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Iranian Journal of Veterinary Research logoLink to Iranian Journal of Veterinary Research
. 2022;23(4):349–357. doi: 10.22099/IJVR.2022.43496.6362

Molecular evidence, risk factors analysis, and hematological alterations associated with Theileria spp. spillover in captive wild mouflon sheep in Punjab, Pakistan

M Naveed 1, M Ijaz 2,*, A Ahmed 3, N Z Ghumman 1, M Ishaq 1, I Muzammil 3, M U Javed 3
PMCID: PMC9984143  PMID: 36874179

Abstract

Background:

Landscape anthropization and interaction between domestic and wild animals are the major contributing factors involved in the emergence of new pathogens in wild animals. Theileriosis is an emerging issue of wild ungulates, especially in the tropical and subtropical areas of the globe.

Aims:

The current study investigated the mouflon sheep for Theileria infection using molecular methods and hematological analysis.

Methods:

This study was conducted on a total of 103 captive wild mouflon sheep present in eight different recreational zoos, and wildlife parks in Punjab, Pakistan to investigate the genotypic prevalence of Theileria spp. by targeting 18S rRNA and molecular evidence for Theileria spillover between domestic and wild mouflon sheep by phylogenetic analysis. The association of assumed risk factors and the effect of Theileria spp. on various hematological parameters were also assessed.

Results:

The results depicted that Theileria spp. was prevalent in 8 (7.77%, CI 95%: 3.99-14.59%), and 11 (10.68%, CI 95%: 06.07-18.12%) animals based on microscopy, and PCR, respectively. The phylogenetic analysis of the 18S rRNA gene of Theileria spp. from mouflon revealed a close resemblance with T. annulata from domestic animals. The risk factor analysis revealed that tick infestation, enclosure hygiene, previous tick infestation history, and the presence of wooden logs in the enclosure were significantly (P<0.05) associated with the occurrence of Theileria spp. infection in the captive mouflon sheep of Pakistan. Furthermore, a significant reduction in blood parameters like PCV, RBCs count, Hb, and platelets was observed in Theileria-positive animals.

Conclusion:

This study is the first evidence at the molecular level to characterize the spillover of Theileria spp. between the captive wild mouflon sheep and domestic animals of Pakistan, and it will be useful in developing control strategies for emerging theileriosis in captive wild animals.

Key Words: Emerging disease, Mouflon sheep, Phylogenetic analysis, Theileriosis, Wildlife

Introduction

Wildlife parks, breeding centers under captivity, and related recreational sanctuaries parks have been designated as key tools to protect the exposed, and threatened wild animal species (Ali et al., 2011). The diseases of wild animals have a huge impact on the national economy, biodiversity, public health, and wildlife conservation (Oludairo et al., 2016). The ecological coexistence of wild and domestic animals elevates the risk of bilateral pathogen transmission among different species (González-Barrio et al., 2022). The population of mouflon sheep (Ovis orientalis), an endangered wild species, is decreasing rapidly due to hunting, stress, deadly infections, parasitic, and hemoparasitic diseases like fasciolosis, muelleriosis, theileriosis, and anaplasmosis (Benfenatki et al., 2016; Adjou Moumouni et al., 2018). Over the last three decades, the mouflon sheep death rate has increased by 30% (Valdez, 2008) while the conflict between human and sympatric impacts as well as the disease load poses a bigger threat to wildlife refuges (Shabbir and Khan, 2010; Posada-Guzm et al., 2015).

In Pakistan, a total of 225 captive mouflon sheep are present in various wildlife parks and recreational zoos in Punjab province according to a recent report by the Wildlife Department. These parks include Safari Zoo Lahore, Wildlife Park Jallo, Lahore Zoo, Wildlife Park Gutwala-Faisalabad, Wildlife Park Changa Manga, Wildlife Park Kamalia, Wildlife Park Lohi Bher Rawalpindi, D.G. Khan Zoo, and Wildlife Park Vehari. Blood-borne parasites are of severe concern in domestic as well as wild animals in Pakistan. Theileria spp., Babesia spp., Ehrlichia spp. and Anaplasma spp. are important transboundary tick-borne pathogens of animals throughout the world (Ito et al., 2013; Batool, 2019; Basit et al., 2022). Among the tick-borne hemoparasitic diseases of mouflon sheep, theileriosis is one of the most prevalent disease of domestic and wild animals living in the subtropical and tropical areas of the globe including Bangladesh, Pakistan, and India. Theileriosis is caused by an obligate intracellular protozoan, belonging to the genus Theileria (Heidarpour Bami et al., 2010; Ghauri et al., 2019; Ali et al., 2020; Abid et al., 2021). In wild ungulates, subclinical infection is caused by the diverse species of Theileria and is transmitted by arthropod vectors between domestic and wild animals (Shabbir and Khan, 2010; Alvarado-Rybak et al., 2016). Theileriosis is mainly transmitted by the Ixodid ticks i. e. Hyalomma anatolicum, H. impeltatum, H. rufipes, H. dromedarii, Rhipicephalus evertsi, R. decoloratus, R. sanguineus, and Amblyomma lepidum (Hussain et al., 2014; Mans et al., 2015; Boucher et al., 2020; Hayati et al., 2020; Ola-Fadunsin et al., 2020). The infected animals exhibit clinical signs including a rise in body temperature, reduced appetite, yellowing of mucous membrane, labored breathing, nasal and conjunctival discharge, coughing, and enlarged lymph nodes (Tanveer et al., 2022). For diagnostic purposes, blood smear examination can be performed. However, the most reliable and expedient tool for the diagnosis of theileriosis is polymerase chain reaction (PCR) (Hassan et al., 2019).

The epidemiological investigation of disease-causing pathogens is required in captive wild animals to minimize the losses in the terms of deteriorating health and mortalities of precious wild animals (da Silveira et al., 2011). Moreover, a pathogen spillover among domestic and wild animals should be considered an important emerging issue that threatens people’s domestic economy based on animal production, public health concerns in zoonosis case, national animal health security in the context of transboundary animal diseases, and the success of integrated conservation and development initiatives in case of wild animal protection scenario (Caron et al., 2013). In Pakistan, a large number of captive wild animals including mouflon sheep suffer from tick-borne diseases every year (Ghafar et al., 2020; AbouLaila et al., 2021). In Pakistan, increased prevalence of theileriosis in various domestic animals including small ruminants (Naz et al., 2012; Zaman et al., 2022), bovines (Farooqi et al., 2017), and equines (Ali et al., 2019; Shah et al., 2020) have been reported, and wildlife practitioners showed concerns regarding hemoparasites and their diagnosis in wild animals (Ishaq et al., 2022). In this regard, this study was designed to investigate the molecular prevalence of Theileria spp. among domestic animals, and captive mouflon sheep of Pakistan.

Materials and Methods

This study was approved by the Advanced Studies and Research Board, University of Veterinary and Animal Sciences, Lahore (Letter No. DAS/8437; Dated 21-11-2019).

Samples collection and initial screening

The present study was conducted on a total of 103 captive wild mouflon sheep kept in eight different recreational zoos and wildlife parks in Punjab, Pakistan (Fig. 1), from March to September 2020. The study was approved by the Advanced Studies and Research Board, University of Veterinary and Animal Sciences, Lahore (Letter No. DAS/8437; Dated 21-11-2019), and consent was taken from the relevant authorities for the sampling process. The mouflon sheep manifesting clinical signs like tick infestation, pyrexia, anorexia, pale mucous membrane, jaundice, or anemia were incorporated in the study. A data capture form was designed to access probable theileriosis risk factors like sex, age, tick infestation, the previous history of tick infestation, the entrance of new wild animals, enclosure hygiene, and the presence of wooden logs. For sampling, animals were captured using a capturing net irrespective of age and sex, and 3 ml of blood was aseptically collected from the jugular vein, and stored in EDTA-coated vacutainers. The blood smears of all samples in triplets were also prepared for the microscopic examination. All samples were transferred to Medicine Research Laboratory, Department of Veterinary Medicine, University of Veterinary and Animal Sciences, Lahore, maintaining a cold chain. For initial screening, Giemsa-staining was performed on blood smears, and all stained smears were observed under the microscope for intra-cytoplasmic inclusion bodies resembling Theileria spp. in blood cells (Radostits et al., 2006). For confirmation, blood samples were subjected to molecular diagnosis protocol.

Fig. 1.

Fig. 1

Map formed by Quantum Geographic Information Systems (QGIS) showing recreational zoos and wildlife parks of Punjab, Pakistan

DNA isolation and PCR

The DNA extraction of all blood samples was performed utilizing a Exgene™ Blood SV DNA extraction kit (GeneAll®, South Korea) as per manufacturer’s recommendations. Extracted DNA from the samples was subjected to purity and concentration measurements using the NanoDrop at 260/280 nm. Forward primer 5´-GAC ACA GGG AGG TAG TGA CAA G-3´ and reverse primer 5´-CTA AGA ATT TCA CCT CTG ACA GT-3´ were utilized for the amplification of 450 bp 18S rRNA gene fragment (Nijhof et al., 2003). The PCR recipe was prepared by mixing master mix (2X PCRTaq Master Mix, Bioshop) (10 μL), DEPC-treated water (6.6 μL), forward primer (0.7 μL), reverse primer (0.7 μL), and sample DNA (2 μL). The conditions for PCR reaction include one step of 5 min of initial denaturation at 95°C, followed by 35 cycles of denaturation (95°C for 30 s), primers annealing (58°C for 30 s), and extension (72°C for 1 min). The final extension was performed with one step at 72°C for 10 min. Gel electrophoresis of 1.5% ethidium bromide-stained agarose gel was conducted at 120 volts, and 200 milliamperes for 35 min. After gel electrophoresis, PCR products were seen on UV illuminator for detecting positive bands at 450 bp position alongside 100 bp molecular mass marker. The PCR positive bands after gel purification using Expin™ Gel SV gel extraction kit (GeneAll®, South Korea) were shipped to 1st BASE sequence lab Singapore, for sequencing.

Molecular evidence for Theileria spp. spillover by phylogenetic analysis

The representative nucleotide sequence of Theileria spp. 18S rRNA gene was evaluated using the basic local alignment search tool (BLAST), and phylogenetically analyzed to find out the pathogen spillover among domestic and wild animals at a molecular level in the country. Firstly, the gene sequence of 18S rRNA of T. annulata reported from domestic cattle, camel, buffalo, goat, and sheep of Pakistan, and from neighboring countries like China, and India were retrived from NCBI database. Then multiple sequence alignment was done by BioEdit Software using the CLUSTAL W method. Similarities or differences among the gene sequences were observed at the nucleotide level to figure out the pathogen resemblance between wild and domestic animals. Finally, phylogenetic tree was constructed using the bootstrap phylogeny testing at 1000 replications by maximum likelihood (ML) method through Mega X Software (Ahmed et al., 2020).

Hematological analysis

To pinpoint the difference in the hematological parameter of Theileria-positive and Theileria-negative mouflon sheep, a comparative hematological analysis was performed. A total of four PCR-based Theileria-positive and four healthy mouflon sheep (Theileria-negative) were selected, and various hematological parameters of them were assessed using an automated hematology analyzer, Abacus Junior Vet (Diatron® Vienna, Austria). The hematological parameters include platelet count, RBCs count, WBCs count, packed cell volume (PCV), and hemoglobin (Hb) to evaluate hematological changes in diseased animals (Penzhorn, 2006; Ahmed et al., 2020).

Statistical analysis

The prevalence of the Theileria spp. infection was calculated by a formula already reported (Thrusfield, 2007). The data of the current study was analyzed by statistical software SPSS (ver. 20) at a 5% probability level. A Chi-square test was performed to evaluate the association of risk factors with Theileria spp. infection. To compare the means of different hematological parameters of Theileria-positive and Theileria-negative mouflon sheep, an independent sample t-test was applied. The variables showing a p-value less than “0.05” were assumed to be significant determinants of disease occurrence.

Results

Prevalence of Theileria spp. infection in mouflon sheep

On microscopic examination, out of 103 mouflon sheep only 8 sheep (7.77%, CI 95%: 3.99-14.59%) were positive for Theileria spp. Molecular test revealed that 11 (10.68%, CI 95%: 06.07-18.12%) out of 103 animals were positive for Theileria spp. from various parks in Pakistan including Jallo Wildlife Park, Lahore Zoo, Changa Manga Forest, and Wildlife Park. Among these 11 animals, eight animals were found positive by both microscopy and PCR while three animals were found positive only by molecular test.

Risk factor analysis

The risk factors assessment revealed that tick-infested animals showed a higher prevalence (19%) of Theileria spp. compared to tick-free animals (4.9%). Similarly, animals with the previous history of tick infestation or any tick-borne disease were at more risk of Theileria spp. infection. Both tick infestation and previous tick infestation history were proved to be significantly associated (P<0.05) with Theileria spp. infection in mouflon sheep. Also, the presence of wooden logs, being a good place for ticks, was assumed a risk factor (P<0.05) for the disease. On the other hand, the animals living in enclosures were at less risk of developing theileriosis significantly (P=0.001).

Risk factors like sex, age, and introduction of new animals were not found statistically significant (P>0.05) associated with the occurrence of disease (Table 1).

Table 1.

Chi-square analysis of assumed risk factors associated with Theileria infection in mouflon sheep

Study variable Category No. samples Positive (%) P-value
Sex Male 43 4 (09.30) 0.7
Female 60 7 (11.67)
Age ≤ 4 Year 25 3 (12.00) 0.8
>4 Year 78 8 (10.26)
Tick infestation Yes 42 8 (19.04) 0.023*
No 61 3 (04.91)
Previous tick history Yes 28 9 (32.14) <0.001*
No 75 2 (02.67)
Enclosure hygiene Yes 59 1 (01.69) 0.001*
No 44 10 (22.72)
Presence of wooden logs Yes 37 7 (18.91) 0.043*
No 66 4 (6.06)
Introduction of new wild animals Yes 33 4 (12.12) 0.7
No 70 7 (10.00)

* Showing P<0.05 which indicates significant results

Molecular evidence of Theileria spp. spillover

The BLAST search of 18S rRNA partial sequences revealed 99% to 100% similarity with T. annulata sequences. These similar T. annulata 18S rRNA sequences were reported from cattle, buffalo, sheep, goat, and ticks (Fig. 2). Furthermore, the phylogenetic tree revealed that the current sequence shares more evolutionary relation with sequences from camel (MW392284), goat (MT318160) (Niaz et al., 2021), buffalo (MN726546) (Ghafar et al., 2020) and sheep (MK838106) (Adegoke et al., 2020) than the sequences from cattle (MT893655, MG599095), and ticks (MZ452896) (Fig. 3).

Fig. 2.

Fig. 2

Clustal W multiple alignments of the study isolate with reported sequences of T. annulata isolated from domestic animals

Fig. 3.

Fig. 3

Phylogenetic tree showing comparison for the study isolate of Theileria spp. with reported sequences isolated from domestic animals

Effect of Theileria spp. infection on hematological parameters

Hematological analysis exhibited a significant (P<0.05) decline in the RBCs, platelets, hemoglobin (Hb), and PCV in Theileria-positive animals in comparison to mouflon sheep negative for Theileria spp. A non-significant (P>0.05) decrease in WBCs was also observed in mouflon sheep positive for Theileria spp. in comparison to the healthy animals (Table 2).

Table 2.

Comparative hematological analysis of Theileria-positive and Theileria-negative mouflon sheep by independent sample t-test

Parameter Unit Theileria-positive animals
(mean±SD)
Theileria-negative animals
(mean±SD)
F-value Mean difference Confidence interval P-value
WBCs × 103/μL 9.67 ± 2.957a 7.5 ± 2.642a 0.228 2.175 2.67-7.02 0.315
RBCs × 106/μL 11.75 ± 2.125a 5.375 ± 1.869b 0.084 6.375 2.91-9.83 0.004*
Hemoglobin g/dL 12.1 ± 2.551a 5.175 ± 1.909b 0.144 6.925 3.02-10.82 0.005*
PCV % 33.575 ± 6.232a 18.625 ± 2.357b 6.56 14.95 6.79-23.10 0.004*
Platelets × 103/μL 513.25 ± 73.89a 319.25 ± 95.45b 1.52 194 46.32-341.68 0.018*

Different lowercase letters indicate significant statistical differences between the mean values of different hematological parameters of Theileria-positive and Theileria-negative mouflon sheep, and * Indicates the significant association of blood parameters with the infection

Discussion

The microscopy-based Theileria spp. prevalence found in the current study was similar to the findings of Zaeemi et al. (2011), and Zobba et al. (2020) who reported the prevalence of 9.2%, and 10.4% in domestic sheep, and goats, respectively. Microscopic evaluation of blood smears revealed a 10% prevalence of theileriosis in Sudanese sheep (Salih et al., 2003). The current findings were not supported by the study conducted on domestic sheep from Khyber Pakhtunkhwa (KPK) province, Pakistan showing a lower molecular prevalence of 4.5% (Saeed et al., 2015). The different molecular studies conducted in Pakistan showed theileriosis prevalence of 7.8%, 13.5%, and 14% in domestic sheep which were in line with the current study findings (Taha et al., 2013; Nasreen et al., 2020; Giangaspero et al., 2015).

Contradictory to the findings of this study, a higher prevalence (35%) of Theileria spp. was reported from domestic sheep in district Lahore (Durrani et al., 2011). Most currently, a prevalence of 6.1% and 1.2% for T. ovis and T. lestoquardi has been reported in the sheep population of Punjab, Pakistan (Tanveer et al., 2022). Another study conducted in Pakistan found a 10.6% molecular prevalence of T. ovis in domestic sheep (Abid et al., 2021). In other countries, two higher prevalence (32.8% and 54.03%) of Theileria spp. were also reported in Iran, and Turkey using nested PCR (Altay et al., 2005; Zaeemi et al., 2011). Also, the high prevalence (15.50%, 57.73%, and 58.79%) of T. ovis in sheep was reported from Turkey (Altay et al., 2004; Aktaş et al., 2005; Chen et al., 2014). Worldwide, the difference in the prevalence of the disease can be associated with the differences in infection status, vector distribution, socioeconomic factors, climate and the type of PCR used to estimate the infection (nested PCR, conventional PCR, and real-time PCR) (Ramos et al., 2009; Cicuttin et al., 2015).

In current study, both tick infestation and previous tick infestation history were proved to be significantly associated (P<0.05) with Theileria spp. infection in mouflon sheep. The risk factor of tick infestation history found in this study were also found in other studies (Al-Fahdi et al., 2017; Boucher et al., 2020; Hayati et al., 2020; Ceylan et al., 2021). Also, the presence of wooden logs, being a good place for ticks, was assumed a risk factor (P<0.05) for the disease. On the other hand, the animals living in enclosures were at less risk of developing theileriosis significantly (P=0.001). The probable reason could be the better hygienic measures for sheep in the enclosures.

In this study, female sheep showed a non-significant increase in prevalence (11.7%) as compared to male sheep (9.3%) which was in agreement with Gebrekidan et al. study (2014), reported a lower infection rate in males (38.1%) in comparison to females (61.9%). The higher infection rate in female sheep may be associated with more stress, particularly during lactation and pregnancy. Similarly, the infection rate in adult sheep (> 4 years) (10.3%) was lower as compared to younger sheep (< 4 years) (12%) which was in agreement with Shabbir and Khan study (2010), reported a non-significantly higher prevalence in younger sheep (17.3%) as compared to adult sheep (16.2%). However, the results of current study were not following a study showed a higher prevalence in adult sheep (79.7%) as compared to younger sheep (20.3%) (Gebrekidan et al., 2014). The findings were also in concurrence with other studies on Theileria spp. infections in sheep and goats (Razmi et al., 2006; Weir et al., 2011). Mouflon sheep with tick infestation was more prone (19%) to diseases as compared to those without tick infestation (4.9%) which was in line with the findings of Saeed et al. study (2015).

The sequence similarity of study isolates with reported isolates suggest the transmission of the pathogen at the livestock/wildlife interface. As a vector-borne disease, the ecological co-existence of the same vector on both domestic and wild animal, and high prevalence of theileriosis among domestic animals in the country could assume as evidences for pathogen transmission between domestic and wild ungulates. The analysis also revealed a similar gene pattern with sequences reported from China and India as neighboring countries, involved in the transboundary trade of animals and related things. However, the minor differences among the gene sequences as compared to reported sequences could be due to the genetic recombination of an organism at the vector-host interface during concurrent infections (Kurtenbach et al., 2006). The possible genetic variation between the extracellular (in ticks) and intracellular (host) phase and the occurrence of more than one Theileria genotype in ticks during infection could be involved in the pathogen’s sequence variation (Al-Hamidhi et al., 2022).

The hematological test is a considerable way to differentiate between healthy and unhealthy animals in veterinary studies (Riaz and Tasawar, 2017). The results obtained in this study were supported by studies showing lower Hb contents, RBCs count, and PCV (%) in Theileria spp. infected sheep (Bell-Sakyi et al., 2004; Nazifi et al., 2011; Razavi et al., 2011). The decline in RBCs number might be due to erythrophagocytosis during theileriosis and resulted in increased oxygen radicals causing anemia (Shiono et al., 2004). Furthermore, various other studies have also supported the current findings (Hussein et al., 2007; Nazifi et al., 2011; Oryan et al., 2013; AL-Mayah and Abdul-Karim, 2020). The considerable decline in Hb, PCV, and total RBCs count reported in the current study agreed with most hematological studies (Hussein et al., 2007; Nazifi et al., 2011; Mohammed et al., 2014; Al-Saad et al., 2015; Kundave et al., 2015). All these changes could occur as a result of persistent loss of blood, and anemia caused by permanent tick feeding (Durrani et al., 2011; Ito et al., 2013). The current study showed an insignificant reduction in total WBCs, as reported by Hussein et al. (2007), and Ramos et al. (2009). The decrease in WBCs count might be explained by the demolition of WBCs especially lymphocytes due to the disease (Sandhu et al., 1998; Omer et al., 2002).

Rapid ecological change, increasing globalization, and the co-existence of domestic and wild animals lead to the re-emergence of old and the emergence of new diseases in wild animals. The current study is the first report regarding the epidemiology, related risk factors, and molecular evidence for Theileria spp. spillover in the mouflon sheep of Punjab, Pakistan. The results proposed that PCR was a more sensitive and specific technique compared to microscopy for the diagnosis of theileriosis in wild mouflon sheep. Phylogenetic analysis revealed similarity among 18S rRNA sequence of T. annulata from domestic animals and wild mouflon sheep which shows possible spillover of the pathogen. Risk factors assessment revealed that tick infestation, enclosure hygiene, previous tick history, and presence of wooden logs were significantly associated with disease occurrence. Furthermore, hematological parameters like RBCs, PCV, Hb, and platelets were significantly decreased in mouflon sheep infected with T. annulata. Further research can be conducted on the tick species involved in the transmission of this disease. The results of this preliminary study could aid in the establishment of better strategies for diagnosis, prevention, and control of theileriosis in the future.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgment

All the authors pay a vote of thanks to the Medicine Research Laboratory, University of Veterinary and Animal Sciences, Lahore for the provision of materials and instruments during the research.

References

  1. Abid K, Bukhari S, Asif M. Molecular detection and prevalence of Theileria ovis and Anaplasma marginale in sheep blood samples collected from Layyah district in Punjab, Pakistan. Trop. Anim. Health Prod. 2021;53:1–9. doi: 10.1007/s11250-021-02870-5. [DOI] [PubMed] [Google Scholar]
  2. AbouLaila M, AbdEl-Aziz AR, Menshawy S, Yokoyama N, Igarashi I, Al-Wabel M, Omar M. Evaluation of the inhibitory effects of coumermycin A1 on the growth of Theileria and Babesia parasites in vitro and in vivo. Pak. Vet. J. 2021;41:469–474. [Google Scholar]
  3. Adegoke A, Kumar D, Bobo C, Rashid MI, Durrani AZ, Sajid MS, Karim S. Tick-borne pathogens shape the native microbiome within tick vectors. Microorganisms. 2020;8:1–16. doi: 10.3390/microorganisms8091299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Adjou Moumouni PF, Guo H, Gao Y, Liu M, Ringo AE, Galon EM, Vudriko P, Umemiya-Shirafuji R, Inoue N, Suzuki H, Xuan X. Identification and genetic characterization of Piroplasmida and Anaplasmataceae agents in feeding Amblyomma variegatum ticks from Benin. Vet. Parasitol. Reg. Stud. Reports. 2018;14:137–143. doi: 10.1016/j.vprsr.2018.10.006. [DOI] [PubMed] [Google Scholar]
  5. Ahmed A, Ijaz M, Ghauri HN, Aziz MU, Ghaffar A, Naveed M, Javed MU. Molecular evidence of Anaplasma infection in naturally affected domestic cats of Pakistan. Comp. Immunol. Microbiol. Infect. Dis. 2020;72:101524. doi: 10.1016/j.cimid.2020.101524. [DOI] [PubMed] [Google Scholar]
  6. Aktaş M, Altay K, Dumanli N. Survey of Theileria parasites of sheep in eastern Turkey using polymerase chain reaction. Small Rumin. Res. 2005;60:289–293. [Google Scholar]
  7. Al-Fahdi A, Alqamashoui B, Al-Hamidhi S, Kose O, Tageldin MH, Bobade P, Johnson EH, Hussain AR, Karagenc T, Tait A. Molecular surveillance of Theileria parasites of livestock in Oman. Ticks Tick Borne Dis. 2017;8:741–748. doi: 10.1016/j.ttbdis.2017.05.008. [DOI] [PubMed] [Google Scholar]
  8. Al-Hamidhi S, Parveen A, Iqbal F, Asif M, Akhtar N, Elshafie EI, Beja-Pereira A, Babiker HA. Diversity and genetic structure of Theileria annulata in Pakistan and other endemic sites. Pathogens. 2022;11:334. doi: 10.3390/pathogens11030334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. AL-Mayah SH, Abdul-Karim AT. Epidemiology and seasonal variation of ixodid ticks and Piroplasmida detection in cattle of Basrah province, Iraq. Indian J. Forensic. Med. Toxicol. 2020;14:1391–1398. [Google Scholar]
  10. Al-Saad KM, AL-Amery MAY, Hamed TAAL, Muhsen RK. Babesiosis caballi in one-humped dromedaries of basrah province. Basrah. J. Vet. Res. 2015;14:207–214. [Google Scholar]
  11. Ali Z, Bibi F, Mahel AQ, Firdous F, Zamaan SU. Captive breeding practices in Pakistan: A review. J. Anim. Plant. Sci. 2011;21:368–371. [Google Scholar]
  12. Ali S, Ijaz M, Farooqi SH, Durrani AZ, Rashid MI, Ghaffar A, Ali A, Rehman A, Aslam S, Khan I. Molecular characterisation of Theileria equi and risk factors associated with the occurrence of theileriosis in horses of Punjab (Pakistan) Equine Vet. Educ. 2019;33:75–83. [Google Scholar]
  13. Ali S, Ijaz M, Ghaffar A, Oneeb M, Masud A, Durrani AZ, Rashid MI. Species distribution and seasonal dynamics of equine tick infestation in two subtropical climate niches in Punjab, Pakistan. Pak. Vet. J. 2020;40:25–30. [Google Scholar]
  14. Altay K, Aktaş M, Dumanli N. Theileria infections in small ruminants in the east and southeast Anatolia. Turkiye Parazitol. Derg. 2004;31:268–271. [PubMed] [Google Scholar]
  15. Altay K, Dumanli N, Holman PJ, Aktas M. Detection of Theileria ovis in naturally infected sheep by nested PCR. Vet. Parasitol. 2005;127:99–104. doi: 10.1016/j.vetpar.2004.09.012. [DOI] [PubMed] [Google Scholar]
  16. Alvarado-Rybak M, Solano-Gallego L, Millán J. A review of piroplasmid infections in wild carnivores worldwide: importance for domestic animal health and wildlife conservation. Parasit. Vectors. 2016;9:538 . doi: 10.1186/s13071-016-1808-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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. [Google Scholar]
  18. Batool M. Prevalence of tick infestation in farm animals from Punjab, Pakistan. Pak. Vet. J. 2019;39:406–410. [Google Scholar]
  19. Bell-Sakyi L, Koney EBM, Dogbey O, Walker AR. Incidence and prevalence of tick-borne haemoparasites in domestic ruminants in Ghana. Vet. Parasitol. 2004;124:25–42. doi: 10.1016/j.vetpar.2004.05.027. [DOI] [PubMed] [Google Scholar]
  20. Benfenatki A, Bouacida NSY, Oudhia KA, Khelef D. Prevalence of Theileria equi infection in Algiers urban area using cELISA and microscopic examination. Asian J. Anim. Vet. Adv. 2016;11:511–515. [Google Scholar]
  21. Boucher F, Moutroifi Y, Peba B, Ali M, Moindjie Y, Ruget AS, Abdouroihamane S, Kassim A, Soulé M, Charafouddine O. Tick-borne diseases in the Union of the Comoros are a hindrance to livestock development: Circulation and associated risk factors. Ticks Tick Borne Dis. 2020;11 doi: 10.1016/j.ttbdis.2019.101283. [DOI] [PubMed] [Google Scholar]
  22. Ceylan O, Uslu A, Ceylan C, Sevinc F. Predominancy of Rhipicephalus turanicus in tick-infested sheep from Turkey: A large-scale survey. Pak. Vet. J. 2021;41:429–433. [Google Scholar]
  23. Chen Z, Liu Q, Jiao FC, Xu BL, Zhou XN. Detection of piroplasms infection in sheep, dogs and hedgehogs in Central China. Infect. Dis. 2014;3:18 . doi: 10.1186/2049-9957-3-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Cicuttin GL, Tarragona EL, De Salvo MN, Mangold AJ, Nava S. Infection with Ehrlichia canis and Anaplasma platys (Rickettsiales: Anaplasmataceae) in two lineages of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) from Argentina. Ticks Tick Borne Dis. 2015;6:724–729. doi: 10.1016/j.ttbdis.2015.06.006. [DOI] [PubMed] [Google Scholar]
  25. da Silveira JAG, Rabelo ÉML, Ribeiro MFB. Detection of Theileria and Babesia in brown brocket deer (Mazama gouazoubira) and marsh deer (Blastocerus dichotomus) in the State of Minas Gerais, Brazil. Vet. Parasitol. 2011;177:61–66. doi: 10.1016/j.vetpar.2010.10.044. [DOI] [PubMed] [Google Scholar]
  26. Durrani AZ, Younus M, Kamal N, Mehmood N, Shakoori AR. Prevalence of ovine Theileria species in district Lahore, Pakistan. Pak. J. Zool. 2011;43:57–60. [Google Scholar]
  27. Farooqi SH, Ijaz M, Saleem MH, Rashid MI, Ahmad SS, Islam S, Aqib AI, Khan A, Hussain K, Khan NU. Prevalence and molecular diagnosis of Theileria annulata in bovine from three distincts zones of Khyber Pakhtunkhwa province, Pakistan. J. Anim. Plant. Sci. 2017;27:1836–1841. [Google Scholar]
  28. Gebrekidan H, Hailu A, Kassahun A, Rohoušová I, Maia C, Talmi-Frank D, Warburg A, Baneth G. Theileria infection in domestic ruminants in northern Ethiopia. Vet. Parasitol. 2014;200:31–38. doi: 10.1016/j.vetpar.2013.11.017. [DOI] [PubMed] [Google Scholar]
  29. Ghafar A, Cabezas-Cruz A, Galon C, Obregon D, Gasser RB, Moutailler S, Jabbar A. Bovine ticks harbour a diverse array of microorganisms in Pakistan Parasit. Vectors. 2020;13:332 . doi: 10.1186/s13071-019-3862-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ghauri HN, Ijaz M, Farooqi SH, Ali A, Ghaffar A, Saleem S, Iqbal MK, Aziz MU, Ghani U, Ullah MR, Ahmad HM. A comprehensive review on past, present and future aspects of canine theileriosis. Microb. Pathogen. 2019;126:116–122. doi: 10.1016/j.micpath.2018.10.033. [DOI] [PubMed] [Google Scholar]
  31. Giangaspero A, Marangi M, Papini R, Paoletti B, Wijnveld M, Jongejan F. Theileria sp OT3 and other tick-borne pathogens in sheep and ticks in Italy: Molecular characterization and phylogeny. Ticks Tick Borne Dis. 2015;6:75–83. doi: 10.1016/j.ttbdis.2014.09.007. [DOI] [PubMed] [Google Scholar]
  32. Hassan S, Skilton RA, Pelle R, Odongo D, Bishop RP, Ahmed J, Seitzer U, Bakheit M, Hassan SM, El Hussein AM. Assessment of the prevalence of Theileria lestoquardi in sheep from the Sudan using serological and molecular methods. Prev. Vet. Med. 2019;169 doi: 10.1016/j.prevetmed.2019.104697. [DOI] [PubMed] [Google Scholar]
  33. Hayati MA, Hassan SM, Ahmed SK, Salih DA. Prevalence of ticks (Acari: Ixodidae) and Theileria annulata infection of cattle in Gezira State, Sudan. Parasite. Epidemiol. Control. 2020;10:e00148. doi: 10.1016/j.parepi.2020.e00148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Hussain MH, Saqib M, Raza F, Muhammad G, Asi MN, Mansoor MK, Saleem M, Jabbar A. Seroprevalence of Babesia caballi and Theileria equi in five draught equine populated metropolises of Punjab, Pakistan. Vet. Parasitol. 2014;202:248–256. doi: 10.1016/j.vetpar.2014.01.026. [DOI] [PubMed] [Google Scholar]
  35. Hussein AH, Mohammed N, Mohammed HK. Theileriosis and babesiosis in cattle: haemogram and some biochemical parameters. Int. Soc. Anim. Hyg. 2007;17:143–150. [Google Scholar]
  36. Ishaq M, Ijaz M, Lateef M, Ahmed A, Muzammil I, Javed MU, Raza A, Ghumman NZ. Molecular characterization of Anaplasma capra infecting captive mouflon (Ovis gmelini) and domestic sheep (Ovis aries) of Pakistan. Small Rum. Res. 2022;216:106837. [Google Scholar]
  37. Ito TY, Lhagvasuren B, Tsunekawa A, Shinoda M, Takatsuki S, Buuveibaatar B, Chimeddorj B. Fragmentation of the habitat of wild ungulates by anthropogenic barriers in Mongolia. PLoS One. 2013;8 doi: 10.1371/journal.pone.0056995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kundave VR, Patel AK, Patel PV, Hasnani JJ, Joshi CG. Detection of theileriosis in cattle and buffaloes by polymerase chain reaction. J. Parasit. Dis. 2015;39:508–513. doi: 10.1007/s12639-013-0386-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kurtenbach K, Hanincová K, Tsao JI, Margos G, Fish D, Ogden NH. Fundamental processes in the evolutionary ecology of Lyme borreliosis. Nat. Rev. Microbiol. 2006;4:660–669. doi: 10.1038/nrmicro1475. [DOI] [PubMed] [Google Scholar]
  40. Mans BJ, Pienaar R, Latif AA. A review of Theileria diagnostics and epidemiology. Int. J. Parasitol. Parasites. Wildl. 2015;4:104–118. doi: 10.1016/j.ijppaw.2014.12.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mohammed OB, Jarelnabi AA, Aljumaah RS, Alshaikh MA, Bakhiet AO, Omer SA, Alagaili AN, Hussein MF. Coxiella burnetii, the causative agent of Q-fever in Saudi Arabia: molecular detection from camel and other domestic livestock. Asian Pac. J. Trop. Med. 2014;7:715–719. [Google Scholar]
  42. Nasreen A, Niaz S, Hassan M, Khan A, Ahmed H, Khattak I, Zeb J, Naeem H, Hassan MA. Molecular detection of small ruminant piroplasmosis and first report of Theileria luwenshuni (Apicomplexa: Theileridae) in small ruminants of Pakistan. Exp. Parasitol. 2020;212:107872. doi: 10.1016/j.exppara.2020.107872. [DOI] [PubMed] [Google Scholar]
  43. Naz S, Maqbool A, Ahmed S, Ashraf K, Ahmed N, Saeed K, Latif M, Iqbal J, Ali Z, Shafi K. Prevalence of theileriosis in small ruminants in Lahore-Pakistan. J. Vet. Anim. Sci. 2012;2:16–20. [Google Scholar]
  44. Nazifi S, Razavi SM, Kianiamin P, Rakhshandehroo E. Evaluation of erythrocyte antioxidant mechanisms: antioxidant enzymes, lipid peroxidation, and serum trace elements associated with progressive anemia in ovine malignant theileriosis. Parasitol. Res. 2011;109:275–281. doi: 10.1007/s00436-010-2248-5. [DOI] [PubMed] [Google Scholar]
  45. Niaz S, Ur Rahman Z, Ali I, Cossío-Bayúgar R, Amaro-Estrada I, Alanazi AD, Khattak I, Zeb J, Nasreen N, Khan A. Molecular prevalence, characterization and associated risk factors of Anaplasma spp and Theileria spp in small ruminants in Northern Pakistan. Parasite. 2021;28:3. doi: 10.1051/parasite/2020075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Nijhof AM, Penzhorn BL, Lynen G, Mollel JO, Morkel P, Bekker CPJ, Jongejan F. Babesia bicornis sp nov and Theileria bicornis sp Tick-borne parasites associated with mortality in the black rhinoceros (Diceros bicornis) J. Clin. Microbiol. 2003;41:2249–2254. doi: 10.1128/JCM.41.5.2249-2254.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ola-Fadunsin SD, Sharma RSK, Abdullah DA, Gimba FI, Jesse FFA, Sani RA. Molecular detection, prevalence and risk factors of Theileria orientalis infection among cattle in Peninsular Malaysia. Prev. Vet. Med. 2020;180:105027. doi: 10.1016/j.prevetmed.2020.105027. [DOI] [PubMed] [Google Scholar]
  48. Oludairo OO, Aiyedun JO, Olorunshola ID, Dibal MA, Gungbias AA, Ayeni AMJ, Adeyi AJ. Transboundary diseases and wildlife management: an overview. Bangladesh J. Vet. Med. 2016;14:123–130. [Google Scholar]
  49. Omer OH, El-Malik KH, Mahmoud OM, Haroun EM, Hawas A, Sweeney D, Magzoub M. Haematological profiles in pure bred cattle naturally infected with Theileria annulata in Saudi Arabia. Vet. Parasitol. 2002;107:161–168. doi: 10.1016/s0304-4017(02)00094-8. [DOI] [PubMed] [Google Scholar]
  50. Oryan A, Namazi F, Sharifiyazdi H, Razavi M, Shahriari R. Clinicopathological findings of a natural outbreak of Theileria annulata in cattle: an emerging disease in southern Iran. Parasitol. Res. 2013;112:123–127. doi: 10.1007/s00436-012-3114-4. [DOI] [PubMed] [Google Scholar]
  51. Penzhorn BL. Babesiosis of wild carnivores and ungulates. Vet. Parasitol. 2006;138:11–21. doi: 10.1016/j.vetpar.2006.01.036. [DOI] [PubMed] [Google Scholar]
  52. Posada-Guzm M, Fernanda G, Romero-Zúñiga JJ. Detection of Babesia caballi and Theileria equi in blood from equines from four indigenous communities in Costa Rica. Vet. Med. Int. 2015;2015:236278. doi: 10.1155/2015/236278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Radostits, OM, OM; Gay. Veterinary medicine E-book: A textbook of the diseases of cattle, horses, sheep, pigs and goats. 11th Edn. Elsevier Health Sciences; 2006. [Google Scholar]
  54. Ramos CA, Ramos RA, Araujo FR, Guedes DS, Souza II, Ono TM, Vieira AS, Pimentel DS, Rosas EO, Faustino MA. Comparison of nested-PCR with blood smear examination in detection of Ehrlichia canis and Anaplasma platys in dogs. Braz. J. Vet. Parasitol. 2009;18:58–62. doi: 10.4322/rbpv.018e1011. [DOI] [PubMed] [Google Scholar]
  55. Razavi SM, Nazifi S, Bateni M, Rakhshandehroo E. Alterations of erythrocyte antioxidant mechanisms: antioxidant enzymes, lipid peroxidation and serum trace elements associated with anemia in bovine tropical theileriosis. Vet. Parasitol. 2011;180:209–214. doi: 10.1016/j.vetpar.2011.03.011. [DOI] [PubMed] [Google Scholar]
  56. Razmi GR, Eshrati H, Rashtibaf M. Prevalence of Theileria spp infection in sheep in South Khorasan province, Iran. Vet. Parasitol. 2006;140:239–243. doi: 10.1016/j.vetpar.2006.04.002. [DOI] [PubMed] [Google Scholar]
  57. Riaz M, Tasawar Z. A study on molecular surveillance of Theileria spp infection and its impact on hematological and biochemical changes in naturally infected small ruminants at Multan, Pakistan. Pure Appl. Biol. 2017;6:1427–1435. [Google Scholar]
  58. Saeed S, Jahangir M, Fatima M, Shaikh RS, Khattak RM, Ali M, Iqbal F. PCR based detection of Theileria lestoquardi in apparently healthy sheep and goats from two districts in Khyber Pukhtoon Khwa (Pakistan) Trop. Biomed. 2015;32:225–232. [PubMed] [Google Scholar]
  59. Salih DA, ElHussein AM, Hayat M, Taha KM. Survey of Theileria lestoquardi antibodies among Sudanese sheep. Vet. Parasitol. 2003;111:361–367. doi: 10.1016/s0304-4017(02)00389-8. [DOI] [PubMed] [Google Scholar]
  60. Sandhu GS, Grewal AS, Singh A, Kondal JK, Singh J, Brar RS. Haematological and biochemical studies on experimental Theileria annulata infection in crossbred calves. Vet. Res. Commun. 1998;22:347–354. doi: 10.1023/a:1006129306093. [DOI] [PubMed] [Google Scholar]
  61. Shabbir MZ, Khan JA. Prevalence of theileriosis in sheep in Okara district, Pakistan. Pak. J. Zool. 2010;42:639–643. [Google Scholar]
  62. Shah MH, Ijaz M, Ahmed A, Aziz MU, Ghaffar A, Ghauri HN, Naveed M. Molecular analysis and risk factors associated with Theileria equi infection in domestic donkeys and mules of Punjab, Pakistan. J. Equine Vet. Sci. 2020;92:103164. doi: 10.1016/j.jevs.2020.103164. [DOI] [PubMed] [Google Scholar]
  63. Shiono H, Yagi Y, Kumar A, Yamanaka M, Chikayama Y. Accelerated binding of autoantibody to red blood cells with increasing anaemia in cattle experimentally infected with Theileria sergenti. J. Vet. Med. Ser. B. 2004;51:39–42. doi: 10.1111/j.1439-0450.2003.00724.x. [DOI] [PubMed] [Google Scholar]
  64. Taha KM, Salih DA, Ali AM, Omer RA, El Hussein AM. Naturally occurring infections of cattle with Theileria lestoquardi and sheep with Theileria annulata in the Sudan. Vet. Parasitol. 2013;191:143–145. doi: 10.1016/j.vetpar.2012.08.003. [DOI] [PubMed] [Google Scholar]
  65. Thrusfield, MV. Veterinary epidemiology. 4th Edn. Oxford, Ames, Iowa: Blackwell Science; 2007. [Google Scholar]
  66. Valdez, R. Ovis orientalis. IUCN red List Threat species. 2008. T15739A5076068. [Google Scholar]
  67. Weir W, Karagenç T, Gharbi M, Simuunza M, Aypak S, Aysul N, Darghouth MA, Shiels B, Tait A. Population diversity and multiplicity of infection in Theileria annulata. Int. J. Parasitol. 2011;41:193–203. doi: 10.1016/j.ijpara.2010.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Zaeemi M, Haddadzadeh H, Khazraiinia P, Kazemi B, Bandehpour M. Identification of different Theileria species (Theileria lestoquardi, Theileria ovis, and Theileria annulata) in naturally infected sheep using nested PCR-RFLP. Parasitol. Res. 2011;108:837–843. doi: 10.1007/s00436-010-2119-0. [DOI] [PubMed] [Google Scholar]
  69. Zaman MA, Rafique A, Mehreen U, Mehnaz S, Atif FA, Abbas A, Hussain K, Raza MA, Altaf S, Siddique F, Masudur RM. Epidemiological investigation and development of loop mediated isothermal amplification for the diagnosis of ovine theileriosis. Pak. Vet. J. 2022;42:370–375. [Google Scholar]
  70. Zobba, R, Chisu, V, Pinna Parpaglia, M, ML; Spezzigu, Spezzigu, A. Molecular characterization and phylogenetic analysis of Babesia and Theileria spp. Sardinian ruminants. Vet. Parasitol. Reg. Stud. Reports. 2020;22:100453. doi: 10.1016/j.vprsr.2020.100453. [DOI] [PubMed] [Google Scholar]

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