Primers for polymerase chain reaction (PCR) were synthesized from clones derived from a Leptospira hardjo (type hardjobovis) library. One pair of synthetic oligonucleotide primers was selected for further analysis. Under experimental conditions an amplification was obtained with DNA of Leptospira interrogans of some serovars belonging to serogroup sejroe. However, very little or no amplification was observed with DNA from other serovars of this group. No amplification was observed with DNA from other serogroups, other bacteria, or eucaryotic organisms. Cattle urine, seeded with hardjobovis, was processed in several ways and subsequently subjected to PCR. Boiling of the samples or treatment with detergents appeared to be most effective. Urine samples containing fewer than 10 leptospires gave a positive result in the PCR assay. Twenty urine samples obtained from a slaughterhouse or farm cows were investigated using the PCR assay, culture isolation, dot and quick blot hybridization, and serological tests. This comparative study suggests that amplification by PCR may be a valuable method for the detection of leptospires in cattle urine.
An improved method of preparing bovine urine samples was developed for the rapid, specific, and sensitive detection of Leptospira interrogans serovar hardjo (subtype hardjobovis) DNA by the polymerase chain reaction (PCR). A total of 100 leptospire-free cows, 4 experimentally infected cows, and 2 negative control cows were used. PCR results were improved by (i) using 10-ml urine samples instead of 1-ml samples, (ii) adding 1 to 108 Leptospira biflexa serovar patoc cells as a carrier to each treated sample, (iii) preventing the loss of pelleted leptospires, and (iv) minimizing the presence of PCR-inhibiting factors in the samples. The preparation method enabled us to use the PCR to reproducibly detect as few as 5 to 10 leptospires per ml of urine without the need for dot blot hybridization. In addition, we were able to estimate the number of leptospires shed by experimentally infected cows.
Recombinant DNA probes derived from genomic libraries of serovars hardjobovis and icterohaemorrhagiae were applied for the characterization of leptospires. Differences in hybridization signals in combination with the banding pattern appear to provide good characteristics for strain typing. The banding patterns were easy to distinguish, since the recombinant DNA probes hybridized with a limited number of fragments. They were also indicative of genomic relationships between serovars. The probes suggested the existence of four subgroups with extensive genomic homology within the serogroup Sejroe. A number of serovars outside the serogroup Sejroe showed genomic homology with these subgroups. Amplification with the polymerase chain reaction showed a correlation with the genomic homologies demonstrated by Southern analysis. Knowledge about genomic relationships between leptospiral strains, as revealed by Southern analysis, may lead to a more rational approach for primer selection for polymerase chain reaction or cloning of particular genes.
Various studies have demonstrated anti-retinal S-antigen (S-ag) antibodies in uveitis sera in assays using bovine S-ag. Because of its molecular similarity and cross-reactivity with human S-ag, reactions with bovine S-ag have been considered a reliable indication of anti-S-ag autoimmunity. To test this assumption, the cross-reactivity of purified human and bovine S-ags was quantitated by ELISA titration of various anti-human and anti-bovine S-ag immune reagents raised in mice, rats and rabbits. Anti-human S-ag reagents appeared to be largely cross-reactive with bovine S-ag, whereas anti-bovine S-ag reagents were 6-10 times less reactive with the cross-reacting human S-ag than with bovine S-ag, thus showing a predominant role of species-specific epitopes on bovine S-ag. Furthermore, a large number of human control and uveitis sera was tested in ELISA with both human S-ag- and bovine S-ag-coated microwells. Both the numbers of positive sera and the levels of anti-S-ag antibodies in the two tests significantly correlated, but many exceptions were found, and the predictive value of reactions with bovine S-ag for the presence and levels of anti-S-ag autoantibodies was low. For individual human sera, assessment of anti-S-ag autoantibodies requires the use of human S-ag in immunoassays.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.