Corrigendum: Acute and subacute toxicity assessment of oxyclozanide in Wistar rats.

[This corrects the article DOI: 10.3389/fvets.2019.00294.].

Virtual Screening and In Vitro Experimental Verification of LuxS Inhibitors for Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli O157:H7 is an important foodborne pathogen that forms biofilms. In this study, three quorum-sensing (QS) inhibitors (M414-3326, 3254-3286, and L413-0180) were obtained through virtual screening, and their in vitro antibiofilm activities were validated. Briefly, the three-dimensional structure model of LuxS was constructed and characterized using the SWISS-MODEL. High-affinity inhibitors were screened from the ChemDiv database (1,535,478 compounds) using LuxS as a ligand. Five compounds (L449-1159, L368-0079, M414-3326, 3254-3286, and L413-0180) with a good inhibitory effect (50% inhibitory concentration <10 µM) on type II QS signal molecule autoinducer-2 (AI-2) were obtained using a AI-2 bioluminescence assay. The absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties predicated that the five compounds had high intestinal absorption levels (high) and plasma protein binding (absorbent strong) and did not inhibit the metabolism of CYP2D6 metabolic enzymes. In addition, molecular dynamics simulation showed that compounds L449-1159 and L368-0079 could not stably bind with LuxS. Thus, these compounds were excluded. Furthermore, surface plasmon resonance results showed that the three compounds could specifically bind to LuxS. IN addition, the three compounds could effectively inhibit the biofilm formation without affecting the growth and metabolism of the bacteria. Finally, the reverse transcription-quantitative PCR results showed that the three compounds downregulated the expression of the LuxS gene. Overall, these results revealed that the three compounds obtained through virtual screening could inhibit biofilm formation of E. coli O157:H7 and are potential LuxS inhibitors that can be used to treat E. coli O157:H7 infections. IMPORTANCE E. coli O157:H7 is a foodborne pathogen of public health importance. Quorum sensing (QS) is a form of bacterial communication that can regulate various group behaviors, including biofilm formation. Here, we identified three QS AI-2 inhibitors (M414-3326, 3254-3286, and L413-0180) that can stably and specifically bind to LuxS protein. The three QS AI-2 inhibitors inhibited biofilm formation without affecting the growth and metabolic activity of E. coli O157:H7. The three QS AI-2 inhibitors are promising agents for treating E. coli O157:H7 infections. Further studies to identify the mechanism of the three QS AI-2 inhibitors are needed to develop new drugs to overcome antibiotic resistance.

Mechanism of Synergy between Piceatannol and Ciprofloxacin against Staphylococcus aureus.

Piceatannol (PIC) is a natural stilbene extracted from grape skins that exhibits biological activities such as antibacterial, antitumor, and antioxidant activities. The present study was carried out to further investigate the effect of PIC on the antibacterial activity of different antibiotics and to reveal the antibacterial mechanism of PIC. We found that PIC had an inhibitory effect against Staphylococcus aureus (S. aureus); its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 128 µg/mL and 256 µg/ mL, respectively. Additionally, we measured the fractional inhibitory concentration (FIC) of PIC combined with antibiotics via the checkerboard method. The results showed that when PIC and ciprofloxacin (CIP) were combined, they displayed a synergistic effect against S. aureus. Moreover, this synergistic effect was verified by time-kill assays. Further, the results of the membrane permeability assay and proton motive force assay revealed that PIC could enhance the sensitivity of S. aureus to CIP by dissipating the bacterial proton motive force (PMF), particularly the ∆ψ component, rather than increasing membrane permeability. PIC also inhibited bacterial adenosine triphosphate (ATP) synthesis and was less likely to induce bacterial resistance but exhibited slight hemolytic activity on mammalian erythrocytes. In summary, the combination of PIC and CIP is expected to become a new drug combination to combat S. aureus.

Prevalence of Multidrug-Resistant CTX-M Extended Spectrum Beta-Lactamase-Producing Escherichia coli From Different Bovine Faeces in China.

CTX-M extended spectrum beta-lactamase-producing Escherichia coli cause severe health hazards in livestock breeding. To date, little is known about antibiotic resistance differences among bacterial isolates from yaks, cows, and beef cattle; therefore, the aims of this study were to analyse the prevalence of CTX-M-producing E. coli in yak, beef cattle, and dairy cattle feces from different provinces in China. A total of 790 fecal samples from yaks, beef cattle, and dairy cows were used. Among all the samples, 523 non duplicate E. coli isolates were identified, and 29.6% of samples harbored CTX-M producers. The results showed that these E. coli strains harbored 15 clusters of CTX-M genes: CTX-M-79, CTX-M-55, CTX-M-15, CTX-M-14, CTX-M-28, CTX-M-179, CTX-M-65, CTX-M-24, CTX-M-27, CTX-M-102, CTX-M-105, CTX-M-173, CTX-M-238, CTX-M-196, and CTX-M-10. The dominant resistance genes were CTX-M-15, CTX-M-14, and CTX-M-55. Moreover, the distribution of CTX-M genes was related to geographical region. Based on the above findings, we reasoned that bovines are potential reservoirs of antibiotic resistance, and this problem should be given adequate attention.

Corrigendum: Prevalence of multidrug-resistant CTX-M extended spectrum beta-lactamase-producing Escherichia coli from different bovine faeces in China.

[This corrects the article DOI: 10.3389/fvets.2022.738904.].

Seasonal diets supersede host species in shaping the distal gut microbiota of Yaks and Tibetan sheep.

Yaks and Tibetan sheep are important and renowned livestock of the Qinghai-Tibetan Plateau (QTP). Both host genetics and environmental factors can shape the composition of gut microbiota, however, there is still no consensus on which is the more dominant factor. To investigate the influence of hosts and seasons on the gut microbiome diversity component, we collected fecal samples from yaks and Tibetan sheep across different seasons (summer and winter), during which they consumed different diets. Using 16S rRNA sequencing, principal component analysis (PCoA) data showed that PCo1 explained 57.4% of the observed variance (P = 0.001) and clearly divided winter samples from summer ones, while PCo2 explained 7.1% of observed variance (P = 0.001) and mainly highlighted differences in host species. Cluster analysis data revealed that the gut microbiota composition displayed a convergence caused by season and not by genetics. Further, we profiled the gut microbial community and found that the more dominant genera in yak and Tibetan sheep microbiota were influenced by seasonal diets factors rather than genetics. This study therefore indicated that seasonal diet can trump host genetics even at higher taxonomic levels, thus providing a cautionary note for the breeding and management of these two species.

The occurrence of antibiotic resistance genes in the microbiota of yak, beef and dairy cattle characterized by a metagenomic approach.

Drug resistance has been partly driven by the overuse of antimicrobials in agricultural animal feed. Better understanding of antibiotic resistance in bovine gut is needed to assess its potential effects based on metagenomic approach and analysis. In this study, we collected 40 fecal samples to explore drug resistance derived from antibiotic use in the bacterial community by an analysis of the diversities and differences of antibiotic-resistant genes (ARGs) in the gut microbiota from yak, beef, and dairy cattle. Overall, 1688 genes were annotated, including 734 ARG subtypes. The ARGs were related to tetracyclines, quinolones, ß-lactam, and aminoglycosides, in accordance with the antibiotics widely used in the clinic for humans or animals. The emergence, prevalence, and differences in resistance genes in the intestines of yaks, beef, and dairy cattle may be caused by the selective pressure of different feeding patterns, where yaks were raised without antibiotics for growth promotion. In addition, the abundance of ARGs in yak was lower than in beef and dairy cattle, whereas the abundance of integron, a kind of mobile genetic elements (MGEs) was higher in yaks than those in beef and dairy cattle. Furthermore, the results of this study could provide the basis for a comprehensive profile of various ARGs among yak, beef, and dairy cattle in future.

In Vitro and In Vivo Activities, Absorption, Tissue Distribution, and Excretion of OBP-4, a Potential Anti-Clostridioides difficile Agent.

Clostridioides difficile infection (CDI) is considered a major concern of the health care system globally, with an increasing need for alternative therapies. OBP-4, a new oxazolidinone-fluoroquinolone hybrid with excellent in vitro activities and good safety, shows promising features as an antibacterial agent. Here, we further evaluated the in vitro and in vivo activities of OBP-4 against C. difficile and its absorption (A), distribution (D), and excretion (E) profiles in rats. In vitro assays indicated that OBP-4 was active against all tested C. difficile strains, with MICs ranging from 0.25 to 1 mg/liter. In addition, OBP-4 showed complete inhibition of spore formation at 0.5× MIC. In the mouse model of CDI, 5-day oral treatment with OBP-4 provided complete protection from death and CDI recurrence in infected mice. However, cadazolid (CZD) and vancomycin (VAN) showed less protection of infected mice than did OBP-4 in terms of diarrhea and weight loss, especially VAN. Subsequently, ADE investigations of OBP-4 with a reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method showed extremely low systemic exposure and predominantly fecal excretion, resulting in a high local concentration of OBP-4 in the intestinal tract-the site of CDI. These results demonstrated that OBP-4 possesses good activity against C. difficile and favorable ADE characteristics for oral treatment of CDI, which support further development of OBP-4 as a potential anti-CDI agent.

Recent advances in microencapsulation of drugs for veterinary applications.

Microencapsulation is a process where very minute droplets or particles of solid or liquid or gas are trapped with a polymer to isolate the internal core material from external environmental hazards. Microencapsulation is applied mostly for flavor masking, fortification, and sustained and control release. It improves palatability, absorption, and bioavailability of drugs with good conformity. Microencapsulation has been widely studied in numerous drug delivery systems for human health. The application of microcapsules in the veterinary pharmaceutical sciences is increasing day by day. The treatment systems for humans and animals are likely to be similar, but more complex in the veterinary field due to the diversity of the species, breeds, body size, biotransformation rate, and other factors associated with animal physiology. Commercially viable, economically profitable, and therapeutically effective microencapsulated vaccine, anthelmintic, antibacterial, and other therapeutics have a great demand for livestock and poultry production. Nowadays, researchers emphasize the controlled and sustained-release dosage form of drugs in the veterinary field. This paper has highlighted the microencapsulation materials, preparation techniques, characteristics, roles, and the application of microcapsules in veterinary medicine.

Detection of Infectious Agents Causing Neonatal Calf Diarrhea on Two Large Dairy Farms in Yangxin County, Shandong Province, China.

Neonatal calf diarrhea (NCD) is one of the most serious health challenges facing the livestock industry and has caused substantial economic losses due to increased morbidity and mortality rates. The present study investigated the main infectious pathogens causing NCD among cattle in Yangxin County, China. Sixty-nine fecal samples were collected from diarrheic newborn cattle and tested for infectious agents, including bovine rotavirus, bovine coronavirus, Escherichia coli K99, Cryptosporidium parvum, and Giardia lamblia, that cause NCD, as determined by rapid kit analysis and polymerase chain reaction (PCR) amplification. The PCR results showed that the percentages of samples that were positive for C. parvum, bovine rotavirus A, bovine coronavirus, and G. lamblia were 44.93, 36.23, 17.39, and 13.04%, respectively. The rapid kit analysis results showed that the prevalence of C. parvum, rotavirus, coronavirus, and G. lamblia was 52.17, 31.88, 28.98, and 18.84%, respectively. No E. coli K99 was detected by either method. The total positivity of the samples, as determined by PCR and rapid kit analysis, was 80.00 and 81.16%, respectively. No significant difference between the two methods was observed. The results of this study may help to establish a foundation for future research investigating the epidemiology of NCD in cattle and may facilitate the implementation of measures to control NCD transmission to cattle in Yangxin County, Shandong Province, China.

Comparative Study on Pharmacokinetics of Four Long-Acting Injectable Formulations of Enrofloxacin in Pigs.

A comparative study on pharmacokinetics of four long-acting enrofloxacin injectable formulations was investigated in 36 healthy pigs after intramuscular injection according to the recommended single dose @ 2.5 mg/kg body weight. The drug concentrations in the plasma were computed using high-performance liquid chromatography (HPLC) with fluorescence detection. WinNonLin5.2.1 software was used to analyze the experimental data and compared it under one-way ANOVA using SPSS software with a 95% confidence interval (CI). The main pharmacokinetic parameters, that is, the maximum plasma concentrations (Cmax), the time to maximum concentration (Tmax), area under the time curve concentration (AUCall) and Terminal half-life (T1/2) were 733.84 ± 129.87, 917.00 ± 240.13, 694.84 ± 163.49, 621.98 ± 227.25 ng/ml, 2.19 ± 0.0.66, 1.50 ± 0.37, 2.89 ± 0.24, 0.34 ± 0.13 h, 7754.43 ± 2887.16, 8084.11 ± 1543.98, 7369.42 ± 2334.99, 4194.10 ± 1186.62 ng h/ml, 10.48 ± 2.72, 10.37 ± 2.38, 10.20 ± 2.81, and 10.61 ± 0.86 h for 10% enrofloxacin (Alkali), 20% enrofloxacin (Acidic), Yangkang and control drug Nuokang® respectively. There were significant differences among Cmax, Tmax, and AUCall of three formulations compare with that of the reference formulation. No significant differences were observed among the T1/2 for tested formulations compare with the reference formulation. The pharmacokinetic parameters showed that the tested formulations were somewhat better compared to the reference one. The calculated PK/PD indices were effective for bacteria such as Actinobacillus pleuropneumoniae and Pasteurella multocida with values higher than the cut-off points (Cmax/MIC90≥10-12 and AUC/MIC90 ≥ 125). However, they were not effective against bacteria like Haemophilus parasuis, Streptococcus suis, E. coli, and Bordetella bronchiseptica where lower values were obtained.

Efficacy of enteric-coated tilmicosin granules in pigs artificially infected with Actinobacillus pleuropneumoniae serotype 2.

BACKGROUND: Porcine infectious pleuropneumonia caused by Actinobacillus pleuropneumoniae (App) is one of the most serious infectious diseases in pigs and has brought huge economic losses to the world pig industry. The aim of this trial was to evaluate the effect of enteric-coated tilmicosin granule in the treatment and control of artificial infection of App. METHODS: Sixty Duroc and Yorkshire crossbred pigs (50 of which were artificially infected) were divided into six groups: BCG (Blank control group), ICG (Infection-only control group), HDG (High-dose enteric-coated tilmicosin granules), MDG (Medium-dose enteric-coated tilmicosin granules), LDG (Low-dose enteric-coated tilmicosin granules) and TPG (Tilmicosin premix drug control group). The cure rate, mortality, clinical respiratory score, body temperature score, weight gain, lung score and so on were recorded. RESULTS: The cure rate of HDG and MDG was as high as 90%, the mortality was 10%, and the clinical signs recovered quickly. CONCLUSION: The results showed that enteric-coated tilmicosin granules had obvious therapeutic effect on artificial infection, which could reduce the damage caused by the disease and reduce the mortality.

Acute and Subacute Toxicity Assessment of Oxyclozanide in Wistar Rats.

Oxyclozanide is an effective anthelmintic and has shown good properties in other ways including anti-adenovirus, anti-biofilm, antifungal, and antibacterial activity. This study aimed to investigate the acute and subacute 28-days repeated dose oral toxicity of an oxyclozanide suspension in Wistar rats. A high oral lethal dose (LD50) of 3,707 mg/kg was observed in the acute toxicity test. During the 28-days time period, no obvious adverse effects or death were detected. Histopathological changes were observed in the heart, liver, and kidney of animals treated with high dose of oxyclozanide. Based on the hematological parameters, there were no statistical differences between the oxyclozanide-treated group and the control group. For biochemistry assays, ALP, AST, GLU, TBIL, GLO, TG, BUN, UA, LDH, and CK were statistically changed in the treatment groups. These data suggested that the LD50 of oxyclozanide was ~3,707 mg/kg body weight (BW), and the lowest observed adverse effect level (LOAEL) of oxyclozanide was at a dose of 74 mg/kg in rats.

Target animal safety testing of an oral salicylanilide suspension, oxyclozanide, for the treatment of fascioliasis in bovine in China.

The aim of this study was to determine the potential toxicity risk of an oxyclozanide suspension to the target animal, bovine. In this experiment, 32 Simmental beef cattle were fattened and fed a full-price diet without antimicrobial agents. The test cattle were divided into 4 groups, which were treated with 0, 1, 3, and 5 times the recommended dosage through continuous intermittent oral administration at intervals of 2 days. The body weight of the cattle was recorded before and after the experiment, and the weight changes were calculated. The safety of the drugs was evaluated by weight gain, observation of clinical toxicity, haematology, clinical chemistry and histopathology. The results showed that the cattle had different degrees of diarrhoea, loss of appetite and depression after administration. The results of clinicopathology had no significant effect. The results of pathological examination showed that there was a certain degree of damage in the 5 times recommended dose group. The recommended dose was safe to use. Thus, the recommended dose should be given by a single oral administration to ensure the safe use of this drug in the clinic.

Genomic Analysis and Resistance Mechanisms in Shigella flexneri 2a Strain 301.

Shigella flexneri is one of the most prominent pathogenic bacteria in developing countries. In the battle against shigellosis and other bacterial diseases, antibiotic resistance has become an increasing global public health threat. Although the serious phenomenon of multidrug resistance (MDR) has been identified as one of the top three burdens on human health, resistance mechanisms are still poorly understood at the molecular level. In this study, we analyzed genomic data and the evolution of resistance in Shigella flexneri under sequential selection stress from three separate antibiotics: ciprofloxacin (CIP), ceftriaxone (CRO), and tetracycline. Through whole-genome sequencing, 82 chromosomal antibiotic resistance genes were identified. Re-sequencing of the evolved populations identified single nucleotide polymorphisms (SNPs) that contributed to MDR and SNPs that were specific to a single drug. A total of 40 SNPs in 8 genes and 3 intergenic regions, including mutations in metG (L582R) and 1538924, 1538924, and 1538924, appeared under each antibiotic. Several nonsynonymous mutations in gyrB (S464Y), ydgA (E378A), rob (R156H), and narX (K75E) were observed under selective pressure from CIP or CRO. Based on a bioinformatic analysis and previous reports, we discuss the contribution of these mutated genes to resistance. Therefore, more circumspect selection and use of antimicrobial drugs for treating shigellosis is necessary.

EFFICACY AND SAFETY OF BAN HUANG ORAL LIQUID FOR TREATING BOVINE RESPIRATORY DISEASES.

BACKGROUND: Ban Huang oral liquid was developed as a veterinary compound preparation by the Lanzhou Institute of Husbandry and Pharmaceutical Sciences of the Chinese Academy of Agricultural Sciences (CAAS). The purpose of this study was to determine whether the oral liquid preparation of traditional Chinese medicine, Ban Huang, is safe and effective for treating respiratory diseases in cattle. MATERIALS AND METHODS: Acute oral toxicity experiments were conducted in Wistar rats and Kunming mice via oral administration. The minimum inhibitory concentration of the drug against Mycoplasma bovis in vitro with the double dilution method was 500 mg/mL, indicating good sensitivity. The results of laboratory pathogen testing, analysis of clinical symptoms, and analysis of pathological anatomy were combined to diagnose bovine respiratory diseases in 147 Simmental cattle caused by mixed infections of M. bovis, bovine respiratory syncytial virus, bovine parainfluenza virus type 3, and Mannheimia haemolytica. These cattle were randomly divided into three groups: drug treatment group 1 (treated via Tilmicosin injection), drug treatment group 2 (treated with Shuang Huang Lian oral liquid combined with Tilmicosin injection), and drug treatment group 3 (treated with Ban Huang oral liquid combined with Tilmicosin injection). Treatment effects were observed within 7 days. RESULTS: The results showed no toxicity and a maximum tolerated dose greater than 20 g/kg BW. For the 87 cattle in drug-treatment group, the cure rate was 90.80%, whereas the response rate was 94.25%. The cure rate of drug treatment group was increased by 14.13% in comparison with that of drug control group 1 and by 7.47% in comparison with that of drug control group 2 (both P < 0.05). CONCLUSION: This study demonstrates that Ban Huang oral liquid is a safe and effective treatment for bovine respiratory diseases, especially for mixed infection caused by M. bovis, bacteria, and viruses.

Determination of antibacterial agent tilmicosin in pig plasma by LC/MS/MS and its application to pharmacokinetics.

A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify tilmicosin in pig plasma. Plasma samples were prepared by liquid-liquid extraction. Chromatographic separation was achieved on a C18 column (2.1 × 30 mm, 3.5 µm) using acetonitrile-water (90:10, v/v; water included 0.1% formic acid) as the mobile phase. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve was linear from 0.5 to 2000 ng/mL (r2 = 0.9998). The intra- and inter-day accuracy and precision were within the acceptable limits of ±10% for all tilmicosin concentrations. The recoveries ranged from 95 to 99% for the three tested concentrations. The LC-MS/MS method described herein was simple, fast and less laborious than other methods, achieved high sensitivity using a small sample volume, and was successfully applied to pharmacokinetic studies of tilmicosin enteric granules after oral delivery to pigs. In comparison with tilmicosin premix, tilmicosin enteric granules slowed the elimination rate of tilmicosin, prolonged its period of action and significantly improved its bioavailability.

[Inhibitory action of Potentilla anserine polysaccharide fraction on H2O2-induced apoptosis of murine splenic lymphocytes].

A water-soluble polysaccharide fraction from root of Potentilla anserine was obtained. Gas chromatogram, FT-IR, physical and chemical characteristics of the Potentilla anserine polysaccharide fraction (PAPF) were analyzed. The protective effects of PAPF against the H2O2 induced process of apoptosis of murine splenic lymphocytes were investigated in vitro. Morphological assessment of apoptosis was performed with light microscope and laser scanning confocal microscope. DNA fragmentation was visualized by agarose gel electrophoresis. The amount of apoptotic cells was measured by flow cytometry. The results showed that PAPF is composed of rhamnose, arabinose glucose and galactose. H2O2 (200 micromol x L(-1)) induced apoptosis of murine splenic lymphocytes with the cell volume reduced, cytoplasm and nuclear shrunk and DNA stained non-uniformly. Condensed chromatin and formation of apoptotic body were observed in the apoptotic cells. Apoptotic bodies in the cells treated with PAPF and H2O2 were less than those in H2O2 treatment alone. DNA fragmentation assay showed that PAPF (50, 100, 200, and 400 microg x mL(-1)) obviously reduced H2O2-induced ladder bands. Flow cytometry analysis showed that H2O2 increased the populations of apoptotic sub-G1 cells from 5.60% (control) to 45.40%, and PAPF decreased H2O2-induced apoptosis to 37.80%, 22.70%, 17.70%, and 8.50%, respectively. In conclusion, PAPF reduced H2O2-induced oxidative damage in a dose dependent manner.