Intraperitoneal administration of a novel chimeric immunogen (rOP) elicits IFN-γ and IL-12p70 protective immune response in BALB/c mice against virulent Brucella.

Recombinant engineering of immunologically active chimeric protein consisting of Omp19 and P39 domains of B. abortus (rOP), was purified under denaturing conditions upon expression in E. coli BL21 (DE3) and refolded to dynamic form.

Confirmed identification and toxin profiling of Campylobacter jejuni using a thermostabilized multiplex PCR formulation.

Cytolethal distending toxin (CDT) producing Campylobacter jejuni species are one of the leading causes of human gastroenteritis worldwide. The main intent of the study was to develop a multiplex PCR assay for the confirmed identification and toxin profiling of C. jejuni.

Development of IgY based sandwich ELISA for the detection of staphylococcal enterotoxin G (SEG), an egc toxin.

Staphylococcal food poisoning (SFP) is a major foodborne illness caused by staphylococcal enterotoxins (SEs). It is a well known fact that foodborne outbreak investigations are solely characterized by commercially available immunoassay kits. However, these assays encompass only few enterotoxins such as SEA-SEE which are renowned as "classical" enterotoxins and unable to detect any other novel enterotoxins even though their involvement is predicted.

Thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic Staphylococcus aureus.

Background/purpose: Among DNA-based techniques, polymerase chain reaction (PCR) is the most widely accepted molecular tool for the detection of pathogens. However, the technique involves several reagents and multiple pipetting steps that often lead to error-prone results. Additionally, the reagents entail a cold-chain facility to maintain their stability during storage and transportation.

Differentiation of entC1 from entC2/entC3 with a single primer pair using simple and rapid SYBR Green-based RT-PCR melt curve analysis.

In spite of their involvement in foodborne illness, the epidemiological relevance of staphylococcal enterotoxin C (SEC) subtypes is poorly documented may be due to high sequence similarity. Among subtypes, SEC1, SEC2, and SEC3 exhibit more than 97 % homology because of which specific detection tools are seldom available to identify and differentiate them. In this study, a SYBR Green-based RT-PCR followed by melt curve analysis was developed for differentiation of entC1 from entC2/entC3 using a single primer pair.

Selection and Characterization of Aptamers Using a Modified Whole Cell Bacterium SELEX for the Detection of Salmonella enterica Serovar Typhimurium.

This study describes the selection of single-stranded DNA (ssDNA) aptamers against Salmonella enterica serovar Typhimurium using a modified whole cell systematic evolution of ligands by exponential enrichment (whole cell SELEX). For evolving specific aptamers, ten rounds of selection to live Salmonella cells, alternating with negative selection against a cocktail of related pathogens, were performed. The resulting highly enriched oligonucleotide pools were sequenced and clustered into eight groups based on primary sequence homology and predicted secondary structure similarity.

Immunogenicity and protective efficacy of Brucella abortus recombinant protein cocktail (rOmp19+rP39) against B. abortus 544 and B. melitensis 16M infection in murine model.

In this study, the immunogenicity and protective efficacy of recombinant proteins Omp19 (rO) and P39 (rP) from Brucella abortus were evaluated individually and compared with the cocktail protein (rO+rP) against B. abortus 544 and Brucella melitensis 16M infection in BALB/c mouse model. Intra-peritoneal (I.

A combinatorial systematic evolution of ligands by exponential enrichment method for selection of aptamer against protein targets.

Aptamers are synthetic DNA recognition elements which form unique conformations that enable them to bind specifically to their targets. In the present study, an attempt was made to standardize a new modified combinatorial method comprising of Ni-NTA affinity Systematic Evolution of Ligands by Exponential Enrichment (SELEX; based on affinity between His tag protein and Ni-NTA), membrane SELEX (based on immobilization of protein on nitrocellulose membrane), and microtiter plate based SELEX (to monitor affinity and to enrich the selected aptamers) for protein targets. For experimental evaluation, staphylococcal interotoxin B was the molecule chosen.

Evaluation of recombinant leukocidin domain of VvhA exotoxin of Vibrio vulnificus as an effective toxoid in mouse model.

Vibrio vulnificus hemolysin A (VvhA) is a pore forming toxin and plays an important role in the pathogenesis. The hemolytic and cytotytic property of VvhA toxin is associated with N-terminal leukocidin domain which triggers apoptotic signaling cascade in epithelial cells. The present study was undertaken to assess the protective efficacy of recombinant VvhA leukocidin domain (rL/VvhA) against VvhA toxin challenge using in vitro and in vivo assays.

Evaluation of recombinant SEA-TSST fusion toxoid for protection against superantigen induced toxicity in mouse model.

Treatment of Staphylococcus aureus infections has become complicated owing to growing antibiotic resistance mechanisms and due to the multitude of virulence factors secreted by this organism. Failures with traditional monovalent vaccines or toxoids have brought a shift towards the use of multivalent formulas and neutralizing antibodies to combat and prevent range of staphylococcal infections. In this study, we evaluated the efficacy of a fusion protein (r-ET) comprising truncated regions of staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin (TSST-1) in generating neutralizing antibodies against superantigen induced toxicity in murine model.

Occurrence of Cellulose-Producing Gluconacetobacter spp. in Fruit Samples and Kombucha Tea, and Production of the Biopolymer.

Cellulose producing bacteria were isolated from fruit samples and kombucha tea (a fermented beverage) using CuSO4 solution in modified Watanabe and Yamanaka medium to inhibit yeasts and molds. Six bacterial strains showing cellulose production were isolated and identified by 16S rRNA gene sequencing as Gluconacetobacter xylinus strain DFBT, Ga. xylinus strain dfr-1, Gluconobacter oxydans strain dfr-2, G.

Studies on recombinant glucokinase (r-glk) protein of Brucella abortus as a candidate vaccine molecule for brucellosis.

Brucellosis is one of the most prevalent zoonotic diseases of worldwide distribution caused by the infection of genus Brucella. Live attenuated vaccines such as B. abortus S19, B.

Development and evaluation of IgY ImmunoCapture PCR ELISA for detection of Staphylococcus aureus enterotoxin A devoid of protein A interference.

In the present study, a sensitive and specific IgY mediated ImmunoCapture-PCR-ELISA (IC-PCR-ELISA) was developed for the detection of staphylococcal enterotoxin A (SEA) from culture supernatants and suspected contaminated samples. Due to the virtue of avian immunoglobulins (IgY) to have the least affinity towards staphylococcal protein A (SpA) responsible for false positives, we employed anti-SEA IgY for capture of SEA toxin and revealed with SEA specific rabbit antibodies conjugated to a 524bp DNA marker. Biotin-11-dUTP was incorporated during PCR amplification and post PCR analysis was performed by PCR-ELISA.

Application of monoclonal antibodies generated against Panton-Valentine Leukocidin (PVL-S) toxin for specific identification of community acquired methicillin resistance Staphylococcus aureus.

Panton-Valentine Leukocidin (PVL) produced by community acquired methicillin Staphylococcus aureus (CA-MRSA) involved in skin and soft-tissue infections and necrotizing pneumonia comprised of two fractions, namely PVL S and PVL F. In the present study, three monoclonal antibodies designated as MAb1, MAb9 and MAb10 were generated against recombinant PVL-S (35kDa) protein of S. aureus.

A recombinant bivalent fusion protein rVE confers active and passive protection against Yersinia enterocolitica infection in mice.

In the present study, a bivalent chimeric protein rVE comprising immunologically active domains of Yersinia pestis LcrV and YopE was assessed for its prophylactic abilities against Yersinia enterocolitica O:8 infection in murine model. Mice immunized with rVE elicited significantly higher antibody titers with substantial contribution from the rV component (3:1 ratio). Robust and significant resistance to Y.

In silico, in vitro and in vivo analysis of binding affinity between N and C-domains of Clostridium perfringens alpha toxin.

Clostridium perfringens alpha toxin/phospholipase C (CP-PLC) is one of the most potent bacterial toxins known to cause soft tissue infections like gas gangrene in humans and animals. It is the first bacterial toxin demonstrated to be an enzyme with phospholipase, sphingomyelinase and lecithinase activities. The toxin is comprised of an enzymatic N-domain and a binding C-domain interconnected by a flexible linker.

Occurrence and molecular detection of toxigenic Aspergillus species in food grain samples from India.

Background: The aim of this study was to detect the presence of different mycotoxigenic Aspergillus species in major food grains from southern states of India, namely maize, paddy, groundnut and sorghum. A total of 200 isolates recovered from 320 grain samples from four southern states were tested for their toxin chemotypes using high-performance liquid chromatography (HPLC), high-performance thin layer chromatography (HPTLC) and polymerase chain reaction (PCR) methods. The diversity and distribution of the isolates were recorded in terms of their frequency, density, importance value index and diversity indices.

Multiplex PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in rice and fingermillet collected from southern India.

Background: The genus Fusarium comprises a diverse group of fungi including several species that produce mycotoxins in food commodities. In the present study, a multiplex PCR was standardised for the group-specific detection of fumonisin-producing and trichothecene-producing strains of Fusarium species. Primers for genus-level recognition of Fusarium spp.

Molecular characterization of Vibrio cholerae isolates from cholera outbreaks in North India.

Vibrio cholerae isolates recovered from cholera outbreaks in Bhind district of Madhya Pradesh and Delhi, Northern India were characterized. The O1 serogroup isolates from Bhind outbreak were of Inaba serotype whereas both Ogawa and Inaba serotypes were recovered from Delhi. PCR analysis revealed that only O1 serogroup V.

Production and characterization of monoclonal antibodies against YopM effector protein of Yersinia pestis.

The YopM is an essential virulence effector produced by the bubonic plague bacterium. Yersinia pestis specific PCR gene was developed using 780 bp fragment of yopM gene. The PCR product was further cloned (in pUC57) an subcloned (pQE32 expression vector) and transformed in SG13009 E.

Utilization of monoclonal antibodies for detection of Plasmodium falciparum antigen in cerebrospinal fluid of cerebral malaria patients.

A uniform protein profile of bands at 34, 43, and 52 kDa was obtained with all the cerebrospinal fluid (CSF) samples of malaria (10 in number) and non-malaria patients (31 in number) by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). An immunoreactive band was observed at 43 kDa in CSF samples of cerebral malaria patients but not in non-malaria cases when tested with rabbit anti-Plasmodium falciparum antibodies by Western blot analysis. Eleven reactive monoclonal antibodies against P.

Generation and characterization of monoclonal antibodies against Klebsiella pneumoniae.

The present work was undertaken with the aim to generate highly specific monoclonal antibodies (MAb) targeted against Klebsiella pneumoniae that could be incorporated into a highly sensitive and simple ELISA (plate and dot) based system for the rapid identification of K. pneumoniae strains. Four murine monoclonal antibodies (KP-1, KP-2, KP-3, and KP-4) reactive to K.

Generation and characterization of murine monoclonal antibodies to recombinant 26-kDa periplasmic protein of Brucella abortus.

In the present study, hybridomas were developed for the production of monoclonal antibodies (MAb) against recombinant 26-kDa periplasmic protein (rBP26) of Brucella abortus. A set of six stabilized hybridoma cell lines were generated. Monoclonal antibodies secreted by all of these clones exhibited reaction for rBP26, as well as with the protein of 26-kDa, derived from whole cell lysate of B.

Development of a real-time PCR assay for the diagnosis of scrub typhus cases in India and evidence of the prevalence of new genotype of O. tsutsugamushi.

A qualitative syber green real-time PCR with primers designed for a truncated portion of the 56kDa major outer membrane antigen gene of Orientia tsutsugamushi was used to diagnose scrub typhus from the blood or serum of suspected patients. Sixty-six blood and/or sera samples from fever cases, either with high index of suspicion for scrub typhus and/or positive by Weil-Felix test (> or = 1:160), were tested with the PCR. Specificity of the PCR was confirmed by end point melt curve analysis and sequencing of the amplicons.

Generation and characterization of murine monoclonal antibodies to genus-specific 31-kilodalton recombinant cell surface protein of Brucella abortus.

In the present study hybridomas were produced from fusion with splenocytes of BALB/c mice immunized with the recombinant 31-kDa cell surface protein (r31CSP) specific for Brucella species. A set of eight stabilized hybridoma cell lines was generated against r31CSP. Monoclonal antibodies (MAbs) produced by all these clones exhibited reactivity for r31CSP as well as with the protein of 31-kDa, derived from whole-cell lysate of 31-kDa Brucella abortus 544.