Bolest Aujeszkoga (BA) je akutna virusna zarazna bolest uzrokovana svinjskim herpes virusom 1 (SHV-1). Najznačajnija mjera iskorjenjivanja BA u domaćih svinja je cijepljenje. Iako se u programima iskorjenjivanja BA mogu koristiti markirana cjepiva koja sadržavaju atenuirani ili inaktivirani SHV-1, kao i subjedinična cjepiva, cjepiva s atenuiranim SHV-1 su se pokazala učinkovitijima u poticanju stanične imunosti. Nadalje, istraživanja su utvrdila da inaktivirani virus Parapoxvirus ovis i kombinacija inaktivirane bakterije Propionibacterium granulosum i detoksificiranih lipopolisaharida (LPS) bakterije Escherichia coli imaju izražena imunomodulacijska svojstva te da njihova primjena s cjepivima može u svinja rezultirati jačim imunosnim odgovorom na razini stanične i humoralne imunosti u odnosu na primjenu samo cjepiva.
Nakon što su svinjama primijenjeni cjepivo (atenuirani SHV-1), odabrani imunomodulatori (inaktivirani virus Parapoxvirus ovis ili kombinacija inaktivirane bakterije P. granulosum i detoksificiranih LPS-a bakterije E. coli) ili cjepivo s pojedinim imunomodulatorom, provedene su hematološke pretrage uzoraka periferne krvi, određena su protutijela za glikoproteine B (gB) i E (gE) SHV-1 u uzorcima seruma te je analiziran fenotip NK stanica, B limfocita i T limfocita, kao i pojedinih populacija i subpopulacija T limfocita u uzorcima mononuklearnih stanica periferne krvi.
Rezultati hematoloških pretraga bili su unutar referentnih vrijednosti u većini skupina tijekom cijelog pokusa. Serumska protutijela za gB SHV-1 utvrđena su nakon jedanaestog dana pokusa samo u cijepljenih svinja, a protutijela za gE SHV-1 nisu utvrđena niti u jedne svinje. Primjena cjepiva i/ili imunomodulatora nije imala značajan učinak na udjele T i B limfocita te NK stanica u limfocitima periferne krvi, kao ni na većinu istraživanih subpopulacija T limfocita periferne krvi. Zajednička primjena cjepiva s kombinacijom inaktivirane bakterije P. granulosum i detoksificiranih LPS bakterije E. coli je sedmi dan pokusa potaknula statistički značajno veći porast udjela CD4+CD8α+ i CD4-CD8α+ αβT limfocita u T limfocitima periferne krvi. Rezultati ukazuju na adjuvantni učinak jednokratne primjene cjepiva i dvokratne primjene kombinacije inaktivirane bakterije P. granulosum i detoksificiranih LPS bakterije E. coli na subpopulacije T limfocita svinja s važnim funkcijama stanične imunosti u zaštiti svinja od infekcije sa SHV-1.
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INTRODUCTION: Aujeszky’s disease (AD) is an infectious disease of many mammalian species. It is caused by suid herpesvirus 1 (SuHV-1) which belongs to the Herpesviridae family. Domestic pigs and wild boar are natural hosts of SuHV-1 because they are the only species that can survive infection. Aujeszky’s disease is economically important because it can cause a high mortality in suckling piglets, slower growth of fattening pigs and reduced fertility in sows. SuHV-1 persists in latent state in pigs that survive infection. That is why many countries completed eradication programs for AD while in others eradication programs are still in process. Although many countries are free of AD in domestic pigs, SuHV-1 circulates in wild boar populations which can be the source of infection for domestic pigs. One of the most important measures for eradication of AD in domestic pigs is vaccination with marker vaccines and the use of appropriate diagnostic tests which can distinguish infected from vaccinated animals (DIVA vaccines, Differentiating Infected from Vaccinated Animals). The most widely used marker vaccines are the ones that lack glycoprotein E of SuHV-1. Marker vaccines that contain the whole virus (attenuated or inactivated) or viral glycoproteins (subunit vaccines) are available. Although vaccines with inactivated SuHV-1 and subunit vaccines are generally considered to be safer, vaccines with attenuated SuHV-1 have shown to be more efficacious in inducing protective immunity in pigs and stronger cell-mediated immune response. Previous studies also showed that SuHV-1 can stimulate different components of innate and adaptive immunity in pigs and that antibodies protect pigs from mortality after SuHV-1 infection, but early protection against clinical signs and reduced virus shedding after infection were related to in vitro T-cell proliferation. Development of monoclonal antibodies directed against porcine leukocyte differentiation antigens have led to immunophenotyping of different T-cell subpopulations and enabled the study of their immune functions and development of more efficacious vaccines. Flow cytometry is one of the most commonly used techniques for analysis of cell phenotype, which also enables quantitative analysis of cell subpopulations. Although much knowledge about phenotype and function of porcine NK cells and different T-cell subpopulations is gained by in vitro studies with SuHV-1, little is known about frequencies of NK cells and different T-cell subpopulations in peripheral blood of pigs vaccinated or infected with SuHV-1. Immunostimulators are biological or chemical substances that stimulate mechanisms of innate and adaptive immunity in a non-specific way. Some immunostimulators, mainly bacteria, components of bacterial cells and plant extracts, are used in veterinary medicine for immunotherapy but can be also
used as adjuvants in veterinary and human vaccines, because they can accelerate, enhance or prolong the immune response to vaccine antigen. The studies have shown that administration of vaccines together with inactivated Parapoxvirus ovis or combination of inactivated Propionibacterium granulosum and detoxified lipopolysacharides (LPS) of Escherichia coli in pigs can induce stronger humoral and cellular immune responses than sole administration of vaccines.
The aim of this research is to determine effects of selected immunomodulators of microbial origin and vaccine against AD on leukocytes, NK, B- and T-cells and T-cell subpopulations in peripheral blood of pigs. Results might provide an insight whether use of investigated immunomodulators can enhance cell-mediated immunity in pigs vaccinated with attenuated SuHV-1 and whether their use at the time of vaccination against AD is justified.
MATERIAL AND METHODS: For the purpose of this research a study using six groups of five, three month old, pigs was conducted. Pigs from five groups were intramuscularly injected with: i) one dose of vaccine (attenuated SuHV-1, strain Bartha K-61 gE-); ii) two doses of inactivated Parapoxvirus ovis; iii) two doses of the combination of an inactivated P. granulosum and detoxified LPS of E. coli; iv) one dose of vaccine together with two doses of Parapoxvirus ovis or v) one dose of vaccine together with two doses of combination of inactivated P. granulosum and detoxified LPS of E. coli. Sixth group was negative control. Samples of peripheral blood were collected by venipuncture on days 1, 7, 11 and 18. Clinical health of animals was observed at least once daily throughout the study. Differential blood count (DBC) and complete blood count (CBC) were analysed in peripheral blood samples by microscopy and automated hematology analyser. Antibodies for SuHV-1 glycoproteins B (gB) and E (gE) in serum samples were analysed by ELISA (Enzyme-linked Immunosorbent Assay). Phenotype of NK cells (CD3-CD16+CD8α+SLA-DR-), B-cells (CD3-CD21+) and T-cells (CD3+CD16-) and phenotype of T-cell subpopulations with respect to expression of TCR-δ, CD4, CD8α and SLA-DR were analysed in samples of peripheral blood mononuclear cells by multicolour flow cytometry.
RESULTS: Local and general reactions were not observed in any group. Results of DBC and CBC were within reference values in most groups during entire study. Serum antibodies for SuHV-1 gB were present only in vaccinated groups from day 11. Serum antibodies for SuHV-1 gE were absent in all groups during entire study. Vaccine and/or immunomodulators did not show a significant effect neither on relative numbers of T-, B- and NK cells in peripheral
blood lymphocytes, nor on the relative numbers of most T-cell subpopulations. On study day 7 group that received vaccine together with the combination of an inactivated P. granulosum and detoxified LPS of E. coli induced significantly higher (p < 0.05) relative numbers of CD4+CD8α+ and CD4-CD8α+ αβ-T cell subpopulations in peripheral blood T-cells compared to the other groups, except the one that received the same immunomodulator alone.
CONCLUSION: Administration of a combination of an inactivated P. granulosum and detoxified LPS of E. coli together with an attenuated SuHV-1 showed a synergistic effect on αβ-T cell subpopulations that contain activated/memory helper T cells and SLA I restricted cytolytic T cells, later of which seem important for early protection of pigs against SuHV-1 infection.
This research suggests that a combination of an inactivated P. granulosum and detoxified LPS of E. coli is a potential adjuvant candidate for vaccines against AD. Further studies should be undertaken to evaluate the fraction of virus specific T cells within CD4+CD8α+ and CD4-CD8α+ αβ-T cell subpopulations.