A Fish Health Survey of Selected Austrian Rivers: Implications for Stocking Practices and a Wide Distri- Bution of Tetracapsuloides Bryosalmonae

Home , Lasberg, Saprolegnia, Spring viraemia of carp, Tetracapsuloides, Tragwein

Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria 105 (2018)

Clinical Division of Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria A fish health survey of selected Austrian rivers: Implications for stocking practices and a wide distribution of Tetracapsuloides bryosalmonae

E. LEWISCH* and M. EL-MATBOULI

received October 10, 2017 accepted February 22, 2018

Keywords: proliferative kidney Schlüsselwörter: Proliferative disease, Tetracapsuloides bryo- Nierenerkrankung, Tetracapsuloides salmonae, monitoring, wild fish, bryosalmonae, Gesundheitsmoni- stocked fish, infectious disease. toring, Wildfische, Fischbesatz, Infektionskrankheit.

Summary Zusammenfassung Dabei gelangten 116 Bachforellen, 22 Regenbogenforellen (Oncor- The aim this study was to as- Untersuchung der Fischgesund- hynchus mykiss), elf Aiteln sess the health status of fish, es- heit in ausgewählten österreich- (Squalius cephalus) und ver- pecially brown trout (Salmo trutta), ischen Gewässern: hohe Präva- schiedene andere Fischarten zur in waters where severe decrease lenz von Tetracapsuloides bryo- Untersuchung. Alle Fische wur- of brown trout catches had been salmonae und Auswirkungen den jeweils einer pathoanatomi- observed. Over the course of one von Besatzmaßnahmen schen, parasitologischen, bak- year, 116 brown trout, 22 rain- teriologischen und virologischen bow trout (Oncorhynchus mykiss), Einleitung Untersuchung unterzogen. eleven European chubs (Squalius In verschiedenen Regionen Euro- cephalus) and several speci- pas wird aktuell eine Änderung Ergebnisse mens of other fish were sampled. der aquatischen Fauna und insbe- Die Studie konnte eine wei- Samples were taken each month sondere ein Rückgang der Bach- te Verbreitung des Erregers der and originated from 22 different forellen (Salmo trutta) beobachtet. Proliferativen Nierenerkrankung sites along 15 rivers. All fish un- Dabei liegen zahlreiche Untersu- (PKD) in den untersuchten derwent necropsy, including par- chungen zum Einfluss von verän- Flüssen nachweisen. Darüber hin- asitological, bacterial, and viral derten Umweltbedingungen und aus konnten der Erreger der Viralen examination. The most relevant dem Besatz mit genetisch unpas- Hämorrhagischen Septikämie aus finding was a wide distribution of senden Fischen vor. Im Gegen- einer Regenbogenforelle sowie das Tetracapsuloides bryosalmonae, satz dazu ist der Beitrag von Virus der Infektiösen Pankreasne- the causative agent of proliferative Infektionskrankheiten zum Rück- krose aus fünf Regenbogen- und kidney disease (PKD) (12/22 sites) gang der Populationen kaum un- einer Bachforelle isoliert wer- with high prevalence in the affect- tersucht. In dieser Studie wurde den. Von drei Bachforellen wurde ed waters. One rainbow trout was der Gesundheitszustand von Bach- Aeromonas salmonicida achromo- found to be infected with viral hem- forellen und anderen Wildfischen in genes aus Nierengewebe kultiviert. orrhagic septicemia virus (VHS-V) betroffenen Gewässern erhoben. Parasiten der Haut und der Kiemen and several rainbow trout and wurden bei 6,9 % der Bach- und brown trout were positive for in- Material und Methode 4,5 % der Regenbogenforellen fectious pancreatic necrosis-virus Im Laufe eines Jahres wurden nachgewiesen. Während die- (IPN-V). Three brown trout were monatlich Fische von 22 Entnah- se Parasiten nicht mit pathologi- positive for Aeromonas salmonici- mestellen aus 15 Flüssen beprobt. schen Erscheinungen assoziiert

*E-mail: [email protected]

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da achromogenes; the bacterium was cultivated from waren, wurden gastrointestinale Läsionen bei mit the head kidney. Infestation with skin and gill parasites Acanthocephalen befallenen Fischen beobachtet. Bei ei- was demonstrated in 6.9 % of brown trout and 4.5 % nigen Bachforellen wurden durch Nematodenbefall her- of rainbow trout. There was no obvious tissue dam- vorgerufene gastrointestinale Granulome nachgewiesen. age caused by these ectoparasites. Infestation with acanthocephales led to gastrointestinal lesions and Schlussfolgerungen even gut perforation in brown trout, European chub, Zum ersten Mal wurde in Österreich eine hohe brook trout (Salvelinus fontinalis) and common barbel Prävalenz von T. bryosalmonae bei Bach- und (Barbus barbus). In several brown trout, granulomas Regenbogenforellen in Freigewässern nachgewie- in the intestinal tract were associated with nematode sen. Darüber hinaus wurden Fälle von Infektionen infection. mit atypischer Furunkulose, VHS und IPN entdeckt. Während für die Verbreitung von T. bryosalmonae Abbreviations: ABOL = Austrian Bar Code of Life; BF = bluegill fry; und somit der PKD Umweltfaktoren eine große Rolle CCB = Cyprinus carpio brain; CHSE = Chinook salmon embryo; COS spielen, ist für die Verbreitung der anderen genann- = Columbia sheep blood; CyHV-3 = Cyprinid herpesvirus-3; IHN = in- ten Infektionserreger der Besatz mit Fischen aus fectious haematopoetic necrosis; IPN = infectious pancreatic necrosis; MALDI-TOF MS = matrix assisted laser desorption ionization-time Aquakultur maßgeblich verantwortlich. Wir empfehlen of flight mass spectrometry; MEM = Minimal Essential Medium; PKD daher für den Besatz von Freigewässern ausschließ- = proliferative kidney disease; SSU rDNA = small subunit ribosomal lich Fische die nachweislich frei von VHS, IHN und IPN DNA; SVCV = spring viraemia of carp virus VHS = viral haemorrhagic sind zu verwenden. septicaemia; wpi = weeks post infection; Materials and methods Introduction Sampling

Brown trout is a widely distributed native species in The sampling period was one year, starting in September 2015. continental Europe with many autochthonous popula- Samples were taken once a month, except for June 2016 when tions. A decline of this fish species has been observed floods occurred. All samples were taken from rivers in the Austrian for several years in many regions (GILES, 1989; ARIAS federal state Upper Austria. In total, 22 sites along 15 rivers were et al., 1995; SCHMIDT et al., 1999; BURKHARDT- sampled. Most of the sampled rivers are situated in a region made of HOLM et al., 2002). Environmental changes, especial- granite and gneiss north to the river Danube (“Mühlviertel”) and enter ly global warming and anthropogenic influence factors, the river Danube, while a few of them drain their waters to the north are discussed as potential causes (BURKHARDT- (Moldova) (details for sampling sites are given in Fig. 1 and Tab. 1). In some rivers, upstream and downstream samples were taken (Fig. 1). HOLM et al., 2002, 2005; BORSUK et al., 2006; Fish were obtained by angling and electro fishing. The number and FILIPE et al., 2013). In Austria, a systematic investiga- species of collected fish as well as frequency of sampling varied con- tion of the anecdotally reported decline of brown trout siderably by site (Fig. 2, Tab. 1) due to the varying fish population in populations has not been carried out. Most rivers are the rivers and organizational issues. Six of the sites were sampled managed by local fishery associations and are stocked regularly, four of which were classical habitats of brown trout, as with trout and other fish on a regular basis. This makes given by history, water temperature and water quality (Tab. 1). Fish it particularly difficult to assess the health state of the were killed by a sharp blow to the head at the collection site and autochthonous fish populations. Reports by the fishery transported to the clinic on ice within 24 hours. managers about a decline of brown trout and the first Gross examination and sampling of fish evidence of Saprolegnia parasitica in Austria (unpub- lished) in the river Feistritz (region "Mühlviertel", feder- Upon arrival, length and weight of each fish were recorded. The al state Upper Austria) prompted fishery managers to body surface was carefully inspected for evidence of skin lesions, initiate the health survey presented in this study. The parasites and fungal infection. Morphological variations were docu- aim of the project was to achieve information about mented. From the dorsolateral surface and the skin region under the left pectoral fin as well as from the dorsal and caudal fins wet mounts the general health condition of wild fish, especially were prepared with a cover glass, spread on a slide together with brown trout. While specific examinations for detection a drop of water and examined with an Olympus BX 53® light micro- of Tetracapsuloides bryosalmonae originally were not scope (Olympus Austria). After removal of the operculum, epithelium part of the project, already the first shipment of sam- from a few gill filaments was scraped and examined as described ples included fish with kidney alterations indicative for for the skin smears. Sex, contents of the gastrointestinal tract and proliferative kidney disease (PKD). Thus, molecular di- any abnormal findings were documented. Cysts, capsules, nodules agnostics for T. bryosalmonae were included for all fol- and other alterations were examined microscopically. The intestinal lowing samples. tract was opened in its entire length and inspected for parasites. Smears from the pyloric caeca region (if applicable), the midgut and the hindgut were prepared. The epiaxal musculature was sliced and inspected for cysts, larval worms and haemorrhages. Other organs were only included in the parasitological examination if suspicious alterations were observed. Parasites were identified according to

82 Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria 105 (2018)

morphological criteria (HOFMANN, 1999). Nematodes and acanthocephales were con- served in 70 % ethanol for molecular species identification. For bacteriological examination, the head kidney was sampled, and the tissue was spread and inoculated on Columbia sheep blood (COS) agar plates (Thermo Fisher Scientific, Oxoid Limited, Cambridge, UK) at 20 °C. From salmonid species, tissues from the anterior kidney, the spleen and the brain were aseptically transferred into Eagle’s Minimum Essential Medium (MEM) (Sigma- Aldrich, Vienna, Austria) for inoculation in cell culture. In cyprinid fish, gill epithelium was additionally sampled for cell culture. From the first shipment, only fish suspected for PKD were tested for the disease. In the next batch- es, kidney tissue from all fish from the same collection site and of the same species were pooled. During the course of the study, brown trout as well as rainbow trout were individually sampled to get a better impression of the real prevalence of the infection. The kidneys of several non-salmonid fish were screened for the presence of DNA from T. bryosalmonae. For this, a 3x3x3 mm piece of the posterior kidney was aseptically transferred into a 2 ml Eppendorf tube and stored at -20 °C.

Bacteriology

Incubated sheep blood agar was checked daily, growing cultures were subcultured and finally identified by gram stain, light microscopy and API identification systems (BioMerieux, Marcy l'Etoile, France). In case of unclear results, a matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Microflex LT Fig. 1: A Map of Austria, the rectangle indicates the region of sampled rivers. B Schematic instrument, Bruker Daltonic GmbH, Leipzig, overview of sampled rivers and sampling sites ( ). White stars ( ) indicate PKD positive Germany) was performed. sites / A Karte von Österreich mit dem Untersuchungsgebiet B Schematischer Überblick Virology über die Beprobungsstellen ( ). Weiße Sterne ( ) bezeichnen PKD positive Stellen.

Salmonid virus isolation attempts for viral haemorrhagic septicaemia virus (VHS-V), infectious haematopoetic necrosis virus (IHN-V) and infectious pancreatic necrosis virus (IPN-V) were carried out according to Commission implementing decision (EU) 2015/1554 (ANONYMOUS, 2015). To isolate cyprinid herpesvirus 3 (CyHV-3) and spring viraemia of carp virus (SVCV), Bluegill fry (BF- 2), Chinook salmon embryo (CHSE-214), and Cyprinus carpio brain (CCB) cells were inoculated with organ suspensions from chubs and incubated at 20 °C in addition to the cell lines Epithelioma papulosum cyprini (EPC). Virus was identified by ELISA (TEST-LINE, Ltd., Brno, The Czech Republic).

PKD molecular screening

® DNeasy Blood and Tissue Kit (Qiagen, Fig. 2: Number of fish species sampled at each site; EB = European bullhead; Hilden, Germany) was used on kidney tissue. CYP = Cyprinids; GR = Grayling, SF = Salvelinus fontinalis, book trout; EC = European chub; After homogenization in 180 μl lysis buffer RT = rainbow trout, BR = brown trout / Anzahl der Fischarten von jeder Beprobungsstelle

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Tab. 1: Details of sampling sites: Names of rivers including abbreviations, latitude and longitude of sampling sites, and number of sampled fish per site. (1) sites sampled regularily, (2) classical brown trout habitats / Informationen zu den Beprobungsstellen: Name des Flusses inkl. verwendeter Abkürzung, Breitengrad und Längengarde der Beprobungsstellen, Anzahl der pro Stelle entnommenen Fische. (1) regelmäßig beprobte Stellen, (2) typische Lebensräume der Bachforelle

Brown Rainbow Other sal- River Latitude Longitude Cyprinids Others trout trout monid fish Große Mühl, Aigen- 1 Leuciscus 1 Cottus Schlägl (1) (2) 48.6449 13.9586111 20 0 0 idus gobio (Große Mühl A-S) Große Mühl, Rohrbach- 1 Thymallus 1 Squalius Berg (1) (2) 48.565 14.0333333 21 1 0 thymallus cephalus (Große Mühl R B) Feistritz Lasberg 1 Salvelinus 5 Gobio 48.4771 14.558701 24 0 0 (Feistritz L) (1) (2) fontinalis gobio Feldaist Apfoltern 1 Squalius 2 Lampetra 48.550214 14.489422 18 0 0 (Feldaist A) (1) (2) cephalus fluviatilis Krems, Kremsmünster 11 Phoxinus 48.05161 14.13232 3 6 0 0 (Krems K) (1) phoxinus Krems, Neuhofen 1 Gobio 48.1525519 14.22995567 3 8 0 0 (Krems N) (1) gobio Feldaist, Kefermarkt- 1 Barbartula 48.481456 14.505391 2 0 0 0 Galgenau (Feldaist KG) barbatula Felberbach Maltsch, 48.583445 14.558331 1 1 0 0 0 Windhaag (Felberbach M) Kleine Gusen 1 Barbus 48.3667 14.469339 0 1 0 0 (Kleine Gusen) barbus Große Gusen 3 Squalius 48.350157 14.41666 3 0 0 0 (Große Gusen) cephalus Reichenthal Kettenbach 1 Barbus 1 Cottus 48.550089 14.394445 2 0 0 (Reichenthal K) barbus gobio 6 Squalius Große Rodl (Große Rodl) 48.470487 14.280763 2 0 0 0 cephalus Steyr Sierninghofen 48.049685 14.339261 0 1 0 0 0 (Steyr Sier) Steyr Steinfeld 48.03379 14.336514 1 0 0 0 0 (Steyr Stein)

Steyr Steyr (Steyr Steyr) 48.065351 14.446161 1 3 0 0 0

Teichl, Spital 2 Salvelinus 47.674843 14.348666 0 0 0 0 (Kohlhofteich) (Teichl) fontinalis Grünbach Offenhausen 48.15 13.8333 3 0 0 0 0 (Grünbach) Haselbach Tragwein 48.389589 14.630549 2 0 0 0 0 (Haselbach) Waldaist St. Leonhard bei 1 Thymallus 48.419484 14.666709 1 0 0 0 Freistadt (Waldaist F) thymallus Waldaist St. Leonhard 1 Salvelinus 48.422309 14.67767 0 0 0 0 Haselmühle (Waldaist H) fontinalis Hängerbach Riemetschlag 48.568316 14.5611241 7 0 0 0 0 (Hängerbach R) Hängerbach Leithenmühle 48.563658 14.5664674 2 1 0 0 0 (Hängerbach L)

84 Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria 105 (2018)

in a bench mixer (Tissue Lyser II, Qiagen) with 1 mm stainless steel beads (Qiagen) for 1 min at 30 Hz, the samples were incubated with Proteinase K at 56 °C and DNA extraction was completed as per the manufacturer’s instructions. DNA was quantified by spectrophotometry (NanodropTechnologies). For detection of T. bryosalmonae, PKD-specific SSU-rDNA primers PKX5f and PKX6r were used according to the protocol by KENT et al. (1998). Amplicons were visualized by gel electrophoresis and purified using a MiniElute gel extraction kit (Qiagen) according to the manufacturer`s instructions. They were se- quenced in a commercial sequencing laboratory (LGC Genomics, Berlin, Germany). Molecular and phylogenetic identification of nematodes and acanthocephales are currently in progress and will be published as a part of the Austrian Bar Code of Life Project (ABOL). Fig. 3: Total length of sampled brown trout in cm from each sampling site (different shades of gray are used for better visibility) / Ganzkörperlänge der untersucht- Results en Bachforellen in cm von jeder Entnahmestelle (Verschiedene Grautöne dienen der Übersichtlichkeit) Morphology and pathoanatomy

Most of the brown trout had a total body length between 17 and 30 cm (Fig. 3). Two rainbow trout had a shortened operculum. In nine brown trout and one rainbow trout the stomach was filled with stones. Five of these brown trout came from the same collecting site (Große Mühl, Aigen Schlägl), while the other four came from three different lo- cations, two of them in the same river (1x Krems, Kremsmünster; 2x Krems, Neuhofen; 1x Feldaist, Apfoltern). In seven brown trout and two European chubs, nodules were found in the wall of the stomach and the anterior intestine (4x Große Mühl, Aigen Schlägl; 1x Große Mühl, Rohrbach Berg; 2x Krems, Neuhofen; 1x Krems, Kremsmünster; 1x Reichenthal, Kettenbach), which were associated with acanthocephales infection in five fish (three trout, both chub). One of the brown trout also had granulomas associated with nematode infection in the liver. Acute intestinal inflammation was mostly noted in connection Fig. 4: A normal kidney of a brown trout, B enlarged, pale kidney of a brown trout in- with intestinal parasitosis. In four brown fected with Tetracapsuloides bryosalmonae / A normale Niere einer Bachforelle, B ver- trout and six rainbow trout, enlarged kid- größerte, blasse Niere einer mit Tetracapsuloides bryosalmonae infizierten Bachforelle ney were observed (Fig. 4). Sporadic findings included fin damages, healed injuries, bleeding in the eyes, exophthalmia, pale gills, Parasitology (excluding T. bryosalmonae) swelling and mucus on the gills, ulceration of the stomach, dilatation of the stomach and intestine, enlarge- A considerable difference in total parasitic load could ment of the liver, discoloration of the liver or haemor- be observed between brown and rainbow trout. While rhages in the intestinal wall. There was no evidence of 17.2 % of the former showed parasitic infection, in rain- fungal infection in any of the fish. bow trout this number was only 4.5 %, which repre-

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sents a single fish with aDactylogyrus sp. infection. Intestinal parasitosis of brown trout was caused by nematodes (6.0 %) and acanthocephales. Nematodes in the intestinal tract were also found in 1/11 (9.1 %) minnows (Phoxinus phoxinus). All these nematode infected fish originated from two sites of the same river (Große Mühl). Heavy infection with acanthocephales was observed in several fish species (brown trout 5/116, 4.3 %; European chub 6/11, 54.5 %; brook trout 2/4, 50 % and barbel 2/2, 100 %) from various sampling sites. Only a few fish showed low numbers of ectoparasites: Trichodina sp. was detected in skin smears of two brown trout and in the gill epithelium of one barbel, Gyrodactylus sp. in the skin of three and in the gills of one brown trout and in the skin of one European chub. Dactylogyrus sp. was found in the skin of one rainbow trout and one brown trout. One leach was found on the skin of a brown trout (Fig. 5).

Bacteriology

From 61 kidney smears (out of 96), no colony growth on sheep blood agar could be observed. In the positive cases, mostly a mixed culture of faculta- Fig. 5: : Number of fish free of parasites and infected fish from each sampling site (ex- tive pathogen or commensal bacteria cluding T. bryosalmonae). A brown trout, B all other species. (species in which no par- was isolated. Bacteria identified were asites were found are not considered); BT- = parasite free brown trout; N = nematode; A = acanthocephales; G = Gyrodactylus sp.; D = Dactylogyrus sp.; T = Trichodina sp.; L Pseudomonas putida, Pseudomonas = leach; RT- = parasite free rainbow trout; RT D = rainbow trout with Dactylogyrus sp.; fluoreszens, Pseudomonas sp., EC- = parasite free European chub; EC A = European chub with acanthocephales; EC G Enterobacter sp., Aeromonas hydro- = European chub with Gyrodactylus sp; SF- = parasite free brook trout (Salvelinus fon- phila, A. sobria, Plesiomonas shig- tinalis); SF A = brook trout with acanthocephales; B- = parasite free barbel; B A = barbel elloides, Klebsiella sp., Actinobacter with acanthocephales; B T = barbel with Trichodina sp.; M- = parasite free minnow; M sp., Serratia sp., Pantoea sp., N = minnow with nematode / Anzahl der parasitenfreien und infizierten Fische (außer T. Shewanella putrefaciens, Citrobacter bryosalmonae) von jeder Entnahmestelle. A Bachforellen, B andere Spezies. (Spezies, in sp., Pasteurella sp., Vibrio sp., and denen gar eine Parasiten gefunden wurden, sind nicht dargestellt). Lactobacilli. From the kidneys of three brown trout Aeromonas salmonicida subsp. achromogenes was isolated (Große Mühl, in the eyes, pale gills and a distended intestinal tract Aigen Schlägl; Große Mühl, Rohrbach-Berg; Große filled with clear mucus). From one sample of another Gusen, Gallneukirchen). rainbow trout showing unspecific symptoms (slightly enlarged spleen, intestinal tract filled with yellow mu- Virology coid contents), viral haemorrhagic septicaemia-virus (VHS-V) and T. bryosalmonae were detected addition- Samples from five rainbow trout and one brown trout ally to IPN-V. were tested positive for IPN-V. These fish were collected from six different sites of three rivers (Große Mühl, PKD molecular screening Rohrbach-Berg; Große Mühl, Aigen Schägl; Krems, Kremsmünster; Krems, Neuhofen; Steyr, Steyr; Steyr, Of the 22 collection sites, twelve sites at ten rivers Sierninghofen). One of the rainbow trout showed symp- were positive for T. bryosalmonae. In rainbow trout, toms indicative for IPN in young fish (haemorrhages 10/18 (55.6 %) samples tested positive and in brown

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Routine bacteriology mostly resulted in mixed cultures of facultatively pathogenic bacteria which are endemic to fish and their environment. Given the fact that the fish had been dead for up to 24 hours, only the evidence of obligate pathogens shall be discussed. Three cases of atypical furunculosis in brown trout (two from the same river but from different sampling sites, one from a river which was sampled only once) were detected. The causative agent of the disease is Aeromonas sal- Fig. 6: Numbers of PKD positive and PKD negative brown and rainbow trout samples from each sampling site. Only fish which were actually sampled for PKD are included. Pools are considered as one monicida subsp. achro- sample. RT = rainbow trout; BT = brown trout / Anzahl an PKD positiven und PKD negativen Bach- mogenes, a bacterium und Regenbogenforellen von jeder Entnahmestelle. Nicht beprobte Fische sind nicht berücksichtigt. affecting a wide variety of Pools sind als Einzelprobe dargestellt. fish species (WIKLUND and DALSGAARD, 1998) in aquaculture and in the wild. trout 36/98 (36.7 %) (Fig. 6). Besides in salmonid fish, According to WIKLUND and DALSGAARD (1998), col- T. bryosalmonae specific PCR resulted in amplification onization of internal organs with atypical A. salmoni- products in three European chubs, one European bull- cida is uncommon and the port of entry are ulcerat- head (Cottus gobio) and one minnow (Phoxinus phox- ed skin lesions. Besides discreet signs of generalized inus). Sequences of European chub and European infection, the three affected fish showed enteritis. bullhead PCR products showed 95 % and 94 % iden- This might suggest an entry of atypical A. salmonici- tity to T. bryosalmonae isolate TbStH 18S ribosomal da via the gastrointestinal tract. Furunculosis, an in- RNA gene, respectively, whereas for the minnow the fection with A. salmonicida subsp. salmonicida, has identity was 100 %. Alignment of the sequences of the been successfully induced by oral infection in Atlantic European chub and the bullhead showed both 91 % salmon (Salmo salar) (ROSE et al., 1989; CIPRIANO identity. et al., 1997). JUTFELT et al. (2006) demonstrated the translocation of viable A. salmonicida across the intestine of rainbow trout. Thus, the intestine as a site Discussion of inapparent infection with atypical A. salmonicida and colonisation of internal organs in fish with an im- Our findings demonstrate the occurrence of various paired health status seem possible. Also, transmission potentially dangerous pathogens in fish populations of of the bacterium from latent carrier fish must be con- stocked rivers in Upper Austria. A possible contribu- sidered. High mortality rates of brown trout caused by tion of the introduced fish and the wide distribution ofT. atypical A. salmonicida have not been reported to the bryosalmonae infected fish in the sampled rivers shall best of our knowledge. Low mortalities however may be discussed below. have remained unnoticed or may have been attribut- While the infection with ectoparasites seems negli- ed to other pathogens (AUSTIN and AUSTIN, 2016). gible, gastrointestinal infestation with nematodes and While the spread of furunculosis from asymptomatic acanthocephales was associated with the formation of wild carrier fish to aquaculture has been acknowledged granulomas in the intestinal tract or the liver. All nema- (McCARTHY, 1977), less is known about a vice versa tode infected fish originated from two sampling sites of infection. the same river. Marked inflammation and ulceration of Virological examination revealed infection of one the anterior intestines up to perforation was observed rainbow trout (from the Große Mühl, Rohrbach-Berg) in European chub, brook trout and common barbel with VHS-V, a notifiable finding regarding EU legisla- heavily infected with acanthocephales. A negative im- tion. The fish was sampled in April, at a temperature of pact of these parasites on health and fitness of the fish approximately 7 °C. A shortened operculum indicated seems compelling. that it was most likely from aquaculture, having been stocked despite carrying VHS-V. In Austria, stocking

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isolated IPN-V from environmental samples in the vicinity of IPN-V infected fish farms but concluded that a reinfection of the farms by environmental reservoirs seems unlikely. The authors did not in- vestigate possible infection of wild fish originating from the infected farms. BUCKE et al. (1979) found that wild fish populations were not affected by IPN disease from the effluents of infected farms. Nevertheless, stocking of rivers with fish from farms with an un- known health status bears the risk of introducing carrier fish and rais- ing the prevalence among salmonid and possibly other fish species (ENZMANN et al., 1987; MEIER et al., 1994). In the present study, all IPN-V positive fish were adult and very likely stocked despite their carrier status. Such carri- Fig. 7: Temperature changes in °C in some of the sampled rivers; data from the other rivers er fish might shed the virus hori- were not available. The annual average temperature from 70 to 100 measurements was tak- zontally under stressful conditions en for each river. The total average was calculated from the annual mean temperatures of (CRANE and HYATT, 2011) or ver- all measured rivers (from ANONYM, 2015a). / Temperaturverlauf in °C einiger der beprobten tically during spawning. Thus, the Flüsse; von den anderen Gewässern waren keine Daten verfügbar. Von jedem Fluss wurde das virus might be propagated locally jeweilige Jahresmittel aus 70-100 Messungen ermittelt. Aus diesen Jahresmitteln wurde ein by fish and over larger distances gemeinsamer Mittelwert dargestellt (Quelle: ANONYM, 2015a). by predatory birds (CRANE and HYATT, 2011). It has been recog- of rivers with fish from category III farms according nized for a long time that IPN associated mortalities to Council Directive 2006/88/EC is widely practiced. among trout younger than six months in aquaculture Official investigations found that the affected river had can reach 10–90 %. While epizootics in farmed and not been stocked six months before the VHS diagno- wild fish cannot be compared because of the different sis. Thus, no further epidemiological investigation was environmental factors, we believe that any disease af- conducted. Besides of being a carrier of VHS-V, this fecting young fish must be considered when a decline trout was also positive for IPN-V and T. bryosalmonae. of indigenous fish populations is discussed. Clinical signs were not indicative, with a stomach filled While for the viral and bacterial pathogens men- with food and mucoid yellow contents in the posteri- tioned above the introduction of infected fish seems or part of the intestines. Infection with IPN-V was also to play a major role, the situation of T. bryosalmonae found in four more rainbow trout and one brown trout. may be different. To assess the significance of our find- The samples originated from six sites at three different ings, the results from four sites at three rivers shall be rivers (Große Mühl, Steyr, Krems). One of them (Steyr) considered first. In these rivers, sampling was carried was only sampled at three occasions. One rainbow out from nine to elven times, and a reasonable number trout showed clinical signs indicative for IPN infection of brown trout was submitted from each site (Tab.1). in young fish. Compared to only one IPN-V positive One upstream site (Große Mühl, Aigen Schlägl) was rainbow trout out of 3400 farmed and feral salmonids negative for T. bryosalmonae throughout the study sampled in Switzerland in 2000/2001 (KNUESEL et al., and it can be assumed that the parasite is not prev- 2003), the finding of six IPN-V and one VHS-V positive alent there. The distance to the T. bryosalmonae pos- in a relatively small sample pool of adult fish might in- itive sampling site at the same river is approximately dicate a notable distribution of IPN infections. Studies 11 km and the sites are separated by fish migration about prevalence of IPN-V in wild fish mostly show a barriers. However, such structures are also found in low prevalence in both salmonid and non-salmonid fish rivers where the parasite is prevalent up- and down- (MUNRO et al., 1976; ANON., 2003). Methods of de- stream. At the other three sites, 26/50 (52.0 %) of the tection and environmental conditions may influence samples were positive for T. bryosalmonae. This does the results of such studies, leading to varying conclu- not necessarily reflect the real number of infected fish, sions (RUANE et al., 2007). GREGORY et al. (2007) since pooled samples were examined at the early state

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of the study. Thus, the incidence might be even high- TOPS et al. ,2009) and food supply through eutrophica- er. At all the other 18 sites, no or only very few brown tion enforced by climate change and locally increased trout were collected, at one or two, in one case at three agricultural activity (WAHLI et al., 2002; ZIMMERLI occasions. Even some of the rivers with only a sin- et al., 2007), the colonies can also be transmitted by gle submission were found to be infected with T. bryo- anthropogenic and animal vectors. Statoblasts are salmonae (Fig. 6). All negative sites were sampled highly tolerant against poor conditions like desicca- only once. Given the wide distribution of this parasite tion and freezing (HENGHERR and SCHILL, 2011) in a region with no history of this disease so far, the and can be dispersed by inanimate as well as ani- question arises if the infection has simply not been no- mate vectors including waterfowl (FIGUEROLA et al., ticed before, or if it is just recently spreading. A simi- 2004). Transmission of PKD through stocking with in- lar observation from Finnish rivers has been published fected fish was suspected to spread T. bryosalmonae (VASEMÄGI et al., 2017). Environmental changes, es- in Norwegian rivers (STERUD et al., 2007). All rivers pecially climate change, are discussed in this context sampled in our study are regularly stocked with brown (WAHLI et al., 2008; OKAMURA et al., 2011). For a trout and rainbow trout. Although it is not always easy part of our sampling sites, temperature data were avail- to track the origin of these fish, a part of them may orig- able from 2005–2016 (Fig. 7). Increased water tem- inate from Italy (PINTER, 2008), where some farms in- peratures do not only influence the thriving of bryozo- tentionally expose trout fry to T. bryosalmonae during ans and increase the proliferation of T. bryosalmone in late summer and autumn to reduce PKD associated the bryozoan host (TOPS et al., 2006), but also impair losses in the following year (OKAMURA et al., 2011). the health status of some fish species. This is espe- The relevance of our findings regarding the detection cially true for brown trout, which is considered a true of T. bryosalmonae in non-salmonid fish (European host of T. bryosalmonae and therefore is adapted to chubs, European bullhead and minnow) with PCR has survive the infection under favourable conditions. In yet to be investigated. The 100 % identity of the sample rainbow trout, which appears to be a dead end host of from the minnow with T. bryosalmonae suggests that the parasite (BUCKE et al., 1991, GRABNER and EL- the parasite has been demonstrated for the first time in MATBOULI, 2008), the innate immune response caus- a cyprinid fish. So far, evidence of T. bryosalmonae in es a rather acute course of the disease (KUMAR et al., non-salmonid fish has only been described in Northern 2013). Among the trout positive for T. bryosalmonae, pike (Esox lucius) (BUCKE et al., 1991). enlargement of the kidney as a typical clinical sign of PKD was observed in 4/36 (11.1 %) brown trout and Acknowledgements 6/18 (33.3 %) rainbow trout. Renal enlargement in- The authors wish to gratefully acknowledge the excel- duced by T. bryosalmonae has been reported in brown lent collaboration with the Fishery Association of Upper trout between 8–17 weeks post infection (wpi) and in Austria (Oberösterreichischer Landesfischereiverband) rainbow trou 4–16 wpi (EL-MATBOULI et al., 2009; who funded the diagnostic examinations. We especially KUMAR et al., 2013). All cases of enlarged kidneys thank their head, Mr. Siegfried Pilgerstorfer. We further except one were observed between September and would like to thank Dr. Andrea Dressler and Dr. Oskar November. This is in accordance with a latency peri- Schachner for their valuable contributions at the bacte- od of four to eight weeks and an infection during the riology and virology labs. summer months. All rainbow trout and brown trout with enlarged kidneys measured between 17.5 and 35 cm. Assumingly, they were stocked fish with no contact to T. bryosalmonae prior to stocking. Other fish found PKD positive might have endured the disease earlier in life and reached the status of asymptomatic carri- ers. The fate of fry and fingerlings in these rivers re- Fazit für die Praxis: mains unclear. PKD may spread via spores, infected Der Besatz österreichischer Gewässer unterliegt bryozoans including their different developmental stag- derzeit nur sehr weit gefassten Bestimmungen, es, and infected brown trout. In an experiment, the vi- welche keinerlei Nachweis bezüglich der Freiheit ability of spores released from fish or bryozoa in wa- von spezifischen Krankheitserregern verlangen. ter was not very high and did not last longer than 24 Aufgrund von veränderten Umweltbedingungen hours (DE KINKELIN et al., 2002). Spread by infected können so gefährliche Pathogene in den bryozoans seems more likely; in fact, the parasite can Fischpopulationen akkumulieren. Diese Studie be passed on by fragmentation of infected colonies zeigt erstmals in Österreich das Vorkommen (MORRIS and ADAMS, 2006), budding, motile zooids solcher Erreger auf. Um die Bestände zu (GORGOGLIONE et al., 2016) and statoblasts (ABD- schützen, sind daher für Besatzfische gezielte ELFATTAH et al., 2014; HILL and OKAMURA, 2007; Untersuchungen auf relevante Erreger zu fordern TATICCHI et al., 2004). While the thriving of bryozoa und durchzuführen. is influenced by temperature (BETTGE et al., 2009;

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References ter bryozoans by waterfowl in Donana (southwest Spain). Can J Zool 82, 835–840. ABD-ELFATTAH, A., FONTES, I., KUMAR, G., SOLIMAN, H., FILIPE, A.F. MARKOVIC, D., PLETTERBAUER, F., TISSEUIL, C., HARTIKAINEN, H., OKAMURA, B., EL-MATBOULI, M. (2014): DE WEVER, A., SCHMUTZ, S., BONADA, N., FREYHOF, J. Vertical transmission of Tetracapsuloides bryosalmonae (2013): Forecasting fish distribution along stream networks: brown (Myxozoa), the causative agent of salmonid Proliferative Kidney trout (Salmo trutta) in Europe. Divers Distrib 19, 1059−1071. Disease. Parasitol 141, 482–490. GILES, N. (1989): Assessing the status of British wild brown trout, ANONYMOUS (2003): IPN in Salmonids: A review. A report prepared Salmo trutta, stocks - a pilot-study utilizing data from game fisher- by the Norwegian Seafood Federation (FHL) and VESO®,115 pp. ies. Freshwater Biol 21, 125–133. ANONYMOUS (2015): Commission implementing decision (EU) GORGOGLIONE, B., KOTOB, M.H., EL-MATBOULI, M. (2016): 2015/1554 of 11 September 2015 laying down rules for the appli- Migrating zooids allow the dispersal of Fredericella sultana cation of Directive 2006/88/EC as regards requirements for sur- (Bryozoa) to escape from unfavourable conditions and further veillance and diagnostic methods. Official Journal of the European spreading of Tetracapsuloides bryosalmonae. J Invertebr Pathol Union L 247/39. 140, 97–102. ARIAS, L., SANCHEZ, L., MARTINEZ, P. (1995): Low stocking inci- GRABNER, D.S., EL-MATBOULI, M. (2008): Transmission of dence in brown trout populations from northwestern Spain moni- Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) to tored by LDH-5* diagnostic marker. J Fish Biol 47, 170–176. Fredericella sultana (Bryozoa: Phylactolaemata) by various fish AUSTIN, B., AUSTIN, D.A. (2016): Bacterial fish pathogens: Disease species. Dis Aquat Org 79, 133–139. of farmed and wild fish. 6th ed., Springer, Switzerland. GREGORY, A., MUNRO, L.A., WALLACE, I.S., BAIN, N., RAYNARD, BETTGE, K., WAHLI, T., SEGNER, H., SCHMIDT-POSTHAUS, H. R.S. (2007): Detection of infectious pancreatic necrosis virus (2009): Proliferative kidney disease in rainbow trout: time- and (IPNV) from the environment in the vicinity of IPNV-infected temperature-related renal pathology and parasite distribution. Dis Atlantic salmon farms in Scotland. J Fish Dis 30, 621–630. Aquat Organ 83, 67–76. HILL, S.L.L., OKAMURA, B. (2007): Endoparasitism in colonial BORSUK, M.E., REICHERT, P., PETER, A., SCHAGER, E., hosts: pattern and processes. Parasitology 134, 841–852. BURKHARDT-HOLM, P. (2006): Assessing the decline of brown HENGHERR, S., SCHILL, R.O. (2011): Dormant stages in freshwa- trout (Salmo trutta) in Swiss rivers using a Bayesian probability ter bryozoans – an adaptation to transcend environmental con- network. Ecol Model 192, 224−244. straints. J Insect Physiol 57, 595–601. BUCKE, D., FINLAY, J., MCGREGOR, D., SEAGRAVE, C. (1979): HOFMANN, G.L. (1999): Parasites of North American freshwater Infectious pancreatic necrosis (IPN) virus: its occurrence in captive fishes. second ed. Comstock Publishing Associates, Ithaka, New and wild fish in England and Wales. J Fish Dis 2, 549–553. York. BUCKE, D., FEIST, S.W., CLIFTON-HADLEY, R.S. (1991): The oc- JUTFELT, F., OLSEN, R.E., GLETTE, J., RINGO, E., SUNDELL, K. currence of proloferative kidney disease (PKD) in cultured and wild (2006): Translocation of viable Aeromonas salmonicida across fish: further investigations. J Fish Dis 14, 583–588. the intestine of rainbow trout, Oncorhynchus mykiss (Walbaum). BURKHARDT-HOLM, P., PETER, A., SEGNER, A. (2002): Decline of J Fish Dis 29, 255–262. fish catch in Switzerland. The project Fishnet – a balance between KENT, M.L., KHATTRA, J., HERVIO, D.M.L., DEVLIN, R.H. (1998): analysis and synthesis. Aquat Sci 64, 36–54. Ribosomal DNA sequence analysis of isolates of the PKX CIPRIANO, R.C., FORD, L.A., SMITH, D.R., SCHACHTE, J.H., myxosporean and their relationship to members of the genus PETRIE, C.J. (1997): Differences in detection of Aeromonas Sphaerospora. J Aquat Anim Health 10, 12–21. salmonicida in covertly infected salmonid fishes by the stress in- KNUESEL, R., SEGNER, H., WAHLI, T. (2003): A survey of viral ducible furunculosis test and culture based assays. J Aquat Anim diseases in farmed and feral salmonids in Switzerland. J Fish Dis Health 9, 108–113. 26, 167–182. CRANE, M., HYATT, A. (2011): Viruses of fish: An overview of signif- KUMAR, G., ABD-ELFATTAH, A., SALEH, M., EL-MATBOULI, icant pathogens. Viruses 3, 2025–2046. M. (2013): Fate of Tetracapsuloides bryosalmonae (Myxozoa) DE KINKELIN, P., GAY, M., FORMAN, S. (2002): The persistence of after infection of brown trout Salmo trutta and rainbow trout infectivity of Tetracapsulabryosalmonae‐infected water for rainbow Oncorhynchus mykiss. Dis Aquat Organ 107, 9–18. trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 25, 477–482. MCCARTHY, D.H. (1977): Some ecological aspects of the bac- EL-MATBOULI, M., MATTES, M., SOLIMAN, H., (2009): Susceptibility terial fish pathogen. In: SKINNER, F.A., SHERVEN, J.M. (eds). of whirling disease (WD) resistance and WD susceptible strains Aeromonas salmonicida in Aquatic microbiology. Academic Press, of rainbow trout Oncorhynchus mykiss to Tetracapsuloides bry- London, New York, 299–324. osalmonae, Yersinia ruckeri and viral haemorrhagic septicaemia MEIER, W., SCHMIDT, M., WAHLI, T. (1994): Viral hemorrhagic sep- virus. Aquaculture 288, 299–304. ticaemia (VHS) of nonsalmonids. Ann Rev Fish Dis 4, 359–373. ENZMANN, P-J., KONRAD, M., PAREY, K., WETZLAR, H. (1987): MORRIS, D.J., ADAMS, A. (2006): Transmission of freshwater myx- Natürliches Wirtsspektrum des Virus der Viralen Hämorrhagischen ozoans during the asexualpropagation of invertebrate hosts. Int J Septikämie der Regenbogenforelle. Tierärztl Umschau 42, Parasitol 36, 371–377 228–230. MUNRO, A.L.S., LIVERSIDGE, J., ELSON, K.G.R. (1976): The dis- FIGUEROLA, J., GREEN, A.J., BLACK, K., OKAMURA, B. (2004): tribution and prevalence of infectious pancreatic necrosis virus in The influence of gut morphology on passive transport of freshwa- wild fish in Loch Awe. P Roy Soc Edinb (B) 75, 221–232.

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OKAMURA, B., HARTIKAINEN, H., SCHMIDT-POSTHAUS, H., TATICCHI, M.I., GUSTINELLI, A., FIORAVANTI, M.L., CAFFARA, WAHLI, T. (2011): Life cycle complexity, environmental change M., PIERONI, G., PREARO, M. (2004): Is the worm-like organ- and the emerging status of salmonid proliferative kidney disease. ism found in the statoblasts of Plumatella fungosa (Bryozoa, Freshwater Biol 56, 735–753. Phylactolaemata) the vermiform phase of Tetracapsuloides bry- PINTER, K. (2008): Rearing and stocking of brown trout, Salmo trutta osalmonae (Myxozoa, Malacosporea)? Ital J Zool 71, 143–146. L.: Literature review and survey of Austrian fish farmers within the VASEMÄGI, A., NOUSIAINEN, I., SAURA, A., VÄHÄ, J-P., VALJUS, frame of the project-initiative TROUTCHECK. Diploma Thesis, J., HUUSKO, A. (2017): First record of proliferative kidney disease University of Natural Resources and Applied Life Sciences, Vienna. agent Tetracapsuloides bryosalmonae in wild brown trout and ROSE, A.S., ELLIS, A.E., MUNRO, A.L.S. (1989): The infectivity by European grayling in Finland. Dis Aquat Organ 125, 73–78. different routes of exposure and shedding rates of Aeromonas sal- WAHLI, T., KNUESEL, R., BERNET, D., SEGNER, H., PUGOVKIN, D., monicida ssp. salmonicida in Atlantic salmon, Salmo salar L., held BURKHARDT-HOLM, P., ESCHER, M., SCHMIDT-POSTHAUS, in sea water. J Fish Dis 12, 573–578. H. (2002): Proliferative kidney disease in Switzerland: current RUANE, N., GEOGHEGAN, F., CINNEIDE, M.O. (2007): Infectious state of knowledge. J Fish Dis 25, 491–500. pancreatic necrosis virus and its impact on the Irish salmon aqua- WAHLI, T., BERNET, D., SEGNER, H., SCHMIDT-POSTHAUS, H. culture and wild fish sector. Marine Institute Oranmore Co. Galway (2008): Role of altitude and water temperature as regulating factors (ed) Marine Environment & Health Series No. 30. for the geographical distribution of Tetracapsuloides bryosalmonae SCHMIDT, H., BERNET, D., WAHLI, T., MEIER, W., BURKHARDT- infected fishes in Switzerland. J Fish Biol 73, 2184–2197. HOLM, P. (1999): Active biomonitoring with brown trout and WIKLUND, T., DALSGAARD, I. (1998): Occurrence and significance rainbow trout in diluted sewage plant effluents. J Fish Biol 54, of atypical Aeromonas salmonicida in non-salmonid and salmonid 585−596. fish species: a review. Dis Aquat Organ 32, 49–69. STERUD, E., FORSETH, T., UGEDAL, O., POPPE, T.T., ZIMMERLI, S., BERNET, D., BURKHARDT-HOLM, P., SCHMIDT- JØRGENSEN, A., BRUHEIM, T., FJELDSTAD, H-P., MO, T.A. POSTHAUS, H., VONLANTHEN, P., WAHLI, T., SEGNER, H. (2007): Severe mortality in wild Atlantic salmon Salmo salar due (2007): Assessment of fish health status in four Swiss rivers show- to proliferative kidney disease (PKD) caused by Tetracapsuloides ing a decline of brown trout catches. Aquat Sci 69, 11–25. bryosalmonae (Myxozoa). Dis Aquat Organ 77, 191–198. TOPS, S., LOCKWOOD, W., OKAMURA, B. (2006): Temperature- Rechtsnormen driven proliferation of Tetracapsuloides bryosalmonae in bryozoan Verordnung des Bundesministers für Gesundheit über hosts portends salmonid declines. Dis Aquat Org 70, 227–236. Gesundheits- und Hygienevorschriften für Tiere der Aquakultur und TOPS, S., HARTIKAINEN, H., OKAMURA, B. (2009): The effects Aquakulturerzeugnisse sowie zur Verhütung und Bekämpfung bes- of infection by Tetracapsuloides bryosalmonae (Myxozoa) and timmter Wassertierkrankheiten (Aquakultur-Seuchenverordnung). temperature on Fredericella sultana (Bryozoa). Int J Parasitol 39, BGBL. II Nr. 315/2009, geändert durch BGBL. II Nr. 55/2013 und 1003–1010. BGBL. II Nr. 2/2015