The first recorded outbreak of avian influenza (AI) in South African chickens (low pathogenicity H6N2) occurred at Camperdown, KwaZulu/Natal Province (KZN) in June 2002. To determine the source of the outbreak, we defined the phylogenetic relationships between various H6N2 isolates, and the previously unpublished gene sequences of an H6N8 virus isolated in 1998 from ostriches in the Leeu Gamka region (A/Ostrich/South Africa/KK98/98). We demonstrated that two distinct genetic H6N2 lineages (sub-lineages I and II) circulated in the Camperdown area, which later spread to other regions. Sub-lineages I and II shared a recent common H6N2 ancestor, which arose from a reassortment event between two South African ostrich isolates A/Ostrich/South Africa/9508103/95 and (H9N2) /Ostrich/South Africa/KK98/98 (H6N8). Furthermore, the H6N2 sub-lineage I viruses had several molecular genetic markers including a 22-amino acid stalk deletion in the neuraminidase (NA) protein gene, a predicted increased Nglycosylation, and a D144 mutation of the HA protein gene, all of which are associated with the adaptation of AI viruses to chickens. The H6N2 NS1 and PB1 genes shared recent common ancestors with those of contemporary Asian HPAI H5N1 viruses. Our results suggest that ostriches are potential mixing vessels for avian influenza viruses (AIV) outbreak strains and support other reports that H6 viruses are capable of forming stable lineages in chickens.

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One hundred serum samples collected from free-ranging white rhinoceros (Ceratotherium simum) in Kruger National Park (KNP) during the 2007 capture season were selected for measurement of antibody levels to several different vector-borne viral agents. These infectious diseases were chosen to compare with an earlier serosurvey that had been conducted in KNP in rhinos during 1987-1997. Positive antibody titers were found against epizootic hemorrhagic disease (EHD) of deer (8%), Bluetongue (BT) (1%), and Rift Valley fever (RVF) (49%). However, none of the 100 animals tested had detected antibody levels to African horse sickness (AHS). These values were in sharp contrast to those measured in the 1987-1997 survey in KNP white rhinos (AHS 60%, EHD 30%, BT 37%, RVF 0%). Vector-borne viral infection prevalence in white rhinos in the same geographical location appears to vary over time and may be important for monitoring presence of pathogens in an ecosystem.

Orbivirus diagnosis in an endemic country presents many diag­nostic complexities. Endemicity is further complicated by mul­tiple serotypes and related viruses which may be cross-reactive in some tests or else be clinically indistinguishable. Some of the cases of either bluetongue (BT) or epizootic haemorrhagic disease (EHD) of the 2008 and 2009 summer seasons are pre­sented to illustrate the challenges. Selection focused on two bovines and three alpacas for which no final diagnoses had been made or confirmed. The two heifers were young, geographi­cally distant but similar in clinical presentation with dysphagia and oesophageal dilations. BTV-6 and 16 were recovered from what were considered to be EHD cases, and no final conclusion was made. The three alpacas formed a geographical cluster, although they were resident on different farms. Two were local and one had been recently imported. In clinical presentation, all three differed and two remained unresolved with no isolate or polymerase chain reaction (PCR) result. The last case, an acute death with severe lung oedema, yielded twin isolates of BTV and EHDV, respectively. Final serotype identification, causal relationship and diagnosis are still pending. These cases amply illustrate the concept that arboviral endemicity can result in a diagnostic dilemma.

This presentation highlights the complexities of making a diag­nosis in a situation where vaccination is taking place, and where there are multiple virus serotypes and related viruses in circula­tion. The main orbiviruses of interest are those of African horse sickness (AHS) and bluetongue (BT) and further complicated by the viruses of equine encephalosis (EE) and epizootic haemor­rhagic disease of deer (EHD). South Africa is an endemic country with all 9 AHS serotypes and 22 of 24 BT serotypes present. Annual vaccination is carried out but a large pool of virus is available in unvaccinated zebra and donkeys for AHS, and indig­enous sheep, cattle and ruminant wildlife for BT. Diagnoses of the two diseases are discussed separately.  AHS had its origin in zebras which, however, do not exhibit the disease, while up to 95% of susceptible horses die. Diagnosis requires recognition of recent infection, and a history of recent vaccination is essential. Diagnostic tests are based on detection of virus and/or antibodies. Use of antibodies is fast and inex­pensive and based on immunoglobulin M or G (IgM or IgG) responses. All horses are seropositive, and paired samples taken 14 days apart are required to demonstrate recent exposure and a rise in titre. IgM enzyme-linked immunosorbent assays (ELISAs) for multiple serotype viruses are not available. The IgG ELISA result is based on a single dilution thus, in paired samples, a rise in titre is a qualitative estimate of exposure. The complement fix­ation test (CFT) is largely an IgM test which uses a dilution series, and paired samples can show a rise in titre. It is slow, old-fash­ioned and labour intensive, but it is the only useful serological option where there are pre-existing antibodies and a rising titre is required. Demonstration of virus or antigen or ribonucleic acid (RNA) presents advantages and disadvantages for each. Virus iso­lation on cells is slow for AHSV but faster intracerebrally in mice. Viral titres are low with circulating antibodies, and serotyping or sequencing to differentiate vaccine and field strains may be needed, an isolate becomes then necessary. Antigen detection by immunohistochemistry is useful, if only formalin material is received, or if tissue tropism is of interest. Polymerase chain reaction (PCR) in its many formats gives a rapid diagnosis but interpretation can be difficult. RNA is present for some months, therefore the time of infection and its significance is difficult to gauge. The antigen-capture ELISA is useful, particularly on tis­sue culture isolates to distinguish AHS and EE, if two PCR tests have not been done initially. As a primary diagnostic tool it lacks sensitivity, and an isolate is often required for additional tests. The best diagnostic option is to get a good history and to do as many different tests as possible in order to make an educated guess. This is because EE mimics AHS clinically, has the same vectors and co-circulates with AHS in the population. Tests do not cross-react, but it is important to focus on both viruses in order to make the proper diagnosis.  Much that has already been mentioned for AHS also applies for BT. The pool of wild-type, infectious virus becomes proportion­ately much larger when BT vaccine coverage is lower, causing the disease to occur widely. Little diagnostic material is received as mortality in sheep is only a low 2–30%; each year, however, at least 2–3 dominant serotypes are present. Similar tests are available in an IgG format in use. All sheep are generally sero­positive because of multiple exposures. Negative sheep need to be sourced from high-altitude cold areas. The very helpful CFT cannot be used as this group-specific test cross-reacts signifi­cantly with EHDV. This problem is addressed in the competitive ELISA with a monoclonal antibody specific for BTV. This test is essential to obtain export certification. In South Africa mortal­ity caused by BT is low and sporadic, and virus spread is slow because of homologous and heterologous background antibod­ies in sheep flocks. Diagnoses become important in the export sector for testing ruminant wildlife going to zoological collec­tions or ovine embryos where donor sheep are tested. BT-free certification is obtained by PCR testing. As mentioned previ­ously, RNA is present for a prolonged period of time. A negative PCR means no exposure, whereas a positive result requires virus isolation to certify that no live whole virus is present, as it could infect vectors. Field, not vaccine strains, require amplification in embryonated eggs first, then isolation from dead embryos on tissue culture. The cross-reactivity between BTV and EHDV in diagnostic tests is not an important consideration except when an isolate cannot be typed. If EHD viruses were as widely preva­lent as those of EE, there would be a diagnostic problem, but EHDV isolates are not found in sheep. From time to time EHDV is found in alpaca and bovine cases. Finally, orbivirus diagnoses are challenging in an endemic country but always interesting and sometimes surprising in the final results obtained.