Protection of sheep against Rift Valley fever virus and sheep poxvirus with a recombinant capripoxvirus vaccine.

Rift Valley fever (RVF) is an epizootic viral disease of sheep that can be transmitted from sheep to humans, particularly by contact with aborted fetuses. A capripoxvirus (CPV) recombinant virus (rKS1/RVFV) was developed, which expressed the Rift Valley fever virus (RVFV) Gn and Gc glycoproteins. These expressed glycoproteins had the correct size and reacted with monoclonal antibodies (MAb) to native glycoproteins. Mice vaccinated with rKS1/RVFV were protected against RVFV challenge. Sheep vaccinated with rKS1/RVFV twice developed neutralizing antibodies and were significantly protected against RVFV and sheep poxvirus challenge. These findings further document the value of CPV recombinants as ruminant vaccine vectors and support the inclusion of RVFV genes encoding glycoproteins in multivalent recombinant vaccines to be used where RVF occurs.

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.

A devastating outbreak of malignant catarrhal fever in a bison feedlot.

In early 2003, an outbreak of malignant catarrhal fever (MCF) occurred in a bison feedlot in southern Idaho. The outbreak resulted in a 51.2% (n = 825) mortality rate among bison, which had been exposed to sheep for 19 days. Diagnosis was made by detection of ovine herpesvirus 2 (sheep-associated MCF virus) DNA in tissues or peripheral blood by polymerase chain reaction (PCR), and by histological examination of tissue lesions. Peak losses occurred between 41 and 55 days postmean exposure time (PME), and reached a maximum of 41 head per day. No known cases of MCF were observed among the 177 head of bison that arrived in the lot 3 1/2 weeks after the departure of the sheep. Of the several thousand head of beef cattle in the lot during the outbreak, only a single case of MCF was identified. This outbreak illustrates the devastating impact the MCF virus can have on bison under certain exposure conditions, the high threat posed by adolescent lambs to susceptible species, the significantly greater susceptibility of bison than beef cattle to MCF, and the lack of horizontal transmission from clinically affected bison to herdmates.

Characterization of Bison bison major histocompatibility complex class IIa haplotypes.

American bison (Bison bison) and domestic cattle (Bos taurus and Bos indicus) evolved from a common ancestor 1-1.4 million years ago. Nevertheless, they show dramatic differences in their susceptibility to infectious diseases, including malignant catarrhal fever (MCF). Although bison are highly susceptible to ovine herpesvirus-2 (OvHV-2) associated MCF, about 20% of healthy domesticated and wild bison are positive for OvHV-2 antibody. We are interested in testing the hypothesis that, within the bison population, the polymorphism of major histocompatibility complex (MHC) class II genes influences resistance to MCF. However, since little was known about the MHC class II genes of bison, it was necessary to first characterize class II haplotypes present in Bi. bison (Bibi). Thus, the MHC class II haplotypes carried by 14 bison were characterized by the PCR-based cloning and sequencing of their DRB3, DQA, and DQB alleles. Twelve MHC class II haplotypes were identified in the 14 bison. These haplotypes comprised six previously reported and six new Bibi-DRB3 alleles, along with 11 Bibi-DQA and 10 Bibi-DQB alleles. For each bison class II allele, it was possible to identify closely related cattle sequences. The closest bison and bovine DQA, DQB, and DRB3 alleles, on average, differed by only 1.3, 3.5, and 5.8 amino acids, respectively. Furthermore, bison MHC haplotypes with both nonduplicated and duplicated DQ genes were identified; these haplotypes appear to have originated from the same ancestral haplotypes as orthologous cattle haplotypes.

A novel subgroup of rhadinoviruses in ruminants.

In the course of investigating the malignant catarrhal fever (MCF) subgroup of rhadinoviruses, seven novel rhadinoviruses were identified in a variety of ruminants, including domestic sheep, bighorn sheep, bison, black-tailed deer, mule deer, fallow deer, elk and addax. Based on the DNA polymerase gene sequences, these newly recognized viruses clustered into a second distinct subgroup in ruminants with three members identified previously in cattle, goats and oryx. Phylogenetic analysis revealed that the currently known ruminant rhadinoviruses appear to comprise three distinct genetic lineages: (i) the MCF subgroup, defined by sequence identity and the presence of the 15A antigenic epitope; (ii) a second distinct subgroup, devoid of the 15A epitope, which contains the previously reported bovine lymphotropic herpesvirus and related viruses; and (iii) a third distinct subgroup represented by Bovine herpesvirus 4. Comparison of phylogenetic trees between the rhadinoviruses and their corresponding hosts further supports the gammaherpesvirus and host co-evolution theory.

Malignant catarrhal fever-like disease in sheep after intranasal inoculation with ovine herpesvirus-2.

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.

Transmission of caprine herpesvirus 2 in domestic goats.

Caprine herpesvirus 2 (CpHV-2) is a recently recognized gammaherpesvirus that is endemic in domestic goats and has been observed to cause clinical malignant catarrhal fever (MCF) in certain species of deer. In this study, transmission of CpHV-2 in goats was examined. A total of 30 kids born to a CpHV-2 positive goat herd were selected and divided into two groups: group 1 (n=16) remained in the positive herd; group 2 (n=14) was separated from the herd at 1 week of age after obtaining colostrum. Peripheral blood samples from each kid were examined regularly by competitive ELISA for MCF viral antibody and by PCR for CpHV-2 DNA. Fifteen out of 16 goats (94%) that remained with the positive herd seroconverted and became PCR-positive for CpHV-2 by 10 months of age. In contrast, all kids (100%) that were separated from the positive herd at 1 week of age remained negative until termination of the experiment at 1 year of age. Additional transmission experiments revealed that all CpHV-2-free adult goats were susceptible to CpHV-2 or ovine herpesvirus 2 (OvHV-2) infection. The data indicate that the transmission pattern of CpHV-2 in goats is similar to the pattern of OvHV-2 in sheep and that CpHV-2-free goats can be established by early separation of kids from positive herds, which has significant implications for MCF control programs.

Experimental infection of sheep with ovine herpesvirus 2 via aerosolization of nasal secretions.

Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever in clinically susceptible ruminants, including cattle, bison and deer. Studies of OvHV-2 have been hampered by the lack of an in vitro propagation system. Here, the use of nasal secretions collected from OvHV-2-infected sheep experiencing intense virus shedding episodes as a source of infectious virus for experimental animal infections was examined. OvHV-2 uninfected sheep were nebulized with nasal secretions containing approximately 10(8) to 10(1) copies of OvHV-2 DNA. The time to detectable viral DNA in peripheral blood leukocytes (7-12 days post-infection) and virus-specific antibody in plasma (9-32 days post-infection) varied with the dose of inocula administered. Here, the use of nasal secretions as a source of infectious OvHV-2 was defined and the minimum infectious dose of a pool of nasal secretions that can be used in further studies of viral pathogenesis and vaccine development was determined.

An outbreak of sheep-associated malignant catarrhal fever in bison (Bison bison) after exposure to sheep at a public auction sale.

An outbreak of malignant catarrhal fever (MCF) among bison sold at an auction market was studied for an 18-month period. Forty-five of 163 bison submitted for sale from 8 different bison farms died on 7 other destination farms. The outbreak began on day 50 after the sale, peaked between days 60 and 70, and ended on day 220. Twenty-one dead bison were confirmed to be MCF cases by clinical histories, pathology, and detection of ovine herpesvirus-2 DNA in their tissues with polymerase chain reaction assays. Twenty-four dead bison were classified as suspect MCF cases from clinical histories. No cases of MCF were observed among bison remaining on originating farms or resident bison mixed with sale bison on destination farms. There were no sheep reported within 3 km of originating or destination farms, limiting bison exposure to sheep to the auction facility, where sheep were present for less than 1 day. The outbreak provides an illustration of the temporal distribution of MCF mortality expected in bison and an estimate of the time from exposure until death from MCF after a single short exposure to sheep. The study provides evidence that bison with MCF do not transmit MCF to other bison.

Shedding of ovine herpesvirus 2 in sheep nasal secretions: the predominant mode for transmission.

Ovine herpesvirus 2 (OvHV-2), the major causative agent of malignant catarrhal fever in ruminant species worldwide, has never been propagated in vitro. Using real-time PCR, a striking, short-lived, peak of viral DNA, ranging from 10(5) to over 10(8) copies/2 microg of DNA, was detected in nasal secretions from over 60.7% of adolescent sheep (n = 56) at some point during the period from 6 to 9 months of age. In contrast, only about 18% of adult sheep (n = 33) experienced a shedding episode during the study period. The general pattern of the appearance of viral DNA in nasal secretions was a dramatic rise and subsequent fall within 24 to 36 h, implying a single cycle of viral replication. These episodes occurred sporadically and infrequently, but over the 3-month period most of the 56 lambs (33, or 60.7%) experienced at least one episode. No corresponding fluctuations in DNA levels were found in either peripheral blood leukocytes or plasma. In a DNase protection assay, complete, enveloped OvHV-2 virions were demonstrated in the nasal secretions of all sheep examined during the time when they were experiencing an intense shedding episode. OvHV-2 infectivity in nasal secretions was also demonstrated by aerosolization of the secretions into OvHV-2-negative sheep. The data herein show that nasal shedding is the major mode of OvHV-2 transmission among domestic sheep and that adolescents represent the highest risk group for transmission.

Demonstration of sheep-associated malignant catarrhal fever virions in sheep nasal secretions.

Ovine herpesvirus-2 (OvHV-2) is the causative agent for sheep-associated malignant catarrhal fever, which has never been propagated in vitro. Previous studies from this laboratory demonstrated significantly high levels of OvHV-2 DNA in sheep nasal secretions, suggesting a likely avenue of transmission. In the present study, real-time PCR was used to identify sheep experiencing an episode of intense OvHV-2 DNA shedding in their nasal secretions. A nuclease-resistance assay was used to examine the secretions for the presence of intact cell-free enveloped OvHV-2 virions. The results revealed that all nasal secretion samples from five selected individuals experiencing intensive shedding events contained cell-free OvHV-2 virions. Virions could not be identified in secretion samples from 11 OvHV-2 infected sheep that were not experiencing a shedding event. This is the first unequivocal demonstration of cell-free OvHV-2 virions. These results suggest that OvHV-2 lytic infection occurs in the epithelium of certain tissues in the upper respiratory tract of the natural host.

The vascular lesions of a cow and bison with sheep-associated malignant catarrhal fever contain ovine herpesvirus 2-infected CD8(+) T lymphocytes.

Malignant catarrhal fever (MCF) is a herpesvirus disease syndrome of ruminants. The microscopic pathology of MCF is characterized by lymphoid proliferation and infiltration, necrotizing vasculitis and epithelial necrosis. Because previous attempts to detect viral antigen or nucleic acids in lesions have been unsuccessful, the pathogenesis of the lesions in acute MCF has been speculated to involve mechanisms of autoimmunity and lymphocyte dysregulation. In this study, the vascular lesions in the brains of a cow and a bison with acute MCF were evaluated by in situ PCR and immunohistochemistry. The results demonstrated that the predominant infiltrating cell type in these lesions was CD8(+) T lymphocytes and that large numbers of these cells were infected with ovine herpesvirus 2. The lesions also contained macrophages, but no detectable CD4(+) or B lymphocytes.

Caprine herpesvirus-2-associated malignant catarrhal fever in white-tailed deer (Odocoileus virginianus).

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.

Evidence of three new members of malignant catarrhal fever virus group in muskox (Ovibos moschatus), Nubian ibex (Capra nubiana), and gemsbok (Oryx gazella).

Six members of the malignant catarrhal fever (MCF) virus group of ruminant rhadinoviruses have been identified to date. Four of these viruses are clearly associated with clinical disease: alcelaphine herpesvirus 1 (AlHV-1) carried by wildebeest (Connochaetes spp.); ovine herpesvirus 2 (OvHV-2), ubiquitous in domestic sheep; caprine herpesvirus 2 (CpHV-2), endemic in domestic goats; and the virus of unknown origin found causing classic MCF in white-tailed deer (Odocoileus virginianus; MCFV-WTD). Using serology and polymerase chain reaction with (degenerate primers targeting a portion of the herpesviral DNA polymerase gene, evidence of three previously unrecognized rhadinoviruses in the MCF virus group was found in muskox (Ovibos moschatus), Nubian ibex (Capra nubiana), and gemsbok (South African oryx, Oryx gazella), respectively. Base on sequence alignment, the viral sequence in the muskox is most closely related to MCFV-WTD (81.5% sequence identity) and that in the Nubian ibex is closest to CpHV-2 (89.3% identity). The viral sequence in the gemsbok is most closely related to AlHV-1 (85.1% identity). No evidence of disease association with these viruses has been found.

Mural folliculitis and alopecia caused by infection with goat-associated malignant catarrhal fever virus in two sika deer.

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.

Serum antibody prevalence of malignant catarrhal fever viruses in seven wildlife species from Alaska.

Blood samples were collected from seven species of free-ranging ungulates in Alaska. Sera were tested for evidence of exposure to malignant catarrhal fever viruses (MCFV) by means of a competitive enzyme-linked immunosorbent assay. Antibody prevalences were as follows: muskox (Ovibos moschatus) 100 positive samples of 104 tested (96%); Dall sheep (Ovis dalli) 212 of 222 (95%); elk (Cervus elaphus) 14 of 51 (27%); bison (Bison bison) 34 of 197 (17%); caribou (Rangifer tarandus) nine of 232 (4%); Sitka black-tailed deer (Odocoileus hemionus sitkensis) one of 49 (2%); and moose (Alces alces) three of 219 (1%). Antibody prevalence in a bison population from the Interior was stable over a 5 yr period. These results indicate that at least one virus in the MCF group is enzootic in Dall sheep and muskox in Alaska. Lower antibody prevalences in the other species in this survey suggest that MCFV are latent or subclinical in these free-ranging ruminants. Whole blood samples were collected from 14 Dall sheep and subjected to a polymerase chain reaction assay. Fragments of ovine herpesvirus-2 DNA were detected in six of the samples. The significance of these findings for the health of free-ranging ungulates in Alaska is unknown.

Effect of passive transfer of maternal immune components on infection with ovine herpesvirus 2 in lambs.

OBJECTIVE: To define the role of passively tranferred immunity in protection against early infection with ovine herpesvirus 2 (OvHV-2) in lambs. ANIMALS: 15 adult sheep and 34 lambs. PROCEDURES: 2 groups of animals were used, including 15 lambs born to OvHV-2-free ewes and 19 lambs born to OvHV-2-positive ewes. After nursing colostrum, all lambs and their dams were introduced into a flock positive for OvHV-2. Blood was obtained from the lambs every 2 weeks and examined by PCR assay and competitive inhibition ELISA. RESULTS: None of the animals had positive results by PCR analysis for samples obtained approximately 2 weeks after introduction into the flock. In the group of lambs from OvHV-2-infected ewes, 5 of 19 had positive results at 1 month of age and 17 of 19 by 5 months of age. In the group of offspring from OvHV-2-negative ewes, only 1 of 15 had positive results at 1 month of age, and the number reached 12 of 15 by 5 months of age. All lambs in both groups had positive results by 6 months. An active antibody response to the virus was detected in animals within 3 weeks after viral DNA became detectable in the blood. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis suggests that passively transferred immunity does not play an important role in the delay of infection with OvHV-2 in lambs. Age also does not seem to influence susceptibility. The rate of infection in young lambs may simply be a reflection of the intensity of viral exposure in their environment.

Recognition of another member of the malignant catarrhal fever virus group: an endemic gammaherpesvirus in domestic goats.

A novel gammaherpesvirus in goats that is herein tentatively designated as caprine herpesvirus-2 was identified based on the sequence of a fragment from the herpesvirus DNA polymerase gene. Sequence alignment analysis revealed that the virus sequence isolated from goats was 67% identical to the homologous sequence from alcelaphine herpesvirus-1, 71% identical to ovine herpesvirus-2 and 73% identical to a recently recognized herpesvirus causing malignant catarrhal fever in white-tailed deer. Combined serological and PCR-survey data demonstrated that this virus is endemic in goats and its transmission pattern may be similar to that of ovine herpesvirus-2 in sheep.

Endothelial cell infection in vivo by equine infectious anaemia virus.

Equine infectious anaemia virus (EIAV) infection of horses is characterized clinically by recurrent episodes of fever, thrombocytopenia and anaemia. In vivo, the only site of virus replication that has been previously demonstrated for EIAV is the tissue macrophage. In this study, in situ hybridization for EIAV was combined with immunohistochemistry for cell-type-specific markers to identify infected endothelial cells. EIAV-infected endothelial cells and macrophages were detected in horses infected with either virulent wild-type or with weakly virulent tissue culture-adapted strains of EIAV. The role of endothelial cell infection in the pathogenesis of EIAV remains undefined, but could contribute to the development of thrombocytopenia. However, endothelial cell infection does not appear to be a determinant of virulence for EIAV.