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Antibiotics are active substances frequently used to treat and prevent diseases in animal husbandry, especially in swine and poultry farms. The use of manure as a fertilizer may lead to the dispersion of antibiotic residue into the environment and consequently the development of antibiotic-resistant bacteria. Most pharmaceutical active ingredients are excreted after administration, in some cases up to 90% of the consumed dose can be found in the feces and/or urine as parent compound. Therefore, due to antibiotic metabolism their residues can be easily detected in manure. This article describes a method for simultaneous analysis of ciprofloxacin, chlortetracycline, doxycycline, enrofloxacin, lincomycin, oxytetracycline, tetracycline, tiamulin, trimethoprim and tylosin in feces, liquid manure and digestate. Antibiotics were extracted from the different matrices with McIlvaine-Na2EDTA buffer solution and the extract was purified by the use two techniques: d-SPE and SPE (Strata-X-CW cartridges) and final eluent was analyzed by LC-MS and LC-MS/MS. The European Commission Decision 2002/657/EC was followed to conduct the validation of the method. Recoveries obtained from spiked pig and poultry feces and liquid manures samples ranged from 63% to 93% depending on analytes. The analysis of 70 samples (feces, liquid manure and digestate) revealed that 18 samples were positive for the presence of doxycycline, oxytetracycline, tetracycline, chlortetracycline, enrofloxacin, tiamulin and lincomycin. The results obtained in the presented study demonstrated that animal feces can be used as a non-invasive method detection antibiotic usage in animal production.

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Increasing evidence exists for the role that cattle play in the epidemiology of campylobacteriosis. In this study, the prevalence and distribution of Campylobacter jejuni were longitudinally examined at the subspecies level in the beef cattle production continuum. Animals were subdivided into two groups: those that were not administered antibiotics and those that were administered the antimicrobial growth promoter chlortetracycline and sulfamethazine (AS700). Samples were longitudinally collected throughout the confined feeding operation (CFO) period and during the slaughter process, and C. jejuni was isolated and genotyped to assess subtype richness and to elucidate transmission dynamics from farm to fork. The bacterium was frequently isolated from cattle, and the bacterial densities shed in feces increased over the CFO period. Campylobacter jejuni was also isolated from digesta, hides, the abattoir environment, and carcasses. The administration of AS700 did not conspicuously reduce the C. jejuni densities in feces or within the intestine but significantly reduced the bacterial densities and the diversity of subtypes on abattoir samples. All cattle carried multiple subtypes, including clinically relevant subtypes known to represent a risk to human health. Instances of intra-animal longitudinal transmission were observed. Although clinically relevant subtypes were transmitted to carcasses via direct contact and aerosols, the bacterium could not be isolated nor could its DNA be detected in ground beef regardless of treatment. Although the evidence indicated that beef cattle represent a significant reservoir for C. jejuni, including high-risk subtypes strongly associated with the bovine host, they do not appear to represent a significant risk for direct foodborne transmission. This implicates alternate routes of human transmission.IMPORTANCE Limited information is available on the transmission of Campylobacter jejuni subtypes in the beef production continuum and the foodborne risk posed to humans. Cattle were colonized by diverse subtypes of C. jejuni, and the densities of the bacterium shed in feces increased during the confined feeding period. Campylobacter jejuni was readily associated with the digesta, feces, and hides of cattle entering the abattoir, as well as the local environment. Moreover, C. jejuni cells were deposited on carcasses via direct contact and aerosols, but the bacterium was not detected in the ground beef generated from contaminated carcasses. We conclude that C. jejuni bacterial cells associated with beef cattle do not represent a significant risk through food consumption and suggest that clinically relevant subtypes are transmitted through alternate routes of exposure.

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OBJECTIVES: The purpose of this study was to evaluate a nonsurgical approach toward peri-implantitis by means of mechanical debridement and local detoxification leaving granulation tissue into the peri-implant pockets. MATERIALS AND METHODS: An analysis was performed on the outcomes of 2 nonsurgical protocols: mechanical debridement plus chemical detoxification with 0.2% chlorhexidine and chlortetracycline hydrochloride (test), and mechanical debridement alone (control). Measures were collected at baseline, 3, 24, and 36 months after treatment. RESULTS: Seventy-five implants with peri-implantitis were included. At a 36-month evaluation, mean pocket probing depth was 3.15 ± 0.32 mm for the test group and 5.97 ± 0.90 mm for the control group, the difference being significant. An improvement of marginal bone level was recorded for the test group, whether the control group showed a further marginal bone loss. Treatment success was 100% in the test group and 31.4% in the control group. CONCLUSION: Nonsurgical implant debridement alone seemed not efficient in the treatment of peri-implantitis. Further longitudinal studies are required to evaluate the efficacy of mechanical debridement with chemical detoxification and removal of granulation tissues against mechanical debridement with chemical detoxification alone.

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BACKGROUND: Sensitivity analysis is an essential step in mathematical modeling because it identifies parameters with a strong influence on model output, due to natural variation or uncertainty in the parameter values. Recently behavior pattern sensitivity analysis has been suggested as a method for sensitivity analyses on models with more than one mode of output behavior. The model output is classified by behavior mode and several behavior pattern measures, defined by the researcher, are calculated for each behavior mode. Significant associations between model inputs and outputs are identified by building linear regression models with the model parameters as independent variables and the behavior pattern measures as the dependent variables. We applied the behavior pattern sensitivity analysis to a mathematical model of tetracycline-resistant enteric bacteria in beef cattle administered chlortetracycline orally. The model included 29 parameters related to bacterial population dynamics, chlortetracycline pharmacokinetics and pharmacodynamics. The prevalence of enteric resistance during and after chlortetracycline administration was the model output. Cox proportional hazard models were used when linear regression assumptions were not met. RESULTS: We have expanded the behavior pattern sensitivity analysis procedure by incorporating model selection techniques to produce parsimonious linear regression models that efficiently prioritize input parameters. We also demonstrate how to address common violations of linear regression model assumptions. Finally, we explore the semi-parametric Cox proportional hazards model as an alternative to linear regression for situations with censored data. In the example mathematical model, the resistant bacteria exhibited three behaviors during the simulation period: (1) increasing, (2) decreasing, and (3) increasing during antimicrobial therapy and decreasing after therapy ceases. The behavior pattern sensitivity analysis identified bacterial population parameters as high importance in determining the trajectory of the resistant bacteria population. CONCLUSIONS: Interventions aimed at the enteric bacterial population ecology, such as diet changes, may be effective at reducing the prevalence of tetracycline-resistant enteric bacteria in beef cattle. Behavior pattern sensitivity analysis is a useful and flexible tool for conducting a sensitivity analysis on models with varied output behavior, enabling prioritization of input parameters via regression model selection techniques. Cox proportional hazard models are an alternative to linear regression when behavior pattern measures are censored or linear regression assumptions cannot be met.

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Antibiotic fitness and acquired resistance are the two critical factors when bacteria respond to antibiotics, and the correlations and mechanisms between these two factors remain largely unknown. In this study, a TMT-labeling-based quantitative proteomics method was used to compare the differential expression of proteins between the fitness and acquired resistance to chlortetracycline in Aeromonas hydrophila biofilm. Bioinformatics analysis showed that translation-related ribosomal proteins, such as 30s ribosome subunits, increased in both factors; fatty acid biosynthesis related proteins, such as FabB, FabD, FabG, AccA, and AccD, increased in biofilm fitness, and some pathways (including propanoate-metabolism-related protein, such as PrpB, AtoB, PflB, AcsA, PrpD, and GabT) displayed decreased abundance in acquired resistance biofilm. The varieties of selected proteins involved in fatty acid biosynthesis and propanoate metabolism were further validated by q-PCR assay or Western blotting. Furthermore, the antibiotic-resistance-function assays showed that fatty-acid biosynthesis should be a protective antibiotics-resistance mechanism and a cocktail of chlortetracycline and triclosan, a fatty-acid-biosynthesis inhibitor, exhibited more efficient antimicrobial capability than did each antibiotic individually on biofilm, specifically on chlortetracycline-sensitive biofilm. We therefore demonstrate that the up-regulation of fatty acid biosynthesis may play an important role in antibiotic resistance and suggest that a cocktail of chlortetracycline and triclosan may be a potential cocktail therapy for pathogenic infections in biofilm.

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BACKGROUND: Dichelobacter nodosus is the main causative agent of ovine footrot, and there are strong indications that the bacterium can be transferred to cattle grazing on the same pasture as sheep. The aim of this study was to investigate if benign and virulent D. nodosus strains isolated from sheep can be transferred to the interdigital skin of cattle under experimental conditions. Further, we wanted to observe the impact of such infection on bovine foot health, and test the effect of topical chlortetracycline (Cyclo spray(®): Eurovet) on the infection. FINDINGS: Six heifers were included in the study. After an initial 18-day maceration period, three heifers were inoculated on one single foot with a benign strain and three with a virulent strain by adding bacterial suspension in a bandage. The bandages were left on for 17 days, and when removed, D. nodosus was isolated from all six heifers. All six heifers developed interdigital dermatitis. In five of the heifers D. nodosus organisms were demonstrated within the epidermis. Twenty-four days after treatment with chlortetracycline all heifers were negative by cultivation, but tested positive for D. nodosus by polymerase chain reaction (PCR). Two of the six heifers still tested positive for D. nodosus by PCR 49 days after treatment. After 70 days, all heifers tested negative for D. nodosus. CONCLUSIONS: This study shows that both virulent and benign D. nodosus strains originating from sheep can be transferred to naïve heifers under experimental conditions. Further, the study supports the hypothesis that infections with virulent D. nodosus in cattle are associated with interdigital dermatitis. No conclusion regarding the treatment of D. nodosus infection with chlortetracycline was possible.

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OBJECTIVE: To observe the effectiveness of chlortetracycline (aureomycin) treatment on vulval white lesions and to explore its possible pathogenesis. MATERIALS AND METHODS: From January 2001 to April 2011, 194 patients with vulvar non-neoplastic epithelial disorders were divided into three groups according to therapy regimens received, ie, chlortetracycline treatment group (72 cases), chlortetracycline + beclomethasone treatment group (66 cases), and beclomethasone treatment group (56 cases); their local changes of vulvar lesions were observed and efficacy of these treatment profiles was evaluated after one year. RESULTS: Effective rates of chlortetracycline group, chlortetracycline + clobetasol group and clobetasol groups were 86.1% (62/72), 87.9% (58/66), and 62.5% (35/56), respectively. There was a significant difference among these three groups (Hc = 10.7766,p = 0.0046), the curative rate of clobetasol group was markedly lower than that of the former two groups (p = 0.0072 and p = 0.0019), but was not statistical significant (p = 0.6077) when compared between the former groups. CONCLUSION: The occurrence of vulvar non-neoplastic epithelial disorders may be associated with chlamydia and mycoplasma infection, the chlortetracycline is an effective drug for this illness, the mechanism of which might be related to killing pathogens directly and inhibiting inflammatory mediators.

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Digital dermatitis (DD) is one of the most important causes of lameness in dairy cattle worldwide. The objective of this study was to evaluate the efficacy of salicylic acid in the treatment of the disease. A total of 201 DD lesions from 173 cows from four commercial dairy herds were evaluated at day 0 during routine hoof trimming and were allocated into two groups, namely, a control group given chlortetracycline spray, and a treatment group given 10 g of salicylic acid powder applied topically within a bandage. Pain, lesion size and clinical appearance (scored M0 to M4) were evaluated on days 3, 14 and 34 post-treatment. A change to M0 was defined as healing, while changes of M2 or M4 to M1 or M3 were classified as clinical improvements. Healing rates did not differ significantly between treatment groups at days 3 and 14. By day 34 the healing rate was fivefold better (P=0.01) for the treatment vs. the control group, with healing rates of 13.6% and 3.1%, respectively. By day 3, the rate of improvement was 2.5-fold better (P=0.02) for the controls. By day 34 the overall positive effect (i.e. healing and improvement) was 1.75-fold better (P=0.05) for the treatment group. Lesions from the control group were 2.2 times more likely (P=0.09) to have a pain score equal to 2 by day 14. The proportion of lesions getting smaller by days 14 and 34 was 2.5 times higher (P<0.08) for the treatment vs. the control group. The findings suggest salicylic acid should be considered as an alternative to chlortetracycline for the treatment of DD as it appears more efficacious and would assist in reducing antibiotic use.

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BACKGROUND: Shoulder ulcers can have a significant impact on the welfare of sows. In Denmark, rubber mats are used for treatment of shoulder ulcers. The objective of the study was to compare a treatment consisting of a combination of rubber mats and zinc ointment with the effect of local antibiotic spray (chlortetracycline) on shoulder ulcers. METHODS: A total of 304 sows with shoulder ulcers were observed shortly after farrowing (day 1) and on days 14 and 21 after the first observation. The sows were paired according to the grade of the shoulder ulcer using the official Danish scoring system. From each pair of sows, one sow was allocated to mat group (M) and the other to an antibiotic group (A) in a random way. In the M group, rubber mats were placed on the floor, and the ulcers were treated with zinc ointment once a day. In the A group, sows were treated with antibiotic spray daily. The size of the shoulder ulcer was measured manually on a continuous scale on days 1, 14 and 21. The data were analysed by use of two multivariable models where the response was the area of the shoulder ulcer on day 14 and day 21, respectively. Explanatory variables were treatment, herd, parity, body condition and size of ulcer on day 1. If a sow had an ulcer on both shoulders, the shoulder with the largest ulcer was selected. RESULTS: The treatment consisting of mats and zinc ointment had a statistical significant effect on the size of the shoulder ulcer on day 14 and day 21 compared to daily treatment with antibiotic spray. For lean sows that were kept on rubber mats and zinc ointment, the average shoulder ulcer was significantly smaller on day 14 (3.8 cm(2) versus 9.5 cm(2) when antibiotic spray was used) and day 21 (3.4 cm(2) versus 6.6 cm(2)) compared to lean sows that were only treated with antibiotic spray. For fat sows this was also the case when using the lesion on day 21 as the response (2.0 cm(2) versus 5.7 cm(2)) but not on day 14. Moreover, the size of the lesion on day 1 was statistically associated with the size of the lesion on day 14 and on day 21. The treatment was equally effective in the three herds. CONCLUSIONS: Rubber mats and daily smearing with zinc ointment slow progression and contribute to the healing of shoulder ulcers compared with housing on slats and daily spraying with antibiotics. It is recommended to place a rubber mat in the farrowing crate at the first sign of shoulder ulcers.

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AIMS: To determine whether therapeutic treatment of pigs with chlortetracycline affects the susceptibility of their Campylobacter isolates for tetracycline, ciprofloxacin and erythromycin. METHODS AND RESULTS: Minimum inhibitory concentrations (MICs) and presence of a tetracycline resistance gene tet(O) were studied in Campylobacter collected before, during and after chlortetracycline treatment. Tetracycline MICs and the presence of tet(O) for additional Campylobacter coli isolates collected previously from seven farrowing farms were also determined. Isolates with ciprofloxacin MICs above the epidemiological cut-off value (ECOFF) were subtyped by flaA restriction fragment length polymorphism (RFLP). Tetracycline MICs of 221 Camp. coli isolates remained under the ECOFF at all sampling stages as well as the MICs for 63 isolates from the other farms. The ciprofloxacin MIC was above the ECOFF for 22% of the isolates, and one Camp. coli isolate had an erythromycin MIC above the ECOFF. None of the studied 300 Campylobacter isolates from nine herds carried tet(O). flaA-RFLP typing revealed the heterogeneity of Camp. coli isolates with high ciprofloxacin MICs. CONCLUSION: Use of chlortetracycline did not increase the MIC values for the antimicrobials studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that susceptibility of Camp. coli isolates is not affected by chlortetracycline therapy if tet(O) is not present in Camp. coli population.

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CASE DESCRIPTION: A sudden onset of extreme dysgalactia in gilts and sows in a 1,000-head farrow-to-wean herd was observed in December 2009. Signs of dysgalactia were identified in sows beginning 1 day after parturition and lasted 4 to 6 days. This resulted in a mean piglet preweaning mortality rate of 18% because of starvation. CLINICAL FINDINGS: Sows were neither off feed nor febrile. Udders were not inflamed or congested. Feed sample analysis did not find ergotamine, mycotoxin contamination, or ration formulation errors. Management practices were acceptable. Piglets attempted to stimulate milk production but none was elicited. Oxytocin (20 U) caused milk ejection but the effect was short-lived. Blood samples from sows with affected litters were positive for Mycoplasma suis (formerly Eperythrozoon suis) by PCR assay, and blood samples from sows with unaffected litters were negative. TREATMENT AND OUTCOME: Chlortetracycline fed to the entire sow herd at 22 mg/kg/d (10 mg/lb/d) for 2 weeks resulted in a near complete absence of dysgalactia in sows farrowing within 5 weeks after the start of treatment. Dysgalactia did occur in sows that received chlortetracycline > 5 weeks prior to farrowing. Currently, gestating sows and gilts receive chlortetracycline in feed at a dosage of 22 mg/kg/d for 2 weeks beginning 3 weeks prior to farrowing. CONCLUSIONS AND CLINICAL RELEVANCE: M suis is spread primarily by blood contact from animal to animal, and diagnosis of infection with this organism can be easily missed by means of standard diagnostic protocols unless PCR assays or specific stains are used. Therefore, its current prevalence and impact are likely to be greatly underestimated.

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The efficacy of chlortetracycline (CTC) in-feed medication to treat pigs with clinical respiratory disease was investigated in a farrow-to-finish pig herd infected with Mycoplasma hyopneumoniae, and with clinical respiratory disease in growing pigs. In total, 533 pigs were included. The animals were vaccinated against M hyopneumoniae and porcine circovirus type 2 at weaning. At onset of clinical respiratory disease, they were randomly allocated to one of the following treatment groups: chlortetracycline 1 (CTC1) (two consecutive weeks, 500 ppm), chlortetracycline 2 (CTC2) (two non-consecutive weeks, with a non-medicated week interval in between, 500 ppm) or tylosin (T) (three consecutive weeks, 100 ppm). Performance (daily weight gain, feed conversion ratio), pneumonia lesions at slaughter and clinical parameters (respiratory disease score) were assessed. Only numeric differences in favour of the CTC2 group were obtained for the performance and the clinical parameters. The prevalence of pneumonia lesions was 20.5, 13.1 and 23.0 per cent (P<0.05) for the CTC1, CTC2 and T groups, respectively. The study demonstrated that CTC, when administered at onset of clinical respiratory disease via the feed at a dose of 500 ppm during two alternative weeks, was able to decrease the prevalence of pneumonia lesions, and numerically reduce performance losses and clinical signs.

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When considering the development of antimicrobial resistance in food animals, comparing gross use estimates of different antimicrobials is of little value due to differences in potencies, duration of activity, relative effect on target and commensal bacteria, and mechanisms of resistance. However, it may be valuable to understand quantities of different antimicrobials used in different ages of swine and for what applications. Therefore, the objective of this project was to construct an estimate of antimicrobial use through the feed in swine production in the United States. Estimates were based on data from the National Animal Health Monitoring System (NAHMS) Swine 2006 Study and from a 2009 survey of swine-exclusive practitioners. Inputs consisted of number of pigs in a production phase, feed intake per day, dose of the antimicrobial in the feed, and duration of administration. Calculations were performed for a total of 102 combinations of antimicrobials (n=17), production phases (n=2), and reasons for use (n=3). Calculations were first conducted on farm-level data, and then extrapolated to the U.S. swine population. Among the nursery phase estimates, chlortetracycline had the largest estimate of use, followed by oxytetracycline and tilmicosin. In the grower/finisher phase, chlortetracycline also had the largest use estimate, followed by tylosin and oxytetracycline. As an annual industry estimate for all phases, chlortetracycline had the highest estimated use at 533,973 kg. The second and third highest estimates were tylosin and oxytetracycline with estimated annual uses of 165,803 kg and 154,956 kg, respectively. The estimates presented here were constructed to accurately reflect available data related to production practices, and to provide an example of a scientific approach to estimating use of compounds in production animals.

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PURPOSE: To determine whether ocular itching associated with ocular demodicosis can be treated by lid massage with 5% tea tree oil ointment (TTOO). METHODS: Twenty-four patients with ocular itching and ocular Demodex were treated with chlortetracycline hydrochloride eye ointment lid massage for 4 weeks and then treatment was switched to TTOO for another 4 weeks. They were examined every 2 weeks and their itching was graded as 0 (none), 1 (mild), 2 (moderate), or 3 (severe). Changes of itching scores and Demodex counts were compared. RESULTS: Before the treatment, all 24 patients reported itching graded as 1 (n = 3), 2 (n = 15), and 3 (n = 6) for a period of 2 weeks to 2 years despite previous topical antibiotics, antiinflammatory medications, or antiallergy medications. Demodex counts per 8 epilated lashes were 5.5 ± 1.6 for all patients but 4.6 ± 1.5, 4.8 ± 1.9, and 7.1 ± 2.1 for patients with grade 1, grade 2, and grade 3 itchiness, respectively (P < 0.05 between grade 2 and grade 3). After 4 weeks of chlortetracycline hydrochloride eye ointment treatment, little changes were observed regarding itching and Demodex counts in all patients (P > 0.05). In contrast, after 5% TTOO treatment, 16 patients were totally free of itching and the remaining 8 patients had different degrees of relief (P < 0.01). The Demodex count decreased to 0.7 ± 0.8 for total (P < 0.01). CONCLUSIONS: There is a strong correlation between ocular itching and Demodex infestation and between symptomatic resolution and reduction of Demodex counts by daily lid massage with 5% TTOO.

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The history of the tetracyclines involves the collective contributions of thousands of dedicated researchers, scientists, clinicians, and business executives over the course of more than 60 years. Discovered as natural products from actinomycetes soil bacteria, the tetracyclines were first reported in the scientific literature in 1948. They were noted for their broad spectrum antibacterial activity and were commercialized with clinical success beginning in the late 1940s to the early 1950s. The second-generation semisynthetic analogs and more recent third-generation compounds show the continued evolution of the tetracycline scaffold toward derivatives with increased potency as well as efficacy against tetracycline-resistant bacteria, with improved pharmacokinetic and chemical properties. Their biologic activity against a wide spectrum of microbial pathogens and their uses in mammalian models of inflammation, neurodegeneration, and other biological systems indicate that the tetracyclines will continue to be successful therapeutics in infectious diseases and as potential therapeutics against inflammation-based mammalian cell diseases.

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Chemosterilization is reported in cattle fed chlortetracycline hydrochloride (CTC) at dosages ranging from 1.1mg/kg for 120 days to 11 mg/kg for 30-60 days. The relationship between plasma CTC drug concentration and carrier clearance has not been described. Chronic carrier status was established in 21 steers with a Virginia isolate of Anaplasma marginale and confirmed by cELISA and an A. marginale-specific RT-PCR. Four negative, splenectomized steers served as active disease transmission sentinels. Steers were randomized to receive 4.4 mg/kg/day (LD); 11 mg/kg/day (MD); or 22 mg/kg/day (HD) of oral chlortetracycline; or placebo (CONTROL) for 80 days. The LD, MD and HD treatment groups consisted of 5 infected steers and 1 splenectomized steer; CONTROL group had six infected steers and 1 splenectomized steer. The daily treatments and ration were divided equally and fed twice daily. Blood samples were collected semi-weekly for determining plasma drug concentration by ultrahigh performance liquid chromatography-mass spectrometry/mass spectrometry method and assessment of disease status by both cELISA and RT-PCR. Mean (CV%) chlortetracycline plasma drug concentrations in the LD, MD, and HD groups were 85.3 (28%), 214.5 (32%) and 518.9 (40%)ng/mL during days 4 through 53 of treatment. A negative RT-PCR assay result was confirmed in all CTC-treated groups within 49 days of treatment; however, cELISA required an additional 49 to 88 days before similar results. Subinoculation of splenectomized steers confirmed chemosterilization. These results are important for influencing future chemosterilization strategies and impacting free trade policy among countries and regions of contrasting endemicity.

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Previously we have shown that experimentally infected swine, fed an antibiotic-free diet, can become colonized and shed Escherichia coli O157:H7 for at least 2 months. However, in epidemiological studies this organism is only rarely recovered from domestic swine and the basis for this discrepancy is not clear. In this report we demonstrate that significantly fewer pigs fed diets containing subtherapeutic levels of either tylosin or chlorotetracycline shed E. coli O157:H7 for longer than 2 weeks compared to those fed an antibiotic-free diet. In contrast to tylosin and chlorotetracycline, the addition of bacitracin methylene disalicylate to the diet did not influence the recovery of E. coli O157:H7. These results suggest that some antibiotics may alter the gastrointestinal tract flora in ways that create a less favorable environment for E. coli O157:H7 in swine.

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