Open Veterinary Journal, (2019), Vol. 9(3): 238–245

Original Research

DOI: http://dx.doi.org/10.4314/ovj.v9i3.8

Molecular prevalence of Anaplasma phagocytophilum in sheep from Iraq

Karrar Jasim Hamzah¹* and Saleem Amin Hasso²

¹Department of Veterinary Internal and Preventive Medicine, College of Veterinary Medicine, Al-Qasim Green University, Babylon, Iraq

²Department of Veterinary Internal and Preventive Medicine, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq

*Corresponding Author: Karrar J. Hamzah. College of Veterinary Medicine, Al-Qasim Green University, Babylon, Iraq. Email: karraraljanabi [at] vet.uoqasim.edu.iq; dr.karraraljanabi41 [at] gmail.com

Submitted: 05/03/2019 Accepted: 17/07/2019 Published: 25/08/2019

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Abstract

Background: Tick-borne diseases are widely distributed among animal populations and are responsible for significant economic losses. However, little attention has been offered for screening such infections world widely. Anaplasma phagocytophilum is among those neglected tick-borne pathogens, particularly in the developing countries.

Aim: This study was conducted to detect A. phagocytophilum infection among sheep in three governorates of Iraq (Babylon, Wasit, and Missan) and try to identify the potential tick vector responsible for A. phagocytophilum transmission among sheep in these analyzed regions.

Methods: A total of 297 blood samples and 103 ticks were collected and examined for A. phagocytophilum by polymerase chain reaction using specific primers amplifying partial sequence for msp4 gene.

Results: The results showed that about 14 out of 297 tested sheep were positive for A. phagocytophilum. There was no difference between A. phygocytophilum prevalence according to animal gender, age, and sampling period. Furthermore, our analysis showed that the main vectors of A. phagocytophilum: Ixodes scapularis, I. pacificus, or I. ricinus were not identified in three regions of Iraq (Rhipicephalus turanicus, Hyalomma anatolicum, and Hyalomma turanicum were identified).

Conclusion: These results highlight the importance of the survey of the tick-borne bacterial infections in Iraq and in the Middle East region in general.

Keywords: Anaplasma phagocytophilum, Iraq, Prevalence, Sheep, Ticks.

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Introduction

Anaplasma phagocytophilum infection is recorded mainly in sheep and a wide range of domestic animals. Anaplasma phagocytophilum also considered as a zoonotic for humans as well. Anaplasma phagocytophilum infection is a tick-borne transmitted disease mainly caused by ixodid tick species (Ogden et al., 2003; Ben Said et al., 2018). The number of clinical cases of human granulocytic anaplasmosis is increasing in Europe and Asia (Park et al., 2003; Ruscio and Cinco, 2003; Ohashi et al., 2005). However, the distribution of A. phagocytophilum is determined by the population of tick vectors and reservoir host species (Stuen et al., 2013). In North America, the common vectors are Ixodes scapularis and I. pacificus (Teglas and Foley, 2006). However, in Europe, the I. ricinus is the main tick vector for A. phagocytophilum (Woldehiwet, 2006). In the Middle East, the Ixodes ricinus is reported as the main possible tick vector for transmitting A. phagocytophilum in Iran and —Turkey (Gokce et al., 2008; Noaman and Shayan, 2009). The detection of A. phagocytophilum using molecular methods like the polymerase chain reaction (PCR) is considering as an effective and sensitive method (Smrdel et al., 2015; Stuen, 2016; Yang et al., 2016).

Tick-borne fever (TBF) caused by the bacterium A. phagocytophilum transmitted by the tick I. ricinus is one of the main challenges for sheep farming industry during the grazing season (Grøva et al., 2011). The infection with A. phagocytophilum is widespread in the USA (Foley et al., 2008), in Europe (Woldehiwet, 2006), and in Asia (Jilintai et al., 2009; Yang et al., 2013). Anaplasma infection has been also reported in some Middle East countries like Turkey (Gokce et al., 2008) and in Iran (Noaman and Shayan, 2009).

This study was aimed to detect A. phagocytophilum in sheep located from three regions of Iraq (Babylon, Wasit, and Missan). Furthermore, this study tried to detect and identify the tick vector responsible for A. phagocytophilum transmission in these three areas.

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Materials and Methods

Animals and data collection

About 400 samples divided into 297 sheep and 103 ticks were involved in this study. Sheep were mixed in gender and the ages were ranging from less than 1 year to more than 5 years. They were randomly selected from three different areas of Iraq (Babylon, Wasit, and Missan) during the period from February to October 2018.

Collection of blood samples

The blood samples were collected from the jugular vein of sheep using EDTA-vacutainer tube. All samples were transferred using cooling boxes. All the laboratory tests were performed at the laboratories of the College of Veterinary Medicine—Al-Qasim Green University.

Collection of ticks

Ticks were collected during the period from February to October 2018. A total of 103 ticks were collected and isolated from the same sheep that were involved in this study from the three aforementioned areas. After collection, the ticks were stored separately in 70% ethanol for classification purposes and for the molecular detection of A. phagocytophilum carriage. A stereomicroscope was used to identify the ticks based on their morphological features, such as mouthparts, scutum, color of legs, festoons, interstitial punctuations, presence or absence of adanal shields, posterior groove, and marginal spots (Hoogstraal, 1956; Hosseini-Chegeni et al., 2013).

DNA extraction

The genomic DNA of A. phagocytophilum from sheep blood samples and ticks was extracted using a DNA mini extraction kit (Geneaid Biotech Ltd, Taiwan) according to the company’s instructions. Extracted genomic DNA of A. phagocytophilum was analyzed for a given concentration range of 15–60 ng/μl. The purity of extracted DNA was analyzed using a Nanodrop spectrophotometer at (260/280 nm).

Polymerase chain reaction (PCR)

The PCR technique was performed for the detection of A. phagocytophilum in the blood samples and ticks according to the method described by Yang et al. (2013). The two versions of extracted DNA (from the sheep blood or ticks) were tested for the presence of A. phagocytophilum using PCR by the amplification of the major surface protein 4 coding gene (msp4). Specific primers were designed and used: the forward primer is F: 5ʹ-ATGCGTCTGATGTTAGCGGT-3ʹ and the reverse primer: R: 5ʹ-AAAACTCGCCCCTAACCCAG-3ʹ. The product size is about 503 bp.

DNA sequencing

In order to confirm that the positive PCR products (503-bp band) belong to the A. phagocytophilum genome and to test the specificity of the primers for detecting A. phagocytophilum in sheep blood and ticks, the products of three positive PCRs were sequenced using Sanger’s sequencing method at Macrogen, Korea. The evolutionary distances were computed using the Maximum Composite Likelihood method by phylogenetic tree UPGMA (MEGA 6.0 version).

Statistical analysis

The results were analyzed using SPSS statistical software. A Chi-square test was used to assess the association between the variable results. p-value of ≤0.05 was considered to be significant.

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Results

Detection of A. phagocytophilum DNA in sheep blood

PCR results showed that 14 out of 297 samples (4.71%) were positive to A. phagocytophilum (Fig. 1). Furthermore, the infection rate was higher in Wasit and Missan (7.81% (5/64) and 7.04% (5/71), respectively) compared to that in Babylon region (2.46% (4/162) (Table 1 and Fig. 2).

However, the results also showed that the infection rate of A. phagocytophilum was not affected by the gender of the examined sheep (Table 2). Indeed, the infection rate was barely equal in both males (2.08%) and females (5.22%) of sheep (Table 2).

Interestingly, results showed that the highest rate of infection was recorded in older sheep (aged 5 years or above) with a rate of 6.25% (Table 3). In contrast, the lowest infection rate was recorded at age less than 2 years (3.57%, Table 3).

Seasonal variation analysis showed that the highest infection rate of Anaplasma phagocytophilum was recorded during the summer season of Iraq (May, June, and July: 3/45 (6.66%), 3/48 (6.25 %), and 4/51 (7.84%), respectively) (Table 4 and Fig. 3). However, there were non-significant variations among these months. On the other hand, all samples that were collected during February, March, and October were negative for A. phagocytophilum (Table 4 and Fig. 3).

Fig. 1. Agarose gel electrophoresis image showing PCR product analysis of the major surface protein (MSP4) gene in A. phagocytophilum positive isolates. Where: M: marker (100–2,000 bp), lane (1–13) positive A. phagocytophilum at (503) bp PCR product, NC negative control.

Table 1. Infection rate of A. phagocytophilum by PCR technique.

Fig. 2. Prevalence of A. phagocytophilum according to regions. Babylon 4/162, Misan 5/71, and Wasit 5/64. Red color representative numbers of infected sheep with A. phagocytophilum, % representative the percentages of infection.

Table 2. Infection and gender relationship of blood samples examined by PCR technique.

Table 3. Infection rates of A. phagocytophilum and age of sheep examined by PCR technique.

Survey of A. phagocytophilum in ticks

PCR result indicated that collected ticks were identified as Rhipicephalus turanicus, Hyalomma anatolicum, and Hyalomma turanicum and none of these examined ticks (total 103) was carrying A. phagocytophilum (Table 5).

Confirmation of PCR products by sequencing

The results of A. phagocytophilum sequencing were analyzed and annotated according to A. phagocytophilum genomes that banked in NCBI GenBank database using Basic Local Alignment Search Tool (BLAST).

Phylogenetic analysis of A. phagocytophilum

To investigate the possible genetic relationship between A. phagocytophilum strains detected in the current study and A. phagocytophilum available in GenBank database at the NCBI, a simple phylogenetic tree was generated based on the partial sequences of major surface protein 4 coding gene (msp4). The partial sequences of msp4 gene of three positive A. phagocytophilum samples (GenBank accession numbers MK423169, MK423170, and MK423171) were shown to be closely related to A. phagocytophilum isolates found in GenBank (Table 6 and Fig. 4).

Table 4. Infection rate of A. phagocytophilum according to months of study by PCR examination.

Fig. 3. Infection rate according to months. Red color representative numbers of infected sheep with A. phagocytophilum, % representative the percentages of infection. February 0/12, March 0/14, April 1/40, May 3/45, June 3/48, July 4/51, August 2/37, September 1/24, and October 0/26.

Table 5. Relationships between infection of A. phagocytophilum with tick infestation.

Table 6. NCBI-BLAST homology sequence identity (%) for msp4 gene in local A. phagocytophilum and NCBI-Blast A. phagocytophilum.

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Discussion

This study targeted the detection of A. phagocytophilum in sheep blood and infesting ticks by amplifying the major surface protein 4 coding gene (msp4). This gene is reported to be conserved for A. phagocytophilum (de la Fuente et al., 2005a; Silaghi et al., 2011; Öter et al., 2015). This consistent infection rates result with what has been found in previous studies conducted by Derdáková et al. (2011)

Fig. 4. Phylogenetic tree of A. phagocytophilum by Maximum Likelihood method based on partial sequence major surface protein 4 (MSP4) and relating the local strain of A. phagocytophilum Gene Bank accession numbers sequence MK423169, MK423170, and MK423171 to other isolated strains, the Gene Bank accession numbers of EF190513.1, LC141078.1, KU497699.1, MH790274.1, LC412081.1, MG283274.1, KY283958.1, HQ456350.1, FJ460452.1, and DQ674246.1 were related to Anaplasma ovis.

Furthermore, Ben Said et al. (2017) demonstrated that in Tunisia, prevalence rates of strains genetically related to A. phagocytophilum in sheep was 3.9%. Using PCR method, Scharf et al. (2011) mentioned that the infection rate of A. phagocytophilum was 4% in sheep.

However, Kiilerich et al. (2009) reported that the infection rates of A. phagocytophilum in Denmark, Italy, and Turkey were higher than those of the current study and of Derdáková et al. (2011), Ben Said et al. (2017), Scharf et al. (2011). Indeed, the infection rates have been reported to be variable between countries and could even vary considerably between neighboring farms (Stuen et al., 2002b). The prevalence of A. phagocytophilum infection in sheep has been reported to range from 0.51% to 80% worldwide (Stuen and Bergström, 2001; de la Fuente et al., 2005b).

Our results showed that infection rates slightly vary according to the sex and conclude that the female sheep have a higher infection rate than males. This could be due to the fact that females are more exposed to different physiological changes such as stresses caused by pregnancies, parturition, and lactation (Weiss and Wardrop, 2011). The age group of more than 5 years showed the highest infection rates, while the sheep aged from 2 to 5 years recorded the lowest rates.

These results are compatible with Chaudhry et al. (2010) who mentioned that the highest rate of infection in adult animals could be due to the chronicity of infection. Ben Said et al. (2015) also reported that the infection rate with A. phagocytophilum-like strains was higher in adult compared to young sheep. Extensive study by (Egenvall et al., 2000) showed that higher infection rates were significant in older ages. These results also agreed with Radostits et al. (2006). Poitout et al. (2005) discussed the seasonality and geographical distribution of A. phagocytophilum infections and found that this due to the feeding habits of each particular tick vector. Most cases were observed between April and July, with fewer cases observed in October. In Berlin (Germany), Kohn et al. (2008) reported that most cases occurred between April and September, and the seasonal distribution of the disease most likely reflects periods of peak tick activities. This finding is in agreement with Beall et al. (2008). The common consequence of the infection with A. phagocytophilum in sheep is the direct and indirect losses and causes immunosuppression status that may lead to secondary infections. A. phagocytophilum infection causes direct losses and mortality rates of 30% lamb in the flock (Stuen and Kjølleberg, 2000). The extent of indirect production loss due to TBF decreases the bodyweight of lamb (Stuen et al., 2002a).

Jensen et al. (2007) indicated that a high infestation with ticks could increase the incidence of A. phagocytophilum infection compared to those with a low rate of tick infestation. Kohn et al. (2011) also mentioned that A. phagocytophilum infection is higher in the months of tick activity. The present results are also supported by Chvostáč et al. (2018) and Grøva (2011) who reported that the seasonal dynamics of tick activity was the highest in April, May, June, and July when correlated with the highest prevalence of A. phagocytophilum infection. Overall, this study is a pioneering study regarding molecular detection of A. phagocytophilum in local Iraqi sheep from three different regions of Iraq. To our knowledge, this study is the first study that reported A. phagocytophilum infection in Iraqi sheep.

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Conclusion

PCR method complemented by sequencing step seems to be accurate and confirmative for the detection and the genetic characterization of A. phagocytophilum in sheep. Furthermore, our results suggested that other tick species might be responsible for the transmission of A. phagocytophilum to Iraqi sheep.

Acknowledgment

We thank our colleagues and the technicians in the laboratory of Veterinary clinical pathology at the College of Veterinary Medicine—Al-Qasim Green University in Iraq for their technical support. Special thanks for Maan M Neamah from Faculty of Veterinary Medicine at the University of Kufa, Iraq, for his kind reviewing and editing of this manuscript. Finally, thanks to Dr.Asaad Mohan, Dr. Adnan Mansour, Dr. Qasim AL-Jebori, Dr. Ameer Dirwal, and Mr. Mohammed Rahman for their great help during the field samples collection. This research is a self-funded project as part of a partially-funded Ph.D. study. No specific grant was obtained from any public, commercial, or not-for-profit funding agencies.

Conflict of interest

We declare no conflicts of interest in relation to this paper.

Author Contributions

Karrar Jasim Hamzah and Saleem Amin Hasso conceived and wrote the study. All authors read and approved the final manuscript.

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