Prevalence of tick-borne haemoparasites in small ruminants in Turkey and diagnostic sensitivity of single-PCR and RLB

Background: Tick-borne haemoparasitic diseases (TBHDs), caused by Theileria, Babesia, Anaplasma and Ehrlichia, are common in regions of the world where the distributions of host, pathogen and vector overlap. Many of these diseases threaten livestock production and some also represent a concern to human public health. The primary aim of this study was to determine the prevalence of the above-mentioned pathogens in a large number of blood samples (n = 1979) collected from sheep (n = 1727) and goats (n = 252) in Turkey. A secondary aim was to assess the diagnostic sensitivity of a number of species-specific polymerase chain reaction (PCR) tests and the reverse line blotting (RLB) assay. DNA samples were screened using species-specific PCR for the presence of Theileria ovis, Theileria sp. MK, T. lestoquardi, T. uilenbergi, T. luwenshuni, Babesia ovis, Anaplasma ovis and A. phagocytophilum while RLB was undertaken to test for the presence of all known Theileria, Babesia, Anaplasma and Ehrlichia species. The diagnostic sensitivity of these two approaches was then compared in terms of their ability to detect single species and mixed infections. Results: Overall, 84 and 74.43% of the small ruminants sampled were identified as hosting one or more pathogen(s) by species-specific PCR and RLB respectively. The presence of Theileria sp. OT1, T. luwenshuni and T. uilenbergi in Turkey was revealed for the first time while the presence of Babesia motasi, B. crassa and T. separata in Turkish small ruminants was confirmed using molecular methods. A high prevalence of mixed infection was evident, with PCR and RLB approaches indicating that 52.24 and 35.42% of animals were co-infected with multiple species, respectively. More than 80% of the mixed infections contained T. ovis and/or A. ovis. The RLB approach was found to be capable of detecting mixed infections with species such as Theileria sp. OT1, Theileria sp. OT3, T. separata, B. crassa and Babesia spp. Conclusion: The results indicated that pathogens causing TBHDs are highly prevalent in sheep and goats in Turkey. The diagnostic sensitivity of species-specific single PCR was generally higher than that of RLB. However, the latter approach was still capable of identifying a high proportion of individuals containing mixed-species infections. The use of species-specific single PCR is recommended to accurately estimate pathogen prevalence and to identify co-infected hosts

Infection dynamics of Theileria annulata over a disease season following cell line vaccination

Tropical theileriosis is a tick-borne haemoparasitic disease of cattle caused by the protozoan parasite Theileria annulata. Globally, the economic impact of the disease is immense and enhanced control measures would improve livestock production in endemic regions. Immunisation with a live attenuated vaccine is an effective and widely used control method, however, the repeated use of live vaccines may have an impact on the field parasite population at a genetic level. Additionally, there has been an increasing number of reports of vaccine breakthrough cases in recent years. Thus, the present study was designed to evaluate the genetic composition of a parasite population over a disease season in a locality where live cell line vaccination is practised. A diverse range of parasite genotypes was identified and every T. annulata positive cattle blood sample harboured multiple parasite genotypes. An alteration in the major genotype and an increasing multiplicity of infection in individual animals was observed over the course of the disease season. Vaccination status was found not to effect within-host multiplicity of infection, while a significantly higher number of genotypes was detected in grazed cattle compared to non-grazed ones. A degree of genetic isolation was evident between parasite populations on a micro-geographic scale, which has not been reported previously for T. annulata. Analysis of parasite genotypes in vaccinated animals suggested only a transient effect of the vaccine genotype on the genetic diversity of the T. annulata population. The vaccine genotype was not detected among clones of two vaccine ‘breakthrough’ isolates and there is no suggestion that it was responsible for disease. The obtained data indicated that in the system studied there is no apparent risk of introducing the vaccine genotype into the population with only a transient effect on the genetic diversity of the parasite population during the disease season

Selection of genotypes harbouring mutations in the cytochrome b gene of Theileria annulata is associated with resistance to buparvaquone

Buparvaquone remains the only effective therapeutic agent for the treatment of tropical theileriosis caused by Theileria annulata. However, an increase in the rate of buparvaquone treatment failures has been observed in recent years, raising the possibility that resistance to this drug is associated with the selection of T. annulata genotypes bearing mutation(s) in the cytochrome b gene (Cyto b). The aim of the present study was: (1) to demonstrate whether there is an association between mutations in the T. annulata Cyto b gene and selection of parasite-infected cells resistant to buparvaquone and (2) to determine the frequency of these mutations in parasites derived from infected cattle in the Aydın region of Türkiye. Susceptibility to buparvaquone was assessed by comparing the proliferative index of schizont-infected cells obtained from cattle with theileriosis before and/or after treatment with various doses of buparvaquone, using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colourimetric assay. The DNA sequence of the parasite Cyto b gene from cell lines identified as resistant or susceptible was determined. A total of six nonsynonymous and six synonymous mutations were identified. Two of the nonsynonymous mutations resulted in the substitutions V135A and P253S which are located at the putative buparvaquone binding regions of cytochrome b. Allele-specific PCR (AS-PCR) analyses detected the V135A and P253S mutations at a frequency of 3.90% and 3.57% respectively in a regional study population and revealed an increase in the frequency of both mutations over the years. The A53P mutation of TaPIN1 of T. annulata, previously suggested as being involved in buparvaquone resistance, was not detected in any of the clonal cell lines examined in the present study. The observed data strongly suggested that the genetic mutations resulting in V135A and P253S detected at the putative binding sites of buparvaquone in cytochrome b play a significant role in conferring, and promoting selection of, T. annulata genotypes resistant to buparvaquone, whereas the role of mutations in TaPIN1 is more equivocal

Agarose gel electrophoresis of AS-PCR.

Gels showing detection of PCR amplicons of isolates with mutations V135A (A) and P253S (B), respectively. Products amplified using drug sensitive and resistance specific forward primers are given at the top and bottom of each gel, respectively. M, 100 bp molecular size marker (Thermo Scientific Corp.); lanes 1–16, products from template DNA of T. annulata samples; lanes S sensitive control, lanes R resistance control, lane X mixed control. (TIF)</p

MTT assay of <i>T</i>. <i>annulata</i> infected cell lines.

A.T. annulata isolates with low IC50 values (2–3 ng/mL). B. T. annulata isolates with medium IC50 values (3–7 ng/mL). C. T. annulata isolates with IC50 values over (7 ng/mL). Numbers on the x axis (-1, 0, 1, 2, 3 and 4) indicates drug concentrations used to treat cell lines at doses of 0, 0.8, 7, 125, 1000 and 1500 ng/mL, respectively.</p

Fig 5 -

MTT assay of cloned cell lines from A10/BT (A) and A21/AT1 (B) isolates. Numbers on the x axis (-1, 0, 1, 2, 3 and 4) indicates drug concentrations used to treat cell lines at doses of 0, 0.8, 7, 125, 1000 and 1500 ng/mL, respectively.</p

Alignments of putative Q_o1 and Q_o2 domains of <i>Cyto b</i>.

Predicted amino acid sequences of Qo1 and Qo2 domains encoded by the Cyto b gene from the published T. annulata / C9 genome sequence and different T. annulata isolates with treatment failure history. Putative Qo1 and Qo2 domains are located between 116–144 and 238–273 amino acids of the protein around the ubiquinone binding site. Predicted amino acid substitutions associated with buparvaquone resistance are marked with ◊.</p

Geographical distribution of the parasite material used in the present study.

The geographical distribution of the provinces where the parasite material used in this study was obtained. The map was prepared using the USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/ with some modification. (TIF)</p

Summary of mutations detected in <i>TaPIN1</i> sequences of <i>T</i>. <i>annulata</i> isolates from Tunisia, Sudan and Turkey.

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