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.

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.

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.

Complete Genome Sequences of the Three African Horse Sickness Virus Strains from a Commercial Trivalent Live Attenuated Vaccine.

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

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.

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.

Equine encephalosis in Thoroughbred foals on a South African stud farm.

Thoroughbred foal body temperature data were collected from shortly after birth until shortly after weaning during the 2007/2008 season on a stud farm in the Western Cape Province of South Africa. Equine encephalosis (EE) caused by EE virus (EEV) serotype 4 (EEV-4) occurred in the foal group during the first autumn after their birth (March and April 2008). A descriptive study was undertaken to provide data on the EEV maternal antibody status, the association between pyrexia and EEV infection, and the incidence of infection amongst the foals prior to and during the episode. This included the frequent capturing of foal body temperature data and regular collection of serum and whole blood during pyretic episodes. Infection by EEV was determined using both virological and serological methods. A high EE incidence of at least 94% occurred amongst the foal cohort, despite the fact that 37% of foals had previously shown maternal antibody to EEV-4. Pyrexia in foals was not directly associated with EE infection and 41% of infected foals showed no detectable pyretic episode. Information obtained from this EE episode showed the high incidence of EEV infection in foals during the first autumn after their birth. Monitoring foal body temperature can alert farmers to outbreaks of infectious disease, such as EE. These results are relevant to the epidemiology of EE and facilitate greater understanding of it as a differential diagnosis of African horse sickness (AHS), given that EE and AHS have similar epidemiologic profiles.

Extraction of nuclear DNA from rhinoceros horn and characterization of DNA profiling systems for white (Ceratotherium simum) and black (Diceros bicornis) rhinoceros.

Rhinoceros horn is now worth more, per unit weight, than gold, diamonds, or cocaine. Rhinoceros horn has been used in traditional Asian medicine as a presumed cure for a wide range of ailments. Rhinoceros poaching in South Africa has, on average, more than doubled each year over the past 5 years with the rapid economic growth in east and southeast Asia being assumed to be the primary factor driving the increased demand for horn. Here we report on the characterization of methods for genomic DNA extraction from rhinoceros horn and on DNA profiling systems for white (Ceratotherium simum) and black (Diceros bicornis) rhinoceros. The DNA profiling system described includes 22 short tandem repeat (STR), or microsatellite, markers and a gender marker (ZF1), which have been used previously in various studies on rhinoceros. Using a θ value of 0.1, a conservative estimate of random match probability in 5 white rhinoceros ranged from 1:7.3x10(6) to 1:3.0x10(8). Given that the total population of white rhinoceros is approximately 20,000 such random match probabilities indicate that the genotyping system described provides data which can be used for evidentiary purposes. Furthermore, the methods are appropriate for use in investigations involving trace amounts of rhinoceros horn and the matching of profiles obtained from seized rhinoceros horn with material collected from live animals or poached carcasses.

Diagnostic accuracy of a duplex real-time reverse transcription quantitative PCR assay for detection of African horse sickness virus.

Blood samples collected from 503 suspect cases of African horse sickness (AHS) and another 503 from uninfected, unvaccinated South African horses, as well as 98 samples from horses from an AHS free country, were tested with an AHS virus (AHSV) specific duplex real-time reverse transcription quantitative PCR (RT-qPCR) assay and virus isolation (VI). The diagnostic sensitivity and specificity of this AHSV RT-qPCR assay and VI were estimated using a 2-test 2-population Bayesian latent class model which made no assumptions about the true infection status of the tested animals and allowed for the possibility of conditional dependence (correlation) in test results. Median diagnostic sensitivity and specificity of the AHSV RT-qPCR were 97.8% and 99.9%, respectively. Median diagnostic specificity of virus isolation was >99% whereas the estimated diagnostic sensitivity was 44.2%. The AHSV RT-qPCR assay provides for rapid, high-throughput analysis of samples, and is both analytically and diagnostically sensitive and specific. This assay is potentially highly useful for demonstrating freedom or infection of horses with AHSV, thus it is appropriate that its reproducibility be evaluated in other laboratories as a global standard for detection of AHSV.

Re-emergence of the apicomplexan Theileria equi in the United States: elimination of persistent infection and transmission risk.

Arthropod-borne apicomplexan pathogens that cause asymptomatic persistent infections present a significant challenge due to their life-long transmission potential. Although anti-microbials have been used to ameliorate acute disease in animals and humans, chemotherapeutic efficacy for apicomplexan pathogen elimination from a persistently infected host and removal of transmission risk is largely unconfirmed. The recent re-emergence of the apicomplexan Theileria equi in U.S. horses prompted testing whether imidocarb dipropionate was able to eliminate T. equi from naturally infected horses and remove transmission risk. Following imidocarb treatment, levels of T. equi declined from a mean of 10(4.9) organisms/ml of blood to undetectable by nested PCR in 24 of 25 naturally infected horses. Further, blood transfer from treated horses that became nested PCR negative failed to transmit to naïve splenectomized horses. Although these results were consistent with elimination of infection in 24 of 25 horses, T. equi-specific antibodies persisted in the majority of imidocarb treated horses. Imidocarb treatment was unsuccessful in one horse which remained infected as measured by nested PCR and retained the ability to infect a naïve recipient via intravenous blood transfer. However, a second round of treatment eliminated T. equi infection. These results support the utility of imidocarb chemotherapy for assistance in the control and eradication of this tick-borne pathogen. Successful imidocarb dipropionate treatment of persistently infected horses provides a tool to aid the global equine industry by removing transmission risk associated with infection and facilitating international movement of equids between endemic and non-endemic regions.

The combination of abundance and infection rates of Culicoides sonorensis estimates risk of subsequent bluetongue virus infection of sentinel cattle on California dairy farms.

Bluetongue (BT) is an important viral disease of ruminants that is transmitted by hematophagous Culicoides midges. We examined the seasonal patterns of abundance and infection of Culicoides sonorensis at four dairy farms in the northern Central Valley of California to develop estimates of risk for bluetongue virus (BTV) transmission to cattle at each farm. These four farms were selected because of their similar meteorological conditions but varying levels of vector abundance and BTV infection of cattle. C. sonorensis midges were collected weekly at each farm during the seasonal transmission period, using three different trapping methods: traps baited with either carbon dioxide (CO(2)) alone or traps with CO(2) and UV light, and by direct aspiration of midges from sentinel cattle. Analysis of BTV-infected midges using group and serotype-specific quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) assays confirmed that BTV serotypes 10, 11, 13 and 17 are all present in the region, but that midge infection rates and the number of BTV serotypes circulating differed markedly among the individual farms. Furthermore, more serotypes of BTV were present in midges than in sentinel cattle at individual farms where BTV circulated, and the virus was detected at each farm in midges prior to detection in cattle. BTV infection rates were remarkably lower among female C. sonorensis midges collected by CO(2) traps with UV light than among midges collected by either animal-baited aspirations or in CO(2) traps without light. A subsample of female midges examined from each collection method showed no overall differences in the proportion of female midges that had previously fed on a host. Findings from this study confirm the importance of using sensitive surveillance methods for both midge collection and virus detection in epidemiological studies of BTV infection, which is especially critical if the data are to be used for development of mathematical models to predict the occurrence of BTV infection of livestock.

Comparison of two trapping methods for Culicoides biting midges and determination of African horse sickness virus prevalence in midge populations at Onderstepoort, South Africa.

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of a variety of pathogens including African horse sickness virus (AHSV), a member of the family Reoviridae, genus Orbivirus. AHSV causes African horse sickness (AHS), an endemic disease of equids with an extremely high mortality rate in horses in sub-Saharan Africa. Culicoides (Avaritia) imicola Kieffer is considered to be the principal vector of AHSV and is the dominant Culicoides species in South Africa. Due to the global distribution of Culicoides vectors, there is a potential risk of AHS spreading from endemic areas to areas traditionally free of the disease, which could have a severe economical impact on the affected equine industry. As part of any risk assessment it is essential to monitor known vectors as well as potential vector species. In the present study, sampling of Culicoides insects was compared using overnight collections in the conventional Onderstepoort light trap and mechanical aspiration of midges at sunset from bait horses. Culicoides imicola was confirmed as the predominant species using both trapping methods. Other species, mainly Culicoides (Avaritia) bolitinos Meiswinkel and Culicoides (Avaritia) gulbenkiani Caeiro, were highly underrepresented in the light trap collections, but made a significant contribution to the mechanical aspiration catches. The time for optimal collection differed between the trapping methods, leading to the conclusion that mechanical aspiration is a useful addition to conventional light trap collection and possibly the better choice when investigating insect vectors. An infection rate of 1.14% was calculated for the midge population based on real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays of collected Culicoides midges, which exceeds previous estimates. This is probably due to the increased sensitivity of the RT-qPCR assay used in this study as compared to the virus isolation assays used in previous studies. RT-qPCR-positive midges were present in midge pools obtained from both light trap and mechanical aspiration. Seven of the positive pools consisted of C. imicola only, four contained mixed species and one pool contained no C. imicola, suggesting the presence of AHSV in midges of other species.

Anthropogenic and meteorological factors influence vector abundance and prevalence of bluetongue virus infection of dairy cattle in California.

Bluetongue is an economically important arboviral disease of ruminants that is transmitted by hematophagous Culicoides midges. In light of dramatic recent changes in the global distribution of bluetongue virus (BTV), the goals of this study were to re-evaluate the prevalence of BTV infection of cattle and abundance of Culicoides midges on individual dairy farms in California. A serosurvey of adult dairy cattle confirmed that BTV infection is prevalent throughout much of the state, although the coastal northwestern region remains free of infection and prevalence varies markedly among farms in the remainder of the state. Intensive sampling for one year of 4 farms in the northern Central Valley of California showed that the abundance of Culicoides midges was markedly different and coincided with the prevalence of BTV infection of sentinel cattle on each farm. Mean maximum and minimum temperatures and other meteorological parameters were similar on all 4 farms, thus we speculate that particular management practices were responsible for both the increased midge abundance and prevalence of BTV infection of cattle at individual farms. Specifically, it is concluded that variation in vector abundance at individual farms most likely is the result of waste-water lagoon and irrigation management practices, leading to higher BTV infection rates among livestock held on farms with more waste-water lagoons and greater acreage of land for waste-water irrigation.