Molecular surveillance of Theileria parasites of livestock in Oman

Background: Theileriosis is one of the most prevalent infectious diseases of livestock in the Arabian Peninsula, and causes high rates of mortality and morbidity in sheep and cattle. However, there is a paucity of information on the distribution of Theileria spp. over the whole region and their impact on different hosts. The present study carried out a country-wide molecular survey for Theileria spp. of livestock in Oman across four governorates. The aim of the survey was to define the prevalence of Theileria spp. in cattle, sheep and goats, highlight risk factors for infection and identify the main tick species involved in parasite transmission. Material and methods: A total of 2020 animals were examined in the survey consisting of sheep [n = 592], goats [n = 981] and cattle [n = 447]. All three species were raised and co-grazed on the same farms. Theileria parasites were detected using PCR-RFLP and RLB of the 18S rRNA gene. Cloning and sequencing of the 18S rRNA was carried out on 11 T. lestoquardi isolates from Ash-Sharqiyah, and Ad-Dhahira governorates, and phylogenetic relationships were inferred using additional sequences of T. lestoquardi, T. annulata and T. ovis available in GenBank. Results: Theileria spp. prevalence was 72.3%, 36.7% and 2.7% among cattle, sheep and goats, respectively. Strong similarity in results was obtained using RLB and PCR-RFLP for detection of Theileria spp. however, RLB detected a higher rate of mixed infection than PCR-RFPL (P < 0.001). Theileria annulata was the only parasite detected in cattle, while sheep and goats carried T. ovis, T. lestoquardi and T. annulata as well as Theileria spp. OT1. Of the four Theileria spp. detected in small ruminants, overall T. ovis was most prevalent (sheep [33.4%], goats [2.0%]), whereas T. lestoquardi was less prevalent (sheep [22.0%], goats [0.5%]). A large proportion of infected sheep (19%) carried mixed infection of T. ovis and T. lestoquardi. However, single T. lestoquardi infections (3.0%) were less prevalent than T. ovis infections (14.5%). Risk of Theileria spp. infection was significantly higher for exotic breeds, relative to native breeds, of cattle (p = 0.00002) and sheep (p = 0.005). Phylogenetic analysis placed T. lestoquardi in Oman in the same clade as other T. lestoquardi strains isolated from the same regional area (Iraq and Iran). The main tick species, identified on the examined animals, Hyalomma anatolicum, was widely distributed and was found in all of the surveyed governorates. Conclusion: Theileria spp. are widespread in Oman with variable prevalence detected in different regions. Two economically important hosts, cattle and sheep are at high risk from virulent T. annulata and T. lestoquardi, respectively. The survey indicates extensive exposure to ticks and transmission of infection that has a significant economic impact. The higher prevalence of T. lestoquardi as mixed rather than single infection requires further investigation

Genetic Diversity and Population Structure of <i>Theileria annulata</i> in Oman

Background: Theileriosis, caused by a number of species within the genus Theileria, is a common disease of livestock in Oman. It is a major constraint to the development of the livestock industry due to a high rate of morbidity and mortality in both cattle and sheep. Since little is currently known about the genetic diversity of the parasites causing theileriosis in Oman, the present study was designed to address this issue with specific regard to T. annulata in cattle. Methods Blood samples were collected from cattle from four geographically distinct regions in Oman for genetic analysis of the Theileria annulata population. Ten genetic markers (micro- and mini-satellites) representing all four chromosomes of T. annulata were applied to these samples using a combination of PCR amplification and fragment analysis. The resultant genetic data was analysed to provide a first insight into the structure of the T. annulata population in Oman. Results: We applied ten micro- and mini-satellite markers to a total of 310 samples obtained from different regions (174 [56%] from Dhofar, 68 [22%] from Dhira, 44 [14.5%] from Batinah and 24 [8%] from Sharqia). A high degree of allelic diversity was observed among the four parasite populations. Expected heterozygosity for each site ranged from 0.816 to 0.854. A high multiplicity of infection was observed in individual hosts, with an average of 3.3 to 3.4 alleles per locus, in samples derived from Batinah, Dhofar and Sharqia regions. In samples from Dhira region, an average of 2.9 alleles per locus was observed. Mild but statistically significant linkage disequilibrium between pairs of markers was observed in populations from three of the four regions. In contrast, when the analysis was performed at farm level, no significant linkage disequilibrium was observed. Finally, no significant genetic differentiation was seen between the four populations, with most pair-wise FST values being less than 0.03. Slightly higher FST values (GST’ = 0.075, θ = 0.07) were detected when the data for T. annulata parasites in Oman was compared with that previously generated for Turkey and Tunisia. Conclusion: Genetic analyses of T. annulata samples representing four geographical regions in Oman revealed a high level of genetic diversity in the parasite population. There was little evidence of genetic differentiation between parasites from different regions, and a high level of genetic diversity was maintained within each sub-population. These findings are consistent with a high parasite transmission rate and frequent movement of animals between different regions in Oman

Box plot of mean number of alleles in four populations in Oman.

<p>Box plot of mean number of alleles in four populations in Oman.</p

Linkage equilibrium among <i>T</i>. <i>annulata</i> populations in Oman and comparison of parasites in Oman, Tunisia and Turkey.

<p>To test the hypothesis of geographical sub-structuring, the I^S_A, V_D and L values were calculated separately for samples in each region. The I^S_A value for Dhira, Sharqia and Dhofar was 0.0219, 0.0337 and 0.023, respectively. The V_D values were greater than the L value, indicating LD in all regions (Dhira, Sharqia and Dhofar) except Batinah.</p><p>Linkage equilibrium among <i>T</i>. <i>annulata</i> populations in Oman and comparison of parasites in Oman, Tunisia and Turkey.</p

Principal Co-ordinate analysis (PCoA) of <i>T</i>. <i>annulata</i> populations from Oman, Turkey and Tunisia.

<p>PCoA was performed on the multi-locus genotype data representing each of the populations sampled. The proportion of the variation in the dataset explained by each axis is indicated in parenthesis.</p

Pair-wise F_ST estimates among <i>T</i>. <i>annulata</i> populations in Oman, as well as between Oman, Tunisia and Turkey.

<p>Pair-wise F_ST estimates among <i>T</i>. <i>annulata</i> populations in Oman, as well as between Oman, Tunisia and Turkey.</p

Locations of collection sites in four regions in Oman; below table represent geographical distance matrix in km between regions.

<p>The study field surveys and samples collection were carried out in accordance with the regulations of the Sultan Qaboos University Committee for Animal Ethics. The field surveys did not involve endangered or protected animal species: blood samples were collected by a veterinarian while animals were manually restrained; no tranquillisers or short-acting anaesthetics were used. Appropriate equipment was used for blood sample collection. No institutional approval was needed, as per Sultan Qaboos University ethics committee such an approval is only required for small animals. The sampling procedures and number of animals to be sampled were approved by the Ministry of Agriculture and Fishery, Oman, as part of obtaining the field permit.</p

The frequency of alleles for three representative markers (TS5, TS15 and TS20) in four regional parasite populations in Oman.

<p>The size of each allele (in bp) is given on the x- axis.</p

Allelic diversity and unbiased heterozygosity (<i>He</i>) at 10 micro- and mini-satellite loci among 310 <i>T</i>. <i>annulata</i> isolates in Oman.

<p>Allelic diversity and unbiased heterozygosity (<i>He</i>) at 10 micro- and mini-satellite loci among 310 <i>T</i>. <i>annulata</i> isolates in Oman.</p