Phylogenetic Analyses of Trypanosoma evansi Isolates and Tabanid Flies with Assessment of their Seasonal Abundance in Southwestern, Saudi Arabia

Abstract

Internal Transcribed Spacer1 (ITS-1) of nuclear ribosomal DNA (rDNA) of Trypanosoma evansi isolates from camel dromedary of King of Saudi Arabia (KSA) used to investigate evolutionary origins and infer phylogenetic relationships with other Global isolates. The study was actually started with microscopic examinations of blood samples on 454 from naturally infected camels in Jazan region, south western, by using wet and thick smear film which revealed 3.1% (14 positive) and 3.5% (16 positive), respectively, however, 100 blood samples were examined using conventional PCR- ITS1 targeting 480 bp, and giving 19% infection +ve. The ITS1 product was subjected to PCR-RoTat 1.2 VSG (151 bp) and yielded 36.8% infection +ve. The molecular characterization and phylogenetic analysis were achieved on product sequences and aligned against the corresponding GenBank sequences of known isolates of T. evansi. Basic Local Alignment Search Tool (BLAST) data of the obtained ITS1 gene sequences showed identity to those of T. evansi, with the homology of 92-99%. Sequence analysis of this gene generated six heterogeneous genotypes of T. evansi in KSA are present, released in GenBank and encoded in following terms of jaz1, jaz2, jaz3, jaz4, jaz5 and jaz6 with accession numbers; MN611173, MN625863, MN625864, MN611174, MN625865 and MN625866, respectively. T. evansi isolates from KSA and from other countries were tightly clustered into the constructed phylogenetic tree based on the ITS-1 rDNA sequence using Maximum Composite Likelihood and neighbour-joining method, revealing apparently genetic diversity among the isolates. In lack of data of identification of tabanids (Horse flies) in KSA, additional to assess their seasonal abundance. The present study was carried out to describe the morphological and molecular characterization of collected tabanid flies with density variation related. Four times a month, a total number of 457 flies’ samples were collected from Jazan region during the period from October 2017 to September 2018, using Nzi traps. More tabanids flies were captured during the wet (67.61%) compared to the dry season (32.39%). Four different species of three Tabanids’ genera were identified morphologically using the standard taxonomic keys; two for the first time namely; Hybomitra lurida and Tabanus par, beside Atylotus agrestis and Tabanus Sufis that were reported before in KSA. Genomic DNA was extracted from Saudi Arabian tabanids, and polymerase chain reaction (PCR) successfully amplified 710 base pairs of mitochondrial cytochrome c oxidase subunit 1 (COI) genes. Sequence analysis was performed, and three out of four species were identified (A. agrestis MW243943, H. lurida MW265638 and T. par MW238412). The product sequences of KSA tabanid flies were aligned each separately against the corresponding GenBank sequences of known specimens and showed that the specimens of A. agrestis and T. par from Saudi Arabia were closely related to that of the Afrotropical region than to the Palaearctic region in the rate of 92-99% and 91-93% respectively, whilst the sp. H. lurida was more similar to that of the Nearctic regions with the homology of 92-94%. This is the first report of investigation of phylogenesis of T. evansi and tabanid flies with assessment of their seasonal abundance in kingdom of Saudi Arabia.