Molecular epidemiology of dourine, equine piroplasmosis and ehrlichiosis from donkeys and horses in South Africa
Abstract
Horses and donkeys are important domestic animals to human beings whereby historically and presently are used for transport and as drought animals. Furthermore, horses are used in different sporting types, and are important animals in police and military services. Therefore their health is of great importance.
Dourine is caused by Trypanosoma equiperdum and is the only trypanosome species that is transmitted sexually from infected to healthy animals, and to foals during birth and from maternal milk. Dourine is known to be present in South Africa with Eastern Cape being the most severely affected province whilst Western Cape, Free State and Limpopo have a comparatively low report case incidents. In addition to observation of clinical signs, laboratory diagnosis of dourine is done only by a serological technique called complement fixation test (CFT) in South Africa. In this study Polymerase chain reaction (PCR) and Loop−mediated isothermal amplification (LAMP) as well as Enzyme immune sorbent assay (ELISA) and Immunochromatographic test (ICT) were used to determine the occurrence of dourine in horses and donkeys in South Africa. The general aim of this study was to determine the occurrence of dourine, equine piroplasmosis, anaplamosis and erhlichiosis from horses and donkeys in provinces of South Africa. In this study, a total of 256 blood samples were collected from equids (32 from donkeys and 224 from horses) from four provinces in South Africa, namely, Free State (FS), Mpumalanga (MP), Northern Cape (NC) and North West (NW). Out of 256 DNA samples screened by PCR, there was an overall prevalence of 14% (36/256) for dourine, with 15% [95% CI = 0.17± 0.29], 17% [95% CI = 1.61 ± 0.33], 11% [95% CI = 0.17 ± 0.21], and 12% [95% CI = 0.22 ± 0.23] for FS (n = 40), MP (n = 94), NC (n = 54) and NW (n = 68). LAMP was used to confirm that PCR positive samples are true positives. PCR positive samples were also sequenced and they matched with other T. equiperdum sequences on NCBI database. A phylogenetic tree constructed with 18S rRNA gene, the T. equiperdum correctly clustered with other trypanosomes of subgenus Trypanozoon. There were 38 genital secretion samples collected by sterile swab in horses in the Free State province and only 1 sample was positive by PCR for the presence of T. equiperdum infections. The overall T. equiperdum prevalence by PCR was 8.8% (3/34) with 8.3% (1/12) and 10% (2/20) in NC and NW provinces in donkeys. There was no T. equiperdum detected in FS province.
The overall seroprevalence of dourine for all sampled equids was 18.4%, 15.6% and 2.4% by recombinant antigen ELISA (rELISA), crude antigen ELISA (caELISA) and ICT respectively. The seroprevalence was 17.6%, 16.6% and 2.8% by rELISA, caELISA and ICT respectively in horses. The seroprevalence was 23.5%, 8.8% and 0% in donkeys by rELISA, caELISA and ICT respectively. Polymerase chain reaction efficiently detected T. equiperdum infections as confirmed by sequencing and rELISA revealed higher detection sensitivity than caELISA. The detection efficiency of ICT for dourine was poor, and needs further improvements. Equine piroplasmosis is one of the most important tick−borne diseases, with an economic worldwide impact on the horse industry and is endemic in equids in most of tropical and sub−tropical regions of the world where tick vectors are present. The disease is caused by the two hemoprotozoan parasites, Theileria equi and Babesia caballi. In South Africa information on the occurrence of equine piroplasmosis based on IFAT and PCR methods is available and recently there is a report based on real time PCR in some provinces such as Free State, Northern Cape, Western Cape, Eastern and KwaZulu−Natal. However there are no records obtained using modern molecular techniques for equine piroplasmosis in other provinces including Mpumalanga and North West. Furthermore, the disease prevalence in other equids such as donkeys in South Africa is not documented. Therefore this study used PCR to detect T. equi and B. caballi infections from blood samples (n = 256) collected from four Provinces of South Africa, namely, FS; MP, NC and NW.
The prevalence obtained by PCR for T. equi in horses for FS, MP, NC and NW was 35% [95% CI = 0.39 ± 0.31], 14.9% [95% CI = 0.39 ± 0.29], 7% [95% CI = 0.11 ± 0.14] and 6% [95% CI = 0.11 ± 0.11] respectively. The χ² = 19.83 (df = 3) and p ˃ 0.05. Babesia caballi prevalence in horses was 22.5% [95% CI = 0.43 ± 042], 5.3% [95% CI = 0.45 ± 0.10], 5.6% [95% CI = 0.15 ± 0.10] and 4.4% [0.15 ± 0.08] respectively [χ² = 16.35 (df = 3) and p ˃ 0.05]. Therefore there is a significant difference observed in the overall prevalence of T. equi and B. caballi in the sampled provinces. Loop mediated isothermal amplification (LAMP) was used to confirm PCR positives. Theileria equi (35%) infections were more prevalent than B. caballi (8.2%) infections. In this study, B. caballi infections were detected in areas where it was not found by PCR previously. Theileria equi and B. caballi parasites appeared to be more prevalent in Free State, 35% (14/40) and 22.9% (9/40), respectively. Horses (14.9%) were more susceptible than donkeys (5.9%) for equine piroplasmosis.
Anaplasmosis and ehrlichisosis are tick−borne diseases caused by obligate intracellular bacteria of the genera Ehrlichia and Anaplasma. These organisms are widespread in nature and the reservoir hosts include numerous wild animals, as well as some domesticated species. In the past decade, ehrlichiosis has been recognized as a new zoonotic disease that is responsible for several thousand cases and several deaths annually. There is currently no information on the prevalence of equine anaplasmosis and ehrlichiosis in South Africa. The current study was aimed at determining the occurrence of equine anaplasmosis and ehrlichiosis by PCR in South African equids. In particular the study focused on detection of Anaplasma phagocytophilum and Neorickettsia risticii across the four sampled provinces. Prevalence of A. phagocytophilum infections obtained was 68.5% [95% CI = 0.16 ± 0.04 in NC, 52.5% [95% CI = 0.24 ± 1.03] in FS, 20.6% [95% CI = 0.43 ± 1.43] in NW and 16.0% [95% CI = 0.17 ± 0.31] in MP {χ² = 57.37 (df = 3) and p < 0.05}. This was for the first time that A. phagocytophilum was detected in horses in selected provinces of South Africa using PCR.
The N. risticii prevalence in horses was 12.5% [95% CI = 0.32 ± 0.22], 3.2% [95% CI = 0.33 ± 0.02] and 1.9% [95% CI = 0.11 ± 0.03] for FS, MP and NC respectively. The χ² = 12.42 (df = 3) and p >0.05. There is a significant difference observed across the sampled provinces. None of the DNA samples from donkeys (Equus asinus) tested positive for N. risticii across all four sampled provinces. This support the fact that equine monocytic ehrlichiosis is the disease that is found in horses only.