Defining the landscape of patient harm after osteopathic manipulative treatment: synthesis of an adverse event model.

BACKGROUND: In the United States, osteopathic manipulative treatment (OMT), is a popular complementary physical health approach for the treatment of neuromusculoskeletal disorders. However, post-OMT adverse events (AEs) are poorly defined in terms of frequency, severity, and temporal evolution. To date, no benchmark for patient safety exists. To improve understanding in this field, we set out to model the landscape of patient harm after OMT. METHODS: We conducted a comprehensive search of all available primary clinical research studies reporting on the occurrence of post-OMT AEs in nonpregnant, adult outpatients treated by an osteopathic physician in the United States. The methodology of eligible studies was then reviewed to select those containing the minimum required dataset to model the post-OMT AEs. The minimum required dataset consisted of four model parameters: 'post-OMT interval', 'OMT encounters with post-OMT interval assessment', 'AEs preceded by an OMT encounter', and 'AE severity.' We used the dataset extracted from selected studies to calculate a patient safety benchmark defined as the incidence rate of AEs per 100 post-OMT interval-days. RESULTS: From 212 manuscripts that we identified, 118 primary clinical research studies were assessed for eligibility. A total of 23 studies met inclusion criteria for methodological review, of which 13 studies passed and were selected for modeling. Mild AEs were the most frequent, accounting for n = 161/165 (98%) of total AEs observed in the literature. The cumulative incidence of mild AEs was also significantly greater (P = 0.01) than both moderate and severe grades. The benchmark incidence rate was 1.0 AEs per 100 post-OMT interval-days. CONCLUSIONS: The majority of post-OMT AEs observed in the primary clinical literature were of mild severity. Modeling of the combined dataset on post-OMT AEs allowed for the derivation of a patient safety benchmark that, to date, has not been established in the field of osteopathic manipulative medicine. Additional research is needed to improve model resolution during the post-OMT period. This work conceptualized a model for identifying and grading post-OMT AEs, which should facilitate future comparisons between institutions in order to continually improve patient safety standards in the field of osteopathic manipulative medicine.

In vitro selection and characterization of ssDNA aptamers by cross-over SELEX and its application for detection of S. Typhimurium.

S. Typhimurium is the most common food-borne pathogen frequently encountered in contaminated food and poses a major threat to public health, animals, and the food industry worldwide. Early and sensitive detection is the prime step to ensuring the microbiological quality of fresh and processed food. We used the cross-over SELEX strategy to select ssDNA aptamers against OM usher protein, FimD that could be employed in the S. Typhimurium detection assay. Altogether, 7 rounds of protein-SELEX, 3 rounds of intermittent counter-SELEX, and 4 rounds of whole bacterial cell-SELEX were undertaken for specific aptamer selection. The aptamer specificity was achieved by performing counter-SELEX with non-target bacterial cells. The enriched pool of aptamers obtained post SELEX process was cloned, sequenced, and further the specificity and affinity were characterized by flow cytometry. The aptamers DFRM-3 and DFRM-10 were specific and showed relatively high binding affinity towards S. Typhimurium with an apparent dissociation constant (Kd value) of 76 nM and 90.01 nM respectively. A dual aptamer-based sandwich assay was developed using biotinylated DFRM-3 aptamer for magnetic capture and AlexaFluor-488 labeled DFRM-10 aptamer for detection of S. Typhimurium. The limit of detection (LOD) of S. Typhimurium by the developed assay was found to be 102 CFU/ml in pure culture and 103 CFU/ml in the artificially contaminated chicken meat samples.

Oral co-administration of bivalent protein r-BL with U-Omp19 elicits mucosal immune responses and reduces S. Typhimurium shedding in BALB/c mice.

The increase in international food trade and travel has dramatically increased the global incidences of Salmonellosis. In the light of widespread resistance to frontline antibiotics, oral vaccines remain the most reliable alternative. In this study, the fusion protein, r-BL was rationally constructed by splicing the Salmonella Typhimurium sseB and ompL genes through G4S linker by over-lap extension PCR. The oral coadministration of r-BL with B. abortus U-Omp19 protein with known protease inhibitor activity resulted in significant increase of mucosal IgA titres to antilog 4.5051 (p < 0.0001) and 4.806 (p < 0.0001) in the fecal samples and intestinal washes respectively. Antibody isotyping of the intestinal washes demonstrated increase in mucosal IgM, IgG1 and IgG2a isotypes also and demonstrated a significant reduction in fecal shedding of S. Typhimurium in challenge study. The r-BL + U-Omp19 treated mice demonstrated a complete termination of Salmonella fecal shedding by the 12th day of challenge as compared to other study groups. In summary, the bivalent protein r-BL when administered with the mucosal adjuvant U-Omp19 was successful in triggering mucosal arm of the immune system which forms the first line of defence in combating the infections caused by the enteric pathogen like Salmonella.

Molecular characterization of B. anthracis isolates from the anthrax outbreak among cattle in Karnataka, India.

BACKGROUND: Anthrax, a zoonotic disease is caused by the Gram positive bacterium Bacillus anthracis. During January 2013, an anthrax outbreak among cattle was reported in Gundlupet Taluk, neighboring Bandipur National Park and tiger reserve, India. The present study aims at the molecular identification and characterization of 12 B. anthracis isolates from this outbreak by 16S rRNA gene sequencing, screening B. anthracis specific prophages and chromosomal markers, protective antigen (pag) gene and canonical single nucleotide polymorphism (canSNP) analysis to subtype the isolates into one of the twelve globally identified clonal sub-lineages of B. anthracis. RESULTS: These isolates had identical 16S rDNA nucleotide sequences with B. anthracis specific dual peaks showing mixed base pair R (G/A) at position 1139 with visual inspection while the automated basecaller software indicated a G. Alternatively the nucleotide A at 1146 position was indicative of the 16S rDNA type 7. Multiple sequence alignment with additional 170 (16S rDNA) sequences of B. cereus sensu lato group from GenBank database revealed 28 new 16S types in addition to eleven 16S types reported earlier. The twelve B. anthracis isolates were found to harbor the four B. anthracis specific prophages (lambdaBa01, lambdaBa02, lambdaBa03, and lambdaBa04) along with its four specific loci markers (dhp 61.183, dhp 77.002, dhp 73.019, and dhp 73.017). The pag gene sequencing identified the isolates as protective antigen (PA) genotype I with phenylalanine-proline-alanine phenotype (FPA phenotype). However, sequence clustering with additional 34 pag sequences from GenBank revealed two additional missense mutations at nucleotide positions 196 bp and 869 bp of the 2294 bp pag sequence among the 5 B. cereus strains with pXO1 like plasmids. The canSNP analysis showed that the isolates belong to A.Br.Aust94 sub-lineage that is distributed geographically in countries of Asia, Africa, Europe and Australia. CONCLUSIONS: The analysis of 16S rDNA sequences reiterated the earlier findings that visual inspection of electropherogram for position 1139 having nucleotide R could be used for B. anthracis identification and not the consensus sequence from base caller. The canSNP results indicated that the anthrax outbreak among cattle was caused by B. anthracis of A.Br.Aust94 sub-lineage.

Functional characterization and evaluation of protective efficacy of EA752-862 monoclonal antibody against B. anthracis vegetative cell and spores.

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.

Immunogenicity and protective efficacy of Burkholderia pseudomallei BLF1-N and BLF1-C terminal domains against BLF1 toxin.

Burkholderia lethal factor 1 (BLF1), a glutamine deamidase, is a key virulence factor that plays significant role in B. pseudomallei pathogenesis. To elucidate the BLF1 immunological responses, two truncated BLF1 structural units, BLF1-C (90-211 amino acids) with structural similarity to T. maritima Chemoreceptor glutamine deamidase (CheD) protein, and BLF1-N (1-89 amino acids) disparate to CheD were identified from the 23 kDa BLF1 protein. Both the components were devoid of toxicity in mice and elicited an antibody titer of 1:16,000 that reacted with the respective truncated proteins and BLF1. A549 cell lines supplemented with anti BLF1-N and BLF1-C antibodies exhibited 73.47% and 83.24% survival when treated with BLF1 toxin. Passive i.p. transfer with antibodies elicited by BLF1-C that contained LSGC active site resulted in 80% protection while anti BLF1-N (devoid of LSGC) antibodies provided 51.4% protection, establishing the role of BLF1-N terminal also in deamidase action. The truncated proteins also elicited cell mediated immune responses through proliferation of CD4+ T cells, IFN-γ and IL-4 cytokines but with bias towards Th2 subsets. BLF1-C and BLF1-N immunization resulted in 80% and 60% active protection when challenged with BLF1 toxin while the sham immunized mice exhibited severe histopathological changes like necrosis in liver, lung, spleen and kidney similar to that observed in melioidosis and were killed within 7 days post challenge. The higher level of active and passive protection by BLF1-C protein could be attributed to the comparatively higher level of immune responses and inclusion of LSGC residues.

A Bivalent Protein r-PAbxpB Comprising PA Domain IV and Exosporium Protein BxpB Confers Protection Against B. anthracis Spores and Toxin.

Anthrax vaccines primarily relying only on protective antigen (PA), the cell binding component in anthrax toxins provide incomplete protection when challenged with spores of virulent encapsulated Bacillus anthracis strains. Alternatively, formaldehyde inactivated spores (FIS) or recombinant spore components generate anti-spore immune responses that inhibit the early stages of infection and augment the PA protective efficacy. In the present study domain IV of PA was spliced with exosporium antigen BxpB via a flexible G4S linker to generate a single functional antigen r-PAbxpB that was further assessed for its protective efficacy against anthrax toxins and spore infection. Immunization of mice with r-PAbxpB elicited significantly high titer antibodies comprising IgG1:IgG2a isotypes in 1:1 ratio, balanced up-regulation of both Th1 (IL2, IL12, IFN-γ) and Th2 (IL4, IL5, IL10) cytokines and high frequencies of CD4+ and CD8+ T cell subsets. The anti-r-PAbxpB antibodies significantly enhanced spore phagocytosis, and killing within macrophages; inhibited their germination to vegetative cells and completely neutralized the anthrax toxins as evidenced by the 100% protection in passive transfer studies. Active immunization with r-PAbxpB provided 100 and 83.3% protection in mice I.P. challenged with 5 × LD100 LD of toxins and 5 × 104 cfu/ml Ames spores, respectively while the sham immunized group succumbed to infection in 48 h. Therefore, the ability of r-PAbxpB to generate protective immune responses against both spores and toxin and provide significant protection suggests it as an efficient vaccine candidate against B. anthracis infection.

A sandwich duplex immuno PCR for rapid and sensitive identification of Clostridium perfringens alpha and enterotoxin.

The prevalence and lethality associated with C. perfringens alpha (CPA) and enterotoxin (CPE) toxaemia necessitate the need for rapid and definitive detection systems to initiate management measures. In the present study, a sandwich duplex immuno-capture PCR (SD-IPCR) was developed by employing IgY antibodies against a bivalent protein r-Cpae derived from CPA and CPE for antigen capture and reporter antibodies against truncated CPA or CPE conjugated to oligomers of distinguishable size for antigen revealing and signal amplification. The avian immunoglobulin's (IgY) were devoid of reactivity with S. aureus protein A (SpA), a commensal that often co-exists with C. perfringens. The assay was specific, had a detection limit (LOD) of 1 pg/ml for both CPA and CPE in PBS and improved the LOD by 104 folds compared to an analogous sandwich ELISA with same set of antibodies. In spiking studies, a ten-fold reduction in LOD was observed in case of intestinal tissue samples (10 pg/ml) however, no change in LOD was observed when SD-IPCR was applied on to faecal, serum or muscle tissue samples. Of the 136 natural samples examined, the SD-IPCR could detect CPA and CPE in 29.4% and 35.3% samples, while the sandwich ELISAs could detect the same in 25.7% and 25% samples respectively owing to the relatively lesser sensitivity. The LOD and specificity of the SD-IPCR demonstrates its applicability as an efficient and rapid platform for direct detection CPA and CPE from diverse samples matrices in clinical microbiological and meat testing laboratories.

Recombinant outer membrane protein 25c from Brucella abortus induces Th1 and Th2 mediated protection against Brucella abortus infection in mouse model.

Development of a safe and efficacious vaccine for brucellosis is a long standing challenge for scientists. Recognizing potential antigens towards developing vaccine candidate is crucial. Omp25c, a porin protein of Brucella, is a paralog of two previously identified promising vaccine candidates namely, Omp25 and Omp31, with notable sequence identity. Also, Omp25c is conserved in all major Brucella species. This highlights the possibility of employing this protein in multivalent subunit vaccine based approach of Brucella management. In this study, we were interested in examining the immunogenicity and protective efficacy of Omp25c against Brucella infections. Recombinant unlipidated form of this antigen (rOmp25c) produced, upon intraperitoneal immunization in BALB/c mice along with Freund's adjuvant, was confirmed to be highly immunogenic; leading to high IgG antibody titers during the study duration. The IgG2a/IgG2b ratio of anti-rOmp25c antibodies revealed elicitation of Th2 based humoral immunity. Lymphocyte proliferation study divulged induction of specific memory response and secretion of both Th1-type (IFN-γ, GM-CSF and TNF-α) and Th2-type cytokine (IL-5) from restimulated splenocytes of rOmp25c immunized mice. CD4 T-cell subpopulation was comparatively increased than total B cell subpopulation in case of immunized mice, indicating the induction of strong cell-mediated (Th1 biased) immunity than humoral (Th2) immunity. The collective Th1 plus Th2 immune response specific to rOmp25c could be the reason for protection against Brucella challenge observed in mice groups that was comparable with S19 vaccine strain. Thus, the study encourages rOmp25c as a potent candidate vaccine against brucellosis.

Highly Sensitive Colorimetric Biosensor for Staphylococcal Enterotoxin B by a Label-Free Aptamer and Gold Nanoparticles.

A simple, sensitive and selective colorimetric biosensor for the detection of Staphylococcal enterotoxin B (SEB) was developed using SEB-binding aptamer (SEB2) as recognition element and unmodified gold nanoparticles (AuNPs) as colorimetric probes. The assay is based on color change from red to purple due to conformational change of aptamer in the presence of SEB, and the phenomenon of salt-induced AuNPs aggregation which could be monitored by naked eye or UV-vis spectrometer. Results showed that the AuNPs can effectively differentiate the SEB induced conformational change of the aptamer in the presence of a given high salt concentration. A linear response in the range of 50 µg/mL to 0.5 ng/mL of SEB concentration was obtained. The assay was highly specific to SEB as compared to other related toxins. The limit of detection (LOD) of SEB achieved within few minutes was 50 ng/mL visually and spectrometric method improved it to 0.5 ng/mL. Robustness of the assay was tested in artificially spiked milk samples and cross-checked using in house developed sandwich ELISA (IgY as capturing and SEB specific monoclonal as revealing antibody) and PCR. This colorimetric assay could be a suitable alternative over existing methods during biological emergencies due to its simplicity, sensitive and cost effectiveness.

Colorimetric DNAzyme Biosensor for Convenience Detection of Enterotoxin B Harboring Staphylococcus aureus from Food Samples.

In the present study, a colorimetric DNAzymes biosensor strategy was devised in combination with immunomagnetic separation for rapid and easy detection of enterotoxin B harboring Staphylococcus aureus from food and clinical samples. The method employs immunocapture of S. aureus and amplification of seb gene by DNAzyme complementary sequence integrated forward primer and with specific reverse primer. The DNAzyme sequence integrated dsDNA PCR products when treated with hemin and TMB (3,3',5,5'-tetramethylbenzidine) in the presence of H2O2 produce colorimetric signal. A linear relationship of optical signal with the initial template of seb was obtained which could be monitored by visually or spectrophotrometrically for qualitative and quantitative detection. The limit of detection for the assay was approximately 102 CFU/mL of seb gene harboring target. This method is convenient compared to gel based and ELISA systems. Further, spiking studies and analysis on natural samples emphasized the robustness and applicability of developed method. Altogether, the established assay could be a reliable alternative, low-cost, viable detection tool for the routine investigation of seb from food and clinical sources.

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. The main aim of the present study was to simplify the utility of a pre-optimized multiplex PCR format that was developed to detect toxigenic strains of Staphylococcus aureus by providing stable, pre-mixed, and ready-to-use master mix in a lyophilized formulation. METHODS: Master mix containing all reagents except the template was lyophilized in the presence of an excipient lyoprotectant to achieve long-term stability without altering the sensitivity, specificity and PCR performance. Bromophenol blue was also included in the master mix to reduce the risk of external contamination during gel loading. The stability of lyophilized master mix was analyzed at different temperatures. The PCR performance was also examined after exposure of master mix to notable temperature fluctuations during transportation. RESULTS: The shelf-life of lyophilized master mix was estimated to be 1.5 months at ambient temperature and 6 months at 4°C. Stability was unaffected by temperature fluctuations during transportation even in cold-chain-free conditions, thus reducing the cost required for cold storage. CONCLUSION: The sensitive, cost-effective, ready-to-use, and ambient temperature stable formulation could be implemented as a detection tool in food analysis and diagnostic laboratories and hospitals and for on-field application outside the laboratories, as well as for detection of toxigenic strains of S. aureus.

Molecular characterization and phylogenetic analysis of Clostridium perfringens from animals and their environments by cpn60 UT sequencing analysis.

Clostridium perfringens an ubiquitous environmental bacterium causes major food borne illnesses, digestive diseases and several soft tissue infections in humans and animals. In the present study, toxin typing of 91 C. perfringens isolates from animals with enteric diseases and their environments revealed the presence of type A and C strains. Enterotoxin gene (cpe), responsible for majority of the food poisoning incidences in humans and enteric infections in animals was present in 60.43 % of the isolates of which 76.3% and 23. 36% were chromosomal and plasmid borne respectively. Neighbour-joining tree inferred from cpn60 UT nucleotide sequences could differentiate the cpe+ve isolates from the cpe-ve isolates, provide clear distinction between the cpe-IS1470 and cpe-IS1151 genotypes and segregate type A and C strains in separate clusters. The present study is the first report on the utilization of cpn60 UT region for C. perfringens phylogeny analysis and demonstrates that cpn60 UT analysis alone, to a greater extent can be a simple, rapid and efficient method for differentiating between cpe+ve and cpe-ve strains or toxin types. The cpb2 gene was observed among 30 isolates of which 16.6% were from porcine sources while the rest were of non-porcine and environmental origin. The cpb2 sequences obtained in the present study though similar among them were diverse both from the consensus and atypical cpb2 sequences reported globally and formed a separate cluster. The study thus reports of novel cpb2 gene variant and warrants its characterization through further studies.

Capture and detection of Staphylococcus aureus with dual labeled aptamers to cell surface components.

In the present study, a high throughput whole cell SELEX method has been applied successfully in selecting specific aptamers against whole cells of Staphylococcus aureus, a potent food poisoning bacterium. A total ten rounds of SELEX and three rounds of intermittent counter SELEX, was performed to obtain specific aptamers. Obtained oligonucleotide pool were cloned, sequenced and then grouped into different families based on their primary sequence homology and secondary structure similarity. FITC labeled sequences from different families were selected for further characterization via flow cytometry analysis. The dissociation constant (Kd) values of specific and higher binders ranged from 34 to 128nM. Binding assays to assess the selectivity of aptamer RAB10, RAB 20, RAB 28 and RAB 35 demonstrated high affinity against S. aureus and low binding affinity for other bacteria. To demonstrate the potential use of the aptamer a sensitive dual labeled sandwich detection system was developed using aptamer RAB10 and RAB 35 with a detection limit of 102CFU/mL. Furthermore detection from mixed cell population and spiked sample emphasized the robustness as well as applicability of the developed method. Altogether, the established assay could be a reliable detection tool for the routine investigation of Staphylococcus aureus in samples from food and clinical sources.

Immuno Affinity SELEX for Simple, Rapid, and Cost-Effective Aptamer Enrichment and Identification against Aflatoxin B1.

Aflatoxins are naturally occurring mycotoxins that contaminate food and agro commodities, leading to acute and chronic health conditions in human and animals. In the present work, an attempt was made to generate high-affinity single stranded DNA aptamers that specifically bind to Aflatoxin B1 (AFB1) by a modified Systemic Evolution of Ligands by Exponential Enrichment (SELEX) procedure with the aid of Immunoaffinity columns. Ten rounds of SELEX and alternating three counter SELEX rounds with a cocktail of related and other mycotoxins were performed to enhance the specificity. Resultant 105 aptamers were clustered into 12 groups according to their primary sequence homology. Candidates with lowest Gibbs free energy (dG value) and unique stem loop structures were selected for further characterization. Aptamers, AFLA5, AFLA53, and AFLA71 exhibiting lower Kd values (50.45 ± 11.06, 48.29 ± 9.45, and 85.02 ± 25.74 nM) were chosen for development of ELONA and determination of purification ability of toxin. The detection limit (LOD) of AFLA5 and AFLA71 was 20 and 40 ng/ml, respectively. HPLC analysis implied that selected aptamers were able to recover and quantify 82.2 to 96.21% (LOQ - 53.74 ng) and 78.3 to 94.22% (LOQ - 66.75 ng) of AFB1 from spiked corn samples, respectively. These findings indicate, immunoaffinity based SELEX can pave an alternative approach to screen aptamers against mycotoxin detection and purification.

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. In this context, the present study involved development of a sandwich ELISA immunoassay for the specific detection of "non-classical" enterotoxin G (SEG). The toxin belongs to enterotoxin gene cluster (egc) which comprises a bunch of five toxin genes that are known to co-express. Thus, the developed assay might indirectly speculate the presence of other toxins in the cluster. The efficiency of ELISA was compared with PCR analysis where all strains possessing seg were found positive for toxin production. Additionally, analogous to other studies which reported the co-occurrence of seg and sei, the PCR analysis accomplished in the study evinced the same. The sandwich format allowed sensitive detection with a detection limit of 1ng/mL. High specificity was achieved in presence of non-target protein as well as bacteria. Likewise, staphylococcal protein A (SpA) interference that is inevitably associated with immunoassays was eliminated by implementation of anti-SEG IgY in our study. Consequently, chicken IgY were used to capture target antigen in developed sandwich ELISA. Further, spiking studies and analysis on natural samples emphasized the robustness as well as applicability of developed method. Altogether, the established assay could be a reliable detection tool for the routine investigation of SEG as well as to predict other egc toxins in samples from food and clinical sources.

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. Nucleotide sequences of all three subtypes were analyzed using Clustal Omega program and the region with significant sequence variation/heterogeneity (where utmost SNPs were closely located and accessible for RT-PCR) was selected for amplification by designing a single primer pair that could amplify all three subtypes. In spite of same amplicon size, entC1 showed distinct melt peak at 76 °C. However, due to high similarity between entC2 and entC3, the developed format was deficient to discriminate between them and both showed melt peak at 82 °C. Reliability of developed RT-PCR was evaluated using various naturally contaminated samples and 91 food and clinical Staphylococcus aureus isolates where satisfactory results were obtained in comparison with commercial immunoassay kit and conventional PCRs using validated primers. To the best of our knowledge, this is the first method being reported to differentiate entC1 from entC2/entC3 using single primer pair which is unachievable by conventional PCR due to same amplicon size. As benefits, the method is sensitive, rapid, and inexpensive with no requirement of fluorescent probes, multiple primers, and post-PCR procedures. Thus, the assay might find its utility as a detection tool in epidemiological survey of foodborne outbreaks for simultaneous identification and differentiation of entC1 from entC2/entC3.

Generation and characterization of recombinant bivalent fusion protein r-Cpib for immunotherapy against Clostridium perfringens beta and iota toxemia.

Clostridium perfringens beta (CPB) and iota (CPI) toxaemias result in some of the most lethal forms of haemorrhagic and necrotic enteritis and sudden death syndrome affecting especially neonates. While CPB enterotoxemia is one of the most common forms of clostridial enterotoxemia, CPI enterotoxemia though putatively considered to be rare is an emerging cause of concern. The similarities in clinical manifestation, gross and histopathology findings of both types of toxaemias coupled to the infrequency of CPI toxaemia might lead to symptomatic misidentification with Type C resulting in therapeutic failure due to habitual administration of CPB anti-toxin which is ineffective against CPI. Therefore in the present study, to generate a composite anti-toxin capable of neutralizing both toxaemias, a novel bivalent chimera r-Cpib was constructed by splicing the non-toxic C terminal binding regions of CPB and CPI, via a flexible glycine linker (G4S) by overlap-extension PCR. The fusion protein was characterized for its therapeutic abilities toward CPI and CPB toxin neutralizations. The r-Cpib was found to be non-toxic and could competitively inhibit binding of CPB to host cell receptors thereby reducing its cytotoxicity. Immunization of mice with r-Cpib generated specific antibodies capable of neutralizing the above toxaemias both in vitro and in vivo. Caco-2 cells exposed to a mixture of anti-r-Cpib sera and native CPI or CPB, displayed significantly superior protection against the respective toxins while passive challenge of mice with a similar mixture resulted in 83 and 91% protection against CPI and CPB respectively. Alternatively, mice exposed to a mixture of sham sera and native toxins died within 2-3 days. This work thus demonstrates r-Cpib as a novel bivalent fusion protein capable of efficient immunotherapy against C. perfringens CPI and CPB toxaemia.

Post-Genomic Analysis of Members of the Family Vibrionaceae.

Similar to other genera and species of bacteria, whole genomic sequencing has revolutionized how we think about and address questions of basic Vibrio biology. In this review we examined 36 completely sequenced and annotated members of the Vibrionaceae family, encompassing 12 different species of the genera Vibrio, Aliivibrio, and Photobacterium. We reconstructed the phylogenetic relationships among representatives of this group of bacteria by using three housekeeping genes and 16S rRNA sequences. With an evolutionary framework in place, we describe the occurrence and distribution of primary and alternative sigma factors, global regulators present in all bacteria. Among Vibrio we show that the number and function of many of these sigma factors differs from species to species. We also describe the role of the Vibrio-specific regulator ToxRS in fitness and survival. Examination of the biochemical capabilities was and still is the foundation of classifying and identifying new Vibrio species. Using comparative genomics, we examine the distribution of carbon utilization patterns among Vibrio species as a possible marker for understanding bacteria-host interactions. Finally, we discuss the significant role that horizontal gene transfer, specifically, the distribution and structure of integrons, has played in Vibrio evolution.

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. The rL/VvhA protein was found to be non-toxic with no significant hemolytic or cytotoxic effects. Intraperitoneal (I.P.) immunization of BALB/c mice with rL/VvhA protein elicited significantly higher specific serum antibody titer with mixed Th1/Th2 mediated immune responses. HeLa cell monolayer supplemented with anti-rL/VvhA antibodies were effectively protected (viability 86.69%) against lethal 5 LD50 toxin challenge. An effective in vitro proliferation of lymphocyte was observed upon re-stimulation of rL/VvhA primed splenocytes with formalin inactivated VvhA toxin (fVvhA). Co-expression of Th1/Th2 polarized cytokines (IFN-γ, IL-12 and IL-4), were seen in the cell culture supernatant. In contrast to sham immunized mice, rL/VvhA immunized mice demonstrated significant protection (90% survival) against native toxin challenge in vitro and in vivo infection models. These results suggested leukocidin domain of the VvhA toxin as protective immunogen for possible protection against V. vulnificus VvhA.