P. Ramadass

A rapid latex agglutination test for detection of leptospiral antibodies.

A rapid semi-quantitative latex agglutination test (LAT) has been standardized for the detection of leptospiral antibodies in serum samples of man and animals. The efficacy of the LAT was compared with the plate enzyme linked immunosorbent assay (ELISA). A total of 276 human serum samples were analyzed by both LAT and ELISA and percentage positives were 84.8 and 85.9%, respectively. Similarly, of 65 animal samples tested, 63.1 and 69.2% positivity were observed in LAT and ELISA, respectively. Even though the ELISA test was slightly more sensitive than LAT, the rapidity, simplicity and economics of the LAT were found to fulfill the requirements of a screening test for leptospiral antibodies.

Recombinant Antigen-based Latex Agglutination Test for Rapid Serodiagnosis of Leptospirosis

A rapid recombinant antigen-based latex agglutination test (LAT) has been developed to detect specific anti-leptospiral antibodies from human and dog sera. The recombinant LipL32 antigen developed and used for detecting the antibodies is specific in detection of the pathogenic serovars of Leptospira as the expression of the LipL32 antigen is restricted only to the pathogenic leptospires. The sensitized latex beads are stable and could be stored at 4°C for more than three months without showing loss of activity for both weakly and strongly positive samples. The test is found to be sensitive, specific and accurate as compared to the standard microscopic agglutination test (MAT). Moreover, the recombinant antigen-coated latex beads could detect the specific anti-leptospiral antibodies in the acute phase of the illness. The test is simple and inexpensive, and is rapid in the management of large numbers of patients.

Recombinant antigen-based dipstick elisa for the diagnosis of leptospirosis in dogs

A recombinant LipL32 antigen-based dipstick elisa was developed as a screening test for the detection of leptospiral antibodies in serum samples from dogs. The antibodies were detected by a change in the colour of the substrate solution when the recombinant antigen-coated dipsticks were dipped into it. The relative sensitivity, specificity and accuracy of the test, compared with the standard microscopic agglutination test, were 95·9 per cent, 93·8 per cent and 94·8 per cent, respectively.

Dipstick enzyme immunoassay for rinderpest antibody in cattle

A dipstick enzyme immunoassay (ELISA) has been standardized for the detection of rinderpest antibodies. One hundred and thirty bovine serum samples were analysed by the dipstick ELISA method and the results compared with the conventional plate ELISA method. The sensitivity was found to be similar in both methods. The dipstick ELISA does not require expensive micro-plates and an ELISA reader, and is recommended for use in field laboratories where the qualitative detection of rinderpest antibodies is required.

Phylogenetic Analysis of LipL32 Gene Sequence of Different Pathogenic Serovars of Leptospira

Leptospirosis is a zoonosis of worldwide significance caused by infection with pathogenic leptospiral species (Levett, 2001). The leptospiral outer membrane proteins may be important in the diagnosis of leptospirosis. The major outer membrane protein, LipL32 is a prominent immunogen in human (Haake et al., 2000) and canine (Dey et al., 2004) leptospirosis. The serovar pomona strain Pomona and serovar icterohaemorrhagiae strain RGA of Leptospira interrogans were chosen for the cloning and sequencing of the LipL32 outer membrane protein gene. In this study, the forward and reverse primers for amplifying 665 bp of the LipL32 gene were based on the LipL32 gene sequence of L. kirschneri species (Haake et al., 2000). The restriction enzyme sites for XbaI and KpnI were incorporated at the 5′ ends of the primers for directional cloning in the pBluescript KS(+) cloning vector (Stratagene, La Jolla, CA, USA). The primers were able to amplify only the pathogenic serovars and not the non-pathogenic serovars of Leptospira. The PCR-amplified LipL32 genes of Pomona and RGA strains were cloned into pBluescript KS(+) cloning vector. The presence of insert was checked by colony PCR using vector specific primer T3 as the forward primer and the reverse gene-specific primer. The restriction enzyme analysis with XbaI and KpnI also confirmed the presence of insert in the recombinant plasmid pKSP4. The portion of the structural gene sequences of LipL32 of the strains Pomona and RGA were submitted to GenBank under accession numbers AY223718 and AY423075, respectively. The nucleotide sequences of LipL32 of these two serovars were stored along with the sequence of the other available pathogenic serovars in FASTA format and used as the input file for the program CLUSTAL X 1.8. The MegAlign package was used to align all the sequences in the worktable using the CLUSTAL V method developed by Higgins and Sharp (1989). MEGA software V1.02 was used to identify the amino acid variations between the serovars. After multiple alignments, a neighbour-joining method (Saitou and Nei, 1987) was employed to reconstruct phylogeny for the putative alignment. The neighbour-joining (NJ) tree file produced three groups (Figure 1). Group I consisted of L. interrogans and L. kirschneri species; group II was represented by L. borgpetersenii