Multiple spillovers from humans and onward transmission of SARS-CoV-2 in white-tailed deer.

Many animal species are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and could act as reservoirs; however, transmission in free-living animals has not been documented. White-tailed deer, the predominant cervid in North America, are susceptible to SARS-CoV-2 infection, and experimentally infected fawns can transmit the virus. To test the hypothesis that SARS-CoV-2 is circulating in deer, 283 retropharyngeal lymph node (RPLN) samples collected from 151 free-living and 132 captive deer in Iowa from April 2020 through January of 2021 were assayed for the presence of SARS-CoV-2 RNA. Ninety-four of the 283 (33.2%) deer samples were positive for SARS-CoV-2 RNA as assessed by RT-PCR. Notably, following the November 2020 peak of human cases in Iowa, and coinciding with the onset of winter and the peak deer hunting season, SARS-CoV-2 RNA was detected in 80 of 97 (82.5%) RPLN samples collected over a 7-wk period. Whole genome sequencing of all 94 positive RPLN samples identified 12 SARS-CoV-2 lineages, with B.1.2 (n = 51; 54.5%) and B.1.311 (n = 19; 20%) accounting for ∼75% of all samples. The geographic distribution and nesting of clusters of deer and human lineages strongly suggest multiple human-to-deer transmission events followed by subsequent deer-to-deer spread. These discoveries have important implications for the long-term persistence of the SARS-CoV-2 pandemic. Our findings highlight an urgent need for a robust and proactive "One Health" approach to obtain enhanced understanding of the ecology, molecular evolution, and dissemination of SARS-CoV-2.

An immuno-chromatographic lateral flow assay (LFA) for rapid on-the-farm detection of classical swine fever virus (CSFV).

Classical swine fever (CSF) is a highly contagious and potentially fatal disease of domestic pigs. Classical swine fever is routinely diagnosed by clinical signs, serology, detection of CSF virus (CSFV) nucleic acid by PCR and virus isolation. Most of the current CSF diagnostic methods are expensive and have an extended turnaround time. In the majority of the CSF endemic countries, lack of easy access to diagnostic facilities is a major problem for swine producers trying to obtain early diagnosis and often results in the entire herd being infected. The acute form of CSF can show non-specific signs of illness, leaving CSF often undiagnosed. Hence there is an urgent need for a rapid and reliable pen side diagnostic assay for the better detection and control of this economically important disease of swine. We developed an immuno-chromatographic lateral flow assay (LFA) for on the farm detection of CSFV. A CSFV isolate [CSFV/AP/TRP2/2009 (TS2)] of genotype 1.1 was used for the production of monoclonal antibodies (mAbs) for the LFA's development. The virus detection level of the LFA device was 36.8 TCID50/ml of CSFV. The sensitivity and specificity of LFA in comparison with PCR were 80.36% and 87.10%, respectively. The positive and negative predictive values of the LFA device were 91.84% and 87.10%, respectively. In conclusion, the CSFV-LFA is a reliable and convenient resource for preliminary on the farm detection of classic swine fever.

Evaluation of Three Bacterial Identification Systems for Species Identification of Bacteria Isolated from Bovine Mastitis and Bulk Tank Milk Samples.

A study was conducted to evaluate Sensititre® Automated Reading and Incubation System 2x System (ARIS), API® (API), and Bruker MALDI-TOF MS (MALDI) bacterial species identification systems using 132 diverse bacterial isolates from bovine milk samples and bulk tank milk received at the Penn State Animal Diagnostic Laboratory. The results were compared with 16S rRNA gene sequence analysis, which served as the reference method for species identification. The ARIS, API, and MALDI identified 0%, 40%, and 33.4% of species classified as Gram-positive rod isolates belonging to genera Arthrobacter, Bacillus, Brachybacterium, Brevibacterium, and Corynebacterium, respectively. It was observed that 76.5%, 93.9%, and 96.9% of catalase-negative, Gram-positive cocci (n = 33; Aerococcus, Enterococcus, Lactococcus, Streptococcus) were correctly identified to the species level by ARIS, API, and MALDI, respectively, while 33.4%, 84.5%, and 97.7% of catalase-positive, Gram-positive cocci (n = 45; Kocuria, Staphylococcus) were correctly identified to their species by ARIS, API, and MALDI, respectively. A total of 48 isolates (Acinetobacter, Citrobacter, Enterobacter, Escherichia, Klebsiella, Pantoea, Pasteurella, Providencia, Pseduomonas, Serratia) of Gram-negative bacteria were examined, of which 85.4%, 93.7%, and 95.8% of the isolates were correctly identified to the species level by ARIS, API, and MALDI, respectively. In our laboratory, the MALDI had the least costs associated with consumables and reagents compared to ARIS, API, and 16S rRNA identification methods. Identification of bacterial species was accomplished in <2 h using MALDI and 24 h for ARIS, API, and 16S rRNA identification systems.

Longitudinal Monitoring of Successive Commercial Layer Flocks for Salmonella enterica Serovar Enteritidis.

The Pennsylvania Egg Quality Assurance Program (EQAP) provided the framework for Salmonella Enteritidis (SE) control programs, including the Food and Drug Administration (FDA) mandated Final Egg Rule, for commercial layer facilities throughout the United States. Although flocks with ≥3000 birds must comply with the FDA Final Egg Rule, smaller flocks are exempted from the rule. As a result, eggs produced by small layer flocks may pose a greater public health risk than those from larger flocks. It is also unknown if the EQAPs developed with large flocks in mind are suitable for small- and medium-sized flocks. Therefore, a study was performed to evaluate the effectiveness of best management practices included in EQAPs in reducing SE contamination of small- and medium-sized flocks by longitudinal monitoring of their environment and eggs. A total of 59 medium-sized (3000 to 50,000 birds) and small-sized (<3000 birds) flocks from two major layer production states of the United States were enrolled and monitored for SE by culturing different types of environmental samples and shell eggs for two consecutive flock cycles. Isolated SE was characterized by phage typing, pulsed-field gel electrophoresis (PFGE), and clustered regularly interspaced short palindromic repeats-multi-virulence-locus sequence typing (CRISPR-MVLST). Fifty-four Salmonella isolates belonging to 17 serovars, 22 of which were SE, were isolated from multiple sample types. Typing revealed that SE isolates belonged to three phage types (PTs), three PFGE fingerprint patterns, and three CRISPR-MVLST SE Sequence Types (ESTs). The PT8 and JEGX01.0004 PFGE pattern, the most predominant SE types associated with foodborne illness in the United States, were represented by a majority (91%) of SE. Of the three ESTs observed, 85% SE were typed as EST4. The proportion of SE-positive hen house environment during flock cycle 2 was significantly less than the flock cycle 1, demonstrating that current EQAP practices were effective in reducing SE contamination of medium and small layer flocks.

Risk Factors Associated With Salmonella in Laying Hen Farms: Systematic Review of Observational Studies.

Salmonella contamination of laying hen flocks and shell eggs is associated with various management and environmental factors. Foodborne outbreaks of human salmonellosis have been traced back to consumption of Salmonella-contaminated shell eggs. In the present study, a systematic literature review was conducted to identify and provide an evidence-based overview of potential risk factors of Salmonella contamination of laying hens, layer premises, and shell eggs. This systematic literature search was conducted using AGRICOLA, CAB Abstracts, and PubMed databases. Observational studies that identified risk factors for Salmonella contamination of layer flocks and shell eggs were selected, and best evidence was synthesized to summarize the results. Altogether, 13 cross-sectional studies and four longitudinal studies published in English were included in the review. Evidence scores were assigned based on the study design and quality of the study to grade the evidence level. The strength of association of a risk factor was determined according to the odds ratios. In this systematic review, the presence of previous Salmonella infection, absence of cleaning and disinfection, presence of rodents, induced molting, larger flock size (>30,000 hens), multiage management, cage housing systems, in-line egg processing, rearing pullets on the floor, pests with access to feed prior to movement to the feed trough, visitors allowed in the layer houses, and trucks near farms and air inlets were identified as the risk factors associated with Salmonella contamination of laying hen premises, whereas high level of manure contamination, middle and late phase of production, high degree of egg-handling equipment contamination, flock size of >30,000, and egg production rate of >96% were identified as the risk factors associated with Salmonella contamination of shell eggs. These risk factors demonstrated strong to moderate evidence of association with Salmonella contamination of laying hens and shell eggs. Eggshells testing positive for Salmonella were 59 times higher when fecal samples were positive and nine times higher when floor dust samples were positive. Risk factors associated with Salmonella Enteritidis infection in laying hens were flock size, housing system, and farms with hens of different ages. As a summary, this systematic review demonstrated that Salmonella contamination of laying hen flocks and shell eggs in layer production systems is multifactorial. This study provides a knowledge base for the implementation of targeted intervention strategies to control Salmonella contamination of laying hen flocks and shell eggs.

Multi-virulence-locus sequence typing of 4b Listeria monocytogenes isolates obtained from different sources in India over a 10-year period.

Listeria monocytogenes is an emerging foodborne pathogen responsible for listeriosis. The incidence of listeriosis has increased during the last 2 decades due to the increase in consumption of ready-to-eat foods and change in food consumption habits. Outbreaks and sporadic cases of listeriosis have been reported in developed countries. These reports have helped determine the safety practices needed to control listeriosis. Although L. monocytogenes has been reported from humans, animals, and a variety of foods in India, limited data exist with respect to prevalence and distribution of L. monocytogenes in the Indian subcontinent. The Indian Listeria Culture Collection Centre in Goa maintains all of the isolates received for subtyping and molecular characterization. Of the listerial isolate collection maintained by this center, three fourths of the isolates are of 4b serotype, while the number of other serotypes is very low. Therefore, we screened L. monocytogenes serotype 4b isolates to determine their relevance to previously defined epidemics and/or outbreaks using multi-virulence-locus sequence typing (MVLST). A total of 25 isolates in serogroup 4b of L. monocytogenes were randomly selected from a repository of 156 L. monocytogenes 4b isolates obtained from different sources in India over a period of 10 years. MVLST sequence types (virulence types, VTs) were compared to known epidemic clones and other known isolates in the L. monocytogenes MVLST database. The 25 isolates were grouped into three clusters. Cluster I comprised 21 isolates including animal (n=9), human (n=4), and food (n=8), which matched Epidemic Clone I (ECI, VT20). Three isolates-two from animal and one from food-formed a cluster while a single animal isolate was placed into two novel VTs (VT98 and VT99), respectively. Based on these findings, it can be inferred that ECI has been isolated from a variety of sources and places and has persisted in India for at least 10 years.

Characterization of Clostridium difficile isolates from human fecal samples and retail meat from Pennsylvania.

A study was conducted to determine the prevalence of Clostridium difficile and characterize C. difficile isolates from human stool and retail grocery meat samples. Human stool samples (n=317) were obtained from a clinical laboratory and meat samples (n=303) were collected from 8 retail grocery stores from October 2011 through September 2012 from Centre County of Pennsylvania and were examined for C. difficile. C. difficile was isolated from 16.7% of stool samples (n=317) and 6.9%, 11.5%, 14.5%, and 7.8% of beef (n=72), pork (n=78), turkey (n=76), and chicken (n=77) samples, respectively. Six different toxin gene profiles were detected in all human and meat isolates of C. difficile based on the presence or absence of toxin genes tcdA, tcdB, and cdtA and cdtB. Interestingly, 75.6% of the human C. difficile isolates lacked any deletion in the tcdC gene (139-bp), whereas a 39-bp deletion was observed in 61.3% of the C. difficile strains isolated from meat samples. C. difficile from meat samples were more susceptible to clindamycin, moxifloxacin, vancomycin, and metronidazole than C. difficile isolates from human samples. Twenty-five different ribotypes were identified in human and meat C. difficile isolates. In conclusion, significant genotypic and phenotypic differences were observed between human and meat isolates of C. difficile; however, a few C. difficile isolates from meat-in particular ribotypes 078, PA01, PA05, PA16, and PA22 with unique profiles (toxin gene, tcdC gene size and antimicrobial resistance profiles)-were similar to human C. difficile isolates.

The Susceptibility of Chickens to Zika Virus: A Comprehensive Study on Age-Dependent Infection Dynamics and Host Responses.

Zika virus (ZIKV) remains a public health concern, with epidemics in endemic regions and sporadic outbreaks in new areas posing significant threats. Several mosquito-borne flaviviruses that can cause human illness, including West Nile, Usutu, and St. Louis encephalitis, have associations with birds. However, the susceptibility of chickens to ZIKV and their role in viral epidemiology is not currently known. We investigated the susceptibility of chickens to experimental ZIKV infection using chickens ranging from 1-day-old chicks to 6-week-old birds. ZIKV caused no clinical signs in chickens of all age groups tested. Viral RNA was detected in the blood and tissues during the first 5 days post-inoculation in 1-day and 4-day-old chicks inoculated with a high viral dose, but ZIKV was undetectable in 6-week-old birds at all timepoints. Minimal antibody responses were observed in 6-week-old birds, and while present in younger chicks, they waned by 28 days post-infection. Innate immune responses varied significantly between age groups. Robust type I interferon and inflammasome responses were measured in older chickens, while limited innate immune activation was observed in younger chicks. Signal transducer and activator of transcription 2 (STAT2) is a major driver of host restriction to ZIKV, and chicken STAT2 is distinct from human STAT2, potentially contributing to the observed resistance to ZIKV infection. The rapid clearance of the virus in older chickens coincided with an effective innate immune response, highlighting age-dependent susceptibility. Our study indicates that chickens are not susceptible to productive ZIKV infection and are unlikely to play a role in the ZIKV epidemiology.

White button mushrooms increase microbial diversity and accelerate the resolution of Citrobacter rodentium infection in mice.

The effect of feeding C57BL/6 mice white button (WB) mushrooms or control (CTRL) diets for 6 wk was determined on the bacterial microflora, urinary metabolome, and resistance to a gastrointestinal (GI) pathogen. Feeding mice a diet containing 1 g WB mushrooms/100 g diet resulted in changes in the microflora that were evident at 2 wk and stabilized after 4 wk of WB feeding. Compared with CTRL-fed mice, WB feeding (1 g/100 g diet) increased the diversity of the microflora and reduced potentially pathogenic (e.g., Clostridia) bacteria in the GI tract. Bacteria from the Bacteroidetes phylum increased and the Firmicutes phylum decreased in mushroom-fed mice compared with CTRL. The changes in the microflora were also reflected in the urinary metabolome that showed a metabolic shift in the WB-fed compared with the CTRL-fed mice. The WB feeding and changes in the microbiome were associated with fewer inflammatory cells and decreased colitis severity in the GI mucosa following Citrobacter rodentium infection compared with CTRL. Paradoxically, the clearance of C. rodentium infection did not differ even though Ifn-γ and Il-17 were higher in the colons of the WB-fed mice compared with CTRL. Adding modest amounts of WB mushrooms (1 g/100 g diet) to the diet changed the composition of the normal flora and the urinary metabolome of mice and these changes resulted in better control of inflammation and resolution of infection with C. rodentium.

Complete Genome Sequence of a Bovine Coronavirus Isolated from a Goat in Pennsylvania, USA.

We report a complete genome sequence of bovine coronavirus (BCoV) isolated from a goat in the state of Pennsylvania in 2022. BCoV often causes calf scours and winter dysentery in cattle.

Rapid detection of the top six non-O157 Shiga toxin-producing Escherichia coli O groups in ground beef by flow cytometry.

Rapid, sensitive, and highly specific flow-cytometric assays were developed for the detection of the top six non-O157 Shiga toxin-producing Escherichia coli (STEC) O groups in ground beef. The analytical sensitivity of the assays was 2 × 10(3) target cells in a bacterial mixture of 10(5) CFU/ml, and the limit of detection in ground beef was 1 to 10 CFU following 8 h of enrichment. The assays may be utilized for rapid detection of STEC O groups in meat.

Detection of the top six non-O157 Shiga toxin-producing Escherichia coli O groups by ELISA.

There is a growing concern of a public health risk associated with non-O157 Shiga toxin-producing Escherichia coli (STEC) since E. coli serogroups O26, O45, O103, O111, O121, and O145 are frequently implicated in outbreaks of human illness worldwide. Recently, the Food Safety and Inspection Service of the U.S. Department of Agriculture declared these six STEC O groups to be adulterants in beef. We describe here a rapid, sensitive, and highly specific enzyme-linked immunosorbent assay (ELISA) for the detection of these top six non-O157 STEC O groups. The assays were tested against 174 reference E. coli O groups, with 60 clinical isolates belonging to the target O groups and 10 non-E coli strains belonging to the family Enterobacteriaceae. Assays for serogroups O103, O111, and O121 exhibited 100% specificity, while assays for serogroups O26 and O45 had 98.2% specificity, and O145 had 99.1% specificity. ELISA conducted using artificially inoculated ground beef samples displayed 100% accuracy. The sensitivity of the assay was 5×10(5) colony-forming unit (CFU)/mL, with limits of detection in the range of 1-10 CFU/25 g of ground beef sample following enrichment. The findings of the study suggest that the assay described is simple and rapid, and can be employed to detect target STEC O groups in beef and other food samples. In addition, the assay provides a conceptual framework that can be adapted for the development of similar tests for the rapid detection of other serogroups of E. coli.

Prevalence of Salmonella cerro in laboratory-based submissions of cattle and comparison with human infections in Pennsylvania, 2005-2010.

The aim of this study was to identify Salmonella serotypes infecting cattle in Pennsylvania, to compare infection rates for the predominant serotype, Salmonella enterica serotype Cerro, with the infection rates for the same serotype in humans, and to study the clonal diversity and antimicrobial resistance for this serotype in cattle from 2005 to 2010. Clonal diversity among the selected isolates was studied using pulsed-field gel electrophoresis (PFGE) and repetitive (rep)-polymerase chain reaction (PCR). Salmonella Cerro showed the single largest increase as a cause of cattle infections over the study period. The proportional distribution of Salmonella Cerro serotype among laboratory-submitted Salmonella positive cases in cattle was 36.1% in the year 2010 compared to 14.3% in 2005. A simultaneous decrease in serotype Newport infections was also observed in cattle (25% in 2005, to 10.1% in 2010). Studies of clonal diversity for cattle and human isolates revealed a predominant PFGE type but showed some variability. All tested isolates (n = 60) were susceptible to sulfamethoxazole-trimethoprim, but 2% of cattle isolates (n = 1/50) and 20% of human isolates (n = 2/10) showed resistance to tetracycline and sulfisoxazole. One human isolate showed additional resistance to ampicillin and gentamicin. This study suggests an increase in Salmonella Cerro infections in the cattle population and a decrease in Salmonella Newport infections. The increase in Cerro infections appears to be restricted to the cattle population, but occasional human infections occur.

Prevalence of Clostridium difficile toxin genes in the feces of veal calves and incidence of ground veal contamination.

A study was conducted in two parts to determine the prevalence of toxigenic Clostridium difficile in veal calves and retail meat. The first part of the study focused on the veal production continuum (farm to abattoir). Fifty calves from 4 veal herds (n=200) were followed for 18-22 weeks from the time of arrival on the veal farm to the time of slaughter. Fecal samples were collected from calves every 4-6 weeks. Half of the calves included in the study (n=100) were followed to the abattoir where carcass swabs were collected post slaughter. Fecal samples and carcass swabs were screened for genes encoding C. difficile toxins TcdA, TcdB, and CDT by using real-time polymerase chain reaction (PCR). Carcass swabs were also screened for toxigenic C. difficile by using traditional culture methods. In the second part of the study, ground veal products (n=50 samples) purchased from local grocery stores were examined for toxigenic C. difficile by using real-time PCR and traditional culture methods. Fecal samples from 56 of 200 (28%) calves tested positive for C. difficile toxin genes at least once over the course of the study. Calf age (p=0.011) influenced prevalence of C. difficile toxin genes in calf feces. Toxin genes of C. difficile were detected in one carcass swab by multiplex real-time PCR only. Toxigenic C. difficile was detected by PCR and culture in four (8%) and three (6%) ground veal samples, respectively. The findings of the study reveal that toxigenic C. difficile was most prevalent in veal calves (12%) just before slaughter, although viable toxigenic C. difficile was not recovered from veal carcasses. On the contrary, viable toxigenic C. difficle was recovered from 6% retail meat, thus suggesting that contamination occurs either during or after veal fabrication.

Transmission history of SARS-CoV-2 in humans and white-tailed deer.

The emergence of a novel pathogen in a susceptible population can cause rapid spread of infection. High prevalence of SARS-CoV-2 infection in white-tailed deer (Odocoileus virginianus) has been reported in multiple locations, likely resulting from several human-to-deer spillover events followed by deer-to-deer transmission. Knowledge of the risk and direction of SARS-CoV-2 transmission between humans and potential reservoir hosts is essential for effective disease control and prioritisation of interventions. Using genomic data, we reconstruct the transmission history of SARS-CoV-2 in humans and deer, estimate the case finding rate and attempt to infer relative rates of transmission between species. We found no evidence of direct or indirect transmission from deer to human. However, with an estimated case finding rate of only 4.2%, spillback to humans cannot be ruled out. The extensive transmission of SARS-CoV-2 within deer populations and the large number of unsampled cases highlights the need for active surveillance at the human-animal interface.

Identification of novel small molecule antimicrobials targeting Mycoplasma bovis.

OBJECTIVES: To screen novel small molecule compounds for inhibition of Mycoplasma bovis growth and to characterize their activity in terms of dose-dependency and ability to function in milk. METHODS: Using a tetrazolium salt cytotoxicity assay, 480 natural compounds were screened to determine which of the small molecules have the potential to become therapeutic options for M. bovis prevention and treatment. The dose response was determined in broth culture and in fresh quarter milk for a subset of compounds shown to be capable of inhibiting M. bovis growth. RESULTS: Data suggest that 32 of the 480 compounds tested were able to inhibit growth of M. bovis using a tetrazolium salt assay. Methanesulphonic acid, 3-[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyloxy](1S,3R,4R,5R)-1,4,5-trihydroxycyclohexane carboxylic acid, S-carboxymethyl-l-cysteine, l-aspartic acid, dihydrotachysterol, eriodictyol and (+)-α-tocopherol acid succinate were selected for further concentration-dependent studies and testing in fresh quarter milk. Each compound demonstrated a dose response in broth culture and at 3 h and 24 h in fresh quarter milk. CONCLUSIONS: Small molecule natural compounds are capable of inhibiting the growth of M. bovis in both a pleuropneumonia-like organism (PPLO) medium and in fresh quarter milk. Results suggest that the compounds are mycoplasmastatic in a dose-dependent manner. By inhibiting M. bovis, small molecule natural compounds offer the potential for prophylactic or therapeutic use on organic and natural farms as a viable alternative to traditional antimicrobial agents.

Subtyping Salmonella enterica serovar enteritidis isolates from different sources by using sequence typing based on virulence genes and clustered regularly interspaced short palindromic repeats (CRISPRs).

Salmonella enterica subsp. enterica serovar Enteritidis is a major cause of food-borne salmonellosis in the United States. Two major food vehicles for S. Enteritidis are contaminated eggs and chicken meat. Improved subtyping methods are needed to accurately track specific strains of S. Enteritidis related to human salmonellosis throughout the chicken and egg food system. A sequence typing scheme based on virulence genes (fimH and sseL) and clustered regularly interspaced short palindromic repeats (CRISPRs)-CRISPR-including multi-virulence-locus sequence typing (designated CRISPR-MVLST)-was used to characterize 35 human clinical isolates, 46 chicken isolates, 24 egg isolates, and 63 hen house environment isolates of S. Enteritidis. A total of 27 sequence types (STs) were identified among the 167 isolates. CRISPR-MVLST identified three persistent and predominate STs circulating among U.S. human clinical isolates and chicken, egg, and hen house environmental isolates in Pennsylvania, and an ST that was found only in eggs and humans. It also identified a potential environment-specific sequence type. Moreover, cluster analysis based on fimH and sseL identified a number of clusters, of which several were found in more than one outbreak, as well as 11 singletons. Further research is needed to determine if CRISPR-MVLST might help identify the ecological origins of S. Enteritidis strains that contaminate chickens and eggs.

Impact of antibiotic use in adult dairy cows on antimicrobial resistance of veterinary and human pathogens: a comprehensive review.

Antibiotics have saved millions of human lives, and their use has contributed significantly to improving human and animal health and well-being. Use of antibiotics in food-producing animals has resulted in healthier, more productive animals; lower disease incidence and reduced morbidity and mortality in humans and animals; and production of abundant quantities of nutritious, high-quality, and low-cost food for human consumption. In spite of these benefits, there is considerable concern from public health, food safety, and regulatory perspectives about the use of antimicrobials in food-producing animals. Over the last two decades, development of antimicrobial resistance resulting from agricultural use of antibiotics that could impact treatment of diseases affecting the human population that require antibiotic intervention has become a significant global public health concern. In the present review, we focus on antibiotic use in lactating and nonlactating cows in U.S. dairy herds, and address four key questions: (1) Are science-based data available to demonstrate antimicrobial resistance in veterinary pathogens that cause disease in dairy cows associated with use of antibiotics in adult dairy cows? (2) Are science-based data available to demonstrate that antimicrobial resistance in veterinary pathogens that cause disease in adult dairy cows impacts pathogens that cause disease in humans? (3) Does antimicrobial resistance impact the outcome of therapy? (4) Are antibiotics used prudently in the dairy industry? On the basis of this review, we conclude that scientific evidence does not support widespread, emerging resistance among pathogens isolated from dairy cows to antibacterial drugs even though many of these antibiotics have been used in the dairy industry for treatment and prevention of disease for several decades. However, it is clear that use of antibiotics in adult dairy cows and other food-producing animals does contribute to increased antimicrobial resistance. Although antimicrobial resistance does occur, we are of the opinion that the advantages of using antibiotics in adult dairy cows far outweigh the disadvantages. Last, as this debate continues, we need to consider the consequences of "what would happen if antibiotics are banned for use in the dairy industry and in other food-producing animals?" The implications of this question are far reaching and include such aspects as animal welfare, health, and well-being, and impacts on food quantity, quality, and food costs, among others. This question should be an important aspect in this ongoing and controversial debate.

Detection of Anaplasma Phagocytophilum in Horses With Suspected Tick-Borne Disease in Northeastern United States by Metagenomic Sequencing.

Metagenomic sequencing of clinical diagnostic specimens has a potential for unbiased detection of infectious agents, diagnosis of polymicrobial infections and discovery of emerging pathogens. Herein, next generation sequencing (NGS)-based metagenomic approach was used to investigate the cause of illness in a subset of horses recruited for a tick-borne disease surveillance study during 2017-2019. Blood samples collected from 10 horses with suspected tick-borne infection and five apparently healthy horses were subjected to metagenomic analysis. Total genomic DNA extracted from the blood samples were enriched for microbial DNA and subjected to shotgun next generation sequencing using Nextera DNA Flex library preparation kit and V2 chemistry sequencing kit on the Illumina MiSeq sequencing platform. Overall, 0.4-0.6 million reads per sample were analyzed using Kraken metagenomic sequence classification program. The taxonomic classification of the reads indicated that bacterial genomes were overrepresented (0.5 to 1%) among the total microbial reads. Most of the bacterial reads (~91%) belonged to phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria and Tenericutes in both groups. Importantly, 10-42.5% of Alphaproteobacterial reads in 5 of 10 animals with suspected tick-borne infection were identified as Anaplasma phagocytophilum. Of the 5 animals positive for A. phagocytophilum sequence reads, four animals tested A. phagocytophilum positive by PCR. Two animals with suspected tick-borne infection and A. phagocytophilum positive by PCR were found negative for any tick-borne microbial reads by metagenomic analysis. The present study demonstrates the usefulness of the NGS-based metagenomic analysis approach for the detection of blood-borne microbes.