Alpacas were first introduced into South Africa during the year 2000. They are valuable because of the fine quality wool they produce which has much better insulation properties than that of merino wool fibres. Alpacas are also used to act as guards of sheep herds against predators. During 2008, blood samples from an alpaca that died acutely with severe lung oedema, respiratory distress and froth exuding from the nose were received at Elsenburg Veterinary Laboratory. The alpaca was from a herd of 23 alpacas of a British veterinarian in the Montagu district in the western Cape. Virus isolation attempts on the blood produced infrequent embryo mortalities. Embryonated chicken egg (ECE) material was send to the Virology Department at the Onderstepoort Veterinary Institute (OVI). A bluetongue virus (BTV) PCR performed at the diagnostic PCR laboratory at OVI on the ECE material was positive. Further intra-venous (IV) inoculations in ECE produced embryo mortalities on two consecutive days, the 8th and 9th November. The dead embryos were harvested separately and named and treated as two separate virus samples, Alp8 and Alp9 which were further passaged on baby hamster kidney (BHK) cells. The BTV virus neutralisation tests (VNT) performed at the Office International des Epizooties (OIE) Laboratory on both Alp8 and Alp9 were negative. Because of the close serological relationship between BTV and epizootic haemorrhagic disease virus (EHDV), an EHDV VNT was also performed and was also negative. In the light of the negative VNT and the positive BTV PCR results, more in-depth molecular analyses were performed. RNA was purified from tissue culture material and agarose gel electrophoresis (AGE) performed. Both Alp8 and Alp9 had a typical orbiviral electrophoretic profile, but their respective profiles were different. A sequence-independent reverse transcriptase PCR amplification method generated ample complementary DNA (cDNA) of both samples for sequencing. Sanger sequencing was used to partially sequence genome segments 5 (NS1) and 2 (VP2). BLAST analysis of the partial information of the genome segments 5 (NS1) of Alp8 confirmed it as being a BTV and Alp9 as being an EHDV. BLAST analysis of the deduced amino acid sequence generated of VP2 of both Alp8 and Alp9 established that these samples were possibly new serotypes of BTV and EHDV respectively. The complete genome of both Alp8 and Alp9 was sequenced with next generation 454 Pyrosequencing. This confirmed the partial sequencing results. BLAST analysis of the complete sequence of S2 (VP2) of Alp8 showed that it has 73 % nucleotide and 77 % deduced amino acid identity to BTV15. In contrast the nucleic acid sequence of genome segment S2 (VP2) of Alp9 had no nucleotide sequence identity to any virus, but its deduced amino acid sequence had 71 % amino acid identity to EHDV2. Hyper immune guinea pig (GP) serum prepared against the putative new BT (Alp8) and EHD (Alp9) virus serotypes were tested for serological cross-reactivity against the 24 OIE reference antigen strains of BTV and the 8 OIE reference antigen strains of EHDV. Alp8 had a neutralising antibody (NAb) titre of > 32 against BTV15. Alp9 did not cross react with any of the OIE BTV and EHDV strains. Six out of the remaining 22 alpacas on the farm had NAbs to a greater or lesser extend against Alp8 (BTV) and Alp9 (EHDV) viruses, which confirmed that the viruses were also present in other alpacas in the herd. Very few cases of EHDV in alpacas have ever been reported in literature. A small scale pilot vector susceptibility study showed that vector competence of C. imicola for both Alp8 and Alp9 was low, below 2 %. The fact that neutralising antibodies to Alp8 and Alp9 were detected in other alpacas in the herd raises the question as to whether there are other Culicoides species circulating in the area that could vector the viruses. In conclusion, the results from the serological and virological analyses as well as the nucleic acid sequence data of the genomes of two virus samples, Alp8 and Alp9, from an alpaca that died in the Montagu district in the western Cape identified Alp9 as a definite new serotype of EHDV and Alp8 as a possible new serotype of BTV most closely related to BTV15. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

Língua Azul (LA) é uma doença causada pelo Vírus da Língua Azul (VLA) e transmitida por vetores do gênero Culicoides. Estudos sorológicos tem demonstrado a ampla presença do vírus no Brasil, entretanto, informações clínicas da LA na América do Sul são limitadas. Este trabalho descreve alterações clínico-patológicas em ovinos acometidos pela LA no Estado do Rio Grande do Sul, 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. Seis ovinos que morreram, apresentaram à necropsia edema subcutâneo na face e região ventral do tórax, secreção nasal de coloração esverdeada, esôfago dilatado e preenchido por grande quantidade de conteúdo alimentar, pulmões não colabados, com áreas de consolidação anteroventrais, e luz da traquéia e brônquios com conteúdo espumoso misturado a conteúdo alimentar. No coração e base da artéria pulmonar observaram-se focos de hemorragia. Histologicamente as principais alterações observadas foram no tecido muscular cardíaco e esquelético, em especial no esôfago, com degeneração hialina e flocular, necrose bifásica e micro-mineralização de miofibras associadas a infiltrado inflamatório mononuclear. Pneumonia aspirativa associada à presença de material vegetal e bactérias na luz de brônquios também foi observada e foi considerada a responsável pela gravidade das manifestações clínicas e pelas mortes. O diagnóstico de LA foi confirmado pelo duplex RT-PCR com a identificação do VLA, sorotipo 12. / Bluetongue (BT) is a disease caused by bluetongue virus (BTV) and transmitted by vectors of the genus Culicoides. Serological studies have demonstrated the widespread distribution of the virus in Brazil, however, clinical information of BT in South America are limited. This paper describes clinical and pathological changes in sheep affected by BTV in southern Brazil. In two outbreaks on different farms, 15 sheep showed clinical signs 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. Six sheep that eventually died, shall necropsy showed subcutaneous edema in the face and ventral region of the chest, runny nose of green color, and dilated esophagus filled with adundant food content, lungs collapsed, with areas of antero ventral consolidation and trachea and bronchi containing foamy contents mixed with food. Heart and base of the pulmonary artery showing 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 duplex RT-PCR to the identification of the BTV, serotype 12.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Bluetongue (BT) is an infectious disease caused by bluetongue virus (BTV), which is transmitted by biting midges of the genus Culicoides, and affects domestic and wild ruminants, but its clinical manifestation is seen basically in sheep. Currently, 26 BTV serotypes are recognized worldwide. However, information about the disease in Brazil are limited, as only two BTV serotypes have been reported. Serological surveys reveal that Rio Grande do Sul (RS) has the lowest prevalence rates of infection among Brazilian states. This article describes the clinical, pathological and epidemiological aspects of 17 outbreaks of BT disease in sheep in the Central Region of RS state, Southern Brazil. Affected farms were visited for clinical examination, necropsy, sample collection and epidemiological investigation. The outbreaks were seasonal and occurred during the summer and fall. Of the 884 sheep in 17 small herds, 180 (20.4%) were affected. All ages of Texel and mixed breed sheep were affected. However, lambs (younger than one year) had higher morbidity than adult sheep. The most frequent clinical signs were anorexia, lethargy, loss of body condition, facial swelling mainly involving the lips, and greenish seromucous nasal discharge. Pulmonary edema, cardiac, skeletal muscle and esophageal striated muscle necrosis were the most prevalent findings. Erosive and ulcerative lesions in the upper gastrointestinal tract, and hemorrhage in the pulmonary artery also were common. The bluetongue virus (BTV) genome was detected by RT-PCR in blood, spleen, and lungs samples of 21 animals from 17 outbreaks. The virus involved in the outbreak 3 was isolated and shown to belong to serotype 17, for the first time reported in Brazil. Serology performed by agar gel immunodiffusion test (AGID) in 20 contact cattle showed seroconversion to BTV in 17 animals. In summary, our data support the BTV as the etiological agent of the outbreaks and indicate that the central region of RS is an area at risk for BT in sheep, a disease previously not recognized in the region. / Língua azul ou bluetongue (BT) é uma doença infecciosa causada pelo vírus da língua azul (BTV), que é transmitido por vetores hematófagos do gênero Culicoides e acomete ruminantes domésticos e selvagens, porém sua manifestação clínica é vista basicamente em ovinos. Atualmente, 26 sorotipos do BTV são conhecidos mundialmente. Entretanto, informações sobre a doença no Brasil são limitadas, com apenas dois sorotipos descritos. Pesquisas sorológicas revelam que o Rio Grande do Sul (RS) possui as menores taxas de prevalência de infecção entre os Estados brasileiros. O objetivo deste trabalho é descrever os aspectos epidemiológicos, clínicos e anatomopatológicos de 17 surtos de BT em ovinos na Região Central do RS. Para isso foram realizadas visitas às propriedades em que ocorreram os surtos para investigação epidemiológica e clínica, realização de necropsias e coleta de amostras. Os surtos foram sazonais e ocorreram durante o verão e outono. Em 17 pequenos rebanhos, de um total de 884 ovinos, 180 adoeceram (20,4%). Ovinos de todas as faixas etárias, da raça Texel e sem raça definida, foram acometidos. Entretanto, ovinos com menos de um ano de idade tiveram taxa de morbidade maior do que ovinos com um ano ou mais. Os sinais clínicos mais frequentes caracterizaram-se por anorexia, apatia, acentuada perda de peso, edema facial, envolvendo principalmente os lábios, e secreção nasal seromucosa e esverdeada. Edema pulmonar, necrose da musculatura cardíaca e esquelética e do músculo estriado do esôfago foram as lesões mais prevalentes. Erosões e úlceras no trato gastrointestinal superior e hemorragia na artéria pulmonar também foram comuns. O genoma do BTV foi detectado por meio da RT-PCR em amostras de sangue, baço e pulmão de 21 animais dos 17 surtos. O vírus envolvido no surto 3 foi isolado e pertence ao sorotipo 17, que pela primeira vez é descrito no Brasil. A sorologia foi realizada pelo método de imunodifusão em gel de ágar e demonstrou que 17 dos 20 bovinos que estavam em contato com os ovinos infectados soroconverteram ao BTV. Em síntese, nossos dados permitem concluir que o BTV é o agente causador dos surtos e indicam que a Região Central do RS é uma área de risco para a ocorrência de BT em ovinos, uma doença, até então, não reconhecida nessa região.

Língua Azul (LA) é uma doença causada pelo Vírus da Língua Azul (VLA) e transmitida por vetores do gênero Culicoides. Estudos sorológicos tem demonstrado a ampla presença do vírus no Brasil, entretanto, informações clínicas da LA na América do Sul são limitadas. Este trabalho descreve alterações clínico-patológicas em ovinos acometidos pela LA no Estado do Rio Grande do Sul, 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. Seis ovinos que morreram, apresentaram à necropsia edema subcutâneo na face e região ventral do tórax, secreção nasal de coloração esverdeada, esôfago dilatado e preenchido por grande quantidade de conteúdo alimentar, pulmões não colabados, com áreas de consolidação anteroventrais, e luz da traquéia e brônquios com conteúdo espumoso misturado a conteúdo alimentar. No coração e base da artéria pulmonar observaram-se focos de hemorragia. Histologicamente as principais alterações observadas foram no tecido muscular cardíaco e esquelético, em especial no esôfago, com degeneração hialina e flocular, necrose bifásica e micro-mineralização de miofibras associadas a infiltrado inflamatório mononuclear. Pneumonia aspirativa associada à presença de material vegetal e bactérias na luz de brônquios também foi observada e foi considerada a responsável pela gravidade das manifestações clínicas e pelas mortes. O diagnóstico de LA foi confirmado pelo duplex RT-PCR com a identificação do VLA, sorotipo 12. / Bluetongue (BT) is a disease caused by bluetongue virus (BTV) and transmitted by vectors of the genus Culicoides. Serological studies have demonstrated the widespread distribution of the virus in Brazil, however, clinical information of BT in South America are limited. This paper describes clinical and pathological changes in sheep affected by BTV in southern Brazil. In two outbreaks on different farms, 15 sheep showed clinical signs 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. Six sheep that eventually died, shall necropsy showed subcutaneous edema in the face and ventral region of the chest, runny nose of green color, and dilated esophagus filled with adundant food content, lungs collapsed, with areas of antero ventral consolidation and trachea and bronchi containing foamy contents mixed with food. Heart and base of the pulmonary artery showing 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 duplex RT-PCR to the identification of the BTV, serotype 12.

Culicoides (Diptera: Ceratopogonidae) are small (<3mm) blood feeding flies. These flies are biological vectors of viruses, protozoa and filarial nematodes affecting birds, humans, and other animals. Among the viruses transmitted those causing bluetongue (BT), African horse sickness (AHS) and epizootic haemorrhagic disease (EHD) are of major veterinary significance. Culicoides (Avaritia) imicola Kieffer, a proven vector of both AHS and BT viruses, is the most abundant and wide spread livestock-associated Culicoides species in South Africa. Field isolations of virus and oral susceptibility studies, however, indicated that a second Avaritia species, C. bolitinos Meiswinkel may be a potential vector of both BT virus (BTV) and AHS virus (AHSV). Differences in oral susceptibility, which are under genetic control, of populations from different geographical areas to viruses may be an indication of genetic differences between these populations, which may be the result of limited contact between these populations. A good knowledge of the distribution, spread and genetic structure of the insect vector is essential in understanding AHS or BT disease epidemiology. In the present study, an effort was made to gather field specimens of both C. imicola and C. bolitinos from different areas within their natural distribution in South Africa. The aim was to partially sequence two mitochondrial genes from these specimens and to analyse the sequence data making use of phylogenetic trees to clarify the genetic relationships between individuals or groups collected from geographically distinct sites. The two species were collected from four geographically separated areas in South Africa viz. Gauteng Province, Eastern Cape Province, Western Cape Province as well as the Free State Province. DNA was extracted from a total of 120 individual midges of the two Culicoides species using DNA extraction kits. Extracted DNA was analysed using PCR, sequencing as well as phylogenetic methods. A total of 117 mitochondrial DNA COI and 104 mitochondrial 16S ribosomal RNA Culidoides</i. sequences were analysed. DNA sequence polymorphism and phylogenetic relationships of various groups of C. imicola and C. bolitinos midges were determined. The results of the phylogenetic analysis of Culicoides populations using mitochondrial COI gene fragment showed that, at least one subpopulation of C. imicola and two distinct genotypes of C. bolitinos species do exist in South Africa, and further analysis is necessary. This study showed that COI has the potential to separate Culicoides midges based on their geography / Dissertation (MSc)--University of Pretoria, 2010. / Veterinary Tropical Diseases / unrestricted