Fatal Diplostomum phoxini infection in captive Atlantic puffin Fratercula arctica chicks following ingestion of infected European minnows Phoxinus phoxinus

: Conservation of endangered animal species is a major task of zoos. Husbandry and breeding of Atlantic puffins Fratercula arctica in captivity is challenging. In 2019, the entire chick population (n = 4 chicks) in Berne Animal Park’s Atlantic puffin colony (Bern, Switzerland) died within 7 d. Due to supply constraints, the chicks had been fed with wild-caught European minnows Phoxinus phoxinus . At necropsy, the main pathological finding in all deceased puffin chicks was a multifocal, moderate to severe subacute heterophilic and granulomatous enteritis with intralesional adult trematodes and eggs. Metacercariae surrounded by few necrotic cells and scattered macrophages were found in the brain and spinal cord of the food fish. Additional microbiological analyses of both the puffin chicks and fish were unremarkable. Diplostomum phoxini DNA could be identified in formalin-fixed paraffin-embedded tissue from the small intestine of all puffin chicks and European minnows following PCR and sequencing of the 18S ribosomal RNA gene and the internal transcribed spacer (ITS1) region. This report illustrates the importance of intensive health checks of food fish for animal species kept in captivity.


INTRODUCTION
Zoos play an important role in the conservation of protected and endangered animal species worldwide within the context of the ongoing global extinction crisis (Conde et al. 2011, Godinez & Fernandez 2019. Good husbandry practices are essential for success. However, several factors can severely im pair conservation efforts, namely poor breeding success, exposure to pathogens in non-adapted species and lack of suitable habitats for reintroduction (IUCN 2013).
The Atlantic puffin Fratercula arctica is a marine bird species belonging to the auk family (Alcidae) that was classified as Endangered by the International Union for Conservation of Nature (IUCN & BirdLife International 2022) due to its rapid decline across Europe. Despite their attractiveness to the public, Atlantic puffins are dif ficult to keep and rear in captivity due to low re production rates and high chick mortality (Tocidlowski et al. 1997). Notably, among Species360 Zoo logical Information Management Software (ZIMS 2022)-affiliated institutions, Atlantic puffins are only kept in 7 European, 5 North American, and 3 Asian zoological institutions, numbering a total of 171 individuals alive and only 11 births in the past 12 mo (data accessed on the 1 June 2022). Another limitation that impairs the maintenance of these animals in captivity is the limited knowledge of threatening diseases. From the 888 Atlantic puffin deaths recorded in ZIMS, the cause of mortality was determined in only 14% (n = 122). In Switzerland, this species is kept and bred at Berne Animal Park (Rosset & Blatter 2019) (at present 13 birds), which is one of 5 zoos worldwide that breed this species at present (ZIMS 2022). The most frequent cause of death since 2009 in the adult animals (n = 32) from the Bernese flock is aspergillosis (47%), followed by avian malaria (25%; Rosset & Blatter 2019, Meister et al. 2021. The Diplostomum genus represents a large group of freshwater parasitic digenean trematodes infecting fish and birds in all major freshwater environments with a complex life cycle (Hoffman & Hundley 1957, Landeryou et al. 2020. Cercariae released from lymnaeid snails (first intermediate host) infect freshwater fish (second intermediate host) and migrate to brain, eyes, or central nervous system and develop into metacercariae. Fish-eating birds (definitive host) are infected upon ingestion of infected fish, and adult flukes live in the intestine (Karvonen et al. 2006). In recent years, several molecular studies revealed a substantial genetic diversity and provided a framework for new insights in the vast taxonomic diversity and distribution of the species of this genus (Georgieva et al. 2013, Blasco-Costa et al. 2014, Landeryou et al. 2020. Although infection with digenean trematodes has been described in different auk species, Diplostomum spp. has only been diagnosed in the common murre Uria aalge, the thick-billed murre U. lomvia, and the razorbill Alca torda (Muzaffar & Jones 2004), without any significant pathogenicity.
In this manuscript, we describe the fatal infection of Atlantic puffin chicks with D. phoxini following ingestion of infected European minnows Phoxinus phoxinus, which led to the loss of the entire 2019 offspring from the Atlantic puffin flock at Berne Animal Park.

Atlantic puffins and European minnows
The total Atlantic puffin offspring of 2019, consisting of 4 males at age 5 d (body weight 60 g), 14 d (98 g), 34 d (280 g) and 45 d (323 g), died within a single week. They were raised as previously de scribed (Rosset & Blatter 2019). Diet consisted of pellets (Ibis-See-(Eis-) Ente-Spezial size 5, Lundi) and thawed sand smelts Atherina boyeri (Parlevliet & Van der Plas). Chicks from earlier successful breeding seasons were additionally fed fresh juvenile captive rainbow trout Oncorhynchus mykiss from a local fish farm starting from the second day following hatching. However, due to supply difficulties of the usual food fish, the 2019 puffin offspring were fed fresh wild minnows Phoxinus phoxinus (1 fish of approximately 5 g bird −1 d −1 ) caught in a local stream (Giesse, 46°53' 49" N, 7°31' 57" E), beginning 3 d before the first death.

Pathological and microbiological analysis of Atlantic puffin chicks and food fish
All dead puffins were submitted for pathological and microbiological analyses. Tissue samples from the intestine, proventriculus, gizzard, crop, pancreas, lung, trachea, heart, eye, skeletal musculature, kidneys, brain, liver, spleen, bursa, and cloaca were collected for histopathological analysis. The intestinal content from 2 chicks was submitted for parasitological flotation analysis. Lung, liver, and kidney samples from the same 2 chicks were submitted for bacteriological analysis.
All Atlantic puffin chicks were tested for avian influenza using cloacal swabs (FASTest ® AIV Ag Testkit; Megacor, Hörbranz); an RT-PCR for Usutu virus according to S. R. R. Pisano et al. (unpubl. data) from brain, liver, and spleen of 2 chicks was also performed. Diagnostic tests for avian influenza and Usutu virus were negative.
Food fish were euthanized using 3-aminobenzoic acid ethyl ester (MS 222 ® , Argent Chemical Laboratories) at a dosage of 150 mg l −1 water buffered with bicarbonate at pH 7. All specimens were tested for the presence of external and intestinal parasites. Parasite exams involved direct microscopic examination of wet mounts of skin, gills, and intestinal content. A standard necropsy was performed. Spleen, liver and kidney were cultured on blood agar plates (Biomerieux) for bacterial growth for 48 h at 22°C and plates examined daily. Skin, brain, liver, spleen and kidney from 5 animals were fixed in RNAlater (Sigma Life Science) prior to fixation and stored at 4°C.
The tissue samples from both the puffin chicks and whole specimens of the European minnows showing macroscopical changes were fixed in 10% neutral buffered formalin, paraffin embedded, cut at 4 μm, stained with haematoxylin and eosin (H&E), and examined under the light microscope.

Molecular identification of trematode species
Formalin-fixed paraffin-embedded (FFPE) tissue and RNAlater-fixed fish brain material were used to identify the metacercariae and adult trematodes detected in the fish and bird tissue, respectively. Two 20 μm sections of FFPE material were prepared. Each section was deparaffinised and lysed, using 200 μl ATL buffer (QIAGEN) and 30 μl Proteinase K at 56°C for 2 h. DNA was extracted from lysed tissue and RNAlater-fixed brain tissue directly using the DNeasy tissue Kit (QIAGEN), according to the manufacturer's protocol. Samples were incubated with Proteinase K at 56°C and shaken at 300 rpm overnight. DNA yield was determined spectrophotometrically (NanoDrop Technologies). Conventional PCR was performed  (95°C for 45 s, 62°C for 30 s, 72°C for 30 s); and 72°C for 5 min. DNAsefree water was used as control. PCR products were purified with WIZARD SV Gel and PCR Clean-Up System (Promega AG), resolved on a 1.5% agarose gel and sent for Sanger sequencing (Microsynth AG). Sequences were deposited in GenBank (www.ncbi. nlm.nih.gov) and analysed with BLAST (http://blast. ncbi.nlm.nih.gov/Blast.cgi). The amplified sequences were aligned with closely related and identical sequences available in NCBI using the MUSCLE algorithm in the Geneious software package. Trimmed 88 or 178 bp products amplified with F1/R1 or F1/R2, respectively, aligned and analysed with jModelTest 2.1.10 (Darriba et al. 2012

Clinical, pathological, and microbiological findings in Atlantic puffin chicks
The youngest chick died peracutely, while the remaining chicks displayed inappetence to anorexia, regurgitation, and weight loss dying within 1 or 2 d after onset of symptoms. Blood chemistry and hematology were within normal limits, microbiological analyses were inconclusive, and the animals did not respond to symptomatic treatments.
All chicks were emaciated. One chick had apparent pericardial visceral gout, 2 chicks had dilated gallbladder and 1 chick a crop dilated with fish and pellets. Histologically, cross and longitudinal sections of adult trematodes, measuring approximately 700 × 300 μm, occasionally attached to the intestinal mucosa with an oral sucker, were found in the lumen of the small intestine of every chick (Fig. 1a). Moderate to high numbers of heterophils and macrophages and occasional multinuclear giant cells, fibroblasts, and collagen deposition infiltrated and expanded the lamina propria (Fig. 1b,d). Additionally, there were varying numbers of non-embryonated and embryonated trematode eggs measuring 50−70 μm in diameter with a smooth, approximately 4 μm thick yellow-brown shell (Fig. 1c) present within the intestinal lumen, which were often intermingled with cellular debris. The morphological diagnosis was moderate to severe, multifocal, subacute heterophilic and granulomatous enteritis with intralesional adult trematodes and eggs and intraluminal cellular debris.
Visceral gout was present in 1 chick, and moderate to severe renal intraluminal minerali deposits with mild tubular degeneration were detected in 2 chicks compatible with dehydration/nutritional impairment secondary to the parasitic infection. Parasitology and bacteriology were negative.

Pathological and microbiological findings in European minnows
Several randomly distributed, brownish to black irregular-sized cutaneous discolorations were observed (Fig. 2a). Direct parasitological examination of the skin and gills revealed a severe infection with Ichthyophthirius multifiliis. Bacteriology showed low numbers of mixed bacteria deemed inconclusive.
Histopathologically, trematode metacercariae were found in the subcutis, muscle (measuring ca. 70 × 200 μm; Fig. 2b), and central nervous system (CNS) but not the eyes. Metacercariae were surrounded by basophilic granulated material, a fibrous capsule, macrophages, and melanocytes, responsible for the brownish cutaneous discolorations seen grossly. In the CNS, metacercariae measured 30 × 100 μm, with a clearly visible apical sucker (Figs. 2c,d). They were surrounded by cellular debris and scattered macrophages.

PCR and sequencing
Puffins' and minnows' FFPE material and directly fixed minnows were PCR positive. Sequences from intestines of the puffins and the brain of the minnows were 100% identical with Diplostomum phoxini (Figs. 3). For the fish skin, all amplicons were 100% identitical with Posthodiplostomum cuticola (Fig. 3a). All sequences from European minnows and puffins were deposited in GenBank under the following

DISCUSSION
The infection with the digenean trematode Diplostomum phoxini of the entire 2019 Atlantic puffin offspring from Berne Animal Park colony probably led to nutrient and energy loss following damage of the intestinal barrier (Schmidt et al. 2006, McKay et al. 2017) and ultimately to peracute or acute death. This infection was possible because the strict Atlantic puffin rearing protocol was adapted by replacement of captive rainbow trout by wild-caught European minnows infested with Posthodipostomum cuticola and D. phoxini metacercariae.
D. phoxini is one of the few species of Diplostomum spp. with a strict secondary intermediate host as the European minnow, even though adult D. phoxini trema todes have been recovered from the small intestine of avian and mammalian hosts (Schwelm et al. 2021). Adult D. phoxini are not known to cause disease in the avian definitive host. However, digenean trematode infections can vary in pathogenicity, depending on infection burden, parasite, and af fected avian species (Huffman 2008). As few as twenty Sphaeridiotrema globulus can lead to death in American coots Fulica americana and mute swans Cygnus olor, whereas in Muscovy ducks Cairina moschata, mallards Anas platyrhynchos and Canada geese Branta canadensis, the fatal worm burden may range from 100 to 3300 parasites (Trainer & Fischer 1963, Campbell & Jackson 1977, Roscoe & Huffman 1982. Experimental infection with only 7 Cyathocotyle bushiensis trematodes may be fatal in ducks (Hoeve & Scott 1988). Interestingly, both Sphaeridiotrema spp. and C. bushiensis are non-native in the midwestern North American states and Canada and were intro-  (Roy & St-Louis 2017). This explains the high susceptibility of some American bird species upon infection with these non-native trematode parasites. In parallel, free-living Atlantic puffins feed on marine fish species and never ingest freshwater European minnows. The living conditions for puffins in Switzerland are very different from those in the wild. It is also possible that the puffin chicks were more susceptible to infection due to their developing immune system.
In conclusion, we hypothesize that the ingestion of European minnows infected with D. phoxini led to the death of all Atlantic puffin offspring hatched in 2019 at Berne Animal Park. To prevent such incidents from recurring in the future, an intensive health check of fresh food fish and a closed supply chain with a known health history is essential, especially for protected, endangered, and vulnerable zoo animals that are particularly difficult to keep and breed in captivity, such as Atlantic puffins.