The In-Feed Antibiotic Carbadox Induces Phage Gene Transcription in the Swine Gut Microbiome.

Carbadox is a quinoxaline-di-N-oxide antibiotic fed to over 40% of young pigs in the United States that has been shown to induce phage DNA transduction in vitro; however, the effects of carbadox on swine microbiome functions are poorly understood. We investigated the in vivo longitudinal effects of carbadox on swine gut microbial gene expression (fecal metatranscriptome) and phage population dynamics (fecal dsDNA viromes). Microbial metagenome, transcriptome, and virome sequences were annotated for taxonomic inference and gene function by using FIGfam (isofunctional homolog sequences) and SEED subsystems databases. When the beta diversities of microbial FIGfam annotations were compared, the control and carbadox communities were distinct 2 days after carbadox introduction. This effect was driven by carbadox-associated lower expression of FIGfams (n = 66) related to microbial respiration, carbohydrate utilization, and RNA metabolism (q < 0.1), suggesting bacteriostatic or bactericidal effects within certain populations. Interestingly, carbadox treatment caused greater expression of FIGfams related to all stages of the phage lytic cycle 2 days following the introduction of carbadox (q ≤0.07), suggesting the carbadox-mediated induction of prophages and phage DNA recombination. These effects were diminished by 7 days of continuous carbadox in the feed, suggesting an acute impact. Additionally, the viromes included a few genes that encoded resistance to tetracycline, aminoglycoside, and beta-lactam antibiotics but these did not change in frequency over time or with treatment. The results show decreased bacterial growth and metabolism, prophage induction, and potential transduction of bacterial fitness genes in swine gut bacterial communities as a result of carbadox administration.IMPORTANCE FDA regulations on agricultural antibiotic use have focused on antibiotics that are important for human medicine. Carbadox is an antibiotic not used in humans but frequently used on U.S. pig farms. It is important to study possible side effects of carbadox use because it has been shown to promote bacterial evolution, which could indirectly impact antibiotic resistance in bacteria of clinical importance. Interestingly, the present study shows greater prophage gene expression in feces from carbadox-fed animals than in feces from nonmedicated animals 2 days after the initiation of in-feed carbadox treatment. Importantly, the phage genetic material isolated in this study contained genes that could provide resistance to antibiotics that are important in human medicine, indicating that human-relevant antibiotic resistance genes are mobile between bacteria via phages. This study highlights the collateral effects of antibiotics and demonstrates the need to consider diverse antibiotic effects whenever antibiotics are being used or new regulations are considered.

Disruption of spindle checkpoint function ahead of facilitation of cell proliferation by repeated administration of hepatocarcinogens in rats.

We aimed to clarify the hepatocarcinogen-specific disruption of cell cycle checkpoint functions and its time course after repeated administration of hepatocarcinogens. Thus, rats were repeatedly administered with hepatocarcinogens (methapyrilene, carbadox and thioacetamide), a marginal hepatocarcinogen (leucomalachite green), hepatocarcinogenic promoters (oxfendazole and ß-naphthoflavone) or non-carcinogenic hepatotoxicants (promethazine and acetaminophen) for 7, 28 or 90 days, and the temporal changes in cell proliferation, expression of G1/S and spindle checkpoint-related molecules, and apoptosis were examined using immunohistochemistry and/or real-time RT-PCR analysis. Hepatocarcinogens facilitating cell proliferation at day 28 of administration also facilitated cell proliferation and apoptosis at day 90. Hepatocarcinogen- or hepatocarcinogenic promoter-specific cellular responses were not detected by immunohistochemical single molecule analysis even after 90 days. Expression of Cdkn1a, Mad2l1, Chek1 and Rbl2 mRNA also lacked specificity to hepatocarcinogens or hepatocarcinogenic promoters. In contrast, all hepatocarcinogens and the marginally hepatocarcinogenic leucomalachite green induced Mdm2 upregulation or increase in the number of phosphorylated MDM2(+) cells from day 28, irrespective of the lack of cell proliferation facilitation by some compounds. However, different Tp53 expression levels suggest different mechanisms of induction or activation of MDM2 among hepatocarcinogens. On the other hand, hepatocarcinogenic methapyrilene and carbadox downregulated the number of both ubiquitin D(+) cells and proliferating cells remaining in M phase at day 28 and/or day 90, irrespective of the lack of cell proliferation facilitation in the latter. These results suggest that hepatocarcinogens disrupt spindle checkpoint function after 28 or 90 days of administration, which may be induced ahead of cell proliferation facilitation.

Lysozyme as an alternative to antibiotics improves growth performance and small intestinal morphology in nursery pigs.

Lysozyme is a 1,4-ß-N-acetylmuramidase that has antimicrobial properties. The objective of this experiment was to determine if lysozyme in nursery diets improved growth performance and gastrointestinal health of pigs weaned from the sow at 24 d of age. Two replicates of 96 pigs (192 total; 96 males, 96 females) were weaned from the sow at 24 d of age, blocked by BW and gender, and then assigned to 1 of 24 pens (4 pigs/pen). Each block was randomly assigned 1 of 3 dietary treatments for 28 d: control (two 14-d phases), control + antibiotics (carbadox/copper sulfate), or control + lysozyme (100 mg/kg diet). Pigs were weighed and blood sampled on d 0, 14, and 28 of treatment. Blood was analyzed for plasma urea nitrogen (PUN) and IgA. At 28 d, pigs were killed, and samples of jejunum and ileum were collected and fixed for intestinal morphology measurements. An additional jejunum sample was taken from the 12 pigs with the median BW per treatment to determine transepithelial electrical resistance (TER). Pigs consuming antibiotics or lysozyme grew at a faster rate than control pigs (0.433 ± 0.009 and 0.421 ± 0.008 vs. 0.398 ± 0.008 kg/d, respectively; P < 0.03), which resulted in heavier ending BW (20.00 ± 0.31, 19.8 ± 0.29, and 18.83 ± 0.32 kg, respectively; P < 0.03). Feed intake was not different (P > 0.48), but G:F was improved in pigs consuming antibiotics or lysozyme (0.756 ± 0.014, 0.750 ± 0.021, and 0.695 ± 0.019 kg/kg; P < 0.05). Immunoglobulin A (P < 0.03) and PUN (P < 0.01) increased during the experiment, regardless of dietary treatment (P > 0.48). Dietary treatment did not affect TER (P > 0.37), but gilts had lower TER compared with barrows (P < 0.04). No differences in villi height or crypt depth were observed in the ileum (P > 0.53). However, jejunum villi height was increased and crypt depth was decreased in pigs consuming antibiotics or lysozyme (P < 0.001), resulting in an increased villi height:crypt depth of 72% (P < 0.001). Thus, we concluded that lysozyme is a suitable alternative to carbadox/copper sulfate diets fed to pigs weaned from the sow at 24 d of age.

Controlling Salmonella infection in weanling pigs through water delivery of direct-fed microbials or organic acids: Part II. Effects on intestinal histology and active nutrient transport.

The objective of this study was to evaluate the effects of water-delivered, direct-fed microbials (DFM) or organic acids on intestinal morphology and active nutrient absorption in weanling pigs after deliberate Salmonella infection. Pigs (n = 88) were weaned at 19 ± 2 d of age and assigned to 1 of the following treatments, which were administered for 14 d: 1) control diet; 2) control diet + DFM (Enterococcus faecium, Bacillus subtilis, and Bacillus licheniformis) in drinking water at 10(9) cfu/L for each strain of bacteria; 3) control diet + organic acid-based blend (predominantly propionic, acetic, and benzoic acids) in drinking water at 2.58 mL/L; and 4) control diet + 55 mg/kg carbadox. Pigs were challenged with 10(10) cfu Salmonella enterica var Typhimurium 6 d after commencement of treatments. Pigs (n = 22/d) were harvested before Salmonella challenge and on d 2, 4, and 8 after challenge. Duodenal, jejunal, and ileal mucosal tissues were sampled for measurement of villus height and crypt depth. Jejunal tissue was sampled for determination of active nutrient absorption in modified Ussing chambers. Duodenal villus height was greater in pigs fed in-feed antibiotic before infection (P < 0.05). Jejunal crypts were deeper in DFM- and acid-treated pigs on d 4 after infection compared with all other treatments (P < 0.05). Salmonella infection resulted in a linear decrease in phosphorus (P < 0.001) and glucose (P < 0.05) active transport, and an increase (P < 0.001) in glutamine uptake immediately after challenge. Salmonella infection reduced basal short-circuit current (I(sc)); however, water-delivered DFM or organic acid treatments caused greater basal I(sc) on d 2 after challenge than did carbadox. Carbachol-induced chloride ion secretion was greatest in negative control pigs before infection (P < 0.01) and DFM-treated pigs (P < 0.05) after infection. In conclusion, both the DFM and acidification treatments induced increases in basal active ion movement and jejunal crypt depth, which could be interpreted as responses consistent with increased Salmonella pathology, but none of the additives markedly affected intestinal absorptive and secretory function in response to Salmonella challenge.

Controlling Salmonella infection in weanling pigs through water delivery of direct-fed microbials or organic acids. Part I: effects on growth performance, microbial populations, and immune status.

Pigs (n = 88) weaned at 19 ± 2 d of age were used in a 14-d study to evaluate the effects of water-delivered direct-fed microbials (DFM) or organic acids on growth, immune status, Salmonella infection and shedding, and intestinal microbial populations after intranasal inoculation of Salmonella Typhimurium (10(10) cfu/pig). Pigs were challenged with Salmonella 6 d after commencement of water treatments. Treatments were 1) control diet; 2) control diet + DFM (Enterococcus faecium, Bacillus subtilis, and Bacillus licheniformis) in drinking water at 10(9) cfu/L for each strain of bacteria; 3) control diet + an organic acid-based blend (predominantly propionic, acetic, and benzoic acid) in drinking water at 2.58 mL/L; and 4) control diet + 55 mg/kg of carbadox. Serum samples were taken on d 6, 8, 10, and 14 for determination of tumor necrosis factor α (TNFα) concentrations. Fecal samples were taken on d 0, 5, 7, and 11 for determination of Salmonella shedding and enumeration of coliforms. Pigs were euthanized on d 6, 8, 10, and 14. Intestinal and cecal tissue and digesta and mesenteric lymph nodes were sampled and analyzed for Salmonella. Duodenal, jejunal, and ileal mucosal scrapings were sampled for measurement of mucosal TNFα concentrations. Water delivery of DFM prevented a decline in ADG on d 2 to 6 postchallenge compared with the negative control (P < 0.05). Coliform counts tended to be greater (P = 0.09) in the cecum of the DFM treatment group on d 2 postinfection compared with the negative control and acid treatment groups. However, Salmonella prevalence in the feces, gastrointestinal tract, or lymph nodes was not affected by water delivery of acids or DFM. Serum and mucosal TNFα concentrations were not affected by treatment throughout the study with the exception of ileal concentrations on d 4 postchallenge, which were greater in the negative control group compared with all other treatments (P < 0.05). The in-feed antibiotic was the only treatment that reduced Salmonella prevalence and this was localized to the cecum on d 8 postinfection. In conclusion, the DFM and organic acid treatments used in this study offered little or no benefits to pigs infected with Salmonella and should not be considered under the constraints of this study as viable alternatives to in-feed antibiotics in a pathogen challenge situation.

Growth performance and gastrointestinal microbial ecology responses of piglets receiving Saccharomyces cerevisiae fermentation products after an oral challenge with Escherichia coli (K88).

The effects of Saccharomyces cerevisiae fermentation products (YFP) on growth performance and gastrointestinal (GIT) microbial ecology in 90 weanling pigs orally challenged with Escherichia coli K88(+) (ETEC) were investigated. The YFP were an original YFP product (XPC) and a water-suspendable yeast fermentation prototype (WSYFP) from a commercial company. Treatments consisted of a negative control (NC, no in-feed or in-water additive), carbadox (AB, 55 mg of carbadox/kg of feed), XPC (in feed, 0.2%), and WSYFP (in water, 0.5, 1, or 2 g/pig per day), and each was allotted to 5 pens (3 pigs/pen). The diets met the 1998 NRC specifications. Pigs were acclimated to treatments for a 7-d period before an ETEC challenge. On d 8, blood was collected from pigs to determine the baseline packed cell volume (PCV) measurement, and pigs were orally challenged with ETEC. At various time points postchallenge, blood samples were taken, performance measures and fecal consistency scores were recorded, and gut digesta and tissue samples were taken to evaluate GIT morphology, microbial ecology, and metabolites. Preplanned contrasts were used for comparison. Pigs receiving YFP had greater ADFI than NC pigs on d 3 (424 vs. 378 g/d; P = 0.01) and d 7 (506 vs. 458 g/d; P = 0.03) postchallenge. This effect of YFP on ADFI was similar to that of AB on d 3, but pigs receiving AB ate more (576 vs. 506 g/d; P = 0.03) at d 7 than pigs receiving YFP. Pigs exhibited reduced (P < 0.001) PCV upon ETEC challenge; however, pigs receiving additives sustained a greater (P < 0.05) PCV at 72 h compared with the NC group. Compared with the NC pigs, pigs receiving YFP showed a smaller (P < 0.05) number of ileal mucosa adherent ETEC and prevalence of the order Enterobacteriales in the ileal digesta, which corresponded to less (5.09 vs. 6.97 mg/dL; P = 0.03) colonic ammonia on d 7 postchallenge. Most of the indices for ileal digesta bacterial richness and diversity were greater (P < 0.01) for YFP pigs compared with NC pigs. However, results also indicated that the influence of YFP on the piglet intestinal microenvironment might differ when given in feed or water during ETEC challenge. In conclusion, pigs receiving YFP showed a better appetite in the presence of ETEC, which, together with the greater ileal digesta bacteria richness and diversity and decreased ETEC adhering to the mucosa and reduced colonic ammonia, indicates a healthier GIT environment.

Effect of supplementing zinc oxide and biotin with or without carbadox on nursery pig performance.

A 28-d nursery experiment was conducted to evaluate the effects of supplementing zinc oxide and biotin with or without a feed-grade antimicrobial agent (carbadox) on nursery pig performance, and plasma and fecal Zn concentrations. One hundred ninety-two crossbred pigs (initial BW = 5.94 +/- 0.03 kg; age = 17 +/- 2 d) were weaned and allotted to 1 of 8 dietary treatments based on BW, sex, and ancestry in a randomized complete block design (3 pigs/pen and 8 replications). Dietary treatments consisted of supplementation of ZnO at 0 or 3,000 mg/kg, d-biotin at 0 or 440 microg/kg, and carbadox at 0 or 55 mg/kg of diets in a 2 x 2 x 2 factorial arrangement of treatments. Phase 1 (d 0 to 14) and phase 2 (d 14 to 28) nursery diets were fed in meal form. Fecal samples were collected weekly, and blood samples were collected at d 0, 14, and 28 to determine fecal and plasma Zn concentrations, respectively. The basal diet contained 165 mg/kg of Zn as ZnSO(4) and 220 microg/kg biotin as d-biotin. Pigs supplemented with 440 microg/kg of d-biotin, independent of antibiotic and ZnO additions, had greater overall ADG (P = 0.02) than pigs fed no supplemental d-biotin postweaning. Overall ADG, ADFI, and G:F were not affected when pigs were supplemented with 3,000 mg/kg of Zn as ZnO or 55 mg/kg of carbadox. When pigs were fed 55 mg/kg of carbadox without supplemental biotin, plasma Zn concentration was less, whereas when biotin and carbadox were supplemented to nursery pig diets, plasma Zn concentrations did not decrease as with feeding carbadox alone (biotin x carbadox, P < 0.001). During wk 2, pigs fed 3,000 mg/kg of Zn as ZnO and 440 microg/kg of d-biotin had greater fecal Zn concentrations than pigs fed diets with only 3,000 mg/kg of Zn as ZnO (Zn x biotin, P = 0.04). In addition, pigs supplemented with 3,000 mg/kg of Zn as ZnO in combination with carbadox and d-biotin had greater fecal Zn concentrations compared with pigs fed diets containing no additional Zn during wk 2 (Zn x biotin x carbadox, P = 0.04). Fecal Zn concentrations increased over time (P < 0.001), and pigs fed supplemental ZnO had greater (P < 0.001) fecal Zn concentrations. These results indicate that feeding nursery pigs 440 microg/kg of d-biotin improved ADG, independent of zinc and carbadox supplementation, as well as increasing Zn excretion.

Effects of water and diet acidification with and without antibiotics on weanling pig growth and microbial shedding.

Two 5-wk experiments were conducted to determine the effects of water and diet acidification with and without antibiotics on weanling pig growth performance and microbial shedding. In Exp. 1, 204 pigs (19.2 d of age) were used in a 3 x 2 factorial, with 3 dietary treatments fed with or without water acidification (2.58 mL/L of a propionic acid blend; KEM SAN, Kemin Americas, Des Moines, IA). Dietary treatments were: 1) control, 2) control + 55 ppm of carbadox (CB), and 3) dietary acid [DA; control + 0.4% organic acid-based blend (fumaric, lactate, citric, propionic, and benzoic acids; Kemin Americas)] on d 0 to 7 followed by 0.2% inorganic acid-based blend (phosphoric, fumaric, lactic, and citric acids; Kemin Americas) on d 7 to 34. In Exp. 2, 210 pigs (average 18.3 d of age) were fed 1 of 3 dietary treatments: 1) control, 2) control + 55 ppm of CB, and 3) control + 38.6 ppm of tiamulin + 441 ppm of chlortetracycline on d 0 to 7 followed by 110 ppm of chlortetracycline on d 7 to 35 (TC) with or without dietary acidification (same as Exp. 1) in a 3 x 2 factorial arrangement of treatments. For both experiments, the pigs were allotted based on genetics, sex, and initial BW [5.5 kg (Exp. 1) or 5.6 kg (Exp. 2)]. Pigs were housed at 6 or 7 (Exp. 1) and 7 (Exp. 2) pigs/pen. Treatments were fed in 3 phases: d 0 to 7, 7 to 21, and 21 to 35 (34 d, Exp. 1). Fecal grab samples were collected from 3 pigs/pen on d 6, 20, and 33 for measurement of pH and Escherichia coli. During phase 3 and overall in Exp. 1, pigs fed CB had greater (P < 0.001) ADG (overall ADG, 389 vs. 348, and 348 g/d, respectively), ADFI (P < 0.007, 608 vs. 559, and 554 g/d, respectively), and d 34 BW (P < 0.001, 18.8 vs. 17.3, and 17.3 kg, respectively) than pigs fed NC and DA. Phase 3 ADG was improved (P < 0.01) by water acidification across all diets. In Exp. 2, pigs fed CB and TC had greater ADG (P < 0.004; 315 and 303 vs. 270 g/d, respectively), ADFI (P < 0.01), and d 35 BW (P < 0.002; 16.7 and 16.2 vs. 15.1 kg, respectively) than pigs fed NC. There was a tendency (P < 0.08) for an improvement in ADG when DA was added to the NC or TC, but decreased ADG when DA was added to CB.

Effects of Acid LAC and Kem-Gest acid blends on growth performance and microbial shedding in weanling pigs.

Weanling pigs with mean initial BW of 6.04 kg (Exp.1) and 5.65 kg (Exp. 2) and mean age at weaning of 18.2 d (Exp. 1) and 17.7 d (Exp. 2) were used in two 5-wk experiments (Exp. 1, n = 180; Exp. 2, n = 300) to evaluate the effects of an organic acid blend (Acid LAC, Kemin Americas Inc., Des Moines, IA) and an inorganic/organic acid blend (Kem-Gest, Kemin Americas Inc.) on weanling pig growth performance and microbial shedding. In Exp. 1, the 5 dietary treatments were 1) negative control, 2) diet 1 + 55 ppm carbadox, 3) diet 1 + 0.4% Acid LAC, 4) diet 1 + 0.2% Kem-Gest, 5) diet 1 + 0.4% Acid LAC and 0.2% Kem-Gest. In Exp. 2, the 6 dietary treatments were diets 1 through 4 corresponding to Exp. 1, plus 5) sequence 1: 0.4% Acid LAC for 7 d followed by 0.2% Kem-Gest for 28 d, and 6) sequence 2: 0.2% Kem-Gest for 7 d followed by 0.4% Acid LAC for 28 d. Pigs were housed at 6 (Exp. 1) or 10 (Exp. 2) pigs/pen. Treatments were fed throughout the experiment in 3 phases: d 0 to 7, d 7 to 21, and d 21 to 35. In Exp. 1, there were no differences (P > 0.05) in ADG, ADFI, or G:F among the dietary treatments at any time during the study. In Exp. 2, throughout the study, pigs fed carbadox (diet 2) and sequence 1 (diet 5) diets had the greatest ADG (d 0 to 35; 262, 294, 257, 257, 292, and 261 g/d, diets 1 through 6, respectively; P < 0.05), greater ADFI than all other acid treatments (P < 0.05), and tended to have greater ADFI than diet 1 (P < 0.10). Fecal pH, Escherichia coli concentrations, and Salmonella presence were determined at d 6, 20, and 34 for Exp. 1, and on d 32 for Exp. 2. For both experiments, there was no effect of treatment on the presence of fecal Salmonella (P > 0.10) at any sampling time. In Exp. 1, fecal E. coli concentrations for pigs fed the carbadox (P < 0.05) diet were greater than for pigs fed the combination diet with 0.4% Acid LAC and 0.2% Kem-Gest on d 34, and the pigs fed the negative control diet tended (P < 0.10) to have greater fecal E. coli concentrations than those fed the combination diet on d 34. In Exp. 2, fecal pH of pigs fed sequence 1 tended to be greater than fecal pH of pigs fed diet 1, diet 4, or sequence 2 (P < 0.10), but there was no dietary effect on fecal E. coli. In Exp. 1, growth performance of pigs fed the Acid LAC and Kem-Gest diets was similar to each other and to that of the carbadox-fed pigs. Adding the combination of 0.4% Acid LAC and 0.2% Kem-Gest to nursery pig diets reduced ADFI and pig growth rate. In Exp. 2, pigs fed the acid sequence of Acid LAC-Kem-Gest had similar growth performance to pigs fed carbadox, and this novel dietary acid sequence may have merit as a replacement for antibiotics in the nursery phase.

Susceptibility of Escherichia coli from growing piglets receiving antimicrobial feed additives.

Concerns regarding an apparent association between the use of antimicrobial feed additives (AFAs) in food animal production and a concomitant increase in antimicrobial drug resistance among zoonotic enteropathogens have provided the impetus to propose cessation of their use. While AFAs have been used in food animal production for nearly 50 years, the future use of AFAs will require an understanding of the effects of different classes of antimicrobials on the antimicrobial resistance of commensal flora. The present study examines the effect of three AFAs (apramycin, carbadox, and chlortetracycline) on the antimicrobial susceptibility of Escherichia coli in growing piglets and on animal performance. Three replicate trials were conducted using growing piglets fed standard diets with and without antimicrobial feed additives (AFAs). Fecal samples were cultured selectively for E. coli at regular intervals from all piglets from birth to market and antimicrobial susceptibility testing of E. coli isolates was performed using a replica-plate screening method and a broth microdilution method. While resistance to tetracycline in E. coli varied widely by sample, group, and trial, a significant increase in the percentage of resistant isolates was observed in piglets receiving AFAs when compared to controls (p < 0.0001). Resistance to apramycin increased in E. coli from piglets fed apramycin when compared to controls (p < 0.0001). However, upon removal of apramycin, resistance in E. coli declined to baseline levels by day 75. Piglets receiving AFAs demonstrated improved feed efficiency during phase 4 (p < 0.001), and higher average daily gains in phases 3 and 4 (p < 0.0001). This study suggests that antimicrobial resistance to AFAs in E. coli is drug-dependent and that some antimicrobials may be suitable for continued use in feeds during specified growth periods without concern for persistence of resistant E. coli populations.

The effect of antimicrobial growth promoter withdrawal on the health of weaned pigs in Finland.

The use of the antimicrobial growth promoters (AGPs) carbadox and olaquindox has been banned in the European Union (EU) since September 1999. We studied the effects of the withdrawal on the health of weaned piglets on two types of piglet-producing farms (farrowing herds and farrow-to-finish herds) from the different regions of Finland. Farms with no major problems with post-weaning diarrhoea were selected for the study to better evaluate the effect of AGPs alone. Data on production, medication and incidence of diarrhoea were collected from 73 farms during 1 year after the withdrawal. On 29 of these farms, the data collection began 4 months before the withdrawal. The health management of the pigs is considered good in Finland, and special attention has been paid to improve the husbandry practices and management of the farms. Eighty-two percent of the farms in the study were free of both Mycoplasma hyopneumoniae and Sarcoptes scabiei infection. Brachyspira hyodysenteriae infection was not detected in any of the farms. The median number of sows in the herds was 56.0 (IQR=43.0; 72.5) in 2000. The level of antimicrobial use in each herd was classified as low, moderate and high when the percentage of weaned pigs treated for diarrhoea during a 4-month period was 0-5%, 6-19% and > or =20%, respectively. Only on four herds (14%), there was an increase in the level of antibiotic use after the AGP withdrawal, when seasonally corresponding 4-month periods were compared. Fourty-one percent of these 29 farms were categorized as low users of antimicrobials, 38% as moderate users and 21% as high users. The level of antimicrobial use for treatment of diarrhoea after weaning (and the incidence of diarrhoea in weaned piglets) did not increase significantly after the withdrawal of AGPs from weaner feeds according to farmers' evaluations. In this study, the Escherichia coli infection was the most-common cause of diarrhoea in weaned pigs. The age at weaning did not change after the withdrawal of AGPs.

Quinoxaline-2-carboxylic acid in pigs: criteria to distinguish between the illegal use of carbadox and environmental contamination.

Carbadox cannot be used in food-producing animals within the European Union following the adoption of Commission Regulation EC 2788/98/EC. Monitoring of the longest remaining residue--quinoxaline-2-car-boxylic acid (QCA)--is the most effective way of enforcing the prohibition on its use. The study was under taken to determine if QCA could be passed from pig to pig following the exposure of unmedicated animals to housing that had previously contained medicated animals. Drug-withdrawal studies were also carried out on medicated animals. Distinction between treated animals and those exposed to QCA might be required by competent national authorities to determine whether a positive result for QCA in tissue is truly 'violative'. Comparison of the ratio concentrations of QCA in tissues and body fluids was made to determine if they, could be used as criteria for discrimination between illegally treated animals and environmental contamination.

Effect of dietary mannanoligosaccharide and sodium chlorate on the growth performance, acute-phase response, and bacterial shedding of weaned pigs challenged with Salmonella enterica serotype Typhimurium.

A 28-d experiment evaluated the growth, acute-phase response, and bacterial shedding patterns in pigs (n = 96; initially 6.8 +/- 1.3 kg) fed mannanoligosaccharides (MANNAN) and sodium chlorate (CHLORATE) before and after oral challenge with Salmonella enterica serotype Typhimurium (ST). The negative control diet contained no antimicrobial (CON), and the positive control contained carbadox (CARB; 55 ppm). Test diets contained (as-fed basis) MANNAN (1,500 ppm) or CHLORATE (800 ppm). Pigs were fed diets for 14 d and then given ST orally. Pigs fed CARB had greater ADG over the entire study than pigs from other treatments (P < 0.05). During wk 1 to 2, before ST challenge, feed intake (as-fed basis) was lower for pigs fed MANNAN and CHLORATE than pigs fed CARB (P < 0.05). During the final 2 wk, pigs fed CARB had greater feed intake than pigs on other treatments (P < 0.05). Gain/feed was greater for pigs fed CARB in the 2 wk before ST (P < 0.05); however, in wk 3 to 4 after ST, gain/feed was reduced for CON pigs compared to pigs on other treatments (P < 0.05). Serum IGF-I was decreased at 2 and 4 d after ST (P < 0.001), and, overall, IGF-I was greater in pigs fed CARB than CON or CHLORATE (P < 0.05). Serum haptoglobin concentrations were greater (P < 0.001) for all treatments at d 6 compared with d 13 after ST. Overall, haptoglobin was greater for MANNAN than for CARB and CHLORATE (P < 0.05) and tended to be increased (P < 0.06) relative to CON. Interleukin-6 was not affected by treatment or day post-ST challenge. Fecal shedding of salmonellae organisms was less for CHLORATE (P < 0.05) than all other treatments at 7 d after ST. Shedding scores decreased from d 7 to 14 after ST (P < 0.05) for the CON, CARB, and MANNAN treatments. We conclude that feeding MANNAN and CHLORATE before acute enteric disease challenge may support improved gut function as evidenced by improved gain/feed, and that CHLORATE may decrease bacterial shedding. But neither MANNAN nor CHLORATE enhanced growth relative to the absence of dietary antimicrobials, nor was either treatment as effective as CARB following ST challenge.

Brewers dried yeast as a source of mannan oligosaccharides for weanling pigs.

Brewers dried yeast, a source of mannan oligosaccharides (MOS), was assessed as an alternative to an antimicrobial agent (carbadox) for young pigs in two experiments. The yeast contained 5.2% MOS. Agglutination tests confirmed adsorption of several serovars of E. coli and Salmonella spp. onto the yeast product. In Exp. 1, seven replicates (five pigs per pen) of 22-d-old pigs were fed a nonmedicated basal diet or the basal diet with carbadox (55 mg/kg), yeast (3%), or a combination of 3% yeast and 2% citric acid for 28 d. Carbadox did not improve growth performance. Growth rate and feed intake were depressed (P < 0.05) in pigs fed yeast alone or in combination with acid. Log counts of total coliforms, Escherichia coli, and Clostridium perfringens in feces were not affected by diet, but Bifidobacteria spp. counts were lower (P < 0.05) in pigs fed the yeast + acid diet and lactobacilli counts were higher (P < 0.05) in pigs fed yeast. Fecal pH and VFA concentrations and intestinal morphological traits were not consistently affected by diet. Serum IgG levels were elevated in the yeast + acid (P < 0.01) group. In Exp. 2, the effects of yeast and carbadox additions to the diet on enteric microbial populations in young pigs housed in isolation units were evaluated. Pigs (n = 24) were weaned at 11 d of age (4.1 kg BW) and placed in isolation chambers (two pigs per chamber) equipped with individual air filtering systems and excrement containers. Treatments were a nonmedicated basal diet and the basal diet with 55 mg/kg of carbadox or with 3% yeast. Diets were fed for 29 d, then each pig was orally dosed with approximately 9.5 x 10(8) CFU of E. coli K88. Daily fecal E. coli K88 counts were not different (P > 0.05) among treatments, but fecal shedding of carbadox-resistant coliforms was higher (P < 0.01) during the 9-d period in pigs fed carbadox. Total fecal coliforms were consistently lower throughout the postinoculation period in pigs fed yeast (P < 0.05). Yeast reduced colonization oftotal coliforms in the duodenum,jejunum, cecum, and colon, but it did not have a consistent effect on colonization of E. coli K88. Pigs fed yeast tended (P < 0.10) to have higher serum IgG levels than controls. In these experiments, brewers dried yeast and carbadox had minimal effects on growth, microbial populations, and intestinal health traits of early-weaned pigs, but certain serum immunological traits were enhanced by feeding yeast.

Changes in plasma renin activity and renal immunohistochemically demonstrated renin in carbadox treated pigs.

Carbadox is known to induce toxic effects on the adrenal cortex, resulting in hypoaldosteronism. To study the involvement of carbadox on the renin-angiotensin system, weaned piglets of five weeks old received feed supplemented with 0 (control group), 50, 100, 150 or 200 ppm carbadox. After four weeks the 100 and 150 ppm groups had significantly higher plasma renin activity levels than the control group and after nine weeks plasma renin activity levels of all treated groups were significantly higher than the control group. Five and 10 weeks after carbadox administration, three and two pigs, respectively, of all groups were necropsied and the kidneys were screened for immunohistochemically demonstrated renin. All dosed pigs demonstrated an increase of immunoreactive renin, which was dose- and time-related. From these results it is concluded that carbadox induces activation of the renin-angiotensin system, secondary to the suppressing effect on mineralocorticoid secretion and that these changes may be responsible for part of the clinical picture.

Persistence of carbadox-induced adrenal lesions in pigs following drug withdrawal and recovery of aldosterone plasma concentrations.

This study was performed to investigate the persistence of carbadox-induced adrenal lesions in pigs after withdrawal of the drug. Six groups (N = 13) received 0 (control group), 25, 50, 100 and 200 ppm carbadox. After 10 weeks, carbadox was withdrawn from the feed. Five and 11 weeks after withdrawal, two pigs per group were necropsied and the adrenals were examined histologically. Five weeks after withdrawal, recovery of lesions was seen in the 25 and 50 ppm groups. In the 100 and 150 ppm groups, adrenal changes were still present. After 11 weeks an incomplete recovery occurred in the 100 ppm group and in one of the pigs from the 150 ppm group; the second pig of this group still demonstrated moderate changes. Pigs from the 200 ppm group showed severe changes and absence of a clear zonal differentiation. Plasma aldosterone values started to recover 2 weeks after withdrawal of carbadox. Histological examination suggested stimulation of the aldosterone-producing glomerular zone, eventually resulting in regressive changes. The mechanisms that possibly induced this continuous stimulation are discussed.

Some pharmacokinetic observations of carbadox medication in pigs.

Concentrations of carbadox and a first metabolite, desoxycarbadox, were measured in contents of the porcine gastrointestinal tract after in-feed administration of carbadox in therapeutic dosages (100-150 ppm). The levels of carbadox in the relevant parts of the gastrointestinal tract were found to be lower than the MIC-values reported for enteropathogenic microorganisms at their sites of action. The presented observations do not provide a pharmacological rationale for the therapeutic use of carbadox in the treatment of dysentery and diarrhoea in swine. The carbadox levels encountered in the proximal part of the gut (stomach, duodenum) however, seem to indicate that in-feed administration of 50 ppm carbadox can provide an effective prophylaxis against Treponema hyodysenteriae, a causative agent in swine dysentery. The timecourse of the blood levels of carbadox and desoxycarbadox after in-feed administration of carbadox (50 ppm) and the concentration profiles in the gastrointestinal tract are discussed with regard to the disposition of this drug in pigs.

Pathological effects of carbadox in pigs with special emphasis on the adrenal.

Weaned pigs, 4-weeks-old, were divided into 6 groups of 13 animals each, which received 0, 25, 50, 100, 150 and 200 ppm (mg per kg) of carbadox medicated feed, respectively. After 5 and 10 weeks of carbadox administration, three and two pigs, respectively, of each group were necropsied. After 5 weeks treatment, gross lesions were seen in pigs receiving 50 ppm or more. The main features were retarded growth, dehydration with dry contents in the intestine, especially in the colon and findings suggestive of pica. The severity of lesions increased with higher dosages. After 10 weeks, the same changes, though much more pronounced, were observed at 100 ppm or higher dosages. After 5 weeks histological changes in the adrenals were found at 50 ppm treatment and upwards. The common feature was a hydropic appearance of the glomerular zone. In the 50 ppm group one out of three and, in the higher dosed groups, all pigs showed these changes. There was a dose-response effect. At 100 ppm or more an enlargement of the glomerular zone was observed, resulting in narrowing of the fascicular layer. The adrenal capsule was slightly thickened and contained cells with PAS-positive granules. After 10 weeks, changes were found at 25 ppm dosage and higher. In the 25 and 50 ppm group half of the pigs had hydropic changes of the glomerular zone. In the higher dosed groups there were also chronic lesions. The outer part of the glomerular zone had become fibrotic. With 150 ppm or more the hydropic changes had extended into the fascicular layer, with development of hyperplastic nodules. This led to disappearance of zonal differentiation. From 100 ppm dosage, many richly granulated PAS-positive cells were present in the thickened capsule, more numerous and more granulated than after 5 weeks treatment. From this study, it can be concluded that carbadox may induce adverse effects on the adrenal in growing pigs at therapeutic (100 to 150 ppm) and feed-additive doses (50 ppm). Even at lower doses (25 ppm), mild lesions were found. The grade of lesions was positively correlated with the duration of exposure to this growth promoter.