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Research Detail

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Md Salim Jahan
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md Mizanur Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Rony Ahmed
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Ajran Kabir
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

ShamsulKaunain Oli
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Jayedul Hassan
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Mahbubul Pratik Siddique
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Md Bahanur Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Duck plague is an enveloped DNA virus that belongs to the Anatid Herpes Virus; the Herpesviridae family is an acute and highly infectious duck, geese, and swan disease that causes tremendous economic losses of duck rearing in Bangladesh and other duck rearing countries. Therefore, we decided to isolate duck plague virus from recent fields’ outbreaks area and performed molecular detection and phylogenetic analysis to find out the similarities between our findings and other isolates around the world. Visceral organs of 13 suspected ducks from recent outbreaks area were collected by post-mortem examination for inoculum preparation. Several passages were performed to harvest into 9-11 old embryonated eggs Chorioallantoic membrane (CAM) route and duck embryo fibroblast (DEF) primary cell culture. DNA polymerase (446bp) and DNA polymerase (UL, 602bp) genes were used for molecular detection by Polymerase chain reaction (PCR). Pathogenicity was done with duckling and TCID50 on DEF. Molecular characterization was performed from extracted DNA of duckling and 2 Positive PCR products were partially sequenced for phylogenetic analysis of their origin and nucleotide variations. Sequenced data was analyzed to reveal genetic relationships among constructed phylogenetic tree for understanding potential transmission with origin of virus and data was then submitted to gene bank and got accession number for DPV-BR1-MN937272 and DPV-BR-2-MN937273. Among 13 samples, 4(30.77%) were found positive by PCR using DNA polymerase at 446 bp and UL at 602 bp gene. Chorioallantoic membrane (CAM) was observed hemorrhagic after 72 days and duck embryo fibroblast (DEF) become round as showed cytopathic characteristics after 48h of infection. Duckling showed that isolated virus was highly pathogenic as characteristics signs of post-mortem examination. Therefore, this has found that recent isolates have similarity with Bangladesh, India and China isolates. Moreover, TCID50 has confirmed the isolates have accepted titer to be a vaccine strain.

  CAM, DEF, DNA, PCR, Phylogenetic tree
  Mohanganj Upazila of Netrokona, and Trishal Upazila of Mymensingh districts, Bangladesh.
  00-06-2018
  00-09-2018
  Animal Health and Management
  Virus, Duck

In the present study, the DPV isolated from the recent field outbreaks using duck embryo and DEF cell culture systems for biological and molecular characterization of isolated DPV to develop a vaccine from the local strains.

 

Study of outbreaks area- The research work has conducted the study from June to September 2018 in two unfamiliar areas of the Mohanganj Upazila of Netrokona (24°34.5′N90°23.5′E), and Trishal Upazila of Mymensingh districts (24°34.5′N90°23.5′E). Where active affected areas have been evaluated by regular observation and contact with veterinary professionals working in veterinary clinics of the district government. Besides this field, we conducted investigations based on information on previous vaccination history and clinical signs where an active outbreak of DPV was reported. During we made sample collection visual inspection to observe typical clinical signs of DPV and did a detailed physical examination on affected ducks. Methods of sample collection- After taking permission from the owners, we collected 13 dead and live ducks from the commercial farms and households. It targeted Ducks showing typical clinical signs of DP including the contact Healthy ducks and the dead birds suspected to be dead of DPV infection to collect different clinical and postmortem samples for the study. Post Mortem (PM) examination was conducted according to (Neher et al., 2019, El'Tholoth et al., 2019) for dead and live infected birds and it scrutinized all internal organs for the presence of typical DP lesions. During the PM review, visceral organs like the heart, esophagus, proventriculus, spleen, liver, and intestine were collected. The post mortem examination was done aseptically and placed the collected organs in sterile falcon tubes with proper labeling. Then the samples were transported to the Virology Laboratory at the Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University (BAU), immediately after collection by maintaining a proper cold chain. The samples were then stored at -20°C till used for molecular identification and molecular characterization of DEV. Preparation of inoculum and sterility test- Each sample was crushed under sterile conditions prior to the analysis of collected samples according to a 10 % suspension using phosphate buffer solution for preparation (PBS)(Manual, 2012). Then the suspension was centrifuged at 6000 rpm for 10 minutes and it collected supernatant in a sterile falcon tube treated with 100 μg/ml antibiotics (Gentamicin). The sterile suspension was examined for sterility in fresh nutrient and blood agar as described at 37°C for 24 hours. Then stored the prepared sterile solution at -20°C for until use(Ahamed et al., 2015a). Propagation of DPV in embryonated duck eggs- It propagated the antibiotic-treated suspension containing viruses in the allantoic cavities of 10-day-old embryonated duck eggs through CAM route (Liu et al., 2011, Manual, 2012, Ahamed et al., 2015a, El-Samadony et al., 2013). Inoculated eggs were incubated at 37°C with properly humidified conditions and observed twice daily for the mortality of the embryo. The embryos that died within 72 hours were discarded as nonspecific deaths(Manual, 2012). After 6-10 days of inoculation, it chilled embryonated eggs at 4°C for overnight and allantoic fluid (AF) and CAM were collected. Isolation of Duck Plague Virus in duck Embryo Fibroblast (DEF) cell culture- Duck embryo fibroblasts (DEFs) cells were prepared freshly as described (Shi et al., 2019, Ager-Wick et al., 2018, Mondal et al., 2010, Zhao et al., 2019) from 11-day-old day-old embryonated duck eggs for isolation of wild strain DPV from the prepared solution. The duck embryo fibroblast (DEF) monolayer cells were grown in 25 cm2 tissue culture flasks incubated at 37°C with 5% CO2 humidified incubator (Liu et al., 2017) with Eagle’s minimal essential medium (EMEM, Sigma, USA) containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY), 100 U/ml penicillin, and 100 μg/ml streptomycin. For infection, it inoculated 1 ml of tissue suspension into 70% confluent cell culture flask with EMEM containing 3% FBS. The inoculated flasks were observed under an inverted microscope (Carl Zeiss, Germany) after 48-72 h postinfection (hpi) when the cytopathic effect (CPE) was over 75% (Lian et al., 2010, You et al., 2018, JING et al., 2017) and harvesting by freeze-thawing cycle and stored at -80° C for further use. Molecular detection of DPV by PCR- The viral DNA from cell suspension was then extracted using a DNA extraction kit (Promega, USA), according to the manufacturer's instructions. For confirmation of the target gene, it used two pairs of primers (DNA polymerase gene, UL gene) as described by (Wu et al., 2011, Zou et al., 2010) and it performed PCR for amplification. For amplification of DNA polymerase gene and UL gene, a total 25 µl PCR mixture was prepared to contain 12.5 µl master mix (Promega-Madison, WI, USA), Nuclease free water 1.5 µl, Primer Forward 2 µl, Primer Reverse 2 µl and Template (Extracted DNA) 7 µl. Thermal condition used for DNA polymerase gene amplification was: initial denaturation at 94°C for 4 min, 30 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 30 sec followed by a final extension at 72°C for 7 min. Further, the PCR condition for UL gene amplification was: initial denaturation at 94°C for 5 min; followed by 30 cycles of reaction denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension 72°C for 30 sec, and a final extension at 70°C for 7 min and stored the amplified PCR products at -20°C until confirmed by gel electrophoresis. Determination of DPV from PCR products by agarose gel electrophoresis- The amplified PCR products were examined by using agarose gel electrophoresis. For UL gene confirmation 1% (Mondal et al., 2010)(Mondal, B., et al.2010) and DNA polymerase (UL30) gene confirmation 1.5% (El'Tholoth et al., 2019)(El'Tholoth, Mohamed, et al.2019) agarose gel were used, respectively. An amount of 7 µL amplified PCR products was mixed well with 1 µL 6X loading dye (Promega, USA), and loaded to the agarose gel well. It stained the DNA with ethidium bromide after electrophoresis and using UV transilluminator (Biometra, Germany) for visualizing stained DNA. Determination of TCID50- The determination of virus titers by infection of duck embryo fibroblasts cell to be a 106.166/mL median tissue culture infective dose (TCID50)/mL. For this purpose, there used 48 well cell culture plate seeding with 100 µl of DEF cells and 500 µl of cell growth media to each well. After 24 hours, the plate observed under the inverted microscope (Carl Zeiss, Germany) for confluent growth and added 100 µl of virus suspension in each wells dilution with 10-1 to 10-8 (You et al., 2018, Li et al., 2016, Mondal et al., 2010, Zhao et al., 2019). Then the plates were incubated at 37ºC for 48 hours and calculated the TCID50/mL titer followed by the Reed &Muench (1938) method. Pathogenicity test in adult duckling- A small group of 4-week-old adult ducks was used for these studies. The ducks were inoculated with 1 mL 106.166 TCID50/mL serum-free minimum essential medium diluted virus through the subcutaneous and intramuscular route under the wings at several sites (Liu et al., 2011). Few adult ducks were kept separately for control and each of them inoculated with a 1 ml culture medium through the same route. After inoculation, ducks also observed for two weeks for clinical signs of disease, and it happened postmortem to re-isolate DPV, which was confirmed by PCR. Sequencing data analysis and construction of phylogenetic tree- For the further experiment, two positive PCR products of UL gene (602 bp) were sent to Invent Technology Ltd. for partial sequencing. The nucleotide sequence generated in this study has been deposited in the GenBank to obtain the accession number. Data analysis and multiple alignments were performed using Codon Code analyzer and MEGA X software. The sequence was compared with the GenBank database using the BLASTn. The evolutionary history was inferred with the Phylogenetic tree using the Neighbor-Joining method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 1000 replicates (Felsenstein, 1985) is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein, 1985). The evolutionary distances were computed using the p-distance method(Nei and Kumar, 2000) and are in the units of the number of base differences per site. This analysis involved 12 nucleotide sequences. All positions containing gaps and missing data were eliminated (complete deletion option). There were a total of 132 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Nei and Kumar, 2000).

  Res. Agric., Livest. Fish.8(1): 125-133, April 2021
  DOI: https://doi.org/10.3329/ralf.v8i1.53274
Funding Source:
1.   Budget:  
  

Our present research reveals the recently affected areas were conducted with DPV, which was confirmed by molecular characterization. The isolation of DP was carried out for the development of vaccines with local strains. DPVs showed the adaptation of embryonated duck eggs and DEFs cell culture with definite characteristics. Moreover, DPVs showed higher pathogenicity in duckling with specific clinical characteristics while infected with titer of TCID50/mL at 10-5 dilution. Our sequenced data and phylogenetic tree analysis reveal that isolated DPV has higher similarity with China, India, Vietnam, and Bangladesh isolates. Further research is needed for the whole genome sequence to reveal proteins sequence for the development of attenuated vaccine of local strain.

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