Hepatozoonosis in Free-Ranging American Mink (Neovison vison) in Pennsylvania, USA: Case Series Open Access

American mink (Neovison vison) are mesocarnivores native throughout North America but are introduced and considered invasive in Europe (Zuberogoitia et al. 2010). They are also commonly farmed throughout the world for their fur and are a model species used in biomedical research to study respiratory viruses (Fenollar et al. 2021; Sun et al. 2021; Peacock and Barclay 2023). As a result, there is abundant literature on pathogens and diseases of these animals in captivity; however, there is a paucity of literature on infectious agents affecting free-ranging American mink in their native range. There is a single report of lesions caused by a Hepatozoon sp. in mink in Canada, but it predated the ability to conduct molecular characterization (Presidente and Karstad 1975). Our study highlights three cases of naturally acquired Hepatozoon sp. infections in free-ranging American mink in Pennsylvania, US, and expands on the limited data of this disease in mustelids in North America.

All cases were adult male mink acquired as submissions of diagnostic cases to the Wildlife Futures Program pathology service at the University of Pennsylvania (Kennett Square, Pennsylvania, USA). The cases originated from Clarion, Chester, and Mercer counties of Pennsylvania in 2023. Case 1 was found lying in a park field in Shippenville, Clarion County, in August with labored breathing; it was minimally responsive before being euthanized and submitted for necropsy. Faint, random, pinpoint, tan to white foci were present throughout the left lung lobes (right lung lobes obscured by postmortem and euthanasia artifacts). Case 2 was found dead in a residential area of Vincent Township, Chester County in February 2023. It had several large wounds on the ribcage, resulting in a diagnosis of trauma, but no other significant gross lesions were noted. Case 3 was euthanized after showing neurological signs at a private residence in Greenville, Mercer County in December 2023. Euthanasia artifact hindered the ability to recognize potential gross lesions in the thoracic cavity.

Representative tissues, including the heart, lung, remaining viscera, and brain, were placed into 10% neutral buffered formalin for fixation, embedded in paraffin wax, cut at 4-µm-thick sections, stained with hematoxylin and eosin, and examined under light microscopy. Immunohistochemistry was performed on lung tissue in all cases for Sarcocystis spp., Neospora caninum, and Toxoplasma gondii according to established protocols at an American Association of Veterinary Laboratory Diagnosticians–accredited laboratory.

Histopathology for Case 1 revealed dozens of poorly demarcated histiocytic foci throughout the lung, forming microgranulomas (Fig. 1A). Many histiocytes had distinct, centralized merozoites with peripheralization of the host nuclei, occasionally forming a crescent shape (Fig. 1A). Occasionally these histiocytic foci also contained larger meronts with peripheralization of nuclei forming a spoke-wheel pattern (termed “Y” schizont by Yanai et al. 1995) or evenly spaced centralized nuclei (termed “X” schizont by Yanai et al. 1995 and “mature meront” by Hodžić et al. 2018; Fig. 1B). The surrounding alveoli rarely had eosinophilic edema, and the pulmonary parenchyma was moderately autolyzed. Small histiocytic aggregates with similar intracytoplasmic merozoites were also present multifocally throughout the myocardium (Fig. 1C) of the left ventricular wall. Case 2 had similar microgranulomas with intracytoplasmic merozoites in the heart and lung, but they were fewer, less severe, and lacked the meront stage. Case 3 had a single similar microgranuloma with intra-cytoplasmic merozoites in the lung. Cases 1 and 3 had variably severe adult nematode burdens preferentially affecting the alveoli (Case 1) or peri-bronchial area (Case 3). These parasites were considered incidental in both cases. Sarcocystis, Neospora, or Toxoplasma spp. immunoreactivity were not detected in any of the tissues.

To characterize the Hepatozoon sp. detected, we conducted PCR and sequence analysis of the 18S rRNA gene. We extracted genomic DNA from lung tissue using the DNeasy Blood and Tissue extraction kit (Qiagen Inc., Germantown, Maryland, USA) following the manufacturer’s instructions. A nested PCR assay was used with the primary primer 5.1/B and secondary primers RLBH-F/RLBH-R to amplify a portion of the Hepatozoon sp. 18S rRNA gene as described (Yabsley et al. 2005). Negative water controls were used in DNA extraction, primary, and nested PCR to detect any contamination. Products were run on a 1.5% agarose gel, and positive bands were extracted and purified using a QIAquick gel extraction kit (Qiagen, Hilden, Germany). Extracted gels were submitted to Genewiz (South Plainfield, New Jersey, USA) for bidirectional sequencing. Sequences were cleaned and analyzed using Geneious (Version 11.5.1, Auckland, New Zealand) to generate a consensus sequence that was compared to other sequences in the NCBI database. Primers Tg58F and Tg348R were also used to detect other apicomplexans (e.g., T. gondii, Neospora, and Sarcocystis spp.; da Silva et al. 2009).

The sequence from Case 1 was identical to a sequence previously detected in mink from Pennsylvania (257 bp overlapping 100% with PP234623) followed by numerous samples from ticks and from other mustelids (99.6% similarity). Case 2 was also PCR positive, and although the sequence was short, it was consistent with a Hepatozoon sp. No samples from Case 3 were tested by PCR. The lack of immunoreactivity on the histology slides and negative PCR results with the Tg58F/Tg348R primer set suggests that T. gondii and Sarcocystis spp. were unlikely to be clinically relevant in these cases.

The lesions of granulomatous pneumonia and myocarditis seen in these free-ranging Pennsylvania mink were similar to those reported in a historical case series from mink in Ontario, Canada (Presidente and Karstad 1975); pine martens (Martes martes) in Scotland (Simpson et al. 2005) and in Bosnia and Herzegovina, and Croatia (Hodžić et al. 2018); stone martens (Martes foina) in Switzerland (Akdesir et al. 2018); and Japanese martens (Martes melampus) in Japan (Yanai et al. 1995). Those other studies reported the heart, skeletal muscle, and adipose tissue being most affected (Yanai et al. 1995; Hodžić et al. 2018). While this tissue distribution may also be true for Hepatozoon spp. infections in North American mink, a systematic collection of tissues to evaluate was not taken and adipose tissue specifically was not always evaluated.

While we speculate that the primary source of severe morbidity in Case 1 was from this Hepatozoon sp., infection in the other two cases was considered incidental or associated with other comorbidities (trauma and canine distemper virus infection). Hepatozoon spp. infections in mustelids are often considered incidental or as part of comorbidity in Europe (Simpson et al. 2005; Akdesir et al. 2018) and Asia (Yanai et al. 1995; Park et al. 2016). Indeed, Yanai et al. (1995) reported a 96% prevalence of lesions or infection in 70 wild-caught free-ranging Japanese martens without overt clinical disease. A thorough review of causes of mortality in mustelids in Switzerland reported rare cases of hepatozoonosis as a cause of mortality (Akdesir et al. 2018). Our Case 3, with canine distemper virus infection, had a mild, focal lesion consistent with Hepatozoon sp. infection, and had more severe nematode infection than the other cases. Nonetheless, this case demonstrated that CDV and hepatozoonosis can co-occur in American mink, similar to a report in yellow-throated martens (Martes flavigula koreana) in Korea (Park et al. 2016).

The Hepatozoon sp. that infects mink has not been formally described and has only recently been genetically characterized (Baker et al. 2024). The sequence from Case 1 was identical to that previously deposited sequence, and they are most similar to Hepatozoon spp. from other mustelids and from ticks, suggesting that this is a typical parasite of mink. While speculative, the same host and identical lesions suggests that the Hepatozoon sp. from the case series in Canada in 1975 (Presidente and Karstad 1975) was the same as that in our study. Additional genetics of Hepatozoon spp. in mink and other wildlife will aid in taxonomic differentiation of this complex protozoan.

The life cycle of this mink Hepatozoon sp. is unknown, but probably involves ingestion of ectoparasites, mainly ticks, or ingestion of prey tissues containing meronts or cystozoites (Thomas et al. 2024). Hepatozoon sp. DNA has been detected in many species of mammal-associated tick genera, including Amblyomma, Dermacentor, Haemaphysalis, Ixodes, and Rhipicephalus (Thomas et al. 2024). Ambylomma and Ixodes spp. have been documented on mink in North America (Bishopp and Trembley 1945; Walker et al. 1998) and thus might provide a route of transmission through ingestion during self-grooming. Additionally, the diet of mink includes rodents (Krawczyk et al. 2013), which can harbor Ixodes and Dermacentor spp. (Bishopp and Trembley 1945). Fleas and lice are also suggested as potential vectors, and they commonly occur on rodents (Thomas et al. 2024). When rodent prey is ingested, these ectoparasites may have the potential to transmit Hepatozoon spp. to mink. Finally, rodent prey may serve as the intermediate host for this Hepatozoon sp. Ultimately the specific route of transmission in these cases is unknown because ectoparasites were not observed or tested for Hepatozoon spp. Our study should help future diagnosticians to recognize this common and often important lesion in free-ranging mustelids and to understand the role of this pathogen as a primary infection or comorbidity. Further work is needed to confirm the taxonomic placement of this and other Hepatozoon spp. in wildlife, as well confirming the life cycle and routes of transmission in their natural hosts.

The authors thank the Pennsylvania Game Commission as well as the members of the public who reported these animals in the field. Diagnostic support was provided by the Histology Laboratory and Molecular Diagnostic Laboratory at New Bolton Center, University of Pennsylvania. Additional funding provided by the Richard King Mellon Foundation.