Ovine Isolates Research Articles

Cryoelectron Microscopy Map of Atadenovirus Reveals Cross-Genus Structural Differences from Human Adenovirus

A three-dimensional (3D) cryoelectron microscopy reconstruction of the prototype Atadenovirus (OAdV [an ovine adenovirus isolate]) showing information at a 10.6-A resolution (0.5 Fourier shell correlation) was derived by single-particle analysis. This is the first 3D structure solved for any adenovirus that is not a Mastadenovirus, allowing cross-genus comparisons between structures and the assignment of genus-specific capsid proteins. Viable OAdV mutants that lacked the genus-specific LH3 and p32k proteins in purified virions were also generated. Negatively stained 3D reconstructions of these mutants were used to identify the location of protein LH3 and infer that of p32k within the capsid. The key finding was that LH3 is a critical protein that holds the outer capsid of the virus together. In its absence, the outer viral capsid is unstable. LH3 is located in the same position among the hexon subunits as its protein IX equivalent from mastadenoviruses but sits on top of the hexon trimers, forming prominent "knobs" on the virion surface that visually distinguish OAdV from other known AdVs. Electron density was also assigned to hexon and penton subunits and to proteins IIIa and VIII. There was good correspondence between OAdV density and human AdV hexon structures, which also validated the significant differences that were observed between the penton base protein structures.

Genetic heterogeneity of bovine viral diarrhoea virus (BVDV) isolates from Turkey: Identification of a new subgroup in BVDV-1

Genetic heterogeneity of Turkish ruminant pestiviruses was investigated by phylogenetic analysis of complete N pro encoding nucleotide sequences. A total of 30 virus isolates obtained from 15 provinces around the country between 1997 and 2005 were included in the phylogenetic analysis. Virus isolates mostly originated from cattle with one isolate from sheep. The bovine isolates all belonged to BVDV-1, the sheep isolate to BVDV-2. Fifteen isolates formed a new subgroup within BVDV-1, tentatively named BVDV-1 l. The remaining bovine isolates were typed as BVDV-1a ( n = 4), BVDV-1b ( n = 4), BVDV-1d ( n = 3), BVDV-1f ( n = 2) and BVDV-1h ( n = 1). The isolates allocated to BVDV-1 l originated from various geographical regions in different years. There was no correlation between genetic grouping and locations where isolates were obtained. Viruses originating from one farm in most cases belonged to the same subgroup ( n = 5). This study indicates that the newly detected subgroup BVDV-1 l is predominant and widespread in Turkey. Moreover, an ovine virus isolate was identified as the first member of BVDV-2 reported in Turkey. A serological survey using samples from western Turkey indicated that BVDV-2 is also present in cattle.

Mouse-Adapted Ovine Scrapie Prion Strains Are Characterized by Different Conformers of PrPSc

The agent responsible for prion disease may exist in different forms, commonly referred to as strains, with each carrying the specific information that determines its own distinct biological properties, such as incubation period and lesion profile. Biological strain typing of ovine scrapie isolates by serial passage in conventional mice has shown some diversity in ovine prion strains. However, this biological diversity remains poorly supported by biochemical prion strain typing. The protein-only hypothesis predicts that variation between different prion strains in the same host is manifest in different conformations adopted by PrPSc. Here we have investigated the molecular properties of PrPSc associated with two principal Prnp(a) mouse-adapted ovine scrapie strains, namely, RML and ME7, in order to establish biochemical prion strain typing strategies that may subsequently be used to discriminate field cases of mouse-passaged ovine scrapie isolates. We used a conformation-dependent immunoassay and a conformational stability assay, together with Western blot analysis, to demonstrate that RML and ME7 PrPSc proteins show distinct biochemical and physicochemical properties. Although RML and ME7 PrPSc proteins showed similar resistance to proteolytic digestion, they differed in their glycoform profiles and levels of proteinase K (PK)-sensitive and PK-resistant isoforms. In addition, the PK-resistant core (PrP27-30) of ME7 was conformationally more stable following exposure to guanidine hydrochloride or Sarkosyl than was RML PrP27-30. Our data show that mouse-adapted ovine scrapie strains can be discriminated by their distinct conformers of PrPSc, which provides a basis to investigate their diversity at the molecular level.

Molecular genotyping of multinational ovine and caprineCorynebacterium pseudotuberculosisisolates using pulsed-field gel electrophoresis

Caseous lymphadenitis (CLA) is a chronic, suppurative disease, with a worldwide distribution, caused by Corynebacterium pseudotuberculosis. The clinical manifestation of CLA is known to vary between different countries, and has been postulated to be due to differences in the strains present in these countries. Forty-two sheep and goat isolates of C. pseudotuberculosis from Australia, Canada, Eire, The Netherlands and Northern Ireland were characterized by pulsed-field gel electrophoresis (PFGE), biotyping, antimicrobial susceptibility, and production of phospholipase D. The PFGE-determined genotypes of this multicentric collection were then compared with representative ovine and caprine isolates from a previously published panel of PFGE profiles of United Kingdom isolates. Digestion with SfiI generated 16-18 bands in the 48.5 and 290 kb range, and differentiated four distinct pulsotypes amongst the 36 ovine and 6 caprine strains which displayed remarkable homogeneity. Based on these results, it would appear that the genome of C. pseudotuberculosis is highly conserved, irrespective of the country of strain origin.

Genetic heterogeneity of Fasciola hepatica isolates in the northwest of Spain

The liver fluke Fasciola hepatica is a worldwide distributed trematode parasite of great veterinary importance. In Spain, the prevalence of the infection is up to 56% in sheep. Its control is mainly based on the use of anthelmitics, although some resistant populations have been described. Genetic studies of the genus Fasciola have been focused on the 28S ribosomal deoxyribonucleic acid (rDNA) gene, with no intraspecific variations and a few interspecific nucleotide differences. We have designed a specific polymerase chain reaction to amplify and sequence a 510-bp-long fragment of the gene to characterize several F. hepatica ovine isolates from the northwest of Spain. All the isolates rendered the expected results on the agarose gel, while sequencing revealed some differences in the 34 site. The anthelmintic-resistant specimens showed the same nucleotide differing of the sensitive ones. Comparing to other studies, our results conclude that there are some genetic variations in F. hepatica (at the level of the 28S rDNA), including heterozygous specimens. In conclusion, a genetic heterogeneity has been found in the F. hepatica populations of ovine origin in the northwest of Spain, using an assay that could be useful for further epidemiology and characterization studies.

At least two genetically distinct large Babesia species infective to sheep and goats in China

A fatal disease of sheep and goats in the northern part of China has been reported to be due to Babesia ovis. However, some characteristics of the causative agent in recent reports are not in accordance with the original attributes ascribed to this parasite. Therefore, the 18S small subunit ribosomal RNA (18S rRNA) genes of a number of Babesia isolates in China were sequenced and compared with that of other Babesia and Theileria species in an attempt to clarify their taxonomic position. In the present study, seven Babesia isolates were collected from distinct areas of northern China, and the 18S rRNA genes were amplified and sequenced. The phylogenetic trees were inferred based on 18S rRNA gene sequences of the Chinese ovine Babesia isolates and some of ovine Babesia and Theileria species available in GenBank. In the phylogenetic tree, Babesia sp. isolates from Madang, Tianzhu, Lintan, Ningxian, Hebei and Liaoning all grouped with B. motasi with 88.2–99.9% identity, while Babesia sp. Xinjiang grouped in a separate clade between B. ovis and B. crassa with 79.7–81.2% identity. The results indicated that there are at least two distinct Babesia species groups—B. motasi and Babesia sp. Xinjiang, the latter was dinstinctly different from other ovine Babesia isolates from China with less than 86.6% identity.

Reclassification of [Pasteurella] trehalosi as Bibersteinia trehalosi gen. nov., comb. nov.

[Pasteurella] trehalosi is an important pathogen of sheep, being primarily associated with serious systemic infections in lambs but also having an association with pneumonia. The aim of the present investigation was to characterize a broad collection of strains tentatively identified as [P.] trehalosi in order to reclassify and rename this taxon to support improvements in our understanding of the pathogenesis and epidemiology of this important organism. The type strain for [P.] trehalosi, strain NCTC 10370(T), was included along with 42 field isolates from sheep (21), cattle (14), goats (1), roe deer (3) and unknown sources (3). An extended phenotypic characterization was performed on all 43 strains. Amplified fragment length polymorphism (AFLP) was also performed on the isolates. Two of the field isolates were subjected to 16S rRNA gene sequencing. These sequences, along with five existing sequences for [P.] trehalosi strains and 12 sequences for other taxa in the family Pasteurellaceae, were subjected to a phylogenetic analysis. All the isolates and the reference strains were identified as [P.] trehalosi. A total of 17 out of 22 ovine isolates produced acid from all glycosides, while only four out of 14 bovine isolates produced acid from all glycosides. All 22 ovine isolates were haemolytic and CAMP-positive, while no other isolate was haemolytic and only two bovine isolates were CAMP-positive. Nineteen AFLP types were found within the [P.] trehalosi isolates. All [P.] trehalosi isolates shared at least 70 % similarity in AFLP patterns. The largest AFLP type included the type strain and 7 ovine field isolates. Phylogenetic analysis indicated that the seven strains studied (two field isolates and the five serovar reference strains) are closely related, with 98.6 % or higher 16S rRNA gene sequence similarity. As both genotypic and phenotypic testing support the separate and distinct nature of these organisms, we propose the transfer of [P.] trehalosi to a new genus, Bibersteinia, as Bibersteinia trehalosi comb. nov. The type strain is NCTC 10370(T) (=ATCC 29703(T)). Bibersteinia trehalosi can be distinguished from the existing genera of the family by the observation of only nine characteristics; catalase, porphyrin, urease, indole, phosphatase, acid from dulcitol, (+)-d-galactose, (+)-d-mannose and (+)-d-trehalose.

Molecular Typing of Staphylococcus aureus Isolated from Cows, Goats and Sheep with Intramammary Infections on the Basis of Gene Polymorphisms and Toxins Genes

We investigated 116 Staphylococcus aureus isolates from cows, goats and sheep with intramammary infections (IMI) in Italy to provide information about the spread of enterotoxigenic strains and to compare strains isolated from different ruminant species. The isolates were typed by restriction fragment length polymorphism (RFLP) analysis of the coagulase (coa) gene, by analysis of polymorphisms of the X region of protein A (spa) gene and by detection of genes encoding enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, sej and sel). Seven different coa types and 12 different spa types were distinguished. On the basis of polymerase chain reaction-RFLP, 29 different coa subtypes were identified. Two different coa subtypes accounted for 49% and 67% of bovine and ovine isolates respectively. Only seven coa subtypes were observed in isolates from more than one host species and no coa subtype was present in isolates from all three ruminant species. Furthermore, 85 of the isolates (73%) harboured at least one enterotoxin gene (se) with a predominance of sea, sed and sej among isolates from bovine IMI, and sec and sel among isolates from caprine and ovine IMI. Comparing the S. aureus isolates on the basis of gene polymorphisms and presence of se genes, significant differences were found in distributions of genotypes among isolates from cows, goats and sheep.

Genetic differences between Tunisian camel and sheep strains of the cestodeEchinococcus granulosusrevealed by SSCP

Ovine and dromedary Echinococcus granulosus isolates from Tunisia were identified as G1 and G6 strains based on polymorphism of the mitochondrial cytochrome C oxydase CO1. Single strand conformation polymorphism (SSCP) was used in order to examine the genetic variation within and between Tunisian G1 and G6 strains and to estimate the extent of selfing. The dromedary isolates are genetically distinct from sheep isolates (high value of genetic variation between populations: Fst= 0.46). No significant deficiency in heterozygotes was found in sheep isolates, whereas heterozygote deficiency (suggesting selfing) was found in a limited number of camel isolates.

Diversity of temperate bacteriophages induced in bovine and ovine Mannheimia haemolytica isolates and identification of a new P2-like phage

The diversity of temperate bacteriophages was examined in 32 Mannheimia haemolytica, six Mannheimia glucosida and four Pasteurella trehalosi isolates. Phage particles were induced and identified by electron microscopy in 24 (75%) M. haemolytica isolates, but in only one (17%) M. glucosida and one (25%) P. trehalosi isolate. The M. haemolytica phages were relatively diverse as seven Siphoviridae, 15 Myoviridae and two Podoviridae-like phages were identified; the Myoviridae-type phages also exhibited structural variation of their tails. The bacteriophages induced in M. glucosida and P. trehalosi were of the Myoviridae type. Restriction endonuclease (RE) analysis identified nine distinct RE types among the M. haemolytica bacteriophages, providing further evidence of their relative diversity. A limited number of phages caused plaques on indicator strains and the phages exhibited a narrow host range. A subgroup of 11 bovine serotype A1 and A6 isolates contained Myoviridae-type phages of the same RE type (type A), but these differed in their abilities to infect and form plaques on the same panel of indicator strains. A P2-like phage (phiPHL213.1), representative of the RE type A phages, was identified from the incomplete M. haemolytica genome sequence. The phiPHL213.1 genome contains previously unidentified genes and represents a new member of the P2 phage family.

Intestinal Shiga toxin-producing Escherichia coli bacteria mitigate bovine leukemia virus infection in experimentally infected sheep.

Ruminants often carry gastrointestinal Shiga toxin (Stx)-producing Escherichia coli (STEC). Stxs belong to a large family of ribosome-inactivating proteins (RIPs), found in many plants and some bacteria. Plant RIPs, secreted into extracellular spaces, limit the spread of viruses through plant tissues by penetrating and killing virally infected cells. Previously, we showed Stx activity against bovine leukemia virus (BLV)-infected cells in vitro and hypothesized that STEC bacteria have antiviral activity in ruminant hosts. Here, we investigated the impact of STEC on the initial phases of BLV infection in sheep. Sheep were treated with biweekly oral doses of E. coli O157:H7 (an STEC) or an isogenic stx mutant strain. A different group of sheep were similarly treated with five naturally occurring ovine STEC isolates or stx-negative E. coli. Intestinal STEC bacteria were enumerated and identified by standard fecal culture and DNA hybridization. Oral STEC treatment did not always result in carriage of STEC, although many animals consistently presented with >10(4) CFU/g feces. BLV viremia was assessed by spontaneous lymphocyte proliferation (SLP) in cultures of blood mononuclear cells and by syncytium formation in cocultures of the same with F-81 indicator cells. SLP was lower (P < 0.05) and syncytia were fewer (P < 0.05) in STEC-treated sheep than in untreated sheep. Both lower SLP and fewer syncytia positively correlated with fecal STEC numbers. Average weight gain post-BLV challenge was higher in STEC-treated sheep than in untreated sheep (P < 0.05). These results support the hypothesis that in ruminants, intestinal STEC bacteria have antiviral activity and mitigate BLV-induced disease.

Acquisition of haemoglobin-bound iron by Histophilus somni

Histophilus somni is an important pathogen of cattle and sheep. H. somni requires iron and can use ruminant transferrins as iron sources for growth. Here, we investigated the abilities of bovine (strains 649 and 2336) and ovine (strains 9L and 3384Y) isolates of H. somni to acquire iron from haemoglobins. Using growth assays, the bovine isolates were shown to acquire iron from bovine haemoglobin, but not from ovine, porcine or human haemoglobins; the ovine isolates, however, failed to use any of these haemoglobins as iron sources for growth. In solid phase binding assays, the bovine isolates, grown under iron-restricted conditions in the presence of bovine haemoglobin, bound not only bovine but also ovine and human haemoglobins. Competition binding assays indicated that all three haemoglobins were bound by the same receptor(s) and SDS-PAGE of membrane fractions revealed that expression of haemoglobin-binding activity was associated with the production of an ∼120-kDa outer membrane protein. PCR approaches allowed the amplification and sequencing of hgbA, and also hugX and hugZ homologues from strains 649, 9L and 3384Y. While hgbA of strain 649 was predicted to encode an HgbA precursor that is processed to yield a mature, 123.9-kDa haemoglobin-binding protein, the hgbA genes of strains 9L and 3384Y were predicted to give rise to truncated products. RT-PCR experiments revealed that in strain 649, hugX, hugZ and hgbA are co-transcribed and iron-regulated and additional sequencing suggested that in strain 2336, expression of HgbA is subject to phase variation involving a poly C tract within hgbA.

Tipificación molecular de cepas de Mycobacterium avium subespecie paratuberculosis de diferentes huéspedes y regiones

The IS1311 polymerase chain reaction-restriction endonuclease analysis was used to detect genetic differences among 38 Mycobacterium avium subsp. paratuberculosis (Map) isolates from cattle, sheep, goats and bison from distinct regions of Spain, India and the United States of America (USA). In Spain, all eight bovine isolates, three out of six caprine isolates and one of ten ovine isolates were of the C type, while the other nine ovine isolates and three caprine isolates were of the S type. In India, all five ovine isolates and six caprine isolates were of the B type, and so were all three isolates from bison (Bison bison) from the USA. These results show that there are genetic differences between Map isolates related to geographic and host factors that have a potential use in the epidemiological tracing of new paratuberculosis isolates.

Evaluation of molecular methods to determine enterotoxigenic status and molecular genotype of bovine, ovine, human and food isolates of Staphylococcus aureus

This study evaluated the use of PFGE and single enzyme AFLP techniques for the determination of the genetic relationships between Staphyloccocus aureus isolates from human, bovine, ovine and food related sources and reports the prevalence of ‘classic’ ( sea to see) and ‘new’ ( seg, seh, sei, sej, sem, sen and seo) staphylococcal enterotoxin ( se) genes in 92 S. aureus strains. A sub-set of the se genotyping results was confirmed by ELISA and the presence of SE toxin determined in isolates from different sources. A 100% correlation was observed, between detection of enterotoxin genes sea– see and expression of corresponding enterotoxin proteins in vitro. The se genotyping data generated from 90 of the S. aureus isolates showed that many of the S. aureus strains producing identical se genotypes correlated with both AFLP and PFGE pattern types. However, single enzyme AFLP technique did not possess the discriminatory power of the PFGE method, but similar clonal relationships were observed by both techniques in many of the isolates tested. Results reported here include the first comprehensive study using a single enzyme AFLP technique to investigate the genetic background of S. aureus isolates from a wide distribution including animal, human and food related sources.

Development of a real time RT-PCR to detect and type ovine pestiviruses

A real time one-step RT-PCR was designed to detect and type border disease virus (BDV), bovine viral diarrhea virus (BVDV) type 1 and BVDV type 2 in ovine samples. The real time RT-PCR was shown to behave in a linear manner and had limits of detection of 100–1000 copies of viral RNA as judged by in vitro transcribed RNA. The real time RT-PCR was validated on 50 clinical samples from UK flocks and was more sensitive than a virus isolation and a classical nested RT-PCR ( nRT-PCR). The results of real time RT-PCR virus typing agreed completely with sequencing. The majority of ovine isolates were BDV; a small proportion were BVDV type 1. BVDV type 2 was not detected in any sample. This test appears reliable and can be used for the typing of ovine pestiviruses in the UK.

Molecular epidemiology of Campylobacter fetus subsp. fetus on bovine artificial insemination stations using pulsed field gel electrophoresis

The presence of Campylobacter fetus subspecies fetus (Cff) on bovine artificial insemination (AI)-stations can have major economical consequences. More knowledge on the epidemiology of C. fetus is needed to control Cff infections at AI-stations. We assessed the epidemiology of Cff on AI-stations and the molecular relationship between Cff strains isolated from outbreaks on AI-stations. Thirteen Cff strains (two Cff strains per outbreak and one sporadic case) isolated from bulls housed on different AI-stations were selected and compared with ten unrelated bovine and ovine Cff isolates from different geographical regions. Molecular typing by pulsed field gel electrophoresis (PFGE) with the restriction enzymes SmaI, SalI and KpnI, yielded unique profiles for most unrelated strains but indistinguishable profiles for all isolates from the same outbreak. Computer aided analysis using a composite data set of SmaI, SalI and KpnI restriction profiles revealed separate clusters for outbreak strains. Thus, PFGE profiling of Cff strains is a valuable tool to discriminate between strains derived from separate outbreaks and to identify routes of infection.

Comparison of Staphylococcus aureus Genotypes Recovered from Cases of Bovine, Ovine, and Caprine Mastitis

Staphylococcus aureus is an important pathogen in domestic ruminants. The main objective of this study was to determine the similarity of epidemiologically unrelated S. aureus isolates from bovine, ovine, and caprine mastitis. By pulsed-field gel electrophoresis, 160 different pulsotypes (PTs) were identified among 905 isolates recovered from 588 herds in 12 counties in Norway. Based on estimates of similarity, using an 80% cluster cutoff, the isolates were assigned to 47 clusters. One cluster included 62% of all the isolates and more than 45% of the isolates from each host species. Twenty-three PTs included isolates from more than one host species; these 23 PTs represented 72% of all the isolates. The six most prevalent PTs included isolates from all host species and contained 45% of the bovine isolates, 54% of the ovine isolates, and 37% of the caprine isolates. Antimicrobial susceptibility testing of 373 of the isolates revealed resistance to penicillin in 2.9% and to streptomycin in 2.4%; only 1.9% were resistant to 1 of the other 11 antimicrobials tested. The results of this study suggest that a small number of closely related genotypes are responsible for a great proportion of S. aureus mastitis cases in cows, ewes, and goats in Norway and that these genotypes exhibit little or no host preference among these species. Selection due to antimicrobial resistance appears not to have contributed to the predominance of these genotypes.

Characterization of Listeria monocytogenes isolates from food animal clinical cases: PFGE pattern similarity to strains from human listeriosis cases

Twenty-one isolates of Listeria monocytogenes from food animal clinical cases that involved meningitis or meningoencephalitis, encephalitis, mastitis and abortion were characterized by serotyping and pulsed-field gel electrophoresis (PFGE) in order to improve our understanding of the genetic links between individual strains and strains recovered from human listeriosis cases. Results showed that five of the isolates were serotype 1/2a, six were 1/2b, nine were 4b, and one was untypeable. A caprine, two bovine and an ovine brain isolate shared identical PFGE patterns indicating that strains of L. monocytogenes are not host specific. Other isolates exhibited distinct patterns that were not shared, indicating a genetic diversity. Dendrogram analysis revealed that PFGE patterns of the isolates clustered primarily according to serotype. We compared the PFGE types obtained for these isolates with PFGE types for human clinical isolates present in the CDC national PulseNet database. Six (29%) of the twenty-one strains had patterns that were indistinguishable from pathogenic human isolates in the database. Our observations offer preliminary evidence that food animals could be significant reservoirs of L. monocytogenes that lead to human infections and support the inclusion of PFGE patterns of veterinary clinical isolates in the national PulseNet database for increased surveillance.

Genetic and antigenic characterization of the matrix protein of two genetically distinct ovine lentiviruses

Small Ruminant Lentiviruses (SRLV) are a group of non-oncogenic retroviruses including Maedi-Visna virus (MVV) and Caprine Arthritis-Encephalitis virus (CAEV), which cause a chronic, multisystemic disease in sheep and goats, respectively. Phylogenetic analyses of SRLV are based in most cases on partial pol sequences. Several reports indicate that the species specificity of these viruses is not as strict as previously thought; MVV-like viruses have been found in goat populations and vice versa. Recently, the sequencing of some Italian ovine isolates has shown the presence of a new cluster more similar to classical caprine isolates (CAEV-like). Few data are available on the variability of structural proteins involved in the antibody response of infected animals. In this study, the gag gene of two genetically distinct ovine isolates, namely the MVV-like It-561 and the CAEV-like It-Pi1, was sequenced and the epitopes of matrix protein (MA) were mapped. Recombinant MAs and their subunits from both ovine aforementioned strains were tested against a panel of sheep and goat sera. Reactive epitopes were found in all three subunits of MA, although the central subunit displayed a more consistent reactivity. Epitope mapping of this subunit demonstrated that the amino acid sequence of at least one immunodominant epitope was quite different in the two strains. This antigenic variability may affect the sensitivity of a single strain-based immunoassay and suggests that both SRLV genotypes should be used in the development of future diagnostic tests, to avoid viral strain selection during the eradication programmes.

Detection of a novel border disease virus subgroup in Tunisian sheep

Nine pestiviruses isolated from different batches of a contaminated Tunisian sheep pox vaccine and one Tunisian field ovine isolate of border disease virus (BDV) were studied at the antigenic and molecular levels. Seroneutralization tests were carried out on three vaccine isolates, the Tunisian field isolate and representative reference strains of the different pestivirus groups. The antigenic study showed that the Tunisian isolates were closer to the two BDV reference strains than to the Alfort-187 and the NADL reference strains. Comparison of the nucleotide sequences of the 5'-non coding regions of all the Tunisian isolates to those of other pestiviruses have shown that these isolates were distinct from the established pestivirus species. The entire N(pro)-E2 coding sequences of four Tunisian isolates were determined and compared to other pestiviruses. Segregation of these pestiviruses based on the N(pro)-E2 region was identical to that obtained with the 5'UTR sequences. The phylogenetic tree obtained with these sequences showed that the Tunisian isolates formed a separate branch between the BDV and CSFV groups, and consequently a possible new species within the pestivirus genus. However, as indicated by the antigenic study and the host origin of the isolates, the Tunisian isolates were assigned to a novel subgroup within the BDV species.