Culturing, Characterization and Identification of Candidate Microorganisms in Cattle Ear, Responsible for Producing Volatile Constituents Attractive to the Brown Ear Tick
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Date
2015-09
Authors
Kogo, Kipchumba Kenneth
Journal Title
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Publisher
Kenyatta University
Abstract
East Coast Fever is a disease of cattle which results in high animal mortality with corresponding economic losses to farmers and to the nation. The disease is caused by a protozoan agent whose vector is Rhipicephalus appendiculatus (Brown Ear Tick). The tick feeds dominantly inside the cattle ear. Studies have shown that the adults of R. appendiculatus exhibit „pull-push‟ effect that guides them to their preferred feeding site. This is linked to the presence of ear volatiles which attracts and anal volatiles which repels the tick. However, there is limited information that describes what produces the volatiles within the cattle ear. This study aims at establishing the role of ear microbes to the brown ear tick behavior while locating its preferred feeding site. In a preliminary observation, release of the ticks on the cattle with one ear sprayed with a broad-spectrum antibiotic showed that most ticks were attracted to the untreated ear, suggesting that the attractant volatiles are derived from specific microbial populations. In the present study where twelve cattle were used, one ear in each cow was treated with vetmycin a broad-spectrum antibiotic while the other ear was left untreated and the orientation behavior of the ticks (R. appendiculatus) placed at six different parts of cattle monitored. Most of the ticks moved to the untreated cattle ear (P<0.001,t-test). Ear swabs were collected and cultured in a general media to obtain a culture of the microbes for in-vitro behavioral tick experiments and genomic studies. In a choice laboratory experiment, ticks were also observed to orient towards cultivable cattle ear microbes relative to a negative control (P< 0.001,t-test). General lab media (Nutrient Agar) was then used to culture different micro-organisms. Sensitivity tests carried out to determine the efficacy of the antibiotic spray used on the cattle ears showed that most microbes were sensitive to the antibiotics contained in the spray according to Kirby Buer calibrations. Profiles of aerobically cultivable microbial communities in each of the twelve cows were investigated using pyrosequencing based analysis. Genomic DNA was extracted and amplified using two sets of primers U1F, U1R which targets band lengths of 490bp and 500bp and primers 27F, 1492R which targets bands lengths of 1490bp. Gene sequences of each ear helped to identify the specific genus of each microorganism and profiles of the candidate microorganisms. Pyro-sequencing blast results revealed that there were diverse communities of microbes majority of which were bacteria. The study lays down significant groundwork in identifying the specific ear microbes responsible for the production of the constituents of ear blend that is responsible for attracting the adults of the Brown Ear Tick. This may open up novel ways of manipulating the production of the blend and in controlling the tick
Description
A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science (Microbiology) in the School of Pure and Applied Sciences, Kenyatta University, September 2015