Camilla T. Weyer

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.

African horse sickness in naturally infected, immunised horses

To determine whether subclinical cases, together with clinical cases, of African horse sickness (AHS) occur in immunised horses in field conditions, whole blood samples were collected and rectal temperatures recorded weekly from 50 Nooitgedacht ponies resident in open camps at the Faculty of Veterinary Science, University of Pretoria, Onderstepoort, during 2008-2010. The samples were tested for the presence of African horse sickness virus (AHSV) RNA by a recently developed real-time RT-PCR. It was shown that 16% of immunised horses in an AHS endemic area were infected with AHSV over a 2 year period, with half of these (8%) being subclinically infected. The potential impact of such cases on the epidemiology of AHS warrants further investigation.

African horse sickness caused by genome reassortment and reversion to virulence of live, attenuated vaccine viruses, South Africa, 2004-2014

Epidemiologic and phylogenetic analyses show repeated outbreaks derived from vaccine viruses.

Occurrence of African horse sickness in a domestic dog without apparent ingestion of horse meat.

This is the first case of African horse sickness (AHS) in a dog where there was no apparent ingestion of horse meat. Significantly, the dog was part of a colony that resides in a Good Clinical Practice and Good Laboratory Practice accredited facility where complete history, weather and feeding records are maintained. The dog died after a week-long illness despite therapy. The principal post-mortem findings were severe hydrothorax and pulmonary consolidation (red hepatisation of the lungs). Histopathology revealed severe oedema and congestion of the lungs, hyaline degeneration of the myocardium and congestion of the liver sinusoids. Immunohistochemistry detected AHS-positive staining granules in the myocardium, whilst a real-time reverse transcription quantitative Polymerase chain reaction assay of tissue samples was strongly positive for African horse sickness virus nucleic acid. Other dogs on the property showed a 43%seroconversion rate to AHS.

The 2011 outbreak of African horse sickness in the African horse sickness controlled area in South Africa

African horse sickness (AHS) is a controlled animal disease in South Africa, and as a result of the high mortality rates experienced, outbreaks in the AHS controlled area in the Western Cape Province have a significant impact on affected properties as well as on the exportation of live horses from the AHS free zone in metropolitan Cape Town. An outbreak of AHS serotype 1 occurred in the surveillance zone of the AHS controlled area of the Western Cape during the summer of 2011. The epicentre of the outbreak was the town of Mamre in the magisterial district of Malmesbury and the outbreak was confined to a defined containment zone within this area by movement control of all equids and a blanket vaccination campaign. A total of 73 cases of AHS were confirmed during this outbreak, which included four confirmed subclinical cases. The morbidity rate for the outbreak was 16%with a mortality rate of 14%and a case fatality rate of 88%. Outbreak disease surveillance relied on agent identification using polymerase chain reaction (PCR)-based assays, which is novel for an AHS outbreak in South Africa. The source of this outbreak was never confirmed although it is believed to be associated with the illegal movement of an infected animal into the Mamre area. This detailed description of the outbreak provides a sound scientific basis to assist decision making in future AHS outbreaks in the AHS controlled area of South Africa and in countries where AHS is an exotic or emerging disease.

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.

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

ABSTRACT 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.

West Nile Virus Lineage 2 in Horses and Other Animals with Neurologic Disease, South Africa, 2008–2015

During 2008–2015 in South Africa, we conducted West Nile virus surveillance in 1,407 animals with neurologic disease and identified mostly lineage 2 cases in horses (7.4%, 79/1,069), livestock (1.5%, 2/132), and wildlife (0.5%, 1/206); 35% were fatal. Geographic correlation of horse cases with seropositive veterinarians suggests disease in horses can predict risk in humans.

Dynamics of African horse sickness virus nucleic acid and antibody in horses following immunization with a commercial polyvalent live attenuated vaccine

African horse sickness (AHS) is a fatal disease of equids relevant to the global equine industry. Detection of AHS virus (AHSV) during outbreaks has become more rapid and efficient with the advent of group specific reverse transcriptase quantitative polymerase chain reaction (GS RT-qPCR) assays to detect AHSV nucleic acid. Use of GS RT-qPCR together with recently described type specific (TS RT-qPCR) assays cannot only expedite diagnosis of AHS but also facilitate further evaluation of the dynamics of AHSV infection in the equine host. A potential limitation to the application of these assays is that they detect viral nucleic acid originating from any AHS live attenuated vaccine (LAV), which is the vaccine type routinely administered to horses in South Africa. The aim of this study was to contrast the dynamics and duration of the RNAaemia to the serological responses of horses following immunization with a commercial polyvalent AHSV-LAV using GS and TS RT-qPCR assays and serum neutralisation tests. The results of the study showed extended RNAemia in vaccinated horses, and that more horses tested positive on GS RT-qPCR with lower Cq values after receiving the AHSV-LAV containing types 1, 3 and 4 prior to the vaccine containing types 2, 6, 7 and 8, rather than when the vaccine combinations were reversed. Furthermore, lower Cq values were obtained when vaccines were administered 4weeks apart as compared with a longer interval or 12weeks apart. These findings are of particular relevance in regions where AHSV-LAVs are used as the use of these vaccines may complicate the accurate interpretation of diagnostic testing results.