Effects exerted by transcriptional regulator PcaU from Acinetobacter sp. strain ADP1.

Protocatechuate degradation is accomplished in a multistep inducible catabolic pathway in Acinetobacter sp. strain ADP1. The induction is brought about by the transcriptional regulator PcaU in concert with the inducer protocatechuate. PcaU, a member of the new IclR family of transcriptional regulators, was shown to play a role in the activation of transcription at the promoter for the structural pca genes, leaving open the participation of additional activators. In this work we show that there is no PcaU-independent transcriptional activation at the pca gene promoter. The minimal inducer concentration leading to an induction response is 10(-5) M protocatechuate. The extent of expression of the pca genes was observed to depend on the nature of the inducing carbon source, and this is assumed to be caused by different internal levels of protocatechuate in the cells. The basal level of expression was shown to be comparatively high and to vary depending on the noninducing carbon source independent of PcaU. In addition to the activating function, in vivo results suggest a repressing function for PcaU at the pca gene promoter in the absence of an elevated inducer concentration. Expression at the pcaU gene promoter is independent of the growth condition but is subject to strong negative autoregulation. We propose a model in which PcaU exerts a repressor function both at its own promoter and at the structural gene promoter and in addition functions as an activator of transcription at the structural gene promoter at elevated inducer concentration.

Immunohistochemical localization of transferrin in the pre- and postnatal bovine brain.

The distribution of transferrin in brains from fetuses (age-range: 1-2 to 8-9 months of gestational age), calves, subadult (between 1 day and 9 months old) and adult cattle (> 12 months old) were examined by immunohistochemistry. In both pre- and postnatal brains transferrin was predominantly found in oligodendrocytes. Furthermore, transferrin immunostaining was present in single to few neurons, within the lumina of vessels, in endothelial cells, in epithelial cells of the choroid plexus and in ependymal cells. A caudo-rostral progress in the appearance of transferrin-positive cells was found. In fetuses, transferrin-positive mature oligodendrocytes and neurons were not detected before 3-4 months of gestational age. Findings in different brain areas of older fetuses suggest an association between the increase of transferrin-positive oligodendrocytes and the process of myelination. In brains from calves and sub-adult cattle a continuous decrease of transferrin-immunoreactive oligodendrocytes and neurons was noted.

Development of myelination in the bovine fetal brain: an immunohistochemical study.

In this study, the development of myelination in the fetal bovine brain (age-range: between 1-2 and 8-9 months) was examined applying: 1. Immunohistochemical staining methods and antibodies against bovine proteolipid protein (PLP), synthetic tridecapeptide of bovine PLP, human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG); and 2. Using the Luxol fast-blue (LFB) technique.

[Urine protein analysis with the sodium-dodecyl-sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE) in healthy cats and cats with kidney diseases]. / Urinoproteinanalyse mit der Sodium-Dodecyl-Sulfat-Polyacrylamid-Gradientengel Elektrophorese (SDS-PAGE) bei gesunden und nierenkranken Katzen.

In this investigation, the value of urine protein analysis by means of molecular-weight related sodium dodecyl-polyacryl gradient gel electrophoresis (SDS-PAGE) was examined with regard to its applicability and diagnostic significance in nephropathy in the cat. A total of 87 cats was included in the study, 30 of them that were clinically healthy served as the control group. The urine protein pattern of this group had, besides the band representing the market albumin, and additional broad band within the size of the marker transferrin. In some cases, weak bands were present within the range of the Tamm-Horsfall-protein and immunoglobulin G. Micromolecular protein bands were not demonstrable. The remaining 57 animals had a histologically proven nephropathy. Thirty-eight cats had elevated urea and/or creatinine values in the plasma (group 1), and 19 animals had values within the reference range (group 2). The urine protein pattern as evidenced by SDS-urine electrophoresis was altered in all cats with histologically proven nephropathy, and it is thus concluded that with this technique a nephropathy can be diagnosed very early and prior to changes of plasma urea and creatinine (group 2). Moreover, in most of the cases, the nephrological changes can be classified as glomerular or tubulo-interstitial (group 1 and group 2). However, it is not possible to draw exact conclusions concerning the underlying morphological changes, nor can the severity of the disease be correctly assessed.

[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.

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.

Variation in neuropathogenicity in sheep fetuses transplacentally infected with non-cytopathogenic and cytopathogenic biotypes of bovine-virus diarrhoea virus.

Pregnant Merino ewes were inoculated intravenously between days 63 and 65 of gestation with a non-cytopathogenic (ncp) bovine-virus diarrhoea-virus (BVDV) isolate (experiment A). The histomorphological findings and the distribution of viral antigen, as revealed by immunohistochemistry in brains of fetuses from experiment A, were compared with those seen in fetal brains from a previous study (experiment B), in which pregnant ewes had been intravenously infected between days 65 and 68 of gestation with the cytopathogenic (cp) BVDV strain Indiana. The two viruses showed remarkable variations concerning their pathogenicity for the developing fetal brain. The cp BVDV had a much higher neuropathogenic potential than the ncp BVDV and induced severe intracranial malformations in most fetuses. In experiment A, exclusively relatively mild leucoencephalomalacic lesions occurred. Between fetuses of the two experiments, significant differences concerning the distribution of viral antigen and the inflammatory response were found. In the majority of fetal brains from experiment B examined at days 10, 14 and 21 post inoculation (p.i.), antigen-containing differentiated brain cells (neurons, astrocytes, oligodendrocytes) and undifferentiated cells in the periventricular germinal zones were seen throughout the different zones of the developing telencephalon and cerebellum. At 21 days p.i., a marked inflammatory response consisting of brain macrophages and other mononuclear cells occurred in the meninges and in the brain parenchyma of fetuses from experiment B. In brain sections of fetuses infected with ncp BVDV, in contrast to fetuses infected with cp BVDV, viral antigen was not detectable during the early stages (days 10 and 20) p.i., and histopathological lesions were not seen at this stage. At days 41 and 47 p.i., antigen-positive astrocytes and oligodendrocytes were found in the developing white matter of the telencephalon and cerebellum. Furthermore, antigen-containing neurons were seen in the developing cerebral cortex. Cellular infiltrations in fetal brains from experiment A were limited to the leucoencephalomalacic areas in the developing cerebral and cerebellar white matter and consisted exclusively of brain macrophages. Immunohistochemical staining in brain sections of fetuses from both experiments revealed that numerous perivascular cells contained viral antigen, whilst positive endothelial cells were exclusively found in fetuses from experiment A. From the findings of this study it was concluded that the cp BVDV stain used in experiment B has a marked tropism for the fetal brain and both its already differentiated and undifferentiated cell populations, and that the resulting brain lesions primarily are the consequence of a direct cytolysis of these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Brain malformations in ovine fetuses associated with the cytopathogenic biotype of bovine viral-diarrhoea virus.

A total of six ewes were intravenously inoculated at between 65 and 68 days of gestation with the Indiana strain of bovine viral diarrhoea virus (BVDV), containing both non-cytopathogenic (ncp) and cytopathogenic (cp) biotypes. Eight transplacentally infected fetuses were sequentially removed from the infected ewes and were found to have inflammatory lesions and malformations of the brain. BVDV RNA was isolated from formalin-fixed, paraffin-embedded brain tissue sections and detected by nested polymerase chain reaction after reverse transcription. The two biotypes of BVDV were distinguished by the fact that a sequence insertion in the RNA of the cp biotype of the inoculum results in larger amplicons. Only RNA from cp BVDV was detected in three of the brains removed up to 14 days post-inoculation (p.i.), and no BVDV RNA was detected after more than 14 days p.i. These findings suggest that, in critical phases of development, cp BVDV may transplacentally infect the ovine fetal brain and cause malformations.

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.

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.

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.

Brain lesions in calves following transplacental infection with bovine-virus diarrhoea virus.

In 33 calves and subadult cattle of the Holstein-Friesian breed ranging from 1 to 210 days of age, the spectrum of brain lesions induced by intra-uterine infection with bovine-virus diarrhoea virus (BVDV) was retrospectively analysed. Of these, 27 animals originated from herds with a long history of BVD. Six calves were derived from dams vaccinated between the 90th and 118th day of gestation with a BVD live vaccine. The most frequent lesion was cerebellar hypoplasia, being present in 25 out of 33 (76%) of the animals. In most of these cases, cerebellar hypoplasia was associated with hydranencephaly, internal hydrocephalus, microencephaly or porencephaly. In cases with hydranencephaly, the fluid-filled cavities were devoid of ependymal lining. The lumina of the lateral ventricles of these cases were surrounded by glial fibrillary acidic protein (GFAP)-positive cells and a dense layer of immunoreactive cell processes. In the white matter adjacent to the dilated ventricular lumina, a reactive astrocytosis was present. Porencephalic cysts were surrounded by astrocytes with increased expression of GFAP and vimentin-positive cells and cell processes. In hydranencephalic brains, staining for neuron-specific enolase (NSE) revealed a marked reduction of NSE-positive nerve cells in cortical areas. In all six experimental cases and in several field cases with hydranencephaly or internal hydrocephalus, small groups of heterotopic NSE-positive neurons were present in the white matter of the cerebral hemispheres. In markedly hypoplastic cerebella, reduction of the cortical cell layers and degenerative changes in, and heterotopia of, Purkinje cells were found. In these cases, NSE- and neurofilament-positive cell processes were either markedly diminished or only remnants of immunoreactive cell processes were present. In five animals without significant gross cerebellar abnormalities, degenerative changes of Purkinje cells were found. Immunohistochemical staining using antibodies against glial and neuron-specific proteins on these brains, which represent postnatal end-stage lesions of BVDV-induced disturbances of the normal brain development, did not provide any insight into the possible pathogenetic mechanisms of these alterations. Application of immunohistochemistry, however, revealed changes, such as reactive astrocytosis and loss of nerve cell processes, which were not obvious on haematoxylin and eosin (H&E) stained sections.

Participation of monocytes and macrophages in canine glomerular disease.

The presence of monocytes/macrophages (MPs) in renal glomeruli was investigated in 86 dogs with different types of glomerulopathies. The identification of MPs in tissue sections was based on cytological criteria and the immunohistochemical demonstration of lysozyme. The highest numbers of MPs in glomeruli were observed in focal and diffuse mesangial proliferative glomerulonephritis (MesPGN), diffuse endocapillary proliferative glomerulonephritis (DEPGN), and diffuse mesangiocapillary glomerulonephritis (DMCGN). In cases of minor glomerular abnormalities (MGA), focal and segmental hyalinosis and sclerosis (FGS), diffuse membranous glomerulonephritis (DMemGN), and diffuse sclerosing glomerulonephritis (DSGN) the presence of glomerular MPs was low. Particularly in MesPGN, the number of MPs was correlated with glomerular hypercellularity and, additionally in DMCGN, with the degree of proteinuria. The results of this study suggest that MPs may be involved in functional and morphological alterations in different types of canine glomerulopathies.

Effect of formalin fixation and long-term storage on the detectability of bovine viral-diarrhoea-virus (BVDV) RNA in archival brain tissue using polymerase chain reaction.

Detection of DNA or RNA in formalin-fixed, paraffin-embedded tissues using polymerase chain reaction (PCR) may be hindered by degradation of nucleic acids during tissue collection, preparation and archivation. This study describes investigations on the effect of formalin fixation and prolonged storage of paraffin-embedded tissues on bovine viral-diarrhoea (BVD)-virus RNA as a model system. Brain tissues from eight persistently BVDV-infected calves containing high amounts of the virus were fixed in 5% neutral-buffered formalin or 10% non-buffered formalin for different fixation times, respectively, and paraffin embedded. Subsequent detection of an 803 bp fragment from single tissue sections using nested PCR after reverse transcription (nested RT-PCR) demonstrated a loss of detectability of viral RNA after more than 10 days (10% non-buffered formalin) and 3 months (5% neutral-buffered formalin) of fixation. Additional studies with 280 initially BVDV-positive brain tissues from 25 persistently BVDV-infected calves after storage of up to 10 years revealed a loss of detectable RNA after more than 1 year of storage. For estimation of the higher sensitivity of nested RT-PCR compared to single step RT-PCR, serially diluted BVD virus suspensions were examined using both methods. Nested RT-PCR was found to be about 100-fold more sensitive than single-step RT-PCR, and is therefore recommended as the appropriate technique for archival studies.

Histological and immunohistological classification of canine glomerular disease.

A histological and immunohistological study of the kidneys of 115 dogs, with and without clinical signs of spontaneous renal disease, was performed to prove the applicability of the WHO criteria for the classification of human glomerulopathy. Aside from the morphological investigation of paraffin and resin semithin sections, deposits of immunoglobulins, the complement component C3, and fibrinogen were observed immunoenzymatically in paraffin-embedded tissue specimens. From this, eight different types of glomerular lesions with various frequencies were identified: minor glomerular abnormalities (28 cases), focal and segmental hyalinosis and sclerosis (12 cases), focal glomerulonephritis (GN; 18 cases), diffuse membranous GN (nine cases), diffuse mesangial proliferative GN (2 cases), diffuse endocapillary proliferative GN (five cases), diffuse mesangiocapillary GN (25 cases), diffuse sclerosing GN (11 cases) und unclassified GN (two cases). In one case, renal dysplasia was diagnosed and two dogs did not present glomerular alterations. The results are discussed with regard to human glomerular diseases and pathogenic mechanisms.

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.

Immunohistochemical localization of glial and neuronal cell markers in the developing bovine brain.

In the present immunohistochemical study, the distribution and differentiation of glial and neuronal cells in bovine fetal brains (age range: between 1-2 and 7-8 months) was examined using antibodies against nervous system-specific proteins, i.e., glial fibrillary acidic protein (GFAP), vimentin, neuron specific enolase (NSE) and a neurofilament protein subunit (NF200kD).

Virological and pathological findings in sheep fetuses following experimental infection of pregnant ewes with cytopathogenic-bovine-virus diarrhoea virus.

Eighteen pregnant Merino ewes were inoculated intravenously between days 65 and 68 of gestation with the unpurified cytopathogenic (cp) bovine virus diarrhoea virus (BVDV) strain Indiana (experiment I). In experiment II, three ewes were inoculated with the same virus after two successive plaque isolations in order to compare its pathogenicity for the fetus with special regard to lesions in the fetal brain. In experiment I, fetal blood and tissue samples, allantoic fluids and placentomes were collected sequentially between 10 and 80 days post-inoculation (p.i.). BVDV was recovered from 6 of 19 fetuses examined during the first 3 weeks after inoculation. From fetuses sampled between 30 and 50 days p.i. virus was isolated from three cases only, and from 60 days p.i. onwards virus was no longer recovered. BVDV was longer detected in the allantoic fluid than in fetal tissues and continued to be present until 80 days post-inoculation. From tissue samples of two fetuses of experiment I, only non-cytopathogenic BVDV was isolated, whilst samples from seven fetuses contained the cp BVDV biotype as revealed by an immunoplaque assay. The cp biotype was also isolated from placentomes. In experiment II, virus was not isolated from any of the tissue samples of two living fetuses collected at 67 days post-inoculation. In both experiments, cp BVDV was recovered from allantoic fluid samples. In contrast to the developing fetal brain, other tissues or organs seemed to be less vulnerable to the cp BVDV strain Indiana. The partial purification of this virus strain did not affect its pathogenicity for the brains of the developing fetuses.

[Pemphigus foliaceus in a foal. A case history]. / Pemphigus foliaceus bei einem Fohlen. Ein kasuistischer Beitrag.

The clinical history, clinical, pathological and immunohistological findings of a four-month-old foal with generalised pemphigus foliaceus are presented. The typical lesions of this autoimmune skin disease are described and discussed.