A role for the Clostridium perfringens beta2 toxin in bovine enterotoxaemia?

Non-enterotoxigenic type A Clostridium perfringens are associated with bovine enterotoxaemia, but the alpha toxin is not regarded as responsible for the production of typical lesions of necrotic and haemorrhagic enteritis. The purpose of this study was to investigate the putative role of the more recently described beta2 toxin. Seven hundred and fourteen non-enterotoxigenic type A C. perfringens isolated from 133 calves with lesions of enterotoxaemia and high clostridial cell counts (study population) and 386 isolated from a control population of 87 calves were tested by a colony hybridisation assay for the beta2 toxin. Two hundred and eighteen (31%) C. perfringens isolated from 83 calves (62%) of the study population and 113 (29%) C. perfringens isolated from 51 calves (59%) of the control population tested positive with the beta2 probe. Pure and mixed cultures of four C. perfringens (one alpha+beta2+, one alpha+enterotoxin+ and two alpha+) were tested in the ligated loop assay in one calf. Macroscopic haemorrhages of the intestinal wall, necrosis and haemorrhages of the intestinal content, and microscopic lesions of necrosis and polymorphonuclear and mononuclear cell infiltration of the intestinal villi were more pronounced in loops inoculated with the alpha and beta2-toxigenic C. perfringens isolate. These results suggest in vivo synergistic role of the alpha and beta2 toxins in the production of necrotic and haemorrhagic lesions of the small intestine in cases of bovine enterotoxaemia. However, isolation of beta2-toxigenic C. perfringens does not confirm the clinical diagnosis of bovine enterotoxaemia and a clostridial cell counts must still be performed.

ELISA and direct immunofluorescence test to detect equine arteritis virus (EAV) using a monoclonal antibody directed to the EAV-N protein.

A monoclonal antibody (mAb) directed against the equine arteritis virus (EAV) nucleocapsid (N) protein was used for indirect enzyme-linked immunosorbent assays (ELISAs) using viral antigen from different sources. The same mAb was labelled with fluorescein isothiocyanate for direct immunofluorescence tests (DIFTs). The N-specific mAb appeared to be suitable for the detection in both ELISA and DIFT of different EAV strains and field isolates from semen and tissue samples after passage in lines of RK-13, Vero and fetal equine kidney cells. The ELISA described is an easy and fast method which can be used in most cases to replace the microneutralization test to prove the EAV specificity of the cytopathic effect of cell cultures. The DIFT, however, is more sensitive than both the ELISA and the microneutralization test because EAV antigen can be detected even in cell cultures without or with very weak cytopathic effect.

Molecular variation between the alpha-toxins from the type strain (NCTC 8237) and clinical isolates of Clostridium perfringens associated with disease in man and animals.

The alpha-toxin produced by the type strain of Clostridium perfringens (NCTC 8237) was shown to differ from the alpha-toxins produced by most strains of C. perfringens isolated from man and from calves with respect to reactivity with a neutralizing monoclonal antibody (DY2F5D11). The difference in antibody binding correlated with three differences in the deduced amino acid sequence (Ala174 to Asp174; Thr177 to Ala177; Ser335 to Pro335) of the alpha-toxins. Using octapeptides synthesized on the basis of the amino acid sequences from these regions of variability, it was shown that the Ala174 to Asp174 change had the greatest effect on reducing the binding of monoclonal antibody DY2F5D11 to the alpha-toxin. These differences did not affect the enzymic or toxic properties of the protein. However, the phospholipase C activity of the alpha-toxin produced by strain NCTC 8237 was more susceptible to inactivation by chymotrypsin. The changes in amino acid sequence did not affect the ability of a C-terminal domain vaccine, derived from the alpha-toxin of strain NCTC 8237, to induce protection against the alpha-toxin from a bovine enteric strain of C. perfringens.

Analysis of T cell receptor V beta regions expressed by rheumatoid synovial T lymphocytes.

The T cell receptor (TCR) V beta gene segment repertoire of T lymphocytes derived from peripheral blood of two healthy individuals and synovial tissue, synovial fluid and peripheral blood of three rheumatoid arthritis (RA) patients was analyzed. A sensitive assay based on the amplification of cDNA by the polymerase chain reaction (PCR) was used to analyze the levels of expression of 20 TCR V beta gene segment families. The relative expression of V beta gene segments in lymphocytes derived from peripheral blood, synovial tissue and synovial fluid was conserved over 155 days in one patient. V beta 9 transcripts were undetectable in the cells of this individual. In the two other patients the frequency of V beta 2 transcripts in synovial T cells of affected joints was significantly higher than in their peripheral blood lymphocytes. Dominance of distinct rearrangements among the V beta 2 transcripts from the synovial cells of these patients support the idea that the synovial T cell response is driven by antigen.

Diagnosis of bovine cryptosporidiosis by an enzyme-linked immunosorbent assay.

This paper describes an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of cryptosporidiosis. A monoclonal antibody with a high affinity against an oocyst antigen was used to set up the test. The efficiency of this assay was compared with that of the flotation test; 275 calf faecal samples were examined by the two methods. There was 96% agreement between the two tests. For the 11 conflicting samples, the two tests were repeated and a modified Ziehl-Neelsen staining was performed on faecal smears. All these 11 samples contained few oocysts, but only five and six of them were shown to be positive by the ELISA and flotation tests, respectively. The degree of sensitivity of the ELISA and flotation tests is comparable; samples heavily or moderately contaminated with oocytes are detected by both methods. This ELISA is reliable and never gives rise to false positive results. Nevertheless, as with the flotation test, the occasional case containing very few oocysts will not always be detected by this test. If necessary, very accurate diagnosis can be made by a staining technique or by a direct immunofluorescent assay. In veterinary medicine, the ELISA seems to be a method of choice; it appears to be a fast and reliable technique which could be used as a routine test for the detection of Cryptosporidium oocysts. Nevertheless the degree of sensitivity must always be borne in mind. There is no need for a microscopic examination, which is an additional advantage.

A field trial in Belgium to control fox rabies by oral immunisation.

Campaigns of fox vaccination against rabies were carried out in Belgium in September 1986 and June and September 1987. The SAD B19 attenuated strain of rabies virus was inserted into baits which were distributed over an area of 2100 km2 at a density of 11 baits/km2. As recommended by the World Health Organisation, the efficacy and the innocuity of the method were controlled in the field and in the laboratory. Samples of blood and brain and jaw were taken from foxes which were shot or found dead in the vaccination area, for the diagnosis of rabies, the titration of antirabies antibody and the detection of tetracycline marker. In rabid animals, the virus strain was characterised by immunofluorescence using monoclonal antibodies. In September 1987, the uptake of the baits had reached 72 per cent by 14 days after distribution. Several wild species competed with foxes in taking the baits. After the last campaign, tetracycline was found in 65 per cent of the healthy foxes collected and rabies virus neutralising antibodies were detected in 77 per cent of them. In 1987, the incidence of rabies decreased markedly in the vaccination area compared with the untreated areas. No vaccine virus was isolated either from rabid animals or from 228 small mammals trapped in the vaccination area.