Linear hoof defects in sheep infected with foot-and-mouth disease.

During the epidemic of foot-and-mouth disease (FMD) in The Netherlands in 2001, a sheep farm was identified that had been subclinically infected with the disease. The FMD virus genome was detected in 12 of 16 probang samples collected from the sheep and the virus was isolated from four of these samples. Linear defects were observed, 1 to 3 cm from the coronary band, in the hooves of several of the sheep. The defects were thought to have been caused by the FMD infection. It was thought that the distance of the defects from the coronary band might be an indication of the time since the animals had been infected. To determine the growth rate of the claws of sheep, the growth of the hoof horn of uninfected lambs and ewes was measured; in the lambs the growth rate was 0.44 mm per day and in the ewes it was 0.29 mm per day.

Native cytokines do not bind to uromodulin (Tamm-Horsfall glycoprotein).

Uromodulin bound with high affinity to human tumour necrosis factor (TNF) coated on microtitre plates. This interaction was not competitively inhibited by native TNF in solution. No interaction was observed between immobilized uromodulin and TNF in the liquid phase unless conditions were chosen which denatured the latter protein. Recombinant interleukin-1 alpha adsorbed on microtitre plates also interacted with uromodulin. However, gel filtration experiments demonstrated no interaction between the proteins in the liquid phase. These and additional results indicate that uromodulin interacts with denatured cytokines, but not with native, soluble cytokines.

Detection of mRNA encoding calcitonin, calcitonin gene related peptide and proopiomelanocortin in human tumors.

Expression of the calcitonin (CT)/calcitonin gene related peptide (CGRP) gene and the proopiomelanocortin (POMC) gene has been demonstrated by Northern blot hybridization analysis of RNA extracted from human medullary thyroid carcinoma (MTC), pheochromocytoma and lung carcinoma. CT mRNA in these tumors could not be distinguished in size from CT mRNA isolated from normal human thyroid tissue. CGRP mRNA (previously demonstrated in 12 out of 12 lung tumor cell lines investigated) could not be detected in 13 primary lung tumors or 10 metastases thereof. The length of POMC mRNA in MTCs (present in all 4 metastases investigated but not in 7 primary tumors) and pheochromocytomas is about 100 nucleotides more than pituitary POMC RNA. In lung tumors 2 POMC RNA species can be detected, one of the same size as in pituitary tissue and one about 100 nucleotides larger.

Lelystad virus belongs to a new virus family, comprising lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus.

Lelystad virus (LV) is an enveloped positive-stranded RNA virus, which causes abortions and respiratory disease in pigs. The complete nucleotide sequence of the genome of LV has been determined. This sequence is 15.1 kb in length and contains a poly(A) tail at the 3' end. Open reading frames that might encode the viral replicases (ORFs 1a and 1b), membrane-associated proteins (ORFs 2 to 6) and the nucleocapsid protein (ORF7) have been identified. Sequence comparisons have indicated that LV is distantly related to the coronaviruses and toroviruses and closely related to lactate dehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV). A 3' nested set of six subgenomic RNAs is produced in LV-infected alveolar lung macrophages. These subgenomic RNAs contain a leader sequence, which is derived from the 5' end of the viral genome. Altogether, these data show that LV is closely related evolutionarily to LDV and EAV, both members of a recently proposed family of positive-stranded RNA viruses, the Arteriviridae.

Validation of a screening liquid phase blocking ELISA for swine vesicular disease.

A direct liquid-phase blocking ELISA (LPBE) was developed for serological examination of swine vesicular disease (SVD). The sensitivity and specificity of the test were assessed on 272 and 365 sera collected on two farms where an outbreak had occurred. The specificity of the direct LPBE was higher than the specificity of the indirect LPBE. The European Community reference serum for SVD (RS 01-04-93), which has been adopted as the threshold for SVD serological examination in the European Community, had a mean titre of 2.19 in the neutralisation test. At a cut-off level of 2.0 in the neutralisation test, the sensitivity of the direct LPBE (screening at 1:432 final dilution) on the two farms was 90% and 99%, respectively. Based on these results, the screening dilution of the direct LPBE was adjusted to 1:160 final dilution, to obtain a sensitivity > or = 98% on both farms. Regression analyses showed a good correlation between the virus neutralisation test and the direct LPBE (r = 0.87). Compared to the indirect LPBE described before, the direct LPBE correlates better with the neutralisation test, has a higher specificity, and is more rapid. Because sera are tested in only one dilution, the test is highly suitable for the examination of large numbers of serum samples.

Development of an ELISA for detection of antibodies to glycoprotein I of Aujeszky's disease virus: a method for the serological differentiation between infected and vaccinated pigs.

A blocking ELISA was developed to distinguish between Aujeszky's disease virus (ADV)-infected and vaccinated pigs, on the basis of presence or absence of serum antibodies to glycoprotein I (gI) of ADV. The gI-ELISA detects antibodies that block the reaction of monoclonal antibodies to one or two epitopes on gI of ADV. The ADV-gI antibody response appeared between one and two weeks post-infection and persisted at a high level for at least seven months. Five of the nine ADV-vaccine strains examined were found to be "gI-negative". Pigs vaccinated with a gI-negative vaccine did not develop an ADV-gI antibody response until they were challenge-exposed to a virulent strain of ADV. The gI-ELISA is highly specific, sensitive and suitable for large-scale sero-epidemiological studies to identify infected pigs in populations vaccinated with gI-negative vaccines. The gI-ELISA provides, therefore, a basis for ADV-eradication programmes, which introduces a novel concept in the control of animal virus diseases.

Detection of carriers of foot-and-mouth disease virus among vaccinated cattle.

To investigate and optimise detection of carriers, we vaccinated 15 calves with an inactivated vaccine based on foot-and-mouth disease virus (FMDV) A Turkey strain and challenged them and two further non-vaccinated calves with the homologous virus four weeks later. To determine transmission to a sensitive animal, we put a sentinel calf among the infected cattle from 60 days post-infection until the end of the experiment at 609 days post-infection. Samples were tested for the presence of FMDV, viral genome, specific IgA antibodies, antibodies against FMDV non-structural (NS) proteins or neutralising antibodies. Virus and viral genome was intermittently isolated from probang samples and the number of isolations decreased over time. During the first 100 days significantly more samples were positive by RT-PCR than by virus isolation (VI), whereas, late after infection more samples were positive by virus isolation. All the inoculated cattle developed high titres of neutralising antibodies that remained high during the entire experiment. An IgA antibody response was intermittently detected in the oropharyngeal fluid of 14 of the 17 calves, while all of them developed detectable levels of antibodies to NS proteins of FMDV in serum, which declined slowly beyond 34 days post-infection. Nevertheless, at 609 days after inoculation, 10 cattle (60%) were still positive by NS ELISA. Of the 17 cattle in our experiment, 16 became carriers. Despite frequent reallocation between a different pair of infected cattle no transmission to the sentinel calf occurred. It remained negative in all assays during the entire experiment. The results of this experiment show that the NS ELISA is currently the most sensitive method to detect carriers in a vaccinated cattle population.

Pathogenesis of swine vesicular disease after exposure of pigs to an infected environment.

The pathogenesis of swine vesicular disease (SVD) has been studied following a natural route of infection. In two experiments groups of ten and eight pigs respectively were introduced into a stable contaminated with SVD virus. At various intervals after stable exposure, pigs were killed and the amount of virus was determined in serum, vesicles (if present), spleen, kidney, and in seven lymph glands representing various parts of the body. One day after the pigs were introduced into the stable, five out of eight pigs were viraemic and virus could be isolated from various tissues. At 2 d after introduction, three out of four pigs killed had vesicular lesions on the feet. The tonsils of all pigs killed between 1 to 7 d after introduction into the stable were virologically positive. Four days after introduction 50% of the pigs were serologically positive and at 7 d all pigs had developed an antibody response. This study shows that contact with a SVD virus contaminated environment can be equally as infectious as injection, or direct contact with SVD infected pigs, causing a rapid spread of the disease. Because the tonsil was shown to be highly efficient in trapping and growing circulating virus, we recommend that in addition to serological examination, virus isolation from pig tonsils should be used to study the epidemiology of SVD on farms where the infection is present.

Singleton reactors in the diagnosis of swine vesicular disease: the role of coxsackievirus B5.

Swine vesicular disease virus (SVDV) and Coxsackie B5 virus (CVB5) are closely related viruses that can infect swine and man and give rise to cross-reacting serum antibodies. It is, therefore, possible that SVD antibodies found in serologic screenings of pigs are induced by CVB5. Single positive animals found in screening programmes are generally referred to as singleton reactors (SR). To determine whether SR in SVDV screenings are induced by CVB5 infection, virus neutralisation tests (VNTs) and radioimmunoprecipitation assays (RIPA) were carried out on sera of SR, sera of pigs experimentally infected with SVDV, and sera from pigs vaccinated with CVB5 isolates. The SR sera reacted repeatedly positive in the SVDV UKG/27/72 VNT, but reacted differently in three other VNTs (SVDV NET/1/92, CVB5A, and CVB5B). The VNT titres obtained with the SR sera revealed a correlation between both SVDV strains, and also between both CVB5 stains, but no correlation was found between SVD and CVB5 VNT titres. Sera of experimentally infected (SVDV) or vaccinated (CVB5) pigs showed titres in all four neutralisation tests. In the RIPA, the reaction patterns of the SR sera varied considerably with all four antigens used, in contrast to sera from pigs experimentally infected with SVDV that reacted with all antigens used, and sera from pigs vaccinated with CVB5 that reacted only with CVB5 antigens. The results presented in this paper show that neither CVB5 nor SVDV infections are the only cause of the SR phenomenon. Testing for CVB5 specific antibodies can reduce the number of SR sera in the serodiagnosis of SVDV.

Radioiodinated Rhodamine-123: a potential cationic hepatobiliary imaging agent.

The labelling of the cationic dye Rhodamine-123 with 125I is described. The biodistribution of the iodinated Rhodamine-123 has been determined at different time intervals after intravenous injection into fasted rats. It turned out that the dye is predominantly cleared by the liver and discharged into the bile. The bile acid taurocholate did not enhance the rate of excretion of 125I-Rhodamine-123.

Immunohistochemical localisation of prostaglandin endoperoxide synthase and prostacyclin synthase in pregnant human myometrium.

The localisation of prostaglandin endoperoxide synthase and prostacyclin synthase in pregnant human myometrium was examined by indirect immunocytofluorescent staining using monoclonal antibodies raised against these enzymes. Both enzymes were demonstrated to be present in myometrial smooth muscle cells. Staining associated with prostacyclin synthase, however, was more intense throughout the myometrium and less circumscript than that associated with prostaglandin endoperoxide synthase. The findings indicate that the uterine smooth muscle cells themselves possess the enzymes necessary for producing prostacyclin from arachidonic acid and that this capacity is not limited to the uterine macro- and microvasculature.

Carriers of foot-and-mouth disease virus: a review.

This review describes current knowledge about persistent foot-and-mouth disease virus (FMDV) infections, the available methods to detect carrier animals, the properties of persisting virus, the immunological mechanisms, and the risk of transmission. In particular, knowledge about the carrier state, the period in which virus can be isolated from animals 28 days or longer post infection, is important, because the risk that animals may carry the virus will influence the diagnostic and preventive measures that need to be taken. Although many years of research have led to much knowledge about foot-and mouth disease and its causative agent, there are still numerous aspects of the virus and the disease that are not yet fully understood. Areas for further research on persistence of FMDV are discussed.

Differentiation and genomic and antigenic variation among fetal, respiratory, and neurological isolates from EHV1 and EHV4 infections in The Netherlands.

Ten monoclonal antibodies (MAbs) were produced against equine herpes virus type 1 (EHV1). Two appeared type-specific, while the other eight were directed against epitopes common to both EHV1 and EHV4. Two MAbs directed against the glycoprotein gp2 recognized linear epitopes, as demonstrated by Western blotting. With pools of type-specific MAbs, 282 field isolates were typed in an immunoperoxidase monolayer assay (IPMA). From a total of 254 fetal or neonatal isolates, 244 (96%) were typed as EHV1, whereas 14 out of 15 (93%) respiratory tract isolates were typed as EHV4. Surprisingly, 3 out of 13 isolates (23%) originating from horses with neurological disease were typed as EHV4. No antigenic differences were found among 75 randomly selected EHV1 field isolates, using the panel of ten MAbs and six additional MAbs, directed against gp2, gB, or gC. Typing by restriction endonuclease analysis with BamHI corresponded completely with that of MAb analysis. There was a remarkable degree of uniformity in BamHI restriction patterns, with 90% of the investigated EHV1 isolates belonging to the 1P electropherotype. Among 30 randomly selected EHV1 isolates we could not identify the EHV1.1B electropherotype, which has been the predominant electropherotype in Kentucky since 1982. Mobility differences were seen in fragments originating from the repeat regions. These differences were not caused by heterologous cell passage, since all viruses were passaged in equine cell systems.

Prognostic value of p53 for high risk superficial bladder cancer with long-term followup.

PURPOSE: The risk of muscle invasive disease in a high risk patient with superficial bladder cancer is up to 50%. Identifying patients at risk for progression remains an unsolved problem. A suggested prognosticator is mutations in the p53 tumor suppressor gene. We determined the value of p53 mutation, as demonstrated by mutation analysis, in a clinically selected group of high risk patients with superficial bladder cancer. MATERIALS AND METHODS: p53 Mutation analysis was performed by automated sequencing of bladder wash samples of 105 patients with high risk superficial bladder cancer. The mutation and WT groups were subsequently compared with regard to mortality, progression, disease worsening and the recurrence-free period. RESULTS: A total of 29 patients had a mutation and 76 had WT. Median followup was 58.3 months (range 3 to 161). A total of 13 patients died of bladder cancer, including 6 of 29 with a mutation and 7 of 76 patients in the WT group. p53 Mutation had no significant prognostic value for decreased survival, progression or disease worsening. Recurrence-free survival was significantly lower in the WT group. CONCLUSIONS: We observed a trend toward a worse clinical outcome in high risk patients with a p53 mutation in the bladder wash. However, no significant differences were seen in clinical outcome parameters. Based on these data we conclude that the prognostic value of a p53 mutation is insufficient for individual policy making.

False-positive lesions detected by fluorescence cystoscopy: any association with p53 and p16 expression?

To determine p53 and p16 status as molecular markers of bladder cancer, in histologically proven benign bladder biopsies, obtained from lesions suspect for malignancy as judged by fluorescence cystoscopy. Immunohistochemical (IHC) staining was performed for p53 and p16, using the antibodies DO-7 and AB-4, respectively. The tissue sections were scored in percentages of nuclear staining for p53 and p16. Of 247 biopsies, 41/49 lesions appeared suspicious on fluorescence cystoscopy, but were histopathologically benign. 2/40 (5%) were > or =20% p53 positive as compared to 7/128 (5.5%) of all histopathologically benign biopsies. 24/37 (64.9%) were p16 negative (<5% positive cells) as compared to 84/125 (67.2%) of all benign biopsies. Most biopsies had a moderate to high degree of chronic cystitis. False positive lesions of fluorescence cystoscopy did not differ from benign lesions detected by standard white light cystoscopy with regard to p53 and p16 immunoreactivity. Little evidence remains for these lesions to be pre-malignant.