Carbadox

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 transductionin vitro; however, the effects of carbadox on swine microbiome functions are poorly understood. We investigated thein vivolongitudinal 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 2days 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.IMPORTANCEFDA 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.

Two experiments were conducted to evaluate the responses to carbadox and Cu additions in the postweaning diet of swine. The first trial contained 470 pigs in five replicates in a 2 X 5 factorial arrangement in a randomized complete block design. Weanling pigs 4-wk of age were fed diets containing 0 or 55 ppm carbadox and 0, 125, 375 or 500 ppm Cu for a 5-wk period. Copper levels of 125 and 250 ppm resulted in improved pig gains and feed intakes, but at 500 ppm, gains and feed intake declined. Carbadox resulted in enhanced gain and feed performance throughout the trial, but most notably during the initial 2-wk period. Copper improved performance measurements only during the latter 3-wk and for the overall period. There was an additive performance response when carbadox and Cu (125 ppm) were provided in combination. When carbadox was not provided, growth responses increased to the 250 ppm dietary Cu level. Liver, kidney cortex, heart and plasma Cu concentrations increased quadratically as dietary Cu levels increased, with the greatest change occurring between 250 and 500 ppm dietary Cu levels. A N and Cu balance trial in group feeding conditions involving 65 pigs was conducted in two replicates of a 2 X 2 X 5 factorial arrangement of a split-block design. Pigs were ad libitum fed diets with or without 250 ppm Cu and carbadox at 0 or 55 ppm for a 5-wk period. A fifth treatment group fed the 250 ppm Cu plus carbadox diet was pair-fed to the pigs fed the basal treatment. Growth rate and N retention increased when carbadox, but not when Cu was provided. When carbadox and Cu were provided in combination, either ad libitum or pair-fed, N retention was greater than when the basal diet was fed. This response was attributed to the carbadox addition. The carbadox addition reduced Cu retention and liver Cu concentrations.

Abused or misused carbadox (CBX) and cyadox (CYA) in animal feed may cause food safety concerns, threatening human health. Here, we describe the design of a novel hapten for preparation of a monoclonal antibody against CBX and CYA simultaneously. Using this antibody, colloidal gold immunochromatographic assay (GICA) was developed for screening of CBX and CYA residues in chicken breast. Under optimal conditions, semiquantitative analysis results were visible by eye, with a visual limit of detection of 8 μg/kg for CBX and CYA, and cut-off values of 20 μg/kg for CBX and 40 μg/kg for CYA in chicken breast. Quantitative analysis could be performed using a hand-held strip scanner, with a calculated limit of detection of 2.92 μg/kg for CBX and 2.68 μg/kg for CYA in chicken breast. Validated by liquid chromatography-MS/MS, the developed GICA provides a useful tool for rapid on-site CBX and CYA residue screening in chicken breast.