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
African horse sickness (AHS) and equine encephalosis (EE) are significant horse diseases in Zimbabwe caused by arthropod-borne viruses belonging to the Reoviridae family, genus
In contrast to AHS, EE is perceived as an emerging infectious disease and was first identified in 1967 by Erasmus et al. (
The distribution of AHS and EE in southern Africa seems to be governed by a number of factors including the prevalence and seasonal incidence of the major arthropod vector(s), which are certain species of
The objectives of this study were to determine the sero-prevalence and sero-incidence of AHSV and EEV in horses and donkeys from sentinel herds established around Harare and Bulawayo in Zimbabwe. The serotypes of AHSV in animals from the sentinel herds and from the samples of suspected clinical cases in horses were also determined.
Donkeys and horses on selected farms around the cities of Bulawayo and Harare, in Zimbabwe, were sampled to determine the sero-prevalence and sero-incidence of AHSV and EEV antibodies. Both locations lie in the central Highveld of Zimbabwe and experience climatic conditions that favour the abundance of the
Venous blood samples (5 mL) were taken from all horses on participating farms using ethylene diamine tetra-acetic acid (EDTA) vacutainers and tested for EEV antibodies to determine the sero-prevalence of EEV in these herds. Blood samples were also taken from all donkeys on participating farms and tested for antibodies against AHSV and EEV to determine the sero-prevalence of AHSV and EEV in these donkey herds. Due to the widespread use of the live, attenuated, AHS vaccine (Onderstepoort Biological Products®) in horses in Zimbabwe, the sero-prevalence of AHSV in horses could not be determined as AHSV-specific antibodies may have been derived from vaccine viruses. The detection of antibodies against AHSV was determined using a competitive enzyme-linked immunosorbent assay (ELISA) following the methodology described by Hamblin et al. (
Sentinel herds of donkeys and horses were established, at the same farms used to determine the sero-prevalence of antibodies to these two viruses during three rainy seasons (1999–2002). These sentinel herds were used to determine the sero-incidence of AHSV antibodies in donkeys and EEV antibodies in horses and donkeys by determining the rates at which sentinel animals seroconverted to AHSV or EEV. The sentinel herds were established using healthy adult horses and donkeys of both sexes that tested as seronegative against AHSV antibodies (donkeys) and EEV antibodies (horses and donkeys). A total of three horse sentinel herds were established around Harare and six around Bulawayo. Two donkey sentinel herds were established on farms around Harare and one around Bulawayo. Venous blood samples from the animals in the sentinel herds were taken using EDTA vacutainers and tested for antibodies against AHSV and EEV every 2 weeks, during each of the three rainy seasons (November–April), until seroconversion was detected.
Private equine veterinary practitioners, working within the Highveld region of Zimbabwe, were asked to monitor for suspected clinical cases and deaths from AHS in horses located within their veterinary practice catchment area over a 5-year period from 1998 to 2003. During this period, spleen samples, blood samples and combined spleen and blood samples were submitted by private equine veterinary practitioners from horses suspected to have died from or suffering from clinical AHS. No clinical cases of EE in horses or donkeys had been reported by practising veterinarians in Zimbabwe to that time although all submitted samples were tested for both AHSV and EEV. The blood samples submitted were collected in EDTA from live patients and the spleen tissue from dead patients following the recommendations outlined by Hamblin et al. (
All submitted blood and spleen samples were washed and lysed using techniques recommended by Hamblin et al. (
Virus isolations were made by intracerebral inoculation of 2-day-old suckling mice (CD-1, UK) and intravenous inoculation of 11-day-old embryonated hens’ eggs (IAH, UK). In each case, 100 μL of a 1 in 10 dilution of lysed blood or homogenated spleen was inoculated. Brains from dead mice and hearts from dead embryos were homogenised as a 1 in 10 suspension and serially blind passaged up to three times in baby hamster kidney (BHK) tissue culture cells to amplify any virus present and to adapt them to growth in cell culture.
The AHS viruses were typed by virus neutralisation, using microtitre plates and BHK cells, following methods similar to those described by Howell (
Data were examined using simple descriptive statistics. Data were tested for normality using the Shapiro–Wilk test. Non-parametric data are presented as median and inter quartile range. The effect of season on prevalence was tested using a Kruskal–Wallis test. All analyses were performed using Stata IC v12 (College Station, TX, USA). The level of significance was set at
Serum decanted from blood samples collected from 279 horses was tested for EEV antibodies over the three rainy seasons to determine the sero-prevalence of EEV in these herds. The small per farm sample size (22; inter quartile range [IQR] 14–28) provided a large between-farm variation in the sero-prevalence of EEV (
The sero-prevalence of antibodies against equine encephalosis virus in horses in Zimbabwe, during three rainy seasons for the period 1999–2002.
Season | Number of farms | Number of horses bled | Number seropositive for antibodies against EEV | Sero-prevalence of EEV (%) |
|
---|---|---|---|---|---|
Median | IQR | ||||
1999/2000 | 6 | 150 | 39 | 28 | 11–62 |
2000/2001 | 3 | 74 | 55 | 76 | 70–70 |
2001/2002 | 3 | 55 | 36 | 64 | 29–100 |
All years | - | 279 | 130 | 63 |
21–73 |
EEV, equine encephalosis virus; IQR, inter quartile range.
Differences between seasons (
Serum decanted from blood samples collected from 155 donkeys was tested for antibodies against AHSV and EEV to determine the sero-prevalence of AHSV and EEV (
The sero-prevalence of antibodies against African horse sickness virus and equine encephalosis virus in donkeys in Zimbabwe during three rainy seasons for the period 1999–2002.
Season | Number of farms | Number of donkeys bled | Number seropositive for antibodies against AHSV | Sero-prevalence of AHSV(%) |
Number seropositive for antibodies against EEV | Sero-prevalence of EEV (%) |
||
---|---|---|---|---|---|---|---|---|
Median | IQR | Median | IQR | |||||
1999/2000 | 2 | 69 | 42 | 64 | 60–67 | 30 | 53 | 39–67 |
2000/2001 | 1 | 35 | 30 | 86 | - | 30 | 86 | - |
2001/2002 | 3 | 51 | 40 | 80 | 69–83 | 45 | 90 | 78–100 |
All years | - | 155 | 112 | 75 | 67–83 | 105 | 80 | 67–90 |
AHSV, African horse sickness virus; EEV, equine encephalosis virus; IQR, inter quartile range.
A total of six horse sentinel herds were established over three rainy seasons to determine the sero-incidence of EEV antibodies in horses. The sentinel herds consisted of a total of 76 horses and a median sero-incidence of EEV of 10.5% (IQR 10–14) was recorded.
One to two donkey sentinel herds were established during each rainy season under study to determine the sero-incidence of AHSV and EEV antibodies in donkeys. The sentinel herds consisted of a total of 18 donkeys, with each sentinel herd in each rainy season comprising 4–5 donkeys. A median sero-incidence of 45% (IQR 40–63) and 50% (IQR 40–60) was recorded for AHSV and EEV, respectively.
During a 5-year period from 1998 to 2003, 22 spleen samples, 92 EDTA blood samples and 11 combined spleen and EDTA blood samples were submitted by private equine veterinary practitioners from 125 horses suspected to have died from or suffering from clinical AHS (
Horse samples submitted by private equine veterinary practitioners for the detection of African horse sickness virus antigens.
Season | Number of samples submitted |
Number positive for AHSV | Prevalence of AHSV (%) | |||
---|---|---|---|---|---|---|
Blood | Spleen | Blood and spleen | Total | |||
1998/1999 | 38 | 16 | 9 | 63 | 52 | 83 |
1999/2000 | 30 | 1 | 1 | 32 | 12 | 38 |
2000/2001 | 7 | 2 | 1 | 10 | 10 | 100 |
2001/2002 | 11 | 1 | 0 | 12 | 4 | 33 |
2002/2003 |
6 |
2 |
0 |
8 |
2 |
25 |
Median(IQR): 38(33–83).
AHSV, African horse sickness virus; IQR, inter quartile range.
The predominant AHSV serotype identified from private equine veterinary practitioners’ horse spleen and blood samples and blood samples from donkey sentinel herd animals showing seroconversion was serotype 7 (19/57, 33%) followed by serotype 2 (15/57, 26%), serotypes 4 and 8 (9/57, 16% each) and serotype 6 (3/57, 5%). Serotypes 3 and 9 (1/57, 2%) were identified for the first time in Zimbabwe. Attempts to serotype EEV were unsuccessful.
This study recorded a high median sero-prevalence of antibodies against AHSV in selected donkey herds and a high median sero-prevalence of antibodies against EEV in selected horse and donkey herds in the Highveld region of Zimbabwe. A sero-prevalence for antibodies against EEV of 56% – 87% in horses and donkeys and a sero-prevalence of AHSV of 44% – 64% in horses were previously reported in Zimbabwe (Blackburn
Because of the annual polyvalent prophylactic immunisation against AHSV practised in horses in Zimbabwe at the time of this study, serotype-specific antibody derived from immunisation, natural challenge or the transfer of colostrum could not have been distinguished by conventional serum viral neutralisation assays (Howell et al.
The median seasonal sero-incidence of EEV antibodies was found to be higher in donkey sentinel herds than in horse sentinel herds. This could be because of the AHS vaccine used in horses conferring some form of protection against EEV in this species but may also reflect the fact that donkeys are rarely stabled at night in Zimbabwe and are thus more at risk of exposure to the
The prevalence and incidence rates, however, show that horses and donkeys face a significant rate of exposure to both AHSV and EEV in Zimbabwe. Previously, EE in southern Africa was thought to be mainly distributed in South Africa and Botswana (Paweska et al.
The sentinel herds were situated in the Highveld of Zimbabwe, which is characterised by high rainfall and temperature that favour the abundance of the
Most sero-conversions to AHSV and EEV in this study occurred from January to April, 3–4 months after the beginning of the annual rainy season. This delay reflects the time taken for vector numbers to increase after the beginning of the annual rains and the time for a viraemia to develop in animals, which then act as sources of infection for the vectors (Mellor
The predominant AHSV serotype recorded in this study was serotype 7, which was identical to the findings reported in the 1980 outbreak of AHS in the north west of Harare (Blackburn & Swanepoel
This study highlighted the significant levels of exposure of horses and donkeys to AHSV and EEV in Zimbabwe. The sero-incidence of AHSV and EEV infection and the clinical cases of AHS were highest at the end of the rainy season in March and April coinciding with peak abundance of the
African horse sickness continues to be a serious problem in Zimbabwe despite the extensive vaccination programme conducted in horses across the country at the time of this study. As EE is largely undiagnosed by equine veterinary practitioners in Zimbabwe, the high reported sero-prevalence and sero-incidence of this disease will hopefully alert Zimbabwean veterinarians to the possibility of its occurrence in both horses and donkeys.
The authors would like to acknowledge the support and cooperation provided by the horse and donkey establishments in Zimbabwe which permitted sentinel herds to be established and monitored over all the rainy seasons under study. The authors are also grateful for the assistance provided by the private equine veterinary practitioners who provided the blood and spleen samples from horses with suspected AHS clinical cases. The authors would also like to acknowledge the support provided by the Biomedical Research and Training Institute and the Wellcome Trust.
The authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article.
S.J.G.G. wrote the manuscript and acted as the project leader. G.M., P.K., P.S.M. and C.H. helped design the project, establish and monitor the sentinel herds, test for serum antibodies and antigens and assist with the virus isolation and identification. C.B., C.W.R. and A.G. made conceptual contributions and assisted with the data analysis, statistical work and the editing of the final manuscript.