Molecular Detection of Anaplasma marginale and Anaplasma ovis (Rickettsiales: Anaplasmataceae) in Ixodid Tick Species in Iran

Introduction

Anaplasma marginale (A. marginale) (Rickettsiales: Anaplasmataceae), the type species of the genus Anaplasma, is the causative agent of bovine anaplasmosis, which is widely distributed in tropical and subtropical regions around the world ( Rar and Golovljova, 2011 ). Anaplasma ovis (A. ovis) (Rickettsiales: Anaplasmataceae), the agent of ovine anaplasmosis, may cause mild to severe diseases in sheep, deer, and goats; however, cattle are not infected with A. ovis ( Aubry and Geale, 2011 ). A. marginale and A. ovis are intraerythrocytic pathogens infecting various domestic and wild ruminants ( Birtles, 2012 ).A. ovis and A. marginale, which are closely related, can be mechanically transmitted by blood-contaminated mouthparts of biting flies and mosquitoes. They can also be biologically transmitted by ticks, which is more efficient, compared to mechanical transmission ( Kocan et al., 2010 ). Boophilus tick is able to transmit A. marginale through the experimental transmission of infection to cattle ( Futse et al., 2003 ). The transmission of A. ovis to humans is yet to be clearly understood ( Chochlakis et al., 2010 ). Approximately 20 species of ticks have been reported capable of transmitting A. marginale, namely Boophilus decoloratus and Rhipicephalus simus in Africa, Boophilus microplus in Australia, and Dermacentor species in the United States ( de la Fuente et al., 2001 , Kocan, ‎2001‎ ). A. marginale and A. ovis infections can be persistent in cattle, goats, and tick hosts, both of which serve as reservoirs ( Brayton, 2012 ). Fatal bovine anaplasmosis is a major problem in endemic countries due to A. marginale ( Hornok et al., 2012 ). Despite mild clinical symptoms of A. ovis infection, it should be considered an important constraint of livestock production due to the high prevalence of A. ovis in such countries ( Renneker et al., 2013 ). Six major surface proteins (MSPs) have been identified on A. marginale related to cattle and ticks ( de la Fuente et al., 2001 ). The major surface proteins 4 (msp4) sequences have been reported as acceptable genetic markers for inferring the phylogeographic patterns of A. marginale on a broad geographic scale ( Kocan et al., 2002 ). Genetic heterogeneity has been shown among the geographical strains of A. marginale explained by tick vector and tick pathogen interactions ( Waner et al., 2010 ). A. marginale and A. ovis have been reported in the blood samples of small ruminants in Iran with cattle and sheep as reservoir hosts of A. marginale ( Noaman et al., 2009 ; Yousefi et al., 2017 ). To date, no studies have focused on detecting a specific polymerase chain reaction (PCR) and sequencing of Anaplasma species within tick specimens. The present study was designed to determine the presence of Anaplasma species in various tick species based on PCR amplifying and sequencing of msp4 gene fragments in different parts of Iran.

Material and Methods

Collection and identification of ticks. A total of 130 tick specimens were collected from Hormozgan, Lorestan, and Guilan, Iran, within 2015 to 2017 (Table 1). The tick specimens were collected by forceps from the hosts. Live specimens were carefully transported to the Laboratory of Razi Herbal Medicines Research Center, Lorestan University of Medical Sciences. The ticks were identified based on species level under a light stereomicroscope (SZX12-Olympus®, Japan) following the identification key of Estrada-Peña et al. (2004). Then, 20 tick specimens were selected from different areas for molecular assays.

Polymerase Chain Reaction. Genomic DNA was extracted using cetyltrimethylammonium bromide according to Doyle and Doyle (1987) . A fragment of msp4 was amplified through PCR. In order to specifically and accurately amplify the target agent in ticks, the primers were newly designed by the authors of the present study, including, Fmsp4: 5ʹ- GTY ARR GGC TAY GRC AAG AG -3ʹ and Rmsp4: 5ʹ- AGT RAA CTG GTA GCT WAT YCC A -3ʹ. The PCR reactions for each gene were carried out in a thermocycler (MyCycler™ Thermal Cycler, Bio-Rad®, USA). The touchdown temperature profile included 5 min at 95 °C, 10X (50 sec at 94 °C, 50 sec at 60-50 °C, and 1 min at 72 °C), followed by 20X (50 sec at 95 °C, 50 sec at 50 °C, and 1 min at 72 °C), and a final extension step (3 min at 72 °C). Each PCR reaction consisted of 12.5 µl RedMaster PCR® (Sinaclon®, Iran) 2X, 1 µl of each primer (10 pM), 4 µl genomic DNA template (50-100 ng/µl), and 6.5 µl distilled water to the final volume of 25 µl. The DNA extracted from PCR positive tick samples with successful sequencing of A. marginale and A. ovis was used as a positive control. In addition, distilled water was utilized instead of target DNA in all PCR reactions as a negative control.

Electrophoresis, Purification, and Sequencing. The PCR products were visualized by 1% agarose gel electrophoresis, and the selective desired bands of different gene fragments from different tick species were purified using GeneAll Expin™ Combo GP Kit (South Korea). Then, purified PCR products were submitted for sequencing to Faza-Biotech® Company (Iran). Subsequently, the sequences were manually edited using FinchTV® software (version 1.4.0). The corrected sequences were compared with the submitted sequences in GenBank using BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Finally, all sequences were submitted to GenBank, and accession numbers were assigned.

Phylogenetic analysis. All sequences were aligned using SeaView software (version 4) ( Gouy et al., 2010 ), and genetic distances among the sequences were calculated using maximum composite likelihood in MEGA software (version 7) ( Kumar et al., 2016 ). Phylogenetic tree were constructed for msp4 sequence data using Bayesian inference methods by BEAST software (version 2.4.8) ( Bouckaert et al., 2014 ). For this purpose, the data of 37 Anaplasmamsp4 sequence, including the sequences from the present study, as well as the sequences from GenBank data, were used to construct an msp4 phylogenetic tree. The constructed clades of the phylogenetic tree were reorganized based on 100% support of posterior probability values and reasonable genetic distance differences within and between the clade members. The sequence of Anaplasmaphagocytophilum (EU857672) was included as an outgroup in the phylogenetic tree.

Results

Tick species, polymerase chain reaction, and sequences. In the present study, Hyalomma asiaticum, Hyalomma dromedarii, Rhipicephalus sanguineus, and Rhipicephalus (Boophilus ) species were identified in different geographical regions. The collected data related to ticks in this study are summarized in Error! Reference source not found.. An amplicon of 464-bp msp4 was amplified in tick species. Three sequences, including one A. marginale from Rhipicephalus (Boophilus ) species and two A. ovis from Rhipicephalus sanguineus, were obtained after sequencing. The accession numbers of MH017204 (A. marginale) and MH017205-6 (A. ovis) were assigned in GenBank.

Major surface protein 4 phylogenetic tree. Phylogenetic trees were constructed using BEAST software, including ingroup and outgroup Anaplasma taxa (Figure 1). The msp4 constructed phylogeny indicating two main clades includes A. marginale and A. ovis with a 10% genetic distance difference observed between the clades. No intraspecies variation in terms of genetic distance was noticed within the members of the two clades. No additional msp4 sequence of Anaplasma taxa was observed in GenBank. The msp4 phylogenetic tree generated from the sequence data consistently supported the monophyly of A. marginale and A. ovis clades. The phylogenetic tree may be observed as a fully resolved tree with high posterior probability values and dichotomous topology (Figure 1).

Figure 1. Phylogenetic tree generated based on major surface proteins 4 sequence data of Anaplasma species in this study and similar sequences from GenBank database constructed using Bayesian inference method; main Anaplasma clades separated in right side of phylogenetic tree; taxa of present study are bold and defined with a name and GenBank accession number; posterior probability values inserted in place of nodes; branch lengths proportional to evolutionary changes; Anaplasma phagocytophilum included as outgroup.

Discussion

In the present study, two economically important pathogen agents, namely A. marginale and A. ovis, were detected in tick samples collected from Iran. Most studies have mainly emphasized animal hosts, such as the ruminants in various provinces of Iran ( Spitalska et al., 2005 ; Ahmadi et al., 2009 ; Noaman et al., 2009 ; Hosseine-Vasoukolaei et al., 2010 ; Jalali et al., 2013 ; Yousefi et al., 2017 ). In northern and northeastern Iran, out of 193 blood samples, 123 samples were A. ovis positive as confirmed by restriction fragment length polymorphism on msp4 gene fragment ( Ahmadi et al., 2009 ). Similar to the present study, a study was conducted in Khuzestan, Iran, where Anaplasma marginale and A. ovis were simultaneously detected in 50% of Anaplasma infected blood samples ( Jalali et al., 2013 ). In a study carried out by Yousefi et al. (2017) , 27.5% of sheep and goats were positive for A. ovis and A. marginale. According to 16S ribosomal ribonucleic acid (rRNA), A. ovis was reported in human and sheep samples in Mazandaran, Iran, ( Hosseine-Vasoukolaei et al., 2010 ), as well as the sheep samples in Fars, Iran ( Spitalska et al., 2005 ). Moreover, positive cases of A. marginale were observed in 75 Anaplasma suspected blood samples in Isfahan, Iran ( Noaman et al., 2009 ). In the present study, Hyalomma asiaticum, Hyalomma dromedarii,Rhipicephalussanguineus, and Rhipicephalus (Boophilus ) species were identified in different geographical regions. Rhipicephalus (Boophilus ) species were identified in northern Iran on Caspian red deer (Cervus elaphus maral). The ticks were collected on the dead marals. In addition, these specimens were positive for a Brucella-like bacterium ( Hosseini-Chegeni et al., 2017 ). The range of maral includes the Caspian provinces of northern Iran, Crimea, Turkey, and Caucasus ( Kiabi et al., 2004 ). Hyalomma asiaticum and Hyalomma dromedarii are the most important ectoparasites of domestic animals in Iran ( Hosseini-Chegeni et al., 2013 ). Rhipicephalus sanguineus is the most prevalent vector species distributed all over Iran ( Telmadarraiy et al., 2015 ). In the present study, three sequences, including one A. marginale from Rhipicephalus (Boophilus ) species and two A. ovis from Rhipicephalus sanguineus, were obtained after sequencing. The occurrence of anaplasmosis due to A. marginale and A. ovis has been reported in different countries adjacent to Iran, namely Turkey ( Bilgic et al., 2017 ), Pakistan ( Jabbar et al., 2015 ), Russia ( Vasilevich et al., 2017 ), and Saudi Arabia ( Al-Khalifa et al., 2009 ). In other countries, A. ovis was more prevalent (40.5%) than other Anaplasma species in the northwest of China ( Yang et al., 2015 ), Ethiopia (5.1%) ( Abdela et al., 2018 ), and co-occurrence of A. ovis with different pathogens in tick species in Serbia (Tomanović et al., 2013). Two similar isolates of A. marginale from cattle and Rhipicephalus tick vector were detected in China ( Cui et al., 2018 ). Five tick species, namely Dermacentor marginatus, Haemaphysalis punctata, Ixodes ricinus, Ixodes ventalloi, and Rhipicephalussanguineus (s.l.) in Portugal, were positive for A. marginale ( Antunes et al., 2016 ). The partial 16S rRNA was used for the detection of A. ovis in tick species of Iran ( Hosseini-Vasoukolaei et al., 2014 ). After the BLAST analysis of 16S rRNA sequences of this study [Acc. Nos.: JF514503-12], it was observed that this is not a reliable gene sequence regarding the occurrence of specific Anaplasma species. Studies conducted on 16S rRNA of Anaplasma species in tick vector encountered similar weaknesses. In the present study, msp4 phylogeny indicating two main clades were A. marginale and A. ovis with a 10% genetic distance difference observed between the clades. The msp4 fully resolved the tree, generated from the sequence data, and consistently supported the monophyly of A. marginale and A. ovis clades. The msp4 may conduce to study genetic diversity in Anaplasma species, especially A. marginale strains in certain regions ( de la Fuente et al., 2005 ). Phylogeny derived from A. ovismsp4 may vary among geographic regions, as well as in sheep and deer hosts, yet less than the variation observed between A. marginale strains ( de la Fuente et al., 2007 ).

The present study was carried out as the first molecular detection of Anaplasma species in tick samples based on the sequencing of msp4 gene fragments in different parts of Iran. It is concluded that bovine and ovine anaplasmosis are present in tick samples in Iran; therefore, it is necessary to develop suitable strategies to detect and breakdown the natural cycle of disease with the emphasis on tick control. Moreover, it is required to perform further studies to determine the presence of Anaplasma agent in the salivary gland and other organs of ticks to determine the vectorial capacity of each tick vector. It is recommended to use the gene families of other MSPs, such as msp1, msp2, and msp5, in different tick species, especially Ixodes ricinus, for the detection of Anaplasma phagocytophilum.

Ethics

We hereby declare all ethical standards have been respected in preparation of the submitted article.

Authors’ Contribution

Study concept and design: Hosseini-Chegeni, A., Telmadarraiy, Z.,

Acquisition of data: Hosseini-Chegeni , A.

Analysis and interpretation of data: Hosseini-Chegeni, A.

Drafting of the manuscript: Hosseini-Chegeni, A., Faghihi, F., Telmadarraiy, Z.

Critical revision of the manuscript for important intellectual content: Faghihi, F.,

Statistical analysis: Hosseini-Chegeni , A.

Administrative, technical, and material support: Hosseini-Chegeni , A., Tavakoli, M., Goudarzi, G. H., Telmadarraiy, Z., Sharifdini, M., Faghihi, F., Ghanbari, M.K.

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