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Food-producing animals such as dairy cattle are potential reservoirs of antimicrobial resistance (AMR), with multidrug-resistant (MDR) organisms such as Escherichia coli observed in higher frequency in young calves compared to older cattle. In this study, we characterized the genomes of enteric MDR E. coli from pre-weaned dairy calves with and without diarrhea and evaluated the influence of host-level factors on genomic composition. Whole genome sequence comparative analysis of E. coli (n = 43) revealed substantial genomic diversity that primarily clustered by sequence type and was minimally driven by calf diarrheal disease status (healthy, diarrheic, or recovered), antimicrobial exposure, and dietary zinc supplementation. Diverse AMR genes (ARGs)—including extended-spectrum beta-lactamase genes and quinolone resistance determinants—were identified (n = 40), with unique sets of ARGs co-occurring in gene clusters with large AMR plasmids IncA/C2 and IncFIB(AP001918). Zinc supplementation was not significantly associated with the selection of individual ARGs in E. coli, however analysis of ARG and metal resistance gene pairs identified positive associations between certain aminoglycoside, beta-lactam, sulfonamide, and trimethoprim ARGs with acid, tellurium and mercury resistance genes. Although E. coli in this study lacked the typical virulence factors of diarrheagenic strains, virulence genes overlapping with those in major pathotypes were identified. Among the 103 virulence genes detected, the highest abundance and diversity of genes corresponded to iron acquisition (siderophores and heme uptake). Our findings indicate that the host-level factors evaluated in this study were not key drivers of genomic variability, but that certain accessory genes in enteric MDR E. coli may be enriched. Collectively, this work provides insight into the genomic diversity and host-microbe interface of MDR E. coli from pre-weaned dairy calves.

Abstract The objective of this study was to evaluate the impact of dietary zinc supplementation in pre-weaned dairy calves on the phenotypic antimicrobial resistance (AMR) of fecal commensal bacteria. A repository of fecal specimens from a random sample of calves block-randomized into placebo (n = 39) and zinc sulfate (n = 28) groups collected over a zinc supplementation clinical trial at the onset of calf diarrhea, calf diarrheal cure, and the last day of 14 cumulative days of zinc or placebo treatment were analyzed. Antimicrobial susceptibility testing was conducted for Enterococcus spp. (n = 167) and E. coli (n = 44), with one representative isolate of each commensal bacteria tested per sample. Parametric survival interval regression models were constructed to evaluate the association between zinc treatment and phenotypic AMR, with exponentiated accelerated failure time (AFT) coefficients adapted for MIC instead of time representing the degree of change in AMR (MIC Ratio, MR). Findings from our study indicated that zinc supplementation did not significantly alter the MIC in Enterococcus spp. for 13 drugs: gentamicin, vancomycin, ciprofloxacin, erythromycin, penicillin, nitrofurantoin, linezolid, quinupristin/dalfopristin, tylosin tartrate, streptomycin, daptomycin, chloramphenicol, and tigecycline (MR = 0.96–2.94, p > 0.05). In E. coli, zinc supplementation was not associated with resistance to azithromycin (MR = 0.80, p > 0.05) and ceftriaxone (MR = 0.95, p > 0.05). However, a significant reduction in E. coli MIC values was observed for ciprofloxacin (MR = 0.17, 95% CI 0.03–0.97) and nalidixic acid (MR = 0.28, 95% CI 0.15–0.53) for zinc-treated compared to placebo-treated calves. Alongside predictions of MIC values generated from these 17 AFT models, findings from this study corroborate the influence of age and antimicrobial exposure on phenotypic AMR.

The incidence of antimicrobial resistance (AMR) in the environment is often overlooked and leads to serious health threats under the One Health paradigm. Infection with extended-spectrum β-lactamase (ESBL) producing bacteria in humans and animals has been widely examined, with the mode of transmission routes such as food, water, and contact with a contaminated environment. The purpose of this study was to determine the occurrence and molecular characteristics of resistant Salmonella enterica (S. enterica) (n = 59) and Escherichia coli (E. coli) (n = 392) isolated from produce commodities collected from fresh markets and supermarkets in Bangkok, Thailand. In this study, the S. enterica isolates exhibited the highest prevalence of resistance to tetracycline (11.9%) and streptomycin (8.5%), while the E. coli isolates were predominantly resistant to tetracycline (22.5%), ampicillin (21.4%), and sulfamethoxazole (11.5%). Among isolates of S. enterica (6.8%) and E. coli (15.3%) were determined as multidrug resistant (MDR). The prevalence of ESBL-producing isolates was 5.1% and 1.0% in S. enterica and E. coli, respectively. A minority of S. enterica isolates, where a single isolate exclusively carried blaCTX-M-55 (n = 1), and another isolate harbored both blaCTX-M-55 and blaTEM-1 (n = 1); similarly, a minority of E. coli isolates contained blaCTX-M-55 (n = 2) and blaCTX-M-15 (n = 1). QnrS (11.9%) and blaTEM (20.2%) were the most common resistant genes found in S. enterica and E. coli, respectively. Nine isolates resistant to ciprofloxacin contained point mutations in gyrA and parC. In addition, the odds of resistance to tetracycline among isolates of S. enterica were positively associated with the co-occurrence of ampicillin resistance and the presence of tetB (P = 0.001), while the E. coli isolates were positively associated with ampicillin resistance, streptomycin resistance, and the presence of tetA (P

California Leafy Green Products Handler Marketing Agreement (LGMA) established food safety metrics with guidance recommendations of 366 m (1,200 feet) and 1,609 m (1 mile) distances between production fields of leafy greens and a concentrated animal feeding operation (CAFO) containing >1,000 and >80,000 head of cattle, respectively. This study evaluated the effect of these distance metrics and environmental factors on the occurrence of airborne Escherichia coli in proximity to seven commercial beef cattle feedlots located in Imperial Valley, California. A total of 168 air samples were collected from seven beef cattle feedlots during March and April 2020, which were the months implicated in the 2018 Yuma Arizona lettuce outbreak of E. coli O157:H7. The distance between air sampling sites and the edge of the feedlot ranged from ∼0 to ∼2,200 m (∼1.3 mile), with each sample comprised of 1,000 L of processed air taken at a 1.2 m elevation over a 10-minute duration. E. coli colonies were enumerated on CHROMagar ECC selective agar and confirmed with conventional PCR. Meteorological data (air temperature, wind speed, wind direction, relative humidity) were collected in situ. The prevalence and mean concentration of E. coli were 6.55% (11/168) and 0.09 CFU per 1,000 L of air, with positive samples limited to within 37 m (120 ft) of the feedlot. Based on logistic regression, the odds of airborne E. coli detection were associated with little to no wind and close proximity to a feedlot. This pilot study found limited dispersal of airborne E. coli in proximity to commercial feedlots in Imperial Valley, with light-to-no wind and proximity within 37 m of a feedlot significant factor-associated airborne E. coli in this produce-growing region of California.

Abstract Background Microbiota play important roles in the gastrointestinal tract (GIT) of dairy cattle as the communities are responsible for host health, growth, and production performance. However, a systematic characterization and comparison of microbial communities in the GIT of cattle housed in different management units on a modern dairy farm are still lacking. We used 16S rRNA gene sequencing to evaluate the fecal bacterial communities of 90 dairy cattle housed in 12 distinctly defined management units on a modern dairy farm. Results We found that cattle from management units 5, 6, 8, and 9 had similar bacterial communities while the other units showed varying levels of differences. Hutch calves had a dramatically different bacterial community than adult cattle, with at least 10 genera exclusively detected in their samples but not in non-neonatal cattle. Moreover, we compared fecal bacteria of cattle from every pair of the management units and detailed the number and relative abundance of the significantly differential genera. Lastly, we identified 181 pairs of strongly correlated taxa in the community, showing possible synergistic or antagonistic relationships. Conclusions This study assesses the fecal microbiota of cattle from 12 distinctly defined management units along the production line on a California dairy farm. The results highlight the similarities and differences of fecal microbiota between cattle from each pair of the management units. Especially, the data indicate that the newborn calves host very different gut bacterial communities than non-neonatal cattle, while non-neonatal cattle adopt one of the two distinct types of gut bacterial communities with subtle differences among the management units. The gut microbial communities of dairy cattle change dramatically in bacterial abundances at different taxonomic levels along the production line. The findings provide a reference for research and practice in modern dairy farm management.

Background Tilapia is a primary aquaculture fish in Thailand, but little is known about the occurrence of antimicrobial resistance (AMR) in Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae colonizing healthy tilapia intended for human consumption and the co-occurrence of these AMR bacteria in the cultivation water. Methods This study determined the phenotype and genotype of AMR, extended-spectrum β-lactamase (ESBL) production, and virulence factors of A. hydrophila, Salmonella spp., and V. cholerae isolated from hybrid red tilapia and cultivation water in Thailand. Standard culture methods such as USFDA’s BAM or ISO procedures were used for the original isolation, with all isolates confirmed by biochemical tests, serotyping, and species-specific gene detection based on PCR. Results A total of 278 isolates consisting of 15 A. hydrophila, 188 Salmonella spp., and 75 V. cholerae isolates were retrieved from a previous study. All isolates of A. hydrophila and Salmonella isolates were resistance to at least one antimicrobial, with 26.7% and 72.3% of the isolates being multidrug resistant (MDR), respectively. All A. hydrophila isolates were resistant to ampicillin (100%), followed by oxytetracycline (26.7%), tetracycline (26.7%), trimethoprim (26.7%), and oxolinic acid (20.0%). The predominant resistance genes in A. hydrophila were mcr-3 (20.0%), followed by 13.3% of isolates having floR, qnrS, sul1, sul2, and dfrA1. Salmonella isolates also exhibited a high prevalence of resistance to ampicillin (79.3%), oxolinic acid (75.5%), oxytetracycline (71.8%), chloramphenicol (62.8%), and florfenicol (55.3%). The most common resistance genes in these Salmonella isolates were qnrS (65.4%), tetA (64.9%), blaTEM (63.8%), and floR (55.9%). All V. cholerae isolates were susceptible to all antimicrobials tested, while the most common resistance gene was sul1 (12.0%). One isolate of A. hydrophila was positive for int1, while all isolates of Salmonella and V. cholerae isolates were negative for integrons and intSXT. None of the bacterial isolates in this study were producing ESBL. The occurrence of mcr-3 (20.0%) in these isolates from tilapia aquaculture may signify a serious occupational and consumer health risk given that colistin is a last resort antimicrobial for treatment of Gram-negative bacteria infections. Conclusions Findings from this study on AMR bacteria in hybrid red tilapia suggest that aquaculture as practiced in Thailand can select for ubiquitous AMR pathogens, mobile genetic elements, and an emerging reservoir of mcr and colistin-resistant bacteria. Resistant and pathogenic bacteria, such as resistance to ampicillin and tetracycline, or MDR Salmonella circulating in aquaculture, together highlight the public health concerns and foodborne risks of zoonotic pathogens in humans from cultured freshwater fish.

This study characterized the effect of distance from beef cattle feedlots, environmental factors, and climate on the occurrence of airborne bacterial indicators and pathogens. Three hundred air samples were collected over 6 months from five feedlots, with each air sample comprising 6000 L of air. Air samples were processed onto TSB-enriched air filters, qPCR-screened, and then qPCR-confirmed for suspect positive colonies of E. coli O157, non-O157-Shiga-toxin-producing E. coli (STEC), Salmonella, and E. coli. Direct enumeration of E. coli was also collected. Although no bacterial pathogens were qPCR-confirmed for the 300 samples, E. coli was detected in 16.7% (50/300) of samples, with an overall mean concentration of 0.17 CFU/6000 L air. Logistic regression analyses revealed a higher odds of E. coli for samples in close proximity compared to >610 m (2000 ft) distance from feedlots, along with significant associations with meteorological factors, sampling hour of day, and the presence of a dust-generating activity such as plowing a field or nearby vehicular traffic. The lack of bacterial pathogen detection suggests airborne deposition from nearby feedlots may not be a significant mechanism of leafy green bacterial pathogen contamination; the result of our study provides data to inform future revisions of produce-safety guidance.

Background The presence of Escherichia coli O157:H7 (E. coli O157:H7) super-shedding cattle in feedlots has the potential to increase the overall number (bio-burden) of E. coli O157:H7 in the environment. It is important to identify factors to reduce the bio-burden of E. coli O157 in feedlots by clarifying practices associated with the occurrence of super-shedders in feedlot cattle. Methods The objective of this study is to (1) identify host, pathogen, and management risk factors associated with naturally infected feedlot cattle excreting high concentrations of E. coli O157:H7 in their feces and (2) to determine whether the ingested dose or the specific strain of E. coli O157:H7 influences a super-shedder infection within experimentally inoculated feedlot cattle. To address this, (1) pen floor fecal samples and herd parameters were collected from four feedlots over a 9-month period, then (2) 6 strains of E. coli O157:H7, 3 strains isolated from normal shedder steers and 3 strains isolated from super-shedder steers, were inoculated into 30 one-year-old feedlot steers. Five steers were assigned to each E. coli O157:H7 strain group and inoculated with targeted numbers of 102, 104, 106, 108, and 1010 CFU of bacteria respectively. Results In the feedlots, prevalence of infection with E. coli O157:H7 for the 890 fecal samples collected was 22.4%, with individual pen prevalence ranging from 0% to 90% and individual feedlot prevalence ranging from 8.4% to 30.2%. Three samples had E. coli O157:H7 levels greater than 104 MPN/g feces, thereby meeting the definition of super-shedder. Lower body weight at entry to the feedlot and higher daily maximum ambient temperature were associated with increased odds of a sample testing positive for E. coli O157:H7. In the experimental inoculation trial, the duration and total environmental shedding load of E. coli O157:H7 suggests that the time post-inoculation and the dose of inoculated E. coli O157:H7 are important while the E. coli O157:H7 strain and shedding characteristic (normal or super-shedder) are not. Discussion Under the conditions of this experiment, super-shedding appears to be the result of cattle ingesting a high dose of any strain of E. coli O157:H7. Therefore strategies that minimize exposure to large numbers of E. coli O157:H7 should be beneficial against the super-shedding of E. coli O157:H7 in feedlots.

Consumption of contaminated food causes 600 million cases, including 420,000 of fatal infections every year. Estimated cost from food-borne illnesses is USD 110 billion per year, which is an economic burden to low- and middle-income countries. Thailand is a leading producer and consumer of seafood, but little is known about bacterial contamination in seafood. In particular, public health agencies need to know the relationship between Salmonella contamination in seafood and risk factors, as assessed with readily available culture-dependent and bacterial phenotyping methods. To address this, levels of indicator bacteria, Salmonella and Vibrio in various seafood products were determined to identify risk factors associated with Salmonella contamination. A total of 335 samples were collected from October 2018 to July 2019 at seafood markets throughout Bangkok, Thailand; overall sample composition was Pacific white shrimp (n = 85), oysters (n = 82), blood cockles (n = 84), and Asian seabass (n = 84). Prevalence was 100% for fecal coliforms and 85% for E. coli. In contrast, prevalence was 59% for V. parahaemolyticus, 49% for V. cholerae, 19% for V. alginolyticus, 18% for V. vulnificus, and 36% for Salmonella. Highest concentrations of fecal coliforms and E. coli were in oysters. Highest concentrations of Salmonella with Matopeni (31%) being the predominant serotype were in shrimp. Salmonella contamination was significantly associated with type of seafood, sampling location, retail conditions, and the presence of E. coli, V. alginolyticus and V. vulnificus. A cutoff value for E. coli concentration of 1.3 × 104 MPN/g predicted contamination of Salmonella, with a sensitivity of 84% and specificity of 61%. Displaying seafood products on ice, presence of E. coli and Vibrio, and seafood derived from Eastern Thailand were associated with an increased risk of Salmonella contamination.

The occurrence of waterborne antimicrobial-resistant (AMR) bacteria in areas of high-density oyster cultivation is an ongoing environmental and public health threat given the popularity of shellfish consumption, water-related human recreation throughout coastal Thailand, and the geographical expansion of Thailand’s shellfish industry. This study characterized the association of phenotypic and genotypic AMR, including extended-spectrum β-lactamase (ESBL) production, and virulence genes isolated from waterborne Escherichia coli (E. coli) (n = 84), Salmonella enterica (S. enterica) subsp. enterica (n = 12), Vibrio parahaemolyticus (V. parahaemolyticus) (n = 249), and Vibrio cholerae (V. cholerae) (n = 39) from Thailand’s coastal aquaculture regions. All Salmonella (100.0%) and half of V. cholerae (51.3%) isolates harbored their unique virulence gene, invA and ompW, respectively. The majority of isolates of V. parahaemolyticus and E. coli, ~25% of S. enterica subsp. enterica, and ~12% of V. cholerae, exhibited phenotypic AMR to multiple antimicrobials, with 8.9% of all coastal water isolates exhibiting multidrug resistance (MDR). Taken together, we recommend that coastal water quality surveillance programs include monitoring for bacterial AMR for food safety and recreational water exposure to water for Thailand’s coastal water resources.

Background Understanding the effects of herd management practices on the prevalence of multidrug-resistant pathogenic Salmonella and commensals Enterococcus spp. and Escherichia coli in dairy cattle is key in reducing antibacterial resistant infections in humans originating from food animals. Our objective was to explore the herd and cow level features associated with the multi-drug resistant, and resistance phenotypes shared between Salmonella, E. coli and Enterococcus spp. using machine learning algorithms. Methods Randomly collected fecal samples from cull dairy cows from six dairy farms in central California were tested for multi-drug resistance phenotypes of Salmonella, E. coli and Enterococcus spp. Using data on herd management practices collected from a questionnaire, we built three machine learning algorithms (decision tree classifier, random forest, and gradient boosting decision trees) to predict the cows shedding multidrug-resistant Salmonella and commensal bacteria. Results The decision tree classifier identified rolling herd average milk production as an important feature for predicting fecal shedding of multi-drug resistance in Salmonella or commensal bacteria. The number of culled animals, monthly culling frequency and percentage, herd size, and proportion of Holstein cows in the herd were found to be influential herd characteristics predicting fecal shedding of multidrug-resistant phenotypes based on random forest models for Salmonella and commensal bacteria. Gradient boosting models showed that higher culling frequency and monthly culling percentages were associated with fecal shedding of multidrug resistant Salmonella or commensal bacteria. In contrast, an overall increase in the number of culled animals on a culling day showed a negative trend with classifying a cow as shedding multidrug-resistant bacteria. Increasing rolling herd average milk production and spring season were positively associated with fecal shedding of multidrug- resistant Salmonella. Only six individual cows were detected sharing tetracycline resistance phenotypes between Salmonella and either of the commensal bacteria. Discussion Percent culled and culling rate reflect the increase in culling over time adjusting for herd size and were associated with shedding multidrug resistant bacteria. In contrast, number culled was negatively associated with shedding multidrug resistant bacteria which may reflect producer decisions to prioritize the culling of otherwise healthy but low-producing cows based on milk or beef prices (with respect to dairy beef), amongst other factors. Using a data-driven suite of machine learning algorithms we identified generalizable and distant associations between antimicrobial resistance in Salmonella and fecal commensal bacteria, that can help develop a producer-friendly and data-informed risk assessment tool to reduce shedding of multidrug-resistant bacteria in cull dairy cows.

Abstract Background There is growing evidence to support the beneficial effects of supplementing direct-fed microbials (DFM) on performance, health status, and immune responses of weaned pigs. Therefore, the objective of this study was to investigate dietary supplementation of Bacillus subtilis (DSM 25841) on growth performance, diarrhea, gut permeability and immunity of weaned pigs experimentally infected with a pathogenic F-18 Escherichia coli (E. coli). Results The F18 E. coli infection reduced (P

Cryptosporidium spp. is a parasite that can infect a wide variety of vertebrate species. The parasite has been detected in sheep worldwide with diverse species and genotypes of various levels of zoonotic potential and public health concern. The purpose of this study was to determine the distribution of genotypes of Cryptosporidium in sheep in California, USA. Microscopic positive samples from individual sheep from central and northern California ranches were genotyped by sequencing a fragment of the 18S rRNA gene and BLAST analysis. Eighty-eight (63.8%) of the microscopic positive samples were genotyped, and multiple genotypes of Cryptosporidium were identified from sheep in the enrolled ranches. Approximately 89% of isolates (n = 78) were C. xiaoi or C. bovis, 10% of isolates (n = 9) were C. ubiquitum, and 1% of isolates (n = 1) were C. parvum. The C. parvum and C. ubiquitum isolates were detected only from lambs and limited to four farms. Given that the majority of Cryptosporidium species (i.e., C. xiaoi and C. bovis) were of minor zoonotic concern, the results of this study suggest that sheep are not a reservoir of major zoonotic Cryptosporidium in California ranches.

Epidemiological studies of low prevalence disease problems are often hindered by the high cost of diagnostic testing. The objective of this study was to evaluate PCR screening of both individual and pooled fecal samples from culled dairy cows for the invA gene of Salmonella followed by culture to determine if the sensitivity and specificity were comparable to the results from traditional culture methods applied to individual samples. Cows from six different dairies were sampled in all four seasons. A total of 240 individual cow fecal samples, 24 fecal pools and 24 pools of 24-hour tetrathionate enrichment broth were tested. Diagnostic sensitivity of PCR screening followed by culture of PCR positive or indeterminate samples (i.e PCR-CUL method) was lower than that of culture (CUL) when applied to individual fecal samples (94.8%, 99.5%), however the specificity was comparable (99.6% and 97.7% respectively). For pools of five fecal samples and pools of five, 24 h tetrathionate broth samples, the specificity of both tests were comparable (∼98%); however, their sensitivity was only comparable in pooled fecal samples (∼93%) but greater for culture compared to PCR-CUL in pooled broth samples (∼99% versus ∼93%). Compared to culture results from testing of individual fecal samples, testing pooled fecal samples by culture had a relative sensitivity of 74% and relative specificity of 96%, testing pooled fecal samples by PCR-CUL resulted in relative sensitivity of 90% and relative specificity of 96%. Testing of pooled 24-hour enrichment broth by PCR-CUL increased the relative sensitivity and specificity to 100%. PCR testing followed by culture of positive or indeterminate samples is a time saving alternative to traditional methods. In addition, pooling of samples may be a useful method for decreasing cost if study aims can accommodate a moderate loss of relative sensitivity.

The objectives of this study were to characterize overall genomic antibiotic resistance profiles of fecal Escherichia coli and Enterococcus spp. from dairy cattle at different production stages using whole-genome sequencing and to determine the association between antimicrobial resistance (AMR) phenotypes and their corresponding genotypes. The Comprehensive Antibiotic Resistance Database (CARD) and ResFinder, two publicly available databases of antimicrobial resistance genes, were used to annotate isolates. Based on the ResFinder database, 27.5% and 20.0% of tested E. coli isolates (n = 40) harbored single and ≥3 antimicrobial resistance genes, respectively; for Enterococcus spp., we observed 87.8% and 8.2%, respectively. The highest prevalence of AMR genes in E. coli was for resistance to tetracycline (27.5%), followed by sulphonamide (22.5%) and aminoglycoside (20.0%); the predominant antimicrobial resistance genes in Enterococcus spp. targeted macrolide drugs (77.6%). Based on the CARD database, resistance to ≥3 antimicrobial classes was observed in all E. coli and 77.6% in Enterococcus spp. isolates. A high degree of agreement existed between the resistance phenotype and the presence of resistance genes for various antimicrobial classes for E. coli but much less so for isolates of Enterococcus. Consistent with prior work, fecal E. coli and Enterococcus spp. isolates from calves harbored a wide spectrum of resistance genes, compared to those from cattle at other production stages, based on the cross-sectional samples from the studied farm.

Background In this study cull dairy cows from six California dairy herds were sampled seasonally over the course of a year. The objectives were to determine the prevalence of antimicrobial resistant (AMR) Salmonella spp. shed in cull cow feces, and the factors associated with fecal shedding of AMR and multidrug resistant (MDR) Salmonella. Methods Six dairy farms located in the San Joaquin Valley of California were identified and enrolled as a convenience sample. On each dairy, and once during each of the four seasons, 10 cull cows were randomly selected for fecal sampling on the day of their removal from the herd. In addition, study personnel completed a survey based on responses of the herd manager to questions related to the previous 4 month’s herd management and the specific cattle sampled. Fecal samples were submitted to the California Animal Health and Food Safety laboratory for Salmonella isolation. Antimicrobial resistance was evaluated using broth microdilution method and a gram-negative assay plate following Clinical Laboratory Standards Institute (CLSI) guidelines and breakpoint references. All statistical models were survey adjusted for number of animals on sampling day. Results A total of 62 Salmonella were isolated from 60 of the 239 fecal samples collected. For 12% (95% confidence interval (CI) [3–20]) of fecal samples a multidrug resistant Salmonella was isolated. The survey-weighted results for the two most common drug classes for which isolates were resistant were tetracycline (39%; 95% CI [27–51]) and ampicillin (18%; 95% CI [9–27]). An important finding was the identification of cephalosporin as the third most common drug class for which isolates were resistant, with ceftriaxone (10%; 95% CI [2–17]) being the most common drug associated with resistance in that class. At the cow-level, reason for culling, prior treatment with antimicrobial drugs as the reason for culling was associated with higher odds of isolating an AMR Salmonella isolate. At the herd-level, percent of animals monthly culled on the farm as well as number of milking cows in the herd were associated with isolation of antimicrobial resistant Salmonella in cull cows. Discussion Salmonella isolated from fecal samples from cull cows were resistant to important antimicrobials, such as ceftriaxone. The most common drug classes for which isolates were resistant were tetracyclines and beta-lactams, with ampicillin, ceftriaxone and ceftiofur being the three most common drugs within the latter. Cow and herd level factors were associated with isolating antimicrobial resistant Salmonella that should be further investigated for their potential role in promoting occurrence of AMR Salmonella. Our results also highlight the importance of monitoring dairy cattle sent to slaughter for shedding of Salmonella resistant to medically important antimicrobial drugs.

Field trials were conducted in July–August and October 2012 to quantify the inactivation rate of Escherichia coli O157:H7 when mixed with fecal slurry and applied to romaine lettuce leaves. Lettuce was grown under commercial conditions in Salinas Valley, California. One-half milliliter of rabbit, chicken, or pig fecal slurry, containing an average of 4.05 × 107 CFU E. coli O157:H7 (C0), was inoculated onto the upper (adaxial) surface of a lower leaf on 288 heads of lettuce per trial immediately following a 2.5 h irrigation event. To estimate the bacterial inactivation rate as a function of time, fecal matrix, irrigation and seasonal climate effects, sets of lettuce heads (n = 28) were sampled each day over 10 days and the concentration of E. coli O157:H7 (Ct) determined. E. coli O157:H7 was detected on 100% of heads during the 10-day duration, with concentrations ranging from ≤340 MPN/head (∼5-log reduction) to >3.45 × 1012 MPN/head (∼5-log growth). Relative to C0, on day 10 (Ct = 12) we observed an overall 2.6-log and 3.2-log mean reduction of E. coli O157:H7 in July and October, respectively. However, we observed relative maximum concentrations due to bacterial growth on day 6 (maximum Ct = 8) apparently stimulated by foliar irrigation on day 5. From this maximum there was a mean 5.3-log and 5.1-log reduction by day 10 (Ct = 12) for the July and October trials, respectively. This study provides insight into the inactivation and growth kinetics of E. coli O157:H7 on romaine lettuce leaves under natural field conditions. This study provides evidence that harvesting within 24 h post irrigation has the potential to increase the concentration of E. coli O157:H7 contamination, if present on heads of romaine lettuce; foliar irrigation can temporarily stimulate substantial regrowth of E. coli O157:H7.

Between October 2013 and May 2016, 506 scat samples were collected from 22 species of wildlife located in a protected watershed of a major municipal water supply in the Pacific Northwest, USA. Overall prevalence of Cryptosporidium in the wildlife scat was 13.8% (70/506), with 15 species of wildlife found positive for Cryptosporidium. Prevalence of Cryptosporidium varied among species of wildlife, with higher prevalences observed in cougars (50.0%), mountain beavers (40.0%), and bobcats (33.3%), but none of these species are riparian-dependent. Genotyping of Cryptosporidium by sequencing PCR amplicons from the 18S rRNA gene were successful for seven species of wildlife, including bobcat, unknown predator, black-tailed deer, deer mouse, snowshoe hare, mountain beaver, and western spotted skunk. BLAST and phylogenetic analyses indicated that multiple species and genotypes of Cryptosporidium were present, with some isolates possibly co-circulating within and between wildlife populations in this protected watershed. Evidence of oocyst exchange between infected prey and their predators was also found. During the study period, several zoonotic Cryptosporidium species and genotypes that are uncommon in humans were detected in bobcat (99.58% identical to Cryptosporidium felis), unknown predator (100% identical to Cryptosporidium canis), snowshoe hare (100% identical to Cryptosporidium sp. skunk genotype), and mountain beaver (100% identical to Cryptosporidium ubiquitum). Novel sequences were also found in mountain beaver. To our knowledge, this is the first published report of a unique genotype or species of Cryptosporidium in mountain beaver (Aplodontia rufa).

Previously we reported the unique Cryptosporidium sp. “c” genotype (e.g., Sbey03c, Sbey05c, Sbld05c, Sltl05c) from three species of Spermophilus ground squirrel (Spermophilus beecheyi, Spermophilus beldingi, Spermophilus lateralis) located throughout California, USA. This follow-up work characterizes the morphology and animal infectivity of this novel genotype as the final step in proposing it as a new species of Cryptosporidium. Analysis of sequences of 18S rRNA, actin, and HSP70 genes of additional Cryptosporidium isolates from recently sampled California ground squirrels (S. beecheyi) confirms the presence of the unique Sbey-c genotype in S. beecheyi. Phylogenetic and BLAST analysis indicates that the c-genotype in Spermophilus ground squirrels is distinct from Cryptosporidium species/genotypes from other host species currently available in GenBank. We propose to name this c-genotype found in Spermophilus ground squirrels as Cryptosporidium rubeyi n. sp. The mean size of C. rubeyi n. sp. oocysts is 4.67 (4.4–5.0) μm × 4.34 (4.0–5.0) μm, with a length/width index of 1.08 (n = 220). Oocysts of C. rubeyi n. sp. are not infectious to neonatal BALB/c mice and Holstein calves. GenBank accession numbers for C. rubeyi n. sp. are DQ295012, AY462233, and KM010224 for the 18S rRNA gene, KM010227 for the actin gene, and KM010229 for the HSP70 gene.

Wildlife are increasingly recognized as important biological reservoirs of zoonotic species of Cryptosporidium that might contaminate water and cause human exposure to this protozoal parasite. The habitat range of the yellow-bellied marmot (Marmota flaviventris) overlaps extensively with the watershed boundaries of municipal water supplies for California communities along the foothills of the Sierra Nevada. We conducted a cross-sectional epidemiological study to estimate the fecal shedding of Cryptosporidium oocysts by yellow-bellied marmots and to quantify the environmental loading rate and determine risk factors for Cryptosporidium fecal shedding in this montane wildlife species. The observed proportion of Cryptosporidium positive fecal samples was 14.7% (33/224, positive number relative to total number samples) and the environmental loading rate was estimated to be 10,693 oocysts animal-1 day-1. Fecal shedding was associated with the elevation and vegetation status of their habitat. Based on a portion of the 18s rRNA gene sequence of 2 isolates, the Cryptosporidium found in Marmota flaviventris were 99.88%–100% match to multiple isolates of C. parvum in the GenBank.

This randomized controlled trial characterized the transfer of E. coli from animal feces and/or furrow water onto adjacent heads of lettuce during foliar irrigation, and the subsequent survival of bacteria on the adaxial surface of lettuce leaves. Two experiments were conducted in Salinas Valley, California: (1) to quantify the transfer of indicator E. coli from chicken and rabbit fecal deposits placed in furrows to surrounding lettuce heads on raised beds, and (2) to quantify the survival of inoculated E. coli on Romaine lettuce over 10 days. E. coli was recovered from 97% (174/180) of lettuce heads to a maximal distance of 162.56 cm (5.33 ft) from feces. Distance from sprinklers to feces, cumulative foliar irrigation, and lettuce being located downwind of the fecal deposit were positively associated, while distance from fecal deposit to lettuce was negatively associated with E. coli transference. E. coli exhibited decimal reduction times of 2.2 and 2.5 days when applied on the adaxial surface of leaves within a chicken or rabbit fecal slurry, respectively. Foliar irrigation can transfer E. coli from feces located in a furrow onto adjacent heads of lettuce, likely due to the kinetic energy of irrigation droplets impacting the fecal surface and/or impacting furrow water contaminated with feces, with the magnitude of E. coli enumerated per head of lettuce influenced by the distance between lettuce and the fecal deposit, cumulative application of foliar irrigation, wind aspect of lettuce relative to feces, and time since final irrigation. Extending the time period between foliar irrigation and harvest, along with a 152.4 cm (5 ft) no-harvest buffer zone when animal fecal material is present, may substantially reduce the level of bacterial contamination on harvested lettuce.

Background Although a wealth of literature points to the importance of social factors on health, a detailed understanding of the complex interplay between social and biological systems is lacking. Social status is one aspect of social life that is made up of multiple structural (humans: income, education; animals: mating system, dominance rank) and relational components (perceived social status, dominance interactions). In a nonhuman primate model we use novel network techniques to decouple two components of social status, dominance rank (a commonly used measure of social status in animal models) and dominance certainty (the relative certainty vs. ambiguity of an individual’s status), allowing for a more complex examination of how social status impacts health. Methods Behavioral observations were conducted on three outdoor captive groups of rhesus macaques (N = 252 subjects). Subjects’ general physical health (diarrhea) was assessed twice weekly, and blood was drawn once to assess biomarkers of inflammation (interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and C-reactive protein (CRP)). Results Dominance rank alone did not fully account for the complex way that social status exerted its effect on health. Instead, dominance certainty modified the impact of rank on biomarkers of inflammation. Specifically, high-ranked animals with more ambiguous status relationships had higher levels of inflammation than low-ranked animals, whereas little effect of rank was seen for animals with more certain status relationships. The impact of status on physical health was more straightforward: individuals with more ambiguous status relationships had more frequent diarrhea; there was marginal evidence that high-ranked animals had less frequent diarrhea. Discussion Social status has a complex and multi-faceted impact on individual health. Our work suggests an important role of uncertainty in one’s social status in status-health research. This work also suggests that in order to fully explore the mechanisms for how social life influences health, more complex metrics of social systems and their dynamics are needed.

Shiga toxin-producing Escherichia coli, like E. coli O157:H7, are important human and animal pathogens. Naturally-acquired E. coli O157:H7 infections occur in numerous species but, particularly, cattle have been identified as a significant reservoir for human cases. E. coli O157:H7 are isolated from a number of domestic and wild animals, including rodents that share a living space with cattle. These Shiga toxin-producing E. coli O157:H7 strains can be highly virulent in humans, but little is known about the sequelae of interspecies transfer. In a group of neonatal calves, we determined the differences in colonization patterns and lesions associated with infection using either a wildlife or bovine E. coli O157:H7 strain. In calves challenged with the wildlife E. coli O157:H7 strain, the large (descending) colon was solely colonized, which differed substantially from the calves inoculated with the bovine E. coli O157:H7 strain, where the spiral colon was the principal target of infection. This study also demonstrated that while both interspecies- and intraspecies-derived E. coli O157:H7 can infect young calves, the distribution and severity differs.

We investigated involvement of feral swine in contamination of agricultural fields and surface waterways with Escherichia coli O157:H7 after a nationwide outbreak traced to bagged spinach from California. Isolates from feral swine, cattle, surface water, sediment, and soil at 1 ranch were matched to the outbreak strain.