Newcastleska bolest (NB) je vrlo kontagiozna zaraza ptica. Preventiva NB se temelji na cijepljenju peradi, a izbijanja NB u cijepljenim jatima upućuju na stalnu potrebu kontrole imunogenosti postojećih i razvoj novih cjepiva. Prethodna istraživanja primjene virusa NB soja ZG1999HDS u pilića ukazala su na imunogenost razvojem humorane imunosti i pripadnost lentogenom patotipu virusa NB. U ovom istraživanju smo usporedno okulonazalno primijenili komercijalno živo cjepivo koje sadrži La Sota soj VNB i suspenziju liofiliziranog soja ZG1999HDS muškim pilićima hibrida komercijalnih nesilica u dobi četiri tjedna. Stanični odgovor odredili smo na temelju proliferacije i razlikovanja subpopulacija leukocita protočnom citometrijom i mikroskopski, brojanjem u komorici za brojanje i na krvnim razmascima, a na razini proizvodnje interferona (IFN) ispoljavanjem gena za IFN-α i IFN- γ u uzorcima slezene pomoću reverzne transkripcije i lančane reakcije polimerazom u stvarnom vremenu (RT-qPCR). Oba primijenjena soja nisu izazvala kliničke znakove u pilića. Imunogenost oba soja potvrđena je tvorbom specifičnih protutijela koja je za čitavog pokusa bila jače potaknuta u pilića imuniziranih sojem ZG1999HDS nego u pilića s primijenjenim cjepnim La Sota sojem, a statistički značajno jače u obje skupine nego u kontrolnoj skupini. Stanična imunost, koja se očitovala jačom proliferacijom B- i T-pomoćničkih limfocita i ispoljavanjem IFN-γ, polučila je bolje rezultate u skupine imunizirane sojem ZG1999HDS nego u skupini cijepljenoj komercijalnim cjepnim La Sota sojem. Na temelju dobivenih rezultata zaključujemo da je soj ZG1999HDS imunogen i pogodan kandidat za daljnja istraživanja i razvoj cjepiva za perad.
|Abstract (english)|| |
Aim: The aim was to determine the ability of Newcastle disease virus (NDV) strain ZG1999HDS to induce protective cell-mediated immune response (CMI) in chickens as measured by interferon response in spleen and proliferation of leucocyte and lymphocyte subpopulations in peripheral blood. The extent of CMI induction achieved was compared to the CMI inducued by commonly used vaccinal La Sota strain and results were used to evaluate justification to consider ZG1999HDS strain as a prospective candidate for poultry vaccine development.
Background: Newcastle disease (ND) is a highly contagious viral infection of birds with significant economic impact on poultry industry. Clinical manifestation of ND varies from unapparent infection to peracute infection with 100% mortality.
Global control of ND is based on prophylactic measures, such as immunisation of poultry, non specific sanitary and biosafety measures, good management practice, depopulation and trade bans in case of outbreaks. Immunisation is performed with inactivated or live attenuated vaccines (LAV) composed of lentogenic or attenuated mesogenic NDV strains. Current commercially available vaccines are successful in disease and mortality prevention, but are unable to prevent infection, replication and shedding of virulent NDV. Predominant circulating strains are viscerotropic, which makes widely used vaccines based on pneumotropic strains, inadequate to protect the animals against ND. Thus, outbreaks of ND in vaccinated flocks occur and indicate the constant need to control immunogenicity of existing and develop new vaccines.
Hemagglutination inhibition (HI) tests or enzyme-linked immunosorbent assay (ELISA) are commonly used 3 to 4 weeks post vaccination for assessment of developed humoral immunity. However, assessment of overall protection against ND requires also assessment of CMI. Available assays for CMI assessment include detection of total number of leukocytes, and different subpopulations (specifically B and T lymphocytes) in blood, and also by their functional assessment i.e. quantitative detection of cytokines and chemokines, antigen specific lymphocyte proliferation, heterofile activation and degranulation and production of oxygen reactive species.
Strain ZG1999HDS of NDV was isolated during the outbreak in July of 1999 at a broiler farm in Croatia. Previous application of this strain in chickens confirmed its
immunogenicity by induction of humoral immunity and lentogenic pathotype. Further characterisation by deduced amino acid sequence at the cleavage site of fusion protein, and in vitro tests in cell cultures revealed its lentogenic nature and oncolytic capacity, respectively.
Material and methods: Trial involved the total of 150 male chickens of light breed type. Chickens were held in cages with water and feed supplied ad libitum. The decision about the trial onset at the age of 28 days was based on HI antibodies titer (i.e. clearance) of maternal antibodies for NDV. Chickens were divided into 3 groups of 50 chickens. Suspension of freeze dried allantoic fluid containing NDV strain ZG1999HDS or freeze dried commercial vaccine of La Sota NDV strain were applied oculonasally to chickens in ZG or LS group, respectively. Control group was treated oculonasally with PBS. Blood and spleen samples (after sacrificing) were collected from randomly selected 6 chickens per group in intervals 6, 12, 24 hours and 2, 3, 5, 7 i 14 days post immunisation (DPI) for real-time polymerase chain reaction (RT-qPCR), and blood samples for immunophenotyping and hemathology assay in intervals before immunisation and 3, 5, 7, and 14 DPI. Blood for separation of sera for seroconversion by HI assay were collected in weekly intervals. Blood samples with addition of heparine were used for hematological testing by microscopy, immunophenotyping of modified ficoll isolated peripheral blood mononuclear cells (PBMC) by flow cytometry. Heparinized blood samples with addition of preservative were intended for subsequent gene expression analysis by RT-qPCR. Total RNA was isolated by TRIzol method from preserved heparinezied full blood and spleen samples and the concentration determined by spectrophotometry. Relative quantification of IFN-α, IFN-γ mRNAs and genomic RNA for NDV strains was performed by RT-qPCR in total RNA samples.
Results and discussion: Application of both NDV strains in our trial didn't cause any adverse reactions in immunised chickens. The induction of humoral immunity was confirmed in both immunized groups with HI antibody titer significantlly higher to PBS treated group and higher in chickens immunised by ZG1999HDS strain compared to La Sota, thus confirming their imunogenicity. Proliferation of B- and T-helper cells in ZG group was significantlly higher compared to LS and control group, providing evidance of better CMI induction by ZG1999HDS strain in addition to higher expression of genes for IFN γ in both experimental groups compared to control. The abscence of both strains from the spleen was confirmed by inability to find viral genomic RNA in spleen samples
which is in accordance of their lentogenic nature and tendancy to localise and replicate at the site of infection.
Conclusion: Based on the results we found that both viral strains induced CMI and the strain ZG1999HDS to be imunogenic and, therefore prospective candidate for further research and vaccine development.