https://orcid.org/0000-0002-1407-2019
Figures
Abstract
Anaplasma is an intracellular alphaproteobacteria that infects diverse blood cell types in animal hosts including small ruminants. Epidemiological and risk factors information on zoonotic anaplasmosis with respect to anaplasmosis in sheep and goats are scarce. Therefore, the objective of the current study was to estimate the prevalence, risk factors of anaplasmosis and phylogenetic investigation of A. capra in sheep and goats from Faisalabad district, Pakistan. Briefly, 384 blood samples were randomly collected from sheep and goats of Faisalabad district, Pakistan, during January to May 2022. The samples were processed for the detection of Anaplasma targeting 16S rRNA gene using PCR. The data regarding disease determinants were collected using a predesigned questionnaire. Out of 384 samples, 131 samples were found positive for Anaplasma spp. with a prevalence rate of 34.11%. The results indicated a significantly higher prevalence of anaplasmosis in goats (41.88%) compared to sheep (22.00%). In addition, the chi square indicated that housing type, tick infestation, gender, tick control practices, age, mix farming, and hygiene were significantly associated with the occurrence of disease. The analysis of multivariate logistic regression expressed gender as the significant risk factor (p = 0.0001, OR = 1.757, CI = 1.305–2.366). The acquired sequences revealed four novel isolates of A. capra (Genbank accession numbers ON834323, ON838209, ON838210, and ON838211). The phylogenetic analysis of the 16S rRNA gene of A. capra revealed three distinct clusters with 99–100% homology with other isolates from different countries. Our isolates showed higher similarity with isolates from China (KM206273, KP314237, MT799937), Pakistan (ON238129, ON238130, ON238131), Angola (MT898988), India (MZ558066), Iran (MW692362), and Turkey (MT632469) isolated from human, sheep, ticks, goats, cattle, Gaddi goat, Persian Onager (Equus hemionus onager), and Turkish goats, respectively. In conclusion, A. capra is endemic in Punjab, Pakistan, there is a need to conduct large scale surveillance studies to assess the status of this pathogen at human-animal interface as well as to develop effective preventive and control strategies to reduce the economic losses associated with anaplasmosis in small ruminants.
Citation: Razzaq MA, Imran M, Atif FA, Abbas RZ, Alvi MA, Swelum AA, et al. (2024) Molecular surveillance based on anaplasmosis in domestic small ruminants: First report on zoonotic Anaplasma capra and phylogenetic insights from Faisalabad, Pakistan. PLoS ONE 19(9): e0305412. https://doi.org/10.1371/journal.pone.0305412
Editor: Faham Khamesipour, Kerman University of Medical Sciences, ISLAMIC REPUBLIC OF IRAN
Received: November 25, 2023; Accepted: May 29, 2024; Published: September 6, 2024
Copyright: © 2024 Razzaq et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The research was partially supported by Higher Education Commission of Pakistan’s project No. 9041/Punjab/NRPU/R&D/HEC/2017; entitled “Molecular epidemiology, concurrent detection and characterization of Anaplasma, Babesia, Theileria and Ehrlichia species from cattle, buffaloes and ixodid ticks” as well as by King Saud University’s project No. RSPD2023R971, Riyadh, Saudi Arabia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: All the authors declare no conflict of interest.
Introduction
Livestock is the major subsector of agriculture, contributing 62.68% to agriculture and 14.36% to the national gross domestic product (GDP) of Pakistan. Over 8 million village families are engaged in livestock rearing, driving 35–40 percent of their earnings from livestock [1]. Pakistan is the third largest goat producing country in the world with 84.7 million heads. Based on sheep production, Pakistan is the 12th largest country with 32.3 million populations [1, 2]. In general, tick-borne diseases (TBDs) are the biggest economic threat to livestock production across the world. Different blood pathogens including Anaplasma, Borrelia, Ehrlichia, Babesia, Theileria and Louping ill virus are the most frequent TBDs in ruminants causing considerable economic losses [3–7].
Anaplasmosis is one of the most prevalent vector-borne illness of domestic and wild animals and humans all over the world including Pakistan [8–12]. There are seven recognized species in the genus Anaplasma viz. A. marginale, A. centrale, A. bovis, A. ovis, A. platys, A. phagocytophilum and A. caudatum [13]. In addition to this, the researchers also proposed additional species of Anaplasma including A. odocoilei, A. capra and Anaplasma sp. ’Omatjenne’ [14]. Of the aforementioned species of Anaplasma, A. ovis, A. capra and A. phagocytophilum are the major species responsible for anaplasmosis in sheep and goats [4, 15].
A new zoonotic tick-borne Anaplasma that gained the attention of veterinary and public health researchers provisionally named as A. capra [16]. This organism was the first identified in goats in central and northern China [17]. Years later, this was detected in human hospitalized patients of Mudanjiang Forestry Central Hospital, Heilongjiang, China. In addition, twenty-eight human cases have been reported in Heilongjiang Province in northeast China. However, this organism is not yet recognized as a separate species. Anaplasma capra is an emerging zoonotic pathogen and phylogenetically distinct from other Anaplasma species [18]. The disease caused by this organism is non-specific signs with fever, malaise, headache, rash, eschar, dizziness, and chills in humans [18, 19]. The disease caused by Anaplasma species exhibit some level of host specificity, but this feature is distorted due to the detection of Anaplasma in various hosts, which further complicates the epidemiology of the disease. A. capra can infect ruminants like sheep, goat, water buffalo [18, 20, 21]; wild animals like Muntjac (Muntiacus muntjak), Japanese Serow (Capricornis crispus), deer, Korean water deer (Hydropotes inermis argyropus), Roe dear (Capreolus capreolus), Persian onegar (Equus hemionus onager) [22–24]; humans [18]; dogs [25]; mouflon sheep-Ovis gmelini [26] and ticks [27, 28].
Although A. capra was first detected in goats (Capra aegagrus hircus) using msp4 and 16S rRNA genes in central and southern China [29] and further studies revealed the occurrence of this pathogen in Pakistan [26], France [23], Turkey [21, 30], Japan [22, 31], Korea [32], Spain [24, 33], South Korea [32, 34], Ghana [28] and Italy [35]. It has also been reported in goats from Sweden [36], Greece [37], Pakistan [38] and Kyrgyzstan [39, 40] by amplifying groEL, gltA and 16S rRNA genes. The infected animals mostly remain asymptomatic and show non-specific signs. These common signs would be very difficult to distinguish clinically from other tick-borne illnesses.
The major ixodid ticks that biologically transmit Anaplasma are Ixodes, Dermacentor, Rhipicephalus, and Amblyomma which transmit the intracellular rickettsiales to their mammalian hosts [38, 41]. The organism is transmitted by tick bite, transplacentally, and blood-contaminated needles [38, 42, 43]. Additionally, the risk of mechanical transmission by the bite of flea and termite bites also exists [44]. Regardless of competent vectors, A. capra has been detected in ticks such as Rhipicephalus microplus, Ixodes persulcatus, Dermacentor abaensis, D. nuttalli, Haemaphysalis longicornis and H. qinghaiensis [45–47]. However, the competent vectors of A. capra are not known yet [30, 46].
Multiple risk factors such as environmental (area, climate, temperature, rainfall, competent vector population, reservoirs, habitat, mechanical insect vectors, season, humidity, altitude, vegetation cover); host (sex, breed, age, gestation, tick infestation, history of disease, body condition score, health status, carriers, drug resistance), and managemental (flock size, housing, floor, use of acaricide, grazing system, hygiene, animal movement, stall feeding, agricultural and animal husbandry practices, contaminated fomites) are associated with the occurrence of anaplasmosis [48–54].
Giemsa-stained blood smears are frequently used to identify Anaplasma infected animals. Although microscopy is affordable and still regarded as the best, yet it is a laborious technique with low accuracy and requires a skilled examiner [55]. Various diagnostic molecular techniques have been utilized for the diagnosis of anaplasmosis such as PCR [18], RFLP-PCR [56], Nested PCR [30], RLB [57] and sequencing [30]. Globally, different genes have been targeted for molecular characterization of A. capra, such as a major surface protein 4 (msp4) [20], groEL [47], gltA [18, 30] and 16S rRNA [18, 26]. Pakistan’s climate, vegetation, and geographic conditions favor the existence and growth of ticks. Defining the regional status of new zoonotic genotypes of tick-borne pathogens is important at disease control standpoint. Epidemiological and risk factors information with respect to zoonotic A. capra in sheep and goats are scarce. There is only one report of A. capra in mouflon (Ovis gmelini) and domestic sheep (Ovis aries) from Pakistan which differ in terms of sample size and study area. Therefore, the present study was aimed to estimate the prevalence, associated determinants on anaplasmosis and phylogenetic investigation of A. capra in sheep and goats from Faisalabad district, Pakistan.
Materials and methods
Study area
Faisalabad district is located between Ravi and Chenab Rivers in the central part of Punjab province, coordinated at 31°25′05.10″N 73°04′39.27″E, and an elevation of 184 meters above sea level (603 feet). The Faisalabad district is indicated in orange color in Fig 1. The raw map was retrieved from https://en.wikipedia.org/wiki/Faisalabad_District#/media/File:Pakistan_-_Punjab_-_Faisalabad.svg. The district has five administrative units/tehsils; namely, Saddar, Sammundari, Tandlianwali, Jaranwala, and Chak Jhumra. The Faisalabad has extended summer with warm (March, November), hot (April, October), sweltering (May to September) and comfortable (January, February, December) weather. The average annual high temperature during summer ranges from 93–104°F (33.9–40°C), while winter is short, dry, cool, and mostly clear with an average low temperature ranging from 44–46°F (6.7–7.8°C). The annual rainfall in Faisalabad is 1.05 inches (26.67 mm) (available at https://weatherspark.com).
Study design
The current study was carried out in the Faisalabad district, Pakistan, from January 2022 to May 2022. The study was approved as per ethical guidelines of Graduate Studies and Research Board, Directorate of Graduate Studies, The University of Agriculture Faisalabad, Pakistan, vide letter no. DGS/10925-28, dated March 28, 2022. A total of 384 blood samples were collected from small ruminants (234 goats and 150 sheep) after consent from animal owners using a simple random sampling method. A sample size of 384 was calculated assuming 50% prevalence at 95% confidence using the formula as described by Thrusfield [58].
Risk factors estimation
For estimation of epidemiological risk factors, a pretested questionnaire was filled to gather information after consent at the time of sample collection from each animal owner/farm manager regarding the biotic and abiotic risk factors of age (<2 years and >2 years), gender (male and female), species (goat and sheep), housing type (concrete and muddy), tick infestation (yes and no), hygiene at farm (poor and good), feeding pattern (animals at farm with zero grazing and free range grazing), previous tick history (yes and no), tick control practices (yes and no) and mix farming (present and absent) associated with anaplasmosis. The questionnaire having close-ended questions was completed on spot at the time of blood sampling for each animal.
Blood smear examination
A 5–7 ml blood was drawn aseptically from the jugular vein of sheep and goats with the sterile syringe and shifted into ethylene diamine tetra acetic acid (EDTA) coated vacutainers for conventional assay. From each blood sample, a thin blood smear was prepared as described by Atif et al. [59] and the smear was examined at 100X under oil immersion lens and at least 50 fields per slide were examined to declare a sample as positive or negative [60].
DNA extraction
One milliliter of blood was collected aseptically from EDTA coated vacutainers individually for DNA extraction. The samples were moved to Laboratory of Veterinary Preventive Medicine and Public Health, Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad under cold chain. The DNA was extracted according to the manufacturer’s instructions using the commercial QIAamp® DNA Mini Kit (Qiagen, Germany; catalog No. 69504). A total of 200 ul of individual blood samples were used to extract DNA. The extracted DNA was kept at -40°C until further testing after being measured for purity and concentration using Nano-drop at 260/280 nm for all samples.
PCR amplification and gel electrophoresis
The PCR was performed targeting 16S rRNA gene of Anaplasma using forward primer HER 16SF (5’-GGTACCYACAGAAGAAGTCC-3’), and reverse primers HER 16SR (5’-TAGCACTCATCGTTTACAGC–3’), as described by Saleem et al. [61]. Final reaction volume of PCR was 20 ul and each reaction contained 10ul Vazyme blue premix (catalog No. P222), A 1 μl forward primer, 1 μl reverse primer, 4 ul water, and 4 ul template DNA was used. Positive and negative controls were also used for the validation of each test. Amplicons were subjected to 2% gel electrophoresis by using 1 μl gel red solution. After solidification, the tray along with the gel was transferred to the electrophoresis tank. The 50 bp DNA ladder (Takara) was loaded in the first well and the samples were loaded into the remaining wells. A 5ul PCR amplicon was loaded in each well of the gel tank, connected to the Power Pac (Bio-Rad, USA) and set to 100–110 volts for electrophoresis at 45–60 minutes. The PCR results were observed with a UV illuminator.
Sequencing and phylogenetic analysis
To validate the presence of Anaplasma specie, four representative PCR positive products were sent for sequencing. Amplified fragments were purified using Gel Extraction Kit (Thermo Scientific) and shipped to Lab Genetix, Lahore for sequencing. Previously published 16S rRNA sequences of A. capra (KM206273, KP314237, KP314238, KU879112, LC432123, LC432126, MF066918, MH762075, MT632469, MT799937, MT898988, MW692362, MW721591, MW930537, MZ558066, OK091152, ON238129, ON238130, ON238131, OQ248254) from china, Argentina, South Korea, Turkey, Angola, Iran, France, Pakistan, India, Portugal and Ghana were retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/]. The selected sequences and isolates from the current study were aligned with MUSCLE. Bootstrap values were determined by using 500 replications. A phylogenetic tree was constructed based on Maximum Likelihood method and Tamura 3-parameter model on MEGA11 [62, 63].
Statistical analysis
Chi square test was performed to determine the association of infection with anaplasmosis and disease determinants. Univariable analysis was used to determine the relationship between anaplasmosis and variables of age (<2 years and >2 years), gender (male and female), species (goat and sheep), housing type (Concrete and muddy), tick infestation (yes and no), hygiene at farm (poor and good), feeding pattern (farmed and free range), previous tick history (yes and no), tick control status (yes and no) and mix farming (present and absent). Multivariable analysis was performed for the variables with values p<0.2 to determine risk factors, odds ratio, and confidence intervals for each variable. The p-value less than or equal to 0.05 was considered as significant. The statistical data was analyzed with IBM SPSS Statistics 26.
Results
Epidemiology
All microscopic positive samples were subjected for further confirmation using PCR. The PCR based overall prevalence of anaplasmosis was 34.11% (131/384). The product of 345bp was perceived on the agarose gel using UV gel illuminator. All the microscopy based positive samples were also found positive from PCR. In the present study, anaplasmosis was more common in goats than sheep. Out of 131 positive animals, 98 goats and 33 sheep were positive for anaplasmosis. The inclusive prevalence of caprine and ovine anaplasmosis was 41.88% and 22%; respectively (Table 1). The Chi square analysis (p<0.05) illustrated that the species-wise prevalence of anaplasmosis was statistically significant (X2 = 59.93, df = 1, P = 0.000). All PCR positive animals were also found positive based on blood smear microscopy and demonstrated intra-erythrocytic inclusion bodies.
The results indicated that Anaplasma is more conjoint in adults than young animals. On the basis of age, animals were categorized into two groups (<2 years and >years). Prevalence was greater in <2 years of age (42.31%; 88/208) than >2 years (24.43%; 43/176) age groups. A significant association was found among different age groups (X2 = 40.29, df = 1, p = 0.000). Gender-wise frequency of disease had a significant association (X2 = 100.10, df = 1, p = 0.000) indicating a higher positivity in females (41.61%; 114/274) than males (15.45%; 17/110), regardless of host type. The animals were screened for tick infestation to assess the role of ticks on disease outcome. The animals infested with ticks had higher disease positivity rates than tick-free animals with significant association (X2 = 88.99, df = 1, p = 0.000). All hypothesized risk factors were statistically significant (Table 1). Furthermore, when compared to adult, young animals experienced the disease more frequently. According to univariate and multivariate analysis gender of small ruminants was a statistically substantial risk factor. The analysis of univariate (p = 0.02, OR = 3.532, CI = 1.201–10.387) and multivariate (p = 0.000, OR = 1.757, CI = 1.305–2.366) logistic regression expressed gender as the significant risk factor (Table 2).
Sequencing and phylogenetic analysis
The PCR results based on amplification of 16S rRNA gene of Anaplasma had revealed 131 positive animals out of 384 samples. The representative sequences (n = 04) were sent for sequencing and displayed 99–100% identity with the Anaplasma species. The acquired sequences revealed novel isolates of A. capra with Genbank accession numbers ON834323, ON838209, ON838210, and ON838211. The phylogenetic analysis of the 16S rRNA gene of A. capra revealed three distinct clusters with 99–100% similarity with other isolates from different countries.
Our isolates are grouped in cluster-I and showed higher similarity with isolates from China (KM206273, KP314237, MT799937), Pakistan (ON238129, ON238130, ON238131), Angola (MT898988), India (MZ558066), Iran (MW692362), and Turkey (MT632469) isolated from human, sheep, ticks, goats, cattle, Gaddi goat, Persian Onager (Equus hemionus onager), and Turkish goats. Cluster-II consisted of Isolates of South Korea (LC432123, LC432126), China (MF066918), France (MW930537) and Turkey (ON763217) isolated from Korean Water Deer, sheep, and water buffalo, respectively. Cluster-III comprised of isolates from Portugal (OK091152), Pakistan (OR643820, OR643666, OR643667) collected from Rhipicephalus sanguineus ticks, goats and sheep, respectively. The isolates of current study are mentioned with red triangles and highlighted in bold with asterisks (Fig 2).
Discussion
The primary sources of financial losses for resource-limited livestock farmers in impoverished nations are vectors and vector-borne diseases. Small ruminants are the major source of income for marginal and small farming communities that are heavily dependent on the environment. Due to global warming and climate change, ectoparasites are spreading their habitat to newer areas, putting the previously unexposed hosts at risk of various tick-transmitted pathogens. Anaplasmosis is one of the most significant vector-borne illnesses at human-animal interface. Recently, A. capra got attention from veterinary and public health researchers, and detected in Asian and European countries [16, 23, 64–66]. The A. capra is a zoonotic pathogen also detected in tick vectors such as Haemaphysalis longicornis, H. qinghaiensis, Dermacentor abaensis, D. nuttalli and Rhipicephalus microplus [37, 38, 41]. They have been isolated from humans and goats inhabiting forests in China [18, 33]. Anaplasma capra has been reported from deer, sheep, and goats in France [23, 67] and Onagers (Equidae) in Iran [65]. In Pakistan, only one study which depicted A. capra in domestic and Mouflon sheep (Ovis gmelini) which differ in terms of small sample size, outcome, and study location [26]. In the first surveillance study, we performed molecular detection, estimated risk factors, and conducted phylogenetic analysis in small ruminants from Faisalabad district, Pakistan.
According to PCR results, the overall prevalence of anaplasmosis in small ruminants was 34.11%. Likewise, earlier reports of anaplasmosis among small ruminants from Korea (10.8%) [34]; Turkey (31.4%) [46], China (30.1 to 59.7%) [68, 69], Pakistan (21.7%) [48] and China (29.1%) [70] were in line with our findings. Additionally, A. capra was found in 10 out of 452 (2.2%) samples from goats in South Korea [34], 79 of 435 (18.2%) samples from sheep in China [68], 13 of 224 (5.8%) from cattle in Malaysia [71], 5 of 155 (3.22%) from sheep, and 0 of 72 from goats in Turkey. Likewise, 40 goats and 46 horses in Malaysian research were found free from A. capra [71]. However, lower prevalence was depicted from Korea, Spain and Turkey ranging from 0.5 to 5.8 percent detected from goats and Cervids [24, 72–74].
The results of the present investigation were similar to Belkahia et al. [75], where they depicted higher disease occurrence in female than male animals. This determinant was proved as the most significant risk factor based on univariate analysis. These findings were also supported by Atif et al. [12] as they mentioned higher infection in females than males. This result can be justified by the fact that immunosuppression in females during pregnancy or lactation could be responsible for higher disease outcome in female animals. Belkahia et al. [75], who mentioned a higher infection rates in ewes than in rams. Furthermore, it was revealed that young animals were more infected than adults. Age-related findings are incoherent with Atif et al. [12], who indicated that adults had higher infectivity than young animals. Similarly, the occurrence of anaplasmosis was significantly higher in sheep than goats. As anticipated, anaplasmosis risk was greater in tick-infected animals in this research than non-tick infested animals. Rahman et al. [76], supported our findings who reported comparable results in Jamnapari breed of goats from Bangladesh. Additionally, animals having a history of tick illness had higher disease positivity rates than those who had no history of disease. Our findings are corroborated by the research of Farooqi et al. [77], conducted on cattle in Northern, Pakistan. The mixed farming yield higher infectivity for Anaplasma infection. This had been the fact that animals housed in the same facility might get tick-borne diseases from various animal species.
The highest prevalence was observed in free-grazing animals (39.6%), followed by stall-fed animals (36.5%), and the lowest infectivity in semi-grazing animals (23.9%). Chi square test specified a significant linking between grazing pattern and disease outcome in small ruminants, while regression analysis revealed a non-significant effect. In addition, Yan and colleagues depicted that animals are more prone to infection and the occurrence of anaplasmosis was associated with free-grazing [43]. The grazing could have augmented the contact between vectors and other animals [78]. Previous studies conducted in Pakistan suggested that grazing animals were at higher risk of infection than their non-grazing counterparts [49].
The acquired sequences revealed four novel isolates of A. capra. The phylogenetic analysis of the 16S rRNA gene of A. capra indicated three distinct clusters with 99–100% similarity with other isolates from different countries. Our isolates showed higher homology with isolates from China (KM206273, KP314237, MT799937), Angola (MT898988), Iran (MW692362.1), India (MZ558066) and Portugal (OK091152.1) isolated from human, tick, goat, cattle, Persian onager (Equus hemionus onager), Gaddi goat and Rhipicephalus microplus ticks, respectively. This is the first report of A. capra in goats from Pakistan. To date, the pathogens remained unnoticed and require large scale surveillance of migratory and non-migratory small ruminants to estimate the gravity of the problem with special reference to vector-transmitted hemoparasites.
Current surveillance studies concluded that zoonotic A. capra is prevalent in small ruminants in Faisalabad district, Pakistan. Gender is the major risk factor for A. capra infection. Phylogenetic analysis indicated novel genotypes. Further studies should be conducted on genetic diversity, host specificity, pathogenicity, vectors competence, and transmission attributes of A. capra zoonotic anaplasmosis using specie specific primers (such as gltA and groEL genes) [79, 80] for effective prevention and control of emerging zoonotic pathogen.
References
- 1. Economic Survey of Pakistan, Government of Pakistan, Finance Division, Economic Advisor Wing, Islamabad. Chapter 2/Agriculture, 2023, p. 27. Available at: https://www.finance.gov.pk/survey/chapters_23/02_Agriculture.pdf. Accessed 24 September, 2023.
- 2. Khan M, Khan M, Ahmad S, Mahmood S. Genetic resources and diversity in Pakistani sheep. Int J Agric Biol. 2007; 9:941–944. https://www.researchgate.net/publication/237318075_Genetic_Resources_and_Diversity_in_Pakistani_Goats.
- 3. Berggoetz M, Schmid M, Ston D, Smith V, Chevillon C, Pretorius AM. Tick-borne pathogens in the blood of wild and domestic ungulates in South Africa, Interplay of game and livestock. Ticks Tick Borne Dis. 2014; 5:166–75. pmid:24418761.
- 4. Khan A, Muhammed AA, Nasreen N, Iqbal F, Cossio-Bayugar R, Ali Sha SS, et al. Tick-borne haemoparasitic diseases in small ruminants in Pakistan: Current knowledge and future perspectives. Saudi J Biol Sci. 2022; 29:2014–2025. pmid:35531246.
- 5. Khan A, Nasreen N, Niaz S, Sajjad Ali Shah S, Mitchell III-RD, Ayaz S, et al. Tick burden and tick species prevalence in small ruminants of different agencies of the Federally Administered Tribal Areas (FATA), Pakistan. Intl J Acarol. 2019; 45:374–80. https://doi.org/10.1080/01647954.2019.1663930.
- 6. Lihou K, Rose Vineer H, Wall R. Distribution and prevalence of ticks and tick-borne disease on sheep and cattle farms in Great Britain. Parasit Vectors. 2020; 13:406. pmid:32778148.
- 7. Basit MA, Ijaz M, Abbas RZ, Khan JA, Ashraf K. First Molecular evidence of Ehrlichia Infection: An emerging pathogen of small ruminants in Pakistan. Pak Vet J. 2022; 42:208–214. http://dx.doi.org/10.29261/pakvetj/2022.012.
- 8. Ceylan O, Uslu A, Oztürk O, Sevinc F. Serological investigation of some vector-borne parasitic and rickettsial agents in dogs in the western part of Turkey. Pak Vet J. 2021; 41:386–392. http://dx.doi.org/10.29261/pakvetj/2021.052.
- 9. Ceylan C, Ekici ÖD. Molecular investigation of ovine and caprine anaplasmosis in south-Eastern Anatolia region of Turkey. Pak Vet J. 2022; 43:139–145. http://dx.doi.org/10.29261/pakvetj/2022.070.
- 10. Abbas SN, Ijaz M, Abbas RZ, Saleem MH, Mahmood AK. Molecular characterization, risk factor analysis and hematological alterations associated with Anaplasma phagocytophilum in domestic cats of Pakistan. Pak Vet J. 2023; 43:493–499. http://dx.doi.org/10.29261/pakvetj/2023.082.
- 11. Atif FA, Abbas RZ, Mehnaz S, Qamar MF, Hussain K, Nazir MU, et al. First report on molecular surveillance based on duplex detection of Anaplasma marginale and Theileria annulata in dairy cattle from Punjab, Pakistan. Trop Anim Health Prod. 2022; 54:155. pmid:35362760.
- 12. Atif FA, Ullah S, Cossío-Bayúgar R, Kashif M, Khan AU, Wu WF. Molecular epidemiology, seasonality and phylogenetic investigations of Anaplasma ovis in small ruminants from diverse agro-climatic regions of Punjab, Pakistan. Microorganisms 2023; 11:2430. pmid:37894088.
- 13. Dumler JS, Barbet AF, Bekker CP, Dasch GA, Palmer GH, Ray SC, et al. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ’HGE agent’as subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol. 2001; 51:2145–2165. pmid:11760958.
- 14. Calchi AC, Vultão JG, Alves MH, Yogui DR, Desbiez AL, De Santi M, et al. Ehrlichia spp. and Anaplasma spp. in Xenarthra mammals from Brazil, with evidence of novel ‘Candidatus Anaplasma spp.’. Sci Rep. 2020; 10:12615. pmid:32724088.
- 15. Dumler Gofton AW, Doggett S, Ratchford A, Oskam CL, Paparini A, Ryan U, et al. Bacterial profiling reveals novel “Ca. Neoehrlichia, Ehrlichia”, and Anaplasma species in Australian human-biting ticks. PloS One 2015; 10:145–449. pmid:26709826.
- 16. Peng Y, Lu C, Yan Y, Shi K, Chen Q, Zhao C, et al. The first detection of Anaplasma capra, an emerging zoonotic Anaplasma sp., in erythrocytes. Emerg Microbes Infect. 2021; 10:226–34. pmid:33446064.
- 17. Liu Z, Ma M, Wang Z, Wang J, Peng Y, Li Y, et al. Molecular survey and genetic identification of Anaplasma species in goats from central and southern China. Appl Environ Microbiol. 2012; 78: 464–470. pmid:22057867.
- 18. Li H, Zheng YC, Ma L, Jia N, Jiang BG, Jiang RR, et al. Human infection with a novel tick-borne Anaplasma species in China: a surveillance study. Lancet. Infect. Dis. 2015; 15:663–670. pmid:25833289.
- 19. Dahlgren FS, Heitman KN, Behravesh CB. Undetermined human ehrlichiosis and anaplasmosis in the United States, 2008–2012: A catch-all for passive surveillance. Am J Trop Med Hyg. 2016; 94:299. pmid:26621564.
- 20. Yang J, Liu Z, Niu Q, Liu J, Han R, Guan G, et al. A novel zoonotic Anaplasma species is prevalent in small ruminants: potential public health implications. Parasit. Vectors 2017; 10:1–6. pmid:28558749.
- 21. Sahin OF, Erol U, Altay K. Buffaloes as new hosts for Anaplasma capra: molecular prevalence and phylogeny based on gtlA, groEL, and 16S rRNA genes. Res Vet Sci. 2022; 152:458–464. pmid:36148715.
- 22. Sato M, Nishizawa I, Fujihara M, Nishimura T, Matsubara K, Harasawa R. Phylogenetic analysis of the 16S rRNA gene of Anaplasma species detected from Japanese serows (Capricornis crispus). J Vet Med Sci. 2009; 71:1677–1679. pmid:20046041.
- 23. Jouglin M, Blanc B, De La Cotte N, Bastian S, Ortiz K, Malandrin L. First detection and molecular identification of the zoonotic Anaplasma capra in deer in France. PLoS One 2019; 14:e0219184. pmid:31276519.
- 24. Remesar S, Prieto A, García‐Dios D, López‐Lorenzo G, Martínez‐Calabuig N, Díaz‐Cao JM, et al. Diversity of Anaplasma species and importance of mixed infections in roe deer from Spain. Transbound Emerg Dis. 2022; 69: e374–e385. pmid:34529897.
- 25. Shi K, Li J, Yan Y, Chen Q, Wang K, Zhou Y, et al. Dogs as new hosts for the emerging zoonotic pathogen Anaplasma capra in China. Front Cell Infect Microbiol. 2019; 9:394. pmid:31850236.
- 26. Ishaq M, Ijaz M, Lateef M, Ahmed A, Muzammil I, Javed MU, et al. Molecular characterization of Anaplasma capra infecting captive mouflon (Ovis gmelini) and domestic sheep (Ovis aries) of Pakistan. Small Rumin Res. 2022; 216, 106837. https://doi.org/10.1016/j.smallrumres.2022.106837.
- 27. Guo WP, Zhang B, Wang YH, Xu G, Wang X, Ni X, et al. Molecular identification and characterization of Anaplasma capra and Anaplasma platys-like in Rhipicephalus microplus in Ankang, Northwest China. BMC Infect Dis. 2019; 19:1–9. pmid:31101084.
- 28. Addo SO, Baako BOA, Bentil RE, Addae CA, Behene E, Asoala V, et al. Molecular survey of Anaplasma and Ehrlichia species in livestock ticks from Kassena-Nankana, Ghana; with a first report of Anaplasma capra and Ehrlichia minasensis. Arch Microbiol. 2023; 205:92. pmid:36795247.
- 29. Peng Y, Wang K, Zhao S, Yan Y, Wang H, Jing J, et al. Detection and phylogenetic characterization of Anaplasma capra: an emerging pathogen in sheep and goats in China. Front Cell Infect Microbiol. 2018; 8:283. pmid:30214896.
- 30. Altay K, Erol U, Sahin O.F. The first molecular detection of Anaplasma capra in domestic ruminants in the central part of Turkey, with genetic diversity and genotyping of Anaplasma capra. Trop Anim Health Prod. 2022; 54:129. pmid:35257219.
- 31. Inokuma H, Brouqui P, Drancourt M, Raoult D. Citrate synthase gene sequence: a new tool for phylogenetic analysis and identification of Ehrlichia. J Clin Microbiol. 2001; 39:3031–3039. pmid:11526124.
- 32. Amer S, Kim S, Yun Y, Na KJ. Novel variants of the newly emerged Anaplasma capra from Korean water deer (Hydropotes inermis argyropus) in South Korea. Parasit. Vectors. 2019; 12:1–9. pmid:31345253.
- 33. Sun XF, Zhao L, Wen HL, Luo LM, Yu XJ. Anaplasma species in China. Lancet Infect Dis. 2015; 15:1263–1264. pmid:26531037.
- 34. Seo HJ, Jin BC, Kim KH, Yoo MS, Seong KW, Jeong SJ, et al. Molecular detection and phylogenetic analysis of Anaplasma spp. in Korean Native goats from Ulsan Metropolitan city. Vector Borne Zoonotic Dis. 2019; 19:773–776. pmid:31355707.
- 35. Rocchigiani G, Ebani VV, Nardoni S, Bertelloni F, Bascherini A, Leoni A, et al. Molecular survey on the occurrence of arthropod-borne pathogens in wild brown hares (Lepus europaeus) from Central Italy. Infect Genet Evol. 2018; 59:142–147. pmid:29421225.
- 36. Lysholm S, Ådén F, Aspán A, Högberg A, Wensman JJ, Omazic A. Presence of Anaplasma spp. and their associated antibodies in the Swedish goat population. Animals. 2023; 13:333. pmid:36766222.
- 37. de Vries MA. Ticks and Tick-borne diseases surveillance with special reference to Anaplasma infections in small ruminants on the island of Lesvos, Greece. Research Project Veterinary Medicine Myrthe de Vries 3833828. 2018. Available from: https://studenttheses.uu.nl/handle/20.500.12932/31293.
- 38. Atif FA. Alpha proteobacteria of genus Anaplasma (Rickettsiales: Anaplasmataceae): Epidemiology and characteristics of Anaplasma species related to veterinary and public health importance. Parasitol. 2016; 143:659–685. pmid:26932580.
- 39. Altay K, Erol U, Sahin OF, Aytmirzakizi A. First molecular detection of Anaplasma species in cattle from Kyrgyzstan; molecular identification of human pathogenic novel genotype Anaplasma capra and Anaplasma phagocytophilum related strain. Ticks Tick Borne Dis 2022; 13:101861. pmid:34773849.
- 40. Altay K, Erol U, Sahin OF, Aytmirzakizi A, Temizel EM, Aydin MF et al. The detection and phylogenetic analysis of Anaplasma phagocytophilum-like 1, A. ovis and A. capra in sheep: A. capra divides into two genogroups. Vet Res Commun 2022; 46:1271–1279. pmid:36167934.
- 41. Younas M, Ashraf K, Rashid MI, Ijaz M, Suleman M, Chohan TA, et al. Expression and purification of recombinant multi-epitope protein of Rhipicephalus microplus tick and its antigenicity in rabbits. Pak Vet J. 2023. http://dx.doi.org/10.29261/pakvetj/2023.086.
- 42. Hosseini-Vasoukolaei N, Oshaghi MA, Shayan P, Vatandoost H, Babamahmoudi F, Yaghoobi-Ershadi MR, et al. Anaplasma infection in ticks, livestock and human in Ghaemshahr, Mazandaran Province, Iran. J. Arthropod Borne Dis. 2014; 8:204–211. https://jad.tums.ac.ir/index.php/jad/article/view/210 pmid:26114134.
- 43. Yang J, Li Y, Liu Z, Liu J, Niu Q, Ren Q, et al. Molecular detection and characterization of Anaplasma spp. in sheep and cattle from Xinjiang, northwest China. Parasit Vectors. 2015; 8:108. pmid:25889906.
- 44. Kocan KM, De La Fuente J, Blouin EF, Garcia-Garcia JC. Anaplasma marginale (Rickettsiales: Anaplasmataceae): recent advances in defining host–pathogen adaptations of a tick-borne rickettsia. Parasitol. 2004; 129:285–300. pmid:15938516.
- 45. Qin XR, Han FJ, Luo LM, Zhao FM, Han HJ, Zhang ZT, et al. Anaplasma species detected in Haemaphysalis longicornis tick from China. Ticks Tick Borne Dis. 2018; 9:840–843. pmid:29567147.
- 46. Han R, Yang JF, Mukhtar MU, Chen Z, Niu QL, Lin YQ, et al. Molecular detection of Anaplasma infections in ixodid ticks from the Qinghai-Tibet Plateau. Infect Dis Poverty. 2019; 8:83–90. pmid:30728069.
- 47. Yang J, Liu Z, Niu Q, Liu J, Han R, Liu G, et al. Molecular survey and characterization of a novel Anaplasma species closely related to Anaplasma capra in ticks, northwestern China. Parasit Vectors. 2016; 9:1–5. pmid:27884197.
- 48. Renneker S, Abdo J, Salih DEA, Karagenç T, Bilgiç H, Torina A, et al. Can Anaplasma ovis in small ruminants be neglected any longer?. Transbound Emerg Dis. 2013; 60:105–112. pmid:24589109.
- 49. Niaz S, Rahman ZU, Ali I, Cossío-Bayúgar R, Amaro-Estrada I, Alanazi AD, et al. Molecular prevalence, characterization and associated risk factors of Anaplasma spp. and Theileria spp. in small ruminants in Northern Pakistan. Parasit. 2021; 28. pmid:33416491.
- 50. Selim A, Attia KA, Alsubki RA, Albohairy F, Kimiko I, Said MB. The first study on the seroprevalence of Anaplasma spp. in small ruminants and assessment of associated risk factors in North Egypt. Vet World. 2022; 15:1221. pmid:35765471.
- 51. Naeem M, Amaro-Estrada I, Taqadus A, Swelum AA, Alqhtani AH, Asif M, et al. Molecular prevalence and associated risk factors of Anaplasma ovis in Pakistani sheep. Front Vet Sci. 2023; 10:1096418. pmid:37065244.
- 52. Noaman V. Epidemiological study on Anaplasma phagocytophilum in cattle: molecular prevalence and risk factors assessment in different ecological zones in Iran. Preventive Vet Med. 2020; 183: 105118. pmid:32891899.
- 53. Noaman V. Factors associated with Anaplasma phagocytophilum infection in sheep in Iran. Small Rumin Res. 2022; 208:106617. https://doi.org/10.1016/j.smallrumres.2022.106617.
- 54. Noaman V, Sazmand A. Anaplasma ovis infection in sheep from Iran: molecular prevalence, associated risk factors, and spatial clustering. Trop Anim Health Prod. 2022; 54: 6. pmid:34890017.
- 55. Silaghi C, Santos AS, Gomes J, Christova I, Matei IA, Walder G, et al. Guidelines for the direct detection of Anaplasma spp. in diagnosis and epidemiological studies. Vector Borne Zoonotic Dis. 2017; 17:12–22. pmid:28055579.
- 56. Noaman V, Shayan P. Comparison of microscopy and PCR-RFLP for detection of Anaplasma marginale in carrier cattle. Iranian J. Microbiol, 2010; 2:89. pmid:22347555.
- 57. Kolo AO, Sibeko-Matjila KP, Maina AN, Richards AL, Knobel DL, Matjila PT. Molecular detection of zoonotic rickettsiae and Anaplasma spp. in domestic dogs and their ectoparasites in Bushbuckridge, South Africa. Vector-Borne Zoonotic Dis. 2016; 16:245–252. pmid:26974185.
- 58.
Thrusfield M. Veterinary epidemiology, John Wiley & Sons; Hoboken, NJ, USA; 2018.
- 59. Atif FA, Khan MS, Iqbal HJ, Roheen T. Prevalence of tick-borne diseases in Punjab (Pakistan) and hematological profile of Anaplasma marginale infection in indigenous and crossbred cattle. Pak J Sci. 2012; 64:11–15. https://pjosr.com/index.php/pjs/article/download/467/350.
- 60. Adam KMG, Paul J, Zaman V. Medical and Veterinary Protozology Churchill living stone Edinburgh and London 1971. pp. 200.
- 61. Saleem S; Ijaz M; Farooqi SH; Rashid MI; Khan A; Masud A, et al. First molecular evidence of equine granulocytic anaplasmosis in Pakistan. Acta Tropica. 2018; 180:18–25. https://pubmed.ncbi.nlm.nih.gov/29306724/ pmid:29306724
- 62. Tamura K. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C-content biases. Mol. Biol. Evol. 1992; 9:678–687. pmid:1630306.
- 63. Tamura K, Stecher G, Kumar S. MEGA11: Molecular evolutionary genetics analysis, Version 11. Mol. Biol. Evol. 2021; 38:3022–3027. pmid:33892491
- 64. Wei W, Li J, Wang YW, Jiang BG, Liu HB, Wei R, et al. Anaplasma platys-Like infection in goats, Beijing, China. Vector Borne Zoonotic Dis. 2020; 20:755–762. pmid:32679008.
- 65. Staji H, Yousefi M, Hamedani MA, Tamai IA, Khaligh SG. Genetic characterization and phylogenetic of Anaplasma capra in Persian onagers (Equus hemionus onager). Vet. Microbiol. 2021; 261:109199. pmid:34385006.
- 66. Remesar S, Diaz P, Prieto A, García‐Dios D, Panadero R, Fernández G, et al. Molecular detection and identification of piroplasms (Babesia spp. and Theileria spp.) and Anaplasma phagocytophilum in questing ticks from northwest Spain. Med. Vet. Entomol. 2021; 35:51–58. pmid:32757238.
- 67. Jouglin M, Rispe C, Grech-Angelini S, Gallois M, Malandrin L. Anaplasma capra in sheep and goats on Corsica Island, France: A European lineage within A. capra clade II?. Ticks Tick Borne Dis. 2022; 13:101934. pmid:35263704.
- 68. Ma M, Liu Z, Sun M, Yang J, Guan G, Li Y, et al. Development and evaluation of a loop-mediated isothermal amplification method for rapid detection of Anaplasma ovis. J Clin Microbiol. 2011; 49: 2143–2146. pmid:21471346.
- 69. Ait Lbacha H, Alali S, Zouagui Z, El Mamoun L, Rhalem A, Petit E, et al. High prevalence of Anaplasma spp. in small ruminants in Morocco. Transbound Emerg Dis. 2017; 64:250–263. pmid:25916245.
- 70. Yang J, Han R, Niu Q, Liu Z, Guan G, Liu G, et al. Occurrence of four Anaplasma species with veterinary and public health significance in sheep, northwestern China. Ticks Tick Borne Dis. 2018; 9:82–85. pmid:29037826.
- 71. Koh FX, Panchadcharam C, Sitam FT, Tay ST. Molecular investigation of Anaplasma spp. in domestic and wildlife animals in Peninsular Malaysia. Vet Parasitol: Reg Stud Reports. 2018; 13:141–147. pmid:31014863.
- 72. Oguz B, Deger MS, Al-Olayan E, El-Ashram S. Molecular Survey of Anaplasma capra in Goats in Van Province, Eastern Türkiye. Acta Parasitol. 2023; 19:1–5. pmid:38112913.
- 73. Seo MG, Ouh IO, Lee H, Geraldino PJ, Rhee MH, Kwon OD, et al. Differential identification of Anaplasma in cattle and potential of cattle to serve as reservoirs of Anaplasma capra, an emerging tick-borne zoonotic pathogen. Vet Microbiol. 2018; 226:15–22. pmid:30389039.
- 74. Miranda EA, Han SW, Cho YK, Choi KS, Chae JS. Co-infection with Anaplasma species and novel genetic variants detected in cattle and goats in the Republic of Korea. Pathogens. 2021; 10:28. pmid:33401478.
- 75. Belkahia H, Said MB, El Hamdi S, Yahiaoui M, Gharbi M, Daaloul-Jedidi M, et al. First molecular identification and genetic characterization of Anaplasma ovis in sheep from Tunisia. Small Rumin. Res. 2014; 121: 404–410. https://doi.org/10.1016/j.smallrumres.2014.07.009.
- 76. Rahman M, Faruque MR, Rahman MM, Chowdhury MYE. Epidemiology and molecular detection of Anaplasma spp. in goats from Chattogram district, Bangladesh. Vet Med Sci. 2022; 8: 1240–1249. pmid:35218684.
- 77. Farooqi SH, Ijaz M, Rashid MI, Nabi H, Islam S, Aqib AI, et al. Molecular epidemiology of bovine anaplasmosis in Khyber Pakhtunkhwa, Pakistan. Trop Anim Health Prod. 2018; 50:1591–1598. https://pubmed.ncbi.nlm.nih.gov/29740781/ pmid:29740781
- 78. Eisawi NM, El Hussein ARM, Hassan DA, Musa AB, Hussien MO, Enan KA, et al. A molecular prevalence survey on Anaplasma infection among domestic ruminants in Khartoum State, Sudan. Trop Anim Health Prod. 2020; 52:1845–1852. pmid:31938957.
- 79. Lin ZT, Ye RZ, Liu JY, Wang XY, Zhu WJ, Li YY, et al. Epidemiological and phylogenetic characteristics of emerging Anaplasma capra: A systematic review with modeling analysis. Infect Genet Evol. 2023; 115:105510. pmid:37778674.
- 80. Altay K, Erol U, Sahin O.F. Anaplasma capra: a new emerging tick-borne zoonotic pathogen. Vet Res Commun, 2024; 10.1007/s11259-024-10337-9. Advance online publication. pmid:38424380.