Vol. 104: 23–34, 2013 DISEASES OF AQUATIC ORGANISMS Published April 29 doi: 10.3354/dao02580 Dis Aquat Org Complex interaction between proliferative kidney disease, water temperature and concurrent nematode infection in brown trout Heike Schmidt-Posthaus1,*, Pascale Steiner2,3, Barbara Müller1, Ayako Casanova-Nakayama1 1Centre for Fish and Wildlife Health, Institute of Animal Pathology, University of Berne, Laenggassstrasse 122, PO Box 8466, 3001 Berne, Switzerland 2Federal Office for the Environment FOEN, 3003 Bern, Switzerland 3Present address: Ecowert GmbH, Denter Tumas 6, 7013 Domat/Ems, Switzerland ABSTRACT: Proliferative kidney disease (PKD) is a temperature-dependent disease caused by the myxozoan Tetracapsuloides bryosalmonae. It is an emerging threat to wild brown trout Salmo trutta fario populations in Switzerland. Here we examined (1) how PKD prevalence and pathology in young-of-the-year (YOY) brown trout relate to water temperature, (2) whether wild brown trout can completely recover from T. bryosalmonae-induced renal lesions and eliminate T. bryo - salmonae over the winter months, and (3) whether this rate and/or extent of the recovery is influ- enced by concurrent infection. A longitudinal field study on a wild brown trout cohort was con- ducted over 16 mo. YOY and age 1+ fish were sampled from 7 different field sites with various temperature regimes, and monitored for infection with T. bryosalmonae and the nematode Raphi- dascaris acus. T. bryosamonae was detectable in brown trout YOY from all sampling sites, with similar renal pathology, independent of water temperature. During winter months, recovery was mainly influenced by the presence or absence of concurrent infection with R. acus larvae. While brown trout without R. acus regenerated completely, concurrently infected brown trout showed incomplete recovery, with chronic renal lesions and incomplete translocation of T. bryosalmonae from the renal interstitium into the tubular lumen. Water temperature seemed to influence com- plete excretion of T. bryosalmonae, with spores remaining in trout from summer-warm rivers, but absent in trout from summer-cool rivers. In the following summer months, we found PKD infec- tions in 1+ brown trout from all investigated river sites. The pathological lesions indicated a re- infection rather than a proliferation of remaining T. bryosalmonae. However, disease prevalence in 1+ trout was lower than in YOY. KEY WORDS: Tetracapsuloides bryosalmonae · PKD · Raphidascaris acus · Water temperature · Salmo trutta fario · Pathology · Recovery Resale or republication not permitted without written consent of the publisher INTRODUCTION biological balance between fish hosts and infectious agents. The aqueous environment exhibits complex inter- Proliferative kidney disease (PKD) is a tempera- play between physical parameters, chemical compo- ture-dependent disease which is considered an sition and biological load. Studying host/pathogen emerging threat to both wild and cultured salmonid interactions in wild fish is one possibility of investi- fish in Europe (Okamura et al. 2011). The spread and gating the influence of environmental factors on the consequences of the disease are suspected to be *Email: [email protected] © Inter-Research 2013 · www.int-res.com 24 Dis Aquat Org 104: 23–34, 2013 enhanced by water temperature (Wahli et al. 2008, depending mainly on water temperature (Moravec Bettge et al. 2009, Okamura et al. 2011). The disease 1970b, 1979, 2004). In the intermediate fish hosts, is caused by Tetracapsuloides bryosalmonae (Myxo- like roach or stone loach, the immune response was zoa: Malacosporea) (Hedrick et al. 1993, Canning et described as granuloma or abscess-like lesions con- al. 2000, Okamura et al. 2001), with bryozoans as taining nematode larvae at different stages of degen- invertebrate hosts (Anderson et al. 1999, Longshaw eration (Valtonen et al. 1994, Koubková et al. 2004). et al. 1999, Okamura et al. 2001) and salmonids as In brown trout serving as intermediate hosts, the vertebrate hosts (Feist & Bucke 1993, Hedrick et al. immune response to third-stage larvae has not yet 1993). T. bryosalmonae infects fish through the skin been described in detail. and gills (Feist et al. 2001, Longshaw et al. 2002). The aims of our study were to examine: (1) how the After invasion, this parasite is distributed systemi- PKD prevalence and pathology in YOY brown trout cally, with the kidney as the main target organ (Kent relate to the level and variation of water temperature & Hedrick 1985). Here, T. bryo salmonae multiplies in the river; (2) whether wild brown trout can com- and differentiates from extra sporogonic stages, pletely recover from Tetracapsuloides bryo sal mo - mainly in the renal interstitium, to sporogonic stages nae− induced renal lesions and eliminate this parasite in the tubular lumen. In young-of-the-year (YOY), over the winter months; (3) if this rate and/or extent proliferative and granulomatous nephritis and necro- of the recovery is influenced by concurrent infec- tizing vasculitis with thrombus formation are host tions, here with Raphidascaris acus; and (4) how dis- reactions to parasite infestation (Hedrick et al. 1993, ease develops in older fish after recurrent contact to El-Matbouli & Hoffmann 1994, Bettge et al. 2009). In T. bryosalmonae. A longitudinal study on a wild laboratory studies, we pre viously showed that surviv- brown trout cohort was conducted over 16 mo. In par- ing fish can recover with complete regeneration of allel to detailed measurements of the water tempera- renal morphology (Schmidt-Posthaus et al. 2012). ture in the respective river system, fish were sampled After complete re generation, no T. bryosalmonae from 7 different river sites at approximately 6, 9, 16 spores were de tected in surviving rainbow trout and 18 mo of age and monitored for PKD and R. acus by histology or immunohistochemistry (Schmidt- infection, the associated pathology and the location Posthaus et al. 2012). However, little is known about of T. bryosalmonae and R. acus. regeneration and the possible influencing factors under field conditions in the natural host in Switzer- land, the brown trout Salmo trutta fario. In addition, MATERIALS AND METHODS the possible development of disease after recurrent contact with T. bryosalmonae in surviving fish in the Study sites and fish sampling following year(s) has not yet been studied. Here, we investigated disease development and the location of The experiments were conducted over a period of T. bryosalmonae in brown trout under the influence 16 mo (June 2008 until September 2009). The river of varying water temperatures and the presence or system examined (Lyssbach) is situated in the west- absence of a concurrent infection, namely infection ern midlands of Switzerland and passes through with Raphidascaris acus larvae. rural and urban areas before feeding into a larger Raphidascaris acus is a widely distributed nema- river, the Alte Aare. The Lyssbach has a number of tode of the digestive tract in predatory fish. Definitive tributaries; 4 of these were included in this study hosts are most frequently pike and brown trout, but (Allenwilbach, Chuelibach, Seebach and Schmide- other salmonids and occasionally other fish species bach) (Fig. 1). Fish can migrate from the tributaries can also be affected (Smith 1984). The life cycle into the Lyssbach and vice versa. In former years, involves obligate intermediate hosts: various fish brown trout Salmo trutta fario were regularly stocked species, cyclostomes and gammarids, and rarely also in the Lyssbach, nevertheless electrofishing revealed amphibians (Moravec 1970a, 1996, 2004, Smith decreasing numbers of fish along the course of the 1984). In the brown trout, nematodes usually mature river (fishing statistics 1993 to 2005, Fishery Inspec- in the gut (Smith 1984). Occasionally, third-stage lar- torate Bern unpubl. data). Stocked fish originated vae are found in the stomach wall, pyloric caeca, from wild mother animals from the Lyssbach. Stock- intestine, or liver of young brown trout (Moravec ing was stopped during the whole experimental 2004). However, the true relevance of brown trout as period, starting a year before until initiation of the intermediate hosts for this nematode is still unclear. experiment, to be certain a single brown trout gener- The development of R. acus is clearly seasonal, ation was investigated originating from the naturally Schmidt-Posthaus et al.: PKD, water temperature and concurrent infection 25 histochemical (IHC) examination. The other half of the kidney was placed in a separate Eppendorf tube and snap frozen in liquid nitrogen for polymerase chain reaction (PCR) examination. Histopathology and immunohistochemistry Fixed samples were paraffin-embedded and rou- tinely processed for histological and IHC examina- tion; sections of 3 µm thickness were cut. For the kid- ney, 2 consecutive sections were prepared. The first slide was examined histologically. The second slide was used for IHC staining. Sections for histology were stained with haema- toxylin-eosin (H&E) and examined by light micro - Fig. 1. The Lyssbach and its tributaries flow (black arrows) scopy. Histopathological changes of the gills, kidney, northwestwards into the Alte Aare near the Swiss city of liver and intestine were graded as 0 (none), 1 (scat- Bern. The 7 sampling sites are marked by dots. Summer- cool tributaries (Chuelibach, Allenwilbach) are labelled in tered), 2 (mild), 3 (mild to moderate), 4 (moderate),