Immunopathological studies in the ovine lung during the course of natural and experimental parainfluenza type 3 virus infection

OF THESIS CARLOS MANUEL GONZALEZ Name of Candidate Address (Regulation 3:5.13) Degree . P.h. :T. : Date . J.L AUGUST . 1.9. 9.5. Title of Thesis IMMUNOPATHOLOGICAL STUDIES IN THE OVINE LUNG DURING THE COURSE OF NATURAL AND EXPERIMENTAL PARAINFLUENZA TYPE 3 VIRUS INFECTION 60,000 No. of words in the mam text of Thesis I lie pulmonary imnuinopalliology of parainfluenza type 3 (I'l V-3) infection in sheep was investigated firstly by isolating ttic virus from field cases of sliccp pneumonia, secondly by experimentally reproducing the disease with the isolated vims and finally try studying changes in lymphocytes subsets and alveolar macrophages, induced by HI V-3 in vivo and in vitro. Throe ovine virus isolates (270-7, 390-10 and 430-7) were obtained and characterised, as l'IV-3. according to virus morphology, transmission electron microscopy (I HM); cylopalhic cll'ccl (CI'H); hacmagglutination, of guinea pig erythrocytes; physicochcmical properties; serological crossrcaclivity with anliscra raised against l'IV-3; and reactivity with monoclonal antibodies to l'IV-3 structural proteins, that crossrcact with human and bovine strains. flic ability of the virus to induce respiratory disease was investigated by experimental inoculation of ovine l'IV-3 isolate, 270-7 in colostrum deprived lambs Clinical, pathological, bacteriological and virological studies were carried out on days 2. 3, 3 and 7 post infection (p i ), l itis I'l V-3 ovine strain was able to induce clinical disease, llistopathological findings were interstitial pneumonia with hyperplasia of bronchiolar associated lymphoid tissue (HALT), degenerative bronchiolar epithelium with lymphocyte inlillration, areas of atelectasis and increased alveolar septa thickness due to proliferation of type II pncmnocytcs, lymphocytes, macrophages and later to Itbrosis. lire large number of lymphocytes, particularly on days 5 and 7 pi, combined with the minimal to moderate cytolvsis of antigen bearing cells suggests that l'IV-3-induced pulmonary disease has an inuuunopathological component. l'IV-3 particles were detected more frequently in bronchiolar epithelium cells than in alveolar septal cell This was correlated with changes of these cell populations in lung lesions, delected by iminunohistochemislry. Alter 7 days p i . virus-induced changes in the leukocyte composition of the lungs were detected using a panel of rnAbs (o ovine lymphocytes and macrophages. Changes in lung tissue were detected by inununohistology and changes in lungwash llnid (LWH) bv llowcylomctry. Association lie I ween lung cells and virus particles was investigated by double immunoslaining. The differential cell count of LWIf from l'IV-3 infected animals was characterised by a significant increase (pet). 03) in lymphocytes and neutrophils. Lymphocy te phcnolyping showed a significant decrease (pet). 05) of C'1711 cells, a significant increase (p<0.05) oT CDK1 cells and a significant inversion (pcO.OO I) or the (171 '/COS1 ratio. Immunoslaining of I'l V-3 infected lung sections showed a remarkable increase of lymphocy tes, particularly in HALT, and most cells were CD81. flic number of macrophages increased in peribronchial and alveolar septa and some were positive lor I'l V-3 particles. Cultures of peripheral blood monocylc-dcrivcd macrophages (MUMf) and alveolar macrophages (AMf) were established, file ability of l'IV-3 lo inlccl these cells was studied by CI'H, and virus particle immtmoslaining. Viral replication was detected by TliM and scanning electron microscopy (SBM). l'IV-3 induced lytic CI'H'w ith inlracyloplastnic eosinophilic inclusion Ixidics and syncy tia formation. I'HM revealed virus budding al the cell membrane, lilamcntous cytoplasmic inclusions and clusters of pleomorphic viral particles in the extracellular space. flic expression of MIIC class I and MIIC class II molecules, which is associated willt antigen presenting function, was studied alter in vitro inlccl ion of MUM and AM with I'l V-3. UK and IX) Ml It. ' class II expression was moderately high (60-80%) on noninl'cclcd fresh monocytes and AM but, allcr 3 days in culture the expression of this molecules was dramatically reduced lo 5%. Stimulation with y-llrN was able to promptly restore MIIC class II expression in cultured noninfcclcd MUM arid AM. This did not occur alter I'l V3 infection. The expression of MIIC class I molecules was not significantly all'cctcd (p>0,05) by culture or I 'I V-3 infection. flic phagocytic activity of macrophages lor IT fC-lnbcllcd/nnlibody-coalcd sheep red blood cclls(SRUC) alter I'l V-3 infection decreased significantly (p< (). ()3) alter . 3 days p i. PGS/ABST/94 xi Use this side only