Distribution and heterogeneity of small ruminant lentivirus envelope subtypes in naturally infected French sheep

Small ruminants lentiviruses (SRLV) nucleotide sequences spanning the V1V2 variable regions of the env gene were amplified by nested-PCR from 38 blood samples collected from 16 naturally infected sheep flocks in France. For the rapid SRLV group determination of field isolates, the PCR-amplified fragments were subjected to a SRLV-adapted heteroduplex mobility assay (HMA). All viral sequences were clearly assignable to the SRLV group B by HMA analysis. Twenty-seven SRLV isolates were selected for DNA sequence analysis. In each case, nucleotide comparison and phylogenetic analyses confirmed the genetic relationships inferred by HMA. Six SRLV isolates belonged to subtype B1, and 21 pertained to subtype B2, one flock being infected with both subtypes. Subtypes B1 and B2 were found with different frequencies and geographic spread, but exhibited similar genetic diversities. These results give a more complete picture of the distribution and heterogeneity of SRLV env subtypes in sheep and confirmed that multiple interspecies transmission occurred in the past. Furthermore, HMA appeared to be a rapid and reliable method to differentiate caprine arthritis encephalitis virus from maedi-visna virus.     

Genomic heterogeneity of small ruminant lentiviruses: existenceof heterogeneous populations in sheep and of the samelentiviral genotypes in sheep and goats

Summary.  We have recently shown that French small ruminant lentiviruses (SRLV) from sheep are more similar to Caprine Arthritis Encephalitis Virus (CAEV) than to visna maedi virus (VMV) in a conserved region of the pol gene. To extend these results, we have examined sequences from a variable region of the env gene in French SRLV. We found that they were nearly equally distant from both CAEV and VMV strains, suggesting a considerable divergence since the initial introduction of the virus. Analysis of separate clones from individual animals showed that some carry a population of variant viruses. The study of further pol gene sequences from both goats and sheep suggests that viral variants show little or no host species specificity. A phylogenetic tree of pol gene sequences confirmed the presence of a novel genotype of SRLV in France. Accepted January 10, 1997 Received August 12, 1996     

Natural transmission and comparative analysis of small ruminant lentiviruses in the Norwegian sheep and goat populations

Serological surveys for small ruminant lentivirus (SRLV) infections have revealed seropositive sheep in several mixed herds, where sheep are kept together with seropositive goats. Here we have examined the genetic relationships in LTR, pol and env surface unit (SU) and the growth patterns in goat (GSM) and sheep (FOS) synovial membrane cell cultures of SRLV isolates obtained from both mixed and single species herds. Phylogenetic analyses of pol and env SU revealed that Norwegian SRLVs derived from both goat and sheep in mixed herds are distributed into group C, while isolates obtained from unmixed sheep flocks cluster in group A, together with maedi-visna-like representatives of the A1 subtype. In this study, the direction of group C virus transmission is proposed to be from goat to sheep. The replication efficiency in GSM and FOS cultures and the cytopathic phenotype induced by the SRLV isolates gave no indication of any species-specific characteristics. No particular nucleotide sequences of the LTR-U3 region or env SU were identified that could be related to cytopathic phenotype. This study shows that sheep in Norway harbour SRLVs belonging to phylogenetic groups A and C, and this provides further evidence for cross-species infection being a regular characteristic of SRLVs, which may represent an important source for viral persistence.     

Seroprevalence,clinical incidence,and molecular and epidemiological characterisation of small ruminant lentivirus in the indigenous Passirian goat in northern Italy

Eight dairy flocks, comprising a total of 323 indigenous Passirian goats from northern Italy, were examined to determine the seroprevalence and clinical incidence of small ruminant lentivirus (SRLV) infections and to identify the SRLV subtypes. The seroprevalence was 81.5% (55-95%). The clinical incidence was 2.5% (0-8.3%) and was apparently low due to the practice of culling clinically affected animals. Phylogenetic analysis of eight PCR fragments (one sample from each flock) revealed that all proviruses belonged to the SRLV subtype B1, which suggests a common source of infection. Subtype B1 being the only circulating SRLV, coupled with the fact that mixed herd systems are very rare in South Tyrol, gives hope that an eradication programme in goats can be successful even without including sheep as long as sheep are kept strictly and permanently isolated.     

Genetic and antigenic characterization of small ruminant lentiviruses circulating in Poland

Small ruminant lentivirus (SRLV) infections are widespread in Poland, but the genetic features of sheep viruses are still lacking and limited to partial gag sequences for goat viruses. In this study, segments from the gag and env genes of Polish SRLV strains screened by heteroduplex mobility assay were subjected to genetic analyses. Subtype A1 was found in both sheep and goats, while subtypes B1 and B2 were found in goats and sheep, respectively. In addition, two novel subtypes (named A12 and A13) were found in sheep. Their close phylogenetic relatedness with SRLV strains previously isolated from Polish goats indicated that these new subtypes are predominant and circulate in both species. The antigenic relationships of subtypes A12 and A13 with other SRLV subtypes were tested in an ELISA assay based on recombinant antigens carrying the immunodominant domains of structural proteins (MA, CA and SU). Antigenic cross-reactivity in the Gag epitopes was evident among genotype A subtypes and, to a lower extent, between genotypes A and B. In contrast, a subtype-specific immunoresponse was detected in the SU epitopes. These results emphasize the broad genetic and antigenic diversity of SRLV strains circulating in Europe and confirmed the need to consider all viral genotypes to choose the antigens in serological tests in order to avoid misdiagnosis in control and eradication programs.     

Molecular characterization and phylogenetic study of Maedi Visna and Caprine Arthritis Encephalitis viral sequences in sheep and goats from Spain

Small ruminant lentiviruses (SRLV) are widely spread in many countries, including Spain. However, little is known about the genetic characteristics of Spanish goat and sheep SRLV. In this study, segments from three genomic regions (pol, gag-p25 and LTR) were amplified using DNA isolated from three Spanish autochthonous sheep (one) and goats (two). Animals (one per flock) belonged to distantly located, single-species flocks (goat or sheep). Sequence analysis showed conservation of regions that are putatively relevant to viral survival. Sequences of Spanish goat and sheep SRLV were allocated into phylogenetic trees (phylograms) with known SRLV groups. The phylograms corresponding to the pol, gag-p25 and LTR regions analyzed presented a compatible topology. This showed that Spanish caprine and ovine SRLV sequences belonged to the A or D phylogenetic groups and were closer to sheep SRLV prototypes (A1 group) than to goat SRLV prototypes (B or C groups), according to the current classification [Shah, C., Boni, J., Huder, J.B., Vogt, H.R., Muhlherr, J., Zanoni, R., Miserez, R., Lutz, H., Schupbach, J., 2004a. Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade. Virology 319 (1), 12-26]. It was not possible to amplify in the three genetic regions the expected fragment in additional Spanish caprine and ovine SRLV proviral DNA sequences with the PCR primers used. This suggests that there is heterogeneity at the primer binding site among Spanish SRLV sequences. It also illustrates the need to develop diagnostic tests that are sensitive in local breeds.     

Compartmentalization of small ruminant lentivirus between blood and colostrum in infected goats

The compartmentalization of small ruminant lentivirus (SRLV) subtype A (Maedi-Visna virus) and B (caprine arthritis-encephalitis virus) variants was analyzed in colostrum and peripheral blood mononuclear cells of four naturally infected goats. Sequence analysis of DNA and RNA encompassing the V4-V5 env regions showed a differential distribution of SRLV variants between the two compartments. Tissue-specific compartmentalization was demonstrated by phylogenetic analysis in three of the four cases. In these animals colostrum proviral sequences were clustered relative to the blood viral sequences. In one goat, the blood and colostrum-derived provirus sequences were intermingled, suggesting trafficking of virus between the two tissues or mirroring a recent infection. Surprisingly, the pattern of free virus variants in the colostrum of all animals corresponded only partially to that of the proviral form, suggesting that free viruses might not derive from infected colostral cells. The compartmentalization of SRLV between peripheral blood and colostrum indicates that lactogenic transmission may involve specific viruses not present in the proviral populations circulating in the blood.     

Evaluation of five enzyme-linked immunosorbent assays and an agar gel immunodiffusion test for detection of antibodies to small ruminant lentiviruses

In the framework of the Dutch control program for small ruminant lentiviral (SRLV) infections, too many drawbacks were encountered with respect to serological testing. To improve the quality of testing, five enzyme-linked immunosorbent assays (ELISAs) and an agar gel immunodiffusion test (AGIDT) were evaluated. The focus was on the sensitivity, specificity, and variances of the commercially available tests. Clear differences were found among the tests in analytical and diagnostic sensitivity and overall diagnostic performance, whereas no significant differences in specificity were found. For serodiagnosis of sheep with clinical symptoms of maedi-visna virus (MVV) (histopathologically confirmed), one ELISA was significantly more sensitive than the other ELISAs and than the AGIDT, while for asymptomatic sheep originating from infected flocks, three ELISAs and the AGIDT demonstrated similar performance. The diagnostic performance appeared to be related to animal species and virus infection (MVV or caprine arthritis encephalitis virus [CAEV]) as well as the phase of infection/progression of disease. Receiver operating characteristic analysis, demonstrating the diagnostic potential of tests irrespective of defined cutoffs, again revealed clear differences between tests with respect to diagnostic performance for detection of antibodies against CAEV or MVV. An indirect ELISA, of which the solid phase is sensitized with a combination of the core protein p27 of MVV produced in Escherichia coli and a peptide derived from the transmembrane protein gp46, appeared to be the test of choice for serodiagnosis of SRLV infections in sheep and goats.     

Phylogenetic analysis of small ruminant lentivirus (SRLV) in Italian flocks reveals the existence of novel genetic subtypes

In order to investigate the genetic heterogeneity of small ruminant lentivirus (SRLV) isolates in Italy, 55 clinical samples collected between 1998 and 2010 were analysed. The phylogenetic study was based on analysis of gag–pol sequences. Our findings revealed that the SRLVs belonged to the subtype A9 (n = 3, sheep), B1 (n = 5, goat), B2 (n = 3, sheep) and E2 (n = 5, goat). Interestingly, 39 isolates from both sheep and goat, significantly differed from all the other SRLVs previously described and formed two separate clusters within genotypes A and B tentatively named A11 (n = 27, goat and sheep) and B3 (n = 12, goat and sheep), which have never been shown before. These results revealed a marked diversity among Italian field SRLV strains which might reflect the absence of any systematic control measures.     

Differential receptor usage of small ruminant lentiviruses in ovine and caprine cells: host range but not cytopathic phenotype is determined by receptor usage

The ovine maedi-visna (MVV) and caprine arthritis-encephalitis (CAEV) small ruminant lentiviruses (SRLV) exhibit differential species tropism and cytopathic effects in vitro. Icelandic MVV-K1514 is a lytic SRLV which can infect cells from many species in addition to ruminants, whereas a lytic North American MVV strain (85/34) as well as nonlytic MVV strain S93 and CAEV can infect only ruminant cells. In the present study, we determined if differential receptor usage in sheep and goat cells is the basis of differential species tropism or cytopathic phenotype of SRLV. Infection interference assays in sheep and goat synovial membrane cells using pseudotyped CAEV vectors showed that North American MVV strains 85/34 and S93 and CAEV use a common receptor (SRLV receptor A), whereas MVV-K1514 uses a different receptor (SRLV receptor B). In addition, human 293T cells expressing CAEV but not MVV-K1514 envelope glycoproteins fused with a goat cell line persistently infected with MVV-K1514, indicating that MVV-K1514 does not use SRLV receptor A for cell-to-cell fusion. Therefore, our results indicate that the differential species tropism of SRLV is determined by receptor usage. However, receptor usage is unrelated to cytopathic phenotype.     

Specific detection of small ruminant lentiviral nucleic acid sequences located in the proviral long terminal repeat and leader-gag regions using real-time polymerase chain reaction

Real-time polymerase chain reaction (RT-PCR) detection of proviral nucleic acid sequences of small ruminant lentiviruses (SRLV) in blood samples was developed and evaluated. Priming oligonucleotides were designed on the highly conserved 5' untranslated leader-gag region while those on the long terminal repeat (LTR) assay were derived from literature. DNA was extracted from the buffycoat interlayer of centrifuged blood samples. Real-time PCR was performed by means of LightCycler technology (Roche Applied Science) using melting temperature analysis (SYBR Green I) for detection. Results were compared with those of serology using samples from Dutch sheep and goat flocks with known SRLV statuses, with sequential samples from a natural transmission experiment and samples from different regions in Norway, France, Spain and Italy. Real-time PCR testing, especially the application of oligonucleotides for priming the leader-gag region appeared promising in detecting SRLV specific proviral DNA in blood samples from both sheep and goats.     

Evaluation of a Caprine Arthritis-Encephalitis Virus/Maedi-Visna Virus Indirect Enzyme-Linked Immunosorbent Assay in the Serological Diagnosis of Ovine Progressive Pneumonia Virus in U.S. Sheep

A caprine arthritis-encephalitis virus (CAEV)/maedi-visna virus (MVV) indirect enzyme-linked immunosorbent assay (iELISA) was validated with samples from U.S. sheep and by the use of radioimmunoprecipitation as the standard for comparison. The sensitivity and the specificity were 86.0% (±5.8%) and 95.9% (±2.9%), respectively. The iELISA format and phylogenetic differences based on the MVV gag sequence contribute to the reduced sensitivity.The diagnosis of small ruminant lentivirus (SRLV) infections in sheep and goats is most commonly determined by the detection of anti-SRLV antibodies in serum by an enzyme-linked immunosorbent assay (ELISA) that is typically created by the use of maedi-visna virus (MVV) or caprine arthritis-encephalitis virus (CAEV) isolates from sheep or goats of a given region or country (1). ELISA formats are typically validated against reference standard tests, including the agar gel immunodiffusion (AGID) assay, the radioimmunoprecipitation (IP) assay, or Western blot analysis. Although most seropositive sheep and goats do not show clinical signs of SRLV disease, they are persistent and potential reservoirs for transmission. Therefore, highly specific and sensitive serological diagnostic assays are essential for the early detection of SRLV.Three hundred ten of 332 serum samples from U.S. sheep from a previous CAEV competitive ELISA (cELISA) validation study (4) were tested in duplicate by using a Chekit CAEV/MVV antibody test kit (IDEXX Laboratories, The Netherlands), according to the manufacturer''s instructions. The CAEV/MVV indirect ELISA (iELISA) results were compared with those of the ovine progressive pneumonia virus (OPPV) WLC1 radio-IP assay, which has been described previously (4). The CAEV/MVV iELISA utilizes whole virus from Swiss MVV strain OLV as the antigen (15, 16). With a value of ≥60% being defined as a CAEV/MVV iELISA-positive serum sample, the sensitivity and the specificity of the CAEV/MVV iELISA were 74.0% (±7.6%) (95% confidence interval) and 98.3% (±2.0%), respectively, compared to the results of the radio-IP assay. Since the sensitivity was less than adequate, we reassessed the cutoff by calculating the mean value (in percent) ± 2 standard deviations for the radio-IP assay-negative serum samples. The results of that analysis placed the cutoff mean value at 33.1%. By using the new cutoff value, the sensitivity of the iELISA improved to 86.0% (±5.8%) and the specificity decreased slightly to 95.9% (±2.9%) compared to the results of the radio-IP assay. However, compared to the CAEV cELISA, which has a sensitivity of 98.6% and a specificity of 96.9% when the results of the radio-IP assay are used as the reference standard, the iELISA had a reduced sensitivity.Since the sera were taken from a number of different U.S. sheep kept under different husbandry and management conditions, we also wanted to test the performance of the CAEV/MVV iELISA with sera from one flock in which the sheep are exposed to the same husbandry and management conditions. Sera from an Idaho sheep flock (n = 405) consisting of sheep of the Rambouillet, Polypay, and Columbia breeds ages 3, 4, 5, and 6 years were tested by the iELISA. The results were compared to those of the CAEV cELISA by using the new iELISA cutoff value of 33.1%, and the discrepant samples were analyzed by Western blotting with OPPV WLC1 and by previously published methods (2). The positive and negative concordances of the CAEV cELISA and the CAEV/MVV iELISA were 92.5% (±3.1%) and 99.3% (±1.4%), respectively. Eighteen of 20 CAEV/MVV iELISA-negative and CAEV cELISA-positive serum samples tested positive by Western blot analysis, and the 2 remaining discrepant serum samples tested negative by Western blot analysis. One CAEV/MVV iELISA-positive and CAEV cELISA-negative sample tested negative by Western blot analysis. The 95% confidence interval for the positive and the negative concordances of the results of the CAEV/MVV iELISA relative to those of the CAEV cELISA for Idaho sheep and U.S. sheep overlapped (data not shown).A difference in the limit of detection between the CAEV/MVV iELISA and the CAEV cELISA may be a major reason for the reduced sensitivity of the CAEV/MVV iELISA (86%) compared to that of the CAEV cELISA (98.6%) with sera from U.S. sheep. Sera require dilution 1:10 for testing by the CAEV/MVV iELISA, whereas undiluted sera are used for the CAEV cELISA. To test whether the limit of detection is greater for the CAEV cELISA than the CAEV/MVV iELISA, 15 Western blot analysis-positive, CAEV cELISA-positive, and CAEV/MVV iELISA-negative serum samples from the Idaho flock were diluted 1:10 and 1:50 with 1× phosphate-buffered saline, pH 7.5, and retested by the CAEV cELISA. Twelve of these 15 serum samples tested positive by the CAEV cELISA at a 1:10 dilution, and 7 of 15 tested positive by the CAEV cELISA at a 1:50 dilution. This indicates that the CAEV cELISA has a higher dilution limit for the detection of anti-SRLV antibodies than the CAEV/MVV iELISA with sera from U.S. sheep. This high dilution limit of detection is likely due to the format of the CAEV cELISA, in which this assay is reliant upon anti-OPPV serum antibodies to inhibit the binding of a peroxidase-labeled monoclonal antibody (monoclonal antibody 74A) to a single, specific epitope on the CAEV-63 surface envelope glycoprotein.A previous report of 95.5% sensitivity and 97.2% specificity has been reported for the iELISA with sera from Swiss sheep (15). Large SRLV strain differences between Swiss MVV and U.S. OPPV strains may account for the lower sensitivity of the iELISA with sera from U.S. sheep. Therefore, gag, which encodes the capsid protein, was evaluated because it is a B-cell-immunodominant viral antigen in sheep naturally infected with MVV and OPPV and is more conserved than other viral genes (2, 7, 17). Unfortunately, no gag sequence is available for the Swiss MVV OLV (the strain used in the iELISA); however, several representative gag sequences from SRLV strains from Swiss goats and sheep that were previously reported for SRLV clades A1, A3, A4, A5, B1, and B2 were utilized (13). Genomic DNA was isolated from peripheral blood leukocytes from nine cELISA- and iELISA-positive Idaho sheep by previously described methods (6). The gag gene encoding the capsid was amplified by PCR with the following primers: primer GAGPSf (5′-TGG-CGA-CGC-AAG-GCT-CAA-A-3′) and primer GAGPSr (5′-GCG-GAC-GGC-ACC-ACA-CG-3′) (Integrated DNA Technology, Coralville, IA). The PCR mixture consisted of 100 ng of genomic DNA from sheep peripheral blood leukocytes or goat synovial membrane cells infected with WLC1, 2.5 mM_f (final concentration) MgCl₂, 0.2 mM_f deoxynucleoside triphosphates, and 1 U of Taq polymerase (Fisher Scientific, Pittsburg, PA). Primers GAGPSf and GAGPSr bind to nucleotides 734 to 752 and 2038 to 2054, respectively, of the sequence with GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AY101611","term_id":"21539308","term_text":"AY101611"}}AY101611. The amplification conditions for the gag PCR were as follows: 95°C for 4 min, followed by 25 cycles of 95°C for 30 s, 58.1°C for 30 s, and 72°C for 2 min, followed by 72°C for 7 min and 4°C indefinitely. The gag PCR products were analyzed, cloned, and sequenced by previously described methods (5). The gag sequences were further refined by hand by using the Se-Al (version 2.0) program (http://tree.bio.edu.ac.uk/software/seal/), and a consensus gag sequence was generated from gag sequences from four sheep and OPPV WLC1.For phylogenetic analysis, the appropriate model of nucleotide substitution was selected by using hierarchical likelihood ratio testing with the MrModelTest2 command block (11) executed in the PAUP program (14). A general time-reversible model (also called the GTR or REV model) (12) with invariant sites and a measure of the rate of heterogeneity determined by use of the gamma distribution was chosen for full Bayesian analysis by using the program MrBayes (8). A 50%-majority-rule consensus tree based on the gag sequences was constructed by using the Mesquite software package (10). The resulting Bayesian phylogenetic tree showed that the sequences from the Idaho sheep formed one clade with OPPV WLC1, OPPV 85/34, and a single Swiss goat SRLV strain (strain 5692 A3) with high posterior probabilities (0.99 to 1.0) (Fig. ​(Fig.1).1). The finding that three gag sequences from Swiss sheep are found in other clades aside from the U.S. sheep OPPV clade (6247 A1, 5720 B2, and 5776 B1) suggests that Swiss sheep SRLV strains are quite different from U.S. OPPV strains, and these differences contribute to the reduced sensitivity of the iELISA with sera from U.S. sheep compared to that achieved with sera from Swiss sheep.Open in a separate windowFIG. 1.A 50%-majority-rule consensus tree of SRLV gag nucleotide sequences obtained by Bayesian phylogenetic analyses with the general time-reversible model with invariant sites and a measure of the rate of heterogeneity. Consensus gag nucleotide sequences from Idaho sheep (sheep 28Oct76, 29Oct10, 28Oct101, 28Oct27, 28Oct83, 28Oct18, 28Oct89, 28Oct68, and 28Oct99) and a consensus gag nucleotide sequence for WLC1 were utilized and compared to the gag nucleotide sequences from other U.S. OPPV strains (85-34), MVV strains, and CAEV strains. The GenBank accession numbers are given in parentheses next to the name or identification number given in previous studies. The numbers on the branches represent the Bayesian posterior probabilities.This combination of information suggests that U.S. sheep maintain strains of small ruminant lentiviruses phylogenetically different (determined on the basis of the gag sequences) from those from most other parts of the world. This suggests that the creation and validation of one serological assay that is based on one viral strain and that is accepted worldwide might not be successful due to the diversity of SRLV strains worldwide (3, 9).     

Lack of trans-activation function for Maedi Visna virus and Caprine arthritis encephalitis virus Tat proteins

All lentiviruses contain an open reading frame located shortly upstream or inside of the env gene and encoding a small protein which has been designated Tat. This designation was mainly with respect to the positional analogy with the first exon of the trans-activator protein of the well studied human immunodeficiency virus type 1 (HIV-1). In this work we comparatively studied the trans- activation activity induced by Tat proteins of the small ruminant Maedi Visna virus (MVV) of sheep and Caprine arthritis encephalitis virus (CAEV) of goats on MVV and CAEV LTRs with that induced by the human lentivirus HIV-1 on its own LTR. The HIV-1 LTR alone weakly expresses the reporter GFP gene except when the HIV-1 Tat protein is coexpressed, the GFP expression is increased 60-fold. In similar conditions only minimal trans-activation increasing two- to three-fold the MVV and CAEV LTR activity was found with MVV Tat protein, and no trans-activation activity was detected in any used cell type or with any virus strain when CAEV Tat was tested. These results indicate that the small ruminant lentiviruses (SRLV) differ from the primate lentiviruses in their control of expression from the viral LTRs and put into question the biological role of the encoded protein named "Tat."     

Small ruminant lentivirus Tat protein induces apoptosis in caprine cells in vitro by the intrinsic pathway

The small ruminant lentiviruses, caprine arthritis-encephalitis virus (CAEV) and maedi visna virus (MVV) naturally cause inflammatory disease in goats and sheep, provoking chronic lesions in several different organs. We have previously demonstrated that in vitro infection of caprine cells by CAEV induces apoptosis through the intrinsic pathway (Rea-Boutrois, A., Pontini, G., Greenland, T., Mehlen, P., Chebloune, Y., Verdier, G. and Legras-Lachuer, C. 2008). In the present study, we used Tat deleted viruses and SLRV Tat-expression vectors to show that the SRLV Tat proteins are responsible for this apoptosis. We have also studied the activation of caspases-3, -8 and -9 by fluorescent assays in caprine cells expressing SRLV Tat proteins, and the effects of transfected dominant negative variants of these caspases, to show that Tat-associated apoptosis depends on activation of caspases-3 and -9, but not -8. A simultaneous disruption of mitochondrial membrane potential indicates an involvement of the mitochondrial pathway.     

Phylogeny of sheep and goat<Emphasis Type="Italic"> Theileria</Emphasis> and<Emphasis Type="Italic"> Babesia</Emphasis> parasites

The phylogenetic relationship of Theileria and Babesia species infecting sheep and goats on the basis of their 18S RNA gene structure was addressed in the present study. For this purpose, the complete sequences of the small ribosomal RNA genes of a panel of sheep and goat piroplasm isolates, including T. lestoquardi, T. ovis, T. separata, B. ovis, B. motasi, B. crassa and several novel species, were sequenced and compared. The classification based on the established phylogenetic tree corresponded with traditional systematics and revealed that sheep/goat piroplasm species are of polyphyletic origin. The independent evolution of almost all sheep/goat piroplasms suggests that speciation may have occurred after transfer of the piroplasm-transmitting tick from a primal wild ruminant host to domestic sheep and goats. In accordance with recent reports, our study confirms the existence of at least two additional sheep/goat piroplasm species, designated Theileria sp. 1 (China) and Theileria sp. 2 (China). The recently reported pathogenic sheep/goat Theileria sp. 1 (China) seems to be identical with a Theileria sp. isolated from Japanese serow. Furthermore, our results suggest that T. ovis represents a single species.     

Comparative analysis of Brucella serotype A and M and Yersinia enterocolitica O:9 polysaccharides for serological diagnosis of brucellosis in cattle, sheep, and goats.

Hapten polysaccharides of Brucella smooth M and A serotypes were prepared from Brucella sp. and Yersinia enterocolitica O:9 by previously described hydrolytic (O chain) or nonhydrolytic (native hapten [NH]) procedures. The purified polysaccharides differed only in the presence (O chain) or absence (NH) of lipopolysaccharide core sugars. The polysaccharides were compared by reverse radial immunodiffusion for the diagnosis of brucellosis in cattle (Brucella abortus biotype 1 [A serotype] and Brucella melitensis biotype 3 [AM serotype]), sheep (B. melitensis biotypes 1 [M serotype] and 3), and goats (B. melitensis biotype 1). The reverse radial immunodiffusion test with the NH from B. melitensis 16 M (serotype M) showed the highest sensitivity (89.6 to 97.3%), regardless of the host species and the serotype of the infecting Brucella sp. Y. enterocolitica O:9 NH (A serotype) was useful for diagnosing disease in cattle infected with B. abortus biotype 1, but not in cattle infected with B. melitensis biotype 3, sheep, or goats. The different results obtained with the serotype M and A polysaccharides and the sera from animals infected with M, A, and AM serotypes of Brucella spp. showed that in naturally infected animals, a large proportion of the antibodies are directed to or react with a previously defined common epitope(s) (J. T. Douglas and D. A. Palmer, J. Clin. Microbiol. 26:1353-1356, 1988) different from the A or M epitopes. By using the radial immunodiffusion test with B. melitensis 16M NH, it was possible to differentiate infected from vaccinated cattle, sheep, and goats with a sensitivity and specificity similar to that of the complement fixation test.     

Comparison and phylogenetic analysis based on the B2L gene of orf virus from goats and sheep in China during 2009-2011

As a zoonotic infectious disease, orf outbreaks have been reported in China in recent years. However, molecular epidemiology analysis has not been performed for Chinese orf virus (ORFV) strains. Here, we have identified 13 ORFVs from goats and sheep in China between 2009 and 2011. Thirty-four complete B2L sequences were used to construct a phylogenetic tree to elucidate the molecular epidemiology of ORFV in China. Nucleotide sequences of B2L genes of clinical samples and attenuated vaccine strains were aligned and compared. Three genotypes were found by molecular epidemiology analysis. Amino acid substitutions were dispersed among B2 polypeptides from wild and attenuated ORFV strains.     

Phylogenetic analysis of the gag region encoding the matrix protein of small ruminant lentiviruses: comparative analysis and molecular epidemiological applications

Little sequence information exists on the matrix-protein (MA) encoding region of small ruminant lentiviruses (SRLV). Fifty-two novel sequences were established and permitted a first phylogenetic analysis of this region of the SRLV genome. The variability of the MA encoding region is higher compared to the gag region encoding the capsid protein and surprisingly close to that reported for the env gene. In contrast to primate lentiviruses, the deduced amino acid sequences of the N- and C-terminal domains of MA are variable. This permitted to pinpoint a basic domain in the N-terminal domain that is conserved in all lentiviruses and likely to play an important functional role. Additionally, a seven amino acid insertion was detected in all MVV strains, which may be used to differentiate CAEV and MVV isolates. A molecular epidemiology analysis based on these sequences indicates that the Italian lentivirus strains are closely related to each other and to the CAEV-CO strain, a prototypic strain isolated three decades ago in the US. This suggests a common origin of the SRLV circulating in the monitored flocks, possibly related to the introduction of infected goats in a negative population. Finally, this study shows that the MA region is suitable for phylogenetic studies and may be applied to monitor SRLV eradication programs.