Abstract
In the present investigation, it was intended to characterize the clinical phases of trypanosomiasis in infected camels, in relation to difference in regulation of cytokines expression. The diagnostic potential of IL-4, IL-6, IL-8, and IL-10 as biomarkers of clinical phase of infections in comparison to other technologies including parasitological, serological, as well as conventional PCR to improve laboratory and/or field diagnosis was studied. The study included 11 localities in Egyptian desert included: North West Coast, Western Desert Oasis, South East Coast, Suez Canal region and Sinai. Blood specimens were collected from 110 camels examined seasonally (4 times/year) from June 2014 to July 2016. trypanozoon parasites were detect in blood by wet blood smears (6.7%), field stained blood film (7.72%), and hematocrit centrifugation (14.66%) within investigated localities in infected camels with high parasitaemia (>= 40 trypanosome/field) and acute infection. On the other hand, serological diagnosis by ELISA; using homemade trypanozoon lysed antigen, as well as, CATT test revealed 44.1% and 41.36% seropositive incidence for surra, respectively. On the other hands, molecular screening for trypanosomiasis was more specific and sensitive. The tryp1 primers pair specifically amplify intragenic spacer I region (its1) of ribosomal DNA of all Trypanosomes; therefore, was the primary target during PCR diagnosis. While, the tbr primers pair could amplify satellite genomic DNA of trypanozoon parasites; thus used as the confirmatory second choice. PCR screening of camels samples utilizing the specific primers of ITSI and SND, revealed 68.3% overall positivity, while seasonal incidence recorded 7.73%, 19.20%, 33.3%, and 8.07% during autumn, spring, summer, and winter, respectively. Positive PCRs were then typed into species/sub-genus level by sequencing then alignment into GenBank database. The annotated fragments revealed infections with T. evansi; the only species of Trypanozoon sub-genus possibly present in Egypt. Sequences alignments show clustering based on the biological origin of the isolates; camel breed local or imported with tendency for clonality that was geographical-dependent. The influence of Trypanosoma evansi on cytokines mRNAs expression of IL-4, IL-6, IL-8 and IL-10 in blood cells were quantified, and then normalized with housekeeping gene (beta-Actin), finally expressed as fold of induction. The qRT-PCR revealed up regulation then down regulation of target cytokine genes in response to acute (high parasitemia) then sub-acute but not with chronic infections, respectively. Despite that sub-acute infected individuals showed fold increase in cytokines genes expression, but were lower than those recorded for acute infections. However, the fold increase was higher for IL-8 and IL-10 than those recorded for IL-4 and IL-6, calculated 11.69 +/- 4.146 and 13.04 +/- 7.103 for IL-8 and IL-10, respectively (Means St. Dev. 2(-) (Delta Delta cT). In conclusion: The results of this study indicate that camels' isolates of T. evansi belong to homologous phylogroup/serogroups/clade that contained a variety of virulence associated traits. On the other hand, the investigation in rodents needs to be completed to evaluate their role as reservoirs of potentially pathogenic trypanosomes in the vicinity of humans and livestock. Finally, a wide range of genes played role in the efficiency of immune reaction associated with surra, however, their inclusion was beyond the scope of the current study.