Development of a loop-mediated isothermal amplification method for detection of Theileria lestoquardi.

A loop-mediated isothermal amplification (LAMP) assay was developed for the diagnosis of Theileria lestoquardi infection. The primers were designed based on the clone-5 sequence of T. lestoquardi. The specificity and sensitivity of the assay were established. Analysis of the specificity showed that the selected LAMP primers amplified the target sequence from T. lestoquardi DNA successfully, while no amplification was seen with DNA from Theileria annulata, Theileria ovis, Babesia ovis, Anaplasma ovis, or ovine genomic DNA. The specificity of the LAMP product was further confirmed by restriction digestion and sequencing. The sensitivity of the LAMP assay was analyzed in comparison to PCR resulting in a detection limit of 10 fg/µl of plasmid DNA containing the clone-5 sequence. The suitability for utilizing the LAMP assay in the field for the diagnosis of T. lestoquardi infection was tested on 100 field samples collected in Sudan and compared with results obtained by PCR. The relative specificity and sensitivity of the established LAMP assay was determined to be 92.1% and 87.5%, respectively, indicating that it may be regarded as an alternative molecular diagnostic tool to PCR which could be used for epidemiological surveys on T. lestoquardi infection.

First confirmed report of outbreak of malignant ovine theileriosis among goats in Sudan.

An outbreak of malignant ovine theileriosis among goats was confirmed and documented. In this outbreak, 16 out of 22 (72.7%) goats died within 4 days showing clinical signs of malignant ovine theileriosis as well as in the postmortem findings. The goats were reared in a mixed flock with sheep in Atbara Town, Northern Sudan. The infection was detected microscopically and confirmed serologically by IFA test and molecularly by PCR technique using specific primer for Theileria lestoquardi. Hyalomma anatolicum was the most prevalent (dominant) tick species found in the farm. It is recommended to undertake future research on the role of goats on the epidemiology of malignant ovine theileriosis.

Population Genetic Analysis and Sub-Structuring of Theileria annulata in Sudan.

Theileria annulata, which causes tropical theileriosis, is a major impediment to improving cattle production in Sudan. Tropical theileriosis disease is prevalent in the north and central regions of Sudan. Outbreaks of the disease have been observed outside the known endemic areas, in east and west regions of the country, due to changes in tick vector distribution and animal movement. A live schizont attenuated vaccination based on tissue culture technology has been developed to control the disease. The parasite in the field as well as the vaccine strain need to be genotyped before the vaccinations are practiced, in order to be able to monitor any breakthrough or breakdown, if any, after the deployment of the vaccine in the field. Nine microsatellite markers were used to genotype 246 field samples positive for T. annulata DNA and the vaccine strain. North and central populations have a higher multiplicity of infection than east and west populations. The examination of principal components showed two sub-structures with a mix of all four populations in both clusters and the vaccine strain used being aligned with left-lower cluster. Only the north population was in linkage equilibrium, while the other populations were in linkage disequilibrium, and linkage equilibrium was found when all samples were regarded as single population. The genetic identity of the vaccine and field samples was 0.62 with the north population and 0.39 with west population. Overall, genetic investigations of four T. annulata populations in Sudan revealed substantial intermixing, with only two groups exhibiting regional origin independence. In the four geographically distant regions analyzed, there was a high level of genetic variation within each population. The findings show that the live schizont attenuated vaccine, Atbara strain may be acceptable for use in all Sudanese regions where tropical theileriosis occurs.

Prevalence of Theileria annulata in dairy cattle in Nyala, South Darfur State, Sudan.

AIM: This study was conducted in dairy cattle in Nyala, South Darfur State, during the period from June to September 2015, to study the prevalence of bovine tropical theileriosis. MATERIALS AND METHODS: Apparently, healthy cattle of different age groups, different breeds, and from both sexes were randomly selected from seven locations. Three age groups of cattle were selected, group one <1 year old, group two 1-3 years old, and group three older than 3 years. These cattle were indigenous and cross (Friesian X zebu). A total of 150 blood samples were collected for blood smears, blood in EDTA tubes, and serum samples as well as ticks infesting cattle. Three diagnostic techniques were used such as blood smear, indirect fluorescent antibody test (IFAT), and polymerase chain reaction (PCR). RESULTS: Of 150 samples, 11 (7.3%, 95% confidence interval [CI]: 9.1-5.5) were positive for Theileria spp. piroplasms in the blood smears, 70 (46.7%, 95% CI: 35.7-57.7) were positive for Theileriaannulata antibodies in the IFAT, and of 100 samples, 39 (39%, 95% CI: 46.6-31.4) were positive for T. annulata using PCR. The prevalence of T. annulata was higher in indigenous breed than cross cattle by the three diagnostic techniques. The highest prevalence of T. annulata was recorded among cattle older than 3 years old. There were three genera and ten species of ticks found feeding on cattle. These were Rhipicephalus evertsi evertsi, Rhipicephalus decoloratus, Rhipicephalus annulatus, Hyalomma dromedrii, Hyalomma impeltatum, Hyalomma rufipes, Hyalomma anatolicum, Hyalomma truncatum, Amblyomma variegatum, and Amblyomma lepidum. CONCLUSION: The study concluded that tropical theileriosis is prevalent among dairy cattle in Nyala. H. anatolicum was found in very low numbers, suggesting other ticks may play a role in the transmission of the disease. Molecular characterization of T. annulata is recommended for accurate mapping of the disease and evaluates the magnitude problem of tropical theileriosis in South Darfur region.

Epidemiology of Theileria annulata infection of dairy cattle in the Sudan using molecular techniques.

This study provides the first epidemiological data regarding T. annulata infection of diary cattle in Sudan using a combination of routine microscopic examination and two molecular techniques, PCR and reverse line blot (RLB).

Influence of subculturing on gene expression in a Theileria lestoquardi-infected cell line.

In this study potential molecular markers for identification of attenuation in a Theileria lestoquardi-infected cell line to be used in vaccination trials were identified. Two markers associated with attenuation in Theileria annulata vaccine strains were analyzed (metalloproteinase activity and TNF? mRNA expression). The result showed a decreased activity of MMP 9 and decreased mRNA expression of TNF? with increasing passage number. Suppression subtractive hybridization was used to identify potential new markers of attenuation. Random screening revealed nine differentially expressed genes, one from the parasite and eight from the host. Quantitative real time-PCR confirmed mRNA expression of the parasite vacuolar H+ATPase to be downregulated at higher passages.

Comparison of the molecular structure of the TaSP gene of Theileria annulata from Sudanese isolates.

The polymorphic region of the Theileria annulata surface protein (TaSP) was cloned and sequenced from different isolates of cattle and cell lines from different areas of Sudan. Amino acid sequence alignment revealed a high diversity showing amino acid and length polymorphism, both within and between parasite isolates. The generation of TaSP diversity may allow the evasion of host immunity by the parasite since TaSP is a highly antigenic parasite protein.