PREPARATION AND EFFICACY OF COMBINED LIVE AND INACTIVATED OIL EMULSION GUMBORO VACCINES IN BROILER

Preparation and use of safe and efficacious vaccines rank amongst the top priorities in medical and veterinary medicine for prevention and control of infectious diseases. Infectious Bursal disease (IBD) known as Gumboro, caused by bima virus is an acute highly contagious, immunosuppressive viral disease of chickens with significant economic importance. There are two serotypes of infectious bursal disease virus (IBDV), serotype I is responsible for infection in commercial poultry. The emergence of new strains (vvIBDV) and variations within the strains of IBDV serotype I has complicated the protection by. vaccines. The present study is about characterization of IBDV isolates prevalent in Pakistan and preparation and use of indigenous live attenuated and inactivated IBD vaccine.\ud \ud In this study fifty field isolates of IBDV were collected from commercial poultry flocks experiencing typical signs and high mortality (10-40 %) of IBD in Karachi, Pakistan during the period 1999- 2005. The IBDV isolates were identified by serological tests; Agar Gel Precipitation (AGP) and Enzyme Linked Immuno Sorbent Assay (ELISA) and virus was isolated using specific pathogen free (SPF) eggs and chickens. Out of fifty, thirty-five samples were found positive to IBDV by AGP and ELISA tests. Five isolates designated as ML-lISPVC/2001, NP2/SPVC/2002, NL-3/SPVC/2003, NK-4/SPVCI2004, and NPK-5/SPVC/2005) were selected on the basis of antigenicity and pathogenicity to evaluate them further and to select a vaccine strain. These isolates were inoculated in susceptible chickens at 4 week of age showed mild to severe gross pathological lesions. The isolates NL-3/SPVC/2003 and NTK5/SPVC/2005 produced haemorrhagic lesions in thigh and pectoral muscles within 24 hours post-virus inoculation; however after 4 days the lesions were more severe and caused atrophy of bursa at days 7 to 9. Other prominent lesions were splenomegaly, nephritis and streaked yellow appearance of liver. The fields isolate NK-4/SPVC/2004 produced almost similar lesions. but the intensity of lesions was less as compared to above-mentioned strains. The isolates ML-lISPVC/2001 and NP- 2/SPVC/2002 showed no hemorrhages in thigh and pectoral muscles during first 24 hours of virus inoculation, but enlargement of bursa during 2-3 days and its atrophy was noticed at day 8 and 9. Splenomegaly was observed at 5 days onwards in all infected chickens. The Mean of the BF (bursa of fabricius): BW (body weight) ratio in the inoculated groups was significantly lower than control. Bursal size was reduced approximately 75% in the. virus inoculated chickens.\ud \ud Epidemiology of diseases in a particular region. understanding of the viral etiology and its characteristic features at molecular level are very important for preparation of an efficacious vaccine and to recommend an effective vaccination program. The selected field isolates and commercial vaccines (D-78, Bursine Plus, Gumboro Forte, 228-E and AK3) were identified and characterized by using the molecular techniques of reverse transcriptase polymerase chain reaction / restriction fragment length polymorphism (RT-PCR/RFLP). Four local isolates and four commercial vaccines were determined to contain IBDV as evidenced by amplification of 743-bp region of VP2 gene of IBDV by RT-PCR. The RT-PCR products were characterized by digestion with three restriction enzymes, BstNl, Mho 1 and Ssp 1. When the amplicons were digested with enzyme Mho 1 the RFLP profile of all field strains were found similar, with a fragment size of 229bp and 362bp. However, when these strains were digested with enzyme BstNl two distinct RFLP profile were obtained. The first profile was detected in field isolate ML-I/SPVC/2001 of IBDV and vaccine strain D78 with four fragments of 119bp, 154bp, 172bp and 209bp that resembled the RFLP profiles of New Zealand, Venezuela and US classic vaccine strains and it was placed in molecular group 4. While the field isolates NL-3/SPVC/2003, NK-4/SPVC/2004, and NPK-5/SPVC/2005 generated a different RFLP profile with fragments of 119, 172 and 424bp and resembled with the profiles of some IBDV strains of Egypt, Puerto Rico, Mexico, El Salvador, South Africa, France, Spain and Taiwanese vvIBDV and they were placed into molecular group 6. However, all the tested field and vaccine strains showed no fragments on digestion with enzyme SsP1 thus indicating that very virulent markers of IBDV are not present in the Pakistani isolates of IBDV. The results demonstrate that Pakistani isolates of IBDV can be placed into molecular group 4 and 6.\ud \ud Studies suggest indigenous vaccines are more effective and can provide better protection due to more antigenic relatedness. In the present study attempts were made to prepare indigenous live attenuated and inactivated oil emulsion IBD vaccine using a local strain NL-3/SPVC/2003 of IBDV. The strain was selected as a vaccine strain on the basis of pathogenic and antigenic and molecular characteristics. For attenuation it was passaged in SPF eggs and chickens and was found attenuated at 35th passage. Master seed virus (MSV) and working seed virus (WSV) were prepared and virus dose containing 103.5 EID50 was injected into embryonated SPF eggs and incubated for 72 hours, chilled for 3-4 hours at 4°C and harvested followed by stabilization. Skimmed milk was added as a stabilizer at the rate of 40% v/v and the stabilized allantoic fluid was dispensed into sterilized glass vials followed by lyophilization. The lyophilization process depended on the number of vials loaded into the machine. The changes in shelf temperature of a fully loaded machine from -20 to -25°C usually took 18 to 24 hours. The freeze dried vaccine was checked for sterility, potency and efficacy was found satisfactory and provided ≥ 80 % protection against challenge. An inactivated IBD vaccine was also prepared using the same vaccine strain. Complete inactivation of the virus was achieved with use of either betapropilactone or formalin (ICN, USA) at 0.3 % and 0.1 % respectively as inactivating agents. Furthermore, the emulsion used for inactivated vaccine was water in oil type. The emulsion contained Tween 80 in aqueous phase and Arlacel 80 or Arlacel A in oil phase. Emulsion vaccine was prepared by homogenization of one volume of antigen that contained 4% (v/v) Tween 80 with four volumes of mineral oil that contained 10% (v/v) Alacel 80. This 1:4 aqueous-oil ratio diluted the antigen by a factor of 1:5. The inactivated vaccine was tested for sterility and potency and it was found satisfactory in vaccine trials.\ud \ud Appropriate vaccination programs are very important for effective control of IBDV since many factors affects vaccination programs particularly maternal antibodies, vaccine strain and flock management practices. Day-old broiler chicks were divided into different groups and vaccinated with various regimens of indigenous live attenuated and inactivated IBDV vaccines. The chickens in group A were immunized with live attenuated vaccine at eight days of age, group B, 8 and 15, group C, 8, 15, and 23 days of age and group D 8 days with live attenuated vaccine followed by in activated vaccine at 21 days, group E was treated as control. The results revealed that the vaccination program adopted group C and D were found to produce significantly higher antibody titer as compared to all other groups by ELISA and AGP tests. The chickens of group B showed higher antibody titer as compared to group A. Furthermore, the level of maternal antibodies (MA) in the sera of broiler chickens at I SI week of age was considerably low. The results of the present study suggested that administration of live attenuated vaccine at 8 days followed by inactivated vaccine at 21 days provides adequate protection against virulent form of IBDV with minimum adverse effects