<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: Food borne pathogens are transmitted to humans through contaminated foods like dairy products, handmade goods and meat. Microbial biofilms are city of microbes in the form of 3D structure and attachment of bacteria with food contact surfaces lead to subsequent development of biofilms that may lead to transmission of disease and food spoilage. This study was designed to evaluate the biofilm production capability of Shiga toxin-producing Escherichia coli (STEC) isolated from milk, milk-based products and hand swabs. 
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Methods: Samples were collected and processed for isolation of E. coli. STEC were detected on the molecular basis through mPCR. Biofilm formation capability of STEC was screened by methods namely Tissue culture plate (TCP), Tube method (TM) and Congo red agar (CRA) assay.&nbsp; 
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Result: The prevalence of E. coli and STEC was found to be 22.50%, 30.55%, 12.5% and 6.25%, 4.86%, 2.08% in retail raw milk, traditional milk products and hand swabs, respectively. The overall prevalence of E. coli, serotype E. coli O157:H7 and STEC was 24.43%, 1.42% and 5.11%, respectively. The biofilm forming STEC revealed by TCP, TM and CRA were 88.88%, 77.77% and 38.88%, respectively.

<div>Evaluation of Biofilm Production Capability of Shiga Toxin Producing Escherichia coli Isolated from Milk, Milk Products and Hand Swabs&nbsp;</div>

<div>Background: The principal cause of caprine enterotoxaemia is Clostridium perfringens type D which is normally resident in digestive tract and soil in fairly low numbers. Overeating of carbohydrate rich diet or sudden change in diet leads to proliferation of the bacteria and release of lethal toxins in intestine of the host. It causes sudden death in affected ruminants of different ages, though lambs of 3 to 10 weeks of age are highly susceptible. It causes sever enterocolitis with or without haemorrhagic diarrhoea and encephalitis resulting in acute nervous signs with a fatal outcome. 
 
Methods: Thirty goats of different breeds under 1-4 years age and both sex were presented to Veterinary Clinical Complex, Veterinary College and Research Institute, Tirunelveli with the neurological signs and diarrhoea were the subject of study. Faecal samples were collected from all these cases and tested on spot by immunochromatographic test kit to identify the Clostridium perfringens Epsilon toxin (ETX). Blood and serum samples were collected and subjected haemato-biochemical analysis. Peripheral blood smears were also collected to rule out haemoprotozoan infections in all the cases. 
 
Result: Epsilon toxin was detected from faecal samples of 36.6 per cent of the goats. Diarrhoea was the most consistent finding which is due to enterocolitis in acute cases. A significant increase (P&lt;0.001) in the neutrophils and leukocytes count and a significant increase (P&lt;0.05) in the levels of BUN, creatinine, glucose, ALP and chloride levels were observed. All the cases were treated with sulphadimidine at recommended dose with supportive therapy but no response was reported due to the fatal encephalitis.</div>

Detection of Epsilon Toxin in Goats Affected with Acute Enterotoxaemia and the Haemato-biochemical Alterations

A comparative study was conducted to evaluate yeast sludge cell wall (YSCW) and commercial toxin binders against different levels of aflatoxins B1 (AFB1) in broilers. A total of 390 one day old (1-d-o) chicks were divided into 13 groups with treatments (three replicates / treatment ten birds in each). Negative control (NC) contained no AFB1 and toxin binder in feed. Positive control (PC) was offered with different AFB1 levels (100, 200 and 300 &micro;g/kg) in feed which were being produced in the laboratory earlier. The remaining treatments included YSCW, bentonites, combination of bentonites and glucomannan (Bent + Gluc) at different AFB1 levels in factorial arrangement from 8th to 28th days of age. Among positive control, 200 and 300 &micro;g/kg AFB1 levels showed 37.42% and 36.38% decrease in weight gain causing 1.41 and 1.35 times increase in feed conversion ratio (FCR) compared to NC. YSCW and Bent + Gluc showed better weight gain (87.9, 83.45 and 75.48% for YSCW and 83.37, 82.83 and 78.35 for Bent + Gluc vs 66.26, 62.6 and 63.6% for PC as compared to NC) at 100, 200 and 300 &micro;g AFB1/kg, respectively. The dietary treatments, aflatoxin levels and toxin binders showed non-significant effect for feed intake, serum alanine aminotransferase (ALAT) and significant on albumin and uric acid. It was concluded that AFB1 affect the growth at different levels significantly. Like other toxin binders, YSCW was found to nullify the deleterious effects of AFB1 and showed non-significant effect among themselves at different AFB1 levels.

Evaluation of yeast sludge cell wall and commercial clay based toxin binders against aflatoxin B1 on growth and serum parameters of broilers

Background: Bacillus thuringiensis (Bt) is widely recognized as a safe and effective bioinsecticide, with Cry toxins targeting pests of various insect orders. The present study focuses on the modeling and validation of the Cry3Aa protein (deduced amino acid sequence of cry3Aa gene cloned from native Bt isolate, T121) and its interaction with midgut receptor proteins (ADAM10 and APN).&nbsp; 
 
Methods: The three-dimensional structures of Cry3Aa protein and the midgut receptors ADAM10 and APN were predicted using the SWISS model server. Functional domain analysis of Cry3Aa revealed three distinct domains: N-terminal (Domain I), Central (Domain II) and C-terminal (Domain III), providing insights into their structural organization. The predicted models of Cry3Aa, ADAM10 and APN were validated using the Ramachandran plot which demonstrated structural integrity.&nbsp; 
 
Result: Primary structure analysis of Cry3Aa revealed a 652 amino acid protein with a theoretical isoelectric point of 5.59, a molecular weight of 74 kDa and stable characteristics. Protein-protein docking analysis using ClusPro 2.0 showed that Cry3Aa exhibited higher level of interaction with the ADAM10 receptor than with the APN receptor. The Cry3Aa-ADAM10 docked complex demonstrated 23 hydrogen bonds, reasoning for its stability and binding affinity. These findings revealed that the Cry3Aa protein has a strong affinity against coleopteran specific midgut receptors and hence Cry3Aa has a potential to be an effective coleopteran specific insecticidal protein.

In silico Analysis and Molecular Docking of Cry3Aa Toxin with Coleopteran Specific Midgut Receptor of ADAM10/APN Receptors

<div>Background: Enterotoxaemia represents one of the major limiting factors in small ruminant farming. Rapid lethal progression of the disease makes treatment impractical in most cases. Thus, suitable immunoprophylactic measures are necessary against this disease. The current study was aimed to test out the ability of novel alum precipitated oil adjuvant vaccine (AOV) for overcoming the difficulties associated with short-term immunity of current enterotoxaemia vaccines. 
 
Methods: A new formulation named, AOV was prepared using a highly toxigenic strain of Clostridium perfringens type D. Immunogenicity and protective efficacy of this vaccine in comparison to presently available epsilon toxoid vaccine (TV) and alum precipitated vaccine (APV) was evaluated in the target species, sheep using indirect ELISA, mouse neutralization test (MNT). 
 
Result: Among the three vaccines tested, AOV produced higher and persistent protective antibody titer up to 150 days post-immunization without any booster dose while APV and TV protected only up to 60th and 45th day respectively. All vaccinated animals remained healthy for the whole duration of the study with no systemic or local reactions. The present study delineates the superiority of AOV over presently available TV and APV for the prevention of an extremely lethal disease of sheep and goats.</div>

A New Alum Precipitated Oil Formulation Vis-a-Vis Alum Precipitated and Toxoid Enterotoxaemia Vaccines for the Enhanced and Sustained Immune Response against Epsilon Toxin in Sheep

<div>Background: In sheep, Escherichia coli infection can occur as asymptomatic entity to septicaemic episode. Chiefly, it causes heavy mortality in lambs and devastates the farm economy. With an objective of documenting the salient epidemiological and clinico-pathological observations in this naturally occurred outbreak of septicaemic colibacillosis, this communication is made herewith.&nbsp; 
 
Methods: In February 2019, sudden death in lambs was observed in three flocks of sheep. Carcasses were subjected for necropsy and histopathological examination. Swab samples from heart, abomasum, intestines, liver and lung were examined for bacteriological and molecular confirmation.&nbsp; 
 
Result: The mean (&plusmn;SE) age of lambs that were affected was 11.00 &plusmn;0.49 days (n=404). Lambs of native Malpura breed (87.5%) and other two crossbred sheep were affected. Overall mortality of lambs was 23.76%. Gross pathological observations were pulmonary edema, ecchymotic lesions in lungs, congestion in kidney and liver and presence soil-mixed ingesta in abomasum. From the morbid materials including stomach and intestinal swabs, 45 E. coli isolates were identified and they were also confirmed on polymerase chain reaction (PCR). Consequently, virulence genes for shiga toxin (stx2) and intimin (eae) were identified from the isolates. Although antibiotics were administered, only probiotics could control the new infection rate.&nbsp;</div>

<div>Soil-borne Septicaemic Colibacillosis in Neonatal Lambs: Salient Observations</div>

Meat is a global vital protein source catering to diverse diets. By 2050, world&rsquo;s population may rise to 9 billion and the demand for meat will exceed supply, necessitating a doubling of meat production. Nutritionally rich composition makes meat, a fertile ground for numerous microbes, leading to spoilage and foodborne illnesses, impacting human health. External factors like poor hygiene, inadequate post-production storage, mishandling and more contribute to meat contamination. The zoonotic strains of E. coli can cause severe food poisoning, with over 700 identified serotypes, hence it is crucial to identify, monitor and control E. coli to ensure food safety. Among E. coli pathotypes, Verocytotoxigenic E. coli (VTEC) stands out as major cause of food poisoning. The VTEC toxin (Shiga toxin), can induce mild diarrhea to severe form of illness like Hemorrhagic Uremic Syndrome and Hemorrhagic Colitis, posing a severe public health risk. The Cattle is a primary reservoir for VTEC, which, by faecal shedding, can pollute food, water and the environment. Furthermore, E. coli can spread through contaminated meat, raw milk and milk products. This bacterium is commonly found in the immediate surroundings of animal reservoir, including soil, grass and manure, leading to potential contamination of milk. E. coli infections not only impact humans but also animals, with cattle, sheep and poultry acting as reservoirs for different E. coli strains. Cattle populations frequently harbor E. coli O157:H7 asymptomatic carriers, contributing to faecal contamination of food and water sources. Educating the public about the risks associated with improper meat handling and contamination is essential to reduce foodborne illnesses. Monitoring and surveillance of meat quality in local markets, as are crucial steps towards ensuring food safety. This underscores the importance of understanding and managing E. coli contamination in meat to safeguard public health and ensure the safety of meat products in the global food supply chain.

Public Health Significance of Verocytotoxigenic Escherichia coli: An Overview

<div style="text-align: justify;">Screening of 105 samples comprising of milk, meat and their products for isolation of Clostridium perfringens revealed a total of 39 (37.14%) samples to be positive for C. perfringens, yielding an equal number of isolates. Among the total isolates, 11 (20.0%) belonged to milk and milk products, of which 10 were isolated from raw cow&rsquo;s milk. The remaining isolate was from paneer sample. Similarly, 28 (56.0%) C. perfringens isolates were recovered from meat and meat products. Majority (37) of the isolates belonged to toxin type A while two isolates recovered each from raw beef and chicken meats were of toxin type C. Among the 39 isolates of C. perfringens, only 5(12.82%) isolates could reveal the presence of cpe (enterotoxin) gene. The protein profile (SDS-PAGE) of both the crude and partially purified enterotoxins prepared from cpe positive C. perfringens isolates (type A and C) revealed a very little difference. Both the partially purified enterotoxins of type A and C of C. perfringens displayed almost similar type of cytopathic effects (CPE) and death of cells in Vero cell line.</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

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

Evaluation of Biofilm Production Capability of Shiga Toxin Producing Escherichia coli Isolated from Milk, Milk Products and Hand Swabs

Detection of Epsilon Toxin in Goats Affected with Acute Enterotoxaemia and the Haemato-biochemical Alterations

Evaluation of yeast sludge cell wall and commercial clay based toxin binders against aflatoxin B₁ on growth and serum parameters of broilers

In silico Analysis and Molecular Docking of Cry3Aa Toxin with Coleopteran Specific Midgut Receptor of ADAM10/APN Receptors

A New Alum Precipitated Oil Formulation Vis-a-Vis Alum Precipitated and Toxoid Enterotoxaemia Vaccines for the Enhanced and Sustained Immune Response against Epsilon Toxin in Sheep

Soil-borne Septicaemic Colibacillosis in Neonatal Lambs: Salient Observations

Public Health Significance of Verocytotoxigenic Escherichia coli: An Overview

Characterization of enterotoxin producing Clostridium perfringens isolated from foods of animal origin