Molecular Detection of Anaplasma and Ehrlichia Infection in Ticks in Borderline of Iran-Afghanistan

Anaplasmosis, a disease caused by various species of Anaplasma, poses important economic constraints to animal breeders. Ehrlichiosis is a worldwide zoonosis illness and mostly occurs in tropical and subtropical regions that are close to the vector’s distribution. Tick-borne pathogens lead to over 100,000 cases of illness in the world each year. Besides the costs of the additional veterinary care, anaplasmosis causes abortion in animals, reduction of milk production, body weight, and frequently leads to death. In this study, we investigated on infection of ticks to Anaplasma and Ehrlichia pathogens in Zabol and Zahak County in Sistan and Baluchestan Province where is bordered with Afghanistan. Totally from June 2013 to May 2014, 369 ticks were caught from goats, cows and sheep. Molecular studies on 53 of these samples which represented all specimens, showed that Ehrlichia’s DNA and Anaplasma’s DNA was found in 14 (26.4%) out of the 53 selected specimens. The results showed the infection of Rhipicephalus sanguineus and Hyalomma anatolicum with Anaplasma ovis. Also we saw infection of H. anatolicum and H. asiaticum ticks to Ehrlichia spp. This study has been intended to do a comprehensive survey of Ehrlichia and Anaplasma distribution in ticks caught from east of Iran; it was designed to investigate the presence of Anaplasma spp. and Ehrlichia spp. in Zabol and Zahak Counties, Iran. These results show that these pathogens should be controlled in such regions. Corresponding author. A. Jafarbekloo et al.


Introduction
Ticks are vectors of bacterial, viral and protozoan agents [1].Investigating on these pathogens in ticks requires the identification of ticks.Ticks are living in most places.There are some investigations about ticks in Iran [2]- [4].Tick-borne pathogens lead to over 100,000 cases of illness in the world each year [5].Ehrlichiachaffeensis, E. canis, E. ewingii, and Anaplasma phagocytophilum are the most important tick-borne pathogens of human and animals belong to the family Anaplasmataceae.Ehrlichiosis is a worldwide zoonosis problem and mostly occurs in tropical and subtropical regions that are close to the vector's distribution [6] [7].
Anaplasmosis, a disease caused by various species of Anaplasma, poses important economic constraints to animal breeders.Besides the costs of the additional veterinary care, anaplasmosis causes abortion in animals, reduction of milk production, body weight, and frequently leads to death [8].Members of the genus Anaplasma are obligatory intracellular gram negative bacteria that infect blood cells of mammals.Six Anaplasma species are currently recognized [9].Vertebrates are main reservoirs of the Anaplasma bacteria, however in many cases bacteria from the genus Anaplasma cause diseases in domestic animals and human.Anaplasma ovis invades and reproduces within erythrocytes.This bacterium induces acute anemia in sheep and goats [10].Anaplasmosis in cattle is caused by A. bovis, A. marginaleand A. centrale infecting monocytes and red blood cells [11] [12].Anaplasma bovis is reported mostly from cattle, but also detected in small ruminantswhich could be a reservoir of this bacterium [13].Ixodid ticks play an important role in maintaining Anaplasma species in nature.It is evidenced that various species of Ixodes, Dermacentor, Rhipicephalus and Amblyomma genera are the main vectors of the Anaplasma bacteria in different regions of the world.Rhipicephalus sanguineus, a common tick vector for Anaplasma, has been reported from India, the United States, all regions of Africa, and around the Mediterranean Basin [14].Animal husbandry is one of the main activities of people in Estern Iran, and anaplasmosis is one of the major veterinary health problems there [15].However, there have been only a few studies to detect tick anaplasmosis infections in the country [16]- [19].We previously studied onanaplasmal infection of human, livestock and ticks in Mazandaran Province, Iran.Our results showed the presence of A. ovis in two Rhipicephalus sanguineus and two Ixodesricinus ticks, one human and 4 sheep samples.Also one Boophilusannulatus tick and one sheep sample were infected with A. bovis.Furthermore, one sample of sheep was infected with A. centrale.That was the first report of tick infection to A. ovis, A. bovis and human infection to A. ovis in Iran [17].Khazeni et al., investigated on the infection of ticks caught from Ardebil Province, Iran and found that Ehrlichia spp.and Anaplasma spp.were found in 43.84% of all the specimens containing Anaplasma ovis and Ehrlichia spp.and Ehrlichiacanis [18] Khazeni et al., reported E. canis in vector from Iran for the first time [18].The aim of the present investigation was to study prevalence and genetic diversity of infection of ixodidae ticks to infectious bacteria due to our observations of ranchers' complaint about their livestock diseases and the lack of documented information about Anaplasma and Ehrlichia species in ticks in eastern Iran.We conducted the present study to distinguish the rate of Anaplasma and Ehrlichia infections in Zabol and Zahak counties which have been located in borderline of Iran-Afghanistan in eastern Iran.

Study Area
This study was conducted in Zabol and Zahak counties in Sistan and Baluchestan Province (Located in Eastern Iran) which is bordered with Afghanistan.Zahak and Zabol Counties with 30˚N latitude and 61˚E longitude are located in the east of the Province.Zabol County has a population about 320,000 and Zahak County has a population about 70,000.

Sample Collection
From June 2013 to May 2014, we collected tick samples from goats, cows and sheep from Hossein Abad, Heydar Abad, Fathollah and Bagher Abad which are located in Zabol County and Khomak, Bonjar and Hassankhoon which are located in Zahak County (Figure 1).Ticks were mostly found on shoulders and ears of the livestock.
Specimens were collected using a forceps and kept in labeled holding tubes individually.Specimens were transferred into the labeled holding tubes individually.In some cases it was impossible to collect all ticks due to lack of time.We did not ask the origin of the sheep, goats and cows.
Specimens were transferred to the Entomology Laboratory, School of Public Health, Tehran University of Medical Sciences.All specimens were identified based on morphological characteristics and the keys given by Janbakhsh (1957) and Walker (2003) based on shape of capitulum, scutum, eyes, festoone and hypostome, spiracle, genital groove, spure of coxa, adanal shield and another characters [20] [21].

DNA Extraction
DNA was extracted using Gspin TM Genomic DNA Extraction kit (iNtRON).Extraction was carried out according to the manufacturer instructions by grinding of individual ticks in an eppendorf microtube after maintaining 5 minutes in the liquid nitrogen tank and using glass pestle.400 μl of G-buffer per 20 -30 mg of tissue was added and incubated at 70˚C for 5 -10 min and then mixed well.At the next step, 400 μl of Binding buffer was added and transferred to the G-spine columns in the next step, centrifuged for 1 min at 13,000 rpm.Then, 500 μl of washing buffer and 100 μl of elution buffer were added respectively and centrifuged for 1 min at 13,000 rpm.After adding elution buffer, samples were incubated for 1 min at room temperature and then they were kept at 4˚C for further use.

Detection of Ehrlichia and Anaplasma by Nested-PCR
By using EHR1, EHR2, EHR3 and EHR4 primers, detection of Ehrlichia and Anaplasma was performed by nested-PCR, 16 s rRNA amplification, [18] [22].These primers are able to detect the infection of both Ehrlichia and Anaplasma.
As positive control we used Anaplasma DNA obtained from Department of Medical Entomology, School of Public Health, Tehran University of Medical Sciences [18], and double distilled water as negative control was used.
First round of PCR amplifications were done in a Maxime PCR premix kit (iTaq).For primary reactions 5 μl of purified DNA was used as a template in mentioned PCR premix kit.PCR cycles were consisted of 5 minutes at 94˚C, 35 cycles at 94˚C for 1 minute (denaturation of DNA), 60˚C for 1 minute (annealing of primers), 72˚C for 1 minute (extension of the primers), and a final extension at 72˚C for 7 minutes.Second round of PCR assay was performed using species-specific primers (Ehr 3, Ehr 4) (Table 1), [18] [23] and 3 μL from the initial PCR product was used as template.The PCR products were loaded in 1% agarose gel, stained with ethidium bromide and then visualized under UV light.

Nucleotide Sequencing
Sequencing was performed using an ABI 3730 sequencer machine.Obtained sequences were checked to correct

Results
The prevalence of ticks was low, so a total of 369 ticks were collected and these ticks were morphologically identified at the level of species.

Detection of Ehrlichia and Anaplasma
Fifty three out of 369 collected ticks were tested for the presence of Anaplasma and Ehrlichia's DNA.Ehrlichia's DNA and Anaplasma's DNA were found in 14 (26.4%)out of the 53 selected specimens (generated characteristic 524 bp products, Figure 2).Nested PCR detected ehrlichial DNA in 14 samples of 53 samples which had been selected by species and region differences and represented the whole regions, whole tick species and whole livestock.Details of positive samples are listed in Table 2. Out of 53 ticks which used for DNA extraction, 14 samples were infected with Anaplasma or Ehrlichia spp.For detection of infection, 7 of these samples were sent for sequencing.The results demonstrated that a female Rhipicephalus sanguineus which had been caught of a sheep from Zabol County was infected with Anaplasma ovis.Two female Hyalomma anatolicum ticks which had been caught from Zahak County were infected with Ehrlichia spp.One of these ticks had been caught from cow and the other had been caught from sheep.A male Hyalomma asiaticum which had been caught from a cow in Zaboldistrict, was also infected with Ehrlichia spp.Two Hyalomma anatolicum which one of them was male and the other was female and were caught from goat, were infected with Anaplasma ovis.Both of these infected ticks were caught from Zahak district.Also a male Hyalomma anatolicum which had been caught from Zabol district was infected with Anaplasma ovis.This tick was caught from a cow.Totally 3 of positive samples were caught from sheep, 3 of them were caught from goats and 8 of them were caught from cows.Two genus of ticks: Rhipicephalus and Hyalomma were found to be infected with Anaplasma or Ehrlichia genus.Eight of positive samples had been caught from Zahak and 6 of them had been caught from Zabol district.Alignment of sequenced Anaplasma ovis samples (KM056396-KM056399) showed 99% identity with each other.Also 3 of the Ehrlichia spp.samples were completely identical to each other (100%).
Comparison of the sequences with available data in GenBank showed that the sequences were highly similar to ITS2 region of Anaplasma ovis and Ehrlichia spp. with 100% identity.Obtained sequences from this study were submitted to the GenBank, under the accession numbersKM056396-KM056402.

Discussion
This study provides primary data, regarding the existence of A. ovis and Ehrlichia spp. in ticks in Zahak and Zabol Counties which is located in east of Iran and bordered with Afghanistan.Nested PCR enhances the sensitivity of detection of target nucleotide sequences [24].This technique has been shown to be sensitive for direct identification of ehrlichiae in ticks [25].Nested PCR with subsequent sequencing had been shown that hard ticks had been containing Anaplasma ovis, Ehrlichia spp.and other anaplasmal or ehrlichial DNA [17] [18] [26].
The main vectors of the Anaplasma bacteria are ticks, especially the genera Ixodes, Dermacentor, Rhipicephalus and Amblyomma [17].Our results demonstrated the presence of A. ovis in Rhipicephalus sanguineus and H. anatolicum; so they might be vectors of A. ovis in this region.We report the infection of R. sanguineus and H. anatolicum to A. ovisin Zahak and Zabol Counties.We also demonstrated the infection of H. anatolicum to Ehrlichia spp. in Zahak and infection of H. asiaticum to Ehrlichia spp. in Zabol County, Iran.Since sheep are reservoirs of A. ovis, human infection with this pathogen may occur, but transmission of A. ovis to human is uncertain.Previously, sequence analysis of PCR products confirmed the presence of A. ovis in Rhipicephalus sanguineus and Ixodesricinus ticks, human and sheep samples in Ghaemshahr, which is located in north of Iran.High level of infestation of R. sanguineus to domestic ruminants, and the prevalence of Anaplasmain these specimens showed that they are the most abundant vector of Anaplasma species in Ghaemshahr county [17].In another study the infection rate of A. ovis in ticks collected from dogs' ears, neck, shoulder and toes reported from Meshkin-Shahr (Ardebil Province, Iran) was as much as 56.6 percent.In addition, 21.17% of nymphs and at least 53.42% of adult ticks were positive for A. ovis and Ehrlichia spp.[18].
Rhipicephalus sanguineus is widely spread not only in Iran but also in all over the world [27].
The results demonstrated the presence of Ehrlichia spp. in H. anatolicum and Hyalomma asiaticum while Rhipicephalus sanguineus was infected with Anaplasma ovis.
Almost 57% of the infected specimens collected on cow, the other belong to sheep or goat.The only specimens which infected with Anaplasma ovis collected on sheep.Based on our results, female ticks were more infected with Anaplasma spp.and Ehrlichia spp. in comparison with male ticks, while in Khazeni et al. investigation, this is contrary [18].
Zahak is more infected with Anaplasma spp.and Ehrlichia spp.than Zabol County.According to our previous study [18] dominant tick species which infected with Ehrlichia was Rhipicephalus sanguineus while Satta et al.Sequence alignments of 3detected Ehrlichia (KM056400-KM056402) in this study showed 100% identity with some of the submitted ones in Genbank.We could find one haplotype in these 3 sequences; but we found 2 haplotypes in A. ovis in our study.Three of sequenced samples (KM056396, KM056398, KM056399) were identical (100%) to each other and to the submitted ones.The other A. ovis sequenced sample (KM056397) which is detected from one H. anatolicum, had an identity of 99% with other samples and the submitted samples in Genbank.The difference between this sequence (KM056397) with the three other sequences (KM056396, KM056398, KM056399) was as much as 2 nucleotides (Figure 3).
In Iran, A. ovis was previously identified in sheep [15] [19]; however R. sanguineus and I. ricinus are dominant tick species in sheep in north part of this country [27] [30].Based on a study which was done in Turkey, A. ovis 16S rRNA gene fragment was detected in two R. sanguineus ticks [31].On the other hand, there are few studies on the infectivity of animal blood samples to Anaplasma in Iran.One of our laboratory studies, demonstrated that 39% of blood samples of humans and livestock were infected with Anaplasma in north of Iran [17].In Khorasan Province, north east of Iran, about 80% of sheep and 38% of goats blood smears were infected with A. ovis [15].In current study, A. ovis DNA was detected in ticks which had been collected from sheep, goats and cows.We could also detect Ehrlichia spp.DNA in ticks which had been collected from cows and sheep.These results make these livestock as potential reservoirs of these pathogens.
This study has been intended to do a comprehensive survey of Anaplasma and Ehrlichia distribution in ticks collected from east of Iran; it was designed to investigate the presence of Anaplasma spp.and Ehrlichia spp. in Zabol and Zahak County, Iran.It is recommended to investigate the competency of vectors to Rhipicephalus sanguineus, Hyalomma asiaticum and H. anatolicum.

Figure 1 .
Figure 1.Geographical location of Sistan and Baluchestan Province and Zabol and Zahak Counties, Iran.Zahak and Zabol Counties are bordered with Afghanistan.
of sequence homologies was done in GenBank by BlastN and aligned with ClustalW was checked using basic local alignment search tool (BLAST) analysis software (www.ncbi.nlm.nih.gov/BLAST).

Figure 2 .
Figure 2. 16 s rRNA amplification of Ehrlichia spp.and Anaplasma spp. in ticks using nested-PCR.Lanes P: positive control, Lane N: Negative control, Lanes 1-14 represent all the positive samples which are infected to Ehrlichia spp.and Anaplasma spp.(524 bp).

Figure 3 .
Figure 3.The differences between 2 A. ovis haplotypes.The sequence "KM056397" has an identity of 99% with three other sequences (KM056396, KM056398, KM056399).demonstratedthat dominant[28], Whereas Meng et al.[29] determined that dominant tick species which infected to Ehrlichia was Hyalomma asiaticum.Khazeni et al.Identified Rhipicephalus sanguineus, Hyalomma asiaticum, Hyalomma marginatum, Hyalomma anatolicum, Dermacentorniveus, Dermacentormarginatus as the infected specimens while in this study we could show Rhipicephalus sanguineus, Hyalomma asiaticum, H. anatolicum and a Hyalomma spp. as the infected specimens.Sequence alignments of 3detected Ehrlichia (KM056400-KM056402) in this study showed 100% identity with some of the submitted ones in Genbank.We could find one haplotype in these 3 sequences; but we found 2 haplotypes in A. ovis in our study.Three of sequenced samples (KM056396, KM056398, KM056399) were identical (100%) to each other and to the submitted ones.The other A. ovis sequenced sample (KM056397) which is detected from one H. anatolicum, had an identity of 99% with other samples and the submitted samples in Genbank.The difference between this sequence (KM056397) with the three other sequences (KM056396, KM056398, KM056399) was as much as 2 nucleotides (Figure3).In Iran, A. ovis was previously identified in sheep[15] [19]; however R. sanguineus and I. ricinus are dominant tick species in sheep in north part of this country[27] [30].Based on a study which was done in Turkey, A. ovis 16S rRNA gene fragment was detected in two R. sanguineus ticks[31].On the other hand, there are few studies on the infectivity of animal blood samples to Anaplasma in Iran.One of our laboratory studies, demonstrated that 39% of blood samples of humans and livestock were infected with Anaplasma in north of Iran[17].In Khorasan Province, north east of Iran, about 80% of sheep and 38% of goats blood smears were infected with A. ovis[15].In current study, A. ovis DNA was detected in ticks which had been collected from sheep, goats and cows.We could also detect Ehrlichia spp.DNA in ticks which had been collected from cows and sheep.These results make these livestock as potential reservoirs of these pathogens.This study has been intended to do a comprehensive survey of Anaplasma and Ehrlichia distribution in ticks collected from east of Iran; it was designed to investigate the presence of Anaplasma spp.and Ehrlichia spp. in Zabol and Zahak County, Iran.It is recommended to investigate the competency of vectors to Rhipicephalus sanguineus, Hyalomma asiaticum and H. anatolicum.

Table 1 .
Details of the primers were used for Ehrlichia spp.and Anaplasma spp.detection in ticks collected on livestock, Zahak and Zabol County, Sistan and Baluchestan Province, Iran.

Table 2 .
Details of infected ticks to Anaplasma/Ehrlichia in two studied districts.