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Research Article
 

Effect of Infectious Bursal Disease Virus on in vitro Propagation of Chicken Embryo Fibroblast Cells



Sarder Nasir Uddin and Sk. A. Hossain
 
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ABSTRACT

The present research was undertaken to study the propagation and observation of cytopathic effect of IBDV in chicken embryo fibroblast cells. Chicken Embryo Fibroblast (CEF) is chicken embryo derived primary cell culture. Infectious bursal disease is an acute, highly contagious viral disease of young chickens caused by a double stranded RNA virus named Infectious Bursal Disease Virus (IBDV). For this purpose, suspected IBDV isolates were collected from the bursas of dead chicken of a particular flock. Local field IBDV isolates was then inoculated in chicken embryo for slight adaptation by several passages. CEF cells was prepared from 9-10 days chicken embryo and transferred in monolayer culture flasks containing maintenance media with 1-2% heat-inactivated fetal calf serum. Adapted IBDV was then inoculated in CEF cell culture for the purpose of propagation. After inoculation characteristics clear and consistent Cytopathic Effects (CPEs) were observed on CEF cell monolayer and after 72 h of infection, the cells were started to change its shape.

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  How to cite this article:

Sarder Nasir Uddin and Sk. A. Hossain , 2006. Effect of Infectious Bursal Disease Virus on in vitro Propagation of Chicken Embryo Fibroblast Cells. Asian Journal of Animal and Veterinary Advances, 1: 55-59.

DOI: 10.3923/ajava.2006.55.59

URL: https://scialert.net/abstract/?doi=ajava.2006.55.59

Introduction

Protein is the most important constituent of cells and its deficiency leads to mental as well as physical abnormalities in human especially among children. Animal protein is the only complete protein, which is required for brain development. Bangladesh is one of the many developing countries facing acute shortage of animal protein. Poultry meat and eggs are two major sources of animal protein.

In the recent years poultry rising has become a growing and prospective industry in Bangladesh. Despite the special emphasis of the Government of Bangladesh on this sector, the development of poultry industry is seriously threatened by the outbreaks of acute, contagious and fatal disease. Although some diseases like Newcastle disease, Mark’s disease, fowl cholera, coccidiosis etc. have been kept under control in most of the commercial poultry farms by vaccination and medication, new emerging diseases like Infectious Brusal Disease (IBD) has virtually brought the progress of poultry industry of Bangladesh to a halt.

Infectious bursal disease, popularly known as Gumboro disease, is a contagious viral disease of young chickens caused by a double stranded RNA virus belonging to the family Birnaviridae. Since the description of the disease by cosgrove, (1962), Gumboro disease has drawn the attention of avian virologists not only because of the high mortality from the disease proper but also due to the profound immunosuppression induced by the virus resulting is subsequent secondary infections and vaccination failure (Allan et al., 1772; Hirari and Shimakura, 1774). In the recent years emergence of a very virulent pathotype of IBD virus with a potential of causing upto 100% mortality has stimulated the resurgence of interests in IBD among the avian virologists. The disease has been occurring in Bangladesh since March 1992 with very high morbidity and mortality (Islam et al., 1994a; Islam et al., 1994b; Rahman, 1994). Viruses are obligatory dependent on host systems. Without the host system, it cannot replicate, propagate or multiply. Host may be bacteria, lower animal or plant, higher animal or plant, insects or even animal cells. Among the host, animal cells are excellent host for viruses and being used for propagation or cultivation for virus.

IBDV can infect and grow on various primary cell culture of avian origin and certain cell line of mammalian origin. Commonly used cell lines to replicate IBDV are Chicken Embryo Fibroblast (CEF) (Sofei et al., 1996). Chicken Embryo Kidney (CEK), Vero (Peilin et al., 1999), Baby Hamster Kidney (BHK) (El-Ebriary et al., 1999), chicken embryo bursa (Lukert and Davis, 1774), normal chicken lymphocytes, B-cell lymphoblastoid, baby grivet monkey kidney (BGM-70) and M4-104 cells (Jackwood et al.,1997) etc. In addition to the above cell line, IBDV can also infect chicken embryo (Sofei et al., 1996).

The present research was undertaken to study the propagation and observation of cytopathic effect of IBDV in chicken embryo fibroblast cells. Considering the economic importance and severity of infectious bursal disease in chickens of Bangladesh. The present study was designed to isolate, propagate and to confirm of the local field strains of IBDV in primary cell culture and to study the cytopathology of the local isolate in chicken embryo fibroblast cell culture.

Materials and Methods

The work was carried on Animal Biotechnology Laboratory of Bangladesh Livestock Research Institute (BLRI), Savar, Bangladesh at early of 2004.

Preparation of Chicken Embryo Fibroblast (CEF) Cells
Eight to ten-day incubated eggs are chosen for the preparation of chick embryo fibroblasts. The eggs are candled and the air space marked. After disinfection with alcohol 70% the shell over the air space is removed. The contents of the egg are poured into a petridish and the embryo is taken out of the amnion sac. The chick embryos are decapitated and the liver and intestines are removed.Then chopping the rest part (excluding liver, intestines and fat) properly by scissors. Then put the carcases into the syringe reservoir, and insert the plunger by pointing the syringe upwards. The carcases are ground by pressing them through the syringe opening. The tissue pulp is washed gently before trypsinization. As the tissue is easily disintegrated, only a few cycles of trypsinization are required. Then 1-2 mL of trypsinized tissue sample solution was inoculated into a 25 cm2 culture flax containing 10 mL of growth medium. Culture flax was placed into a CO2 incubator for 20-25 min. After 20-25 min, culture flax was replaced by 10 mL of maintenance media. The Infectious bursal disease viruses were isolated from IBDV suspected dead chickens to propagate them on CEF cells.

Preparation of Inoculum with the IBDV Suspected Bursal Samples
Each bursal sample was cut into small pieces and triturated by a pestle and mortar. PBS was added to the tissue homogenate as to make a 10% weight/volume (w/v) suspension of bursal tissue. The suspension was then centrifuged at 3000 rpm for 15 min. The supernatant of the centrifuged tube was collected from the suspension. Penicillin and Streptomycin at the dose rate of 10,000 IU mL-1 and 10,000 μL mL-1 were added to the collected supernatant respectively. After adding antibiotics the suspension was kept at room temperature for 45 min and shacked gently for every 10 min. The suspension was then inoculated into sterile blood agar media for bacteriological sterility of the antibiotic treated suspension. The inoculated blood agar media was incubated at 37°C for 24 h. Bacteriologically sterile suspension was used as an inoculum for the isolation of virus from the bursal suspension.

Inoculation of Virus in Cell Culture
Confluent monolayer of CEF grown in 25 cm3 culture flasks shown in the photograph 5. Within 48 h after seeding when the cells were fully confluent, the growth medium was removed from the culture flasks with a pipette and 0.3 to 0.5 mL field virus (adapted in chicken embryo) isolates were then inoculated in each culture flask for the purpose of propagation of field IBDV isolates. The flasks were incubated at 37°C in a humidified incubator for one hour to allow the virus to adsorb. After that, one mL of maintenance medium was added to each flask and the flasks were taken back to the incubator. The cells were daily under an inverted microscope for the appearance of any Cytopathic Effect (CPE). On day 5 infection (p.i) the cells in the flasks were frozen at -20°C irrespective of the appearance of CPE.

Results and Discussion

Formation of Confluent Monolayer of Chicken Embryo Fibroblast (CEF) Cells
Culture flax containing tissue culture sample were placed in a CO2 incubator. During first day observation under inverted microscope, cells were shown to grow to form monolayer culture. Confluent monolayer of Chicken Embryo Fibroblast (CEF) cells (Fig. 1) was found during second day observation under inverted microscope.

Propagation of IBDV on CEF Cell Culture
Isolated slightly adapted IBDV is inoculated in confluent monolayer of chicken embryo fibroblast cells for the purpose to propagate. After 144 h of inoculation Cytopathic Effects (CPEs) were observed. Consistent and clear CPEs indicate optimum propagation of IBDV (Fig. 2).

The infectious bursal disease viruses were isolated from IBDV suspected dead chickens to propagate on Chicken Embryo Fibroblast (CEF) cells. Suspected IBDV was inoculated through injection into live birds of the experimental flock of Bangladesh Livestock Research Institute. Live birds became dead after 13-14 days following infection. The results indicated the confirm identity of IBDV, collected from suspected field samples of bursa of dead chickens of 33-37 days age group.

To propagate IBDV on Chicken Embryo Fibroblast (CEF) cells, suspected IBDV were inoculated in chicken embryo for the purpose of adaptation. Field isolates IBDV can never propagate directly after inoculation in primary chicken embryo fibroblast cell culture. Therefore, suspected field isolates IBDV need to be adapted in chicken embryo. For this purpose, field isolates IBDV were inoculated in chicken embryo by several passages. After several passages (7 passages) IBDV was slightly adapted in chicken embryo. These virus samples were used for the propagation in Chicken Embryo Fibroblast (CEF) cells.

Cytopathic effects involved rounding, aggregation of CEF cells monolayer due to infection by virus. The CEF cell monolayer was examined under inverted microscope twice a day for observing CPEs. Following 48 h of infection no CPEs was found, the cells were like as confluent monolayer (Fig. 1). After 72 h of infection, the cells were just started to change its shape.

Image for - Effect of Infectious Bursal Disease Virus on in vitro Propagation of Chicken Embryo Fibroblast Cells
Fig. 1: Confluent monolayer of CEF cells

Image for - Effect of Infectious Bursal Disease Virus on in vitro Propagation of Chicken Embryo Fibroblast Cells
Fig. 2: Cytopathic Effect (CPEs) on CEF cell culture

At this stage, few rounding were observed. The cells gradually started to change its shape in order to produce CPEs following 96 to 120 h of infection. CPEs were characterized by formation of rounding cells. Aggregation of rounding cells was formed during 120 to 144 h of incubation following infection. Clear and optimum CPEs were formed after 144 h of incubation following infection (Fig. 2). Formation of clear and optimum CPEs on CEF cell monolayer shows optimum propagation of infectious bursal disease virus.

Acknowledgments

The study was carried on Animal Biotechnology Laboratory of Bangladesh Livestock Research Institute, Savar, Bangladesh. So the authors are thankful to the personnel of that Labrotry for their help.

REFERENCES

1:  Allan, W.H., J.T. Faragher and G.A. Cullen, 1972. Immunoppression of infectious bursal agent in chicks immunised against newcastle disease. Vet. Record, 90: 511-512.

2:  El-Ebriary, E.A., S.S. El-Mahidy and M.H. Khodeir, 1999. Trials of using cell cultures of evaluation of infectious bursal disease virus vaccines in egypt. Assiut. Vet. Med. J., 36: 105-123.

3:  Hirari, K. and S. Shimakura, 1974. Structure of infectious bursal disease virus. J. Virol., 14: 957-964.

4:  Islam, M.R., P.M. Das, E.H. Chowdhury and M.L. Dewan, 1994. Very virulent infectious bursal disease virus a challenge for poultry industry in bangladesh. Proceedings of the 12th Annual Conferance of Bangladesh Society of Microbiologists, BARC, Dhaka, January 19 and February 11, 1994.

5:  Islam, M.R., P.M. Das, E.H. Chowdhury and M.L. Dewan, 1994. Some observations on infectious bursal disease of chickens reproduced experimentally with a highly virulent local isolate. Proceedings of the 18th Bangladesh Science Conference, Bangladesh Agricultural University, Mymensingh, 22-24 June, 1994.

6:  Jackwood, D.H., Y.M. Saif and J.H. Hughes, 1997. Replication of infectious bursal disease virus in continuous cell line. Avian Dis., 31: 370-375.

7:  Lukert, P.D. and R.B. Davis, 1974. Infectious bursal disease virus growth and characterization in cell cultures. Avian Dis., 18: 243-250.

8:  Peilin, W., C. Baoxiang and L. Jinsong, 1999. Adaptation and propagation of infectious bursal disease virus in vero cells. Chinese J. Vet. Sci. Technol., 27: 23-24.

9:  Rahman, M.M., 1994. Gumboro disease and some observations on its outbreaks in a poultry breeding farm. Proceedings of the Symposium on Gumboro Disease Sponsored by Intervet International, Dhaka and 19th October 1994.

10:  Sofei, D.M., E. Avran, M. Danes, S. Plamadeala, E. Bucur and E. Vior, 1996. Gumbovivac cc live vaccine against infectious bursal disease prepared on cell cultures. Studies Res. Vet. Med., 4: 122-126.

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