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The global emergence of antimicrobial resistance (AMR) needs no emphasis. In this study, the in vitro stability, safety, and antimicrobial efficacy of nanosilver-entrapped cinnamaldehyde (AgC) against multi-drug-resistant (MDR) strains of enteroaggregative

Severe Acute Respiratory Syndrome Corona Virus- 2 (SARS-CoV-2) or coronavirus disease (COVID-19) has been identified as the cause of a pandemic of respiratory illness. It has been declared as a Public Health Emergency of International Concern by the World Health Organization. The reported symptoms include fever, cough, fatigue, pneumonia, headache, diarrhea, hemoptysis, and dyspnea. The human-to-human transmission has been described via droplets, contaminated hands or surfaces. The incubation times ranges between 2-14 days. Early diagnosis, quarantine, and supportive treatments are required to cure patients. Treatments, including antiviral agents, chloroquine and hydroxychloroquine, monoclonal antibodies, and convalescent plasma transfusion have been used for successful treatment of the patients. Preventive measures such as masks, hand hygiene practices, avoidance of public contact, case detection, contact tracing, and quarantines have been discussed as ways to reduce transmission. The pandemic has impacted on the meat industry. The event demands urgent implementation of a multidisciplinary team in a One Health approach to address the present-day challenges at the human-animal-environment interface. We reviewed the literature on the epidemiology, the disease, diagnosis, therapeutics and mitigation strategies of COVID-19. Although many studies are relevant to control the current public emergency, more research is needed to provide valid and reliable ways to manage this kind of public health emergency in both the short- and long-term.

The seasonal occurrence of Coxiella burnetii infection in the slaughtered buffaloes was determined. A total of 424 samples were collected from 212 buffaloes in winter [serum (n = 105) and uterine tissue (n = 105)] and summer [serum (n = 107) and uterine tissue (n = 107)]. The serum and uterine tissue samples were analysed for the detection of C. burnetii using trans-PCR (IS1111 gene) assay, while the serum samples were also screened for the detection of anti-C. burnetii IgG antibodies using commercial indirect-ELISA kit. In PCR assay, 1.90% serum and 0.95% uterine tissue samples collected during winter season were positive for the IS1111 gene. All the samples collected during summer season were negative in the PCR assay. In ELISA, 10.47% and 8.41% of serum samples collected during winter and summer season were positive for anti-C. burnetii antibodies, respectively. Overall, the per cent positivity showed a seasonal variation with higher positivity in winter (11.43%) as compared to summer (8.41%) season. Albeit, significant difference was not observed between the two seasons; however, more extensive systematic temporal epidemiological studies need to be conducted in order to confirm the seasonal association of the pathogen.

The occurrence of Listeria monocytogenes was studied by using cultural and serological methods in cattle housed in a particular gaushala (cattle shelter) and organized dairy farm.A total of 1201 samples from cattle comprising blood (n = 207), milk (n = 203), vaginal swabs (n = 210), and serum (n = 207) from an organized farm (n = 210) and blood (n = 100), milk (n = 74), vaginal swabs (n = 100), and serum (n = 100) from a gaushala (n = 100) were collected and analyzed for L. monocytogenes. All samples excluding serum were analyzed for isolation and identification of L. monocytogenes, while the serum samples were screened for seropositivity. The isolates were further subjected to assess their virulence potential (in vitro and in vivo), biofilm formation ability, and antibiotic susceptibility patterns.Four L. monocytogenes strains were isolated from the cattle; three (0.48%) from the organized farm and one (0.36%) from the gaushala. On serological screening of cattle from the organized dairy farm, 16.42% were found to be positive for antibodies against listeriolysin O, while cattle from the gaushala revealed 36% seropositivity. Furthermore, on characterization of the isolates for their pathogenic potential and biofilm-forming ability, all were found to be pathogenic by both in vitro and in vivo assays and were weak to moderate biofilm formers. The minimum inhibition concentration (MIC) of recovered isolates revealed resistance for ampicillin by two L. monocytogenes isolates (MIC256 μg/mL), whereas three L. monocytogenes isolates were intermediately resistant (MIC4 μg/mL) and one resistant against amoxicillin (MIC8 μg/mL). However, all four isolates were susceptible to gentamicin, cotrimoxazole, and erythromycin.Isolation of virulent and antibiotic-resistant strains of L. monocytogenes warrants the need for epidemiological surveillance, antimicrobial susceptibility, and implementation of control measures to combat the occurrence of L. monocytogenes infection in animals as well as humans.

Background: The present investigation of Coxiella burnetii infection in cattle and farm workers on an organized cattle dairy farm, which appears to be the first of its kind in India, was undertaken to assess the status of this largely neglected and masked zoonosis. Methods: A total of 665 samples comprising of serum (n = 224), milk (n = 217) and vaginal swabs (n = 224) collected from milch animals (n = 224) with a history of reproductive disorders were screened. Besides these, ticks (n = 114); animal feed (n = 4) and environmental samples (n = 13) as well as serum (n = 19) of farm workers were also collected. The animal sera and milk samples as well as human sera were tested for antibodies against C. burnetii by commercial ELISA kit, whereas, all the collected samples were subjected to trans-PCR targeting the IS1111 gene of C. burnetii. Results: A high positivity for coxiellosis was detected in sera (29.91%) and milk (26.73%) samples of dairy cattle as well as sera from human contacts (84.21%) by ELISA. The trans-PCR detected the pathogen in 12.94% sera, 14.73% vaginal swabs and 5.53% milk samples of cattle, and in one soil sample, however, the sera of the farm workers and tick were tested negative. Conclusions: The high positivity for coxiellosis among cattle and farm workers highlight the need to undertake extensive epidemiological studies to unravel the trends of C. burnetii infection in India. Keywords: Coxiella burnetii, Coxiellosis, Farm workers, Organized farm, Zoonosis

We developed the com1 gene based loop-mediated isothermal amplification (LAMP) assay for the detection of Coxiella burnetii and validated it by screening DNA isolated from serum samples collected from animals and humans. The detection of Coxiella by LAMP assay was comparable with the com1 based-PCR.

High throughput in vivo laboratory models is need for screening and identification of effective therapeutic agents to overcome microbial drug-resistance. This study was undertaken to evaluate in vivo antimicrobial efficacy of short-chain antimicrobial peptide- Cecropin A (1–7)-Melittin (CAMA) against three multi-drug resistant enteroaggregative Escherichia coli (MDR-EAEC) field isolates in a Galleria mellonella larval model. The minimum inhibitory concentration (MIC; 2.0 mg/L) and minimum bactericidal concentration (MBC; 4.0 mg/L) of CAMA were determined by microdilution assay. CAMA was found to be stable at high temperatures, physiological concentration of cationic salts and proteases; safe with sheep erythrocytes, secondary cell lines and commensal lactobacilli at lower MICs; and exhibited membrane permeabilization. In vitro time-kill assay revealed concentration- and time-dependent clearance of MDR-EAEC in CAMA-treated groups at 30 min. CAMA- treated G. mellonella larvae exhibited an increased survival rate, reduced MDR-EAEC counts, immunomodulatory effect and proved non-toxic which concurred with histopathological findings. CAMA exhibited either an equal or better efficacy than the tested antibiotic control, meropenem. This study highlights the possibility of G. mellonella larvae as an excellent in vivo model for investigating the host-pathogen interaction, including the efficacy of antimicrobials against MDR-EAEC strains.

Q fever caused by Coxiella burnetii is an important zoonosis and has great public health significance. A total of 905 clinical samples from 387 cattle [serum (n = 387); vaginal swabs (n = 387); milk (n = 131)] and 59 serum samples from humans were collected from gaushala (cattle shelter) and screened for anti-C. burnetii IgG antibodies in the sera using an indirect-ELISA kit. Further, the samples were tested for C. burnetii DNA employing TaqMan real-time and conventional PCR assays targeting the com1 gene. In ELISA, 9.56% and 6.78% of animal and human sera samples were positive for anti-C. burnetii antibodies, respectively. Upon pathogen detection, 3.87% sera, 1.81% vaginal swabs, and 6.87% milk samples from cattle tested positive in TaqMan real-time PCR and 1.55% sera, 0.52% vaginal swabs, and 3.05% milk samples were found positive in conventional PCR. In humans, one serum sample was positive in both the PCR assays. The PCR positive samples (n = 12) were partially sequenced and the phylogenetic tree was constructed using com1 gene sequences (n = 42) from a different host and geographical areas. The study highlights infection of cattle and their human contacts in gaushala and identifies relationships between strains identified in the gaushala and those in other parts of the globe.

Brucellosis, caused by Brucella species, is a neglected, under reported and under recognized infection of worldwide distribution. Owing to its greater economic and zoonotic importance, control of brucellosis is a priority target set forth by the WHO and development agencies. The genus Brucella is comprised of 12 species. The four main species infecting humans and livestock populations include B. melitensis, B. abortus, B. suis, and B. canis. Brucellosis is a cause of acute febrile illness and a potentially debilitating chronic infection in humans. Brucellosis is a granulomatous disease and can affect any organ. Thus, it requires long-term treatment to achieve clinical cure. In order to reduce the burden of human brucellosis, it is a must to control the infection of livestock. More research on novel control strategies, diagnostics and vaccines including cost-effectiveness studies is required particularly in the developing world. Here, we reviewed the general description of Brucella organisms, epidemiology of brucellosis and advances in immunodiagnostics.

The present study for the first time evaluates the serodiagnostic efficacy of two recombinant antigens namely, listeriolysin O (rLLO) and phosphatidyl-inositol phospholipase C (rPI-PLC). Indirect ELISA with the above recombinant antigens was used on samples collected from bovines (n=106), goats (n=138) and pigs (n=92) having either a history of abortion, emaciation and/or apparently healthy animals. Isolation of Listeria was attempted from the blood samples using USDA-FSIS method. On screening of test sera by rLLO-based ELISA, antibodies against anti-listeriolysin O (ALLO) were observed in goats (22.46%), bovines (15.10%) and pigs (16.31%). As advocated, after adsorption of positive serum samples with streptolysin O (SLO), the seropositivity for ALLO was marginally reduced (p>0.05) in goats (21.73%) and bovines (10.38%), whereas, in pigs the reduction (5.43%) was significant (p

A novel multiplex PCR assay was developed to identify genus Listeria, and discriminate Listeria monocytogenes and its major lineages (LI, LII, LIII). This assay is a rapid and inexpensive subtyping method for screening and characterization of L. monocytogenes.

The objectives of the study were to isolate and identify livestock associated methicillin resistant Staphylococcus aureus (LA-MRSA) and methicillin sensitive S. aureus (LA-MSSA) from clinical mastitis cases and to compare their antibiotic susceptibility, biofilm formation and in vivo pathogenicity in Galleria mellonella larva model. A total of 60 milk samples were collected from cows suffering from mastitis and processed for isolation and identification of S. aureus using standard conventional methods. All the recovered S. aureas isolates were subjected for detection of MRSA and/or MSSA employing phenotypic (Cefoxitin disc assay) and genotypic (the mecA gene PCR) assays. Antibiotic susceptibility pattern of LA-MRSA and LA-MSSA test isolates was determined using disc diffusion method, biofilm formation by 96 well microtiter plate assay and pathogenicity testing in G. mellonella larvae. On microbiological, biochemical and PCR analyses, 14 S. aureus isolates were confirmed. Of these, 4 were tested as LA-MRSA and the remaining 10 isolates were LA-MSSA. Comparative evaluation suggested that MRSA isolates were resistant to different classes of antibiotics and were equally lethal to G. mellonella larvae. However, bioflim forming ability was significantly higher (p < 0.001) in the MSSA test isolates. An association of biofilm formation and pahogenicity testing was not observed between LA-MRSA and LA-MSSA test isolates. Further, LA-MRSA were resistant to different classes of antibiotic and were more lethal to G. mellonella larvae. These preliminary observations are of great concern as the LA-MRSA infections in the community have been documented and warrant in depth research for such pathogens.

The present study examined the anti-biofilm efficacy of two short-chain antimicrobial peptides (AMPs), namely, indolicidin and cecropin A (1-7)-melittin (CAMA) against biofilm-forming multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC) isolates. The typical EAEC isolates re-validated by PCR and confirmed using HEp-2 cell adherence assay was subjected to antibiotic susceptibility testing to confirm its MDR status. The biofilm-forming ability of MDR-EAEC isolates was assessed by Congo red binding, microtitre plate assays and hydrophobicity index; broth microdilution technique was employed to determine minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs). The obtained MIC and MBEC values for both AMPs were evaluated alone and in combination against MDR-EAEC biofilms using crystal violet (CV) staining and confocal microscopy-based live/dead cell quantification methods. All the three MDR-EAEC strains revealed weak to strong biofilm-forming ability and were found to be electron-donating and weakly electron-accepting (hydrophobicity index). Also, highly significant (P

A novel Com1 synthetic peptide-based latex agglutination test (LAT) was developed and evaluated against commercial ELISA kit for sero-screening of coxiellosis in cattle. The developed test is economical, has field applicability and can serve as an important rapid tool for sero-screening of coxiellosis in cattle.

We report a novel, rapid, economical and species-specific DNA-based assay for the authentication of pork. The technique specifically amplified porcine mitochondrial D loop region by combining Alkaline Lysis (AL) method of DNA extraction and Loop Mediated Isothermal Amplification (LAMP). Visual detection of the reaction was accomplished by color development in the reaction with the addition of SYBR Green I dye. Dependable amplification was possible in thermally processed meat samples heated up to 121 °C for 30 min. The assay was able to detect pork in beef up to the level of 0.1% admixture and limit of detection of DNA was at 0.5 ng/μL. Cross-amplification of related species like cattle, buffalo, sheep, goat and chicken was excluded by incorporating their DNA in the reaction assay. The novel approach (AL-LAMP technique) was found to be robust and handy, suitable even for resource compromised laboratories engaged in the food analysis.

The in-house developed DnaK and Com1 synthetic peptide-based Latex Agglutination Tests (LATs) were comparatively evaluated with commercial indirect-ELISA kit, to provide a rapid, economical and onsite field applicable test for seroscreening of coxiellosis in bovines.

Two Listeria -like isolates obtained from mangrove swamps in Goa, India were characterized using polyphasic combinations of phenotypic, chemotaxonomic and whole-genome sequence (WGS)-based approaches. The isolates presented as short, non-spore-forming, Gram-positive rods, that were non-motile, oxidase-negative, catalase-positive and exhibited α-haemolysis on 5 % sheep- and horse-blood agar plates. The 16S rRNA gene sequences exhibited 93.7–99.7 % nucleotide identity to other Listeria species and had less than 92 % nucleotide identity to species of closely related genera, indicating that the isolates are de facto members of the genus Listeria . Their overall fatty acid composition resembled that of other Listeria species, with quantitative differences in iso C15 : 0, anteiso C15 : 0, iso C16 : 0, C16 : 0, iso C17 : 0 and anteiso C17 : 0 fatty acid profiles. Phylogeny based on 406 core coding DNA sequences grouped these two isolates in a monophyletic clade within the genus Listeria . WGS-based average nucleotide identity and in silico DNA–DNA hybridization values were lower than the recommended cut-off values of 95 and 70 %, respectively, to the other Listeria species, indicating that they are founding members of a novel Listeria species. We suggest the name Listeria goaensis sp. nov. be created and the type strain is ILCC801T (=KCTC 33909;=DSM 29886;=MCC 3285).

Coxiella burnetii is one of the most contagious pathogen associated with Q fever in humans, while, ruminants act as important source of infection for humans. In the present cross sectional study, a total of 464 samples were collected from 218 goats comprising of 218 sera, 218 blood and 28 milk from various parts of Chhattisgarh and Odisha region, India. Besides, environmental (33; soil- 4, faecal- 10, feed-6, drainage water- 6, drinking water- 7) and rodent (38) samples were also collected from the premises of the animals. Human sera samples (93) were collected from same sampling area comprised of workers at an organized dairy farm (43), and farmers (50). The samples were subjected to PCR targeting the trans and com1 genes and detection of antibodies using commercial ELISA kits. An overall 14.22% (95% CI: 10.2-19.47%) of the goat samples were positive using either PCR or ELISA. While, by using PCR and ELISA, 11.93% (26/218) and 9.63% (21/218) of the samples were positive for C. burnetii. A higher seropositivity (46.24%; 95% CI: 36.46-56.32%) was observed for antibodies against C. burnetii in samples collected from humans. None of the human, environmental and rodent samples were positive for C. burnetii using PCR. This seems to be the first cross sectional study to focus the hidden threat of coxiellosis among goat population and associated risk factors in India.

Bacillus cereus infection-associated suppurative pneumonia was recorded in an adult African grey parrot (Psittacus erithacus erithacus). Grossly, the diaphragmatic lung lobes showed presence of multiple, irregular, creamy colored areas over the surface. The adjacent parenchyma appeared congested and pneumonic. Histopathologically, multifocal areas of abscesses characterized by presence of necrotic cell shreds, fibrin, and collection of heterophils with presence of bacterial aggregates were observed in the lungs. Taylor’s staining revealed presence of multiple aggregates of Gram-positive bacteria within fibrin shreds in the necrotic debris in the lungs. Other vital organs such as heart, liver, and brain revealed variably engorged and dilated blood vasculature, and the intestine showed hemorrhagic enteritis. Thus, based on above pathological findings and the partial sequence analysis report of 16S rRNA gene (919 bp) amplified from a DNA extracted from the lung tissue, B. cereus was predicted as a causative agent for the death of the psittacine bird. Thus, to the best of our knowledge, B. cereus-associated suppurative pneumonia in African grey parrot appears to be the first report from India.

Enteroaggregative Escherichia coli (EAEC) is an important pathotype that causes infection in humans and animals. EAEC isolates ( n = 86) recovered from diarrhoeal cases in human infants (37) and young animals (49) were characterized as ‘typical’ and/or ‘atypical’ EAEC strains employing PCR for virulence associated genes ( cvd 432, aai A, ast A, pil S, irp 2, ecp , pic , aggR , aaf A, agg A, and agg 3A). Besides, biofilm formation ability of human and animal EAEC isolates was assessed using microtiter plate assay. In addition, the transcriptional profile of biofilm associated genes ( fis and ecp ) was also evaluated and correlated with biofilm formation assay for few selected EAEC isolates of human and animal origins. Overall, a diverse virulence gene profile was observed for the EAEC isolates of human and animal origins as none of the EAEC isolates revealed the presence of all the genes that were targeted. Nine ‘typical’ EAEC isolates were identified (6 from humans and 3 from animals) while, the majority of the isolates were ‘atypical’ EAEC strains. Isolation and identification of three ‘typical’ EAEC isolates from animals (canines) appears to be the first report globally. Further, based on the observations of the biofilm formation assay, the study suggested that human EAEC isolates in particular were comparatively more biofilm producers than that of the animal EAEC isolates. The fis gene was highly expressed in majority of ‘typical’ EAEC isolates and the ecp gene in ‘atypical’ EAEC isolates.

Chicken infectious anaemia (CIA) is an economically important and emerging poultry disease reported worldwide. Current CIA vaccines have limitations like, the inability of the virus to grow to high titres in embryos/cell cultures, possession of residual pathogenicity and a risk of reversion to virulence. In the present study, a DNA vaccine, encoding chicken infectious anaemia virus (CIAV) VP1 and VP2 genes, was developed and co-administered with truncated chicken high mobility group box 1 (HMGB1ΔC) protein in young chicks for the evaluation of vaccine immune response. CIAV VP1 and VP2 genes were cloned in pTARGET while HMGB1ΔC in PET32b vector. In vitro expression of these gene constructs was evaluated by Western blotting. Further, recombinant HMGB1ΔC was evaluated for its biological activity. The CIAV DNA vaccine administration in specific pathogen free chicks resulted in moderately protective ELISA antibody titres in the range of 4322.87 ± 359.72 to 8288.19 ± 136.38, increased CD8(+) cells, and a higher titre was observed by co-administration of novel adjuvant (HMGB1ΔC) and booster immunizations. The use of vaccine with adjuvant showed achieving antibody titres nearly 8500, titre considered as highly protective, which indicates that co-immunization of HMGB1ΔC may have a strong adjuvant activity on CIAV DNA vaccine induced immune responses. The able potential of HMGB1 protein holding strong adjuvant activity could be exploited further with trials with vaccines for other important pathogens for achieving the required protective immune responses.

The study describes a rapid approach for detection of common enteric bacterial pathogens, which involves partial amplification of the 16S rRNA gene by PCR using a colony from selective medium followed by restriction enzyme (RE) digestion using the EcoRI, HindIII and SalI enzymes. On the basis of RE digestion analysis different genera namely, Escherichia, Salmonella, Shigella, Vibrio, Campylobacter, Arcobacter, Yesinia and Listeria were differentiated.

Introduction : Infectious diarrhoea particularly due to pathogenic bacteria is a major health problem in developing countries, including India. Despite significant reports of diarrhoeagenic Escherichia coli (DEC) pathotypes around the globe, studies which address genetic relatedness, antibiogram profile and their correlation with respect to their isolation from different sources are sparse. The present study determines isolation and identification of DEC pathotypes from different sources, their genetic characterisation, antibiogram profile and their correlation if any. Materials and methods : A total of 336 samples comprising diarrhoeic stool samples from infants ( n =103), young animal ( n =106), foods ( n =68) and associated environmental sources ( n =59) were collected from Bareilly region of India. All the samples were screened by using standard microbiological methods for the detection of E. coli . The identified E. coli were then confirmed as DEC pathotypes using polymerase chain reaction–based assays. Those DEC pathotypes identified as Enteroaggregative E. coli (EAEC) were further confirmed using HEp-2 adherence assay. All the isolated DEC pathotypes were studied for their genetic diversity using pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing was performed by using disc diffusion method as per Clinical Laboratory Standards Institute guidelines. Results and discussion : Of the four DEC pathotypes investigated, EAEC was found to be the predominant pathogen with an isolation rate of 16.5% from infants, 17.9% from young animals, 16.2% from foods and 3.4% from the associated environmental sources. These EAEC isolates, on further characterisation, revealed predominance of ‘atypical’ EAEC, with an isolation rate of 10.7% from infants, 15.1% from young animals, 16.2% from foods, and 3.4% from the associated environmental sources. On PFGE analysis, discrimination was evident within DEC pathotypes as 52 unique pulsotypes were observed for 59 recovered DEC pathotypes. However, a few EAEC isolates were found to be clonal (clusters A, B, C, D, F, G, and H) irrespective of their source of isolation, suggests sharing and/or circulation among different sources. Further, a high antibiotic resistance pattern was observed among isolated DEC pathotypes as almost 86.4% of isolates were found to be resistant against ≥3 tested drugs. Keywords: antibiotic resistance; ‘Atypical’ enteroaggregative Escherichia coli; diarrhoeagenic Escherichia coli; genetic diversity; pulsed-field gel electrophoresis (Published: 18 May 2016) Citation: Infection Ecology and Epidemiology 2016, 6: 31055 - http://dx.doi.org/10.3402/iee.v6.31055

The in vitro and in vivo antimicrobial effects of Lactobacillus plantarum and Lactobacillus acidophilus were evaluated individually and synergistically against multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC). In vitro evaluation of each probiotic strain when co-cultured with MDR-EAEC isolates revealed a reduction in MDR-EAEC counts (eosin-methylene blue agar) in a dose- and time-dependent manner: probiotics at a dose rate of 10(10) CFU inhibited MDR-EAEC isolates at 72 h post-inoculation (PI), whereas at lower concentrations (10(8) and 10(9) CFU) MDR-EAEC isolates were inhibited at 96 h PI. The synergistic antimicrobial effect of both probiotic strains (each at 10(10) CFU) was highly significant (P

Francisella tularensis is a category A biothreat agent for which there is no approved vaccine and the correlates of protection are not well understood. In particular, the relationship between the humoral and cellular immune response to F. tularensis and the relative importance of each in protection is controversial. Yet, understanding this relationship will be crucial to the development of an effective vaccine against this organism. We demonstrate, for the first time, a differential requirement for humoral vs cellular immunity in vaccine-induced protection against F. tularensis infection, and that the requirement for Ab observed in some protection studies, may be overcome through the induction of enhanced cellular immunity. Specifically, following intranasal/mucosal immunization of mice with inactivated F. tularensis organisms plus the cholera toxin B subunit, we observe increased production of IgG2a/2c vs IgG1 Ab, as well as IFN-γ, indicating induction of a Th1 response. In addition, the requirement for F. tularensis-specific IgA Ab production, observed in studies following immunization with inactivated F. tularensis alone, is eliminated. Thus, these data indicate that enhanced Th1 responses can supersede the requirement for anti-F. tularensis-specific IgA. This observation also has important ramifications for vaccine development against this organism.

A study was carried out to compare phosphatidylinositol specific phospholipase C (PI-PLC) assay, chromogenic medium ALOA and a PCR for virulence associated genes (the plcA , hlyA and prfA ) with mice and chick embryo inoculation for detection of pathogenic Listeria monocytogenes from milk and ready-to-eat (RTE) indigenous milk products. Eighteen strains of L. monocytogenes were isolated. A good correlation was observed among all the assays. ALOA exhibited distinct results (bluish green colonies with halo formation) within 24–48 h however; enzymatic activity was expressed on PI-PLC assay by 4 days of inoculation. About 93% hemolytic L. monocytogenes isolates were positive for genotypic and phenotypic expression of plcA gene and also proved lethal to mice and chick embryo. The detection of PI-PLC activity on ALOA with PCR targeting the hlyA and plcA genes would be reliable in vitro alternatives to in vivo assays for detecting pathogenic L. monocytogenes.

Numerous studies have demonstrated that targeting Ag to Fc receptors (FcR) on APCs can enhance humoral and cellular immunity. However, studies are lacking that examine both the use of FcR-targeting in generating immune protection against infectious agents and the use of FcRs in the induction of mucosal immunity. Francisella tularensis is a category A intracellular mucosal pathogen. Thus, intense efforts are underway to develop a vaccine against this organism. We hypothesized that protection against mucosal infection with F. tularensis would be significantly enhanced by targeting inactivated F. tularensis live vaccine strain (iFt) to FcRs at mucosal sites, via intranasal immunization with mAb-iFt complexes. These studies demonstrate for the first time that: 1) FcR-targeted immunogen enhances immunogen-specific IgA production and protection against subsequent infection in an IgA-dependent manner, 2) FcγR and neonatal FcR are crucial to this protection, and 3) inactivated F. tularensis, when targeted to FcRs, enhances protection against the highly virulent SchuS4 strain of F. tularensis, a category A biothreat agent. In summary, these studies show for the first time the use of FcRs as a highly effective vaccination strategy against a highly virulent mucosal intracellular pathogen.

Listeria monocytogenes isolates (n = 36) recovered from human and animal clinical cases and foods from different geographical regions of India were characterized using multiplex PCR-based serotyping, pulsed field gel electrophoresis (PFGE), in vitro and in vivo pathogenicity tests and antibiogram profiling. Multiplex PCR-based serotyping distributed L. monocytogenes isolates into 3 serogroups, of which 91.67% belonged to 4b, 4d, 4e serogroup, followed by 5.56% to 1/2a, 3a and 2.78% to 1/2b, 3b serogroups. PFGE analysis using ApaI and AscI restriction enzymes revealed 17 pulsotypes among 36 L. monocytogenes isolates with 6 major clusters having similar fingerprint profile within their cluster and 11 unique fingerprint profiles. Interestingly, PFGE analysis inferred that foods of animal origin could be a significant source of infection for spread of listeriosis among human populations. Furthermore, on comparison of in vitro and in vivo pathogenicity tests, an overall good correlation was observed between hemolytic titer assay and chick embryo inoculation test as most of the isolates with a hemolytic titer of ≥ 16 were found to be lethal to chick embryo. All the isolates were found to be susceptible to tested antimicrobials except for one animal isolate which showed resistance towards co-trimoxazole.

In the present study, Salmonella isolates (n=40) recovered from clinical, food, poultry and environmental sources were characterized for serotype identification, genetic diversity and biofilm formation capability. Serotype identification using multiplex PCR assay revealed six isolates to be Salmonella Typhimurium, 14 as Salmonella Enteritidis, 11 as Salmonella Typhi, and the remaining nine isolates unidentified were considered as other Salmonella spp. Most of the Salmonella isolates (85%) produced biofilm on polystyrene surfaces as assessed by microtitre plate assay. About 67.5% isolates were weak biofilm producers and 17.5% were moderate biofilm producers. There was no significant difference in biofilm-forming ability among the Salmonella isolates recovered from different geographical regions or different sources. Among the genetic methods, Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR revealed greater discriminatory power (DI, 0.943) followed by pulsed field gel electrophoresis (PFGE) (DI, 0.899) and random amplification of polymorphic DNA (RAPD) PCR (DI, 0.873). However, composite analysis revealed the highest discrimination index (0.957). Greater discrimination of S. Typhimurium and S. Typhi was achieved using PFGE, while ERIC PCR was better for S. Enteritidis and other Salmonella serotypes. A strong positive correlation (r=0.992) was observed between biofilm formation trait and clustered Salmonella isolates in composite genetic analysis.

Aim: This study was carried out to determine the prevalence, distribution, and identification of Salmonella serotypes in diarrheagenic infants and young animals, including sewage waste and fresh vegetables. Materials and Methods: A total of 550 samples were processed for the isolation of Salmonella spp., using standard microbiological and biochemical tests. Further polymerase chain reaction (PCR) detection of Salmonella genus was carried out using self-designed primers targeting invA gene and thereafter identification of important serotypes namely Salmonella Enterica serovar Typhimurium, Salmonella Enterica serovar Enteritidis, Salmonella Enterica serovar Typhi was performed using published standardized multiplex PCR. Results: An overall low prevalence of 2.5% (14/550) was observed. The observed prevalence of Salmonella spp. in diarrheagenic infants was 1.2% (05/400), diarrheagenic young animals 4% (02/50), sewage waste 10% (05/50), and fresh vegetables 4% (02/50), respectively. In diarrheagenic infants, of the five Salmonella isolates identified, two were Salmonella Typhimurium, two Salmonella Enteritidis, and one was unidentified and hence designated as other Salmonella serovar. All the Salmonella isolates identified from diarrheagenic young animals and sewage waste belonged to other Salmonella serovar, whereas, of the two isolates recovered from fresh vegetables, one was identified as other Salmonella serovar, and one as Salmonella Typhimurium, respectively. Conclusion: Isolation of Salmonella spp. especially from sewage waste and fresh vegetable is a matter of great concern from public health point of view because these sources can accidentally serve as a potential vehicle for transmission of Salmonella spp. to animals and human beings.

The present study describes the utility of a chromosomal associated fimbrial subunit gene ( fimA ) for screening ‘typical’ as well as ‘atypical’ Enteroaggregative Escherichia coli (EAEC) by PCR and its possible role as a promising molecular marker for rapid detection of ‘typical’ and ‘atypical’ EAEC pathotype.

A total of 98 previously characterized and serotyped L. monocytogenes strains, comprising 32 of 1/2a; 20 of 1/2b and 46 of 4b serotype, from clinical and food sources were studied for their capability to form a biofilm. The microtiter plate assay revealed 62 (63.26%) strains as weak, 27 (27.55%) strains as moderate, and 9 (9.18%) strains as strong biofilm formers. Among the strong biofilm formers, 6 strains were of serotype 1/2a and 3 strains were of serotype 1/2b. None of the strain from 4b serotype exhibited strong biofilm formation. No firm correlation (p = 0.015) was noticed between any serotype and respective biofilm formation ability. Electron microscopic studies showed that strong biofilm forming isolates could synthesize a biofilm within 24 h on surfaces important in food industries such as stainless steel, ceramic tiles, high-density polyethylene plastics, polyvinyl chloride pipes, and glass. Cell enumeration of strong, moderate, and weak biofilm was performed to determine if the number of cells correlated with the biofilm-forming capabilities of the isolates. Strong, moderate, and weak biofilm showed 570±127× 103 cells/cm2, 33±26× 103 cells/cm2, 5±3× 103 cells/cm2, respectively, indicating that the number of cells was directly proportional to the strength of the biofilm. The hydrophobicity index (HI) analysis revealed higher hydrophobicity with an increased biofilm formation. Fatty acid methyl esterase analysis revealed the amount of certain fatty acids such as iso-C15:0, anteiso-C15:0, and anteiso-C17:0 fatty acids correlated with the biofilm-forming capability of L. monocytogenes. This study showed that different strains of L. monocytogenes form biofilm of different intensities which did not completely correlate with their serotype; however, it correlated with the number of cells, hydrophobicity, and amount of certain fatty acids.