This document describes a proposed study to detect Clostridium perfringens types in goats in Bannu district, Pakistan using multiplex PCR. The study aims to 1) detect C. perfringens in goat blood samples using PCR, 2) identify the toxin types produced, and 3) determine the epidemiological characteristics of C. perfringens in the district. Blood samples will be collected from clinically suspected goats and tested microscopically, through DNA extraction and PCR amplification to detect C. perfringens. Gel electrophoresis will also be used to detect the bacteria. Statistical analysis will be conducted to analyze the results.

1. Molecular and Epidemiological of prevalence of Clostridium perfringens
types in Goats by Multiplex PCR in district Bannu
By
Muhammad Moaz Ashraf
(ZO220172032)
Supervisor-I Dr.Kalim Ullah Department of Zoology ……………
KUST, Kohat signature
Supervisor-II Dr. abcaccacacacac Department of Biotechnology ……………
KUST, Kohat signature
Chairman Dr. Shaid Niaz khan Department of Zoology ……………
KUST, Kohat signature
Department of Zoology
Kohat University of Science and Technology, Kohat-26000
Khyber Pakhtunkhwa, Pakistan

2. Introduction
Clostridium perfringens is an important anaerobic spore-forming, Gram-positive, non-motile
and rods bacteria that are affecting humans and animals. The organism is the normal inhabitant
of the alimentary tract of goats [1]. The environment in the GIT is altered by sudden change in
diet or other factors (high concentrations of grains or animals are heavily parasitized with
gastrointestinal parasites or less immune system). C. perfringens proliferates rapidly and
generates large amount of toxins producing the disease [2]. C. perfringens produce 5 major
types (A-E) based on the types of toxins it produces. Alpha (α) toxin is produced by all
toxinotype of C. perfringens. Beta (β) toxin is produce by ‘B’ and ‘C’ types. Epsilon (ε) toxin
is produced by ‘B’ and ‘D’ type of C. perfringens while iota (ι) toxin is produced only by type
‘E’ along with alpha (α) toxin [3].
Enterotoxaemia in goats is caused by different toxin types of C. perfringens with case fatality
rates leading to considerable economic losses to the farmers [4]. It is one of the most frequently
occurring diseases of sheeps and goats worldwide with reported prevalence rates ranging
between 24.13% and 100% [5]. Enterotoxaemia is one of the major endemic diseases of small
ruminants in Pakistan. The disease mainly occurs in sheeps and goats and rarely in large
animals [6].
The diagnosis of C.perfringens is usually based on clinical signs and pathological findings, but
identification of toxins in GIT contents is necessary to confirm the diagnosis. The most widely
used method for toxin detection is the serological and molecular techniques. The Indirect
ELISA Test (IELISA) is also used for the detection of antibodies to C. perforinges, but
serological cross reactions make species diagnosis difficult. The polymerase chain reaction is
the most sensitive and specific technique used to detect C.perfringens. The present study is
designed to identify the C.perfringens through PCR in goats of study area.
Objectives
1. To detect C.perfringens in goats through Polymerase Chain Reaction.
2. To find out the different types of toxins produced by C.perfringens.
3. To determine the epidemiological characteristics of C.perfringens in district Bannu.

3. Materials and Methods
Study Area and Samples Collection
This present study will be conducted in district Bannu. Geographically it is a temperate region
situated on the Southern part of Khyber Pakhtunkhwa, having 877 km2 area. It lies between
32∘ -43 and 33∘ -06N latitude and 73∘ - 20 and 70∘ -07E longitude. Approximately 5 ml of
blood samples will be collected from clinically suspected goats through jugular vein in EDTA
tubes after taking consent form from the Owners and blood will be keep at 4°C for further
analysis.
Microscopic Examination
Blood smears will be prepared and stained with Giemsa stain according to the prescribed
protocol. Slides will be examined for the presence or absence of C.perfringens through
microscope.
DNA Extraction and Amplification (PCR)
DNA will be extracted from the collected blood samples under the standard protocol. The target
DNA will be amplified through Polymerase chain reaction as described elsewhere [7].
Gel Electrophoresis
The PCR reaction will be run on gel electrophoresis for further detection of C. perfringens.
Statistical Analysis
Data will be statistically analyzed through appropriate statistical tests using SPSS version 20.
Time frame
S. No Research component Time required
1 Review of literature Two months
2 Research work Five months
3 Data interpretation Two months
4 Thesis writing Three months

4. REFRANCES
[1] Uzal, F.AVidal, J.E., McClane, B.A. and Guraj, A.A. (2014). “C. perfringens toxins
involved in mammalian veterinary diseases”, Open Toxicol. J, 2: 24-42.
[2] Netherwood, T., Binns, M., Townsend, H., Wood, J. l., Mumford, J. A. & Chanter, N.
(1998) "The C. perfringens enterotoxin factors from equine isolates; its
characterization, sequence and role in foal diarrhea”. Epidemiology and Infection
120:193-200.
[3] Petit L, Gilbert M, Popoff MR (1999) “Clostridium perfringens Toxinotype and
genotype”, Trends Microbiol, 7: 104-110.
[4] Rood, J.I. and Cole, S.T. (1991) “Molecular genetics and pathogenesis of Clostridium
perfringes”. Microbiol. Rev., 55(4): 621-648.
[5] “Evaluation of enzyme-linked immunosorbent assay for diagnosis of C. perfringens
enterotoxemias”. Vet. Microbiol. 31: 389-396.
[6] Javed MT, M Irfan, N Mukhtar, S Rahman and R Hussain, (2009) “An outbreak of
enterotoxaemia at livestock farm during subtropical summer”. Acta Trop, 112: 225-
227.
[7] Van Asten AJ, Van der Wiel CW, (2009) “A multiplex PCR for toxin typing of
Clostridium perfringens isolates”. Vet Microbiol, 136: 411-412,