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, Marks 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.
|| Confluent monolayer of CEF cells
|| 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.
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.