Profiling of Brevibacillus borstelensis transcriptome exposed to high temperature shock.

To understand the molecular mechanisms underlying the ability of the bacteria to survive at high temperature, gene expression profile of Brevibacillusborstelensis at 55°C during 5 and 10min heat shock period was carried out by high-throughput sequencing technology. A total of 2555 non-redundant transcripts were annotated. A total of 575 genes at 5min and 400 genes at 10min exhibited significant differential expression in response to temperature upshift from 50 to 55°C. Genes up-regulated under heat shock were associated with metabolism (mtnE), membrane transport, signal transduction, transcriptional regulation (ycxD, codY) and folding and sorting (hsp90). A larger number of genes encoding hypothetical proteins were identified. RT-PCR experimental results carried out on genes expressed under heat shock were found to be consistent with transcriptome data. The results enhance our understanding of adaptation strategy of thermophilic bacteria thereby providing a strong background for in depth research in thermophiles.

Assessment of genetic diversity of Bacillus spp. isolated from eutrophic fish culture pond.

The genus Bacillus comprises of a diverse group with a wide range of nutritional requirements and physiological and metabolic diversity. Their role in nutrient cycle is well documented. 16S rDNA sequences do not always allow the species to be discriminated. In this study 40 Bacillus spp. obtained from fish culture pond and 10 culture type strains were analysed for their genomic diversity by PCR-RFLP of intergenic spacer region of 16S-23S and HSP60 genes. TaqI digestion of PCR products amplified by ITS PCR did not render distinctive RFLP patterns. Numerical analysis of ITS PCR-RFLP pattern differentiated the isolates into 11 clusters. Same species were found to be grouped in different clusters. But PstI digested PCR products amplified from HSP60 gene of the isolates showed distinctive RFLP patterns. The dendrogram constructed from HSP60 PCR-RFLP delineated the isolates into 11 clusters also. All the clusters, except cluster I grouped only one type of species. The results showed that Bacillus spp. could be clearly distinguished by PCR-RFLP of HSP60 gene. Therefore, the HSP60 gene is proposed as an additional molecular marker for discrimination of Bacillus group.

Survey of the transcriptome of Brevibacillus borstelensis exposed to low temperature shock.

Molecular mechanisms underlying the ability of Brevibacillus borstelensis to survive and adapt to various environmentally relevant stresses are poorly understood. To define organism's molecular response to low temperature, gene expression profile of B. borstelensis at 20 °C was carried out by high-throughput sequencing technology. A total of 4579 transcripts with a maximum transcript length of 9919 bp were annotated. Gene expression profiling identified 712 genes that were significantly up- or down-regulated during cold shock. Functional categorization of the differentially expressed genes revealed that response to stress, regulation of transcription, transport, signal transduction and cytoplasm were the differentially regulated processes. The microbial stress responsive genes (hsp90, hslU, grpE, dnaK, dnaJ, hslV) and genes under regulatory adaptive responses (rpoN) were identified. The gene encoding cold shock protein purine nucleoside phosphorylase was found to be remarkably up-regulated. RT-PCR experiments carried out on genes expressed under cold shock independently verified the transcriptome data results. In addition, a large number of genes encoding hypothetical protein were identified. The brief survey of the transcripts obtained in response to cold shock underlines the survival strategy of thermophilic bacteria exposed to low temperature environment, which is further helpful in generating genetic information associated with this bacteria.

Sequence polymorphism of GroEL gene in natural population of Bacillus and Brevibacillus spp. that showed variation in thermal tolerance capacity and mRNA expression.

GroEL, a class I chaperonin, plays an important role in the thermal adaptation of the cell and helps to maintain the viability of the cell under heat shock condition. Function of groEL in vivo depends on the maintenance of proper structure of the protein which in turn depends on the nucleotide and amino acid sequence of the gene. In this study, we investigated the changes in nucleotide and amino acid sequences of the partial groEL gene that may affect the thermotolerance capacity as well as mRNA expression of bacterial isolates. Sequences among the same species having differences in the amino acid level were identified as different alleles. The effect of allelic variation on the groEL gene expression was analyzed by comparison and relative quantification in each allele under thermal shock condition by RT-PCR. Evaluation of K a/K s ratio among the strains of same species showed that the groEL gene of all the species had undergone similar functional constrain during evolution. The strains showing similar thermotolerance capacity was found to carry same allele of groEL gene. The isolates carrying allele having amino acid substitution inside the highly ATP/ADP or Mg(2+)-binding region could not tolerate thermal stress and showed lower expression of the groEL gene. Our results indicate that during evolution of these bacterial species the groEL gene has undergone the process of natural selection, and the isolates have evolved with the groEL allelic sequences that help them to withstand the thermal stress during their interaction with the environment.

Upregulation of transcripts for metabolism in diverse environments is a shared response associated with survival and adaptation of Klebsiella pneumoniae in response to temperature extremes.

Klebsiella pneumoniae being ubiquitous in nature encounters wide differences in environmental condition. The organism's abundance in natural water reservoirs exposed to temperature variation forms the basis of its persistence and spread in the soil and other farm produce. In order to investigate the effect of temperature changes on the survival and adaptation of the bacteria, the transcriptional response of K. pneumoniae subjected to low (20 °C) and high (50 °C) temperature shock were executed using Applied Biosystems SOLiD platform. Approximately, 33 and 34% of protein coding genes expressed in response to 20 and 50 °C, respectively, displayed significant up- or downregulation (p < 0.01). Most of the significantly expressed transcripts mapped to metabolism, membrane transport, and cell motility were downregulated at 50 °C, except for protein folding, sorting, and degradation, suggesting that heat stress causes general downregulation of gene expression together with induction of heat shock proteins. While at 20 °C, the transcripts of carbohydrate, lipid, and amino acid metabolism were highly upregulated. Hypothetical proteins as well as canonical heat and cold shock proteins, viz. grpE, clpX, recA, and deaD were upregulated commonly in response to 20 and 50 °C. Significant upregulation of genes encoding ribosomal proteins at 20 and 50 °C possibly suggest their role in the survival of K. pneumoniae cells under low- and high-temperature stress.

Construction of Geobacillus thermoglucosidasius cDNA library and analysis of genes expressed in response to heat stress.

Thermophiles exhibit various kinds of molecular mechanisms to survive in extreme environment, but their behavioral responses to long duration stress is poorly understood until date. In the present study, we have prospected for the genes differentially expressed in response to long duration heat stress in thermophilic bacteria. A cDNA library was constructed from Geobacillus thermoglucosidasius grown with a temperature upshift of 10 °C from optimum growth temperature of 45 °C for 16 h. A total of 451 clones from the library were sequenced with accurate base calling that generated 257 high quality sequences with an average read length of 350 bp. We queried our collection of single pass sequences against the NCBI non-redundant database using the BLASTX algorithm and obtained sequences that showed significant similarity (>60%) with heat shock proteins, metabolic proteins and hypothetical proteins. The expressed sequence tags (ESTs) expressed in response to heat stress were annotated that further commuted a strong interaction network among one another. The ESTs based on the best hits were validated by RT-PCR. Di- and tri-nucleotide repeat motifs were also found to be associated with 17 genes involved in heat shock response, metabolism, transport and transcriptional regulation. The present results provide the novel identification of the putative genes responsible for imparting tolerance to bacteria under heat stress and unveil their role for survival of life in environmental extremes.

Specific oligonucleotide primers for detection of endoglucanase positive Bacillus subtilis by PCR.

A polymerase chain reaction (PCR) assay was developed for discrimination of Bacillus subtilis from other members of B. subtilis group as well as rapid identification from environmental samples. Primers ENIF and EN1R from endoglucanase gene were used to amplify a1311 bp DNA fragment. The specificity of the primers was tested with seven reference strains and 28 locally isolated strains of endoglucanase positive Bacillus species. The PCR product was only produced from B. subtilis. The results demonstrated high specificity of two oligonucleotides for B. subtilis. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of B. subtilis. To our knowledge this is the first report of a B. subtilis specific primer set.

Inductive expression of toll-like receptor 5 (TLR5) and associated downstream signaling molecules following ligand exposure and bacterial infection in the Indian major carp, mrigal (Cirrhinus mrigala).

Toll-like receptors (TLRs) are one of the key components of innate immunity. Among various types of TLRs, TLR5 is involved in recognizing bacterial flagellin and after binding, it triggers myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway to induce pro-inflammatory cytokines. In this report, we analyzed the expression profile of TLR5 and its associated downstream signaling molecules like MyD88 and tumor necrosis factor (TNF) receptor-associated factor (TRAF) 6 in the Indian major carp (IMC), mrigal (Cirrhinus mrigala) which is highly commercially important fish species in the Indian subcontinent. Ontogeny analysis of TLR5, MyD88 and TRAF6 revealed constitutive expression of these genes in all embryonic developmental stages, and highlighted the importance of embryonic innate immune defense system in fish. Tissue specific expression analysis of these genes by quantitative real-time PCR (qRT-PCR) revealed their wide distribution in various organs and tissues; highest expression of TLR5 and MyD88 was in liver and TRAF6 was in kidney. Modulation of TLR5, MyD88 and TRAF6 gene expression, and the induction of interleukin (IL)-8 and TNF-α were analyzed in various organs by qRT-PCR following flagellin stimulation, and Aeromonas hydrophila and Edwardsiella tarda infection. In the treated fish, majority of the tested tissues exhibited significant induction of these genes, although with varied intensity among the tissues and with the types of treatments. Among the examined tissues, a significant relationship of TLR5 induction, MyD88 and TRAF6 up-regulation, and enhanced expression of IL-8 and TNF-α gene transcripts was observed in the blood and intestine of both flagellin stimulated and bacteria infected fish. These findings may indicate the involvement of TLR5 in inducing IL-8 and TNF-α, and suggest the important role of TLR5 in augmenting innate immunity in fish in response to pathogenic invasion. This study will enrich the information in understanding the innate immune mechanism in fish and may be helpful in developing preventive measures against infectious diseases in fish.

Molecular characterization of nucleotide binding and oligomerization domain (NOD)-2, analysis of its inductive expression and down-stream signaling following ligands exposure and bacterial infection in rohu (Labeo rohita).

Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic pattern recognition receptor (PRR) and is a member of NOD like receptor (NLR) family. It senses a wide range of bacteria and viruses or their products and is involved in innate immune responses. In this report, NOD-2 gene was cloned and characterized from rohu (Labeo rohita) which is highly commercially important fish species in the Indian subcontinent. The full length rohu NOD-2 (rNOD-2) cDNA comprised of 3176 bp with a single open reading frame (ORF) of 2949 bp encoding a polypeptide of 982 amino acids (aa) with an estimated molecular mass of 109.65 kDa. The rNOD-2 comprised two N-terminal CARD domains (at 4-91 aa and 111-200 aa), one NACHT domain (at 271-441 aa) and seven C-terminal leucine rich repeat (LRR) regions. Phylogenetically, rNOD-2 was closely related to grass carp NOD-2 (gcNOD2) and exhibited significant similarity (94.2%) and identity (88.6%) in their amino acids. Ontogeny analysis of rNOD-2 showed its constitutive expression across the developmental stages, and highlighted the embryonic innate defense system in fish. Tissue specific analysis of rNOD-2 by quantitative real-time PCR (qRT-PCR) revealed its wide distribution; highest expression was in liver followed by blood. In response to PGN and LTA stimulation, Aeromonas hydrophila and Edwardsiella tarda infection, and poly I:C treatment, expression of rNOD-2 and its associated downstream molecules RICK and IFN-γ were significantly enhanced in the treated fish compared to control. These findings suggested the key role of NOD-2 in augmenting innate immunity in fish in response to bacterial and viral infection. This study may be helpful for the development of preventive measures against infectious diseases in fish.

Characterization of Vibrio species isolated from freshwater fishes by ribotyping.

Three Vibrio species from the resident microflora of gastrointestinal tract of freshwater carps and prawns were isolated and confirmed biochemically as V. fluvialis from Cyprinus carpio/Labeo rohita; V. parahaemolyticus from Macrobrachium rosenbergii and V. harveyi from Macrobrachium malcomsoni. The genetic relationship among these Vibrio species was carried out by polymerase chain reaction (PCR) amplification of 16S rRNA gene followed by restriction digestion with Hae III, Bam HI and Pst I. Dendogram based on ribotyping showed the isolated Vibrios were differentiated into three clusters. V. harveyi was closely related to V. vulnificus (reference Microbial type Culture Collection (MTCC) strain) and distantly related to V. parahaemolyticus as well as V. fluvialis.

Phenotypic and genetic characterization of Edwardsiella tarda isolated from pond sediments.

The extent of genotypic and phenotypic diversity of Edwardsiella tarda isolated from pond sediment was assessed by SDS-PAGE, Plasmid Profiling and ERIC-PCR. SDS-PAGE of whole cell protein extracts reveals 20-23 discrete bands with molecular wt of 14-110 kDa. Several bands with molecular weight range of 38-83 kDa were present in all the isolates. Numerical analysis of protein electrophoregram delineated the isolates into four clusters. Two different types of plasmids having molecular mass of 23 kDa and 29 kDa were obtained by plasmid profiling. About 51% of the isolates carried both the plasmids. ERIC-PCR generates 3-7 bands with molecular mass of 14-1013 bp. Numerical analysis differentiated the ERIC pattern into 5 clusters at 60% similarly level. It was concluded that out of three methods ERIC-PCR was found to be more sensitive for intraspecific typing of E. tarda and can be used as a potential tool for epidemiological studies in the future.

Effect of endotoxin on the immunity of Indian major carp, Labeo rohita.

Endotoxin, a lipopolysaccharide component of outer cell wall membrane of the Gram-negative bacteria is a factor responsible for a number of biological effects including immunostimulatory activities in different animal species including fish. In this study, L. rohita yearlings of weight ranging from 80 to 100g were injected intraperitoneally with 0.5, 1, 2, 5, 10 and 20 EU/fish dose of endotoxin to find out its effect on the immunity. The L. rohita yearlings were found to resist the endotoxin dose up to 20 EU/fish and at the lower doses, i.e., at 1 and 2 EU/fish; it acted as an immune potentiator. Different serum and immune parameters like protein, globulin, lysozyme, respiratory burst activity, myeloperoxidase activity, natural agglutination titre were found to be significantly high (p<0.01) at a dose of 1 EU/fish. While at 10 and 20 EU/fish, most of these parameters were lower thereby indicating the immuno-suppressive nature of the endotoxin at these higher doses.

Characterisation of Pseudomonas aeruginosa isolated from freshwater culture systems.

Members of the genus Pseudomonas are important phytopathogens and agents of human infections, while other strains and species exhibit bioremediation and biocontrol activities. Species-specific detection of Pseudomonas species in the environment may help to gain a more complete understanding of the ecological significance of these microorganisms. The objective of present study was comparative analysis of biochemically and PCR based confirmed 10 isolates of Pseudomonas aeruginosa (6 from fish intestine and 4 from pond sediment). PCR-ribotyping and PAGE revealed that there was extensive heterogeneity at the genetic and protein levels. Both genetic and phenotypic heterogeneity were more in the sediment isolates compared to the fish isolates. SDS-PAGE clearly demonstrated the differences between fish and sediment isolates as evident from the higher range of protein profiling. In antibiotic sensitivity test no habitat specific antibiogram was obtained. Zinc adversely affected the DNA of all the isolates to be amplified by PCR as DNA banding pattern was different from normal DNA in stressed DNA. Thus stress, particularly, zinc may interfere monitoring of Pseudomonas by PCR.

Genotyping of Edwardsiella tarda isolated from freshwater fish culture system.

The applicability of PCR-RFLP of 16S rDNA and conventional phenotypic methods for differentiation of Edwardsiella tarda associated in freshwater fish culture system was studied. In this study, by conventional biochemical tests and antibiotic resistant patterns 2 and 14 groups were obtained. But these methods failed to discriminate the isolates habitat wise. However, PCR-RFLP of 16S rDNA was found to be specific to detect habitat-specific isolates. All the fish isolates belonging to particular genotypes were found only in fish, not in water or sediment. Some of the genotypes were exclusively present in water and sediment. This study indicates the prevalence of site-specific genotypes in freshwater ecosystems. Molecular method is found to be superior to discriminate the E. tarda habitat wise to conventional typing methods.

Effect of fowl adenovirus-1 (IBH isolate) on humoral and cellular immune competency of broiler chicks.

Fowl adenovirus-1 (FAV-1), isolated from field outbreaks of inclusion body hepatitis (IBH), was administered orally to 3-week-old disease-free broiler chicks. Humoral immune competency was evaluated by determining the antibody response of infected chicks to sheep red blood cells (SRBC) and Brucella abortus. FAV-1 infection significantly decreased the antibody response of chicks to B. abortus (T-cell-independent antigen) by decreasing IgM responses, however, the decreased antibody response to SRBC (T-cell-dependent antigen) was statistically non-significant. Bursal index was also found lowered in infected chicks as compared to the control chicks. A significant decrease was seen in blastogenesis response of peripheral blood lymphocytes to phytohaemagglutinin (PHA-P) in FAV-1-infected chicks on 2 and 3 weeks post-infection (WPI). These results indicated that FAV-1 affects humoral as well as cellular immune competency of infected chicks.

Detection of bovine herpesvirus-1 infection in breeding bull semen by virus isolation and polymerase chain reaction.

Serum samples from 51 apparently healthy breeding bulls were screened for bovine herpesvirus-1 (BHV-1) antibodies using an avidin-biotin enzyme-linked immunosorbent assay, revealing a sero-positive prevalence rate of 45.09%. Semen samples were then collected from 12 of the sero-positive and 12 of the sero-negative bulls and tested for BHV-1 antigen using both a virus isolation assay and a polymerase chain reaction (PCR) assay; PCR was applied to detect BHV-1 deoxyribonucleic acid by using primers selected from the relatively conserved sequence of the gl glycoprotein gene to amplify a 468 base pair fragment. The PCR-amplified products were confirmed as BHV-1 by restriction enzyme, Dde 1, which produced fragments of predictable sizes, namely 340 and 128 base pairs. Positive virus isolation test results, confirmed by virus neutralisation, found BHV-1 antigen in the semen of five sero-positive and six sero-negative bulls. In comparison, positive PCR results found BHV-1 genome in the semen of six sero-positive and eight sero-negative bulls. From the 24 semen samples tested, 14 were shown to be positive by PCR and 11 by virus isolation. The sensitivity and specificity of virus isolation were 57.14% and 70% respectively, and were significantly lower than PCR. In the semen samples taken from sero-negative bulls, BHV-1 was detected more often by PCR methods than by virus-isolation, suggesting that PCR is a more sensitive method for BHV-1 screening in bulls.

Genomic diversity and prevalence of Rotavirus in cow and buffalo calves in northern India.

Faecal samples were collected from seventy-eight diarrhoeic cow and buffalo calves between November 1998 and February 1999 to study the genomic diversity and prevalence of Rotavirus infection by ribonucleic acid polyacrylamide gel electrophoresis (RNA-PAGE) and enzyme-linked immunosorbent assay (ELISA). In the organised dairy farm (where daily production and health records were maintained), the overall prevalence of infection with Rotavirus, recorded by RNA-PAGE and ELISA, was 27.02% (10/37) in both cow and buffalo calves. In unorganised dairy herds (where no production or health records were maintained), RNA-PAGE and ELISA detected infection with Rotavirus in 26.8% (11/41) of cow and 19.5% (8/41) of buffalo calves. Five distinct electropherotypes were found to circulate in cow and buffalo calves. All were short electropherotypes except the single long electropherotype observed in a buffalo calf in an unorganised dairy herd. Some differences in RNA migration pattern were observed when these electropherotypes were compared with the neonatal calf diarrhoea virus strain of Rotavirus. Some electropherotypes were restricted to one farm while others were found in both organised and unorganised dairy herds and in both cow and buffalo calves.

Structural polypeptides of different clinical strains of avian adenovirus type-1.

The polypeptides of two liver strains, two ovarian strains and one standard strain of avian adenovirus-1 (AAV-1) were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot using polyclonal antibodies to all the strains and isoelectric focusing (IEF). All the strains had 11 polypeptides and molecular weight varied between 9 and 100 kDa. The polypeptide pattern was similar in all the strains as revealed by SDS-PAGE and Western blot. However, some differences among the strains on the basis of isoelectric point of polypeptide were observed by IEF.

Humoral immune response of chicks to different clinical isolates of avian adenovirus type-1.

The humoral immune response of 7-day-old chicks to different strains of avian adenovirus (AAV) type-1 was determined by counter immunoelectrophoresis (CIE) and enzyme-linked immunosorbent assay (ELISA). In CIE antibody was detected 14 days post-infection (PI) whereas in ELISA antibody was detected 7 days PI. The ELISA titre reached its peak at 21 days PI in all groups. The pattern of immune response among the groups was found to be similar.

An improved dot ELISA to detect fowl adenovirus type-1 antigen.

An improved dot immunobinding assay to detect fowl adenovirus type-1 is described. The method consists of spotting of antigen on nitrocellulose membrane sheet, blocking with either 5% acetic acid or with 5% defatted milk powder and fixation of either antigen or antigen-antibody complex, with either 50% methanol or 0.25% glutaraldehyde or 0.2% tannic acid. The results revealed that fixation of both antigen and antigen-antibody complex resulted in 4-fold increase in sensitivity when acetic acid was used as blocking agent. The use of two substrates simultaneously resulted in more colour intensity and clarity than using both substrates separately.