Institute for Medical Microbiology, University of Munich, Veterinaerstr. 13, 80539 Munich, Germany1
Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, California, USA2
Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa3
Author for correspondence: Uwe Truyen. Fax +49 89 2180 2155.e-mail uwe.truyen{at}micro.vetmed.uni-muenchen.de
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Abstract |
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Introduction |
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Methods |
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The bat-eared fox (Otocyon megalotis) sample was collected in Namibia. The animal was found as an orphaned kit, and was then raised and brought to a veterinary clinic for a routine health check at the age of 4 months. Some days later the animal developed fever (41 °C), bloody diarrhoea and vomiting. A faecal sample was taken for further analysis.
During a behavioural study of honey badgers (Mellivora capensis) in the Kalahari Gemsbok National Park, South Africa, faecal samples of two free-ranging animals showing clinical signs of diarrhoea were collected.
Two 18-month-old cheetahs (Acinonyx jubatus), kept on a private farm in Namibia, developed fever, anorexia and diarrhoea a few days after being hospitalized in a private veterinary clinic. Faecal samples from both animals were analysed.
Five cases from zoological gardens in the United States were submitted for viral PCR because histopathological lesions indicative of parvoviral enteritis were detected during the routine pathology surveillance that is part of the American Zoo and Aquarium Associations Cheetah Species Survival Plan. Three cheetahs had subacute-to-chronic crypt-necrotizing enteritis. Ante mortem enteric clinical signs included chronic diarrhoea over the preceding year in one cheetah with mild lesions and loose stools for 3 months before euthanasia in a second cheetah. The two other cheetahs had no clinical signs referable to the intestines, although one of these cheetahs had severe necrotizing enteritis histologically. All five cheetahs had chronic vomiting, but three cheetahs from which stomach was available for histopathology had gastritis and all had some degree of renal failure. None of the cheetahs had leukopenia in the weeks preceding death.
Formalin-fixed tissues were also obtained from three cheetahs, from the De Wildt animal rescue centre, South Africa, which died after showing clinical signs of enteritis.
Two free-ranging African wild cats (Felis lybica) were brought to the Animal Rescue Centre (ARC) in Pretoria, South Africa, and developed severe enteritis and died soon after arrival. Tissue samples from the small intestine were taken, formalin-fixed and examined.
In the Schwerin zoological garden, Germany, anorexia and diarrhoea were observed in several large cats in 1998. A faecal sample from one male Siberian tiger (Panthera tigris altaica) was available for viral examination.
Faecal samples from two free-ranging wild dogs (Lycaon pictus) from Lapallala wilderness game reserve, South Africa, were also examined.
Preparation of DNA.
Either about 1 g of faeces or tissue material or two 10 µm slides of paraffin-embedded small intestines were used for viral DNA preparation. Paraffin-embedded samples were first deparaffinized with xylene and washed with ethanol (98·9%) as described by Truyen et al. (1994). To the faecal or tissue material 2 ml of binding buffer [3 M guanide thiocyanate, 10 mM Tris, 5% (v/v) 99·8% ethanol, pH 6·6] was added; the mixture was then incubated for 24 h at room temperature and centrifuged at 15000 r.p.m. for 10 min. Finally, viral DNA in the supernatant was purified using the Boehringer Mannheim High Pure PCR Product Purification Kit.
PCR.
Viral DNA was amplified by PCR with Taq polymerase (3·75 U per reaction), MgCl2 concentration 1·5 mM, nucleotide concentration 0·1 mM, and various primer pairs (M1 & M2, M1 & M41, M10 & M11 and #19 & M5; primer concentration 100 pmol/µl). Together the sequences amplified over approximately 85% of the virus gene encoding the VP2 coat protein. Important nucleotide positions determining antigenicity and host range are located in regions of this capsid protein (Fig. 1). Primer pairs M13 & M14 and #34 & #63 were used for nested PCR only if no amplification was obtained with any of the primer pairs. The primer sequences and annealing temperature (Tm) are listed in Table 1
; the programme details are summarized in Table 2
. PCR products were sequenced by automated sequencing (MWG Biotech) using Taq polymerase either directly or after being cloned into the vector pCR 2.1 (Invitrogen). Finally, the sequences were aligned to the sequence of a CPV-2 strain (CPV-d; Parrish et al., 1991
b) for the analysis of subtype-specific and evolutionarily important nucleotide differences. Nucleotides 3025, 3045, 3065, 3094, 3685, 3699, 3753, 4062, 4449, 4477 and 4489 define the various antigenic types of CPV and FPV (Parrish et al., 1991
; Truyen et al., 1995
a, 1998b
) (Fig. 1
).
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Results |
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From one honey badger sample a 201 bp DNA sequence (primers M1 & M2) was amplified. The nucleotides at positions 3025, 3065 and 3094 were characteristic for an FPV-type virus. The other sample was negative both in FASTest Parvo and PCR.
In the formalin-fixed intestinal tissue of one African wild cat, parvovirus DNA sequences were obtained using primer pairs M1 & M2, M10 & M11 and #19 & M5. The DNA sequences showed features typical for FPV viruses. The sample from the second animal was negative.
From the single faecal sample of a Namibian cheetah amplicons of 482 and 372 bp were sequenced after nested PCR reaction with primer pairs M13 & M14 and #34 & #63. After analysis of the phylogenetically important nucleotides 3685, 3699, 3753, 4062, 4449, 4477 and 4489 the sequences were classified as new antigenic type CPV-2b sequences. The sample from the second Namibian cheetah revealed an FPV sequence after PCR with primer pair #19 & M5 and analysis of nucleotides 4062, 4449, 4477 and 4489. Both faecal samples were positive in FASTest Parvo.
From each of the sections collected from four captive cheetahs in the United States a 538 bp PCR product was obtained using primer pair M10 & M11. Additionally, from two of these samples 901 bp amplicons were obtained using primer pair #19 & M5. The sequences analysed from the four cheetahs were identical to CPV-2b-like viruses. The sample from the fifth cheetah was negative.
Parvovirus-specific DNA could not be amplified by PCR from any of the three cheetahs from South Africa.
After examination of the PCR products obtained with primer pairs M10 & M11 and #19 & M5 the sample from the Siberian tiger from a German zoo showed sequences characteristic for CPV-2a-like viruses. In this case the faeces were negative in FASTest Parvo.
Both faecal samples from wild dogs were negative in FASTest Parvo and PCR.
All PCR results are summarized in Table 3.
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Discussion |
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All the sequences described were either similar to sequences of typical FPV DNA or to sequences of the new antigenic types CPV-2a and CPV-2b. No original CPV-2 sequence could be detected, which further indicates a worldwide replacement of CPV-2 by its new antigenic types, as has been shown for the domestic dog population in different parts of the world (Parrish et al., 1991 ; Truyen et al., 1996b
; Steinel et al., 1998
). Also, no sequences intermediate between FPV and CPV were seen, which is of importance because of a possible involvement of wild carnivores in the emergence of CPV-2. An intermediate sequence has been described for a virus isolated from a red fox in Germany (Truyen et al., 1998a
), but intermediate viruses may be restricted to fox-like canids.
For the first time a parvovirus infection was demonstrated in bat-eared fox and honey badger. The sequences obtained from the bat-eared fox, covering 85% of the capsid protein VP2, allowed the virus to be classified as a typical CPV-2b-type. As the bat-eared fox is a member of the family Canidae, susceptibility to CPV-2a and CPV-2b would be expected. No advantage for one or other of the two new antigenic types of CPV-2 is obvious and animals susceptible to one virus type can be infected by the other type.
From one honey badger sample a 201 bp amplicon was obtained. The faecal sample from this free-ranging animal, from the Kalahari Gemsbok Park in South Africa, was exposed for days to high ultraviolet radiation and kept for weeks unfrozen at varying temperatures. This might have caused partial destruction of the viral particles and DNA, making PCR more difficult, and may have hindered the amplification of longer amplicons (>>300 bp). The small sequence showed typical FPV-like nucleotides at positions 3025, 3065 and 3094. Mink enteritis virus (MEV) has been known to infect members of the family Mustelidae since 1947 (Schofield, 1949 ), but its taxonomic status is not clear. However, sequence analyses of both FPV and MEV could not demonstrate any definitive conserved nucleotide differences (Truyen et al., 1995
b). As a member of the family Mustelidae the honey badger probably shows the same susceptibility to these very similar, possibly identical viruses.
Three different species of the family Felidae were examined: one African wild cat, six cheetahs and one Siberian tiger. The African wild cat, belonging to the genus Felis, is one of the closest relatives of the domestic cat (Felis catus; Wayne et al., 1989 ). The domestic cat is a host for CPV-2a and CPV-2b as well as for FPV but the main cause of parvoviral infection in domestic cats remains FPV, with an incidence of 95% (Truyen et al., 1996b
). The African wild cat examined was infected with FPV, but in this study CPV-2a and CPV-2b seem to be the predominant infectious agents in large cats. FPV was found only in one cheetah from Namibia whereas the other five cheetahs from Namibia and the United States were infected with a CPV-2b virus. Also, the sequences obtained from the Siberian tiger were from a CPV-2a-like virus. The reason for the much higher incidence of CPV-2a/2b infections in large cats compared to domestic cats is unknown. Perhaps they are more susceptible to canine parvovirus infection, reminiscent of the higher susceptibility of large cats to canine distemper virus (Roelke-Parker et al., 1996
). The high incidence of CPV-2a/2b in large cats has to be considered especially when dealing with valuable and endangered species, such as cheetahs and tigers. At places with high, unavoidable parvovirus contamination such as veterinary clinics, zoos or animal orphanages, the risk of infection is high for unprotected animals. In zoological gardens, virus carriers such as stray cats, visitors dogs, martens or others may play a role in contaminating the environment with the highly resistant feline parvoviruses. The source of infection for the large cats is unknown; however, it appears likely that they acquire the virus from dogs, as CPV-2a and CPV-2b are the prevalent viruses in dog populations. The parvoviruses obtained from the cheetah samples analysed from the United States and Namibia were all CPV-2b viruses, which is consistent with the fact that 7090% of all canine parvoviruses isolated from dogs in these countries are CPV-2b viruses (Parrish et al., 1991
; Steinel et al., 1998
). In contrast, the Siberian tiger from Germany was infected with a CPV-2a virus, which correlates with the predominance of this virus type in Germany (Truyen et al., 1996b
).
The two Namibian hand-raised cheetahs as well as the captured African wild cat and the captive Siberian tiger had never been vaccinated. However, all five captive cheetahs from United States zoos had recently been vaccinated with a killed multivalent vaccine. Two of these cheetahs had been vaccinated 1 month before death, one cheetah was vaccinated 1 year before death and the fourth cheetah was vaccinated 2 years previously, suggesting that inactivated FPV vaccines may not always produce a protective antibody response against challenge infections with canine parvovirus. Modified-live FPV virus vaccines have been shown to protect domestic cats against a challenge infection with a CPV-2b isolate (Chalmers et al., 1999 ) but experiments with an inactivated vaccine have never been reported. Vaccination of Felidae in Basel Zoo, Switzerland, with an inactivated CPV-2 vaccine has been described and the vaccine was well tolerated (Gutzwiller et al., 1984
). In our study six out of seven (86%) cases of parvovirus infections in large cats were caused by the new antigenic types of canine parvovirus. Vaccination of large cats in zoos against feline parvovirus is therefore strongly recommended and the development of inactivated CPV-2a or CPV-2b vaccines for use in large cats should be considered.
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Acknowledgments |
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References |
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Received 27 July 1999;
accepted 12 October 1999.