Robert J. Callan, DVM, MS, PhD, DACVIM
Malignant catarrhal fever (MCF) is a severe lymphoproliferative disease affecting several ruminant species including cattle, bison, deer, and elk. It has a worldwide distribution. The disease is caused by a group of gammaherpesviruses1 collectively referred to as malignant catarrhal fever viruses (MCFV). MCF is observed in both domestic and wild ruminants. There are two etiological forms of MCF that are determined by the host reservoir of the virus. Wildebeest Associated MCF (WA-MCF) is caused by alcelaphine herpesvirus-1 (AlHV-1). The principal reservoir of AlHV-1 is the blue or white-bearded wildebeest (Connochaetes taurins). WA-MCF is seen primarily in Africa or in zoological parks where other ruminants have contact with Wildebeest. Sheep associated MCF (SA-MCF) is caused by ovine herpesvirus type 2 (OvHV-2). Domestic and wild sheep and goats are asymptomatic reservoirs of the virus. SA-MCF is the primary form of MCF that is observed outside of Africa.
Cattle and bison are the primary clinical hosts for SA-MCF. Common clinical signs of MCF include persistent fever, lymph node enlargement, mucosal ulceration, keratitis and corneal opacity, mucopurulent nasal discharge, ocular discharge, diarrhea, and hematuria. In all cases, the disease is characterized by widespread lymphocytic vasculitis. Cases are generally sporadic with a high mortality rate although chronic and recovered cases are observed. There is no known curative treatment or prophylaxis.
While virus culture techniques exist for AlHV-1, efforts to isolate and grow OvHV-2 in cell culture have been unsuccessful. However, the development of competitive inhibition ELISA (CI-ELISA)2,3 and polymerase chain reaction (PCR)4,5 assays have greatly aided the study of OvHV-2 in both asymptomatic and clinically affected hosts. The CI-ELISA test is based on identification of serum antibodies directed against a homologous glycoprotein epitope that is conserved on both AlHV-1 and OvHV-2. This test utilizes a monoclonal antibody, 15-A, that reacts with antigen prepared from a MCFV isolated from a cow in Minnesota. This virus isolate has since been determined to be a variant of AlHV-1. The CI-ELISA test readily detects seroconversion to AlHV-1 or OvHV-2 but can not distinguish between the two. MCF CI-ELISA can be useful for detecting persistently infected asymptomatic carriers in sheep, cattle, and bison. It can also be used to confirm clinical cases of MCF, however, seroconversion is not always present in acute cases of MCF.
PCR is an extremely sensitive and rapid test that will detect AlHV-1 or OvHV-2 infection in blood and tissues. Differentiation between AlHV-1 and OvHV-2 can be obtained by proper selection of primers. The PCR tests readily detect infection in clinical cases of MCF and have a very high sensitivity for confirming clinical infection. However, viral DNA in blood may be below detection limits when screening for asymptomatic infections in cattle and bison.
Epidemiology of OvHV-2
The prevalence of OvHV-2 infection determined by PCR in healthy adult North American domestic sheep has been reported as high as 99%.6 This value correlates very well with the seroprevalence in the same population (94%). Positive CI-ELISA tests have been observed in bison (2-23%), domestic goats (74%), elk (9%), mule deer (2%), white-tailed deer (3%), pronghorn antelope (25%), bighorn sheep (37%), muskox (40%), and mouflon sheep (62). Black-tailed deer, llama, and mountain goats have been tested and were not found to be seropositive for MCF.7,8
The transmission of OvHV-2 in lambs has been studied.9 The prevalence of viral infection in pre-suckling lambs is less than 5%. The majority of lambs remain uninfected up to 2.5 months of age. After 3.5 months of age, the lambs develop infections through close contact with adult sheep approaching 100% prevalence by 5.5 months. Shedding and transmission of OvHV-2 is believed to be through nasal and ocular secretions. Seroconversion and detection of OvHV-2 DNA in nasal secretions lags the detection of viral DNA in blood by several months. Separation of lambs from adults at 2 months of age reliably produces OvHV-2-free sheep.10 These findings indicate that newborn lambs are not a major source of virus infection for cattle. Peak nasal viral shedding occurs around 7-8 months of age and then declines to lower levels as adults. Adult sheep continue to shed virus at variable levels throughout their life. OvHV-2 shedding from adult sheep may be higher during the periparturient period or while on feed in feedlots, resulting in a higher risk to commingled cattle. OvHV-2 is readily transmitted between in contact sheep, however, transmission by inoculation of whole blood or PBMCs is inconsistent.11 This suggests that infectious virus is primarily produced and shed by nasal or ocular secretions and that infection of blood cells may be largely non-productive.
Studies of four Colorado dairies show that endemic OvHV-2 infections can occur within individual herds.12 Dairies with a history of clinical MCF also have known history of sheep exposure. One dairy studied is located within 70 meters of a sheep feedlot and observes sporadic cases of MCF every year with an annual incidence of 0.1 to 0.6%. In this herd MCF seroprevalence increased from 0.8% in heifers <15 months of age to nearly 10% by 21 months of age. Seroprevalence in adult cattle ranged from 20-30%. Seroprevalence was associated with proximity to the sheep feedlot. The prevalence of asymptomatic OvHV-2 infections in adult lactating cattle from this dairy is 21.3%. Thirty adult cows were monitored monthly by CI-ELISA and OvHV-2 PCR for a 20 month period.13 At the initial sampling 8/30 animals were seropositive. By the end of the 20 month period 21 of the 30 animals had at least one positive CI-ELISA or PCR test result. None of the animals developed clinical signs of MCF.
MCF can be a particularly devastating disease in bison.7,14-16 MCF was observed in 45 of 163 bison exposed to sheep at an auction yard for a period of only 1 day.15 Clinical disease occurred between 50 and 220 days after exposure and peaked around 60-70 days. No cases of MCF were observed among exposed bison on the destination farms indicating that bison with MCF do not transmit MCF to other bison. MCF was also observed in 51.2% (N=825) of bison exposed to sheep in a feedlot for 19 days. During that same time, only 1 of several thousand cattle at the feedlot developed MCF. This outbreak demonstrates the difference in susceptibility of bison and cattle to MCF and the high threat of OvHV-2 transmission from juvenile lambs in a feedlot environment.
Clinical Presentation of MCF
Records of 32 cases of MCF were evaluated from a single dairy in close proximity to a sheep feedlot from 1996 to 2006. Parameters recorded included age, year of illness, month of illness, clinical signs, bloodwork, and necropsy results. The age distribution was from 4 months to 6 years with a mean of 1.9 years. Twenty of 32 cases occurred in animals less than 2 years of age. The number of cases per year ranged from no cases up to 8 cases per year. The majority of cases occurred in the months of July through October. The most common clinical signs recorded were corneal opacity followed by fever, lymphadenopathy, nasal discharge, ocular discharge, dyspnea, and hematuria. The complete blood count showed no consistent findings and of particular note is that the animals showed no or minimal inflammatory changes in the CBC in spite of the high fever. The serum chemistry showed no consistent abnormalities.
The clinical findings in these cases are consistent with what is reported in acute outbreaks. The lack of changes in the leukogram in spite of the high fever is a significant negative finding that should lead the clinician to suspect MCF. Corneal opacity and lymphadenopathy are consistent signs of MCF. The observation of the majority of cases in the late summer to fall is different than what has been previously reported in acute outbreaks in feedlots. However, this seasonal finding is consistent with peak viral shedding observed in juvenile lambs. It is likely that high viral exposure from the neighboring sheep feedlot is associated with the seasonal case prevalence.
Treatment and Viral Kinetics of MCF
The literature reports 50-90% case mortality for cattle with MCF. Clinical treatment was attempted in 9 cases of MCF in adult Holstein cattle. Some of these cattle were treated with thimerosol as a potential treatment for MCF. Additional supportive treatment included flunixin meglumine, oral kaopectate, rumen fluid transfaunation, and antibiotics if there was indication of secondary bacterial infection. Five of nine cases survived and returned to normal production. There was no indication of a change in outcome related to treatment with thimerosol. Animals that were pregnant at the time of disease and recovered carried the pregnancy to term and delivered normal viable calves.
Appetite was observed to be a strong indicator of a positive prognosis. Necropsy of animals that died or were euthanized consistently showed severe gastrointestinal erosions. Daily quantitative real time PCR was performed on whole blood samples of clinical cases. There was no difference in viral DNA kinetics in the blood of survivors and fatal cases. There was also no difference in the viral DNA kinetics in the blood of animals treated with thimerosol.
Cellular Tropism of OvHV-2
The pathogenesis of MCF is still poorly understood. Histologically, the disease presents as a lymphoproliferative disease. It is not understood why sheep and goats develop asymptomatic infections and some cattle and bison develop sever and often fatal disease. It is also not understood what factors contribute the development of clinical signs in a small percentage of infected cattle. One theory is that the cellular tropism of OvHV-2 is different between species and may also be different in individual animals. Whole blood samples were obtained from infected asymptomatic sheep, sub-clinically infected cattle, clinically affected cattle, and recovered cattle for evaluation of OvHV-2 DNA copies in different phenotypic subsets of leukocytes by quantitative real time PCR. Clinical cases had the highest viral DNA copies followed by sheep and recovered cases. OvHV-2 DNA copies were below detection limits in sub-clinically infected cattle. T-cells were preferentially infected over B-cells and monocytes in sheep, clinically affected cattle, and recovered cattle. B-cells were negative in all groups except clinical cases. There was a dramatic decrease in viral DNA copies following recovery in clinically affected animals.
Recovery of Lymphoblastoid Cell Lines
Stable lymphoblastoid cell lines (LCL) were developed from whole blood of four clinical cases of MCF in cattle. On LCL consisted of gamma-delta T-lymphocytes and the other three cell lines were CD8 T-lymphocytes. The gamma-delta cell line spontaneously become IL-2 independent and the other LCLs were IL-2 dependent. Cellular replication kinetics varied between the cell lines. In addition, the cell lines expressed differing OvHV-2 DNA replication kinetics. DNAse protected OvHV-2 DNA could be detected in supernatants of all 4 cell lines at low copy numbers which could indicate virion particles. Expression of ORF 75, an OvHV-2 structural gene, was observed in all four cell lines. This also suggests late gene expression and the potential for virion production. Cells, and cell culture supernatants from one of the cell lines was injected into rabbits and no evidence of OvHV-2 infection was detected.
Newly Published Research
Discovering the pathophysiological basis of clinical malignant catarrhal fever has been a long sought after goal. Study of SA-MCF is still difficult as OvHV-2 remains an unculturable agent. Studies comparing the genome sequence of OvHV-2 genomes isolated from sheep and cattle indicate that they are virtually identical.17 Thus, it does not appear that specific virus mutation is the cause of clinical disease in cattle. Recent studies have been able to induce clinical MCF in cattle and bison by aerosol or intranasal inoculation of OvHV-2 infected nasal secretions from juvenile sheep at peak shedding.18-20 These studies are extremely important as they provide the opportunity to study the pathogenesis of MCF from the time of infection through the development of clinical disease. Further, they open the opportunity to investigate potential antiviral treatments or vaccination as a preventative measure.21 One very interesting study examined expression of the OvHV-2 major capsid protein gene (ORF 25). In this study, ORF 25 expression was predominantly observed in turbinate epithelium in sheep while it occurs in all tissues of cattle and bison with SA-MCF.22 This finding is counter-intuitive as one would not expect late gene expression in cattle or bison since these hosts are not believe to produce intact or infectious virions. Further evaluation of OvHV-2 gene expression in infected sheep, cattle and bison is warranted in order to evaluate important virus-host interactions. Such gene expression studies will be greatly aided by the recent sequencing of the complete OvHV-2 genome.23 Of particular interest is the recent finding that resistance to SA-MCF in some bison is associated with MHC class IIa polymorphisms.24
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