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Bovine viral diarrhoea (BVD) control/eradication programmes based on the test and removal of persistently infected cattle without use of vaccination were first introduced by the Scandinavian countries in the early 1990s. Within the last 10 years the programmes have proven to be very successful and have served as a blueprint for several other European regions. However, in areas with high cattle densities, intense animal trade and high BVD prevalence this control approach is risky, because there is a high probability that herds, which have been cleared of persistently infected (PI) animals and have become partly or fully susceptible to reintroduction of the virus, will come in contact with a BVD virus (BVDV) infected animal. A combination of the test and removal strategy with subsequent systematic vaccination of cattle could overcome this problem. The goals of vaccination in such a programme is protection against reintroduction of BVDV into herds free from PI cattle and foetal protection of pregnant animals accidentally exposed to the virus. Two-step vaccination is based on the use of inactivated BVDV-1 vaccine for priming followed by a live attenuated vaccine booster 4 weeks later. The immune response elicited by such a vaccination scheme has proven to be long lasting and foetal infection after challenge with BVDV-1 and BVDV-2 was prevented in pregnant animals 5 months after vaccination. These findings suggest that the implementation of a two-step vaccination in the initial phase of control programmes in addition to test and removal of PI animals in areas with high cattle densities and endemic BVD is practical and efficacious.

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The aim of the study was the assessment of rise and persistence of neutralizing antibodies (nAb) to bovine viral diarrhea virus (BVDV) and border disease virus (BDV) after a two step vaccination using an inactivated BVDV/BDV (Mucobovin) and a modified live BVDV vaccine (Vacoviron). In a first experiment eight heifers were kept in isolation and were serologically surveyed regularly over a three year period after vaccination. The same experiment was done with 80 vaccinated cattle kept under field conditions. Neutralizing antibody titres were monitored using homologous as well as heterologous BVDV and one BDV strain, respectively. Maximum titres were obtained two to three months after vaccination. During the three years of monitoring the antibody titres decreased but never fell below the detection limit. This slow antibody regression demonstrates that a single two step vaccination elicited high nAb titres which persist over at least three years. These results might serve as a decision tool when considering the necessity and time of revaccination of cattle, which have been vaccinated using the two step method.

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In order to assess the efficacy of a two-step vaccination protocol with respect to foetal protection against transplacental infections with bovine virus diarrhoea virus (BVDV) with special attention to BVDV-2 seronegative heifers were vaccinated with an inactivated BVDV-1 vaccine and boostered with a modified live BVDV-1 vaccine after 4 weeks. A second group was left unvaccinated as control. Between days 30 and 120 of pregnancy the heifers of both groups were intranasally challenged with a mixture of BVDV-1 and -2. All heifers of the vaccinated group gave birth to nine clinically healthy, seronegative (precolostral) and BVDV-free calves. In contrast in the control group four BVDV viraemic underdeveloped calves were born. Additionally, one calf was stillborn and another viraemic calf was not viable and died 2 days after birth. All six calves of the control group were viraemic with BVDV-2. This study demonstrated for the first time that two-step vaccination of breeding cattle with a modified live BVDV vaccine 4 weeks after application of an inactivated BVDV vaccine was capable of providing a foetal protection against transplacental infection with BVDV-2.

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During recent years neutralizing antibodies against Border Disease Virus (BDV) were found repeatedly in German pig herds. Consequently there was a demand for a differential diagnostic system. A permanent sheep cell line and BDV reference strain Moredun were chosen and were applied in a could be used case study. A pestivirus could be isolated from piglets on a mixed farm and was characterised as 'non-Classical Swine Fever' (CSF) by using monoclonal antibodies. Due to a CSF suspicion the pig herd was destroyed immediately. Serum samples of sheep from the same farm were used for further characterisation of the new virus isolate. A neutralization test of the sheep sera was performed against different pestiviruses and the new isolate. Neutralizing antibody titres against the new virus pig isolate were significantly higher than against all other pestiviruses. BDV strain Moredun recognised the antibodies clearly, whereas CSF viral strain Alfort 187 and several isolates of bovine viral diarrhoea virus (BVDV) strains scored the lowest cross reaction.

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The aim of this study was to find whether an antigenic drift had occurred in Lower Saxony in the past 40 years. For this, the genetic diversity of bovine viral diarrhea virus (BVDV) isolates mainly from Lower Saxony was estimated by RT-PCR and sequencing of a 420 bp fragment of the E2 glycoprotein gene. Sixty-one field virus isolates collected during routine diagnostics between 1960 and 2000 in Lower Saxony, Northern Germany, were analyzed. Phylogenetic analysis allowed discrimination of genotypes BVDV 1 and 2. Excepting two isolates, which were of BVDV type 2, most of the isolates were classified as BVDV type 1. This group could be further subdivided into four subgroups and one disparate isolate. Independent of the year of isolation and geographical localization, 54 isolates clustered in two subtypes (BVDV subtypes 1b and 1d). Only one isolate was classified as BVDV type 1a, thus being similar to the North American NADL strain, and to the vaccine strain Oregon C24V, which was extensively used for vaccination in Germany. The remaining isolates belonged to new clusters tentatively designated as BVDV subtypes 1g and 1f. To compare the cluster designation with that of other studies, phylogenetic analysis of representatives of each of the subgroups based on the 5' untranslated region (5'UTR) was performed. It grouped the viruses similarly. The results indicate that the BVDV population seems to be relatively stable over 40 years in Lower Saxony.

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The RT-PCR is an in vitro technique that is increasingly being used for diagnosis of viral animal pathogens. Due to its high sensitivity it is considered as an alternative to current standard methods for detecting BVDV especially in pooled samples, e.g. from bulk tank milk. A prerequisite for the performance of RT-PCR is an efficient and simple method for sample preparation. The aim of this work was to compare the efficiency of three commercially available kits for RNA extraction, and their suitability for sample preparation for the detection of the BVDV genome by RT-PCR in blood, milk and tissue samples. The kits were based on different methods for extraction of RNA and differed in costs, labour and time consumption. The most sensitive RT-PCRs (exception: heparinised blood) were obtained when sample preparation was performed by acidic guanidinium-isothiocyanate-phenol-chloroform extraction with the Trizol (Gibco) reagent. Using a kit based on the binding of RNA to silica membrane in a spin column, positive results in RT-PCR were obtained from all samples, but with lower sensitivity. The advantage of the column-based kits is that they are less time-consuming, easier to handle and suitable for automatisation of sample preparation. A kit using salt precipitation of the desoxribose nucleic acid (DNA) and proteins was unsuitable for the isolation of viral RNA from the samples.

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Vaccination with live cytopathogenic (cp) bovine viral diarrhoea virus (BVDV) is often used for control of this disease. In animals which are persistently infected with noncytopathogenic (ncp) BVDV this can lead to the outbreak of mucosal disease (MD). To simulate vaccination of such animals and to monitor the clinical-virological course after superinfection, nine clinically healthy calves which were persistently viremic were superinfected with different cp BVDV strains. One animal succumbed to early onset MD within three weeks after superinfection. During the observation period of 18 months four animals developed severe clinical signs. While two animals developed late onset MD, the other two had to be euthanized due to clinical signs which could not be related to the superinfecting BVDV. These results indicated that after superinfection or vaccination of persistently infected calves with cp BVDV the probability of developing early and/or late onset MD is significantly increased. The risks arising from uncritical vaccination of herds with unknown virological status in relation with the control of BVDV conforming to the actual official guidelines are discussed.

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Mucosal disease (MD) can be induced in cattle persistently infected with noncytopathogenic bovine viral diarrhea virus (ncp BVD virus) by superinfecting them with antigenically related cytopathogenic (cp) BVD virus strains. While some of these animals succumb to early onset MD after 2 to 3 weeks post infectionem (p.i.), others only react by producing neutralizing antibodies against the cp BVD virus strain and may develop late onset MD after longer incubation periods. The aim of this study was to determine if an increasing degree of antigenic homology between the ncp and the superinfecting cp BVD virus strains as determined by their comparative reactivity with E2 glycoprotein specific monoclonal antibodies (mabs) increases the probability of inducing early or late onset MD, respectively. For this, each two of eight clinically healthy animals from the same herd and persistently infected with the same ncp BVD viruses were superinfected with four different cp BVD virus strains. As only two of these animals developed late onset MD, one animal from a different herd that developed early onset MD was included in the study. Besides clinical observation and testing for antibody production, virus isolation and characterization of the cp BVD virus isolates were performed. The results indicate that antigenic similarity as determined by comparative mab analysis alone is not sufficient to allow prediction of the outcome of the disease.

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Bronchiectasis is pathologically defined as an abnormal and permanent dilatation of one or several bronchi. There are localized and generalized types of bronchiectasis. A vicious circle hypothesis, including an initial insult to the lower airways, impaired mucociliary clearance, microbial colonization/infection, bronchial obstruction and a local inflammatory response, has been proposed to explain the damage to the bronchial tree and the adjacent lung parenchyma. The clinical picture is variable and affected individuals might be asymptomatic or suffer from severe respiratory failure. Daily sputum production is the most common, though unspecific symptom of bronchiectasis. Other common symptoms are hemoptysis and recurrent episodes of sputum purulence, fever and pleurisy. Occasionally, major, life-threatening hemoptysis from a ruptured bronchial artery occurs. Infectious complications, e.g. lung abscess, empyema, brain abscess, and secondary amyloidosis are rarely seen today. The chest radiograph reveals changes suggestive of bronchiectasis in the majority of patients with clinically important disease. High resolution computed tomography of the lung has almost completely replaced bronchography for diagnosis, the latter rarely being of value if surgery is contemplated. No etiology is identified in about one- to two-thirds of the patients, although there are many diseases eventually associated with bronchiectasis. Prevention and therapy of underlying diseases are most important. Traditionally, the therapy of symptomatic bronchiectasis is based on antibiotics, antibronchoobstructive medication, and chest physical therapy. Surgical resection is the treatment of choice for localized symptomatic disease. Bilateral lung transplantation should be considered in younger patients with severe, generalized bronchiectasis and respiratory failure. Prospective, randomized, largescale trials supporting any of the different treatment strategies are not available, but antibiotics and surgery probably have improved the long-term outcome of many patients with bronchiectasis. In this review, some recent findings regarding the classification, pathogenesis, pathology, etiology, diagnosis, treatment, and prognosis of bronchiectasis are discussed.

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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.

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The frequency of bronchial anastomotic complications following lung transplantation has decreased in recent years, but continues to be a potential cause of morbidity and mortality. We have, therefore, reviewed the results of 67 consecutive bronchial anastomoses at risk in 43 patients surviving more than 7 days following lung transplantation. The bronchial anastomoses were performed using a standardized technique, without direct or indirect revascularization. Regular triple immunosuppressive therapy was given, including prednisone (0.5 mg x kg(-1) daily) starting on the day of surgery. Bronchial healing was graded using the Couraud classification. The median follow-up time was 14 months (range 1-45 months). No major airway complications occurred. On 236 serial bronchoscopic examinations, no anastomotic stenoses were observed. One anastomosis showed limited focal necrosis (2 mm) (Couraud 3a), and two anastomoses had partial primary mucosal healing without necrosis (Couraud 2a). In all other anastomoses, primary mucosal healing (Couraud 1) was observed. Carefully performed bronchial anastomosis according to the technique described enables reliable bronchial healing and yields a low complication rate. Additional measures, such as direct revascularization, forced telescoping, omentum wrap and interruption of steroid therapy, are not necessary.

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In a 45-year-old woman presenting with subacute liver failure and portal hypertension the diagnostic workup revealed portal vein thrombosis and occlusion of small hepatic veins. An occult myeloproliferative syndrome was assumed. During full-dose heparin therapy the thrombotic process progressed to segmental venous small bowel infarctions, liver failure and death. In-vitro culture of mononuclear blood cells showed spontaneous growth of erythroid precursor cells. Necropsy demonstrated acute hemorrhagic necrosis of the liver, thrombotic material within the portal and mesenteric veins, thrombosis, dilatation, sclerosis, and partial obliteration of small portal vein branches, and obliterative fibrosis and thrombosis of small intrahepatic veins. The bone marrow and spleen findings support the diagnosis of a myeloproliferative disorder.

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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)

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In a 63-year-old woman admitted to the emergency ward because of coma of unknown origin, clinical examination, analysis of blood and cerebrospinal fluid, electroencephalography, and computed tomography of the brain were unrevealing. On the fourth hospital day the coma was followed by an apathetic state and amnesic syndrome, suggesting bilateral thalamic infarction. Magnetic resonance imaging of the brain demonstrated bilateral thalamic infarction of the paramedian territories. The role of the thalamus for functions of vigilance and cognition is emphasized.

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A pestivirus isolated from a healthy pig out of a mixed pig-cattle holding was identified by use of monoclonal antibodies as a porcine virus related or identical to bovine viral diarrhoea (BVD) virus. About 7% of the pigs of this herd showed neutralizing antibodies (nab) against BVD and Border Disease (BD) virus and against the homologous porcine nonclassical swine fever (CSF) pestivirus isolate 10421/Han94. The nab titres against this virus were clearly higher than against CSF virus strain Alfort/187. Amongst the cattle kept in the farm no BVD viraemic animal was detected. About 13% of them showed nab to BVD virus with higher titres against the BVD virus strain NADL than against the porcine pestivirus virus isolate 10421/Han94. There was no of a BVD virus transmission from the cattle to the pigs within this herd. The problem of misinterpretation of non-CSF virus isolation from pigs is discussed. The necessity of identifying pestiviruses of porcine origin by use of species-specific monoclonal antibodies is stressed in order to prevent erroneous declarations of CSF outbreaks.

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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.

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Murine monoclonal antibodies (MAbs) were generated against phocid herpesviruses (PhHV 2557/Han88 and 7848/Han90) isolated from European harbour seals (Phoca vitulina), and against strains of both felid (FHV strain FVR 605) and canid herpesviruses (CHV isolate 5105/Han89). MAbs were characterized with respect to certain biological properties and used to outline antigenicity profiles of isolates of PhHV (n = 8), FHV (n = 7) and CHV (n = 3) in enzyme immunoassays employing fixed infected cells. A close antigenic relationship between herpesviruses derived from pinnipeds and terrestrial carnivores became evident: The majority of the MAbs was directed against epitopes which were expressed by at least two of the viral species tested. A number of MAbs detected epitopes which were conserved between all isolates of PhHV, FHV and CHV. A few MAbs recognized type-specific B-cell epitopes and facilitated the identification of single viral species. Moreover, the PhHV isolate 7848/Han90 was antigenically distinguishable both from seven other phocid herpesvirus isolates and from FHV or CHV. PhHV 7848/Han90 proved to be antigenically distinct from all other viruses tested when examined by cross neutralization utilizing various reconvalescent and hyperimmune sera. Although more data are needed to ensure that PhHV 7848/Han90 indeed is a new genuine seal herpesvirus, the preliminary clustering of two groups of phocid herpesvirus isolates, tentatively designated PhHV-1 (type isolate 2557/Han88) and PhHV-2 (represented by 7848/Han90), seems to be justified. By using selected MAbs an unambiguous identification and typing of herpesvirus isolates derived from marine mammals and terrestrial carnivores is significantly facilitated.

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Bovine viral diarrhoea (BVD)/mucosal disease (MD) is an economically important infectious disease of cattle, and persistently infected animals are of central epidemiological significance. The identification of these animals is a prerequisite for further sanitary measures in affected herds. In order to process large numbers of samples an antigen capture enzyme immunoassay (AC-ELISA) for the detection of bovine viral diarrhoea viral antigen in peripheral blood leukocytes of cattle of all ages has been developed. In this communication an improved method for the preparation of samples is described. A total of 563 blood samples was tested using the AC-ELISA and a routine virus isolation procedure on susceptible bovine cells. When compared to virus isolation in cell culture, the sensitivity of the AC-ELISA was 97%. From the samples that were virus negative in cell culture, 99.1% were also negative in the AC-ELISA. There is some evidence that the AC-ELISA discriminated between transiently and persistently infected animals.

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