Intra-nasal inoculation of American bison (Bison bison) with ovine herpesvirus-2 (OvHV-2) reliably reproduces malignant catarrhal fever

Sheep-associated malignant catarrhal fever (MCF) due to infection with ovine herpesvirus 2 (OvHV-2) is common in commercial herds of American bison ( Bison bison). Inability to propagate OvHV-2 in vitro has been a constraint on experimental studies of the disease. We sought to establish whether nasal secretions from sheep that shed OvHV-2 might induce the disease in bison and to define a minimum challenge dose. Fourteen bison were nebulized with sheep nasal sections containing 10(3)-10(7) OvHV-2 deoxyribonucleic acid (DNA) copies. Most challenged bison (11/14, 78.6%) developed clinical signs at 29-52 days postnebulization (DPN). The mean incubation time was 42.18 (+/-7.33 SD) DPN. Using real-time polymerase chain reaction, we detected OvHV-2 DNA in peripheral blood leukocytes at 21-31 DPN. All bison that developed MCF had antibodies against the MCF group viruses. Gross and histologic lesions were typical of the acute disease. There was no morphologic evidence of a dose-related difference in the severity or distribution of lesions. This is the first successful reproduction of MCF in bison using a nasal route of exposure. Experimentally challenged bison are more susceptible to MCF, compared with experimentally challenged domestic cattle in a previous experiment. Bison are a pertinent ruminant species in which the pathogenesis of the disease can be investigated.     

Experimental aerosol infection of cattle (Bos taurus) with ovine herpesvirus 2 using nasal secretions from infected sheep

Infection of clinically susceptible ruminants, including domesticated cattle and American bison, with ovine herpesvirus 2 (OvHV-2) can result in the fatal lymphoproliferative and vasculitis syndrome known as malignant catarrhal fever (MCF). A reliable experimental infection model is needed to study the pathogenesis of MCF and to develop effective vaccination strategies to control the disease. An experimental aerosol infection model using sheep, the natural carriers of OvHV-2, has been developed (Taus et al., 2005). Using the protocol and OvHV-2 inoculum established in the previous study, eight calves were nebulized with four different doses of OvHV-2 in nasal secretions from infected sheep. Two control calves were nebulized with nasal secretions from uninfected sheep. Infection status of all calves was monitored using competitive inhibition ELISA, PCR and clinical parameters. Six of eight nebulized calves became infected with OvHV-2. One calf receiving the highest dose of virus developed typical clinical, gross and histological changes of MCF. This study showed that nasal secretions collected from sheep experiencing OvHV-2 shedding episodes were infectious for cattle and capable of inducing MCF. The data also indicate that cattle are relatively resistant to disease following infection. The use of more susceptible species as experimental animal models, such as bison and selected cervid species should be examined.     

Malignant catarrhal fever: a review

Malignant catarrhal fever (MCF) is a fatal lymphoproliferative disease of cattle and other ungulates caused by the ruminant γ-herpesviruses alcelaphine herpesvirus 1 (AlHV-1) and ovine herpesvirus 2 (OvHV-2). These viruses cause inapparent infection in their reservoir hosts (wildebeest for AlHV-1 and sheep for OvHV-2), but fatal lymphoproliferative disease when they infect MCF-susceptible hosts, including cattle, deer, bison, water buffalo and pigs. MCF is an important disease wherever reservoir and MCF-susceptible species mix and currently is a particular problem in Bali cattle in Indonesia, bison in the USA and in pastoralist cattle herds in Eastern and Southern Africa.MCF is characterised by the accumulation of lymphocytes (predominantly CD8⁺ T lymphocytes) in a variety of organs, often associated with tissue necrosis. Only a small proportion of these lymphocytes appear to contain virus, although recent results with virus gene-specific probes indicate that more infected cells may be present than previously thought. The tissue damage in MCF is hypothesised to be caused by the indiscriminate activity of MHC-unrestricted cytotoxic T/natural killer cells. The pathogenesis of MCF and the virus life cycle are poorly understood and, currently, there is no effective disease control.Recent sequencing of the OvHV-2 genome and construction of an AlHV-1 bacterial artificial chromosome (BAC) are facilitating studies to understand the pathogenesis of this extraordinary disease. Furthermore, new and improved methods of disease diagnosis have been developed and promising vaccine strategies are being tested. The next few years are likely to be exciting and productive for MCF research.     

Malignant catarrhal fever in a bison (Bison bison) feedlot, 1993-2000.

A fatal enteric syndrome was identified in American bison (Bison bison) at a large feedlot in the American Midwest in early 1998. An estimated 150 bison died of the syndrome between January 1998 and December 1999. The syndrome was identified as malignant catarrhal fever (MCF), primarily the alimentary form. Clinical onset was acute, and most affected bison died within 1-3 days; none recovered. Consistent lesions were hemorrhagic cystitis, ulcerative enterotyphlocolitis, and arteritis-phlebitis. Vasculitis was milder and more localized than that in cattle with MCF, and in contrast to the situation in cattle, lymphadenomegaly was minimal. Virtually all affected bison examined were positive for ovine herpesvirus 2 (OvHV-2) by polymerase chain reaction (PCR) assay. A retrospective study of archived tissues established that MCF occurred in the yard as early as 1993. A prospective study was undertaken to establish the importance of MCF relative to other fatal diseases at the feedlot. The fate of a group of 300 healthy male bison in a consignment of 1,101 animals was followed for up to 7 months to slaughter. At entry, 23% (71/300) of bison were seropositive for MCF viruses, and 11% (8/71) of these seropositive bison were PCR positive for OvHV-2. Forty seronegative bison were selected at random from the group, and all were PCR negative for OvHV-2. There was no change in seroprevalence in the group during the investigation. The minimum infection rate for MCF virus was 36.3% (93/256). Twenty-two (7.3%) of the 300 bison in the feedlot died. Of these, 15 had MCF, 4 had acute or chronic pneumonia, and 3 were unexamined. Losses in the entire consignment were higher (98/1,101; 8.8% death loss); 76% of deaths were attributable to MCF. The study failed to reveal a relationship between subclinical infection and development of clinical disease.     

Experimental induction of malignant catarrhal fever in pigs with ovine herpesvirus 2 by intranasal nebulization

Malignant catarrhal fever (MCF), a frequently fatal herpesviral disease primarily of ruminant species, has been sporadically reported in pigs. All cases of naturally occurring porcine MCF reported to date have been linked to ovine herpesvirus 2 (OvHV-2), a gammaherpesvirus in the genus Macavirus carried by sheep. Experimental induction of MCF by aerosolization of the virus in nasal secretions collected from infected sheep has been successful in bison, cattle and rabbits. The goals of this study were to determine the susceptibility of pigs to MCF following experimental intranasal inoculation of OvHV-2, and to characterize the disease. Twelve pigs in four groups were nebulized with 10(5), 10(6), 10(7), or 10(8) DNA copies of OvHV-2 from sheep nasal secretions. Three control pigs were nebulized with nasal secretions from uninfected sheep. Three additional pigs were inoculated intravenously with 10(7) DNA copies of OvHV-2 to evaluate this route of infection with cell-free virus. Seven of twelve intranasally challenged pigs became infected with OvHV-2. Five of these seven, all in higher dose groups, developed MCF. Lesions resembled those reported in natural cases of porcine MCF. The most striking and consistent histological lesions were in trachea, lung, kidney and brain. These comprised mucopurulent tracheitis, interstitial pneumonia, necrotizing arteritis-periarteritis, and nonpurulent meningoencephalitis. No infection was established in the intravenously challenged or control groups. The study showed that MCF can be experimentally induced in pigs by aerosol challenge using sheep nasal secretions containing OvHV-2. Domestic pigs are a natural clinically susceptible host for sheep-associated MCF. They represent a useful, cost-effective model for MCF research.     

Molecular epidemiology of ovine herpesvirus type 2 infection in Kashmir, India

The aims of this investigation were to determine the prevalence of ovine herpesvirus type 2 (OvHV-2) (the causative agent of malignant catarrhal fever) infection in cattle, the carrier status of sheep and goats, and to define the pattern of acquisition of OvHV-2 in lambs under natural flock conditions in Kashmir, India. None of the buffy coat samples from 21 lambs contained OvHV-2 DNA sequences up to 28 days after birth, only one lamb had sequences of OvHV-2 DNA as early as 29 days after birth, and they were detected in the other 20 lambs when they were between 43 and 94 days of age. Sequences of OvHV-2 DNA were detected in buffy coat samples from 28 (85 per cent) of 33 adult sheep and in 16 (61 per cent) of 26 samples from adult goats by hemi-nested PCR. Seventeen (31 per cent) of 55 cattle with malignant catarrhal fever-like clinical signs had sequences of OvHV-2 DNA in their blood, and nine of the 17 died, all of them during the months of April to November, between November 2002 and March 2004. No clinical cases of sheep-associated malignant catarrhal fever was recorded during the months of December to March. The overall prevalence of OvHV-2 infection in the cattle in the region was estimated to be less than 1 per cent.     

Virus in sheep's skin

In a double sense, the ovine gamma herpesvirus type 2 (OvHV-2) is a virus in sheep's skin. Not only is it present world wide in all sheep breeds but also it causes malignant catarrhal fever (MCF) in cattle pigs, elk, and bison. OvHV-2 cannot be propagated in cell culture. Therefore, new results from OvHV-2 research are based on molecular techniques and may be summarized as follows. OvHV-2 is transmitted by respiratory routes as well as by sexual intercourse. Lambs are infected within the first few months of life. Leucocytes, primarily latently infected lymphocytes, are responsible for disseminating the virus over the entire organism. On rare occasions, virus particles could be visualized by electron microscopy in explanted lymphocyte cultures. Structural antigens were detected by immunohistology in M-cells of diseased rabbits. Immunologically and cell biologically active genes have been detected on the viral genome.The products arising from those are thought to fine balance, the number of latently infected cells in sheep and to keep them alive without causing harm. Thus, it seems that this balance has been found through co-evolution, favoring both virus and natural host. In contrast, other host species that were exclude from the process of co-evolution, are bound to fall from MCF.     

A simpler, more sensitive competitive inhibition enzyme-linked immunosorbent assay for detection of antibody to malignant catarrhal fever viruses.

An earlier competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) was developed for detection of specific antibody against malignant catarrhal fever (MCF) viruses (MCFV) in ruminants. In this study, the indirect CI-ELISA was improved by conjugating the monoclonal antibody 15-A directly with horseradish peroxidase and by developing a method of producing precoated, dried antigen plates. This new test is referred to as a direct CI-ELISA. The reformatted test yielded a significantly improved sensitivity, and the time required was reduced to about one-sixth of the previous time. Of 37 MCF cases in cattle that were confirmed by histopathology and polymerase chain reaction (PCR) assay, 37 (100%) were positive by the new test, whereas the indirect CI-ELISA detected only 23 (62%). The direct CI-ELISA detected antibody to MCFV in 100% of 48 sheep that had been defined as infected with ovine herpesvirus 2 (OvHV-2) by PCR, whereas the indirect CI-ELISA detected only 41 (85%). Comparison of antibody titers measured by the 2 assays for sera collected from OvHV-2-infected sheep and from cattle, bison, and deer with clinical sheep-associated MCF revealed that the direct CI-ELISA offered a 4-fold increase in analytical sensitivity over the indirect format. The number of seropositive animals detected by the direct CI-ELISA among apparently normal cattle and bison was 2-3 times greater than the number detected by the indirect CI-ELISA, indicating that a significant percentage of normal cattle and bison are subclincally infected with MCFV.     

Long distance spread of malignant catarrhal fever virus from feedlot lambs to ranch bison

Malignant catarrhal fever (MCF) caused by OvHV-2 occurred in ranch bison herds separated by significant distances from feedlot lambs. Mortality rates correlated with distances: 17.5%, 6.1%, and 0.43% at approximately 1.6, 4.2, and 5.1 km, respectively. The study further defines the importance of distance of species separation for MCF control.     

Malignant catarrhal fever in pigs and a genetic comparison of porcine and ruminant virus isolates in Finland

An outbreak of the sheep-associated form of malignant catarrhal fever (MCF) in a Finnish sow herd was diagnosed by histopathology and confirmed by PCR. Several gilts and sows were suffering from high fever and anorexia and had aborted, and six of them had died. Typical signs of lymphoproliferation and vasculitis were observed histologically in several tissues, including the uterus. Ovine herpesvirus-2 (OvHV-2) was detected by PCR in two sows. Sequences of the OvHV-2 tegument protein gene obtained from the sows and from three cases of sheep-associated mcf in Finnish cattle were compared and found to be identical. These are the first confirmed cases of mcf in pigs in Finland.     

Molecular detection and characterization of ovine herpesvirus-2 using heminested PCR in Pakistan

BackgroundMalignant catarrhal fever (MCF) is a highly fatal lymphoproliferative disease of cattle, deer, bison, water buffalo, and pigs caused by the gamma-herpesviruses alcelaphine herpesvirus-1 (AlHV-1) and ovine herpesvirus-2 (OvHV-2).ObjectivesThis study aimed to determine the prevalence of OvHV-2 in sheep, goats, cattle, and buffalo in Rawalpindi and Islamabad, Pakistan, by applying molecular and phylogenetic methods.MethodsBlood samples were aspirated from sheep (n = 54), goat (n = 50), cattle (n = 46) and buffalo (n= 50) at a slaughterhouse and several farms. The samples were subjected to heminested polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis of the OvHV-2 POL gene and the OvHV-2 ORF75 tegument protein gene.ResultsThe highest percentage of MCF positive samples was in sheep (13%), whereas goat, cattle, and buffalo had lower positive percentages, 11%, 9%, and 6.5%, respectively. Four OvHV-2-positive PCR products obtained from sheep samples were sequenced. The sequences obtained were submitted to the NCBI GenBank database ({"type":"entrez-nucleotide","attrs":{"text":"MK852173","term_id":"1694854431","term_text":"MK852173"}}MK852173 for the POL gene; {"type":"entrez-nucleotide","attrs":{"text":"MK840962","term_id":"1686143390","term_text":"MK840962"}}MK840962, {"type":"entrez-nucleotide","attrs":{"text":"MK852171","term_id":"1694854427","term_text":"MK852171"}}MK852171, and {"type":"entrez-nucleotide","attrs":{"text":"MK852172","term_id":"1694854429","term_text":"MK852172"}}MK852172 for the ORF75 tegument protein gene). Phylogenetic analysis revealed a close similarity of study sequences with those of worldwide samples.ConclusionsThis study is the first cross-sectional study on the prevalence and molecular detection of OvHV-2 in apparently healthy cattle and buffalo that could be carrying OvHV-2 acquired from OvHV-2-positive sheep and goats. The results indicate that OvHV-2 is circulating in Pakistan. Further studies are needed to characterize OvHV-2 and elucidate further its prevalence.     

Detection and molecular characterization of naturally transmitted sheep associated malignant catarrhal fever in cattle in India

Malignant catarrhal fever (MCF) is a fatal herpesvirus infection of domestic and wild ruminants, with a short and dramatic clinical course characterized primarily by high fever, severe depression, swollen lymph nodes, salivation, diarrhea, dermatitis, neurological disorders, and ocular lesions often leading to blindness. In the present study, fatal clinical cases of sheep associated malignant catarrhal fever (SA-MCF) were identified in cattle in the state of Karnataka. These cases were initially presented with symptoms of diarrhea, respiratory distress, conjunctivitis, and nasal discharges. Laboratory diagnosis confirmed the detection of ovine herpesvirus-2 (OvHV-2) genome in the peripheral blood samples of two ailing animals. The blood samples collected subsequently from sheep of the neighboring areas also showed presence of OvHV-2 genome indicating a nidus of infection in the region. The positive test results were further confirmed by nucleotide sequencing of the OIE approved portion of tegument gene as well as complete ORF8 region of the OvHV-2 genome. Phylogenetic analysis based on the sequence of the latter region indicated close genetic relationship with other OvHV-2 reported elsewhere in the world.     

Experimental transmission of sheep-associated malignant catarrhal fever from sheep to Japanese deer (Cervus nippon) and cattle

The assumption that sheep carry ovine herpesvirus-2 (OvHV-2), the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), is widely accepted, albeit OvHV-2 has not been isolated. We attempted experimental contact transmission of MCF from Japanese sheep persistently infected with OvHV-2 to Japanese deer (Cervus nippon) and cattle. In Experiment 1, a deer was kept in close quarters with an infected ewe. In Experiment 2, a second deer was kept with the same ewe. In Experiment 3, two cows were each kept with two infected wethers. In Experiment 1, the deer developed clinical signs at 138 days after first contact and then died. OvHV-2 genes by polymerase chain reaction (PCR) and fluorescent antibodies to Alcelaphine herpesvirus-1 were detected in the affected deer. Moreover, sequences of PCR products (423bp), obtained by amplification of materials from the sheep and from the affected deer, coincided. These results clearly confirmed that the sheep was a carrier of OvHV-2, and that this virus had induced SA-MCF in a deer. In other experiments, no OvHV-2 infection occurred in deer and cattle during the 6-18 months periods of contact, though viral genes were detected in the nasal swabs and white blood cells of the sheep. To our knowledge, this is the first report on successful experimental transmission of MCF from OvHV-2-infected sheep to deer.     

Antibodies to ovine herpesvirus 2 glycoproteins decrease virus infectivity and prevent malignant catarrhal fever in rabbits

Ovine herpesvirus-2 (OvHV-2) is the etiological agent of sheep-associated malignant catarrhal fever (SA-MCF), a fatal lymphoproliferative disease of many species in the order Artiodactyla. Development of a vaccine is critical to prevent mortality. Because OvHV-2 has not been cultured in vitro, SA-MCF research is hindered by the lack of in vitro tools to study viral constituents and specific host immune responses. As an alternative, in this study the neutralizing activity of antibodies against OvHV-2 glycoproteins gB and gH/gL was evaluated in vivo using rabbits. OvHV-2-specific antibodies were developed in rabbits by immunization using biolistic delivery of plasmids expressing the genes of interest. A lethal dose of OvHV-2 was incubated with the antisera and then nebulized into rabbits. Virus neutralization was assessed by measuring infection parameters associated with the virus infectious dose. Anti-gB or anti-gH/gL antibodies alone blocked infection in five out of six rabbits (83%), while a combination of anti-gB and anti-gH/gL antibodies protected all six rabbits (100%) from infection. These results indicate that antibodies to OvHV-2 gB and gH/gL are capable of neutralizing virions, and consequently, reduce virus infectivity and prevent SA-MCF in rabbits. Thus, OvHV-2 gB and gH/gL are suitable targets to be tested in a SA-MCF vaccine aimed at stimulating neutralizing antibody responses.     

The biology of bovine herpesvirus-4 infection of cattle

The biology of bovine herpesvirus-4 (BHV-4) infection of cattle is reviewed. The infection is distributed worldwide. Most of isolated viruses are non-pathogenic in cattle; some of them are able to produce a genital disease. Twenty-nine structural polypeptides were described; ten of them are glycosylated. Two major glycoproteins were characterized by monoclonal antibodies. Restriction maps of BHV-4 DNA are available for the enzymes EcoRI, BamHi and HindIII. The strain variations studied by restriction analysis are very weak. The virus is able to persist in a latent state after primary infection. The identified sites of latency are nervous ganglia and mononuclear blood cells. The immune response of cattle after BHV-4 infection is characterized by low or undetectable levels of neutralizing antibodies. Four envelope proteins are recognized by convalescent sera and are the main antigenic components. Skin test remains negative in immunized cattle. Bovine herpesvirus-4 is not strictly species-specific: infection was proved in American bison (Bison bison), African buffalo (Syncerus caffer), sheep and probably cat, because feline herpesvirus-2 is in fact a BHV-4 strain. Finally BHV-4 shares antigenic and genomic relationships with alcelaphine herpesvirus-1, the causal agent of the African form of malignant catarrhal fever.     

The prevalence of ovine herpesvirus-2 in 4 sheep breeds from different regions in South Africa

About 90% of bovine malignant catarrhal fever (BMCF) PCR-positive cases in South Africa are caused by alcelaphine herpesvirus-1 (AlHV-1) and the other 10% by ovine herpesvirus-2 (OvHV-2). The prevalence of OvHV-2 in different sheep breeds in South Africa was determined in order to investigate whether the lower incidence of BMCF caused by OvHV-2 in comparison with AlHV-1 can be ascribed to a low incidence of the virus in sheep. A single-tube hemi-nested PCR was developed, evaluated and applied to detect OvHV-2 DNA. The prevalence of the virus in 4 sheep breeds from various regions in South Africa was shown to be 77%. No statistically significant difference was found amongst the sheep breeds tested.     

Characterization of ovine herpesvirus 2-induced malignant catarrhal fever in rabbits

Malignant catarrhal fever (MCF) is a frequently fatal lymphoproliferative disease syndrome primarily of ruminant species, caused by gammaherpesviruses in the genus Macavirus. Ovine herpesvirus 2 (OvHV-2), carried by sheep, causes sheep-associated MCF worldwide, while Alcelaphine herpesvirus 1 (AlHV-1), carried by wildebeest, causes wildebeest-associated MCF, mainly in Africa. Diseases in rabbits can be induced by both viruses, which are clinically and pathologically similar; however, recent studies revealed different expression of viral genes associated with latency or lytic replication during clinical disease between the two viruses. In this study, we further characterized experimentally induced MCF in rabbits by nebulization with OvHV-2 from sheep nasal secretions to elucidate the course of viral replication, along with in vivo incorporation of 5-Bromo-2'-Deoxyuridine (BrdU), to evaluate lymphoproliferation. All six rabbits nebulized with OvHV-2 developed MCF between 24 and 29 days post infection. OvHV-2 DNA levels in peripheral blood leukocytes (PBL) remained undetectable during the incubation period and increased dramatically a few days before onset of clinical signs. During the clinical stage, we found that predominantly lytic gene expression was detected in PBL and tissues, and both T and B cells were proliferating. The data showed that the viral gene expression profile and lymphoproliferation in rabbits with OvHV-2 induced MCF were different from that in rabbits with AlHV-1 induced MCF, suggesting that OvHV-2 and AlHV-1 may play a different role in MCF pathogenesis.