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The unexpected pandemic set off by the novel coronavirus (SARS-CoV2) has spread to more than 210 countries across the globe, including India. In the current pandemic situation, various steps have been taken by the Indian government to prevent and control the spread of the SARS-CoV2 infection. To date, there are no proven vaccines or effective therapeutic interventions against the virus. Current clinical management includes infection prevention and control, symptom-specific relief and supportive care. Physicians and scientists across the country have been tirelessly working on developing effective diagnostic and therapeutic strategies and to combat and control this infection. As the demand for diagnostics and therapeutics continues to rise in India and around the globe, it is essential to rapidly develop various algorithms to successfully identify and contain the virus. This review discusses the updates on the recent developments in COVID-19 diagnostics and therapeutics in India.

In the present study, we have generated a murine monoclonal antibody (mAb) named Sal-06 by using the crude outer membrane protein preparation of Salmonella enteric subsp. enterica serovar Typhimurium ATCC 14028 strain as antigen. Sal-06mAb belonging to IgG1 isotype demonstrated broad cross-reactivity to standard and isolated strains of genus Salmonella and others such as Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis. Cross-reactivity across several bacterial genera indicated that the epitopes reactive to Sal-06mAb are conserved among these members. Neutralizing effects of Sal-06mAb on Salmonella growth and survival was evaluated in vitro using bacteriostatic and bactericidal activity with and without complement and bacterial invasion inhibition assay. Sal-06mAb demonstrated a bacteriostatic effect on the growth of S. typhimurium ATCC 14028 strain which is both time and concentration (of mAb) dependent. It was also found that the bacterial growth inhibition was complement independent. When the bacterial cells were preincubated with Sal-06mAb, it reduced the adherence and invasion of bacterial cells into A549 epithelial cell line. This was confirmed by CFU count analysis, phase contrast, and fluorescence microscopy. Scanning electron microscope (SEM) imaging confirmed the antimicrobial effects of Sal-06mAb on S. typhimurium ATCC 14028. The development of broadly reactive and cross protective Sal-06mAb opens new possibilities for immunotherapy of sepsis caused by Gram-negative Enterobacteriaceae members.

The milder form of infection and higher rates of recovery witnessed among COVID-19 patients in India is indicative of the potential intervention of other “unconventional” biological mechanisms. The recently established similarity between beta-coronavirus strains in animals and humans led us to hypothesize that previous contact with infected dogs or cattle could shield humans from the circulating SARS-CoV-2 virus. We further believe that our hypothesis, if confirmed by further studies, could be used as a potential vaccine strategy.

Salmonella enterica, enterohemorrhagic Escherichia coli, and Staphylococcus aureus are major pathogens contaminating low moisture foods. E. coli and Shigella spp. may acquire significance in future since cross-contamination plays a critical role in outbreaks involving low moisture foods. This study investigated a PMA-IAC-mPCR for rapid, reliable and simultaneous detection of viable Salmonella spp., E. coli, Staphylococcus aureus, E. coli O157:H7, and Shigella spp., in low moisture foods. Propidium monoazide (PMA) was applied to detect only viable cells by eliminating PCR signal from dead cells. In addition, an internal amplification control (IAC) was included in the multiplex PCR as an indicator of false negative results arising due to inhibitors in low moisture foods. The sensitivity of the assay for viable cells with PMA treatment was 102–103 CFU/mL for all the target pathogens reflecting the non-influence of PMA treatment on sensitivity. After 10 h enrichment, the PMA-IAC-mPCR could detect 101 CFU/g of Salmonella spp., E. coli, Staphylococcus aureus, E. coli O157:H7, and Shigella spp., in artificially inoculated low moisture foods (peanut butter, chocolate, milk powder and egg powder). This PMA-IAC-mPCR assay would find its promising application in simultaneous detection of these viable target pathogens in low moisture foods.

The most promising means of controlling anthrax, a lethal zoonotic disease during the early infection stages, entail restricting the resilient infectious form, i.e., the spores from proliferating to replicating bacilli in the host. The extractible antigen (EA1), a major S-layer protein present on the vegetative cells and spores of Bacillus anthracis, is highly immunogenic and protects mice against lethal challenge upon immunization. In the present study, mice were immunized with r-EA1C, the C terminal crystallization domain of EA1, to generate a neutralizing monoclonal antibody EA752–862, that was evaluated for its anti-spore and anti-bacterial properties. The monoclonal antibody EA752–862 had a minimum inhibitory concentration of 0.08 mg/ml, was bactericidal at a concentration of 0.1 mg/ml and resulted in 100% survival of mice against challenge with B. anthracis vegetative cells. Bacterial cell lysis as observed by scanning electron microscopy and nucleic acid leakage assay could be attributed as a possible mechanism for the bactericidal property. The association of mAb EA752–862 with spores inhibits their subsequent germination to vegetative cells in vitro, enhances phagocytosis of the spores and killing of the vegetative cells within the macrophage, and subsequently resulted in 90% survival of mice upon B. anthracis Ames spore challenge. Therefore, owing to its anti-spore and bactericidal properties, the present study demonstrates mAb EA752–862 as an efficient neutralizing antibody that hinders the establishment of early infection before massive multiplication and toxin release takes place.

The current study reports the preparation and stabilization of novel functional drinks based on fruit and vegetable juices incorporating bacterial cellulose from Acetobacter xylinum. Pineapple, musk melon, carrot, tomato, beet root and a blend juice containing 20 % each of carrot and tomato juice with 60 % beet root juice has been studied. These juices have been stabilized over a storage period of 90 days at 28 °C, by the use of nisin and maintaining a low pH circumventing the need for any chemical preservatives or refrigeration. Instrumental color values have been correlated with the pigment concentrations present in the fresh as well as stored juices. There was 36, 72 and 60 % loss of total carotenoids in the case of carrot, pineapple and musk melon juices respectively while the lycopene content remained unchanged after 90 days of storage. The betanin content decreased 37 % in the case of beetroot juice and 25 % in the case of beetroot juice blended with carrot and tomato juices. Sensory analysis has revealed a clear preference for the beetroot blended mixed juice.

Staphylococcal protein A (Spa) secreted by all Staphylococcus aureus strains is the major hindrance in development of specific immunoassays for detecting S. aureus antigens, because of its characteristic feature of binding to Fc region of most mammalian immunoglobulins and also to Fab region of certain classes of mammalian immunoglobulins. Immunoglobulin Y (IgY) is the avian equivalent of mammalian IgG which does not have any affinity to Spa. In the present study we report that using chicken egg yolk IgY over mammalian IgG as capture antibody prevents both soluble and surface bound protein A from causing false positives quantified by chicken anti-protein A antibodies. This was demonstrated by development of sandwich ELISA for detection of alpha hemolysin toxin from culture supernatants of S. aureus strains with anti alpha hemolysin IgY as capture and rabbit anti alpha hemolysin IgG as revealing antibody. This indirect sandwich ELISA was evaluated onto a large number of S. aureus isolates recovered from clinical sources for alpha hemolysin secretion. Results of sandwich ELISA were compared with PCR and Western blot analysis. The immunoassay is highly specific and has high sensitivity of detecting less than 1 ng/ml. This procedure is highly effective in eliminating Spa interference and can be extended to detection of other important superantigen toxins of S. aureus.

The aim of the present study was to develop a multiplex PCR (mPCR) assay for the simultaneous detection of five major metabolic pathway genes viz. aflr (Aflatoxin), pks (Ochratoxin A), tri5 (Trichothecene), pks13 (Zearalenone) and fum13 (Fumonisin), producing Aspergillus, Penicillium and Fusarium species. The mPCR assay with competitive internal amplification control to eliminate false negative results employing specific primers for each of the above mentioned five genes was optimized and validated using standard strains. The standardized mPCR assay detected all five major mycotoxin metabolic genes along with artificially inoculated maize seeds with mycotoxigenic Fusarium, Penicillium and Aspergillus spores. The detection limit of this mPCR assay was 1 × 103 spores per gram of artificially inoculated samples upon 48 h of incubation at room temperature. When the developed mPCR assay was applied on to 177 contaminated maize, paddy and sorghum, many of the samples (100 out of 177) were contaminated with either one or more mycotoxins. The mPCR results were further evaluated with high performance liquid chromatography and in general both the methods provided unequivocal results. The developed mPCR based assay was found to be rapid, cost effective and user friendly and can be used for diagnosis of major mycotoxigenic fungi.

Aim: To evaluate an inter-generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. Methods and Results: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap-extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r-αCS specifically detected 42-kDa and 33-kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild-type toxins in vitro. Conclusions: The r-αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild-type toxins in vitro. Significance of the Study: The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co-infections like gangrenous ischaemia, gangrenous mastitis, etc.

The aim of this study is to develop simple and rapid diagnostic method which can be utilized routinely for detection of Klebsiella pneumoniae. This study was done in India at DRDE, Gwalior. We have collected standard K.pneumoniae strain 3296 from Yamaguchi University, Japan and total fifty-nine clinical isolates of Klebsiella species from Armed Force Medical College, Pune, and Patel chest hospital, New Delhi. Bacteria were grown on trypticase soy broth and two Newzealand wistar white rabbit were immunized subcutaneously with purified capsular polysaccharide (CPS) of K.pneumoniae. Specificity of hyper immune sera raised against CPS was tested by counter current immunoelectrophoresis. We have generated four monoclonal antibodies, out of which two IgM producing clones (KP-1 & KP-2) and hyper immune sera against CPS antigen were used for agglutination test. KP-1, KP-2 and CPS hyperimmune sera showed different reactions. Out of the twenty biochemically confirmed K.pneumoniae clinical isolates, hyperimmune sera to CPS detected sixteen as positive whereas KP-1 reacted to eleven and KP-2 to thirteen of these isolates. One clinical Klebsiella species biochemically negative for K.pneumoniae and other Enterobacteriaceae organisms were negative by agglutination test to all the three antibody reagents. K.aerogenes, however, reacted to KP-2 and CPS hyperimmune sera. Standard K.pneumoniae was positive to all the three antibodies. The rapidity and ease of performance merited the hyper immune sera to CPS and IgM monoclonal antibody based agglutination test as the preliminary identification system for K.pneumoniae.

Aeromonas hydrophila and other aeromonads are ubiquitous organisms found in meat, vegetables, drinking water and various other food items. They cause diarrhea and extra-intestinal infections in normal and immunocompromised patients. The aim of the study was to develop a multiplex PCR assay for the detection of virulence-associated genes of Aeromonas including hemolysin (hlyA), aerolysin (aerA), glycerophospholipid-cholesterol acyl transferase (GCAT) alongwith a 16S rRNA gene. Internal amplification control (IAC), which was coamplified with aerA primers, was also included in this study. The results showed that all cultures of Aeromonas were accurately identified by the assay without showing non-specificity. A. hydrophila could be detected at a range of 10-50 CFU ml(-1) from experimentally spiked fish, chicken and milk samples following overnight enrichment in alkaline peptone water supplemented with 10 μg/ml ampicillin (APW-A) by this multiplex PCR (mPCR). When evaluated on a total of 74 naturally occurring food samples, four samples were identified to contain Aeromonas by mPCR. All these results were compared to the conventional culture, isolation and biochemical identification procedures. The high throughput and cost-effective mPCR method developed in this study could provide a powerful tool for detection of pathogenic Aeromonas spp. from food and environmental samples and in addition, the method has advantages in terms of specificity, sensitivity and ease of use compared to other reported PCR methods and DNA hybridization assays.

The lactic acid bacteria (LAB) play an important role in the fermentation of vegetables to improve nutritive value, palatability, acceptability, microbial quality and shelf life of the fermented produce. The LAB associated with beetroot and carrot fermentation were identified and characterized using different molecular tools. Amplified ribosomal DNA restriction analysis (ARDRA) provided similar DNA profile for the 16 LAB strains isolated from beetroot and carrot fermentation while repetitive extragenic palindromic PCR (rep-PCR) genotyping could differentiate the LAB strains into eight genotypes. Thirteen strains represented by five genotypes could be clustered in five distinct groups while three LAB strains exhibiting distinct genotypes remained ungrouped. These genotypes could be identified to be belonging to L. plantarum group by 16S rDNA sequencing. The recAnested multiplex PCR employing species-specific primers for the L. plantarum group members identified the LAB strains of six genotypes to be L. paraplantarum and the other two genotypes to be L. pentosus. Three genotypes of L. paraplantarum were consistently found on the third and sixth day of beetroot fermentation whereas a distinct genotype of L. paraplantarum and L. pentosus appeared predominant on the tenth day. From carrot Kanji two distinct genotypes of L. paraplantarum and one genotype of L. pentosus were identified. REP-PCR DNA fingerprinting coupled with 16S rDNA sequencing and recA-nested multiplex PCR could clearly identify as well as differentiate the diverse L. plantarum group strains involved in the fermentation.

Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are the major concerns for the food safety in terms of frequency and/or seriousness of the disease. Being members of the same group and sharing DNA homology to a larger extent, they do create problems when their specific detection/identification is attempted from different food and environmental sources. Numerous individual polymerase chain reaction (PCR) and few multiplex PCR (mPCR) methods have been employed to detect these organisms by targeting toxin genes but with lack of internal amplification control (IAC). Therefore, we attempted a mPCR with IAC for the detection of enterotoxic B. cereus group strains by selecting hbl A, nhe A and cyt K genes from B. cereus, indicative of the diarrheal potential and cry I A and pag genes, the plasmid borne phenotypic markers specific to B. thuringiensis and B. anthracis strains, respectively. Multiplex PCR assay validation was performed by simultaneous comparison with the results of single-target PCR assays and correlated to the classical conventional and biochemical identification of the organisms. The mPCR was able to detect as low as 10(1)-10(2) organisms per ml following overnight enrichment of spiked food samples (vegetable biriyani and milk) in buffered peptone water (BPW). The presence of these organisms could also be detected by mPCR in naturally contaminated samples of rice based dishes and milk. The high throughput and cost-effective mPCR method described could provide a powerful tool for simultaneous, rapid and reliable detection of enterotoxic B. cereus group organisms.

Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus are of major concern for food safety in terms of frequency and seriousness of the disease. The occurrence these three important pathogens and their coexistence in food matrices are predominant. Moreover, symptoms associated with B. cereus and S. aureus food poisoning not only closely resembles each other but can also be overlapping with other foodborne infections. In this context, detection of these three pathogens simultaneously in food samples by a single multiplex PCR (mPCR) would have advantages in terms of rapidity and cost saving, when compared with single organism specific PCRs. mPCR has been standardized by targeting three major diarrheal enterotoxin genes hbl A, cyt K and nhe A of B. cereus, virulence associated nuc and Ent B genes of S. aureus and virulence associated hly and iap genes of L. monocytogenes along with internal amplification control (IAC). The results showed that mPCR accurately identified all the three organisms individually or in combination without non-specificity. The mPCR was able to detect as low as 10 to 100 organisms per ml of growth following overnight enrichment of spiked food samples (vegetable biriyani and milk) and their presence in naturally contaminated samples also. The high throughput and cost effective multiplex PCR method developed in this study could provide a powerful tool for simultaneous, rapid and reliable detection of B. cereus, S. aureus and L. monocytogenes in food samples.

The aim of the work was to specifically differentiate S. typhimurium from other closely related Salmonella serovars by monoplex or multiplex PCR and to detect it from water and food samples. Genes targeted were invA, iroB, STM4497, STM2755, fliC, fljB and rfbJ and evaluated on 58 Salmonella standard serovars/strains including 9 S. typhimurium strains, 7 suspected Salmonella isolates and 8 other organisms as negative controls. Both invA and iroB showed a uniform amplification with all serovars of S. enterica group. STM2755 and STM4497 gene based PCR’s specifically exhibited amplification in all the nine confirmed S. typhimurium strains. The rfbJ PCR produced amplification with confirmed S. typhimurium strains, in addition showed reaction with S. abony. Both STM4497, STM2755 PCR’s and rfbJ could identify two of the seven biochemically suspected Salmonella isolates that were later confirmed to be S. typhimurium on the basis of sequence data. PCR for fliC genes had amplification exhibited by a large number of serovars of the S. enterica group, including S. typhimurium strains but not to S. brunei,S. newporti, S. abony and S. weltevreden. fljB was detected in all strains of S. enterica and E. coli with the exception of S. typhi. fljB and fliC were amplified in 6/7 and 5/7 presumptive Salmonella isolates. The same PCR’s were converted into two multiplex formats for simultaneous identification of the Salmonella genus, S. enterica group and S. typhimurium as a species. The first multiplex set comprised on invA, iroB, STM4497, STM2755 and the IAC. The second multiplex set comprised of invA, iroB, fljB, fliC, rfbJ along with IAC. The detection limit for S. typhimurium in the two multiplex PCR sets was in the range of 350–400 cfu/PCR reaction and that of DNA around 2 pg. The multiplex PCR (format 1) was first evaluated on spiked water, chicken and mutton samples and the detection limit for S. typhimurium was in the range of 100 cfu/100 ml

The genotype and antibiotic resistance pattern of the toxigenic Vibrio cholerae strains associated with cholera outbreaks vary frequently. Fifty-one V. cholerae strains isolated from cholera outbreaks in Chennai (2002–2005) were screened for the presence of virulence and regulatory genes by multiplex polymerase chain reaction (PCR) assay. Genotyping of the isolates was done by VC1 primers derived from enterobacterial repetitive intergenic consensus (ERIC)-related sequence in V. cholerae. All the isolates possessed toxigenic genes, such as ctxA, ctxB, tcpA, ace, ompU, toxR and zot. Two different El Tor genotypes and one O139 genotype could be delineated by VC1-PCR. One of the El Tor genotypes was similar to the El Tor strains isolated from Bhind district and Delhi during 2004. Antibiotic susceptibility testing revealed greater variability among the isolates tested. All the isolates were found to be susceptible to norfloxacin, ciprofloxacin and tetracycline. Thiry-three per cent of the isolates were found to be resistant to more than 4 antibiotics and could be termed as multiple antibiotic resistant. Coexistence of O139 serogroup along with the El Tor biotype could be identified among the strains recovered during the period 2002–2004. The O139 isolates were found to be more susceptible to the antibiotics tested when compared to the El Tor isolates.

The three strains of non-pathogenic Proteus species namely, Proteus vulgaris OX2, P. vulgaris OX19 and Proteus mirabilis OXK used in the Weil-Felix test are the group-specific cross-reactive antigens for Rickettsia and Orientia species. Earlier studies have revealed that the group specific and cross-reactive antigens responsible for the Weil-Felix test lie mostly in the lipopolysaccharide (LPS) moiety of the bacterial cell wall [Amano et al. (1993a) Infect Immun 61:4350-4355, (1993b) Microbiol Immunol 37:927-933, (1998) Infect Immun 66:923-926]. The three Proteus strains (OX2, OX19 and OXK) were used to raise murine monoclonal antibodies (MAbs) by hybridoma technology. Several MAb-producing hybridomas could be stabilized following limiting dilution. Affinity and specificity of these MAbs were checked by indirect ELISA using a battery of homologous and heterologous antigens including LPS. Amongst these, one MAb was found to be specific for P. vulgaris OX19 LPS. Since the Weil-Felix reaction is based on the cross-reactivity between the LPS based epitopes, this MAb could be of potential use in mapping of epitopes on the cross-reactive LPS and may also be useful as a potential diagnostic reagent.

YopM, an effector, YopB, a translator, and LcrV, a regulator, are proteins forming important componants of type III secretion system of Yersinia pestis. Recombinant truncated YopM of 32 kDa, YopB of 28 kDa and LcrV of 31 kDa sizes were utilized for priming BALB/c mice for the generation of monoclonal antibodies following standard poly-ethylene glycol (PEG) fusion protocol. Nine, 10 and 6 stabilized hybridoma cell lines could be generated against YopM, YopB and LcrV proteins, respectively. All these monoclonal antibodies were found reactive to Y. pestis strain A1122 and did not show any cross-reactivity to Y. enterocolitica, Y. pseudotuberculosis, Y. kristensenii, Y. frederiksenii, Y. intermedia, Klebsiella pneumoniae, Escherichia coli, Salmonella typhi, Salmonella abortus-equi and Staphylococcus aureus tested by ELISA and Western blotting. Monoclonal antibodies also exhibited reactivity to their corressponding native protein antigens in Y. pestis i.e. 42 kDa for YopM, 41 kDa for YopB and 37 kDa for LcrV in immunoblotting. Reactivity of monoclonal antibodies was further assessed on 26 Y. pestis isolates including 18 from 1994 plague outbreak regions (11 from pneumonic patients, 7 from rodents) and 8 from rodents of Deccan plateau of Southern India by Western blotting as well as by sandwich ELISA. The monoclonal antibodies could specifically locate the expression of yopM, yopB and lcrV genes among these Indian Y. pestis strains as well. Results obtained with sandwich ELISA and Western blot were identical to those observed by PCR. Monoclonal antibodies to Yops, therefore, can be employed for an early and reliable identification of virulent Y. pestis strains.

A combination of immunomagnetic separation and polymerase chain reaction (IMS-PCR) was used to detect Salmonella typhi in food and water samples. IMS was found to be an effective method for specific capture of S. typhi from artificially inoculated meat rinse samples. The bacteria could be detected within 6 h by IMS-PCR with a sensitivity of 105 cells. However, when tested in milk samples, the method was less effective. In comparison to conventional culture method, IMS-PCR is a rapid and specific method for detection of S. typhi and could be useful in outbreak situations for tracing the source of infection.

Monoclonal antibodies (MAbs) were generated against the recombinant plasminogen activator (Pla) protein of Yersinia pestis. These MAbs detected Pla in all the 18 isolates of Y. pestis obtained from the sputum of pneumonic plague patients and from the liver and spleen of rodents from plague-affected areas of India during 1994–1995 as well as in seven of the eight isolates obtained from rodents in the surveillance regions of Hosur and Palmner in India during 1998 by simple dot-ELISA. In immunoblotting, the MAbs reacted with the Pla antigen only in Y. pestis isolates at 37 and 35 kDa region. These monoclonal antibodies, being strictly specific, can be used for detecting Y. pestis isolates that are Fraction 1 antigen-negative. Also, the radiolabelled pla fragment hybridized specifically to the representative DNA samples of Y. pestis isolates.

Salmonella enterica and Shigella species are commonly associated with food and water borne infections leading to gastrointestinal diseases. The present work was undertaken to develop a sensitive and reliable PCR based detection system for simultaneous detection of Salmonella enterica and Shigella at species level. For this the conserved regions of specific genes namely ipaH1, ipaH, wbgZ, wzy and invA were targeted for detection of Shigella genus, S. flexneri, S. sonnei, S. boydii and Salmonella enterica respectively along with an internal amplification control (IAC). The results showed that twenty Salmonella and eleven Shigella spp., were accurately identified by the assay without showing non-specificity against closely related other Enterobacteriaceae organisms and also against other pathogens. Further evaluation of multiplex PCR was undertaken on 50 natural samples of chicken, eggs and poultry litter and results compared with conventional culture isolation and identification procedure. The multiplex PCR identified the presence of Salmonella and Shigella strains with a short pre-enrichment step of 5 h in peptone water and the same samples were processed by conventional procedures for comparison. Therefore, this reported multiplex PCR can serve as an alternative to the tedious time-consuming procedure of culture and identification in food safety laboratories.

Aims To develop a multiplex PCR assay coupled with selective enrichment step to detect major virulence-associated genes of enterotoxigenic Stap-hylococcus aureus and evaluate the same directly on contaminated food samples. Methods and Results The most important virulence-associated genes of Staph. aureus, which are commonly related to food safety issues, are targeted in this study. They include five major enterotoxigenic genes-sea, seb, sec, seg and sei, tst-which encodes TSST-1, mecA-which confer methicillin resistance and coa-for the enzyme coagulase along with an internal amplification control (IAC) to rule out false-negative result. A modified mannitol salt broth (MSB) supplemented with sodium pyruvate was used for selective enrichment of Staph. aureus from food samples prior to PCR. Evaluation of efficiency of different media revealed that enrichment of samples in modified MSB followed by PCR resulted in specific, sensitive and effective amplification of the targeted genes in comparison with other enrichment media. Incorporation of bovine serum albumin (BSA) as PCR enhancer improved the intensity of amplicons. The standardized multiplex PCR (mPCR) format was able to detect all the target genes at a bacterial load of 106 CFU ml−1 in any sample. The PCR results were unequivocally correlated with the conventional methods when the mPCR format was assessed on a total of 91 Staph. aureus isolates. The entire assay was found to be effectual when evaluated on naturally contaminated food samples. Conclusions The combinatorial approach involving selective enrichment followed by mPCR developed in this study was found to be effective for the detection of toxigenic Staph. aureus directly from various food sources. Significance and Impact of the Study The developed format would find a promising application in early detection of food contaminations as well as in the diagnosis of food poisoning due to Staph. aureus.

Hundred Fusarium culmorum strains, isolated from freshly harvested maize grain samples from Southern parts of India, were incubated in czapek-dox medium and analyzed for trichothecene (DON/NIV) production. The mPCR assay was standardized targeting trichothecene metabolic pathway genes viz., Tri6, Tri7, Tri13 for detection of trichothecene (DON/NIV) chemotypes and rDNA gene for specific detection of F. culmorum species. Primers for targeted genes were designed and used to predict whether these isolates could produce deoxynivalenol/nivalenol, 94 isolates were able to produce DON/NIV by mPCR assay. Chemical analysis of DON/NIV was carried out for mPCR positive isolates by high performance-thin layer chromatography (HPTLC). To check the practical usefulness of developed mPCR assay, 150 field samples of maize were evaluated and results were compared with conventional HPTLC method. Out of 150 samples, 34% samples stayed as a positive for NIV contamination whereas 44% were found to have deoxynivalenol contamination. Moreover, mPCR results are equivocally matched with the HPTLC chemical analysis for field samples. Chemotyping of F. culmorum isolates were reported for the first time from India, and highlights the important potential of F. culmorum to contaminate maize with DON/NIV.

To evaluate an inter-generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap-extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r-αCS specifically detected 42-kDa and 33-kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild-type toxins in vitro. The r-αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild-type toxins in vitro. The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co-infections like gangrenous ischaemia, gangrenous mastitis, etc.

Previous studies in UK subjects suggested that the 19 kDa protein antigen of Mycobacterium tuberculosis might be valuable in the serodiagnosis of paucibacillary tuberculosis. In this study, antibody titres for the 19 kDa antigen were higher in healthy controls in India than in the UK. Consequently, the diagnostic sensitivity of this antigen and its TB23 epitope was negligible in Indian patients with tuberculosis. However, a diagnostic sensitivity of 50% was achieved in patients with skin tuberculosis on the basis of a high ratio between antibody titres for the whole antigen and its TB23 epitope.

Haemolysin BL (HBL) of Bacillus cereus, listeriolysin O (LLO) of Listeria monocytogenes and enterotoxin B (SEB) of Staphylococcus aureus are among the major toxin components contributing to the pathogenicity of these organisms in foodborne illnesses. In this study, an intergenus non-toxic multidomain fusion protein (r-HLE) was generated with specificity for HBL, LLO and SEB. The fusion gene (r-hle) comprising the conserved regions of hblD and the hly and entB genes was codon-optimized for expression in Escherichia coli and encoded a 50 kDa recombinant multidomain chimeric protein (r-HLE). Hyperimmune antiserum raised against r-HLE specifically reacted with the L1 (38 kDa) component of the HBL complex of B. cereus, LLO (58 kDa) of L. monocytogenes and SEB (28 kDa) of S. aureus during Western blot analysis when tested on standard strains. During testing on isolates, the antiserum again identified the appropriate toxin molecules and was highly specific to the relevant bacterial species. The antigenicity of the SEB component of the r-HLE protein was also confirmed using a commercially available TECRA kit. The described procedure of creating a single antigenic molecule carrying components of three different toxins whilst still retaining the original antigenic determinants of individual toxins will be highly advantageous in the development of rapid, reliable and cost-effective immunoassays.

The PhoP-PhoQ two-component system of Yersinia pseudotuberculosis, a Gram-negative enteric pathogen which causes a variety of gastrointestinal and extraintestinal infections in humans, has been shown to be necessary for virulence. A phoP-phoQ null mutant of a strain of Y. pseudotuberculosis cured of its native plasmid pYV was obtained and studied for generation of immune response in mouse model following intravenous inoculation. The phoP-phoQ null mutant elicited much weaker IgG antibody response to whole cell sonicated (WCS) antigen, in particular that of IgG2a isotype. Interferon-gamma levels were also significantly reduced in cultured splenocytes of mice immunized with phoP-phoQ null mutant. The null mutant was found to be about 72-fold less virulent than the parent isogenic strain of Y. pseudotuberculosis. Average counts in spleen of mice inoculated with the null mutant were observed to reduce by at least four logs when compared with the counts in the spleen of mice inoculated with parent isogenic strain. We can thus suggest that the Th1-type immune response of the phoP-phoQ null mutant of Y. pseudotuberculosis is diminished in mice.

Monoclonal antibodies (MAbs) were produced by fusing SP2/0 myeloma cells with spleen cells of BALB/c mice that were immunized with live whole cells of the four most prevalent Leptospira serovars--namely, autumnalis, australis, grippotyphosa, and icterohaemorrhagiae. A total of 26 MAbs (10 autumnalis, 5 australis, 4 grippotyphosa, and 7 icterohaemorrhagiae) were produced that showed specific, restricted, or broad cross-reactivity when tested with 19 standard pathogenic and 3 standard saprophytic serovars by MAT and dot-ELISA. Monoclonal antibodies like AT4 and AT5 against serovar autumnalis; AS1 and AS2 against serovar australis; GR1, GR3, and GR4 raised against serovar grippotyphosa; and also the MAbs IC3 to IC7 against serovar icterohaemorrhagiae were all usable as typing reagents in a rapid dot-ELISA. Selected MAbs were subsequently utilized in a rapid sandwich dot-ELISA for identification of Leptospira serovars as well as for antigen detection in experimentally infected mice and guinea pigs. Results of rapid sandwich dot-ELISA were compared with dark field microscopy, culture, and PCR in experimentally infected animals and sandwich dot-ELISA detected the presence of Leptospira antigen during the bacteremia stage in all experimental animals. Besides detecting antigens in animals infected with homologous serovars, the sandwich dot-ELISA employing pooled capture and revealing antibodies also detected Leptospira in the group of animals infected separately with the serovars australis and icterohaemorrhagiae. Results showed PCR to be a reliable and rapid test for demonstration of Leptospira in the plasma samples. The rapid sandwich dot-ELISA appeared more advantageous over PCR in being simple, rapid, field based, and economical. This method shows better promise of being used as a bedside test for routine diagnostic purposes.

Among the many potential virulence factors of B. cereus , Haemolysin BL is a unique and potent three component pore forming toxin composed of a binding component, B, and two lytic components, L 1 and L 2 . Heterogeneity in nucleic acid and protein sequences of HBL components and problems during expression of L 1 and L 2 proteins in recombinant host due to their toxicity causes problems for development of specific detection systems based on PCR and Immunoassay, respectively. Commercially available kit (BCET RPLA, Oxoid) is useful for detection of L 2 component of HBL, but detection of only one component is insufficient to give comprehensive view on HBL toxin producing strains as some strains produced only one or two of the three HBL components. To address above mentioned problems, in this study, we cloned conserved domains of B, L 1 and L 2 components together as single fusion gene and expressed as recombinant multidomain chimeric protein in E. coli . The resultant protein having L 1 , B and L 2 components in the form of single protein had no toxicity towards E. coli as we followed truncated protein approach. The hyperimmune antisera raised in mice against r-chimeric protein reacted with all the three components of HBL toxin of B. cereus (ATCC 14579) and provided three reaction bands at ~ 40 kDa to ~ 50 kDa regions during Western blot analysis. The hyperimmune sera of r-chimeric protein also notably neutralized the hemolytic activity of native HBL toxin. These results demonstrated that the obtained chimeric protein is correct and retained the antigenicity of native HBL toxin components. Therefore, it has better application in the development of a comprehensive HBL detection immunoassay and may also be a potential candidate molecule for vaccine studies.

Epidemic leptospirosis is increasingly being reported from northern Kerala during the monsoon months. We investigated the risk factors for mortality during the 2002 epidemic.Three hundred and forty patients suspected to have leptospirosis during the epidemic were studied by clinical examination, laboratory investigations and Leptospira serology (microscopic agglutination test). Two hundred and eighty-two seropositive cases were analysed for the clinical and laboratory profile, and risk factors for mortality using univariate and logistic regression analysis.Of the 282 seropositive cases, 58.9% were men. No significant association with occupational risk factors was seen; 62.9% had wounds on the feet. The majority had Weil syndrome with hepatic (69.8%) and renal (56.3%) involvement. Thrombocytopenia (65.8%) was common. Transient hyperglycaemia was observed in 10.3% of cases. Pulmonary haemorrhage (4.7%) and meningism (4.3%) were less common. Jaundice occurred in 46% of cases in the first week. The mortality rate was 6.03%. Hyperkalaemia (OR= 27.3), meningism (OR= 10.6), oliguria (OR=8.2), haemoptysis (OR= 5.4), bilirubin15 mg/dl (OR= 5.4), disorientation (OR=5), tachycardia (OR=4.1) and muscle tenderness (p=0.03) were the predictors of high mortality in univariate analysis. Only involvement of the lung and central nervous system were significant predictors of death in logistic regression.Leptospirosis is no more a mere occupational hazard in Kerala. Early occurrence of complications such as hepatitis mandates caution in the primary care setting. Lung and central nervous system involvement are significant predictors of mortality.

A new dot-ELISA kit for detection of Brucella antibodies in human sera was developed and compared with that of serum agglutination test, Rose Bengal plate test, rapid slide agglutination and Coomb's antiglobulin test. Following testing of 120 human sera from suspected patients of occupational risk, 25 gave positive reaction in Rose Bengal plate test, 25 in rapid slide agglutination test, 26 in serum agglutination test, 27 in Coomb's antiglobulin test and 28 in dot-ELISA kit. Dot-ELISA kit picked up more positive than any other Serological test, indicating its superiority over the other laboratory tests for the diagnosis of brucellosis.

Monoclonal antibodies (MoAbs) were generated following immunization of BALB/c mice with protective antigen (PA) of B. anthracis. Five clones reactive to this protein were stabilized and preserved. These MoAbs could detect nanogram levels of PA when tested in ELISA. In Western blotting, they reacted with all PA preparations tested and no cross-reactivity was observed with lethal factor, edema factor of B. anthracis and with other organisms. These MoAbs could detect PA from 22 confirmed clinical isolates of B. anthracis on Western blotting and hold promise for direct detection of PA in clinical samples for diagnosing anthrax.

Anthrax has been reported from almost every country and India is endemic for this disease. There is considerable under reporting of the disease because of lack of microbiological facilities and diagnostic reagents. In India only conventional methods which have limitations, are being used to diagnose the disease. Hence the aim of this study was to isolate and purify protective antigen (PA) using different protocols and to use this PA for detection of anti-PA antibodies from sera samples.Protective antigen was isolated and purified from the Sterne strain of Bacillus anthracis. B. anthracis lacking pXO1 and pXO2 transformed with pYS5 (B. anthracis pYS5) and recombinant Escherichia coli transformed with pQE30 containing PA gene using hydroxyapatite (HA), Q-sepharose fast protein liquid chromatography (FPLC) and nickel-nitrilotriacetic acid (Ni-NTA) chromatographic methods, respectively. A mixture of PA and edema factor (EF) was injected subcutaneously into rabbits to test the biological activity of PA. The immunogenicity of PA was tested by inoculating the protein into rabbits along with adjuvant. Using this PA, 20 bovine sera samples (pre- and post-vaccinated) were tested by Western blotting (WB) for the presence of anti-PA antibodies.The 83 kDa PA protein was obtained from all the bacteria with the yields of 13, 50 and 9.0 mg/l from Sterne B. anthracis, B. anthracis pYS5 and recombinant Esch. coli, respectively. Formation of edematous ulcers at the site of PA+EF injection clearly confirmed the retention of biological activity of the proteins. Of the 10 post-vaccination sera tested, 9 showed clear positive by WB whereas none of the pre-vaccination sera showed the reaction.The purified PA preparations obtained in the present study may possibly be utilized for detection of anti-PA antibodies in the sera of anthrax patients for timely diagnosis of the disease and, might also be tested for their efficacy and use as human anthrax vaccine.

PCR has been reported for amplification of the 482 bp genus specific region in 23S rRNA gene of Leptospira species. The sequence of this region was analyzed for specific restriction sites that could have yielded digestion products expected to provide differentiating banding profile for the pathogenic and the saprophytic group of leptospires.Sixteen standard serovars of pathogenic group, two standard serovars of saprophytic group, 12 Leptospira isolates recovered from hospitalized patients with fever and jaundice or pyrexia of unknown origin and 23 isolates from different water sources were studied. Conventional tests, PCR methods and restriction digestion were used for confirming the identity of these isolates.All 12 isolates from patients and 1 from tap water source were identified as pathogenic and 22 isolates from water sources as saprophytic by the conventional tests and PCR. Of the 5 restriction endonuclease enzymes, viz, Apa I, Ban II, Hae III, Pst I and Sin I analyzed for digestion of PCR amplified 482 bp product, Apa I, Ban II, Pst I and Sin I provided fragments of different sizes providing distinct patterns for saprophytic and pathogenic leptospires.The identity of a strain to genus Leptospira could be confirmed by PCR amplification of 482 bp region of 23S rRNA and with further restriction digestion a clear distinction into pathogenic or saprophytic group was achieved with the use of any of these 4 restriction enzymes.

Monoclonal antibodies (MoAbs) were generated following immunization of Balb/C mice with adenovirus type 5 grown in Hep2 cell line. Six clones reactive to hexon antigen of the virus were stabilized, of which 4 had mu-heavy chain specificity and 2 were of gamma-heavy chain type. Three of the clones (ADV-1, ADV-3 and ADV-5) had a high ELISA reactivity to the hexon antigen but exhibited differential specificity to the adenovirus types tested. In Western blotting, ADV-1 and ADV-3 reacted with all the adenovirus types tested (types 3,4,5,7 and 8) with reactions at 116 kDa region (hexon antigen), in addition, ADV-3 also had reactivity at 80 kDa region, the penton antigen. Reactivity to these adenoviral types by the 2 MoAbs was demonstrable by dot ELISA. ADV-5 had a type specific reaction only to adenovirus type 5 in dot ELISA with specificity in the hexon region in Western blotting. The reactivity of these 3 clones was not observed to the normal Hep2 tissue culture antigens and to the 3 enteroviruses tested (polio, coxsackie A9 and echo 4).

A saprophytic Leptospira isolate recovered from tap water was utilized for serological testing. One hundred-twenty Serum samples comprising 55 cases from PUO/febrile jaundice and 65 samples from apparently healthy individuals were tested by MAT and HA using this environmental saprophytic strain and the results compared with that of Leptospira biflexa semaranga patoc, the standard saprophytic strain commonly employed for sero-diagnosis of leptospirosis. The MAT data showed 96.4 per cent correlation between the two strains. Similarly, the HA results were matching to the extent of 94.5 per cent. Results, therefore, suggest that local saprophytic Leptospira strain may serve as a substitute to serovar patoc for serodiagnosis of leptospirosis.

A serological survey was performed in groups of patients with active sputum smear-positive or smear-negative pulmonary tuberculosis, healthy household contacts, and controls. Sera were tested for titers of antibodies which bound to each of five purified mycobacterial antigens by enzyme immunoassay and for competition of binding to single epitopes, using six radiolabeled monoclonal antibodies directed toward corresponding molecules. The evaluation of diagnostic specificity was based on a positive score represented by titers above the cutoff point of 2 standard deviations above the mean titer of a control group. For smear-positive samples, the best sensitivity (83%) was achieved by exclusive use of the 38-kilodalton (kDa) antigen or its corresponding monoclonal antibodies. For smear-negative samples, levels of antibodies binding to the 19-kDa antigen showed a lower sensitivity of 62% compared with the control group or 38% compared with the contact group. Titers of antibody binding to the 14-kDa antigen were raised in Mycobacterium bovis BCG-vaccinated contacts, indicating that the greatest potential of this antigen may be in the detection of infection in a population for which tuberculin testing is unreliable. The results demonstrated the differing antibody responses to each of the tested antigens and distinct associations with the stage of infection or disease.

A dot-enzyme linked immunosorbent assay (dot-ELISA) for the detection of antibodies in bovine brucellosis using soluble antigens extracted from Brucella abortus S-99 is described in which antigen was deposited on a nitrocellulose sheet (0.5 x 0.5 cm) bound to a plastic strip and the unsaturated sites blocked by a solution of spray-dried milk powder. Sera were tested at dilutions of 1:800 and 1:1600, using rabbit anti-bovine-immunoglobulin or protein-A coupled to peroxidase as conjugates and diamino-benzidine as substrate. A positive reaction was clearly indicated by a brown dot on the nitrocellulose sheet. Using antigen-coated, pre-blocked strips, the test could be completed within 45 minutes.