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Conventional Pcr Based Detection of Brucella Abortus Infected Cattle in Some Selected Areas of Bangladesh

M. A. S. Sarker
Department of Livestock Services, Dhaka, Bangladesh

M. M. Begum
Youth Training Centre, Munshigonj, Ministry of Youth and Sports, Dhaka, Bangladesh

M. F. Rahman
Commandant Armed Forces Medical College, Dhaka Cantonment, Dhaka

M. T. Islam
Department of Medicine, Bangladesh Agricultural University, Mymensingh2202, Bangladesh

M. A. Rahman
Department of Surgery and Theriogenology, Jhenidah Government Veterinary College, Jhenidah, Bangladesh

L. Yasmin
Department of Livestock Services, Dhaka, Bangladesh

M. A. Ehsan
Commandant Armed Forces Medical College, Dhaka Cantonment, Dhaka

M. S. Rahman
Commandant Armed Forces Medical College, Dhaka Cantonment, Dhaka

Abstract/ Executive Summary:

Brucella spp. are facultative intracellular bacteria causing chronic disease which may persist for the whole life of the affected organism. In animals brucellosis affects reproduction, fertility and reduces newborns survival and also milk production. The present research was carried out to estimate the prevalence and to identify the risk factors of brucellosis along with determination of genetic diversity in Bangladesh. In CCBS&DF (government farm) out of 191 cows the MRT prevalence (positive) was 7.85% and RBT prevalence (positive) was 7.33%. In Rangpur out of 238 cows the MRT prevalence (positive) was 1.88% and RBT prevalence is 1.56%. In Jamalpur out of 201 cows the MRT prevalence was 1.49% and RBT prevalence 0.05%. In Gaibandha out of 93 cows the MRT prevalence was 1.07% and RBT prevalence 0%. In Mymensingh out of 320 cows the MRT prevalence was 1.88% and RBT prevalence 1.56%. Among the five groups of cows the high prevalence of MRT and RBT prevalence was in Government Farm 7.85% and 7.33%. On the other hand the lower prevalence in cows of Gaibandha which was MRT and RBT 1.07% and 0% respectively and followed by Jamalpur MRT 1.49% and RBT 0.50%, in Rangpur MRT 1.68% and RBT 1.26%, and in Mymensingh district MRT 1.88% and RBT 1.56%. The prevalence of brucellosis was significantly (p<0.01) higher in CCBS&DF than all district (Table 8). Out of 14 MRT and RBT positive milk samples of CCBS&DF (21.43%) were PCR positive but all other 9 such samples originated from Jamalpur, Rangpur, Gaibandha and Mymensingh districts were PCR negative.

Keywords: Cattle, MRT, RBT, PCR, Prevalence, Area, Brucellosis

Location: Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Cattle, Diseases

Objectives:

To know the area wise prevalence of brucellosis in cattle and conventional PCR based detection of Brucella abortus in organized and smallholder dairy farms in some selected areas of Bangladesh.

Methodology:

The molecular tests were performed like conventional PCR at Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh. DNA extracted from milk samples by using Wizard Genomic DNA Purification Kit (Promega, USA) according to manufacturer’s instruction and then PCR was performed. Briefly, at first, the required number of PCR tubes were labeled and kept on ice. Then 23 µl of reaction mixture was dispensed into each of the PCR tubes and 2 µl of DNA template from each sample was added to that respective tube and mixed well with the help of the micropipette. The tubes were placed in a twenty-four wells thermo cycler (Eppendorf, Germany). Then the temperature of the thermo cycler was set according to the thermal profile mentioned below. After completion of PCR, PCR products were separated by electrophoresis in a (1X TAE) 1% agarose gel stained by ethidium bromide. The band was then visualized with a medium wavelength UV light. In this study required clinical, epidemiological, environmental and reproductive information recorded. During the study period a questionnaire based data on age sex, geographical area, status of pregnancy, disease history, hygroma, reproductive disorder such as abnormal abdominal uterine discharge, abortion, retention of placenta, and reproductive diseases were recorded. The RBT was used as a screening test to identify the infected animal and human with Brucella abortus strain1119-3 (Dae Sung Microbiological lab, South Korea). iELISA (Svanova Biotech AB, Uppsala Sweden) RBT and SAT were performed according to the procedure described by OIE (2009). The RBT positive sera were re-tested with SAT CFT, ELISA and qRT-PCR. For the qRT-PCR, DNA was isolated from 200 µL of seropositive serum using the high pure PCR template Preparation Kit (Roche Diagnostic, Mannheim, Germany) according to manufacturer’s instructions. Brucella IS711 targeting genus specific qRT-PCR was done according to the established and routine protocol (Tomaso et al., 2010) on a light cycler 2.0 instrument (Roche, Mannheim, Germany). Cycler threshold values (CT) ≤ 40 were interpreted as positive. Positive samples were then typed with the Brucella IS711 species specific qRT-PCRs for Brucella abortus and Brucella melitensis according to Probert et al. (2004). CT values were calculated by the instrument’s software MxPro3000P v 4.01. CT values ≤ 42 were interpreted as positive. iELISA and CFT were performed according to protocol provided by the iELISA and CFT kits manufacturer company. Data analysis: The Chi-square test (χ2) was performed to find out the relationship between the prevalence of brucellosis and demographic variables of cows. The questionnaire based data was processed by Microsoft Excel and MSTATC, the results were statistically analyzed for interpretation by using Chi-square tests. Significance was determined at 1 to 5% level where applicable.

Source of Information: Bangl. J. Vet. Med. (2018). 16(1): 39–44 ISSN: 1729-7893 (Print), 2308-0922 (Online)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In CCBS&DF (government farm) out of 191 cows the MRT prevalence (positive) was 7.85% and RBT prevalence (positive) was 7.33%. In Rangpur out of 238 cows the MRT prevalence (positive) was 1.88% and RBT prevalence is 1.56%. In Jamalpur out of 201 cows the MRT prevalence was 1.49% and RBT prevalence 0.05%. In Gaibandha out of 93 cows the MRT prevalence was 1.07% and RBT prevalence 0%. In Mymensingh out of 320 cows the MRT prevalence was 1.88% and RBT prevalence 1.56%. Among the five groups of cows the high prevalence of MRT and RBT prevalence was in Government Farm 7.85% and 7.33%. On the other hand the lower prevalence in cows of Gaibandha which was MRT and RBT 1.07% and 0% respectively and followed by Jamalpur MRT 1.49% and RBT 0.50%, in Rangpur MRT 1.68% and RBT 1.26% , and in Mymensingh district MRT 1.88% and RBT 1.56%. The prevalence of brucellosis was significantly (p<0.01) higher in CCBS&DF than all district (Table 8). Out of 14 MRT and RBT positive milk samples of CCBS&DF (21.43%) were PCR positive but all other 9 such samples originated from Jamalpur, Rangpur, Gaibandha and Mymensingh districts were PCR negative. Currently veterinary diagnostic laboratories utilize Milk Ring Test (MRT) for diagnosis of bovine milk samples, which indirectly identifies Brucella spp. in the host (Chimana et al., 2010).Just MRT positivity does indicate acute infection. To declare acute infection evidence of Brucella organism or detection of Brucella DNA in animal sample is essential (Bricker, 2002; Gupta et al., 2006; Hamdy and Amin, 2002). Acute infection of brucellosis in dairy cattle can be determined by MRT, RBT and conventional PCR techniques. This finding will help to cull dairy cattle acutely infected with brucellosis having serious public health hazard.

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