African horse sickness (AHS) is a life-threatening disease of equids caused by African horse
sickness virus (AHSV), a member of the genus Orbivirus in the family Reoviridae. The virus
is transmitted by midges (Culicoides spp.) and the disease is most prevalent during the time
of year, and in areas where vector Culicoides spp. are most abundant, namely in late
summer in the summer rainfall areas of endemic regions. The disease is of importance to
health and international trade in horses worldwide. Effective surveillance is critical in order to
establish transparent criteria for animal trade from a country or region where AHS occurs.
The 2011 outbreak of African horse sickness in the African horse sickness controlled
area in South Africa: An outbreak of AHS caused by AHSV type one (AHSV1) 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
through movement control of all equids and a blanket vaccination campaign. A total of 73
confirmed cases of AHS were reported during this outbreak, which included four subclinical
cases confirmed by virus isolation (VI). The estimated morbidity rate for the outbreak was
16% with an estimated mortality rate of 14% and a case fatality rate of 88% based on the
figures above. Outbreak disease surveillance relied on agent identification using AHSV
group specific reverse transcriptase quantitative polymerase chain reaction (GS RT-qPCR)
based assays, which was novel for an AHS outbreak in South Africa. The source of this
outbreak was not confirmed at the time, but was believed to be associated with an illegal
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movement of an infected animal into the Mamre area. A detailed description of the outbreak
is given in Chapter 2, and the outbreak provided an opportunity to assess 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. This outbreak further highlighted deficiencies and
complications of available AHSV diagnostic testing and surveillance methods, and the need
for further refinement of these assays and strategies.
Development of three triplex real-time reverse transcription PCR assays for the
qualitative molecular typing of the nine types of African horse sickness virus: The
typing of the specific AHSV involved in the Mamre outbreak was initially done by partial,
direct sequencing of the S10 gene (encoding the non-structural protein NS3) and the L2
gene (encoding the type-specific outer capsid protein VP2) which confirmed the virus to be
AHSV1. This process is time consuming and it became evident that a faster alternative was
needed. This led to the development of type specific RT-qPCR (TS RT-qPCR) assays to
supplement the GS RT-qPCR assay that had already been developed, characterized and
validated. Blood samples collected during routine diagnostic investigations from South
African horses with clinical signs suggestive of AHS were subjected to analysis with the GS
RT-qPCR assay and VI with subsequent serotyping by plaque inhibition (PI) assays using
AHSV type-specific antisera. Blood samples that tested positive by AHSV GS RT-qPCR
were then selected for analysis using 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 types, as well as recently derived stocks of these same viruses. Of the 503
horse blood samples tested, 156 were positive by both AHSV GS RT-qPCR and VI assays,
whereas 135 samples that were VI negative were positive by AHSV GS RT-qPCR assay.
The virus isolates made from the various blood samples included all 9 AHSV types, 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 this study confirmed
that the AHSV TS RT-qPCR assays for the identification of individual AHSV types 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.
Evaluation of the use of foals for active surveillance in an AHS containment zone
during the season following an AHS outbreak: In order to further evaluate the AHS status
of horses in the Mamre area after the outbreak of 2011, a targeted surveillance strategy was
developed. Serial serum and whole blood samples were collected on a monthly basis from
January to June, 2012 from foals (identified by microchip) that were born in the Mamre
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district after the end of the outbreak. Sera were evaluated using traditional serological
methods and the results were compared to the results obtained using the newly developed
molecular assays for virus detection and identification. This study confirmed that AHSV was
eradicated in the Mamre area after the outbreak and, therefore, that the control measures
implemented in the area by the State Veterinary Authorities were effective.
Characterization of the dynamics of African horse sickness virus in horses by
assessing the RNAaemia and serological responses following immunisation with a
commercial polyvalent live attenuated vaccine: As was shown in the 2011 Mamre
outbreak, detection of AHSV during outbreaks has become more rapid and efficient with the
recent development of quantitative GS RT-qPCR assays to detect AHSV nucleic acid. Use
of this assay together with the TS RT-qPCR assays described in Chapter 3, will not 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 AHSV live attenuated vaccine (AHSVLAV),
which is the vaccine type routinely administered to horses in South Africa. A study
was, therefore, designed to characterize the dynamics and duration of the RNAaemia as
compared to the serological responses of horses following vaccination with a commercial
AHSV-LAV, using GS and TS RT-qPCR assays and serum neutralisation tests. This study
provided baseline data on the GS and TS nucleic acid dynamics in weanling foals
vaccinated for the first time, yearlings vaccinated for a second time and adult mares
following a booster to multiple previous vaccinations. These data are fundamental to
interpreting results of AHSV GS RT-qPCR testing of vaccinated horses within an area where
virological surveillance is being applied.
African horse sickness caused by genome reassortment and reversion to virulence of
live, attenuated vaccine viruses, South Africa, 2004 - 2014: In 2014 a further outbreak of
AHS caused by AHSV1 occurred in the Porterville area of the AHS protection zone (PZ),
spreading into the Wellington area in the AHS surveillance zone (SZ). Further involvement of
the Robertson area (AHS PZ) subsequently also occurred. The case fatality rate was much
lower than that of the Mamre outbreak. The clinical signs in infected horses were also
generally milder in the 2014 outbreak, as compared to the 2011 outbreak. Whole genome
sequencing of samples from the Porterville outbreak confirmed that causative virus was a
recombination (reassortant) of AHSV types 1 and 4, with genes derived from the relevant
vaccine strains contained in OBP comb1 of the commercial polyvalent AHSV-LAV used in
South Africa. This led to further analysis of 39 AHSV strains from field cases of AHS that
originated from outbreaks within the controlled area, which confirmed reversion to virulence
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of AHSV type 1 vaccine in two outbreaks (2004 and 2011) and multiple reassortment events
in two outbreaks (2004 and 2014) with genes derived from all three AHSV vaccine strains
(types 1, 3 and 4). This study provided a molecular and epidemiological comparison of the
five unique AHSV type 1 outbreaks in the AHS controlled area. It was shown that all the
outbreaks in the AHS controlled area attributed to AHSV type 1 since the inception of the
area in 1997, have been due either to reversion to virulence of the AHSV type 1 vaccine
strain, or recombination of AHSV type 1 vaccine strain with one or both of the other vaccine
strains in OBP comb1 of the commercial AHSV-LAV.
