Effect of long-term dietary probiotic Lactobacillus reuteri 1 or antibiotics on meat quality, muscular amino acids and fatty acids in pigs.

This study investigated effects of 175-d dietary treatment with Lactobacillus reuteri 1 (LR1) or antibiotics (olaquindox and aureomycin) on the longissimus thoracis (LT) of pigs. Results showed that antibiotics decreased pork quality by increasing drip loss, shear force, and altering myofiber characteristics including diameter, cross-sectional area and myosin heavy chain isoforms compared to LR1. Pigs fed antibiotics had lower muscle contents of free glutamic acid, inosinic acid, and higher glutamine compared to pigs fed the controls and LR1 diets (P ≤ .05). Furthermore, antibiotics decreased free isoleucine, leucine, methionine in LT compared to the control (P ≤ .05). Compared to antibiotics, LR1 likely improved protein synthesis by modulating expression of amino acid transport and ribosomal protein S6 kinase 1 (S6K1) genes, and altered fatty acid profile by regulating metabolic pathways. Overall, LR1 improved pork quality compared to antibiotics by decreasing drip loss and shear force, increasing inosinic acid and glutamic acid that may improve flavor, and altering muscle fiber characteristics.

Impact of added copper, alone or in combination with chlortetracycline, on growth performance and antimicrobial resistance of fecal enterococci of weaned piglets.

Studies suggest a link between added copper (Cu) and co-selection of antimicrobial resistance (AMR) in Enterococcus spp., but data are inconsistent. This study aimed to assess the impact of added Cu, alone or with a feed-grade antimicrobial, on growth performance, transferable Cu resistance gene (tcrB) prevalence, abundance of tcrB in fecal community DNA, and AMR in fecal enterococci in weaned piglets. A total of 320 barrows (DNA 200 × 400, DNA Genetics) weaned at approximately 21 d of age with 7.4 kg (7.4 ±â€…0.06 kg) BW were used in a 28-d study. Piglets were fed a common non-medicated diet for 7 d of acclimation. Treatments were arranged in a 2 × 2 factorial design with main effects of added Cu (0 vs. 200 mg/kg Cu from Cu sulfate) and chlortetracycline (0 vs. 440 mg/kg CTC). Growth performance and fecal samples were obtained on days 0, 14, and 28. There was no evidence (P > 0.05) for Cu and CTC interaction in growth performance. Pigs fed diets with added Cu had increased (P < 0.05) ADG and ADFI from days 0 to 14, with no evidence for differences (P > 0.05) from days 15 to 28 and 0 to 28. Pigs fed diets with CTC had improved (P < 0.01) ADG, ADFI, and G:F from days 0 to 28. Prevalence of tcrB-positive enterococci was not affected by the addition of Cu and/or CTC (P > 0.05). Prevalence of tcrB-positive enterococci was higher on day 14 than other sampling days (P = 0.002). Prevalence of tetracycline resistance gene [tet(M)]-positive enterococci was not affected by treatments or day (P > 0.05). Prevalence of macrolide resistance gene [erm(B)]-positive enterococci had a significant treatment and sampling day interaction (P = 0.021). The abundance of the tcrB gene in feces, quantified by PCR, was not affected by Cu treatment. The median Cu minimum inhibitory concentrations (MIC) of tcrB-negative and -positive isolates were 3 and 20 mM, respectively (P < 0.001). For day 0 and day 28, all Enterococcus isolates were susceptible to gentamicin, kanamycin, streptomycin, daptomycin, and tigecycline, with a majority of isolates resistant to chloramphenicol, erythromycin, lincomycin, linezolid, tetracycline, tylosin tartrate, and Synercid. In conclusion, 200 mg/kg added Cu or 440 mg/kg CTC in nursery diets improved growth performance of nursery pigs. Added Cu, with or without a selection pressure of CTC, did not increase Cu-resistant enterococci and did not co-select resistance to antibiotics.

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.

Effects of palygorskite composites on growth performance and antioxidant status in broiler chickens.

This work aimed to investigate the effects of the palygorskite (PAL) composites on the growth performance and antioxidant status in broiler chickens. A total of 192 one-day-old Ross 308 broilers were randomly divided into 3 treatment groups. Broilers were fed basal diets supplemented with either 50 mg/kg chlortetracycline (CTC group), 1 g/kg ZnO/PAL (ZnO/PAL group), or 1 g/kg chitooligosaccharides/ZnO/PAL (COS/ZnO/PAL group), respectively. The results showed that PAL composites were found to exhibit similar effects on growth performance as CTC (P > 0.05). ZnO/PAL and COS/ZnO/PAL enhanced the activity of serum glutathione peroxidase (GSH-Px) compared with CTC both at 21 and 42 d (P < 0.05). Compared with the CTC group, COS/ZnO/PAL enhanced serum catalase (CAT) activity at 21 d (P < 0.05), and decreased serum malondialdehyde (MDA) content at 42 d (P < 0.05). Compared with the CTC group, ZnO/PAL decreased duodenal mucous MDA content at 21 d, while ZnO/PAL did not affect activities of superoxide dismutase (SOD) and GSH-Px in the duodenum (P > 0.05). The duodenal mucous activities of SOD and GSH-Px were the highest in the COS/ZnO/PAL group at 42 d (P < 0.05). At 21 d, broilers in the COS/ZnO/PAL group had the lowest MDA content and the highest total antioxidant capacity (T-AOC) in the jejunum (P < 0.05). Palygorskite composites decreased ileum mucous MDA content compared with CTC treated broilers at 21 d (P < 0.05). At 42 d, ileum mucous T-AOC was increased both in the ZnO/PAL and COS/ZnO/PAL groups compared with the CTC group (P < 0.05). The ileum mucous GSH-Px activities both in the ZnO/PAL and COS/ZnO/PAL groups were increased compared with the CTC group (P < 0.05). In conclusion, the broilers given the basal diet supplemented with the PAL composites exhibited similar growth performance to their counterparts in the AGP group. Additionally, the PAL composites improved the antioxidant status of broilers and the beneficial effects of COS/ZnO/PAL on the antioxidant status are more pronounced.

Effects of antibiotic on microflora in ileum and cecum for broilers by 16S rRNA sequence analysis.

An experiment was conducted to analyze and compare the microbial composition, abundance, dynamic distribution, and functions without and with antibiotic fed to broilers. A 16S rRNA-sequencing approach was used to evaluate the bacterial composition of the gut of male broilers under different groups. A total of 240 1-day old AA male broilers were randomly assigned to two groups, with 120 broilers per group. The treatment group was administered an antibiotic with their feed, while the control group was not administered antibiotic (control group). A total of 10 replicates were assessed per treatment. The control group was fed a basal diet containing corn, soybean meal, and cottonseed meal and met the nutritional requirement. The antibiotic group was fed 100 mg/kg aureomycin (based on the basal diet). The trial lasted 42 days. Operational taxonomic unit partition and classification, alpha diversity, taxonomic composition, beta diversity, and microflora comparative analyses along with key species screening were performed for all of the treatment groups. Our data indicate that aureomycin treatment in broilers is directly correlated with variations of the gut content of specific bacterial taxa, and herein provide insights into the impact of antibiotic on microbial communities in cecum and ileum of broiler chickens.

Effects of chlortetracycline alone or in combination with direct fed microbials on nursery pig growth performance and antimicrobial resistance of fecal Escherichia coli.

A total of 300 nursery pigs (initially 5.9 ± 0.05 kg BW) were used in a 42-d growth trial to evaluate the effects of feeding a therapeutic level of chlortetracycline (CTC) with or without direct fed microbials (DFM) on growth performance and antimicrobial resistance (AMR) of fecal Escherichia coli. CTC is a broad-spectrum in-feed antibiotic commonly used in the swine industry. Weaned pigs (~21 d of age) were allotted to pens based on initial BW and fed a common starter diet for 4 d. Pens were then blocked by BW and allotted to dietary treatments in a completely randomized block design. Dietary treatments were arranged in a 2 × 3 factorial consisting of combinations of CTC (none vs. 400 mg/kg from days 0 to 42) and DFM (0 vs. 0.05% DFM 1 vs. 0.05% DFM 2). Fecal samples were collected from three randomly selected pigs from each pen on days 0, 21, and 42 for E. coli isolation and AMR determination. Overall, pigs fed diets containing CTC had improved (P < 0.001) ADG, ADFI, and BW compared to those not fed CTC with no evidence for any effect of either DFM 1 or DFM 2. Regardless of CTC, inclusion of DFM 2 in diets improved (P < 0.05) ADFI from days 0 to 14 and on day 14 BW compared to diets that did not include DFM 2. The addition of CTC with or without DFMs to nursery pig diets increased (P < 0.05) the probability of AMR to tetracycline and ceftiofur of fecal E. coli isolates, but this resistance generally decreased (P < 0.05) over time. A decrease (P < 0.05) in AMR to ampicillin and tetracycline (TET) throughout the trial was observed, while resistance to ceftriaxone decreased (P < 0.020) from days 0 to 21 and increased from days 21 to 42 amongst dietary treatments regardless of CTC or DFM inclusion in the diet. A CTC × DFM × day interaction (P < 0.015) was observed for streptomycin, whereby from days 21 to 42 AMR increased in diets containing either CTC or DFM 1 alone, but the combination decreased resistance. There was no evidence for any effect of DFMs on AMR of fecal E. coli isolates to any other antibiotics evaluated. In conclusion, therapeutic levels of added CTC with or without DFM inclusion improved nursery pig performance, but increased AMR of fecal E. coli isolates to TET and ceftiofur. A moderate improvement in intake and day 14 BW was observed when DFM 2 was included in the diet with or without CTC, but, except for streptomycin, there was no evidence that added dietary DFMs affected resistance of fecal E. coli to antibiotics.

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.

Effect of composting and soil type on dissipation of veterinary antibiotics in land-applied manures.

The objective of this study was to determine the fate of commonly used veterinary antibiotics in their naturally excreted form when manure-based amendments are applied to soil. Beef cattle were administered sulfamethazine, tylosin, and chlortetracycline and dairy cows were treated with pirlimycin. The resulting manure was composted for 42 d under static or turned conditions and applied at agronomic N rates to sandy, silt, and silty clay loam soils and compared with amendment with corresponding raw manures in sacrificial microcosms over a 120-day period. Antibiotic dissipation in the raw manure-amended soils followed bi-phasic first order kinetics. The first phase half-lives for sulfamethazine, tylosin, chlortetracycline, and pirlimycin ranged from 6.0 to 18, 2.7 to 3.7, 23 to 25, and 5.5-8.2 d, respectively. During the second phase, dissipation of sulfamethazine was negligible, while the half-lives for tylosin, chlortetracycline, and pirlimycin ranged from 41 to 44, 75 to 144, and 87-142 d, respectively. By contrast, antibiotic dissipation in the compost-amended soils followed single-phase first order kinetics with negligible dissipation of sulfamethazine and half-lives of tylosin and chlortetracycline ranging from 15 to 16 and 49-104 d, respectively. Pirlimycin was below the detection limit in the compost-amended soils. After incubating 120 d, antibiotics in compost-amended soils (up to 3.1 µg kg-1) were significantly lower than in manure-amended soils (up to 19 µg kg-1, p < .0001), with no major effect of soil type on the dissipation. Risk assessment suggested that composting can reduce antibiotic resistance selection potential in manure-amended soils.

Pharmacokinetics of chlortetracycline in maternal plasma and in fetal tissues following oral administration to pregnant ewes.

The purpose of this study was to determine if concentrations of chlortetracycline could be detected in fetal plasma or tissues after administering an oral dose of chlortetracycline (CTC; 500 mg/head/day) reported to be effective in controlling Campylobacter spp. abortions. Five pregnant ewes were administered 250 mg/head twice a day (total dose 500 mg/hd/d) for 7 days. On the beginning of day 7, intravenous catheters were surgically implanted or inserted into the fetus and dam. Plasma samples were collected from the ewe and fetus at various time points before and up to 36 hr after the last dose of CTC. All ewes were then sacrificed, and tissues were harvested from the fetus for drug analysis. Concentrations of CTC in maternal plasma were consistent with our previous study and below the minimum inhibitory concentration of Campylobacter abortion isolates. Concentrations of CTC were below the limit of detection in three of five fetal plasma samples and all of the placenta, amniotic fluid, and fetal stomach contents. Low concentrations were detectable in fetal kidney and liver, suggesting that CTC reaches the fetus, although at a variable and low ratio when compared to maternal concentrations.

Models of antimicrobial pressure on intestinal bacteria of the treated host populations.

Antimicrobial drugs are used to treat pathogenic bacterial infections in animals and humans. The by-stander enteric bacteria of the treated host's intestine can become exposed to the drug or its metabolites reaching the intestine in antimicrobially active form. We consider which processes and variables need to be accounted for to project the antimicrobial concentrations in the host's intestine. Those include: the drug's fraction (inclusive of any active metabolites) excreted in bile; the drug's fractions and intestinal segments of excretion via other mechanisms; the rates and intestinal segments of the drug's absorption and re-absorption; the rates and intestinal segments of the drug's abiotic and biotic degradation in the intestine; the digesta passage time through the intestinal segments; the rates, mechanisms, and reversibility of the drug's sorption to the digesta and enteric microbiome; and the volume of luminal contents in the intestinal segments. For certain antimicrobials, the antimicrobial activity can further depend on the aeration and chemical conditions in the intestine. Model forms that incorporate the inter-individual variation in those relevant variables can support projections of the intestinal antimicrobial concentrations in populations of treated host, such as food animals. To illustrate the proposed modeling framework, we develop two examples of treatments of bovine respiratory disease in beef steers by oral chlortetracycline and injectable third-generation cephalosporin ceftiofur. The host's diet influences the digesta passage time, volume, and digesta and microbiome composition, and may influence the antimicrobial loss due to degradation and sorption in the intestine. We consider two diet compositions in the illustrative simulations. The examples highlight the extent of current ignorance and need for empirical data on the variables influencing the selective pressures imposed by antimicrobial treatments on the host's intestinal bacteria.

Efficacy of tiamulin alone or in combination with chlortetracycline against experimental Mycoplasma gallisepticum infection in chickens.

Mycoplasma gallisepticum (M. gallisepticum) remains one of the most important diseases in poultry production. Controlling the impact of the disease is done by eradication of positive breeder flocks or by vaccination and medication. A widely used molecule in medication programs is tiamulin, a pleuromutilin antibiotic. Since recent data on the in vivo efficacy of this molecule are scarce, 2 challenge studies were conducted using a recently isolated M. gallisepticum strain belonging to the wildtype population with regard to its tiamulin and tetracycline minimum inhibitory concentration (MIC). In the first challenge study, the dose rate of tiamulin was tested. For this, broilers were infected with M. gallisepticum and treated with 10 mg or 25 mg tiamulin hydrogen fumarate (hf)/kg body weight (BW) for 5 successive days. In a second challenge study, the dose rate of tiamulin combined with chlortetracycline was tested. For this, broilers were infected with M. gallisepticum and treated with 6.25 mg tiamulin hf/18.75 mg chlortetracycline hydrochloride (hcl)/kg BW or 12.5 mg tiamulin hf/37.5 mg chlortetracycline hcl/kg BW for 5 successive days. Clinical scoring of respiratory signs, macroscopic scoring of respiratory tract lesions, M. gallisepticum isolation from the respiratory organs, weight gain, and mortality were the monitored efficacy parameters. The first study demonstrated that a 5-day 10 mg/kg BW tiamulin hf treatment provided significant protection against the M. gallisepticum infection. However, since the 5-day 25 mg/kg BW group was significantly better than the 10 mg/kg BW for reducing the post-treatment clinical signs and the M. gallisepticum numbers in the respiratory organs, the 25 mg/kg BW treatment is recommended for clinical M. gallisepticum infections. In the second study, the combined 12.5 mg tiamulin hf/37.5 mg chlortetracycline hcl/kg BW resulted in a significant reduction of the severity of clinical respiratory disease post treatment and a significant reduction of the M. gallisepticum numbers in the respiratory tract.

Antimicrobial growth promoters modulate host responses in mice with a defined intestinal microbiota.

Antibiotics can promote growth in livestock (antimicrobial growth promoters, AGPs), however lack of knowledge regarding mechanisms has hampered the development of effective non-antibiotic alternatives. Antibiotics affect eukaryotic cells at therapeutic concentrations, yet effects of AGPs on host physiology are relatively understudied, partially due to the complexity of host-microorganism interactions within the gastrointestinal tract. To determine the direct effects of AGPs on the host, we generated Altered Schaedler Flora (ASF) mice, and administered chlortetracycline (CTC) and tylosin phosphate (TYL) in feed. Mice were challenged with Citrobacter rodentium to determine how AGPs alter host responses to physiological stress. Although CTC and TYL had inconsistent effects on the ASF taxa, AGPs protected mice from weight loss following C. rodentium inoculation. Mice treated with either CTC or TYL had lower expression of ßd1 and Il17a in the intestine and had a robust induction of Il17a and Il10. Furthermore, AGP administration resulted in a lower hepatic expression of acute phase proteins (Saa1, Hp, and Cp) in liver tissue, and ameliorated C. rodentium-induced reductions in the expression of genes involved in lipogenesis (Hmgcl and Fabp1). Collectively, this indicates that AGPs directly affect host physiology, and highlights important considerations in the development of non-antibiotic alternatives.

Changes in the diversity and composition of gut microbiota of weaned piglets after oral administration of Lactobacillus or an antibiotic.

The gut microbiota plays important roles in the health and well-being of animals, and high-throughput sequencing facilitates exploration of microbial populations in the animal gut. However, previous studies have focused on fecal samples instead of the gastrointestinal tract. In this study, we compared the microbiota diversity and composition of intestinal contents of weaned piglets treated with Lactobacillus reuteri or chlortetracycline (aureomycin) using high-throughput sequencing. Nine weaned piglets were randomly divided into three groups and supplemented with L. reuteri, chlortetracycline, or saline for 10 days, and then the contents of three intestinal segments (jejunum, colon, and cecum) were obtained and used for sequencing of the V3-V4 hypervariable region of the 16S rRNA gene. The microbiota diversity and composition in the jejunum were different from those in the colon and cecum among the three treatments. In the jejunum, treatment with L. reuteri increased the species richness of the microbiota, as indicated by the ACE and Chao1 indexes, compared with the chlortetracycline group, in which several taxa were eliminated. In the colon and cecum, relative abundances of the phylum Firmicutes and the genus Prevotella were higher in the chlortetracycline group than in the other groups. Distances between clustered samples revealed that the L. reuteri group was closer to the chlortetracycline group than the control group for jejunum samples, while colon and cecum samples of the L. reuteri group were clustered with those of the control group. This study provides fundamental knowledge for future studies such as the development of alternatives to antibiotics.

Meta-analysis of the effects of laidlomycin propionate, fed alone or in combination with chlortetracycline, compared with monensin sodium, fed alone or in combination with tylosin, on growth performance, health, and carcass outcomes in finishing steers in North America.

The objective of this research was to use data from multiple studies to comprehensively quantify the effects of feeding 1) laidlomycin propionate (LP), alone and/or in combination with chlortetracycline, compared with 2) monensin sodium (MS), alone and/or in combination with tylosin, at commercially approved dosages, on ADG, DMI, feed efficiency (FE), mortality, and carcass characteristics (HCW and liver abscesses). A secondary objective was to explore potential sources of heterogeneity among the comparative effectiveness studies. A systematic review of peer-reviewed literature and industry reports was used to identify studies that included direct comparisons of these treatments in finishing steers in North America. Random-effects meta-analysis models of performance, carcass, and health-related outcomes were fitted with extracted data, consisting of a total of 17 data sets comprising a total of 135 pens and 13,603 steers. Results showed that pens of steers fed LP had increased ADG (live and carcass adjusted), DMI, and HCW compared with those fed monensin ( < 0.05). However, liver abscesses were more common in LP-fed cattle than in MS-fed cattle ( < 0.05), presumably because of differences in the concurrently fed antimicrobials. No significant effects ( > 0.05) were identified for FE or for health-related outcomes (overall and cause-specific mortality). There was a substantial amount of heterogeneity in outcomes among studies, and when pen size and type of production setting were included in mixed-effects meta-regression models, they accounted for only a small proportion of the between-study heterogeneity found in the meta-analysis models. Therefore, caution should be exercised when interpreting summary estimates in the presence of substantial heterogeneity. However, these results provide comprehensive information on the comparative effects of different ionophores across multiple studies and multiple years, states, and production settings. These unique results can enable quantitative and informed decisions by potential end users of these feed additives that are widely used in the U.S. beef industry for reducing the costs of beef production through enhanced cattle performance.

Impact of medicated feed along with clay mineral supplementation on Escherichia coli resistance to antimicrobial agents in pigs after weaning in field conditions.

The aim of this study was to examine changes in antimicrobial resistance (AMR) phenotype and virulence and AMR gene profiles in Escherichia coli from pigs receiving in-feed antimicrobial medication following weaning and the effect of feed supplementation with a clay mineral, clinoptilolite, on this dynamic. Eighty E. coli strains isolated from fecal samples of pigs receiving a diet containing chlortetracycline and penicillin, with or without 2% clinoptilolite, were examined for antimicrobial resistance to 15 antimicrobial agents. Overall, an increased resistance to 10 antimicrobials was observed with time. Supplementation with clinoptilolite was associated with an early increase but later decrease in blaCMY-2, in isolates, as shown by DNA probe. Concurrently, a later increase in the frequency of blaCMY-2 and the virulence genes iucD and tsh was observed in the control pig isolates, being significantly greater than in the supplemented pigs at day 28. Our results suggest that, in the long term, supplementation with clinoptilolite could decrease the prevalence of E. coli carrying certain antimicrobial resistance and virulence genes.

Comparison of feed additive technologies for preconditioning of weaned beef calves.

Our objective was to evaluate the response of weaned calves to different supplemental feed additives in a supplement to affect calf performance and mitigate stress response observed during weaning and preconditioning. At weaning in each of 2 yr, 160 Angus and Brangus calves (203 and 227 ± 2.3 and 2.5 kg) were stratified by BW, sex, and breed and were randomly allotted to 1 of 4 treatments ( = 40 calves/treatment): 1) supplement without feed additives (control, CON), 2) supplemented with chlortetracycline, 350 mg/d (CTC), 3) supplemented with monensin, 175 mg/d (RUM), and 4) supplemented with rumen modifier, 5 g/d (ACT). Calves were held by treatment in 1 of 4 drylot pens for 7 d after weaning and were offered ad libitum access to hay and 2.27 kg/d of supplement before placement in one of thirty-two 0.8-ha pastures (5 calves/pasture). On pasture calves were supplemented with 2.27 kg/d (yr 1) or supplemented at 1.0% BW (yr 2). Calf BW and blood samples were collected following weaning (d 0, 1, 4, 7, 11 in yr 1; d 0, 1, 3, 7, 14 in yr 2), at the conclusion of the preconditioning period (d 50, 51 in yr 2), and after transportation (d 52, 55, 59, 65 in yr 2) for analysis of acute phase protein (APP) concentrations. In yr 2, after 44 d on pasture, calves were loaded on 2 semitrucks and transported for 24 h. On return, calves were placed in 4 pastures with hay and fed their respective supplements for 14 d. For each year, data were analyzed with the MIXED procedure of SAS. The model included the main effect of treatment, and pasture was the experimental unit. All variables quantified by day were analyzed using repeated measures. In yr 1, ACT and CTC had greater (P <0.05) 52-d ADG than RUM, whereas CON was intermediate. However, in yr 2, over the 50-d postweaning period there was no difference (P = 0.20; 0.52 kg/d) in ADG response among treatments. After transportation, 7- and 14-d ADG were improved (P < 0.05) for ACT and CTC compared with CON and RUM. In both years, postweaning plasma concentrations of haptoglobin were similar (P > 0.05) among treatments; however an effect of day after transport (P < 0.001) was observed. Feed cost of gain and income over production cost (P ≥ 0.15; mean = $0.51/kg and $73.51, respectively) were not different among treatments. Use of supplemental additives may improve calf performance during a preconditioning period of this duration, but no additive was effective at mitigating stress postweaning. Additives were equally effective in supporting calf growth performance during a posttransportation period.

Effect of oxytocin and PGF2α on chlortetracycline absorption from the uterus of early postpartum camels (Camelus dromedarius).

Fifteen parturient camels given chlortetracycline (CTC) as intrauterine pessaries (3 g/head) were divided into the control group (n = 5), which remained untreated, oxytocin-treated group (50 IU, intramuscular; n = 5), and cloprostenol-treated group (Estrumate, 500 µg, intramuscular; n = 5). Serum samples were collected at 0, 1, 2, 4, 8, 24, 48, and 72 hours after treatment and CTC was determined. The CTC appeared in blood within 1 hour. The maximum concentration of CTC was detected in blood after 72 (543.58 ± 117.85 µg/L), 8 (520.48 ± 13.65 µg/L), and 1 hour (831.98 ± 111.01 µg/L) of administration in control, oxytocin-, and PGF2α-treated camels, respectively. There was a high significant effect of time (P < 0.001) and treatment-by-time interaction (P < 0.001) on serum CTC concentration. In the control group, there was a significant (P < 0.05) increase in CTC concentrations at 72 hours compared to the other times. In the oxytocin group, there was a significant (P < 0.05) decrease in CTC concentrations at 24, 48, and 72 hours compared to its level after 1 or 8 hours. In PGF2α, there was a significant (P < 0.001) decrease in CTC concentrations at 2, 4, 8, 24, 48, and 72 hours compared to its level after 1 hour. Treatment contrast at different time points showed a significant (P < 0.001) increase in CTC concentration after 1 hour in the PGF2α-treated group compared to oxytocin and control groups. By 72 hours, CTC concentrations were significantly (P < 0.05) lower in PGF2α and oxytocin groups than in the control group. In conclusion, serum CTC concentration in dromedary camels increases within 1 hour after intrauterine administration and remains elevated for at least 72 hours in control, oxytocin-, and PGF2α-treated animals.

6-Iso-chlortetracycline or keto form of chlortetracycline? Need for clarification for relevant monitoring of chlortetracycline residues in food.

Chlortetracycline (CTC) is a broad-spectrum antibiotic used in veterinary medicine for pulmonary or digestive infections and having a regulatory maximum residue limit (MRL) necessitating an official analytical control method. The purpose of this study was to clarify the identification of different forms of CTC observed in standard solution, in spiked muscle samples and in naturally incurred muscle samples of pigs analysed by LC-MS/MS and to demonstrate the in vivo formation of 6-iso-chlortetracycline and 4-epi-6-iso-CTC as a metabolite of CTC and 4-epi-CTC in muscle. The six following forms were identified, all being isobaric with a protonated molecule at m/z 479 (precursor ion): the keto-enol forms of CTC and the keto-enol forms of 4-epi-chlortetracycline (4-epi-CTC), 6-iso-chlortetracycline (6-iso-CTC) and 4-epi-6-iso-chlortetracycline (4-epi-6-iso-CTC). The 6-iso-CTC and 4-epi-6-iso-CTC were observed only in incurred pig samples so were identified for the first time as metabolites of CTC and 4-epi-CTC. Identification of the different forms was obtained by comparing incurred muscle samples with standard solutions and with spiked samples. Then the differences between the features of the chromatograms obtained by LC-TQ-MS and the fragmentation study of the different forms of CTC obtained by LC-Q-TOF-MS helped us to support this identification. The extraction steps and the LC-MS/MS conditions developed to analyse muscle tissue samples are described. This clarification concerning the rigorous identification of chromatographic peaks allowed us to evaluate the relevance of our monitoring method with regard to the regulations in place in the European Union and could be of help to laboratories involved in official control of antibiotic residues in food of animal origin. Additional results are also presented highlighting the transformation of the CTC when prepared in a mixture with other antibiotics.

Effects of chlortetracycline and copper supplementation on the prevalence, distribution, and quantity of antimicrobial resistance genes in the fecal metagenome of weaned pigs.

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.

Effects of Clostridium butyricum on growth performance, antioxidation, and immune function of broilers.

To investigate the effects of Clostridium butyricum on growth performance, antioxidation, and immune function of broilers, 320 one-day-old Arbor Acres commercial male chicks were assigned to one of 5 treatments with 8 replicates in a completely randomized design for 42 d. The 5 treatments were basal diet (control), basal diet supplemented with 2.5×10(8) cfu C. butyricum/kg (CB1), basal diet supplemented with 5×10(8) cfu C. butyricum/kg (CB2), basal diet supplemented with 1×10(9) cfu C. butyricum/kg (CB3), and basal diet supplemented with 150 mg aureomycin/kg (antibiotic). The results showed that all C. butyricum-supplemented groups during d 1 to 21 and the CB2 group during d 22 to 42 had higher ADG compared with the control (P<0.05). Chicks fed the CB3 diet had higher glutathione S-transferase (GST) activity (P<0.05), and chicks fed the CB2 diet had a higher glutathione (GSH) concentration in duodenal and ileal mucosa at 21 d of age than those in the control group (P<0.05). Chicks fed the CB3 diet had a lower malondialdehyde (MDA) concentration in duodenal mucosa than those in the control and CB1 groups (P<0.05). Chicks fed the CB2, CB3, and antibiotic diets had a lower MDA concentration in ileal mucosa than those in the control and CB1 groups (P<0.05). Broilers fed the CB3 diet had greater superoxide dismutase (SOD) activity in the ileal mucosa on d 21 and in jejunal mucosa on d 42 than those in the other groups (P<0.05). Chicks fed the CB2, CB3, and antibiotic diets had a higher GSH concentration in duodenal and jejunal mucosa on d 42 than those in the control group (P<0.05). Broilers fed the CB2 and CB3 diets had a lower MDA concentration in the jejunal mucosa on d 42 than those in the control and CB1 groups. Chicks fed diets supplemented with C. butyricum had a higher IgM concentration than those in the control group at 21 and 42 d of age (P<0.05). The results indicate that C. butyricum improves broilers' growth performance, antioxidation, and immune function.