No impact of hygienic behavior and viral coinfection on the development of European foulbrood in honey bee (Apis mellifera) colonies during blueberry pollination in Michigan.

European foulbrood (EFB) is a severe disease of honey bee (Apis mellifera) larvae caused by the bacterium Linnaeus [Hymenoptera: Apidae]) Melissococcus plutonius (ex White) Bailey and Collins (Lactobacillales: Enterococcaceae). Many beekeepers in North America report severe EFB following blueberry pollination, but it is not clear what factors during pollination are related to clinical disease. Additionally, the impact that other factors such as viral load and hygienic behavior have on EFB has not been studied. In Spring of 2020 we enrolled 60 commercial honey bee colonies in a prospective cohort study. Colonies were inspected 3 times over the season with hive metrics and samples taken for viral testing. Each colony was tested for hygienic behavior twice and the score was averaged. Viral loads were determined by qPCR for deformed wing virus (DWV) A and B. We found no statistical difference in the EFB prevalence or severity between the 2 yards at any timepoint; 50% (n = 16) of the colonies in the holding yard and 63% (n = 17) in blueberry developed moderate to severe EFB over the study period. When colonies from both yards were pooled, we found no relationship between viral load or hygienic behavior and development of EFB. These results suggest that other factors may be responsible for driving EFB virulence and hygienic behavior is not likely helpful in managing this disease.

Sacbrood viruses and select Lake Sinai virus variants dominated Apis mellifera colonies symptomatic for European foulbrood.

European foulbrood (EFB) is a prevalent disease in the European honey bee (Apis mellifera) in the United States, which can lead to colony decline and collapse. The bacterial components of EFB are well-studied, but the diversity of viral infections within infected colonies has not been explored. In this study, we use meta-transcriptomics sequencing of 12 honey bee hives, symptomatic (+, n = 6) and asymptomatic (-, n = 6) for EFB, to investigate viral infection associated with the disease. We assembled 41 viral genomes, belonging to three families (Iflaviridae, Dicistroviridae, and Sinhaliviridae), all previously reported in honey bees, including Lake Sinai virus, deformed wing virus, sacbrood virus, Black queen cell virus, and Israeli acute paralysis virus. In colonies with severe EFB, we observed a higher occurrence of viral genomes (34 genomes) in contrast to fewer recovered from healthy colonies (seven genomes) and a complete absence of Dicistroviridae genomes.We observed specific Lake Sinai virus clades associated exclusively with EFB + or EFB - colonies, in addition to EFB-afflicted colonies that exhibited an increase in relative abundance of sacbrood viruses. Multivariate analyses highlighted that a combination of site and EFB disease status influenced RNA virome composition, while EFB status alone did not significantly impact it, presenting a challenge for comparisons between colonies kept in different yards. These findings contribute to the understanding of viral dynamics in honey bee colonies compromised by EFB and underscore the need for future investigations to consider viral composition when investigating EFB.IMPORTANCEThis study on the viromes of honey bee colonies affected by European foulbrood (EFB) sheds light on the dynamics of viral populations in bee colonies in the context of a prevalent bacterial brood disease. The identification of distinct Lake Sinai virus and sacbrood virus clades associated with colonies affected by severe EFB suggests a potential connection between viral composition and disease status, emphasizing the need for further investigation into the role of viruses during EFB infection. The observed increase in sacbrood viruses during EFB infection suggests a potential viral dysbiosis, with potential implications for honey bee brood health. These findings contribute valuable insights related to beekeeping practices, offering a foundation for future research aimed at understanding and mitigating the impact of bacterial and viral infection in commercial honey bee operations and the management of EFB.

Northward Expansion of Amblyomma americanum (Acari: Ixodidae) into Southwestern Michigan.

Amblyomma americanum (Linnaeus) (Acari: Ixodidae) (lone star tick) is an aggressive, generalist parasite that vectors numerous important human and animal pathogens. In recent decades its geographic range has expanded northwards from endemic regions in the southeastern and southcentral United States. In 2019 five questing A. americanum ticks, comprising two life stages were detected at one site in southwestern Michigan, satisfying one CDC criterium for an established population for the first time in recent history in the state. To better characterize the extent of emerging A. americanum, we conducted active surveillance (i.e., drag sampling) in summer 2020 throughout Michigan's southern counties and detected one adult A. americanum from each of six widespread sites, including where they had been detected in 2019. A larger established population was identified at another site in Berrien County, which yielded 691 A. americanum comprising three life stages, and questing phenologies here were similar to that reported for other endemic regions. Statewide surveillance in 2021 revealed no A. americanum outside of Berrien County, but establishment criteria were met again at the two sites where established populations were first detected respectively in 2019 and 2020. These observations may represent the successful invasion of A. americanum into Michigan. Data from passive (1999-2020) and active surveillance (2004-2021) efforts, including a domestic animal sentinel program (2015-2018), are reported to provide context for this nascent invasion. Continued active surveillance is needed to help inform the public, medical professionals, and public health officials of the health risks associated with this vector.

Antimicrobial-resistant non-typhoidal Salmonella enterica prevalence among poultry farms and slaughterhouses in Chitwan, Nepal.

BACKGROUND AND AIM: Antibiotic-resistant infections are one of the leading threats to public health globally. Indiscriminate use of antibiotics in food animal production is an important driver of resistance, particularly among foodborne pathogens such as non-typhoidal Salmonella enterica (NTS). While there has been extensive research on antimicrobial-resistant (AMR) S. enterica in India and China, there have been few studies in countries in South Asia, including Nepal. This is particularly important with the rise of commercial poultry farming in Nepal as a means of economic development and nutritional subsistence. This descriptive study seeks to identify the prevalence and resistance patterns of NTS serotypes focusing on Chitwan, Nepal's leading poultry producing district. MATERIALS AND METHODS: A mixture of purposive and judgment sampling of 18 poultry farms and 20 slaughterhouses representing a broad geographic distribution across multiple municipalities in Chitwan was conducted in May 2019. Environmental samples taken from poultry farms included: Water, litter, feces, feed, farm swabs, and eggshell swabs. Biological samples taken from nearby slaughterhouses included: Muscle, heart, liver, skin, cecum, crop, and spleen. Samples were cultured and tested for the presence of NTS. Positive isolates were serotyped and tested for antimicrobial susceptibility to seven antibiotics known to be important to both human and animal health regionally. Farm practices were also characterized through a survey, the results of which are detailed in the accompanying paper. RESULTS: Out of 708 samples (288 environmental and 420 biological), 103 (15%) tested positive for NTS (9% of environmental; n=26, 18% of biological; n=77). The percentage of positive environmental and biological samples varied by source. Environmental sample positive rates were water (27.5%), feces (10.6%), litter (8.6%), farm swabs (5%), feed (1.8%), and eggshells (0%). Biological sample positive rates were skin (28%), heart (23%), crop (20%), muscle (15%), liver (15%), spleen (15%), and cecum (12%). Out of 103 positive S. enterica isolates, 48.5% were identified as Salmonella Typhimurium, 35% Salmonella Enteritidis, 7.8% Salmonella Gallinarum, 4.9% Salmonella Virchow, and 3.9% were Salmonella Agona. Of the 103 positive isolates, 80 (78%) were resistant to at least one antibiotic, and 21 (20%) were multidrug-resistant (MDR). CONCLUSION: NTS is highly prevalent among Chitwan's growing poultry industry with higher rates of positivity found in slaughterhouse samples compared with environmental samples from farms. In addition, a high rate of AMR (78%) was revealed, and an extremely concerning number of those were shown to be MDR (20%). This baseline data has important implications for poultry production and consumption in the region. Further research will elucidate the extent to which this contamination and drug resistance is impacting the health of the local population and help inform treatment and management strategies.Note: The characterization of the poultry industry and practices that might be linked to NTS contamination in the Chitwan district are detailed in the previous paper in this series (www.veterinaryworld.org/Vol.14/February-2021/14.pdf).

Prevalence of non-typhoidal Salmonella and risk factors on poultry farms in Chitwan, Nepal.

BACKGROUND AND AIM: Poultry is becoming an increasingly important source of protein in the Nepalese diet. The Chitwan region of Nepal is the hub of the emerging poultry industry. Little is known about the prevalence of non-typhoidal Salmonella (NTS) on poultry farms or the role of farm management practices that may contribute to the presence of NTS on farms. The role of poultry in the transmission of Salmonella enterica to humans is also poorly defined. This descriptive study seeks establish baseline data through estimation of the prevalence of NTS on broiler and layer operations in various farms of the Chitwan district of Nepal. MATERIALS AND METHODS: Based on district documents on poultry production and meat marketing, a purposive sampling of 18 commercial poultry farms comprising ten broilers farms and eight layers farms was conducted. Environmental samples including water, litter, feces, feed, farm, and eggshell swabs were randomly collected from each farm. Samples were cultured and tested for the presence of NTS; positives were serotyped, and antimicrobial susceptibility determined. A comprehensive farm and practice questionnaire was administered to each farm manager. RESULTS: The farm level point prevalence rate was 55% (10 of 18 farms) for S. enterica. Of the total 288 farm environmental samples collected, 26 samples (9%) were positive. The rate of isolation varied according to the origin of samples: Water (27.5%), feces (10.6%), litter (8.6%), farm swabs (5%), feed (1.8%), and eggshells (0%). Farm management variables/risk factors are summarized and categorized as non-modifiable and modifiable for analysis. Broiler operations were more likely to be positive than layer operations as were poultry houses with two or less open sides. All-in/all-out management style was found to be protective. Due to the small sample size (18 farms), no associations reached statistical significance. CONCLUSION: Based on environmental sampling results, NTS is highly prevalent on the poultry farms in the Chitwan district of Nepal. Certain risk factors are associated with finding NTS on farms. Our findings are generally in agreement with other studies in similar countries with rapidly emerging poultry industries. The identification of risk factors provides owners, technicians, and veterinarians with some guidance to help reduce the prevalence of NTS on farms. This baseline data are critical to understanding the epidemiology of zoonotic strain of NTS in the region and are necessary for the design of future studies and mitigation plans and underlines the need for a one-health approach to protect public health-related to Salmonella spp. from poultry farms.