Echocardiographic evaluation of four giant Aldabra tortoises (Aldabrachelys gigantea).

OBJECTIVES: In recent years echocardiography has become a good diagnostic tool in Zoo Medicine but in some cases, it is still a challenge. In Aldabra giant tortoise (Aldabrachelys gigantea) the big size of animals and the few individuals hosted in Zoo are critical points for the application of this diagnostic technique.The purposes of this research were: to evaluate the feasibility of the diagnostic imaging technique on big-sized turtles; to define the echographic parameters for this species; and to describe the morphofunctional and physiological echographic characteristics of their cardiovascular system. DESIGN: Repeated measures in vivo. SETTING: Ultrasonography systematic description and Doppler analysis of the cardiovascular system of Aldabra giant tortoise were carried out; B-mode examination allowed the evaluation of the kinetics of the ventricle, the atria and the atrioventricular valves. PARTICIPANTS: 4 Aldabra giant tortoises (two adult males and two young females) hosted in two zoological gardens. INTERVENTIONS: Echocardiography was performed placing the animals in ventral on a restraining platform raised from the floor, to provide adequate accessibility to the thoracic windows where the probe was placed. No chemical restraint was used. PRIMARY AND SECONDARY OUTCOME MEASURES: Heart rate, systolic and diastolic areas and volumes, vessel diameters and blood flow velocity were measured. RESULTS: Heart rate was 21±4 bpm (range 14-25 bpm). The averages of the diastolic and systolic area indexes linked to the subject weight were: 21±3 cm2 and 9±1 cm2.The aortic annulus diameter in female specimens measured 11.2±0.8 mm, while it measured 21.5±0.3 mm in male species. CONCLUSION: Results confirm the effectiveness of echocardiography as a means to study and evaluate the cardiovascular system of this species even if more studies on a bigger number of patients would be necessary to develop the echocardiography technique.

Use of Mini-FLOTAC and Fill-FLOTAC for rapidly diagnosing parasitic infections in zoo mammals / Utilização do Mini-FLOTAC e Fill-FLOTAC no diagnóstico rápido de infecções parasitárias de mamíferos em zoológicos

Abstract Animals reared in restricted environments are highly susceptible to gastrointestinal infection by helminths and protozoa and therefore zoos are characterized as being parasite-rich environments. Successful implementation of control programs of these parasites in zoo environment depends upon precise and rapid diagnosing of gastrointestinal infections. The aim of this study was to demonstrate the role of the Mini-FLOTAC technique in combination with Fill-FLOTAC for rapidly diagnosing parasitic infections in zoo mammals. Fecal samples were collected from 70 animals in four different zoos located in central and southern Italy. All the samples were analyzed using Mini-FLOTAC in combination with Fill-FLOTAC. Out of the 70 pooled samples examined, 80% (24/30) were positive for at least one parasite. Among the gastrointestinal nematodes, Strongyles were the most frequent (40%), followed by Trichuris spp. (23.3%), Parascaris spp. (13.3%) and Capillaria spp. (3.3%). Among the protozoa, Blastocystis spp., Giardia spp. and Eimeria spp. were detected in 6.6%, 3.3% and 3.3%, respectively. These results show that Mini-FLOTAC in combination with Fill-FLOTAC can be used, not only for rapidly diagnosing parasitic infections in zoo mammals, but also for monitoring control programs in which large numbers of fecal samples need to be examined rapidly and reliably.

Resumo Animais criados em ambiente restritos são altamente suscetíveis a infecção gastrointestinal por helmintos e protozoários, constituindo os zoológicos em ambientes com alta contaminação por parasitos. O sucesso da implementação de programas de controle contra estes parasitos em zoológicos depende do rápido diagnóstico das infecções por parasitas gastrointestinais. O objetivo deste estudo foi demonstrar o papel da técnica do Mini-FLOTAC em combinação com o Fill-FLOTAC no diagnóstico rápido das infecções parasitárias em mamíferos em zoológicos. Amostras de fezes foram coletadas de 70 animais de quatro diferentes zoológicos no centro e sudoeste da Itália. Todas as amostras foram analisadas pela técnica do Mini-FLOTAC em combinação com o Fill-FLOTAC. Do total de 70 pools de fezes examinadas, 80% (24/30) foram positivas para pelo menos um parasito. Entre os nematoides gastrointestinais a maior frequência foi observada para estrongilídeos (40%), seguida por Trichuris spp. (23,3%), Parascaris spp. (13,3%) e Capillaria spp. (3,3%). Entre os protozoários Blastocystis spp., Giardia spp. e Eimeria spp. foram detectados em 6,6%, 3,3% e 3,3%, respectivamente. Estes resultados demonstram que a técnica do Mini-FLOTAC em combinação com o Fill-FLOTAC pode ser utilizada não somente para o diagnóstico rápido das infecções parasitárias em mamíferos em zoológicos, mas também no monitoramento de programas de controle onde grande número de amostras fecais devem ser examinadas de forma rápida e confiável.

Use of Mini-FLOTAC and Fill-FLOTAC for rapidly diagnosing parasitic infections in zoo mammals.

Animals reared in restricted environments are highly susceptible to gastrointestinal infection by helminths and protozoa and therefore zoos are characterized as being parasite-rich environments. Successful implementation of control programs of these parasites in zoo environment depends upon precise and rapid diagnosing of gastrointestinal infections. The aim of this study was to demonstrate the role of the Mini-FLOTAC technique in combination with Fill-FLOTAC for rapidly diagnosing parasitic infections in zoo mammals. Fecal samples were collected from 70 animals in four different zoos located in central and southern Italy. All the samples were analyzed using Mini-FLOTAC in combination with Fill-FLOTAC. Out of the 70 pooled samples examined, 80% (24/30) were positive for at least one parasite. Among the gastrointestinal nematodes, Strongyles were the most frequent (40%), followed by Trichuris spp. (23.3%), Parascaris spp. (13.3%) and Capillaria spp. (3.3%). Among the protozoa, Blastocystis spp., Giardia spp. and Eimeria spp. were detected in 6.6%, 3.3% and 3.3%, respectively. These results show that Mini-FLOTAC in combination with Fill-FLOTAC can be used, not only for rapidly diagnosing parasitic infections in zoo mammals, but also for monitoring control programs in which large numbers of fecal samples need to be examined rapidly and reliably.

Multiple Organ Dysfunction Syndrome (MODS) induced by Candida krusei in an Aldabra giant tortoise (Aldabrachelys gigantea) and confirmed by electron microscopy analysis.

A young female Aldabra giant tortoise (Adabrachelys gigantea) was presented with anorexia, ataxia, severe constipation and bloating. Analysis revealed liver disease and collected biopsy diagnosed Candida krusei infection. Despite Itraconazole treatment, the tortoise got worse and died. Full necropsy was performed; microbiology showed Candida krusei presence in liver, but histopathology didn't confirm fungal presence with special stains, so scanning electron microscopy was essential to prove a detailed diagnosis of extensive mycosis.

Development of a serum neutralization assay to detect Pteropine Orthoreovirus Indonesia/2010 neutralizing antibodies.

Pteropine Orthoreoviruses (PRVs) are fusogenic bat-borne orthoreoviruses that cause flu-like upper respiratory tract infections in humans. The presence of this group of viruses in bats and humans has been well documented in areas where their biological reservoirs - fruit bats (family Pteropodidae) - live densely. In the present study, a serum neutralization (SN) assay to detect neutralizing antibodies against PRV Indonesia/2010 isolate was set up and used to assess the seroprevalence of this virus in Italian domestic animals. The new developed assay was able of detecting PRV neutralizing antibodies in the hyper-immune polyclonal serum produced in rabbits (titer of 1:160). The negative serum was negative at all tested dilutions. No cross-reactions have been evidenced neither against reference MRVs nor against their respective hyper-immune sera. Eight hundred and fifty-three serum samples collected from 524 bovines, 271 small ruminants, and 58 horses (all used as sentinel animals in the Bluetongue and West Nile disease National surveillance program) were also tested with the new developed SN assay. According to the results of this survey, neither PRV nor PRV cross- reacting viruses antibodies have been demonstrated in Italian domestic animals. However, the new developed SN assay could be a very valuable diagnostic tool to detect infection in animals and humans.

Circovirus in domestic and wild carnivores: An important opportunistic agent?

Circoviruses are relatively novel pathogens with increased importance in canids. In this study, we first screened the presence of dog circovirus (DogCV) by molecular methods from a total number of 389 internal organ samples originating from 277 individuals of domestic dogs and wild animals including wolves, foxes and badgers. All the animals originated from Central-Southern Italy, specifically from Abruzzi and Molise regions, areas hosting several natural parks. DogCV was detected in 9/34 wolves (P=26.4%; IC 95%: 14.6-43.1%), 8/209 dogs (P=3.8%; IC 95%: 1.9-7.3%), 0/24 foxes (P=0%; IC 95%: 0-13.8%), 1/10 badgers (P=10%; IC 95%: 1.79-40.4%). However, all DogCV positive animals were shown to be infected at least by an additional key pathogen, including canine distemper virus (CDV) and canine parvovirus type 2. All wolves, but one, presenting DogCV in the internal tissues suffered from CDV infection. The DNA purified from 17 DogCV infected organs was used for whole genome sequencing and phylogenetic analysis.

Diagnosis and treatment considerations in a case of malignant mesenchymoma in an African fur seal (Arctocephalus pusillus).

A 20-yr-old African fur seal (Arctocephalus pusillus) presented with a slowly growing mass located on the dorsum at the level of the last thoracic vertebrae. The mass was hard, 10 cm in diameter, and not adherent to the underlying tissues. Multiple biopsies were collected for histopathology and revealed extensive areas of necrosis, small nodules of malignant mesenchymal proliferation with areas of chondroid metaplasia, and atypical cells in vessel walls. The morphologic diagnosis was suggestive of malignant mesenchymal neoplasia originating from the vascular wall. The mass was removed 1 mo later due to ulceration and infection. Histologically, based on the World Health Organization's classification of neoplastic processes in domestic animals, the tumor was consistent with malignant mesenchymoma. The margins of resection revealed the presence of neoplastic cells. Based on these results, the particular species involved, the high local invasiveness, and the high metastatic index of this malignant tumor in domestic mammals and humans, the prognosis was poor. The animal died 6 mo later with metatastic disease.

Evaluation of a butorphanol, detomidine, and midazolam combination for immobilization of captive Nile lechwe antelopes (Kobus magaceros).

Field immobilization of captive antelope may be required for medical examination, blood sample collection, and animal identification. The aim of this study was to evaluate the effects of a combination of butorphanol, detomidine, and midazolam (BDM) and its partial reversibility in Nile lechwe antelope (Kobus megaceros). Nine captive lechwes, weighing 28-64 kg, were immobilized, in February 2011, with butorphanol 0.20 ± 0.05 (mean ± SD) mg/kg, detomidine 0.20 ± 0.05 mg/kg, and midazolam 0.31 ± 0.08 mg/kg administered intramuscularly (IM) with a blowpipe. Physiologic parameters and depth of anesthesia were recorded when the animals became recumbent at 19.55 ± 8.36 min after darting (T0) and after 10 (T10), 20 (T20), and 30 (T30) min. An arterial blood sample was collected at T20. At the end of the procedures, immobilization was partially reversed with atipamezole 0.25 mg/kg IM. Quality of induction, immobilization, and recovery was scored. The BDM combination induced immobilization and lateral recumbency in 13.44 ± 5.61 min. Median induction score (scored 1 [excellent] to 4 [poor]) was 1 (range 1-2). Heart rate varied 40-104 beats/min, respiratory rate 16-108 breaths/min, and rectal temperature 36.5-40.3 C. Hyperthermia was observed and rapidly treated in three animals that demonstrated insufficient immobilization after darting. Arterial blood gas analyses revealed a mean pH of 7.43 ± 0.07, partial arterial pressure of CO(2) of 44.1 ± 6.0 mmHg, partial arterial pressure of O(2) of 74.0 ± 13.5 mmHg, and an arterial O(2) saturation of 94.77 ± 3.96%. Recovery was smooth and animals were walking in 13.44 ± 7.85 min. Median recovery score (1 = excellent to 4 = poor) was 1 (range 1-2). The BDM was effective in immobilizing captive healthy lechwes with minimal cardiorespiratory changes.

Immunity after natural exposure to enteric canine coronavirus does not provide complete protection against infection with the new pantropic CB/05 strain.

Recently, an outbreak of fatal infection caused by a pantropic variant (strain CB/05) of canine coronavirus (CCoV) has been reported. In this study, evidence is provided that immunity induced by natural exposure to enteric CCoV is not fully protective against strain CB/05. Twenty-two, 10-week-old beagles with a recent natural infection by enteric CCoV were randomly distributed in two experimental groups of eight (groups A and B) and one control group of six (group C) dogs. Dogs in groups A and B were inoculated oronasally with different doses (4 x 10(5) or 4 x 10(3)TCID(50)) of the pantropic strain CB/05, whereas dogs in group C were used as negative controls. Clinical, post-mortem and virological investigations showed that, despite the high serum antibody titres induced by the prior natural infection with enteric CCoV, dogs were susceptible to experimental infection with strain CB/05. This was shown by the occurrence of faecal shedding, and dogs displaying moderate clinical signs, mainly vomiting and diarrhoea. Involvement of the lymphoid tissues was evident as demonstrated by the acute lymphopenia (below 70% of the initial counts), gross lesions in spleen and lymph nodes and detection of CB/05 RNA in thymus, spleen and lymph nodes of some infected dogs. The presence of viral RNA in lymphoid tissues was observed only in dogs euthanised in the early stages of infection and the clinical course of the infection was unrelated to the viral dose administered. The present study demonstrates that strain CB/05 is able to induce infection and disease in dogs seropositive to enteric CCoV, thus highlighting the need for extensive epidemiological investigation and for the possible development of novel antigenically relevant vaccines.

Detection of canine parvovirus type 2c by a commercially available in-house rapid test.

Diagnosis of canine parvovirus (CPV) infection is usually carried out by means of rapid immunochromatographic assays, but the ability of these tests to detect all CPV variants, including the recently identified CPV-2c, is still debated. To determine if the assays detect the different CPV variants, 201 CPV PCR-positive faecal samples or rectal swabs were tested using a commercially available in-house test. Specimens (CPV-2a, n=51; CPV-2b, n=50; CPV-2c, n=100), containing CPV DNA loads >10(5) DNA copies/mg faeces, as determined by real-time PCR, were selected from previous studies. The percentage of positive in-house tests was 80.4%, 78.0% and 77.0% for CPV types 2a, 2b and 2c, respectively, confirming the ability of the test to detect the new variant CPV-2c. However, considering the sensitivity limits of the in-house tests that have been observed previously, negative results from the in-house test kit should be confirmed by PCR-based methods.

A candidate modified-live bovine coronavirus vaccine: safety and immunogenicity evaluation.

A modified-live vaccine against the respiratory form of bovine coronavirus (BCoV) infection was developed by progressive attenuation of a respiratory strain (438/06-TN). The vaccine was found to be safe as four colostrum-deprived newborn calves remained healthy after oronasal administration of ten doses of the vaccine. The immunogenicity of the vaccine was assessed by intramuscular injection of one vaccine dose to 30 BCoV-antibody negative 2-3-month-old calves. At 30 days post-vaccination, all vaccinated calves displayed high antibody titres against BCoV. Sequence analysis of the S gene of wild-type and cell-adapted 438/06-TN strain detected 10 nucleotide changes, 9 of which were non-synonymous.

Molecular characterization of a canine respiratory coronavirus strain detected in Italy.

Coronaviruses (CoVs) are positive-stranded, non-segmented RNA viruses generally responsible for the emergence of respiratory and enteric disease in humans, companion animals and livestock. Their aptitude to evolve by genetic recombination and/or point mutation is recognized, thus giving rise to new viral genotypes and mutants with different tissues or host tropism. In particular, a probable origin from the strictly related bovine coronavirus (BCoV) or, alternatively, from a common ancestor has been suggested for some group 2a CoVs, including canine respiratory coronavirus (CRCoV). In this study, we report the sequence analysis of the viral RNA 3'-end of an Italian CRCoV, strain 240/05, together with the sequence comparison with extant bovine-like viruses including the sole CRCoV strain 4182 previously described. Interestingly, although the structural proteins show the same features of CRCoV 4182, the genomic region between the spike and the envelope protein genes of CRCoV 240/05 encodes for three distinct products, including the equivalent bovine 4.9 kDa non-structural protein and a truncated form of the 4.8 kDa protein, whereas CRCoV 4182 has a unique 8.8 kDa protein.

Recombinant canine coronaviruses related to transmissible gastroenteritis virus of Swine are circulating in dogs.

Four canine coronavirus type II (CCoV-II) strains were identified in the guts and internal organs of pups which had died of acute gastroenteritis. The CCoV-II strains were strictly related to porcine transmissible gastroenteritis virus (TGEV) in the N-terminal domain of the spike protein, whereas in the other parts of the genome, a higher genetic relatedness to recent CCoV-II isolates was observed. Experimental infection of dogs with a TGEV-like isolate induced mild gastroenteritis without any systemic involvement. By virus neutralization tests, antigenic differences between reference and TGEV-like CCoVs were found. Our data support the potential recombinant origin of the TGEV-like CCoVs.

Reversible immobilization of asiatic black bear (Ursus thibetanus) with detomidine-tiletamine-zolazepam and atipamezole.

Chemical immobilization of free-ranging and captive wildlife is often required in many clinical situations. In this trial, tiletamine-zolazepam was combined with the alpha2-agonist, detomidine, in order to use the least amount of anesthetic drug possible to achieve a rapid immobilization; to ensure safety for animals and operators; and to be easily reversible with specific antagonists for a fast recovery. Twelve captive Asiatic black bears were anesthetized for clinical procedures, including clinical examination and blood sample collection, and for electrocardiographic and echocardiographic procedures. The combination detomidine-tiletamine-zolazepam, at the dosages of 0.03 mg/kg for detomidine and 1.5 mg/kg for tiletamine-zolazepam, proved to be reliable and effective in immobilizing Asiatic black bears for a 1-hr handling period for routine clinical procedures. Minimal or no respiratory and/or cardiopulmonary adverse side effects were observed, even with dosages calculated on the basis of an estimated body weight. The respiratory rate, pulse rate, and hemoglobin-oxygen saturation remained stable for the entire duration of anesthesia. Cardiac rhythm was always sinusal in all animals. Small injection volumes and darts for blowpipe use were utilized to minimize tissue damage at the site of injection. Induction and recovery were smooth and predictable, and provided for the safety of operators who could observe the bears' activities from a safe distance. Furthermore, the availability of the alpha2-antagonist atipamezole to counteract the effects of detomidine made this anesthetic regimen easily controllable and reversible. Moreover, the recovery time can be shortened by intravenous administration of this antagonist drug.

Malignant catarrhal fever in a captive American bison (Bison bison) in Italy.

Malignant catarrhal fever (MCF) is a fatal, systemic disease of cattle and other domestic and wild ruminants that, in Europe, is caused by Ovine herpesvirus 2 (OvHV-2). American bison (Bison bison) are highly susceptible to the disease. An adult American bison, housed in a zoo in southern Italy in close cohabitation with a group of domestic sheep (Ovis aries aries) displayed clinical signs that resembled the acute form of MCF. By real-time polymerase chain reaction, OvHV-2 DNA was detected intravitam in blood, in nasal and ocular swabs, and postmortem in tissue samples of the bison. By indirect fluorescent antibody test, high MCF antibody titers were found in the bison serum. Ovine herpesvirus 2 DNA and antibodies were also found in blood samples from the domestic sheep, thus suggesting a potential role of these animals as a source of the infection. To the authors' knowledge, this is the first MCF case in captive ruminants in Italy and the second confirmed case in captive bison of European zoos.

Detection of bovine coronavirus using a TaqMan-based real-time RT-PCR assay.

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of bovine coronavirus (BCoV) RNA in clinical samples is described. The assay is based on TaqMan technology, consisting of two primers and one probe labeled with the reporter dye 6-carboxyfluorescein that binds selectively to the transmembrane-protein gene of BCoV. The BCoV real-time RT-PCR assay was able to detect the tested BCoV and BCoV-like viruses (canine respiratory coronavirus and bubaline coronavirus), whereas other common viral pathogens of cattle were not recognised by the established oligonucleotide set, thus showing that the test was specific for bovine-like CoVs. The detection limit of the assay was 20 BCoV RNA copies (1-log higher with respect to traditional gel-based RT-PCR) and the reproducibility was satisfactory, thus allowing for a sensitive and accurate measurement of the viral RNA load in clinical samples. Two hundred and twenty clinical specimens (92 rectal, 82 nasal and 46 ocular swabs) were subjected to gel-based and real-time RT-PCR. By conventional amplification, 43 rectal, 54 nasal and 34 ocular samples tested positive, whereas the TaqMan assay was able to detect the BCoV nucleic acid in 49 rectal, 60 nasal and 37 ocular swabs. The rapidity and high throughput of the BCoV TaqMan assay makes this method a powerful tool for a sensitive and specific diagnosis of BCoV infection in cattle.

Respiratory disease associated with bovine coronavirus infection in cattle herds in Southern Italy.

Four outbreaks of bovine respiratory disease (BRD) associated with bovine coronavirus (BCoV) infection in Italian cattle herds were reported. In 3 outbreaks, BRD was observed only in 2-3-month-old feedlot calves, whereas in the remaining outbreak, lactating cows, heifers, and calves were simultaneously affected. By using reverse transcription polymerase chain reaction (RT-PCR), BCoV RNA was detected in all outbreaks without evidence of concurrent viral pathogens (i.e., bovine respiratory syncytial virus, bovine herpesvirus type 1, bovine viral diarrhea virus, bovine parainfluenza virus). Common bacteria of cattle were recovered only from 2 outbreaks of BRD: Staphylococcus spp. and Proteus mirabilis (outbreak 1) and Mannheimia haemolytica (outbreak 4). A recently established real-time RT-PCR assay showed that viral RNA loads in nasal secretions ranged between 3.10 x 10(2) and 7.50 x 10(7) RNA copies/microl of template. Bovine coronavirus was isolated from respiratory specimens from all outbreaks except outbreak 1, in which real-time RT-PCR found very low viral titers in nasal swabs.

Development of a real-time PCR for the detection and quantitation of caprine herpesvirus 1 in goats.

Caprine herpesvirus 1 (CpHV-1) is an alphaherpesvirus interfering with goat reproductive performances. The virus is associated with neonatal mortality in kids and reproductive failure in adults. A real-time PCR assay based on TaqMan technology and targeting the gene encoding for glycoprotein C (gC) was developed for detection and quantitation of CpHV-1 in samples collected from infected goats. The detection limit of the assay was 1 x 10(2) standard DNA copies, with a sensitivity of 1-2 logs higher than the conventional gel-based PCR assay targeting the same gene. The real-time PCR was reproducible, as shown by satisfactory low intra-assay and interassay coefficients of variation. The quantitative assay was validated on clinical samples, including genital swabs and various tissue samples collected from goats either infected naturally or experimentally with CpHV-1. The high sensitivity, simplicity and reproducibility of the CpHV-1 fluorogenic PCR assay, combined with its wide dynamic range and high throughput, make this method especially suitable for studies on the pathogenesis and for trials with experimental vaccines and antiviral drugs.

Experimental infection of dogs with a novel strain of canine coronavirus causing systemic disease and lymphopenia.

A pantropic canine coronavirus (CCoV) strain (CB/05) has been recently associated to a fatal outbreak of systemic disease in young dogs. We report the clinical, virological and serological findings in dogs experimentally infected with strain CB/05. The dogs, three 2.5-month-old and two 6-month-old pups, were successfully infected, shedding viral RNA with their faeces for the entire observation period (21 days) and displaying systemic clinical signs resembling those observed during the course of natural infection. Leucopenia (acute lymphopenia) occurred in all infected dogs, with values dropping below 60% of the initial counts. Considering the severity of the CB/05-induced disease, two of the youngest pups were euthanized for ethical reasons at days 8-9 postinfection, whereas the other pups underwent a slow but progressive improvement of their clinical status with complete recovery. At postmortem examination, remarkable lesions were observed in the internal organs of the euthanized pups, that tested positive for CCoV by real-time RT-PCR and virus isolation on cell cultures. All pups seroconverted for CCoV, as shown by the high optical density values and antibody titres detected by ELISA and virusneutralisation tests, respectively. The present study confirms that strain CB/05 is highly pathogenic for dogs, being able to induce a severe disease (and in some cases the death) even in experimental conditions.

Biological and genetic analysis of a bovine-like coronavirus isolated from water buffalo (Bubalus bubalis) calves.

We describe the isolation, biological and genetic characterization of a host-range variant of bovine coronavirus (BCoV) detected in water buffalo (Bubalus bubalis). By conventional and real-time RT-PCR assays, the virus was demonstrated in the intestinal contents of two 20-day-old buffalo calves dead of a severe form of enteritis and in the feces of additional 17 buffalo calves with diarrhea. Virus isolation, hemagglutination and receptor-destroying enzyme activity showed that the buffalo coronavirus (BuCoV) is closely related to BCoV but possesses some different biological properties. Sequence and phylogenetic analyses of the 3' end (9.6 kb) of the BuCoV RNA revealed a genomic organization typical of group 2 coronaviruses. Moreover, the genetic distance between BuCoV and BCoV was proven to be the same or even higher than the distance between other ruminant coronaviruses and BCoV. In conclusion, our data support the existence of a host-range variant of BCoV associated with enteritis in buffaloes.