Molecular diagnosis and genetic diversity of tick-borne Anaplasmataceae agents infecting the African buffalo Syncerus caffer from Marromeu Reserve in Mozambique

Machado et al. Parasites & Vectors (2016) 9:454 DOI 10.1186/s13071-016-1715-y RESEARCH Open Access Molecular diagnosis and genetic diversity of tick-borne Anaplasmataceae agents infecting the African buffalo Syncerus caffer from Marromeu Reserve in Mozambique Rosangela Zacarias Machado1*, Marta Maria Geraldes Teixeira2, Adriana Carlos Rodrigues2, Marcos Rogério André1, Luiz Ricardo Gonçalves1, Jenevaldo Barbosa da Silva1 and Carlos Lopes Pereira3 Abstract Background: Tick-borne diseases (TBDs) are very important in relation to domestic ruminants, but their occurrence among wild ruminants, mainly in the African buffalo Syncerus caffer, remains little known. Methods: Molecular diagnostic methods were applied to detect Anaplasma marginale, Anaplasma centrale, Anaplasma phagocytophilum, Ehrlichia ruminantium and Ehrlichia chaffeensis in 97 blood samples of African buffalo captured at the Marromeu Reserve in Mozambique. Molecular detection of agents belonging to the family Anaplasmataceae were based on conventional and qPCR assays based on msp5, groEL, 16S rRNA, msp2, pCS20 and vlpt genes. Phylogenetic reconstruction of new Anaplasma isolates detected in African buffalo was evaluated based on msp5, groEL and 16S rRNA genes. Results: All the animals evaluated were negative for specific PCR assays for A. phagocytophilum, E. ruminantium and E. chaffeensis, but 70 animals were positive for A. marginale, showing 2.69 × 100 up to 2.00 × 105 msp1β copies/μl. This result overcomes the conventional PCR for A. marginale based on msp5 gene that detected only 65 positive samples. Sequencing and phylogenetic analyses were performed for selected positive samples based on the genes msp5, groEL and 16S rRNA. Trees inferred using different methods separated the 29 msp5 sequences from buffalo in two distinct groups, assigned to A. centrale and A. marginale. The groEL sequences determined for African buffalo samples revealed to be more heterogeneous and inferred trees could not assign them to any species of Anaplasma despite being more related to A. marginale and A. centrale. The highly conserved 16S rRNA gene sequences suggested a close relationship of the new 16 sequences with A. centrale/A. marginale, A. platys and A. phagocytophilum. Conclusions: Our analysis suggests that different species of Anaplasma are simultaneously present in the African buffalo. To the best of our knowledge, this is the first study that diagnosed Anaplasma spp. in the African buffalo and inferred the taxonomic status of new isolates with different gene sequences. The small fragment of msp5 sequences revealed to be a good target for phylogenetic positioning of new Anaplasma spp. isolates. Keywords: African buffalo, Anaplasma marginale, Anaplasma centrale, Anaplasma platys, Anaplasma phagocytophilum, Genotyping, groEL, msp5, 16S rRNA, Mozambique * Correspondence: 1 Department of Veterinary Pathology, School of Agrarian and Veterinary Sciences (FCAV/UNESP), Jaboticabal, SP, Brazil Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Machado et al. Parasites & Vectors (2016) 9:454 Background Tick-borne agents (TBAs) form one of the main groups of pathogens infecting both domestic and wild ruminants in sub-Saharan Africa and in tropical and subtropical regions. In Mozambique, theileriosis, ehrlichiosis, anaplasmosis and babesiosis are the most important tick-borne diseases (TBDs), causing significant economic losses to the national cattle industry [1]. Wild ruminants may play a role as hosts and reservoirs for several tick-borne pathogens, especially Anaplasmataceae agents and piroplasms. For instance, Anaplasma phagocytophilum, Ehrlichia chaffeensis and Ehrlichia ruminantium are of major concern due to their importance in veterinary and/or human medicine. Anaplasma phagocytophilum is transmitted by ticks of the genus Ixodes and causes tick-borne fever in sheep, goats and cattle in Europe and granulocytic anaplasmosis in humans [2, 3]. This pathogen has been recognized as the causal agent of illnesses in ruminants in Scotland (United Kingdom), Ireland and Scandinavia [4]. Ehrlichia chaffeensis, which is transmitted by the tick Amblyomma americanum in the USA, is the causative agent of human monocytic ehrlichiosis [5]. White-tailed deer (Odocoileus virginianus) are considered the natural reservoirs for both pathogens in wildlife in the USA [4]. In turn, E. ruminantium has been reported in Africa and can be transmitted by ticks of the genus Amblyomma (especially A. variegatum and A. hebraeum). Currently, ehrlichiosis is considered to be one of the most important diseases of domestic ruminants in sub-Saharan Africa [6], with a high mortality rate among susceptible sheep, goats and cattle. Although the African buffalo is considered to act as wild reservoir for this agent, clinical signs and prevalence in this animal species remain little known [7, 8]. Although several tick-borne agents (TBA) may affect buffalo, special attention needs to be paid to A. marginale, since this is an important pathogen that is responsible for significant economic losses relating to cattle-rearing in South America and Africa [9]. In these regions, this bacterium can be transmitted mechanically by hematophagous dipteran insects, including various species of Tabanus and Stomoxys, and by some mosquito species in the genera Culex and Aedes [10]. Although A. marginale has already been detected in several wild ruminant species, such as Odocoileus virginianus, O. hemionus hemionus, O. hemionus columbianus, Antilocapra americana, Cervus elaphus nelson and Ovis canadensis canadensis in North America and Connochaetes gnou, Damaliscus dorcas phillipsi and Sylvicapra grimmia grimmi in Africa [11], most of the studies on the occurrence, seroepidemiology and molecular characterization of these agents have been conducted among cattle [12, 13]. When A. marginale infects Page 2 of 9 ruminant species other than cattle, the infection is generally of a chronic nature [11]. In Mozambique, large numbers of African buffalo are maintained in national parks under the protection of the country’s legislation. However, there are still no studies on the prevalence of tick-borne pathogens circulating in this group of animals. In the present study Anaplasma species was detected in African buffalo in the Marromeu Reserve (Mozambique) and characterize (...truncated)