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.

Department of Ecology and Diseases of Zoo Animals Game Fish and Bees Faculty of Veterinary Hygiene and Ecology University of Veterinary and Pharmaceutical Sciences Brno Brno Czech Republic

Department of Infectious Diseases and Preventive Medicine Veterinary Research Institute Brno Czech Republic

Department of Pathobiology Centre for Fish and Wildlife Health University of Bern Bern Switzerland

Department of Zoology Fisheries Hydrobiology and Apiculture Mendel University in Brno Brno Czech Republic

Institute of Vertebrate Biology Czech Academy of Sciences Brno Czech Republic

Zobrazit více v PubMed

Abd‐Elfattah, A., Kumar, G., Soliman, H., & El‐Matbouli, M. (2014). Persistence of Tetracapsuloides bryosalmonae (Myxozoa) in chronically infected brown trout Salmo trutta . Diseases of Aquatic Organisms, 111(1), 41–49. 10.3354/dao02768 PubMed DOI PMC

Adams, A., Richards, R. H., & de Mateo, M. M. (1992). Development of monoclonal antibodies to PK ‘X’, the causative agent of proliferative kidney disease. Journal of Fish Diseases, 15(6), 515–521. 10.1111/j.1365-2761.1992.tb00683.x DOI

Bettge, K., Segner, H., Burki, R., Schmidt‐Posthaus, H., & Wahli, T. (2009). Proliferative kidney disease (PKD) of rainbow trout: Temperature‐and time‐related changes of Tetracapsuloides bryosalmonae DNA in the kidney. Parasitology, 136(6), 615–625. PubMed

Bettge, K., Wahli, T., Segner, H., & Schmidt‐Posthaus, H. (2009). Proliferative kidney disease in rainbow trout: Time‐and temperature‐related renal pathology and parasite distribution. Diseases of Aquatic Organisms, 83(1), 67–76. 10.3354/dao01989 PubMed DOI

Bonin, S., Petrera, F., Niccolini, B., & Stanta, G. (2003). PCR analysis in archival postmortem tissues. Molecular Pathology, 56(3), 184. 10.1136/mp.56.3.184 PubMed DOI PMC

Bramwell, N. H., & Burns, B. F. (1988). The effects of fixative type and fixation time on the quantity and quality of extractable DNA for hybridization studies on lymphoid tissue. Experimental Hematology, 16(8), 730–732. PubMed

Canning, E. U., Curry, A., Feist, S. W., Longshaw, M., & Okamura, B. (2000). A new class and order of myxozoans to accommodate parasites of bryozoans with ultrastructural observations on Tetracapsula bryosalmonae (PKX organism). Journal of Eukaryotic Microbiology, 47(5), 456–468. 10.1111/j.1550-7408.2000.tb00075.x PubMed DOI

Castagnaro, M., Marin, M., Ghittino, C., & Hedrick, R. P. (1991). Lectin histochemistry and ultrastructure of rainbow trout Oncorhynchus mykiss kidneys affected by proliferative kidney disease. Diseases of Aquatic Organisms, 10(3), 173–183. 10.3354/dao010173 DOI

Chilmonczyk, S., Monge, D., & De Kinkelin, P. (2002). Proliferative kidney disease: Cellular aspects of the rainbow trout, Oncorhynchus mykiss (Walbaum), response to parasitic infection. Journal of Fish Diseases, 25(4), 217–226. 10.1046/j.1365-2761.2002.00362.x DOI

Clifton‐Hadley, R. S., Bucke, D., & Richards, R. H. (1986). Economic importance of proliferative kidney disease of salmonid fish in England and Wales. Veterinary Record, 119(12), 305–306. 10.1136/vr.119.12.305 PubMed DOI

Clifton‐Hadley, R. S., Richards, R. H., & Bucke, D. (1986). Proliferative kidney disease (PKD) in rainbow trout Salmo gairdneri: Further observations on the effects of water temperature. Aquaculture, 55(3), 165–171. 10.1016/0044-8486(86)90112-2 DOI

Feist, S. W., Longshaw, M., Canning, E. U., & Okamura, B. (2001). Induction of proliferative kidney disease (PKD) in rainbow trout Oncorhynchus mykiss via the bryozoan Fredericella sultana infected with Tetracapsula bryosalmonae . Diseases of Aquatic Organisms, 45(1), 61–68. 10.3354/dao045061 PubMed DOI

Ferguson, H. W. (1981). The effects of water temperature on the development of proliferative kidney disease in rainbow trout, Salmo gairdneri Richardson. Journal of Fish Diseases, 4(2), 175–177. 10.1111/j.1365-2761.1981.tb01122.x DOI

Ferguson, H. W., & Ball, H. J. (1979). Epidemiological aspects of proliferative kidney disease amongst rainbow trout Salmo gairdneri Richardson in Northern Ireland. Journal of Fish Diseases, 2(3), 219–225. 10.1111/j.1365-2761.1979.tb00161.x DOI

Ferguson, H. W., & Needham, E. A. (1978). Proliferative kidney disease in rainbow trout Salmo gairdneri Richardson. Journal of Fish Diseases, 1(1), 91–108. 10.1111/j.1365-2761.1978.tb00008.x DOI

Gay, M., Okamura, B., & De Kinkelin, P. (2001). Evidence that infectious stages of Tetracapsula bryosalmonae for rainbow trout Oncorhynchus mykiss are present throughout the year. Diseases of Aquatic Organisms, 46(1), 31–40. 10.3354/dao046031 PubMed DOI

Grabner, D. S., & El‐Matbouli, M. (2010). Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) portal of entry into the fish host. Diseases of Aquatic Organisms, 90(3), 197–206. 10.3354/dao02236 PubMed DOI

Hedrick, R. P., MacConnell, E., & De Kinkelin, P. (1993). Proliferative kidney disease of salmonid fish. Annual Review of Fish Diseases, 3, 277–290. 10.1016/0959-8030(93)90039-E DOI

Hedrick, R. P., Marin, M., Castagnaro, M., Monge, D., & De Kinkelin, P. (1992). Rapid lectin‐based staining procedure for the detection of the myxosporean causing proliferative kidney disease in salmonid fish. Diseases of Aquatic Organisms, 13(2), 129–132. 10.3354/dao013129 DOI

Henderson, M., & Okamura, B. (2004). The phylogeography of salmonid proliferative kidney disease in Europe and North America. Proceedings of the Royal Society of London. Series B: Biological Sciences, 271(1549), 1729–1736. 10.1098/rspb.2004.2677 PubMed DOI PMC

Kent, M. L., & Hedrick, R. P. (1985). PKX, the causative agent of proliferative kidney disease (PKD) in Pacific salmonid fishes and its affinities with the myxozoa 1. The Journal of Protozoology, 32(2), 254–260. 10.1111/j.1550-7408.1985.tb03047.x PubMed DOI

Kent, M. L., Khattra, J., Hervio, D. M. L., & Devlin, R. H. (1998). Ribosomal DNA sequence analysis of isolates of the PKX myxosporean and their relationship to members of the genus Sphaerospora. Journal of Aquatic and Animal Health, 10, 12–21. 10.1577/1548-8667(1998)010<0012:RDSAOI>2.0.CO;2 DOI

Klontz, G. W., & Chacko, A. J. (1983). Methods to detect the organism causing proliferative kidney disease in salmonids. Bulletin of the European Association of Fish Pathologists, 3, 33–36.

Kopp, R., Palíková, M., Papežíková, I., Mareš, J., Navrátil, S., Pikula, J., & Pohanka, M. (2018). Oxidative stress response of rainbow trout (Oncorhynchus mykiss) to multiple stressors. Acta Veterinaria Brno, 87(1), 55–64. 10.2754/avb201887010055 DOI

Lewisch, E., Unfer, G., Pinter, K., Bechter, T., & El‐Matbouli, M. (2018). Distribution and prevalence of T. bryosalmonae in Austria: A first survey of trout from rivers with a shrinking population. Journal of Fish Diseases, 41(10), 1549–1557. PubMed

Longshaw, M., Feist, S. W., Canning, E. U., & Okamura, B. (1999). First identification of PKX in bryozoans from the United Kingdom‐molecular evidence. Bulletin‐European Association of Fish Pathologists, 19, 146–148.

Longshaw, M., Le Deuff, R. M., Harris, A. F., & Feist, S. W. (2002). Development of proliferative kidney disease in rainbow trout, Oncorhynchus mykiss (Walbaum), following short‐term exposure to Tetracapsula bryosalmonae infected bryozoans. Journal of Fish Diseases, 25(8), 443–449. 10.1046/j.1365-2761.2002.00353.x DOI

Morris, D. C., Morris, D. J., & Adams, A. (2002). Molecular evidence of release of Tetracapsula bryosalmonae, the causative organism of proliferative kidney disease from infected salmonids into the environment. Journal of Fish Diseases, 25(8), 501–504. 10.1046/j.1365-2761.2002.00352.x DOI

Nowak, B., Mueffling, T. V., Caspari, K., & Hartung, J. (2006). Validation of a method for the detection of virulent Yersinia enterocolitica and their distribution in slaughter pigs from conventional and alternative housing systems. Veterinary Microbiology, 117(2–4), 219–228. 10.1016/j.vetmic.2006.06.002 PubMed DOI

Okamura, B., Anderson, C. L., Longshaw, M., Feist, S. W., & Canning, E. U. (2001). Patterns of occurrence and 18S rDNA sequence variation of PKX (Tetracapsula bryosalmonae), the causative agent of salmonid proliferative kidney disease. Journal of Parasitology, 87(2), 379–385. PubMed

Palikova, M., Papezikova, I., Markova, Z., Navratil, S., Mares, J., Mares, L., Vojtek, L., Hyrsl, P., Jelinkova, E., & Schmidt‐Posthaus, H. (2017). Proliferative kidney disease in rainbow trout (Oncorhynchus mykiss) under intensive breeding conditions: Pathogenesis and haematological and immune parameters. Veterinary Parasitology, 238, 5–16. PubMed

Pojezdal, Ľ., Adamek, M., Syrová, E., Steinhagen, D., Minářová, H., Papežíková, I., Seidlová, V., Reschová, S., & Palíková, M. (2020). Health surveillance of wild brown trout (Salmo trutta fario) in the Czech Republic revealed a coexistence of proliferative kidney disease and Piscine Orthoreovirus‐3 infection. Pathogens, 9(8), 604. PubMed PMC

Ribeiro, C. N. M., Peres, L. C., & Pina‐Neto, J. M. (2004). DNA extraction and quantification from touch and scrape preparations obtained from autopsy liver cells. Brazilian journal of medical and biological research, 37(5), 635–642. PubMed

Rüssmann, H., Kempf, V. A., Koletzko, S., Heesemann, J., & Autenrieth, I. B. (2001). Comparison of fluorescent in situ hybridization and conventional culturing for detection of Helicobacter pylori in gastric biopsy specimens. Journal of Clinical Microbiology, 39(1), 304–308. 10.1128/JCM.39.1.304-308.2001 PubMed DOI PMC

Schmidt‐Posthaus, H., Bettge, K., Forster, U., Segner, H., & Wahli, T. (2012). Kidney pathology and parasite intensity in rainbow trout Oncorhynchus mykiss surviving proliferative kidney disease: Time course and influence of temperature. Diseases of Aquatic Organisms, 97(3), 207–218. 10.3354/dao02417 PubMed DOI

Sengüven, B., Baris, E., Oygur, T., & Berktas, M. (2014). Comparison of methods for the extraction of DNA from formalin‐fixed, paraffin‐embedded archival tissues. International Journal of Medical Sciences, 11(5), 494. 10.7150/ijms.8842 PubMed DOI PMC

Skovgaard, A., & Buchmann, K. (2012). Tetracapsuloides bryosalmonae and PKD in juvenile wild salmonids in Denmark. Diseases of Aquatic Organisms, 101(1), 33–42. 10.3354/dao02502 PubMed DOI

Sterud, E., Forseth, T., Ugedal, O., Poppe, T. T., Jørgensen, A., Bruheim, T., Fjeldstad, H.‐P., & Mo, T. A. (2007). Severe mortality in wild Atlantic salmon Salmo salar due to proliferative kidney disease (PKD) caused by Tetracapsuloides bryosalmonae (Myxozoa). Diseases of Aquatic Organisms, 77(3), 191–198. PubMed

Suresh, K., & Smith, H. (2004). Comparison of methods for detecting Blastocystis hominis . European Journal of Clinical Microbiology and Infectious Diseases, 23(6), 509–511. 10.1007/s10096-004-1123-7 PubMed DOI

Syrová, E., Palíková, M., Mendel, J., Seidlová, V., Papežíková, I., Schmidt‐Posthaus, H., Somerlíková, K., Minářová, H., Mareš, L., Mikulíková, I., Pikula, J., & Mareš, J. (2020). Field study indicating susceptibility differences between salmonid species and their lineages to proliferative kidney disease. Journal of Fish Diseases, 43(10), 1201–1211. 10.1111/jfd.13221 PubMed DOI

Tops, S., Lockwood, W., & Okamura, B. (2006). Temperature‐driven proliferation of Tetracapsuloides bryosalmonae in bryozoan hosts portends salmonid declines. Diseases of Aquatic Organisms, 70(3), 227–236. 10.3354/dao070227 PubMed DOI

Vasemägi, A., Nousiainen, I., Saura, A., Vähä, J. P., Valjus, J., & Huusko, A. (2017). First record of proliferative kidney disease agent Tetracapsuloides bryosalmonae in wild brown trout and European grayling in Finland. Diseases of Aquatic Organisms, 125(1), 73–78. 10.3354/dao03126 PubMed DOI

Whyte, P., Mc Gill, K., Collins, J. D., & Gormley, E. (2002). The prevalence and PCR detection of salmonella contamination in raw poultry. Veterinary Microbiology, 89(1), 53–60. 10.1016/S0378-1135(02)00160-8 PubMed DOI

Nephrocalcinosis in farmed salmonids: diagnostic challenges associated with low performance and sporadic mortality