First detection of two novel malignant catarrhal fever-related gammaherpesviruses associated with sudden deaths of dorcas gazelles (Gazella Dorcas osiris)
Magdalena Larska, Wojciech Socha, Paweł Kulik, Wojciech Paszta, Aleksandra Jarosz, Tomasz Grenda, Anna Kycko
TL;DR
Two new viruses were found in dorcas gazelles that died suddenly, possibly causing a deadly disease similar to malignant catarrhal fever.
Contribution
First detection of two novel gammaherpesviruses associated with fatal disease in dorcas gazelles.
Findings
Two new gammaherpesviruses were identified in dorcas gazelles linked to sudden deaths.
The viruses showed similarity to known gammaherpesviruses in other ruminants.
Histopathological changes in organs of gazelles suggest MCF-like disease.
Abstract
Malignant catarrhal fever (MCF) is a sporadic infectious disease of domesticated and non-domesticated ruminants including: cattle, sheep, bison, cervids, antelope and buffaloes, and is caused by gammaherpesviruses. Between March and May 2023, several cases of sudden death were observed in endangered species of dorcas gazelle (Gazella dorcas osiris) in zoological garden in southeastern Poland. Given the rapid course of disease, MCF was suspected. During the epizootic investigation, other ruminants that could be a source of infection and routes of spread were examined, also taking into account the possibility of air-born and via rodents transmission. In total, tissue, blood, nasal swab and fecal samples were collected from 52 animals kept in the zoo between March 2023 and August 2025. Samples came from 10 ruminants species: dorcas gazelle (Gazella dorcas), blackbuck (Antilope cervicapra),…
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
Click any figure to enlarge with its caption.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5- —National Science Centre of Ministry of Education and Science of Poland
Peer Reviews
No public reviews on file for this paper yet. If you reviewed it on a platform where reviews are public (OpenReview, ICLR, NeurIPS, ICML), you can paste yours below so the community can read it here.
Videos
No videos yet. Explain this paper in a talk, walkthrough, or lecture? Add one.
Taxonomy
TopicsHerpesvirus Infections and Treatments · Poxvirus research and outbreaks · Vector-Borne Animal Diseases
Background
Malignant catarrhal fever (MCF) is a frequently fatal infectious disease caused by gammaherpesvirus infection, affecting various species of wild and farmed members of the Artiodactyla order such as cattle (Bos taurus), sheep (Ovis aries), American (Bos bison) and European bison (Bison bonasus), water buffalo (Bubalus bubalis) or pigs (Sus domesticus) and various deer, antelopes and gazelles [1–3]. Historically, two major causative infectious agents associated with this syndrome were distinguished: ovine herpesvirus 2 (OvHV-2) and alcelaphine herpesvirus 1 (AlHV-1). The viruses, belong to Macavirus genus of the highly diverse Gammaherpesvirinae subfamily. The reservoir and asymptomatic carriers of the two viruses are: domestic sheep for OvHV-2 in most continents and blue wildebeest (Connochaetes taurinus) and white-tailed gnu (Connochaetes gnou) antelopes for AlHV-1 in Africa [4]. Depending on the reservoir species and causative viral agent, disease is described as either sheep - associated MCF (SA-MCF) or wildebeest - associated MCF (WA-MCF). In the recent years, however, MCF-like cases have been associated with five additional related gammaherpesviruses: caprine gammaherpesvirus-2 (CpGHV-2), MCFV-white-tailed deer/caprine gammaherpesvirus 3 (MCF-WTD/CpGHV-3), Ibex-MCFV, alcelaphine herpesvirus 2 (AlHV-2) and alcelaphine herpesvirus 3 (AlHV-3) [5–7]. After the exposure to MCFVs from latently but subclinically infected reservoir species, the susceptible species of Bovidae, Cervidae and Giraffidae may develop clinical signs of diverse outcomes ranging from peracute to chronic lymphoproliferative disease. In the peracute MCF, susceptible animals develop minimal clinical signs such as transient diarrhoea 12–24 h before their sudden death. Infection could also be accompanied with fever, inappetence, ocular and nasal discharge, erosions and ulcerations of the buccal cavity and muzzle, lymphoadenomegaly, diarrhoea and depression [8–10]. In some cases, neurological signs such as: limb weakness, incoordination, a demented appearance, muscle tremor, nystagmus, head-pressing, paralysis and convulsions; as well as cutaneous abnormalities (hyperkeratosis, alopecia, dermatitis etc.) may be observed [11, 12]. Acute forms of infections are associated with a very high (over 90%) mortality rate in susceptible species [1, 13]. Chronic form of disease could occur in animals that survived initial phase of infection with lesions in various organs such as: proliferative concentric fibrointimal plaques, disrupted inner elastic lamina, focally atrophic tunica media, and vasculitis of variable severity in cattle [14]. However observed clinical signs and course of infection may vary between species [9].
Transmission of virus usually occurs by direct contact or aerosol shed from the asymptomatically infected reservoir species to susceptible one but transmission on longer distances have also been reported (1 km and more) both for OvHV-2 and AlHV-1. Transmission on longer distance is most probably facilitated by wind but involvement of birds as mechanical vectors or transfer through common fomites was also speculated [15, 16]. Additionally transplacental transmission has been described for OvHV-2 in cattle [17]. While study and control of transmission between reservoir and susceptible species is relatively easy in case of farm animals, it becomes more challenging when wildlife is involved. In Europe, SA-MCF is sporadically observed in cattle, while in Africa, outbreaks of WA-MCF in domestic indigenous African bovids occur quite frequently due to the great migration (nearly 3000 km) of blue wildebeest (Connochaetes taurinus***)*** herds [8]. During migrations, the herds come in contact with wide variety of farmed and wild artiodactyl species, however, data on the possibility of interspecies gammaherpesvirus transmission and impact on other free ranging animal species inhabiting the sub-Saharan plains of Africa is scarce. In contrast, testing animals from zoological gardens that house multiple species from distant geographical regions allow for observation of occurring interspecies pathogen transmission, even those that has not been yet observed in nature. One glaring example is the case of MCFs in eastern bongo (Tragelaphus eurycerus) associated with the West Caucasian tur macavirus infection [18]. The knowledge acquired under ex situ breeding conditions can contribute to better conservation of endangered species but also provide data on the pathogenesis, therapy and prevention of infections in controlled environments [1]. The occurrence of SA-MCF, WB-MCF and other forms of MCF have been described in zoological gardens around the world with fatal cases involving species such as banteng (Bos javanicus), Himalayan tahr (Hemitragus jemlahicus), Nile lechwe (Kobus megaceros), sika deer (Cervus nippon), European bison (Bison bonasus), bongo antelope (Tragelaphus eurycerus), white-tailed deer (Odocoileus virginianus) and pudu (Pudu puda) [3, 4, 19–23]. Although, gammaherpesvirus infections are cited as one of the more common threats to zoological collections, access to MCF diagnostics in Europe is limited due to the lack of universal antibody diagnostic tests reliable in various animal species, therefore, it is very likely that many infections may remain undiagnosed [24].
Our research was launched following cases of sudden death of several dorcas gazelles (Gazella dorcas osiris, Blaine, 1913) at Zamość Zoological Garden in Poland. The limited size of dorcas population (approx. 40,000 as estimated in 1999 and declining), which survive dry climates of northern Africa makes them vulnerable species included in the Red List of Threatened Species of the International Union for Conservation of Nature [25, 26]. Zamość Zoo participates in the conservation and reintroduction programme of the G. dorcas osiris subspecies supervised by the Barcelona Zoo in collaboration with the Spanish National Research Council [27]. Aims of our study were to identify causes of observed fatal cases with emphasis of possible involvement of gammaherpesvirus infections. The findings of this study expand knowledge of gammaherpesvirus variation in Bovidae and interactions between reservoir and susceptible species. They also indicate the need to revalidate and update the animal health procedures in zoos and rehabilitation centres, especially those involved in ex situ breeding of endangered species.
Materials and methods
Sample collection
In total, 72 ruminants of 15 species were present in the Zamość Zoological Garden at the beginning of 2023. From this group, samples were collected, from 52 ruminants of 10 species: nine dorcas gazelles, eight blackbuck/Indian antelopes (Antilope cervicapra), two lechwe (Kobus leche), two Defassa waterbucks (Kobus ellipsiprymnus defassa), one Reeves’s muntjac (Muntiacus reevesi), 14 Kirk’s dik-dik antelopes (Madoqua kirkii), one scimitar oryx (Oryx dammah), five Barbary sheep/aoudad (Ammotragus lervia), two mhorr gazelles (Nanger dama) and six domestic goats (Capra hircus) between March 2023 and August 2025. The study began after a female dorcas gazelle was found to have suddenly collapsed at the zoological garden in Zamość in South-Eastern Poland in March 2023 (Fig. 1A). Shortly afterwards, animal keepers reported the presence of bloody faeces on the floor of the dorcas enclosure and watery diarrhoea in blackbucks and waterbucks (Fig. 1B). Parasitological and microbiological tests of faecal samples in a commercial laboratory ruled out the presence of internal parasites, including Giardia spp. and Cryptosporidium spp., as well as bacteria such as pathogenic E. coli spp., Shigella spp., and Salmonella spp. Over the following two months, five further dorcas gazelles died mostly without any apparent clinical signs, in quite good condition, including four adults (Fig. 1A) and one newborn (Fig. 1C). No displacements or other stressful conditions that could have caused the deaths were identified. Due to the suspicion of MCF, domestic goats as the reservoir species were sampled first and then the subsequent species were examined (Table 2). When possible, dead animals were necropsied and sampled on site or in case of newborns were send to National Veterinary Research Institute (NVRI) for post mortem examination and sampling. In total, tissue samples were collected from 3 dorcas gazelle, 5 Kirk’s dik-dik, 4 blackbucks, 3 Barbary sheep and mhorr. The rest of the ruminants, which were apparently healthy were chemically immobilized and sampled (blood, nasal swabs and faecal samples) as a part of epidemiological investigation. The sedation was performed using a combination of xylazine (0.2–0.25 mg/kg) and ketamine (2 mg/kg) in blackbucks and oryx, medetomidine (0.1–0.2 mg/kg) and ketamine (2–3 mg/kg) in kobs, lechwe, mhorr and Barbary sheep, and medetomidine 0.2 mg/kg) and ketamine (2–3 mg/kg) and butorfanol (0.2 mg/kg) in muntjac [28–30]. The anaesthetics were administered using air pressure dart rifle (Fig. 2). In some animals like dik-diks and Barbary sheep, only nasal swabs were taken by animal keepers to minimise their stress. Whole blood, nasal swab samples, tissue fragments of lungs, spleen, liver, kidney and/or intestines were collected from ruminants during post-mortem examination. In parallel to MCF-testing, all animals were confirmed to be serologically negative to bovine viral diarrhea virus (BVDV), bovine herpesvirus 1 (BoHV-1), bluetongue virus (BTV), epizootic haemorrhagic virus (EHDV) and Schmallenberg virus (SBV) using ELISA tests as previously described (Table S1) [31, 32]. Tissue samples (spleen, liver and intestines) were also collected from 3 rats (Rattus norvegicus) caught at the zoo as potential MCFV vectors [33]. Additionally, environmental swabs were collected from surfaces and equipment (drinkers and feeders) at the enclosures housing ruminants including: dorcas, Barbary sheep, dik-diks, domestic goats, blackbucks, Defassa waterbuck, mhorr/giraffe **(**Giraffa camelopardalis rothschildi), Javan rusa (Rusa timorensis)/lechwe and Thorold’s deer (Cervus albirostris) (Figure S1). For collection of nasal and environmental samples, dry or Virocult (MWE, Wiltshire, England) swabs with liquid transport medium were used. The animal feed used for all even-toed ungulates examined at a time when gazelle mortality was observed was also sampled and tested for presence of C. perfringens contamination.
Fig. 1. Adult (A) and newborn (1-2-days-old) (C) dorcas gazelles found dead at the zoo in Zamość and bloody diarrhoeal feces (B) observed at the dorcas gazelle enclosure
Fig. 2. Chemical immobilization of scimitar oryx using Telinject air-rifle Vario 13 mm loaded with 5 mL lightweight syringe dart containing aesthetic for sampling for epizootic investigation
Gammaherpesvirus molecular detection
Prior to DNA extraction, the samples were prepared as follows: the whole blood was diluted in PBS without calcium and magnesium (MP Biomedicals) in proportion 1:3; „dry” nasal and environmental swabs were soaked in 1 mL Minimum Essential Medium Eagle (MEM) (Sigma-Aldrich, Irvine, UK) for minimum of 6 h in RT and mixed. In case of tissue and fecal samples, 10% homogenates were prepared in MEM with 1.4 mm ceramic beads (Lysing Matrix D, MP Biomedicals, Irvine, CA, USA) using a TissueLyser LT (Qiagen). DNA was extracted from 200 µL of samples using a IndiMag Pathogen Kit w/o plastics (INDICAL Bioscience, Leipzig, Germany) according to the manufacturer’s guidelines. The genetic material was eluted in 90 µL of elution buffer and stored at -65 °C or directly used for nested PCR using primers flanking a fragments of viral DNA polymerase (DPOL) gene designed by Van Devanter et al., 1996 [34] described in Table 1. The first reaction was run in 28 µL of reaction mix that comprised 10 µL of water, 13 µL of ALLin™ HS Red Taq Mastermix 2x (HighQu, Kraichtal, Germany), 1 µL of each MCFV - specific forward (DFA, ILK) and reverse primers (KG1) (20 µM) and 2 µL of DNA sample. After 5 min of incubation at 94 °C, 45 cycles of amplification were run, each consisting of 30 s of denaturation at 94 °C, 60 s of annealing at 46 °C, and 60 s of elongation at 72 °C. An incubation of 72 °C for 7 min ended reaction. The second reaction was run in 28 µL of reaction mix that comprised 10 µL of water, 13 µL of ALLinTM HS Red Taq Mastermix 2x (HighQu, Kraichtal, Germany), 1 µL of each MCFV - specific forward (TGV) and reverse primer (IYG) (20 µM) and 3 µL of PCR product (from the first reaction). After 5 min of incubation at 94 °C, 45 cycles of amplification were run each consisting of 30 s of denaturation at 94 °C, 60 s of annealing at 46 °C, and 60 s of elongation at 72 °C. An incubation of 72 °C for 7 min ended reaction. 6 µl of each PCR product was visualised on 3% agarose in 1× Tris-acetate-EDTA (TAE). Electrophoretic separation was carried out for 1 h at 55 V. The molecular weight of the obtained products was assessed using a GeneRuler Low Range DNA Ladder (Thermo Fisher Scientific, Waltham, MA, USA). Positive samples were visible at approximately 200 bp.
Table 1. Primers used for panherpesvirus nested PCR reactionTarget genePrimerSequence (5’-3’)Amplicon size (bp)ReferenceHerpesvirusDNA polymerase (DPOL)DFAGAYTTYGCNAGYYTNTAYCC474–736 [34]ILKTCCTGGACAAGCAGCARNYSGCNMTNAAKG1GTCTTGCTCACCAGNTCNACNCCYTTTGVTGTAACTCGGTGTAYGGNTTYACNGGNGT215–315IYGCAC AGA GTC CGT RTC NCC RTA DAT
Gammaherpesvirus positive samples were purified and sequenced using Sanger method with previously described primers TGV and IYG (Table 1) by external company Genomed S.A. (Warsaw, Poland). Partial sequences of polymerase gene were aligned, using MEGA software (Version 11.0.13), with sequences of previously identified gammaherpesviruses of artiodactyls, available in GenBank. Alignment was used to construct a neighbour-joining phylogenetic tree [35].
Virus isolation in cell culture
An attempt was made to isolate live virus from MCFV PCR positive samples. Blood, tissue homogenate, and nasal swabs were tested using two cell lines: rabbit kidney cells 13 (RK-13, ATCC Cat# CCL-37, RRID: CVCL_3155, Lot: 3993687 sex: unspecified) and Madin-Darby bovine kidney (MDBK(NBL-1), ATCC Cat# CCL-22, RRID: CVCL_0421, Lot: 3752721, sex: male). Both cell lines are tested on regular bases for Mycoplasma spp. infection freedom by specific PCR [36]. Briefly, cell monolayers were overlaid with tested material and left to adsorb for an hour, then mounted with MEM with antibiotics and incubated at 37 °C in 5% CO2 with humidity. The cell monolayers were monitored microscopically for the appearance of cytopathic effects (CPEs) for up to 5–7 days. If no CPE was observed, subsequent passage in the cell culture was performed. Positive CPE or negative cell culture results for virus isolation were confirmed after each cell culture passage by aforementioned PCR.
Clostridium perfringens detection
For the differential diagnostic, each sample with a mass/volume of 1 g or 1 mL (depending on the state of aggregation) was inoculated into Wrzosek’s broth media, in two replicates. One replicate was heated at 70 °C for 15 min. Subsequently, the prepared inocula were subjected to the incubation at 37 °C for 48 h under anaerobic conditions using anaerobic atmosphere generating sachets (AnaeroGen, Thermo Fisher Scientific, Waltham, MA, USA). After the incubation period, bacterial growth was observed on the liquid medium by characteristic turbidity, grey sediment at the bottom of the test tubes, as well as gas in the Durham tubes placed in the test tubes. In the next step, 10 µL of liquid cultures were taken from the tubes and spread on Willis-Hobbs differentiation agar (10 g/L peptic digest of animal tissue, 10 g/L meat extract, 5 g/L sodium chloride, 12 g/L lactose, 0.032 g/L neutral red, 10 g/L skim milk powder, 2 g egg yolk powder, and 10 g/L agar, with a final pH of 7.0 ± 0.2 at 25 °C). The prepared plates were incubated under anaerobic conditions at 37 °C for 48 h. The obtained colonies were evaluated for their shape, surface area, size, lecithinolytic features. Lemon-yellow colonies with regular edges and a distinct zone of lecithinolysis, approximately 2–3 mm in size, were considered as suspected of belonging to the species C. perfringens. Genomic DNA for confirmation of the presence and typing of Clostridium perfringens species was isolated from 1 mL of liquid cultures, and several colonies were obtained from agar plates. The DNA was extracted with a Genomic Mini AX Bacteria kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s instructions. The amount of DNA used in the PCR reaction varied between 1 and 25 ng. The DNA amount was estimated using a Nicolet Evolution 300 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The extracted DNA was frozen at − 20 °C or directly subjected to multiplex PCR analysis. To confirm the presence and to determine the toxic type of suspected strains of belonging to the C. perfringens species, mutliplex PCR was run according to procedure described by Rood et al. (2018) [37]. This method, enabled the simultaneous detection of genes determining the production of the main toxins, i.e. plc or cpa (α-toxin); cpb (β-toxin); etx (ε-toxin); iap and ibp (ι-toxin); cpe (CPE); netB (NetB). The assignment to the appropriate toxotypes was made according to the ability of individual strains to produce specific main toxins, according to the scheme presented by Rood et al. (2018) [37]. Agarose gels were prepared at a concentration of 2% in 1× Tris-acetate-EDTA (TAE) and visualized with SimplySafe nuclease dye (EURx, Gdansk, Poland). Electrophoretic separation was carried out for 1.5 h at 100 V. The molecular weight of the obtained products was assessed using a GeneRulerTM 100 bp DNA Ladder Plus (Thermo Fisher Scientific, Waltham, MA, USA).
Histopathological analysis
Tissues submitted for histopathology included lungs, liver, kidney and intestines from animals with the signs that may have been associated with MCF. All of them originated from adult animals including: two gammaherpesvirus positive dorcas gazelles and one dorcas gazelle being additionally positive for Clostridium perfringens type D. It was not possible to obtain fresh samples for histopathological examination from all animals in time, as some of the deaths occurred at night or over the weekend and were unsuitable for obtaining reliable slides due to tissue decomposition. The tissues were collected into 10% neutral-buffered formalin during necropsy. In histopathological unit at NVRI, the tissues were cut into fragments not thicker than 5 mm, placed in histological cassettes and submitted to standard tissue processing by transferring through series of alcohols of increasing concentrations, xylene and paraffin (tissue processor Leica TP 1020, Leica Biosystems, USA) then embedded in paraffin blocks. The paraffin-embedded tissues were cut on a microtome (HM 350 S, Mikrom, Germany) into 4 μm thick slides which were stained using a routine hematoxylin-eosin (HE) staining method. Finally, the stained slides were examined and photographed using a light microscope (Axioskop 2, Zeiss, Germany) equipped with a colour camera (Axiocam 208 colour, Zeiss, Germany). Upon initial microscopic examination, tissues from the dorcas coinfected with gammaherpesvirus and Clostridium perfingens were excluded from further analysis due to extensive autolysis obscuring the tissues’ architecture.
Results
In total 14 of 48 (29.2%) tested ruminants were found to be positive by herpesvirus specific PCR (Table 2). Genetic material of the herpesviruses was detected in blood, nasal swabs and tissue samples. Gammaherpesvirus infection was confirmed in all five tested adult dorcas, while no virus was present in the blood of a newborn. Additionally, C. perfringens type D was detected in the tissues of the last dorcas that died in June 2023. Subsequent PCR testing of remaining two healthy dorcas showed no gammaherpesvirus infection when tested two years later (Table 2). Other infected animals included blackbucks and dik-diks, whereas no virus was detected in the samples collected from the other ruminants (Table 3). The virus was detected in the blood or spleen of a newborn and adult blackbuck, while it was absent in the nasal swabs collected from the same animals. The situation was different with dik-diks, in which gammaherpesvirus DNA was detected in the nasal swabs, and due to the lack of available samples, we were unable to confirm its presence in their blood (Table 2). No virus was found in any of the diarrhoeic faecal samples, however, the samples from dorcas and waterbuck enclosures contained Clostridium perfringens type D and type A, respectively (Table 2).
Table 2. List of animal species from zoo Zamość, Poland sampled and tested in the course of the study in chronological orderNoSpeciesSexAgeOrigin^b^Samplingdate (d/m/y)Clinical signsPanherpesvirus PCR^a^C. perfringens PCR/typeB^c^Lu^c^S^c^Li^c^I^c^Ns^c^F^c^1dorcasF3 monthslocal14.03.2023sudden death+n/t^d^n/tn/tn/t+n/t+/D2dorcasM6 yearsintroduced20.03.2023sudden death++++n/tn/tn/t-3dorcasF6 yearslocal02.05.2023sudden death+n/tn/tn/t++n/t-4dorcasF4 yearslocal11.05.2023sudden death+++n/tn/tn/tn/t-5dorcasF1 yearlocal11.06.2023sudden death+n/tn/tn/tn/t-n/t+/D6dorcasU^e^newbornU02.05.2023newborn death-n/tn/tn/tn/tn/tn/t-7dorcas^f^UUU18.03.2023bloody diarrhoean/tn/tn/tn/tn/tn/t-+/D8dorcasM5 yearsintroduced15.08.2025--n/tn/tn/tn/tn/tn/t9dorcasM6 monthslocal15.08.2025--n/tn/tn/tn/tn/tn/t10dik-dikF7 yearsintroduced13.06.2024pneumonia+n/t+n/tn/tn/t-11dik-dikF7 yearsintroduced15.04.2024-n/tn/tn/tn/tn/t+n/tn/t12dik-dikF2 yearslocal15.04.2024-n/tn/tn/tn/tn/t+n/tn/t13dik-dikM1 yearlocal15.04.2024-n/tn/tn/tn/tn/t+n/tn/t14dik-dikM2 yearslocal15.04.2024-n/tn/tn/tn/tn/t+n/tn/t15dik-dikF1 yearlocal15.04.2024-n/tn/tn/tn/tn/t+n/tn/t16dik-dikM11 monthslocal15.04.2024-n/tn/tn/tn/tn/t+n/tn/t17dik-dikFnewbornlocal13.04.2024newborn deathn/tn/t-n/tn/tn/tn/t-18dik-dikF5 yearsintroduced15.04.2024-n/tn/tn/tn/tn/t-n/tn/t19dik-dikM1 yearlocal15.04.2024-n/tn/tn/tn/tn/t-n/tn/t20dik-dikM1 yearlocal18.04.2024-n/tn/t-n/tn/tn/tn/tn/t21dik-dikM11 monthslocal01.10.2024-n/tn/t-n/tn/tn/tn/t22dik-dikMnewbornlocal13.05.2024-n/tn/tn/tn/tn/t-n/t23dik-dikUFetuslocal25.10.2024--n/t-n/tn/tn/tn/t24blackbuck^f^UUU18.03.2023diarrhoean/tn/tn/tn/tn/tn/t--25blackbuckFnewbornlocal04.04.2023newborn deathn/t-+n/tn/t-n/t-26blackbuckMnewbornlocal21.05.2023newborn deathn/tn/tn/t-n/tn/tn/t-27blackbuckMnewbornlocal21.05.2023newborn deathn/tn/tn/t-n/tn/tn/t-28blackbuckF2 yearslocal04.03.2024-+n/tn/tn/tn/t-n/t-29blackbuckFNewbornlocal14.11.2024newborn death-n/t-n/tn/tn/tn/tn/t30blackbuckMNewbornlocal14.11.2024newborn death-n/t-n/tn/tn/tn/tn/t31blackbuckF3 monthslocal08.05.2024--n/tn/tn/tn/t-n/t-32blackbuckF1 yearlocal04.03.2024--n/tn/tn/tn/t-n/t-33blackbuckFnewbornlocal24.08.2024--n/t-n/tn/t-n/tn/t34waterbuck^f^UUU18.03.2023diarrhoean/tn/tn/tn/tn/tn/t-+/A35waterbuckF14 yearsintroduced12.06.2024--n/tn/tn/tn/t-n/t-36Barbary sheepMnewbornlocal07.04.2024newborn deathn/tn/t-n/tn/t-n/t-37Barbary sheepMnewbornlocal15.04.2024diarrhoean/tn/t-n/tn/tn/tn/t-38Barbary sheepMNewbornlocal14.11.2024newborn death-n/t-n/tn/tn/tn/tn/t39Barbary sheepFUU15.04.2024-n/tn/tn/tn/tn/t-n/tn/t40Barbary sheepFnewbornlocal19.04.2024--n/tn/tn/tn/t-n/t-41goatF5 yearslocal20.03.2023--n/tn/tn/tn/t-n/t-42goatM3 yearslocal20.03.2023--n/tn/tn/tn/tn/tn/t-43goatF6 yearslocal20.03.2023--n/tn/tn/tn/t-n/t-44goatU7 yearsintroduced20.03.2023--n/tn/tn/tn/t-n/t-45goatF6 yearslocal20.03.2023--n/tn/tn/tn/t-n/t-46goatM2 yearslocal20.03.2023--n/tn/tn/tn/t-n/t-47ratUUlocal03.09.2024-n/t----n/tn/tn/t48ratUUlocal03.09.2024-n/t----n/tn/tn/t49ratUUlocal03.09.2024-n/t----n/tn/tn/t50MhorrF1 yearintroduced13.05.2024--n/tn/tn/tn/t-n/t-51MhorrM2 monthslocal15.07.2024--n/t-n/tn/t-n/t-52LechweFnewbornlocal12.06.2024--n/tn/tn/tn/t-n/t-53LechweMnewbornlocal21.06.2024--n/tn/tn/tn/t-n/t-54muntjac deerF11 monthslocal14.03.2024--n/tn/tn/tn/t-n/t-55scimitar oryxM12 yearsintroduced12.06.2024--n/tn/tn/tn/t-n/t-^a^panherpesvirus PCR result defined as: (+) gammaherpesvirus positive, (-) gammaherpesvirus negative^b^local – born in Zamość Zoo, introduced – transferred from other zoological garden^c^type of tissue: B-blood, Lu-lung, S-spleen, Li- liver, I – intestines, Ns-nasal swabs, F – faeces^d^not tested^e^unknown^f^samples collected from the ground (individual unidentified). The animals tested by histopathology are marked with a star
Sequencing of the DPOL partial sequences enabled identification of four distinct genotypes. Two of those genotypes showed high, over 90%, degree of homology with previously described Bovidae gammaherpesvirus 2 – detected in two blackbucks and Rusa unicolor equina gammaherpesvirus 1 – in six Kirk’s dik-diks (Table 3) [5]. Additionally, infections with previously unknown gammaherpesviruses were identified in all five fallen adult dorcas gazelles and one Kirk’s dik-dik. They represented two distinct genotypes sharing less than 60% nucleotide identity in DNA polymerase fragment, with each other. First of them, was detected in 4 dorcas and showed over 80% identity of nucleotide sequence with Bovidae gammaherpesvirus. Second one was present in 3 animals (two dorcas gazelles and a dik-dik), showed 78% homology to Rusa unicolor equina gammaherpesvirus 1. Interestingly, both viruses were simultaneously detected in one individual dorcas at the same time (Table 3).
Table 3. Genetic characterization of the gammaherpesvirus strains by species and their similarity to homologous reference strainshost species(latin name)n/N^a^%^b^Closest matchVirus nameGenBank IDnt_query_/nt_ref_^e, f^Identity %^e, f^dorcas gazelle(Gazella dorcas)2/9^g^22.2MN599444^c^100/12878.1Gazella dorcas GHV-2^h^4/9^g^44.4PP346678^d^140–141/16982.2–84.4Gazella dorcas GHV-1^h^blackbuck (Antilope cervicapra) 2/1020.0PP346678173–174/17499.4–100Bovidae GHV-2Barbary sheep (Ammotragus lervia) 0/50.0--Kirk’s dik-dik gazelle (Madoqua kirki) 6/1442.9MN599444170–172/17597.1–98.3Rusa unicolor equina GHV-11/147.1MN599444100/12878.1Gazella dorcas GHV-2mhorr gazelle(Nanger dama)0/20.0--lechwe(kobus leche)0/20.0--Defassa waterbuck(Kobus ellipsiprumnus defassa)0/20.0--goat(Capra hircus)0/60.0--Reeves’s muntjac(Muntiacus reevesi)0/10.0--Scimitar oryx(Oryx dammah)0/10.0--Total14/5226.9^a^number of positives/total number of tested^b^percentage of positives^c^Rusa unicolor equina gammaherpesvirus 1 isolate DPOL-36^d^Bovidae gammaherpesvirus 2 isolate BB-2^e^nucleotides matching reference sequence/total length of reference (query by NCBI BLAST❩^f^refers to available fragment of virus DNA polymerase (DPOL)^g^in total 5/7 dorcas gazelles were infected, including one individual (nr 14 in Table 2) coinfected with two viruses^h^provisional names given in this study
None of the viruses showed growth in RK-13 and MDBK cell cultures. Gammaherpesvirus positive animals were found in three separate enclosures located in different parts of the Zamość Zoological Garden. At the same time, all environmental samples and samples from rats collected during pest control in the animal enclosures were negative to gammaherpesviruses (Figure S1).
Additionally, the presence of Clostridium perfringens was detected in 3 out of 33 (9.1%) animals tested alongside to the gammaherpesvirus. Two of them infected with C. perfringens type D were adult dorcas which suddenly died and were simultaneously infected with Gazella dorcas gammaherpesvirus 1 (Table 2). The same bacteria were also isolated from the bloody stool collected at the dorcas enclosure, while C. perfringens type A in the diarrhoeic faeces of waterbuck. C. perfringens type D was also detected in the scrapings from the feeder surface in the dik-dik’s in-house enclosure, however, was not present in the feed samples. The bacteria were not found in any other environmental samples.
Neighbour-joining phylogenetic tree constructed based on the sequences acquired in the study and other available in the GenBank confirmed that all viruses isolated from animals in Zamość Zoological Garden grouped into Macavirus genus of gammaherpesvirinae subfamily (Fig. 3).
Fig. 3. Neighbour-joining phylogenetic tree of DNA polymerase gene of gammaherpesviruses. Strains isolated in this study are marked with shapes depending on host: ● – blackbuck, ■ – dorcas gazelle, ♦ - Kirk’s dik-dik. Caprinae, Alcelaphinae/Hippotraginae and MCFs groups are defined according to Bianchessi et al. 2024 [5]. All sequences acquired in this study are available in GenBank under accession numbers PV963081-PV963095
The post-mortem examination revealed no macroscopic changes in adult dorcas, probably due to the hyperacute process and sudden death. Based on the autopsy of newborns, no changes indicating viral infection were found or rather indicating food deprivation. Histopathological analysis revealed moderate to severe congestion of the blood vessels multifocally in the liver, kidney and lungs in the two dorcas gazelles (one infected with Gazella dorcas GHV-1 and one coinfected with GHV-2). Due to their decomposition, the histopathological examination of the tissues collected from the dorcas coinfected with Clostridium perfringens was unfortunately unsuccessful. All the intestine tissues showed mostly extensive autolysis and faded structure, with the exception of one, C. perfringens negative, in which blood vessel congestion and edema were visible in preserved tissue segments. The changes in the lungs included hyperaemia and microhemorrhages in the interstitial areas and in the interalveolar spaces. In one case (animal no. 4), mild vasculitis and perivascular lymphocytic infiltrations were additionally present (Fig. 4A). The kidneys displayed multifocal congestion of the blood vessels accompanied by blood extravasations and occasionally interstitial lymphoplasmacytic infiltrations and multifocal degeneration of ductal epithelium (Fig. 4B, animal no. 3). In the liver, there was hyperaemia dilating portal and central veins and in one case zonal haemorrhages in several hepatic lobules. Occasionally, there were mild lymphoplasmacytic periportal infiltrates present and mild vasculitis visible (Fig. 4C-D, animal no. 3).
Fig. 4. Histopathological changes observed in tissue samples from fallen gammaherpesvirus-positive adult female dorcas gazelles (No. 3 and 4, Table 2). Lung, (animal no. 4): congested blood vessels in alveolar septa. Mild vasculitis characterised by infiltration of the vascular wall by single lymphocytes (insert) (A); Kidney, (animal no. 3): congestion of blood vessels (black arrows) and multifocal degeneration of ductal epithelium visible as sloughed epithelial cells (white arrows)(B); Liver, (animal no. 3): extensive hyperaemia and mild periportal lymphocytic infiltration (insert)(C); Liver, (animal no. 3): vasculitis characterised by portal vein wall thickened by fibrinous exudate (black arrows) (D). HE, objective magnification: (A,** B)**: 20x, (C): 10x, (D): 40x
Discussion
Malignant catarrhal fever is considered one of the re-emerging yet neglected threats which impact on animal health depends on the geo-epidemiological context. The pattern for the occurrence of the disease is quite complex and involves an interplay between gammaherpesvirus diversity, reservoir competence, presence of susceptible species and diagnostic capacity [38]. Until now, in Poland, the only observation on MCF concerned subclinical OvHV-2 infections in two sheep herds from Wielkopolska region sampled two decades ago, however, confirming the presence of the infections in farmed animals [39]. In contrast, although many species known to be potential carriers of viruses involved in MCF are kept in zoological gardens in Poland, this population have not been studied yet. In our study, for the first time, we have described cases of peracute illness resulting in sudden death of five adult dorcas gazelles in zoological garden in Poland; associated with the presence of gammaherpesvirus infection. The retrospective inventory of animal mortality has revealed four other deaths of adult dorcas between May 2022 and January 2023 at the zoo. In 2024, only a pair of adult dorcas remained alive from a herd consisting of 11 dorcas, thus the overall mortality was almost 82%. In the study, we have shown that 83.3% (5/6) of these animals’ deaths were related to the presences of gammaherpesviruses that might cause MCF. Infections with MCF related gammaherpesviruses has been described previously in multiple species of artiodactyls in zoological gardens around the world [5, 18, 20]. For example in 2011 in Rome, introduction of OvHV-2 infected sheep led to infection and death of 11 animals including Banteng, Sika deer, Himalayan tahr and Nile lechwe [20]. Recently, infection with a novel gammaherpesvirus related to AlHV-1 was described in captive roan antelopes (Hippotragus equinus) in German zoo [6]. Significant loss observed in our study is comparable to the impact that MCF has on survival of the most susceptible species such as bantengs (Bos javanicus), Bison species and some deer [15, 16, 19, 20, 23]. Although, no gross changes were observed at the necropsy, histopathological lesions such as lymphocytic infiltration and vasculitis considered characteristic for MCFV infection were confirmed microscopically in two gammaherpesvirus infected dorcas, in which no co-infection with Clostridium was confirmed [3, 6, 23]. Furthermore, no signs of diseases were observed in two last dorcas that remained at the same enclosure of the zoological garden after an outbreak, which were recently confirmed to be uninfected with gammaherpesviruses. The absence of obvious symptoms and macroscopic changes can be explained by the rapid course of the infection and the likely high species sensitivity. Similar observations were made in bison and deer where the disease can be preacute and rapid with either no clinical signs, or depression followed by diarrhoea and dysentery 12–24 h prior to death [40]. Whereas in cattle, the disease progresses far enough for more severe symptoms are developed, with the emblematic head and eye form. The virus causes inflammation and damage to blood vessels (vasculitis) accumulation of activated, dysregulated cytotoxic lymphocytes in various tissues leading to multiorgan failure [41]. It seems that the more resistant species usually have a protracted infection and more pronounced lesions, while in more susceptible species the course of the disease is shorter and the clinical symptoms less emphasised but with fast and high mortality [40].
To exclude other pathogenic factors, animals were also tested for other infectious agents like BTV, EHDV, BVDV, BoHV-1 and Schmallenberg virus. First and foremost, we wanted to rule out anaerobic bacterial infections as Clostridium spp. are considered one of the most significant threats to animals in zoos, which, in addition to typical symptoms of enterocolitis [42, 43], occur as a peracute form with sudden mortality. Moreover, identified hereby in two dorcas Clostridium perfringens toxinotype D has been described to be responsible for fatalities in other gazelle species (chinkara) before [44]. It was also observed that reactivation of certain gammaherpesviruses might be correlated with shedding of C. perfringens [45]. In elephants, co-infections with C. perfringens type C and betaherpesvirus lead to fatal disease, while the macroscopic lesions were indistinguishable in the case of single infections with one of these pathogens. Moreover, frequent co-occurrence gave cumulative morbid outcome [46]. Therefore, it is possible that the observed coinfection with C. perfringens type D and Gazella dorcas gammaherpesviruses 1 and 2 triggered fatality in two cases of dorcas in our study. The type D C. perfingens has been described as responsible for enterotoxemia in sheep, goats, cervids and camelids [47, 48]. As the bacteria was also detected on the surface of one of the feeders, the infection could be associated to the ineffective biosecurity measures at the zoo, which were verified and subsequently improved by disinfection, removal of bedding and replacement of wooden feeders with plastic ones. Nevertheless, the other gammaherpesvirus infected dorcas did not become infected with clostridia, and the outcome was similarly fatal. We could link this to histopathological changes in their organs, but we were unable to verify what lesions occurred in the co-infected with C. perfringens individuals. Other non-infectious causes like trauma or stress-caused mortality were excluded due to lack of evidence at the necropsy, i.e. mechanical damage or myopathy. We have also rejected poisoning, since the affected and unaffected ruminants were fed with same feeder, while the deliberate action was very unlikely since the access of unauthorised outsiders is impossible.
However, we should also take into account that gazelles kept in zoos may be more susceptible to infection owing to their inbred and chronic stress associated with captivity or overcrowding [49, 50]. The latter seems to confirm the observed neonatal mortality in the newborn Barbary sheep, blackbucks, dik-diks and dorcas (Fig. 1C), which were most probably connected to the husbandry problems in captivity such as maternal neglect, trauma, pneumonia and stress [50]. None of the newborns tested positive for the gammaherpesvirus, which confirms observations made in sheep and wildebeest, which become infected several months after the birth [40]. In the recent study, we have also demonstrated that infection and subsequent transmission of the porcine lymphotropic herpesvirus in pigs occurs in the first few weeks after the birth [51]. Once infected, the reservoir species becomes latently infected trough the life without developing clinical symptoms.
Infections with gammaherpesviruses were previously observed in other Gazella species. However, our study was the first one that describes gammaherpesvirus fatal infection in dorcas [18, 52]. The cases were associated with two different virus types, both previously unknown but with high homology to Bovidae gammaherpesvirus 2 (provisional named Gazella dorcas gammaherpesvirus 1) and Rusa unicolor equina gammaherpesvirus 1 (Gazella dorcas gammaherpesvirus 2), respectively. In one individual dorcas, both viruses were detected, first in blood sample and the second one in nasal swab and intestine samples. Coinfections with different gammaherpesvirus has already been described in zoological gardens so they were not unexpected [18]. However in this case, coinfection with two potentially pathogenic gammaherpesviruses in the susceptible species with signs of MCF makes it a unique finding [18, 53]. Perhaps more cases of such co-infections could have been identified, but the methodology based on pan-herpes PCR followed by sequencing does not always allow for the simultaneous detection of individual herpesvirus strains [5, 54]. The inability to propagate most gammaherpesviruses in cell culture also hinders diagnosis. The only MCF virus that can be propagated remains AlHV-1 [40]. Further testing also revealed the presence of Gazella dorcas gammaherpesvirus 2 in one dik-dik. In that case no clinical signs were observed which suggests dik-dik as potential reservoir and the possible source of infection. Although, Kirk’s dik-diks were kept separately from dorcas gazelles, their enclosures were located nearby (~ 50 m) (Figure S1). In addition, all ruminants at the zoo are manned by the same staff. Moreover, interestingly enough, two years earlier, dorcas brought from the zoo in Barcelona were placed next to an dik-dik’s enclosure, with which they also shared a pen. At that time, the first deaths of dorcas were observed, however, no epizootic investigation was undertaken at the time. We can suspect that at least one of the newly identified gammaherpesviruses - Gazella dorcas gammaherpesvirus 2, may have been carried by the imported dik-diks already when those animals were introduced to Zamość Zoological Garden. Those infections remained unnoticed until susceptible dorcas were introduced later. The potential source of Gazella dorcas GHV-1 is less clear as no asymptomatic carrier was identified. The virus showed relation to Bovidae GHV-2 that was found in two blackbucks in the enclosure separate from both dik-diks and dorcas. While we cannot exclude some degree of viral adaptation to new host species, the observed differences of 28–29 nucleotide substitutions in DNA polymerase fragment (82.2–84.2% identity) seems too significant to attribute it to strain to strain variation which previously was shown to not exceed two substitutions for the analysed gene fragment in other gammaherpesviruses such as EHV-2 and EHV-5 [55].
While it was speculated that MCF viruses are transmitted mainly by direct contact or shared pastures, long distance transmission is not unusual and has been described previously [15]. In our study, we have not confirmed the presence of gammaherpesviruses in the environmental samples. Additionally, although previous studies showed that rodents are susceptible to MCFV, making them potential vectors, all tested rats were negative for gammaherpesviruses in our study [33]. However, since all ungulates at the zoo were handled by the same personnel, therefore, except for air-borne transmission, gammaherpesviruses might have spread via the clothing or vehicles [9, 56]. We are currently verifying the hypothesis of virus transmission by insects [57]. Tightening of biosecurity measures at the zoo probably prevented the death of remaining dorcas, which, despite sharing the same enclosure, were shown to be uninfected with gammaherpesvirus.
In the environment, the risk of MCF is connected to the number of reservoir species including the emergence of invasive or alien species, i.e. mouflon (Ovis aries musimon), sika deer (Cervus nippon) and fallow deer (Dama dama); the intensification of animal and human movements which may influence the number of susceptible species and other factors; like climate change, which affects, for instance, animal susceptibility to infection due to heat stress, changing grazing conditions or wildlife movements [38, 58–61]. Historically, first reports of occurrence MCF come from Africa. Already in prehistoric times in the eastern part of the continent, herdsmen linked the occurrence of MCF in cattle to the perinatal period in wildebeest coinciding with the start of the rainy season [9]. However it is unlikely if the same relation could exist between dik-diks and Dorcas in natural conditions. In the environment, both infected species populate different regions with dorcas present mostly in Northern Africa and middle-east, and Kirk’s dik-diks inhabiting eastern and south-western Africa (Fig. 5). In addition to this, blackbucks, which have also been suspected source of infection, are found on another continent. Therefore, its direct contact seems to be unlikely in the natural habitat [62, 63]. However, there are other, closely related species of dik-diks: Guenther and Salt dik-diks (Madoqua guentheri and Madoqua saltiana, respectively), populating northern parts of Ethiopia, Somalia and Erythrea – regions that overlap with the south-eastern edge of the dorcas gazelle range [63–65].
Fig. 5. The geographical ranges of three ruminant species: Dorcas gazelle (red), Kirk’s dik-dik (green) and blackbuck (blue) involved in the interspecies transmission of MCFV at the zoo drawn up on the basis of data from the IUCN [66]
While antibodies to MCF related viruses were detected in dorcas gazelle before, our study is the first describing actual cases of the illness in this species [64, 67]. It is probable, that previously described cases were associated with different MCF viruses, to which this species possess natural resistance, while conditions in zoological garden allowed for the contact with gammaherpesvirus that could causes symptomatic infection. Earlier studies described all the MCF-associated viruses to be closely related forming two separate clads within gammaherpesvirinae subfamily: Caprinae group and Alcelaphinae/Hippotraginae group [5]. However, in case of Gazelle dorcas GHV-1 and Gazelle dorcas GHV-2, it was not proven as the DNA polymerase gene showed less than 60% nucleotide sequence homology in comparison to any gammaherpesvirus identified previously as associated with MCF cases. The risks of gammaherpesvirus transmission should also be taken into account when selecting areas for the reintroduction into wild and welfare protocols for captive dorcas [68, 69].
Since subclinical gammaherpesvirus infections were detected also in blackbucks (Bovidae gammaherpesvirus 2) and Kirk’s dik-diks (Rusa unicolor equina gammaherpesvirus 1 related virus), suggests again their carrier state. It is consistent with earlier reports of Bovidae gammaherpesvirus 2 infection in captive blackbucks in zoological garden in UK in 2016 and in San Diego zoo in 2006–2008. Similarly to our study, the infections were not associated with any apparent signs of disease in both cases [5, 18]. Furthermore, the sequences of gammaherpesviruses derived from dik-diks showed very high reciprocal homology (98.8%-100%) and high similarity to previously described Rusa unicolor equina gammaherpesvirus 1 (97.1–98.3%), Madaqua kirkii gammaherpesvirus 1 (95–97,5%), Bovidae gammaherespvirus 5 (98.6–100%) and Bovidae gammaherpevirus 9 (98.6–100%) detected in various ruminants species [5, 18]. It is likely, that all of this sequences in fact represented various strains of this same gammaherpesvirus, however to confirm this, sequencing of larger fragments of viral genomes would be required. Nevertheless, none of the above mentioned gammaherpesviruses is known to be associated with MCF.
Conclusions
Due to few data on MCFV transmission between sylvatic and synanthropic environments, studies in wildlife centres, enclosures and zoos can be considered as models. Our study for the first time confirmed the presences of gammaherpesviruses associated with MCF in undomesticated animals in Polish zoological garden. Additionally, two novel MCF viruses were identified associated with fatal disease in dorcas gazelles, the species not previously known to be susceptible to MCF. Finally, our results suggested Kirk’s dik-dik as a potential reservoir species of Gazella dorcas gammaherpesvirus 2. While the association between dorcas mortality and the infection with a second strain referred as Gazella dorcas gammaherpesvirus 1 should be verified by extended molecular analysis of the viral genome. It should also be noted that the pathogenic potential of gammaherpesviruses in gazelles may be exacerbated by environmental contamination by other factors such as Clostridium perfringens.
Supplementary Information
Supplementary Material 1.
Supplementary Material 2.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
- 1Zhu H, Huang Q, Hu X, Chu W, Zhang J, Jiang L, et al. Caprine herpesvirus 2-associated malignant catarrhal fever of captive Sika deer (Cervus nippon) in an intensive management system. BMC Vet Res. 2018;14. 10.1186/s 12917-018-1365-8.10.1186/s 12917-018-1365-8PMC 579658929391011 · doi ↗ · pubmed ↗
- 2Flach E. Part 2: Husbandary Guidelines. 12. Handling. In: The Biology, Husbandry and Conservation of the Scimitar-horned Oryx (Oryx dammah). 2nd edition. Winchester, Hampshire, United Kingdom: Marwell preservation trust; 2004. pp. 35–7.
- 3World Organisation for Animal Health. WOAH Terrestrial Manual. 2018, Malignant catarrhal fever. https://www.woah.org/fileadmin/Home/fr/Health_standards/tahm/3.04.13_MCF.pdf. Accessed 7 Nov 2025.
- 4Hagos F. Ecological distribution, abundance and conservation status of the three indigenous species of gazelles: Nanger soemmerringii, Gazella dorcas and Eudorcas tilonura in Eritrea. Ph D thesis. Universita di Pavia; 2023.
- 5The IUCN Red List of threatened species. https://www.iucnredlist.org/. Accessed 22 Sep 2025.