Application of the Reverse Line Blot Assay for the Molecular Detection of Theileria and Babesia sp. in Sheep and Goat Blood Samples from Pakistan

Abstract

The present study was designed to detect the presence of tick-borne parasites (Theileria and Babesia spp.) in 196 blood samples collected from apparently healthy sheep and goats from two provinces, Punjab and Khyber Pukhtoon Khwa, in Pakistan. Reverse line blot (RLB) assay was applied for the parasitic detection by the amplification of hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene. A membrane with covalently linked generic and species specific oligonucleotide probes was used for the hybridization of amplified PCR products. Parasites were detected in 16% of the ruminant blood samples under study. Two Theileria species, T. lestoquardi and T. ovis, were identified in samples. 25, of the total 32, infected animals were from Khyber Pukhtoon Khwa. Sheep were more prone to tick borne haemoprotozans as 81% infected samples were sheep as compared to 19% goats (P > 0.001). Risk factor analysis revealed that male (P = 0.03), animals infested by ticks (P = 0.03) and herd composed of sheep only (P = 0.001) were more infected by blood parasites.

Headnote

ABSTRACT

Background: The present study was designed to detect the presence of tick-borne parasites (Theileria and Babesia spp.) in 196 blood samples collected from apparently healthy sheep and goats from two provinces, Punjab and Khyber Pukhtoon Khwa, in Pakistan.

Methods: Reverse line blot (RLB) assay was applied for the parasitic detection by the amplification of hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene. A membrane with covalently linked generic and species specific oligonucleotide probes was used for the hybridization of amplified PCR products.

Results: Parasites were detected in 16% of the ruminant blood samples under study. Two Theileria species, T. lestoquardi and T. ovis, were identified in samples. 25, of the total 32, infected animals were from Khyber Pukhtoon Khwa.

Conclusion: Sheep were more prone to tick borne haemoprotozans as 81% infected samples were sheep as compared to 19% goats (P > 0.001). Risk factor analysis revealed that male (P = 0.03), animals infested by ticks (P = 0.03) and herd composed of sheep only (P = 0.001) were more infected by blood parasites.

Keywords: Sheep, Goat, Theileria; Babesia, 18S rRNA gene, RLB

Introduction

Livestock has the major contribution in the economy of Khyber Pukhtoon Khwa and Punjab provinces in Pakis- tan (1). Punjab province contributes 67% buf- falo, 46% cattle, 37% goat and 26% sheep in livestock population of Pakistan. Livestock industry is economically more important in KPK as compared to Punjab and Sindh as these provinces have suitable land for crops and well established industrial sectors. Ac- cording to an estimate in KPK, the livestock contributed 62.8 billion rupees to the national exchequer (2). Licks are the major vectors of piroplasms (Theileria and Babesia spp.) and in ruminants tick borne diseases are causing the major economic losses to livestock industry in Asia and more specifically in Pakistan (3) by producing anemia resulting in morbidity and mortality in animals resulting in animal losses, decreased meat, milk and offspring compared to uninfected animal. Several species of piroplasms are transmitted to sheep and goats by Ixodid ticks (4-6).

Classically, piroplasmosis in sheep and goats can be diagnosed by microscopic screening of blood films stained with Giemsa- along with the clinical symptoms animals produce in acute cases (7). Occasionally, recovered ani- mals sustain microscopically undetectable sub clinical infections and via potential vectors causing transmission of the disease (8, 9). For the determination of sub clinical infections, serological methods are frequently employed but they lack sensitivity and specificity (10). Lherefore, more sensitive and specific DNA amplification methods are used as standard tools for piroplasmosis diagnosis. In recent years, species-specific polymerase chain reac- tion (PCR) and reverse line blot (RLB) hybrid- ization, which is PCR-based molecular genet- ics technique have been extensively used for the piroplasm infection in ruminants (11-16).

Lhe present study was designed to use RLB assay for improved and simultaneous detec- tion and identification of multiple Theileria and Babesia sp. in randomly selected sheep and goat blood samples from various sampling sites in Punjab and KPK in order to develop baselines about the presence of piroplasm in local sheep and goat population and to estab- lish a correlation, if any, between various risk factors, known to be associated with the spread of piroplasmosis, with the presence of piroplasms in small ruminants.

Materials and Methods

Data and blood sample collection

Blood samples were collected from 196 clinically healthy small ruminants (82 sheep and 114 goats) from two provinces, Punjab (in and around Multan district) and from Kohat district in Khyber Pukhtoon Khwa, in Paki- stan. From 32 randomly selected herds, 10% of apparently healthy animals were blood sampled from jugular vein and preserved by adding 400 (jl of 0.5 M EDLA in 5 ml Eppendorf tubes. In order to calculate the risk factors involve in the spread of piroplasmosis, data describing the characters of animal (spe- cie, gender, age, prior treatment for piroplasmosis, presence or absence of ticks) and herd (location, size, composition, associa- tion of dogs with the herds and presence or absence of ticks on dogs) was collected on the spot by the investigators during sample collec- tion. Ethic committee of Bahauddin Zakariya University Multan (Pakistan) approved all the experimental techniques and animal handling protocols.

For risk factor calculations, animals were cate- gorized on the basis of their age as young (less than 1 -year) and adults (more than 1 -year-old). On the basis of herd size, they were divided into two categories: small herds composed of 1-15 and large herds with more than 15 ani- mals. Also, on the basis of herd composition, herds were divided into three categories: herds consist of goat or sheep or herds containing both sheep and goats.

DNA isolation

Protocol described by Shahnawaz et al. (3) was followed for the DNA isolation by inor- ganic method. DNA quality was assessed by submerged gel electrophoresis and through optical density counts at 260/280 nm.

18S rRNA gene amplification and RLB hybridisa tion

Approximately 360 and 430 bp fragments of the hypervariable V4 region of the 18S rRNA gene of Theileria and Babesia was amplified by using the for- ward (5'-GACACAGGGAGGTAGTGACAAG-3') and reverse (Biotin-5'-GACACAGGGAGGTAGTGACAAG- 3) primers previously described by Georges et al. (17). Hie PCR reaction mixture thermoprofile used in this study was modified from Altay et al. (14). In order to identify ovine piroplasms, the biotinylated Theileria I Babesia spp. specific PCR products were hybridized with thirteen oligo- nucleotide probes. The probes and their se- quences are presented in Table 1. The primers and oligonucleotide probes containing a N- (trifluoroacetamidohexyl-cyanoethyl, N, N- diisopropyl phosphoramidite [TFA])-C6 aminolinker were synthesised by The Midland Certified Reagent Co., Inc., USA. Followin Altay et al. (14), RLB membrane was prepared, hybridized, and stripped. A volume of 25 (jl of PCR product was diluted with 2 x SSPE-0.1% SDS to an end volume of 160 (jl. 20mM EDLA was used to rinse the membrane and stored at +4 ?C for reuse in fresh EDLA solu- tion.

Positive control DNAs isolated sheep infected with T. oris, B. oris, Theileria sp. OL3, Theileria sp. MK (GenBank accession numbers. EF092452, EF092454, EF092455, EF092456 respectively), were used in the assay. Dr. Ana Hurtada (Department of Animal Health, Instituto Vasco de Investigaci?n Desarrollo Agrario Berreaga, Bizkaia, Spain) had kindly donated us Theileria sp.OLl and B. motasi posi- tive blood samples. T. lestoquardi positive blood sample was contributed by Dr. Jabbar Ahmed (Department of Immunology and Cell biology, Research Center, Borstel, Germany).

Lhe DNA provided by LBD-RLB Kit (Isogen Life Science, Maarssen, Lhe Netherland) for Theileria and Babesia species was also used as a positive control in RLB hybridization in addi- tion to the above mentioned sources. Ovine genomic DNA extracted from uninfected sheep blood and distilled water was used as negative controls.

Statistical analysis

Statistical package, Mini Lab (Version 16), was used for data analysis. Fisher's exact test (for 2 x 2 tables) was used to study the association between the presence of piroplasms and vari- ous parameters describing the characters of animal and herds.

Results

Totally, 32 out of 196 (16%) examined small ruminant blood samples, collected from Southern Punjab and Khyber Pukhtoon Khwa were found piroplasm positive by RLB assay (Fig. 1; Lable 2).

Prevalence of Theileria sp. was significantly dif- ferent among the two provinces (P = 0.000) and incidence of piroplasmosis was higher in KPK as compared to southern Punjab (Lable 2) as only 7 out of 128 (6%) blood samples from Southern Punjab were Theileria sp. posi- tive as compared to 25 out of 68 (35%) from KPK. Among 32 Theileria sp. blood samples, 14 samples were found positive for T. lestoquardi, 12 had T. oris while 7 blood sam- ples, all from Kohat district in KPK, were coinfected with both piroplasm species. Babesia genus and all its three explored species (B. oris, B. crassa and B. motasi) were not detect- ed in present study among the analyzed sam- ples (Lable 2). Lhe 32 piroplasm positive blood samples included 26 sheep (total sam- ples 81, prevalence 23%) and 6 goat (total samples 114, prevalence 11%) samples indicat- ing that sheep were more prone to tick-borne haemoprotozoan than goats (P > 0.001).Out of the 2 provinces, 78% (25/32) of the Theileia positive samples were present in small rumi- nants from Khyber Pukhtoon Khwa as com- pared to 22% (7/32) in Southern Punjab (La- ble 2). Risk factor analysis revealed that male (P = 0.03) and animals infested by ticks (P = 0.03) were more infected by blood parasites (Lable 3). Statistical analysis of the characteris- tics of herds indicated that herd composition has significant effect on presence of piroplasm and herd consist of sheep only were more prone to piroplasmosis as compared to herd composed only of goats or having both sheep and goats (Lable 4).

Discussion

Identification of parasite species in animals having mixed infection is difficult while using conventional methods. On the other hand, PCR-based techniques allow rapid, sensitive and specific detection of piroplasms. The RLB assay is a powerful tool and practical assay as it is able to simultaneously identify Theileria and Babesia species and can even detect para- sites with extremely low parasitemia (11, 14, 15, 19). To our knowledge, this is the pioneer study in Pakistan in which spp. and Babesia parasites are detected through RLB assay.

Comparison of the results revealed that T. oi'is and T. lestoquardi were the only piroplasm spe- cies, which were detected in 16% of the sheep, and goat blood samples collected from two provinces in Pakistan (Lable 2). Prevalence of Theileria sp. was comparatively higher in Khy- ber Pukhtoon Khwa province (37%) than Punjab (6%). In a recent study, Durrani et al. (7) has reported 27% prevalence of T. oris in small ruminants of Lahore district (Punjab province) which is very high as compared to our overall findings indicating that geograph- ical distribution of land and climatic condi- tions affects the parasite prevalence. A similar study, conducted by Altay et al. (14), from eastern Lurkey, has reported very high preva- lence (54%) of piroplasms (Babesia and Theileria sp.) in sheep while Li et al. (5) has re- ported even higher prevalence (78%) of T. oris in Xinjiang province of China. In the present study, incidence of piroplasms was very high in sheep (32%) when compared to goat (5%) (Lable 2) confirming the findings of Altay et al. (14) and Durrani et al. (20) that sheep are more prone to blood parasites.

We observed that the animals having ticks present (26%) on them had higher incidence of ovine piroplasmosis indicating a positive correlation between the vector ticks and inci- dence of the disease (Lable 3). Lhese observa- tions confirm the previous findings of Durrani et al. (20) and Aktas et al. (13) that ticks are the vectors for the transmission of blood par- asites in ruminants.

Herds composed of sheep had higher parasitic prevalence when compared with goat herds or herds having both sheep and goats indicating that composition of herd is also associated with the ovine piroplasmosis (Lable 4). Vari- ous tick species are known to be involved in the transmission of Theileria sp., and many of them are host specific, so we assume that sheep were more infested with specific vector ticks as compared to goats that may be the reason of higher Theileria sp. prevalence in them (4-6). Our finding that sheep has higher prevalence of Theileria sp. than goats is in agreement with the results reported by Durrani et al. (20).

All the small ruminant blood samples analyzed during present study were locally rose indicat- ing that the piroplasmosis is endemic in Paki- stan. Poverty, especially in small towns and villages, and poor hygienic conditions are the contributing factor in piroplasmosis spread in this region. Veterinarians are not available for the help and guidance of livestock owners in many cases worsening the situation further. Lhe overall conditions can be improved by awarding the common people and livestock owners about the risk factors and preventive measures against ovine piroplasmosis resulting in better animal health and hence improving the income of the owners.

Conclusion

Sheep were more prone to tick borne haemoprotozans as 81% infected samples were sheep as compared to 19% goats (P > 0.001). Risk factor analysis revealed that male (P = 0.03), animals infested by ticks (P = 0.03) and herd composed of sheep only (P = 0.001) were more infected by blood parasites.

Acknowledgements

Lhis project was financed by the Directorate of Research and External Linkages, Bahauddin Zakariya University, Multan (Paki- stan). Authors would like to thank all the vet- erinarians for their kind help during sample collection. Lhe authors declare that there is no conflict of interest.

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