Detection of Leptospira species in environmental samples by amplification of 16S rRNA and rpoβ genes

This study attempted to identify and determine distribution of Leptospira spp. in environmental samples using 16S rRNA and rpoβ genes amplification. The samples were collected from high risk areas in Selangor, Malaysia. A total of 105 environmental samples consisting of soil and water were subjected to direct DNA extraction and PCR reaction. PCR products were analysed using gel electrophoresis and subjected to sequence analysis. Thirteen out of 105 (12.38%) samples were amplified for 16S rRNA with an expected amplicon size of 330 bp, while 50 out of 105 (47.62%) samples showed amplification using rpoβ primers, but were not of expected size. Of the 13 16S rRNA amplified samples, only 5 were identified as Leptospira in the gene sequence analysis and clustered under uncultured group via phylogenetic tree. This study showed the DNA-based approach using PCR and sequence analysis is able to detect the presence of Leptospira, although environmental samples may contain diverse microbial populations that may complicate the detection. Overall, the study suggested the importance of surveillance for Leptospira from environmental samples

Verification of H5 gene expression from H5-recombinant fowlpox viruses co-expressing host cytokine using indirect immunofluorescence antibody test (IFAT)

Fowlpox virus (FWPV) has been used in vaccine development against avian influenza virus (AIV) by the expression of the AIV haemagglutinin (HA) gene. Recombinant FWPV (rFWPV) vaccines can be improved by co-expression of interleukins (IL) that can act as immunostimulatory molecule. Prior to any field applications, rFWPV ability to stably express HA and IL genes in cells and its efficacy to induce immune response needs to be verified. Thus, the objective of this study is to verify the H5 gene expression from rFWPV-H5 and rFWPV-H5-IL-15 via indirect immunofluorescence antibody test (IFAT) method. Firstly, freshly prepared chicken embryonic fibroblast (CEF) cells were infected with recombinant viruses and wild type fowlpox virus served as a negative control. After overnight incubation, the cells were overlaid with H5 primary antibodies raised in rabbits for 2 hours. Counter-staining was done using fluorescein (FITC)- conjugated anti-rabbit secondary antibody raised in goats. Phase-contrast fluorescence microscopy showed faint green fluorescence signals in rFWPV-H5 and false positive in rFWPV-H5-IL-15 when ab21292 H5 primary antibody was used. Bright green fluorescence signals were exhibited in both recombinants, but not negative controls, when ab62587 H5 primary antibody was used, indicative of successful H5 recombinant protein expression

Detection of pathogenic Leptospira in rats and phylogenetic analysis using outer membrane lipoprotein Lipl32 gene at two major public markets

Introduction: Limited studies have been documented on the presence of pathogenic Leptospira in public markets serving the community in sub-districts of Selangor. The aim of this study was to detect the presence of pathogenic Leptospira in rats using a gene encoding an outer membrane lipoprotein LipL32. Methods: Polymerase chain reaction (PCR) was performed using LipL32 primers on sixty kidney samples of rats trapped at two locations of study; Pasar Borong Selangor in Seri Kembangan and Pasar Basah Bandar Baru Bangi in Bangi. Results: Out of 60 samples analysed, 36.7% were positive for the presence of LipL32. All positive samples highly matched (>94%) nucleotide sequence for LipL32 of pathogenic Leptospira and related to the pathogens through phylogenetic analysis. Conclusion: The detection of LipL32 indicates the potential presence of pathogenic Leptospira species at public markets. Although only 60 rats were successfully trapped, the rats are mobile and might further transmit the pathogenic organisms to other areas