Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (β-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.
Animals ; B-Lymphocytes/parasitology ; Cattle ; Cell Line ; Cells/*parasitology ; Cells, Cultured ; Gene Expression Profiling ; Host-Parasite Interactions/*genetics ; Real-Time Polymerase Chain Reaction/*veterinary ; Reproducibility of Results ; Signal Transduction/*genetics ; Theileria annulata/physiology ; Theileriasis/*physiopathology

This study was carried out to investigate fifteen cases of acute lethal infection of calves (< or = 4 months of age) by the protozoan parasite Theileria (T.) annulata in the south of Portugal. Calves developed multifocal to coalescent nodular skin lesions, similar to multicentric malignant lymphoma. Infestation with ticks (genus Hyalomma) was intense. Theileria was seen in blood and lymph node smears, and T. annulata infection was confirmed by isolation of schizont-transformed cells and sequencing of hypervariable region 4 of the 18S rRNA gene. At necropsy, hemorrhagic nodules or nodules with a hemorrhagic halo were seen, particularly in the skin, subcutaneous tissue, skeletal and cardiac muscles, pharynx, trachea and intestinal serosa. Histologically, nodules were formed by large, round, lymphoblastoid neoplastic-like cells. Immunohistochemistry (IHC) identified these cells as mostly CD3 positive T lymphocytes and MAC387 positive macrophages. A marker for B lymphocytes (CD79alphacy) labeled very few cells. T. annulata infected cells in these nodules were also identified by IHC through the use of two monoclonal antibodies (1C7 and 1C12) which are diagnostic for the parasite. It was concluded that the pathological changes observed in the different organs and tissues were caused by proliferation of schizont-infected macrophages, which subsequently stimulate a severe uncontrolled proliferation of uninfected T lymphocytes.
Animals ; Base Sequence ; Cattle ; Cattle Diseases/epidemiology/*parasitology/pathology ; Cell Growth Processes/physiology ; DNA, Protozoan/chemistry/genetics ; Disease Outbreaks/*veterinary ; Female ; Immunohistochemistry/veterinary ; Lymphocytes/parasitology/*pathology ; Male ; Molecular Sequence Data ; Polymerase Chain Reaction/veterinary ; Portugal/epidemiology ; RNA, Ribosomal, 18S/chemistry/genetics ; Sequence Analysis, DNA ; Skin Diseases/epidemiology/parasitology/pathology/*veterinary ; Theileria annulata/*isolation & purification ; Theileriasis/epidemiology/parasitology/*pathology