Abortion and various associated risk factors in small ruminants in Algeria.

Identification of the causes of abortion among the huge population of small ruminants in Algeria (≈31 millions heads), is an important task for the control of livestock productivity and viability scourges to the small ruminants industry. Optimal production and utilization is constrained by a number of factors: disease, poor feeding and low management skills. Therefore, in the present study the prevalence of abortion in Algerian small ruminant's flocks was estimated and its possible association was correlated with infectious (PPR, BT and Brucellosis seropositivity) and managerial (flock size, grazing system, type of farming, and contact with other flocks) risk factors. The present study showed an overall flock prevalence of small ruminant's abortion as 75.33% (113/150) [95% CI 71.72-78.94%]. The risk factor analysis using multivariable logistic regression recognized the north-western and the steppe region as well as PPR positivity as a risk factor for abortion in Algerian small ruminant's flocks. The odds of flock abortion was 11.47 [95% CI 2.39-54.88; P=0.002] and 10.31 [95% CI 1.28-82.88; P=0.028] times higher in north-western and steppe regions respectively compared to other region. Also the presence of PPRV infection in small ruminant flocks amplified the odds by 6 times [95% CI 2.221-17.427; P=0.001].Surprisingly, the univariate analysis for the other risk factors associated with abortions in Algerian small ruminant flocks indicated no statistically significant links with bluetongue (P=1.000) and brucellosis seropositivity (P=0.334). Flock size (P=0.574), type of farming (P=0.443), grazing system (P=0.117) and contact with other flocks (P=0.245) was also not statistically significant. Our results revealed that abortion in small ruminants is a challenge to farmers and PPR was chiefly linked to it. Therefore an effective vaccination and control programme is advocated for small ruminants in Algeria.

Alterações clínicas e patológicas em ovinos infectados naturalmente pelo vírus da língua azul no Rio Grande do Sul / Clinical and pathological changes in sheep naturally infected with bluetongue virus in Rio Grande do Sul, Brazil

Língua azul (LA) é uma doença causada pelo vírus da língua azul (VLA) e transmitida por vetores do gênero Culicoides. Estudos sorológicos têm demonstrado a ampla presença do vírus no Brasil; entretanto, informações clínicas da LA na América do Sul são limitadas. Esse trabalho descreve alterações clínico-patológicas em ovinos acometidos pela LA no Sul do Brasil. Em dois surtos, em propriedades distintas, 15 ovinos apresentaram como principais sinais clínicos hipertermia, apatia, aumento de volume da face e região submandibular, dificuldade de deglutição com regurgitação, secreção nasal mucopurulenta esverdeada, alterações respiratórias, além de acentuada perda de peso e erosões na mucosa oral. Os achados de necropsia em seis ovinos afetados incluíram edema subcutâneo na face e região ventral do tórax, secreção nasal esverdeada, esôfago dilatado preenchido por grande quantidade de conteúdo alimentar, pulmões não colabados com áreas consolidadas anteroventrais, bem como luz da traquéia e brônquios preenchida por espuma misturada com conteúdo alimentar. No coração e base da artéria pulmonar, havia focos de hemorragia. Histologicamente, as principais alterações observadas ocorriam no tecido muscular cardíaco e esquelético, especialmente no esôfago e consistiam de lesões bifásicas caracterizadas por degeneração/necrose hialina e flocular de miofibras associadas com micro-calcificação e infiltrado inflamatório mononuclear. Pneumonia aspirativa associada à presença de material vegetal e bactérias na luz de brônquios também foi observada. O diagnóstico de LA foi confirmado pela detecção do genoma viral por duplex RT-PCR em amostras de sangue de animais afetados, seguido da identificação do VLA, sorotipo 12 por sequenciamento.

Bluetongue (BT) is a disease caused by bluetongue virus (BTV) and transmitted by vectors of the genus Culicoides. Serological studies have demonstrated the widespread presence of the virus in Brazil, however, clinical information of BT in South America are limited. This article describes clinical and pathological changes observed in sheep naturally infected by BTV in southern Brazil. In two outbreaks on different farms, 15 sheep showed clinical signs such as severe hyperthermia, apathy, swelling of the face and submandibular area, difficulty in swallowing with regurgitation, greenish mucopurulent nasal secretion, severe weight loss, and erosions in the oral mucosa. Necropsy findings in six sheep included subcutaneous edema of the face and ventral region of the chest, greenish nasal discharge, and dilated esophagus filled with abundant food contents, collapsed lungs with areas of anteroventral consolidation, and trachea and bronchi filled by foamy material mixed with food. In the heart and base of the pulmonary artery there were foci of hemorrhage. Histologically, the main changes were in cardiac and skeletal muscles and consisted of biphasic lesions characterized by hyaline and floccular degeneration/necrosis of myofibers associated with micro-mineralization and mononuclear cell infiltration. Pneumonia associated with the presence of organic matter and bacteria in the lumen of the bronchi was also observed. The diagnosis of BT was confirmed by detection of the viral genome by duplex RT-PCR in blood of affected animals, followed by the identification of BTV, serotype 12 by nucleotide sequencing.

Some epidemiological and economic aspects of a bluetongue-like disease in cattle in South Africa--1995/96 and 1997.

In December 1995 to March 1996 and the early summer of 1997 South Africa experienced above average rainfall which favoured the occurrence of Culicoides transmitted diseases. During this period several outbreaks of an uncommon disease of cattle occurred over a large part of the country. The clinical signs were similar to those of infection with the viruses of bluetongue (BT) and epizootic haemorrhagic disease of deer (EHD). Virus isolation from cattle and Culicoides yielded both viruses. Dual infections occurred on several farms. Typing of BT isolates yielded types 2, 3, 6 and 8. On at least two farms more than one BT virus serotype was involved. On one farm only EHD virus could be isolated from cattle and Culicoides. Serological tests confirmed that on this farm the disease was caused by EHD. In 1932/33, when a similar disease was reported conditions were vastly different. Rainfall figures show that the 1932/33 season was exceptionally dry. Techniques available at that time could not identify EHD and the cause was reported to be BT. The occurrence of BT in a dry season and over a much wider area than the distribution in South Africa of Culicoides imicola, the only proven vector for BT, is a clear indication that other species less dependent on high rainfall are involved. The present isolation of BT virus from three of five pools of parous C. bolitinos is evidence that this species, which breeds in cattle dung, may be an additional vector for BT.

Virulence-associated antigenic and genetic characteristics of bluetongue virus-17 isolates.

Bluetongue virus (BLU), an orbivirus, is of importance to the sheep and cattle industries. We have obtained 5 United States BLU-17 isolates which have been tested for virulence in sheep and 16 BLU-17 field isolates from the Caribbean and Central America. Using a panel of 15 monoclonal antibodies (MAb) against an avirulent BLU-17, we observed that 6 MAbs had negligible or very low neutralization titers for the virulent isolates in contrast to moderate to high titers for the avirulent isolates. These MAbs also differentiated the field isolates into two groups--inadequate vs effective neutralization. All 6 MAbs immunoprecipitated the outer capsid protein, VP2. Electropherotyping of genomic RNA from all 21 viruses identified an increase in RNA segment 3 mobility for those isolates which were not neutralized by the 6 specific MAbs. RNA segment 3 codes for the inner core protein, VP3. There were no detectable electrophoretic differences for RNA segment 2, which encodes VP2. In summary, the virulent BLU-17 isolates differed from the avirulent isolates in both the antigenicity of the outer capsid protein, VP2, and the electrophoretic mobility of RNA segment 3, and we hypothesize that one or both of these changes may result in BLU virulence.

Phylogenetic characterisation of bluetongue viruses from naturally-infected insects, cattle and sheep in Australia.

The polymerase chain reaction was used to detect the presence of bluetongue virus (BTV) in a number of clinical and insect samples collected in the Northern Territory of Australia. Sequence analyses of the amplified BTV genes differentiated endemic Australian and exotic viruses. Two potential exotic BTV were detected as a result of PCR analyses of blood from sentinel animals and of the insect vector, Culicoides wadai. The detection of BTV in C wadai was the first direct demonstration of the presence of BTV in this potential vector. This new technology can significantly reduce the time taken for a diagnosis from a clinical sample and increase the amount of useful information obtained on a BTV isolate by using rapid sequencing techniques. Sequence data were used to differentiate between BTV20 isolated in 1975 and two isolates of the same serotype, isolated in 1992, and indicated that the latter were probably a recent incursion into Australia from Indonesia due to their greater VP3 sequence homology to the BTV9 (Java) than to Australian BTV isolates.

Bluetongue virus infection in sheep: haematological changes and detection by polymerase chain reaction.

Eight sheep vaccinated with 10(6) pfu of attenuated Australian bluetongue virus serotype 23 (BTV 23A) and eight BTV-free sheep were challenged with virulent BTV 23. There was little subsequent variation in the mean clinical score, or in the mean lymphocyte and platelet concentrations in the peripheral blood of the eight vaccinated sheep. There was a marked thrombocytopenia and lymphopenia in the naive sheep as the mean lymphocyte and platelet concentrations fell to a minimum at days 8 and 11 after inoculation, respectively. Similar changes were observed in three other naive sheep inoculated with field isolates of BTV 1,9 or 23. BTV was detected by nested polymerase chain reaction in whole blood of these sheep between days 6 and 28, in mononuclear leukocytes between days 3 and 14, and in platelets between days 6 and 21.

Diagnostic analysis of the prolonged bluetongue virus RNA presence found in the blood of naturally infected cattle and experimentally infected sheep.

Bluetongue virus (BTV) RNA was detected by the polymerase chain reaction (PCR) in the blood of 24 naturally infected cattle as long as 160 days after the estimated date of infection. Blood samples from these animals and from 10 experimentally BTV-infected sheep, which also exhibited a prolonged hematologic BTV RNA presence, were concurrently evaluated for viral infectivity. Infectivity analyses were conducted using the sentinel sheep inoculation and embryonated chicken egg inoculation procedures. Blood specimens from the experimental sheep 50, 56, 71, and 89 days after BTV inoculation were uniformly negative for viral infectivity despite their uniformly positive status with PCR evaluation. Three collections of blood from the naturally infected cattle at least 100, 135, and 160 days after infection also revealed no recoverable viral infectivity but an initially high and progressively decreasing prevalence of BTV with the PCR technique. These retrospective epidemiologic and prospective experimental approaches were concordant in that both studies demonstrated consistent discrepancies between the viral infectivity and the PCR diagnostic data. The significance of these discrepancies is discussed with respect to Koch's postulates and with respect to the possibility that the biological vector of BTV (Culicoides variipennis) may recover BTV infectivity from PCR-positive but virus isolation-negative blood.

Bluetongue virus isolations from vectors and ruminants in Central America and the Caribbean. Interamerican Bluetongue Team.

A regional prospective study of the epidemiology of bluetongue virus (BTV) serotypes covering 11 countries in Central America and the Caribbean took place between 1987 and 1992. Active surveillance revealed BTV infection to be endemic in the absence of confirmed indigenous cases of bluetongue. During the 6-year span of the study, over 300 BTV isolations were obtained from cattle and sheep. Results of the earlier years of the study were summarized, and surveillance activities in the concluding months of the study from November 1990 to February 1992 were evaluated. Forty-five BTV isolations were made during this time, 44 from sentinel cattle and 1 from a ram with clinical signs compatible with contagious ecthyma. Virus isolation from potential vectors also was attempted, yielding a further 9 BTV isolates from parous Culicoides insignis and C pusillus, 2 BTV isolates from blood-engorged C filarifer, and 1 epizootic hemorrhagic disease virus type-2 isolate from parous C pusillus. Our extensive network of sentinel herds in the region detected BTV-1 as the predominant serotype in Central America in 1991, after an apparent absence of 1 year in the sentinel animals. Other serotypes in Central America at that time included BTV-3 and BTV-6. In Puerto Rico and the Dominican Republic, BTV-4 became the predominant serotype, without detection of BTV-8 and BTV-17, which were common in recent years of the study. The serotypes found in the Caribbean Basin continued to have marked differences from those in North America. The importance of viewing bluetongue as an infection, the distribution of which is determined principally by ecologic factors, is emphasized.

Association of bluetongue virus gene segment 5 with neuroinvasiveness.

Two strains (UC-2 and UC-8) of bluetongue virus were used to determine genetic factors influencing neuroinvasiveness. Reassortants were produced in vitro, and the parental origins of their genes were determined by polyacrylamide gel electrophoresis profiles and restriction endonuclease digestion. Gene segment 5 of UC-8 correlated with neuroinvasiveness of reassortants when inoculated subcutaneously into newborn mice.

Detection of bluetongue virus in the blood of inoculated calves: comparison of virus isolation, PCR assay, and in vitro feeding of Culicoides variipennis.

The interval after infection when bluetongue virus (BTV) was present in the blood of calves inoculated with BTV serotype 10 (BTV 10) was evaluated by virus isolation (VI) in embryonated chicken eggs (ECE), BTV-specific polymerase chain reaction (PCR), and in vitro blood feeding of vector Culicoides variipennis (C.v.) sonorensis. BTV nucleic acid was detected by PCR in blood cells for 16 to 20 weeks after infection whereas infectious virus was detected by VI in ECE for 2 to 8 weeks. BTV was detected in calf blood by in vitro feeding of C.v. sonorensis for only 0 to 2 weeks after inoculation of calves with BTV 10. Selected bloods which were positive by PCR analysis but not by VI in ECE were not infectious for sheep. The data are consistent with the hypothesis that prolonged viremia in BTV-infected cattle results from association of the virus with blood cells, especially erythrocytes. The fact that calf blood that contained viral nucleic acid as determined by PCR analysis, but not infectious virus as determined by VI in ECE, was not infectious for either the insect vector or sheep suggests that cattle whose blood contains BTV nucleic acid but not infectious virus are unimportant to the epidemiology of BTV infection.

The use of discriminant analysis in predicting the distribution of bluetongue virus in Queensland, Australia.

The climatic variables that were most useful in classifying the infection status of Queensland cattle herds with bluetongue virus were assessed using stepwise linear discriminant analysis. A discriminant function that included average annual rainfall and average daily maximum temperature was found to correctly classify 82.6% of uninfected herds and 72.4% of infected herds. Overall, the infection status of 74.1% of herds was correctly classified. The spatial distribution of infected herds was found to parallel that of the suspected vector, Culicoides brevitarsis. This evidence supports the role of this arthropod species as a vector of bluetongue viruses in Queensland. The effect of potential changes in temperature and rainfall (the so-called 'global warming' scenario) on the distribution of bluetongue virus infection of cattle herds in Queensland was then investigated. With an increase in both rainfall and temperature, the area of endemic bluetongue virus infection was predicted to extend a further 150 km in and in southern Queensland. The implications of this for sheep-raising in Queensland are discussed.

Heterogeneity of the L2 gene of field isolates of bluetongue virus serotype 17 from the San Joaquin Valley of California.

Genome segment 2 (L2) from six field isolates of bluetongue virus (BTV) serotype 17 was sequenced by cycling sequencing after the amplification of the viral cDNA by the polymerase chain reaction. The viruses were isolated from sheep, cattle and a goat in the San Joaquin Valley of California during the years 1981 and 1990. These viruses exhibit divergent patterns of neutralization with BTV 17-specific monoclonal antibodies. The six L2 genes of the BTV 17 field isolates all encode a protein of 955 amino acids. Similarity of the nucleotide sequences of the L2 genes with respect to the prototype strain ranges between 93.8% and 95.1%, whereas the similarity between the field isolates ranges from 96.8% to 99.1%. Although very closely related, the L2 gene of each virus is distinct. Furthermore, mutations in the L2 gene of field isolates of BTV do not consistently follow a linear pattern of accumulation over time. Some amino acid changes in the VP2 protein of field strains were conserved over time, whereas others were not correlated with the year of isolation and some substitutions were unique to individual viruses. The predicted VP2s constitute a group of non-identical, but closely related proteins. Phylogenetic analyses suggest that the viral variants which co-circulate in the San Joaquin Valley could evolve by different evolutionary pathways.

Prevalence of bluetongue virus expression in leukocytes from experimentally infected ruminants.

Replication of bluetongue virus (BTV) in leukocytes from the blood of sheep, cattle, elk, and mule deer inoculated with BTV serotype 10 or 17 was assessed by immunocytochemical staining and dot blot northern hybridization to determine if differences in the prevalence of infection in this blood fraction might account for the differences in clinical disease among these species. Viremia was confirmed by virus isolation in all inoculated animals. Analysis of leukocytes with monoclonal antibodies specific for BTV proteins revealed low numbers of infected leukocytes in only 2 sheep 8 days after inoculation with BTV serotype 10. Most of the cells expressing BTV were identified morphologically as monocytes; approximately 10% of infected cells were lymphocytes. Bluetongue virus was not detected by use of dot-blot hybridization on samples of blood. Our results suggest that differential infection of leukocytes does not account for the pronounced differences in clinical signs and pathologic changes among ruminants.

Neutralization determinants of United States bluetongue virus serotype ten.

A panel of five neutralization-resistant escape mutant (EM) viruses was used to investigate the neutralization determinants of the U.S. prototype strain of bluetongue virus serotype 10 (BTV-10). The phenotypic properties of each EM virus were characterized by neutralization and immuneprecipitation assays with a panel of four monoclonal antibodies (MAbs). These MAbs were used to select the various EM viruses and together the MAbs define four distinct neutralizing epitopes on the prototype strain of BTV-10 (Heidner, H.W., Rositto, P.V., and MacLachlan, N.J., Virology 176, 658-661 (1990)). Sequencing of the L2 gene identified mutations responsible for the altered phenotypic properties exhibited by each EM virus. The L2 gene encodes BTV outer capsid protein VP2 which is responsible for virus neutralization. Four amino acids in three distinct regions of VP2 are critical to expression of the epitopes recognized by the MAb panel. Both amino acid 208 and 211 can affect the binding of MAb 039 and MAb 045, amino acid 327 affects binding of MAb 041, and amino acids 327 and 402 cooperatively interact to affect binding by MAb 034. The location of two of these critical regions on VP2 of BTV-10 is identical to two of those which affect neutralization of Australian BTV-1, despite the fact that these two viruses are antigenically distinct and have divergent L2 gene sequences (Gould, A.R., and Eaton, B.T., Virus Res. 17, 161-172 (1990)). The four individual neutralizing epitopes on VP2 of BTV-10 are interactive (Heidner, H.W., Rositto, P.V., and MacLachlan, N.J., Virology 176, 658-661 (1990)) and at least two are conformationally dependent.

Detection of bluetongue virus RNA in cell cultures and in the central nervous system of experimentally infected mice using in situ hybridization.

Two radiolabelled complementary DNA (cDNA) probes (1663 bp and 200 bp respectively) were prepared from the genome segment that encodes the non-structural protein 1 (NS1) of bluetongue virus serotype 4 (BTV4). The probes were used to optimize the in situ hybridization (ISH) method on baby hamster kidney-21 (BHK-21) cells and to investigate the use of the technique as a diagnostic procedure. Cells were infected with BTV4 at a multiplicity of infection of 0.5 PFU/cell. An intense cytoplasmic hybridization signal could be detected from 3 hours post-infection onwards, reaching a peak at 17 hours. The ISH procedure has potential use as a diagnostic technique, but will probably find a wider application in pathogenesis studies. An in situ hybridization method was also developed for the detection of BTV RNA in the central nervous system of newborn mice after intracranial inoculation with BTV10. Viral RNA-positive cells were detected from Day 3 onwards, predominantly in areas where the virus caused necrotic encephalitis.

Clinical pathology of Australian bluetongue virus serotype 16 infection in merino sheep.

Viraemic blood from an ox naturally infected with Australian bluetongue (BLU) virus serotype 16 was passaged twice in sheep. Twelve 2- to 4-years-old Merino ewes, negative in a bluetongue agar gel immunodiffusion test, were inoculated with viraemic blood from the second sheep passage. They were examined for 18 days and compared with a control group. Significant changes in haematological measurements, namely packed cell volume, total white cell count and lymphocyte count, and in plasma enzyme concentrations, namely aspartate transaminase and creatine kinase, occurred in the infected sheep. All infected sheep became sick. The antibody response, and clinical and necropsy findings were consistent with other reports of mild to moderate disease with Australian BLU serotypes.

Comparison of slot blot nucleic acid hybridization, immunofluorescence, and virus isolation techniques to detect bluetongue virus in blood mononuclear cells from cattle with experimentally induced infection.

A slot blot hybridization technique was applied for detection of bluetongue virus (BTV) in blood mononuclear cells (BMNC) obtained from cattle with experimentally induced infection. This technique lacked sensitivity to detect the viral nucleic acid directly in clinical specimens. When aliquots of mononuclear cells from these cattle were cultivated in vitro for 10 days to amplify virus titer, only 33.3% of the samples collected during viremia gave a positive signal in the slot blot hybridization format. By contrast, results for 34.3% of noncultured and 63.3% of cultured mononuclear cell samples collected during viremia were positive by immunofluorescence. The average number of infected cells, as detected by immunofluorescence in the noncultured mononuclear cell samples, was 1 to 5/300,000, and was usually > 10/300,000 in the cultured cell samples. Virus was isolated from all postinoculation blood samples obtained from 4 heifers that were seronegative at the time of inoculation, but was not isolated from any of the preinoculation samples, or from any of the postinoculation samples obtained from 2 heifers that were seropositive at the time of inoculation. When virus isolation was attempted from separated mononuclear cells in 2 heifers, 43.7% of the noncultured and 87.5% of the cultured samples had positive results.

Experimental bluetongue and epizootic hemorrhagic disease virus infection in California black-tailed deer.

Four adult black-tailed deer (Odocoileus hemioneus columbianus) and five fawns were inoculated with bluetongue virus (BTV) and one adult deer was inoculated with epizootic hemorrhagic disease (EHD) virus to produce clinical signs and lesions of hemorrhagic disease. Serologic response was monitored using the agar gel immunodiffusion (AGID) test and the competitive enzyme-linked immunosorbent assay (C-ELISA). Embryonating chicken eggs and vero cells were used to detect viremia. No animal exhibited clinical or pathologic signs of hemorrhagic disease. Bluetongue viremia was detected as early as 2 days post-inoculation (DPI-2) and in some animals, persisted until at least DPI-12. The earliest detection of BTV antibodies using the AGID was DPI-8. Two adult deer remained seropositive for BTV antibodies for > 9 mo and 1 yr, respectively, using both the AGID and C-ELISA tests. We observed cross reactions between BT and EHD antibodies using the AGID tests. Also, the AGID test did not consistently detect exposure to BTV. Viremia was not detected in the deer inoculated with EHD although this animal was AGID positive between DPI-6 and DPI-49.