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The family Herpesviridae includes viruses identified in mammals, birds and reptiles. All herpesviruses share a similar structure, consisting of a large linear double-stranded DNA genome surrounded by a proteic icosahedral capsid further contained within a lipidic bilayer envelope. The continuous rise of genetic variability and the evolutionary selective pressure underlie the appearance and consolidation of novel viral strains. This applies also to several gamma(γ)-herpesviruses, whose role as primary pathogen has been often neglected and, among these to newly emerged viruses or virus variants responsible for the development of Malignant Catarrhal Fever (MCF) or MCF-like disease. The identification of γ-herpesviruses adapted to new zoological hosts requires specific molecular tools for detection and characterization. These viruses can cause MCF in livestock and wild animals, a disease generally sporadic but with serious welfare implications and which, in many cases, leads to death within a few days from the appearance of the clinical signs. In the absence of a vaccine, the first step to improve disease control is based on the improvement of molecular tools to identify and characterize these viruses, their phylogenetic relationships and evolutionary interaction with the host species. A Panherpes PCR-specific test, based on the conserved DNA polymerase gene, employing consensus/degenerate and deoxyinosine-substituted primers followed by sequencing, is still the preferred diagnostic test to confirm and characterize herpesviral infections. The drawback of this test is the amplification of a relatively short sequence, which makes phylogenetic analysis less stringent. Based on these diagnostic requirements, and with a specific focus on γ-herpesviruses, the present review aims to critically analyze the currently available methods to identify and characterize novel MCFV strains, to highlight advantages and drawbacks and to identify the gaps to be filled in order to address research priorities. Possible approaches for improving or further developing these molecular tools are also suggested.

Sheep-associated malignant catarrhal fever (SA-MCF) is a severe, frequently fatal, lymphoproliferative disease that affects a wide variety of ruminants and is caused by ovine gammaherpesvirus 2 (OvHV-2), a member of the MCF virus (MCFV) complex. The typical clinical manifestations of SA-MCF are well known and easily recognized by veterinarians, resulting in clinical diagnosis of MCF when characteristic clinical signs are present. This article describes the findings observed in cattle infected with OvHV-2 but without typical clinical manifestations of SA-MCF. Three calves with episodes of diarrhea before death and a yearling that died suddenly were investigated. Gross alterations were not suggestive of SA-MCF. Histopathology revealed a combination of proliferating vascular lesions (PVLs) and necrotizing vasculitis in three animals (two calves and the yearling); with PVLs being identified only at the carotid rete mirabile of two calves infected with OvHV-2. Additional significant histopathologic lesions included atrophic enteritis, portal lymphocytic hepatitis, interstitial pneumonia, suppurative bacterial bronchopneumonia, and pulmonary hemorrhage. An immunohistochemical assay designed to identify only antigens of MCFV revealed, positive, intralesional, intracytoplasmic immunoreactivity within epithelial cells of multiple tissues of all animals with PVLs. PCR assays amplified OvHV-2 DNA from multiple tissues of the animals that contained MCFV proteins, confirming the MCFV identified as OvHV-2. Additionally, bovine coronavirus (BCoV) nucleic acids were amplified from tissues of all animals, including the animal not infected by OvHV-2. Collectively, these findings confirmed the participation of OvHV-2 in the development of the disease patterns observed in these animals that were concomitantly infected by BCoV and provide additional confirmation that cattle can be subclinically infected with OvHV-2. Consequently, the real occurrence of OvHV-2-related disease may be more elevated than reported, since asymptomatic or subclinically infected animals are not likely to be investigated for OvHV-2. Furthermore, PVLs should be included as possible histologic indicators of OvHV-2-related diseases in ruminants.

The Macavirus , ovine gammaherpesvirus 2 (OvGHV2), is the cause of sheep-associated malignant catarrhal fever (SA-MCF). Although SA-MCF occurs in a wide range of mammalian hosts, there are few descriptions of this disease and/or infection in goats. This report describes the findings observed in a goat that was infected by OvGHV2 and adds to the rare description of this infection in this animal species. A 6.5-year-old, female, Anglo Nubian goat, with a neurological syndrome, that was euthanized after severe esophageal obstruction was investigated to determine the cause of the brain disease. Histopathology revealed cerebral cortical edema, hemorrhagic rhombencephalitis, severe hepatic necrosis, and atrophic enteritis. An immunohistochemical (IHC) assay identified intracytoplasmic antigens of a malignant catarrhal fever virus (MCFV) within epithelial cells of the intestine, liver, lungs, and kidneys. A semi-nested PCR assay amplified the partial fragment of the OvGHV2 tegument protein gene from the intestine, confirming that the MCFV identified by IHC was OvGHV2. A qPCR assay that targeted the OvGHV2 polymerase gene revealed an elevated quantification cycle ( Cq ), while nanoplate-based digital PCR (dPCR) detected low viral copy load within the OvGHV2 DNA. Furthermore, the nucleic acids of several disease pathogens associated with diseases in ruminants were not amplified. However, the exact cause of the neurological syndrome remained obscure since nucleic acids of neurological disease pathogens such as bovine viral diarrhea virus, bovine alphaherpesvirus 1 and 5, Histophilus somni , and OvGHV2 were not detected from the brain. Collectively, the results of the Cq and dPCR confirmed that this goat was infected with a low viral load of OvGHV2, which probably was insufficient to induce the typical histopathological alterations and subsequent clinical manifestations associated with SA-MCF and/or infections by OvGHV2. Therefore, elevated viral loads of OvGHV2 would have been required for the development of histological lesions and/or clinical manifestations of SA-MCF in this goat. Furthermore, the dPCR methodology can be used for the efficient detection and quantification of OvGHV2 DNA in animals with or without clinical and/or histopathological evidence of SA-MCF. Additionally, since previous cases of OvGHV2 infections in goats did not have the typical clinical manifestations of SA-MCF, one wonders if this Macavirus can induce SA-MCF in goats.

Malignant catarrhal fever (MCF) presents a sporadic yet significant threat to livestock and wildlife. A comprehensive investigation in Karnataka, India into the prevalence and transmission patterns of sheep-associated MCF (SA-MCF) was conducted. A total of 507 sheep peripheral blood leukocyte samples from 13 districts along with 27 cows and 10 buffalo samples from various regions in Karnataka were tested for SA-MCF infection i.e. Ovine gammaherpesvirus 2 (OvHV-2) using heminested PCR. Furthermore, serum samples collected from 73 cows and 15 buffalo suspected of MCF were tested using a commercially available ELISA kit. Additionally, histopathological examinations of affected tissues and phylogenetic analysis of viral tegument protein sequences were conducted. Our findings indicated a 20.11%, 33.33% and 20% positivity for OvHV-2 in sheep, cows and buffalo respectively by PCR. Statistical analysis revealed a significant association between the age of sheep and the detection of OvHV-2. Seven cows and one buffalo serum samples tested positive for ELISA. Clinical findings in bovids were consistent with typical MCF signs, and histopathological results revealed multi-organ involvement characterised by necrotising vasculitis and lymphoid hyperplasia. The nucleotide pairwise identity matrix revealed 99.5% identity between the sequences obtained in the study with sequences from other states. The phylogenetic analysis of partial tegument protein sequences from bovid and sheep samples suggested a close genetic relationship between the local OvHV-2 strains and those from various global regions. Crucially, this study underscores the widespread presence of SA-MCF in Karnataka, with significant implications for both livestock management and wildlife conservation.

Bovine gammaherpesvirus 6 (BoGHV6), previously known as bovine lymphotropic virus, is a member of the Macavirus genus, subfamily Gammaherpesvirinae. Other members of the genus Macavirus include viruses that produce malignant catarrhal fever (MCF) in mammalian hosts, collectively referred to as the MCF virus (MCFV) complex, and the porcine lymphotropic herpesvirus (PLHV). However, the current role of BoGHV6 in the development of diseases and/or disease syndromes remains uncertain and controversial. This paper investigated the participation of BoGHV6 in the development of pulmonary disease in a cow with interstitial pneumonia by histopathology and molecular testing. Tissue antigens of common viral agents of respiratory diseases and Mycoplasma bovis were not identified by immunohistochemistry. Additionally, molecular assays designed to amplify common bacterial and viral pathogens of pulmonary disease did not amplify the nucleic acids of these agents. However, a pan-PCR assay amplified the DNA of the herpesvirus polymerase gene, while the specific BoGHV6 nested-PCR assay amplified the partial fragment of the BoGHV6 polymerase gene derived from the pulmonary tissue with interstitial pneumonia. Phylogenetic analysis revealed that the BoGHV6 strain herein identified had 99.8% nucleotide (nt) sequence identity with reference strains of BoGHV6, but only 72.2–73.5% and 67.9–68.6% nt identity with reference strains of MCFV and PLHV, respectively. Consequently, these results suggest that BoGHV6 was associated with the pulmonary disease observed in this cow.

This study investigated the possible occurrence of pulmonary disease in beef cattle from 13 municipalities within the State of Mato Grosso (MT), Brazil. The state of MT is a leading player in beef cattle production in Brazil, but with comparatively few data relative to the occurrence of pulmonary disease or ovine gammaherpesvirus 2 (OvGHV2)-related infections in cattle. Pulmonary samples from 44 beef cattle, with ages ranging between 18 and 28 months, were collected during slaughter and processed to determine the patterns of pulmonary lesions. Additionally, duplicate fragments were used in immunohistochemical (IHC) assays designed to detect malignant catarrhal fever (MCFV) antigens and in molecular assays to amplify 10 agents associated with the development of bovine respiratory disease (BRD). Interstitial pneumonia (IP) was diagnosed in most of the lungs (98%; 43/44) evaluated from all municipalities. MCFV antigens were detected in 37% (16/43) of the animals with IP. Only four pathogens were amplified by molecular assays within the lungs of cattle with IP: OvGHV2 (23%; 10/43), bovine viral diarrhea virus (12%; 5/43), bovine coronavirus (7%; 3/43), and Mannheimia haemolytica (2%; 1/43). The nucleic acids of bovine respiratory syncytial virus, bovine alphaherpesvirus 1, bovine parainfluenza virus 3, Pasteurella multocida, Histophilus somni, and Mycoplasmopsis (Mycoplasma) bovis were not amplified. Singular infections in cattle from municipalities were associated with MCFV (n = 3) and OvGHV2 (n = 2), while OvGHV2 occurred in all dual and triple infections. However, in four animals with IP, none of the disease pathogens identified were detected. Statistically, MCFV antigens were more frequently (p = 0.048) detected in the lungs of female (75%; 12/16) cattle with IP relative to males (25%; 4/16). Additionally, there was a positive correlation (p < 0.001) between the IHC detection of MCFV antigens within the bronchial epithelium and the epithelium of peribronchial glands of cattle with IP. This is the first study to statistically demonstrate that female cattle are at greater risk of developing MCFV-related infections as compared to male animals. The detection of OvGHV2 in singular and multiple infections during this investigation supports earlier studies that associate this pathogen with the development of pulmonary disease in cattle, indicating that OvGHV2 can contribute to the etiology of IP. Additionally, the detection of OvGHV2-induced infections in asymptomatic cattle suggests that all animals were subclinically infected, confirming that subclinically OvGHV2-induced infections may be widespread in ruminants from Brazil. Furthermore, the occurrence of atypical interstitial pneumonia cannot be discarded, particularly in animals with IP but without any associated pathogen. These initial findings suggest the need for a more elaborate investigation to understand the dynamics of pulmonary disease within this state.

Background Wildebeest associated malignant catarrhal fever (WA-MCF) is a fatal disease of cattle. Outbreaks are seasonal and associated with close interaction between cattle and calving wildebeest. In Kenya, WA-MCF has a dramatic effect on cattle-keepers who lose up to 10% of their cattle herds per year. The objective of this study was to report the impact of WA-MCF on a commercial ranch and assess the performance of clinical diagnosis compared to laboratory diagnosis as a disease management tool. A retrospective study of WA-MCF in cattle was conducted from 2014 to 2016 at Kapiti Plains Ranch Ltd., Kenya. During this period, 325 animals showed clinical signs of WA-MCF and of these, 123 were opportunistically sampled. In addition, 51 clinically healthy animals were sampled. Nested polymerase chain reaction (PCR) and indirect enzyme linked immunosorbent assay (ELISA) were used to confirm clinically diagnosed cases of WA-MCF. A latent class model (LCM) was used to evaluate the diagnostic parameters of clinical diagnosis and the tests in the absence of a gold standard. Results By PCR, 94% (95% C.I. 89–97%) of clinically affected animals were positive to WA-MCF while 63% (95% C.I. 54–71%) were positive by indirect ELISA. The LCM demonstrated the indirect ELISA had poor sensitivity 63.3% (95% PCI 54.4–71.7%) and specificity 62.6% (95% PCI 39.2–84.9%) while the nested PCR performed better with sensitivity 96.1% (95% PCI 90.7–99.7%) and specificity 92.9% (95% PCI 76.1–99.8%). The sensitivity and specificity of clinical diagnosis were 99.1% (95% PCI 96.8–100.0%) and 71.5% (95% PCI 48.0–97.2%) respectively. Conclusions Clinical diagnosis was demonstrated to be an effective method to identify affected animals although animals may be incorrectly classified resulting in financial loss. The study revealed indirect ELISA as a poor test and nested PCR to be a more appropriate confirmatory test for diagnosing acute WA-MCF. However, the logistics of PCR make it unsuitable for field diagnosis of WA-MCF. The future of WA-MCF diagnosis should be aimed at development of penside techniques, which will allow for fast detection in the field.

Ovine gammaherpesvirus-2 (OvHV-2) causes a lethal disease in cattle and some wild ruminants called malignant catarrhal fever (MCF), which affects the epithelial and lymphoid tissues of the respiratory and digestive tracts and has an important impact on the livestock industry. In this study, MCF was diagnosed in 18 of 427 cattle from different sites in Egypt by its typical clinical signs, found in all 18 animals: corneal opacity, fever, erosions in the buccal cavity, lymphadenitis, and purulent nasal discharge. All affected cattle had been reared in contact with clinically inconspicuous sheep. Of the 18 clinically ill cattle, 13 succumbed to the disease, resulting in estimated morbidity and case fatality rates of 4.2% and 72.2%, respectively. Five samples collected from the affected cattle were positive for OvHV-2 by real-time PCR and were used for sequencing of an 832-bp fragment of the ORF27/gp48 gene. The ORF27 nucleotide sequence of all Egyptian samples was identical, but distinct from viruses found in other parts of Africa and the Mediterranean.

Malignant catarrhal fever (MCF) can affect both domestic and wild artiodactyls. In a zoological setting, in which subclinical carriers and susceptible species are often housed in close proximity, the disease can prove fatal. This report describes a case of goat-associated MCF in a captive moose (Alces alces). The diagnosis was confirmed by histopathology, which showed lymphocytic vasculitis in the brain and panuveitis, and by detection of caprine herpesvirus 2 DNA in tissues. Identical viral DNA sequences amplified from the clinically affected moose and from domestic, petting goats (Capra aegagrus hircus) housed in the zoo suggest that the goats were the source for the virus transmutation. This is the first report, to our knowledge, of confirmed goat-associated MCF in any moose in North America and of the surveillance measures and procedures put in place to prevent additional spread of the disease.

An outbreak of sheep associated malignant catarrhal fever in crossbred cattle in a village of Andhra Pradesh, southern India, affected thirteen adult cows and two calves from a population of forty animals. All the affected animals were died between December and January 2013–14. The clinical and gross postmortem findings were typical of MCF in Indian crossbred cattle. Migrating sheep flocks were suspected source of infection for the cattle. The diagnosis was confirmed by heminested PCR in all the affected cattle and the suspected sheep flock. The PCR provided evidence of ovine herpes virus type 2.