Recent progress and major gaps in the vaccine development for African swine fever.

The swine industry across the globe is recently facing a devastating situation imparted by a highly contagious and deadly viral disease, African swine fever. The disease is caused by a DNA virus, the African swine fever virus (ASFV) of the genus Asfivirus. ASFV affects both wild boars and domestic pigs resulting in an acute form of hemorrhagic fever. Since the first report in 1921, the disease remains endemic in some of the African countries. However, the recent occurrence of ASF outbreaks in Asia led to a fresh and formidable challenge to the global swine production industry. Culling of the infected animals along with the implementation of strict sanitary measures remains the only options to control this devastating disease. Efforts to develop an effective and safe vaccine against ASF began as early as in the mid-1960s. Different approaches have been employed for the development of effective ASF vaccines including inactivated vaccines, subunit vaccines, DNA vaccines, virus-vectored vaccines, and live attenuated vaccines (LAVs). Inactivated vaccines are a non-feasible strategy against ASF due to their inability to generate a complete cellular immune response. However genetically engineered vaccines, such as subunit vaccines, DNA vaccines, and virus vector vaccines, represent tailored approaches with minimal adverse effects and enhanced safety profiles. As per the available data, gene deleted LAVs appear to be the most potential vaccine candidates. Currently, a gene deleted LAV (ASFV-G-∆I177L), developed in Vietnam, stands as the sole commercially available vaccine against ASF. The major barrier to the goal of developing an effective vaccine is the critical gaps in the knowledge of ASFV biology and the immune response induced by ASFV infection. The precise contribution of various hosts, vectors, and environmental factors in the virus transmission must also be investigated in depth to unravel the disease epidemiology. In this review, we mainly focus on the recent progress in vaccine development against ASF and the major gaps associated with it.

Comparative genomics of Brucella abortus and Brucella melitensis unravels the gene sharing, virulence factors and SNP diversity among the standard, vaccine and field strains / La genómica comparada de Brucella abortus y Brucella melitensis revela el intercambio de genes, los factores de virulencia y la diversidad de SNP entre las cepas estándar, vacunales y de campo

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.(AU)

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.

In vitro virulotyping, antifungal susceptibility testing and DNA fingerprinting of Microsporum canis strains of canine and feline origin.

Microsporum canis is considered the common dermatophyte agent associated with ringworm in felines and canines. In the present study, we sampled n = 548 felines and canines for the probable isolation of M. canis. The rate of isolation from the cats and dogs was 70.27 % (52/74) and 1.68 % (8/474), respectively and Persian cats were found to be highly susceptible to M. canis infection. The strains were evaluated for their production of phospholipase, lipase, catalase, and hemolysis and their ability to grow at 35 â„ƒ. All the strains were identified as low producers of catalase and n = 17 strains exhibited high thermotolerance ability. Terbinafine was found to be the most effective antifungal drug and fluconazole was the least effective, in vitro. AFLP analysis revealed three genotypes of M. canis with 15 sub-clusters showing ≥ 90 % similarity and 7 sub-clusters exhibiting 100 % similarity. However, the phenotypic characters cannot be attributed based on the AFLP profiles.

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).

Molecular epidemiology of Trichophyton infections among canines from Northern India.

Dermatophytes are keratinophilic fungi that cause skin infections in both humans and animals. Recently, the incidence rates of fungal infections associated with Trichophyton spp. have been considered endemic in many locations. The aim of this study was to isolate and characterize Trichophyton spp. from canines and felines. In the present study, screened 442 canine (n = 386) and feline (n = 56) samples for dermatophytes. Among all the samples, ten isolates were identified as Trichophyton spp. based on micro-morphological features. For comparative analysis, we included three human strains of Trichophyton mentagrophytes complex. In vitro susceptibility of antifungal drugs indicated the highest sensitivity except for fluconazole. The canine and human strains were genetically characterized by sequencing three genes: the internal transcribed spacer region of rDNA, translation elongation factor 1- gene, and beta-tubulin. Based on sequence homology and phylogenetic analysis, the ten canine strains belonged to four different species/ genotypes such as T. mentagrophytes genotype VIII (T. indotineae) (n = 5), T. interdigitale (n = 2), T. simii (n = 2) and T. quinckeanum (n = 1). The three human strains used for comparative analysis were identified as T. mentagrophytes genotype VIII (n = 2) and T. benhamiae (n = 1). The study hence indicates that the T. mentagrophytes genotype VIII, considered as an endemic and emerging human pathogenic clone in India, is also the prevalent in animals.

Dermatophytosis caused by Nannizzia nana (Microsporum nanum): a comprehensive review on a novel pathogen.

Keratinophilic fungi are mostly soil-inhabiting organisms with occasional infections in humans and animals. Even though most dermatophytes are host-adapted, cross-species infections are common by zoophilic and geophilic dermatophytes. N. nana is considered an etiological agent of ringworm in pigs but has also been isolated from other animals, including humans. However, it also possesses many characteristics of geophilic dermatophytes including the ability to grow in soil. N. nana produces characteristic pear-shaped macroconidia and usually exhibits an ectothrix pattern of hair infection. It has been isolated from dermatitis lesions as well as from soil. N. nana infections in pigs are not of much concern as far as economy or health is concerned. But it has been associated with onychomycosis and gonathritis in humans, which are significant in human medicine. The shift in the predominance of dermatophytes in humans and the ability to evolve into a potential tinea pathogen necessitates more understanding of the physiology and genetics of N. nana. In this review, we have attempted a detailed analysis of the studies about N. nana, emphasizing growth and cultural characters, physiology, isolation, infection in humans and animals, molecular characterization and antifungal susceptibility.

Fusarium: more than a node or a foot-shaped basal cell.

Recent publications have argued that there are potentially serious consequences for researchers in recognising distinct genera in the terminal fusarioid clade of the family Nectriaceae. Thus, an alternate hypothesis, namely a very broad concept of the genus Fusarium was proposed. In doing so, however, a significant body of data that supports distinct genera in Nectriaceae based on morphology, biology, and phylogeny is disregarded. A DNA phylogeny based on 19 orthologous protein-coding genes was presented to support a very broad concept of Fusarium at the F1 node in Nectriaceae. Here, we demonstrate that re-analyses of this dataset show that all 19 genes support the F3 node that represents Fusarium sensu stricto as defined by F. sambucinum (sexual morph synonym Gibberella pulicaris). The backbone of the phylogeny is resolved by the concatenated alignment, but only six of the 19 genes fully support the F1 node, representing the broad circumscription of Fusarium. Furthermore, a re-analysis of the concatenated dataset revealed alternate topologies in different phylogenetic algorithms, highlighting the deep divergence and unresolved placement of various Nectriaceae lineages proposed as members of Fusarium. Species of Fusarium s. str. are characterised by Gibberella sexual morphs, asexual morphs with thin- or thick-walled macroconidia that have variously shaped apical and basal cells, and trichothecene mycotoxin production, which separates them from other fusarioid genera. Here we show that the Wollenweber concept of Fusarium presently accounts for 20 segregate genera with clear-cut synapomorphic traits, and that fusarioid macroconidia represent a character that has been gained or lost multiple times throughout Nectriaceae. Thus, the very broad circumscription of Fusarium is blurry and without apparent synapomorphies, and does not include all genera with fusarium-like macroconidia, which are spread throughout Nectriaceae (e.g., Cosmosporella, Macroconia, Microcera). In this study four new genera are introduced, along with 18 new species and 16 new combinations. These names convey information about relationships, morphology, and ecological preference that would otherwise be lost in a broader definition of Fusarium. To assist users to correctly identify fusarioid genera and species, we introduce a new online identification database, Fusarioid-ID, accessible at www.fusarium.org. The database comprises partial sequences from multiple genes commonly used to identify fusarioid taxa (act1, CaM, his3, rpb1, rpb2, tef1, tub2, ITS, and LSU). In this paper, we also present a nomenclator of names that have been introduced in Fusarium up to January 2021 as well as their current status, types, and diagnostic DNA barcode data. In this study, researchers from 46 countries, representing taxonomists, plant pathologists, medical mycologists, quarantine officials, regulatory agencies, and students, strongly support the application and use of a more precisely delimited Fusarium (= Gibberella) concept to accommodate taxa from the robust monophyletic node F3 on the basis of a well-defined and unique combination of morphological and biochemical features. This F3 node includes, among others, species of the F. fujikuroi, F. incarnatum-equiseti, F. oxysporum, and F. sambucinum species complexes, but not species of Bisifusarium [F. dimerum species complex (SC)], Cyanonectria (F. buxicola SC), Geejayessia (F. staphyleae SC), Neocosmospora (F. solani SC) or Rectifusarium (F. ventricosum SC). The present study represents the first step to generating a new online monograph of Fusarium and allied fusarioid genera (www.fusarium.org).

A novel bicistronic DNA vaccine with enhanced protective immune response against Bacillus anthracis through DNA prime-protein boost vaccination approach.

Anthrax, by Bacillus anthracis, remains a dreadful fatal hazard worldwide. The currently used anthrax vaccines are plagued by numerous issues that limit their widespread use. As an immunization approach targeting both extracellular antigens and toxins of B. anthracis may achieve better sterile immunity, the present investigation designed a bicistronic secretory anti-anthrax DNA vaccine targeting immune response against toxin and cells. The efficacy of the vaccine was compared with monocistronic DNA vaccines and the currently used anthrax vaccine. For this, mice were immunized with the developed vaccine containing pag (encoding protective antigen to block toxin) and eag genes (encoding EA1 to target cells) of B. anthracis through DNA-prime/Protein-boost (D/P) and DNA prime/DNA-boost (D/D) approaches. There was a >2 and > 5 fold increase in specific antibody level by D/D and D/P approaches respectively, on 42nd days post-immunization (dpi). Serum cytokine profiling showed that both Th1 and Th2 immune responses were elicited, with more Th2 responses in D/P strategy. More importantly, challenge with 100 times LD50 of B. anthracis at 42nd dpi exhibited maximum cumulative survival (83.33 %) by bicistronic D/P approach. Remarkably, immunization with EA1 delayed mortality onset in infection. The study forms the first report on complement-dependent bactericidal activity of antiEA1 antibodies. In short, co-immunization of PA and EA1 through the developed bicistronic DNA vaccine would be an effective immunization approach in anthrax vaccination. Further, D/P strategy could enhance vaccine-induced immunity against B. anthracis. Altogether, the study generates certain critical insights having direct applications in next-generation vaccine development against anthrax.

Development of a visible loop mediated isothermal amplification assay for rapid detection of Bacillus anthracis.

Distressing effects on animal and human health with lethal progression, being used as bioweapon and shared features with non-pathogenic bacteria demands sensitive, specific, safe, cost effective and rapid detection methods for anthrax causing organisms. Conventional microbiology based diagnostics for anthrax are time consuming and need sophisticated equipment, while molecular diagnostics require less time and labor. The Loop mediated isothermal amplification assay (LAMP) is rapid, sensitive and specific assay and requires no specialized equipment. In the present study, we developed a LAMP assay for rapid as well as specific detection of Bacillus anthracis. The optimized assay produced positive results with the Sterne strain and one field isolate of B. anthracis and, negative results with other bacteria of the same and different genera within 2 h. Sensitivity was 500 fg of total DNA of B. anthracis, which was 100 times more sensitive than conventional PCR. The present study also demonstrated that the simple method of total DNA extraction by repeated boiling and freezing will not adversely affect the LAMP results. In conclusion, the optimized LAMP assay is a promising tool for the specific, sensitive, less time-consuming diagnosis for anthrax causing bacteria and also, for detecting the virulence of suspected B. anthracis cultures.

A Rapid and Automated Sample-to-Result Candida auris Real-Time PCR Assay for High-Throughput Testing of Surveillance Samples with the BD Max Open System.

The multidrug-resistant yeast pathogen Candida auris continues to cause outbreaks and clusters of clinical cases worldwide. Previously, we developed a real-time PCR assay for the detection of C. auris from surveillance samples (L. Leach, Y. Zhu, and S. Chaturvedi, J Clin Microbiol 56:e01223-17, 2018, https://doi.org/10.1128/JCM.01223-17). The assay played a crucial role in the ongoing investigations of the C. auris outbreak in New York City. To ease the implementation of the assay in other laboratories, we developed an automated sample-to-result real-time C. auris PCR assay using the BD Max open system. We optimized sample extraction at three different temperatures and four incubation periods. Sensitivity was determined using eight pools of patient samples, and specificity was calculated using four clades of C. auris and closely and distantly related yeasts. Three independent extractions and testing of two patient sample pools in quadruplicate yielded assay precision. BD Max optimum assay conditions were as follows: DNA extraction at 75°C for 20 min and the use of PerfeCTa multiplex quantitative PCR (qPCR) ToughMix. The limit of detection (LOD) of the assay was one C. auris CFU/PCR. We detected all four clades of C. auris without cross-reactivity to other yeasts. Of the 110 patient surveillance samples tested, 50 were positive for C. auris using the BD Max system with 96% clinical sensitivity and 94% accuracy compared to the results of the manual assay. The BD Max assay allows high-throughput C. auris screening of 180 surveillance samples in a 12-h workday.

Rapid visual isothermal nucleic acid-based detection assay of Brucella species by polymerase spiral reaction.

AIM: The aim of this study was to develop polymerase spiral reaction (PSR) for rapid, sensitive and specific detection of Brucella sp. METHODS AND RESULTS: Polymerase spiral reaction assay was developed using specifically designed primers targeting the conserved multicopy IS711 gene of Brucella sp. The assay could be performed within 60 min at an isothermal temperature of 64°C. The lower limit of detection of PSR was 11·8 fg and conventional PCR was 1·18 pg of Brucella abortus genomic DNA. Thus, PSR was found to be 100-fold more sensitive than conventional PCR and was comparable to real-time PCR. The specificity of PSR was tested with other non-Brucella bacteria and also with some bacterial and viral pathogens causing abortions. The assay was found to be specific as it did not detect any putative pathogens other than Brucella sp. Fifty-six clinical samples suspected for brucellosis (aborted fetal stomach content) were screened with PSR to validate the applicability of the test to detect Brucella DNA. The same samples were also screened with conventional PCR and real-time PCR. Of 56 samples, 25 samples were found to be positive with both PSR as well as real-time PCR, whereas only 20 samples were found positive with conventional PCR. CONCLUSIONS: The results of this study indicated that the PSR assay is a simple, rapid, sensitive and specific method for the detection of Brucella sp. that may improve diagnostic potential in clinical laboratories or can be used at diagnostic laboratories with minimal infrastructure. SIGNIFICANCE AND IMPACT OF THE STUDY: The PSR assay, because of its simplicity and low cost, can be preferred to other molecular methods in the diagnosis of infectious diseases.

Effect of feeding of blend of essential oils on methane production, growth, and nutrient utilization in growing buffaloes.

OBJECTIVE: An experiment was conducted to study the effect of a blend of essential oils (BEO) on enteric methane emission and growth performance of buffaloes (Bubalus bubalis). METHODS: Twenty one growing male buffaloes (average body weight of 279±9.3 kg) were divided in to three groups. The animals of all the three groups were fed on a ration consisting of wheat straw and concentrate mixture targeting 500 g daily live weight gain. The three dietary groups were; Group 1, control without additive; Group 2 and 3, supplemented with BEO at 0.15 and 0.30 mL/kg of dry matter intake (DMI), respectively. RESULTS: During six months feeding trial, the intake and digestibility of dry matter and nutrients (organic matter, crude protein, ether extract, neutral detergent fibre, and acid detergent fibre) were similar in all the groups. The average body weight gain was tended to improve (p = 0.084) in Group 2 and Group 3 as compared to control animals. Feeding of BEO did not affect feed conversion efficiency of the animals. The calves of all the three groups were in positive nitrogen balance with no difference in nitrogen metabolism. During respiration chamber studies the methane production (L/kg DMI and L/kg digestible dry matter intake was significantly (p<0.001) lower in Group 2 and Group 3 as compared to control animals. CONCLUSION: The results indicated that the BEO tested in the present study have shown potential to reduce enteric methane production without compromising the nutrient utilization and animal performance and could be further explored for its use as feed additive to mitigate enteric methane production in livestock.

Effect of high and low roughage total mixed ration diets on rumen metabolites and enzymatic profiles in crossbred cattle and buffaloes.

AIM: A comparative study was conducted on crossbred cattle and buffaloes to investigate the effect of feeding high and low roughage total mixed ration (TMR) diets on rumen metabolites and enzymatic profiles. MATERIALS AND METHODS: Three rumen-fistulated crossbred cattle and buffalo were randomly assigned as per 3×3 switch over design for 21-days. Three TMR diets consisting of concentrate mixture, wheat straw and green maize fodder in the ratios of (T1) 60:20:20, (T2) 40:30:30, and (T3) 20:40:40, respectively, were fed to the animals ad libitum. Rumen liquor samples were collected at 0, 2, 4, 6, and 8 h post feeding for the estimation of rumen biochemical parameters on 2 consecutive days in each trial. RESULTS: The lactic acid concentration and pH value were comparable in both species and treatments. Feed intake (99.77±2.51 g/kg body weight), ruminal ammonia nitrogen, and total nitrogen were significantly (p<0.05) higher in buffalo and in treatment group fed with high concentrate diet. Production of total volatile fatty acids (VFAs) was non-significant (p>0.05) among treatments and significantly (p<0.05) greater in crossbred cattle than buffaloes. Molar proportions of individual VFAs propionate (C3), propionate:butyrate (C3:C4), and (acetate+butyrate):propionate ([C2+C4]:C3) ratio in both crossbred cattle and buffalo were not affected by high or low roughage diet, but percentage of acetate and butyrate varied significantly (p<0.05) among treatment groups. Activities of microbial enzymes were comparable among species and different treatment groups. A total number of rumen protozoa were significantly (p<0.05) higher in crossbred cattle than buffaloes along with significantly (p<0.05) higher population in animal fed with high concentrate diet (T1). CONCLUSION: Rumen microbial population and fermentation depend on constituents of the treatment diet. However, microbial enzyme activity remains similar among species and different treatments. High concentrate diet increases number of rumen protozoa, and the number is higher in crossbred cattle than buffaloes.

Effect of condensed tannins from Ficus infectoria and Psidium guajava leaf meal mixture on nutrient metabolism, methane emission and performance of lambs.

OBJECTIVE: The study examined the effect of condensed tannins (CT) containing Ficus infectoria and Psidium guajava leaf meal mixture (LMM) supplementation on nutrient metabolism, methane emission and performance of lambs. METHODS: Twenty four lambs of ~6 months age (average body weight 10.1±0.60 kg) were randomly divided into 4 dietary treatments (CT-0, CT-1, CT-1.5, and CT-2 containing 0, 1.0, 1.5, and 2.0 percent CT through LMM, respectively) consisting of 6 lambs each in a completely randomized design. All the lambs were offered a basal diet of wheat straw ad libitum, oat hay (100 g/d) along with required amount of concentrate mixture to meet their nutrient requirements for a period of 6 months. After 3 months of experimental feeding, a metabolism trial of 6 days duration was conducted on all 24 lambs to determine nutrient digestibility and nitrogen balance. Urinary excretion of purine derivatives and microbial protein synthesis were determined using high performance liquid chromatography. Respiration chamber study was started at the mid of 5th month of experimental feeding trial. Whole energy balance trials were conducted on individual lamb one after the other, in an open circuit respiration calorimeter. RESULTS: Intake of dry matter and organic matter (g/d) was significantly (p<0.05) higher in CT-1.5 than control. Digestibility of various nutrients did not differ irrespective of treatments. Nitrogen retention and microbial nitrogen synthesis (g/d) was significantly (p<0.01) higher in CT-1.5 and CT-2 groups relative to CT-0. Total body weight gain (kg) and average daily gain (g) were significantly (linear, p<0.01) higher in CT-1.5 followed by CT-1 and CT-0, respectively. Feed conversion ratio (FCR) by lambs was significantly (linear, p<0.01) better in CT-1.5 followed by CT-2 and CT-0, respectively. Total wool yield (g; g/d) was linearly (p<0.05) higher for CT-1.5 than CT-0. Methane emission was linearly decreased (p<0.05) in CT groups and reduction was highest (p<0.01) in CT-2 followed by CT-1.5 and CT-1. Methane energy (kcal/d) was linearly decreased (p<0.05) in CT groups. CONCLUSION: The CT supplementation at 1% to 2% of the diet through Ficus infectoria and Psidium guajava LMM significantly improved nitrogen metabolism, growth performance, wool yield, FCR and reduced methane emission by lambs.

Urinary haptoglobin, alpha-1 anti-chymotrypsin and retinol binding protein identified by proteomics as potential biomarkers for lupus nephritis.

The study was aimed at identification by proteomics and validation by enzyme-linked immunosorbent assay (ELISA) of potential urinary biomarkers for lupus nephritis. Study subjects comprised 88 systemic lupus erythematosus (SLE) patients and 60 controls (rheumatoid arthritis, diabetes mellitus and healthy individuals). Based on the SLE disease activity index (SLEDAI), patients were classified as active renal (AR), active non-renal (ANR) or inactive disease (ID). Urinary proteins from a group of patients with AR or ID were resolved by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS/MS). The selected biomarkers were validated by ELISA using samples from all patients and controls. AR patients were followed-up for 12 months after start of therapy. Three urinary proteins, alpha-1 anti-chymotrypsin (ACT), haptoglobin (HAP) and retinol binding protein (RBP), were detected in patients with AR and not ID. Upon validation, ACT levels were higher in AR patients than the other groups (P < 0·001) and showed good correlation with renal SLEDAI (r = 0·577, P < 0·001) as well as SLEDAI (r = 0·461, P < 0·001). Similarly, HAP levels were > 10-fold higher in AR than other groups (P < 0·001) and correlated well with renal SLEDAI (r = 0·594, P < 0·001) and SLEDAI (r = 0·371, P < 0·01). RBP levels were also higher in AR patients than in other groups (P < 0·05), except diabetes, and showed moderate correlation with renal SLEDAI (r = 0·284, P < 0·008) and SLEDAI (r = 0·316, P < 0·003). Upon follow-up with treatment, levels of all three proteins declined at 6 and 12 months (P < 0·01). Multiple logistic regression identified ACT as the best marker to differentiate AR from ANR. Urinary HAP, ACT and RBP are potential biomarkers for lupus nephritis activity.

Additional synthesis on thiophene-containing trisubstituted methanes (TRSMs) as inhibitors of M. tuberculosis and 3D-QSAR studies.

We earlier reported thiophene-containing trisubstituted methanes (TRSMs) as novel cores carrying anti-tubercular activity, and identified S006-830 as the phenotypic lead with potent bactericidal activity against single- and multi-drug resistant clinical isolates of Mycobacterium tuberculosis (M. tb). In this work, we carried out additional synthesis of several TRSMs. The reaction scheme essentially followed the Grignard reaction and Friedel-Crafts alkylation, followed by insertion of a dialkylaminoethyl chain. We also performed microbiological evaluations including in vitro screening against the virulent strain M. tb H37Rv, cytotoxicity assessment in the Vero C-1008 cell line, and 3D-QSAR studies with comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA). CoMFA and CoMSIA models yielded good statistical results in terms of q2 and r2 values, suggesting the validity of the models. It was concluded that a para-substituted benzene ring with bulkier electron-donating groups and aminoalkyl chains are required for higher inhibitory capacity against M. tuberculosis. We believe that these insights will rationally guide the design of newer, optimal, TRSMs.

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.