Two hundred and four strains of S. zooepidemicus were isolated from the genital sites of mares diagnosed with metritis. The isolates were differentiated by PCR-RFLP analysis of the szp gene. The 30 kinds of restriction fragment profiles were defined by 4 to 6 sizes of DNA fragments ranging from approximately 65 to 390 bp. Although 16 of the 30 PCR-RFLP profiles consisted of 149 isolates, 73% were identical to the szp of the organisms from horses with respiratory disease. The remaining 14 consisted of 55 isolates of which 27% were newly found. Ten genital swabs which were positive for isolation of the organisms were used to investigate whether the 5 clones of the organisms from a single sample were genetically identical to each other or not. Results showed each of 5 clones to be indistinguishable, expect for 1 clone from a single swab. In conclusion, the usefulness of PCR-RFLP analysis in discriminating between strains was demonstrated on genital isolates in the same way as for respiratory isolates, suggesting that the major isolates were szp genotypes that are present at both genital and respiratory sites, even though it was suspected that some genotypes specifically infect the genital site or the respiratory site.
A shuttle PCR test targeting the conserved region of the SeM gene of Streptococcus equi was developed as a method for detecting this bacterium from horses. An amplicon 289 bp in size was detected from 137 strains of S. equi, but was not seen in 517 strains of other bacteria, including S. zooepidemicus and in 227 nasal mucosal swabs taken from horses which S. equi had not been isolated. A comparative examination using 12 strains of S. equi in serial dilution and 73 swabs taken from the nasal mucosa of horses from which S. equi had been isolated showed the sensitivity of our PCR was slightly inferior to conventional PCR described in a previous report, even though it was enough to use as a diagnostic tool. In conclusion, we suggest that the PCR method described here has advantages with respect to specificity and sensitivity as a diagnostic test for strangles.
The purpose of this investigation was to establish the accuracy and precision of bone mineral content (BMC) measurements made by the total radiographic bone aluminum equivalence (RBAE) method in the evaluation of the horse skeleton. To study the accuracies and precisions of the total RBAE method, the analogue ortho system and the digital computed radiography (CR) system were used. For investigation of the correlation with the bone ash weight and total RBAE value, the clinically healthy left third metacarpal bone (McIII) of 30 post-mortem horses were used. In the evaluation of the accuracies and precisions of total RBAE method, three different values were measured using an aluminum stepwedge. A high positive correlation was noted between the bone ash weight of the measured region and the total RBAE value. The precisions of the total RBAE method were high when measurements with a low total RBAE value by the ortho system and a high total RBAE value by the CR system were excluded, use of the development-fixing solution was limited to within 3 days in the ortho system. The RBAE value changed when the X-ray irradiation angle was altered in the horizontal and vertical directions in both systems, thus an accurate dorsopalmar view is demanded. Based on these findings, the total RBAE method of McIII in horses using the ortho and CR systems had high precision, suggesting it is a reliable tool for the measurement of BMC in horses.
Ferritin-binding proteins (FBPs) in horse serum were characterized by immunoblotting and ferritin-binding experiments. FBPs purified from horse serum by horse spleen ferritin-Sepharose 4B affinity chromatography were separated into two fractions by Sephacryl S-300 gel chromatography: FBP 1 and FBP 2. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, FBP 1 separated into 79.0- and 25.3-kDa bands, and FBP 2 separated into 62.7- and 25.3-kDa bands. Immunoblotting analysis using antibodies specific for horse IgM and IgA heavy chains and IgG F(c) fragment showed that the 79.0- and 62.7-kDa bands were IgM and IgG heavy chains, respectively. After forming complexes of horse FBPs with horse spleen ferritin, rabbit anti-horse ferritin antibody was used to form immune complexes against ferritin, allowing co-precipitation and subsequent identification by monoclonal antibodies to horse immunoglobulin (IgM, IgA, IgGa and IgGb). Horse serum was positive for IgM, IgGa, IgGb and IgA complexes and affinity-purified FBPs for IgM and IgGb complexes. FBP 1 was identified as an IgM complex that forms with ferritin but FBP 2 did not form any complexes with ferritin. These results suggest that circulating horse serum ferritin is in the form of IgM, IgG (IgGa and IgGb) and IgA complexes.