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Molecular Epidemiologic Study on Avian Rotavirus Prevailing in Bangladesh

M. R. Karim
Department of Animal Breeding and Genetics, Barisal Government Veterinary College, Barisal, Bangladesh.

F. I. Rume
Department of Animal Breeding and Genetics, Barisal Government Veterinary College, Barisal, Bangladesh.

M. M. Alam
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh..

M. U. Ahmed
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Abstract/ Executive Summary:

The present study on molecular epidemiologic study on avian rotavirus prevailing in Bangladesh was attempted to know the present status of avian rotavirus in Bangladesh. A total of 210 faecal samples of chickens were collected from August 2006 to March 2007 and examined by polyacrylamide gel electrophoresis and silver staining (PAGE-ss) technique for the presence of rotavirus dsRNA. The prevalence of avian rotavirus infection was 13.81% in chickens. The prevalence of avian rotavirus infection in Chittagong, Mymensingh, Gazipur and Barisal were 2.86%, 21.25%, 5% and 22.5%, respectively. The highest prevalence was recorded in Barisal (22.5%) and the lowest in Chittagong (2.86%). The highest prevalence (40%) was observed in 12 day-old birds and the lowest (5.71%) in 22 days old birds. The highest percentage (18%) of avian rotavirus was detected in the summer season. The migration patterns of all detected positive strains were similar in gel electrophoresis and their migration speed was same as previously designated avian rotavirus group D.

Keywords: Epidemiology, PAGE, Rotavirus, Chickens

Location: a) Chittagong: Pahartoli Zonal Poultry Farm (PZPF), Nazma Poultry Farm; b) Barisal: Ahmed Ali Poultry Farm, Kader Poultry Farm; c) Mymensingh: Tania Poultry Farm, Soma Poultry Farm and d) Gazipur: Shaikat Poultry Farm.

Start Date: 00-08-2006

End Date: 00-03-2007

Program Area: Animal Health and Management

Commodity: Chicken

Objectives:

To identify the molecular epidemiologic study on avian rotavirus prevailing in Bangladesh.

Methodology:

Faecal samples were collected from 210 diarrhoeic and nondiarrhoeic poultry birds. The faecal samples were collected from the cloaca of the birds and from the litter (bedding) immediately after voiding by the poultry birds (layer and broiler). For each bird, 5-10 g sample was collected. Precautions were taken to avoid contamination of one sample with other. The date of collection, age, clinical signs and environmental history were recorded for each case. After collection, the samples were transported to the laboratory of the Department of Medicine, BAU, Mymensingh with ice pack and stored at -20⁰C until used for electrophoresis. After thawing the stored samples, 1 g of each sample was taken in a test tube and 9 ml of PBS of PH 7.4 was added. Faeces and PBS were thoroughly suspended by using a spinmix. After thawing, the faecal suspension was thoroughly mixed and vortexed and centrifuged at 15000 rpm for 15 minutes and then at 10000 rpm for 10 minutes. 300 μl of supernatant was collected separately in eppendorf tube and 60 μl of disrupting solution (6% Sodium Dodecyl Sulphate, 0.6% 2-Mercaptoethanol and 0.036 M EDTA) was added in each eppendorf tube and vortexed for 30 seconds. Then the eppendorf tubes were incubated at 37⁰C for 30 minutes. After incubation, 500 μl of saturated phenol was added to each eppendorf tube and vortexed for 30 seconds. The eppendorf tubes were then centrifuged at 10000 rpm for 1 minute in eppendorf microcentrifuge. The upper clear aqueous phase was collected separately in another eppendorf tube. A volume of 1:10 of 5 M sodium chloride and 3 volume of chilled ethanol were added to each eppendorf tube. RNA was precipitated at –20⁰C for overnight. After thawing, RNA was pelleted by centrifugation the eppendorf tube at 10000 rpm for 3 minutes. The liquid from the eppendorf tube was discarded carefully. The RNA pellet was dried at 37⁰C. The dried RNA was suspended in 10 μl of sample buffer (0.12 M Tris-hydrochloride, 15% glycerol and 0.001% bromophenol blue). Electrophoresis of the viral RNA was carried out in 10% polyacrylamide slab gels. After preparing the gels in slabs, the two gel slabs were set on the electrophoresis chamber. Electrode buffer PH 8.3 (0.25 M Tris-hydrochloride, 0.192 M glycine, 0.001 M EDTA) was pored on the chamber and in between the two slabs. Thereafter, 10 μl of diluted RNA was loaded separately on the wells of the gels and the corresponding well was recorded. The electrophoresis was performed at 40 mAmp (for double gels) for 16 hours. After electrophoresis, the gels were stained with staining solution (0.011 M silver nitrate) for 2 hours with continuous shaking. Then, the gels were washed three times in distilled water for 5 minutes. Finally the reduction step was performed by adding developing solution (0.75 M sodium hydroxide, 0.1 M formaldehyde) in gel trays and with continuous shaking. The double-stranded 11 RNA segments of avian rotavirus were divided into 4 groups for better classification. RNA segment bands 1-5 are denoted as group I, bands 6 and 7 as group II, bands 8 and 9 as group III and bands 10 and 11 as group IV.

Source of Information: Bangl. J. Vet. Med. (2007). 5 (1 & 2): 43–48

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In this study, all rotavirus infected birds were found diarrhoeic, dehydrated, anorectic and low body weight. These observations are reported that in field conditions, rotavirus infections in poultry might induce subclinical manifestations, or they might be associated with enteritis, dehydration, anorexia, unrest, litter ingestion, low weight gain and increased mortality. From the findings, it may be concluded that the prevalence of avian rotavirus infection was 13.81% in chickens in Bangladesh. Rotavirus infection was high in young birds and gradually became low in older birds. The prevalence of avian rotavirus infection was high in Barisal district and low in Chittagong district. Avian rotavirus infection was high in summer season. Only group D rotavirus was found during the study period in Bangladesh. Therefore, there is a possibility of happening higher rate of rotavirus infection in chickens of Bangladesh in near future.

Knowledge Management: Journal