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

Drug resistant Mycobacterium tuberculosis in Oman: resistance-conferring mutations and lineage diversity

Background The Sultanate of Oman is country a low TB-incidence, with less than seven cases per 105 population detected in 2020. Recent years have witnessed a persistence in TB cases, with sustained incidence rate among expatriates and limited reduction among Omanis. This pattern suggests transmission from the migrant population. The present study examined the genetic profile and drug resistance-conferring mutations in Mycobacterium tuberculosis collected from Omanis and expatriates to recognise possible causes of disease transmission. Methods We examined M. tuberculosis cultured positive samples, collected from Omanis (n = 1,344) and expatriates (n = 1,203) between 2009 and 2018. These isolates had a known in vitro susceptibility profile to first line anti-TB, Streptomycin (SM), Isoniazid (INH), Rifampicin (RIF), Ethambutol (EMB) and Pyrazinamide (PZA). The diversity of the isolates was assessed by spacer oligo-typing (spoligotyping). Drug resistance-conferring mutations resulted from full-length sequence of nine genes (katG, inhA, ahpc, rpoB, rpsL, rrs, embB, embC, pncA) and their phenotypic relationship were analysed. Results In total, 341/2192 (13.4%), M. tuberculosis strains showed resistance to any drug, comprising mono-resistance (MR) (242, 71%), poly-resistance (PR) (40, 11.7%) and multi-drug resistance (MDR) (59, 17.3%). The overall rate of resistance among Omanis and expatriates was similar; however, MDR and PZAR were significantly higher among Omanis, while INHR was greater among expatriates. Mutations rpsL K43R and rpoB S450L were linked to Streptomycin (SMR) and Rifampicin resistance (RIFR) respectively. Whereas, katG S315T and inhA –C15T/G–17T were associated with Isoniazid resistance (INHR). The resistance patterns (mono-resistant, poly-resistant and MDR) and drug resistance-conferring mutations were found in different spoligo-lineages. rpsL K43R, katG S315T and rpoB S450L mutations were significantly higher in Beijing strains. Conclusions Diverse drug resistant M. tuberculosis strains exist in Oman, with drug resistance-conferring mutations widespread in multiple spoligo-lineages, indicative of a large resistance reservoir. Beijing’s M. tuberculosis lineage was associated with MDR, and multiple drug resistance-conferring mutations, favouring the hypothesis of migration as a possible source of resistant lineages in Oman

Dynamics of Mycobacterium tuberculosis Lineages in Oman, 2009 to 2018

Study aim. Effective Tuberculosis (TB) control measures in Oman have reduced the annual incidence of tuberculosis cases by 92% between 1981 and 2016. However, the current incidence remains above the program control target of <1 TB case per 100,000 population. This has been partly attributed to a high influx of migrants from countries with high TB burdens. The present study aimed to elucidate Mycobacterium tuberculosis infection dynamics among nationals and foreigners over a period of 10 years. Methods. The study examined TB cases reported between 2009 and 2018 and examined the spatial heterogeneity of TB cases and the distribution of M. tuberculosis genotypes defined by spoligotypes and MIRU-VNTR among Omanis and foreigners. Results. A total of 484 spoligoprofiles were detected among the examined isolates (n = 1295). These include 943 (72.8%) clustered and 352 (27.2%) unique isolates. Diverse M. tuberculosis lineages exist in all provinces in Oman, with most lineages shared between Omanis and foreigners. The most frequent spoligotypes were found to belong to EAI (318, 30.9%), CAS (310, 30.1%), T (154, 14.9%), and Beijing (88, 8.5%) lineages. However, the frequencies of these lineages differed between Omanis and foreigners. Of the clustered strains, 192 MTB isolates were further analysed via MIRU-VNTR. Each isolate exhibited a unique MIRU-VNTR profile, indicative of absence of ongoing transmission. Conclusions. TB incidence exhibits spatial heterogeneity across Oman, with high levels of diversity of M. tuberculosis lineages among Omanis and foreigners and sub-lineages shared between the two groups. However, MIRU-VNTR analysis ruled out ongoing transmission

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

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

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

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

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

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