Examination of presence of specific antibodies against avian influenza virus in some species of wild birds

Abstract

Infections caused by the avian influenza virus have been known for a long time and they are present, to a smaller or greater extent, in both extensive and intensive poultry production in many parts of the world. Epidemiological investigations have established a definite significance of the population of wild birds in maintaining and spreading this infection. Avian influenza is a zoonosis, and the virus has a great potential for causing mortality in humans, in particular its subtypes H5 and H7, which is why it has lately been provoking much attention among scientists and experts, as well as the general public. The objective of the work was to catch a certain number of wild birds in several locations in the Republic of Serbia, to identify them, and to collect samples of their blood serum for the determination of specific antibodies against the avian influenza virus. Birds were caught in ten locations in a manner that was safe for the birds themselves, as well as for the staff that did the catching. The birds were caught in especially produced nets, and in some cases in special traps. The caught wild birds were identified using the methods described in reference literature. All the names of the wild birds were coordinated with the valid Serbian nomenclature of European birds, prepared by prominent ornithologist and bird lover Milan Ružić. Following catching and identification, blood samples were taken from the birds from the wing vein (in bigger birds) or from the leg vein (in smaller birds). In taking blood samples from wild birds, all the principles of asepsis and antisepsis were followed in order to prevent any possibility of infection. After that, the birds were returned to their natural habitat, to the same locations in which they were caught. Serums were isolated from the taken blood samples and they were stored at -20ºC until the final examinations. A total of 46 species of wild birds were identified among a total of 259 birds from which 259 samples of blood serum were isolated. The following were used for the detection of the presence of specific antibodies against the avian influenza virus in blood serum samples of wild birds: agar gel precipitation (AGP), the hemagglutination inhibition test (HI) for subtypes H5 and H7, the cELISA test with antigen for the A type avian infleunza virus, and the cELISA test with antigen for subtype H5 of the avian influenza virus. Due to the fact that about 360 different species of wild birds live in the Republic of Serbia, the number of 46 identified species covered by these investigations account for 12.77% of the total number of bird species present in Serbia, which is considered a good sample. Specific antibodies against the A type avian influenza virus were established in serum samples of only 9 of the 259 birds covered by examinations using the cELISA test. Of the 46 identified wild bird species, 6 belonged to birds that live exclusively in water habitats and are considered a reservoir of the avian influenza virus (white stork, mallard, mute swan, common pochard, common goldeneye, and Eurasian coot). Among the listed species, particular attention was drawn to 4 species of wild birds of the order Anseriformes and the family Anatidae (mallard, mute swan, common pochard, common goldeneye) of which there were 30 birds among the total of 259 examined. In the 30 blood serum samples of the listed bird species, specific antibodies against the A type avian influenza virus were established in 9 (30%) serum samples using cELISA. Specific antibodies against the avian influenza virus subtype H5 were established in 3 serum samples of mute swans (one serum sample originated from a mute swan which was tagged in Poland) and in one blood serum sample of a common pochard, or a total of 4 (13.33%) serum samples, using the hemagglutination inhibition test. Specific antibodies against the avian inluenza virus subtype H7 were established in 3 (10%) blood serum samples, in two serum samples from mallards and one sample from a mute swan, using the hemagglutination inhibition test. Specific antibodies against the avian influenza virus type A were not established in any examined bird species using the AGP test. In the population of wild bird species in the Republic of Serbia covered by these investigations, specific antibodies against the avian influenza virus were established only in serum samples of birds of the family Anatidae. Specific antibodies against the avian influenza virus type A established in 3 (6.52%) species of wild birds, and against subtypes H5 and H7 in 2 (4.34%) of the total of 46 examined species. The sensitivity of the cELISA test for the avian influenza virus subtype H5 and the hemagglutination inhibition test for subtype H5 amounted to 100%.

Infekcije izazvane virusom avijarne influence, su već odavno poznate i prisutne u manjem ili većem obimu, kako u ekstenzivnoj, tako i u intenzivnoj živinarskoj proizvodnji, u mnogim delovima sveta. Epidemiološkim ispitivanjima je utvrđen nesumnjiv značaj populacije divljih ptica u očuvanju i širenju ove infekcije. Avijarna influenca je zoonoza, a virus ima veliki potencijal da izazove visoku smrtnost kod ljudi, posebno njegovi podtipovi H5 i H7, tako da u novije vreme izaziva veliku pažnju, kako naučne i stručne, tako i najšire javnosti. Cilj ovog rada je bio da se na nekoliko lokacija u Republici Srbiji uhvati određeni broj divljih ptica, izvrši njihova identifikacija i prikupe uzorci krvnog seruma radi otkrivanja specifičnih antitela protiv virusa avijarne influence. Hvatanje ptica vršeno je na deset lokacija na bezbedan način, kako za same ptice, tako i za osoblje koje ih je hvatalo. Hvatanje ptica obavljano je posebnim za te svrhe proizvedenim mrežama, a u nekim slučajevima i posebnim zamkama (klopkama). Za identifikaciju uhvaćenih divljih ptica korišćene su metode koje su opisane u stručnoj literaturi. Svi nazivi divljih ptica usklađeni su sa važećom srpskom nomenklaturom ptica Evrope. Nakon hvatanja i identifikacije, pticama je uzimana krv iz krilne vene (kod većih ptica) ili iz nožne vene (kod malih ptica). Prilikom uzimanja krvi od divljih ptica poštovani su svi principi asepse i antisepse, kako bi se sprečila svaka mogućnost infekcije. Nakon toga, ptice su vraćane u prirodu, na iste lokacije na kojima su i uhvaćene. Od uzetih uzoraka krvi izdvojeni su serumi koji su ostavljani na - 20ºC i čuvani do konačnog ispitivanja. Identifikovano je 46 vrsta divljih ptica sa ukupno 259 jedinki od kojih je izdvojeno 259 uzoraka krvnog seruma. Za otkrivanje prisustva specifičnih antitela protiv virusa avijarne influence u uzorcima krvnog seruma divljih ptica korišćeni su agar gel precipitacija (AGP), test inhibicije hemaglutinacije (IH) za podtipove H5 i H7, cELISA test sa antigenom A tipa virusa avijarne influence i cELISA sa antigenom podtipa H5 virusa avijarne influence. S obzirom na činjenicu da na teritoriji Republike Srbije živi oko 360 različitih vrsta divljih ptica, broj od 46 identifikovanih vrsta obuhvaćenih ispitivanjem, činio je 12,77% od ukupnog broja prisutnih vrsta ptica u Srbiji, što se smatra dobrim uzorkom. Specifična antitela protiv A tipa virusa avijarne influence ustanovljena su u uzorcima seruma samo 9 od 259 jedinki koje su bile obuhvaćene ispitivanjem primenom cELISA testa. U identifikovanih 46 vrsta divljih ptica 6 je pripadalo pticama koje žive isključivo u vodenim staništima i smatraju se rezervoarom virusa avijarne influence (bela roda, patka gluvara, labud grbac, riđoglava patka, patka dupljašica i liska). Od navedenih vrsta posebnu pažnju privukle su 4 vrste divljih ptica iz reda Anseriformes i familije Anatidae (patka gluvara, labud grbac, riđoglava patka, patka dupljašica) kojima je od ukupno 259 ptica pripadalo 30 jedinki. U 30 uzoraka krvnog seruma navedenih vrsta ptica, specifična antitela protiv A tipa virusa avijarne influence utvrđena su u 9 (30%) uzoraka seruma, primenom cELISA. Specifična antitela protiv podtipa H5 virusa avijarne influence su ustanovljena u 3 uzorka seruma labudova grbaca (jedan uzorak seruma je poticao od labuda grbca koji je prstenovan u Poljskoj) i u jednom uzorku krvnog seruma riđoglave patke, ili ukupno u 4 (13,33%) uzorka seruma, primenom testa inhibicije hemaglutinacije. Specifična antitela protiv podtipa H7 virusa avijarne influence utvrđena su u 3 (10%) uzorka krvnog seruma i to u dva seruma pataka gluvara i u jednom serumu labuda grbca, primenom inhibicije hemaglutinacije. Specifična antitela protiv A tipa virusa avijarne influence nisu ustanovljena ni kod jedne ispitivane vrste ptice, primenom AGP testa. U populaciji divljih vrta ptica u Republici Srbiji obuhvaćenih ovim ispitivanjem, specifična antitela protiv virusa avijarne influence ustanovljena su samo u uzorcima seruma ptica iz familije Anatidae. Specifična antitela protiv A tipa virusa avijarne influence su otkrivena kod 3 (6,52%) vrste divljih ptica, a protiv podtipova H5 i H7 kod 2 (4,34%) od ukupno 46 vrsta koje su ispitivane. Senzitivnost cELISA testa za podtip H5 virusa avijarne influence i testa inhibicije hemaglutinacije za isti podtip iznosila je 100%.