Studies on development and storage stability of dehydrated pumpkin based instant soup mix

The study was carried out to develop and standardize Instant Soup Mix (ISM) from dehydrated pumpkin powder and to evaluate nutritional (moisture, sugars, protein, ?-carotene, fat, fibre and water activity) and sensory qualities (colour, texture, flavour and overall acceptability) for determining its shelf-life during a period of six month. It was packed in aluminium laminated pouches and was analyzed periodically for changes in quality. Among various recipes optimized for the development of soup mix, soup mix containing 20g pumpkin powder, 5g moong dhal, 15g tomato powder, 11.4g spices (salt and black pepper) and condiments (onion, garlic and ginger powder), 2g dried pea, 2g dried spinach, and 2g dried carrot was selected as base recipe for addition of different starch source viz. rice, corn and potato @ 10 g. From the nutritional analysis, it was observed that corn based Instant Soup Mix had higher ?-carotene (7.01 mg/100 g) and protein (12.65 %) content, while fibre (2.09 %) was higher in soup mix containing rice starch. It was observed that on the basis of sensory evaluation corn based soup mix was more acceptable. Therefore, results of nutrition and sensory evaluation indicated that a good quality ISM can be prepared by using corn starch. During the six month of storage, there was about 5.49 per cent increase in moisture, 3.16 and 5.27 per cent decrease in protein and ?-carotene, respectively, along with slight losses in total sugars, fat and sensory quality. Further, the product was stable for 6 months under ambient condition

JJo, a recombinant dimer of conformationally restricted peptide elicits protective response against Group A Streptococcus (GAS) isolates from a GAS-endemic region

A peptide (J14) containing conformationally restricted epitopes from the M protein of Group A Streptococcus (GAS) is capable of eliciting protective immune response against GAS infection. However, the protective response may be lost possibly due to its weak secondary-structure when the antigen is fused with other antigens in a recombinant polyepitope vaccine construct. We previously showed that JJo, a conformationally stabilized derivative of dimeric J14, overcomes this problem. We now show that anti JJo antibodies react with diverse GAS isolates found in the Indian sub-continent and that these antibodies are opsonic for GAS. The GAS strains used in this study were isolated from throat and skin swabs from Mumbai, Chennai and Vellore. Sera from mice immunized with recombinant JJo peptide were tested by ELISA, immunofluorescence, flow-cytometry, indirect bactericidal assay and mouse challenge assays to determine specific immunogenicity, opsonic functions and protection against an Indian isolate. We propose that JJo is a robust antigen suitable for inclusion in recombinant multi-epitope vaccines which are potentially affordable option for the pediatric population of developing countries

SARS Coronavirus-2 microneutralisation and commercial serological assays correlated closely for some but not all enzyme immunoassays

Serological testing for SARS-CoV-2-specific antibodies provides important research and diagnostic information relating to COVID-19 prevalence, incidence and host immune response. A greater understanding of the relationship between functionally neutralising antibodies detected using microneutralisation assays and binding antibodies detected using scalable enzyme immunoassays (EIA) is needed in order to address protective immunity post-infection or vaccination, and assess EIA suitability as a surrogate test for screening of convalescent plasma donors. We assessed whether neutralising antibody titres correlated with signal cut-off ratios in five commercially available EIAs, and one in-house assay based on expressed spike protein targets. Sera from recovered patients or convalescent plasma donors who reported laboratory-confirmed SARS-CoV-2 infection (n = 200), and negative control sera collected prior to the COVID-19 pandemic (n = 100), were assessed in parallel. Performance was assessed by calculating EIA sensitivity and specificity with reference to microneutralisation. Neutralising antibodies were detected in 166 (83%) samples. Compared with this, the most sensitive EIAs were the Cobas Elecsys Anti-SARS-CoV-2 (98%) and Vitros Immunodiagnostic Anti-SARS-CoV-2 (100%), which detect total antibody targeting the N and S1 antigens, respectively. The assay with the best quantitative relationship with microneutralisation was the Euroimmun IgG. These results suggest the marker used (total Ab vs. IgG vs. IgA) and the target antigen are important determinants of assay performance. The strong correlation between microneutralisation and some commercially available assays demonstrates their potential for clinical and research use in assessing protection following infection or vaccination, and use as a surrogate test to assess donor suitability for convalescent plasma donation

Characteristion of virulence genes (Shigatoxins 1 and 2, and intimin) in Shiga toxin producing Escherichia coli (STEC) of ovine origin and an assessment of the role of these STEC in human pathogenesis

Enterohaemorrhagic Escherichia coli (EHEC), represent a subset of Shiga toxin-producing Escherichia coli (STEC), which cause diarrhoea, haemorrhagic colitis (HC) and haemorrhagic uraemic syndrome (HUS) in humans worldwide. STEC are part of the normal gastro-intestinal flora of ruminants, especially cattle and sheep, and commonly enter the food chain by the faecal contamination of carcasses at slaughter. Most studies of STEC in ruminants have focused on the bovine reservoir. This study examines the genetic characteristics of key virulence genes (stXl, SlX2 and eae) in STEC of ovine origin and a subset of STEC of human origin that possess same serotypes as those commonly recovered from sheep but not cattle. Shiga toxins 1 and 2 are essential virulence attributes of STEC required for the induction of HUS and HC, and may play an important role in infections leading to milder gastrointestinal diseases such as diarrhoea. In this study a PCR restriction fragment length polymorphism (PCR-RFLP) assay was developed that differentiates SIXlc from other common SIXl subtypes. The SIXlc was the most common subtype identified (133 of 203; 65.5%) in STEC of ovine origin and was associated with 40 serotypes. Some serotypes, particularly 075:H8 (14 of 21 isolates) were shown to simultaneously possess both common SIXl and SIXlc subtypes. Furthermore, STEC isolates of serotypes commonly found in sheep and recovered from both symptomatic and healthy humans also contained SIXlc (12 of 34; 35.3%). These data suggest that these 12 isolates from humans may have had an ovine origin. The predominance of SIXlc among STEC isolated from ovine faeces suggests that the bacteriophage encoding this subtype preferentially inhabits the gastro-intestinal tract of sheep and/or shows a host range restricted to E. coli serotypes that colonise sheep but not cattle. For the genetic characterisation of stX2 variants several previously published PCR-RFLP assays were used. The stX2d (stX2d-OuntJOIlllOX3a) subtypes, representing 13 serotypes, were almost exclusively identified (141 of 146; 96.6%) in STEC recovered from ovine faeces. These subtypes were predominantly associated with serotypes (05:H-, 091:H-, 0123 :Hand 0128:H2) commonly recovered from healthy sheep and rarely from healthy cattle. Furthermore, STEC isolates with serotypes predominantly associated with sheep and recovered from both symptomatic and healthy humans also contained these stX2d (11 of 21 ; 52.4%) subtypes suggesting that they probably had an ovine origin. A single isolate of serotype 091 :H21 recovered from a human with diarrhoea simultaneously possessed two stX2 variants (stX2 and stx2vhb). Ovine STEC possessing stXlc and StX2d subtypes were never associated with the typical EHEC serogroups 026, 0103 and 0157. Recent clinical studies have demonstrated that StXlc and StX2d subtypes are rarely associated with STEC recovered from patients with HUS or He. However, examples of human STEC isolates of serotypes 05:H- and OX3:H8 associated with HUS that do possess this combination of virulence factor subtypes were identified in this study. Intimin (lnt), encoded by the eae gene, is a well characterised outer membrane protein adhesin involved in the intimate adherence of STEC to the host epithelial membrane leading to the formation of the characteristic attaching and effacing lesions. A PCR-RFLP subtyping assay capable of simultaneously differentiating all 10 recognised intimin subtypes is reported for the first time in this study. This assay was also used to identify and type two previously unreported intimin subtypes identified as Int-£2 and Int-t2. Int-~ was the most commonly identified intimin subtype (58 of 153; 13.7%) and was associated with the greatest number ofserotypes (n=16), followed by Int-s (21 of 153; 13.7%; 7 serotypes), Int-£l (18 of 153; 11.7%; 5 serotypes), Int-y(13 of 153; 8.5%; 6 serotypes) and Int-8 (6 of 153; 4%; 5 serotypes). Intimin subtypes aI, a2, A, 8 and t1 were infrequently identified. None of the 153 eae-containing isolates simultaneously harboured more than one inti min subtype. However, intimin genes from 19 of 153 (12.4%) ovine E. coli isolates representing 16 different serotypes were untypeable suggesting an even greater variety of intimin subtypes in STEC derived from ovine faeces. Phylogenetic analyses of the C-terminal 280 amino acids (lnt280) using the Phylip package confirmed the previous division of the family of intimin proteins into the six distinct clusters represented by subtypes a, ~, y, 8, £ and 8. In addition, the inti min subtypes S, t and A submitted to GenBank, also resolved as distinct groups but their relationship to other intimin subtypes remain unclear. The clonal relationships of ovine and human STEC isolates of serotypes (05:H-, 091 :Hand 0128:H2) were assessed by pulsed-field gel electrophoresis (PFGE) and repetitive sequence based PCR (REP and ERIC PCR). PFGE differentiated 11, 13 and 13 groups among the sixteen 05:H-, seventeen 091:H- and eighteen 0128:H2 strains respectively suggesting that STEC with these serotypes represent genetically heterogenous groups. There were no matches observed in the PFGE profiles between strains of STEC isolated from sheep and those isolated from humans. However, one 091 :H- isolate obtained from mettwurst sausage and an 091 :H- isolate from a patient with diarrhoea produced identical PFGE patterns and both the isolates were associated with a food poisoning outbreak in South Australia. This suggests that PFGE may be a useful epidemiological tool for tracing non-O 157 STEC infections. Genetic fingerprinting using REP and ERIC PCR showed a low discriminative ability for these isolates and appears unsuitable for this purpose. Serotypes 05:H-, 091:H-, 0103:H2, 0123:H-, 0128:H2, 0157:H-1H7 and OX3:H8 have been isolated from sheep faeces and have been recovered from patients with HC and HUS. With the exception of 0157:H-1H7, the other serotypes have never been associated with outbreaks of these diseases and are only rarely recovered from sporadic cases of HC and HUS. The data obtained in this study show that sheep are a reservoir of STEC that possess Shiga toxin subtypes (stXlc and stX2d) that are rarely associated with HC and HUS. While EHEC serogroups such as 026, 0103, 0111 and 0157 are important causal serogroups in HUS, the role of other less common non-O 157 serogroups remains to be clearly defined but is probably underestimated . The subtyping assays developed in this study will play an important role in the future characterisation of STEC and will be useful tools to clarify the role ofnon-0157 STEC in human disease

stx(1c) Is the Most Common Shiga Toxin 1 Subtype among Shiga Toxin-Producing Escherichia coli Isolates from Sheep but Not among Isolates from Cattle

Unlike Shiga toxin 2 (stx(2)) genes, most nucleotide sequences of Shiga toxin 1 (stx(1)) genes from Shiga toxin-producing Escherichia coli (STEC), Shigella dysenteriae, and several bacteriophages (H19B, 933J, and H30) are highly conserved. Consequently, there has been little incentive to investigate variants of stx(1) among STEC isolates derived from human or animal sources. However stx(1OX3), originally identified in an OX3:H8 isolate from a healthy sheep in Germany, differs from other stx(1) subtypes by 43 nucleotides, resulting in changes to 12 amino acid residues, and has been renamed stx(1c). In this study we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that distinguishes stx(1c) from other stx(1) subtypes. The PCR-RFLP assay was used to study 378 stx(1)-containing STEC isolates. Of these, 207 were isolated from sheep, 104 from cattle, 45 from humans, 11 from meat, 5 from swine, 5 from unknown sources, and 1 from a cattle water trough. Three hundred fifty-five of the 378 isolates (93.9%) also possessed at least one other associated virulence gene (ehxA, eaeA, and/or stx(2)); the combination stx(1), stx(2), and ehxA was the most common (175 of 355 [49.3%]), and 90 of 355 (25.4%) isolates possessed eaeA. One hundred thirty-six of 207 (65.7%) ovine isolates possessed stx(1c) alone and belonged to 41 serotypes. Seventy-one of 136 (52.2%) comprised the common ovine serotypes O5:H(−), O128:H2, and O123:H(−). Fifty-two of 207 isolates (25.1%) possessed an stx(1) subtype; 27 (51.9%) of these belonged to serotype O91:H(−). Nineteen of 207 isolates (9.2%) contained both stx(1c) and stx(1) subtypes, and 14 belonged to serotype O75:H8. In marked contrast, 97 of 104 (93.3%) bovine isolates comprising 44 serotypes possessed an stx(1) subtype, 6 isolates possessed stx(1c), and the remaining isolate possessed both stx(1c) and stx(1) subtypes. Ten of 11 (91%) isolates cultured from meat in New Zealand possessed stx(1c) (serotypes O5:H(−), O75:H8/H40, O81:H26, O88:H25, O104:H(−)/H7, O123:H(−)/H10, and O128:H2); most of these serotypes are commonly recovered from the feces of healthy sheep. Serotypes containing stx(1) recovered from cattle rarely were the same as those isolated from sheep. Although an stx(1c) subtype was never associated with the typical enterohemorrhagic E. coli serogroups O26, O103, O111, O113, and O157, 13 human isolates possessed stx(1c). Of these, six isolates with serotype O128:H2 (from patients with diarrhea), four O5:H(−) isolates (from patients with hemolytic-uremic syndrome), and three isolates with serotypes O123:H(−) (diarrhea), OX3:H8 (hemolytic-uremic syndrome), and O81:H6 (unknown health status) represent serotypes that are commonly isolated from sheep

Distribution of Intimin Subtypes among Escherichia coli Isolates from Ruminant and Human Sources

The intimin gene eae, located within the locus of enterocyte effacement pathogenicity island, distinguishes enteropathogenic Escherichia coli (EPEC) and some Shiga toxin-producing E. coli (STEC) strains from all other pathotypes of diarrheagenic E. coli. EPEC is a leading cause of infantile diarrhea in developing countries, and intimin-positive STEC isolates are typically associated with life-threatening diseases such as hemolytic-uremic syndrome and hemorrhagic colitis. Here we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that reliably differentiates all 11 known intimin types (α1, α2, β, γ, κ, ɛ, η, ι, λ, θ, and ζ) and three new intimin genes that show less than 95% nucleotide sequence identity with existing intimin types. We designated these new intimin genes Int-μ, Int-ν, and Int-ξ. The PCR-RFLP assay was used to screen 213 eae-positive E. coli isolates derived from ovine, bovine, and human sources comprising 60 serotypes. Of these, 82 were STEC isolates, 89 were stx-negative (stx(−)) and ehxA-positive (ehxA(+)) isolates, and 42 were stx(−) and ehxA-negative isolates. Int-β, the most commonly identified eae subtype (82 of 213 [38.5%] isolates), was associated with 21 serotypes, followed by Int-ζ (39 of 213 [18.3%] isolates; 11 serotypes), Int-θ (25 of 213 [11.7%] isolates; 15 serotypes), Int-γ (19 of 213 [8.9%] isolates; 9 serotypes), and Int-ɛ (21 of 213 [9.9%] isolates; 5 serotypes). Intimin subtypes α1, α2, κ, λ, ξ, μ, ν, and ι were infrequently identified; and Int-η was not detected. Phylogenetic analyses with the Phylip package of programs clustered the intimin subtypes into nine distinct families (α, β-ξ, γ, κ, ɛ-η-ν, ι-μ, λ, θ, and ζ). Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-containing E. coli strains

Allelic variants of streptokinase from Streptococcus pyogenes display functional differences in plasminogen activation

A common mammalian defense mechanism employed to prevent systemic dissemination of invasive bacteria involves occlusion of local microvasculature and encapsulation of bacteria within fibrin networks. Acquisition of plasmin activity at the bacterial cell surface circumvents this defense mechanism, allowing invasive disease initiation. To facilitate this process, S. pyogenes secretes streptokinase, a plasminogen-activating protein. Streptokinase polymorphism exhibited by S. pyogenes isolates is well characterized. However, the functional differences displayed by these variants and the biological significance of this variation has not been elucidated. Phylogenetic analysis of ska sequences from 28 S. pyogenes isolates revealed 2 main sequence clusters (clusters 1 and 2). All strains secreted streptokinase, as determined by Western blotting, and were capable of acquiring cell surface plasmin activity after incubation in human plasma. Whereas culture supernatants from strains containing cluster 1 ska alleles also displayed soluble plasminogen activation activity, supernatants from strains containing cluster 2 ska alleles did not. Furthermore, plasminogen activation activity in culture supernatants from strains containing cluster 2 ska alleles could only be detected when plasminogen was prebound with fibrinogen. This study indicates that variant streptokinase proteins secreted by S. pyogenes isolates display differing plasminogen activation characteristics and may therefore play distinct roles in disease pathogenesis

Conserved anchorless surface proteins as group A streptococcal vaccine candidates

Streptococcus pyogenes (group A Streptococcus (GAS)) causes similar to 700 million human infections each year, resulting in over 500,000 deaths. The development of a commercial GAS vaccine is hampered by the occurrence of many unique GAS serotypes, antigenic variation within the same serotype, differences in serotype geographical distribution, and the production of antibodies cross-reactive with human tissue that may lead to autoimmune disease. Several independent studies have documented a number of GAS cell wall-associated or secreted metabolic enzymes that contain neither N-terminal leader sequences nor C-terminal cell wall anchors. Here, we applied a proteomic analysis of serotype M1T1 GAS cell wall extracts for the purpose of vaccine development. This approach catalogued several anchorless proteins and identified two protective vaccine candidates, arginine deiminase and trigger factor. These surface-exposed enzymes are expressed across multiple GAS serotypes exhibiting >= 99% amino acid sequence identity. Vaccine safety concerns are alleviated by the observation that these vaccine candidates lack human homologs, while sera from human populations suffering repeated GAS infections and high levels of autoimmune complications do not recognize these enzymes. Our study demonstrates anchorless cell surface antigens as promising vaccine candidates for the prevention of GAS disease