Poultry industry plays a vital role in the economy of Pakistan and is responsible for 12% GDP. It also contributes 651 tones poultry meat and 11258 millions number of eggs produced per anum (Anonymous, 2008-09). Its contribution in agriculture growth is 4.81% and in Livestock growth 9.84%. Poultry meat contributes 19% of the total meat production in the country.
Hydro-pericardium has caused huge economic losses to poultry industry in Pakistan since 1987 when it was first reported at Angara Goth, a broiler producing area in Karachi (Jaffery, 1988). In order to control Hydro-pericardium syndrome various vaccine formulations are being used in the field but none of them fulfill the criteria of eliciting a prompt and long lasting immune response against the natural outbreaks of disease. There is an extensive use of liver homogenates in which the virus is inactivated by formalin (Afzal et al., 1989; Ahmad and Naz, 1990; Hussain et. al., 1996; Kataria et al., 1997; Zia et al., 2001). The immune response provoked by these vaccines is not always consistent and predictable. Secondly, by using these liver homogenates there is a greater risk of secondary bacterial infection as well.
There should be sufficient attempts to propagate the virus on specific pathogen free embryonated eggs and cell culture cultures for the production of killed and live attenuated vaccines.
- Isolation and identification of HPS virus from field at various places in Punjab.
- Identification and confirmation of virus by agar gel precipitation test (AGPT).
- Adaptation of hydro-pericardium syndrome virus (HPSV) on chicken embryo liver cells (CELs)
- Adaptation and attenuation of field HPS virus through continuous passage on chicken embryo liver cells (CELs).
- Pathogencity of adapted and attenuated HPSV.
Collection of samples
Infected liver samples were collected from HPS outbreaks farms at different places of the Punjab. Complete history of the each outbreak was recorded. The samples were labeled and stored at –20°C till further use.
Processing of samples
A 20% homogenate of infected liver samples were prepared in phosphate buffered saline (PBS) having antibiotics. Homogenized material was centrifuged to sediment tissue debris. A clear layer of supernatant was stored in the sterilized screw capped test tubes at –20°C till further use.
The supernatant was treated with chloroform to obtain clear supernatant. A ratio of 1:2 (chloroform: sample) was subjected to centrifugation at 5000 RPM for 20 min (Reddy et al., 1997). The chloroform treatment was helpful for effective precipitation of liver protein.
IDENTIFICATION OF VIRUS
Hyper-immune serum: -
For the production of hyper-immune serum against HPSV three rabbits were used according the method as described by Hussain et al. (2004). After collection of serum it has been inactivated at 56oC for 30 min. in water bath and stored at –20oC for further use.
Agar gel precipitation test: -
AGPT was conducted on hyper-immune serum samples and prepared liver homogenates, to detect HPS-associated adenovirus antibodies and virus presence respectively following protocol (Muhammad. 1995).
ADAPTATION OF HPSV ON CHICKEN EMBRYO LIVER CELLS (CELs)
Propagation of Primary cell monolayer
The flasks having primary cell cultures were processed for further propagation by adopting the protocol given below
- The exhausted medium was removed.
- The monolayer was washed once with sterile PBS (pH= 7.2)
- 5-10 ml of 0,05 % trypsin solution was added in the flasks incubated at room temperature for 3-5 minutes and was gently shaken to disintegrate the cells of monolayer.
- 13-14 ml M199 with 9-12 % fetal calf serum was poured into each of the flasks. These suspensions were transferred further in 12 well cell culture plates.
- The plates were incubated at 37oC for 24-48 hours and after incubation period cell monolayer was observed under inverted microscope.
Infection of monolayer with HPSV
Samples were processed as described earlier.
- The growth medium of the flasks was removed and the monolayers were washed with pre warmed sterilized Dulbecco’s PBS.
- A 0.2 ml of HPSV inoculum was dispensed over the each monolayer using 0.2 μm pore size filter (Whatman Int. Ltd., England) along with negative pressure.
- The inoculum was spread uniformly over each monolayer and flasks were incubated at 37oC for 1 hour with intermittent rotation to allow the virus to adsorb on the surface of CELs.
- Five ml of sterilized pre-warmed maintenance medium was added in each flask and incubated them at 37oC in presence of 5% CO2 in CO2 incubator.
- The mono layers were examined twice daily under inverted microscope for cytopathic effects (CPEs).
Harvesting of virus
The culture fluid was harvested by three freeze-thaw cycles and clarified by centrifugation at 5000 rpm for 5 minutes (Peilin et al., 1997).
Adaptation and attenuation
The passage 1 (P1) virus was inoculated again to fresh, healthy and semi confluent mono layers of CELs using the same technique and observed for CPEs. The virus was harvested by three freeze-thaw cycles, clarified and labeled as passage 2 (P2). In this way HPSV was adapted to CELs when the CPEs were clear and consistent. The adapted virus was serially passaged until it became attenuated (non-pathogenic).
Pathogenicity testing of virus
The original indigenous HPSV, passage-1, passage-3, passage-5 and Passage-7 of HPS viruses derived from chicken embryo liver cell (CELs) culture were selected and subjected to pathogenicity testing in chicken. Sixty 3-week old chickens free from antibodies against HPSV were divided into six groups i.e. 1, 2, 3, 4, 5, and 6 of 10 birds each. The chicken of 1 to 5 group were inoculated with 0.2 ml of original HPSV (group-1) and passage no 1, 3, 5 and 7 (Group, 2, 3, 4, and 5), respectively through intraocular (0.1 ml) and intranasal (0.1 ml) routes. The control birds (group 6) were inoculated similarly with PBS. After inoculation, chicken were examined for clinical signs. At day 5 and 10 post-inoculation (PI), five birds from each group were slaughtered. The bursae, spleen and liver of these birds were weighed separately and the percentage ratio of these organs to body weight was calculated.
TITRATION OF ATTENUATED VIRUS/VIRUS ASSAY
The CELs adapted and attenuated HPSV was titrated by plaque assay and tissue culture infective dose 50 (TCID50). (Skeeles et al., 1979, Reed and Muench, 1938).
No cytopathic effects (CPEs) were observed in the first passage even up to 5 days (120 hours) post-infection. The culture supernatant was harvested by three freeze-thaw cycles and inoculated on the fresh mono layers. Slight aggregation of CLEs was observed at 72 to 120 hours post infection in second passage. However clear and consistent cytopathic effects of HPSV on CLEs were observed in third passage. In third passage, CPEs appeared 120 hours post-infection and cytopathic effects involved the rounding and aggregation of cells.
After 3rd passage HPS virus was adapted on CLEs. The CPEs continued to appear with same intensity afterward. The CPEs appeared fully at 72 hours post-infection after adaptation till 7th passage. The seventh passage virus was completely attenuated (non-pathogenic) and was further grown on more fresh mono layers to produce the attenuated HPSV in large amount for vaccine production.