Molecular Epidemiology of Theileria lestoquardi in Small Ruminants in District Charsadda, Khyber Pakhtunkhwa, Pakistan

Shahab-ud-Din1, Mashal Khan1, Shad Muhammad1, Said Sajjad Ali Shah2*, Hameed Ullah3, Arab Hussain1, Amjad Ullah1, Ahmad Yar1, Sohaib ul Hassan4 and Naveed Ullah4

1Government Postgraduate College, Charsadda

2Parasitology Laboratory, Veterinary Research Institute, Peshawar

3Foot and Mouth Disease Research Centre, Veterinary Research Institute, Peshawar

4College of Veterinary Sciences, The University of Agriculture, Peshawar

ABSTRACT

Small ruminant theileriosis is an economically significant disease in tropical and subtropical countries caused by Theileria lestoquardi, Theileria ovis, and Theileria luwenshuni, of which T. lestoquardi is very pathogenic. The goal of the current study was to investigate the molecular epidemiology of T. lestoquardi and T. ovis in small ruminants in the Charsadda district of Khyber Pakhtunkhwa. A total of 150 whole blood samples (sheep, n=104, and goats, n=46) were collected for this study in various locations throughout the district of Charsadda from June to August 2021. Microscopically, Theileria piroplasms were observed in 10 samples (6.6%) while T. lestoquardi was identified through PCR in 37 samples (24.6%) and none of the samples was positive for T. ovis. T. lestoquardi target gene (i.e., 18srRNA) was sequenced and the results showed close homology with Iranian isolates of T. lestoquardi, indicating the possibility of cross-border transmission of the disease. Tick infestation and age were significant (p<0.05) among the several risk factors examined, however, species and sex were deemed to be non-significant (p>0.05). Analysis of clinical manifestation revealed that fever and lymphadenopathy were moderately related to small ruminant theileriosis, whereas anorexia was weakly associated with the disease. According to the current study results, T. lestoquardi is responsible for causing theileriosis in small ruminants in Charsadda, Khyber Pakhtunkhwa whereas PCR is a sensitive diagnostic test as compared to microscopy.

Article Information

Received 09 November 2023

Revised 25 January 2024

Accepted 10 February 2024

Available online 05 June 2024

(early access)

Published 02 July 2025

Authors’ Contribution

SSAS, AH, AU, AY designed the study. SUD, MK, and SM collected the field samples. HU, SUH, and NU, assisted in processing the samples in the lab. SSAS conducted the statistical analysis and phylogenetic analysis. All authors contributed to the writing of the manuscript.

Key words

Small ruminants, Theileriosis, Clinical signs, Risk factors, PCR, Phylogenetic analysis

DOI: https://dx.doi.org/10.17582/journal.pjz/20231109090656

* Corresponding author: [email protected]

0030-9923/2025/0004-1907 $ 9.00/00

Copyright 2025 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Pakistan is home to a huge population of small ruminants (78.2 and 30.9 million goats and sheep, respectively), and currently, 3rd largest country for goat production and whereas 12th largest sheep producing country (Niaz et al., 2021). There are about 34 goat and 28 sheep breeds in Pakistan (Razzaq et al., 2015), and most of these breeds are meat breeds whereas only a few i.e., Beetal, Damani, Nachi, and Dera Din Pannah are considered milk breeds (Khan and Ashfaq, 2010). The livestock industry of Pakistan as a whole especially small ruminants is under continued threat because of prevailing diseases (bacterial, viral, and/or parasitic) and inadequate disease control measures (Shah et al., 2017b). Parasitic diseases especially hemoparasitic diseases, implicit heavy economic losses in small ruminants in the form of morbidity and mortality (Demessie and Derso, 2015). Important hemoparasitic diseases of livestock with special reference to small ruminants are; theileriosis, anaplasmosis, and babesiosis (Razzaq et al., 2015). Theileriosis in small ruminants is caused by different species but Theileria lestoquardi, causing malignant ovine theileriosis, is extremely pathogenic (Hakimi et al., 2019) and is equivalent to T. annulata infection in cattle whereas T. ovis is slightly pathogenic in small ruminants (Rjeibi et al., 2014) and all other species (T. separate, T. uilenbergi, T. recondite) are almost avirulent (Aydin et al., 2013).

Theileriosis is a tick-borne disease, transmitted mainly by tick species of Hyalomma and Haemphysalis in small ruminants (Yin et al., 2008). The epidemiology of theileriosis is dependent on the climatic conditions of the country and the presence of a suitable tick vector (Morrison, 2015). The disease is prevalent in tropical and sub-tropical regions of the world including Pakistan, India, Bangladesh, and Western China (Riaz and Tasawar, 2017b). Theileriosis in small ruminants, caused by T. lestoquardi, is characterized by high fever, lymphadenopathy, pale mucous membrane, anorexia, and emaciation (Razzaq et al., 2015) whereas T. ovis infection is characterized by reduced production, continuous weight loss, fever and eventually death of the infected animals (Shahzad et al., 2013).

In the earlier work, the greatest consideration was given to bovine piroplasmosis while tick-borne diseases of small ruminants are not much focused. Due to the economic importance of small ruminants in several countries, interest has recently risen in small ruminant’s piroplasmosis, especially infections with Theileria species (Ahmed et al., 2018). The present study aimed to determine the molecular epidemiology of T. lestoquardi in small ruminants of district Charsadda, and to sequence the target gene for phylogenetic analysis. Moreover, the study also established the correlation between various risk factors and clinical manifestations with the onset of theileriosis in small ruminants.

MATERIALS AND METHODS

Study area

The present study was conducted in district Charsadda which lies between 34.14° North latitude and 71.74° east longitude and is located about 18 miles northeast of the city of Peshawar. According to the Koppen climatic classification, Charsadda has a humid subtropical, no dry climate and is classified as Cfa, having 47-50% humidity (Khan, 2019).

Sampling

The sample size for this study was determined with the help of the formula given by Thrusfield (2018), which is based on prevalence, absolute precision, and confidence interval which were set as prevalence (10%), absolute precision (5%), and 95% confidence interval. The sample size according to this formula was calculated as 150, which were collected from the suspected small ruminants in the study area. A total of 150 whole blood samples (Sheep, n=104, goats, n=46) were collected in EDTA added vacutainer from different villages of district Charsadda for molecular detection of theileriosis whereas thin blood smears were prepared for microscopy. During collection, the history of the animals i.e., species, age, gender, presence of ticks, signs, and symptoms related to the disease was recorded on a predesigned questionnaire. Animals were grouped into three categories based on age i.e., less than 1 year, 1-2 years, and more than 2 years.

Microscopic examination

For microscopic examination, thin blood smears were prepared at the time of collection and were air-dried. The slides were rinsed in 100% methanol, air-dried, and stained in 10% Giemsa stain for 25 min. Slides were rinsed by running tap water to remove extra stains and air-dried (Altay et al., 2008). Dried smears were examined through a 100× objective (oil immersion lens) for the presence of theileria piroplasm in the erythrocytes, and a total of about 30-40 microscopic fields were observed (Hegab et al., 2016).

Extraction of DNA and PCR

DNA was extracted from whole blood samples according to the kit instructions (Innu PREP DNA Mini Kit, Analytic Jena Life Science). The DNA extracted was quantified through Nanodrop to determine the concentration and purity of DNA (A260/280). The concentration of DNA was in the range 40-60ng/µl and purity (A260/280) was 1.6-1.8. The PCR reaction mixture was prepared by adding 1µl each primer (10pmol), 12.5µl Sso fast Evagreen Supermix, 2 µl DNA template, and 8.5µl PCR water. The reaction was performed in Bio-Rad thermocycler (CFX-96) and conditions were first optimized as; initial denaturation for 3 min at 94°C, 34 cycles of cyclic denaturation at 94°C for 30 sec, annealing at 53°C for 30 sec, cyclic extension at 72°C for 30 sec, followed by a final extension at 72°C for 5 min. PCR primers used in this study are given in Table I. The PCR product (8µl) was mixed with DNA loading dye (2µl) and loaded in the wells of 1.5% agarose gel and bands were compared with DNA ladder (100bp).

DNA sequencing and phylogenetic analysis

PCR product (30µl) of two positive samples were sent to the BGI sequencing lab for DNA sequencing through the Sanger sequencing method (Sanger and Coulson, 1978). The acquired sequences were edited for redundant sequences before being submitted to Genbank (NCBI), where accession numbers (OP712457, OP712458) were assigned. The isolates were confirmed using the basic local alignment search tool (BLAST), and sequences with high homology were downloaded from the National Center for Biotechnology Information (NCBI) database for evolutionary analyses. DNA sequences with homology ranging from 96-100% that had previously been submitted to the NCBI database were downloaded with accession

 

Table I. PCR primers used in this study for the detection of Theileira lestoquardi, and Theileria ovis in small ruminants.

Species	Target gene	Sequence (5`→3`)	Amplicon size (bp)	Reference
T. lestoquardi	18s rRNA	F-GTGCCGCAAGTGAGTCA	785	(Li et al., 2014)
		R- GGACTGATGAGACGATGA
T. ovis	18s rRNA	F-TCGAGACCTTCGGGT	520
		R-TCCGGACATTGTAAAACAAA

 

numbers; EF092916 (Iran), LC430944 (Iran), LC430944 (Iran), LC430945 (Iran), KY965145 (Sudan), MF765610 (Sudan), LC430946 (Iran), MZ541894 (Pakistan), LC430948 (Iran), AJ006448 (Iran), AF004775 (Kenya), ON408247 (India), KY965146 (Sudan), MK941606 (Pakistan), EF092917 (Iran), AJ006447 (Iran), LC430947 (Iran), MZ150568 (India), MZ074324 (India). In contrast, T. annulata (AF214883) was selected as an outgroup. All sequences were aligned through Bioedit and the phylogenetic tree (Neighbor-joining method) was prepared by using MEGA X (Kumar et al., 2018; Tamura et al., 2011).

Data analysis

Data regarding the molecular prevalence of theileriosis in small ruminants was compiled in Microsoft Excel and analyzed through univariate logistic regression to establish the significance of different risk factors and contingency coefficients for clinical signs and symptoms of the disease.

RESULTS

Out of 150 blood samples examined, Theileria piroplasms (Fig. 1) were observed in 10 samples (6.6%) through microscopy while 37 samples (24.6%) were positive for T. lestoquardi on PCR whereas none of the samples tested positive for T. ovis. Samples positive on PCR gave amplicon sizes 785 bp for T. lestoquardi.

Sequencing and phylogenetic analysis

Using Bioedit software, the sequences acquired were edited to remove any redundant sequences before being subjected to BLASTn analysis by NCBI. T. lestoquardi SSRU gene amplification was validated using sequence analysis, and to investigate the evolutionary relationships, sequences were retrieved from the NCBI database. A total of 18 sequences of T. lestoquardi with high homology from neighboring countries were retrieved, and one sequence of T. annulata was used as an outgroup. The current study isolates shared a significant degree of similarity with Iranian isolates (LC430945, LC430943, and LC430944), which led to their grouping into the same clade (Fig. 2).

 

 

Risk factors associated with theileriosis in small ruminants

The prevalence of theileriosis in small ruminants was determined with respect to different risk factors i.e., species, age, sex, and presence of ticks. Species-wise prevalence of theileriosis was 26.9% in sheep and 19.5% in goats whereas in different age groups, the higher prevalence was observed in the 1-2 years age group (38.3%), followed by less than 1 year age group (20.4%) and more than 2 years age group (16.9%). Similarly, 26.5% prevalence was recorded in females (ewes, and goats) and 20% in male animals (ram and bucks), and 40.7% of animals infested with ticks were found positive for theileria. The difference in the prevalence of theileriosis was significant (P<0.05) for age, and tick infestation whereas a non-significant (p>0.05) difference was observed for species and sex (Table II).

 

Table II. Relationship between T. lestoquardi prevalence in sheep and goats and the studied variables describing animal characteristics based on PCR.

Variables	Categories	Positive (%)	OR	CI 95% (Lower- Upper)	P value
Species	Sheep (n=104)	26.9	0.301	0.15-0.77	0.34
	Goat (n=46)	19.5
Age	<1 year (n=44)	20.4	1.04	0.58-1.8	0.03
	1-2 years (n=47)	38.3
	>2 years (n=59)	16.9
Sex	Female (n=115)	26.08	0.69	0.2-2.01	0.46
	Male (n=35)	20
Ticks infestation	Yes (n=76)	40.7	1.7	1.1-2.35	0.00
	No (n=74)	8.1

 

P value ≤ 0.05 indicate significance, OR, Odd ratio.

 

Table III. Association/relationship of different clinical signs and symptoms with small ruminant theileriosis in small ruminants in the district Charsadda.

Sign symptoms	Positive (%)	Contingency co-efficient value	Relationship/ Association
Temperature	34/37(91.89)	0.49	Moderate
Lymphadenopathy	35/37(94.6)	0.499	Moderate
Anorexia	37/37(100)	0.389	Weak
Abortion	3/37(8.1)	0.187	Very weak

 

Clinical signs observed

A brief clinical history of animals was recorded during the collection of samples and the association of these clinical signs/symptoms with theileriosis in small ruminants was determined through contingency coefficient values. Out of a total of 37 positive samples, 91.89% of animals were having temperature whereas only 8.1% were not suffering from temperature, and the contingency coefficient value was 0.49 which is an indication of a moderate relationship. Similarly, contingency coefficient values were 0.499 for lymphadenopathy, 0.389 for anorexia, and 0.187 for abortion, which indicated a moderate, weak, and very weak relationship with theileriosis, respectively (Table III).

DISCUSSION

Small ruminant theileriosis is caused by many species of theileria but T. lestoquardi is the most pathogenic species and is almost similar to T. annulata infection in large ruminants (Fatima et al., 2015). In the current investigation, 150 blood samples were examined, and theileria piroplasms were found in 6.6% samples using microscopy. This statement is congruent with the findings of Shah et al. (2017a), and Naz et al. (2012) who also reported the same prevalence of theileriosis in small ruminants through microscopy in districts Peshawar and Lahore, respectively. Durrani et al. (2011) and some other researchers have reported a higher prevalence of theileriosis in small ruminants through microscopy and there may be various reasons for this gap, one may be that microscopy is not a sensitive technique and artifacts, stage of disease, etc may make the diagnosis of theileriosis difficult through microscopy.

T. lestoquardi was confirmed in 24.6% of the samples by PCR, but no samples tested positive for T. ovis. Several researchers in different parts of the country did similar investigations and so far have reported the occurrence of T. lestoquardi, and T. ovis in different parts of the country (Fatima et al., 2015; Niaz et al., 2021; Riaz and Tasawar, 2017b). A similar occurrence of T. lestoquardi was reported by Durrani et al. (2011), whereas a higher prevalence was reported by Riaz and Tasawar (2017a, b) and a lower prevalence by Fatima et al. (2015), Niaz et al. (2021) and Saeed et al. (2015). The topographical makeup of the study areas and the inclusion criteria of sampled animals differ in each study, which could explain the variation in T. lestoquardi occurrence. Microscopy and PCR were used for the detection of small ruminant theileriosis in the current study however it is challenging to detect piroplasms in carrier animals or in cases of mixed infections when using traditional diagnostic procedures, such as microscopy (Altay et al., 2012).

T. lestoquardi 18srRNA gene sequences from the current study were highly similar to those from Iran (LC430945, LC430943, and LC430944) and were placed in the same clade. Balochistan province of Pakistan shares a long border with the Iranian region, and there is extensive animal migration across the border, especially for small ruminants. Many sheep and goats are raised by nomads in Pakistan, where animals roam consistently in search of food and occasionally move across the border areas of Pakistan, Iran, and Afghanistan. This could explain why there is a significant degree of similarity between this study’s isolates and Iranian isolates.

T. lestoquardi was more common in sheep than in goats, which is consistent with the findings of other researchers (Altay et al., 2012; Niaz et al., 2021; Riaz and Tasawar, 2017b). A possible explanation for this could be the difference in the nature of the skin of sheep and goats. Ticks find it more challenging to attach to goat skin than to sheep skin because it is smoother, whereas ticks may readily twine themselves in sheep’s wool and transmit disease (Niaz et al., 2021; Shabana et al., 2018). The tendency of goats to graze in rugged, difficult to access places, where there are fewer opportunities for contact with ticks that feed on other animals during their life cycle, may also contribute to the lower prevalence in goats than in sheep (Alessandra and Santo, 2012). Age-wise occurrence of theileriosis in small ruminants was almost similar to the findings of other researchers (Durrani et al., 2012; Hegab et al., 2016; Iqbal et al., 2013). Young animals are more susceptible to theileriosis than older animals, which may be explained by their lack of disease immunity; nonetheless, once infected, they develop lifelong immunity (Ahmed et al., 2008). T. lestoquardi was more common in females than in males, which is consistent with the results of several researchers throughout the world (Khan et al., 2017; Magzoub et al., 2021; Naz et al., 2012; Osman et al., 2017; Taha et al., 2015). Different hormonal changes and stress involved in pregnancy and lactation may predispose the female to the onset of theileriosis. Since T. lestoquardi is a vector-borne pathogen, ticks infestation is thought to be a significant risk factor for the development of T. lestoquardi infection in small ruminants (Niaz et al., 2021).

High temperature and swollen lymph nodes were the two main clinical manifestations seen in the study animals, along with other more general symptoms such as anorexia, diarrhea, lacrimation, and weakness. Different clinical signs of theileriosis in cattle are believed to be caused by the overproduction of proinflammatory cytokines, including TNF-α, IL-1, and IL-6. The intracellular stage of theileria directly contributes to the severe clinical alterations observed in animals by promoting the growth of naive T lymphocytes, and these aberrantly activated T cells generate substantial levels of IFN-γ (Agina et al., 2020). The draining lymph node enlarges in size as a result of the multiplication of both infected cells and T lymphocytes (Glass et al., 2005). The association of abortion with small ruminants theileriosis was explained by Esmaeilnejad et al. (2018), as T. lestoquardi can, however, be transmitted transplacentally, which increases the chance of abortion and neonatal death. They further stated that Theileria may obstruct or even cross the placental barrier, which could have an impact on fetal development and growth as well as cause abortion.

Conclusions

The present study confirmed that T. lestoquardi is mainly responsible for causing small ruminant theileriosis in the study area. Among the diagnostic techniques tested, PCR is a sensitive and specific diagnostic test for theileriosis in small ruminants whereas microscopy is only useful in the acute stage of the disease when parasitemia is high. The fact that Iran, Afghanistan, and Pakistan share borders and most people travel across the border freely as nomads, can help to explain that the same strain of T. lestoquardi may circulate along the areas adjacent to the border.

Declarations

Acknowledgments

We are very appreciative of the local veterinarians assistance in sample collection and grateful to the Veterinary Research Institute, Peshawar, for giving us the chance to conduct lab work.

Funding

This study was partially supported by Pakistan Science Foundation (PSF) via project No. PSF/NSLP/KP-VRI (759) for provision of chemicals/reagents, and glassware’s/plasticwares.

Ethical statement

Blood samples were collected from target animal species with the help of trained veterinarian to ensure humane method for blood collection. Before samples collection, verbal consent was obtained from the animal owners, and they were informed about the purpose of blood collection.

Statement of conflict of interest

The authors declared no conflict of interest.

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