The seroprevalence of African horse sickness virus, and risk factors to exposure, in domestic dogs in Tshwane, South Africa.

Dogs are the only non-equid species to develop the fatal form of African horse sickness (AHS). Research conducted in 2013 questioned the long-held belief that naturally occurring cases of AHS in dogs were contracted exclusively through the ingestion of contaminated horse meat. Culicoides midges, the vector of AHS virus (AHSV) for horses, have an aversion to dog blood meals and dogs were believed to be dead-end or incidental hosts. More recently, dog mortalities have occurred in the absence of horse meat consumption and vector transmission has been suspected. The current study is a retrospective serological survey of AHSV exposure in dogs from an endemic area. Dog sera collected from dogs (n = 366) living in the city of Tshwane, Gauteng Province, South Africa, were randomly selected from a biobank at a veterinary teaching hospital, corresponding to the years 2014-2019. The study used a laboratory in-house indirect recombinant VP7 antigen-based enzyme-linked immunosorbent assay (iELISA) with a test cut-off calculated from AHSV exposure-free dog sera (n = 32). Study AHSV seroprevalence was 6 % (22/366) with an estimated true prevalence of 4.1 % (95 % confidence interval (CI) = 1.3-8.1 %). Incidence was estimated for dogs with multiple serological results with seroconversion occurring at a rate of 2.3 seroconversions per 10 dog years at risk (95 % CI = 0.6-6.2). A subsection of the study sera was tested with AHSV viral neutralisation test (VN) (n = 42) for serotype determination. Antibodies to AHSV serotype 6 were most prevalent (90 %) in VN seropositive dogs (n = 20) with most dogs seemingly subclinically infected (>95 %). Seroprevalence descriptively varied by year and identified risk factors were annual rainfall > 754 mm (odds ratio (OR) = 5.76; 95 % CI = 2.22 - 14.95; p < 0.001), medium human population densities, 783-1663 people/km2 (OR = 7.14; 95 % CI = 1.39 - 36.73; p = 0.019) and 1664-2029 people/km2 (OR = 6.74; 95 % CI = 1.40 - 32.56; p = 0.018), and the month of March (OR = 5.12; 95 % CI = 1.41 - 18.61; p = 0.013). All identified risk factors were consistent with midge-borne transmission to dogs. The relatively high seroprevalence and seroconversion rates suggest frequent exposure of dogs to AHSV and indicates the need to investigate the role dogs might play in the overall epidemiology and transmission of AHSV.

Clinical Course of Infection and Cross-Species Detection of Equine Parvovirus-Hepatitis.

Since its first discovery by Arnold Theiler in 1918, serum hepatitis also known as Theiler's disease has been reported worldwide, causing idiopathic acute hepatitis and liver failure in horses. Recent studies have suggested a novel parvovirus, named equine parvovirus hepatitis (EqPV-H), to be associated with Theiler's disease. Despite the severity and potential fatality of EqPV-H infection, little is known about the possibility of developing chronic infections and putative cross-species infection of equine sister species. In the present longitudinal study, we employed qPCR analysis, serology, and biochemical testing as well as pathology examination of liver biopsies and sequence analysis to investigate potential chronic EqPV-H infection in an isolated study cohort of in total 124 horses from Germany over five years (2013-2018). Importantly, our data suggest that EqPV-H viremia can become chronic in infected horses that do not show biochemical and pathological signs of liver disease. Phylogenetic analysis by maximum likelihood model also confirms high sequence similarity and nucleotide conservation of the multidomain nuclear phosphoprotein NS1 sequences from equine serum samples collected between 2013-2018. Moreover, by examining human, zebra, and donkey sera for the presence of EqPV-H DNA and VP1 capsid protein antibodies, we found evidence for cross-species infection in donkey, but not to human and zebra. In conclusion, this study provides proof for the occurrence of persistent EqPV-H infection in asymptomatic horses and cross-species EqPV-H detection in donkeys.

Complete Genome Sequences of Virus Strains Isolated from Bottle A of the South African Live Attenuated Bluetongue Virus Vaccine.

This is a report of the complete genome sequences of plaque-selected isolates of five virus strains included in bottle A of the South African Onderstepoort Biological Products commercial live attenuated bluetongue virus vaccine.

Direct culture-independent sequence typing of Taylorella equigenitalis obtained from genital swabs and frozen semen samples from South African horses.

We report herein the use of crude extracts obtained from samples of Taylorella equigenitalis-infected horses for the purpose of multi-locus sequence typing (MLST). Samples (n = 36) were collected from horses in South Africa from 1996 to 2017: 34 from genital swabs (stored at -20°C for 2-3 y) and 2 from cryopreserved raw semen aliquots (stored at -70°C for 18 y) prior to assay. The MLST assay showed a single sequence type (ST), designated ST4, that supported a point introduction and thus a common source for the South African outbreak of contagious equine metritis.

Evaluating African horse sickness virus in horses and field-caught Culicoides biting midges on the East Rand, Gauteng Province, South Africa.

A prospective study was undertaken during 2013 and 2014, to determine the prevalence of African horse sickness virus (AHSV) in Culicoides midges and the incidence of infection caused by the virus in 28 resident horses on two equine establishments on the East Rand, Gauteng Province, South Africa. Field caught Culicoides midges together with whole blood samples from participating horses were collected every two weeks at each establishment. Culicoides midges and blood samples were tested for the presence of AHSV RNA by real-time quantitative reverse transcription polymerase chain reaction. Nine immunised horses became infected with AHSV during the study period, although infections were subclinical. African horse sickness virus was also identified from a field-collected midge pool. The observations recapitulate previously published data in another setting, where further investigation is warranted to determine what role subclinical infection plays in the diseases epidemiology.

A field investigation of an African horse sickness outbreak in the controlled area of South Africa in 2016.

An outbreak of African horse sickness (AHS) caused by AHS virus type 1 occurred within the South African AHS surveillance zone during April and May 2016. The index case was detected by a private veterinarian through passive surveillance. There were 21 cases in total, which is relatively low compared to case totals during prior AHS outbreaks in the same region (and of the same AHS virus type) in 2004, 2011 and 2014. The affected proportion of horses on affected properties was 0.07 (95% CI 0.04, 0.11). Weather conditions were conducive to high midge activity immediately prior to the outbreak but midge numbers decreased rapidly with the advent of winter. The outbreak was localized, with 18 of the 21 cases occurring within 8 km of the index property and the three remaining cases on two properties within 21 km of the index property, with direction of spread consistent with wind-borne dispersion of infected midges. Control measures included implementation of a containment zone with movement restrictions on equids. The outbreak was attributed to a reversion to virulence of a live attenuated vaccine used extensively in South Africa. Outbreaks in the AHS control zones have a major detrimental impact on the direct export of horses from South Africa, notably to the European Union.

First detection and frequent occurrence of Equine Hepacivirus in horses on the African continent.

Since the discovery of equine hepacivirus (EqHV) in 2011, the virus has been detected in horse populations from more than twelve countries across five continents. EqHV seroprevalence has been reported to be as high as 61.8% and EqHV ribonucleic acid (RNA) prevalence to range between 0.9% and 34.1%. Molecular and serological indications of EqHV infection have never been reported in equids on the African continent. Therefore, investigation of EqHV prevalence in South African horses and subsequent viral genetic characterization contribute to a better understanding of the global epidemiology of this virus. In a cross-sectional study, serum samples from 454 Thoroughbred foals (aged 58-183 days) were analysed for anti-EqHV non-structural protein 3 (NS3)-specific antibodies (abs) with a luciferase immunoprecipitation system (LIPS) and for EqHV RNA by quantitative real-time polymerase chain reaction (qRT-PCR). Farms of origin (n = 26) were situated in South Africa's Western Cape Province. The associations between EqHV infection state and farm of origin, foal gender and foal age were subsequently described. Furthermore, nested PCRs were performed on parts of the 5'UTR, NS3 and NS5B genes of 17 samples. Samples were sequenced and phylogenetic analyses were conducted. The population's seroprevalence was 83.70% and RNA was detected in 7.93% of samples. Increasing foal age was associated with decreasing ab prevalence and increasing prevalence of EqHV RNA. Sequences from South African EqHV strains did not show in-depth clustering with published sequences of EqHV isolates from particular continents. In conclusion, EqHV is present in the South African Thoroughbred population and appears more prevalent than reported in other horse populations worldwide.

Robust forensic matching of confiscated horns to individual poached African rhinoceros.

Black and white rhinoceros (Diceros bicornis and Ceratotherium simum) are iconic African species that are classified by the International Union for the Conservation of Nature (IUCN) as Critically Endangered and Near Threatened (http://www.iucnredlist.org/), respectively [1]. At the end of the 19th century, Southern white rhinoceros (Ceratotherium simum simum) numbers had declined to fewer than 50 animals in the Hluhluwe-iMfolozi region of the KwaZulu-Natal (KZN) province of South Africa, mainly due to uncontrolled hunting [2,3]. Efforts by the Natal Parks Board facilitated an increase in population to over 20,000 in 2015 through aggressive conservation management [2]. Black rhinoceros (Diceros bicornis) populations declined from several hundred thousand in the early 19th century to ∼65,000 in 1970 and to ∼2,400 by 1995 [1] with subsequent genetic reduction, also due to hunting, land clearances and later poaching [4]. In South Africa, rhinoceros poaching incidents have increased from 13 in 2007 to 1,215 in 2014 [1]. This has occurred despite strict trade bans on rhinoceros products and strict enforcement in recent years.

Immunogenicity of plant-produced African horse sickness virus-like particles: implications for a novel vaccine.

African horse sickness (AHS) is a debilitating and often fatal viral disease affecting horses in much of Africa, caused by the dsRNA orbivirus African horse sickness virus (AHSV). Vaccination remains the single most effective weapon in combatting AHS, as there is no treatment for the disease apart from good animal husbandry. However, the only commercially available vaccine is a live-attenuated version of the virus (LAV). The threat of outbreaks of the disease outside its endemic region and the fact that the LAV is not licensed for use elsewhere in the world, have spurred attempts to develop an alternative safer, yet cost-effective recombinant vaccine. Here, we report the plant-based production of a virus-like particle (VLP) AHSV serotype five candidate vaccine by Agrobacterium tumefaciens-mediated transient expression of all four capsid proteins in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant expression vector system. The production process is fast and simple, scalable, economically viable, and most importantly, guinea pig antiserum raised against the vaccine was shown to neutralize live virus in cell-based assays. To our knowledge, this is the first report of AHSV VLPs produced in plants, which has important implications for the containment of, and fight against the spread of, this deadly disease.

A serosurvey of bluetongue and epizootic haemorrhagic disease in a convenience sample of sheep and cattle herds in Zimbabwe.

A convenience sample of sheep and cattle herds around the cities of Harare, Kwekwe and Bulawayo, located in the Highveld region of Zimbabwe, was used to estimate the seroprevalence and sero-incidence of bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) antibodies. A competitive enzyme-linked immunosorbent assay was used to identify serum antibodies against BTV and EHDV across three rainy seasons. The median sero-prevalence of BTV and EHDV antibodies in cattle was 62% (interquartile range [IQR]: 30-89) and 56% (IQR: 5-77), respectively. In sheep, the median sero-prevalence of BTV and EHDV was 41% (IQR: 19-63) and 0% (IQR: 0-21), respectively. Median sero-incidences of BTV and EHDV antibodies in cattle of 43% (IQR: 22-67) and 27% (IQR: 9-57) respectively were recorded. The median sero-incidence of BTV in sheep was 14% (IQR: 6-23). Based on these preliminary findings, animal health workers in Zimbabwe should continue to monitor the exposure rates of cattle and sheep to BTV and consider the possibility of strains emerging with increased pathogenicity. There are no previous published reports of antibodies against EHDV in Zimbabwe so the possibility of epizootic haemorrhagic disease existing in domestic livestock should now be considered by Zimbabwean animal health officials. Seroconversions to BTV and EHDV occurred predominantly at the end of each rainy season (March and April), which generally corresponds to high numbers of the Culicoides vectors. BTV isolations were made from three individual cows in two of the sentinel herds and all three were identified as serotype 3. This is the first time BTV serotype 3 has been recorded in Zimbabwe, although its presence in neighbouring South Africa is well documented.

The effect of alphacypermethrin-treated mesh protection against African horse sickness virus vectors on jet stall microclimate, clinical variables and faecal glucocorticoid metabolites of horses.

BACKGROUND: African horse sickness (AHS) is of importance to health and international trade in horses worldwide. During export from and transit through AHS endemic countries or zones, physical and chemical measures to protect horses from the vectors of AHS virus (AHSV) are recommended by the World Organization for Animal Health. Protection of containerized air transport systems for horses (jet stalls) with alphacypermethrin insecticide-treated high density polyethylene mesh is effective in reducing the Culicoides midge vector attack rate. In order to determine the effect of this mesh on jet stall ventilation and horse welfare under temperate climatic conditions, jet stall microclimate, clinical variables and faecal glucocorticoid metabolite (FGM) levels of 12 horses were monitored during overnight housing in either a treated or untreated stall in two blocks of a 2 × 3 randomized crossover design. RESULTS: Temperature difference between the treated stall and outside was significantly higher than the difference between the untreated stall and outside at 1/15 time points only (P = 0.045, r = 0.70). Relative humidity (RH) difference between the treated stall and outside did not differ from the untreated stall and outside. Temperature and RH in the treated stall were highly and significantly correlated with outside temperature (r = 0.96, P < 0.001) and RH (r = 0.95, P < 0.001), respectively. No significant differences were detected between rectal temperatures, pulse and respiratory rates of horses in the treated stall compared to the untreated stall. Mean FGM concentrations for horses housed in the treated stall peaked earlier (24 h) and at a higher concentration than horses housed in the untreated stall (48 h), but were not significantly different from baseline. No significant difference was detected in FGM concentrations when the treated and untreated stall groups were compared at individual time points up to 72 h after exiting the jet stall. CONCLUSIONS: Alphacypermethrin-treated HDPE mesh could be used under temperate climatic conditions to protect horses in jet stalls against AHSV vectors, without compromising jet stall microclimate and horse welfare.

The sero-prevalence and sero-incidence of African horse sickness and equine encephalosis in selected horse and donkey populations in Zimbabwe.

Sentinel herds and samples submitted by private equine practitioners were used to determine the sero-prevalence and sero-incidence of African horse sickness virus (AHSV) and equine encephalosis virus (EEV) in horse and donkey populations in the Highveld region of Zimbabwe. The sero-prevalence and sero-incidence of antibodies against these viruses were determined using the competitive enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies. In donkeys, the median sero-prevalence of AHSV antibodies, across the three rainy seasons under study, was 75% (inter quartile range [IQR] 67-83), with a seasonal median sero-incidence of 45% (IQR 40-63). In horses, the median sero-prevalence of EEV antibodies was 63% (IQR 21-73), with a median seasonal sero-incidence of 10.5% (IQR 10-14), while in donkeys the median sero-prevalence of EEV antibodies was 80% (IQR 67-90), with a median seasonal sero-incidence of 50% (IQR 40-60). This study highlighted the significant levels of exposure of donkeys to AHSV and horses and donkeys to EEV in Zimbabwe despite equine encephalosis remaining unreported by Zimbabwean veterinarians to date. Most seroconversions in sentinel herd animals to AHSV and EEV occurred towards the end of the rainy season in March, April and May corresponding to the time of the year when the Culicoides vectors are in high abundance. In order to determine the clinical significance of these infections, blood and spleen samples, submitted by private equine veterinary practitioners over a 5-year period, from horses showing characteristic clinical signs of African horse sickness were tested for the presence of viral antigen using the antigen capture ELISA. The median sero-prevalence of AHSV antigen in horses recorded from these samples was 38% (IQR 33-88). The predominant AHSV antigen from these samples was serotype 7 (33%) followed by serotype 2 (26%) and serotypes 4 and 8 (16% each). African horse sickness virus serotypes 3 and 9, identified in this study, had not been previously reported in Zimbabwe.

African Horse Sickness Caused by Genome Reassortment and Reversion to Virulence of Live, Attenuated Vaccine Viruses, South Africa, 2004-2014.

African horse sickness (AHS) is a hemorrhagic viral fever of horses. It is the only equine disease for which the World Organization for Animal Health has introduced specific guidelines for member countries seeking official recognition of disease-free status. Since 1997, South Africa has maintained an AHS controlled area; however, sporadic outbreaks of AHS have occurred in this area. We compared the whole genome sequences of 39 AHS viruses (AHSVs) from field AHS cases to determine the source of 3 such outbreaks. Our analysis confirmed that individual outbreaks were caused by virulent revertants of AHSV type 1 live, attenuated vaccine (LAV) and reassortants with genome segments derived from AHSV types 1, 3, and 4 from a LAV used in South Africa. These findings show that despite effective protection of vaccinated horses, polyvalent LAV may, paradoxically, place susceptible horses at risk for AHS.

Complete Genome Sequences of Five Bluetongue Virus (BTV) Vaccine Strains from a Commercial Live Attenuated Vaccine, a BTV-4 Field Strain from South Africa, and a Reassortant Strain Isolated from Experimentally Vaccinated Cattle.

This is a report of the complete genome sequences of plaque-selected isolates of each of the five virus strains included in a South African commercial trivalent bluetongue virus (BTV) attenuated live virus vaccine, a BTV-4 field strain isolated from Rustenburg, South Africa, in 2011, and a bluetongue reassortant (bluetongue virus 4 strain 4/O. aries-tc/ZAF/11/OBP-115) isolated from experimentally vaccinated cattle. Full-genome sequencing and phylogenetic analyses show that the bluetongue virus 9 strain 9/B. taurus-tc/ZAF/15/Onderstepoort_B02b is a reassortant virus containing segments from both BTV-9 and BTV-8.

Quantitative Risk Assessment for African Horse Sickness in Live Horses Exported from South Africa.

African horse sickness (AHS) is a severe, often fatal, arbovirus infection of horses, transmitted by Culicoides spp. midges. AHS occurs in most of sub-Saharan Africa and is a significant impediment to export of live horses from infected countries, such as South Africa. A stochastic risk model was developed to estimate the probability of exporting an undetected AHS-infected horse through a vector protected pre-export quarantine facility, in accordance with OIE recommendations for trade from an infected country. The model also allows for additional risk management measures, including multiple PCR tests prior to and during pre-export quarantine and optionally during post-arrival quarantine, as well as for comparison of risk associated with exports from a demonstrated low-risk area for AHS and an area where AHS is endemic. If 1 million horses were exported from the low-risk area with no post-arrival quarantine we estimate the median number of infected horses to be 5.4 (95% prediction interval 0.5 to 41). This equates to an annual probability of 0.0016 (95% PI: 0.00015 to 0.012) assuming 300 horses exported per year. An additional PCR test while in vector-protected post-arrival quarantine reduced these probabilities by approximately 12-fold. Probabilities for horses exported from an area where AHS is endemic were approximately 15 to 17 times higher than for horses exported from the low-risk area under comparable scenarios. The probability of undetected AHS infection in horses exported from an infected country can be minimised by appropriate risk management measures. The final choice of risk management measures depends on the level of risk acceptable to the importing country.

Complete Genome Sequences of Four African Horse Sickness Virus Strains from a Commercial Tetravalent Live Attenuated Vaccine.

This is a report of the complete genome sequences of plaque-selected isolates of each of the four virus strains included in a South African commercial tetravalent African horse sickness attenuated live virus vaccine.

Draft Genome Sequence of Taylorella equigenitalis Strain ERC_G2224 Isolated from the Semen of a Lipizzaner Stallion in South Africa.

Taylorella equigenitalis is the causative agent of contagious equine metritis (CEM), a sexually transmitted infection of horses. We report here the genome sequence of T. equigenitalis strain ERC_G2224, isolated in 2015 from a semen sample collected in 1996 from a Lipizzaner stallion in South Africa.

The occurrence of Culicoides species, the vectors of arboviruses, at selected trap sites in Zimbabwe.

A study of the distribution of Culicoides species was conducted by establishing 12 light trap sites over five rainy seasons between 1998 and 2003 covering all the geo-climatic natural regions of Zimbabwe. In total, 279 919 specimens of Culicoides were trapped over a total of 163 trapping nights. The highest median counts of Culicoides per trapping night were recorded in natural region III, which has climatic conditions conducive to the successful development of the larvae. Culicoides imicola, the major vector of bluetongue and African horse sickness viruses in Africa, was found to be the most abundant species (80.4%), followed by Culicoides enderleini (5.9%) and Culicoides milnei (5.2%). This study identified 10 species of Culicoides that had not been previously described in Zimbabwe, including Culicoides loxodontis and Culicoides miombo, which are members of the C. imicola complex. A total of 23 994 Culicoides midges were collected from five trap sites in Harare, Zimbabwe, with the dominant species, C. imicola, representing 91.6% of the total collection. Seventeen arboviruses were isolated from these midges, 15 of which were bluetongue virus. The predominant bluetongue virus serotype was serotype 11, followed by serotypes 1, 8, 12 and 15. Bluetongue virus serotypes 1, 2, 8, 10, 12, 15, 16 and 18, detected in this study, had not been previously reported in Zimbabwe.

Development of three triplex real-time reverse transcription PCR assays for the qualitative molecular typing of the nine serotypes of African horse sickness virus.

Blood samples collected as part of routine diagnostic investigations from South African horses with clinical signs suggestive of African horse sickness (AHS) were subjected to analysis with an AHS virus (AHSV) group specific reverse transcription quantitative polymerase chain reaction (AHSV RT-qPCR) assay and virus isolation (VI) with subsequent serotyping by plaque inhibition (PI) assays using AHSV serotype-specific antisera. Blood samples that tested positive by AHSV RT-qPCR were then selected for analysis using AHSV type specific RT-qPCR (AHSV TS RT-qPCR) assays. The TS RT-qPCR assays were evaluated using both historic stocks of the South African reference strains of each of the 9 AHSV serotypes, as well as recently derived stocks of these same viruses. Of the 503 horse blood samples tested, 156 were positive by both AHSV RT-qPCR and VI assays, whereas 135 samples that were VI negative were positive by AHSV RT-qPCR assay. The virus isolates made from the various blood samples included all 9 AHSV serotypes, and there was 100% agreement between the results of conventional serotyping of individual virus isolates by PI assay and AHSV TS RT-qPCR typing results. Results of the current study confirm that the AHSV TS RT-qPCR assays for the identification of individual AHSV serotypes are applicable and practicable and therefore are potentially highly useful and appropriate for virus typing in AHS outbreak situations in endemic or sporadic incursion areas, which can be crucial in determining appropriate and timely vaccination and control strategies.