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Determination of antibody titres of sera collected from poultry flock to diagnose IBDV infected chickens by developing single serum working dilution ELISA (SSD-ELISA) was made. The indirect ELISA standard procedure was adopted with standard positive and negative serum controls. The standard IBDV antigen was prepared using the Tamil Nadu isolate of IBDV. The relative sensitivity and specificity of a single serum dilution ELISA test in qualitative comparison with the commercial kit were 82% and 100% respectively and the accuracy was 82.1%. This single serum dilution assay gave reproducible results and allowed considerable savings on the time and cost of reagents compared with indirect ELISA. Based on these results a SDS-ELISA kits have been developed in this study to replace the commercial kit. The IBDV antigen coated ELISA plates can be stored under refrigeration and the test can be performed rapidly under field conditions by trained personnel., Titar protutijela za virus zarazne bolesti burze određen je u uzorcima seruma pilića razvijenim imunoenzimnim testom s jednostrukim razrjeđenjem uzoraka seruma. Standardni imunoenzimni test prilagođen je uz uporabu standardnih pozitivnih i negativnih kontrolnih uzoraka seruma. Standardni antigen za virus zarazne bolesti burze bio je pripravljen od izolata Tamil Nadu virusa. U usporedbi s komercijalnim kompletom, osjetljivost testa iznosila je 82%, a specifičnost 100%. Točnost mu je bila 82,1%. Razvijenim testom postignuti su reproducibilni rezultati uz znatnu uštedu vremena i materijala pa se može uporabiti umjesto komercijalnih kompleta. Antigen se može pohraniti u hladnjaku, a uvježbano osoblje može brzo izvesti test u terenskim uvjetima.

The current trends for pursuing research programmes warrant abundant, non-invasive experimentation techniques avoiding the use of laboratory animals have led to a search for alternate sources of commercial antibodies for various immunodiagnostic and prophylactic assays. The present experiment exploited the diagnostic property of egg immunoglobulins (IgY) against infectious bursal disease (IBD) as a novel, alternative approach. The anti-IBD -IgY was harvested by the water dilution method, purified by salt precipitation, characterized by SDS - PAGE and assessed for its purity by immunoelectrophoresis. An average 200 - 250 mg. of immunoglobulins were harvested from an egg with 57-60% recovery of specific immunoglobulins. The anti - IBD-IgY thus harvested was found to possess immunodiagnostic potency as assessed by agar gel precipitation test and counter immunoelectrophoresis for replacing the use of conventional antibody., Sadašnje smjernice u provođenju istraživačkih projekata zahtijevaju neinvazivne tehnike u provođenju pokusa, izbjegavanje korištenja laboratorijskih životinja te pronalazak alternativnih izvora komercijalnih protutijela za različite imunodijagnostičke i profilaktičke postupke. Ovaj pokus temeljio se na dijagnostičkim osobinama imunoglobulina jajeta (IgY) protiv zarazne bolesti burze (ZBB) kao novom alternativnom pristupu. Anti-ZBB-IgY dobiveni su metodom razrjeđivanja vodom, pročišćeni pomoću precipitacije solima, obilježeni pomoću SDS-PAGE i pretraženi na čistoću imunoelektroforezom. Prosječna količina od 200-250 mg imunoglobulina dobivenih po jednom jajetu sadržavala je 57-60% specifičnih imunoglobulina. Na ovaj način dobiveni anti-ZBB-IgY posjeduju imunodijagnostičku sposobnost i mogu zamjeniti konvencionalna protutijela, što je i potvrđeno GDP testom i imunoelektroforezom.

Reverse transcription-polymerase chain reaction (RT-PCR) followed by restriction fragment-length polymorphism (RFLP) analysis was tested in order to determine its accuracy and sensitivity for differentiation of infectious bursal disease virus (IBDV) strains. Two commercial vaccine strains and two Croatian field isolates were analysed. RT-PCR assay was used for amplification of a variable part of VP2 gene (422 bp) encoding for major neutralizing epitopes. Amplified VP2 fragments were further characterized by RFLP analysis using the SacI and TaqI restriction endonucleases. Both field isolates showed RFLP pattern similar to highly virulent European and Asian strains. Both vaccine strains had RFLP profiles different from that of the field isolates. Vaccinal strain 2512 had RFLP pattern similar to mild vaccinal strain pbg-98, and strain VMG-91 was similar to other classical vaccinal strains of intermediate pathogenicity., Procijenjena je točnost i osjetljivost metode temeljene na razlici dužina restrikcijskih odsječaka DNA (RFLP, restriction fragment lenght polymorphism) za razlikovanje sojeva virusa zarazne bolesti burze (VZBB). Lančanom reakcijom polimerazom (PCR, polymerase chain reaction) uz prethodnu reverznu transkripciju (RT, reverse transcription) umnožen je dio promjenljivog dijela VP2 gena koji kodira glavne virusne neutralizacijske epitope. Nastali produkti veličine 422 parova baza (bp, base pair) pocijepani su restrikcijskim endonukleazama SacI i TaqI. Restrikcijski profili nastali enzimskim cijepanjem uspoređeni su s podatcima navedenim u banci gena. Metoda je testirana na dva komercijalna cjepiva proizvedena u Hrvatskoj (Gumbokal® SPF, soj 2512 i Gumbokal® IM SPF, soj VMG 91) te dva hrvatska terenska izolata VZBB (IBDV/96/01 i IBDV/ 97/02). Oba su terenska izolata pokazala RFLP profil jednak vrlo virulentnim europskim i azijskim sojevima VZBB. Oba cijepna soja imala su različit RFLP profil od terenskih izolata. Cijepni soj 2512 ima RFLP profil jednak blagim cijepnim sojem pbg-98, a VMG 91 je sličan poznatim intermedijarnim sojevima VZBB.

Infectious bursal disease (IBD), also known as Gumboro disease, hit the poultry production twice in the last several decades. The first time in 1962 when the disease was recognised, causing around 5% mortality in chickens aged three weeks and older, and severe, prolonged immunosuppression of chickens infected at an early age. The vaccines became available and during following decades the disease turned to mainly subclinical infection causing less than 1% mortality. The second stroke occurred in the late 80's in Western Europe changing dramatically IBD situation. The emerged highly virulent IBD (hvIBDV) virus soon spread all around world causing mortality up to 25% in broilers and 60 % in layers. Moreover, clinical signs as well as mortality were observed in very young birds, even as young as seven days. Mild live vaccines, which were in wide use, were not able to protect against this infection. Isolated virus showed to be of classical type, but mentioned biological characteristics of hvIBDV forced poultry industry to switch from mild to more invasive vaccinal strains, almost overnight. The vaccination programmes required some adjustment, too. IBD virus is very stable in the environment, thus once introduced in poultry house will continue to cause disease in flock after flock, despite vigorous disinfection. So, in contrast to some other viral diseases, vaccination is the principal method used for the control of IBD infection in chickens. Vaccination programmes have to be carefully designed considering the actual IBD situation in particular farm, level and uniformity of maternally derived antibodies, type of birds as well as type of vaccine. Also, protection of young birds by vaccination is directed in two ways: hyperimmunization of parent flocks using inactivated vaccines and vaccination of progeny using live vaccines. There are a lot of types of available live vaccines ranging from mild to so called 'hot' vaccines. Generally, immunogenicity of the live vaccines ie. serologic response correlate with adverse effect regarding bursa damage. According to own experiences, hvIBD can be well controlled if timing for vaccination and live intermediate vaccines in progeny are used. It is important to mention that satisfactory result may not be expected in the first vaccinated flock, but after several consequently vaccinated flocks in the same poultry house.