Poultry production has made an astounding progress over the past few years in Pakistan. Despite being well established and well organized, the poultry industry in Pakistan is still confronted with many acute and fatal diseases. Of these inclusion body hepatitis-hydropericardium syndrome (IBH-HPS) causes huge economic losses to poultry industry in Pakistan since 1987. The disease is also called ‘Angara’ disease in Pakistan, after the place Angara Goth, near Karachi (Akthar, 1994), ‘Leechy disease’ or ‘Litchi disease’ in India, after the peculiar appearance of the heart floating in pericardial fluid, which appear similar to the deshelled leechy (lichee) fruit (Gowda and Satyanarayana, 1994; Gowda, 1994) or inclusion body hepatitis-hydro-pericardium syndrome (IBH-HPS) (Abdul-Aziz and Attar,1991; Jadhao et al., 1997; Balamurugan et al., 2002).
IBH-HPS has been observed in broiler chickens of either sex, aged 3 to 6 weeks or over 5 weeks of age and occasionally in layer and breeder pullets aged to 20 weeks. Rare outbreaks of IBH-HPS in older birds/ broiler and in other species of poultry including pigeons, quail (Kumar et al., 1997). The disease is characterized by its sudden occurrence and high morbidity, with a high mortality of up to 80% in broiler and low mortality of below 10% in layers, associated with hydro-pericardium (Shane, 1996).
In this project, an egg adapted montanide adjuvanted IBH-HPS vaccine developed scientifically which was previously tested and applied in the field for the benefit of poultry farmers and technology transfer purposes.
- The scientifically developed montanide-adjuvanted IBH-HPS vaccine produced and tested previously was introduced in various villages given in the list below
- The improved montanide-adjuvanted IBH-HPS vaccine produced using indigenous virus seed.
- The following villages surveyed and selected
- Application of treatment done in the selected areas and data collected
- Farmers day conducted on the basis of application of treatment and data collection for the feedback of the farmers
For the scientifically development of egg adapted montanide-adjuvanted IBH-HPS vaccine in the laboratory, following steps were taken
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 infectious livers
About 20 gram of liver samples was chopped with a sterilized pair of scissors. The chopped material was homogenized, making 30% W/V suspension of infected livers in PBS containing antibiotics (Pencilline-G 10,000 IU and streptomycin 1000ug/ml). The homogenized suspension was centrifuged at 5000 RPM for 20 minutes, and supernatant was filtered through 0.2 u PPD and collected for further purification (Reddy et al., 1997).
Sonication of Infected and Normal Isolates
The clarified supernatant was sonicated using the sonifier (Rapidis, 75 watts/cm2). Samples were placed in an ice bucket subsequently to lower the temperature of the sonified homogenate. Two shocks, of 30 second each, were given with 15 second interval (Schleider and Hoas, 1969).
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. The clear supernatant above the band of precipitated liver protein was collected and stored in 3 ml plastic vials at -20oC in deep freezer until use.
QUANTIFICATION / IDENTIFICATION OF ANTIGEN
The infected liver samples were be processed for qualitative and quantitative estimation of the virus on the basis of reverse passive haemagglutination test (RPHA) (Manzoor et al 2003) and Agar gel precipitation test (Beard, 1970).
CHARACTERIZATION OF THE VACCINE
The characterization of the viral proteins was determined with the help of sodium dodecyl poly-acrylamide gel electrophoresis (SDS PAGE).
ADAPTATION AND ATTENTION OF VIRUS
The virus was passaged using the same method until it loses its pathogenicity. Eggs were opened and allantoic fluid was harvested. The embryos were examined for any gross pathological changes on the body surface and the internal organs. To confirm that the adapted virus is HPSV, the allantoic fluid was subjected to agar gel precipitation test using know polyclonal hyper-immune serum.
In this way the indigenous virus was isolated from field outbreak and then passaged into the embryonated eggs and propagated for vaccine preparation and about 4000 ml of seed culture has been collected.
The stability of vaccines was checked by observing the vaccines and by keeping them at temperature of 25oC, and 37oC.
The prepared vaccines were tested for sterility by inoculating a small amount of them on to different agars and broths.
- The vaccines were streaked on the MacConkey agar and nutrient agar plates. A loopful material was transferred to each of the set of 5 tubes containing nutrient broth and mycoplasma broth.
- The inoculated media were incubated for 48 hrs while the Mycoplasma broth incubated at 37oC for at least 10 days and examined daily for microbial growth.
- The Mycoplasma broth was transferred to fresh tubes.
- The media were observed for observable bacterial growth and other contaminants in the vaccine.
The broth was checked for the presence of any turbidity.
CALCULATION OF EID50
To determine EID50 of the stock virus ten fold dilutions were prepared in phosphate buffer saline (PBS) (pH 7.2). Thirty 9 days old embryonating eggs were taken and divided into 5 different g groups of five eggs that were marked from 10-1 to 10-5. Fertile eggs were inoculated via chorio-allantoic route and were again incubated. The infectivity was checked up to 96 hrs and EID50 was calculated (Reed and Muench, 1938).
Collection of Serum Samples
The Serum samples from 10 chickens out of each farm were randomly selected weekly after vaccination. The birds were taken and blood was collected in the cleaned Petri-plates and kept for clotting in the refrigerator. After 10 minutes the clots were agitated to the depth of the plates at many places and were again kept in the refrigerator for 30-45 minutes at tilting position. Then serum was collected from lower part of the Petri-plates in dropping bottles (plastic vials of 3 ml capacity) The vial were stored in the deep freezer until qualitative and quantitative analysis for antibodies.
- The selected farms recieving improved montanide-adjuvanted IBH-HPS vaccine as compared to conventional vaccine had significantly higher antibody Titre
- The improved montanide-adjuvanted IBH-HPS vaccine requires single shot in comparison with conventional vaccines which save time, money and stress