Molecular characterisation of Anaplasma species from African buffalo (Syncerus caffer) in Kruger National Park, South Africa

Abstract

Bovine anaplasmosis is a tick-borne disease, mainly caused by Anaplasma marginale and A. centrale and is distributed in tropical and sub-tropical areas. This study aimed to characterise A. marginale and A. centrale from African buffaloes in Kruger National Park (KNP), South Africa, using the DNA sequences of the genes coding for major surface protein (msp1β) and heat shock protein (groEL), respectively. A total of 747 blood samples were collected from February 2014 to August 2016 from African buffaloes kept in KNP, and DNAs were tested using a molecular-phylogenetic approach. Out of 747 samples tested, 129 (17.3%) and 98 (13.1%) were positive for single infection with A. marginale and A. centrale, respectively; whereas 113 (15.1%) were positive for both Anaplasma spp. Pairwise difference of 1.6–8.5% was observed in msp1β sequences of A. marginale whereas that was only 0.3–2.4% for groEL sequences of A. centrale. Separate phylogenetic analyses of msp1β and groEL sequences of A. marginale and A. centrale, respectively, revealed that sequences of Anaplasma spp. from African buffaloes were unique and they grouped separately when compared with previously published sequences of both species. This is the first study to characterise A. marginale and A. centrale from African buffalo using species specific molecular markers. This study will pave the way for future studies to assess genetic variation among Anaplasma spp. from wild ruminants using molecular markers that are better at differentiating between species and strains than the more commonly used 16S rRNA gene, and help to undertake health and fitness studies and host-parasite dynamics using quantitative molecular tools.

Introduction

Anaplasmosis is an important tick-borne disease (TBDs) of domestic and wild animals in tropical and subtropical regions of the world, and is caused by obligate intracellular bacteria of the genus Anaplasma (Rickettsiales: Anaplasmataceae). It is one of the four most detrimental tick-borne diseases (TBDs) of bovines in sub-Saharan Africa (others: babesiosis, cowdriosis and theileriosis) (Debeila, 2012), and is estimated to be responsible for 3% of all cattle mortalities in South Africa (Aubry and Geale, 2011, Brown, 2012, De Waal, 2000, Eygelaar et al., 2015). In South Africa, anaplasmosis is mainly transmitted by Amblyomma spp. and Rhipicephalus spp. (Gallivan et al., 2011, Horak et al., 2007, Horak et al., 2011). Bovine anaplasmosis is mainly caused by Anaplasma marginale and, to a lesser extent, by A. centrale. The disease is usually transmitted by ticks, but it can also be transmitted transplacentally, or mechanically by biting flies or blood-contaminated fomites (Aubry and Geale, 2011, Potgieter and Stoltz, 2004). Major clinical signs in infected cattle include pyrexia, progressive anaemia, jaundice, anorexia, depression, reduced milk production, abortion in pregnant animals, and death, particularly in exotic breeds (Aubry and Geale, 2011, Debeila, 2012, Kocan et al., 2010, Potgieter and Stoltz, 2004).

While anaplasmosis is well documented in cattle, very little is known about the disease and its impact in wild bovids such as the African buffalo (Syncerus caffer). Tick-borne infections are common in buffalo, which serve as a reservoir for bovine theileriosis (Theileria parva) (Debeila, 2012, Henrichs et al., 2016). Buffalo appear to be only mildly affected by Anaplasma infections (Berggoetz et al., 2014, Debeila, 2012, Kuttler, 1984), which raises the question whether they might also function as reservoirs for this group of parasites. To date, A. marginale, A. centrale, A. sp. Omatjenne, A. bovis and A. phagocytophilum have been detected from African buffaloes using the 16S rRNA gene, though A. phagocytophilum has only been found once in one animal (Fyumagwa et al., 2013, Henrichs et al., 2016). African buffalo are a useful wildlife species in which to study Anaplasma species dynamics as they are known to be wildlife reservoirs for numerous diseases such as foot-and-mouth disease, bovine tuberculosis and theileriosis, yet their role in maintaining Anaplasma species is unknown (Debeila, 2012). Identification of wildlife reservoirs is essential to controlling infection in livestock which may have a direct or indirect contact with the reservoir population (Haydon et al., 2002).

Various methods have been used to diagnose anaplasmosis, including microscopy, serology (complement fixation, rapid card agglutination, indirect immunofluorescent antibody tests, capillary tube agglutination tests, enzyme-linked immunosorbent assays, latex agglutination and radioimmunassays) and molecular methods (Aubry and Geale, 2011, Potgieter and Stoltz, 2004). For molecular methods utilising polymerase chain reaction (PCR), a number of markers such as the 16S rRNA, major surface protein (msp1α, msp1β, msp2, msp3, msp4 and msp5), citrate synthase gltA and the heat shock protein groEL genes have been used for the detection of Anaplasma spp. (Carelli et al., 2007, Ceci et al., 2008, de la Fuente et al., 2001, Lew et al., 2002, Lew et al., 2003, Molad et al., 2006); whereas, msp1α, msp1β, msp4 and groEL have been used for finer scale differentiation of various Anaplasma species and strains.

Very little is known about the diversity of Anaplasma spp. from African buffaloes and only one marker, the 16S rRNA gene, has been used so far to characterise them (Debeila, 2012, Henrichs et al., 2016). However, the most commonly used markers for characterisation of these species, such as genes for major surface proteins and heat shock protein groEL, allow for better resolution which might reveal significant genetic differences among Anaplasma spp. found in domestic and wild ruminants, and can be used in quantitative molecular methods. Therefore, the aim of this study was to characterise the two important species of Anaplasma (A. marginale and A. centrale) from African buffaloes in South Africa using DNA sequences from the genes coding for a major surface protein and heat shock protein groEL, respectively. This study will allow future research into buffalo as a reservoir host of Anaplasma spp.