Apoptosis in Bovine viral diarrhea virus (BVDV)-induced mucosal disease lesions: a histological, immunohistological, and virological investigation.

Cattle persistently infected with a noncytopathic Bovine viral diarrhea virus (BVDV) are at risk of developing fatal "mucosal disease" (MD). The authors investigated the role of various apoptosis pathways in the pathogenesis of lesions in animals suffering from MD. Therefore, they compared the expression of caspase-3, caspase-8, caspase-9, and Bcl-2L1 (Bcl-x) in tissues of 6 BVDV-free control animals, 7 persistently infected (PI) animals that showed no signs of MD (non-MD PI animals), and 11 animals with MD and correlated the staining with the localization of mucosal lesions. Caspase-3 and -9 staining were markedly stronger in MD cases and were associated with mucosal lesions, even though non-MD PI animals and negative controls also expressed caspase-9. Conversely, caspase-8 was not elevated in any of the animals analyzed. Interestingly, Bcl-x also colocalized with mucosal lesions in the MD cases. However, Bcl-x was similarly expressed in tissues from all 3 groups, and thus, its role in apoptosis needs to be clarified. This study clearly illustrates ex vivo that the activation of the intrinsic, but not the extrinsic, apoptosis pathway is a key element in the pathogenesis of MD lesions observed in cattle persistently infected with BVDV. However, whether direct induction of apoptosis in infected cells or indirect effects induced by the virus are responsible for the lesions observed remains to be established.

Distribution of Borna disease virus antigen and RNA in tissues of naturally infected bicolored white-toothed shrews, Crocidura leucodon, supporting their role as reservoir host species.

Borna disease is a severe viral-induced disorder of the central nervous system of horses, sheep, and a few other animal species, occurring in certain areas of central Europe. Pathogenesis and epidemiology of natural Borna disease virus (BDV) infections are still not fully understood; several unique epidemiologic features, however, point toward the existence of BDV reservoir populations other than the final hosts. In this study, 69 mice and 12 shrews were trapped and examined. The virus distribution was investigated in detail in 2 BDV-positive bicolored white-toothed shrews, Crocidura leucodon, by immunohistochemistry and TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR). RT-PCR amplification products were sequenced, and the sequences were compared. These shrews had been collected in a BDV-endemic geographical region using live traps and did not show obvious clinical or pathological disease signs. BDV antigen and nucleic acid were identified in several organs, including the brain, mainly in nerve tissue and neurons, respectively, but also in parenchymal cells (eg, hepatocytes, Leydig cells) and epithelial cells, particularly of the respiratory and urogenital tract.

Mucosal lesions in a sheep infected with the Border Disease Virus (BDV).

A 28-week-old sheep was presented at the animal hospital because of chronic emaciation, anemia and slight diarrhea. Due to poor general condition and bad prognosis the animal was euthanized and submitted for postmortem investigation. Multiple erosions and ulcerations were found in the dorsal region of the tongue, the pharynx, the hard palate, in the esophagus and the ruminal pillars. Histologically, these lesions consisted of necrosuppurative inflammation. The animal was tested positive for pestivirus antigen both by immunohistochemical and by virological examination (cell culture, antigen capture ELISA and RT-PCR). A non-cytopathic Border Disease Virus was identified, and sequencing revealed a virus belonging to the BDV-3 cluster. Based on the macroscopical, histological, immunohistological and virological results this case was diagnosed as Border Disease with mucosal lesions. This is the first report of such a case in Switzerland.

Does surface anodisation of titanium implants change osseointegration and make their extraction from bone any easier?

OBJECTIVES: Titanium implants have a tendency for high bone-implant bonding, and, in comparison to stainless steel implants are more difficult to remove. The current study was carried out to evaluate, i) the release strength of three selected anodized titanium surfaces with increased nanohardness and low roughness, and ii) bone-implant bonding in vivo. These modified surfaces were intended to give improved anchorage while facilitating easier removal of temporary implants. MATERIAL AND METHODS: The new surfaces were referenced to a stainless steel implant and a standard titanium implant surface (TiMAX). In a sheep limb model, healing period was 3 months. Bone-implant bonding was evaluated either biomechanically or histologically. RESULTS: The new surface anodized screws demonstrated similar or slightly higher bone-implant-contact (BIC) and torque release forces than the titanium reference. The BIC of the stainless steel implants was significant lower than two of the anodized surfaces (p = 0.04), but differences between stainless steel and all titanium implants in torque release forces were not significant (p = 0.06). CONCLUSION: The new anodized titanium surfaces showed good bone-implant bonding despite a smooth surface and increased nanohardness. However, they failed to facilitate implant removal at 3 months.

Immunohistochemical diagnosis of persistent infection with Bovine Viral Diarrhea Virus (BVDV) on skin biopsies.

Detection of persistent infection with BovineViral Diarrhea Virus (BVDV) is essential for both epidemiological and clinical reasons. In addition to the classical virological methods such as virus isolation in tissue culture, ELISA and RT-PCR, immunohistochemistry of skin biopsies has become a useful and reliable tool. Assuming that the presence of BVDV antigen in skin structures is restricted to persistent infection, this method could differentiate from transient infection. In order to answer this question, 6 calves were experimentally infected orally with a non-cytopathic genotype 1 BVDV strain belonging to the subtype k.The calves developed fever, mucopurulent nasal discharge, coughing and leucopenia with relative lymphopenia. Immunohistochemistry of skin biopsies taken daily up to day 13-post infection did not reveal any evidence of BVDV infection. BVDV was, however, isolated from blood samples on cell cultures. Anti-NS3-antibody-ELISA and serum neutralization tests showed that all six calves seroconverted. We conclude that in acute BVDV infections, with genotype 1 and the subtypes found in Switzerland (b, e, h and k) viral antigen is not found in epidermal structures of the skin. In contrast, persistently infected animals test positive for BVD viral antigen by immunohistochemistry of the skin.

Testicular hypoplasia in a bull persistently infected with bovine diarrhoea virus.

A bull aged 16 months with bilateral testicular hypoplasia and azoospermia was persistently infected with bovine viral diarrhoea virus (BVDV). Viral antigen was detected in serum and semen by ELISA, but the animal was serologically negative. After slaughter, the genital tract was examined histopathologically and by immunohistochemistry, including double immunolabelling with BVDV antibody and either S-100 antibody (for Sertoli cells) or ferritin antibody (for Leydig cells). The seminiferous tubules of both testes were lined by a single layer of Sertoli cells and the germinal epithelium was completely absent except for a few remaining spermatogonia. BVDV antigen was demonstrated (1) in the media of arterial vessel walls of the testis, epididymis, urethra, prostate, and vesicular and bulbourethral glands, (2) in epithelial cells of the ductus epididymidis, the accessory glands and the urethra, and (3) in the testis, mainly in Sertoli cells and to a lesser extent in the spermatogonia that remained, but not in Leydig cells. The testicular hypoplasia was possibly linked to the BVDV infection.

Reverse transcription real-time PCR assays for detection and quantification of Borna disease virus in diseased hosts.

Borna disease is a severe, immunopathological disorder of the central nervous system caused by the infection with the Borna disease virus (BDV). The detection of BDV in diseased animals, mainly sheep and horses, is achieved by histological, immunohistochemical and serological approaches and/or PCR-based technologies. In the present study, reverse transcription, real-time PCR assays were established for the detection of BDV in the brain tissue from sheep and horses, using loci for the p40 (nucleoprotein) and the p24 (phosphoprotein) genes. The PCRs were equally specific and sensitive, detecting 10 target molecules per reaction and one BDV-infected cell among 10(6) non-infected cells. In tissues from BDV-diseased sheep and horses, the p24 target was detected at higher abundance than for p40. Therefore, the p24 test is suggested to be of higher value in the diagnostic laboratory. However, both assays should be useful for addressing questions in pathogenesis and for detecting BDV reservoirs in endemic areas.

Mosaicplasty with photooxidized, mushroom shaped, bovine, osteochondral xenografts in experimental sheep.

The goal was to study the performance of mushroom shaped, photooxidized, osteochondral grafts in mosaicplasty focusing on graft stability and survival. Mushroom shaped, photooxidized grafts (6 mm for the cartilaginous head of the mushroom, 3 mm for the stem) were implanted in the medial femoral condyle of 10 sheep. Four transplants were inserted per condyle in an overlapping fashion using the pressfit technique (n=40 grafts in 10 condyles). The grafts were followed for 6 and 12 months. Semi-quantitative evaluation of graft performance was performed using a validated score system. All grafts were mechanically stable at 6 and 12 months with one exception, where the mushroom head broke off. The formation of cystic lesions in the subchondral bone area was minimal. Repopulation of the old photooxidized cartilage was noticed with cells invading the matrix from the subchondral bone area and also from the pannus on the surface. Fusion between host and graft cartilage was observed in some of the grafts at 12 months, while remodeling of the calcified cartilage zone and tidemark was noticed in all grafts. Results scored significantly better for the 6 months compared to the 12 months group if cartilage surface integrity was compared (p<0.05). In all other variables no significant differences were found between groups. Despite moderate graft recession in the 12 months group partial fusion of grafts and functional results were satisfactory. The photooxidized mushroom shaped osteochondral transplants may be a suitable type of graft for functional results in cartilage resurfacing if stable anchorage of the grafts can be achieved.

Histomorphological and immunohistochemical findings in testes, bulbourethral glands and brain of immunologically castrated male piglets.

The aim of this study was the histological and immunohistochemical evaluation and comparison of testicular, bulbourethral and brain tissue in immunized and intact control boars. Fourteen male piglets, aged between 10 and 16 weeks, were vaccinated twice subcutaneously 4 to 5 weeks apart with Improvac, an anti-GnRH vaccine. The pigs were sacrificed 1 to 16 weeks following the second injection. Testicular weight was recorded and various tissue samples were collected and fixed in formalin and Bouin's fixative for histological examination. In addition, 2 boars were immunized five times and slaughtered 60 weeks after the last injection. Histological and immunohistological studies performed on testes and epididymes showed clear signs of atrophy in the immunized animals and a significant reduction in paired testes weight was seen in treated boars. Microscopically, the mean diameter of the seminiferous tubules was markedly reduced. Spermatogonia as well as few spermatocytes were visible between the Sertoli cells and Leydig cells were atrophic. None or only few spermatozoa were detected in the epididymis. The bulbourethral glands of immunocastrated pigs were smaller than in control pigs and showed histological evidence of atrophy. Immunohistological detection of LH and FSH in the pituitary gland of treated and control boars showed no quantifiable difference in the amount of these two gonadotropins and no lesions were visible in the hypothalamus and the pituitary gland. From our findings it can be concluded that the anti-GnRH vaccine Improvac induces severe atrophy of testes and bulbourethral glands in immunized pigs. This effect appears to be reversible, depending on the immune response of each animal and the time elapsed after the last booster injection.

[Investigation about the clinical use of brushite- and hydroxylapatite-cement in sheep]. / Untersuchungen uber den klinischen Einsatz von Brushite- und Hydroxylapatit-Zement beim Schaf.

For future clinical use as synthetic bone replacement, an injectable brushite-(chronOS-Inject) and hydroxylapatite-(Biobon) cement were compared in a drill hole model in 10 sheep over time at 2, 4, 6, 8, 16 and 24 weeks. Results were compared regarding their practical use, biocompatibiliy, resorption mechanism and subsequent new bone formation. The cements were filled into drill holes (psi 8 x 13mm) of the proximal and distal humerus, and femur and the samples evaluated macroscopically, radiologically and microscopically including histomorphometrical quantification of percentages of new bone, fibrous tissue and remnants of cements. The cement area decreased continuously from 2 to 24 weeks with chronOS-Inject, as well as the area of granules. Inversely, the subsequent new bone formation increased from 2-24 weeks accordingly. With Biobon the cement area decreased slower between 2 and 24 weeks, and the new bone formation was less. Both cements were well integrated into the bone in long bones. chronOS-Inject demonstrated good biocompatibility and was almost completely replaced through bone within 24 weeks. Biobon was resorbed considerably slower and initially a slight inflammatory reaction including bone resorption was observed within the adjacent host bone.

The use of photooxidized, mushroom-structured osteochondral grafts for cartilage resurfacing--a comparison to photooxidized cylindrical grafts in an experimental study in sheep.

OBJECTIVE: This article addresses the problem of structural design with osteochondral grafts used for cartilage resurfacing. METHODS: Photooxidized cylindrical or mushroom-shaped grafts were surgically implanted in the weight bearing area of the medial and lateral femoral condyles of eight sheep (condyles: N=8/group). Both types of photooxidized grafts contained no viable chondrocytes at the time of implantation. Results were evaluated at 2 and 6 months after surgical implantation of the grafts. Qualitative and quantitative evaluation of the subchondral bone area was performed using plastic embedded sections of non-decalcified bone and cartilage specimens and placing emphasis on graft anchorage, cyst-like lesions at the base of the cartilage junction and at the base of the graft in the subchondral bone region. Cartilage morphology was studied qualitatively focusing on viability of the graft and adjacent host cartilage, while a score system was developed for semi-quantitative evaluation of the overall articular cartilage performance. The semiquantitative scores and histomorphometrical measurements were subjected to statistical analysis using a factorial analysis of variance (ANOVA-test). RESULTS: The photooxidized mushroom-shaped grafts developed less fibrous tissue and cyst-like lesions in the subchondral bone area at 2 and 6 months compared to the cylindrical grafts. Areas of endochondral ossification and bone remodeling were noticeable in the mushroom structured grafts at 2 months, and also bone remodeling was more complete at 6 months than with the cylindrical grafts. Increased numbers of cells were seen in the basal remodeling zones of both graft types increased from the 2 months to the 6 months specimens, but mushroom structured grafts showed better results. In both graft types, however, the midzone of the cartilage matrix was still acellular at 6 months. Cells from the subchondral bone area started to penetrate the calcified cartilage zone and tide mark at 2 months and repopulated the old photooxidized cartilage matrix already at 6 months after implantation. Cartilage repopulation was dependent on a stable subchondral bone area in both types of grafts. Matrix degradation of the adjacent host cartilage was minimal at 2 and 6 months. At 6 months a junction between host and graft cartilage was already noticed in some of the mushroom-shaped grafts. CONCLUSION: This study confirmed the importance of the subchondral bone area for osteochondral graft survival. In addition it demonstrated that the structure of the graft influences considerably the architecture of the subchondral bone, and with this the possibility for the repopulation of the old cartilage matrix including the junction between the host and graft cartilage matrix.

In vivo behavior of three different injectable hydraulic calcium phosphate cements.

Two dicalcium phosphate dihydrate (DCPD) hydraulic cements and one apatite hydraulic cement were implanted in epiphyseal and metaphyseal, cylindrical bone defects of sheep. The in vivo study was performed to assess the biocompatibility of the DCPD cements, using the apatite cement as control. After time periods of 2, 4 and 6 months the cement samples were clinically and histologically evaluated. Histomorphometrically the amount of new bone formation, fibrous tissue and the area of remaining cement were measured over time. In all specimens, no signs of inflammation were detectable either macroscopically or microscopically. All cements were replaced by different amounts of new bone. The two DCPD-cements showed the highest new bone formation and least cement remnants at 6 months, whereas the apatite was almost unchanged over all time periods.

Changes in subchondral bone in cartilage resurfacing--an experimental study in sheep using different types of osteochondral grafts.

OBJECTIVE: This article addresses the subchondral bone integrity in cartilage resurfacing by comparing fresh, untreated auto-, xeno-, and photooxidized osteochondral allo- and xenografts. Photooxidation was expected to improve mechanical stability of the osteochondral grafts through an improved linkage of the collagen fibers within the bone matrix. DESIGN: Untreated auto- and xenografts and with photooxidation pretreated allo- and xenografts were surgically implanted in femoral condyles of sheep (n=40). After 2, 6, 12 and 18 months results were evaluated histologically using non-decalcified bone embedded in acrylic resin. Qualitative evaluation was performed with emphasis on bone matrix, biomechanical stability of graft anchorage, formation of cystic lesions, and bone resorption and formation. Quantitative evaluation of the total subchondral bone area was conducted histomorphometrically. Statistical analysis (factorial ANOVA test) was used to compare differences between groups with respect to the percentage of bone matrix and fibrous tissue per section. RESULTS: Subchondral bone resorption was fastest in untreated, fresh autografts, followed by photooxidized allografts, untreated, fresh xenografts and last pretreated photooxidized xenografts. Cystic lesions were seen in all types of grafts, but were most pronounced at 6 months in autografts and least in photooxidized grafts. Cyst-like lesions had subsided substantially in the untreated auto- and photooxidized xenografts, if no graft dislocation occurred during the healing period. Mononuclear cell infiltration and an increase in the presence of multinuclear cells were observed at 2 months, mostly in untreated autografts, followed by photooxidized allo- and untreated xenografts. They were much higher in numbers compared to photooxidized grafts, at least in the early specimens at 2 months. Graft stability was linked to the rate of bone resorption. CONCLUSION: Substantial resorption of the subchondral bone, involving the development of cyst-like lesions, lead to dislocation and finally to cartilage matrix degradation of the grafts. The process of photooxidation decreased the speed of bone resorption in osteochondral grafts and, thus, improved graft stability and cartilage survival. These results suggest that the remodeling of the subchondral bone of the host and the graft within the first 6 months is an important factor in graft stability and overall results of cartilage resurfacing.

Haemangiosarcoma with a metastasis of a malignant mixed mammary gland tumour in a dog.

An 11-year-old dog with a history of weight loss, anorexia, anaemia, epistaxis and a nodule in the mammary gland showed multifocal lung masses during radiography. At necropsy as well as in the histological examination two different tumours were identified in several organs including the right heart auricle. By immunohistochemistry, using a double-staining DAKO kit (DAKO Diagnostics, Zug, Switzerland), it was possible to identify the two components as haemangiosarcoma and metastasizing malignant mixed mammary gland tumour.

In vitro studies of a photo-oxidized bovine articular cartilage.

Bovine articular cartilage was photo-oxidized and cultured with native articular bovine cartilage and synovial membrane to study the interaction between these tissues mimicking the physiological situation in the joint. The photo-oxidation was applied as a pretreatment of cartilage for future use in cartilage resurfacing procedures in joints. Properties of the transplant were assessed by testing the production of local mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and neutral metalloproteinase activities under normal conditions and after stimulation with various stimulants representative of inflammatory changes in pathophysiological conditions. Unlike normal cartilage photo-oxidized cartilage did not release significant amounts of NO and PGE2 and showed less gelatinolytic and caseinolytic activity compared to native bovine articular cartilage. Enzyme activity of the combined cultures was at a level intermediate between that of photo-oxidized cartilage and native cartilage cultures alone. In contrast to normal cartilage, living chondrocytes were not visible in photo-oxidized cartilage using live/dead staining. These results indicate, that the photo-oxidized cartilage may have a beneficial effect on adjacent native host cartilage and therefore be a suitable transplant for use in in vivo experiments.

Long term in-vivo studies of a photo-oxidized bovine osteochondral transplant in sheep.

BACKGROUND: Articular cartilage has limited capacity to repair. Defects greater than 3 mm heal with formation of inferior fibrous cartilage. Therefore, many attempts have been made to find the ideal graft for larger cartilage lesions. Different grafts, such as untreated or cryopreserved osteochondral transplants, have been used with variable success. METHODS: Photo-oxidized osteochondral grafts were implanted in both femoral condyles of one ovine knee. Untreated xenogeneic and autogeneic grafts served as controls. Three groups of 8 sheep each were formed and they were sacrificed 6, 12 or 18 months after surgery. RESULTS: The macroscopic evaluation of the condyle and graft showed a well-maintained cartilage surface in most grafts at all time points. However, the host cartilage matrix deteriorated considerably in all xenogeneic, most autogeneic and fewer of the photo-oxidized grafts at 12 and 18 months, respectively. The blue colour of the photo-oxidized grafts resulting from the process of photo-oxidation was visible in all grafts at 6 months, had diminished at 12 months and had completely disappeared at 18 months after surgery. Histologically a loss of matrix staining was almost never noticed in untreated xenografts, transiently at 6 months in photo-oxidized grafts and increased at 12 and 18 months. Fusion between graft and host cartilage could be seen in photo-oxidized grafts at 12 and 18 months, but was never seen in autografts and xenografts. CONCLUSIONS: The photo-oxidation of osteochondral grafts and its use as transplant appears to have a beneficial effect on cartilage and bone remodelling. Osteochondral grafts pre-treated with photo-oxidation may be considered for articular cartilage replacement and therefore may delay artificial joint replacements in human patients.

[Borna disease in Switzerland and in the principality of Liechtenstein]. / Die "Bornasche Krankheit" in der Schweiz und im Fürstentum Liechtenstein.

Borna disease (BD) is a rare immunopathological disorder of the central nervous system (CNS) caused by infection with Borna disease virus (BDV) and histologically characterized by mononuclear encephalomyelitis. BD primarily affects equines and sheep in well defined endemic areas of central Europe, but BDV infections have also been reported in other host species including humans, as well as in non endemic regions. In this paper recent data on the pathogenesis of BD are reviewed and the current situation in Switzerland and the Principality of Liechtenstein is summarized.

[Pestivirus as causative agent of abortion and perinatal mortality in cattle and sheep in Switzerland]. / Ursächliche Beteiligung von Pestiviren an Aborten und perinatalen Todesfällen bei Rindern und Schafen in der Schweiz.

The causal involvement of bovine viral diarrhoea virus (BVDV) and border disease virus (BDV) infection in bovine and ovine abortion and perinatal mortality remain unclear. From 1992 until 1994, 213 bovine and 31 ovine foetuses as well as 36 calves and 25 lambs which had died perinatally were investigated. Tissue samples were tested for the presence of pestiviruses and serum or fluid from the body cavities were analysed for the presence of pestivirus antibodies. Detection of pestiviruses was performed by (i) cell culture isolation, (ii) antigen ELISA and (iii) immunohistochemical staining for viral antigen. For antibody-testing an indirect ELISA was used. In nine bovine foetuses and in two calves BVDV was isolated. Pestiviruses, most likely BDV were detected in one ovine foetus and three lambs. In 6% of the bovine and 11% of the ovine foetuses anti-pestivirus antibodies were detected. However, clinical features and history of bovine cases did not show a correlation with the diagnostic results, In contrast, the presence of central nervous system signs in neonatal lambs and the detection of BDV was correlated.

Immunohistochemical detection of bovine viral diarrhea virus in skin biopsies: a reliable and fast diagnostic tool.

Skin biopsies from 184 cattle were immunohistochemically tested for bovine viral diarrhea virus (BVDV) infection. BVDV infection was detected sensitively and specifically by visceral organ immunohistochemistry or by standard virological methods.