Persistent effects of intramammary ceftiofur treatment on the gut microbiome and antibiotic resistance in dairy cattle.

BACKGROUND: Intramammary (IMM) ceftiofur treatment is commonly used in dairy farms to prevent mastitis, though its impact on the cattle gut microbiome and selection of antibiotic-resistant bacteria has not been elucidated. Herein, we enrolled 40 dairy (Holstein) cows at the end of the lactation phase for dry-cow therapy: 20 were treated with IMM ceftiofur (Spectramast®DC) and a non-antibiotic internal teat sealant (bismuth subnitrate) and 20 (controls) received only bismuth subnitrate. Fecal grab samples were collected before and after treatment (weeks 1, 2, 3, 5, 7, and 9) for bacterial quantification and metagenomic next-generation sequencing. RESULTS: Overall, 90% and 24% of the 278 samples had Gram-negative bacteria with resistance to ampicillin and ceftiofur, respectively. Most of the cows treated with ceftiofur did not have an increase in the number of resistant bacteria; however, a subset (25%) shed higher levels of ceftiofur-resistant bacteria for up to 2 weeks post-treatment. At week 5, the antibiotic-treated cows had lower microbiota abundance and richness, whereas a greater abundance of genes encoding extended-spectrum ß-lactamases (ESBLs), CfxA, ACI-1, and CMY, was observed at weeks 1, 5 and 9. Moreover, the contig and network analyses detected associations between ß-lactam resistance genes and phages, mobile genetic elements, and specific genera. Commensal bacterial populations belonging to Bacteroidetes most commonly possessed ESBL genes followed by members of Enterobacteriaceae. CONCLUSION: This study highlights variable, persistent effects of IMM ceftiofur treatment on the gut microbiome and resistome in dairy cattle. Antibiotic-treated cattle had an increased abundance of specific taxa and genes encoding ESBL production that persisted for 9 weeks. Fecal shedding of ESBL-producing Enterobacteriaceae, which was classified as a serious public health threat, varied across animals. Together, these findings highlight the need for additional studies aimed at identifying factors associated with shedding levels and the dissemination and persistence of antibiotic resistance determinants on dairy farms across geographic locations.

The effect of bovine leukemia virus on dairy cow longevity.

Bovine leukemia virus (BLV) is a retrovirus of cattle that infects approximately 45% of all US dairy cattle, with about 90% of US dairy herds having at least one infected animal. Studies have found BLV infection to be associated with multiple measures of decreased immune function, which may explain the observed economic losses from milk production, decreased cow longevity, and predisposition to lymphoma and other diseases. Our objective was to measure the association between BLV infection and cow longevity in dairy cow operations. Ninety-one dairy herds from 9 US states volunteered to participate in this study. Milking dairy cows (n = 3,611) were tested for BLV antibodies using an ELISA milk test, and their presence in the herd was monitored for an average of 29 mo. The survival analysis controlled for herd and lactation number. Cows sold for dairy purposes were excluded, and individual cow results were not shared with producers so as not to influence culling decisions. Overall, 47.1% (1,701/3,611) of cows were BLV-positive by ELISA. The significant hazard ratio of 1.30 indicated that positive cows were 30% more likely than their negative herdmates to die or be culled during the monitoring period. These results are consistent with other studies in finding a negative effect of BLV infection on cow lifespan.

Characterizing the Cattle Gut Microbiome in Farms with a High and Low Prevalence of Shiga Toxin Producing Escherichia coli.

Cattle are the main reservoirs of Shiga toxin producing Escherichia coli (STEC), a major foodborne pathogen associated with acute enteric disease and hemolytic-uremic syndrome in humans. A total of 397 beef and dairy cattle from 5 farms were included in this study, of which 660 samples were collected for 16S rRNA gene sequencing. The microbiota of farms with a high-STEC prevalence (HSP) had greater richness compared to those of farms with a low-STEC prevalence (LSP). Longitudinal analyses showed STEC-shedders from LSP farms had higher microbiome diversity; meanwhile, changes in the microbiome composition in HSP farms were independent of the STEC shedding status. Most of the bacterial genera associated with STEC shedding in dairy farms were also correlated with differences in the percentage of forage in diet and risk factors of STEC carriage such as days in milk, number of lactations, and warm temperatures. Identifying factors that alter the gut microbiota and enable STEC colonization in livestock could lead to novel strategies to prevent fecal shedding and the subsequent transmission to humans.

Diagnostic Measures of Disease Progression in Cattle Following Natural Infection with Bovine Leukemia Virus.

This study describes the longitudinal changes in bovine leukemia virus (BLV) ELISA antibodies, proviral load (PVL), and blood lymphocyte counts (LC) observed over a 2.5-year period in naturally infected cattle. The dataset utilized was from a BLV intervention field trial on three Midwestern dairy herds. Our analysis showed ELISA false negatives were more likely to occur in cattle with low PVL and normal LC. On average, negligible changes in LC were observed during six-month intervals. Periods of lymphocytosis, defined as >10,000 lymphocytes per uL of blood, were observed in 31.5% (68/216) of BLV test-positive cattle. In BLV test-positive cows, an average increase of 2900 to 3100 proviral copies per 100,000 cells was observed during each subsequent six-month sampling interval. The difference between the minimum and maximum PVL observed for an ELISA-positive cow with 3 or more observations ranged from 0 to 115,600 copies per 100,000 cells (median: 12,900; mean: 19,200). Therefore, following the identification of ELISA-positive cattle and the assessment of PVL and LC, subsequent semiannual tests to assess disease progression may not be needed. Further work is needed to determine how available diagnostic tests can be optimized to design cost-effective testing schemes for BLV control programs.

Long-read sequencing revealed cooccurrence, host range, and potential mobility of antibiotic resistome in cow feces.

While it is well recognized that the environmental resistome is global, diverse, and augmented by human activities, it has been difficult to assess risk because of the inability to culture many environmental organisms, and it is difficult to evaluate risk from current sequence-based environmental methods. The four most important criteria to determine risk are whether the antibiotic-resistance genes (ARGs) are a complete, potentially functional complement; if they are linked with other resistances; whether they are mobile; and the identity of their host. Long-read sequencing fills this important gap between culture and short sequence-based methods. To address these criteria, we collected feces from a ceftiofur-treated cow, enriched the samples in the presence of antibiotics to favor ARG functionality, and sequenced long reads using Nanopore and PacBio technologies. Multidrug-resistance genes comprised 58% of resistome abundance, but only 0.8% of them were plasmid associated; fluroquinolone-, aminoglycoside-, macrolide-lincosamide-streptogramin (MLS)-, and ß-lactam-resistance genes accounted for 2.7 to 12.3% of resistome abundance but with 19 to 78% located on plasmids. A variety of plasmid types were assembled, some of which share low similarity to plasmids in current databases. Enterobacteriaceae were dominant hosts of antibiotic-resistant plasmids; physical linkage of extended-spectrum ß-lactamase genes (CTX-M, TEM, CMY, and CARB) was largely found with aminoglycoside-, MLS-, tetracycline-, trimethoprim-, phenicol-, sulfonamide-, and mercury-resistance genes. A draft circular chromosome of Vagococcus lutrae was assembled; it carries MLS-, tetracycline- (including tetM and tetL on an integrative conjugative element), and trimethoprim-resistance genes flanked by many transposase genes and insertion sequences, implying that they remain transferrable.

Antimicrobial Resistance Hidden within Multiserovar Salmonella Populations.

Salmonella enterica can exist in food animals as multiserovar populations, and different serovars can harbor diverse antimicrobial resistance (AMR) profiles. Conventional Salmonella isolation assesses AMR only in the most abundant members of a multiserovar population, which typically reflects their relative abundance in the initial sample. Therefore, AMR in underlying serovars is an undetected reservoir that can readily be expanded upon antimicrobial use. CRISPR-SeroSeq profiling demonstrated that 60% of cattle fecal samples harbored multiple serovars, including low levels of Salmonella serovar Reading in 11% of samples, which were not found by culture-based Salmonella isolation. An in vitro challenge revealed that Salmonella serovar Reading was tetracycline resistant, while more abundant serovars were susceptible. This study highlights the importance of AMR surveillance in multiserovar populations.

Current Developments in the Epidemiology and Control of Enzootic Bovine Leukosis as Caused by Bovine Leukemia Virus.

Enzootic Bovine Leukosis (EBL) caused by the bovine leukemia virus (BLV) has been eradicated in over 20 countries. In contrast, the U.S. and many other nations are experiencing increasing prevalence in the absence of efforts to control transmission. Recent studies have shown that BLV infection in dairy cattle has a greater impact beyond the long-recognized lymphoma development that occurs in <5% of infected cattle. Like other retroviruses, BLV appears to cause multiple immune system disruptions, affecting both cellular and humoral immunity, which are likely responsible for increasingly documented associations with decreased dairy production and decreased productive lifespan. Realization of these economic losses has increased interest in controlling BLV using technology that was unavailable decades ago, when many nations eradicated BLV via traditional antibody testing and slaughter methods. This traditional control is not economically feasible for many nations where the average herd antibody prevalence is rapidly approaching 50%. The ELISA screening of cattle with follow-up testing via qPCR for proviral load helps prioritize the most infectious cattle for segregation or culling. The efficacy of this approach has been demonstrated in at least four herds. Breeding cattle for resistance to BLV disease progression also appears to hold promise, and several laboratories are working on BLV vaccines. There are many research priorities for a wide variety of disciplines, especially including the need to investigate the reports linking BLV and human breast cancer.

Bovine leukemia virus detection and dynamics following experimental inoculation.

Bovine leukemia virus (BLV) infects more than 40% of the United States cattle population and impacts animal health and production. Control programs aiming to reduce disease prevalence and incidence depend on the ability to detect the BLV provirus, anti-BLV antibodies, and differences in blood lymphocyte counts following infection. These disease parameters also can be indicative of long-term disease progression. The objectives of this study were to determine the timing and to describe early fluctuations of BLV-detection by qPCR, ELISA, and lymphocyte counts. Fifteen Holstein steers were experimentally inoculated with 100 µL of a blood saline inoculum. Three steers served as in-pen negative controls and were housed with the experimentally infected steers to observe the potential for contract transmission. Five additional negative controls were housed separately. Steers were followed for 147 days post-inoculation (DPI). Infections were detected in experimentally infected steers by qPCR and ELISA an average of 24- and 36 DPI, respectively. Significant differences in lymphocyte counts between experimentally infected and control steers were observed from 30 to 45 DPI. Furthermore, a wide variation in peak proviral load and establishment was observed between experimentally infected steers. The results of this study can be used to inform control programs focused on the detection and removal of infectious cattle.

Impact of bovine leukemia virus infection on beef cow longevity.

Bovine leukosis is a chronic lymphoproliferative disorder caused by bovine leukemia virus (BLV). Previous studies estimate that 38 % of cow-calf beef herds and 10.3 % of individual beef cows in the US are BLV seropositive. About 70 % of BLV infected animals are asymptomatic carriers of the virus, while less than 5% develop lymphosarcoma, the leading reason for carcass condemnation at the US slaughterhouses. Studies provide evidence that BLV infection leads to decreased immune function making animals more vulnerable to other diseases, which could shorten their productive lifespan and increase economic losses in the cattle industry. BLV seropositive dairy cows are reportedly more likely to be culled sooner compared with their uninfected herd mates. Beyond simple prevalence studies, little is known about the impact of BLV infection in beef cattle production or specifically on beef cow longevity. Our objective was to determine the association between BLV infection and cow longevity in beef cow-calf operations. Twenty-seven cow-calf herds from the Upper Midwest volunteered to participate in this study. Female beef cattle (n = 3146) were tested for serum BLV antibodies by ELISA. A subsample of 648 cows were also tested for BLV proviral load (PVL). Culling data was collected for the subsequent 24 months. Twenty-one herds (77.7 %) had at least one BLV-infected animal, and 29.2 % (930/3146) of tested animals were BLV seropositive. Of the BLV-positive cows, 33.7 % (318/943) were culled compared with 32.1 % (541/1682) of the seronegative cows. BLV status did not affect cows' longevity within herds (P = 0.062). However, cows with high BLV PVL had decreased survival within the herd compared with ELISA- negative cows (P = 0.01). Overall, infection with BLV did not impact beef cow longevity unless the disease had progressed to a point of high BLV PVL.

Effect of proximal abducting ulnar osteotomy (PAUL) on frontal plane thoracic limb alignment: An ex vivo canine study.

OBJECTIVE: To determine the effect of proximal abducting ulnar osteotomy (PAUL) on frontal plane thoracic limb alignment in standing and recumbent positions. STUDY DESIGN: Ex vivo cadaveric study. SAMPLE POPULATION: Canine thoracic limbs (n = 15 limb pairs). METHODS: Limbs were acquired from healthy Labrador retrievers that had been euthanized for reasons unrelated to this study. A limb press was used to obtain standing and recumbent caudocranial radiographs before and after PAUL. Foot lateralization and rotation were directly measured in standing position. Mechanical joint angles were determined using full limb radiographic montages and the center of rotation of angulation (CORA) method for pre-PAUL (Pre), 2-mm PAUL (PAUL2), and 3-mm PAUL (PAUL3). Data are reported as mean ± SD and 95% CI. Mixed linear modeling was used to identify differences in limb alignment values and foot position, with significance established at P ≤ .004. RESULTS: There were differences in five of 12 limb alignment values pre-PAUL and post-PAUL in standing and recumbent positions. In the standing position, there was an increase in mechanical medial proximal radioulnar angle (Pre, 80.6° ± 2.5°; PAUL2, 82.6° ± 2.4°; PAUL3, 84° ± 2.4°) and a decrease in elbow compression angle (Pre, 1.4° ± 1.3°; PAUL2, 1° ± 0.9°; PAUL3, 0.8° ± 1°). There was a movement of mechanical humeral radioulnar angle (Pre, -8.9° ± 2.8°; PAUL2, -6.1° ± 2.7°; PAUL3, -5.2 ± 2.7°), mechanical thoracic humeral angle (Pre, 3.9° ± 1.7°; PAUL2, 2.4° ± 1.4°; PAUL3, 2.6° ± 1.5°), and elbow mechanical axis deviation (Pre, 1.9% ± 1.1%; PAUL2, 0.9% ± 1.1%; PAUL3, 0.4% ± 1.4%) toward a value of "0" representing coaxial alignment of the limb. The foot underwent lateralization (Pre, 1.4 ± 0.6 cm; PAUL2, 1.8 ± 0.7 cm; PAUL3, 2.3 ± 0.7 cm) and external rotation (Pre, 10.5° ± 4.7°; PAUL2, 13.7° ± 5.1°; PAUL3, 16° ± 6.6°). CONCLUSION: In the ex vivo setting, PAUL resulted in translation of the mechanical axis of the thoracic limb from a medial to lateral direction through alterations in limb alignment values associated with the elbow, humerus, and proximal radius/ulna. CLINICAL SIGNIFICANCE: Additional studies are required to determine whether PAUL alters thoracic limb alignment in client-owned dogs.

Quantitative dynamics of Salmonella and E. coli in feces of feedlot cattle treated with ceftiofur and chlortetracycline.

Antibiotic use in beef cattle is a risk factor for the expansion of antimicrobial-resistant Salmonella populations. However, actual changes in the quantity of Salmonella in cattle feces following antibiotic use have not been investigated. Previously, we observed an overall reduction in Salmonella prevalence in cattle feces associated with both ceftiofur crystalline-free acid (CCFA) and chlortetracycline (CTC) use; however, during the same time frame the prevalence of multidrug-resistant Salmonella increased. The purpose of this analysis was to quantify the dynamics of Salmonella using colony counting (via a spiral-plating method) and hydrolysis probe-based qPCR (TaqMan® qPCR). Additionally, we quantified antibiotic-resistant Salmonella by plating to agar containing antibiotics at Clinical & Laboratory Standards Institute breakpoint concentrations. Cattle were randomly assigned to 4 treatment groups across 16 pens in 2 replicates consisting of 88 cattle each. Fecal samples from Days 0, 4, 8, 14, 20, and 26 were subjected to quantification assays. Duplicate qPCR assays targeting the Salmonella invA gene were performed on total community DNA for 1,040 samples. Diluted fecal samples were spiral plated on plain Brilliant Green Agar (BGA) and BGA with ceftriaxone (4 µg/ml) or tetracycline (16 µg/ml). For comparison purposes, indicator non-type-specific (NTS) E. coli were also quantified by direct spiral plating. Quantity of NTS E. coli and Salmonella significantly decreased immediately following CCFA treatment. CTC treatment further decreased the quantity of Salmonella but not NTS E. coli. Effects of antibiotics on the imputed log10 quantity of Salmonella were analyzed via a multi-level mixed linear regression model. The invA gene copies decreased with CCFA treatment by approximately 2 log10 gene copies/g feces and remained low following additional CTC treatment. The quantities of tetracycline or ceftriaxone-resistant Salmonella were approximately 4 log10 CFU/g feces; however, most of the samples were under the quantification limit. The results of this study demonstrate that antibiotic use decreases the overall quantity of Salmonella in cattle feces in the short term; however, the overall quantities of antimicrobial-resistant NTS E. coli and Salmonella tend to remain at a constant level throughout.

Breeding bulls as a potential source of bovine leukemia virus transmission in beef herds.

OBJECTIVE: To determine the prevalence of bovine leukemia virus (BLV) in beef bulls; evaluate the presence of BLV provirus DNA in blood, smegma, and semen samples; and analyze whether blood BLV proviral load was associated with differential blood cell counts. DESIGN: Observational cross-sectional study. ANIMALS: 121 beef bulls ≥ 2 years old from 39 Michigan herds. PROCEDURES: Blood, smegma, and semen samples were collected from each bull during a routine breeding soundness examination. An ELISA was used to detect serum anti-BLV antibodies. A coordination of common motifs-quantitative PCR assay was used to detect BLV provirus DNA in blood, smegma, and semen samples. Bulls with positive results on both the BLV serum ELISA and coordination of common motifs-quantitative PCR assay were considered infected with BLV. RESULTS: 19 of 39 (48.7%) herds and 54 of 121 (44.6%) bulls were infected with BLV. Provirus DNA was detected in the blood of all 54 and in smegma of 4 BLV-infected bulls but was not detected in any semen sample. Lymphocyte count was significantly greater in BLV-infected bulls than in uninfected bulls. The proportion of BLV-infected bulls with lymphocytosis (16/54 [29.6%]) was greater than the proportion of uninfected bulls with lymphocytosis (6/67 [9%]). Lymphocyte count was positively associated with BLV proviral load in BLV-infected bulls. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that almost half of beef bulls and herds were infected with BLV, and BLV provirus DNA was detected in the smegma of some BLV-infected bulls. Bulls may have an important role in BLV transmission in beef herds.

Lack of Bovine leukemia virus transmission during natural breeding of cattle.

Bovine leukosis is a chronic lymphoproliferative disorder that leads to significant economic losses in the beef and dairy industries. The major route of virus transmission is believed to be iatrogenic through the transfer of blood containing infected lymphocytes. In addition, BLV proviral DNA has been identified in nasal secretions, saliva, milk, colostrum, semen and smegma; however, natural transmission of BLV through these secretions has not been clearly demonstrated. The use of bulls for natural breeding has been identified as a risk factor in BLV infected dairy herds. However, the risk of BLV-infected bulls transmitting the virus is unknown. The objective of this study was to evaluate the potential for BLV transmission during natural breeding between a BLV-infected bull and uninfected heifers. Forty healthy, BLV seronegative, and proviral-negative beef heifers were randomly assigned to one of two groups: control heifers (n = 20) exposed to a BLV seronegative and proviral negative bull and challenged heifers (n = 20) exposed to a BLV seropositive and proviral-positive bull. Each group was housed with the bull for a period of 38 days in a 5-acre pasture to replicate the housing of commercial beef cattle during the breeding season. Blood samples were collected from heifers at -60, -30 and 0 days prior to breeding and day 30, 60 and 90 after the breeding period ended. Blood samples were tested for BLV antibodies by ELISA and BLV proviral DNA by CoCoMo-qPCR. New infection was not detected by ELISA or CoCoMo-qPCR in any of the challenge or control heifers at any time point during the study. Based on these results, BLV infected bulls that are healthy and aleukemic may not be a significant risk of BLV transmission during a defined breeding season.

Epidemiological Study of Mycobacterium bovis Infection in Buffalo and Cattle in Amazonas, Brazil.

Bovine Tuberculosis (BTB) is an endemic disease in about one hundred countries, affecting the economy causing a decrease in productivity, condemnation of meat, and damaging the credibility on international trade. Additionally, Mycobacterium bovis the major causative agent for BTB can also infect humans causing a variety of clinical presentations. The aim of this study was to determine BTB prevalence and the main risk factors for the Mycobacterium bovis prevalence in cattle and buffalos in Amazonas State, Brazil. Tissue samples from 151 animals (45 buffalo and 106 cattle from five herds with buffalo only, 22 herds with cattle only, and 12 herds with buffalo and cattle) were obtained from slaughterhouses under State Veterinary Inspection. M. bovis were isolated on Stonebrink medium. The positive cultures were confirmed by polymerase chain reaction (PCR) testing. The apparent herd and animal prevalence rates were 56.4 and 5.40%, respectively. Regarding animal species, the apparent prevalence rates were 3% in cattle and 11.8% in buffalo. Generalized Linear Mixed Models (GLMM) with random effect were used to assess the association with risk factors on the prevalence. Species (buffalo), herds size (>100 animals) and the presence of both species (buffalo and cattle) in the herd were the major risk factors for the infection by Mycobacterium bovis in the region. The findings reveal an urgent need for evidence-based effective intervention to reduce BTB prevalence in cattle and buffalo and prevent its spread to the human population. Studies are needed to understand why buffalo are more likely to be infected by M. bovis than cattle in Amazon. Recommendations for zoning, use of data from the inspection services to generate information regarding BTB focus, adoption of epidemiological tools, and discouragement of practices that promote the mixing of cattle and buffalo, were made.

Prospects for predictive modeling of transition cow diseases.

Transition cow diseases can negatively impact animal welfare and reduce dairy herd profitability. Transition cow disease incidence has remained relatively stable over time despite monitoring and management efforts aimed to reduce the risk of developing diseases. Dairy cattle disease risk is monitored by assessing multiple factors, including certain biomarker test results, health records, feed intake, body condition score, and milk production. However, these factors, which are used to make herd management decisions, are often reviewed separately without considering the correlation between them. In addition, the biomarkers that are currently used for monitoring may not be representative of the complex physiological changes that occur during the transition period. Predictive modeling, which uses data to predict future or current outcomes, is a method that can be used to combine the most predictive variables and their interactions efficiently. The use of an effective predictive model with relevant predictors for transition cow diseases will result in better targeted interventions, and therefore lower disease incidence. This review will discuss predictive modeling methods and candidate variables in the context of transition cow diseases. The next step is to investigate novel biomarkers and statistical methods that are best suited for the prediction of transition cow diseases.

Prevalence of Bovine Leukemia Virus Antibodies in US Dairy Cattle.

OBJECTIVE: To estimate current US herd-level and animal-level prevalence of bovine leukemia virus (BLV) in dairy cows and characterize epidemiologic features. DESIGN: Cross-sectional observational study design and survey. ANIMALS: 4120 dairy cows from 103 commercial dairy herds in 11 states across the US. PROCEDURES: Milk samples were collected from dairy cows through routine commercial sampling and tested for anti-BLV antibodies by antibody capture ELISA. Based on the ELISA results of a sample of an average of 40 cows per herd, within-herd apparent prevalence (AP) was estimated by a directly standardized method and by a lactation-weighted method for each herd. Within-herd AP estimates were summarized to give estimates of US herd-level and animal-level AP. Differences in AP by lactation, region, state, breed, and herd size were examined to characterize basic epidemiologic features of BLV infection. RESULTS: 94.2% of herds had at least one BLV antibody positive cow detected. The average within-herd standardized AP was 46.5%. Lactation-specific AP increased with increasing lactation number, from 29.7% in first lactation cows to 58.9% in 4th and greater lactation cows. Significant differences were not observed based on region, state, breed, or herd size. CONCLUSIONS AND CLINICAL RELEVANCE: These results are consistent with a historical trend of increasing prevalence of BLV among US dairy cattle. Given the findings of other studies on the negative impacts of BLV infection on milk production and cow longevity, these findings are clinically relevant for veterinarians counseling dairy clients on the risks of BLV to their herds.

Effects of Ceftiofur and Chlortetracycline on the Resistomes of Feedlot Cattle.

Treatment of food-producing animals with antimicrobial drugs (AMD) is controversial because of concerns regarding promotion of antimicrobial resistance (AMR). To investigate this concern, resistance genes in metagenomic bovine fecal samples during a clinical trial were analyzed to assess the impacts of treatment on beef feedlot cattle resistomes. Four groups of cattle were exposed, using a 2-by-2 factorial design, to different regimens of antimicrobial treatment. Injections of ceftiofur crystalline-free acid (a third-generation cephalosporin) were used to treat all cattle in treatment pens or only a single animal, and either chlortetracycline was included in the feed of all cattle in a pen or the feed was untreated. On days 0 and 26, respectively, pre- and posttrial fecal samples were collected, and resistance genes were characterized using shotgun metagenomics. Treatment with ceftiofur was not associated with changes to ß-lactam resistance genes. However, cattle fed chlortetracycline had a significant increase in relative abundance of tetracycline resistance genes. There was also an increase of an AMR class not administered during the study, which is a possible indicator of coselection of resistance genes. Samples analyzed in this study had previously been evaluated by culture characterization (Escherichia coli and Salmonella) and quantitative PCR (qPCR) of metagenomic fecal DNA, which allowed comparison of results with this study. In the majority of samples, genes that were selectively enriched through culture and qPCR were not identified through shotgun metagenomic sequencing in this study, suggesting that changes previously documented did not reflect changes affecting the majority of bacterial genetic elements found in the predominant fecal resistome.IMPORTANCE Despite significant concerns about public health implications of AMR in relation to use of AMD in food animals, there are many unknowns about the long- and short-term impact of common uses of AMD for treatment, control, and prevention of disease. Additionally, questions commonly arise regarding how to best measure and quantify AMR genes in relation to public health risks and how to determine which genes are most important. These data provide an introductory view of the utility of using shotgun metagenomic sequencing data as an outcome for clinical trials evaluating the impact of using AMD in food animals.

Population dynamics of enteric Salmonella in response to antimicrobial use in beef feedlot cattle.

A randomized controlled longitudinal field trial was undertaken to assess the effects of injectable ceftiofur crystalline-free acid (CCFA) versus in-feed chlortetracycline on the temporal dynamics of Salmonella enterica spp. enterica in feedlot cattle. Two replicates of 8 pens (total 176 steers) received one of 4 different regimens. All, or one, out of 11 steers were treated with CCFA on day 0 in 8 pens, with half of the pens later receiving three 5-day regimens of chlortetracycline from day 4 to day 20. Salmonella was isolated from faecal samples and antimicrobial susceptibility was analysed via microbroth dilution. Serotype was determined by whole-genome sequencing. On day 0, mean Salmonella prevalence was 75.0% and the vast majority of isolates were pansusceptible. Both antimicrobials reduced overall prevalence of Salmonella; however, these treatments increased the proportion of multi-drug resistant (MDR) Salmonella from day 4 through day 26, which was the last day of faecal collection. Only six Salmonella serotypes were detected. Salmonella serotype Reading isolates were extensively MDR, suggesting a strong association between serotype and resistance. Our study demonstrates that the selection pressures of a 3rd generation cephalosporin and chlortetracycline during the feeding period contribute to dynamic population shifts between antimicrobial susceptible and resistant Salmonella.

The Effect of Sliding Humeral Osteotomy (SHO) on Frontal Plane Thoracic Limb Alignment: An Ex Vivo Canine Cadaveric Study.

OBJECTIVE: To determine the effect of sliding humeral osteotomy (SHO) on frontal plane thoracic limb alignment in standing and recumbent limb positions. STUDY DESIGN: Canine cadaveric study. SAMPLE POPULATION: Canine thoracic limbs (n=15 limb pairs). METHODS: Limbs acquired from healthy Labrador Retrievers euthanatized for reasons unrelated to this study were mounted in a limb press and aligned in a standing position followed by axial loading at 30% body weight. Frontal plane radiography was performed in standing and recumbent positions pre- and post-SHO. In the standing position, lateralization of the foot was measured pre- and post-SHO using a textured grid secured to the limb press base plate. Twelve thoracic limb alignment values (mean ± SD and 95% CI) were determined using the center of rotation of angulation (CORA) method were compared using linear mixed models to determine if significant differences existed between limb alignment values pre- or post-SHO, controlling for dog, limb, and limb position. RESULTS: Six of 12 standing or recumbent alignment values were significantly different pre- and post-SHO. SHO resulted in decreased mechanical lateral distal humeral angle and movement of the mechanical humeral radio-ulnar angle, radio-ulnar metacarpal angle, thoracic humeral angle, and elbow mechanical axis deviation toward coaxial limb alignment. In the standing position, the foot underwent significant lateralization post-SHO. CONCLUSION: SHO resulted in significant alteration in frontal plane thoracic limb alignment. Additional studies are necessary to determine if the changes reported using our ex vivo model occur following SHO in vivo.

Factors Associated with Shiga Toxin-Producing Escherichia coli Shedding by Dairy and Beef Cattle.

UNLABELLED: Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that can cause hemorrhagic colitis and hemolytic-uremic syndrome. Cattle are the primary reservoir for STEC, and food or water contaminated with cattle feces is the most common source of infections in humans. Consequently, we conducted a cross-sectional study of 1,096 cattle in six dairy herds (n = 718 animals) and five beef herds (n = 378 animals) in the summers of 2011 and 2012 to identify epidemiological factors associated with shedding. Fecal samples were obtained from each animal and cultured for STEC. Multivariate analyses were performed to identify risk factors associated with STEC positivity. The prevalence of STEC was higher in beef cattle (21%) than dairy cattle (13%) (odds ratio [OR], 1.76; 95% confidence interval [CI], 1.25, 2.47), with considerable variation occurring across herds (range, 6% to 54%). Dairy cattle were significantly more likely to shed STEC when the average temperature was >28.9°C 1 to 5 days prior to sampling (OR, 2.5; 95% CI, 1.25, 4.91), during their first lactation (OR, 1.8; 95% CI, 1.1, 2.8), and when they were <30 days in milk (OR, 3.9; 95% CI, 2.1, 7.2). These data suggest that the stress or the negative energy balance associated with lactation may result in increased STEC shedding frequencies in Michigan during the warm summer months. Future prevention strategies aimed at reducing stress during lactation or isolating high-risk animals could be implemented to reduce herd-level shedding levels and avoid transmission of STEC to susceptible animals and people. IMPORTANCE: STEC shedding frequencies vary considerably across cattle herds in Michigan, and the shedding frequency of strains belonging to non-O157 serotypes far exceeds the shedding frequency of O157 strains, which is congruent with human infections in the state. Dairy cattle sampled at higher temperatures, in their first lactation, and early in the milk production stage were significantly more likely to shed STEC, which could be due to stress or a negative energy balance. Future studies should focus on the isolation of high-risk animals to decrease herd shedding levels and the potential for contamination of the food supply.