Virulence Genotyping of Pasteurella multocida

In this study, 108 P. multocida isolates recovered from various host animals such as cattle, buffalo, swine, poultry (chicken, duck, and emu) and rabbits were screened for carriage of 8 virulence associated genes. The results revealed some unique information on the prevalence of virulence associated genes among Indian isolates. With the exception of toxA gene, all other virulence associated genes were found to be regularly distributed among host species. Association study between capsule type and virulence genes suggested that pfhA, nanB, and nanH genes were regularly distributed among all serotypes with the exception of CapD, whereas toxA gene was found to be positively associated with CapD and CapA. The frequency of hgbA and nanH genes among swine isolates of Indian origin was found to be less in comparison to its equivalents around the globe. Interestingly, very high prevalence of tbpA gene was observed among poultry, swine, and rabbit isolates. Likewise, very high prevalence of pfhA gene (95.3%) was observed among Indian isolates, irrespective of host species origin

Cloning and sequence analysis of hyaluronoglucosaminidase (nagH) gene of Clostridium chauvoei

Aim: Blackleg disease is caused by Clostridium chauvoei in ruminants. Although virulence factors such as C. chauvoei toxin A, sialidase, and flagellin are well characterized, hyaluronidases of C. chauvoei are not characterized. The present study was aimed at cloning and sequence analysis of hyaluronoglucosaminidase (nagH) gene of C. chauvoei. Materials and Methods: C. chauvoei strain ATCC 10092 was grown in ATCC 2107 media and confirmed by polymerase chain reaction (PCR) using the primers specific for 16-23S rDNA spacer region. nagH gene of C. chauvoei was amplified and cloned into pRham-SUMO vector and transformed into Escherichia cloni 10G cells. The construct was then transformed into E. cloni cells. Colony PCR was carried out to screen the colonies followed by sequencing of nagH gene in the construct. Results: PCR amplification yielded nagH gene of 1143 bp product, which was cloned in prokaryotic expression system. Colony PCR, as well as sequencing of nagH gene, confirmed the presence of insert. Sequence was then subjected to BLAST analysis of NCBI, which confirmed that the sequence was indeed of nagH gene of C. chauvoei. Phylogenetic analysis of the sequence showed that it is closely related to Clostridium perfringens and Clostridium paraputrificum. Conclusion: The gene for virulence factor nagH was cloned into a prokaryotic expression vector and confirmed by sequencing

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Not AvailableClostridium chauvoei causes fatal black quarter infection in cattle and buffaloes. The quorum sensing (QS) system, a bacterial cell to cell communication process, of the pathogen was characterized in the current study. The results indicated that C. chauvoei lacked luxS (autoinducer-2) based quorum sensing as detected by the sensor strain Vibrio harveyi BB170. This was supported by absence of luxS gene in C. chauvoei genome. However, the genomic analysis indicated the presence of agrBD system in all three genomes of C. chauvoei available at the NCBI database. The AgrD, which synthesizes QS messenger autoinducing peptide, was a 44 amino acid protein which shared 59% identity and 75% similarity with AgrD of C. perfringens strain 13 and 56% identity (20% coverage) with Staphylococcus aureus N315. The functional cysteine amino acid was conserved in all the strains. The genomic organisation further suggests the presence of diguanylate cyclase, a gene responsible for synthesis of secondary messenger cyclic di-GMP, at 3’ immediate downstream of agrD gene. The real time expression analysis for agrD gene indicated that expression was better at 37 C (1.9e3.7 fold increase) compared to a higher temperature of 40 C. However, stable expression was observed at different growth stages (log and early stationary phase) with 0.8e1.4 fold changes in expression pattern. The results indicate the presence of a constitutively expressed agrBD quorum sensing system in C. chauvoei.Not Availabl

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Not AvailableTreatment of bovine mastitis caused by Staphylococcus (S.) aureus is becoming very difficult due to the emergence of multidrug-resistant strains. Hence, the search for novel therapeutic alternatives has become of great importance. Consequently, bacteriophages and their endolysins have been identified as potential therapeutic alternatives to antibiotic therapy against S. aureus. In the present study, the gene encoding lysin (LysSA4) in S. aureus phage SA4 was cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clone revealed a single 802-bp open reading frame encoding a partial protein with a calculated mass of 30 kDa. Results of this analysis also indicated that the LysSA4 sequence shared a high homology with endolysin of the GH15 phage and other reported phages. The LysSA4 gene of the SA4 phage was subsequently expressed in Escherichia coli. Recombinant LysSA4 induced the lysis of host bacteria in a spot inoculation test, indicating that the protein was expressed and functionally active. Furthermore, recombinant lysin was found to have lytic activity, albeit a low level, against mastitogenic Staphylococcus isolates of bovine origin. Data from the current study can be used to develop therapeutic tools for treating diseases caused by drug-resistant S. aureus strains.Not Availabl

Cloning and expression of P67 protein of Mycoplasma leachii

Aim: The present study was undertaken to clone, express and study the immunogenicity of P67 protein of Mycoplasma leachii. Materials and Methods: P67 gene was amplified from genomic DNA of M. leachii. The polymerase chain reaction (PCR) product was inserted in pRham N-His SUMO Kan vector and was used to transform competent Escherichia cloni 10G cells. Recombinant protein expression was done by inducing cells with 0.2% Rhamnose. Purification was done using nickel nitrilotriacetic acid affinity chromatography. Western blot and dot blot analysis were performed to assess the immunoreactivity of P67 protein. Results: PCR amplicon size of P67 gene was found to be 1500 base pair. The size of the fusion protein with SUMO tag was 79 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The recombinant P67 fusion protein expressed in pRham N-His SUMO Kan vector was found to be immunogenic in both western blot and dot blot analysis. Conclusion: Western blot and dot blot analysis of P67 protein of M. leachii revealed that the protein is immunogenic. Further work is needed to evaluate the role of P67 antigen of M. leachii as an immunodiagnostic agent

Expression of canine relaxin precursor protein in <i>Pichia pastoris </i> host: A possible tool for early pregnancy diagnosis

44-49In canines, a physical examination and ultrasound technique remain the most common methods of pregnancy diagnosis. The rapid detection of pregnancy is important in canines. There are several reports of peptide hormone relaxin to be exclusively produced in pregnant bitches and not by non-pregnant or pseudo pregnant bitches. We present here the expression of canine relaxin in Pichaia pastoris GS115 host. The cDNA prepared using canine placental total RNA was used to amplify 432 bprelaxin gene, which was subsequently cloned in pPICZ-alphaA vector and followed by transformation into Pichia pastoris GS115 strain by electroporation. The zeocin resistant recombinant clones were confirmed by colony PCR and sequencing. One of the positive clones was selected for protein expression in culture medium. The supernatant was taken and precipitated using 20% TCA in acetone. The recombinant relaxin precursor protein showed a mol wt of 24 kDa, which is higher than the expected 19 kDa and that could be due to glycosylation in the Pichia pastoris. The 24 kDa recombinant protein was detected up to 48 h of post-induction. This is the first report describing the expression of recombinant canine relaxin in Pichia pastoris. The polyclonal antiserum against canine relaxin would be useful for detection of pregnancy in bitches and also in assessing the prognosis of canine mammary tumors

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Not AvailableWe report here the genome sequence of Vibrio campbellii LB102, isolated from the broodstock rearing system of a shrimp hatchery in India. Sequence analysis revealed the presence of effector toxins of the type III (YopT, sharing 39% identity with Yersinia pestis) and type VI (VgrG-3 and hemolysin coregulated protein of V. cholerae) secretion systems.Not Availabl

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Not AvailableGenome wide host gene expression analysis in mice experimentally infected with Pasteurella multocid

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Not AvailableAims: 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.Not Availabl

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Not AvailableSalmonella 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 incomparison to the conventional serotyping which involves more random application of serum.Not Availabl