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: [email protected] 1Department 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 Page 2 of 9 Background ruminant species other than cattle, the infection is Tick-borne agents (TBAs) form one of the main groups generally of a chronic nature [11]. of pathogens infecting both domestic and wild rumi- In Mozambique, large numbers of African buffalo are nants in sub-Saharan Africa and in tropical and subtrop- maintained in national parks under the protection of the ical regions. In Mozambique, theileriosis, ehrlichiosis, country’s legislation. However, there are still no studies anaplasmosis and babesiosis are the most important on the prevalence of tick-borne pathogens circulating in tick-borne diseases (TBDs), causing significant economic this group of animals. In the present study Anaplasma losses to the national cattle industry [1]. species was detected in African buffalo in the Marromeu Wild ruminants may play a role as hosts and reser- Reserve (Mozambique) and characterized based on voirs for several tick-borne pathogens, especially Ana- msp5, groEL and 16S rRNA genes for comparison and plasmataceae agents and piroplasms. For instance, phylogenetic inferences. Anaplasma phagocytophilum, Ehrlichia chaffeensis and Ehrlichia ruminantium are of major concern due to Methods their importance in veterinary and/or human medicine. Experimental area Anaplasma phagocytophilum is transmitted by ticks of In 2011, blood samples were collected from 97 African the genus Ixodes and causes tick-borne fever in sheep, buffalo (Syncerus caffer) in Mozambique, Marromeu goats and cattle in Europe and granulocytic anaplasmo- Reserve. This reserve is a special buffalo protection area sis in humans [2, 3]. This pathogen has been recognized located in the Marromeu district (Sofala Province), with as the causal agent of illnesses in ruminants in Scotland an area of 1,500 km2 (www.jenmansafaris.com). Sampled (United Kingdom), Ireland and Scandinavia [4]. Ehrli- animals were apparently healthy young male and female chia chaffeensis, which is transmitted by the tick individuals. Blood samples had been collected before the Amblyomma americanum in the USA, is the causative animals were transferred from Marromeu Reserve to the agent of human monocytic ehrlichiosis [5]. White-tailed Gorongosa Reserve, a distance of c.300 kilometers. deer (Odocoileus virginianus) are considered the natural reservoirs for both pathogens in wildlife in the USA [4]. Samples and DNA extraction In turn, E. ruminantium has been reported in Africa and Blood samples were collected from the buffalo using can be transmitted by ticks of the genus Amblyomma EDTA and were mixed (v/v) with ethanol for further (especially A. variegatum and A. hebraeum). Currently, DNA extraction. In Brazil, the blood samples from these ehrlichiosis is considered to be one of the most import- naturally infected buffalo were incubated in a lysis buffer ant diseases of domestic ruminants in sub-Saharan Af- (1 % SDS, 100 mM EDTA at pH 8.0, 20 mM Tris-HCl at rica [6], with a high mortality rate among susceptible pH 8.0 and 350 mg/ml of proteinase K) at 37 °C for 18 h sheep, goats and cattle. Although the African buffalo is and centrifuged at 14,000× g for 5 min. The DNA was considered to act as wild reservoir for this agent, clinical purified using Wizard Purification Systems (Promega). signs and prevalence in this animal species remain little The concentration of each DNA sample was determined known [7, 8]. in a NanoDrop 2000c spectrophotometer (Thermo Sci- Although several tick-borne agents (TBA) may affect buf- entific, San Jose, CA, USA). falo, special attention needs to be paid to A. marginale, since this is an important pathogen that is responsible for PCR screening for tick-borne pathogens significant economic losses relating to cattle-rearing in qPCR South America and Africa [9]. In these regions, this bacter- A quantitative real-time PCR, based on a fragment of ium can be transmitted mechanically by hematophagous msp1β gene of A. marginale and previously described by dipteran insects, including various species of Tabanus and Carelli et al. [14], was used aiming to estimate the para- Stomoxys, and by some mosquito species in the genera sitemia by means of absolute quantification (number of Culex and Aedes [10]. Although A. marginale has copies/μl). Additionally, a multiplex qPCR for A. phago- already been detected in several wild ruminant spe- cytophilum (msp2 gene) and E. chaffeensis (vlpt gene) cies, such as Odocoileus virginianus, O. hemionus was performed [15]. Serial dilutions of plasmid DNA hemionus, O. hemionus columbianus, Antilocapra containing the target sequence were performed aiming americana, Cervus elaphus nelson and Ovis canaden- to construct standards with different concentrations of sis canadensis in North America and Connochaetes the target sequence (2.0 × 107 copies/μl to 2.0 × 100 gnou, Damaliscus dorcas phillipsi and Sylvicapra copies/μl) of studied agents. The number of plasmid grimmia grimmi in Africa [11], most of the studies copies was determined in accordance with the formula on the occurrence, seroepidemiology and molecular (Xg/μlDNA/[plasmidsize(bp)×660])×6.022×1023 ×plas- characterization of these agents have been conducted mid copies/μl. The amplification reactions were per- among cattle [12, 13]. When A. marginale infects formed using a final total reaction volume of 10 μl, Machado et al. Parasites & Vectors (2016) 9:454 Page 3 of 9 containing a mixture of 1.0 μl of sample DNA, 0.2 μlof The phylogenetic analyses were carried out using differ- probe, 0.9 μl of each primer, 5.0 μl of PCR buffer (IQ ent methods: the neighbour-joining (NJ) algorithm was Multiplex Power Mix®, BioRad) and 2.0 μl of ultra-pure run in the Mega 4 software [25]; maximum parsimony sterile water (Nuclease-Free Water®, Promega). (MP) and bootstrap analyses were carried out using PAUP* 4.0b10 [26] with 100 replicates