Detecção e caracterização molecular de Theileria orientalis em búfalos (Bubalus bubalis) do Pará, Brasil

Abstract

In the present study, we investigated the genetic diversity of Theileria orientalis circulating among buffalo populations from nine herds with and without a history of lymphoma in the state of Pará, in the Amazon region, Brazil. Blood samples from 500 animals were molecularly evaluated, over the years 2011 and 2020, from Murrah, Mediterranean and Jafarabadi breeds, of both sexes, aged between 2 and 15 years. Samples from 135 animals were also evaluated using the direct parasitological techniques of blood smear, Buffy coat, Woo and Brener technique for direct investigation of hemopathogens. Six animals with suspected lymphoma were also evaluated through necropsy, histopathological analysis, molecular analysis of tumor masses and lymph nodes, cytological analysis of lymph nodes from four animals and differential diagnosis for viruses that cause lymphoma of the species Bovine leukemia virus, Bovine herpes virus 6 and Bovine immunodeficiency virus and hemopathogens of the Anaplasma genera; Ehrlichia; Mycoplasma; Babesia and Trypanosoma. No hemopathogens were detected through direct parasitological techniques. Of the 500 blood samples submitted to molecular analyzes directed to the 18S rRNA gene of the genus Theileria, 15.4% (77/500) were positive. These same samples were positive when evaluated for ITS and MPSP genes, specific for T. orientalis. It was possible to obtain 21 quality sequences referring to the MPSP gene (~770pb) for phylogenetic analysis. Nucleotide sequence analysis of MPSP of T. orientalis gene fragments amplified from T. orientalis positive blood DNA samples revealed the presence of three genotypes. Of these, the present study described for the first time the presence of a new parasite genotype of the T. orientalis complex, namely type N4. The genotypes consisted of types N1, N2 and N4. Also included in the present study were six cases of lymphomas in buffaloes from two properties located in Pará, where a clinical, anatomopathological and etiological study of these lymphomas was carried out. Due to the anatomopathological characteristics, the lymphomas were characterized as multicentric, affecting animals aged between ten and 11 years. The presence of genetic material from viruses and hemopathogens was not detected in the lymphomas and blood of these animals, however, the presence of T. orientalis DNA was verified by the ITS gene, 18S rRNA and MPSP. By the phylogenetic inferences of the MPSP gene, the sequences isolated from the lymphomas presented the genotypes N2 and N4. The new N4 genotype was the most frequent in the populations studied and seems to be involved in the etiology of lymphomas in the evaluated animals. This is the first report describing the genetic diversity of T. orientalis in water buffaloes in South America and its possible etiological involvement with lymphomas in buffaloes.