Ispitivanje imunofenotipa mononuklearnog ćelijskog infiltrata u jetri svinja prirodno inficiranih virusom hepatitisa E

Abstract

U ovoj disertaciji ispitano je 233 rektalna brisa od klinički zdravih svinja, različitih uzrasnih kategorija, poreklom sa četiri farme, na prisustvo nukleinske kiseline virusa hepatitisa E metodom RT-PCR. Pored toga, RT-PCR, bakteriološkom i PCR analizom su ispitani uzorci jetre od 12 svinja kod kojih je RT-PCR nalaz iz uzorka rektalnog brisa pokazao prisustvo HEV RNK. Kod 12 svinja kod kojih je metodom RT-PCR u uzorku rektalnog brisa utvrđena nukleinska kiselina virusa hepatitisa E, izvršena je punkcija vena cava cranialis i uzeta je krv za biohemijska ispitivanja. Nakon koagulisanja krvi i izdvajanja seruma uz pomoć poluautomatskog biohemijskog spektrofotometra određeni su sledeći parametri: ukupni proteini, albumini, aspartat-aminotransferaza (AST), alanin-aminotransferaza (ALT), γ- glutamil-transpeptidaza (GGT) i ukupni bilirubin. Histopatološka i imunohistohemijska analiza urađena je na uzorcima jetre koji su fiksirani u 10% neutralnom formalinu, obrađeni u automatskom tkivnom procesoru i uklopljeni u parafin. Parafinski isečci debljine oko 3-5μm bojeni su hematoksilineozin i Masson trihrom metodom. Imunofenotipizacija ćelija mononuklearnog infiltrata izvršena je streptavidin-biotin (LSAB2) metodom i tehnikom dvostrukog imunohistohemijskog bojenja, uz upotrebu antitela za CD3 i CD79. Ekspresija TGFβ1 molekula postignuta je streptavidin-biotin (LSAB2) metodom. Za procenu gustine i distribucije limfocitno – plazmaćelijskog infiltrata u jetri korišćena je semikvantitativna analiza. Broj CD3 i CD79 pozitivnih ćelija u jetri određen je morfometrijskom analizom. Pored deskriptivnih statističkih parametara upotrebljeni su ANOVA i Tukey test. Ukupno je ispitano 233 svinje, od kojih je kod dvanaest u uzorku rektalnog brisa dokazana HEV RNK (5,15%). Istovremeno prisustvo HEV RNK u uzorku rektalnog brisa i jetre je ustanovljeno kod pet svinja (41,67%). U dvanaest uzoraka jetre nije dokazano prisustvo aerobnih i anaerobnih bakterija i genoma svinjskog cirkovirusa tip 2...

In this thesis from clinically healthy pigs of different ages, originating from 4 farms, a total of 233 rectal swabs were tested for the presence of HEV RNA by RTPCR. In addition, liver samples of 12 pigs which were RT-PCR positive to HEV RNA were tested by RT-PCR, bacteriological and PCR analysis. A total of 12 pigs which rectal swabs tested positive by RT-PCR analysis were further on sampled by puncture of vena cava cranialis, as well. After coagulation blood sera were analyzed on a biochemical spectrophotometer and the following parameters were determined: total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ – glutamil transpeptidase (GGT) and total bilirubin. Histopathological and immunohistochemical analyses were performed on liver samples fixed in 10% neutral formalin, processed in an automated tissue processor and embedded in paraffin. Paraffin sections about 3-5μm thick were stained with hematoxilin-eosin and by Masson trichrome method. Immunophenotypisation of mononuclear infiltrate cells was carried out by the streptavidin – biotin method (LSAB2) and by the double immunohistochemical staining technique with antibodies for CD3 and CD79. Expression of TGF-β1 molecule was obtained by the streptavidin – biotin method (LSAB2). Estimation of the density and distribution of lymphocyticplasmacytic infiltrate in the liver was done by semiquantitative analysis. The number of CD3 and CD79 positive cells in the liver was determined by morphometric analysis. In addition to descriptive statistical parameters ANOVA and Tukey test were used, as well. A total of 233 pigs were tested. Out of this number in 12 rectal swabs HEV RNA (5.15%) was confirmed. Concurrent presence of HEV RNA in both rectal and liver samples was determined in 5 pigs (41.67%). In 12 liver samples the presence of aerobic and anaerobic bacteria, as well as circovirus type 2 genome were not confirmed. Results of blood serum biochemical testing did not show statistical significance between values for total proteins, albumins, and AST between the infected pigs and the control group. A statistically very significant difference (p<0.01) between the infected and the control group was determined for ALT, and a significant difference (p<0.05) was recorded for GGT and total bilirubin...