Molecular characterization of Anaplasma and Ehrlichia in ixodid ticks and reservoir hosts from Palestine: a pilot survey

Abstract

Tick-borne anaplasmosis and ehrlichiosis are clinically important emerging zoonoses usually overlooked by veterinarians and physicians alike. This study aimed at detecting and genetically characterizing Ehrlichia and Anaplasma species in ixodid ticks and their animal hosts from the West Bank, Palestine. A total of 723 ixodid ticks belonging to three genera (Rhipicephalus, Hyalomma, Haemaphysalis) were collected from dogs, sheep, goats and camels. In addition, 189 blood samples were collected from dogs, sheep, camels, horses and a goat from the West Bank, Palestine. All tick and blood samples were investigated for the presence of Anaplasma and Ehrlichia targeting a 345 bp fragment of the 16S rRNA gene followed by sequence analysis. The infection rate of Anaplasma spp. in ticks was 6.5% (47/723). Anaplasma platys was identified in 28% (13/47) of them. Whereas, based on a partial sequence (851 bp) of msp4 gene, 38% (18/47) were identified as A. ovis. The species of the remaining 16 positive samples (16/47, 34%) could not be identified. Simultaneously, the infection rate of Ehrlichia spp. in the ticks was 0.6% (4/723). Three of which were E. canis and one was Ehrlichia spp. The infection rate of A. platys in dogs' blood samples was 10% (13/135), while it was 1.5% (2/135) for E. canis. The infection rate of Anaplasma in sheep blood samples was 40% (19/47), out of which 26% (5/19) were caused by A. ovis as revealed by msp4-PCR. Implementation of purely-spatial analysis by saTScan for all cases of Anaplasma revealed two statistically significant clusters in two districts; Tubas town and Majdal-Bani-Fadil village on the western hills of the Jordan Valley. Most cases of Anaplasma (83%) were from rural areas where life cycle components (vector, host and reservoir) abundantly interact. This study is the first in Palestine to reveal the presence of Anaplasma and Ehrlichia in ticks, dogs and sheep providing crucial platform for future epidemiological surveys and control strategies in the country and region.

Keywords: Anaplasma; Ehrlichia; Ixodid ticks; Palestine; dogs; sheep.

Figure 1

Spot maps of cases of Anaplasma (red circles) and Ehrlichia (blue circles) isolated from ticks (a), dogs (b) and sheep (c). The number within the circles indicates cases spotted in the area while those without numbers indicate one case.

Figure 2

Phylogenetic analysis of Anaplasma and Ehrlichia based on partial sequences of (a) 16S rRNA and (b) msp4 genes. Phylogenetic analysis were constructed using the maximum likelihood method used on MEGA X program, with the complete deletion option, based on Jukes‐Cantor model for nucleotide sequences. Initial trees for the heuristic search were automatically obtained by applying the Nearest‐Neighbor‐Interchange (NNI) algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. Statistical support for internal branches of the trees was evaluated using bootstrap of 1000 replications. (a) Based on 16srRNA sequences (345 bp): cluster I represents Ehrlichia strains. Cluster II represents Anaplasma spp. and Cluster III represents Anaplasma platys. The DNA sequence of Neorickettsia sennetsu (NR_044746.1) was used as an out‐group to produce rooted tree. (b): 851 bp msp4 DNA Anaplasma sequences detected in this study compared to Anaplasma reference sequences deposited in the NCBI GenBank. The Species, GenBank accession numbers and country of origin from which the sequences were derived are included for each sequence. Sequences derived from this study are marked by star (*). The number of identical sequences is in brackets. Selected reference Anaplasma spp. sequences from GenBank are also shown.

Figure 3

Geographical distribution of statistically significant foci of Anaplasma cases in Palestine (excluding Gaza strip) on implementing purely spatial analysis by SaTScan: red circles indicate positive cases while blue circles indicate negative cases. The numbers within the circles indicate the number of cases.