Detection of Theileria species infecting equine ...

Indian J. Anim. Res.,

AGRICULTURAL RESEARCH COMMUNICATION CENTRE

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Detection of Theileria species infecting equine population in Punjab by 18S rRNA PCR Deepak Sumbria, L.D.Singla*, Amrita Sharma and Paramjit Kaur Department of Veterinary Parasitology, College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, India Received: 12-03-2015 Accepted: 29-09-2015

DOI:10.18805/ijar.7490

ABSTRACT Present study demonstrates the prevalence and risk factors of infection to equines with Theileria species in Punjab, (India). The prevalence by thin blood film examination and Polymerase chain reactions based molecular assay was 4.17 and 33.33%, respectively. PCR targeting 18S rRNA gene of Theileria sp. produced high fidelity 1,100 bp amplification products with 100% sensitivity to blood films. The assessment of various risk factors revealed the prevalence of Theileria sp. to be uniformly distributed in equine population; the age of host animal being apparently the most influential factor for infection (Odds ratio= 1.6154, P= 0.41). The multiple sequence alignment revealed that the primer pair used could perceive the presence of wide range of species of Theileria genus; however the phylogenetic analysis of the custom sequenced amplicons clustered in one node with diverse Theileria equi isolates indicating the evolutionary homology. Key words: Equines, Molecular detection, Risk factors, Theileriosis. INTRODUCTION Equine piroplasmosis (EP) is a tick-borne disease caused by the obligatory intracellular haemoprotozoan parasites (Babesia (Theileria) equi and Babesia caballi) of phylum Apicomplexa, order Piroplasmida (Mehlhorn and Schein, 1998). The distribution of piroplasmosis depends on the presence of ixodid tick vectors and the disease is endemic in tropical and subtropical regions (Bruning, 1996). These haemoprotozoan parasites are mainly transmitted by ticks of the genera Dermacentor, Hyalomma and Rhipicephalus (De Waal, 1992). Equine theileriosis caused by Babesia equi (reclassified as Theileria equi) is endemic in India. Reports on isolated clinical cases (Sharma et al., 1982; Chhabra et al., 2011), and outbreaks of the disease are available in literature (Gautam and Dwivedi, 1976). Overall impact of equines on Indian economy has not been assessed yet but in US, the total impact of equines alone has been estimated to about $102 billion (http:// www.horsecouncil.org/national-economic-impact-ushorse-industry) which are at risk due to haemoprotozoan infections. Traditionally, the microscopic examination of stained blood smears is the tool of choice for the detection and identification of haemoprotozoans in infected horses (De Waal, 1992). However, due to the low sensitivity of microscopy in subclinical or carrier animals, molecular assay of polymerase chain reaction (PCR) targeting small subunit (18S) ribosomal ribonucleic acid (rRNA) sequence, equine merozoites antigen-1 gene, rhoptryassociated protein 1, and 16S-rRNA have been developed *Corresponding author’s e-mail: [email protected].

and employed in the recent past for the detection of low level piroplasmosis (Allsopp et al., 2007). Nuclear ribosomal rRNA genes have been shown to provide appropriate targets to assist in the identification of piroplasms (Katzer et al., 1998; Chae et al., 1999). Although a high degree of 18S rRNA gene sequence conservation has been reported between Babesia and Theileria species, it has been recommended that the complete 18S rRNA gene of these parasites should be determined, particularly when dealing with new organisms, to ensure that genetic variation is not overlooked (Hunfeld et al., 2008). Though several PCRs, based on the 18S rRNA gene, have been developed for the detection of the parasites that cause equine piroplasmosis around the globe (Bashiruddin et al., 1999; Nicolaiewsky et al., 2001; Battsetseg et al., 2002; Rampersad et al., 2003; De Waal and Van Heerden, 2004; Alhassan et al., 2007), the local isolates of equine piroplasmosis in Punjab region of India have not yet been studied. The paucity of enough information in the available literature regarding the infection status of latent equine piroplasmosis in Punjab (India) has prompted authors to undertake this investigation on molecular diagnosis of T. equi particularly targeting 18S rRNA gene. MATERIALS AND METHODS Ethical aspects: The Ethics Committee for Animal Experiments of the Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana granted an approval for the conduction of this research work. For the collection of blood prior consent was taken from the owners of the equines. Complete care and measures were taken to avoid any accidental injury to the equines while collecting the blood samples.

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INDIAN JOURNAL OF ANIMAL RESEARCH

Study areas and sampling frame: A total of 96 samples (60 from male and 36 from female) were randomly collected from central plain zone of Punjab, India for the present study to screen the equines for the presence of Theileria sp. infection. To study the status of molecular prevalence of the infection, the expected prevalence of 50% with confidence limits of 95% and a desired absolute precision of 5% to collect maximum number of samples was considered (Thrusfield, 2005). Blood (~3ml) was collected from jugular vein of each animal into anticoagulant-coated vacutainers for microscopy and nucleic acid extraction. During sampling an epidemiological questionnaire covering the horse breed, age, sex, drugs/deworming/vaccination, management status of the farm, presence of ticks on the animals, keeping of pets by the owner, etc was also maintained to calculate the risk associated with EP transmission. Blood film: Thin blood smears were prepared immediately after the blood sample collection and were stained with Giemsa stain (Coles, 1986).The smears were observed for the presence of EP in erythrocytes. Morphological characteristics of EP was conducted according to key described by Soulsby (2005). DNA Extraction and PCR assay: Genomic DNA was extracted from the blood samples by using the protocol of DNeasy Blood & Tissue Kit (QIAGEN). Primers: 989 AGTTTCTGACCTATCAG and 990 TTGCCTTAAACTTCCTTG specifically targeting 18S rRNA gene of Theileria sp. (Ananyutthawongese et al., 1999) were procured from Bangalore Genei, India Pvt. Ltd. PCR reaction mixture (25 l) was constituted by 12.5l of KAPA2G® Fast HotStart Ready Mix (2X containing KAPA2G® Fast HotStart DNA polymerase, KAPA2G® Fast HotStart PCR buffer, 0.2 mM dNTP each, 1.5mM MgCl2), 1.5 l of 10 pmol 989/990 primers and 5l of DNA template suspended in 4.5 l of nuclease-free water. The reaction was set in automated thermocycler with the following program: initial denaturation at 95°C (5 min), 33 cycles of denaturation at 94°C (45 sec), annealing at 57°C (1 min), extension at 72° C (1 min) and final extension at 72°C (10 min). The 1,100-bp amplified PCR products (Figure 2) were separated by electrophoresis on 1.5% agarose gel and visualized under UV Transilluminator. Nucleotide analysis: Amplicons from PCR product targeting 18S rRNA gene specifically were custom sequenced from Xcelris Genomics, Ahmedabad, India. The nucleotide sequences were subjected to BLASTn analysis (Altschul et al., 1990) for determining the similarity with the sequences present in the nucleotide database. The homologous sequences of 18S rRNA belonging to different strains of Theileria sp. were retrieved from database using highly similar BLASTn. The Tajima test statistics (Tajima, 1989) with phylogenetic analyses were conducted in MEGA4

(Tamura et al., 2007). All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). The abbreviations used were as follows: m = number of sites, S = number of segregating sites, ps = S/m,  = ps/a1, and  = nucleotide diversity. D is the Tajima test statistic. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length= 15.63738773 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches (Felsenstein, 1985). The evolutionary distances were computed using the Poisson correction method (Zuckerkandi and Pauling, 1965) and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 980 positions in the final dataset. Statistical analysis: The prevalence of Theileria sp. according to the blood film and PCR in each province was statistically analyzed employing Pearson’s chi-square test P0.05 as the values representing a significant difference. The statistical significance in this study was defined as P0.05. RESULTS AND DISCUSSION Out of 96 samples screened, the molecular detection method [PCR] (Fig. 2) revealed 33.33% (32/96) infection among equine population of Punjab supporting the fact that in latent infection, microscopic examinations does not produce promising results as it could detect only 4.17% cases (4/96) as positive (Table-1, Fig. 1). Herbert and Lumsden (1976) pointed out the unfeasibility of microscopic detection until 2.5 X 106 parasites per ml of blood are present. The low level parasitaemia in subclinical infection makes the detection by Giemsa-stained blood smears cumbersome (Bose et al., 1995). In PCR positive equines, the parasites

FIG.1: Different morphological forms (comma-shaped, Maltese cross) of T. equi

Vol. Issue , () may be in their early stages of multiplication hence may not be visualized in peripheral blood stream; however, they might be present in spleen cells (Ribeiro et al., 2013). The assessment of Odds ratio revealed the prevalence of Theileria sp. to be uniformly distributed among equine population with respect to various risk factors (Table-1). The age of the host animal was apparently the most influential factor for the infection with Odds ratio of 1.6154 (P= 0.41). This unapparent difference in the two age groups of equines is because of the fact that like other parts of world (Karatepe et al., 2009; Mujica et al., 2011; Steinman et al., 2012), utmost care of equines is taken in Punjab state as they are primarily utilized for breeding and recreational purposes (Table-1). A contrary scenario has been reported by Rüegg et al. (2007) and Kouam et al. (2010). Among male and female, the infection levels differed non significantly as also reported by Kouam et al. (2010). The lack of association of infection risk with gender is in agreement with other studies (Botteon et al., 2002; Kouam et al., 2010). In present study only a small number of donkeys/ mules were examined as compared to horses; so, the degree of alliance between Theileria sp. infection and animal species can not be generalized until a larger number of donkeys/ mules is investigated in further studies. The non-momentous increase in infection of the equids reared in unorganized sector (36%, n = 25) can be explained by the fact that these animals have closer contact with other domestic animals and inevitably when compared to those reared in organized system (32.39%, n= 71). Moreover, the chances of direct contact with tick vectors in unorganized farm is more and it may be attributed to unhygienic open grazing, non grooming practices and use of equines for transportation purposes (Kouam et al., 2010; Moretti et al., 2010; Abutarbush et al., 2012; Steinman et al., 2012; Peckle et al., 2013).

Equines kept with pets showed relatively higher prevalence of Theileria equi in concordance with the findings of Peckle et al. (2013) and Garcia-Bocanegra et al. (2013). In Punjab, equines are generally immunized against equine influenza virus and tetanus, but not against EP (as no vaccine is available). Other diseases create immuno-suppressive affect in unvaccinated animals, thus may enhance the prospect of infection (Garcia-Bocanegra et al., 2013). During blood sampling only one animal showed the presence of ticks so the actual rate of infection related to tick vector cannot be assessed. Phylogenetic analysis using the neighbor-joining, maximum-likelihood revealed the closest homology with EU642508.1 of South Africa (Fig. 3). The significantly high positive value of Tajima’s D (m=36, S=977, ps=1.00  =0.241  =0.71, D=7.33) indicated extensive immigration,

FIG. 2: Agarose gel electrophoresis (1.5%) showing amplified DNA of 1,100 bp for Theileria sp. Lane M: 100 bp plus DNA ladder; Lane P: positive control; Lane N: negative control; Lane 1–5: Tested field samples.

TABLE 1: Distribution of variables identified to determine the risk factors associated with Theileria sp. prevalence in equids in Punjab, India

Factor Age Sex Species Tick vector Farm management Deworming/ vaccination Pets on farm Use of equines

Total Samples

PCR positive

Percentage

95% CI

Odds ratio (P value)

Less than 2 years More than 2 years Male

14 82 60

6 26 19

42.86 31.71 31.67

20.51-65.21 23.02-40.39 21.52-41.82

1.6154 (0.41)

Female Horse Donkey/Mule Present Absent Organized Unorganized Done Not done Present Absent Recreation use Commercial use

36 90 6 1 95 71 25 38 58 14 82 72 24

13 29 3 0 32 23 9 11 21 5 27 24 8

36.11 32.22 50 0 33.68 32.39 36 28.95 36.21 35.71 32.93 33.33 33.33

22.58-49.64 23.90-40.55 15.50-84.50 0-0 25.49-41.90 23.01-41.78 19.78-52.22 16.51-41.38 25.54-46.87 14.07-57.36 24.16-41.70 23.94-42.72 17.07-49.60

1.2197 (0.6549) 1.0517 (0.9458) 1.5354 (0.7946) 1.1739 (0.7424) 1.3931 (0.4614) 1.1317 (0.8380) 1.00 (1.00)

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FIG. 3: Phylogenetic tree of Theileria sp. This tree was constructed by Neighbor-Joining method with MEGA 4 program. Numbers shown at branch nodes indicate bootstrap values. This phylogenetic analysis showed high genetic diversity of 18S rRNA gene among different strains of T. equi. Consensus sequences obtained in this study are indicated as ‘‘PresentStudy”.

balancing selection (favouring increase in diversity) and/or population subdivision (a few alleles at high frequency). The sequence retrieved in the present study showed the conserved primer binding sites for diverse species of genus Theileria upon multiple sequence alignment in ClustalW, including Theileria sp. (AB000270.1) T. sinensis (EU274472.1), T. buffeli (AF236094.1), T. orientalis (AB520955.1), T. sergenti (GU143087.1), T. cervi (HQ184406.1), T. equi (JX177673.1) and B. equi (Z15105.1) (Fig. 4a, b). In conclusion, although the multiple sequence alignment revealed that the primer pair used could perceive the presence of wide range of species of Theileria genus; the phylogenetic

analysis of the custom sequenced amplicons clustered in one node with diverse T. equi isolates indicated the evolutionary homology. ACKNOWLEDGEMENTS Thanks are due to the Director of Research, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana for the facilities provided. The funds for conducting this work were obtained by UGC project entitled “Development of control strategies based on molecular epidemiology and drug efficacy for equine piroplasmosis in Punjab” (C-VPS-UGC24).

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FIG. 4: (a) Sequence from various Theileria sp. isolates aligned for forward primer used in the present study. (b) Sequence from various Theileria sp. isolates aligned for reverse primer used in the present study.

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