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.

The pulmonary involvement in Theileria lestoquardi naturally infected sheep.

Malignant Ovine Theileriosis (MOT) caused by Theileria lestoquardi is considered a major constraint for sheep production in many areas of the world including Sudan. Pulmonary oedema is thought to be the main cause of animal death, but the mechanism, the cell types involved and/or the probable cause of this pneumonia has yet to be defined. The present study was carried out to investigate the pulmonary involvement post T. lestoquardi infection and to identify the cell types involved in pneumonia. Apparently healthy sheep were exposed to ticks challenge in T. lestoquardi endemic area. Lungs impression smears and tissue sections for histopathology were processed. At necropsy, fifteen infected sheep revealed severe pneumonia associated with oedema and accumulation of creamy-grayish frothy exudates. The microscopic findings of examined lungs showed emphysema, congestion, collapse and proliferation of immense amount of different kinds of cells. The current study indicates that T. lestoquardi infections are accompanied with remarkable pulmonary involvements and may lead to respiratory failure and death.

Molecular identification of different Theileria and Babesia species infecting sheep in Sudan.

The epidemiological aspects of sheep piroplasmosis in Sudan are poorly studied, and further investigations using sensitive and precise techniques are required. In this study, the Reverse Line Blot (RLB) hybridization assay was used to detect and simultaneously differentiate between Theileria and Babesia species. DNA was extracted from blood collected on filter paper (n=219) from apparently healthy sheep from six different geographical localities in Sudan. Results indicated that Theileria ovis (88.6%), T. separata (20.1%), T. lestoquardi (16.4%) and T. annulata (16.4%) DNA could be detected in the blood samples. Single and mixed Theileria infections were detected in 74 (33.8%) and 124 (56.6%) respectively and T. ovis being the most prevalent species in the country. T. ovis and T. separata were reported for the first time in sheep in Sudan.