Ceftiofur in swine manure contributes to reducing pathogens and antibiotic resistance genes during composting.

Aerobic composting is a common way for the disposal of feces produced in animal husbandry, and can reduce the release of antibiotic resistance genes (ARGs) from feces into the environment. In this study, we collected samples from two distinct treatments of swine manure compost with and without ceftiofur (CEF), and identified the ARGs, mobile genetic elements (MGEs), and bacterial community by metagenomic sequencing. The impacts of CEF on the bacterial community composition and fate of ARGs and MGEs were investigated. With increasing composting temperature and pH, the concentration of CEF in the manure decreased rapidly, with a degradation half-life of 1.12 d and a 100% removal rate after 10 d of aerobic composting. Metagenomics demonstrated that CEF in the manure might inhibit the growth of Firmicutes and Proteobacteria, thereby reducing some ARGs and MGEs hosted by these two bacteria, which was further confirmed by the variations of ARGs and MGEs. A further redundancy analysis suggested that pH and temperature are key environmental factors affecting ARG removal during composting, and intI1 and bacterial communities also have significant influence on ARG abundance. These results are of great significance for promoting the removal of some ARGs from animal manure by controlling some key environmental factors and the type of antibiotics used in animals.

Clinical Breakpoint of Apramycin to Swine Salmonella and Its Effect on Ileum Flora.

The purpose of this study was to establish the clinical breakpoint (CBP) of apramycin (APR) against Salmonella in swine and evaluate its effect on intestinal microbiota. The CBP was established based on three cutoff values of wild-type cutoff value (COWT), pharmacokinetic-pharmadynamic (PK/PD) cutoff value (COPD) and clinical cutoff value (COCL). The effect of the optimized dose regimen based on ex vivo PK/PD study. The evolution of the ileum flora was determined by the 16rRNA gene sequencing and bioinformatics. This study firstly established the COWT, COPD in ileum, and COCL of APR against swine Salmonella, the value of these cutoffs were 32 µg/mL, 32 µg/mL and 8 µg/mL, respectively. According to the guiding principle of the Clinical Laboratory Standards Institute (CLSI), the final CBP in ileum was 32 µg/mL. Our results revealed the main evolution route in the composition of ileum microbiota of diarrheic piglets treated by APR. The change of the abundances of Bacteroidetes and Euryarchaeota was the most obvious during the evolution process. Methanobrevibacter, Prevotella, S24-7 and Ruminococcaceae were obtained as the highest abundance genus. The abundance of Methanobrevibacter increased significantly when APR treatment carried and decreased in cure and withdrawal period groups. The abundance of Prevotella in the tested groups was significantly lower than that in the healthy group. A decreased of abundance in S24-7 was observed after Salmonella infection and increased slightly after cure. Ruminococcaceae increased significantly after Salmonella infection and decreased significantly after APR treatment. In addition, the genera of Methanobrevibacter and Prevotella were defined as the key node. Valine, leucine and isoleucine biosynthesis, D-Glutamine and D-glutamate metabolism, D-Alanine metabolism, Peptidoglycan and amino acids biosynthesis were the top five Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the ileum microbiota of piglets during the Salmonella infection and APR treatment process. Our study extended the understanding of dynamic shift of gut microbes during diarrheic piglets treated by APR.

The adsorption-desorption characteristics and degradation kinetics of ceftiofur in different agricultural soils.

Cephalosporins are one of the most widely used antibiotics. When cephalosporins are discharged into the environment, they not only induce the production of antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARBs) but also cause toxic effects on animals and plants. Due to their complicated environmental behavior and lack of relevant data, the environmental behavior remains unclear. In this study, the adsorption-desorption and degradation characteristics of the third-generation cephalosporin drug ceftiofur (CEF) were investigated in three agricultural soils (sandy loam, loam and clay). According to the relevant parameters of the Freundlich adsorption isotherm (the Kf range was 57.63-122.44 µg1-1/n L1/n kg-1), CEF was adsorbed moderately in the soils and had the potential to migrate into groundwater. CEF exhibited low persistence in the soils and faster degradation than other antibiotics, such as tetracyclines and fluoroquinolones. The degradation half-lives (DT50) of CEF in soils ranged from 0.76 days to 4.31 days. Adding feces, increasing the water content, providing light and increasing the temperature significantly accelerated the degradation of CEF in soils. The DT50 values of CEF in soils were significantly prolonged when the soils were sterilized, indicating that both physical degradation and biodegradation played important roles in the degradation of CEF in soils. The DT50 values of CEF in soils were significantly prolonged at high concentrations, indicating that the degradability of CEF in soils was affected by the initial concentration. No significant differences were observed in the DT50 values for the different soil types (p > 0.05). This study provides useful information about the environmental behavior of CEF and improves the environmental risk assessment of CEF.

Adsorption/desorption and degradation of doxycycline in three agricultural soils.

Veterinary antibiotics are widely used in animal agriculture. Owing to its good absorption in the gastrointestinal tract, strong tissue permeability, and long biological half-life, doxycycline (DOX) is widely used to treat bacterial infections; however, this use can pose an environmental risk. The adsorption/desorption and degradation of DOX in three agricultural soils were investigated. DOX rapidly adsorbed to the soils, with an adsorption equilibrium time of 12 h for the three soils. The Freundlich equation was used to fit the adsorption and desorption of DOX in soils. A high Freundlich affinity coefficient (KF) was obtained from Freundlich isotherms, indicating strong sorption of DOX to agricultural soils and weak mobility to aquatic environment. Soil organic matter, the clay ratio and the cation exchange capacity were significantly positively correlated with KF (P < 0.05). The half-life (DT50) of DOX degradation in the soils ranged from 2.51 to 25.52 d. Soil microorganisms, soil moisture, temperature, the initial concentration, illumination and soil texture all significantly affected the degradation of DOX in soil (P < 0.05). When 8% (w/w) manure was added, DOX degradation was significantly accelerated (P < 0.05). Biotic and abiotic factors affected the degradation of DOX in soils. These results indicated that soil properties and environmental conditions greatly affected the fate and transport of DOX into agricultural soils.

The Evolution of Fluoroquinolone Resistance in Salmonella under Exposure to Sub-Inhibitory Concentration of Enrofloxacin.

The evolution of resistance in Salmonella to fluoroquinolones (FQs) under a broad range of sub-inhibitory concentrations (sub-MICs) has not been systematically studied. This study investigated the mechanism of resistance development in Salmonella enterica serovar Enteritidis (S. Enteritidis) under sub-MICs of 1/128×MIC to 1/2×MIC of enrofloxacin (ENR), a widely used veterinary FQ. It was shown that the resistance rate and resistance level of S. Enteritidis varied with the increase in ENR concentration and duration of selection. qRT-PCR results demonstrated that the expression of outer membrane porin (OMP) genes, ompC, ompD and ompF, were down-regulated first to rapidly adapt and develop the resistance of 4×MIC, and as the resistance level increased (≥8×MIC), the up-regulated expression of efflux pump genes, acrB, emrB amd mdfA, along with mutations in quinolone resistance-determining region (QRDR) gradually played a decisive role. Cytohubba analysis based on transcriptomic profiles demonstrated that purB, purC, purD, purF, purH, purK, purL, purM, purN and purT were the hub genes for the FQs resistance. The 'de novo' IMP biosynthetic process, purine ribonucleoside monophosphate biosynthetic process and purine ribonucleotide biosynthetic process were the top three biological processes screened by MCODE. This study first described the dynamics of FQ resistance evolution in Salmonella under a long-term selection of sub-MICs of ENR in vitro. In addition, this work offers greater insight into the transcriptome changes of S. Enteritidis under the selection of ENR and provides a framework for FQs resistance of Salmonella for further studies.

Single and ternary competitive adsorption-desorption and degradation of amphenicol antibiotics in three agricultural soils.

The widespread usage of veterinary antibiotics results in antibiotic contamination and increases environmental risks. This study was evaluated the single and ternary competitive adsorption-desorption and degradation of three amphenicol antibiotics (AMs): chloramphenicol (CAP), thiamphenicol (TAP), and florfenicol (FF) in three agricultural soils. The adsorption capacity of amphenicol antibiotics in the soil was weak, and the Kf value was in the range of 0.15-3.59 µg1-1/nL1/n kg-1. In the single adsorption-desorption experiment, the ranked order of adsorption capacity was TAP > FF > CAP. However, in the ternary competitive adsorption experiment, the order was changed to be CAP > FF > TAP. The degradation of AMs in soils was performed at various conditions. All AMs were vulnerable to microbial degradation in soils. A higher initial concentration would reduce the degradation rate and enhance the persistence of AMs in soil. The degradation of AMs was positively influenced by changes in soil moisture content and culture temperatures up to 30 °C and decreased at higher temperatures. An equation was used to predict the leachability of AMs in soils and assess their risk to the water environment. The weak adsorption capacity and poor persistence of FF indicated that it may have a strong effect on groundwater based on the equation. It is imperative to further assess the biological impacts of FF at environmentally relevant concentrations given its mobility and extensive use in the livestock industry.

The Effects of Natural Products and Environmental Conditions on Antimicrobial Resistance.

Due to the extensive application of antibiotics in medical and farming practices, the continued diversification and development of antimicrobial resistance (AMR) has attracted serious public concern. With the emergence of AMR and the failure to treat bacterial infections, it has led to an increased interest in searching for novel antibacterial substances such as natural antimicrobial substances, including microbial volatile compounds (MVCs), plant-derived compounds, and antimicrobial peptides. However, increasing observations have revealed that AMR is associated not only with the use of antibacterial substances but also with tolerance to heavy metals existing in nature and being used in agriculture practice. Additionally, bacteria respond to environmental stresses, e.g., nutrients, oxidative stress, envelope stress, by employing various adaptive strategies that contribute to the development of AMR and the survival of bacteria. Therefore, we need to elucidate thoroughly the factors and conditions affecting AMR to take comprehensive measures to control the development of AMR.

Construction of an Electrochemical Receptor Sensor Based on Graphene/Thionine for the Sensitive Determination of ß-Lactam Antibiotics Content in Milk.

In antibiotics, ß-lactam is one kind of major concern acknowledged as an unavoidable contaminant in milk. Thus, a facile and sensitive method is essential for rapid ß-lactam antibiotics detection. In our work, a specific electrochemical receptor sensor based on the graphene/thionine (GO/TH) composite was established. The mechanism of the electrochemical receptor sensor was a direct competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP-AMP) to the mutant BlaR-CTD protein by free ß-lactam antibiotics. Then, horseradish peroxidase (HRP) catalyzed the hydrolysis of the substrate hydrogen peroxide (H2O2), which produced an electrochemical signal. Under optimal experimental conditions, this method could quantitatively detect cefquinome from 0.1 to 8 µg L-1 and with the limit of detection (LOD) of 0.16 µg L-1, much lower than the maximum residue limit (MRL) of 5 µg L-1 set by the European Union. In addition, the LOD of spiked milk samples with cefalexin, cefquinoxime, cefotafur, penicillin G and ampicillin were 14.88 µg L-1, 2.46 µg L-1, 17.16 µg L-1, 0.06 µg L-1, 0.21 µg L-1 and the limits of quantitation (LOQ) were 36.09 µg L-1, 5.40 µg L-1, 41.45 µg L-1, 0.13 µg L-1, 0.42 µg L-1, respectively. The sensor showed a favorable recovery of 84.89-102.44%. Moreover, the electrochemical receptor sensor was successfully applied to assay ß-lactam antibiotics in milk, which showed good correlation with the results obtained from liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Resistance and Virulence Mechanisms of Escherichia coli Selected by Enrofloxacin in Chicken.

This study aimed to investigate the genetic characteristics, antibiotic resistance patterns, and novel mechanisms involved in fluoroquinolone (FQ) resistance in commensal Escherichia coli isolates. The E. coli isolates were recovered from a previous clinical study and subjected to antimicrobial susceptibility testing and molecular typing. Known mechanisms of FQ resistance (target site mutations, plasmid-mediated quinolone resistance [PMQR] genes, relative expression levels of efflux pumps and porins) were detected using DNA sequencing of PCR products and real-time quantitative PCR. Whole-genome shotgun sequencing was performed on 11 representative strains to screen for single nucleotide polymorphisms (SNPs). The function of a key SNP (A1541G) was investigated by site-directed mutagenesis and allelic exchange. The results showed that long-term enrofloxacin treatment selected multidrug-resistant (MDR) E. coli isolates in the chicken gut and that these E. coli isolates had diverse genetic backgrounds. Multiple genetic alterations, including double mutations on GyrA (S83L and D87N), a single mutation on ParC (S80I) and ParE (S458E), activation of efflux pumps, and the presence of the QnrS1 protein, contributed to the high-level FQ resistance (enrofloxacin MIC [MICENR] ≥ 128 µg/ml), while the relatively low-level FQ resistance (MICENR = 8 or 16 µg/ml) was commonly mediated by decreased expression of the porin OmpF, besides enhancement of the efflux pumps. No significant relationship was observed between resistance mechanisms and virulence genes. Introduction of the A1541G mutation on aegA was able to increase FQ susceptibility by 2-fold. This study contributes to a better understanding of the development of MDR and the differences underlying the mechanisms of high-level and low-level FQ resistance in E. coli.

Magnetic solid-phase extraction based on carbon nanotubes for the determination of polyether antibiotic and s-triazine drug residues in animal food with LC-MS/MS.

Carbon nanotubes-magnetic nanoparticles, comprising ferroferric oxide nanoparticles and carbon nanotubes, were prepared through a simple one-step synthesis method and subsequently applied to magnetic solid-phase extraction for the determination of polyether antibiotic and s-triazine drug residues in animal food coupled with liquid chromatography with tandem mass spectrometry. The nanocomposites were characterized by transmission electron microscopy, X-ray diffraction, and vibrating sample magnetometry. The components within the nanocomposites endowed the material with high extraction performance and manipulative convenience. Compared with carbon nanotubes, the as-prepared carbon nanotubes-magnetic nanoparticles showed better extraction and separation efficiencies for polyether antibiotics and s-triazine drugs thanks to the contribution of the iron-containing magnetic nanoparticles. Various experimental parameters affecting the extraction efficiency had been investigated in detail. Under the optimal conditions, the good linearity ranging from 1 to 200 µg/kg for diclazuril, toltrazuril, toltrazuril sulfone, lasalocid, monensin, salinomycin, narasin, nanchangmycin, and maduramicin, low limits of detection ranging from 1 to 5 µg/kg, and satisfactory spiked recoveries (77.1-91.2%, with the inter relative standard deviation values from 4.0 to 12.2%) were shown. It was confirmed that this novel method was an efficient pretreatment and enrichment procedure and could be successfully applied for extraction and determination of polyether and s-triazine drug residues in complex matrices.

The critical role of oxidative stress in the toxicity and metabolism of quinoxaline 1,4-di-N-oxides in vitro and in vivo.

Quinoxaline 1,4-dioxide derivatives (QdNOs) have been widely used as growth promoters and antibacterial agents. Carbadox (CBX), olaquindox (OLA), quinocetone (QCT), cyadox (CYA) and mequindox (MEQ) are the classical members of QdNOs. Some members of QdNOs are known to cause a variety of toxic effects. To date, however, almost no review has addressed the toxicity and metabolism of QdNOs in relation to oxidative stress. This review focused on the research progress associated with oxidative stress as a plausible mechanism for QdNO-induced toxicity and metabolism. The present review documented that the studies were performed over the past 10 years to interpret the generation of reactive oxygen species (ROS) and oxidative stress as the results of QdNO treatment and have correlated them with various types of QdNO toxicity, suggesting that oxidative stress plays critical roles in their toxicities. The major metabolic pathways of QdNOs are N→O group reduction and hydroxylation. Xanthine oxidoreductase (XOR), aldehyde oxidase (SsAOX1), carbonyl reductase (CBR1) and cytochrome P450 (CYP) enzymes were involved in the QdNOs metabolism. Further understanding the role of oxidative stress in QdNOs-induced toxicity will throw new light onto the use of antioxidants and scavengers of ROS as well as onto the blind spots of metabolism and the metabolizing enzymes of QdNOs. The present review might contribute to revealing the QdNOs toxicity, protecting against oxidative damage and helping to improve the rational use of concurrent drugs, while developing novel QdNO compounds with more efficient potentials and less toxic effects.

Autocatalytic activation of a thermostable glutamyl endopeptidase capable of hydrolyzing proteins at high temperatures.

Glutamyl endopeptidases (GSEs) specifically hydrolyze peptide bonds formed by α-carboxyl groups of Glu and Asp residues. We cloned the gene for a thermophilic GSE (designated TS-GSE) from Thermoactinomyces sp. CDF. A proform of TS-GSE that contained a 61-amino acid N-terminal propeptide and a 218-amino acid mature domain was produced in Escherichia coli. We found that the proform possessed two processing sites and was capable of autocatalytic activation via multiple pathways. The N-terminal propeptide could be autoprocessed at the Glu-1-Ser1 bond to directly generate the mature enzyme. It could also be autoprocessed at the Glu-12-Lys-11 bond to yield an intermediate, which was then converted into the mature form after removal of the remaining part of the propeptide. The segment surrounding the two processing sites was flexible, which allowed the proform and the intermediate form to be trans-processed into the mature form by either active TS-GSE or heterogeneous proteases. Deletion analysis revealed that the N-terminal propeptide is important for the correct folding and maturation of TS-GSE. The propeptide, even its last 11-amino acid peptide segment, could inhibit the activity of its cognate mature domain. The mature TS-GSE displayed a temperature optimum of 85 °C and retained approximately 90 % of its original activity after incubation at 70 °C for 6 h, representing the most thermostable GSE reported to date. Mutational analysis suggested that the disulfide bonds Cys32-Cys48 and Cys180-Cys183 cumulatively contributed to the thermostability of TS-GSE.

In vitro antimicrobial activities of animal-used quinoxaline 1,4-di-N-oxides against mycobacteria, mycoplasma and fungi.

BACKGROUND: The quinoxaline 1,4-di-N-oxides (QdNOs) were known as potent antibacterial agents. For the purpose of evaluating the bioactivity of existing animal-used QdNOs drugs against representative pathogenic microorganism, the representative drugs of quinoxalines including cyadox, mequindox, quinocetone and their metabolites were submitted to the in vitro evaluation for antituberculosis, antimycoplasma, antifungal and antiviral activities. RESULTS: In antituberculosis assays, the prototype compounds were active (MIC = 4 ~ 8 µg/mL) against Mycobacterium tuberculosis H37Rv and M. bovis. Combined antimicrobial susceptibility test indicated that cyadox, mequindox and quinocetone combined with rifampicin had additive effect against M. tuberculosis complex with Fractional Inhibitory Concentration Index (FIC) of 0.75. Results of antifungal assays showed that quinocetone was active against Microsporum canis with MIC of 8 µg/mL. Antimycoplasma screening showed a generally good activity of quinocetone against Mycoplasma gallisepticum and Mycoplasma hyopneumoniae, with MIC between 8 and 16 µg/mL. As shown from the combined antimicrobial susceptibility test, cyadox, mequindox and quinocetone combined with tetracycline had additive effect against Mycoplasma gallisepticum with FIC of 0.75. These compounds were also submitted to antiviral assay against infectious bursal disease virus, porcine reproductive and respiratory syndrome virus, porcine parvovirus and classical swine fever virus. The results obtained showed that these QdNOs and their metabolites have no inhibitory activity against these viruses in vitro. CONCLUSIONS: QdNOs exhibit antimicrobial activities against mycobacteria, mycoplasma and fungi. This study gives new insight in further application of QdNOs and offers a way to promote the healthcare of animal husbandry.

Evaluation of the safety of primary metabolites of cyadox: Acute and sub-chronic toxicology studies and genotoxicity assessment.

Cyadox (CYA) is a synthetic antimicrobial agent, belonging to quinoxaline (QdNO) family. Cy1 (bidesoxy cyadox), Cy2 (N4-desoxycyadox) and Cy10 (N1-desoxycyadox) are the primary metabolites of CYA. In our present study, an acute toxicity test, a sub-chronic toxicity test, and a battery of three genotoxicity tests were carried out according to standard protocols. The LD50 of the metabolites were above 5000 mg/kg b.w. The maximum tolerated dose (MTD) of Cy1 and Cy-M (mixture of Cy2 and Cy10) in rats, and the MTD of Cy1, Cy2 and Cy10 in mice were above 6000 mg/kg b.w./day. In subchronic study, rats were separately administered Cy1 and Cy-M at the dose levels of 0, 50, 150 and 2500 mg/kg diet for 90 days, with CYA (2500 mg/kg) as a control. Significant decreases in body weight and changes in clinical serum biochemistry were observed in the high-dose group of Cy1 and Cy-M, as well as CYA. Significant changes in relative weights of organs at 150 and 2500 mg/kg diet of Cy1 and CYA were noted. Additionally, the high-dose groups of Cy1, Cy-M and CYA showed pathological changes near the hepatic portal area. There was no evidence for genotoxic activity of any of the three metabolites in the bacterial reverse mutation test, mouse bone marrow micronucleus assay or an in vitro assay for clastogenicity. Based on the subchronic study, the target organ of the primary metabolites was the liver, and the no-observed-adverse-effect level for Cy1 and Cy-M was 150 mg/kg diet.

Deoxidation rates play a critical role in DNA damage mediated by important synthetic drugs, quinoxaline 1,4-dioxides.

Quinoxaline 1,4-dioxides (QdNOs) are synthetic agents with a wide range of biological activities. However, the mechanism of DNA damage mediated by QdNOs is far from clear. Five classical QdNOs, quinocetone (QCT), mequindox (MEQ), carbadox (CBX), olaquindox (OLA), and cyadox (CYA), were used to investigate the genotoxicity of QdNOs. The deoxidation rate of QdNOs was presumed to play a role in their genotoxicity. Deoxidation rates of QdNOs in both rat and pig liver microsomes were investigated using LC/MS-IT/TOF, and their relative quantification was achieved with HPLC. To reveal the relationships between the deoxidation rate and genotoxicity, cell damage, oxidative stress, and DNA damage were detected. Under low oxygen conditions, the rank order of the desoxy and bidesoxy rates in rat and pig liver microsomes was QCT < CBX < MEQ < OLA < CYA and QCT < MEQ < CBX < OLA < CYA, respectively. Only desoxy-quinoxalines were detected under aerobic conditions. The concentrations of deoxidized metabolites under low oxygen conditions were at least 6 times higher than those under aerobic conditions. In rats, porcine primary hepatocytes, and HepG2 cells, oxidative stress indices and DNA damage showed inverse relationships with the deoxidation rate, indicating that the deoxidation rate of QdNOs, especially bidesoxy rates, might play a critical role in mediating their ability to promote DNA damage. These results indicated that faster deoxidation of QdNOs results in lower DNA-damage-induced toxicity. Our results shed new light on the prevention of DNA damage mediated by QdNOs and help to understand the relationships among the chemical structures, metabolism, and DNA damage of QdNOs.

Degradation of intact chicken feathers by Thermoactinomyces sp. CDF and characterization of its keratinolytic protease.

Thermoactinomyces is known for its resistance to extreme environmental conditions and its ability to digest a wide range of hard-to-degrade compounds. Here, Thermoactinomyces sp. strain CDF isolated from soil was found to completely degrade intact chicken feathers at 55 °C, with the resulting degradation products sufficient to support growth as the primary source of both carbon and nitrogen. Although feathers were not essential for the expression of keratinase, the use of this substrate led to a further 50-300 % increase in enzyme production level under different nutrition conditions, with extracellular keratinolytic activity reaching its highest level (∼400 U/mL) during the late-log phase. Full degradation of feathers required the presence of living cells, which are thought to supply reducing agents necessary for the cleavage of keratin disulfide bonds. Direct contact between the hyphae and substrate may enhance the reducing power and protease concentrations present in the local microenvironment, thereby facilitating keratin degradation. The gene encoding the major keratinolytic protease (protease C2) of strain CDF was cloned, revealing an amino acid sequence identical to that of subtilisin-like E79 protease from Thermoactinomyces sp. E79, albeit with significant differences in the upstream flanking region. Exogenous expression of protease C2 in Escherichia coli resulted in the production of inclusion bodies with proteolytic activity, which could be solubilized to an alkaline solution to produce mature protease C2. Purified protease C2 was able to efficiently hydrolyze α- and ß-keratins at 60-80 °C and pH 11.0, representing a promising candidate for enzymatic processing of hard-to-degrade proteins such as keratinous wastes.

Acute and sub-chronic toxicity study of diaveridine in Wistar rats.

Diaveridine, a developed dihydrofolate reductase inhibitor, has been widely used as anticoccidial drug and antibacterial synergist. However, few studies have been performed to investigate its toxicity. To provide detailed toxicity with a wide spectrum of doses for diaveridine, acute and sub-chronic toxicity studies were conducted. Calculated LD50 was 2330 mg/kg b.w. in females and 3100 mg/kg b.w. in males, and chromodacryorrhea was noted in some females before their death. In the sub-chronic study, diaveridine was fed to Wistar rats during 90 days at dietary levels of 0, 23, 230, 1150 and 2000 mg/kg, which were about 0, 2.0-2.3, 21.0-23.5, 115.2-126.9 and 212.4-217.9 mg/kg b.w., respectively. Significant decrease in body weights in both genders at 1150 and 2000 mg/kg groups and significant increases in relative weights of brain in both genders, liver in females, kidneys and testis in males, alkaline phosphatase and potassium in both genders at 2000 mg/kg diet were noted. Significant decrease in absolute weights of several organs, hemoglobin and red blood cell count in both genders, albumin and total protein in females were observed at 2000 mg/kg diet. Fibroblasts in the kidneys, cell swelling of the glomerular zone in the adrenals and inflammation in the liver were found at 2000 mg/kg group. The no-observed-adverse-effect level of diaveridine was 230 mg/kg diet (21.0-23.5 mg/kg b.w./day).

Assessment of thirteen-week subchronic oral toxicity of cyadox in Beagle dogs.

Cyadox (2-formylquinoxaline-N(1),N(4)-dioxide cyanocetylhydrazone) is a new antimicrobial agent and growth-promoter to be used in food-producing animals. Although its toxicity has been clearly documented in rodents, no study is available in non-rodent animals. Therefore, we studied the subchronic effects of cyadox in Beagle dogs to provide additional information with which to establish safety criteria for human exposure. For this purpose, 36 Beagle dogs, 18 males and 18 females, were divided into four groups and fed diets containing 0, 100, 450 and 2500 mg/kg of cyadox, respectively, for 13 weeks. It was found that there were no significant changes among the examined parameters, except for an increase in the level of serum potassium (K(+)) in 2500 mg/kg cyadox group in males at week 13 of the study. However, the K(+) level returned to normal during the recovery period. In conclusion, cyadox showed slight effects in Beagle dogs in the subchronic oral toxicity study. The no-observed-adverse-effect level of cyadox was considered to be 450 mg/kg diet, which equates to approximately 15.3-15.4 mg/kg b.w./day. The study provided subchronic effects of cyadox in Beagle dogs, suggesting that cyadox might present mild toxicity in non-rodents.

Two-generation reproduction and teratology studies of feeding aditoprim in Wistar rats.

Aditoprim, a new bacteriostatic agent that belongs to diaminopyrimidines, has a broad antimicrobial spectrum, good antibacterial activity and excellent pharmacokinetics. To evaluate the reproductive toxicity and teratogenic potential of aditoprim, different concentrations of aditoprim were administered to Wistar rats by feeding diets containing 0, 20, 100 and 1000 mg kg(-1) , respectively. Each group consisting of 18 males and 25 females (F0 ) was treated with different concentrations of aditoprim through a 13-week period before mating and during mating, gestation, parturition and lactation. At weaning, 20 males and 25 females of the F1 generation weanlings per group were selected randomly as parents for the F2 generation. Selected F1 weanlings were exposed to the same diet and treatment as their parents. At 1000 mg kg(-1) dose group, body weights in F0 and F1 rats, fetal body weight on day 21 (0, 4 and 21) after birth and number of viable fetuses in the F0 and F1 generation significantly decreased. Teratogenicity study was performed in combination with the F1 generation of a two-generation reproduction study. F1 parents of the reproduction study were mated after weaning of the F2a pups. Pregnant female rats were subjected to cesarean section on gestational day 20 for teratogenic examination. At 1000 mg kg(-1) group, body weights, fetal body lengths, tail lengths, litter weights and number of viable fetuses were significantly decreased. No obvious external, skeletal or visceral malformations in fetuses were noted in any groups in the teratogenic test. The no-observed-adverse-effect level for reproduction/development toxicity of aditoprim was 100 mg kg(-1) diet (about 7.89-9.25 mg kg(-1) body weight day(-1) ).

Safety assessment of aditoprim acute, subchronic toxicity and mutagenicity studies.

Aditoprim (ADP), a new developed dihydrofolate reductase (DHFR) inhibitor, has great potential in clinical veterinary medicine because of its greater pharmacokinetic properties than structural analogs. Preclinical toxicology studies were performed to assess the safety of ADP including an acute oral toxicity test, a subchronic toxicity test and five mutagenicity tests. In the acute oral toxicity test, ADP was administered singly by oral gavage to Wistar rats and Kunming mice. The LD50 calculated was 1400 mg kg(-1) body weight (BW) day(-1) in rats and 1130 mg kg(-1) BW day(-1) in mice. In a subchronic study, Wistar rats were administered ADP at dose levels of 0, 20, 100 and 1000 mg kg(-1) diet for 90 days. Significant decreases were observed on body weight and food efficiency in the high-dose group. Treatment-related changes in clinical serum biochemistry were found in the medium- and high-dose groups. Significant increases in the relative weights of livers and kidneys in females and testis in males in the 1000 mg kg(-1) diet, and significant decrease in relative weights of livers in males in the 100 mg kg(-1) diet were noted. Histopathological observations revealed that the 1000 mg kg(-1) ADP diet could induce lymphocytic infiltration and hepatocytic necrosis near the hepatic portal area. The genotoxicity of ADP was negative in tests, such as the bacterial reverse mutation assay, mice bone marrow erythrocyte micronucleus assay, in vitro chromosomal aberration test, in vitro cho/hgprt mammalian cell mutagenesis assay and mice testicle cells chromosome aberration. Based on the subchronic study, the no-observed-adverse-effect level for ADP was a 20 mg kg(-1) diet, which is about 1.44-1.53 mg kg(-1) BW day(-1) in rats.