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Bluetongue is primarily a disease of sheep in South Africa, while cattle and goats are mostly subclinically infected. The viraemia of bluetongue virus in cattle lasts much longer than in sheep and the role of cattle in the epidemiology of bluetongue in South Africa is poorly understood. Bluetongue virus has a segmented double-stranded ribonucleic acid genome and reassortment of genomes is a common feature. The aim of the study was to investigate whether reassortment occurs between vaccine and field strains when simultaneously administered to cattle. Six cattle between the ages of 6 and 12 months were infected with five strains of modified live vaccine bluetongue virus and a virulent field isolate of bluetongue virus 4. Blood samples were subsequently collected daily from these animals from day 1 to day 39 post-inoculation. Viruses were directly isolated during viraemia from the buffy coat on Vero cells using the plaque forming unit method. Analysis of plaques indicated that no reassortants between virulent field and vaccine strains occurred and the virulent bluetongue virus 4 was identified as the predominant virus strain. However, a reassortant virus between two bluetongue virus vaccine strains was isolated from the buffy coat. Whole genome sequences from the vaccine viruses were compared to the suspected reassortant and it was found that segment 8 exchanged between the bluetongue virus 8 and bluetongue virus 9 vaccine strains. The use of the live-attenuated bluetongue virus multivalent vaccine in South Africa causes circulation of different vaccine serotypes in Culicoides spp. and susceptible hosts and cattle might provide the ideal host for reassortment to occur.

Several studies have demonstrated the ability of certain viruses to overwinter in arthropod vectors. The over-wintering mechanism of bluetongue virus (BTV) is unknown. One hypothesis is over-wintering within adult Culicoides midges (Diptera; Ceratopogonidae) that survive mild winters where temperatures seldom drop below 10 °C. The reduced activity of midges and the absence of outbreaks during winter may create the impression that the virus has disappeared from an area. Light traps were used in close association with horses to collect Culicoides midges from July 2010 to September 2011 in the Onderstepoort area, in Gauteng Province, South Africa. More than 500 000 Culicoides midges were collected from 88 collections and sorted to species level, revealing 26 different Culicoides species. Culicoides midges were present throughout the 15 month study. Nine Culicoides species potentially capable of transmitting BTV were present during the winter months. Midges were screened for the presence of BTV ribonucleic acid (RNA) with the aid of a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. In total 91.2% of midge pools tested positive for BTV RNA. PCR results were compared with previous virus isolation results (VI) that demonstrated the presence of viruses in summer and autumn months. The results indicate that BTV-infected Culicoides vectors are present throughout the year in the study area. Viral RNA-positive midges were also found throughout the year with VI positive midge pools only in summer and early autumn. Midges that survive mild winter temperatures could therefore harbour BTV but with a decreased vector capacity. When the population size, biting rate and viral replication decrease, it could stop BTV transmission. Over-wintering of BTV in the Onderstepoort region could therefore result in re-emergence because of increased vector activity rather than reintroduction from outside the region.

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 (FAdV1to 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 multifocalto 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 FAdV isolates indicated homology (99%) with prototype strains P7-A for FAdV-2, as well as for FAdV-8b.

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.

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 106 egg infective doses (EID50) FAdV 2 were 80% - 87% and mortality rates for embryos challenged with 105.95 (EID50) FAdV 8b were 65% - 80%.

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.

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.

In 2008, a suspected outbreak of Rift Valley fever (RVF) was reported on a farm in the Bela-Bela area, Limpopo Province, South Africa. Seven calves died on the affected dairy farm, where no RVF vaccination programme was practised. No apparent clinical disease was reported in the other 300 cattle (33 calves included) or 200 sheep on the farm. During the outbreak, blood samples from 77.7% (233/300) of the cattle and 36.5% (73/200) of the sheep were collected on the affected farm and 55 blood samples were taken from cattle on a neighbouring farm. Eight weeks later, 78% of the cattle (234/300) and 42.5% of the sheep (85/200) were bled on the affected farm only. All sera were tested by an Immunoglobulin M (IgM)-capture Enzymelinked immunosorbent assay (ELISA) and by an indirect Immunoglobulin G (IgG) ELISA. Selected IgM-positive (n = 14), IgG-positive (n = 23) and samples negative for both IgM and IgG-specific antibodies against RVF virus (n = 19) were tested using the serum neutralisation test (SNT). Sera from IgM-positive (n = 14) and negative (n = 20) animals were also tested by a TaqMan polymerase chain reaction (PCR). On the affected farm, 7% (16/233) of the cattle were IgM-positive and 13.7% (32/233) IgG-positive at the first bleed and 2% were IgM-positive at the second bleed, whilst the number of cattle positive for IgG-specific antibodies increased by 21.3% compared with the first bleed. Only 1.4% of sheep were positive for both IgM and IgG antibodies at the first collection; at the second bleed, IgM-positive cases decreased to 1.2%, whilst IgG-positive cases increased to 2.4%. Whilst no IgM-positive cattle were found on the neighbouring farm, 5.5% of cattle were IgG-positive. The SNT confirmed most of the ELISA results, whilst PCR results were all negative. Although serology results indicated virus circulation on both farms, the negative PCR results demonstrated that the animals were not viraemic at the time they were sampled. The movement of infected mosquito vectors by wind over long distances into a low-lying area that favoured their breeding on the Bela-Bela farm may have led to an outbreak of the disease there, but the reason for the low level of virus circulation amongst susceptible animals remains unclear.

Recent outbreaks of bluetongue virus (BTV) serotypes 2 and 8 in many European countries provided an opportunity to investigate the possibility of improving the safety of the modified live vaccines administered mainly in South Africa. Modified live vaccines (MLV) released at a titre of 5 x 104 PFU/mL, raised concerns and prompted the need to determine the minimum titre which will still be protective and also safe. The BTV serotypes 2 and 8 vaccines were produced at the following titres: 102 PFU/mL, 103 PFU/mL and 104 PFU/mL, and were injected into 24 sheep which were then monitored. Blood was collected on days 0, 3, 6, 9, 12, 15, 18, 21, 25, 28 and 4 months post vaccination, for seroconversion and viraemia studies. These sheep were later challenged at 4 months post vaccination using BTV infected cell culture material, they were then observed and bled and again tested for viraemia. There was no viraemia post vaccination, however, a febrile reaction did occur and seroconversion was demonstrated at low titres for both BTV 2 and 8. Although viraemia was demonstrated post challenge, sheep vaccinated with the low titre BTV 2 vaccine showed more than a 90% protection index at a lower titre of 103 PFU/mL, compared with BTV 8 that showed a protection index above 90% at all the titres used. It is recommended that for BTV 2 vaccine, sheep should be vaccinated at a titre of 103 PFU/mL and at a titre of 102 PFU/mL with BTV 8 vaccine.