Host responses and bacterial colonization following inoculation of Pasteurella multocida P52 strain into unvaccinated and aluminum hydroxide gel hemorrhagic septicemia vaccinated mice.

Hemorrhagic septicemia is a highly infectious and contagious disease caused by Pasteurella multocida serogroup B:2 in tropical Asian and African countries. The acute inflammatory responses induced by Pasteurella multocida are the main cause of death in hemorrhagic septicemia. Therefore, present study was undertaken to examine the blood cytokine expression profiles (TNF-α, IL-1ß, and IL-6), bacterial colonization and histopathological changes of intraperitoneally and subcutaneously challenged vaccinated and unvaccinated mice with 102 CFU of P. multocida P52. The observations were made at 6, 12, 18, 24 h and 48 h intervals. Real-time PCR based blood cytokine profiles (TNF-α, IL-1ß, and IL-6) measurement revealed a significantly higher amount of pro-inflammatory cytokines expression in the unvaccinated challenged group of mice than the vaccinated challenged group. There was heavy bacterial load in all organs of mice viz. trachea, lung, spleen, within 6 h of challenge in both vaccinated and unvaccinated group of mice, but bacterial load increased in the unvaccinated challenged group of mice with respect to time whereas the load were constant in the vaccinated challenged group. Histopathological changes were mild in the vaccinated challenged group of mice in comparison to the unvaccinated challenged group. There was no significant difference in the bacterial load, histopathological changes and cytokines expression when challenged through different routes.

First report of whole genome sequence of septicemic Pasteurella multocida serovar B:2 'Soron' strain isolated from swine.

Pig pasteurellosis, caused by Pasteurella multocida, is an acute infection that also has economic implications for pig farmers. We report the complete genome sequence of a P. multocida, serovar B:2 'Soron' strain isolated from the blood of a pig that had died of pasteurellosis in India. The isolate was not found to be haemorrhagic septicaemia (HS) specific B:2 by the PCR assay. The genome of 'Soron' strain is a single circular chromosome of 2,272,124 base pairs in length and contains 2014 predicted coding regions, 4 ribosomal RNA operons, and 52 tRNAs. It has 1812 protein-coding genes that were also found in reference sequence PmP52Vac. Phylogenetic analysis revealed that Pm_P52VAc and P. multocida 'Soron' serovar B:2 were clustered in different clades. Pasteurella multocida 'Soron' serovar B:2 was found to cluster with the same ancestor of Pm70, which is of avian origin. The genome was found to contain regions that encode proteins which may confer resistance to various antibiotics including cephalosporin, which is used to treat pasteurellosis. The isolate was also found to harbour a phage region. This strain represents a novel multi-locus sequence type (MLST) that has not been previously identified, as all of the alleles used for MLST were found, but did not match any of the alleles in the database with 100% nucleotide identity. The most closely related ST was ST221. This is the first whole-genome sequence from P. multocida serovar B:2 of pig origin.

Molecular characterization of Indian rabies virus isolates by partial sequencing of nucleoprotein (N) and phosphoprotein (P) genes.

Rabies is endemic and an important zoonosis in India. There are very few reports available on molecular epidemiology of rabies virus of Indian origin. In this study to know the dynamics of rabies virus, a total of 41 rabies positive brain samples from dogs, cats, domestic animals, wildlife, and humans from 11 states were subjected to RT-PCR amplification of N gene between nucleotide N521-N1262 (742 bp) and P gene between nucleotide P239-P750 (512 bp). The N gene could be amplified from 30, while P gene from 41 samples, using specific sets of primers. The N gene-based phylogenetic analysis indicated that all Indian virus isolates are genetically closely related with a single cluster under arctic/arctic-like viruses. However, two distinct clusters were realized in P gene-based phylogeny viz., Rabies virus isolates of Punjab and Rabies virus isolates of remaining parts of India (other than Punjab). All the Indian rabies virus isolates were closely related to geography (>95% homology), but not to host species.

Detection of Mycobacterium avium & M. tuberculosis from human sputum cultures by PCR-RFLP analysis of hsp65 gene & pncA PCR.

BACKGROUND & OBJECTIVE: Identification of mycobacteria by conventional methods is slow, labour intensive and may at times fail to produce precise results. Molecular techniques developed in the recent past, overcome these disadvantages facilitating rapid identification of most species. We undertook this study to characterize mycobacteria isolated from sputa of human patients suspected to have tuberculosis by conventional methods and later, by polymerase chain reaction-restriction fragment length polymorphism analysis (PRA) of hsp65 gene and pncA PCR. METHODS: Twenty two mycobacteria isolated from 30 sputum samples were identified based on growth rate, pigmentation, cultural and biochemical properties and subjected to PRA of hsp65 gene involving amplification of hsp65 gene and digestion of the product with BstEII and HaeIII in separate reactions and analysis of digests by 3 per cent agarose gel electrophoresis. The mycobacteria were simultaneously evaluated by M. tuberculosis-specific and M. bovis-specific pncA PCR assays in separate reactions. RESULTS: With the conventional biochemical tests, the 22 sputum culture isolates were identified as M. tuberculosis (19) and M. avium complex (MAC) (3). PCR of hsp65 gene yielded 439 bp product in all the mycobacteria tested. The RFLP patterns of three MAC isolates with BstEII and HaeIII were identical to reference M. avium strain with two fragments in each of the digest. M. intracellulare reference strain showed a distinct pattern with 3 fragments each in both enzyme digests. The PRA of hsp65 confirmed MAC isolates as M. avium. M. tuberculosis isolates including H37Rv and M. bovis strains could not be discriminated by PRA of hsp65. The two pncA PCR assays (M. bovis-specific and M. tuberculosis-specific) detected specifically the respective organisms with an amplification product of 185 bp. The MAC strains yielded no amplification product in both the pncA PCR assays. INTERPRETATION & CONCLUSION: PRA profiles of hsp65 could differentiate MAC isolates into M. avium and M. intracellulare but could not distinguish between M. tuberculosis and M. bovis. pncA PCR assays were found specific in detecting the respective mycobacterial species. The study confirms utility of pncA PCR assays in differential identification of M. tuberculosis and M. bovis and that of PRA of hsp65 in the identification of M. avium.

Random amplified polymorphic DNA (RAPD) analysis of Mycobacterium tuberculosis strains in India.

The usefulness of random amplification of polymorphic DNA (RAPD) analysis for typing Indian strains of M. tuberculosis was investigated. M. tuberculosis H37Rv, M. tuberculosis DT and 42 clinical isolates of M. tuberculosis were subjected to RAPD-PCR using 7 random decamer primers. All 7 primers were found to be differentiated and produced specific RAPD profiles. The polymorphic amplicons served as RAPD markers for M. tuberculosis. The dendrograms, obtained by different primers, showed the discriminatory ability of the primers. RAPD analysis provided a rapid and easy means of identifying polymorphism in M. tuberculosis isolates, and it was found to be a valuable alternative epidemiological tool. In addition, the results of the present study showed heterogeneity in the M. tuberculosis strains in the population studied.

Rapid differentiation of Mycobacterium bovis and Mycobacterium tuberculosis based on a 12.7-kb fragment by a single tube multiplex-PCR.

The aim of this work was the design and validation of a rapid and easy single tube multiplex-PCR (m-PCR) assay for the unequivocal differential detection of Mycobacterium bovis and Mycobacterium tuberculosis. Oligonucleotide primers were based on the uninterrupted 229-bp sequence in the M. bovis genome and a unique 12.7-kb insertion sequence from the M. tuberculosis genome, which is responsible for species-specific genomic polymorphism between these two closely related pathogens. The m-PCR assay was optimized and validated using 22 M. bovis and 36 M. tuberculosis clinical strains isolated from diverse host species and 9 other non-tuberculous mycobacterial (NTM) strains. The designed primers invariably amplified a unique 168-bp (M. bovis-specific) and 337-bp (M. tuberculosis-specific) amplicon from M. bovis and M. tuberculosis strains, respectively. The accuracy of the assay, in terms of specificity, was 100%, as none of the NTM strains tested revealed any amplification product. As little as 20 pg of genomic DNA could be detected, justifying the sensitivity of the method. The m-PCR assay is an extremely useful, simple, reliable and rapid method for routine differential identification of cultures of M. bovis and M. tuberculosis. This m-PCR may be a valuable diagnostic tool in areas of endemicity, where bovine and human tuberculosis coexist, and the distinction of M. bovis from M. tuberculosis is required for monitoring the spread of M. bovis to humans.

A multiplex-PCR for the differentiation of Mycobacterium bovis and Mycobacterium tuberculosis.

A multiplex-polymerase chain reaction (PCR) assay based on one-step amplification and detection of two different mycobacterial genomic fragments was designed for differentiation of Mycobacterium bovis and Mycobacterium tuberculosis. The oligonucleotide primers were chosen from a 500-bp genomic fragment which is well conserved in M. bovis and the pncA gene (based on M. tuberculosis-specific nucleotide polymorphism, a cytosine residue at position 169), specific for M. tuberculosis. The multiplex-PCR allowed detection of a single product of 500 bp in M. bovis isolates while M. tuberculosis isolates generated a single product of 185 bp, with or without an additional product of 500 bp. None of the atypical mycobacterial isolates revealed any amplification products. The method was found to be highly specific and could detect as little as 20 pg of pure DNA. This multiplex-PCR assay, based on the 500-bp fragment and the pncA gene, may be very useful for the rapid and specific differentiation of these two closely related mycobacteria and easy to use in medical and veterinary microbiological laboratories.

Molecular fingerprinting of clinical isolates of Mycobacterium bovis and Mycobacterium tuberculosis from India by restriction fragment length polymorphism (RFLP).

Forty mycobacterial strains comprising clinical Indian isolates of Mycobacterium tuberculosis (28 field isolates +1H37 Rv) and Mycobacterium bovis (10 field isolates +1 AN5) were subjected to restriction fragment length polymorphism analysis (RFLP) using IS6110 and IS1081 probes. Most of these strains originated from dairy cattle herd and human patients from Indian Veterinary research Institute (IVRI) campus isolated from the period of 1986 to 2000. Our study showed presence of 8 copies of IS6110 in most of the M.tuberculosis (96.6%) strains irrespective of their origin with the exception of one M.tuberculosis strain with presence of an extra copy (3.4%). All M.bovis strains showed a single copy of IS6110 on the characteristic 1.9 kb restriction fragment. RFLP analysis with IS1081 invariably showed the presence of 5 copies in all isolates of M.bovis and M.tuberculosis at the same chromosomal location. Similarity of IS6110 RFLP fingerprints of M.tuberculosis strains from animals and human suggested the possibility of dissemination of single M.tuberculosis strain among animals as well as human. It was not possible to discriminate within the isolates of either M.tuberculosis or M.bovis, when IS1081 was used as target sequence. The IS6110 RFLP is a valuable tool for disclosing transmission chain of M. tuberculosis and M. bovis among humans as well as animals.

Classical swine fever in pigs: recent developments and future perspectives.

Classical swine fever (CSF) is one of the most devastating epizootic diseases of pigs, causing high morbidity and mortality worldwide. The diversity of clinical signs and similarity in disease manifestations to other diseases make CSF difficult to diagnose with certainty. The disease is further complicated by the presence of a number of different strains belonging to three phylogenetic groups. Advanced diagnostic techniques allow detection of antigens or antibodies in clinical samples, leading to implementation of proper and effective control programs. Polymerase chain reaction (PCR)-based methods, including portable real-time PCR, provide diagnosis in a few hours with precision and accuracy, even at the point of care. The disease is controlled by following a stamping out policy in countries where vaccination is not practiced, whereas immunization with live attenuated vaccines containing the 'C' strain is effectively used to control the disease in endemic countries. To overcome the problem of differentiation of infected from vaccinated animals, different types of marker vaccines, with variable degrees of efficacy, along with companion diagnostic assays have been developed and may be useful in controlling and even eradicating the disease in the foreseeable future. The present review aims to provide an overview and status of CSF as a whole with special reference to swine husbandry in India.

Evaluation and comparison of native and recombinant LipL21 protein-based ELISAs for diagnosis of bovine leptospirosis.

A 21-kDa leptospiral lipoprotein (LipL21) was evaluated for its diagnostic potential to detect bovine leptospirosis by ELISA. Both native LipL21 (nLipL21) and recombinant LipL21 (rLipL21) proteins were tested and compared regarding diagnostic efficiency, and no statistically significant difference was observed. The sensitivity of rLipL21 ELISA for 62 microscopic agglutination test (MAT) positive sera was 100% and the specificity with 378 MAT negative sera was 97.09%. Thus, rLipL21 protein-based ELISA could be used as an alternative to MAT for the diagnosis of bovine leptospirosis.

Cloning and molecular characterization of outer membrane protein A (ompA) gene from Mycobacterium bovis.

The ompA gene, 981 bp in size and identified in the Mycobacterium bovis genome, was cloned and sequenced. Predicted amino acid sequences showed a protein of molecular weight 33.5 kDa with an isoelectric point of 7.28 and an OmpA conserved domain at the carboxy terminal with signal sequence at the N-terminal. Homology search showed complete homology with the Mycobacterium tuberculosis ompA gene with varying degree of homology with omp genes of other bacteria, so it was predicted that it may act like porin. The ompA gene of M. bovis was expressed in the pProEX HTb expression vector. Recombinant protein OmpA, was purified by using a nickel affinity column. The expressed recombinant OmpA (32 kDa) was confirmed by western blot with Ni-NTA horseradish peroxidase (HRP) conjugate and with specific antiserum raised in rabbits. The effect of low pH (pH 6.0, 5.5 and 5) on the transcription of the ompA gene in M. bovis using real-time PCR showed that there was an increase in transcription of ompA in M. bovis cultured at low pH (maximum at pH 5.5).

Occurrence of RD9 region and 500 bp fragment among clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis.

The present investigation dealt with the identification of Mycobacterium tuberculosis and M. bovis by RD9 region and 500 bp fragment PCR assays. Eight M. tuberculosis and 5 M. bovis characterized and identified from 40 human sputum and 41 bovine lung specimens and 20 M. tuberculosis and 9 M. bovis strains maintained at Mycobacteria Laboratory, Indian Veterinary Research Institute were included in this study. In this way, 28 M. tuberculosis and 14 M. bovis strains and, for comparison and control purpose, M. tuberculosis H37Rv, M. bovis BCG, M. canetti, M. smegmatis, M. phlei, M. chelonae, M. kansasii, M. xenopi and M. avium were subjected to RD9 and 500 bp amplification by PCR. All M. tuberculosis strains, M. tuberculosis H37 Rv and M. canetti yielded a product of 333 bp which showed presence of RD9 region in these strains, whereas all M. bovis yielded a product of 206 bp with RD9 PCR assay. There was no ampli-fication product found in M. bovis BCG, M. xenopi, M. smegmatis, M. phlei, M. chelonae, M. kansasii, and M. avium. PCR based on 500 bp fragment showed a product of 500 bp in all M. bovis strains and M. bovis BCG. There was no amplification product of 500 bp found in M. canetti, M. smegmatis, M. phlei, M. chelonae, M. avium, M. kansasii, M. xenopi and was absent in all M. tuberculosis strains. The PCR assay results correlated 100% with the culture and biochemical results of the isolates. Our study suggested that PCR based on RD9 and 500 bp may effectively identify two closely related species of M. tuberculosis and M. bovis.