Conference 17 - 2024 Case: 03 20250122

Wednesday Slide Conference, Conference 17, Case 3

Signalment:

Juvenile, female Angus cow, bovine (Bos taurus)

History:
This cow was one of 45 presenting with cutaneous lumps when mustered from a herd of 95 animals. Rectal temperatures ranged from 40-41ºC and some of the affected cows appeared sick. Some cows were panting with increased lung sounds. The mucous membranes were pink. There was no evidence of typical photosensitization lesions in predisposed locations.

Gross Pathology:
Nodules were distributed over the neck, brisket, thorax and to a lesser extent abdomen and forelimbs. Nodules ranged from 10-30mm diameter and were not ulcerated, although the surface of some could be scratched off.

Laboratory Results:
A Pan-Herpesvirus nested PCR was performed, and the product was sent for Sanger sequencing. A BLAST query of the product sequence found it was 100% identical to Bovine alphaherpesvirus 2 [MT862164] 181 nt partial sequence. A ruminant biochemistry panel found elevation in GLDH 96 U/L (0-30 U/L), mild hyperglobulinaemia 48.4 g/L (30.0-45.0 g/L) and mild elevation in serum hemoglobin 0.25 g/dL (0.00-0.20 g/dL). A Capripoxvirus TaqMan assay on the EDTA blood and a skin scab was negative, and a Capripox double antigen multispecies ELISA performed on the serum sample was negative.

Microscopic Description:
Haired skin (x3): Within the sections of haired skin, multifocally within the epidermis, there are hypereosinophilic necrotic keratinocytes, numerous small expansions containing eosinophilic fluid (vesicles) and necrotic cell debris and degenerate neutrophils, sometimes forming small aggregates (micropustules). Multifocally, epithelial cells within the stratum basale, stratum spinosum and stratum granulosum are sloughed, replaced with clumps of multinucleated epithelial cells with hypereosinophilic cytoplasm (syncytia). Moderate numbers of epithelial cells and syncytia within the surface epithelium, and occasionally within epithelium lining hair follicles, contain 1-6µm diameter intranuclear eosinophilic inclusion bodies with margination of chromatin. Multifocally, keratinocytes are swollen with clearing of cytoplasm (ballooning degeneration). There is mild, multifocal orthokeratotic hyperkeratosis, with mild to moderate extravasated erythrocytes adjacent to the epidermal surface (hemorrhage). Throughout the superficial and deep dermis, there are multifocal, moderate infiltrates of neutrophils, lymphocytes, plasma cells, eosinophils and macrophages associated with cell debris, and multifocal mild to moderate separation of dermal collagen fibres with clear space and/or eosinophilic fluid (edema). Occasionally sebaceous glands are surrounded and infiltrated by neutrophils and apocrine glands are dilated. Few lymphatic vessels in the deep dermis are dilated. Within the dermis of one section, adjacent to the cut margin, there is a focal aggregate of refractile material, surrounded by eosinophilic necrotic material, and subsequently surrounded by a moderate margin of leukocytes with squash artefact.

Contributor’s Morphologic Diagnosis:
Haired skin, dermatitis, necrosuppurative and, lymphoplasmacytic multifocal to widespread, chronic-active, with edema, syncytial cells, pustules, vesicles and intranuclear epithelial eosinophilic viral inclusion bodies, Bos taurus.

Contributor’s Comment:
Infection with Bovine Herpesvirus 2 (BoHV-2) in cattle can cause two distinct clinical conditions: one is an acute onset, mild generalized skin infection called Pseudo Lumpy Skin Disease (PLSD) which manifests as cutaneous nodules which develop a central depression; the second is Bovine Herpesvirus Mammillitis (BHM), a localized ulcerative condition affecting the teats and udder of cattle. The disease is found in many parts of the world and is likely transmitted by biting insects or mechanically by equipment used on farms. The gross appearance of the generalized skin lesions can closely resemble Lumpy Skin Disease, which has trade implications in many countries.²¹

Bovine herpesvirus 2 (BoHV-2) is an enveloped double stranded DNA virus approximately 150-200nm in size, belonging to the family Orthoherpesviridae, subfamily Alphaherpesvirinae, genus simplexvirus.

Bovine herpesvirus 2 infection was first identified in Southern and Eastern Africa. Since its discovery in the 1950’s, it has been reported almost globally with case reports from Europe, Japan, Australia, Brazil, the United States and Israel.^{2,3,8–10,13,16}

In the wild, BoHV-2 infects cattle, wild ruminants and pseudo-ruminants (hippopotamus).¹³ Sheep have been experimentally infected with BoHV-2 producing clinical disease similar to Bovine Herpesvirus Mammillitis.¹⁷ Rabbits, guinea pigs and mice have also been experimentally infected with BoHV-2.⁸

Transmission of BoHV-2 most likely occurs via intradermal injection of the virus by biting insects, or via contaminated equipment coming into contact with wounds and cuts. Biting flies have been demonstrated to be able to transmit the infection between cell cultures in the lab, and the distribution and seasonality of outbreaks of Pseudo Lumpy Skin Disease are thought to be related to the distribution and peaks in biting insect activity.^6,9,12 Milking equipment is suspected of being able to transmit the virus within milking herds if clinically affected cattle are present.^5,10

Serological surveys indicate that circulation of BoHV-2 in cattle can occur without any episodes of clinical disease suggesting most infections may be subclinical.^8,9,15

In experiments where BoHV-2 was intradermally inoculated into the flank of cattle, a discrete raised swelling was observed at the injection site by day 3 post inoculation. Generalized discrete swellings appeared over the head, neck and dorsal body by day 6 which developed crusts which gradually lifted over the course of 3 weeks to reveal greyish-white areas of skin with reduced numbers of hairs (alopecia). Lymph node enlargement was observed by day 5 and persisted to day 10 post inoculation.¹⁶

In the field, the incubation period of BoHV-2 is considered to be around 1-2 weeks, with skin lesions resolving 2-4 weeks after first appearing. Neutralizing antibodies appear 7-14 days after initial exposure.^16,18 Outbreaks can occur seasonally or sporadically depending on the activity of biting insects and the presence of naive hosts.^11,19

Post infection, like other herpesviruses, BoHV-2 becomes latent with the virus likely retreating to the neuronal tissue such as the trigeminal ganglion and possibly lymphoid tissue where it may lay dormant until infection is reactivated during periods of stress.^3,17

Generalized infection with BoHV-2 induces Pseudo Lumpy Skin Disease, which is characterized by:^1,4,16

Circumscribed cutaneous nodules over the body, frequently on the head, neck, shoulders, back and perineum.
Around 4-6 days after the appearance of gross lesions, the cutaneous lesions begin to crust over. The crusts eventually fall off leaving areas of alopecia.
Lesions disappear when the hair regrows.
Lymphadenitis may be observed during early stages of infection.
Animals make a full recovery without treatment.

Localized infection with BoHV-2 of the udder and teats is called Bovine Herpesvirus Mammillitis, and is characterized by:^11,20

Variable sized tender edematous swellings on the udder or teats.
Vesicles may appear on affected areas
The overlying skin then ulcerates and becomes painful, taking 3-10 weeks to heal

Histopathological changes of BoHV-2 infection are characterized by the formation of epithelial syncytia initially within the stratum basale and inner stratum spinosum with prominent intranuclear eosinophilic inclusion bodies.¹¹ Intranuclear inclusions are classified as Cowdry type A inclusions and have central eosinophilic or amphophilic deposits surrounded by a clear halo and the nuclear chromatin is pushed to the margin of the nucleus.¹⁹ Intranuclear inclusions are most numerous after the appearance of the gross nodules but before the appearance of crusts (around 4-6 days later),^1,11 which coincides approximately with the time when virus can be isolated or detected by PCR (see below). When the lesions crust over, the epithelium becomes necrotic and infiltrated by large numbers of neutrophils. Loss of the necrotic epithelium results in an ulcer which becomes covered with haemorrhage, fibrinous exudate and numerous degenerate neutrophils. Deeper in the dermis, there is edema and a mononuclear infiltrate. As the lesion heals, there is fibrosis in the superficial dermis with perivascular infiltrates of mononuclear cells.¹¹

The following assays have been reported in the literature to support the diagnosis of BoHV-2 infection.

Test	Required Samples	Notes
PCR^1,12,19	Fresh skin lesions	Most likely to yield detectable virus DNA up to 8 days post infection or before cutaneous lesions crust over or ulcerate
Cytology¹¹	Early skin lesions (<5 days after initial appearance)	Presence of syncytia with intranuclear inclusions
Serology - ELISA¹⁵	Serum
Serology – Serum neutralization^9,18	Serum	Antibodies detected as early as 10 days post inoculation or about 5-7 days after appearance of cutaneous lesions
Serology – Immunofluorescent antibody test (IFAT)¹⁰	Serum
Negative staining electron microscopy¹⁹	Fresh skin lesions	Herpesvirus particles may be seen
Virus culture^6,9,10,16,18	Blood, Fresh skin lesions up to around day 6-7 post infection	Vero cells Calf kidney cells Fetal bovine muscle cells Madin Darby Bovine Kidney (MDBK)

The following conditions should be considered as potential differentials for Bovine Herpesvirus-2 infection based on their clinical appearance.^4,11,20

Disease	Clinical Presentation	Tests to diagnose/ differentiate	Key differentiating histological features
Viral
Early-stage Lumpy Skin Disease (Capripoxvirus)	Firm circumscribed cutaneous nodules which develop central areas of necrosis and scarring	Capripox PCR, biopsy + histopathology, Electron microscopy demonstrating pox virus particles	Epidermal vacuolar swelling with intracytoplasmic inclusions. Vasculitis
Bovine Papillomavirus	Papillomas or warts of varying size on body. Mostly frequently seen in younger cattle	Biopsy + histopathology, PCR of skin lesions	Unencapsulated, proliferative cutaneous neoplasm accompanied by basal cell hyperplasia, acanthosis and hyperkeratosis
Bovine papular stomatitis (Parapoxvirus)	Raised red papules, erosions or ulcers around muzzle and nose, occasionally elsewhere on the body	Electron microscopy of tissue, virus culture, biopsy + histopathology, ELISA/Agar gel precipitation tests to look for antibodies	Epidermal vacuolar swelling with intracytoplasmic inclusions, epidermal proliferation
Pseudocowpox (Parapoxvirus)	Raised red sores, scabs or vesicles on teats, thighs or perineum	PCR on skin lesions, scabs or blood, biopsy + histopathology.	Epidermal vacuolar swelling with intracytoplasmic inclusions, epidermal proliferation
Cowpox (Orthopoxvirus)	Papules or ulcers on the teat or udder	PCR of skin lesions, electron microscopy of scabs demonstrating pox virus particles¹⁴, biopsy + histopathology	Epidermal vacuolar swelling with intracytoplasmic inclusions, focal epidermal hyperplasia.
BVDV dermatitis	Patchy hyperkeratosis around neck, shoulder and perineum	Bovine Viral Diarrhea Virus Antigen ELISA or PCR on ear notch, blood, hair or visceral organs
Bovine Herpesvirus 4	Vesicles, pustules and ulcers on udder. Teats are usually not involved	PCR on skin lesions, serological testing, biopsy + histopathology	Intraepithelial pustular dermatitis
Bacterial
Dermatophilosis (Dermatophilus congolensis)	Papules with crusts entrapped in hair and areas of alopecia often over the dorsal aspect of head, neck and body	Bacterial cultures, cytology of crusts, biopsy + histopathology	Multiple parallel rows of cocci, trapped within alternating layers of hyperkeratosis and serous fluid and degenerate neutrophils. Purulent folliculitis
Cutaneous tuberculosis (Mycobacterium bovis)	Single or multiple nodules, abscesses or ulcers	PCR, mycobacterial culture, biopsy + histopathology	Pyogranulomatous dermatitis with central caseous necrosis with acid fast bacilli present.
Fungal
Dermatophytosis (Ringworm, Trichophyton verrucosum)	Expanding circular hairless lesions mostly on head and neck. May appear red and moist to scaly and grey.	Dermatophyte cultures, biopsy + histopathology, PCR	Fungal hyphae clustering within hair shafts and arthrospores around the outside of hairs.
Parasites
Mites (multiple species)	Nodules, alopecia and crusts which may be itchy	Superficial or deep skin scrapings
Onchocercosis	Cutaneous nodules over brisket, abdomen and udder	Biopsy + histopathology	Presence of microfilaria, surrounded by eosinophilic inflammation
Besnoitiosis (Besnoitia sp.)	Alopecia, scaling and thickening of skin over neck ,shoulders and rump	biopsy + histopathology, PCR, serology	Presence of protozoal tissue cysts
Hypoderma sp. (warbles)	Firm raised nodules in the skin frequently along the back and shoulders	Clinical examination and presence of breathing holes Serological tests
Neoplasia
Cutaneous Lymphoma	Multifocal subcutaneous/intracutaneous nodules, often accompanied by alopecia, lymphadenopathy	biopsy + histopathology, haematology may reveal lymphocytosis. May arise sporadically and not necessarily be associated with bovine leukosis virus infection	Sheets of neoplastic lymphocytes.
Mast cell tumor	Mulltifocal cutaneous or subcutaneous nodules over body +/- alopecia +/- pigmented +/- ulceration	biopsy + histopathology	Sheets of neoplastic mast cells often accompanied by numerous eosinophils
Other
Photosensitization	Alopecia, erythema and blistering of non-pigmented skin	History – exposure to toxic plants, drug administration, biochemistry – liver function, biopsy + histopathology	Coagulative necrosis of epidermis, dermal edema, vascular necrosis
Skin allergies/urticaria	Haired wheals +/- crusts anywhere on the body, angioedema	History of recent drug administration, insect or plant exposure, response to glucocorticoid administration, biopsy + histopathology	Dermal edema, perivascular infiltrates of granulocytes, mast cells and lymphocytes inconsistently present in upper to mid dermis.

Contributing Institution:
Elizabeth Macarthur Agricultural Institute, Department of Primary Industries and Regional Development

JPC Diagnosis:
Haired skin: Dermatitis, necrotizing, subacute, diffuse, severe, with epithelial viral syncytia, intranuclear viral inclusions, corneal pustules, mixed dermal infiltrates, and edema.

JPC Comment:
The contributor provides an excellent section to accompany an thorough writeup on the topic. The slide has generous viral inclusions and syncytia along with raised vesicles to establish an etiological diagnosis. Conference participants did touch on the nature of intracellular and intercellular edema in this case the lack of overt cellular swelling and intracytoplasmic inclusions lowered suspicion for a poxviral etiology.

One ancillary finding in section was a focal granuloma centered on foreign material (possibly plant material). We speculated that this might be secondary to necrosis of the overlying epithelium and entry into the dermis, though the adjacent portions of skin lack significant viral changes. This lesion is at the edge of the section and is only on one of the three sections of skin provided which limits further interpretation of this incidental observation.

References:

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