<div>Background: The present study assessed the occurrence and biofilm forming potential of ESBL-producing Escherichia coli isolates recovered from raw salad vegetables collected from 32 retail vegetable shops of Mirzapur district, Uttar Pradesh, India (n=224). 
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Methods: Standard bacteriological culture methods using cefotaxime-supplemented EMB agar were used for initial isolation of cefotaxime-resistant E. coli which were subjected to phenotypic detection of ESBL production by Kirby-Bauer disc diffusion test. Subsequently, all the phenotypically positive ESBL-producing E. coli isolates were subjected to determine their biofilm-producing ability by Congo red agar (CRA) assay and microtiter plate assay (MPA) followed by genotypic confirmation of biofilm production by PCR assay targeted at fim-H, Sfa and papC genes. Additionally, the isolates were genotypically explored for a potential presence of Shiga-like toxin genes (stx1 and stx2). 
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Result: Altogether, 276 cefotaxime-resistant E. coli isolates were recovered, of which, 99.27% (274/276) were observed to be phenotypically positive for ESBL production. The brown, dry and rough (BDAR) and red, dry and rough (RDAR) colony morphotypes were observed by 70.80% (194/274) and 29.19% (80/274) of the isolates, respectively by CRA method revealing their potential to form biofilms. Biofilm formation was evident in 94.16% (258/274) of the isolates by MPA method, of which 79.45% (205/258), 17.05% (44/258) and 3.48% (9/258) were considered as weak, moderate and strong biofilm producers, respectively.&nbsp; The fimH gene was found to be the predominant genetic determinant of biofilm formation which was detected in 77% (211/274) of the tested isolates followed by Sfa gene (11.31%) and papC gene (4.01%). A significant difference (p&lt;0.05) was evident between CRA and MPA methods as well as phenotypic and genotypic methods for detection of biofilm formation among the recovered isolates. In addition, significant differences (p&lt;0.05) were also observed upon vegetable-wise comparison of ESBL-producing E. coli isolates and the presence of biofilm producing genes. The stx1 and stx2 genes were found to be present in one isolate recovered from a carrot sample and two isolates from coriander samples, respectively. . The findings of this study highlight the widespread occurrence of ESBL-producing E. coli from raw salad vegetables with significant biofilm-forming potential posing a public health risk. The detection of Shiga-like toxin genes in some isolates further underscores the need for stringent surveillance and improved hygiene practices to mitigate contamination risk in fresh produce.</div>

Assessment of Biofilm Formation and Shiga-like Toxin Genes in ESBL-producing E. coli Isolates Recovered from Retail Salad Vegetables in Mirzapur District, Uttar Pradesh, India

<div style="text-align:justify">Background: Shiga toxin-producing Escherichia coli (STEC) strains are considered to be most common food-borne enteric zoonotic pathogen, causing various disease conditions in both animals and humans and are highly pathogenic to human in low infectious doses. Resistance against antibiotics by STEC is also a big concern now a days. Hence in view of the public health significance of STEC, present work was planned to know the combination of phenotypic and genotypic resistance patterns against certain most commonly used antibiotics by using disk diffusion method and multiplex PCR respectively. 
 
Methods: A total of 426 PCR confirmed STEC isolates isolated from pooled samples (animal faecal(179), farm water(122) and human faecal samples (125) of different livestock farms in and around Proddatur andhra Pradesh, were subjected to antibiotic sensitivity test by using disc diffusion method. The isolates that showed resistance for tetracycline, streptomycin, sulphonamides and ampicillin were selected and subjected to multiplex PCR for the detection of resistance genes. 
 
Result: Disk diffusion assay revealed highest phenotypic resistance for STEC isolates against Cepahlothin (100%), followed by Ampicillin (99.06%), Tetracycline (97.2%), Streptomycin (94.3%), Sulphonamides (90.8%) and Trimethoprim (84.5%). Pooled samples also revealed the presence of antimicrobial resistance genes like tetA(59.2%), tetB(43.5%), tetC (9.2%), strA (39.3%), strB(54.1%), sul1 (40.8%), sul2 (58.7%), sul3 (3.8%) and blaTEM(83.4%). These findings indicate the highest prevalence of antimicrobial resistance among the STEC isolates, which alarms indiscriminate use of antibiotics both for therapeutic purpose and as growth promoters. Strict hygienic, sanitation and HACCP programmes should be applied to counter STEC prevalence.</div>

<div style="text-align:justify">&nbsp;</div>

Detection of Antimicrobial Resistance Genes from Shiga Toxin Producing Escherichia coli by Multiplex PCR

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Background: In vitro fertilization (IVF) the technique is useful for understanding early mammalian development and maximizing and speeding up genetic improvement in livestock. Genes that function as genetic markers and are involved in the pre-implantation development of embryos may be used to evaluate the quality of an embryo and the expression of these genes is correlated with the timing of embryonic genomic activation.&nbsp; 
 
Methods: Maturing the oocytes of bovine, then fertilizing these matured oocytes and finally culturing the fertilized oocytes to develop into embryos. Determined the gene expression of different certain genes HSP70, OCT4, GLUT1, BAX and DNMT1 in these different cell stages of embryos that were produced.&nbsp; 
 
Result: The mRNA expression of these genes was estimated after collecting the embryos listed above that were produced by the in vitro technique. The results showed statistically significant differences in the relative abundance between some of the genes mentioned above in each embryo stage. The timing of the zygotic genome activation process (ZGA) can result in differences in gene expression levels between the early embryonic genes in each developmental stage of in vitro-produced bovine embryos.

Comparison of Gene Expression for Preimplantation Genes in Bovine Embryos Fertilized in vitro

<div>Background: Pasteurella multocida is the causative agent of many economically important diseases in a wide range of hosts. The mechanisms by which these bacteria can invade the mucosa, evade innate immunity and cause systemic disease are slowly being elucidated. Many key virulence factors are yet to be identified, including those required for initial attachment and invasion of host cells and for persistence in a relatively nutrient poor and hostile environment. This has led to intensive research to understand host adaptation mechanisms and virulence factors in order to develop effective vaccines.&nbsp; 
 
Methods: The present study was carried out to know the distribution of virulence genes viz., haemoglobin binding proteins (hgbA and hgbB), outer membrane protein (ompH), fimbrial antigen (ptfA), filamentous haemagglutinin (pfhA) and transferrin binding protein (tbpA) by PCR in P. multocida CapA isolates from apparently healthy or carrier animals and CapB isolates from field Haemorrhagic septicemia (HS) cases to monitor the epidemiological associations of virulence genes in Cap A and Cap B isolates. 
 
Result: The study revealed that all the six virulence associated genes were present in Cap B isolates. None of the Cap A isolates harboured tbpA and pfhA genes. These two genes were closely related to serotype B causing Haemorrhagic septicemia and were epidemiologically associated with disease status.</div>

<div>Virulence Gene Pattern of Pasteurella multocida Isolates of Buffalo in Association to Capsule Biosynthesis Genes</div>

<div>Present investigation was undertaken to detect and characterize the PPR virus from different clinical tissue samples of 14 sheep and 17 goats with respiratory disease from Maharashtra, India. All animals were tested by Sandwich ELISA, of which 70.96% were found positive carrying high PPR virus inwhich 12 were sheep and 10 were goats respectively. For confirmation of PPR, molecular detection was performed with RT-PCR using F gene and N gene specific primers. Intestine samples accounted for highest percent positivity (75%) followed by blood (66.66%) and lymph node (62.5%) for presence of virus. Unusually higher positivity was observed in heart, liver and Kidney (60%, each) than normal predilection sites such as lungs (57.84%) and spleen (50%). While nasal swabs and blood were individually processed with F gene and N gene specific PCR, the triturated organs were pooled for processing into &lsquo;Sample A&rsquo; comprised of heart, kidney, liver and intestine combined together and &lsquo;Sample B&rsquo; comprising of lung, spleen and lymph nodes combined for the molecular detection of PPR yielding the products each of 372bp and 463bp sizes, respectively. Out of total 40 samples tested, 09/12 each from both sample A and B, while 02/10 nasal swabs resulted positive, respectively and all 06 blood samples remained negative. (F as well as N gene PCR methods were found best suitable for detection of PPR virus from tissue samples of small ruminants).&nbsp;</div>

<div>F Gene and N Gene based Reverse Transcription PCR for Molecular Characterization of Peste des Petits Ruminants Virus</div>

<div>Background: Salmonella Typhimurium (S.Typhimurium) adapts to the broad fluctuations of oxygen concentrations encountered in the host. The transition from aerobic to microaerobic/anaerobic condition encountered in the intestine is mainly regulated by fumarate and nitrate reductase (fnr) regulatory gene and aerobic respiratory control A (arcA) gene. Aim is to appraise the role of fnr gene under anaerobic conditions. 
 
Methods: In this study, we deleted fnr gene from S.Typhimurium using lambda red-recombinase mediated gene knockout protocol. Further carried out in vitro characterization and analyzed the differential protein expression in wild type (WT) and isogenic &Delta;fnr null mutant (&Delta;fnr) using SDS-PAGE and MALDI-TOF mass spectrometry under anaerobic conditions. 
 
Result: In growth competition, WT strain outcompeted the &Delta;fnr and biofilm-forming ability of &Delta;fnr was significantly reduced compared to WT strain. Swimming motility was reduced in &Delta;fnr strain. Besides, differential protein expression revealed the global changes in the expression of many proteins in fnr strain. One differentially expressed protein was identified as TolA, an inner membrane envelope protein. It points out that fnr may regulate the genes responsible for motility and biofilm formation. FNR protein positively regulates TolA, which is important for bacterial virulence, maintenance of membrane integrity, LPS production and replication of bacteria.</div>

<div>Regulatory Role of fnr Gene in Growth and tolA Gene Expression in Salmonella Typhimurium</div>

Background: MicroRNA-449b (miR-449b) is a ~22-base pair long nucleotide, that post-transcriptionally affects processes like proliferation, differentiation and apoptosis by directly targeting the HDAC and DNMTs family genes that regulate DNA methylation. 
 
Methods: This study aimed to investigate the effect of miR-449b mimic, inhibitor and scrambled sequence on buffalo fibroblast cells containing the human insulin (hINS) gene. A transgenic buffalo cell line containing human insulin gene (hINS) was treated with 40 nM miR-449b mimic and inhibitor by transfection method (lipofectamine-3000). 
 
Result: miR-449b mimic treated cells showed a significant decrease (P&lt;0.05) in the expression of epigenetic-related genes and found a significant reduction (P&lt;0.05) in the expression of HDAC1, DNMT3A and DNMT3B genes. In contrast, DNMT1 showed no significant change (P&lt;0.05) in their expression level as compared to control whereas miR-449b inhibitor-treated cells found significantly increased (P&lt;0.05) expression levels of HDAC1, DNMT 3A, DNMT 3B and DNMT1 genes as compared to control. In apoptosis-related genes, miR-449b mimic significantly increased (P&lt;0.05) the expression of BCL-XL and MCL-1, while miR-449b inhibitor significantly increased (P&lt;0.05) the expression of MCL-1 gene whereas there was no significant change (P&lt;0.05) in the expression of BCL-XL. These findings indicate that treatment of miR-449b mimic on transgenic cells reduces the DNA methylation and apoptosis levels. This reduced methylation level helps in the nuclear reprogramming of transgenic cells. Thus, these cells can be a better choice as donor somatic cells for efficient handmade transgenic cloned embryo production.

Microrna-449b Reduces Methylation and Apoptosis-related Gene Expression in Fibroblast Cells Containing Human Insulin Gene

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<div>The objective of the study was to identify suitable reference genes that can be used for quantitative real-time PCR (qPCR) analysis in mung bean (Vigna radiata). Therefore, 10 potential reference genes were selected and the results showed that ubiquitin-conjugating enzyme was suitable as reference under drought and pathogen infection stress; elongation factor 1-&aacute; was the most stable gene under waterlogging; and actin performed the best under saline stress. These selected reference genes were further confirmed by analysis of the expression profiles of catalase and peroxidase under waterlogging. Our results will contribute to the improvement of the accuracy of gene expression evaluation in mung bean.</div>

<div>Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Mung Bean under Abiotic Stress and Cercospora canescens Infection</div>

<div>Background: E. coli is a Gram negative bacterium commonly found in the intestine of animals and humans. Most E. coli are harmless but some can cause infection. Antimicrobial resistance is a public health problem, hence this study was carried out to estimate the antimicrobial resistance pattern and virulotyping of&nbsp;E. coli isolates. 
 
Methods: During the period 2018-19, a total of 100 faecal samples from goats (n=50) and sheep (n=50) were processed for isolation and identification of E.coli. All the E. coli isolates were tested for resistance against 10 different antibiotics. The prevalence of various antibiotic resistance genes viz bla TEM, tetA and tetB and virulence genes Stx1, Stx2 and hlyA in all E. coli isolates was determined by PCR. 
 
Result: Out of 100 samples, 40 isolates of E.coli (24 from goats and 16 from sheep) were isolated, identified biochemically and by PCR. Majority of the E.coli isolates were found resistant to penicillin (100%) followed by ampicillin (93%), tetracycline, (68.8%), gentamicin (37.4%), ceftriazone (18.8%), amikacin (16.7%), chloramphenicol (4.2%), co- trimoxazole (4.2%) and ofloxacin (4.2%). Out of all the isolates, 12 isolates were positive for blaTEM, 10 for tetB, 19 for tetA, five for Stx1 and hlyA and none carried Stx2 gene.</div>

Molecular Detection of Antimicrobial Resistance Genes and Virulence Genes in E. coli Isolated from Sheep and Goat Faecal Samples

<div>Background:&nbsp;The major mastitis-producing organisms are Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli. Molecular characterization of the major bacterial pathogens for some genes responsible for their virulence should be considered the reduction of risk factors responsible for the presence and spread of contagious pathogens through milk. 
 
Methods: The two hundred milk samples were collected from cows and buffaloes of four different tehsils of the Sirohi district of Southern Rajasthan. Out of 200 milk samples, 74 milk samples that were found positive for SCM were cultured for primary isolation of predominant bacterial pathogens. Out of these 74 positive samples, total of 97 isolates were recovered from these milk samples either as a single or mixed infection. To genotypically characterize&nbsp;S. aureus&nbsp;isolates, genes encoding virulence determinants (spa-IgG-binding and Coa), Staphylococ cal enterotoxins (bac and bca) and E. coli Shiga toxins (stx1 and stx2) were investigated. 
 
Result: The etiological prevalence of SCM caused by different bacteria was,&nbsp;S. aureus, (27%),&nbsp;Streptococcus spp. (15%) and E. coli (6.5%) respectively either as single and or as mixed infections. All S. aureus isolates were tested by PCR for the presence of the spa gene and coa gene results revealed that 40 isolates (74.0%) carried both spa (IgG-binding) and coa gene. 11 isolates of S. agalactiae (36.6%) carried the bca gene. The bca gene codes for Alpha-C protein, a surface protein that helps the bacteria to enter the host cells. E. coli isolates were also screened for the presence of virulence gene stx1&nbsp;and stx2 gene. Out of 13 isolates tested, 6 isolates harboured both stx1 and stx2 genes.</div>