Evaluation of cross-protection of bluetongue virus serotype 4 with other serotypes in sheep

Bluetongue (BT) is a non-contagious disease of sheep and other domestic and wild ruminants caused by the bluetongue virus (BTV). Currently 26 serotypes of the virus have been identified. In South Africa, 22 serotypes have been identified and BT is controlled mainly by annual vaccinations using a freeze-dried live attenuated polyvalent BTV vaccine. The vaccine is constituted of 15 BTV serotypes divided into three separate bottles and the aim is to develop a vaccine using fewer serotypes without compromising the immunity against the disease. This study is based on previously reported cross-neutralisation of specific BTV serotypes in in vitro studies. Bluetongue virus serotype 4 was selected for this trial and was tested for cross-protection against serotype 4 (control), 1 (unrelated serotype), 9, 10 and 11 in sheep using the serum neutralisation test. The purpose of the study was to determine possible cross-protection of different serotypes in sheep. Of those vaccinated with BTV-4 and challenged with BTV-1, which is not directly related to BTV-4, 20% were completely protected and 80% showed clinical signs, but the reaction was not as severe as amongst the unvaccinated animals. In the group challenged with BTV-10, some showed good protection and some became very sick. Those challenged with BTV-9 and BTV-11 had good protection. The results showed that BTV-4 does not only elicit a specific immune response but can also protect against other serotypes

Seroprevalence of Rift Valley fever and lumpy skin disease in African buffalo (Syncerus caffer) in the Kruger National Park and Hluhluwe-iMfolozi Park, South Africa

Rift Valley fever and lumpy skin disease are transboundary viral diseases endemic in Africa and some parts of the Middle East, but with increasing potential for global emergence. Wild ruminants, such as the African buffalo (Syncerus caffer), are thought to play a role in the epidemiology of these diseases. This study sought to expand the understanding of the role of buffalo in the maintenance of Rift Valley fever virus (RVFV) and lumpy skin disease virus (LSDV) by determining seroprevalence to these viruses during an inter-epidemic period. Buffaloes from the Kruger National Park (n = 138) and Hluhluwe-iMfolozi Park (n = 110) in South Africa were sampled and tested for immunoglobulin G (IgG) and neutralising antibodies against LSDV and RVFV using an indirect enzyme-linked immunosorbent assay (I-ELISA) and the serum neutralisation test (SNT). The I-ELISA for LSDV and RVFV detected IgG antibodies in 70 of 248 (28.2%) and 15 of 248 (6.1%) buffaloes, respectively. Using the SNT, LSDV and RVFV neutralising antibodies were found in 5 of 66 (7.6%) and 12 of 57 (21.1%), respectively, of samples tested. The RVFV I-ELISA and SNT results correlated well with previously reported results. Of the 12 SNT RVFV-positive sera, three (25.0%) had very high SNT titres of 1:640. Neutralising antibody titres of more than 1:80 were found in 80.0% of the positive sera tested. The LSDV SNT results did not correlate with results obtained by the I-ELISA and neutralising antibody titres detected were low, with the highest (1:20) recorded in only two buffaloes, whilst 11 buffaloes (4.4%) had evidence of co-infection with both viruses. Results obtained in this study complement other reports suggesting a role for buffaloes in the epidemiology of these diseases during inter-epidemic periods

Insights into the pathogenesis of viral haemorrhagic fever based on virus tropism and tissue lesions of natural Rift Valley fever

Rift Valley fever phlebovirus (RVFV) infects humans and a wide range of ungulates and historically has caused devastating epidemics in Africa and the Arabian Peninsula. Lesions of naturally infected cases of Rift Valley fever (RVF) have only been described in detail in sheep with a few reports concerning cattle and humans. The most frequently observed lesion in both ruminants and humans is randomly distributed necrosis, particularly in the liver. Lesions supportive of vascular endothelial injury are also present and include mild hydropericardium, hydrothorax and ascites; marked pulmonary congestion and oedema; lymph node congestion and oedema; and haemorrhages in many tissues. Although a complete understanding of RVF pathogenesis is still lacking, antigen-presenting cells in the skin are likely the early targets of the virus. Following suppression of type I IFN production and necrosis of dermal cells, RVFV spreads systemically, resulting in infection and necrosis of other cells in a variety of organs. Failure of both the innate and adaptive immune responses to control infection is exacerbated by apoptosis of lymphocytes. An excessive proinflammatory cytokine and chemokine response leads to microcirculatory dysfunction. Additionally, impairment of the coagulation system results in widespread haemorrhages. Fatal outcomes result from multiorgan failure, oedema in many organs (including the lungs and brain), hypotension, and circulatory shock. Here, we summarize current understanding of RVF cellular tropism as informed by lesions caused by natural infections. We specifically examine how extant knowledge informs current understanding regarding pathogenesis of the haemorrhagic fever form of RVF, identifying opportunities for future research

An investigation into the possibility of bluetongue virus transmission by transfer of infected ovine embryos

Bluetongue (BT), a disease that affects mainly sheep, causes economic losses owing to not only its deleterious effects on animals but also its associated impact on the restriction of movement of livestock and livestock germplasm. The causative agent, bluetongue virus (BTV), can occur in the semen of rams and bulls at the time of peak viraemia and be transferred to a developing foetus. The risk of the transmission of BTV by bovine embryos is negligible if the embryos are washed according to the International Embryo Transfer Society (IETS) protocol. Two experiments were undertaken to determine whether this holds for ovine embryos that had been exposed to BTV. Firstly, the oestrus cycles of 12 ewes were synchronised and the 59 embryos that were obtained were exposed in vitro to BTV-2 and BTV-4 at a dilution of 1 x 10(2.88) and 1 x 10(3.5) respectively. In the second experiment, embryos were recovered from sheep at the peak of viraemia. A total of 96 embryos were collected from BTV-infected sheep 21 days after infection. In both experiments half the embryos were washed and treated with trypsin according to the IETS protocol while the remaining embryos were neither washed nor treated. All were tested for the presence of BTV using cell culture techniques. The virus was detected after three passages in BHK-21 cells only in one wash bath in the first experiment and two unwashed embryos exposed to BTV-4 at a titre of 1 x 10(3.5). No embryos or uterine flush fluids obtained from viraemic donors used in the second experiment were positive for BTV after the standard washing procedure had been followed. The washing procedure of the IETS protocol can thus clear sheep embryos infected with BTV either in vitro or in vivo

An investigation into the possibility of bluetongue virus transmission by transfer of infected ovine embryos

Bluetongue (BT), a disease that affects mainly sheep, causes economic losses owing to not only its deleterious effects on animals but also its associated impact on the restriction of movement of livestock and livestock germplasm. The causative agent, bluetongue virus (BTV), can occur in the semen of rams and bulls at the time of peak viraemia and be transferred to a developing foetus. The risk of the transmission of BTV by bovine embryos is negligible if the embryos are washed according to the International Embryo Transfer Society (IETS) protocol. Two experiments were undertaken to determine whether this holds for ovine embryos that had been exposed to BTV. Firstly, the oestrus cycles of 12 ewes were synchronised and the 59 embryos that were obtained were exposed in vitro to BTV-2 and BTV-4 at a dilution of 1 x 102.88 and 1 x 103.5 respectively. In the second experiment, embryos were recovered from sheep at the peak of viraemia. A total of 96 embryos were collected from BTV-infected sheep 21 days after infection. In both experiments half the embryos were washed and treated with trypsin according to the IETS protocol while the remaining embryos were neither washed nor treated. All were tested for the presence of BTV using cell culture techniques. The virus was detected after three passages in BHK-21 cells only in one wash bath in the first experiment and two unwashed embryos exposed to BTV-4 at a titre of 1 x 103.5. No embryos or uterine flush fluids obtained from viraemic donors used in the second experiment were positive for BTV after the standard washing procedure had been followed. The washing procedure of the IETS protocol can thus clear sheep embryos infected with BTV either in vitro or in vivo

The effect of Rift Valley fever virus Clone 13 vaccine on semen quality in rams

Rift Valley fever (RVF) is an arthropod-borne viral disease of importance in livestock and humans. Epidemics occur periodically in domestic ruminants. People in contact with infected livestock may develop disease that varies from mild flu-like symptoms to fatal viraemia. Livestock vaccination may assist in disease control. Rift Valley fever virus (RVFV) Clone 13 is a relatively new vaccine against RVF, derived from an avirulent natural mutant strain of RVFV, and has been shown to confer protective immunity against experimental infection with RVFV. The hypothesis tested in the current trial was that rams vaccinated with RVFV Clone 13 vaccine would not experience a reduction in semen quality (measured by evaluating the percentage progressively motile and percentage morphologically normal spermatozoa in successive ejaculates) relative to unvaccinated control animals. Ram lambs were screened for antibodies to RVFV using a serum neutralisation test. Animals without detectable antibodies (n = 23) were randomly allocated to either a test group (n = 12) or a control group (n = 11). Animals in the test group were vaccinated with RVFV Clone 13 vaccine. Daily rectal temperature measurements and weekly semen and blood samples were taken from all animals. Seven animals were eliminated from the statistical analysis because of potential confounding factors. Logistic regression analysis was performed on data gathered from the remaining animals to determine whether an association existed between animal group, rectal temperature and semen quality parameters. No correlation existed between the treatment group and values obtained for the semen quality parameters measured. There was no statistically significant post-vaccination decline in the percentage of live morphologically normal spermatozoa, or the percentage of progressively motile spermatozoa, either when assessed amongst all animals or when assessed within individual groups. A repeat study with a larger sample size and a more comprehensive pre-screening process may be indicated to avoid the inclusion of unsuitable animals

Molecular differentiation and pathogenicity of Aviadenoviruses isolated during an outbreak of inclusion body hepatitis in South Africa

Fowl adenovirus (FAdV) is a member of the genus Aviadenovirus and causes a number of economically important poultry diseases. One of these diseases, inclusion body hepatitis (IBH), has a worldwide distribution and is characterised by acute mortality (5% - 20%) in production chickens. The disease was first described in the United States of America in 1963 and has also been reported in Canada, the United Kingdom, Australia, France and Ireland, but until now, not in South Africa. Adenoviruses isolated from the first outbreak of IBH in South Africa were able to reproduce the disease in chicken embryo livers. The aim of the present study was to characterise the viruses and determine the pathogenicity of the FAdV strains responsible for the first reported case of IBH in South Africa. Polymerase chain reaction (PCR) amplification of the L1 loop region of the fowl adenovirus hexon gene using degenerate primer pair hexon A/B was used to identify the viruses that were isolated. Restriction fragment length polymorphism (RFLP) of the amplification products was used for the differentiation of 14 isolates of fowl adenovirus. Sequencing of the PCR products followed by amino acid comparison and phylogenetic analysis using the L1 loop region of the hexon protein was done to determine the identity of the isolates. Amino acid sequences of the hexon genes of all the South African isolates were compared with those of reference strains representing FAdV species. Amino acid comparison of 12 South Africa field isolates to FAdV reference strains revealed a high sequence identity (> 93.33%) with reference strains T8-A and 764. Two of the isolates had high sequence identity (93.40%) with reference strains P7-A, C2B and SR48. Phylogenetic analysis of the L1 loop region of the hexon protein of all 14 South African isolates was consistent with their RFLP clusters. The mortality rates of embryos challenged with 10(6) egg infective doses (EID50) FAdV 2 were 80% - 87% and mortality rates for embryos challenged with 10(5.95) (EID50) FAdV 8b were 65% - 80%

Inclusion body hepatitis associated with an outbreak of fowl adenovirus type 2 and type 8b in broiler flocks in South Africa

Inclusion body hepatitis is an acute disease of chickens ascribed to viruses of the genus Aviadenovirus and referred to as fowl adenovirus (FAdV). There are 12 FAdV types (FAdV1 to FAdV8a and FAdV8b to FAdV11), classified into five species based on their genotype (designated FAdVA to FAdVE). A total of 218 000 chickens, 2–29 days of age, were affected over a 1-year period, all testing positive by microscopy, virus isolation and confirmation with polymerase chain reaction (PCR). Affected birds were depressed, lost body weight, were weak and had watery droppings. Pathological changes observed during necropsy indicated consistent changes in the liver, characterised by hepatomegaly, cholestasis and hepatitis. Lesions were also discernible in the spleen, kidney and gizzard wall and were characterised by splenomegaly, pinpoint haemorrhages, nephritis with haemorrhage,visceral gout and serosal ecchymosis of the gizzard wall. Histopathological lesions were most consistently observed in the liver but could also be seen in renal and splenic tissue. Virus isolation was achieved in embryonated eggs and most embryos revealed multifocal to diffuse hepatic necrosis, with a mixed cellular infiltrate of macrophages and heterophils (necro-granulomas), even in the absence of macroscopic pathology. Virus isolation results were verified by histopathology and PCR on embryonic material and further characterised by nucleotide sequence analysis. Two infectious bursal disease virus isolates were also made from the Klerksdorp flock. Nucleotide sequence analysis of the L1 hexon loop of all the FAdVisolates indicated homology (99%) with prototype strains P7-A for FAdV-2, as well as for FAdV-8b

The prevalence of Culicoides spp.in 3 geographic areas of South Africa

The seasonal abundance of Culicoides midges, the vector of Bluetongue and African horse sickness viruses (BTV/AHSV) and the presence of viruses in midges were determined in 3 geographic areas in South Africa. In the Onderstepoort area, more than 500,000 Culicoides midges belonging to 27 species were collected. Eighteen midge species were collected throughout Winter and the presence of AHSV and BTV RNA in midges was detected using real time reverse transcription quantitative polymerase chain reaction. The nucleic acid of AHSV was found in 12 pools out of total pools of 35 Culicoides. Twenty‑five Culicoides species were detected in the Mnisi area. The RNA of BTV was detected in 75.9% of the midge pools collected during Winter and 51.2% of those collected during Autumn. Antibodies for BTV were detected in 95% of cattle sampled using a competitive enzyme‑linked immunosorbent assay (cELISA). The dominant species in these 2 areas was Culicoides imicola. Eight Culicoides species were collected in Namaqualand. Culicoides imicola represented the 0.9% and Culicoides bolitinos the 1.5% of total catches, respectively. Antibodies for AHSV were detected in 4.4% of 874 equines tested using an indirect ELISA. Results showed that transmission of AHSV and BTV can carry on throughout Winter and the outbreak may begin as soon as Culicoides populations reach a certain critical level. [Abstract] I Culicoides sono noti vettori del virus della Bluetongue (BTV) e del virus della Peste equina africana (AHSV). Il lavoro riporta i risultati sull'abbondanza stagionale di Culicoides e sulla presenza di BTV e AHSV in diversi vettori, in tre aree geografiche del Sudafrica. Nell'area di Onderstepoort sono stati individuati più di 500.000 esemplari di Culicoides appartenenti a 27 specie diverse. Durante la stagione invernale sono state individuate 18 specie. Frammenti di RNA di AHSV e BTV sono stati rilevati mediante specifiche RT-qPCR. L'RNA del virus AHSV è stato individuato in 12 pool di Culicoides su 35 esaminati. Nell'area di Mnisi sono state identificate 25 specie di Culicoides. L'RNA di BTV è stato rilevato nel 75,9% dei pool di Culicoides catturati durante la stagione invernale e nel 51,2% di quelli catturati durante la stagione autunnale. Anticorpi contro BTV sono stati osservati nel 95% dei sieri prelevati da bovini dell'area di Mnisi ed esaminati con il metodo c‑ELISA. La specie dominante in queste due aree è risultata Culicoides imicola. Nella regione di Namaqualand sono state individuate otto specie, C. imicola ha rappresentato lo 0,9% delle specie catturate e Culicoides bolitinos l'1,5%. Anticorpi contro il virus AHSV sono stati individuati mediante ELISA nel 4,4% degli 874 equini esaminati. I risultati ottenuti hanno dimostrato che in Sudafrica la trasmissione di BTV e AHSV può continuare durante la stagione invernale mentre, con ogni probabilità, si ha la comparsa dei primi focolai quando le diverse popolazioni di Culicoides raggiungono un livello riproduttivo critico

Culicoides species abundance and potential overwintering of African horse sickness virus in the Onderstepoort area, Gauteng, South Africa

In South Africa, outbreaks of African horse sickness (AHS) occur in summer; no cases are reported in winter, from July to September. The AHS virus (AHSV) is transmitted almost exclusively by Culicoides midges (Diptera: Ceratopogonidae), of which Culicoides imicola is considered to be the most important vector. The over-wintering mechanism of AHSV is unknown. In this study, more than 500 000 Culicoides midges belonging to at least 26 species were collected in 88 light traps at weekly intervals between July 2010 and September 2011 near horses in the Onderstepoort area of South Africa. The dominant species was C. imicola. Despite relatively low temperatures and frost, at least 17 species, including C. imicola, were collected throughout winter (June–August). Although the mean number of midges per night fell from > 50 000 (March) to < 100 (July and August), no midge-free periods were found. This study,using virus isolation on cell cultures and a reverse transcriptase polymerase chain reaction (RT-PCR) assay, confirmed low infection prevalence in field midges and that the detection of virus correlated to high numbers. Although no virus was detected during this winter period,continuous adult activity indicated that transmission can potentially occur. The absence of AHSV in the midges during winter can be ascribed to the relatively low numbers collected coupled to low infection prevalence, low virus replication rates and low virus titres in the potentially infected midges. Cases of AHS in susceptible animals are likely to start as soon as Culicoides populations reach a critical level