Malignant catarrhal fever–like clinical disease was diagnosed in a free-ranging bighorn sheep (Ovis canadensis) from Alberta, Canada, in June 2015. Antemortem and gross pathology findings included muscle atrophy, marked weight loss, and bilaterally symmetric alopecia with hyperpigmentation and crusting over the face, medial surfaces of the pinnae, dorsal trunk, distal limbs, perineal area, and tail. Histologically, the skin lesions were characterized by granulomatous mural folliculitis with numerous multinucleated giant cells and fewer lymphocytes and eosinophils consistent with previous reports of chronic ovine herpesvirus-2 (OvHV-2) infection. Multiple skin samples were positive for OvHV-2 DNA on PCR, and on partial sequencing of the viral DNA, there was 94% homology with reference GenBank OvHV-2. Quantitative PCR confirmed an increased level of OvHV-2 DNA in the lesional skin tissues. Based on exclusion of other disease processes, gross and histological lesions, PCR, and viral DNA sequencing results, a diagnosis of OvHV-2–mediated malignant catarrhal fever–like dermatitis was made.
Malignant catarrhal fever (MCF) is a disease caused by several herpesviruses in the Gammaherpesvirinae subfamily that infect susceptible artiodactylid species (O'Toole and Li 2014). These viruses are maintained in reservoir host species, typically as subclinical infections and periodically infect nonnatural hosts (Heuschele and Reid 2001). Free-ranging North American artiodactylids are susceptible to several strains, with ovine herpesvirus 2 (OvHV-2) and caprine herpesvirus 2 being the most important, and are implicated in clinical disease in many North American cervids and bison (O'Toole and Li 2014). Clinical disease in bighorn sheep (BHS; Ovis canadensis) caused by OvHV-2 has not been reported, with BHS being considered a natural reservoir of infection (O'Toole and Li 2014).
In June 2015, a free-ranging, adult female BHS was observed in Banff National Park (51°7′N, 115°44′W) with marked weight loss and muscle atrophy and bilaterally symmetric alopecia, hyperpigmentation, and crusting over the face, dorsal trunk, and distal limbs (Fig. 1a). Based on a clinical suspicion of psoroptic mange (Psoroptes ovis), the animal was euthanized and submitted for a complete necropsy. The BHS was in poor body condition with mild to moderate generalized muscle atrophy. There was extensive bilaterally symmetric alopecia, crusting and hyperpigmentation of the medial pinnae, occipital area, forehead, bridge of the nose, cheeks, rostral aspect of carpi, caudal aspect of metacarpi, rostral aspect of metatarsi, tail, hip, and perineum (Fig. 1a–d). There was focal alopecia on the ventral thorax. Crusting was particularly severe on the medial surfaces of the pinnae, tail, around the coronary bands and fetlocks. Other findings included excessive wear of the molar dentition with a focal tooth root abscess and associated osteomyelitis of the right ramus. A retropharyngeal abscess was present, and the left axillary lymph node was markedly enlarged and edematous with multifocal, firm, white foci. Bullae of emphysema were present bilaterally in the cranial lung lobes, and there were multifocal nodules in the lungs, in both liver lobes, and on the gallbladder mucosa. Several tapeworm cysts were attached to the greater omentum and to the capsule of the liver.
Scrapings, digests, and histopathology on multiple skin samples failed to demonstrate P. ovis or other mite species infestation. Multiple skin sections were examined histologically for pathologic agents using H&E and Gram, periodic acid–Schiff, Fite-Faraco (acid fast), and Gomori methenamine silver stains. The main histologic pattern in all sections was a granulomatous to granulomatous and eosinophilic mural folliculitis (Fig. 2a). Many multinucleated histiocytic giant cells were infiltrating the walls of hair follicles (Fig. 2b) with a few that surrounded keratin and follicular fragments in areas of furunculosis. Multifocally, in the epidermis there were multilevel apoptotic keratinocytes occasionally surrounded by mononuclear cells (satellitosis) and occasional intracorneal pustules (Fig. 2c). Multinucleated histiocytic giant cells were occasionally noted within the epidermis. There was mild diffuse hyperplasia (acanthosis) of the epidermis with hyperpigmentation and marked orthokeratotic hyperkeratosis with variable serocellular crusting. Within the dermis there was a moderate perivascular and perifollicular infiltrate of predominantly histiocytes, lymphocytes, and eosinophils (Fig. 2a). No pathogenic agents were detected using special stains. Moderate to severe, bronchointerstitial pneumonia was associated with adult nematodes, larvae, and embryonated eggs, with morphology consistent with protostrongylid parasites. In the remaining pulmonary parenchyma there was variable congestion, edema, emphysema, and focal hemorrhage. Additional histology findings included severe diffuse splenic lymphoid depletion and hemosiderosis; mild, granulomatous inflammation of the liver; mild, interstitial myocarditis; mild, membranous glomerulonephritis; and low numbers of Sarcocystis-like cysts in the myocardium and skeletal muscle.
Ancillary testing by PCR on lymph node, lung, heart, kidney, liver, and spleen and bacterial culture on lung were performed using standard procedures (Animal Health Laboratory, University of Guelph, Guelph, Ontario, Canada). Samples were negative for bovine herpesvirus-1, bovine parainfluenzavirus-3, bovine respiratory syncytial virus, and mycoplasma. PCR testing on skin samples was negative for parapox, bovine viral diarrhea, and border disease viruses (Animal Health Center, Abottsford, British Columbia, Canada). Bacterial cultures were performed on the retropharyngeal abscess, retropharyngeal lymph node, and lung using standard procedures according to previously published methods (Prairie Diagnostic Services, Saskatoon, Saskatchewan, Canada; Songer and Post 2005; Quinn et al. 2011). The retropharyngeal abscess had heavy growth of Trueperella pyogenes, while the retropharyngeal lymph node produced a heavy growth of Bibersteinia trehalosi, low numbers of T. pyogenes, and a few Staphylococcus aureus bacteria. Trueperella pyogenes was isolated in low numbers from the lung. The moderate to marked lungworm infection consisted of adult nematodes (350 μm in diameter), larvae (20–30 μm in diameter), and embryonated eggs with morphological characteristics of a Protostrongylus sp. infection. A Wisconsin double centrifugation fecal floatation test found 236 oocysts/g of Eimeria sp., eggs of Marshallagia sp., and strongylate eggs. A routine Baermann larval count was performed, and 21 larvae of Protostrongylus sp./g of feces were noted.
The gross and histopathologic findings in the skin of this BHS, in particular the alopecia and granulomatous and eosinophilic mural folliculitis with multinucleated histiocytic giant cells, have been previously reported in other species chronically infected with OvHV-2 or caprine herpesvirus 2 and represent a known, chronic manifestation of MCF (Crawford et al. 2002; Foster et al. 2010). To support the histologic evidence of chronic dermatitis associated with MCF-like disease, DNA was extracted from the skin of the coronary bands, head, and pinna using QiaAMP® DNA Mini kit (Qiagen Inc., Toronto, Ontario, Canada) and used in a previously published conventional PCR targeting the OvHV-2 tegument protein gene (Baxter et al. 1993) with modifications (Animal Health Centre, Abbotsford). The PCR assay specifically amplified the expected 423 base pair amplicons from DNA extracted from all epidermal sites. Positive PCR results were confirmed by direct sequencing of the PCR product using the Big Dye® Terminator V3.1 Cycle Sequencing Kit (Life Technologies, Burlington, Ontario, Canada) and ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) that yielded a 362 base pair long consensus sequence. The BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) analyses revealed that this sequence shared 94% homology with similar sequences from OvHV-2 tegument protein gene deposited in GenBank. This DNA sequence was deposited in GenBank (accession number: KX060582).
To further support a role of OvHV-2 in skin lesion development, OvHV-2 DNA levels in tissues were investigated using a quantitative PCR (qPCR; Animal Disease Research Unit, US Department of Agriculture, Agricultural Research Service, Pullman, Washington, USA). DNA from various tissue samples was extracted using the FastDNA kit (QBiogene, Irvine, California, USA) as described by the manufacturer and quantified using a fluorometer (Qubit, Invitrogen, Carlsbad, California, USA). The tissue samples tested included skin (head, limbs, and pelvis), tonsil, lymph node, respiratory mucosa, and colon. Archived tissues from six other BHS (adult male, n=1; adult female, n=2; juvenile female, n=2; fetus, n=1) originating in the Rocky Mountains of Alberta included skin, lung, mesenteric lymph node, tonsil, and prescapular lymph node. The cause of death for each of these control BHS was trauma, with one juvenile female diagnosed with parapox virus. The qPCR was performed as previously described (Traul et al. 2007), and the results reported as OvHV-2 genome copies per 50 ng of total DNA. From the diseased BHS, 2,060 OvHV-2 genome copies were quantified in skin samples, 1,130 copies in the respiratory mucosa, and a few copies in the colon; lymph node and tonsil samples did not yield any amplification. None of the tissues from the six control BHS had detectable OvHV-2 DNA by qPCR. Previous studies in OvHV-2-induced MCF in bison and rabbits showed that the increased viral DNA levels are observed as the disease progresses, and lesion severity is positively associated with the increased levels of viral DNA in tissues (Cunha et al. 2012, 2013). Taken together, the increased level of OvHV-2 DNA in affected skin samples from the diseased BHS further supports the conclusion that the virus played a key role in the development of the chronic dermatitis.
Bighorn sheep are considered a natural reservoir for OvHV-2, with seropositive herds identified in North America (Zarnke et al. 2002; O'Toole and Li 2014). To date, there have been no reports of OvHV-2 causing clinical disease in BHS. Only under experimental conditions have OvHV-2 naïve domestic sheep developed clinical disease with lesions primarily occurring in the lung, but without chronic dermatitis (Li et al. 2005). Currently, the seroprevalence of OvHV-2 in BHS in western Canada is unknown, but seronegative populations of wild BHS occur, indicating that naïve BHS populations exist (Li et al. 1996; O'Toole and Li 2014). In this case, poor body condition and immunosuppression, as suggested by the severe splenic lymphoid depletion and concurrent parasitic and bacterial infections, coupled with the other incidental lesions noted on histology, may have facilitated activation of latent OvHV-2, leading to the chronic skin lesions. The OvHV-2 should be a differential diagnosis for dermatological diseases in BHS populations. Further research is required to determine the seroprevalence of OvHV-2 in western Canada and to determine the incidence of OvHV-2 infections and clinical disease in wild sheep and other wildlife populations.
The authors thank Mike Grande for assistance in the field, Resource Conservation staff of Banff National Park, and Donal O'Toole for their assistance with this case.