AMAM Zonaed Siddiki*
Department of Pathology and Parasitology, Chittagong Veterinary and Animal Sciences University, Chittagong-4225, Bangladesh
Sohana Akter Mina
Department of Genetic Engineering and Biotechnology, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh
Md. Anayet Hasan
Department of Genetic Engineering and Biotechnology, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh
Mohammed Touaha Akbar
Department of Genetic Engineering and Biotechnology, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh
Rashel Alam
Department of Genetic Engineering and Biotechnology, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh
Israt Zahan Ira
Department of Clinical Pharmacy and Pharmacology, Noakhali Science and Technology University, Noakhali, Bangladesh
Bibi Ayesa
Department of Genetic Engineering and Biotechnology, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh
Md. Ashraful Islam
Department of Genetic Engineering and Biotechnology, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh
Eimeria , PCR, SCAR, Coccidiosis, Bangladesh
Pest Management
Isolation and washing down of Eimeria oocysts from litter samples This research work was conducted with litter samples from broiler farms where coccidiosis is suspected based on bloody droppings. Total 18 litter samples were collected manually from different poultry farms located in different parts of Chittagong Metropolitan area in Southern Bangladesh. Fresh litters were collected by random sampling from 18 broiler chickens flocks with or without previous exposure to anticoccidial vaccines. Oocysts were excreted from the host via the feces. For this study the previously established protocol (Collected from Royal Veterinary College, London, UK) were used. The protocol describes the collection of fecal material, together with the purification of oocysts from it through soaking in water, mixing with a blender and separation of coarse materials by sieving. A combined centrifugation and salt floatation step was incorporated to separate the oocysts from fecal materials based upon a second criterion: specific gravity. The recovered oocysts were then washed out of salt and sporulated. After purification, the oocysts were preserved in 2.5% potassium dichromate at 28º. The potassium dichromate was removed before these oocysts were used for DNA extraction for subsequent use for PCR analyses.
Parasitologic examination Using a calibrated ocular micrometer at 400x magnification, a modified saturated salt floatation technique was used to isolate oocysts for length measurements. Fifty random oocysts from each sample were examined by measuring their length and width with light microscopy, armed with calibrate ocular lens as well as determination of the oocysts shape and index (Length/Width). Sporocysts’ diameters were also determined for more accurate diagnosis.
Genomic DNA preparation Purified oocysts were washed in PBS, disrupted using 0.5 mm glass beads and DNA was extracted from the lysate using the Qiagen Mini Stool DNA Kit (Qiagen, Germany) according to the manufacturer’s instructions and stored at -20°C. Extracted DNA concentration was measured by Thermo scientific Nano drop 2000 spectrophotometer (USA)
PCR amplification PCR targets were designed using the SCARdb database of species-specific SCARs described before [4]. The markers were designed in non-repetitive regions of each SCAR identified using Tandem repeats finder version 4.00 as a screen. The length of each PCR target was chosen to be between 100 and 150 bp. Primers and probes were designed using Primer3 software. Reactions were performed using GoTaq Green Mastermix (Promega, USA), 500uM forward and reverse primers and 5 ul template DNA in a 20ul volume and run using a Applied Biosystem PCR thermal cycler (USA). For positive controls, Paracox® a commercial vaccine, which included all the eight pathogenic Eimeria species of chicken was used and water was used as negative control in PCR. The thermal cycling program consisted of initial denaturation at 950C for 4 min followed by 40 cycles of denaturation at 940C for 30s, annealing at 580C for 30s and extension at 680C for 2 min. The final extension was set at 680C for 5 min. To verify the results, 10 µl of each PCR product was electrophoresed in a 1.5% agarose gel (Cat no. # DV3123, Promega®), stained with SYBR Green Fuorescent Dye (Sigma, USA), and visualized on a UV transilluminator (Biometra, Germany). The PCR products were identified by size using a 100 base pair ladder (Cat. no# RD002, RBC Bioscience, Taiwan).
Microscopic examination and oocyst count The aim of the study was to use modern molecular biological tools to diagnose Eimeria species causing chicken coccidiosis in Bangladesh. Following standard protocol, the oocysts were collected from selected poultry farms with history of bloody diarrhea and moist litter. A modified saturated salt flotation technique was used to isolate oocysts for micrometry using a calibrated ocular micrometer at 400x magnification. Using a camera fitted with microscope, images of different oocysts were captured where three species of Eimeria including E. tenella, E. acervulina, E. maxima were ovserved clearly. Several other oocysts were not possible to identify based on size itself. Among three observed species E. tenella was predominant. The numbers of oocysts were counted using McMaster technique as found in different isolates The concentration of oocysts were different in each isolate which were from 1600-300,000.
IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS) e-ISSN: 2319-2380, p-ISSN: 2319-2372.Volume 7, Issue 1 Ver. I (Jan. 2014), PP 13-17
Journal