Abstract

Malignant catarrhal fever (MCF) is caused by one of two gamma herpesviruses; alcelaphine herpesvirus 1 (AHV-1), first isolated from blue wildebeest (Connochaetes taurinus), and ovine herpesvirus 2 (OHV-2) of sheep which cannot be grown in tissue culture and thus is incompletely characterized. These viruses are non-pathogenic in their true hosts, but infection of other species causes a lymphoproliferative disease with very high mortality. Both the wildebeest-associated and sheep-associated forms of MCF have been responsible for deaths in zoos.³

Serologic surveys of artiodactyl collections using AHV-1 antigens have revealed that there are species whose populations have a high prevalence of sero-positives,¹ suggesting that they carry their own endemic gamma herpesvirus. Two of these related viruses have been isolated and named; alcelaphine herpesvirus 2 (AHV-2) from topi (Damaliscus lunatus) and hartebeest (Alcelaphus buselaphus),² and hippotragine herpesvirus 1 (HiHV-1) from a roan antelope (Hippotragus equinus).⁴ Species of the genus Oryx are also potential carriers of their own gamma herpesvirus and indeed an MCF-like virus has been reported from a neonatal scimitar-horned oryx (SH oryx; Oryx dammah).¹

In order to investigate the carrier status of the breeding herds of SH oryx and gemsbok (Oryx gazella gazella) at Whipsnade Wild Animal Park, a study was carried out during 1998. The study material consisted of: a) serum samples collected during the previous 10 yr from animals anesthetized for veterinary or management reasons, b) blood samples from 11 SH oryx and 4 gemsbok calves born during 1998 taken within 24 hr of birth, c) tissue samples from the three SH oryx neonates and one gemsbok which died, d) further blood samples from the eight surviving SH oryx calves at 4–5 mo of age, and e) blood samples from two adult SH oryx and four gemsbok anesthetized during the year for veterinary or management reasons. Serum samples were tested for antibodies to AHV-1 in an indirect immunofluorescent antibody test (IFAT) and a virus neutralization test (VNT). The polymerase chain reaction (PCR) was used to amplify DNA derived from peripheral blood leukocytes (PBL) or tissues of calves, using as primers OHV-2 (PCR/1) or two amplifications of a mixture of the non-specific gamma herpesvirus primers POL1 and POL2, on its own (PCR/2), or followed by primers for AHV-1 (PCR/3) or OHV-2 (PCR/4).

Thirty-two out of 34 (94.1%) stored SH oryx sera and all 16 stored gemsbok sera were positive in the IFAT to AHV-1, but only four SH oryx and 10 gemsbok sera had neutralizing antibody to the virus.

Eight of the 11 newborn SH oryx calves had detectable gamma herpesvirus DNA, six had a band at 800 base-pairs (bp) (PCR/2), two responded to AHV-1 POL amplification (PCR/3) and six responded to OHV-2 POL (PCR/4) (Table 1). Two newborn gemsbok calves were positive, one gave a band at 388bp in PCR/2 and one a band at 173 bp after OHV-2 POL amplification in PCR/4 (Table 2). A selection of these PCR products were tested by Southern blot hybridization against a specific AHV-1 probe, but none reacted. All the serum samples from newborn calves which were tested (eight SH oryx and four gemsbok) were positive in the IFAT and four of the SH oryx and two of the gemsbok samples were also positive in the virus neutralization test against AHV-1 (Tables 1 and 2).

When the eight surviving SH oryx calves were re-sampled at 4–5 mo of ages, all eight reacted in PCR/2, none to AHV-1 POL (PCR/3) and six to OHV-2 POL (PCR/4) (Table 1). However, only one animal was still positive in the IFAT to AHV-1. One of the seronegative animals was re-tested 3 mo later and was still PCR/4 positive, but IFAT negative. Pooled PBL from the eight calves were added to a monolayer of kidney cells derived from one of the SH oryx calves that died. Following two sub-passages and 83 days in culture a dense multinucleate focus was observed. Similar foci appeared and increased in number over the next few days. Cells from these cultures were co-cultivated with further SH oryx kidney cells and with cultured bovine turbinate cells. Similar cytopathic effects developed in these cultures. DNA from these cells were positive by the PCR/1, 2, and 4. In addition, when cells from one of the affected cultures were inoculated into a rabbit it developed a rectal temperature of 41/C on day 13 and at necropsy lymphoproliferative lesions, characteristic of malignant catarrhal fever, were observed grossly and histologically.

All of the adult SH oryx and gemsbok were found to have viral DNA in their PBL (Tables 1 and 2).

These results confirm the suspicion that oryx carry their own gamma herpesvirus which is related to, but distinct from, AHV-1 and OHV-2. On the basis of the PCRs it would appear that the viruses of both the SH oryx and the gemsbok are more closely related to OHV-2, which may explain the relative lack of neutralizing antibodies to AHV-1. Interestingly, a higher proportion of gemsbok than SH oryx had virus neutralizing antibodies, suggesting a difference between their respective viruses. Virus has now been isolated from the SH oryx which should allow a more detailed analysis of the viral DNA and a more specific diagnostic test in scimitar-horned, and other, oryx. That a rabbit developed MCF following inoculation with this virus demonstrates its capability of inducing disease, though there is no evidence for transmission to susceptible species in zoos. The probable designation of the virus will be hippotragine herpesvirus-2, after the subfamily to which SH oryx are ascribed. It would appear likely that the virus is transmitted in utero, because so many of the neonates had virus DNA in their PBL before they were 24 hr old. The antibodies detected were probably maternal and in all but one case had disappeared by 4–5 mo of age. It is hoped to retest the animals to find out when they mount their own antibody response.

Table 1. Summary of serologic and PCR results from scimitar-horned oryx.

^aIndirect immunofluorescent antibody test against AHV-1 infected cell culture
^bVirus neutralization test. Positive if serum protected more than 50% of cell culture wells at a dilution of 1:10 or greater.
^cPCR/1 OHV-2 PCR
PCR/2 POL1/POL2 PCR (2 amplifications)
PCR/3 POL1/POL2 (2 amplifications) then POL2/AHV POL
PCR/4POL1/POL2 (2 amplifications) then POL2/OHV POL
^dPCR/1 not tested, PCR/3 and PCR/4 not tested on calves 1,2, and 8

Table 2. Summary of serologic and PCR results from gemsbok.

^aIndirect immunofluorescent antibody test against AHV-1 infected cell culture
^bVirus neutralization test. Positive if serum protected more than 50% of cell culture wells at a dilution of 1:10 or greater.
^cPCR/1 OHV-2 PCR
PCR/2 POL1/POL2 PCR (2 amplifications)
PCR/3 POL1/POL2 (2 amplifications) then POL2/AHV POL
PCR/4 POL1/POL2 (2 amplifications) then POL2/OHV POL
^dPCR/1 not tested on calves or adult 2

Literature Cited

1.  Heuschele,W.P. and H.R. Fletcher. 1984. Recent findings on the epidemiology of malignant catarrhal fever in exotic ruminants. Proceedings of the American Association of Zoo Veterinarians Annual Meeting, October 1984, Louisville, Kentucky. 95–96.

2.  Heuschele,W.P., H.R. Fletcher, J. Oosterhuis, et al. 1984. Epidemiologic aspects of malignant catarrhal fever in the USA. Proceedings of the 88th Annual Meeting of the U.S. Animal Health Association. 88; 640–651.

3.  Heuschele, W.P 1993. Malignant catarrhal fever. In: Zoo and Wild Animal Medicine; Current Therapy 3. Ed. M.E. Fowler. W.B. Saunders Co., Philadelphia. 504–506.

4.  Reid,H.W. and Bridgen. 1991. Recovery of a herpesvirus from a roan antelope (Hippotragus equinus). Veterinary Microbiology, 28; 269–278.