Immune mechanisms in the pathogenesis of viral diseases: a review.

Three immunopathological mechanisms may determine the pathogenesis of viral diseases in animals. (1) A variety of viruses causes transient or prolonged immunosuppression by infecting lymphoreticular tissues and interacting with components of the immune system. (2) In persistent viral infections effective immune responses may result in tissue damage. The mechanisms involved are T-cell-mediated destruction of infected cells and delayed-type hypersensitivity. (3) In a number of viral diseases pathogenic immune complexes are formed when antibodies are produced and react with viral antigen molecules persisting in the host. The selected examples of immune dysfunction are the focus of this review.

Infection of ovine fetal brain cell cultures with cytopathogenic and non-cytopathogenic bovine viral diarrhoea virus.

The in vitro cell tropism of non-cytopathogenic (ncp) and cytopathogenic (cp) bovine viral diarrhoea virus (BVDV) was studied in primary dissociated brain cell cultures derived from ovine fetuses of different gestational ages. The cell types infected were identified by double immunofluorescence using antibodies against BVDV and cell type-specific markers. In cultures infected with ncp BVDV viral antigen was present in neurofilament (NF 200 kDa)-positive neurons, glial fibrillary acidic protein (GFAP)-positive astrocytes and fibronectin-expressing cells. Estimation of the percentages of individual cell types infected with ncp BVDV indicated a tropism for NF 200-positive neurons. In cultures infected with cp BVD virus cytopathic changes were observed beginning at 40 hours post infection. Viral antigen was present in vacuolated NF 200-, GFAP- and fibronectin-positive cells. In comparison with non-infected control cultures a considerable reduction of the number of the different cell types was seen.

Application of the indirect enzyme immunoassay for the detection of antibodies against Erysipelothrix rhusiopathiae.

Sera from swine and rats experimentally infected with Erysipelothrix rhusiopathiae and field sera from swine were investigated for antibodies against E. rhusiopathiae using the microtiter enzyme immunoassay (EIA) and, for comparison, the growth test (GT) and the agglutination test (AT). In principle there was a good correspondence between the results of EIA and those of the two other methods, but EIA and GT were more sensitive than AT. On the basis of the evaluation pattern of GT and AT on swine sera, EIA titers of 1/320 were considered as "chronic erysipelas titers". Compared with GT and AT, the EIA has some advantages: it is not influenced by contamination of the test sera, it takes only a few hours and using the microtiter system it is easy and economical to perform.

Immunohistological detection of bovine viral diarrhoea virus antigen in the central nervous system of persistently infected cattle using monoclonal antibodies.

In a total of 25 cattle persistently infected with bovine viral diarrhoea virus (BVDV) the distribution of viral antigens in the central nervous system was studied. Using a panel of monoclonal antibodies (anti pestivirus C16; anti cytophathic BVDV C38; anti cytopathic and non-cytopathic BVDV C42; anti gp53 BVDV CA-1 and CA-3) and the indirect immunoperoxidase technique, BVDV antigen was located exclusively in neurons. Predilection sites for viral persistence were cerebral cortex and hippocampus. Morphological cellular alterations were not seen. Reactive perivascular lymphocytic infiltrations were occasional findings.

Pathomorphological and immunohistological findings in cattle experimentally infected with rinderpest virus isolates of different pathogenicity.

Experimental infection of nine cattle with seven rinderpest virus strains of different pathogenicity resulted in significant variations of clinical signs, morphological lesions and distribution of viral antigen in tissues. The severity of clinical disease was correlated with the extent of tissue alterations and the amount of immunohistologically detectable viral antigen. Both mild and virulent strains of rinderpest share essentially the same tissue tropisms in vivo, i.e. epithelio- and lympho-tropism. However, rinderpest virus isolates of higher pathogenicity showed a more rapid and wider distribution with more extensive lesions than milder strains, which probably accounts for the higher mortality.

Effects of trypsinization and microwave treatment on lectin labelling of microglial cells in paraffin-embedded sections from pre- and postnatal bovine brains.

The effects of microwave heat treatment on lectin histochemical staining of microglial cells with Griffonia simplicifolia B4 isolectin (GSA I-B4) and Ricinus communis agglutinin-I (RCA-I) in paraffin-embedded pre- and postnatal bovine brain tissue fixed in two different fixatives (Bouin's fluid and 4% neutral buffered formaldehyde) were examined, and the results compared with lectin labelling obtained in untreated and trypsinized serial sections. The results indicate that lectin labelling of bovine microglial cells depends on the kind of lectin applied, the fixative used for tissue preservation, the isotype of microglia to be labelled, and the pretreatment of tissue sections. In brain tissue fixed in Bouin's fluid, GSA I-B4 staining of both microglial isotypes, i.e., amoeboid and ramified microglial cells, was achieved without trypsinization. Staining of sections with RCA-I, however, yielded negative results both on untreated and on trypsinized sections. These findings suggest that species-specific differences in the density of binding sites accessible to GSA I-B4 and RCA-I, respectively, may exist. Pretreatment of sections by microwave irradiation had different effects depending on the lectin and fixative used and on the microglial isotype to be stained. Microwave heat treatment of sections prior to incubation with RCA-I enabled the labelling of amoeboid and ramified microglial cells. The latter cell type, however, was exclusively stained in brain tissue fixed in Bouin's fluid. With GSA I-B4, exclusively the labelling of ramified microglial cells in sections fixed in Bouin's fluid was improved. It is assumed that by microwave pretreatment of sections from bovine brain the access of both lectins to their receptors, i.e., D-galactose residues on microglial cells, may be facilitated.

Persistence of acquired immunity to Sarcocystis miescheriana infection in growing pigs.

Sixteen 8-week-old pigs were each experimentally immunized by subclinical infections with 10(3) Sarcocystis miescheriana (syn. S. suicanis) sporocysts and 8 other pigs served as non-immunized controls. Four groups of pigs (each consisting of 4 immunized plus 2 control pigs) were then challenged by infection with 3 X 10(6) sporocysts at either 40, 80, 120 or 160 days post-immunization (dpi) to determine the persistence of the protective immunity against acute sarcocystosis. Pigs challenged 40 dpi demonstrated a solid immunity to lethal challenge and disease. They survived challenge following a mild fever phase whereas both controls died from acute disease. This immunity however, did not prevent the further establishment of parasitic cysts within the host musculature following challenge. The protective immunity against acute disease persisted to 80 dpi, but was not evident thereafter. At necropsy, the clinical and pathological findings in all pigs which had been subjected to challenge were consistent with anamnestic responses of sensitized hosts to re-infection.

An immunohistochemical study of the fetal sheep neocortex and cerebellum with antibodies against nervous system-specific proteins.

The topographical distribution of glial fibrillary acidic protein (GFAP), vimentin, neuron-specific enolase (NSE) and neurofilament (NF) proteins in the developing neocortex and cerebellum of sheep fetuses of different gestational ages (60-149 days) was described. For comparison, brain tissues from a lamb and two adult sheep were included in this study. In the walls of the developing cerebral hemispheres GFAP- and vimentin-immunoreactive radial glial fibres were demonstrated. From 80 days of gestation onwards a continuous decrease of radial fibres occurred which was accompanied by an increase of GFAP-positive mature astrocytes. In Bergmann glial fibres of the cerebellum, which are the equivalent of radial fibres in the telencephalon, both GFAP and vimentin were detectable in fetuses and adult sheep. With polyclonal antibodies against NSE and NF proteins (NF-M, NF-H) prominent staining of neuronal fibre tracts was seen in fetuses of all gestational ages studied. In the neocortex, staining for NF-L did not occur before day 80 of gestation. With monoclonal antibodies against phosphorylated NF-H (clone SMI 31), however, reaction of neocortical fibre tracts was first seen at 85 days of gestation, and cytoplasmic staining of single neocortical neurons was first found in a 149-day-old fetus. Several fixatives and proteolytic pretreatment were examined for their effects on preservation and re-establishment of marker protein expression, respectively. GFAP and vimentin in radial glial fibres were not demonstrable without pretrypsinization of tissue sections. The most intensive staining of NF proteins with polyclonal antisera was seen in brains fixed in Bouin's fluid.

A canine nephropathy resembling minimal change nephrotic syndrome in man.

In the dog, massive proteinuria and/or the nephrotic syndrome have been commonly associated with renal amyloidosis and membranous glomerulonephritis. Primary glomerulopathies associated with the nephrotic syndrome in man also include minimal change nephrotic syndrome and focal glomerular sclerosis. A 4-year-old Collie dog is described with clinical, histological, immunohistological, and ultrastructural findings similar to those which characterize the minimal change nephrotic syndrome (MCNS) in man.

Immunohistochemical identification and crossreactions of amyloid-A fibril protein in man and eleven other species.

Antisera were prepared in rabbits, sheep or chicken against purified amyloid fibril protein AA from man, mouse, stone marten, dog, cow and hamster. These antisera were tested by immunodiffusion against all purified antigens and applied to tissue sections containing amyloid from man, mouse, hamster, guinea pig, rabbit, cat, dog, mink, stone marten, pine marten, cow and horse. The binding of the antibodies to amyloid in tissue sections was assessed by the indirect immunoperoxidase method. The strongest reactions in the immunodiffusion and immunohistochemical methods were found between amyloid deposits of members of a given species and an antibody raised against protein AA from the same species. In contrast to the lack of cross-reactivity in immunodiffusion (except in the mouse-man relationship), extensive cross-reactions were observed immunohistochemically in phylogenetically related species, e.g. between stone marten, pine marten and mink, or between hamster and mouse. However, cross-reactions were also observed in combinations such as man-mouse, man-dog, man-cat, mouse-horse, and dog-cow. In addition, individual antisera showed variations in immunohistochemical reactivity with amyloid deposits of different members of one given species. Moreover, antisera prepared in rabbits reacted more restrictedly than those prepared in sheep, while rabbit antisera against any AA-protein did not react with rabbit amyloid. Finally, the widest degree of cross-reactivity including almost all mammalian species investigated was observed with a chicken antiserum to human amyloid AA protein.

Expression of class II major histocompatibility complex molecules in renal tubular epithelial cells of canine kidneys affected with tubulointerstitial nephritis.

Class II major histocompatibility complex (MHC) products are important molecules on various antigen-presenting cells and induce a T cell-specific immune response. The distribution of class II MHC molecules in the normal canine kidneys of dogs with tubulointerstitial nephritis was investigated by using a sensitive immunocytochemical method. In the normal canine kidney, class II MHC molecules were detected in interstitial 'dendritic' cells. In cases of tubulointerstitial nephritis, however, the expression of class II MHC molecules extended to other renal elements such as the epithelial cells of cortical and medullary tubules and, in some cases, the endothelial cells of peritubular capillaries. The tubular expression of class II MHC molecules was enhanced in dogs with higher levels of proteinuria. The results suggest that heavy proteinuria may be one triggering factor in canine tubulointerstitial damage, probably mediated by the reabsorption of filtered cytokines and immunogenic peptides which induce tubular epithelial cells to behave as immune accessory cells.

Evidence of cytokeratin expression in canine visceral glomerular epithelial cells in vivo.

Visceral glomerular epithelial cells (vGECs) originate from a mesenchymal blastema and transiently express cytokeratin during embryogenesis. There are no reports of cytokeratin expression in vGECs of mature, normal or damaged, human or other mammalian kidneys in vivo, but in vitro studies have provided evidence of the synthesis of cytokeratin in cultured vGECs. Cytokeratin expression was observed in vGECs in the damaged kidneys of four dogs with spontaneous renal diseases and, by using monoclonal antibodies, type 18 cytokeratin was identified. vGECs are apparently able to (re-) activate in vivo a mechanism for switching on the synthesis of cytokeratin in damaged glomeruli.

Ultrastructural co-localisation of vimentin and cytokeratin in visceral glomerular epithelial cells of dogs with glomerulonephritis.

The expression of cytokeratin and vimentin was studied in the glomerular epithelial cells of canine kidneys with and without glomerular abnormalities. Using ultrastructural, immunogold single and double labelling techniques, cytokeratin and vimentin were found together in the visceral glomerular epithelial cells (vGECs) of abnormal kidneys. In normal kidneys, the vGECs expressed only vimentin, and cytokeratin was found exclusively in parietal glomerular epithelial cells (pGECs). These results confirm previous findings in the same animals, obtained by immunohistological staining techniques.

Demonstration of amoeboid and ramified microglial cells in pre- and postnatal bovine brains by lectin histochemistry.

In embryonic, fetal and postnatal bovine brains the development and distribution of microglial cells was examined by lectin histochemistry, using the isolectin B4 from Griffonia simplicifolia (GSA I-B4), the lectin from Ricinus communis (RCA-I), and mistletoe lectin (ML I). With GSA I-B4 and ML I, different types of microglial cells, i.e., amoeboid, intermediate and ramified cells, were specifically stained. On sections fixed in Bouin's fluid significantly higher numbers of microglial cells were labelled than on sections fixed in formalin. On the latter, proteolytic pretreatment was required. With RCA-I, no staining of microglial cells was achieved. This finding may indicate the presence of very low concentrations of beta-D-galactose residues on bovine microglial cells in comparison with other species studied so far. In the fetal telencephalon, the highest numbers of amoeboid microglial cells were found in transitory structures (subependymal regions of the lateral ventricles, cavum septi pellucidi, intermediate zone) and in areas of developing axon tracts (corpus callosum, internal and external capsules) between three and five months of gestational age. From 3-4 months of gestational age onward, the appearance of ramified microglial cells was noted. In 7-8 month-old fetuses, a complete change of the microglial cell picture occurred. Ramified cells clearly predominated, whereas amoeboid cells had markedly decreased. In 8-9 month-old fetuses, amoeboid microglial cells had almost disappeared from fetal brains. In brains from subadult and adult cattle, lectin-positive ramified microglial cells with up to five cellular processes were seen in all brain areas, located adjacent to vessels or surrounding neuronal perikarya.

Sequential study of vasculitis in MRL mice.

The frequency, age of onset and organ distribution of spontaneously occurring vasculitis was examined in a sequential study with 170 MRL mice of both substrains. Necrotizing vasculitis was seen in 55.8% of MRL/Mp-lpr/lpr mice studied, beginning at the age of 3 months. The kidney and urinary bladder were most frequently involved. In MRL/Mp- +/+ mice necrotizing vasculitis was much less frequently present (7.6%), beginning at the age of 18 months, and was seen only in the kidney, stomach and testes. In both substrains mononuclear infiltration of pulmonary vessel walls preceded the occurrence of necrotizing arteritis in other organs. The immunofluorescence study revealed the presence of immune complex components (immunoglobulin G, C3, murine leukaemia virus antigen gp71) in the vessel walls of the renal arteries of six out of 36 lpr/lpr mice with necrotizing arteritis.

Mastitis in mink due to Staphylococcus aureus and Escherichia coli.

An epizootic of mastitis in mink due to Staphylococcus aureus and Escherichia coli associated with food poisoning was studied on a Connecticut ranch with 3,500 mink. In the course of the epizootic, approximately 2,000 mink kits and 480 adult mink, mostly nursing females, died within 10 days. Affected females had swollen mammary glands due to acute mastitis; S. aureus was isolated in pure culture from 2 mink and E. coli in pure culture from a 3rd. Staphylococcus aureus was isolated from the organs of 1 mink kit, S. aureus and E. coli from a 2nd kit, and E. coli from a 3rd. The organs of the remaining 7 kits examined did not contain bacteria. Both isolates were pathogenic when inoculated intraperitoneally into mice and mink, causing fatal septicemia within 16 to 24 hours. The meat from a septicemic bovine carcass fed prior to the epizootic was considered a possible source of infection, since it was found to be heavily contaminated with E. coli, S. aureus, and Streptococcus spp.

Detection of canine distemper viral antigen in formalin-fixed and paraffin-embedded tissue of a fitch (Mustela putorius), using an immunoperoxidase technique.

The present study describes how a naturally infected fitch (Mustela putorius) caused an outbreak of canine distemper in a colony of insufficiently vaccinated dogs. The detection of canine distemper virus (CDV) on paraffin sections of different organs of the fitch and one of the dead dogs was achieved using a monoclonal antibody against the nucleocapsid protein (NP) of CDV and the avidin-biotin-peroxidase complex (ABC) technique.

[Immunohistochemical studies on organ tropism of different biotypes of BVD virus in experimentally infected sheep fetuses]. / Immunhistochemische Untersuchungen zum Organtropismus unterschiedlicher Biotypen des BVD-Virus bei experimentell infizierten Schaffeten.

Paraffin sections from various organs of sheep fetuses following transplacental infection with non-cytopathogenic (ncp) bovine viral diarrhoea virus (BVDV) or cytopathogenic (cp) BVDV were stained immunohistochemically with BVDV-specific monoclonal antibodies. Comparison of the distribution of viral antigen in sections from fetuses of experiment A revealed that in organs such as parotid, thyroid, thymus, lung, spleen, kidney, liver and skin from 20 days post inoculation (p.i.) onwards numerous antigen-containing cells were present. In organs of fetuses infected with cp BVDV, however, antigen-positive cells were only detectable until days 10 and 14 p.i. These findings suggest that the ncp BVDV used in experiment A replicated considerably faster and more efficient than the cp BVDV used in experiment B and that the two virus biotypes differ considerably concerning their tropism for fetal ovine organs.