Comparative genomics of Brucella abortus and Brucella melitensis unravels the gene sharing, virulence factors and SNP diversity among the standard, vaccine and field strains.

Brucella abortus and Brucella melitensis are the primary etiological agents of brucellosis in large and small ruminants, respectively. There are limited comparative genomic studies involving Brucella strains that explore the relatedness among both species. In this study, we involved strains (n=44) representing standard, vaccine and Indian field origin for pangenome, single nucleotide polymorphism (SNP) and phylogenetic analysis. Both species shared a common gene pool representing 2884 genes out of a total 3244 genes. SNP-based phylogenetic analysis indicated higher SNP diversity among B. melitensis (3824) strains in comparison to B. abortus (540) strains, and a clear demarcation was identified between standard/vaccine and field strains. The analysis for virulence genes revealed that virB3, virB7, ricA, virB5, ipx5, wbkC, wbkB, and acpXL genes were highly conserved in most of the Brucella strains. Interestingly, virB10 gene was found to have high variability among the B. abortus strains. The cgMLST analysis revealed distinct sequence types for the standard/vaccine and field strains. B. abortus strains from north-eastern India fall within similar sequence type differing from other strains. In conclusion, the analysis revealed a highly shared core genome among two Brucella species. SNP analysis revealed B. melitensis strains exhibit high diversity as compared to B. abortus strains. Strains with absence or high polymorphism of virulence genes can be exploited for the development of novel vaccine candidates effective against both B. abortus and B. melitensis.

Outer membrane vesicles as nanovaccine candidates against pathogenic Leptospira in experimental Guinea pig model.

Leptospira interrogans serovar Hardjo is a long slender bacterium of size 0.1-0.3 µm × 5-50 µm. It is one of the major causes of bovine leptospirosis and is of economical importance because of the reproductive failure, still birth, abortion, and reduced productivity in cattle. It is also a zoonotic disease-causing infection in humans characterized by headaches, fever, chills, sweats and myalgia, lethargy, aching joints, pulmonary haemorrhages, and death in severe cases. Control of the disease involves antibiotic therapy, management and vaccination, of which immunization is the cheapest and effective means of disease prevention. The present study was developed to isolate and characterize the outer membrane vesicles of Leptospira interrogans serovar Hardjo and to evaluate their vaccine potential in guinea pig model. The OMVs were isolated from the culture by sonication and ultracentrifugation. In transmission electron microscopy, the isolated OMVs appeared as small spherical structures of 50-200 nm size. In Western blot and indirect ELISA, antibodies specific to OMVs were observed as indicative of a good humoral immune response elicited by L. interrogans serovar Hardjo OMV. The OMV-based Leptospira vaccine was able to prevent kidney lesions and renal colonization compared to the control and bacterin vaccinated group as proven by histopathology and PCR.

Isolation and characterization of bacteriophage Ib_pec2 against shigatoxigenic Escherichia coli.

This study was aimed to isolate and characterize bacteriophage against drug-resistant, shigatoxigenic Escherichia coli (STEC), one of the zoonotic, food-borne organisms associated with ruminants, mainly cattle. STEC were isolated (n = 35) from neonatal calves, dairy workers, and the surrounding environment and their antimicrobial resistance pattern was studied. Out of the 35 isolates tested, 17 isolates were found to be multidrug resistant to important antibiotics like ampicillin, amoxicillin-clavulanate, ciprofloxacin, streptomycin, and tetracycline. Bacteriophage namely Ib_pec2 was isolated against one of the STEC isolates and its morphology, genetic and proteomic characterization was done. Morphological analysis by TEM revealed bacteriophages belonging to myoviridae family. The genetic characterization of g23 gene revealed that the bacteriophage belonged to Tequatrovirus of myoviridae family. Proteomic analysis was able to identify five proteins identical to Tequatrovirus of myoviridae family. One-step growth curve experiment revealed a latency period of 40 min and a burst size of 893 pfu/bacteria. Temperature and pH ranging from 40°C to 50°C, pH 6-8, respectively. Phage could able to lyse majority of the STEC isolates. STEC are commensal organisms in the gastrointestinal tract of ruminants but are pathogenic in humans. Bacteriophages can be used as alternatives to antibiotics to control bacterial growth in ruminants and prevent its further spillage in the environment.

Core Genome Multilocus Sequence Typing Scheme for Improved Characterization and Epidemiological Surveillance of Pathogenic Brucella.

Brucellosis poses a significant burden to human and animal health worldwide. Robust and harmonized molecular epidemiological approaches and population studies that include routine disease screening are needed to efficiently track the origin and spread of Brucella strains. Core genome multilocus sequence typing (cgMLST) is a powerful genotyping system commonly used to delineate pathogen transmission routes for disease surveillance and control. Except for Brucella melitensis, cgMLST schemes for Brucella species are currently not established. Here, we describe a novel cgMLST scheme that covers multiple Brucella species. We first determined the phylogenetic breadth of the genus using 612 Brucella genomes. We selected 1,764 genes that were particularly well conserved and typeable in at least 98% of these genomes. We tested the new scheme on 600 genomes and found high agreement with the whole-genome-based single nucleotide polymorphism (SNP) analysis. Next, we applied the scheme to reanalyze the genome of Brucella strains from epidemiologically linked outbreaks. We demonstrated the applicability of the new scheme for high-resolution typing required in outbreak investigations as previously reported with whole-genome SNP methods. We also used the novel scheme to define the global population structure of the genus using 1,322 Brucella genomes. Finally, we demonstrated the possibility of tracing distribution of Brucella strains by performing cluster analysis of cgMLST profiles and found nearly identical cgMLST profiles in different countries. Our results show that sequencing depth of more than 40-fold is optimal for allele calling with this scheme. In summary, this study describes a novel Brucella-wide cgMLST scheme that is applicable in Brucella molecular epidemiology and helps in accurately tracking and thus controlling the sources of infection. The scheme is publicly accessible and should represent a valuable resource for laboratories with limited computational resources and bioinformatics expertise.

A comparative genomics approach for identifying genetic factors in Escherichia coli isolates associated with bovine diseases.

AIMS: E. coli are ubiquitously present bacterial pathogens that cause septicaemia, diarrhoea and other clinical illness in farm animals. Many pathogen factors can be associated with disease conditions. Currently, studies inferring E. coli genetic factors associated with infection in bovines are limited. Hence, the present study envisaged to determine the pathogen genetic factors associated with bovine disease conditions. METHOD AND RESULTS: The comparative genomic analysis involved genome sequence data of 135 diseased and 145 healthy bovine origin E. coli strains. Phylogroups A and C, as well as pathotypes ExPEC and EPEC, were found to have a strong connection with bovine disease strains. STEC strains, including EHEC, seem to play a less important role in bovine disease. Sequence types (STs) predominant among strains from diarrhoeal origin were ST 301 (CC 165) and ST 342. Correlation of core genome phylogeny with accessory gene-based clustering, phylogroups and pathotypes indicated lineage-specific virulence factors mostly associated with disease conditions. CONCLUSIONS: Comparative genomic analysis was applied to infer genetic factors significant in bovine disease origin E. coli strains. Isolates from bovine disease origin were enriched for the phylogroups A and C, and for the pathotypes ExPEC and EPEC. However, there was minimal evidence of STEC involvement. The study also indicated predominant genetic lineages and virulence genes (pap, sfa and afa) associated with disease origin strains. SIGNIFICANCE AND IMPACT OF STUDY: The study revealed significant pathotypes, phylogroups, serotypes and sequence types associated with bovine disease conditions. These identified genetic factors can be applied for disease diagnosis, implementing vaccines and therapeutic measures. In addition, E. coli isolates from the bovine species revealed a complex pattern of disease epidemiology.

Microcapillary LAMP for rapid and sensitive detection of pathogen in bovine semen.

A microcapillary-based loop-mediated isothermal amplification (µcLAMP) has been described for specific detection of infectious reproductive pathogens in semen samples of cattle without sophisticated instrumentation. Brucella abortus, Leptospira interrogans serovar Pomona and bovine herpesvirus 1 (BoHV-1) cultures were mixed in bovine semen samples. The µcLAMP assay is portable, user-friendly, cost-effective, and suitable to be performed as a POC diagnostic test. We have demonstrated high sensitivity and specificity of µcLAMP for detection of Brucella, Leptospira, and BoHV-1 in bovine semen samples comparable to PCR and qPCR assays. Thus, µcLAMP would be a promising field-based test for monitoring various infectious pathogens in biological samples.HighlightsDetect infectious organism in bovines semenReduction in carryover contamination is an important attribute, which may reduce the false-positive reaction.µcLAMP is a miniaturized form, which could be performed with a minimum volume of reagents.The µcLAMP assay is portable, user-friendly, and suitable to be performed as a POC diagnostic test.

Multilocus sequence typing of pathogenic Mycoplasma mycoides subsp. capri reveals the predominance of a novel clonal complex among isolates from goats in India.

Mycoplasma mycoides subsp. capri (Mmc) typically causes pneumonia, mastitis, arthritis, keratitis and septicaemia in goats. Mortality associated with Mmc in goat flocks is lower compared to Mycoplasma capricolum subsp. capripneumoniae-associated respiratory infections. Case fatality rates associated with Mmc ranged from 9.8 to 26.8% among several states in India. Molecular epidemiology approaches aimed at genotyping help to identify the diversity of isolates involved in a disease. Ten clinical pathogenic Mmc isolates were analysed by multilocus sequence typing (MLST) for studying genotypic relationships with 50 isolates available from public databases. The MLST analysis indicates high genetic diversity among Mmc isolates. From a total number of 60 isolates, 43 six sequence types (STs) were recognized comprising of six STs from India and 37 STs from other geographical regions. MLST profiles of isolates revealed none of the STs observed in Indian isolates were shared with global isolates. Some of the STs representing Indian isolates (four STs) were clustered into a novel clonal complex 1 (CC1). Maintenance of genetically related STs forming CCs among the goat population in India for longer periods indicates disease causing potentiality of these isolates. Based on various recombination analysis, weak clonal relationship among Mmc isolates were identified. The present study has enlightened further steps in disease investigations and to design future control measures by employing prevalent genotypes as vaccine candidates against Mmc infections.

Brucella abortus S19 rfbD mutant is highly attenuated, DIVA enable and confers protection against virulent challenge in mice.

Brucella abortus S19 is an important tool for controlling bovine brucellosis across the globe. However, vaccination with S19 suffers critical shortcomings such as, presence of residual virulence, induction of abortion and sero-diagnostic interference. In this study, rfbD gene deleted mutant S19 was developed. The mutant strain designated S19ΔR displayed rough LPS phenotype, which was confirmed by acriflavine dye-agglutination and LPS-SDS-PAGE analysis. The virulence was amply reduced as suggested by increased sensitivity to complement killing; reduction in splenic-bacterial load and the recovery time RT50 as validated in mice model. Anti-brucella humoral response was significantly lower as compared to S19 immunization. The minimal induction of Brucella specific IgG1, IgG2a & IgG2b, and IgG3 resulted in no apparent reactivity to RBPT antigen. S19ΔR showed protective index of 1.90 against virulent challenge. S19ΔR being highly attenuated and DIVA compatible may facilitate a platform for developing a safer bovine adulthood vaccine.

Differential expression of cytokines in PBMC of Bos indicus and Bos taurus × Bos indicus cattle due to Brucella abortus S19 antigen.

Brucellosis is the most dreadful disease among bovines, although breed differences have been observed in prevalence of disease, worldwide. In present study, antibody response and relative expression of proinflammatory cytokines was compared in Bos indicus (zebu) and Bos taurus × Bos indicus (crossbred) cattle vaccinated by live attenuated Brucella abortus S19 antigen. Six female calves (4-6 months age) of both groups were vaccinated with B.abortus S19 strain. Blood samples were collected before vaccination (0d) and 7th (7d), 14th (14d) and 28th (28d) days after vaccination. Indirect ELISA showed high (p < .05) anti-Brucella antibody level after vaccination; with no significant difference between the groups. During Real-time expression, IFNγ, TNFα, IL6 and IL10 genes initially showed down regulation followed by upregulation in both the groups; however, the trend was much prominent in crossbreds. The expressions of IFNγ, TNFα and IL6, proinflammatory molecules important for initial containment of the Brucella were significantly (p < .01) higher in crossbred. The study showed that the Sahiwal cattle were less responsive to B.abortus S19 antigen than crossbreds, indicating its lower sensitivity to the Brucella, comparatively. In contrary, higher expression of the proinflammatory molecules in crossbreds could be important for containment of the organism during initial stage of infection.

Multilocus sequence typing of Clostridium perfringens strains from neonatal calves, dairy workers and associated environment in India.

Clostridium perfringens is a globally recognized zoonotic pathogen. We report isolation and genotyping of C. perfringens from neonatal calves, dairy workers and their associated environment in India. A total of 103 fecal samples from neonatal calves, 25 stool swabs from the dairy workers and 50 samples from their associated environment were collected from two dairy farms. C. perfringens was detected in 26 out of 103 (25.2%) neonatal calf samples, 7 out of 25 (28%) human stool samples and 17 out of 50 (34%) environmental samples. C. perfringens type A strains were predominant in neonatal calves (24/26; 92.3%) and associated environment (15/17; 88.2%). In contrast, strains from dairy workers mostly belonged to type F (5/7; 71.4%), which also carried the beta2 toxin gene. Seventeen strains were analyzed by multilocus sequence typing (MLST) for studying genotypic relationship along with 188 C. perfringens strains available from public databases. A total of 112 sequence types (STs) were identified from 205 C. perfringens strains analyzed. A Clonal complex (CC) represented by three STs (ST 98, ST 41 and ST 110) representing predominantly type F (18/20 strains) were mostly associated with human illnesses. Among predominant STs, ST 54 was associated with enteritis cases in foals and dogs and ST 58 associated with necrotic enteritis in poultry. Seventeen Indian strains were assigned to 13 STs. Genetic relatedness among strains of calves, dairy worker and associated environments indicate inter-host transfers and zoonotic spreads.

Rapid and visual detection of Leptospira in urine by LigB-LAMP assay with pre-addition of dye.

Leptospirosis is considered to be the most widespread zoonotic disease caused by pathogenic species of Leptospira. The present study reports a novel set of primers targeting LigB gene for visual detection of pathogenic Leptospira in urine samples through Loop-mediated isothermal amplification (LAMP). The results were recorded by using Hydroxyl napthol blue (HNB), SYBR GREEN I and calcein. Analytical sensitivity of LAMP was as few as 10 leptospiral organisms in spiked urine samples from cattle and dog. LigB gene based LAMP, termed as LigB-LAMP, was found 10 times more sensitive than conventional PCR. The diagnostic specificity of LAMP was 100% when compared to SYBR green qPCR for detection of Leptospira in urine samples. Though qPCR was found more sensitive, the rapidity and simplicity in setting LAMP test followed by visual detection of Leptospira infection in clinical samples makes LigB-LAMP an alternative and favourable diagnostic tool in resource poor setting.

Proteomic analysis and identification of cell surface-associated proteins of Clostridium chauvoei.

Blackleg is a highly fatal disease of cattle and sheep, caused by Clostridium chauvoei, a Gram positive, anaerobic, spore forming bacteria. Cell surface-associated proteins play a major role in inducing the protective immunity. However, the identity of a majority of cell surface-associated proteins of C. chauvoei is not known. In the present investigation, we have used SDS-PAGE, 2D-gel electrophoresis and Western blotting followed by mass spectrometry to identify cell surface-associated proteins of C. chauvoei. Among the identified proteins, which have shown to offer protective antigencity in other bacteria, Enolase, Chaperonin, Ribosomal protein L10, Glycosyl Hydrolase and Flavoprotein were characterized by sequencing and their overexpression in Escherichia coli. In conclusion, cell surface-associated proteins were identified using proteomic approach and the genes for the immunoreactive proteins were expressed, which may prove to be potential diagnostic or vaccine candidates.

Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay.

Several pathogens including Brucella spp. are shed in semen of infected bulls and can be transmitted to cows through contaminated semen during artificial insemination. The present study reports omp2a and bcsp31 gene based loop-mediated isothermal amplification (LAMP) assays for detection of Brucella genomic DNA in semen from infected bulls. The positive results could be interpreted visually by change in colour of reaction mixture containing hydroxyl naphthol blue (HNB) dye from violet to sky blue. LAMP assays based on omp2a and bcsp31 could detect as little as 10 and 100 fg of B. abortus S19 genomic DNA, respectively. Sensitivity of omp2a and bcsp31 LAMP assays for direct detection of organisms in bovine semen was 2.28 × 10(1) CFU and 2.28 × 10(2) CFU of B. abortus S19 in spiked bovine semen, respectively. The omp2a LAMP assay was found equally sensitive to TaqMan probe based real-time PCR and 100 times more sensitive than conventional PCR in identifying Brucella in spiked semen. The diagnostic applicability of the omp2a LAMP assay was evaluated with seventy-nine bovine semen samples and results were re-evaluated through TaqMan probe based real-time PCR and conventional PCR. Taken together, the omp2a LAMP assay is easy to perform, rapid and sensitive in diagnosis of Brucella spp. in bovine semen.

Evaluation of recombinant LigB antigen-based indirect ELISA and latex agglutination test for the serodiagnosis of bovine leptospirosis in India.

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of the genus Leptospira, causing febrile infection characterized by multi-organ failure in humans and animals. Leptospiral Ig-like protein B (LigB) is a surface-expressed antigen that mediates host cell invasion or attachment. In this study, N-terminal conserved region of LigB protein (46 kDa) was evaluated for its diagnostic potential to detect anti-leptospiral antibodies in the sera of various animal species. Dot blot analysis revealed immunoreactivity of Leptospira-positive sera of cattle, buffalo, dog, sheep and goat to purified LigB protein. We have analyzed 1126 bovine serum samples, collected from Northern and Eastern part of India, by microscopic agglutination test (MAT) and recombinant LigB (rLigB) based ELISA and latex agglutination test (LAT). The sensitivity of rLigB based ELISA for 554 MAT positive sera was 96.9% and the specificity with 572 MAT negative sera was 91.08% whereas LAT showed sensitivity and specificity of 93.68% and 92.31%, respectively. Kappa values of 0.879 and 0.860 for recombinant antigen based ELISA and LAT indicate excellent agreement with the gold standard serological test, MAT, for the detection of anti-leptospiral antibodies in sera. Further, LAT based on rLigB antigen is a simple and rapid test, suitable for serodiagnosis of leptospirosis under field conditions, owing to its portability and longer shelf life.

Diagnostic and prophylactic potential of a stabilized foot-and-mouth disease serotype Asia1 virus like particles designed through a structure guided approach.

Intact capsids of foot-and-mouth disease virus (FMDV) play a vital role in eliciting a protective immune response. Any change in the physico-chemical environment of the capsids results in dissociation and poor immunogenicity. Structural bioinfomatics studies have been carried out to predict the amino acids at the interpentameric region that resulted in the identification of mutant virus-like particles(VLPs) of FMDV serotype Asia1/IND/63/1972. The insect cell expressed VLPs were evaluated for their stability by sandwich ELISA. Among 10 mutants, S93H showed maximum retention of antigenicity at different temperatures, indicating its higher thermal stability as revealed by the in-silico analysis and retained the antigenic sites of the virus demonstrated by Sandwich ELISA. The concordant results of the liquid phase blocking ELISA for estimation of antibody titre of known sera with stable mutant VLP as antigen in place of virus antigen demonstrate its diagnostic potential. The stable mutant VLP elicited a robust immune response with 85.6 % protection in guinea pigs against virus challenge. The stabilized VLP based antigen requires minimum biosafety and cold storage for production and transit besides, complying with differentiation of infected from vaccinated animals. It can effectively replace the conventional virus handling during antigen production for prophylactic and diagnostic use.

Genome-wide DNA methylation profiles regulate distinct heat stress response in zebu (Bos indicus) and crossbred (Bos indicus × Bos taurus) cattle.

Epigenetic variations result from long-term adaptation to environmental factors. The Bos indicus (zebu) adapted to tropical conditions, whereas Bos taurus adapted to temperate conditions; hence native zebu cattle and its crossbred (B indicus × B taurus) show differences in responses to heat stress. The present study evaluated genome-wide DNA methylation profiles of these two breeds of cattle that may explain distinct heat stress responses. Physiological responses to heat stress and estimated values of Iberia heat tolerance coefficient and Benezra's coefficient of adaptability revealed better relative thermotolerance of Hariana compared to the Vrindavani cattle. Genome-wide DNA methylation patterns were different for Hariana and Vrindavani cattle. The comparison between breeds indicated the presence of 4599 significant differentially methylated CpGs with 756 hypermethylated and 3845 hypomethylated in Hariana compared to the Vrindavani cattle. Further, we found 79 genes that showed both differential methylation and differential expression that are involved in cellular stress response functions. Differential methylations in the microRNA coding sequences also revealed their functions in heat stress responses. Taken together, epigenetic differences represent the potential regulation of long-term adaptation of Hariana (B indicus) cattle to the tropical environment and relative thermotolerance.

Isolation and characterization of vB_SenS_Ib_psk2 bacteriophage against drug-resistant Salmonella enterica serovar Kentucky.

Salmonella enterica serovar Kentucky is one of the food-borne zoonotic pathogens which is isolated in high frequency from poultry meat in the recent decades and is known for its multidrug resistance. The current study was aimed to isolate and characterize a bacteriophage against S. enterica serovar Kentucky isolate, 5925, which showed resistance to at least seven antibiotics and to study its efficiency to decontaminate S. Kentucky from chicken skin. The bacteriophage against S. enterica serovar Kentucky was isolated and was named vB_SenS_Ib_psk2 representing the place, source, and host. Electron microscopy revealed that the phage possesses isometric head and contractile tail, indicative of Siphoviridae family. Molecular detection of major capsid protein E gene yielded 511 bp, and NCBI blast analysis revealed that the phage belonged to the genus chivirus. The optimum temperature and pH for phage survival and multiplication were found to be - 20 to 42 °C and 6-10, respectively. One-step growth curve experiment of vB_SenS_Ib_psk2 revealed a latent period of 20 min and burst size of 253 phages/bacterial cell. The host susceptibility studies revealed that 83% of MDR isolates of S. enterica were susceptible to vB_SenS_Ib_psk2. Artificial spiking studies on chicken skin revealed that high multiplicity of infection (MOI) of phages of 106 pfu/mL is required for significant reduction (p ≤ 0.01) of bacterial concentration (0.14 ± 0.04) after 24-h incubation at 8 °C compared to group 1 (2.55 ± 0.89 cfu/mL).

Early-Life Intervention of Lactoferrin and Probiotic in Suckling Piglets: Effects on Immunoglobulins, Intestinal Integrity, and Neonatal Mortality.

The aim of this study was to determine the effects of early-life bovine lactoferrin and host specific probiotic interventions on growth performance, mortality, and concentrations of immunoglobulin A and immunoglobulin G and transforming growth factor beta 1 (a marker of intestinal integrity) in serum of neonatal piglets. A total of eight piglet litters from parity matched sows were randomly divided into four groups and assigned to one of the four interventions: control (sterile normal saline), bovine lactoferrin (100 mg bovine lactoferrin), probiotic (1 × 109 colony forming unit (cfu) of swine origin Pediococcus acidilactici FT28 probiotic), and bovine lactoferrin + probiotic (100 mg bovine lactoferrin and 1 × 109 CFU of P. acidilactici FT28 probiotic). All the interventions were given once daily through oral route for first 7 days of life. The average daily gain (p = 0.0004) and weaning weight (p < 0.0001) were significantly improved in the probiotic group. The piglet survivability was significantly higher in bovine lactoferrin and probiotic groups than control group in Log-rank (Mantel-Cox) test. The concentrations of immunoglobulin A on day 21 in bovine lactoferrin, probiotic, and bovine lactoferrin + probiotic groups increased significantly (p < 0.05). Immunoglobulin G concentrations on day 7 and 15 in bovine lactoferrin and bovine lactoferrin + probiotic groups and on day 15 in probiotic group were significantly (p < 0.05) elevated, whereas, the concentration of transforming growth factor-ß1 was significantly (p < 0.05) increased from day 7 to 21 in all the supplemented groups. In conclusion, the early-life bovine lactoferrin and P. acidilactici FT28 probiotic interventions reduced the mortality in the suckling piglets by promoting the systemic immunity and enhancing the intestinal integrity.

Development and validation of multiplex PCR based molecular serotyping of Salmonella serovars associated with poultry in India.

Salmonella species are Gram-negative bacteria with more than 2600 serovars. Among these serovars, many are associated with various diseases in livestock and humans. White Kauffman Le-Minor (WKL) serotyping scheme applies specific serum to determine the serovars of Salmonella. Recent studies have applied molecular methods for serovar predictions. These methods include PCR, hybridization and sequence data to detect/predict serovar-specific genetic elements. Among these, PCR is a robust method if the unique genetic element is already known. Within this context, also involving novel primers, two multiplex PCR assays were standardized to detect six important Salmonella serovars viz. Typhimurium, Enteritidis, Kentucky, Infantis, Virchow and Gallinarum associated with poultry in India. The developed PCR assays showed targeted serovar specificity. Serial dilution experiments of both kit-based and crude lysate DNA preparations indicated similar applicability of both methods for testing from pure cultures. Further the developed assays were validated with 25 recent field isolates to confirm the applicability in routine diagnosis. The PCR assay could predict all the targeted serovars (17/25) with 100% specificity (CI-95%; 0.63-1). Molecular serotyping can reduce the number of serum used in comparison to the conventional serotyping which involves more random application of serum.

Antimicrobial resistance in Indian isolates of non typhoidal Salmonella of livestock, poultry and environmental origin from 1990 to 2017.

A retrospective antimicrobial resistance study of nontyphoidal Salmonella enterica isolates from India during 1990-2017 was conducted to study the microbial susceptibility to antibiotics. A total of 271 Salmonella enterica isolates from poultry (n = 146), farm animals (n = 55) and environmental sources (n = 70) were tested for susceptibility using 15 antimicrobial drugs. The drug classes include aminoglycosides, phenicols, cephalosporins, penicillins, carbapenems, fluoroquinolones, and sulphonamide-trimethoprim. Study revealed that overall, 133 (49.08%) of 271 isolates were resistant to ≥ 1 antimicrobial drugs and 81 (29.89%) out of 271 isolates were multidrug resistant (resistance to ≥ 3 drugs). Majority (68.96%) of Typhimurium serovars (n = 87) were susceptible to all antibiotics tested, whereas only 5% Kentucky serovars (n = 40) were pan susceptible. All the drugs revealed decreasing trend of susceptibility from 1990 towards 2017 except cephalosporins and carbapenems. Statistical analysis of association between time period and antimicrobial resistance revealed a significance of < 0.05. Molecular detection of genetic determinants associated with antimicrobial resistance revealed the presence of genes like class I integrons, sul1, sul2, catIII, cmlA, dfrA, blaTEM, blaAmpC in the resistant isolates. Furthermore, plasmid mediated quinolone resistant determinants like qnrD and qnrS were also reported in the current study.