This document describes a study to identify pathogens in Hyalomma ticks collected from cattle in various regions of Khyber Pakhtunkhwa, Pakistan. 70 engorged female ticks will be collected and identified to species. Genomic DNA will be extracted from the ticks and tested using PCR and sequencing to detect Theileria, Babesia, Borrelia burgdorferi, Rickettsia, and Coxiella burnetii pathogens. Sequence results will be compared to the GenBank database to identify the pathogens present. This will help determine the occurrence of tick-borne pathogens affecting cattle in Khyber Pakhtunkhwa.

1. MOLECULER IDENTIFICATION OF PATHOGENS IN HYALOMMA TICKS
COLLECTED FROM VARIOUS REGIONS OF KHYBER PAKHTUNKHWA,
PAKISTAN.
By
Shams Ullah
(Reg. No. …………)
Supervisor-I: Dr. ABC
Department of Zoology Signature
KUST, Kohat
Supervisor-II: Dr. XYZ
Department of Biotechnology Signature
KUST, Kohat
Chairman: Dr. Shaid Niaz Khan
Department of Zoology Signature
KUST, Kohat
Department of Zoology
University of Science and Technology, Kohat-26000
Khyber Pakhtunkhwa, Pakistan.

2. Introduction
Ticks are ectoparasite infesting livestock’s in every geographic area in the world and also the main
vectors of different pathogenic organisms including protozoans, bacteria and viruses. Among tick-
borne bacteria, Borrelia burgdorferi sensu lato, the causative agent of Lyme disease, is mainly
distributed in northern China [1], and can be transmitted to humans or animals by the bite of
infected Ixodes ticks, causing multi-organ damage, such as carditis, arthritis, and neurological
manifestations [2]. Rickettsiae are gram-negative, obligate intracellular bacteria which have the
ability to cause infection in human as well as in other vertebrates [3]. Q fever is a cosmopolitent
zoonotic disease caused by an obligate Gram-negative bacterium Coxiella burnetii [4]. Domestic
animals such as sheep, cattle and goats are well reservoirs of C. burnetii [5]. The continuous
exploitation of environmental resources and increase in human external activities, which becomes
entitle for the contact with tick vectors normally present in the field, assist the emergence and
resurgence of ticks borne pathogens [6]. However, while vectorial capacity is affected by
behavioral and environmental determinants influencing variables such as vector density, durability
and competence, vector competence is a component of vectorial capacity that depends on genetic
factors influencing the capability of a vector to transmit a pathogen [7].
Objectives:
The aims of the is to determine the occurrence of pathogens in Hyalomma species collected from
cattle, in various region of KPK Pakistan.

3. Material and methods
Ticks collection and DNA extraction:
70 engorged female ticks will collected from cattle from various region of KPK Pakistan. All of
the ticks will identified as Hyalomma species according to standard taxonomic keys [8], and will
have been evaluated the infection with piroplasms [9]. Ticks will disinfected in 75 % ethanol for
15 min, rinsed with sterilized distilled water for three times, and dried on a filter paper.
Molecular detection of pathogens:
Extracted genomic DNA will used as a template for PCR amplification. For the amplification
of Theileria and Babesia species in ticks, a touchdown PCR will performed. For the
amplification of an approximately 360–430-bp fragment of the hypervariable V4 region of the
18S rRNA gene, one set of primers [(RLB)-R2 (Biotin-5′-
CTAAGAATTTCACCTCTGACAGT-3′) and RLB-F2 (5′-
GACACAGGGAGGTAGTGACAAG-3′)] will used [10]. Six microliters of PCR product will
visualized by UV transillumination in a 2 % agarose gel after electrophoresis and stained with
ethidium bromide.
Sequencing analysis:
The PCR products Will purified from the agarose gel using a commercial PCR Clean up System
(MinElute PCR Purification Kit, 28004) and directly sequenced. Sequencing results will
compared with other sequences present in NCBI database
(http://www.ncbi.nlm.nih.gov/nuccore) and submitted to GenBank.

4. References
[1]. Hao Q, Hou X, Geng Z, Wan K (2011) Distribution of Borrelia burgdorferi sensu lato in
China. J Clin Microbiol 49:647–650.
[2]. Biesiada G, Czepiel J, Lesniak MR, Garlicki A, Mach T (2012) Lyme disease: review.
Arch Med Sci 8:978–982.
[3]. Raoult D, Roux V (1997) Rickettsioses as paradigms of new or emerging infectious
diseases. Clin Microbiol Rev 10:694–719.
[4]. Chmielewski T, Tylewska-Weirzbanowska S. Q fever at the turn of the century. Polish. J.
Microbiol. 2012; 61: 81–93.
[5]. Berri M, Arricau-Bouvery N, Rodolakis A. PCR-based detection of Coxiella burnetii from
clinical samples. In: Sachse K, Frey J, editors. Methods in Molecular Biology. Totowa:
Humana Press Inc; 2003. pp. 153–161.
[6]. Jongejan, F, and Uilenberg, G. (2004). The global importance of ticks. Parasitology129
(Suppl.), S3–S14. doi: 10.1017/S0031182004005967.
[7]. Beerntsen, B. T., James, A. A., and Christensen, B. M. (2000). Genetics of mosquito vector
competence. Microbiol. Mol. Biol. Rev. 64, 115–137. doi: 10.1128/MMBR.64.1.115-137.2000.
[8]. Teng K, Jiang Z (1991) Economic insect fauna of China Fasc 39 Acari: Ixodidae.
Science, Beijing, pp 52–349
[9]. Abdallah M, Niu Q, Yang J, Hassan M, Yu P, Guan G, Chen Z, Liu G, Luo J, Yin H
(2017) Identification of 12 piroplasms infecting ten tick species in China using reverse
line blot hybridization. J Parasitol. doi: 10.1645/16-161.
[10]. Georges K, Loria GR, Riili S, Greco A, Caracappa S, Jongejan F, Sparagano O (2001)
Detection of haemoparasites in cattle by reverse line blot hybridisation with a note on the
distribution of ticks in Sicily. Vet Parasitol 99(4):273–286.