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Reverse Transcription Polymerase Chain Reaction (rt-pcr) Based Detection and Serotyping of Fmd Virus from Field Samples of Gazipur, Bangladesh, and Adaptation of the Virus in Bhk-21 Cell

Mohammad Ashraful Alam
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Marzia Rahman
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Liakot Hossen
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Sultan Ahmed
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Shafiullah Parvej
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Mohammad Ferdousur Rahman Khan
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. Bahanur Rahman
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

The study aimed for the detection and serotyping of Foot and Mouth Disease virus (FMDV) circulating in Kapasia Upazila, Gazipur district of Bangladesh during 2013. Twelve samples comprising of tongue epithelium (n=8) and inter digital tissue (n=4) were collected from suspected cattle, and inocula were prepared. The inocula were inoculated into confluent BHK-21 cell line for virus propagation. After 3 subsequent passages; progressive cytopathic effects (CPE) specific for FMDV i.e., rounding and flattening of cells, breaking down of the intercellular bridge and finally cell death (almost 100%) were observed; these were indicative of successful virus propagation in the cells. Viral RNA was extracted, and Reverse Transcription Polymerase Chain Reaction (RT-PCR) was performed using three sets of primers corresponding to the serotype ‘O’, ‘Asia-1’ and ‘A’, respectively. Out of the 12 samples, 10 (83.33%) were found to be positive for FMDV, and all of those were of serotype ‘O’. It is concluded that FMDV serotype ‘O’ is circulating among the cattle of Gazipur district, Bangladesh.

Keywords: BHK-21 cell; cytopathic effects; FMDV; RT-PCR

Location: Kapasia Upazila, Gazipur district of Bangladesh

Start Date: 00-00-2013

End Date: 00-00-2013

Program Area: Animal Health and Management

Commodity: Cattle

Objectives:

To identify the types of FMDV that are currently circulating in Gazipur, Bangladesh, so that the findings of the study can be used to adopt effective disease management and control strategies in Bangladesh.

Methodology:

Study area: FMDV samples i.e., tongue epithelium (T.E), and foot tissues (F.T) were collected from different villages of Kapasia Upazila (sub-district) under the district of Gazipur, during the period of January to November 2013.
Sample collection and processing: A total of 12 samples comprising of 8 tongue epithelia and 4 foot tissue from inter-digital space were collected aseptically from FMD affected cattle. The samples were collected in virus transport medium (VTM) containing 10,000 μg Streptomycin, 10,000 IU Penicillin and 25 μg Amphotericin B, and immediately transported in cool condition to the Virology Laboratory, Department of Microbiology and Hygiene, Bangladesh Agricultural University for analysis. The field samples were then homogenized with mortar and pestle separately, and 10% suspensions were prepared by adding sterile phosphate buffered saline (PBS). The suspension was then centrifuged at 5000 rpm for 15 min at 4ºC, and the supernatant was collected. The collected supernatant was treated with 10,000 μg Streptomycin, 10,000 IU Penicillin and 25 μg Amphotericin B for 1 h (at 37ºC), and then filtered with 0.22 μm filter. Sterility of the inocula was tested in fresh blood agar media and stored at -80ºC for future use.
Adaptation and propagation of virus in BHK-21 cell line: The cells those were found as complete and confluent monolayer in the culture flask within 24 h of incubation were selected for infection with viruses. The growth media from the flask containing BHK-21 cell was removed and then the monolayer cells were washed with sterile PBS for 2 times. The inoculum was spread over the cell sheet by tilting at 37ºC for about 60 min for better adsorption. Then, 5 mL of the maintenance media (2% heat inactivated FBS) was added in a 25 cm2 flask and it was then returned to the incubator and kept at 37ºC. The cells were examined twice daily under inverted microscope (Carlt Zeiss®, Germany) until showing characteristic cytopathic effects (CPE) caused by FMDV. Various kinds of characteristic changes of cell rounding, swelling, breaking down of intercellular bridge and finally cell death indicated the presence of FMDV in the sample. In this way, all the 12 (100%) samples were successfully passaged in BHK-21 cell line, and the infectious fluid (IF) could be harvested after 48 to 72 h of post-infection for further investigation for detection and serotyping of the FMDV by RT-PCR.
Viral RNA extraction and RT-PCR: Viral RNA was extracted from cell culture fluid using SV Total RNA Isolation System (Promega, USA), according to the instructions of the manufacturer. RT-PCR was carried out by Access RT-PCR system (Promega, USA) according to the manufacturer?s protocol using specific primers (Table 2). The thermal profile used for cDNA synthesis was at 45ºC for 45 min and at 94ºC for 2 min for one cycle. For PCR amplification, thermal profile was used as initial denaturation at 94ºC for 5 min, followed by denaturation at 94ºC for 30 sec, annealing at 60ºC for 1 min, extension at 68ºC for 2 min, for 40 cycles, and a final extension at 68ºC for 7 min. After amplification, the amplicon was then visualized in 2% agarose gel stained with ethidium bromide after electrophoresis.
Agarose gel electrophoresis: The PCR products were analyzed in 2% agarose gel, stained with ethidium bromide and examined against UV light using an UV Solo-transilluminator (Biometra®, Germany). The positive sample was recorded based on the appearance of expected size of band in the gel.

Source of Information: J Adv Vet Anim Res. 2015; 2(3): 291-295; eISSN: 2311-7710;

Article Link (Online Journal): doi: 10.5455/javar.2015.b88

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Tongue epithelium is found to be better as compared to foot tissue as a source of FMD virus for isolation. This study also confirms that BHK-21 cell line is highly sensitive for FMD virus isolation. Besides, RT-PCR can be used as an effective method for the detection of FMD virus serotype(s). Through this study, FMD virus serotype „O? is confirmed to be circulated among cattle of Gazipur, Bangladesh.

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