Polymorphism of cytokine and innate immunity genes associated with bovine brucellosis in cattle.

Genetic susceptibility to brucellosis is multifactorial, and it is known that impairment of the immune system could contribute to risk for getting brucellosis. The aim of the study was to find association of bovine brucellosis with 20 SNPs pertaining to bovine cytokine (IFNG, IFNGR1, IFNGR2, TNFA) and innate immunity (SLC11A1, TLR1, TLR4, and TLR9) genes using PCR-RFLP genotyping technique and it was observed that SLC11A1 (+1066 C/G), TLR1 (+1446 C/A), TLR1 (+1380 G/A), TLR4 (+10 C/T) and TLR4 (+399 C/T) loci were significantly (P≤0.05) associated with bovine brucellosis. The odds ratios (OR) of CG and CC genotypes versus GG genotype were 0.31 (0.12-0.82; 95% CI) and 0.18 (0.03-1.06; 95% CI) at SLC11A1 (+1066 C/G) locus in cases of brucellosis affected cattle. For TLR1 (+1380 G/A) locus, the OR for AG and AA genotypes versus GG genotypes were 0.15 (0.05-0.44; 95% CI) and 0.26 (0.04-1.47; 95% CI) which indicated that proportion of GG homozygote was significantly higher in brucellosis affected animals as compared to control. At TLR1 (+1446 C/A) locus the OR of AC genotype versus CC genotype was 0.24 (0.08-0.68; 95% CI) which revealed that relative proportion CC genotypes was significantly higher in case population. The TLR4 (+10 C/T) locus had three genotypes (TT, CT and CC) where OR of CT and CC genotypes versus TT genotype were near to zero. The OR of CT genotypes versus CC genotypes was 8.25 (0.94-71.92; 95% CI) at TLR4 (+399 C/T) locus and indicated that CT genotype had higher odds of bovine brucellosis than control animals.

Ethoxyresorufin-O-deethylase (EROD) activity modulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,3',4,4',5-pentachlorobiphenyl (PCB 126) in the presence of aqueous suspensions of nano-C60.

The increase in commercial production and inevitable release of fullerenes into the environment accelerates concerns about their potential toxicity. Furthermore, the concomitant release of xenobiotics poses a health hazard to humans, and might present potential long-term risks to human health. In the present study, we found that an aqueous suspension of buckminsterfullerene (aqu-nC60) does not result in the induction of ethoxyresorufin-O-deethylase (EROD) activity in H4IIE rat liver cells in vitro. The simultaneous and sequential exposure of aqu-nC60 and the dioxin TCDD induces EROD activity to the same extent as TCDD alone (i.e. in the absence of fullerene), in spite of the high affinity of C60 for TCDD. However, the co-exposure of aqu-nC60 and PCB 126 induces elevated EROD activity, and sequential exposure increases responses 2-fold compared to the control samples. Our in vitro observations suggest a potential source of drug-drug type interaction of fullerene with xenobiotics, particularly after a sequential exposure.

Adhesive assisted TiB2 coating effects on friction stir welded joints.

Friction stir welding is a novel technique for joining ferrous and non-ferrous materials in a solid state. The groove fill techniques are most popular and generally used by researchers to dope reinforcement in the FSWed zone to improve the properties of joints. The main drawback of this technique is that a few amounts of reinforcement material come out from the groove during the fabrication of the joint. In the present work, the adhesive-assisted reinforcement technique was used to overcome this problem for the fabrication of particulates reinforced friction stirred weld joint. In the present work, the aluminum alloy plate edges were coated with a thin layer of TiB2. The coated and non-coated edge plates were joined using friction stir welding at the rotational speed of 1400 and 2240 rpm, and welding speed of 32 mm/min using a taper threaded pin tool. The tensile strength of coated edge plate welded joints was found highest in comparison to non-coated joints which was 39.74% superior. The percentage elongation of coated edge joint was observed about 1.5 times lower than the non-coated edge plate joint. The flexure strength of TiB2 reinforced coated edge joint was found about 1.5 times higher. However, the impact strength of coated edge plate was found nearly three times lower than the uncoated edge joints. The TiB2 coated edge joints reveal 22.75% higher hardness than the non-coated edge plate joints welded at the rotational speed of 2240.

Friend or Foe: Draft Genome Sequence of Bradyrhizobium sp. Strain SRS-191.

We report the genomic features of Bradyrhizobium sp. strain SRS-191, which was isolated from a former nuclear legacy site in Aiken, South Carolina, USA. With a genome size of 7,621,400 bp, the strain harbored genes not only for environmentally beneficial traits (e.g., heavy metal resistance, nitrogen fixation, and aromatic biodegradation) but also for antimicrobial resistance.

A Rapid and High Throughput MIC Determination Method to Screen Uranium Resistant Microorganisms.

The assessment of minimum inhibitory concentration (MIC) is a conventional technique used for the screening of microbial resistance against antibiotics, biocides, and contaminants such as heavy metals. However, as part of our ongoing work, we have observed biases associated with using traditional liquid MIC method to screen microbial heavy metal resistance, including both bacterial and fungal strains. Specifically, the addition of uranium into synthetic media causes immediate precipitation prior to the initiation of microbial growth, thus hampering the optical density measurements, and the obtained MIC values are thus flawed and inaccurate. To address this discrepancy, we report the optimization and development of a serial-dilution-based MIC method conducted on solid growth media supplemented with uranium, which is more accurate, relative to the testing of MICs performed in liquid cultures. Notably, we report on the efficacy of this method to screen not only bacteria that are resistant to uranium but also demonstrate the successful application to yeast and fungal isolates, for their ability to resist uranium, is more accurate and sensitive relative to the liquid method. We believe that this newly developed method to screen heavy metal resistance, such as uranium, is far superior to the existing liquid MIC method and propose replacing the liquid assay with the solid plate MIC reported herein.

Draft Genome Sequence of Mercury-Resistant Serratia sp. Strain SRS-8-S-2018.

A mercury (Hg)-resistant Serratia sp. strain, SRS-8-S-2018, was isolated, followed by generation of its draft genome sequence, which indicated a genomic size of 5,323,630 bp composed of 5,261 coding sequences. A suite of genomic functions in strain SRS-8-S-2018 was identified, and these likely facilitate survival in a metalliferous soil habitat.

Is Long-Term Heavy Metal Exposure Driving Carriage of Antibiotic Resistance in Environmental Opportunistic Pathogens: A Comprehensive Phenomic and Genomic Assessment Using Serratia sp. SRS-8-S-2018.

The carriage of both, heavy metal and antibiotic resistance appears to be a common trait in bacterial communities native to long-term contaminated habitats, including the Savannah River Site (SRS). There is widespread soil contamination at the SRS; a United States Department of Energy (DOE) facility with long-term contamination from past industrial and nuclear weapons production activities. To further evaluate the genomic and metabolic traits that underpin metal and antibiotic resistance, a robust mercury (Hg) and uranium (U)-resistant strain- SRS-8-S-2018, was isolated. Minimum inhibitory concentration of this strain revealed resistance to Hg (10 µg/ml) and U (5 mM), the two main heavy metal contaminants at the SRS. Metabolic assessment of strain SRS-8-S-2018 using Biolog metabolic fingerprinting analysis revealed preference for carbohydrate utilization followed by polymers, amino acids, carboxy acids, and esters; this physiological activity diminished when Hg stress was provided at 1 and 3 µg/ml and completely ceased at 5 µg/ml Hg, indicating that continued release of Hg will have negative metabolic impacts to even those microorganisms that possess high resistance ability. Development of antibiotic resistance in strain SRS-8-S-2018 was evaluated at a functional level using phenomics, which confirmed broad resistance against 70.8% of the 48 antibiotics tested. Evolutionary and adaptive traits of strain SRS-8-S-2018 were further assessed using genomics, which revealed the strain to taxonomically affiliate with Serratia marcescens species, possessing a genome size of 5,323,630 bp, 5,261 proteins (CDS), 55 genes for transfer RNA (tRNA), and an average G + C content of 59.48. Comparative genomics with closest taxonomic relatives revealed 360 distinct genes in SRS-8-S-2018, with multiple functions related to both, antibiotic and heavy metal resistance, which likely facilitates the strain's survival in a metalliferous soil habitat. Comparisons drawn between the environmentally isolated Serratia SRS-8-S-2018 with 31 other strains revealed a closer functional association with medically relevant isolates suggesting that propensity of environmental Serratia isolates in acquiring virulence traits, as a function of long-term exposure to heavy metals, which is facilitating development, recruitment and proliferation of not only metal resistant genes (MRGs) but antibiotic resistant genes (ARGs), which can potentially trigger future bacterial pathogen outbreaks emanating from contaminated environmental habitats.

Announcing the Availability of a Culture Collection of Uranium-Resistant Microbial Assemblages (CURMA) Obtained from Metalliferous Soils of the Savannah River Site, USA.

Metagenomic assessment provides a comprehensive survey of soil microbiota; however, isolation and characterization of functionally relevant microbiota are required prior to their application(s), such as for metal remediation. Toward this end, we report the availability of a culture collection comprising uranium (U)-resistant microbial assemblages (CURMA) to the scientific community.

Characterization of Bacterial and Fungal Assemblages From Historically Contaminated Metalliferous Soils Using Metagenomics Coupled With Diffusion Chambers and Microbial Traps.

The majority of environmental microbiomes are not amenable to cultivation under standard laboratory growth conditions and hence remain uncharacterized. For environmental applications, such as bioremediation, it is necessary to isolate microbes performing the desired function, which may not necessarily be the fast growing or the copiotroph microbiota. Toward this end, cultivation and isolation of microbial strains using diffusion chambers (DC) and/or microbial traps (MT) have both been recently demonstrated to be effective strategies because microbial enrichment is facilitated by soil nutrients and not by synthetically defined media, thus simulating their native habitat. In this study, DC/MT chambers were established using soils collected from two US Department of Energy (DOE) sites with long-term history of heavy metal contamination, including mercury (Hg). To characterize the contamination levels and nutrient status, soils were first analyzed for total mercury (THg), methylmercury (MeHg), total carbon (TC), total nitrogen (TN), and total phosphorus (TP). Multivariate statistical analysis on these measurements facilitated binning of soils under high, medium and low levels of contamination. Bacterial and fungal microbiomes that developed within the DC and MT chambers were evaluated using comparative metagenomics, revealing Chthoniobacter, Burkholderia and Bradyrhizobium spp., as the predominant bacteria while Penicillium, Thielavia, and Trichoderma predominated among fungi. Many of these core microbiomes were also retrieved as axenic isolates. Furthermore, canonical correspondence analysis (CCA) of biogeochemical measurements, metal concentrations and bacterial communities revealed a positive correlation of Chthoniobacter/Bradyrhizobium spp., to THg whereas Burkholderia spp., correlated with MeHg. Penicillium spp., correlated with THg whereas Trichoderma spp., and Aspergillus spp., correlated with MeHg, from the MT approach. This is the first metagenomics-based assessment, isolation and characterization of soil-borne bacterial and fungal communities colonizing the diffusion chambers (DC) and microbial traps (MT) established with long-term metal contaminated soils. Overall, this study provides proof-of-concept for the successful application of DC/MT based assessment of mercury resistant (HgR) microbiomes in legacy metal-contaminated soils, having complex contamination issues. Overall, this study brings out the significance of microbial communities and their relevance in context to heavy metal cycling for better stewardship and restoration of such historically contaminated systems.

Gene Determinants for Mercury Bioremediation as Revealed by Draft Genome Sequence Analysis of Stenotrophomonas sp. Strain MA5.

A soilborne Stenotrophomonas sp. strain (MA5) that is resistant to mercury was isolated. A draft genome sequence-based analysis revealed a suite of gene determinants to resist mercury and other heavy metals, multidrug efflux, stress response, and membrane transport, and these provide cues to a suite of mechanisms that underpin cellular survival in contaminated soil.

Multiple Lines of Evidences Reveal Mechanisms Underpinning Mercury Resistance and Volatilization by Stenotrophomonas sp. MA5 Isolated from the Savannah River Site (SRS), USA.

A largely understudied microbially mediated mercury (Hg) bioremediative pathway includes the volatilization of Hg2+ to Hg°. Therefore, studies on Hg resistant bacteria (HgR), isolated from historically long-term contaminated environments, can serve as models to understand mechanisms underpinning Hg cycling. Towards this end, a mercury resistant bacterial strain, identified as Stenotrophomonas sp., strain MA5, was isolated from Mill Branch on the Savannah River Site (SRS); an Hg-impacted ecosystem. Minimum inhibitory concentration (MIC) analysis showed Hg resistance of up to 20 µg/mL by MA5 with 95% of cells retaining viability. Microcosm studies showed that the strain depleted more than 90% of spiked Hg2+ within the first 24 h of growth and the detection of volatilized mercury indicated that the strain was able to reduce Hg2+ to Hg°. To understand molecular mechanisms of Hg volatilization, a draft whole genome sequence was obtained, annotated and analyzed, which revealed the presence of a transposon-derived mer operon (merRTPADE) in MA5, known to transport and reduce Hg2+ into Hg°. Based on the whole genome sequence of strain MA5, qRT-PCR assays were designed on merRTPADE, we found a ~40-fold higher transcription of merT, P, A, D and E when cells were exposed to 5 µg/mL Hg2+. Interestingly, strain MA5 increased cellular size as a function of increasing Hg concentrations, which is likely an evolutionary response mechanism to cope with Hg stress. Moreover, metal contaminated environments are shown to co-select for antibiotic resistance. When MA5 was screened for antibiotic resistance, broad resistance against penicillin, streptomycin, tetracycline, ampicillin, rifampicin, and erythromycin was found; this correlated with the presence of multiple gene determinants for antibiotic resistance within the whole genome sequence of MA5. Overall, this study provides an in-depth understanding of the underpinnings of Stenotrophomonas-mercury interactions that facilitate cellular survival in a contaminated soil habitat.

Simultaneous determination of hydrazinocurcumin and phenol red in samples from rat intestinal permeability studies: HPLC method development and validation.

Hydrazinocurcumin (HZC) is a patented multiactivity compound and is a potent derivative of curcumin which is not yet explored for further development as formulation and requires the determination of biopharmaceutical suitability of the molecule. Intestinal permeability and logP of a compound are two vital biopharmaceutical properties by which, any "hit" molecule proceeds towards NCE (new chemical entity) and govern formulation design of bioactive molecules. In this report, a simple, precise and accurate isocratic reverse phase (RP) liquid chromatography method for simultaneous analysis of HZC and phenol red, for the application in rat in situ single-pass intestinal perfusion (SPIP) was developed and validated using FDA bioanalytical guidelines. RP-HPLC method was developed on C-18 column with UV detection at 332 nm for both the compounds. Isocratic run with the mobile phase consisting of organic phase (methanol:acetonitrile:: 50:20 v/v) and water in the ratio of 80:20 v/v provided a short run time of 7 min with resolution of more then two without interference of blank matrix. The working/expected concentration range for HZC and phenol red were 0.5-50 and 2-200 microg/ml. LOQs for HZC and phenol red of the method was found to be 0.167 and 0.271 microg/ml respectively. The validation parameters indicate that method was suitable for the intended purpose. Permeability, considering water flux with the help of non-permeable marker phenol red was calculated to be 0.34 x 10(-4)cm/s. Along with other descriptors, logP (1.78) and MW (<500) of HZC makes it a potential candidate for oral formulation.

Proteogenomic Analysis of Burkholderia Species Strains 25 and 46 Isolated from Uraniferous Soils Reveals Multiple Mechanisms to Cope with Uranium Stress.

Two Burkholderia spp. (strains SRS-25 and SRS-46) were isolated from high concentrations of uranium (U) from the U.S. Department of Energy (DOE)-managed Savannah River Site (SRS). SRS contains soil gradients that remain co-contaminated by heavy metals from previous nuclear weapons production activities. Uranium (U) is one of the dominant contaminants within the SRS impacted soils, which can be microbially transformed into less toxic forms. We established microcosms containing strains SRS-25 and SRS-46 spiked with U and evaluated the microbially-mediated depletion with concomitant genomic and proteomic analysis. Both strains showed a rapid depletion of U; draft genome sequences revealed SRS-25 genome to be of approximately 8,152,324 bp, a G + C content of 66.5, containing a total 7604 coding sequences with 77 total RNA genes. Similarly, strain SRS-46 contained a genome size of 8,587,429 bp with a G + C content of 67.1, 7895 coding sequences, with 73 total RNA genes, respectively. An in-depth, genome-wide comparisons between strains 25, 46 and a previously isolated strain from our research (Burkholderia sp. strain SRS-W-2-2016), revealed a common pool of 3128 genes; many were found to be homologues to previously characterized metal resistance genes (e.g., for cadmium, cobalt, and zinc), as well as for transporter, stress/detoxification, cytochromes, and drug resistance functions. Furthermore, proteomic analysis of strains with or without U stress, revealed the increased expression of 34 proteins from strain SRS-25 and 52 proteins from strain SRS-46; similar to the genomic analyses, many of these proteins have previously been shown to function in stress response, DNA repair, protein biosynthesis and metabolism. Overall, this comparative proteogenomics study confirms the repertoire of metabolic and stress response functions likely rendering the ecological competitiveness to the isolated strains for colonization and survival in the heavy metals contaminated SRS soil habitat.

Development and evaluation of probe based real time loop mediated isothermal amplification for Salmonella: A new tool for DNA quantification.

A one step, single tube, accelerated probe based real time loop mediated isothermal amplification (RT LAMP) assay was developed for detecting the invasion gene (InvA) of Salmonella. The probe based RT LAMP is a novel method of gene amplification that amplifies nucleic acid with high specificity and rapidity under isothermal conditions with a set of six primers. The whole procedure is very simple and rapid, and amplification can be obtained in 20min. Detection of gene amplification was accomplished by amplification curve, turbidity and addition of DNA binding dye at the end of the reaction results in colour difference and can be visualized under normal day light and in UV. The sensitivity of developed assay was found 10 fold higher than taqman based qPCR. The specificity of the RT LAMP assay was validated by the absence of any cross reaction with other members of enterobacteriaceae family and other gram negative bacteria. These results indicate that the probe based RT LAMP assay is extremely rapid, cost effective, highly specific and sensitivity and has potential usefulness for rapid Salmonella surveillance.

New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination.

Loop mediated isothermal amplification (LAMP) assay, a promising diagnostic test, has been developed for detection of different pathogens of human as well as animals. Various positive points support its use as a field level test but the major problem is product cross contamination leading to false positive results. Different methods were adopted by various researchers to control this false positive amplification due to cross contamination but all have their own advantages and disadvantages. A new closed tube LAMP assay based on agar dye capsule was developed in the present study and this technique has some advantages over the other closed tube technique.•Agar at the concentration of 1.5% was used to sandwich SYBR green dye I with the aid of intradermal syringe. This agar dye capsule was placed over the LAMP reaction mixture before it was amplified.•To eliminate the hazardous nature of Ultra Violet (UV) light during result visualization of LAMP products, the present study demonstrates the use of Light Emitting Diode (LED) lights for result visualization.•LAMP was carried out for Brucella species detection using this modified techniques yielding good results without any cross contamination and LED showed similar fluorescence compared to UV.

Loop-mediated isothermal amplification (LAMP) test for specific and rapid detection of Brucella abortus in cattle.

BACKGROUND: Brucella abortus, the major causative agent of abortion in cattle and a zoonotic pathogen, needs to be diagnosed at an early stage. Loop-mediated isothermal amplification (LAMP) test is easy to perform and also promising to be adapted at field level. OBJECTIVE: To develop a LAMP assay for specific and rapid detection of B. abortus from clinical samples of cattle. METHODS: LAMP primers were designed targeting BruAb2_0168 region using specific software tool and LAMP was optimized. The developed LAMP was tested for its specificity with 3 Brucella spp. and 11 other non-Brucella spp. Sensitivity of the developed LAMP was also carried out with known quantity of DNA. Cattle whole blood samples and aborted fetal stomach contents were collected and used for testing with developed LAMP assay and results were compared with polymerase chain reaction (PCR). RESULTS: The developed LAMP assay works at 61 °C for 60 min and the detection limit was observed to be 100-fold more than the conventional PCR that is commonly used for diagnosis of B. abortus. Clinical sensitivity and specificity of the developed LAMP assay was 100% when compared with Rose Bengal plate test and standard tube agglutination test. SYB® green dye I was used to visualize the result with naked eye. CONCLUSION: The novelty of the developed LAMP assay for specifically detecting B. abortus infection in cattle along with its inherent rapidness and high sensitivity can be employed for detecting this economically important pathogen of cattle at field level as well be exploited for screening of human infections.

Immunomodulators in day to day life: a review.

There are ongoing trends of immunomodulation to combat a vast range of human and animal diseases including the incurable diseases like viral diseases, cancers, autoimmune diseases and inflammatory conditions. Animate as well as non-animate factors, surrounding us are interacting with our immune system. A balanced diet should contain all essential components from energy to vitamin and trace minerals. Each of these constituent has a very special effect on the immune system starting from their development to active role in immunity therefore, the outcome of their deficiency often ends in disease. Edible items which we consume like various vegetables, spices, herbs, fruits etc., are also equally responsible in manipulation of our system either in positive or negative way. Water has biggest share in our body and acts as the main medium to support the activities of the different system of body without exception of immune system. Proper environmental temperature is essential to maintain body's functions and experiments carried out regarding the effect of temperature suggest that extremes of the temperature are often cause immunosuppression directly by acting on the cells of immunity or indirectly through inducing stress and thereby increasing production of catecholamine which are potent anti-immune molecules. Various pathogenic as well as non-pathogenic bacteria cause immune suppression and immune potentiation, respectively. Proper exercise hold a prime position in the healthy life as it supports immunity and keeps disease away. The present review deals with all these immunomodulators having both positive and negative impact on the health status of an individual.