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Malignant catarrhal fever (MCF), caused by ovine herpesvirus 2 (OvHV2; Orthoherpesviridae , Macavirus ovinegamma2 ), has sheep as natural hosts. OvHV2 is an important macavirus globally that induces fatal disease in dead-end hosts. Goats, which can be infected subclinically with OvHV2, rarely develop MCF. A 28-wk-old female goat was presented with fever and multifocal crusty skin lesions. Histologic examination of a skin biopsy suggested erythema multiforme (EM), with pyoderma and dermal vasculitis. The doe was euthanized and subjected to postmortem and histologic examination. MCF was suspected and PCR assays for macaviruses were performed, followed by immunohistochemistry (IHC) for OvHV2 latency-associated nuclear antigen (oLANA), RNA in situ hybridization for Ov2.5 mRNA, and IHC to characterize infiltrating leukocytes. The main postmortem finding was severe multifocal ulcerative dermatitis with macrophage- and T cell-mediated arteritis. The latter was also detected in kidney, spleen, heart, and intestinal wall. The PCR assay detected high loads of OvHV2 in tissues. OvHV2 oLANA and Ov2.5 mRNA were expressed within the lesions in leukocytes, endothelial cells, fibroblasts, and/or keratinocytes. Our case confirms that MCF can initially manifest clinically as a skin disease in goats and as EM with confirmed viral etiology., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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.

In this study, positive blood and organ samples were obtained from different mixed herds of sheep and cattle against ovine herpesvirus 2 (OvHV-2) infection. Target-positive DNA was sequenced and compared with worldwide distributed OvHV-2 sequences. Tegument gene (422 base pairs) and glycoprotein B (gB) gene (2800 base pairs) amplicons of OvHV-2 genome were used for understanding of epidemiology of malignant catarrhal fever (MCF) infection in Turkey. The results of nucleotide sequencing of polymerase chain reaction (PCR) products indicated presence of sheep-associated form for MCF infection in Turkey. Although the obtained sequences were genetically different from each other, it was found that genetic variations were limited.

A case of malignant catarrhal fever (MCF) occurred in a 4‑month‑old calf housed in a semi‑intensive herd in central Italy is described. The herd was in strict cohabitation with a group of domestic sheep. The calf displayed clinical signs that resembled the acute form of MCF and, after a few days of antibiotic and anti inflammatory therapy, died in September 2016. The diagnosis was confirmed in vivo in blood by detection of ovine herpesvirus type 2 DNA through real‑time PCR. At necropsy, the gross post‑mortem findings were typical of MCF and the histological and molecular assays confirmed the presence of the virus. The sheep flock was suspected to be the source of the infection. In Italy, as well as in Europe, there is little data regarding the epidemiology and the recurrence of the disease in herds of cattle, due to the lack of an active surveillance plan and to a major consideration of MCF between differential diagnosis.

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.

Domestic and wild sheep are the natural reservoirs for ovine gammaherpesvirus 2 (OvHV-2), the causative agent of sheep-associated malignant catarrhal fever (SA-MCF). Virtually all adult sheep are infected with OvHV-2 under natural flock conditions, and infection is normally subclinical. MCF-like clinical signs and typical histologic lesions in sheep have been linked during case investigations at veterinary diagnostic laboratories; however, the confirmation of naturally occurring MCF in sheep is problematic. To date, the assays for detection of OvHV-2-specific antibodies or DNA are usually positive in sheep, regardless of health status, so mere detection of antibodies or the agent is of minimal diagnostic significance in this species. We document herein a naturally occurring MCF case in a 4-mo-old domestic lamb and demonstrate that the affected animal had 100-1,000 times more OvHV-2 copy numbers in tissues than in healthy adult and age-matched sheep. These results indicate that high copy numbers of viral DNA in tissues associated with characteristic lesions can be used to confirm the diagnosis of MCF in sheep.

A 10-y-old Watusi ( Bos taurus africanus) steer housed at a drive-through game park in Winston, Oregon developed severe clinical illness including fever, marked nasal discharge, injected scleral and conjunctival membranes, plus oral hemorrhages and erosions. The animal responded poorly to supportive treatment and was euthanized. Additional gross findings at postmortem examination included papules and erosive lesions on the tongue, hemorrhagic large intestine, and multifocal cardiac hemorrhages. Histopathologic findings included multifocal lymphoplasmacytic vasculitis plus fibrin exudation in heart and tongue. Total DNA obtained from the splenic samples was positive for alcelaphine gammaherpesvirus 1 (AlHV-1) as tested by a multiplex PCR for malignant catarrhal fever (MCF) viruses. The AlHV-1 detection was further confirmed by amplification and sequencing of a viral DNA polymerase gene fragment, which was identical to AlHV-1 sequences in GenBank. This was the first diagnosis of clinical wildebeest-associated MCF on these premises, although wildebeest have been held at the park for over 25 y. This disease is sporadic in North America and should be considered as a differential diagnosis for febrile illness with ulcerative oral lesions in ruminants.

Malignant Catarrhal Fever (MCF) was investigated in the central nervous system of cattle with neurological syndrome. Two-hundred-ninety samples were analyzed by histology, and molecular methods to detect ovine herpesvirus type 2 (OvHV-2) were optimized and validated. The qualitative polymerase chain reaction (qualitative PCR) analytical sensitivity was 10 1 DNA copies/μL and found 4.8% (14/290) positive for OvHV-2. The quantitative polymerase chain reaction (qPCR) analytical sensitivity was 10 0 DNA copy/μL and 5.9% (17/290) positivity, with 47.1% (8/17) of the positive samples presenting histological evidence of non-purulent meningo-encephalitis. The qualitative PCR products (422bp of the ORF75 region) were sequenced and submitted to phylogenetic analysis. Identity matrices showed 100% similarity in OvHV-2 samples obtained in this study and those recovered from GenBank, corroborating other studies., (Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

A 3-year-old cow was presented with bilateral corneal edema, increased respiratory effort, nasal discharge, and pyrexia. Ovine herpesvirus-2 was detected, confirming malignant catarrhal fever (MCF). The findings from this case suggest that MCF should be included in the differential diagnosis of mature cattle with ocular and nasal lesions, especially when sheep are present on the farm.

Background: Malignant catarrhal fever (MCF) is a fatal herpesvirus infection, affecting various wild and domestic ruminants all over the world. Water buffaloes were reported to be particularly susceptible for the ovine herpesvirus-2 (OvHV-2) causing the sheep-associated form of MCF (SA-MCF). This report describes the first case of possibly caprine-associated malignant catarrhal fever symptoms in a domestic water buffalo in Switzerland., Case Presentation: The buffalo cow presented with persistent fever, dyspnoea, nasal bleeding and haematuria. Despite symptomatic therapy, the buffalo died and was submitted to post mortem examination. Major findings were an abomasal ulceration, a mild haemorrhagic cystitis and multifocal haemorrhages on the epicardium and on serosal and mucosal surfaces. Eyes and oral cavity were not affected. Histopathology revealed a mild to moderate lymphohistiocytic vasculitis limited to the brain and the urinary bladder. Although these findings are typical for MCF, OvHV-2 DNA was not detected in peripheral blood lymphocytes or in paraffin-embedded brain, using an OvHV-2 specific real time PCR. With the aid of a panherpesvirus PCR, a caprine herpesvirus-2 (CpHV-2) sequence could be amplified from both samples., Conclusions: To our knowledge, this is the first report of malignant catarrhal fever in the subfamily Bovinae, where the presence of CpHV-2 could be demonstrated. The etiological context has yet to be evaluated.

Alcelaphine herpesvirus 1 (AlHV-1), carried by wildebeest asymptomatically, causes malignant catarrhal fever (WD-MCF) when cross-species transmitted to a variety of susceptible species of the Artiodactyla order. Experimentally, WD-MCF can be reproduced in rabbits. WD-MCF is described as a combination of lymphoproliferation and degenerative lesions in virtually all organs and is caused by unknown mechanisms. Recently, we demonstrated that WD-MCF is associated with the proliferation of CD8(+) cells supporting a latent type of infection in lymphoid tissues. Here, we investigated the macroscopic distribution of AlHV-1 infection using ex vivo bioluminescence imaging in rabbit to determine whether it correlates with the distribution of lesions in lymphoid and nonlymphoid organs. To reach that goal, a recombinant AlHV-1 strain was produced by insertion of a luciferase expression cassette (luc) in an intergenic region. In vitro, the reconstituted AlHV-1 luc(+) strain replicated comparably to the parental strain, and luciferase activity was detected by bioluminescence imaging. In vivo, rabbits infected with the AlHV-1 luc(+) strain developed WD-MCF comparably to rabbits infected with the parental wild-type strain, with hyperthermia and increases of both CD8(+) T cell frequencies and viral genomic charge over time in peripheral blood mononuclear cells and in lymph nodes at time of euthanasia. Bioluminescent imaging revealed that AlHV-1 infection could be detected ex vivo in lymphoid organs but also in lung, liver, and kidney during WD-MCF, demonstrating that AlHV-1 infection is prevalent in tissue lesions. Finally, we show that the infiltrating mononuclear leukocytes in nonlymphoid organs are mainly CD8(+) T cells and that latency is predominant during WD-MCF.

Malignant catarrhal fever (MCF) is a frequently fatal disease, primarily of ruminants, caused by a group of gammaherpesviruses. Due to complexities of pathogenesis and epidemiology in various species, which are either clinically-susceptible or reservoir hosts, veterinary clinicians face significant challenges in laboratory diagnostics. The recent development of specific assays for viral DNA and antibodies has expanded and improved the inventory of laboratory tests and opened new opportunities for use of MCF diagnostics. Issues related to understanding and implementing appropriate assays for specific diagnostic needs must be addressed in order to take advantage of molecular diagnostics in the laboratory.

Two cases of sheep-associated malignant catarrhal fever (MCF) in pigs were diagnosed on a small farm in New York, and in Kentucky. In both cases, the initial diagnosis was based on histopathological changes representing typical lymphoproliferative vasculitis in multiple tissues of the affected pigs. Ovine herpesvirus 2 (OvHV-2) DNA was detected by polymerase chain reaction in the tissues of affected pigs in both cases. The amplified sequences were identical between the clinically affected pigs and the OvHV-2-infected sheep. Additional virological and bacteriological examination showed that the affected pigs were negative for agents that cause Aujeszky's disease, classical swine fever, porcine enterovirus, and rabies. An antibody against a conserved epitope among MCF viruses was detected in 1 clinically affected pig and 2 unaffected cohort pigs, as well as in all the associated sheep. To the authors' knowledge, this is the first report of porcine MCF in North America, and suggests that OvHV-2 is associated with clinical MCF in pigs.

Malignant catarrhal fever (MCF) is a fatal, systemic disease of cattle and other domestic and wild ruminants that, in Europe, is caused by Ovine herpesvirus 2 (OvHV-2). American bison (Bison bison) are highly susceptible to the disease. An adult American bison, housed in a zoo in southern Italy in close cohabitation with a group of domestic sheep (Ovis aries aries) displayed clinical signs that resembled the acute form of MCF. By real-time polymerase chain reaction, OvHV-2 DNA was detected intravitam in blood, in nasal and ocular swabs, and postmortem in tissue samples of the bison. By indirect fluorescent antibody test, high MCF antibody titers were found in the bison serum. Ovine herpesvirus 2 DNA and antibodies were also found in blood samples from the domestic sheep, thus suggesting a potential role of these animals as a source of the infection. To the authors' knowledge, this is the first MCF case in captive ruminants in Italy and the second confirmed case in captive bison of European zoos.

A 4-yr-old male bongo antelope (Tragelaphus euryceros) died after an acute clinical course involving a febrile illness, anorexia, lethargy, minor oculonasal discharge, and diarrhea. Histologic lesions were compatible with malignant catarrhal fever (MCF). Polymerase chain reaction (PCR) revealed an amplified region of a herpesviral DNA polymerase gene sequence nearly identical to that of a MCF virus previously identified in Nubian ibex (Capra nubiana). The bongo had been housed across from an exhibit containing Nubian ibex that tested positive for MCF viral antibodies by competitive inhibition enzyme-linked immunosorbent assay. Further testing of the zoo's ibex via PCR also revealed viral DNA sequences nearly identical to those found in the bongo's tissues.

Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain ruminants, is caused by ovine herpesvirus 2 (OvHV-2). Molecular diagnosis of SA-MCF in affected animals has relied on detection of OvHV-2 DNA using a nested PCR, which has significant potential for amplicon contamination as a routine method in diagnostic laboratories. In this report, a nonnested and a previously developed real-time PCR were validated for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of blood or tissue samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of clinical SA-MCF; 2) 200 samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples defined by nested PCR from clinically affected animals, 95 (98%) were positive by nonnested PCR and 93 (96%) were positive by real-time PCR, respectively. One hundred percent of the samples from the animals with experimentally induced MCF were positive by real-time PCR, while 99% were positive by nonnested PCR. Neither nonnested PCR nor real-time PCR yielded a positive result on any of the 100 nested PCR-negative samples from animals without evidence of clinical MCF. The data confirmed that both nonnested and real-time PCR maintained high specificity and sensitivity for the detection of OvHV-2 DNA in clinical samples.

A single-tube duplex nested polymerase chain reaction (sdn-PCR) was developed for the detection of and discrimination between ovine herpesvirus-2 (OvHV-2) and alcelaphine herpesvirus-1 (AIHV-1). These viruses respectively cause sheep- and wildebeest-associated malignant catarrhal fever (SA-MCF and WA-MCF). In the first step of the sdn-PCR, two primers with high annealing temperatures based on conserved regions of the tegument genes were used for DNA amplification. In the second step, two primer sets based on variable regions of the respective OvHV-2 and AIHV-1 genes and with annealing temperatures > 11 degrees C below the primers used in the first step, were used. Internal regions of different sizes from amplicons produced in the first step were amplified. This single-tube test obviates the need for two separate assays to detect both viral types, thereby reducing time, labour and cost.

Objective: To monitor ovine herpesvirus type 2 (OvHV-2) infection status and the association between OvHV-2 infection and development of clinical signs of malignant catarrhal fever (MCF) in cattle., Design: Longitudinal study., Animals: 30 mature adult cows and 18 cattle submitted for necropsy., Procedure: Blood and milk samples were collected at monthly intervals from 30 adult cows for 20 consecutive months. Nasal and ocular swab specimens were also collected during months 9 through 20. Polymerase chain reaction (PCR) assay for detection of OvHV-2 was performed on blood, milk, nasal swab, and ocular swab specimens. Competitive inhibition ELISA (CI-ELISA) for detection of antibodies against MCF viruses was performed on serum samples obtained prior to study initiation and monthly during the last 12 months. Tissues obtained from herdmates without clinical signs of MCF that were submitted for necropsy were analyzed for OvHV-2 DNA via PCR assay for possible sites of latency., Results: Initially, 8 of 30 cows had positive CI-ELISA results. Seroconversion was detected in 4 cows. Ovine herpesvirus type 2 DNA was intermittently detected in blood, milk, nasal secretions, or ocular secretions from 17 of 30 cows. Twenty-one cows had positive CI-ELISA or PCR assay results. No cattle in the study developed clinical signs of MCF. Results of PCR assays performed on tissue samples from 2 of 18 animals submitted for necropsy were positive for OvHV-2. CONCLUSIONS AND CLINICAL RELEVANCE; OvHV-2 infection can occur in cattle without concurrent development of clinical MCF. Ovine herpesvirus type 2 DNA was detected intermittently, suggesting fluctuating viral DNA loads or reinfection in subclinical cattle. A definitive site of latency was not identified from tissues obtained during necropsy.

A malignant catarrhal fever (MCF)-like disease was induced experimentally in 3 sheep after aerosol inoculation with ovine herpesvirus-2 (OvHV-2). Each of 3 OvHV-2-negative sheep was nebulized with 2 ml of nasal secretions containing approximately 3.07 X 10(9) OvHV-2 DNA copies from a sheep experiencing an intensive viral-shedding episode. Ovine herpesvirus-2 DNA became detectable by polymerase chain reaction in the peripheral blood leukocytes of all 3 sheep within 3 days, and all 3 seroconverted between 6 and 8 days postinfection (PI). The sheep developed clinical signs, with copious mucopurulent nasal discharge and fever around 14 days PI. One of the 3 clinically affected sheep was euthanized at 18 days PI. Major lesions at necropsy were multifocal linear erosions and ulcers in mucosa of the cheeks, tongue, pharynx, and proximal esophagus and mild disseminated pneumonia. Microscopically, there was extensive moderate superficial histiocytic-lymphocytic rhinitis with epithelial dissociation and degeneration. Moderate multifocal histiocytic bronchointerstitial pneumonia was associated with loss of terminal bronchiolar epithelium. Lymphocytic vasculitis was present only in the lung. The remaining 2 sheep recovered clinically, approximately 25 days PI. The study revealed that clinical signs and lesions resembling MCF can develop when uninfected sheep are exposed to a high dose of aerosolized OvHV-2.

For the first time Ovine Herpesvirus 2 (OvHV-2) was identified in Swiss pigs as the causative agent of Porcine Malignant Catarrhal Fever (MCF). Diseased animals from two farms were observed to show weakness, anorexia, fever up to 41 degrees C, and neurological symptoms, i.e. ataxia, convulsions and hyperesthesia, erosion on the snout and in the oral and nasal mucosa, as well as multiple skin lesions. Histopathological findings included severe non-purulent inflammation with mononuclear cell infiltration in several organs. Most dominant were meningo-encephalitis, disseminated nephritis as well as purulent catarrhalic bronchopneumonia. The findings were quite reminiscent of the lesions due to MCF in cattle and give therefore substantial proof to use Porcine Malignant Catarrhal Fever as the term for the disease. Identification of the causative agent was done with a quantitative PCR specific for OvHV-2. Different tissues from diseased animals were positive. Furthermore, one animal which had been ill for more than five days tested positive for antibodies against an epitope conserved among MCF viruses. Serum samples from diseased animals reacted negative towards Classical Swine Fever- and Pseudorabies virus antigen. A weakly positive reaction against porcine enterovirus type I argued against the involvement of enteroviruses in the observed disease. Moreover, by means of different conventional PCRs, we detected the newly discovered porcine lymphotropic herpesviruses for the first time in Switzerland and could at the same time exclude their involvement in Porcine Malignant Catarrhal Fever.

A subacute disease presenting primarily as alopecia and weight loss occurred in 2 white-tailed deer (Odocoileus virginianus) on farms in Minnesota and in Texas. A presumptive diagnosis of malignant catarrhal fever (MCF) was made on the basis of histological lesions. Antibody against an epitope conserved among the MCF group viruses was detected in the serum of both deer. DNA samples from the deer were subjected to a variety of PCR amplifications. Alignment of the amplified sequences from the diseased animals revealed that they were 100% identical to each other and to the same DNA fragment from the newly recognized member of the MCF virus group endemic in domestic goats (Capra hircus), provisionally named caprine herpesvirus 2 (CpHV-2). A seroprevalence survey from one of the deer farms showed a high rate of subclincal infection in the deer population. This study provides further confirmation that CpHV-2 is a pathogen, at least for deer, and emphasizes the risk of loss from MCF when mixing cervids with goats.

Two sika deer from a zoo in Florida were examined because of chronic hair loss and skin lesions. No common causes of alopecia were identified in either deer. One deer was treated with prednisone, but the condition worsened when the dosage was decreased. Both deer were euthanatized after several months because of continued disease. The predominant histologic lesion in skin specimens was granulomatous mural folliculitis. Serologic testing and sequencing of fragments produced with a consensus polymerase chain reaction assay indicated that both deer were infected with caprine herpesvirus-2, a newly recognized member of the malignant catarrhal fever group of viruses. Disease in these deer was substantially different from that typically seen following infection with ovine herpesvirus-2, the sheep-associated malignant catarrhal fever virus. Findings in these deer establish the pathogenicity of caprine herpesvirus-2 in sika deer and illustrate the ability of this group of complex herpesviruses to cause a wide variety of clinical abnormalities in diverse species.

A previously described polymerase chain reaction (PCR) assay (amplification of a 238-bp fragment of ovine herpesvirus 2 [OHV-2] genomic DNA) for diagnosis of sheep-associated malignant catarrhal fever (MCF) was adapted for use on formalin-fixed, paraffin-embedded tissues. Variables affecting its use were examined. Archived tissues from cattle, white-tailed deer (Odocoileus virginianus), and bison (Bison bison) diagnosed with MCF by clinical signs or histologic lesions were obtained from 2 veterinary diagnostic laboratories. Tissues from healthy animals and from animals diagnosed with other common bovine viral diseases were examined as controls. A total of 86 blocks from 37 suspect MCF cases were examined. Forty-one blocks from healthy animals and animals with unrelated viral diseases were examined as controls. The assay was specific for sheep-associated MCF and did not yield false-positive signals from healthy animals or from cases of infectious bovine rhinotracheitis, bovine virus diarrhea, mucosal disease, or parainfluenza-3 virus infection. A wide variety of tissues were suitable substrates, including spleen, lymph node, intestine, brain, lung, and kidney. Extracted DNA provided a more suitable target than did unextracted tissue lysate. The highest levels of viral DNA were present in lymphoid organs and intestine, but the data indicate that in acute clinical cases, most organs contain sufficient viral DNA to serve as a suitable diagnostic specimen. Fixation of 0.5-cm3 blocks of tissue in 10% neutral buffered formalin was deleterious to the target DNA, and PCR signals progressively diminished after fixation for >45 days. Detection of genomic DNA of OHV-2 by PCR was successful for archived tissues that were 15 years old.

Until recently, sheep-associated malignant catarrhal fever (SA-MCF) was diagnosed mainly on the basis of clinical presentation and histopathological changes. Using clinically diagnosed field cases, we have evaluated a seminested PCR and a competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) and compared these assays in the diagnosis of SA-MCF in cattle with histopathology as a provisional "gold standard." Samples from 44 cattle with clinical signs suggestive of SA-MCF were examined by histopathology, PCR, and CI-ELISA. In addition, samples from healthy cattle were evaluated by PCR (n = 96) and CI-ELISA (n = 75). Based on histopathology, 38 of the 44 clinical cases were classified as SA-MCF positive, 3 were classified as inconclusive, and 3 were classified as SA-MCF negative. The sensitivity of PCR was 95 to 97%, whereas the specificity ranged between 94 and 100%. The CI-ELISA showed a sensitivity of 56 to 87% and a specificity between 91 and 100%. In the field, there is good correlation between the diagnoses of SA-MCF by histopathology, PCR, and CI-ELISA. These data also confirm the close association of ovine herpesvirus 2 with SA-MCF in Switzerland.

Oligonucleotide primers derived from alcelaphine herpesvirus 1 (AHV-1) isolate WC11 DNA, the first identified agent of malignant catarrhal fever (MCF), were used to assay blood lymphocyte DNA using the polymerase chain reaction (PCR). Multiple species of exotic ruminants were examined to determine the suitability of this technique for detecting animals that may be latently infected. To correlate the PCR results with those of serology, serum samples were obtained from each animal concurrently with lymphocyte collection and subjected to an AHV-1 virus-neutralization assay (VNA). A total of 86 MCF-susceptible animals were tested, and the results of the VNA and PCR assays were compared. PCR results were positive for 44 animals. Of these, 13 were positive by VNA. Animals positive by both VNA and PCR were all wildebeest, the asymptomatic carriers of AHV-1, confirming the ability of the primers to amplify AHV-1 sequence. Positive PCR results from species other than wildebeest may represent sequence amplified from viruses related to AHV-1, which may not induce antibodies capable of neutralizing the WC11 isolate used in the VNA. This study demonstrates that PCR is capable of detecting the presence of MCF agents in various populations of captive ruminants prior to the appearance of clinical MCF so that the sources of infection can be more reliably ascertained.

We derived sequence information from cloned HindIII fragment "D" of alcelaphine herpesvirus 1 strain WC11, an agent of malignant catarrhal fever (MCF). Based on this sequence, oligonucleotide primers were selected and synthesized for use in a polymerase chain reaction amplification assay. These primers were used to test samples of total nucleic acids isolated from multiple tissues taken from an Indian gaur (Bos guarus gaurus) at the San Diego Wild Animal Park in San Diego, California (USA) which had clinical signs of a natural infection of MCF. Six of eight tissue samples examined had amplifiable sequences present. A nucleic acid probe complementary to the sequence of the original clone between the primer sites also was synthesized and used to confirm the identity of the amplified viral sequences, thus providing a diagnosis of MCF at the molecular level.

A natural non-fatal case of sheep-associated malignant catarrhal fever in a cow is reported. Viral DNA confirmed by nucleic acid hybridisation and polymerase chain reaction persisted in peripheral blood leukocytes for at least 3 months after the clinical onset of the disease. Concultivation of leukocytes with various cell cultures failed to result in isolation of the virus either by cytopathic effect or by nucleic acid hybridisation.

Alcelaphine herpesvirus 1 (AHV-1) genomic DNA was analyzed using restriction enzymes having recognition sequences both low in guanine-cytosine content (BamHI, KpnI, HindIII) and high in guanine-cytosine content (SmaI, AvaI, ApaI). The results from the restriction enzyme analyses along with exonuclease treatment demonstrated that the termini of AHV-1 DNA are likely composed of polyrepetitive sequences high in guanine-cytosine content similar to those found in Herpesvirus saimiri DNA. Cleaving AHV-1 DNA with the restriction enzyme SmaI produced polyrepetitive sequences that appeared as low molecular weight supermolar bands less than 1 kilobase pairs (kb) in size. Analysis of AHV-1 DNA cleaved with HindIII indicated that the polyrepetitive sequences are approximately 29.5 kb in size. The total molecular weight for AHV-1 DNA was determined to be approximately 118 kb. Seventy-five percent of the AHV-1 genome was cloned into the plasmids pAT153 and pBR322. Cloned DNA fragments representing unique sequences of the AHV-1 genome hybridized with AHV-1 genomic DNA but showed no appreciable hybridization with AHV-2 DNA or bovine herpesvirus 1, 2, or 4 DNA.

Nilgiri tahr (Hemitragus hylocrius) are native to India and are a rare zoo exhibit. This report describes an acute respiratory disease in tahr that caused the death of 15 of 16 animals in an extensive exhibit of about 35 acres where they were housed together with a variety of other exotic species of ruminants. The deaths occurred in two separate outbreaks and were associated with losses from malignant catarrhal fever in other ruminants in the exhibit. The most prominent clinical sign was severe dyspnea, and death occurred within five days. The principal lesions were an acute nonsuppurative inflammation of the respiratory tract and pulmonary vessels, lymphadenopathy and lymphoid cell infiltration in the organs of some animals. It was conjectured that the tahr died of a unique pneumonic form of malignant catarrhal fever. Attempts at viral isolation were negative.