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Isolation and Molecular Detection of Fowl Pox and Pigeon Pox Viruses for the Development of Live Attenuated Vaccine Seeds from the Local Isolates

Rebeka Sultana
Department of Microbiology & Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

KHM Nazmul Hossain Nazir
Department of Microbiology & Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Tanvir Rahman
Department of Microbiology & Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Shamim Ara Nipa
Department of Microbiology & Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Mizanur Rahman
Department of Microbiology & Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Sabiha Sultana Soma
Department of Microbiology & Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Bahanur Rahman
Department of Microbiology & Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Avipox is a viral disease of fowl and pigeon which is characterized by proliferative and nodular lesions in the feather-free parts of the skin or fibro-necrotic and proliferating part in the mouth, esophagus, and mucous membrane of the upper respiratory tract. This investigation was carried out with an aim to isolate and molecular detection of Fowl pox virus (FPV) and Pigeon pox virus (PPV) for development of live attenuated vaccine seeds from the local virus isolates. In this study, nodular lesions were collected from seven pigeons and four chickens from different areas of Mymensingh in Bangladesh which were affected by pox. Viral inoculums were prepared and DNA materials were extracted for PCR-based identification of P4b genes. Detection of virus was confirmed by PCR following propagation into 9-11 days old embryonated chicken egg (ECE) and also chicken embryo fibroblast (CEF) cell culture All the field samples were found positive for FPV and PPV by PCRR. These field isolates were propagated and attenuated in duck embryo through CAM route and duck embryo fibroblast (DEF) cell culture for the development of live attenuated vaccine seeds. Attenuation of both FPV and PPV were successful in duck embryo through CAM route and duck embryo fibroblast (DEF) cell culture after serial six passages. Attenuation of the virus was confirmed by inoculation into experimental birds. Inoculation of attenuated FPV and PPV in chicken and pigeon respectively exhibited no pox lesions whereas control chicken and pigeon inoculated with field isolates develop nodular lesions. Both FPV and PPV were confirmed from both groups of birds by PCR. These attenuated local isolates of FPV and PPV could be used as potential vaccine candidates for the prevention and control of fowl pox and pigeon pox in Bangladesh.

Keywords: Fowl pox virus, Pigeon pox virus, PCR, CEF, DEF, Propagation, Attenuation

Location: Different areas of Mymensingh in Bangladesh

Start Date: 00-01-2017

End Date: 00-11-2018

Program Area: Animal Health and Management

Commodity: Pigeon

Objectives:

The aim of this study was to isolate and identify Fowl pox and Pigeon pox viruses by molecular techniques from chickens and pigeons and development of live attenuated vaccine seeds.

Methodology:

Sample Collection A total of 11 samples (Fowl Pox=4 and Pigeon Pox=7) were collected from Mymensingh district during the period of January, 2017 to November, 2018. Nodular lesions were screened using sterile blade from Fowl pox (FP) and Pigeon pox infected sick birds and placed in eppendorf tube containing 0.5 ml sterile Virus transport medium (VTM). Samples were immediately transported in the laboratory of Department of Microbiology and Hygiene in cool box containing ice packs. The experimental protocols involving the use of birds and cell cultures were approved by the institutional ethical committee (AWEEC/BAU/2019(5). Inoculum Preparation Both FP and PP suspected birds samples (nodules) were subjected to grinding and mixed with phosphate buffered saline (PBS) for the preparation of 10% (w/v) viral suspension. The viral suspensions were then treated by broad spectrum antibiotic gentamycin (Gentin® 20, Opsonin Pharma. Ltd., Bangladesh) at 300µl/ml for 60 minutes followed by spreading on blood agar supplemented with 5% bovine blood (HI media, India) and nutrient agar (HI media, India), incubate for 24 hours. Molecular detection of Fowl pox virus (FPV) and Pigeon pox virus (PPV) DNA was extracted by chemical method using SV total RNA isolation system (Promega, USA) according to the protocol described by the manufacturer. P4b gene specific PCR was performed in a thermal cycler (Mastercycler, Eppendorf, Germany) using the condition described by Roy et al. (2013) with slide modification. T A 25µl of PCR mixture was prepared by mixing green PCR master mix (Promega®, USA) (12.5 µl), forward primer (1µl), reverse primer (1 µl), nuclease free water (3.5 µl) and template DNA (7 µl). Thermal profile used for the amplification of P4b gene was: initial denaturation at 94°C for 5 min; followed by 35 cycles of reaction consisting of 94°C for 45 seconds, 48°C for 1.5 min, 60°C for 2 min, along with final extension at 60°C for 10 min. The PCR products were analyzed using 1.5% agarose gel (Sigma-Aldrich, USA). Isolation, Propagation and PCR detection of FPV and PPV The inoculums were inoculated into 9-11 days-old embryonated chicken eggs (ECE) at the doses of 0.2 ml through chorioallantoic membrane (CAM) route and 1 ml in chicken embryo fibroblast (CEF) cell culture for primary propagation and isolation of viruses. Simultaneously DNA was extracted directly using kit (SV Total RNA isolation system, Promega, USA) from the inoculum for performing PCR targeting P4b gene to detect the positive samples. Attenuation of Fowl pox virus (FPV) and Pigeon pox virus (PPV) After primary propagation, and isolation of the viruses in ECE and CEF cell culture and confirmation by PCR, these propagated viruses were further inoculated into embryonated duck egg (EDE) at the dose of 0.2 ml through CAM route and also 1ml in duck embryo fibroblast (DEF) cell culture for adaptation and subsequent attenuation. Adaptation of viruses in DEF cell culture was confirmed by observing characteristics cytopathic effect (CPE) using inverted microscope (Carl Zeiss, Germany) and finally confirmed by P4b gene specific PCR. After adaptation of viruses both in EDE and DEF cell culture system, the viruses were serially passaged for six times in the systems for live attenuated vaccine seeds preparation. Determination of TCID50 of Fowl pox virus (FPV) and Pigeon pox virus (PPV) From original virus sample, 100 µl virus samples were taken and a series of dilutions at 1:10 of the virus was made. Diluted virus inoculums of 100 µl from each dilution were added to each column of the quadruplicate wells. The plates of virus were tapped tightly and tilting was done carefully for 90 minutes. Then 500 µl of infection media was added to each well. The plate was then kept at 37ºC for 48 hours and then monitoring of CPE using the inverted microscope. The number of positive and negative wells was recorded. Result of the TCID50 titer was calculated by the Reed and Muench (1938) method. Testing of attenuation in experimental birds Attenuation of both Fowl pox and Pigeon pox viruses were checked by inoculating them in experimental birds. For this purpose six chicks and six squabs were bought from local market of Mymensingh. Those birds were divided into two groups A and B. In case of chicks, two birds in group A and four birds in group B and similarly, in case of squabs, two squabs in group A and four squabs in group B. Attenuation was confirmed by inoculating the attenuated inoculum of FPV and PPV into group B of both chicks and squabs separately through nasal route at the dose of 0.5 ml (TCID50106.67/ml) and was observed for two weeks. Group A of both chicks and squabs kept as control and was inoculated by ECE propagated FP and PP virus inoculum through nasal route at the dose of 0.5 ml (TCID50106.67/ml) and was also observed for two weeks.

Source of Information: J Bangladesh Agril Univ 17(2): 211–219, 2019 ISSN 1810-3030 (Print) 2408-8684 (Online)

Article Link (Online Journal): https://doi.org/10.3329/jbau.v17i2.41971

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

All the four Fowl pox field samples and seven Pigeon pox field samples were found positive for the presence of FPV and PPV respectively by PCR. Propagation of FPV and PPV was successfully done in chicken embryo both on CAM route and CEF cell culture. Attenuation of FPV and PPV was also done successfully in duck embryo both on CAM route and DEF cell culture for the development of live attenuated vaccine seeds after six serial passages. The attenuation of the developed live attenuated vaccine seeds were checked in experimental birds. These attenuated FPV and PPV can be used as potential vaccine candidate for the eradication of avipox outbreaks in Bangladesh.

Knowledge Management: Journal