Situation Analysis and Recommendations for the Biosecurity Status of Dairy Farms in Punjab, India: A Cross-Sectional Survey.

Farm biosecurity is an important herd management strategy to assure infection prevention and animal health. The present study aimed to evaluate the implementation of biosecurity measures on dairy farms in Punjab, India, using the standardized Biocheck.UGent™ scoring system. Convenience sampling was used to select 94 dairy farms, comprising both cattle and buffaloes, with a mean herd size of 74.8 animals. The study found that the mean external and internal biosecurity scores for the selected dairy farms were 45.4% and 43.7%, respectively. Among the subcategories, the highest external biosecurity score was observed for 'vermin control and other animals' (63%), and the highest internal biosecurity score was observed for 'adult cattle management' (76.6%). Whereas the lowest score for external biosecurity was observed for 'purchase and reproduction' (30.6%), and the lowest score for internal biosecurity was observed for 'health management' (33.6%). The overall mean biosecurity score of the present study was 44.8%, which was lower than the overall mean global score of 52%. The correlation analysis indicated a positive correlation between herd size and overall biosecurity scores, indicating that larger farms had, on average, higher biosecurity scores. Based on these results, it can be concluded that the implementation of biosecurity measures on dairy farms in Punjab needs improvement, especially in the subcategories of 'purchase and reproduction' and 'health management'. Further research to identify the factors influencing the implementation of biosecurity measures on dairy farms in the region is recommended.

Can Improved Farm Biosecurity Reduce the Need for Antimicrobials in Food Animals? A Scoping Review.

Limited and judicious antimicrobial usage (AMU) is considered the key to saving the success of human and veterinary medicine in treating infections. With the limited alternatives for antimicrobials, farm biosecurity (and herd management) is considered a promising tool to mitigate the non-judicious AMU and to maintain animal health, production, and welfare. The present scoping review aims to analyse the effect of farm biosecurity on AMU in livestock systems and formulate recommendations. Peer-reviewed manuscripts published between 2001-2022 were analyzed using the PRISMA framework using PubMed, Scopus, and Science Direct databases. After applying the inclusion criteria, 27 studies were found to assess the effect of farm biosecurity (or management practices) on AMU at the herd/farm level in quantitative/semi-quantitative terms. These studies were carried out in 16 countries, of which 74.1% (20/27) were from 11 European countries. The highest number of studies were from pig farms [51.8% (14/27)], followed by poultry (chicken) farms [25.9% (7/27)], cattle farms [11.1% (3/27)], and a single study from a turkey farm. Two studies include both pig and poultry farms. Most of the studies were cross-sectional [70.4% (19/27)], seven were longitudinal, and one was a case-control study. Complex interactions were observed among factors influencing AMU, such as biosecurity measures, farm characteristics, farmers' attitudes, availability of animal health services, stewardship, etc. A positive association between farm biosecurity and reduction in AMU was observed in 51.8% (14/27) of the studies, and 18.5% (5/27) showed that improvement in farm management practices was associated with a reduction in AMU. Two studies highlighted that coaching and awareness among farmers might lead to a decrease in AMU. A single study on economic assessment concluded biosecurity practices as a cost-effective way to reduce AMU. On the other hand, five studies showed an uncertain or spurious association between farm biosecurity and AMU. We recommend the reinforcement of the concept of farm biosecurity, especially in lower- and middle-income countries (LMICs). Further, there is a need to strengthen the evidence on the association between farm biosecurity and AMU in region- and species-specific farm settings.

Monitoring of antimicrobial usage among adult bovines in dairy herds of Punjab, India: A quantitative analysis of pattern and frequency.

The present study aimed to evaluate the antimicrobial usage (AMU) pattern in dairy herds of Punjab, India. The on-farm quantification of AMU in adult bovine animals by the manual collection of empty drug containers ("bin method") along with the records of the treatment was carried out in 38 dairy farms involving 1010 adult bovines for 1 year from July 2020 to June 2021. The farm owners were asked to record the antibiotic treatments as well as to deposit empty antibiotic packaging/vials into the provided bins placed at the farms. A total of 14 different antibiotic agents in 265 commercial antibiotic products were administered to the dairy herds during the study. A total of 179 (67.55%) administered products contained antimicrobials of "critical importance" as per the World Health Organization (WHO). Mastitis (54.72%), followed by the treatment of fever (19.62%), reproductive problems (15.47%), and diarrhea (3.40%) accounted for the majority of drugs administered in the herds during the study period. The most commonly used antibiotics were enrofloxacin (89.47% herds; 21.51% products), followed by ceftriaxone (50% herds; 12.83% products), amoxicillin (50% herds; 12.83% products), oxytetracycline (55.26% herds; 11.70% products), and procaine penicillin (47.37% herds; 12.83% products). The highest quantity of AMU [in terms of antimicrobial drug use rate (ADUR)] was observed for ceftiofur, followed by ceftriaxone, procaine benzyl penicillin ceftizoxime, enrofloxacin, cefoperazone, amoxicillin and ampicillin. A total of 125 (47.17%) products contained "highest priority critically important antimicrobials" (HPCIA) and 54 (20.37%) products contained "high priority critically important antimicrobials". In terms of overall number of animal daily doses (nADD), the highest priority critically important antimicrobials (HPCIA) of the WHO such as third-generation cephalosporins and quinolones, respectively accounted for 44.64 and 22.35% of the total antibiotic use in the herds. The bin method offers an alternative to monitoring AMU as a more accessible approach for recording the actual consumption of antimicrobials. The present study, to the best of our knowledge, is the first of its kind to provide an overview of the qualitative and quantitative estimate of AMU among adult bovines from India.

Prevalence, molecular characterization, and antimicrobial resistance profile of Clostridium perfringens from India: A scoping review.

Clostridium perfringens is one of the most important foodborne pathogens that causes histotoxic diseases and intestinal infections in both humans and animals. The present scoping review has been designed to analyze the literature published during 2000-2021 from India on the prevalence, molecular characterization, and antimicrobial resistance profile of C. perfringens isolates recovered from humans, animals, animal-based foods, and associated environmental samples. The peer-reviewed articles retrieved from four electronic databases (Google Scholar, PubMed, Science Direct, and Web of Science) were assessed using PRISMA-ScR guidelines. A total of 32 studies from India were selected on the basis of their relevance and inclusion criteria. The overall prevalence of C. perfringens among domestic animals having history of clinical symptoms and among healthy animals was found to be 65.8% (508/772) and 42.8% (493/1152), respectively. The pathogen was also detected in clinically affected wild animals (75%), healthy wild animals (35.4%), and captive birds (24.5%). The detection of C. perfringens among poultry having necrotic enteritis and among healthy birds was found to be 66.8% (321/480) and 25.6% (80/312), respectively. The detection of pathogen among animal-based foods (i.e., meat, milk, and fish and their products) and environmental samples depicted a prevalence of 20.8% (325/1562) and 30.2% (23/76), respectively. However, the prevalence of C. perfringens among humans having history of diarrhea and among healthy humans was found to be 25% (70/280) and 23.2% (36/155), respectively. The genotyping of C. perfringens isolates revealed that toxin type A was found to be the most prevalent genotype. Along with the alpha toxin gene (cpa), beta (cpb), epsilon (etx), iota (itx), enterotoxin (cpe), beta-2 toxin (cpb2), and NetB (netB) toxins were also detected in different combinations. Antimicrobial resistance profile of C. perfringens isolates recovered from different sources demonstrated that the highest resistance was detected against sulphonamides (76.8%) and tetracycline (41.3%) by phenotypic and genotypic detection methods, respectively. Comprehensive scientific studies covering different geographical areas at the human-animal-environment interface are crucial to generalize the real magnitude of C. perfringens-associated problem in India and for establishing a reliable database.

Qualitative Study on Antimicrobial Usage and Resistance in the Dairy Chain: A Situation Analysis and Solutions by Stakeholders from Punjab, India.

The rising prevalence of antimicrobial resistance in animal foods and injudicious antibiotic use in the dairy sector pose significant threats to public health. Focus group discussions (FGDs) and key informant interviews (KIIs) were conducted with a strategic sample of four stakeholder groups (114 participants) associated with antibiotic usage in the dairy sector of Punjab. The FGDs were conducted among veterinarians (n = 56), para-veterinarians (n = 28), and KIIs were conducted among chemists (n = 18) and dairy quality managers (n = 12) during 2020-2021. FGDs and qualitative interviews of various stakeholders depict existing risk practices in the fields that may promote antimicrobial resistance. The present study revealed that widely prevalent quackery (treatment practices carried out by unauthorized persons without any recognized diploma/degree) and self-treatment by farmers, over-the-counter availability of antibiotics, low veterinarian per animal ratio, and lack of awareness among the society about the potential public health effects of antimicrobial resistance were the main risk factors for injudicious antibiotic use in the dairy sector. The present study involved a comprehensive approach targeting the stakeholders in the dairy sector and their reflections on judicious antimicrobial usage and antimicrobial resistance adapted to the dairy farming of Punjab. There is an urgent need for the advocation of policies that consider the specific challenges faced by the dairy sector to simultaneously improve access to veterinary services as well as strengthen antibiotic stewardship.

Biofilms as a microbial hazard in the food industry: A scoping review.

Biofilms pose a serious public health hazard with a significant economic impact on the food industry. The present scoping review is designed to analyse the literature published during 2001-2020 on biofilm formation of microbes, their detection methods, and association with antimicrobial resistance (if any). The peer-reviewed articles retrieved from 04 electronic databases were assessed using PRISMA-ScR guidelines. From the 978 preliminary search results, a total of 88 publications were included in the study. On analysis, the commonly isolated pathogens were Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Escherichia coli, Bacillus spp., Vibrio spp., Campylobacter jejuni and Clostridium perfringens. The biofilm-forming ability of microbes was found to be influenced by various factors such as attachment surfaces, temperature, presence of other species, nutrient availability etc. A total of 18 studies characterized the biofilm-forming genes, particularly for S. aureus, Salmonella spp., and E. coli. In most studies, polystyrene plate and/or stainless-steel coupons were used for biofilm formation, and the detection was carried out by crystal violet assays and/or by plate counting method. The strain-specific significant differences in biofilm formation were observed in many studies, and few studies carried out analysis of multi-species biofilms. The association between biofilm formation and antimicrobial resistance was not clearly defined. Further, viable but non-culturable form of the foodborne pathogens is posing an unseen (by conventional cultivation techniques) but potent threat to the food safety. The present review recommends the need for carrying out systematic surveys and risk analysis of biofilms in food chain to highlight the evidence-based public health concerns, especially in regions where microbiological food hazards are quite prevalent.

Emergency preparedness for public health threats, surveillance, modelling & forecasting.

In the interconnected world, safeguarding global health security is vital for maintaining public health and economic upliftment of any nation. Emergency preparedness is considered as the key to control the emerging public health challenges at both national as well as international levels. Further, the predictive information systems based on routine surveillance, disease modelling and forecasting play a pivotal role in both policy building and community participation to detect, prevent and respond to potential health threats. Therefore, reliable and timely forecasts of these untoward events could mobilize swift and effective public health responses and mitigation efforts. The present review focuses on the various aspects of emergency preparedness with special emphasis on public health surveillance, epidemiological modelling and capacity building approaches. Global coordination and capacity building, funding and commitment at the national and international levels, under the One Health framework, are crucial in combating global public health threats in a holistic manner.

Knowledge, Attitude, and Practices (KAP) Survey among Veterinarians, and Risk Factors Relating to Antimicrobial Use and Treatment Failure in Dairy Herds of India.

The indiscriminate usage of antimicrobials in the animal health sector contributes immensely to antimicrobial resistance (AMR). The present study aims to assess the antimicrobial usage pattern and risk factors for AMR in animal husbandry sector of India. A cross-sectional survey about Knowledge, Attitude, and Practices (KAP) among veterinarians was carried out using a questionnaire comprising of 52 parameters associated with antibiotic use and the emergence of AMR in dairy herds. Respondents' KAP scores were estimated to rank their level of knowledge, attitude, and practice. Furthermore, risk factors associated with treatment failure were analyzed by univariable and multivariable analyses. Out of a total of 466 respondents, the majority had average knowledge (69.5%), neutral attitude (93.2%), and moderate practice (51.3%) scores toward judicious antibiotic usage. Veterinarians reported mastitis (88.0%), reproductive disorders (76.6%), and hemoprotozoan infections (49.6%) as the top three disease conditions that require antibiotic usage. Most of the veterinarians (90.6%) resorted to their "own experience" as the main criteria for antibiotic choice. The use of the highest priority critically important antimicrobials (HPCIA) listed by the World Health Organization (WHO) in animals, particularly quinolones (76.8%) and third-generation cephalosporins (47.8%), has been reported. On multivariable regression analysis of the risk factors, the lack of cooperation of the dairy farmers in the completion of a prescribed antibiotic course by the veterinarian and the demand for antibiotic use even in conditions not requiring antibiotic use were found to be significantly associated with the outcome variable "treatment failure" having respective odds of 1.8 (95%CI: 1.1-3.0) and 3.6 (95%CI: 2.3-5.8) (p < 0.05). The average KAP score of veterinarians, poor farm management practices, lack of awareness among farmers on prudent antibiotic use, and lack of antibiotic stewardship are the significant factors that need attention to combat the rising AMR in veterinary sector in India.

Molecular characterization and antimicrobial resistance profile of Clostridium perfringens type A isolates from humans, animals, fish and their environment.

The study was aimed to characterize, and determine antibiogram of C. perfringens type A isolated from the feces of human and animal diarrhoeal cases, as well as healthy animals, meat of pigs and goats, gills and intestine of fish and samples from fish pond. A total of 460 samples, including human diarrhoeal cases (n = 130); diarrhoeal cases of pig (n = 52) and goat (n = 50); fecal samples from healthy pig (n = 50) and goat (n = 50); meat samples viz. pork meat (n = 52); goat meat (n = 50) and fish including their environmental sources (n = 26) were used for isolation and identification of C. perfringens type A. All the biochemically confirmed isolates were positive for species-specific 16S rRNA and cpa genes by PCR assays. Toxinotyping of C. perfringens type A isolates showed that overall prevalence of C. perfringens type A with only cpa+ gene was 43.2%; with cpa+ and cpb2+ genes was 45.4%; with cpa+ and cpe+ genes was 4.9%; however, with cpa+, cpb2+and cpe+ genes was 6.6%. Antimicrobial susceptibility testing revealed that 83.7% of isolates were resistant to three or more antibiotics.

Antimicrobial effects of Lactobacillus plantarum and Lactobacillus acidophilus against multidrug-resistant enteroaggregative Escherichia coli.

The in vitro and in vivo antimicrobial effects of Lactobacillus plantarum and Lactobacillus acidophilus were evaluated individually and synergistically against multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC). In vitro evaluation of each probiotic strain when co-cultured with MDR-EAEC isolates revealed a reduction in MDR-EAEC counts (eosin-methylene blue agar) in a dose- and time-dependent manner: probiotics at a dose rate of 10(10) CFU inhibited MDR-EAEC isolates at 72 h post-inoculation (PI), whereas at lower concentrations (10(8) and 10(9) CFU) MDR-EAEC isolates were inhibited at 96 h PI. The synergistic antimicrobial effect of both probiotic strains (each at 10(10) CFU) was highly significant (P < 0.01) and inhibited the growth of MDR-EAEC isolates at 24 h PI. For in vivo evaluation, weaned mice were fed orally with 10(7) CFU of MDR-EAEC. At Day 3 post-infection, treated mice were fed orally with the probiotic strains (each at 10(10) CFU). Compared with the control, post-treatment a significant (P < 0.01) reduction in MDR-EAEC counts was observed in faeces by Day 2 and in intestinal tissues of treated mice by Days 3 and 4 as evidenced by plate count (mean 2.71 log and 2.27 log, respectively) and real-time PCR (mean 1.62 log and 1.57 log, respectively) methods. Histopathologically, comparatively mild changes were observed in the ileum and colon from Days 3 to 5 post-treatment with probiotics; however, from Day 6 the changes were regenerative or normal. These observations suggest that these probiotic strains can serve as alternative therapeutics against MDR-EAEC-associated infections in humans and animals.

Genetic diversity and antibiogram profile of diarrhoeagenic Escherichia coli pathotypes isolated from human, animal, foods and associated environmental sources.

INTRODUCTION: Infectious diarrhoea particularly due to pathogenic bacteria is a major health problem in developing countries, including India. Despite significant reports of diarrhoeagenic Escherichia coli (DEC) pathotypes around the globe, studies which address genetic relatedness, antibiogram profile and their correlation with respect to their isolation from different sources are sparse. The present study determines isolation and identification of DEC pathotypes from different sources, their genetic characterisation, antibiogram profile and their correlation if any. MATERIALS AND METHODS: A total of 336 samples comprising diarrhoeic stool samples from infants (n=103), young animal (n=106), foods (n=68) and associated environmental sources (n=59) were collected from Bareilly region of India. All the samples were screened by using standard microbiological methods for the detection of E. coli. The identified E. coli were then confirmed as DEC pathotypes using polymerase chain reaction-based assays. Those DEC pathotypes identified as Enteroaggregative E. coli (EAEC) were further confirmed using HEp-2 adherence assay. All the isolated DEC pathotypes were studied for their genetic diversity using pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing was performed by using disc diffusion method as per Clinical Laboratory Standards Institute guidelines. RESULTS AND DISCUSSION: Of the four DEC pathotypes investigated, EAEC was found to be the predominant pathogen with an isolation rate of 16.5% from infants, 17.9% from young animals, 16.2% from foods and 3.4% from the associated environmental sources. These EAEC isolates, on further characterisation, revealed predominance of 'atypical' EAEC, with an isolation rate of 10.7% from infants, 15.1% from young animals, 16.2% from foods, and 3.4% from the associated environmental sources. On PFGE analysis, discrimination was evident within DEC pathotypes as 52 unique pulsotypes were observed for 59 recovered DEC pathotypes. However, a few EAEC isolates were found to be clonal (clusters A, B, C, D, F, G, and H) irrespective of their source of isolation, suggests sharing and/or circulation among different sources. Further, a high antibiotic resistance pattern was observed among isolated DEC pathotypes as almost 86.4% of isolates were found to be resistant against ≥3 tested drugs.

Genetic diversity, virulence potential and antimicrobial susceptibility of Listeria monocytogenes recovered from different sources in India.

Listeria monocytogenes isolates (n = 36) recovered from human and animal clinical cases and foods from different geographical regions of India were characterized using multiplex PCR-based serotyping, pulsed field gel electrophoresis (PFGE), in vitro and in vivo pathogenicity tests and antibiogram profiling. Multiplex PCR-based serotyping distributed L. monocytogenes isolates into 3 serogroups, of which 91.67% belonged to 4b, 4d, 4e serogroup, followed by 5.56% to 1/2a, 3a and 2.78% to 1/2b, 3b serogroups. PFGE analysis using ApaI and AscI restriction enzymes revealed 17 pulsotypes among 36 L. monocytogenes isolates with 6 major clusters having similar fingerprint profile within their cluster and 11 unique fingerprint profiles. Interestingly, PFGE analysis inferred that foods of animal origin could be a significant source of infection for spread of listeriosis among human populations. Furthermore, on comparison of in vitro and in vivo pathogenicity tests, an overall good correlation was observed between hemolytic titer assay and chick embryo inoculation test as most of the isolates with a hemolytic titer of ≥ 16 were found to be lethal to chick embryo. All the isolates were found to be susceptible to tested antimicrobials except for one animal isolate which showed resistance towards co-trimoxazole.

Evaluation of a PCR targeting fimbrial subunit gene (fimA) for rapid and reliable detection of Enteroaggregative Escherichia coli recovered from human and animal diarrhoeal cases.

The present study describes the utility of a chromosomal associated fimbrial subunit gene (fimA) for screening 'typical' as well as 'atypical' Enteroaggregative Escherichia coli (EAEC) by PCR and its possible role as a promising molecular marker for rapid detection of 'typical' and 'atypical' EAEC pathotype.

Characterization and biofilm forming ability of diarrhoeagenic enteroaggregative Escherichia coli isolates recovered from human infants and young animals.

Enteroaggregative Escherichia coli (EAEC) is an important pathotype that causes infection in humans and animals. EAEC isolates (n=86) recovered from diarrhoeal cases in human infants (37) and young animals (49) were characterized as 'typical' and/or 'atypical' EAEC strains employing PCR for virulence associated genes (cvd432, aaiA, astA, pilS, irp2, ecp, pic, aggR, aafA, aggA, and agg3A). Besides, biofilm formation ability of human and animal EAEC isolates was assessed using microtiter plate assay. In addition, the transcriptional profile of biofilm associated genes (fis and ecp) was also evaluated and correlated with biofilm formation assay for few selected EAEC isolates of human and animal origins. Overall, a diverse virulence gene profile was observed for the EAEC isolates of human and animal origins as none of the EAEC isolates revealed the presence of all the genes that were targeted. Nine 'typical' EAEC isolates were identified (6 from humans and 3 from animals) while, the majority of the isolates were 'atypical' EAEC strains. Isolation and identification of three 'typical' EAEC isolates from animals (canines) appears to be the first report globally. Further, based on the observations of the biofilm formation assay, the study suggested that human EAEC isolates in particular were comparatively more biofilm producers than that of the animal EAEC isolates. The fis gene was highly expressed in majority of 'typical' EAEC isolates and the ecp gene in 'atypical' EAEC isolates.