Outbreaks of bovine herpesvirus 2 infections in calves causing ear and facial skin lesions

704480VDIXXX10.1177/1040638717704480Watanabe et al.bohv-2 infection causing facial and ear crusts and alopecia research-article2017 Brief Communication Outbreaks of bovine herpesvirus 2 infections in calves causing ear and facial skin lesions Journal of Veterinary Diagnostic Investigation 2017, Vol. 29(5) 686 690 2017 The Author(s) Reprints and permissions: sagepub.com/journalspermissions.nav https://doi.org/10.1177/1040638717704480 DOI: jvdi.sagepub.com Tatiane T. Negrão Watanabe, Robert B. Moeller Jr, 1 Beate M. Crossley, Patricia C. Blanchard Abstract. We describe 3 outbreaks of superficial dermatitis caused by bovine herpesvirus 2 (BoHV-2) in dairy breed calves. Clinically, all of the affected calves were 12 26 d of age, had alopecia and crusts on the face and ears, and were non-pruritic and afebrile. Affected animals recovered spontaneously without any treatment within 2 4 wk after onset of clinical signs based on 1 herd with follow up. Histologic examination of all skin crust or tissue samples identified neutrophilic inflammation, mild hyperkeratosis, multinucleate syncytial cells, and intranuclear inclusion bodies in the syncytial cells. Real-time PCR testing on affected surface crusts or tissue provided evidence of BoHV-2, and testing, where performed, was negative for parapoxvirus including bovine papular stomatitis virus and the ovine form of malignant catarrhal fever tested in EDTA blood samples. Bovine viral diarrhea virus also was negative by ELISA, as well as bovine herpesvirus 1 by immunohistochemistry. Direct electron microscopy of infected tissues in the first outbreak revealed herpesvirus-like particles. Key words: Bovine herpesvirus 2; bovine mammillitis virus; calves; polymerase chain reaction; pseudo-lumpy skin disease. Latent infection within specific tissues is a common feature of all herpesviruses. 2,4,15 Bovine alphaherpesvirus 2 species (BoHV-2; order Herpesvirales, family Herpesviridae, subfamily Alphaherpesvirinae, genus Simplexvirus) is the cause of ulcerative mammillitis, which is usually a self-limiting cutaneous disease of the udder and teats. 12,16 BoHV-2 is also responsible for pseudo-lumpy skin disease (PLSD), which causes generalized superficial cutaneous nodules with central depressions over the entire body of affected animals. 8 We describe 3 outbreaks of BoHV-2 causing head and ear skin lesions in young calves from dairy farms and calf-raising facilities located in the Tulare County region of the Central Valley of California. The outbreaks occurred on 3 separate premises over a 10-y period. The first outbreak occurred in 2005 on a 5,000- head calf ranch with 95 100% of 2- to 3-wk-old calves affected over a 1-mo period in August. According to the referring veterinarian ~200 calves were affected on the day of sample submission. The second outbreak was on a 3,500 lactating cow dairy farm in September 2012 where nearly 100% of Jersey and Holstein calves developed ear skin lesions at ~3 wk of age, leading to alopecia; 150 calves were affected on the day of sample submission. The third outbreak occurred in July 2015 on a 5,000-head calf ranch where 50% of calves at 12 26 d of age (~100 calves affected on day of sample submission) developed face and ear lesions. Calves affected were Holsteins (premises 1 and 3) and Holstein and Jersey calves (premises 2). Affected animals showed alopecia and surface crust formation with skin peeling from the ears. The most common sites of involvement were the dorsal portion of the head, periocular skin, and edges of the ears. Some areas demonstrated complete loss of hair (Fig. 1), whereas other calves had well-circumscribed hair loss. In all cases, calves were in good body condition. Animals were bright, alert, afebrile, non-pruritic, did not have increased sensitivity in affected areas, and ate and drank without any difficulty. It was reported that lesions spontaneously regressed within 2 4 wk after onset (premises 1). The submitted case material consisted of 9 EDTA blood samples (outbreak 1) and 17 skin crusts or tissue (outbreaks 1 3). No animals were submitted for postmortem examination. Samples from the skin crust or tissue were received fresh with a portion of the sample fixed in 10% neutralbuffered formalin, processed routinely, embedded in paraffin, and sectioned at 5 µm for staining with hematoxylin and eosin (H&E) for histologic examination and testing for BoHV-1 immunohistochemistry [IHC] using a specific California Animal Health and Food Safety Laboratory, San Bernardino Laboratory (Negrão Watanabe), Tulare Laboratory (Moeller Jr, Blanchard), and Davis Laboratory (Crossley), School of Veterinary Medicine, University of California, Davis, CA. 1 Corresponding author: Robert B. Moeller Jr, California Animal Health and Food Safety Laboratory, University of California, Tulare Branch, 18830 Road 112, Tulare, CA 93274. rbmoeller@ucdavis.edu

BoHV-2 infection causing facial and ear crusts and alopecia 687 Figure 1. Facial skin lesions (outbreak 1). (A) Mild alopecia with crust formation above the left eye and on the left ear pinna. (B) Severe alopecia of dorsum of ear bilaterally, periocular hair loss, and numerous flies on face and body of calf. polyclonal antibody (VMRD, Pullman, WA). The fresh skin crusts were submitted for: 1) polymerase chain reaction (PCR) testing at the California Animal Health and Food Safety (CAHFS) laboratory Davis branch for BoHV-2 (1 sample for parapoxvirus including bovine papular stomatitis virus [BPSV]), and 2) bovine viral diarrhea virus (BVDV) antigen ELISA (BVDV PI X2, IDEXX, Westbrook, ME), following the manufacturer s recommendation. For extraction of total nucleic acid, fresh skin samples were vortexed in viral transport medium and pulse spun. The supernatant was extracted using magnetic beads (Mag- Max 96 Viral RNA isolation well kit, Life Technologies, Carlsbad, CA), according to the manufacturer s recommendation. Extracted DNA was subjected to real-time PCR (qpcr; primer and probe information for BoHV-2 targeting glycoprotein B by DNA sequencing, pan-parapox agents, and BPSV was kindly provided by Dr. K. Toohey-Kurth, WVDL, Madison, WI). All qpcr reactions utilized the Path-ID multiplex one-step RT-PCR kit (Life Technologies) and were performed with the 7500 Fast real-time PCR system (Life Technologies) under the following conditions: stage 1 at 50 C for 10 min; stage 2 at 95 C for 10 min; stage 3 at 95 C for 15 s; followed by stage 4 at 60 C for 1 min; stages 3 4 were repeated for 40 cycles. Blood samples tested for ovine herpesvirus 2 (OvHV-2) and 2 of the BoHV-2 tests were outsourced to the USDA-ARS laboratory in Pullman, Washington; all other testing was performed at CAHFS. For direct electron microscopy via negative staining, a small section of fresh skin crust or tissue was processed in 2% phosphotungstic acid, and centrifuged at 2,300 g for ~20 min at 30 C. The supernatant was filtered through syringe filters of descending pore size ending with 0.8 µm. Ultrafiltrate was centrifuged (Beckman 70.1 Ti rotor, Fullerton, CA) at 277,000 g for a minimum of 45 min at 5 C. Figure 2. Syncytial cells in skin crust containing basophilicto-amphophilic intranuclear inclusion bodies (arrows) are admixed with abundant neutrophilic inflammatory infiltrate and cellular debris. H&E. 600. The pellet was suspended in double-distilled water and mixed with 2% neutralized phosphotungstic acid at a ratio of ~1:10 and then applied to a formvar-coated, carbonbacked grid. Grids were examined in a transmission electron microscope (Zeiss 906E, Oberkochen, Germany) at 80 kv accelerating voltage. Given that the lesions were taken from sites with surface crusts, the skin tissue examined was usually nonviable epidermal tissue and debris. Microscopically, the epidermal crusts consisted of faintly stained epithelial cells, mixed with numerous multinucleate syncytial cells that contained lightly basophilic-to-amphophilic intranuclear inclusions filling and distending nuclei. Abundant neutrophils often infiltrated among the syncytial cells and epithelial cells, disrupting the epidermal architecture (Fig. 2). Some affected epithelial

688 Watanabe et al. Figure 3. A nucleoid (round particle in the nucleus of the cell) is surrounded by a dense ring in this electron micrograph of a skin crust. cells had undergone ballooning degeneration with irregular granularity. In some sections examined, abundant cellular debris was noted in the ostia of associated hair follicles (outbreak 1). Orthokeratotic hyperkeratosis (outbreak 1) and perivascular lymphocytic dermatitis (outbreak 3) were also noted histologically. Histologic examination of the skin lesions showed serocellular suppurative epidermitis with intranuclear inclusions within syncytia. There was no evidence of coccal bacterial colonies (except for 1 sample in outbreak 3), fungal hyphae including dermatophytes, Dermatophilus sp., or ectoparasite structures. Lesions noted during histologic examination were highly suggestive of a herpesvirus, and BoHV-2 was detected by molecular assays in 15 calves. In addition, direct electron microscopy of skin crusts in 2 additional calves revealed herpesviral particles (Fig. 3). No histologic lesions compatible with parapox were seen, parapox was not detected by electron microscopy, and a submitted sample from outbreak 2 had no detection by qpcr of BPSV, or of pan-parapoxvirus (Table 1). All 9 calves tested also were negative by IHC for BoHV-1. In addition, BVDV antigen was negative on ear tissue by antigen ELISA in all 3 calves tested (Table 1). The EDTA blood samples from all 5 calves tested (outbreak 1) were negative by qpcr for OvHV-2 (cause of sheep-associated malignant catarrhal fever). The gross appearance and location (around the eyes and on the ears) of these skin lesions with hair loss were the main clinical findings. Based on the estimated age of the lesions on the ear, it appeared that the lesions may have started on the dorsal pinnae along the edge and later spread over the entire ear. BoHV-2 infection is usually associated with vesicles, crusts, and ulcers affecting 1 or more teats of lactating cows. Rarely is this virus identified in oral and muzzle lesions of nursing calves. 14,19 A second syndrome associated with BoHV-2, known as PLSD or Allerton virus infection, is characterized by generalized nodular skin lesions, often accompanied by mild fever and depression. 8,10 In our report, BoHV-2 cases did not show fever or depression at the time of, or prior to, the onset of the skin lesions. PCR was essential to confirm the infectious agent in these cases, given its high specificity and sensitivity in detecting and identifying viral nucleic acid. 1,6,7,11 In one case, positivity for BoHV-2 virus was identified in a 3.5-y-old animal presented at a slaughterhouse. 20 The lesions characterized by lumps and scattered circular areas of alopecia were identified on the head, neck, shoulders, and on the perineum, representing the chronic pattern of PLSD. The location of the lesions and high attack rate in the affected herds (affecting almost all calves in 2 of the outbreaks) suggest biting face flies as the mode of virus transmission to these calves. 5 At the time of these outbreaks (July September), biting face flies were very common. In cases of PLSD, the virus is known to spread from animal to animal mechanically by biting flies. 3,8,9,13 The incubation period for BoHV-2 infections is considered to be 1 2 wk. 8 Based on the age of the calves affected (12 26 d), the infection probably occurred during the first week of life. Interestingly, inclusion bodies are only observed during the exudative crust phase. 9 When the surface crust fell off, the underlying skin was alopecic. Coinfection with BoHV-2 and other bovine herpesviruses might be possible 2 ; however, in the cases described herein, BoHV-1 was not detected using IHC. Where follow-up was available (outbreak 1), spontaneous recovery occurred in 2 4 wk after the detection of skin lesions. It is assumed that, after seroconversion, clinical signs tend to disappear, but there would be latent virus in the skin, nervous system, and lymph nodes. 4,17,18 Potential differential diagnoses should include ringworm (Trichophyton verrucosum), contact dermatitis, photosensitization, poxvirus and parapoxvirus (e.g., bovine papular stomatitis virus and cowpox virus) skin diseases, and BVDV dermatitis. In the present case, ringworm was not detected. In addition, the animals had not been fed or exposed to any known photosensitizing agents or hepatotoxins. Infection by BoHV-2 should be included in the list of differential diagnoses in cases of skin lesions in cattle. The definitive diagnosis was based on gross and histologic

BoHV-2 infection causing facial and ear crusts and alopecia 689 Table 1. Identification of bovine herpesvirus 2 outbreaks and ancillary tests performed. Outbreak Type production Animal ID Breed Sex Age (d) IHC qpcr Direct electron ELISA BoHV-1 OvHV-2 BoHV-2 BPSV Pan-PPV microscopy BVDV 1 Calf ranch 1 Holstein M 14 21 Negative ND Positive ND ND ND Negative 2 Negative ND ND ND ND Positive Negative 3 Negative ND ND ND ND Positive Negative 4 Negative ND Positive ND ND ND ND 5 Negative Negative Positive ND ND ND ND 6 Negative Negative Positive ND ND ND ND 7 Negative Negative Positive ND ND ND ND 8 Negative Negative Positive ND ND ND ND 9 Negative Negative Positive ND ND ND ND 2 Dairy farm 1 Jersey F 21 ND ND Positive Negative Negative ND ND 3 Calf ranch 1 Holstein M 12 21 ND ND Positive ND ND ND ND 2 ND ND Positive ND ND ND ND 3 ND ND Positive ND ND ND ND 4 ND ND Positive ND ND ND ND 5 ND ND Positive ND ND ND ND 6 ND ND Positive ND ND ND ND 7 ND ND Positive ND ND ND ND BoHV-1 and -2 = bovine herpesvirus 1 and 2; BPSV = bovine papular stomatitis virus; BVDV = bovine viral diarrhea virus; ELISA = enzyme-linked immunosorbent assay; F = female; IHC = immunohistochemistry; M = male; ND = not determined; OvHV-2 = ovine herpesvirus 2; Pan-PPV = panparapoxvirus; qpcr = real-time polymerase chain reaction. qpcr performed on whole blood at the USDA-ARS laboratory, Pullman, WA. Sequence confirmation at USDA ARS laboratory, Pullman, WA. examinations, virus detection by qpcr, DNA sequencing, and virus particle identification by direct electronic microscopy. Acknowledgments We thank Dr. Jim Reynolds and Mr. Roger Blanchard (Visalia, CA), Dr. Ralph Walton (Tulare, CA), Drs. Hong Li and Naomi Taus (USDA-ARS Research Laboratory, Pullman, WA). Declaration of conflicting interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The authors received no financial support for the research, authorship, and/or publication of this article. References 1. Brenner J, et al. Herpesvirus type 2 in biopsy of a cow with possible pseudo-lumpy-skin disease. Vet Rec 2009;165:539 540. 2. Campos FS, et al. Detection of bovine herpesvirus 2 and bovine herpesvirus 4 DNA in trigeminal ganglia of naturally infected cattle by polymerase chain reaction. Vet Microbiol 2014;171:182 188. 3. Carn VM. The role of dipterous insects in the mechanical transmission of animal viruses. Br Vet J 1996;152:377 393. 4. Castrucci G, et al. Reactivation in calves of Bovid herpesvirus 2 latent infection. Arch Virol 1982;72:75 81. 5. Connor RJ, Mukangi DJ. Concurrent outbreak of pseudolumpy skin disease and acute Trypanosoma vivax infection in cattle. Trop Anim Health Prod 1986;18:127 132. 6. De-Giuli L, et al. Development of a polymerase chain reaction and restriction typing assay for the diagnosis of bovine herpesvirus 1, bovine herpesvirus 2, and bovine herpesvirus 4 infections. J Vet Diagn Invest 2002;14:353 356. 7. d Offay JM, et al. Use of polymerase chain reaction assay to detect bovine herpesvirus type 2 DNA in skin lesions from cattle suspected to have pseudo-lumpy skin disease. J Am Vet Med Assoc 2003;222:1404 1407. 8. Gibbs EPJ, Rweyemamu MM. Bovine herpesvirus. Part II. Bovine herpesvirus 2 and 3. Vet Bull 1977;47:411 425. 9. Gigstad DC, Stone SS. Clinical, serologic, and cross-challenge response and virus isolation in cattle infected with three bovine dermatotropic herpesviruses. Am J Vet Res 1977;38:753 757. 10. Ginn PE, et al. Skin and appendages. In: Maxie MG, ed. Jubb, Kennedy, and Palmer s Pathology of Domestic Animals. 5th ed. Vol. 1. Philadelphia, PA: Saunders, 2007:672 676. 11. Imai K, et al. Development of a shuttle polymerase chain reaction for the detection of bovine herpesvirus 2. J Vet Med Sci 2002;64:953 956. 12. Kemp R, et al. Atypical bovine herpes mammillitis affecting cows and calves. Vet Rec 2008;163:119 121. 13. Letchworth GJ, et al. Bovid herpesvirus 2: natural spread among breeding bulls. Cornell Vet 1982;72:200 210. 14. Letchworth GJ, La Due R. Bovine herpes mammillitis in two New York dairy herds. J Am Vet Med Assoc 1982;180:902 907.

690 Watanabe et al. 15. Martin WB, Scott FMM. Latent infection of cattle with Bovid herpesvirus 2. Arch Virol 1979;60:51 58. 16. Petrovski KR. An acute outbreak of teat lesions affecting 38% of a dairy herd in Northland. N Z Vet J 2005;53:162 163. 17. Torres FD, et al. Distribution of latent bovine herpesvirus 2 DNA in tissues of experimentally infected sheep. Res Vet Sci 2009;87:161 166. 18. Torres FD, et al. Acute and latent infection by bovine herpesvirus type 2 in a guinea pig model. Microb Pathog 2010;48:69 73. 19. Wellenberg GJ, et al. Viral infections and bovine mastitis: a review. Vet Microbiol 2002;88:27 45. 20. Woods JA, et al. Isolation of bovine herpesvirus-2 (BHV- 2) from a case of pseudo-lumpy skin disease in the United Kingdom. Vet Rec 1996;138:113 114.