Hypoderma bovis Hypodermin Rekombinant Antijenlerinin Karakterizasyonu Ekspresyon Profilleri ve Serolojik Teşhisteki Etkinliklerinin Değerlendirilmesi

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Thesis Type: Doctorate

Institution Of The Thesis: Erciyes University, Veteriner Fakültesi, Klinik Öncesi Bilimler, Turkey

Approval Date: 2020

Thesis Language: Turkish

Student: Mübeccel OKUR

Co-Supervisor: Alparslan Yıldırım

Supervisor: Abdullah İnci

Open Archive Collection: AVESIS Open Access Collection

Abstract:

An ELISA methodology was developed using recombinant antigens and peptides designed from amino acid sequences of the related genes and their efficiencies were evaluated. Afterward, the seroprevalence of hypodermosis in the sera of cattle from the field was investigated. The third stage Hypoderma larvae (L3) collected from infested cattle during abattoir inspections were constituted the material of the study. Whole blood samples were got from the animals prior to slaughter. Genomic DNA and total RNA isolations were performed followed by cDNA synthesis. After morphological identifications, species identification of H. bovis was confirmed by sequence analyses of the barcode mitochondrial cytochrome c oxidase subunit I (COI) DNA region of the larvae. Hypodermin genes were amplified in H. bovis cDNA libraries with originally designed primers. In order to obtain nucleotide and amino acid sequences, recombinant plasmid purifications were utilized after cloning the amplicons into appropriative bacterial plasmids and transformation steps. Recombinant plasmids were bidirectionally sequenced, and consensus sequences were obtained for each gene region by processing the sequences with genetic software. The open reading frame (ORF) sequences of HA, HB and HC gene were determined as in the length of 705 bp, 771 bp, 783 bp, and their coding protein aminoacid lengths were 234 aa, 256 aa, and 260 aa, respectively. There were no intraspecific differences in each Hypodermin gene, while 29.5%, 57.9%, and 53.9% interspecific differences were determined between HA and HB, HA and HC, HB and HC, respectively. Hypoderma bovis HA, HB and HC genes expressed sizes of 25.744 kDa, 27.795 kDa and 28.516 kDa proteins based on in-silico analyses. In the next step, expression studies were conducted on the Hypodermin genes that were cloned to bacterial expression system and expression efficiencies were determined by SDS-PAGE and Western Blot analyses. HA was not expressed in E. coli expression system, while purified recombinant HB (rHB), and HC (rHC) antigens were found as immune reactive in Western Blot analyses using sera dilutions of positive cattle. Protein sizes were also consistent with the result of in-silico analyses. rHC antigen and pHC peptide were found as the most effective with giving similar results to each other for ELISA in the standardization studies on purified rHB, rHC, and peptides (pHA, pHB, pHC). In the ELISA base on rHC and pHC, a seroprevalence of 62.0% of hypodermosis was determined in the examined 200 cattle, and the effects of several parameters such as age, gender, and breed on the distribution of the infection were evaluated. In conclusion, first molecular characterization data was obtained on the genes encoding HA, HB and HC proteins that are high antigenic importance and have an active role in immunomodulation of H. bovis which is an important endo-ectoparasite of cattle, and cause high economic losses with this study. Unique data were also obtained on the immune reactivities of these proteins and a novel ELISA methodology was developed with recombinant technology in order to the serological diagnosis of hypodermosis. Key words: Expression, Hypoderma bovis, hypodermin A, hypodermin B, hypodermin C, molecular characterization, ELISA, cattle