Prevalence, Risk Factors, and Molecular Epidemiology of Anaplasma phagocytophilum in Sheep Raising in Khuzestan Province, Iran

Introduction
The genus Anaplasma belongs to the family Anaplasmataceae and order Rickettsiales. The important species of this genus in ruminants are as follows: Anaplasma marginale, Anaplasma centrale, Anaplasma ovis, Anaplasma bovis, and Anaplasma phagocytophilum (common species between humans and livestock). They are transmitted through hard ticks and cause anaplasmosis in ruminants (Abdullah et al., 2020; Kocan et al., 2015; Noaman, 2019). Anaplasmosis has resulted in a lot of losses for the livestock industry around the world so that in the United States, the annual losses in the cattle industry are estimated to be 300 million dollars, and in Latin America, the losses are estimated to be 800 million dollars (da Silva et al., 2018). In Iran, bovine anaplasmosis is a significant disease that causes a lot of damage to the livestock industry. Still, sheep anaplasmosis usually does not cause severe illness, and only in some cases does the sheep exposed to stress or contributing factors show clinical signs of anaplasmosis (Noaman et al., 2016; Noaman et al., 2017). The infections commonly have no observable symptoms, but anemia, icterus, fever, lethargy, and weight loss are rarely seen in infected animals (Stuen et al., 2009; Stuen et al., 2010). 
Microscopic examination of Giemsa-stained blood slides can only confirm acute anaplasmosis and fails to diagnose persistent infection and disease reservoirs (Aubry et al., 2011; Tabrizchi et al., 2023). In serological diagnosis methods, the differentiation of Anaplasma sp. is impossible due to cross-species interactions; these methods lack sensitivity and reliability compared to molecular tests. To identify Anaplasma sp. in infected animals, polymerase chain reaction (PCR), nested polymerase chain reaction (nPCR), and restriction fragment length polymorphism (RFLP) is used (Atif, 2015).
Additionally, many studies have been conducted on the molecular identification of Anaplasma spp. and the differentiation of species in Iran so that A. marginale (Jalali et al., 2013; Noaman & Shayan, 2010a; Noaman et al., 2016), A. phagocytophilum (Jalali et al., 2013; Noaman and Shayan, 2009), A. bovis (Noaman and Shayan, 2009), A. ovis (Jalali et al., 2013; Noaman, 2012), and A. centrale of amorphous strain in cattle and sheep have been reported in Iran (Noaman, 2012).
Considering the economic losses caused by the disease and its unnoticeable clinical symptoms in sheep, it is necessary to investigate the risk factors in the distribution of infection in different areas for its control and prevention. Despite the distribution of different species of hard mite in Khuzestan Province, Iran, this study aimed to report the molecular identification of A. phagocytophilum in sheep. We intended to determine the environmental and risk factors involved in its prevalence in southwestern Iran, which has a tropical climate.

Materials and Methods

Sample collection
A random sampling of 200 sheep from semi-industrial and traditional farms in Khuzestan Province (hot and humid climate in the southwest of Iran: 31° 32ʹ 73ʺ N, 48° 69ʹ 40ʺ E) was conducted in 22 cities with two climate types: mountain and plain regions. Five milliliters of blood were taken from the jugular veins of apparently healthy sheep and collected in tubes containing an anticoagulant (EDTA), which was sent to the laboratory in ice at 4°C. 

DNA extraction 
According to the manufacturer’s instructions, genomic DNA was extracted using an extraction kit (MBST Iran). The purification of the extracted DNA was conducted by OD260/280 ratio.
PCR and nested PCR: 
The Anaplasma sp. all primer was used; the nucleotide sequence was found in all Anaplasma species (Table 1).

The first product amplified the 16S rRNA gene (1468 bp) of the Anaplasma sp. The PCR solution was prepared based on the following instruction and with a final volume of 25 μL: 2.5 μL of DNA, 2.5 μL of PCR 10X buffer, 0.75 μL of MgCl2 solution at a concentration of 50 µM, 0.5 μL of dNTP at a concentration of 10 µM, 0.5 μL of each primer at a concentration of 20 µM, 0.5 μL of Taq DNA polymerase at a concentration of 5 U/μL, and 17.625 μL of distilled water. After preparing the solutions, frequent DNA amplification was conducted: primary denaturation step at 95°C for 5 minutes, denaturation step at 94°C for 45 seconds, primer connection step at 55°C for 45 seconds, chain lengthening step at 72°C for 90 seconds. Each step was conducted for 35 cycles and then examined in 1.5% agarose gel of electrophoresis with ethidium bromide staining. Specific internal primer sets targeting the V1 region of the 16S rRNA (962 bp) were used to detect A. phagocytophilum. Specific nPCR reactions were performed directly with 1 μL of the primary PCR product separately. The nPCR for A. phagocytophilum was performed in 25 μL total volume (Kawahara et al., 2006). The nested-PCR solution, with a total volume of 20 μL, was prepared as follows: 0.5 μL of the sample (from primary PCR), 2 μL of PCR 10X buffer, 0.6 μL of MgCl2 solution at a concentration of 50 µM, 0.4 μL of dNTP at a concentration of 10 µM, 0.4 μL of each primer at a concentration of 20 µM, 0.1 μL of Taq DNA polymerase at a concentration of 5 U/μL, and 15.6 μL of distilled water. After preparing the solutions, the frequent DNA amplification was conducted under the following program: primary denaturation step at 95°C for 5 minutes, denaturation step at 94°C for 45 seconds, primer connection step at 56°C for 45 seconds, and chain lengthening step at 72°C for 45 seconds. Also, each step was conducted for 35-40 cycles and then was examined in 1.5% agarose gel of electrophoresis with ethidium bromide staining.

Statistical analysis
The chi-square (χ2) test was used to compare the variables: Climate, altitude, season, farm type, hygiene, distance from other farms, and farm density. Factors such as vectors (mosquito, tick), use of acaricide, age, and sex were performed for analysis by using SPSS software (SPSS Inc, Chicago, USA), version 18 in the sheep infected with A. phagocytophilum (P≤0.05).

Results
After DNA extraction, 200 sheep blood samples from Khuzestan Province, located southwest of Iran, were amplified with the 16S rRNA gene (1468 bp) in the primary PCR with Anaplasma sp. Of 200 sheep, 154 samples (77%) were positive for infection with Anaplasma sp. detected by PCR (Figure 1).

Nested PCR was used to confirm the detection of A. phagocytophilum. The amplification of primary PCR products with this primer pair produced a 926 bp, entirely consistent with the size of the expected product (Figure 2). Of 200 samples, 34(17%) were positive for A. phagocytophilum with nPCR. As a result, the prevalence of A. phagocytophilum infections in sheep in Khuzestan Province was 17%.

Based on the statistical analysis of distribution risk factors (Table 2) of infection in different regions, it was determined that factors such as age, hygiene, farm density, use of acaricide, and season were significantly influential in the prevalence of anaplasmosis. The prevalence of A. phagocytophilum was significant in sheep aged 3-5 years (P=0.027). Low hygienic farms were significantly affected (P=0.001) compared to good and normal hygienic farms. In the statistical study of the use of acaracide on the farm, all herds that did not use it were infected (P<0.0001). Farms with high density were significantly infected (P<0.0001) compared to low-density farms. The warm season was another factor that showed a statistically significant difference in the prevalence of infection (P=0.32). There was no significant association between the occurrence of A. phagocytophilum infection and the variables of altitude, farm type, vectors, distance from other farms, and sex (Table 2).

Discussion 
A. phagocytophilum is recognized as an emerging tick-borne pathogen that is important for animals and humans (Atif, 2016; Rar et al., 2021). Various tick vectors and reservoir hosts are responsible for this pathogen’s geographic distribution and emergence (Woldehiwet, 2010). Epidemiological studies have demonstrated that climate changes and temperature levels can lead to changes in the geographic distribution of ticks and, as a result, the diseases they transmit (Atif, 2016; Stuen et al., 2013). The absence of clinical signs of ruminants infected with A. phagocytophilum highlights the need for molecular techniques to discriminate this species from other species. In determining the genus Anaplasma sp., it has been shown that the 16SrRNA gene is highly capable of detecting the genus (Atif, 2015). Various genes, such as gltA, msp4, and groEL, have been used to detect A. phagocytophilum in sheep (Kang et al., 2011). The molecular results presented the high frequency (19.4%) of Candidatus Anaplasma camelii in camels in the south of Iran (Moradi et al., 2021). In this study, the presence of Anaplasma sp. was determined in 77% of samples collected from sheep by PCR based on 16SrRNA. Positive samples were genetically characterized by A. phagocytophilum and 34(17%) of 154 positive samples gave positivity with nPCR based on the V1 region of the 16S rRNA (962 bp). 
The epidemiological study of risk factors of A. phagocytophilum in the region showed that factors such as age, hygiene, farm density, use of acaricide, and season play essential roles in the prevalence of the infection. Infection was more common in sheep raised over 3 years of age in poor hygienic conditions and high-density farms. There was also a significant difference in the infection prevalence in herds that did not use acaricide. The warm season has also been another important factor in the prevalence of infection in the region.
In the study of the distribution of infection of A. phagocytophilum in cattle in different regions of Iran with various climatic conditions, it was found that the highest infection has been reported in the Caspian zone (33%) located in the north of Iran (Noaman, 2020). The zone has high rainfall, high humidity, vast forests, and grasslands, which are suitable conditions for the growth and reproduction of ticks and, as a result, the development of tick-borne diseases. In Northern and Central Europe, the tick-borne disease usually appears from early April until mid-November. However, most cases are seen between the middle of May and the middle of June due to suitable conditions for the growth and reproduction of vector ticks. Although in this study there is no statistically significant difference in latitude and longitude of different regions, research on tick-borne diseases such as Anaplasma sp. has shown that prevalent in mountainous areas are more likely compared to plain areas (Khaki et al., 2015; Noaman, 2020; Dantas-Torres, 2015).
Another factor in increasing the prevalence of Anaplasma sp. is poor hygiene and old facilities on the farm, which are good places to increase ticks and tick-borne diseases. In the study of anaplasmosis infection in Pakistan, the prevalence was higher (26.05%) on farms where cleaning was done weekly than on farms cleaned once or twice daily (Shaukat et al., 2019).
All age groups of lambs are of epidemiological importance for maintaining A. phagocytophilum in tick populations. In the present study, the infection in sheep over 3 years of age had a statistically significant difference with other ages. In research by Noaman and Moradi (2019), the prevalence of A. phagocytophilum infections was 3% in dairy cattle in Southwest Iran. Regarding the risk factors of the infection epidemiology, cattle <1 year of age, with low milk yield, and low hygienic farms were significantly at lower risk, versus cattle raised in mountain regions, which were significantly at higher risk.
In this study, the blood smears were taken from 160 cattle, 391 sheep, and 385 goats and examined for various Anaplasma spp. The results showed that 19.37% of cattle were infected with A. marginale, 80.3% of sheep, and 38.92% of goats were infected with A. ovis. In a study by Jalali et al. (2013), the contamination of Ahvaz sheep in Iran was reported to be 33%. The species detected by RFLP revealed that all PCR-positive samples were A. ovis. A mixed infection with A. marginale was seen in 50% of Anaplasma sp. infected samples. According to the review of Soosaraei et al. (2020), the most prevalent infection among Anaplasma spp. was related to A. ovis and A. marginale infection rates, and the lowest prevalence to A. phagocytophilum. Noaman and Shayan (2009), in a study on Anaplasma sp. in cattle around Isfahan City, Iran, showed that out of 150 extracted DNA samples, 58(38.67%) were positive for A. marginale in primary PCR, semi-nested PCR, and RFLP-PCR. In a study performed by Noaman and Shayan (2010b) on sheep in Isfahan Province based on the 16S rRNA gene, it was found that 33% of the samples were positive for PCR-RFLP. In the above research, no clinical signs were recorded in PCR-positive sheep. In the analysis of blood smears of livestock, no inclusion was observed in neutrophils, indicating that the measured livestock are vectors. The results of this research are consistent with the research conducted. The report of genotyping and phylogenetic analysis of Anaplasma capra in Europe, in domestic, endemic, and wild ruminants, shows the wide host range previously described for this species in Asian countries (Jouglin et al., 2022). Noaman (2020), in an epidemiological study of infection in different parts of Iran cattle, showed that the highest prevalence was found in the Caspian zone (18%) North of Iran, followed by Central (16.8%), Zagros (16%), and Persian-Gulf zone (3%). Prevalence rates of A. marginale, Theileria ovis, and Theileria lestoquardi in sheep in Pakistan were 07%, 06%, and 1.2%, respectively. Phylogenetic analysis revealed that these isolates were closely related to what was found in Iran (Tanveer et al., 2022).
To control diseases such as anaplasmosis, with its zoonotic potential and ticks-born nature, it is necessary to study the critical risk factors in the epidemiology of the infection. Based on the present study and other research, the temperature during the season, mountain climate, farm sanitary conditions, use of acaricides, long-term breeding, and increasing herd age are risk factors that contribute to the prevalence of infection and should be considered in the design controlling and prevention programs.

Conclusion
The present study shows that in Khuzestan Province the tropical region of Iran and the prevalence of anaplasmosis was 17% with no clinical sign. It can be a guide to strategic control programs for anaplasmosis in this area. Further studies are needed on the identification of biological and mechanical vectors of Anaplasma species in this region.

Ethical Considerations

Compliance with ethical guidelines
The animal study was reviewed and approved by Ethical Committee of Razi Vaccine & Serum Research Institute. Written informed consent was obtained from the owners for the participation of their animals in this study.

Funding
This study was supported by Razi Vaccine & Serum Research Institute, Agricul­tural Research, Education and Extension Organization (AREEO) (Project No.: 0-38-18-90016/2).

Authors' contributions
Conceptualization and supervision: Vahid Noaman; Methodology, investigation, data collection and data analysis: Vahid Noaman and Razieh Heidari, Writing: All authors.

Conflict of interest
All authors declared no conflict of interest.

Acknowledgments
All authors appreciate the farmers for their cooperation. This study was funded by the Razi Vaccine and Serum Research Institute and Agricultural Research, Education, and Extension Organization (AREEO). 

References

Abdullah, D. A., Ali, F. F., Jasim, A. Y., Ola-Fadunsin, S. D., Gimba, F. I., & Ali, M. S. (2020). Clinical signs, prevalence, and hematobiochemical profiles associated with Anaplasma infections in sheep of North Iraq. Veterinary World, 13(8), 1524-1527. [DOI:10.14202/vetworld.2020.1524-1527][PMID]

Atif, F.A. (2015). Anaplasma marginale and Anaplasma phagocytophilum: Rickettsiales pathogens of veterinary and public health significance. Parasitology Research, 114(11), 3941–3957.[DOI:10.1007/s00436-015-4698-2][PMID]

Atif, F. A. (2016). Alpha proteobacteria of genusAnaplasma (Rickettsiales: Anaplasmataceae): Epidemiology and characteristics of Anaplasma species related to veterinary and public health importance. Parasitology, 143(6), 659–685. [DOI:10.1017/S0031182016000238][PMID]

Aubry, P., & Geale, D. W. (2011). A review of bovine anaplasmosis. Transboundary and Emerging Diseases, 58(1), 1–30.[DOI:10.1111/j.1865-1682.2010.01173.x][PMID]

Dantas-Torres, F. (2015). Climate change, biodiversity, ticks and tick-borne diseases: The butterfly effect. International Journal for Parasitology. Parasites and wildlife, 4(3), 452–461.[DOI:10.1016/j.ijppaw.2015.07.001][PMID]

da Silva, N. B., Taus, N. S., Johnson, W. C., Mira, A., Schnittger, L., & Valente, J. D. M., et al. (2018). First report of Anaplasma marginale infection in goats, Brazil. Plos One, 13(8), e0202140. [DOI:10.1371/journal.pone.0202140][PMID]

Jalali, S. M., Khaki, Z., Kazemi, B., Bandehpour, M., Rahbari, S., & RaziJalali, M., et al. (2013). Molecular detection and identification of Anaplasma species in sheep from Ahvaz, Iran. Iranian Journal of Veterinary Research, 14(1), 50 -56. [Link]

Jouglin, M., Rispe, C., Grech-Angelini, S., Gallois, M., & Malandrin, L. (2022). Anaplasma capra in sheep and goats on Corsica Island, France: A European lineage within A. capra clade II? Ticks and Tick-borne Disease, 13(3), 101934. [DOI:10.1016/j.ttbdis.2022.101934][PMID]

Kang, J. G., Ko, S., Kim, Y. J., Yang, H. J., Lee, H., & Shin, N. S., et al. (2011). New genetic variants of Anaplasma phagocytophilum and Anaplasma bovis from Korean water deer (Hydropotes inermis argyropus). Vector Borne and Zoonotic Diseases (Larchmont, N.Y.), 11(7), 929–938. [DOI:10.1089/vbz.2010.0214][PMID]

Kawahara, M., Rikihisa, Y., Lin, Q., Isogai, E., Tahara, K., & Itagaki, A., et al. (2006). Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp. In wild deer and ticks on two major islands in Japan. Applied and Environmental Microbiology, 72(2), 1102–1109. [DOI:10.1128/AEM.72.2.1102-1109.2006][PMID]

Khaki, Z., Jalali, S. M., Kazemi, B., Razi Jalali, M., & Yasini, S. P. (2015). A study of hematological changes in sheep naturally infected with Anaplasma spp. and Theileria ovis: Molecular diagnosis. Iranian Journal of Veterinary Medicine, 9(1), 19-26. [DOI:10.22059/IJVM.2015.53228]

Kocan, K. M., de la Fuente, J., & Cabezas-Cruz, A. (2015). The genus Anaplasma: New challenges after reclassification. Revue scientifique et technique, 34(2), 577-586. [DOI:10.20506/rst.34.2.2381][PMID]

Moradi, Z., Ebrahimzadeh, E., Shayan, P., & Zarghami, F. (2021). Morphological and molecular investigation of anaplasma infection in dromedary camel (camelus dromedarius) in Bushehr Province, Iran. Iranian Journal of Veterinary Medicine, 15(3), 295-300. [DOI:10.22059/IJVM.2020.306095.1005109]

Noaman, V. (2012). Identification of hard ticks collected from sheep naturally infected with Anaplasma ovis in Isfahan province, central Iran. Comparative Clinical Pathology, 21, 367-369. [DOI:10.1007/s00580-012-1438-1]

Noaman, V. (2013). Discrimination between Anaplasma marginale and Anaplasma ovis by PCR-RFLP. World Applied Sciences Journal, 21(2), 190-195. [Link]

Noaman, V. (2019). [A review on Anaplasma phagocytophilum as a zoonotic agent (Persian)]. Tehran University of Medical Sciences Journal, 76(12), 778-785. [Link]

Noaman, V. (2020). Epidemiological study on Anaplasma phagocytophilum in cattle: Molecular prevalence and risk factors assessment in different ecological zones in Iran. Preventive Veterinary Medicine, 183, 105118. [DOI:10.1016/j.prevetmed.2020.105118][PMID]

Noaman, V., Allameh, S. K., & Nabavi, R. (2017). Anaplasmosis in Ruminants of Iran: An overview. Advanced Technology in Clinical Microbiology, 1(2), 13. [Link]

Noaman, V., Bastani, D. (2016). Molecular study on infection rates of Anaplasma ovis and Anaplasma marginale in sheep and cattle in West-Azerbaijan province, Iran. Veterinary Research Forum, 7(2), 163–167. [PMID]

Noaman, V., & Moradi, M. (2019). Molecular epidemiology and risk factors assessment of Anaplasma spp. on dairy cattle in southwest of Iran. Acta Veterinaria Eurasia, 45(1), 30-36. [DOI:10.26650/actavet.2019.18014]   

Noaman, V., Nabinejad, A., Shahmoradi, A., & Esmaeilkhanian, S. (2016). Molecular detection of bovine leukocytic Anaplasma species in Isfahan, Iran. Research in Molecular Medicine, 4(2), 47-51 [DOI:10.18869/acadpub.rmm.4.2.47]

Noaman, V., & Shayan, P. (2009). Molecular detection of Anaplasma phagocytophilum in carrier cattle of Iran-first documented report. Iranian Journal of Microbiolog, 1(2), 37-42. [Link]

Noaman, V., & Shayan, P. (2010). Molecular detection of Anaplasma bovis in cattle from central part of Iran. Veterinary Research Forum, 1(2), 117-122. [Link]

Noaman, V., & Shayan, P. (2010). Comparison of microscopic methods and PCR-RFLP for detection of Anaplasma marginale in carrier cattle. Iranian Journal of Microbiology, 2(2), 89–94. [PMID]

Rar, V., Tkachev, S., & Tikunova, N. (2021). Genetic diversity of Anaplasma bacteria: Twenty years later. Infection, Genetics and Evolution, 91, 104833. [DOI:10.1016/j.meegid.2021.104833][PMID]

Shaukat, A., Mehmood, K., Shaukat, I., Naeem, M. A., Mehfooz, A., & Saleem, M. I., et al (2019). Prevalence, haematological alterations and chemotherapy of bovine anaplasmosis in Sahiwal and crossbred cattle of District Faisalabad, Punjab Pakistan. Pakistan Journal of Zoology, 51(6), 2023-2032. [Link]

Soosaraei, M., Haghi, M. M., Etemadifar, F., Fakhar, M., Teshnizi, S. H., & Asfaram, S., et al. (2020) Status of Anaplasma spp. infection in domestic ruminants from Iran: A systematic review with meta-analysis. Parasite Epidemiology Control, 11, e00173. [DOI:10.1016/j.parepi.2020.e00173][PMID]

Stuen, S., Granquist, E. G., & Silaghi, C. (2013). Anaplasma phagocytophilum—a widespread multi-host pathogen with highly adaptive strategies. Frontiers in Cellular and Infection Microbiology, 3, 31. [DOI: 10.3389/fcimb.2013.00031]

Stuen, S., Scharf, W., Schauer, S., Freyburger, F., Bergström, K., & von Loewenich, F. D. (2010). Experimental infection in lambs with a red deer (Cervus elaphus) isolate of Anaplasma phagocytophilum. Journal of Wildlife Diseases, 46(3), 803–809.[DOI:10.7589/0090-3558-46.3.803][PMID]

Stuen, S., Torsteinbø, W. O., Bergström, K., & Bårdsen, K. (2009). Superinfection occurs in Anaplasma phagocytophilum infected sheep irrespective of infection phase and protection status. Acta veterinaria Scandinavica, 51(1), 41. [DOI:10.1186/1751-0147-51-41][PMID]

Tabrizchi, M. R., & Ahmadi-hamedani, M. (2023). Effects of blood storage time and temperature on Döhle body or döhle body-like inclusions in feline neutrophils. Iranian Journal of Veterinary Medicine, 17(1), 47-52. [DOI:10.32598/IJVM.17.1.1005253]

Tanveer, M., Farooq, M., Amjad, M., Asif, M., Kashif, M., & Latif, M., et al. (2022). Molecular prevalence, associated risk factors and phylogeny of Anaplasma marginale, Theileria ovis and T. lestoquardi in sheep from Pakistan. Comparative Immunology, Microbiology and Infectious Diseases, 86, 101822.[DOI:10.1016/j.cimid.2022.101822][PMID]

Woldehiwet, Z. (2010). The natural history of Anaplasma phagocytophilum. Veterinary Parasitology, 167(2-4), 108–122.[DOI:10.1016/j.vetpar.2009.09.013][PMID]