salmonids, which is exacerbating with increasing water temperature. Its causative agent, the myxozoan parasite Tetracapsuloides bryosalmonae, exploits freshwater bryozoans as primary hosts and salmonids as intermediate hosts. Our experiments showed that the manipulation of exposure concentrations of infective malacospores had relatively minor impacts for the disease outcomes in the fish host. In this study, brown trout (Salmo trutta) were exposed to three different exposure concentrations of T. bryosalmonae malacospores: (a) a single low parasite concentration (LC), (b) a single high parasite concentration (HC) and (c) three times a low concentration (repeat exposure, RE). Parasite dynamics in the fish host and release of fish malacospores were quantified and fish kidney histopathology was evaluated to determine PKD pathogenesis. Infection prevalence was always lower in the LC group than in the other groups over the course of the study. While the parasite proliferation phase was slower in the LC group, the maximum parasite burden did not differ significantly amongst treatments. The onset of fish malacospore release (day 45 post-exposure), indicated by detection of T. bryosalmonae DNA in the tank water, occurred at the same time point for all groups. Reduced intensity of kidney pathological development was observed in the LC treatment indicating lower disease severity. While the LC treatment resulted in reduced outcomes across several infection parameters (infection prevalence, parasite proliferation, total fish malacospores released), the overall differences were small. The RE and HC treatment outcomes were for most parameters comparable. Our results suggest that repeated exposure, as is likely to occur in the wild during the summer months, might play a more important role in the dynamics of PKD as an emerging infectious disease than the actual concentration of spores.

BACKGROUND: Renibacterium salmoninarum and Mycobacterium sp. are important bacterial pathogens of fish. R. salmoninarum is the causative agent of bacterial kidney disease, a Gram-positive bacterium mostly known for causing chronic infections in salmonid fish, while multiple species belonging to the Mycobacterium genus have been associated with mycobacteriosis in fish as well as in human. The objective of this study was to determine the prevalence of these two bacterial pathogens in populations of wild brown trout (Salmo trutta fario) in four rivers (Kamp, Wulka, Traun and Ybbs) in Austria.
RESULTS: A total of 457 kidney samples were examined for both bacterial agents using nested and conventional PCR as well as bacterial cultivation on KDM-2, histological examination and immunohistochemistry. Molecular evidence showed an estimated prevalence level of 0.94% for R. salmoninarum in 2017 while the bacterium could not be detected in 2018 and histology showed signs consistent with a low-level chronic inflammation in the kidney of infected fish. Similarly, no fish were found positive for Mycobacterium in 2017 but in 2018, the prevalence was found to be 37.03% in the Kamp river (4.08% across all rivers). The sequencing data confirmed that these fish carried Mycobacterium sp. although the precise species of Mycobacterium could not be ascertained.
CONCLUSIONS: This survey constitutes the first insight into the prevalence rate of R. salmoninarum and Mycobacterium sp. in wild brown trout (Salmo trutta fario) populations in Austria. Both of these pathogens were only detected in the summer months (June and July), which might suggest that the stress linked to increased water temperature could act as stressor factor and contribute to the outbreak of these diseases. The age of the fish might also play a role, especially in the case of Mycobacterium sp. as all the infected fish were in their first summer (June).

Tetracapsuloides bryosalmonae is a myxozoan parasite responsible for proliferative kidney disease (PKD) in a wide range of salmonids. PKD, characterized by high mortality and morbidity, is well known for affecting aquaculture operations and wild salmonid populations across Europe and North America. The life cycle of T. bryosalmonae revolves around freshwater bryozoan and salmonid fish hosts. In recent years, T. bryosalmonae has been reported among wild salmonids from the European countries where it has not been reported previously. T. bryosalmonae is believed to be a possible reason for the diminishing wild salmonid populations in the natural water bodies of many European countries. Climate crisis driven rising water temperature can further accelerate the distribution of T. bryosalmonae. Expansion of the geographical distribution of T. bryosalmonae may further advocate the decline of wild salmonid populations, especially brown trout (Salmo trutta) in their habitats. Mathematical models are used to understand the pattern and distribution of T. bryosalmonae among the host in the natural water bodies. The present manuscript not only summarizes the incidences of T. bryosalmonae among the wild salmonid populations, but also discusses the contemporary understanding about the development of T. bryosalmonae in its hosts and the influences of various factors in the spread of the disease in the wild.

The number of fish as pets far exceeds the populations of any other companion animal. As our knowledge of aquatic animal species and aquatic animal medicine continues to expand, veterinary expertise is becoming more critical to the client, researcher, fisheries biologist, aquarist, farmer, and fish hobbyist. Similar to other vertebrates, fish are susceptible to infectious and noninfectious renal disease. This article compares vertebrate renal anatomy and physiology and highlights some renal disease examples.

Proliferative kidney disease (PKD) is an emerging disease of salmonids caused by the myxozoan parasite Tetracapsuloides bryosalmonae, which plays a major role in the decrease of wild brown trout (Salmo trutta) populations in Switzerland. Strong evidence demonstrated that water temperature modulates parasite infection. However, less knowledge exists on how seasonal water temperature fluctuations influence PKD manifestation under field conditions, how further environmental factors such as water quality may modulate the disease, and whether these factors coalesce with temperatures role possibly giving rise to cumulative effects on PKD. The aims of this study were to (1) determine the correlation between seasonal course of water temperature and PKD prevalence and intensity in wild brown trout populations, (2) assess if other factors such as water quality or ecomorphology correlate with the infection, and (3) quantitatively predict the implication of these factors on PKD prevalence with a statistical model. Young-of-the-year brown trout were sampled in 45 sites through the Canton of Vaud (Switzerland). For each site, longitudinal time series of water temperature, water quality (macroinvertebrate community index, presence of wastewater treatment plant effluent) and ecomorphological data were collected and correlated with PKD prevalence and intensity. 251 T. bryosalmonae-infected trout of 1,118 were found (overall prevalence 22.5%) at 19 of 45 study sites (42.2%). Relation between PKD infection and seasonal water temperature underlined that the mean water temperature for June and the number of days with mean temperature ≥15°C were the most significantly correlated parameters with parasite prevalence and intensity. The presence of a wastewater treatment plant effluent was significantly correlated with the prevalence and infection intensity. In contrast, macroinvertebrate diversity and river ecomorphology were shown to have little impact on disease parameters. Linear and logistic regressions highlighted quantitatively the prediction of PKD prevalence depending on environmental parameters at a given site and its possible increase due to rising temperatures. The model developed within this study could serve as a useful tool for identifying and predicting disease hot spots. These results support the importance of temperature for PKD in salmonids and provides evidence for a modulating influence of additional environmental stress factors.