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Salmonella contamination of beef products, specifically from the incorporation of bovine lymph nodes into ground beef, remains a food safety concern. Current postharvest Salmonella mitigation techniques do not address Salmonella bacteria that are harbored in the lymph nodes, nor is it well understood how Salmonella invades the lymph nodes.

Age and diet are among the factors that influence the community composition of the fecal microbiome. Additionally, antimicrobial use can alter the composition of bacterial communities. An 86-d study with finisher pigs aimed to evaluate age-related dynamics (day 98 to 177 of age), effects of types and levels of dietary fiber, and injectable antimicrobials on the fecal microbiome and antimicrobial resistance (AMR) was conducted. A total of 287 pigs, housed in 36 pens, with 7 to 8 pigs per pen, fed a corn grain and soybean meal-based basal diet, formulated to contain 8.7% neutral detergent fiber (NDF), were randomly assigned to one of three treatments: 1) basal diet with no supplement, 2) basal diet supplemented with 20% distillers dried grains with solubles (DDGS) formulated to contain 13.6% NDF, or 3) basal diet supplemented with 14.5% sugar beet pulp (SBP) formulated to contain 13.6% NDF. Five finisher pigs from each treatment group were selected randomly, and fecal samples were collected on days 98, 110, 144, and 177 of age. In addition, fecal samples were collected from pigs that were injected intramuscularly ceftiofur hydrochloride or penicillin G on days 1 and 3 along with pen-mate-untreated controls on day 1. Fecal samples were subjected to 16S rRNA amplicon-based microbiome analysis and culture methods to quantify the abundance of total AMR coliforms and enterococci populations. The alpha-diversity, such as species richness, increased with age, and the overall bacterial composition changed with age (P =0.001) and diet (P = 0.001). Diet-associated shifts in the specific bacterial taxa were observed. The richness, diversity, and evenness of bacterial taxa did not differ between pigs that were injected with ceftiofur vs. their untreated pen mates or by dietary treatments but differed in pigs that received penicillin G injection. Both antimicrobial treatments contributed to changes in the overall fecal bacterial composition at the genus level. Collectively, the data demonstrate that both age and the diet (control vs. DDGS-, control vs. SBP-, or DDGS- vs. SBP-based diets) were associated with the overall bacterial community composition, and the impact of age on variations in fecal microbiome composition was greater than the diet. Antibiotic treatment had minimal effect on bacterial diversity and relative abundance of taxa. Furthermore, diets and antimicrobial treatment had minimal impact on the overall counts of AMR coliforms and enterococci populations in feces.Bacterial communities in the gut and the feces are strongly influenced by a number of factors, particularly the age of the animal and the diet. In addition, antibiotic administration routinely used to treat bacterial diseases can also affect the community composition. A study with finisher pigs was conducted to evaluate age-related changes, effects of types—distiller’s dried grains with solubles (DGGS) or sugar beet pulp (SBP)—and levels of dietary fiber, and injectable antibiotics on the fecal bacterial composition and antibiotic resistance in fecal bacteria. Fecal samples were collected from five pigs in each of the three dietary treatment groups, control diet with no supplement or supplemented with DDGS or SBP, on days 98, 110, 144, and 177 of age and on days 1 and 3 after the first injection of antibiotics, ceftiofur or penicillin G. Samples were analyzed to identify the bacterial community composition and prevalence of antibiotic resistance in fecal bacteria. Data generated suggested that the overall bacterial composition changed with age and diet, and age appeared to have a greater impact than diet. Antibiotics had only a modest impact on the bacterial community and had minimum impact on antibiotic resistance of fecal bacteria.

The use of antibiotics to treat dairy calves may result in multidrug-resistant extended-spectrum beta-lactamase (ESBL)-producing

Salmonella enterica is a major foodborne pathogen, and contaminated beef products have been identified as the primary source of Salmonella-related outbreaks. Pathogenicity and antibiotic resistance of Salmonella are highly serotype- and subpopulation-specific, which makes it essential to understand high-resolution Salmonella population dynamics in cattle. Time of year, source of cattle, pen, and sample type(i.e., feces, hide or lymph nodes) have previously been identified as important factors influencing the serotype distribution of Salmonella (e.g., Anatum, Lubbock, Cerro, Montevideo, Kentucky, Newport, and Norwich) that were isolated from a longitudinal sampling design in a research feedlot. In this study, we performed high-resolution genomic comparisons of Salmonella isolates within each serotype using both single-nucleotide polymorphism (SNP)-based maximum likelihood phylogeny and hierarchical clustering of core-genome multi-locus sequence typing. The importance of the aforementioned features on clonal Salmonella expansion was further explored using a supervised machine learning algorithm. In addition, we identified and compared the resistance genes, plasmids, and pathogenicity island profiles of the isolates within each sub-population. Our findings indicate that clonal expansion of Salmonella strains in cattle was mainly influenced by the randomization of block and pen, as well as the origin/source of the cattle; that is, regardless of sampling time and sample type (i.e., feces, lymph node or hide). Further research is needed concerning the role of the feedlot pen environment prior to cattle placement to better understand carry-over contributions of existing strains of Salmonella and their bacteriophages.ImportanceSalmonella serotypes isolated from outbreaks in humans can also be found in beef cattle and feedlots. Virulence factors and antibiotic resistance are among the primary defense mechanisms of Salmonella, and are often associated with clonal expansion. This makes understanding the subpopulation dynamics of Salmonella in cattle critical for effective mitigation. There remains a gap in the literature concerning subpopulation dynamics within Salmonella serotypes in feedlot cattle from the beginning of feeding up until slaughter. Here, we explore Salmonella population dynamics within each serotype using core genome phylogeny and hierarchical classifications. We used machine-learning to quantitatively parse the relative importance of both hierarchical and longitudinal clustering among cattle host samples. Our results reveal that Salmonella populations in cattle are highly clonal over a 6-month study period, and that clonal dissemination of Salmonella in cattle is mainly influenced spatially by experimental block and pen, as well by the geographical origin of the cattle.

The transfer of antimicrobial resistance genes commonly occurs via vertical and horizontal gene transfer, as such genes are often found on the same mobile genetic element. This occurrence can lead to the co-selection of resistance to antimicrobials without their application. Dairy cattle located in the south-western United States were enrolled in a matched-pair longitudinal study to evaluate the effects of a two-dose ceftiofur treatment for metritis on levels of third-generation cephalosporin resistance among faecal Escherichia coli temporally. Escherichia coli chosen for further investigation were isolated on selective media, harboured extended-spectrum beta-lactam, fluoroquinolone and macrolide resistance genes. This combination has previously been unreported; importantly, it included genes encoding for resistance to antibiotics that can only be used in dairy cattle less than 20 months of age. Fluoroquinolones, macrolides and third and higher generation cephalosporins are considered critically important and highest priority for human medicine by the World Health Organization.

Though carbapenems are not licensed for use in food animals in the U.S., carbapenem resistance among Enterobacteriaceae has been identified in farm animals and their environments. The objective of our study was to determine the extent to which older-generation β-lactam antibiotics approved for use in food animals in the U.S. might differentially select for resistance to antibiotics of critical importance to human health, such as carbapenems.Escherichia coli (E.coli)strains from humans, food animals, or the environment bearing a single β-lactamase gene (n = 20 each) forblaTEM-1,blaCMY-2, andblaCTX-M-*or elseblaKPC/IMP/NDM(due to limited availability, often in combination with otherblagenes), were identified, along with 20E.colistrains lacking any known beta-lactamase genes. Baseline estimates of intrinsic bacterial fitness were derived from the population growth curves. Effects of ampicillin (32 μg/mL), ceftriaxone (4 μg/mL) and meropenem (4 μg/mL) on each strain and resistance-group also were assessed. Further,in vitrobatch cultures were prepared by mixing equal concentrations of 10 representativeE.colistrains (two from each resistance gene group), and each mixture was incubated at 37°C for 24 hours in non-antibiotic cation-adjusted Mueller-Hinton II (CAMH-2) broth, ampicillin + CAMH-2 broth (at 2, 4, 8, 16, and 32 μg/mL) and ceftiofur + CAMH-2 broth (at 0.5, 1, 2, 4, and 8μg/mL). Relative and absolute abundance of resistance-groups were estimated phenotypically. Line plots of the raw data were generated, and non-linear Gompertz models and multilevel mixed-effect linear regression models were fitted to the data. The observed strain growth rate distributions were significantly different across the groups. AmpC strains (i.e.,blaCMY-2) had distinctly less robust (p < 0.05) growth in ceftriaxone (4 μg/mL) compared to extended-spectrum beta-lactamase (ESBL) producers harboringblaCTX-M-*variants. With increasing beta-lactam antibiotic concentrations, relative proportions of ESBLs and CREs were over-represented in the mixed bacterial communities; importantly, this was more pronounced with ceftiofur than with ampicillin. These results indicate that aminopenicillins and extended-spectrum cephalosporins would be expected to propagate carbapenemase-producing Enterobacteriaceae in food animals if and when Enterobacteriaceae from human health care settings enter the food animal environment.

Urinary tract infections (UTIs) affect nearly half of women and an estimated 14 % of the canine companion animal population at least once in their lifetime. As with humans, Escherichia coli is the most commonly isolated bacteria from canine UTIs and infections are dominated by specific phylogenetic groups with notable virulence attributes. In this study, we evaluated uropathogenic E. coli (UPEC) (n = 69) isolated from canine UTIs phenotypically and genotypically for virulence factors, biofilm formation and antimicrobial resistance profiles. Biofilm formation in UPEC strains was positively associated with common virulence factors including papG (p = 0.006), fimH (p < 0.0001), sfaS (p = 0.004), focA (p = 0.004), cnf-1 (p = 0.009) and hlyA (p = 0.006). There was a negative association between biofilm formation and phenotypic antimicrobial resistance for ampicillin (p < 0.0004), ciprofloxacin (p < 0.0001), and trimethoprim-sulfamethoxazole (p < 0.02), as well as multidrug resistance (isolates resistant to ≥ 3 classes of antimicrobials) (p < 0.0002), and the presence of extended spectrum beta-lactamase (ESBL)-producing genes (p < 0.05). In conclusion, UPECs isolated from clinical cases of canine UTIs show a broad negative association between antimicrobial resistance and biofilm formation, and this observation is supported both by phenotypic and genotypic endpoints. As the biofilm formation may result in antimicrobial tolerance, this could be a secondary evasive tactic of UPEC lacking traditional antimicrobial resistance traits. This observation is important for veterinary practitioners to consider when treating puzzling chronic intractable and/or recurrent cases of UTI that appear to be susceptible to antimicrobial therapy via traditional antimicrobial susceptibility testing (AST) methods.

Antibiotic use in cattle can select for multidrug-resistant Salmonella enterica, which is considered a serious threat by the U.S. Centers for Disease Control and Prevention. A randomized controlled longitudinal field trial was designed to determine the long-term effects of a single dose of ceftiofur or tulathromycin on Salmonella population characteristics in cattle feces and peripheral lymph nodes and on hides. A total of 134 beef cattle from two sources were divided among 12 pens, with cattle in each of the 3-pen blocks receiving a single dose of either ceftiofur or tulathromycin or neither (control) on day 0. Fecal samples were collected before treatment (day 0) and repeatedly following treatment until slaughter (day 99+). Hide and lymph node samples were collected at slaughter age. Salmonella prevalence, phenotypic antimicrobial resistance, serotype, and phylogenetic relationships were examined. Multilevel mixed logistic regression models indicated no significant effects (P ≥ 0.218) of metaphylactic antibiotics on the prevalence of Salmonella across sample types. However, there was a significant time effect observed, with prevalence increasing from spring through the midsummer months (P IMPORTANCESalmonella is a leading bacterial foodborne pathogen, causing a significant number of human infections and deaths every year in the United States. Macrolides and 3rd-generation cephalosporins play critical roles in the treatment of human salmonellosis. Use of these antibiotics in beef cattle can select for resistant bacteria that may enter the food chain or spread from the farm via manure. There is a lack of longitudinal research concerning the long-term effects of metaphylactic antibiotic administration. Here, we assessed Salmonella population dynamics during the feeding period until slaughter following single-dose antibiotic treatment. We found no long-term effects of antibiotic use early in the cattle-feeding period on Salmonella prevalence and antimicrobial resistance at slaughter. We identified the pens in which cattle were housed as the factor that contributed most to Salmonella serotypes being shared; importantly, the dominant strain in each pen changed repeatedly over the entire feeding period.

Antibiotic use in cattle can select for multidrug-resistant Salmonella enterica, which is considered a serious threat by the U.S. Centers for Disease Control and Prevention. A randomized controlled longitudinal field trial was designed to determine the long-term effects of a single dose of ceftiofur or tulathromycin on Salmonella population characteristics in cattle feces and peripheral lymph nodes and on hides. A total of 134 beef cattle from two sources were divided among 12 pens, with cattle in each of the 3-pen blocks receiving a single dose of either ceftiofur or tulathromycin or neither (control) on day 0. Fecal samples were collected before treatment (day 0) and repeatedly following treatment until slaughter (day 99+). Hide and lymph node samples were collected at slaughter age. Salmonella prevalence, phenotypic antimicrobial resistance, serotype, and phylogenetic relationships were examined. Multilevel mixed logistic regression models indicated no significant effects (P ≥ 0.218) of metaphylactic antibiotics on the prevalence of Salmonella across sample types. However, there was a significant time effect observed, with prevalence increasing from spring through the midsummer months (P < 0.0001) in feces. The majority of Salmonella isolates were pansusceptible to a panel of 14 antibiotics both before and after treatment. Highly prevalent Salmonella serotypes were Salmonella enterica serovar Montevideo, Salmonella enterica serovar Anatum, Salmonella enterica serovar Cerro, and Salmonella enterica serovar Lubbock across all sample types. Strong pen and cattle source serotype clustering effects were observed among Salmonella isolates originating from fecal, lymph node, and hide samples; however, the potential role of Salmonella isolates from the pen environment prior to animal placement was not assessed in this study. IMPORTANCE Salmonella is a leading bacterial foodborne pathogen, causing a significant number of human infections and deaths every year in the United States. Macrolides and 3rd-generation cephalosporins play critical roles in the treatment of human salmonellosis. Use of these antibiotics in beef cattle can select for resistant bacteria that may enter the food chain or spread from the farm via manure. There is a lack of longitudinal research concerning the long-term effects of metaphylactic antibiotic administration. Here, we assessed Salmonella population dynamics during the feeding period until slaughter following single-dose antibiotic treatment. We found no long-term effects of antibiotic use early in the cattle-feeding period on Salmonella prevalence and antimicrobial resistance at slaughter. We identified the pens in which cattle were housed as the factor that contributed most to Salmonella serotypes being shared; importantly, the dominant strain in each pen changed repeatedly over the entire feeding period.

Salmonella enterica (non-Typhi) is one of the top five pathogens causing enteric infections worldwide. Draft whole-genome sequences of multidrug-resistant (MDR) Salmonella enterica serovar I 4,[5],12:i:− isolates from swine tissue samples collected at slaughter were evaluated for antimicrobial resistance genotypes., Salmonella enterica (non-Typhi) is one of the top five pathogens causing enteric infections worldwide. Draft whole-genome sequences of multidrug-resistant (MDR) Salmonella enterica serovar I 4,[5],12:i:− isolates from swine tissue samples collected at slaughter were evaluated for antimicrobial resistance genotypes. In recent years, Salmonella enterica serovar I 4,[5],12:i:− has increased in prevalence in humans and animals and has been linked to the consumption of contaminated pork.

The Romeral shear zone marks the geological boundary between the physiographic Central and Western cordilleras of the Colombian Andes. It demarcates and encompasses the main locus of deformation associated with the amalgamation and geological-structural evolution of the Northern Andean region during the Meso-Cenozoic, although as an ancient plate boundary, its origins may be traced into the Late Paleozoic. Today, Romeral corresponds to a km-scale shear zone containing poly-deformed, structurally juxtaposed fragments and slivers of a wide range of rock types, of varying ages and diverse origins, including metaigneous and metasedimentary blocks of the disjointed and reworked paleo-autochthon, fragments of pericratonic and possibly exotic oceanic margin, and late, autochthonous siliciclastic and volcanic sequences, each reflecting distinct development stages in the complex plate dynamics of the Northern Andean paleo-margin.

Although Salmonella enterica can produce life-threatening colitis in horses, certain serotypes are more commonly associated with clinical disease. Our aim was to evaluate the proportional morbidity attributed to different serotypes, as well as the phenotypic and genotypic antimicrobial resistance (AMR) of Salmonella isolates from patients at an equine referral hospital in the southern United States. A total of 255 Salmonella isolates was obtained from clinical samples of patients admitted to the hospital between 2007 and 2015. Phenotypic resistance to 14 antibiotics surveilled by the U.S. National Antimicrobial Resistance Monitoring System was determined using a commercially available panel. Whole-genome sequencing was used to identify serotypes and genotypic AMR. The most common serotypes were Salmonella enterica serotype Newport (18%), Salmonella enterica serotype Anatum (15.2%), and Salmonella enterica serotype Braenderup (11.8%). Most ( n = 219) of the isolates were pansusceptible, while 25 were multidrug resistant (≥3 antimicrobial classes). Genes encoding beta-lactam resistance, such as bla CMY-2 , bla SHV-12 , bla CTX-M-27 , and bla TEM-1B , were detected. The qnr B2 and aac(6′)-Ib-cr genes were present in isolates with reduced susceptibility to ciprofloxacin. Genes encoding resistance to gentamicin ( aph(3′)-Ia , aac(6′)-IIc ), streptomycin ( str A and str B), sulfonamides ( sul1 ), trimethoprim ( dfrA ), phenicols ( catA ), tetracyclines [ tet (A) and tet (E)], and macrolides [ ere (A)] were also identified. The main predicted incompatibility plasmid type was I1 (10%). Core genome-based analyses revealed phylogenetic associations between isolates of common serotypes. The presence of AMR Salmonella in equine patients increases the risk of unsuccessful treatment and causes concern for potential zoonotic transmission to attending veterinary personnel, animal caretakers, and horse owners. Understanding the epidemiology of Salmonella in horses admitted to referral hospitals is important for the prevention, control, and treatment of salmonellosis. IMPORTANCE In horses, salmonellosis is a leading cause of life-threatening colitis. At veterinary teaching hospitals, nosocomial outbreaks can increase the risk of zoonotic transmission, lead to restrictions on admissions, impact hospital reputation, and interrupt educational activities. The antimicrobials most often used in horses are included in the 5th revision of the World Health Organization's list of critically important antimicrobials for human medicine. Recent studies have demonstrated a trend of increasing bacterial resistance to drugs commonly used to treat Salmonella infections. In this study, we identify temporal trends in the distribution of Salmonella serotypes and their mechanisms of antimicrobial resistance; furthermore, we are able to determine the likely origin of several temporal clusters of infection by using whole-genome sequencing. These data can be used to focus strategies to better contain the dissemination and enhance the mitigation of Salmonella infections and to provide evidence-based policies and guidelines to steward antimicrobial use in veterinary medicine.

Heavy metals, such as copper, are increasingly supplemented in swine diets as an alternative to antibiotics to promote growth. Enterococci, a common gut commensal, acquire plasmid-borne, transferable copper resistance (tcrB) gene-mediated resistance to copper. The plasmid also carried resistance genes to tetracyclines and macrolides. The potential genetic link between copper and antibiotic resistance suggests that copper supplementation may exert a selection pressure for antimicrobial resistance. Therefore, a longitudinal study was conducted to investigate the effects of in-feed copper, chlortetracycline, and tylosin alone or in combination on the selection and co-selection of antimicrobial-resistant enterococci. The study included 240 weaned piglets assigned randomly to 6 dietary treatment groups: control, copper, chlortetracycline, tylosin, copper and chlortetracycline, and copper and tylosin. Feces were collected before (day 0), during (days 7, 14, 21), and after (days 28 and 35) initiating treatment, and enterococcal isolates were obtained from each fecal sample and tested for genotypic and phenotypic resistance to copper and antibiotics. A total of 2592 enterococcal isolates were tested for tcrB by polymerase chain reaction. The overall prevalence of tcrB-positive enterococci was 14.3% (372/2592). Among the tcrB-positive isolates, 331 were Enterococcus faecium and 41 were E. faecalis. All tcrB-positive isolates contained both erm(B) and tet(M) genes. The median minimum inhibitory concentration of copper for tcrB-negative and tcrB-positive enterococci was 6 and 18 mM, respectively. The majority of isolates (95/100) were resistant to multiple antibiotics. In conclusion, supplementing copper or antibiotics alone did not increase copper-resistant enterococci; however, supplementing antibiotics with copper increased the prevalence of the tcrB gene among fecal enterococci of piglets.

Zinc (Zn) is often supplemented at elevated concentrations in swine diets, particularly in piglets, to prevent enteric infections and promote growth. Previous studies from Denmark have suggested a genetic linkage and a phenotypic association between Zn resistance, encoded by czrC, and methicillin-resistance conferred by mecA in Staphylococcus aureus. Such an association has not been reported in the U.S. swine population. We conducted an analysis of the effects of Zn, supplemented as zinc oxide (ZnO), on the nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) in nursery (n=40) and finisher pigs (n=40) enrolled in a nutritional study. Nasal swabs, collected from nursery and finisher pigs, were inoculated onto MRSA CHROMagar and presumptive MRSA colonies were tested for the presence of mecA and czrC genes by polymerase chain reaction. Zinc susceptibility was determined by the agar dilution method. The prevalence of mecA-positive MRSA was 10% (4/40) and 20% (8/40) among nursery and finisher pigs, respectively. Of the 12 mecA-positive S. aureus isolates, 7 had the czrC gene (58.3%) compared to none among the 68 mecA-negative isolates. The presence of both mecA (p=0.002) and czrC (p=0.006) genes were positively associated with higher levels of Zn supplementation. The median minimum inhibitory concentrations of Zn for czrC-positive and czrC-negative isolates were 12 and 2 mM, respectively (p0.0001). The link between czrC and mecA genes suggests the importance of elevated Zn supplementation in the co-selection and propagation of methicillin resistance among S. aureus in pigs.

Feed-grade chlortetracycline (CTC) and copper are both widely utilized in U.S. pig production. Cluster randomized experiment was conducted to evaluate the effects of CTC and copper supplementation in weaned pigs on antimicrobial resistance (AMR) among fecal Escherichia coli. Four treatment groups: control, copper, CTC, or copper plus CTC were randomly allocated to 32 pens with five pigs per pen. Fecal samples were collected weekly from three pigs per pen for six weeks. Two E. coli isolates per fecal sample were tested for phenotypic and genotypic resistance against antibiotics and copper. Data were analyzed with multilevel mixed effects logistic regression, multivariate probit analysis and discrete time survival analysis. CTC-supplementation was significantly (99% [95% CI=98-100%]) associated with increased tetracycline resistance compared to the control group (95% [95% CI=94-97%]). Copper supplementation was associated with decreased resistance to most of the antibiotics tested, including cephalosporins, over the treatment period. Overall, 91% of the E. coli isolates were multidrug resistant (MDR) (resistant to ≥3 antimicrobial classes). tetA and blaCMY-2 genes were positively associated (P

Copper, an essential micronutrient, is supplemented in the diet at elevated levels to reduce morbidity and mortality and to promote growth in feedlot cattle. Gut bacteria exposed to copper can acquire resistance, which among enterococci is conferred by a transferable copper resistance gene ( tcrB ) borne on a plasmid. The present study was undertaken to investigate whether the feeding of copper at levels sufficient to promote growth increases the prevalence of the tcrB gene among the fecal enterococci of feedlot cattle. The study was performed with 261 crossbred yearling heifers housed in 24 pens, with pens assigned randomly to a 2�2 factorial arrangement of treatments consisting of dietary copper and a commercial linseed meal-based energy protein supplement. A total of 22 isolates, each identified as Enterococcus faecium , were positive for tcrB with an overall prevalence of 3.8% (22/576). The prevalence was higher among the cattle fed diets supplemented with copper (6.9%) compared to normal copper levels (0.7%). The tcrB -positive isolates always contained both erm (B) and tet (M) genes. Median copper MICs for tcrB -positive and tcrB -negative enterococci were 22 and 4 mM, respectively. The transferability of the tcrB gene was demonstrated via a filter-mating assay. Multilocus variable number tandem repeat analysis revealed a genetically diverse population of enterococci. The finding of a strong association between the copper resistance gene and other antibiotic (tetracycline and tylosin) resistance determinants is significant because enterococci remain potential pathogens and have the propensity to transfer resistance genes to other bacteria in the gut.

Copper, as copper sulfate, is increasingly used as an alternative to in-feed antibiotics for growth promotion in weaned piglets. Acquired copper resistance, conferred by a plasmid-borne, transferable copper resistance ( tcrB ) gene, has been reported in Enterococcus faecium and E. faecalis . A longitudinal field study was undertaken to determine the relationship between copper supplementation and the prevalence of tcrB- positive enterococci in piglets. The study was done with weaned piglets, housed in 10 pens with 6 piglets per pen, fed diets supplemented with a normal (16.5 ppm; control) or an elevated (125 ppm) level of copper. Fecal samples were randomly collected from three piglets per pen on days 0, 14, 28, and 42 and plated on M- Enterococcus agar, and three enterococcal isolates were obtained from each sample. The overall prevalence of tcrB- positive enterococci was 21.1% (38/180) in piglets fed elevated copper and 2.8% (5/180) in the control. Among the 43 tcrB- positive isolates, 35 were E. faecium and 8 were E. faecalis . The mean MICs of copper for tcrB- negative and tcrB- positive enterococci were 6.2 and 22.2 mM, respectively. The restriction digestion of the genomic DNA of E. faecium or E. faecalis with S1 nuclease yielded a band of ∼194-kbp size to which both tcrB and the erm (B) gene probes hybridized. A conjugation assay demonstrated cotransfer of tcrB and erm (B) genes between E. faecium and E. faecalis strains. The higher prevalence of tcrB- positive enterococci in piglets fed elevated copper compared to that in piglets fed normal copper suggests that supplementation of copper in swine diets selected for resistance.

Blood samples from dogs with clinical signs compatible with ehrlichiosis were examined for infection of Ehrlichia canis using PCR, multiplex real-time PCR, and DNA sequencing analysis. Eleven of 25 samples were positive for a new strain of E. canis . This is the first molecular identification of E. canis infection in dogs from Peru.

The shape-preferred orientation (SPO) is a technique, which allows the study of the fabric of deformed rocks and understanding its dominant deformational style and partitioning. This work presents SPO results and methodological aspects for one hand specimen of rock and a highresolution composed image of an outcrop through digital image processing. The technique involves imaging of three semi-orthogonal oriented sections for digital processing and subsequent implementation in structural software. The image processing consists of digital separation of a phase of interest, i.e. defining the deformational fabric. The processed images are then implemented in the SPO2003® software for acquisition of sectional ellipses and finally implemented in the Ellipsoid2003® software to obtain the characteristic ellipsoid of deformational fabric. For outcrops, due to the difficulty to finding three appropriate sections for photography, it was only obtained a sectional ellipse, characteristic of the rock fabric based on contrasting deformed quartz segregates in mylonitic schists. Los estudios de orientación Preferencial de Forma (OPF) permiten estudiar la fábrica de una roca deformada y comprender su estilo deformacional y partición. Presentamos estudios de OPF y aspectos metodológicos para secciones pulidas de roca en muestra de mano y afloramiento a través de tratamiento digital de imagen. La técnica consiste en la captura de imágenes de tres cortes de roca semi-ortogonales orientadas para tratamiento digital y posterior implementación en software estructural. El tratamiento de imagen consiste en la separación digital de una fase mineral que defina la fábrica deformacional. Las imágenes tratadas, son implementadas en el software SPO2003® para la obtención de elipses seccionales e implementadas en el software Ellipsoid2003® para la obtención del elipsoide característico de la fábrica deformacional. Para el caso de afloramiento, se presentan resultados para una sección vertical métrica. Se obtiene la elipse representativa de la fábrica bidimensional definida por los segregados milonitizados de cuarzo.

Experimental autoimmune myasthenia gravis can be induced in some strains of mice and rats by immunizing with acetylcholine receptor. Also, epidemiologic studies demonstrate an MHC linkage of myasthenia gravis in the man. In order to obtain direct experimental evidence for the influence of the genes of the MHC complex in the development of myasthenia gravis, we used mice transgenic to individual HLA molecules. We observed an increased susceptibility to the disease in HLA DQ8 transgenic mice compared to HLA DQ6 transgenic mice ( J. Immunol. 160:4169; 1998). These mice lacked endogenous mouse class II molecules. In the present study we mapped the cryptic and dominant sequences on the extra cellular region of human acetylcholine receptor. Although some epitopes ( e.g. , α11-30, α141-160, α171-190) were common between DQ8 and DQ6 transgenic mice, several others were disparately recognized. We also found a functional dichotomy in T cells from mice differing by one MHC molecule (HLA DQ8 or DQ6) when primed by sequences immunodominant in DQ8 and DQ6 tg mice. Differential disease manifestation in the two different HLA transgenic mice could be explained not only by differential recognition of peptides by these antigen presenting molecules, but also by the difference in the functional profile of T cells generated when primed by promiscuous sequence regions.

Aims The objective was to investigate whether in-feed supplementation of copper, at elevated level, co-selects for macrolide resistance in faecal enterococci. Methods and Results The study was conducted in cattle (n = 80) with a 2 × 2 factorial design of copper (10 or 100 mg kg−1 of feed) and tylosin (0 or 10 mg kg−1 of feed). Thirty-seven isolates (4·6%; 37/800) of faecal enterococci were positive for the tcrB and all were Enterococcus faecium. The prevalence was higher among cattle fed diets with copper and tylosin (8·5%) compared to control (2·0%), copper (4·5%) and tylosin (3·5%) alone. All tcrB-positive isolates were positive for erm(B) and tet(M) genes. Median copper minimum inhibitory concentrations (MICs) for tcrB-positive and tcrB-negative enterococci were 20 and 4 mmol l−1, respectively. Conclusions Feeding of elevated dietary copper and tylosin alone or in combination resulted in an increased prevalence of tcrB and erm(B)-mediated copper and tylosin-resistant faecal enterococci in feedlot cattle. Significance and Impact of the Study In-feed supplementation of elevated dietary copper has the potential to co-select for macrolide resistance. Further studies are warranted to investigate the factors involved in maintenance and dissemination of the resistance determinants and their co-selection mechanism in relation to feed-grade antimicrobials' usage in feedlot cattle.

Use of in-feed antibiotics such as chlortetracycline (CTC) in food animals is fiercely debated as a cause of antimicrobial resistance in human pathogens; as a result, alternatives to antibiotics such as heavy metals have been proposed. We used a total community DNA approach to experimentally investigate the effects of CTC and copper supplementation on the presence and quantity of antimicrobial resistance elements in the gut microbial ecology of pigs. Total community DNA was extracted from 569 fecal samples collected weekly over a 6-week period from groups of 5 pigs housed in 32 pens that were randomized to receive either control, CTC, copper, or copper plus CTC regimens. Qualitative and quantitative PCR were used to detect the presence of 14 tetracycline resistance (tet) genes and to quantify gene copies of tetA, tetB, blaCMY-2 (a 3rd generation cephalosporin resistance gene), and pcoD (a copper resistance gene), respectively. The detection of tetA and tetB decreased over the subsequent sampling periods, whereas the prevalence of tetC and tetP increased. CTC and copper plus CTC supplementation increased both the prevalence and gene copy numbers of tetA, while decreasing both the prevalence and gene copies of tetB. In summary, tet gene presence was initially very diverse in the gut bacterial community of weaned pigs; thereafter, copper and CTC supplementation differentially impacted the prevalence and quantity of the various tetracycline, ceftiofur and copper resistance genes resulting in a less diverse gene population.

Medium-grade metabasites and metapelites from the Cajamarca Complex (Central Cordillera of Colombia) are in fault contact with the Jurassic Ibague batholith and show a penetrative foliation, locally mylonitic, suggesting intense dynamic–thermal metamorphism. The amphibolites are composed of calcic amphibole + epidote + plagioclase + quartz plus rutile + titanite + apatite + carbonate as accessory phases. Chlorite and albite appear as retrograde replacements. The metapelites are mainly composed of phengite + quartz + garnet + chlorite, plus epidote + albite + apatite + titanite + haematite as accessory phases. Bulk geochemistry of the amphibolites indicates basaltic protoliths with a mid-ocean ridge basalt (MORB) signature, although enrichment in the mobile large-ion lithophile elements compared to MORB suggests pre- and/or syn-metamorphic alteration by fluids. Peak pressure–temperature determinations for both types of rocks are similar, ranging 550–580°C and 8 kbar (approximately 26 km depth and an appare...

TICKS are the second most important group of arthropod vectors of diseases transmissible to animals and human beings. They are obligate parasites, and while feeding on a host may transmit a range of pathogens, such as bacteria, spirochaetes, rickettsiae, protozoa, viruses, nematodes, and toxins. The

A randomized controlled field trial was conducted to evaluate the effects of two sets of treatment strategies on ceftiofur and tetracycline resistance in feedlot cattle. The strategies consisted of ceftiofur crystalline-free acid (CCFA) administered to either one or all of the steers within a pen, followed by feeding or not feeding a therapeutic dose of chlortetracycline (CTC). Eighty-eight steers were randomly allocated to eight pens of 11 steers each. Both treatment regimens were randomly assigned to the pens in a two-way full factorial design. Non-type-specific (NTS) E. coli (n = 1,050) were isolated from fecal samples gathered on Days 0, 4, 12, and 26. Antimicrobial susceptibility profiles were determined using a microbroth dilution technique. PCR was used to detect tet(A), tet(B), and bla CMY-2 genes within each isolate. Chlortetracycline administration greatly exacerbated the already increased levels of both phenotypic and genotypic ceftiofur resistance conferred by prior CCFA treatment (P

Objective. To determine prevalence of MAP in intestinal and nodal tissue from dogs and cats at necropsy at Kansas State University and to determine if an association existed between presence of MAP and gastrointestinal inflammation, clinical signs, or rural exposure. Procedures. Tissue samples were collected from the duodenum, ileum, and mesenteric and colic nodes of adult dogs (73) and cats (37) undergoing necropsy for various reasons. DNA was extracted and analyzed for insertion sequence 900 using nested PCR. Positive samples were confirmed with DNA sequencing. An online mapping system was used to determine if patients lived in an urban or rural environment based on the home address. Medical records were reviewed for clinical signs and histological findings at necropsy. Results. MAP was identified from 3/73 (4.1%) dogs and 3/37 (8.1%) cats. There was no documented association between presence of MAP and identification of histologic-confirmed gastrointestinal inflammation, gastrointestinal clinical signs, or exposure to a rural environment. Conclusion and Clinical Relevance. MAP-specific DNA can be identified within the intestinal and nodal tissue of dogs and cats that do not have pathological lesions or clinical signs consistent with gastrointestinal disease. The significance of this organism’s presence without associated gastrointestinal pathology is unknown.

Ixodes scapularis (Say) is the vector for Borrelia burgdorferi (Bb) the causative agent of Lyme disease (LD). The increased number and presence of ticks in the environment pose a significant health risk to people and many domestic animals including dogs, cats, and horses. This study characterized the distribution and expansion of I. scapularis and Bb and identified areas of increased risk of LD transmission in Indiana using geographical information systems (GIS) and spatial analysis. A cross-sectional sampling was performed for 3 consecutive years (2005-2007). A total of 3,412 harvested white-tailed deer (Odocoileus virginianus) were searched for ticks at Department of Natural Resources manned deer check-in stations. Hunters were asked for verbal permission to search the deer and to indicate on a road atlas where the deer was killed. All deer points were digitized into a GIS database. Identification of clustering in space and time for these organisms was performed using geostatistical software. Multiple spatial clusters of I. scapularis-infested deer were identified in western Indiana. B. burgdorferi was isolated from tick pools in 11 counties. In addition to the I. scapularis clusters, one spatial cluster of Bb-infected ticks was identified. Our current survey results and cluster analysis indicate that the western geographic regions of Indiana should be considered by the healthcare community to be at increased risk of LD compared with the rest of Indiana.

Summary Two independent vaccination trials using a hybrid synthetic poly pep-tide containing epitopes from four proteins of Plasmodium falciparum were performed. In the first trial 63 and in the second 122 volunteers were vaccinated, using different immunization schedules. The analysis of the humoral response to the vaccine, measured by IgG antibody titres to the polypeptide showed a bimodal distribution in both cases suggesting genetic control of the immune response to this protein. There was a small group of low or non-responders and a large group of good responders. HLA phenotyping of the two groups disclosed an association of the low responders to HLA-DR4 antigens with chi-square P value of 0.00039 when compared with the good responders group. These findings provide evidence for the genetic control of the immune response to the synthetic vaccine by the association of this response with particular alleles of the HLA class II antigens; such findings may lead to an explanation of the mechanism involved in disease susceptibility and need to be used in the design of a totally effective vaccine.

Active coinfection of Borrelia burgdorferi and Babesia microti has been suggested to result in an increased disease severity due to changes in immune response patterns. However, the immunological mechanisms involved in this processes are still unknown. The aim of this study is to characterize the cell immune responses during the progression of B. burgdorferi and B. microti coinfection. To address this issue, C3H.HeN and BALB/c mice cohorts were coinfected or singly infected and monitored for 1, 7, 10, 14, 21 and 60 days post-infection. Spleens and lymph nodes from infected and non-infected mice were removed for tissue culture experiments. Mononuclear cells were analyzed by flow cytometry assays using monoclonal antibodies. Interestingly, we found minimum changes among CD4+/CD3+, CD8+/CD3+, gamma-delta and NKT cells in C3H and BALB/c strains. In contrast, B cell populations were significantly augmented in BALB/c than C3H mice after infection. Furthermore, significant increased levels of B cells were also detected in coinfected BALB/c mice at days 14, 21 and 60. Spleen macrophage populations reached a peak at day 7 in both C3H and BALB/c strains for singly infected mice, in contrast with coinfected mice that showed significant decreased population of these cells. The reduction of spleen macrophages in coinfected mice may suggest an impair immune response during B.burgdorferi and B. microti coinfection resulting in an increased disease severity and persistence of Lyme disease. NIH P20RR016443-04.

Lyme disease is caused by Borrelia burgdorferi and represents a worldwide public health problem. This multisystemic and chronic disease may affect several organs including the heart. However, little is known about the molecular mechanisms that control and/or facilitate the development of Lyme carditis. Signal transducer and activator of transcription 3 (STAT3) is critical in mediating cytokine signaling and it has been involved in diverse pathological processes and also implicated in a variety of immune responses. The aim of this study is to examine the role of STAT3 in the initiation and progression of Lyme carditis using a murine model. Six week-old C3H and BALB/c mice were inoculated with B. burgdorferi. Hearts were excised at defined time intervals that reflected the progression of Lyme disease in the mouse model: 7, 14, 21, 30, and 60 days post-inoculation. Infected cardiac tissues were screened for expression of STAT3 and STAT3-regulated proteins by Western blot analyses, immnunoprecipitation and immunofluorescence studies. We show that STAT3, pSTAT3 and STAT3 regulated proteins were upregulated in cardiac tissues infected with B. burgdorferi. Furthermore, we found a direct correlation between upregulation of STAT3/pSTAT3 and progression of the disease during the acute phase of Lyme carditis. Our results suggest that STAT3 plays a key role in Lyme carditis and may represent a molecular target for monitoring the initiation and progression of Lyme carditis. NIH P20RR016443-04 .

The most commonly recognized tick-borne coinfection in most of the eastern USA is Lyme borreliosis and babesiosis, caused by the spirochete Borrelia burgdorferi and the erythrocytic parasite Babesia microti respectively, accounting for ~80% of coinfections. Studies of clinical outcomes indicate that coinfected patients have more severe symptoms and longer duration of illness than patients with Lyme borreliosis alone. Little is known about the immune related mechanisms during tick-borne coinfections. This study used a murine model to examine whether defined disease conditions differed in severity in mice infected solely with B. burgdorferi and in mice coinfected with B. microti and B. burgdorferi. C3H.HeN and BALB/c mice cohorts were coinfected or singly infected and then monitored experimentally for 1, 7, 10, 14, 21 and 60 days after inoculation. Carditis and arthritis was determined by blinded histopathological evaluation of myocardium and tibiotarsal joints. Cytokine measurements were made on lymph node and spleen supernatants for IFN-γ, interleukin (IL-4), IL-10 and TNF-α. Coinfected C3H and BALB/c mice had a significant increase in carditis severity at day 7. In addition, a significant increase in arthritis severity was observed at day 21 and 60 for BALB/c mice. This clinical observation was correlated with a significant increase in the expression of IFN-γ and TNF-α and with a significant reduction of IL-4 and IL-10. NIH P20RR016443-04