Tuberculosis remains a major infectious disease with over 8 million new cases and 2 million deaths annually. Therefore, a vaccine more potent than BCG is desperately needed. In this regard, an approximately 800 bp DNA encoding a mycobacterial synthetic gene designated as VacIII (containing ubiquitin gene UbGR and four immunogenic mycobacterial epitopes or genes of ESAT-6, Phos1, Hsp 16.3, and Mtb8.4) was sub-cloned into a bacterial expression vector of pRSET-B resulting in a 6 x His-VacIII fusion gene construction. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea and the recombinant protein was purified by Ni-NTA column and dialyzed by urea gradient dialysis. This method produced a relatively high yield of recombinant VacIII protein and the cloned VacIII gene offers the potential development of other vaccine formats such as DNA vaccine and recombinant vaccine.
There is a need for identification of new infection markers against common Leptospira isolates in Malaysia. To achieve this goal, seven-day-old cultures of Leptospira interrogans serogroup Icterohemorrhagiae (L44) and Leptospira interrogans serogroup Javanica (L55) were used for antigen preparation by sequential extraction method using 40 mM Tris, 8M Urea and 2M thiourea. Immunoblot analysis of the antigens were performed using serum samples from 46 local patients with confirmed acute leptospirosis, 28 patients with other infections and 14 healthy controls. The patients serum samples used in this study contained heterologous antibody against a number of different leptospira serovars. A strong IgM reactivity to a broad diffuse band of 10-15 kDa was observed. Combining results using L44 and L55 antigens showed sensitivity of 80.4% and specificity of 95.2% for detection of leptospirosis. Proteinase K and periodate treatment indicated that the band is likely to be lipopolysaccharide (LPS) in nature. This study showed that the 10-15 kDa antigen could potentially be useful for serodiagnosis of acute leptospirosis in Malaysia.
Leptospirosis is a widespread zoonotic disease and a public health problem, particularly in tropical and subtropical countries. Varied clinical manifestations of the disease frequently lead to misdiagnosis resulting in life-threatening multi-organ complications. Therefore, early laboratory investigation using an appropriate diagnostic approach is crucial. In the present study, a potential protein marker was identified and evaluated for its usefulness in the serodiagnosis of acute leptospirosis.
Vibrio cholerae is the causative agent of the infectious disease, cholera. The bacteria adhere to the mucosal membrane and release cholera toxin, leading to watery diarrhea. There are >100 serovars of V. cholerae, but the O1 and O139 serovars are the main causative agents of cholera. The present study aimed to compare the severity of intestinal mucosal infection caused by O1 El Tor and O139 V. cholerae in a rabbit ileal loop model. The results showed that although the fluid accumulation was similar in the loops inoculated with O1 and O139 V. cholerae, the presence of blood was detected only in the loops inoculated with the O139 serovar. Serosal hemorrhage was confirmed by histopathological examination and the loops inoculated with O139 showed massive destruction of villi and loss of intestinal glands. The submucosa and muscularis mucosa of the ileum showed the presence of edema with congested blood vessels, while severe hemorrhage was seen in the muscularis propria layer. The loops inoculated with O1 El Tor showed only minimal damage, with intact intestinal villi and glands. Diffuse colonies of the O139 serovar were seen to have infiltrated deep into the submucosal layer of the intestine. Although the infection caused by the O1 serovar was focal and invasive, it was more superficial than that due to O139, and involved only the villi. These observations were confirmed by immunostaining with O1 and O139 V. cholerae-specific monoclonal antibodies. The peroxidase reaction demonstrated involvement of tissues down to the submucosal layer in O139 V. cholerae infection, while in O1 El Tor infection, the reaction was confined mainly to the villi, and was greatly reduced in the submucosal region. This is the first reported study to clearly demonstrate the histopathological differences between infections caused by the O139 Bengal and O1 El Tor pathogenic serovars of V. cholerae.
A PCR assay has been developed based on a lolB (hemM) gene, which was found to be highly conserved among the Vibrio cholerae species but non-conserved among the other enteric bacteria. The lolB PCR detected all O1, O139 and non-O1/non-O139 serogroup and biotypes of V. cholerae. The analytical specificity of this assay was 100% while the analytical sensitivity was 10 pg/microL and 10(3) CFU/mL at DNA and bacterial level respectively. The diagnostic sensitivity and specificity was 98.5% and 100% respectively.
The sensitivity and specificity of 18S rRNA polymerase chain reaction (PCR) in the detection of fungal aetiology of microbial keratitis was determined in thirty patients with clinical diagnosis of microbial keratitis.
In this paper, we describe the development of VCUSM2, a live metabolic auxotroph of Vibrio cholerae O139. Auxotrophy was achieved by mutating a house keeping gene, hemA, that encodes for glutamyl-tRNA reductase, an important enzyme in the C5 pathway for delta-aminolevulenic acid (ALA) biosynthesis, which renders this strain dependent on exogenous ALA for survival. Experiments using the infant mouse and adult rabbit models show that VCUSM2 is a good colonizer of the small intestine and elicits greater than a four-fold rise in vibriocidal antibodies in vaccinated rabbits. Rabbits vaccinated with VCUSM2 were fully protected against subsequent challenge with 1 x 10(11) CFU of the virulent wild type (WT) strain. Experiments using ligated ileal loops of rabbits show that VCUSM2 is 2.5-fold less toxic at the dose of 1 x 10(6) CFU compared to the WT strain. Shedding of VCUSM2 in rabbits were found to occur for no longer than 4 days and its maximum survival rate in environmental waters is 8 days compared to the greater than 20 days for the WT strain. VCUSM2 is thus a potential vaccine candidate against infection by V. cholerae O139.