Vector competence of Swedish Culex pipiens mosquitoes for Japanese encephalitis virus.

BACKGROUND: Japanese encephalitis virus (JEV) is an emerging mosquito-borne Orthoflavivirus that poses a significant public health risk in many temperate and tropical regions in Asia. Since the climate in some endemic countries is similar to temperate climates observed in Europe, understanding the role of specific mosquito species in the transmission of JEV is essential for predicting and effectively controlling the potential for the introduction and establishment of JEV in Europe. METHODS: This study aimed to investigate the vector competence of colonized Culex pipiens biotype molestus mosquitoes for JEV. The mosquitoes were initially collected from the field in southern Sweden. The mosquitoes were offered a blood meal containing the Nakayama strain of JEV (genotype III), and infection rates, dissemination rates, and transmission rates were evaluated at 14, 21, and 28 days post-feeding. RESULTS: The study revealed that colonized Swedish Cx. pipiens are susceptible to JEV infection, with a stable infection rate of around 10% at all timepoints. However, the virus was only detected in the legs of one mosquito at 21 days post-feeding, and no mosquito saliva contained JEV. CONCLUSIONS: Overall, this research shows that Swedish Cx. pipiens can become infected with JEV, and emphasizes the importance of further understanding of the thresholds and barriers for JEV dissemination in mosquitoes.

Understanding Vietnamese chicken farmers' knowledge and practices related to antimicrobial resistance using an item response theory approach.

Introduction: Antimicrobial resistance (AMR) poses a threat to animal and human health, as well as food security and nutrition. Development of AMR is accelerated by over- and misuse of antimicrobials as seen in many livestock systems, including poultry production. In Vietnam, high AMR levels have been reported previously within poultry production, a sector which is dominated by small-scale farming, even though it is intensifying. This study focuses on understanding small- and medium-scale chicken farmers' knowledge and practices related to AMR by applying an item response theory (IRT) approach, which has several advantages over simpler statistical methods. Methods: Farmers representing 305 farms in Thai Nguyen province were interviewed from November 2021 to January 2022, using a structured questionnaire. Results generated with IRT were used in regression models to find associations between farm characteristics, and knowledge and practice levels. Results: Descriptive results showed that almost all farmers could buy veterinary drugs without prescription in the local community, that only one third of the farmers received veterinary professional advice or services, and that the majority of farmers gave antibiotics as a disease preventive measure. Regression analysis showed that multiple farm characteristics were significantly associated to farmers' knowledge and practice scores. Conclusion: The study highlights the complexity when tailoring interventions to move towards more medically rational antibiotic use at farms in a setting with high access to over-the-counter veterinary drugs and low access to veterinary services, since many on-farm factors relevant for the specific context need to be considered.

Knowledge and practices related to antibiotics among poultry producers and veterinarians in two Indian states.

Background: Antibiotics are frequently utilized in livestock, particularly poultry, for therapy and growth promotion, resulting in antimicrobial resistance. Multidrug-resistant bacteria are frequent in poultry samples from India. The purpose of this study was to better understand main antibiotic consumption patterns in poultry value chains, as well as antibiotic knowledge and practices among the stakeholders. Methods: A cross-sectional survey was conducted in Assam and Karnataka, India. The poultry farmers were interviewed on antibiotic usage, antibiotic knowledge, feeding practices, and preventive measures on the farm. Poultry farmers reported their veterinarians, and we also interviewed them on knowledge and practices related to antimicrobial use in poultry and antimicrobial resistance. Item response theory (IRT) was used to assess the association between the answers and demographic factors. Results: This survey interviewed 62 poultry farmers and 11 veterinarians. Small poultry farms with fewer than 4000 birds were owned by 51.6% of farmers. Most poultry farmers had heard about antibiotics, and 62.9% thought they cured all diseases. If one chicken is sick, 72.6% said others should be given antibiotics to prevent the disease. All veterinarians utilized tetracyclines, aminoglycosides, and cephalexin on the poultry farms. Over half (54.5%) stated antibiotics prevent diseases, and 72.7% said they treat and prevent diseases. Some (45.5%) said antibiotics boost growth. IRT analysis showed that 8 questions assessed a knowledge scale well. Univariable analysis showed that Assam farmers and women were likely to have have more knowledge. Conclusion: The poultry farmers were mostly unaware of the relation between antibiotic use and antimicrobial resistance. Despite being aware, the veterinarians agreed with use antibiotics as a prophylactic measure. It is vital that these stakeholders understand the repercussions of such widespread antibiotic use. In order to increase knowledge, frequent trainings and antimicrobial stewardship programmes with effective communication and incentives for behaviour change should be conducted.

Antibiotics in semen extenders - a multiplicity of paradoxes.

Addition of antibiotics to semen extenders was taken for granted for many years, from the time that commercial artificial insemination in livestock first began many decades ago. However, there is now a growing realisation that this non-therapeutic utilisation of antibacterial agents is contrary to current recommendations for prudent use that medical and veterinary professionals are advised to follow. Furthermore, antibiotics are not benign, having negative effects on sperm samples, the inseminated female, personnel and potentially the environment. The purpose of this review is three-fold: to highlight the fact that antibiotics are used in semen extenders, with the result that considerable amounts are used globally in animal breeding, to review recent studies on the negative aspects of using antibiotics for this purpose, and to look at possible alternatives. Recent changes in the legislation regarding semen extenders occurred in some, but not all, countries, leaving question marks for semen producers as to whether antibiotics should be added to semen extenders or not.

Limiting the spillover of zoonotic pathogens from traditional food markets in developing countries and a new market design for risk-proofing.

Traditional food markets are age-old systems that primarily serve the food supply needs of society's less affluent sectors, often operating with minimal infrastructure. These markets are prevalent in low and middle-income countries. However, their hygienic conditions are frequently suboptimal, potentially fostering the emergence and spread of presumptive zoonotic diseases. The recent emergence of zoonotic or potentially zoonotic diseases and their possible links to traditional food markets underscore the need for focused attention on this overlooked issue. The socioeconomic characteristics of traditional food markets reveal that despite the risk of zoonotic pathogen spread, these markets play a crucial role for large segments of the population. These individuals rely on such markets for their livelihood, food, and nutrition. Therefore, a comprehensive set of measures addressing various aspects of traditional food markets is necessary to manage and mitigate the risks of potential zoonotic disease emergence. In this article, we explore various facets of traditional food markets, paying special attention to the risks of zoonotic diseases that urgently require stakeholder attention. We also propose a new market design to prevent the risk of zoonotic spillover and advocate for the development of a Market Hygiene Index for these markets.

Analyses of Extended-Spectrum-ß-Lactamase, Metallo-ß-Lactamase, and AmpC-ß-Lactamase Producing Enterobacteriaceae from the Dairy Value Chain in India.

The consumption of milk contaminated with antibiotic-resistant bacteria poses a significant health threat to humans. This study aimed to investigate the prevalence of Enterobacteriaceae producing ß-lactamases (ESBL, MBL, and AmpC) in cow and buffalo milk samples from two Indian states, Haryana and Assam. A total of 401 milk samples were collected from dairy farmers and vendors in the specified districts. Microbiological assays, antibiotic susceptibility testing, and PCR-based genotyping were employed to analyze 421 Gram-negative bacterial isolates. The overall prevalence of ß-lactamase genes was 10% (confidence interval (CI) (7-13)), with higher rates in Haryana (13%, CI (9-19)) compared to Assam (7%, CI (4-11)). The identified ß-lactamase genes in isolates were blaCMY, blaMOX, blaFOX, blaEBC, and blaDHA, associated with AmpC production. Additionally, blaCTX-M1, blaSHV, and blaTEM were detected as ESBL producers, while blaVIM, blaIMP, blaSPM, blaSIM, and blaGIM were identified as MBL producers. Notably, Shigella spp. were the dominant ß-lactamase producers among identified Enterobacteriaceae. This study highlights the presence of various prevalent ß-lactamase genes in milk isolates, indicating the potential risk of antimicrobial-resistant bacteria in dairy products. The presence of ß-lactam resistance raises concern as this could restrict antibiotic options for treatment. The discordance between genotypic and phenotypic methods emphasizes the necessity for comprehensive approaches that integrate both techniques to accurately assess antibiotic resistance. Urgent collaborative action incorporating rational and regulated use of antibiotics across the dairy value chain is required to address the global challenge of ß-lactam resistance.

Effects of Aflatoxins and Fumonisins, Alone or in Combination, on Performance, Health, and Safety of Food Products of Broiler Chickens, and Mitigation Efficacy of Bentonite and Fumonisin Esterase.

The current study evaluated the effects of feeding diets contaminated with aflatoxin B1 (AFB1), fumonisins (FBs), or both on the performance and health of broiler chickens and the safety of their food products as well as the efficacy of bentonite and fumonisin esterase to mitigate the effects of these mycotoxins under conditions representative for sub-Saharan Africa (SSA). Four hundred one-day-old Cobb 500 broiler chickens were randomly assigned to 20 treatments with either a control diet, a diet with moderate AFB1 (60 µg/kg feed) or high AFB1 (220 µg/kg feed), or FBs (17,430 µg FB1+FB2/kg feed), alone or in combination, a diet containing AFB1 (either 60 or 220 µg/kg) and/or FBs (17,430 µg FB1+FB2/kg) and bentonite or fumonisin esterase or both, or a diet with bentonite or fumonisin esterase only. The experimental diets were given to the birds from day 1 to day 35 of age, and the effects of the different treatments on production performance were assessed by feed intake (FI), body weight gain (BWG), and feed conversion ratio (FCR). Possible health effects were evaluated through blood biochemistry, organ weights, mortality, liver gross pathological changes, and vaccine response. Residues of aflatoxins (AFB1, B2, G1, G2, M1 and M2) were determined in plasma, muscle, and liver tissues using validated UHPLC-MS/MS methods. The results obtained indicated that broiler chickens fed high AFB1 alone had poor FCR when compared to a diet with both high AFB1 and FBs (p = 0.0063). Serum total protein and albumin from birds fed FBs only or in combination with moderate or high AFB1 or detoxifiers increased when compared to the control (p < 0.05). Liver gross pathological changes were more pronounced in birds fed contaminated diets when compared to birds fed the control or diets supplemented with mycotoxin detoxifiers. The relative weight of the heart was significantly higher in birds fed high AFB1 and FBs when compared to the control or high AFB1 only diets (p < 0.05), indicating interactions between the mycotoxins. Inclusion of bentonite in AFB1-contaminated diets offered a protective effect on the change in weights of the liver, heart and spleen (p < 0.05). Residues of AFB1 were detected above the limit of quantification (max: 0.12 ± 0.03 µg/kg) in liver samples only, from birds fed a diet with high AFB1 only or with FBs or the detoxifiers. Supplementing bentonite into these AFB1-contaminated diets reduced the levels of the liver AFB1 residues by up to 50%. Bentonite or fumonisin esterase, alone, did not affect the performance and health of broiler chickens. Thus, at the doses tested, both detoxifiers were safe and efficient for use as valid means of counteracting the negative effects of AFB1 and FBs as well as transfer of AFB1 to food products (liver) of broiler chickens.

Effect of light-touch intervention and associated factors to microbial contamination at small-scale pig slaughterhouses and traditional pork shops in Vietnam.

Traditional pork value chains dominate the production and distribution of pork in Vietnam; however, the high level of microbiological contamination in pork may increase the risk of food-borne disease for consumers. There is limited evidence about how to feasibly and scalably reduce microbial contamination in pork sold in traditional markets. This study aimed to assess the effectiveness of light-touch interventions for changing worker behaviour in small-scale slaughterhouses and vendors at traditional pork shops, as well as to identify risk factors for pork contamination. The intervention packages consisted of providing hygiene tools and delivering a food safety training which had been designed in a participatory way and covered 10 small-scale slaughterhouses and 29 pork shops. Pig carcasses, retailed pork, contact surfaces, and hands were sampled to measure the total bacterial count (TBC) and Salmonella contamination before, three and six weeks after the intervention, and trainee practices were observed at the same time. Linear and generalized linear mixed effects models were constructed to identify risk factors for TBC and Salmonella contamination at the slaughterhouses and pork shops. The interventions at slaughterhouses and pork shops both showed a slight reduction of TBC contamination in pig carcasses and Salmonella prevalence in retailed pork, while the TBC in retailed pork decreased only marginally. For slaughterhouses, the regression model indicated that smoking or eating during slaughtering (indicating poor hygienic practices) was associated with TBC increasing, while cleaning floors and wearing boots reduced TBC contamination. For pork shops, using rough materials (cardboard or wood) to display pork was the only factor increasing TBC contamination in pork, whereas cleaning knives was associated with lower TBC. Besides, the presence of supporters and wearing aprons reduced the probability of Salmonella contamination in pork. The findings highlight the effectiveness of light-touch interventions in reducing microbial contamination in pig carcasses at small-scale slaughterhouses and pork at traditional shops over the study period.

Prevalence of bovine tuberculosis and analysis of risk factors among the dairy farms in and around Guwahati metropolitan city, India.

Bovine tuberculosis (bTB), a neglected zoonotic disease caused by Mycobacterium bovis is being reported worldwide. The present work was carried out from December 2020 to November 2021 to assess the prevalence and risk factors of bTB in peri-urban and urban dairy farms of Guwahati, Assam, India. A questionnaire was used to collect data on knowledge about bTB on 36 farms, and ten animals per farm were screened by single intradermal comparative cervical tuberculin test (SICCT) to determine the prevalence of bTB, giving a total of 360 animals. The demographic data of the farmers revealed that 61.1% respondents were illiterate, 66.7% had no awareness about bovine tuberculosis and 41.7% consumed unpasteurised milk and milk products. SICCT showed that 38 cattle from 18 of the farms were positive reactors for bTB, yielding an overall animal level prevalence of 10.55% (95% confidence interval (CI = 7.58-14.2%) and a 50% herd prevalence (95% CI 32.9-67.1%). Animals 5 years and above were found to be more likely to be positive for bTB (17.18%). The study highlighted the widespread prevalence of bovine tuberculosis in peri-urban and urban dairy farms of Guwahati which gives a picture also about other major cities of India. Hence, it is of utmost importance to undertake a comprehensive epidemiological study in such cities for effective control and prevention of bTB in a one health approach.

Implementing a one health approach to strengthen the management of zoonoses in Ethiopia.

In East Africa, a region with many endemic and emerging zoonoses, and in countries such as Ethiopia in particular, One Health (OH) approaches are increasingly seen as effective ways, to mitigate the risk of zoonoses at the interface between human, animal and the environment. The OH approach promotes interdisciplinary cooperation and collaboration between researchers and practitioners from the disciplines of human, animal and environmental health. Moreover, it advocates for the establishment of a public health sector model which recognises the imperative to holistically address diseases that occur in the human, animal and environmental health arena. Key informant interviews were conducted with human and animal health practitioners and academic researchers in Ethiopia to collect data on the implementation of the OH approach to manage zoonotic diseases at the human and animal health interface. Participants' observations were undertaken within animal and human health clinics and government laboratories to gather additional data. Environmental health was not considered in this study as it is not yet fully integrated into the OH approach in Ethiopia. The results reveal a lack of interdisciplinary cooperation, collaboration, and coordination between animal and human health practitioners in operationalising the OH framework in Ethiopia. Professionals in academic and non-academic institutions and organisations are interested in implementing the OH approach, however, an organisational "silo" culture constrains collaboration between institutions dealing with animal and human health. Understaffing and underfunding of institutions were also cited as major challenges to the implementation of a OH approach. Lack of interdisciplinary training for animal and human health practitioners hinders collaboration in the management of zoonoses. Policymakers need to go beyond the rhetoric to a genuine focus on reform of health management and implement policies that bridge human, animal and environmental health. There is a need for multidisciplinary and transdisciplinary training in human, animal and environmental health and collaborative research for the management of zoonoses.

From farm to table: exploring food handling and hygiene practices of meat and milk value chain actors in Ethiopia.

Livestock value chains constitute a source of livelihood for meat and milk value chain actors in Ethiopia, from dairy farmers to other associated value chain actors such as milk traders, abattoir workers, public health officials, veterinarians, butcheries selling meats, milk cooperatives, artisanal milk processors, and transporters. The development of these livestock value chains, however, is constrained by poor food safety and quality, while consumers are also exposed to public health risks due to milk and meat value chain actors' food handling and hygiene practices.This study used Photovoice and participant observation to explore meat and milk value chain actors' food handling and hygiene practices in suburban areas of Addis Ababa and neighbouring Oromia in Ethiopia. The results of this study reveal that milk and meat value chain actors' food handling practices are not aligned with the recommended Ethiopian food safety and quality standards. Low compliance with food safety and quality standards reflected a combination of factors such as lack of incentives, poor road infrastructure and low enforcement of food safety standards.Participatory and visual research methods enable a researcher to collect context-aware data that can lead to the development of policies and intervention strategies that reflect local needs and priorities. The results of this study affirm the need to identify socially acceptable and economically viable policies and intervention strategies that are acceptable to all chain actors; and suggest there is an imperative to train milk and meat value chain actors on good hygiene handling practices, improve road infrastructure, and facilitate access equipment such as fridges and freezers that can contribute to maintaining food safety and quality.

Multidrug-resistant non-typhoidal Salmonella of public health significance recovered from migratory birds in Bangladesh.

Non-typhoidal Salmonella provides an exemplar for the One Health approach as it encompasses public and animal health, food safety, and environmental considerations. The contribution of environmental aspects is currently less well-defined. The purpose of this study was to determine the carriage occurrence of non-typhoidal Salmonella in migratory birds in Bangladesh and assess the potential significance to public and animal health. Cloacal swabs (N = 453) were collected in the years 2018-2020 from Tanguar and Hakaluki Haors, important wetland ecosystems in Northeastern Bangladesh. The prevalence of Salmonella was 13.5% (61 positive swabs). Classical serotyping identified six serovars: Salmonella enterica subsp. enterica serovars Perth, Kentucky, Albany, Infantis, Weltevreden, and Brancaster. Resistance towards 14 antimicrobials was assessed by broth microdilution minimum inhibitory concentration determination and the antimicrobial resistance (AMR) genotype established by whole-genome sequencing. S. Perth and S. Weltevreden isolates were susceptible and harbored no acquired AMR genes. Isolates from the remaining serovars were multidrug resistant, commonly possessing resistance to tetracycline, ampicillin, chloramphenicol, sulfamethoxazole, trimethoprim, and ciprofloxacin. Salmonella resistant to ciprofloxacin meets WHO criteria for priority pathogens. There was excellent concordance between resistance phenotype and the presence of corresponding AMR genes, many of which reside on Salmonella Genomic Islands. High-level ciprofloxacin resistance correlated with the presence of mutations in the chromosomal gyrB and/or parC genes. The S. Kentucky isolates were ST198, a widely distributed multidrug-resistant lineage reported in humans and animals, and constituting an ongoing risk to public health worldwide. We have demonstrated that multidrug-resistant non-typhoidal Salmonella of public health significance can be recovered from migratory birds. A potential for risk can manifest through direct interaction, transmission to food-producing livestock on farms, and dissemination via the long range migratory movements of birds. Risks can be mitigated by measures including continued surveillance and implementation of good farm biosecurity practices.

Microbial Contamination and Antibiotic Resistance in Marketed Food in Bangladesh: Current Situation and Possible Improvements.

Antimicrobial resistance (AMR) is a public health problem worldwide. Bangladesh, like its neighboring countries, faces many public health challenges, including access to safe food, inadequate food surveillance, as well as increasing AMR. This study investigated bacterial contamination and the AMR profile of pathogens in marketed food in Bangladesh and explored barriers to reducing AMR in the country. We collected 366 tomatoes, 359 chicken and 249 fish samples from 732 vendors in traditional markets in urban, peri-urban and rural areas in Bangladesh, as well as from 121 modern retails in Dhaka capital to analyse Vibrio cholerae and Escherichia coli in fish, Salmonella in chicken, and Salmonella and E. coli in tomatoes. Antibiotic susceptibility against 11 antibiotics was tested using a disc diffusion test and interpreted by an automated zone inhibition reader. In addition, a qualitative study using key informant interviews was conducted to explore antimicrobial use and AMR reduction potential in Bangladesh. We found E. coli in 14.21% of tomatoes and 26.91% of fish samples, while 7.38% of tomatoes and 17.27% of chicken were positive for Salmonella, and 44.98% of fish were positive for Vibrio cholerae. In total 231/319 (72.4%) of all pathogens isolated were multidrug-resistant (MDR) (resistant to three or more antibiotic groups). Qualitative interviews revealed an inadequate surveillance system for antibiotic use and AMR in Bangladesh, especially in the agriculture sector. To be able to fully understand the human health risks from bacterial hazards in the food and the AMR situation in Bangladesh, a nationwide study with a one health approach should be conducted, within all sectors, including AMR testing as well as assessment of the antimicrobial use and its drivers.

Antimicrobial Resistance in Vaginal Bacteria in Inseminated Mares.

Antimicrobials are added to semen extenders to inhibit the growth of bacteria that are transferred to the semen during collection. However, this non-therapeutic use of antimicrobials could contribute to the development of antimicrobial resistance. The objective of this study was to determine changes in the antibiotic susceptibility of vaginal microbiota after artificial insemination. Swabs were taken from the vagina of 26 mares immediately before artificial insemination and again 3 days later. Bacteria isolated from the vagina at both time points were subjected to antibiotic susceptibility testing and whole-genome sequencing. In total, 32 bacterial species were identified. There were increases in the resistance of Escherichia coli to trimethoprim (p = 0.0006), chloramphenicol and (p = 0.012) tetracycline (p = 0.03) between day 0 and day 3. However, there was no significant effect of exposure to antibiotics in semen extenders with respect to the resistance of Staphylococcus simulans and Streptococcus equisimilis (p > 0.05). Whole-genome sequencing indicated that most phenotypic resistance was associated with genes for resistance. These results indicate that the resistance patterns of vaginal bacteria may be affected by exposure to antibiotics; therefore, it would be prudent to minimize, or preferably, avoid using antibiotics in semen extenders.

Molecular Characterization of Methicillin-Resistant Staphylococci from the Dairy Value Chain in Two Indian States.

Bovine milk and milk products may contain pathogens, antimicrobial resistant bacteria, and antibiotic residues that could harm consumers. We analyzed 282 gram-positive isolates from milk samples from dairy farmers and vendors in Haryana and Assam, India, to assess the prevalence of methicillin-resistant staphylococci using microbiological tests, antibiotic susceptibility testing, and genotyping by PCR. The prevalence of genotypic methicillin resistance in isolates from raw milk samples was 5% [95% confidence interval, CI (3-8)], with 7% [CI (3-10)] in Haryana, in contrast to 2% [CI (0.2-6)] in Assam. The prevalence was the same in isolates from milk samples collected from farmers [5% (n = 6), CI (2-11)] and vendors [5% (n = 7), CI (2-10)]. Methicillin resistance was also observed in 15% of the isolates from pasteurized milk [(n = 3), CI (3-38)]. Two staphylococci harboring a novel mecC gene were identified for the first time in Indian dairy products. The only SCCmec type identified was Type V. The staphylococci with the mecA (n = 11) gene in raw milk were commonly resistant to oxacillin [92%, CI (59-100)] and cefoxitin [74%, CI (39-94)], while the isolates with mecC (n = 2) were resistant to oxacillin (100%) only. All the staphylococci with the mecA (n = 3) gene in pasteurized milk were resistant to both oxacillin and cefoxitin. Our results provided evidence that methicillin-resistant staphylococci occur in dairy products in India with potential public health implications. The state with more intensive dairy systems (Haryana) had higher levels of methicillin-resistant bacteria in milk.

Efficacy of Bentonite and Fumonisin Esterase in Mitigating the Effects of Aflatoxins and Fumonisins in Two Kenyan Cattle Breeds.

The objective of the study was to investigate the efficacy of bentonite and fumonisin esterase, separately or combined, in mitigating the effects of aflatoxins (AF) and fumonisins (FUM) in Boran and Friesian-Boran crossbreed cattle. These effects were studied by measuring mycotoxins, their metabolites, and biomarkers that relate to animal health, productivity, and food safety. The study was divided into three experiments each lasting for 2 weeks. Cows in experiment 1 received in random order aflatoxin B1 (AFB1) [788 µg/cow/day (69.7 µg/kg dry matter intake (DMI)) for Borans and 2,310 µg/cow/day (154 µg/kg DMI) for crossbreeds], bentonite (60 g/cow/day), or both AFB1 and bentonite. Boran cows in experiment 2 received in random order FUM (12.4 mg/cow/day (1.1 mg/kg DMI)), fumonisin esterase (120 U/cow/day), or both FUM and fumonisin esterase. Boran cows in experiment 3 received in random order AFB1 (952 µg/cow/day (84.2 µg/kg DMI)) + FUM (30.4 mg/cow/day (2.7 mg/kg DMI)), bentonite (60 g/cow/day) + fumonisin esterase (120 U/cow/day), or both AFB1 + FUM and bentonite + fumonisin esterase. Feeding AFB1 and/or FUM contaminated feed with or without the addition of the detoxifiers for 14 days did not affect DMI, milk composition, hematology, and blood biochemical parameters. The addition of bentonite in a diet contaminated with AFB1 led to a decrease in milk aflatoxin M1 (AFM1) concentration of 30% and 43%, with the carry-over subsequently decreasing from 0.35% to 0.20% and 0.08% to 0.06% for crosses and Borans, respectively. No significant change was observed in the sphinganine/sphingosine (Sa/So) ratio following feeding with FUM alone or in combination with fumonisin esterase; however, the ability of fumonisin esterase to hydrolyze FUM into less toxic fully hydrolyzed FUM and partially hydrolyzed FUM was evident in the rumen fluid and feces. These results indicate bentonite was effective in decreasing AFM1 concentration in milk, and AFB1 and AFM1 in plasma, while fumonisin esterase can convert FUM into less toxic metabolites and can be a suitable addition to feed cocontaminated with AFB1 and FUM.

Development of High-Throughput Sample Preparation Procedures for the Quantitative Determination of Aflatoxins in Biological Matrices of Chickens and Cattle Using UHPLC-MS/MS.

Aflatoxins (AFs) frequently contaminate food and animal feeds, especially in (sub) tropical countries. If animals consume contaminated feeds, AFs (mainly aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2) and their major metabolites aflatoxin M1 (AFM1) and M2 (AFM2)) can be transferred to edible tissues and products, such as eggs, liver and muscle tissue and milk, which ultimately can reach the human food chain. Currently, the European Union has established a maximum level for AFM1 in milk (0.05 µg kg-1). Dietary adsorbents, such as bentonite clay, have been used to reduce AFs exposure in animal husbandry and carry over to edible tissues and products. To investigate the efficacy of adding bentonite clay to animal diets in reducing the concentration of AFB1, AFB2, AFG1, AFG2, and the metabolites AFM1 and AFM2 in animal-derived foods (chicken muscle and liver, eggs, and cattle milk), chicken and cattle plasma and cattle ruminal fluid, a sensitive and selective ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed. High-throughput sample preparation procedures were optimized, allowing the analysis of 96 samples per analytical batch and consisted of a liquid extraction using 1% formic acid in acetonitrile, followed by a further clean-up using QuEChERS (muscle tissue), QuEChERS in combination with Oasis® Ostro (liver tissue), Oasis® Ostro (egg, plasma), and Oasis® PRiME HLB (milk, ruminal fluid). The different procedures were validated in accordance with European guidelines. As a proof-of-concept, the final methods were used to successfully determine AFs concentrations in chicken and cattle samples collected during feeding trials for efficacy and safety evaluation of mycotoxin detoxifiers to protect against AFs as well as their carry-over to animal products.

Emergence of Highly Pathogenic Avian Influenza A Virus (H5N1) of Clade 2.3.4.4b in Egypt, 2021-2022.

Wild migratory birds have the capability to spread avian influenza virus (AIV) over long distances as well as transmit the virus to domestic birds. In this study, swab and tissue samples were obtained from 190 migratory birds during close surveillance in Egypt in response to the recent outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus. The collected samples were tested for a variety of AIV subtypes (H5N1, H9N2, H5N8, and H6N2) as well as other pathogens such as NDV, IBV, ILT, IBDV, and WNV. Among all of the tested samples, the HPAI H5N1 virus was found in six samples; the other samples were found to be negative for all of the tested pathogens. The Egyptian HPAI H5N1 strains shared genetic traits with the HPAI H5N1 strains that are currently being reported in Europe, North America, Asia, and Africa in 2021-2022. Whole genome sequencing revealed markers associated with mammalian adaption and virulence traits among different gene segments, similar to those found in HPAI H5N1 strains detected in Europe and Africa. The detection of the HPAI H5N1 strain of clade 2.3.4.4b in wild birds in Egypt underlines the risk of the introduction of this strain into the local poultry population. Hence, there is reason to be vigilant and continue epidemiological and molecular monitoring of the AIV in close proximity to the domestic-wild bird interface.

Maximizing Laboratory Production of Aflatoxins and Fumonisins for Use in Experimental Animal Feeds.

Warm and humid climatic conditions coupled with poor agricultural practices in sub-Saharan Africa favor the contamination of food and feed by Aspergillus flavus and Fusarium verticillioides fungi, which subsequently may produce aflatoxins (AFs) and fumonisins (FBs), respectively. The growth of fungi and the production of mycotoxins are influenced by physical (temperature, pH, water activity, light and aeration), nutritional, and biological factors. This study aimed at optimizing the conditions for the laboratory production of large quantities of AFs and FBs for use in the animal experiments. A. flavus and F. verticillioides strains, previously isolated from maize in Kenya, were used. Levels of AFB1 and total FBs (FB1, FB2, and FB3) in different growth substrates were screened using ELISA methods. Maize kernels inoculated with three different strains of A. flavus simultaneously and incubated at 29 °C for 21 days had the highest AFB1 level of 12,550 ± 3397 µg/kg of substrate. The highest level of total FBs (386,533 ± 153,302 µg/kg of substrate) was detected in cracked maize inoculated with three different strains of F. verticillioides and incubated for 21 days at temperatures of 22-25 °C in a growth chamber fitted with yellow light. These two methods are recommended for the mass production of AFB1 and FBs for animal feeding trials.

Farmer's knowledge and suggested approaches for controlling aflatoxin contamination of raw milk in Pakistan.

Monitoring of aflatoxin levels in milk is often complicated in developing countries due to the dominance of informal markets channeling milk in raw form. Farmer's awareness and voluntary participation in aflatoxin mitigation can be critical in such scenarios. Therefore, the present study was conducted to understand the perceptions of dairy farmers about aflatoxins and link it with aflatoxin mitigation programs on milk in Pakistan. Information was collected from 450 peri-urban dairy farmers in seven cities using questionnaires. Majority (77.9%) of the farmers were aware of the negative impact of moldy feed on animal health. However, only 40.6% of the farmers were aware of the transferability of the toxins from moldy feed to milk. The farmers had almost no awareness of aflatoxins as 95% never heard of the term. After receiving an onsite briefing on effects of the toxin on animal and human health, and its transferability to milk, 98.3% farmers showed willingness to buy aflatoxin-safe feedstuffs, while 88.5% showed willingness to control aflatoxin in milk. Around half of the farmers considered aflatoxin control programs as affordable. On average, farmers agreed to pay 10.1% higher price for aflatoxin certified oilseed cakes. Availability of feedstuffs certified of low aflatoxin content was suggested by 22% of the participants as the critical step in reducing aflatoxins in milk. Other important suggestions included; subsidy on quality feeds (18%), raising awareness (18%), and legislation and monitoring (16%). The present results suggest that the current practice of milk monitoring in the country can yield desirable results only if it is coupled with feed certification programs ensuing availability of aflatoxin-safe feeds. Further, awareness can positively impact participation of producers in aflatoxin control programs. In this regard, awareness about effects of aflatoxins on animal health was found to be a more powerful trigger of voluntary control compared with the awareness of the toxin's transferability to milk.