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Gamma herpesvirus carrier status of captive artiodactyls
Research in Veterinary Science 2002, 73, 93–99 doi: 10.1016/S0034-5288(02)00094-2, available online at http://www.idealibrary.com on
Gamma herpesvirus carrier status of captive artiodactyls E. J. FLACH ,*, H. REIDà, I. POWà, A. KLEMT
Institute of Zoology, Whipsnade Wild Animal Park, Dunstable, Bedfordshire LU6 2LF, UK, àMoredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, UK SUMMARY Between 1998 and 2000, 103 individuals of 19 species of the order Artiodactyla at Whipsnade Wild Animal Park were tested for evidence of infection with gamma herpesviruses in order to distinguish between species which are susceptible to malignant catarrhal fever (MCF), caused by alcelaphine herpesvirus-1 (AlHV-1) of wildebeest (Connochaetes sp.) or ovine herpesvirus-2 (OvHV-2) of domestic sheep, and species which carry related viruses sub-clinically. Gamma herpesvirus DNA was detected in the known, or suspected, carrier species: roan antelope (Hippotragus equinus), scimitarhorned oryx (Oryx dammah), gemsbok (Oryx gazella), musk ox (Ovibos muschatus) and mouflon (Ovis musimon). In six other species: lowland anoa (Bubalus depressicornis) yak (Bos grunniens), sitatunga (Tragelaphus spekei), greater kudu (Tragelaphus strepsiceros), waterbuck (Kobus ellipsiprymnus) and Nile lechwe (Kobus megaceros), DNA was present in some newborn calves and over 30% of adults, strongly suggesting a carrier state. In contrast five P ere David’s deer (Elaphurus davidianus) and two swamp deer (Cervus duvauceli) died of MCF during the study. A virus isolated from scimitar-horned oryx calves produced cytopathic effects in scimitar-horned oryx kidney cell-culture and caused MCF in a rabbit. Ó 2002 Elsevier Science Ltd. All rights reserved.
GAMMA herpesviruses are a group of double-stranded deoxyribonucleic acid (DNA) viruses which are envelopped, icosahedral (typical of the Herpesviridae), and highly cell-associated, predominantly infecting lymphocytes. The importance of gamma herpesviruses to ungulates of the order Artiodactyla lies in the capacity of two of them, alcelaphine herpesvirus-1 (AlHV-1), found naturally in wildebeest (Connochaetes species) (Plowright et al 1960), and ovine herpesvirus-2 of domestic sheep (OvHV-2), to cause malignant catarrhal fever (MCF), a lymphoproliferative disease with very high mortality, in domestic cattle and buffalo, farmed deer and a range of wild artiodactyl species in zoos, game parks and other collections (Heuschele and Reid 2001). These viruses are nonpathogenic in their true hosts. Two related viruses, which are carried sub-clinically by antelope hosts, have been isolated and named; alcelaphine herpesvirus-2 (AlHV-2) from hartebeest (Alcelaphus buselaphus) (Reid and Rowe 1973) and topi (Damaliscus lunatus ¼ D.korrigum) (Mushi et al 1981a), and hippotragine herpesvirus-1 (HipHV-1) from a roan antelope (Hippotragus equinus) (Reid and Bridgen 1991). There has never been any evidence of these causing MCF in the same or other species. Transmission of AlHV-1 in free-living wildebeest occurs in the first few months of life. Plowright (1965a,b, 1967) showed that virus could be isolated from a proportion of foetuses, that the majority of 1–3 month-old calves (88 of 97) had antibodies, and that
*
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cattle housed with viraemic wildebeest calves under three months of age contracted MCF, but a further group of cattle housed with the calves when they were older than three months did not succumb. Virus was isolated from nasal or ocular secretions of 50% (six of 12) of six to eight-week-old wildebeest calves (Mushi et al 1980), suggesting that the virus spreads by these secretions, and possibly aerosols. All adult wildebeest had neutralizing antibodies to AlHV-1, and carried the virus latently. Wildebeest cows may become viraemic in late pregnancy (Plowright 1965a), if stressed by confinement or abrupt changes in diet, or if injected with corticosteroids for a week (Rweyemamu et al 1974). In the study of Rweyemamu et al the animals also excreted virus. There is less direct evidence concerning the transmission of OvHV-2. All adult sheep have antibodies cross-reacting with AlHV-1 in serological tests, unless delivered by caesarian section and reared in isolation, and even some animals reared in this way became sero-positive (Rossiter 1981). Baxter et al (1997) monitored nine lambs from birth and found OvHV-2 DNA in nasal secretions of all of them between the ages of one and seven weeks (median four). The viral DNA was most prevalent in the cornea and turbinates, but also commonly in the pharyngeal and nasal epithelium, lacrimal gland and soft palate. It seems likely that the virus is transmitted to some foetuses in utero and infects all others soon after birth by nasal and ocular secretions and by aerosols, as in the case of wildebeest. Some other artiodactyl species are assumed to be carriers of gamma herpesviruses because populations in the wild and captivity have been shown to have high prevalences of antibodies to AlHV-1 and they appear Ó 2002 Elsevier Science Ltd. All rights reserved.
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E. J. Flach, H. Reid, I. Pow, A. Klemt
to be resistant to clinical disease (Reid et al 1975, Mushi et al 1981b, Heuschele et al 1984). Examples are antelope of the genus Oryx and members of the Caprinae (sheep, goats and relatives). North American species of the latter sub-family also have high prevalences of seropositives against OvHV-2 using a competitive-inhibition enzyme-linked immunosorbent assay (C-I ELISA) (Li et al 1996). The isolation of a gamma herpesvirus from a scimitar-horned oryx was reported by Heuschele et al (1984), but no details were given. Malignant catarrhal fever has occurred sporadically at Whipsnade Wild Animal Park. Cases were diagnosed in P ere David’s deer (Elaphurus davidianus) in the 1950s (Tong et al 1961), and in sitatunga (Tragelaphus spekei) and red deer (Cervus elaphus) in the late 1970s (Ashton 1982). A serological study in the early 1980s (Ashton 1982) indicated a high prevalence of sero-positive scimitar-horned oryx (Oryx dammah) and sitatunga, some animals having high antibody titres. All of the sitatunga with significant titres were clinically affected with MCF. A limited study was carried out between May and August 1998 to investigate the occurrence of gamma herpesvirus DNA in Oryx species and other artiodactyls kept at Whipsnade (Klemt 1998). Viral DNA was detected on several occasions, prompting a continuation and extension of the study from October 1998 to December 2000, with the following aims: (a) to investigate the carrier status of scimitar-horned oryx and gemsbok (O. gazella), and (b) to determine whether other artiodactyl species carry gamma herpesviruses.
MATERIAL AND METHODS Animals and test samples Whipsnade Wild Animal Park maintained 37 species of the Order Artiodactyla (including domestic pigs, cattle, sheep and goats) during the years of the study (1998–2000). They were kept either as single-species in grass paddocks with associated concrete yards and housings, or as mixed-species in a large, grass parkland which visitors drove through in cars. The latter exhibit held P ere David’s deer mixed with herds of Bactrian camels (Camelus bactrianus) and yak (Bos grunniens) in one enclosure, and swamp deer (Cervus duvauceli), Chinese water deer (Hydropotes inermis), fallow deer (Dama dama), axis deer (Axis axis), hog deer (Axis porcinus), Formosan sika deer (Cervus nippon), nilgai (Boselaphus tragocamelus) and blackbuck (Antilope cervicapra) in another. Venous blood samples were collected when animals were handled for veterinary procedures. These procedures included clinical investigations, health examinations and elective procedures, such as hoof trimming. For two species, the scimitar-horned oryx and gemsbok, health checks were performed on all calves born in 1998; when less than 24 h old (at the time of eartagging), at 4–5 months of age (when tested for, and treated against, gastrointestinal parasites; scimitar-
horned oryx only), and at one year old (for assessment of body condition and state of hooves, and removal of males from the breeding herd). Serum was separated from clotted blood by centrifugation, and then removed and stored frozen at )20 °C. Blood in ethylenediamine tetra-acetic acid (EDTA) was sent on the day of collection by first-class post to the Moredun Research Institute (MRI) where the peripheral blood leukocytes (PBL) were removed and stored frozen until PCR testing. The EDTA plasma was also stored frozen. A full post mortem examination was performed on all animals which died during the study period. Blood, if available, and samples of cornea, buccal mucosa, peripheral lymph node and kidney, placed in 25 ml phosphate buffered saline (PBS) with added antibiotics (30 mg benzyl penicillin sodium [Britannian Pharmaceuticals] and 12.5 mg gentamycin [Bimeda] per sample), were sent on the day of collection to the MRI. Tests Sera and EDTA plasma were tested for the presence of antibodies against AlHV-1 using an indirect fluorescent antibody test (IFAT). Briefly, the test serum (or plasma) was diluted 1/10 in PBS, and 80 ll was then added to an acetone-fixed monolayer of AlHV-1 infected bovine turbinate cells on a glass coverslip. After washing off excess, bound antibodies were detected with a 1/40 dilution in PBS of donkey anti-ovine gamma globulin conjugated to the fluorescent dye fluorescine isothiocyanate. The coverslip was examined under ultra-violet light for cellular fluorescence, and compared to positive and negative controls. The samples from the newborn oryx were also tested in a virus neutralisation assay. Test sera (or plasma) were diluted in a 10-fold dilution series, and then 100 ll was incubated for 2 h at 37 °C with 100 ll of a fixed concentration of AlHV-1. One hundred microlitre of this mixture were then added to wells of a 96-well, flatbottomed, microtitre plate containing monolayers of bovine turbinate cells. Plates were incubated for 7 days at 37 °C. The wells were examined microscopically to assess the degree of cytopathic effect. The endpoint of the assay was the dilution of serum which protected 50% of wells, and if equal to, or greater than, 1/10 the sample was considered to be positive. DNA extracted from PBL or from tissues collected post mortem, were tested for the presence of gamma herpesvirus sequences, using polymerase chain reactions (PCRs). Briefly, PBL were incubated with an extraction buffer comprising sodium chloride, hydrochloric acid–Tris (pH 8.0), EDTA and sodium dodecyl sulphate (SDS), and then extracted into phenol and chloroform (6:4). DNA was precipitated by adding ammonium acetate in ethanol, washed in ethanol and then stored frozen in water until tested. Tissue samples were snap frozen in liquid nitrogen and ground up before extraction, using the same technique, but with a longer incubation in an extraction buffer with added proteinase K in Tris. The PCR for OvHV-2 used the
Gamma herpesvirus carrier status of captive artiodactyls
TABLE 1:
PCR primers described in the text
POL1 50 -GGC (CT) CA (CT) AA (CT) CT ATG CTA CTC CAC-30 POL2 50 -ATT (AG) TC CAC AAA CTG TTT TGT-30 AlHV-POL 50 -CCA AAA TGA AGA CCA TCT TA-30 OvHV-POL 50 -AAA AAC TCA GGG CCA TTC TG-30
same conditions and primers as Baxter et al (1993). The product size from the first amplification using primers 755/556 was 422 basepairs (bp), and from the second amplification with primers 555/556 was 238 bp, but only the results of the second amplification are recorded (OvHV PCR). In addition, PCR amplifications were carried out using the non-specific herpesvirus primers POL1 and POL2 (Table 1; first amplification product size 386 bp ¼ POL-1/2 PCR), and following this with a final amplification with either AlHV-POL/POL2 (product size 172 bp ¼ POL-A PCR) or OvHV-POL/ POL2 (product size 172 bp ¼ POL-O PCR) (Table 1; POL primers from MWG-Biotech GmbH, Ebersberg, Germany). For the POL1/POL2 reactions there was a total volume of 50 ll containing 10 mM Tris–HCl, 1.5 mM MgCl2 , 50 mM KCl, 200 lM dATP, dCTP, dGTP and dTTP, 1:0 lM of each primer and 2 units of Taq DNA polymerase and 2 lg DNA. This mixture was placed in a thermal cycler for a pre-cycle incubation at 95 °C for 5 min, then dNTP and enzyme mix were added followed by 24 cycles of 1 min each at three temperatures, 94, 45 and 72 °C, and a final cycle of 94 °C for 1 min, 45 °C for 1 min and 72 °C for 5 min. For the second amplification, 2 ll of the primary amplification product was transferred to a new amplification mixture and the process repeated. The final product was loaded onto an agarose gel and subjected to electrophoresis. The gel was then stained with ethidium bromide and photographed under UV light. Any DNA bands took up the stain and were visualised. Positive virus controls were added to each PCR and a molecular weight marker was included on the gel. Tissues from a one-day-old scimitar-horned oryx calf which died from aspiration pneumonia were sent to the MRI and trypsin-dispersed kidney cell monolayer cultures were prepared. These were subsequently passaged using standard techniques. Pooled PBL from the eight scimitar-horned oryx calves at 4–5 months of age were added to a monolayer of these kidney cells and cultured routinely. Further details of virus isolation techniques are described with the results below.
RESULTS Serological and PCR testing One hundred and three individuals of 19 species of artiodactyls were sampled during the study period (Table 2). All five P ere David’s deer were tested post mortem, having shown clinical signs (profound depression and anorexia) and gross lesions (widespread congestion and
95
petechiation, lymphadenopathy and haemorrhagic gastroenteritis) consistent with MCF in deer. The five died, or were euthanased, between February and May 2000. Histopathological findings in four of them included lymphoid infiltration and vasculitis in multiple organs (the other animal was not examined). They all had OvHV-2 DNA in blood or tissues (OvHV PCR) and tested positively in one or more of the POL PCR series. The three which were tested serologically had antibodies cross-reacting with AlHV-1. One animal also had a Pasteurella multocida pneumonia, two had mild parasitic gastritis and a fourth was found to have Clostridium perfringens epsilon toxin in small intestinal contents, and a Campylobacter species in faeces. Three adult swamp deer died between January and June 2000, and were found to have haemorrhagic gastroenteritis post mortem. Histopathological findings from one (widespread vascular lymphoid infiltration) was consistent with MCF, and this was one of two animals which tested positive for OvHV-2 DNA (histopathology was not performed on the other). The third animal had no histopathological evidence of MCF and, further, an Aeromonas species was cultured from the lung, liver and kidney, and a Campylobacter species from the faeces, but although negative for OvHV-2 it did have gamma herpesvirus DNA (POL-O PCR). A neonatal fawn was negative for DNA, but one out of three adults tested during a routine immobilisation was positive in the POL-A PCR. The single hog deer died of pneumonia, but also had a haemorrhagic gastroenteritis, from which a Campylobacter species was isolated. It reacted in POL-O PCR and was sero-positive, whereas the alpaca (Lama pacos) and four giraffe (Giraffa camelopardalis) were negative for both DNA and antibodies. Among the members of the sub-family Bovinae both a female anoa (Bubalus depressicornis) and her newborn calf had DNA reacting in the POL-A PCR, and the mother also reacted in the POL-O PCR. One adult and one newborn yak (Bos grunniens) had gamma herpesvirus DNA, both reacting in the POL-O PCR. The two congo buffalo (Synceros caffer) were negative in all tests, but the American bison (Bison bison) reacted in POL-A PCR. Both tragelaphid species, sitatunga and greater kudu (Tragelaphus strepsiceros) had a high prevalence of gamma herpesvirus DNA detected in the POL series, but absence of antibodies cross-reacting to AlHV-1. Most sitatunga calves (80%) and all five kudu were positive. One of the sitatunga calves, a 12-day-old female, also reacted in the specific OvHV PCR. The animal has not shown any clinical signs up to the time of writing. Similarly, there were adult and juvenile (newborn) waterbuck (Kobus ellipsiprymnus) and Nile lechwe (K. megaceros) which had gamma herpesvirus DNA, but antibodies to AlHV-1 were only detected in one calf of each species. Four out of six lechwe reacted in the POL PCR series, and one also had specific OHV2 DNA (OvHV PCR). This individual is still alive and showing no clinical signs. Roan antelope, scimitarhorned oryx and gemsbok had high prevalences of gamma herpesvirus DNA reacting in the POL series,
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TABLE 2:
E. J. Flach, H. Reid, I. Pow, A. Klemt
Gamma herpesvirus results from species of Artiodactyla at Whipsnade
Species
IFAT
No. tested
No. positive
Calves ð<1 yrÞ Adults (1 yr +)
Camelidae Alpaca (Lama pacos) Cervidae Hog deer (Axis porcinus) Swamp deer (Cervus duvauceli) re David’s deer Pe (Elaphurus davidianus) Giraffidae Giraffe (Giraffa camelopardalis) Bovidae—Bovinae Lowland anoa (Bubalus depressicornis) Yak (Bos grunniens) Congo buffalo (Synceros caffer) American bison (Bison bison) Sitatunga (Tragelaphus spekei) Greater kudu (T. strepsiceros) Bovidae—Hippotraginae Waterbuck (Kobus ellipsiprymnus) Nile lechwe (K. megaceros) Roan antelope (Hippotragus equinus) SH oryxc (Oryx dammah) Gemsbokc (O. gazella) Bovidae—Caprinae Musk ox (Ovibos moschatus) Domestic goat (Capra hircus) Mouflon (Ovis musimon)
1
0/1
0/1
0/1
1
1/1
0/1
0/1
0/1
1/1
1/1
4
ND 1/4 1/1b ð1Þ 2/2b ð2Þ
0/1 2/6b ð2Þ 1/1b ð1Þ 4/4b ð4Þ
0/1 0/6 1/1b ð1Þ 2/4b ð2Þ
0/1 1/6 0/1 1/4b ð1Þ
0/1 2/6bð1Þ 1/1bð1Þ 3/4bð3Þ
0/1 3/6b ð1Þ 1/1b ð1Þ 4/4b ð4Þ
3
ND 0/3
0/1 0/3
0/1 0/3
0/1 0/3
0/1 0/3
0/1 0/3
3 2
0/1 0/1 0/4 0/1 0/1
0/1 0/1 0/4 0/3 0/2
0/1 0/1 0/4 0/3 0/2
1/1 1/1 1/4 0/3 0/2
0/1 1/1 1/4 1/3 0/2
1/1 1/1 1/4 1/3 0/2
1
0/1
0/1
0/1
1/1
0/1
1/1
0/5 0/13 0/3 0/2
1/5 0/13 0/3 0/2
1/5 4/12 2/3 2/2
2/5 4/12 3/3 1/2
3/5 2/12 2/3 2/2
4/5 5/12 3/3 2/2
8
1/3 0/2 1/2 0/5 1/2 1/5 1/8 4/6 4/4 7/8
0/4 0/2 0/2 1/6 0/2 0/2 0/8 0/6 0/3 0/8
0/4 0/2 1/2 4/6 1/2 1/5 8/8 3/6 1/3 3/6
1/4 0/2 1/2 2/6 2/2 1/5 0/8 3/6 0/3 2/6
1/4 1/2 0/2 2/6 1/2 1/5 6/8 3/6 1/3 5/6
1/4 1/2 1/2 4/6 2/2 2/5 8/8 6/6 2/3 5/6
1
1/1
0/1
0/1
1/1
1/1
1/1
1
0/1
0/1
0/1
0/1
0/1
0/1
1
0/1
0/1
0/1
1/1
0/1
1/1
1a 1 a
a
5 (1 ) 13
4 2
a
POL series
0/1
1 (foetus)
a
POL-O
0/1
1
3
POL-A
0/1
a
a
POL-1/2
1
6
4
PCR OvHV
2 2 6 2a 5 8d 6 4a
Notes: ND not done. Newborn calves (<72 h). b Died of malignant catarrhal fever (number of animals dying in parentheses). c Selected results; complete results below. d Calves 4–5 months of age. a
and many had antibodies to AlHV-1. The adult roan had a lower prevalence of DNA (40%) and antibodies (20%) than the scimitar-horned oryx (100% and 67%) and gemsbok (83% and 88%). The effect of age in the latter two species is reported below. Only single individuals of three species of the subfamily Caprinae were tested. None had specific OHV-2 DNA (OvHV PCR), but the musk ox (Ovibos moschatus) had gamma herpesvirus DNA reacting in both POL-A and POL-O PCRs and antibodies to AlHV-1, and the mouflon had DNA reacting in the POL-A PCR, but no detectable antibodies. Influence of age Eight of the 11 newborn scimitar-horned oryx calves had detectable gamma herpesvirus DNA, six reacted in
the POL PCR, two in POL-A and six in POL-O (Table 3). The eight sera tested were positive in the IFAT, and four in the VNT. When the eight surviving calves were re-sampled at between four and five months of age, all eight reacted in the POL PCR, none in POL-A and six in POL-O. However, only one animal was still positive in the IFAT to AlHV-1. One calf died three months later and was still POL-O PCR positive, but IFAT negative. The remaining seven were re-examined at one year of age (12–15 months), but the blood sample from one was unsuitable for PCR testing. Five of the six tested were positive to one of the POL series, and the highest number, four, reacted to POL-A. Four out of the seven were strongly positive in the IFAT, and a further two showed a weak or doubtful reaction. All of the adult scimitar-horned oryx adults tested positive in one of the POL PCRs, with an equal number (three)
Gamma herpesvirus carrier status of captive artiodactyls
TABLE 3:
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Effect of age on gamma herpesvirus carrier status of scimitar-horned oryx Number positive/number tested
Age
IFAT
< 1 Day 4–5 Months 1 Year Adult 5–17 years
8/8 1/8 4/7a 4/6
VNT
4/8 NT NT NT
PCR OvHV
POL-1/2
POL-A
POL-O
POL series
NT 0/8 0/7 0/6
6/11 8/8 3/6 3/6
2/8 0/8 4/6 3/6
6/8 6/8 2/6 3/6
8/11 8/8 5/6 6/6
Notes. NT: not tested. a 2/7 doubtful reaction, only 1/7 negative.
reacting in each test, but only four out of six (67%) were seropositive. Two newborn gemsbok calves were positive for gamma herpesvirus DNA, one reacted in POL-1/2 PCR and one in POL-O PCR (Table 4). All four serum samples from newborn calves were positive in the IFAT and two out of three tested were also positive in the VNT. The three surviving gemsbok calves were retested at 13 months of age. All three reacted in the POL-A PCR, and one also reacted in POL-O. However, only one animal had detectable antibodies to AlHV-1 in the IFAT. Five out of six adults (83%) had detectable gamma herpesvirus DNA, the majority being positive in the POL-O PCR, and seven out of eight (88%) were seropositive in the IFAT.
lymphoproliferative lesions, characteristic of malignant catarrhal fever, were observed grossly and histologically.
DISCUSSION Our results support the theory that scimitar-horned oryx and gemsbok carry their own gamma herpesviruses. None of the animals tested had OvHV-2 DNA, but there was a high prevalence of gamma herpesvirus DNA detected by the POL series. The lack of consistency in the POL-A and POL-O PCRs suggests that their viruses are related to, but distinct from, AlHV-1 and OvHV-2. Cross-reactivity between herpesviruses is common in serological tests, particularly the IFAT, and so the presence, in many of the individual’s sera, of antibodies to AlHV-1 does not contradict this theory. The virus isolated from the scimitar-horned oryx, however, did react in the specific OvHV-2 PCR, but since OvHV-2 has never been isolated in culture it is highly unlikely that they are one and the same. Research is continuing to compare it directly to AlHV-1, OvHV-2 and the roan antelope virus, HipHV-1 and, if it proved to be a new species, the designation is likely to be hippotragine herpesvirus-2. An analysis of viral DNA may help to unravel the genetic make-up of the gamma herpesviruses and, possibly, the differences in genetic structure may help explain differences in infectivity and pathogenesis. Oryx have never been implicated in transmitting MCF to other species, but the experimental infection in rabbits show that the scimitarhorned oryx virus has the capacity to do so. Finally, it should be possible in the future to produce more specific PCR and serological tests for oryx and other bovids. Gamma herpesvirus DNA was detected in most of the newborn scimitar-horned oryx calves (possibly all, be-
Virus isolation Cytopathogenic effects characteristic of a gamma herpesvirus were observed in the monolayer cultures of the neonatal scimitar-horned oryx kidney when inoculated with the pooled PBL from the eight scimitarhorned oryx calves aged 4–5 months. Following two subpassages 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 cocultivated with further scimitarhorned oryx kidney cells and similar cytopathic effects developed. Cocultivation with cultured bovine turbinate cells was initially unsuccessful, but after addition of 5 mg/ml triphenylalanine (TPA) to the medium, extensive cytopathic effects were seen, although the virus remained cell-associated. Extracts of DNA from these cells were positive by the OvHV, POL-1/2 and POL-O PCRs. 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 TABLE 4:
Effect of age on gamma herpesvirus carrier status of gemsbok Number positive/number tested
Age
<1 Day 1 Year Adult 2–9 years Notes. NT: not tested.
IFAT
4/4 1/3 7/8
VNT
2/3 NT NT
PCR OvHV
POL-1/2
POL-A
POL-O
POL series
0/3 0/3 0/8
1/3 0/3 3/6
0/3 3/3 2/6
1/3 1/3 5/6
2/3 3/3 5/6
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E. J. Flach, H. Reid, I. Pow, A. Klemt
cause POL-A and POL-O PCRs were not performed on three calves) and two out of three gemsbok, strongly suggesting that they were infected in utero. The third gemsbok was positive when re-tested at one year of age, but one of the scimitar-horned oryx failed to react at this age, despite having done so at four to five months. All newborn oryx had antibodies to AlHV-1 in the IFAT. These were probably maternal because most of the scimitar-horned oryx calves had lost them by four to five months of age. Thereafter, active antibody production appeared to be slow, despite the presence of viral DNA, because even at one year old only four of seven scimitarhorned oryx and one of three gemsbok had seroconverted. By adulthood, however, all except one individual (a gemsbok) were sero-positive. There was a lower proportion of newborn calves with neutralising antibodies against AlHV-1, probably indicating a lower degree of cross-reaction than in the IFAT, but this proportion was higher amongst the gemsbok (2/3) than the scimitar-horned oryx (4/8). Mushi and Karstad (1981b) found a similar age-related pattern in fringe-eared oryx calves (Oryx gazella beisa ¼ O. beisa callotis). Neutralising antibody titre declined from birth to six months of age, and then increased after eight months of age. The results from the waterbuck and Nile lechwe were also consistent with a sub-clinical carrier state. There were neonates and adults with both gamma herpesvirus DNA and antibodies, and one adult Nile lechwe reacted specifically to OvHV-2 in the PCR series. The roan antelope were expected to be carriers of their own virus, HipHV-1, but despite both newborn calves being positive, only two out of five adults had detectable gamma herpesvirus DNA. This may indicate that not all individuals become infected, or carry infection to adulthood, or alternatively that viruses are present, but not always detectable. Not all of the scimitar-horned oryx and gemsbok calves were positive on each occasion they were tested, so there may indeed be variable detectability of viral DNA. Among the other bovids, gamma herpesvirus DNA was detected in the single American bison and both adult and newborn yak, anoa, sitatunga and greater kudu, but all were sero-negative. Sero-positives have been detected by the IFAT in these species before, so either the anti-ovine immunoglobulin conjugate was not binding to their antibodies or, more probably, they were truly sero-negative. Bison, sitatunga and greater kudu have all been affected by MCF in other collections, and an outbreak occurred in the sitatunga herd at Whipsnade in the late 1970s and early 1980s (Ashton 1982). Interestingly, one of the sitatunga calves tested positive in the specific OvHV-2 PCR (PCR/1) suggesting that it was carrying OvHV-2 itself. These findings could be taken to show that some individual sitatunga infected by OvHV-2 recover and continue to carry and even transmit the virus to calves in utero without apparent antibody response. O’Toole et al (1997) showed that recovery and carriage of OvHV-2 does occur in cattle that recover from MCF, but that they continued to show chronic lesions of the eyes and blood vessels, and most (eight out of 11) were seropositive in the C-I ELISA. Alternatively, sitatunga
may carry their own virus which is closely related to OvHV-2, but under certain circumstances succumb to the full disease, especially if caused by AlHV-1, and, indeed, the earlier cases did occur when they were mixed with brindled wildebeest (C. Tack, personnal communication, 2001). Testing stored histopathological blocks could prove whether or not this was the case. Further support for this latter theory of incomplete resistance to other gamma herpesviruses lies in the fact that roan antelope, which may carry HipHV-1, can be susceptible to MCF caused by OvHV-2 (Gulland et al 1989). In contrast, none of the giraffe, the alpaca or the deer, except for one individual, had any evidence of sub-clinical carriage of gamma herpesvirus. In addition, sheep-associated MCF, alone or in combination with other diseases, was diagnosed in five Pere David’s deer and two swamp deer; underlying the susceptibility of these species compared to other species in the mixed exhibits, including sika deer, axis deer, Chinese water deer, nilgai and blackbuck, all of which have contracted MCF in other collections. There had been no contact with sheep or goats, or with any of the suspected carrier species, and the sheep and goats at Whipsnade were not breeding at the time so transmission was suspected to have occurred via aerosol or fomites from nearby farms on which lambing was taking place. The musk ox and mouflon were found to have gamma herpesvirus DNA, although they were not positive in the OvHV-2 specific PCR. They have been implicated as OvHV-2 carriers (Li et al 1996, 1999), but it is possible that they carry related, but not identical viruses. Despite the limitations of opportunistic sampling, our results suggest that there may be a large number of gamma herpesviruses infecting artiodactyl hosts without clinical disease in the natural host, and with no evidence of spread to other, susceptible species. It is hoped that sequence data from the amplified fragments will indicate the number and relatedness of these. One explanation for lack of spread would be if these viruses infect all foetuses in utero and have lost the ability to survive outside the host as free virus. Alternatively, they may spread widely, but have very low inherent pathogenicity compared to AlHV-1 and OvHV-2. However, this appears unlikely because the scimitarhorned oryx virus was pathogenic to a rabbit.
ACKNOWLEDGMENTS We are extremely grateful to Jo Dodds for her unstinting help with collecting and packaging samples, the keepers of the Africa, Asia and Europe regions, Whipsnade Wild Animal Park for their assistance with handling and sampling animals, veterinary colleagues in the Institute of Zoology who dealt with some of the clinical cases, and Shaheed Macgregor for parasitological and bacteriological examinations. Histopathological examinations of most of the deer were performed by Cosme Sanchez-Miguel for his M.Sc. project in Veterinary Pathology at the Royal Veterinary College, all other post mortem tissues were ex-
Gamma herpesvirus carrier status of captive artiodactyls
amined by Idexx Laboratory, Wetherby. Testing for clostridial enterotoxins was carried out by the Veterinary Laboratory Agency, Bury St. Edmunds.
REFERENCES ASHTON, D. G. (1982) Veterinary Clinical Report; Whipsnade. Scientific Report of the Zoological Society of London 1979–1981. Journal of Zoology 197, 82–83 BAXTER, S. I. F., POW, I., BRIDGEN, A. & REID, H. W. (1993) PCR detection of the sheep-associated agent of malignant catarrhal fever. Archives of Virology 132, 145–159 BAXTER, S. I. F., WIYONO, A., POW, I. & REID, H. W. (1997) Identification of ovine herpesvirus-2 infection in sheep. Archives of Virology 142, 823–831 GULLAND, F. M. D., REID, H. W., BUXTON, D., LEWIS, J. C. M., KOCK, R. A. & KIRKWOOD, J. K. (1989) Malignant catarrhal fever in a roan antelope (Hippotragus equinus) at Regent’s Park. Veterinary Record 124, 42–43 HEUSCHELE, W. P., FLETCHER, H. R., OOSTERHUIS, J., JANSSEN, D. & ROBINSON, P. T. (1984) Epidemiologic aspects of malignant catarrhal fever in the USA. Proceedings of the 88th Annual Meeting of the US Animal Health Association (88)640–651 HEUSCHELE, W. P. & REID, H. W. (2001) Malignant catarrhal fever. In: Infectious Diseases of Wild Mammals. Eds: Williams, E. S. & Barker, I. K. London, Manson Publishing Ltd, pp. 157–164 KLEMT, A. (1998) A study of malignant catarrhal fever in several ruminants from Whipsnade Wild Animal Park. M.Sc. Thesis, The Royal Veterinary College, University of London LI, H., SHEN, D. T., JESSUP, D. A., KNOWLES, D. P., GORHAM, J. R., THORNE, T., O’TOOLE, D. & CRAWFORD, T. B. (1996) Prevalence of antibody to malignant catarrhal fever virus in wild and domestic ruminants by competitive-inhibition ELISA. Journal of Wildlife Diseases 32, 437–443 MUSHI, E. Z., KARSTAD, L. & JESSETT, D. M. (1980) Isolation of bovine malignant catarrhal fever virus from ocular and nasal secretions of wildebeest calves. Research in Veterinary Science 29, 168–171
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MUSHI, E. Z., ROSSITER, P. B., JESSETT, D. M. & KARSTAD, L. (1981a) Isolation and characterization of a herpesvirus from topi (Damaliscus korrigum, Ogilby). Journal of Comparative Pathology 91, 63–68 MUSHI, E. Z. & KARSTAD, L. (1981b) Prevalence of virus neutralising antibodies to malignant catarrhal fever virus in oryx (Oryx beisa callotis). Journal of Wildlife Diseases 17, 467–470 O’TOOLE, D., LI, H., MILLER, D., WILLIAMS, W. R. & CRAWFORD, T. B. (1997) Chronic and recovered cases of sheep-associated malignant catarrhal fever in cattle. Veterinary Record 140, 519–524 PLOWRIGHT, W. (1965a) Malignant catarrhal fever in East Africa. I. Behaviour of the virus in free-living populations of blue wildebeest (Gorgon taurinus taurinus, Burchell). Research in Veterinary Science 6, 56–68 PLOWRIGHT, W. (1965b) Malignant catarrhal fever in East Africa. II. Observations on wildebeest calves in the laboratory and contact transmission of the infection to cattle. Research in Veterinary Science 6, 69–83 PLOWRIGHT, W. (1967) Malignant catarrhal fever in East Africa. III. Neutralising antibody in free-living wildebeest. Research in Veterinary Science 8, 129–136 PLOWRIGHT, W., FERRIS, R. D. & SCOTT, G. R. (1960) Blue wildebeests and the aetiological agent of bovine malignant catarrhal fever. Nature 188, 1167–1169 REID, H. W. & ROWE, L. (1973) The attenuation of a herpes virus (malignant catarrhal fever virus) isolated from hartebeest (Alcelaphus buselaphus cokei, Gunther). Research in Veterinary Science 15, 144–146 REID, H. W., PLOWRIGHT, W. & ROWE, L. W. (1975) Neutralising antibody to herpesvirus derived from wildebeest and hartebeest in wild animals in East Africa. Research in Veterinary Science 18, 269–273 REID, H. W. & BRIDGEN, (1991) Recovery of a herpesvirus from a roan antelope (Hippotragus equinus). Veterinary Microbiology 28, 269–278 ROSSITER, P. B. (1981) Antibodies to malignant catarrhal fever virus in sheep sera. Journal of Comparative Pathology 91, 303–311 RWEYEMAMU, M. M., KARSTAD, L., MUSHI, E. Z., OTEMA, J. C., JESSETT, D. M., ROWE, L., DREVEMO, S. & GROOTENHUIS, J. G. (1974) Malignant catarrhal fever virus in nasal secretions of wildebeest: a probable mechanism for virus transmission. Journal of Wildlife Diseases 10, 478–487 TONG, E. H., SENIOR, M. & HALMAN, C. R. E. (1961) Whipsnade Park. Proceedings of the Zoological Society of London 136, 477
Accepted 21 May 2002
Gamma herpesvirus carrier status of captive artiodactyls E. J. FLACH ,*, H. REIDà, I. POWà, A. KLEMT
Institute of Zoology, Whipsnade Wild Animal Park, Dunstable, Bedfordshire LU6 2LF, UK, àMoredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, UK SUMMARY Between 1998 and 2000, 103 individuals of 19 species of the order Artiodactyla at Whipsnade Wild Animal Park were tested for evidence of infection with gamma herpesviruses in order to distinguish between species which are susceptible to malignant catarrhal fever (MCF), caused by alcelaphine herpesvirus-1 (AlHV-1) of wildebeest (Connochaetes sp.) or ovine herpesvirus-2 (OvHV-2) of domestic sheep, and species which carry related viruses sub-clinically. Gamma herpesvirus DNA was detected in the known, or suspected, carrier species: roan antelope (Hippotragus equinus), scimitarhorned oryx (Oryx dammah), gemsbok (Oryx gazella), musk ox (Ovibos muschatus) and mouflon (Ovis musimon). In six other species: lowland anoa (Bubalus depressicornis) yak (Bos grunniens), sitatunga (Tragelaphus spekei), greater kudu (Tragelaphus strepsiceros), waterbuck (Kobus ellipsiprymnus) and Nile lechwe (Kobus megaceros), DNA was present in some newborn calves and over 30% of adults, strongly suggesting a carrier state. In contrast five P ere David’s deer (Elaphurus davidianus) and two swamp deer (Cervus duvauceli) died of MCF during the study. A virus isolated from scimitar-horned oryx calves produced cytopathic effects in scimitar-horned oryx kidney cell-culture and caused MCF in a rabbit. Ó 2002 Elsevier Science Ltd. All rights reserved.
GAMMA herpesviruses are a group of double-stranded deoxyribonucleic acid (DNA) viruses which are envelopped, icosahedral (typical of the Herpesviridae), and highly cell-associated, predominantly infecting lymphocytes. The importance of gamma herpesviruses to ungulates of the order Artiodactyla lies in the capacity of two of them, alcelaphine herpesvirus-1 (AlHV-1), found naturally in wildebeest (Connochaetes species) (Plowright et al 1960), and ovine herpesvirus-2 of domestic sheep (OvHV-2), to cause malignant catarrhal fever (MCF), a lymphoproliferative disease with very high mortality, in domestic cattle and buffalo, farmed deer and a range of wild artiodactyl species in zoos, game parks and other collections (Heuschele and Reid 2001). These viruses are nonpathogenic in their true hosts. Two related viruses, which are carried sub-clinically by antelope hosts, have been isolated and named; alcelaphine herpesvirus-2 (AlHV-2) from hartebeest (Alcelaphus buselaphus) (Reid and Rowe 1973) and topi (Damaliscus lunatus ¼ D.korrigum) (Mushi et al 1981a), and hippotragine herpesvirus-1 (HipHV-1) from a roan antelope (Hippotragus equinus) (Reid and Bridgen 1991). There has never been any evidence of these causing MCF in the same or other species. Transmission of AlHV-1 in free-living wildebeest occurs in the first few months of life. Plowright (1965a,b, 1967) showed that virus could be isolated from a proportion of foetuses, that the majority of 1–3 month-old calves (88 of 97) had antibodies, and that
*
Corresponding author. Fax: +44-1582-873989; E-mail: [email protected] 0034-5288/02/$ - see front matter
cattle housed with viraemic wildebeest calves under three months of age contracted MCF, but a further group of cattle housed with the calves when they were older than three months did not succumb. Virus was isolated from nasal or ocular secretions of 50% (six of 12) of six to eight-week-old wildebeest calves (Mushi et al 1980), suggesting that the virus spreads by these secretions, and possibly aerosols. All adult wildebeest had neutralizing antibodies to AlHV-1, and carried the virus latently. Wildebeest cows may become viraemic in late pregnancy (Plowright 1965a), if stressed by confinement or abrupt changes in diet, or if injected with corticosteroids for a week (Rweyemamu et al 1974). In the study of Rweyemamu et al the animals also excreted virus. There is less direct evidence concerning the transmission of OvHV-2. All adult sheep have antibodies cross-reacting with AlHV-1 in serological tests, unless delivered by caesarian section and reared in isolation, and even some animals reared in this way became sero-positive (Rossiter 1981). Baxter et al (1997) monitored nine lambs from birth and found OvHV-2 DNA in nasal secretions of all of them between the ages of one and seven weeks (median four). The viral DNA was most prevalent in the cornea and turbinates, but also commonly in the pharyngeal and nasal epithelium, lacrimal gland and soft palate. It seems likely that the virus is transmitted to some foetuses in utero and infects all others soon after birth by nasal and ocular secretions and by aerosols, as in the case of wildebeest. Some other artiodactyl species are assumed to be carriers of gamma herpesviruses because populations in the wild and captivity have been shown to have high prevalences of antibodies to AlHV-1 and they appear Ó 2002 Elsevier Science Ltd. All rights reserved.
94
E. J. Flach, H. Reid, I. Pow, A. Klemt
to be resistant to clinical disease (Reid et al 1975, Mushi et al 1981b, Heuschele et al 1984). Examples are antelope of the genus Oryx and members of the Caprinae (sheep, goats and relatives). North American species of the latter sub-family also have high prevalences of seropositives against OvHV-2 using a competitive-inhibition enzyme-linked immunosorbent assay (C-I ELISA) (Li et al 1996). The isolation of a gamma herpesvirus from a scimitar-horned oryx was reported by Heuschele et al (1984), but no details were given. Malignant catarrhal fever has occurred sporadically at Whipsnade Wild Animal Park. Cases were diagnosed in P ere David’s deer (Elaphurus davidianus) in the 1950s (Tong et al 1961), and in sitatunga (Tragelaphus spekei) and red deer (Cervus elaphus) in the late 1970s (Ashton 1982). A serological study in the early 1980s (Ashton 1982) indicated a high prevalence of sero-positive scimitar-horned oryx (Oryx dammah) and sitatunga, some animals having high antibody titres. All of the sitatunga with significant titres were clinically affected with MCF. A limited study was carried out between May and August 1998 to investigate the occurrence of gamma herpesvirus DNA in Oryx species and other artiodactyls kept at Whipsnade (Klemt 1998). Viral DNA was detected on several occasions, prompting a continuation and extension of the study from October 1998 to December 2000, with the following aims: (a) to investigate the carrier status of scimitar-horned oryx and gemsbok (O. gazella), and (b) to determine whether other artiodactyl species carry gamma herpesviruses.
MATERIAL AND METHODS Animals and test samples Whipsnade Wild Animal Park maintained 37 species of the Order Artiodactyla (including domestic pigs, cattle, sheep and goats) during the years of the study (1998–2000). They were kept either as single-species in grass paddocks with associated concrete yards and housings, or as mixed-species in a large, grass parkland which visitors drove through in cars. The latter exhibit held P ere David’s deer mixed with herds of Bactrian camels (Camelus bactrianus) and yak (Bos grunniens) in one enclosure, and swamp deer (Cervus duvauceli), Chinese water deer (Hydropotes inermis), fallow deer (Dama dama), axis deer (Axis axis), hog deer (Axis porcinus), Formosan sika deer (Cervus nippon), nilgai (Boselaphus tragocamelus) and blackbuck (Antilope cervicapra) in another. Venous blood samples were collected when animals were handled for veterinary procedures. These procedures included clinical investigations, health examinations and elective procedures, such as hoof trimming. For two species, the scimitar-horned oryx and gemsbok, health checks were performed on all calves born in 1998; when less than 24 h old (at the time of eartagging), at 4–5 months of age (when tested for, and treated against, gastrointestinal parasites; scimitar-
horned oryx only), and at one year old (for assessment of body condition and state of hooves, and removal of males from the breeding herd). Serum was separated from clotted blood by centrifugation, and then removed and stored frozen at )20 °C. Blood in ethylenediamine tetra-acetic acid (EDTA) was sent on the day of collection by first-class post to the Moredun Research Institute (MRI) where the peripheral blood leukocytes (PBL) were removed and stored frozen until PCR testing. The EDTA plasma was also stored frozen. A full post mortem examination was performed on all animals which died during the study period. Blood, if available, and samples of cornea, buccal mucosa, peripheral lymph node and kidney, placed in 25 ml phosphate buffered saline (PBS) with added antibiotics (30 mg benzyl penicillin sodium [Britannian Pharmaceuticals] and 12.5 mg gentamycin [Bimeda] per sample), were sent on the day of collection to the MRI. Tests Sera and EDTA plasma were tested for the presence of antibodies against AlHV-1 using an indirect fluorescent antibody test (IFAT). Briefly, the test serum (or plasma) was diluted 1/10 in PBS, and 80 ll was then added to an acetone-fixed monolayer of AlHV-1 infected bovine turbinate cells on a glass coverslip. After washing off excess, bound antibodies were detected with a 1/40 dilution in PBS of donkey anti-ovine gamma globulin conjugated to the fluorescent dye fluorescine isothiocyanate. The coverslip was examined under ultra-violet light for cellular fluorescence, and compared to positive and negative controls. The samples from the newborn oryx were also tested in a virus neutralisation assay. Test sera (or plasma) were diluted in a 10-fold dilution series, and then 100 ll was incubated for 2 h at 37 °C with 100 ll of a fixed concentration of AlHV-1. One hundred microlitre of this mixture were then added to wells of a 96-well, flatbottomed, microtitre plate containing monolayers of bovine turbinate cells. Plates were incubated for 7 days at 37 °C. The wells were examined microscopically to assess the degree of cytopathic effect. The endpoint of the assay was the dilution of serum which protected 50% of wells, and if equal to, or greater than, 1/10 the sample was considered to be positive. DNA extracted from PBL or from tissues collected post mortem, were tested for the presence of gamma herpesvirus sequences, using polymerase chain reactions (PCRs). Briefly, PBL were incubated with an extraction buffer comprising sodium chloride, hydrochloric acid–Tris (pH 8.0), EDTA and sodium dodecyl sulphate (SDS), and then extracted into phenol and chloroform (6:4). DNA was precipitated by adding ammonium acetate in ethanol, washed in ethanol and then stored frozen in water until tested. Tissue samples were snap frozen in liquid nitrogen and ground up before extraction, using the same technique, but with a longer incubation in an extraction buffer with added proteinase K in Tris. The PCR for OvHV-2 used the
Gamma herpesvirus carrier status of captive artiodactyls
TABLE 1:
PCR primers described in the text
POL1 50 -GGC (CT) CA (CT) AA (CT) CT ATG CTA CTC CAC-30 POL2 50 -ATT (AG) TC CAC AAA CTG TTT TGT-30 AlHV-POL 50 -CCA AAA TGA AGA CCA TCT TA-30 OvHV-POL 50 -AAA AAC TCA GGG CCA TTC TG-30
same conditions and primers as Baxter et al (1993). The product size from the first amplification using primers 755/556 was 422 basepairs (bp), and from the second amplification with primers 555/556 was 238 bp, but only the results of the second amplification are recorded (OvHV PCR). In addition, PCR amplifications were carried out using the non-specific herpesvirus primers POL1 and POL2 (Table 1; first amplification product size 386 bp ¼ POL-1/2 PCR), and following this with a final amplification with either AlHV-POL/POL2 (product size 172 bp ¼ POL-A PCR) or OvHV-POL/ POL2 (product size 172 bp ¼ POL-O PCR) (Table 1; POL primers from MWG-Biotech GmbH, Ebersberg, Germany). For the POL1/POL2 reactions there was a total volume of 50 ll containing 10 mM Tris–HCl, 1.5 mM MgCl2 , 50 mM KCl, 200 lM dATP, dCTP, dGTP and dTTP, 1:0 lM of each primer and 2 units of Taq DNA polymerase and 2 lg DNA. This mixture was placed in a thermal cycler for a pre-cycle incubation at 95 °C for 5 min, then dNTP and enzyme mix were added followed by 24 cycles of 1 min each at three temperatures, 94, 45 and 72 °C, and a final cycle of 94 °C for 1 min, 45 °C for 1 min and 72 °C for 5 min. For the second amplification, 2 ll of the primary amplification product was transferred to a new amplification mixture and the process repeated. The final product was loaded onto an agarose gel and subjected to electrophoresis. The gel was then stained with ethidium bromide and photographed under UV light. Any DNA bands took up the stain and were visualised. Positive virus controls were added to each PCR and a molecular weight marker was included on the gel. Tissues from a one-day-old scimitar-horned oryx calf which died from aspiration pneumonia were sent to the MRI and trypsin-dispersed kidney cell monolayer cultures were prepared. These were subsequently passaged using standard techniques. Pooled PBL from the eight scimitar-horned oryx calves at 4–5 months of age were added to a monolayer of these kidney cells and cultured routinely. Further details of virus isolation techniques are described with the results below.
RESULTS Serological and PCR testing One hundred and three individuals of 19 species of artiodactyls were sampled during the study period (Table 2). All five P ere David’s deer were tested post mortem, having shown clinical signs (profound depression and anorexia) and gross lesions (widespread congestion and
95
petechiation, lymphadenopathy and haemorrhagic gastroenteritis) consistent with MCF in deer. The five died, or were euthanased, between February and May 2000. Histopathological findings in four of them included lymphoid infiltration and vasculitis in multiple organs (the other animal was not examined). They all had OvHV-2 DNA in blood or tissues (OvHV PCR) and tested positively in one or more of the POL PCR series. The three which were tested serologically had antibodies cross-reacting with AlHV-1. One animal also had a Pasteurella multocida pneumonia, two had mild parasitic gastritis and a fourth was found to have Clostridium perfringens epsilon toxin in small intestinal contents, and a Campylobacter species in faeces. Three adult swamp deer died between January and June 2000, and were found to have haemorrhagic gastroenteritis post mortem. Histopathological findings from one (widespread vascular lymphoid infiltration) was consistent with MCF, and this was one of two animals which tested positive for OvHV-2 DNA (histopathology was not performed on the other). The third animal had no histopathological evidence of MCF and, further, an Aeromonas species was cultured from the lung, liver and kidney, and a Campylobacter species from the faeces, but although negative for OvHV-2 it did have gamma herpesvirus DNA (POL-O PCR). A neonatal fawn was negative for DNA, but one out of three adults tested during a routine immobilisation was positive in the POL-A PCR. The single hog deer died of pneumonia, but also had a haemorrhagic gastroenteritis, from which a Campylobacter species was isolated. It reacted in POL-O PCR and was sero-positive, whereas the alpaca (Lama pacos) and four giraffe (Giraffa camelopardalis) were negative for both DNA and antibodies. Among the members of the sub-family Bovinae both a female anoa (Bubalus depressicornis) and her newborn calf had DNA reacting in the POL-A PCR, and the mother also reacted in the POL-O PCR. One adult and one newborn yak (Bos grunniens) had gamma herpesvirus DNA, both reacting in the POL-O PCR. The two congo buffalo (Synceros caffer) were negative in all tests, but the American bison (Bison bison) reacted in POL-A PCR. Both tragelaphid species, sitatunga and greater kudu (Tragelaphus strepsiceros) had a high prevalence of gamma herpesvirus DNA detected in the POL series, but absence of antibodies cross-reacting to AlHV-1. Most sitatunga calves (80%) and all five kudu were positive. One of the sitatunga calves, a 12-day-old female, also reacted in the specific OvHV PCR. The animal has not shown any clinical signs up to the time of writing. Similarly, there were adult and juvenile (newborn) waterbuck (Kobus ellipsiprymnus) and Nile lechwe (K. megaceros) which had gamma herpesvirus DNA, but antibodies to AlHV-1 were only detected in one calf of each species. Four out of six lechwe reacted in the POL PCR series, and one also had specific OHV2 DNA (OvHV PCR). This individual is still alive and showing no clinical signs. Roan antelope, scimitarhorned oryx and gemsbok had high prevalences of gamma herpesvirus DNA reacting in the POL series,
96
TABLE 2:
E. J. Flach, H. Reid, I. Pow, A. Klemt
Gamma herpesvirus results from species of Artiodactyla at Whipsnade
Species
IFAT
No. tested
No. positive
Calves ð<1 yrÞ Adults (1 yr +)
Camelidae Alpaca (Lama pacos) Cervidae Hog deer (Axis porcinus) Swamp deer (Cervus duvauceli) re David’s deer Pe (Elaphurus davidianus) Giraffidae Giraffe (Giraffa camelopardalis) Bovidae—Bovinae Lowland anoa (Bubalus depressicornis) Yak (Bos grunniens) Congo buffalo (Synceros caffer) American bison (Bison bison) Sitatunga (Tragelaphus spekei) Greater kudu (T. strepsiceros) Bovidae—Hippotraginae Waterbuck (Kobus ellipsiprymnus) Nile lechwe (K. megaceros) Roan antelope (Hippotragus equinus) SH oryxc (Oryx dammah) Gemsbokc (O. gazella) Bovidae—Caprinae Musk ox (Ovibos moschatus) Domestic goat (Capra hircus) Mouflon (Ovis musimon)
1
0/1
0/1
0/1
1
1/1
0/1
0/1
0/1
1/1
1/1
4
ND 1/4 1/1b ð1Þ 2/2b ð2Þ
0/1 2/6b ð2Þ 1/1b ð1Þ 4/4b ð4Þ
0/1 0/6 1/1b ð1Þ 2/4b ð2Þ
0/1 1/6 0/1 1/4b ð1Þ
0/1 2/6bð1Þ 1/1bð1Þ 3/4bð3Þ
0/1 3/6b ð1Þ 1/1b ð1Þ 4/4b ð4Þ
3
ND 0/3
0/1 0/3
0/1 0/3
0/1 0/3
0/1 0/3
0/1 0/3
3 2
0/1 0/1 0/4 0/1 0/1
0/1 0/1 0/4 0/3 0/2
0/1 0/1 0/4 0/3 0/2
1/1 1/1 1/4 0/3 0/2
0/1 1/1 1/4 1/3 0/2
1/1 1/1 1/4 1/3 0/2
1
0/1
0/1
0/1
1/1
0/1
1/1
0/5 0/13 0/3 0/2
1/5 0/13 0/3 0/2
1/5 4/12 2/3 2/2
2/5 4/12 3/3 1/2
3/5 2/12 2/3 2/2
4/5 5/12 3/3 2/2
8
1/3 0/2 1/2 0/5 1/2 1/5 1/8 4/6 4/4 7/8
0/4 0/2 0/2 1/6 0/2 0/2 0/8 0/6 0/3 0/8
0/4 0/2 1/2 4/6 1/2 1/5 8/8 3/6 1/3 3/6
1/4 0/2 1/2 2/6 2/2 1/5 0/8 3/6 0/3 2/6
1/4 1/2 0/2 2/6 1/2 1/5 6/8 3/6 1/3 5/6
1/4 1/2 1/2 4/6 2/2 2/5 8/8 6/6 2/3 5/6
1
1/1
0/1
0/1
1/1
1/1
1/1
1
0/1
0/1
0/1
0/1
0/1
0/1
1
0/1
0/1
0/1
1/1
0/1
1/1
1a 1 a
a
5 (1 ) 13
4 2
a
POL series
0/1
1 (foetus)
a
POL-O
0/1
1
3
POL-A
0/1
a
a
POL-1/2
1
6
4
PCR OvHV
2 2 6 2a 5 8d 6 4a
Notes: ND not done. Newborn calves (<72 h). b Died of malignant catarrhal fever (number of animals dying in parentheses). c Selected results; complete results below. d Calves 4–5 months of age. a
and many had antibodies to AlHV-1. The adult roan had a lower prevalence of DNA (40%) and antibodies (20%) than the scimitar-horned oryx (100% and 67%) and gemsbok (83% and 88%). The effect of age in the latter two species is reported below. Only single individuals of three species of the subfamily Caprinae were tested. None had specific OHV-2 DNA (OvHV PCR), but the musk ox (Ovibos moschatus) had gamma herpesvirus DNA reacting in both POL-A and POL-O PCRs and antibodies to AlHV-1, and the mouflon had DNA reacting in the POL-A PCR, but no detectable antibodies. Influence of age Eight of the 11 newborn scimitar-horned oryx calves had detectable gamma herpesvirus DNA, six reacted in
the POL PCR, two in POL-A and six in POL-O (Table 3). The eight sera tested were positive in the IFAT, and four in the VNT. When the eight surviving calves were re-sampled at between four and five months of age, all eight reacted in the POL PCR, none in POL-A and six in POL-O. However, only one animal was still positive in the IFAT to AlHV-1. One calf died three months later and was still POL-O PCR positive, but IFAT negative. The remaining seven were re-examined at one year of age (12–15 months), but the blood sample from one was unsuitable for PCR testing. Five of the six tested were positive to one of the POL series, and the highest number, four, reacted to POL-A. Four out of the seven were strongly positive in the IFAT, and a further two showed a weak or doubtful reaction. All of the adult scimitar-horned oryx adults tested positive in one of the POL PCRs, with an equal number (three)
Gamma herpesvirus carrier status of captive artiodactyls
TABLE 3:
97
Effect of age on gamma herpesvirus carrier status of scimitar-horned oryx Number positive/number tested
Age
IFAT
< 1 Day 4–5 Months 1 Year Adult 5–17 years
8/8 1/8 4/7a 4/6
VNT
4/8 NT NT NT
PCR OvHV
POL-1/2
POL-A
POL-O
POL series
NT 0/8 0/7 0/6
6/11 8/8 3/6 3/6
2/8 0/8 4/6 3/6
6/8 6/8 2/6 3/6
8/11 8/8 5/6 6/6
Notes. NT: not tested. a 2/7 doubtful reaction, only 1/7 negative.
reacting in each test, but only four out of six (67%) were seropositive. Two newborn gemsbok calves were positive for gamma herpesvirus DNA, one reacted in POL-1/2 PCR and one in POL-O PCR (Table 4). All four serum samples from newborn calves were positive in the IFAT and two out of three tested were also positive in the VNT. The three surviving gemsbok calves were retested at 13 months of age. All three reacted in the POL-A PCR, and one also reacted in POL-O. However, only one animal had detectable antibodies to AlHV-1 in the IFAT. Five out of six adults (83%) had detectable gamma herpesvirus DNA, the majority being positive in the POL-O PCR, and seven out of eight (88%) were seropositive in the IFAT.
lymphoproliferative lesions, characteristic of malignant catarrhal fever, were observed grossly and histologically.
DISCUSSION Our results support the theory that scimitar-horned oryx and gemsbok carry their own gamma herpesviruses. None of the animals tested had OvHV-2 DNA, but there was a high prevalence of gamma herpesvirus DNA detected by the POL series. The lack of consistency in the POL-A and POL-O PCRs suggests that their viruses are related to, but distinct from, AlHV-1 and OvHV-2. Cross-reactivity between herpesviruses is common in serological tests, particularly the IFAT, and so the presence, in many of the individual’s sera, of antibodies to AlHV-1 does not contradict this theory. The virus isolated from the scimitar-horned oryx, however, did react in the specific OvHV-2 PCR, but since OvHV-2 has never been isolated in culture it is highly unlikely that they are one and the same. Research is continuing to compare it directly to AlHV-1, OvHV-2 and the roan antelope virus, HipHV-1 and, if it proved to be a new species, the designation is likely to be hippotragine herpesvirus-2. An analysis of viral DNA may help to unravel the genetic make-up of the gamma herpesviruses and, possibly, the differences in genetic structure may help explain differences in infectivity and pathogenesis. Oryx have never been implicated in transmitting MCF to other species, but the experimental infection in rabbits show that the scimitarhorned oryx virus has the capacity to do so. Finally, it should be possible in the future to produce more specific PCR and serological tests for oryx and other bovids. Gamma herpesvirus DNA was detected in most of the newborn scimitar-horned oryx calves (possibly all, be-
Virus isolation Cytopathogenic effects characteristic of a gamma herpesvirus were observed in the monolayer cultures of the neonatal scimitar-horned oryx kidney when inoculated with the pooled PBL from the eight scimitarhorned oryx calves aged 4–5 months. Following two subpassages 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 cocultivated with further scimitarhorned oryx kidney cells and similar cytopathic effects developed. Cocultivation with cultured bovine turbinate cells was initially unsuccessful, but after addition of 5 mg/ml triphenylalanine (TPA) to the medium, extensive cytopathic effects were seen, although the virus remained cell-associated. Extracts of DNA from these cells were positive by the OvHV, POL-1/2 and POL-O PCRs. 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 TABLE 4:
Effect of age on gamma herpesvirus carrier status of gemsbok Number positive/number tested
Age
<1 Day 1 Year Adult 2–9 years Notes. NT: not tested.
IFAT
4/4 1/3 7/8
VNT
2/3 NT NT
PCR OvHV
POL-1/2
POL-A
POL-O
POL series
0/3 0/3 0/8
1/3 0/3 3/6
0/3 3/3 2/6
1/3 1/3 5/6
2/3 3/3 5/6
98
E. J. Flach, H. Reid, I. Pow, A. Klemt
cause POL-A and POL-O PCRs were not performed on three calves) and two out of three gemsbok, strongly suggesting that they were infected in utero. The third gemsbok was positive when re-tested at one year of age, but one of the scimitar-horned oryx failed to react at this age, despite having done so at four to five months. All newborn oryx had antibodies to AlHV-1 in the IFAT. These were probably maternal because most of the scimitar-horned oryx calves had lost them by four to five months of age. Thereafter, active antibody production appeared to be slow, despite the presence of viral DNA, because even at one year old only four of seven scimitarhorned oryx and one of three gemsbok had seroconverted. By adulthood, however, all except one individual (a gemsbok) were sero-positive. There was a lower proportion of newborn calves with neutralising antibodies against AlHV-1, probably indicating a lower degree of cross-reaction than in the IFAT, but this proportion was higher amongst the gemsbok (2/3) than the scimitar-horned oryx (4/8). Mushi and Karstad (1981b) found a similar age-related pattern in fringe-eared oryx calves (Oryx gazella beisa ¼ O. beisa callotis). Neutralising antibody titre declined from birth to six months of age, and then increased after eight months of age. The results from the waterbuck and Nile lechwe were also consistent with a sub-clinical carrier state. There were neonates and adults with both gamma herpesvirus DNA and antibodies, and one adult Nile lechwe reacted specifically to OvHV-2 in the PCR series. The roan antelope were expected to be carriers of their own virus, HipHV-1, but despite both newborn calves being positive, only two out of five adults had detectable gamma herpesvirus DNA. This may indicate that not all individuals become infected, or carry infection to adulthood, or alternatively that viruses are present, but not always detectable. Not all of the scimitar-horned oryx and gemsbok calves were positive on each occasion they were tested, so there may indeed be variable detectability of viral DNA. Among the other bovids, gamma herpesvirus DNA was detected in the single American bison and both adult and newborn yak, anoa, sitatunga and greater kudu, but all were sero-negative. Sero-positives have been detected by the IFAT in these species before, so either the anti-ovine immunoglobulin conjugate was not binding to their antibodies or, more probably, they were truly sero-negative. Bison, sitatunga and greater kudu have all been affected by MCF in other collections, and an outbreak occurred in the sitatunga herd at Whipsnade in the late 1970s and early 1980s (Ashton 1982). Interestingly, one of the sitatunga calves tested positive in the specific OvHV-2 PCR (PCR/1) suggesting that it was carrying OvHV-2 itself. These findings could be taken to show that some individual sitatunga infected by OvHV-2 recover and continue to carry and even transmit the virus to calves in utero without apparent antibody response. O’Toole et al (1997) showed that recovery and carriage of OvHV-2 does occur in cattle that recover from MCF, but that they continued to show chronic lesions of the eyes and blood vessels, and most (eight out of 11) were seropositive in the C-I ELISA. Alternatively, sitatunga
may carry their own virus which is closely related to OvHV-2, but under certain circumstances succumb to the full disease, especially if caused by AlHV-1, and, indeed, the earlier cases did occur when they were mixed with brindled wildebeest (C. Tack, personnal communication, 2001). Testing stored histopathological blocks could prove whether or not this was the case. Further support for this latter theory of incomplete resistance to other gamma herpesviruses lies in the fact that roan antelope, which may carry HipHV-1, can be susceptible to MCF caused by OvHV-2 (Gulland et al 1989). In contrast, none of the giraffe, the alpaca or the deer, except for one individual, had any evidence of sub-clinical carriage of gamma herpesvirus. In addition, sheep-associated MCF, alone or in combination with other diseases, was diagnosed in five Pere David’s deer and two swamp deer; underlying the susceptibility of these species compared to other species in the mixed exhibits, including sika deer, axis deer, Chinese water deer, nilgai and blackbuck, all of which have contracted MCF in other collections. There had been no contact with sheep or goats, or with any of the suspected carrier species, and the sheep and goats at Whipsnade were not breeding at the time so transmission was suspected to have occurred via aerosol or fomites from nearby farms on which lambing was taking place. The musk ox and mouflon were found to have gamma herpesvirus DNA, although they were not positive in the OvHV-2 specific PCR. They have been implicated as OvHV-2 carriers (Li et al 1996, 1999), but it is possible that they carry related, but not identical viruses. Despite the limitations of opportunistic sampling, our results suggest that there may be a large number of gamma herpesviruses infecting artiodactyl hosts without clinical disease in the natural host, and with no evidence of spread to other, susceptible species. It is hoped that sequence data from the amplified fragments will indicate the number and relatedness of these. One explanation for lack of spread would be if these viruses infect all foetuses in utero and have lost the ability to survive outside the host as free virus. Alternatively, they may spread widely, but have very low inherent pathogenicity compared to AlHV-1 and OvHV-2. However, this appears unlikely because the scimitarhorned oryx virus was pathogenic to a rabbit.
ACKNOWLEDGMENTS We are extremely grateful to Jo Dodds for her unstinting help with collecting and packaging samples, the keepers of the Africa, Asia and Europe regions, Whipsnade Wild Animal Park for their assistance with handling and sampling animals, veterinary colleagues in the Institute of Zoology who dealt with some of the clinical cases, and Shaheed Macgregor for parasitological and bacteriological examinations. Histopathological examinations of most of the deer were performed by Cosme Sanchez-Miguel for his M.Sc. project in Veterinary Pathology at the Royal Veterinary College, all other post mortem tissues were ex-
Gamma herpesvirus carrier status of captive artiodactyls
amined by Idexx Laboratory, Wetherby. Testing for clostridial enterotoxins was carried out by the Veterinary Laboratory Agency, Bury St. Edmunds.
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Accepted 21 May 2002