Toxicological Implications and Inflammatory Response in Human Lymphocytes Challenged with Oxytetracycline

Antibiotics are widely used in zoo technical and veterinary practices as feed supplementation to ensure wellness of farmed animals and livestock. Several evidences have been suggesting both the toxic role for tetracyclines, particularly for oxytetracycline (OTC). This potential toxicity appears of great relevance for human nutrition and for domestic animals. This study aimed to extend the evaluation of such toxicity. The biologic impact of the drug was assessed by evaluating the proinflammatory effect of OTC and their bone residues on cytokine secretion by in vitro human peripheral blood lymphocytes. Our results showed that both OTC and OTC‐bone residues significantly induced the T lymphocyte and non‐T cell secretion of interferon (IFN)‐γ, as cytokine involved in inflammatory responses in humans as well as in animals. These results may suggest a possible implication for new potential human and animal health risks depending on the entry of tetracyclines in the food‐processing chain.     

Pesticide Residues in Agricultural Produce in Hubei Province,PR China

Pesticide residues in agricultural produce were examined in two villages of Qianjiang municipality, Hubei province, PR China. Six food groups were sampled from the fields prior to harvesting from the farmers' market and from farmers' homes. Organochlorine residues were detected in almost all examined food groups, with residue levels significantly lower than the national tolerances (MRL). Total BHC residues were on average 31.7?µg/kg and DDT 102.5 (µg/kg. Organophosphorus residues were detected in vegetable samples. Mean residue levels of phoxim and methamidophos in vegetables were 89.9?µg/kg and 36.5?µg/kg, respectively, both exceeding the MRL. Estimated daily intakes (EDIs) of pesticide residues per person, based on the six food groups sampled, were 4.88?mg for total DDT, 2.04 (µg for total BHC, and 19.33 (µg for methamidophos. The overall results show that the increased use and misuse of pesticides for crop protection in Qianjiang municipality, notably in vegetable production, have led to worrisome levels of pesticide contamination of agricultural produce that may carry certain risks for human health.     

韭菜上五种有机磷农药的残留监测与安全性评价

【目的】2013—2016年,在北京、天津、河北和山东采集韭菜样品,采用超高效液相色谱-三重四级杆串联质谱法检测甲胺磷、甲拌磷、甲基对硫磷、对硫磷和毒死蜱的残留量,并对其残留安全性进行评价。【方法】韭菜样品用乙腈提取,石墨碳固相萃取小柱净化,超高效反相液相色谱分离,三重四级杆质谱检测,基质匹配标准溶液的外标法定量,在添加浓度为0.01~5.0 mg/kg时,方法的准确度、灵敏度和精密度满足农药残留检测的要求。【结果】甲胺磷的最高检出浓度由70.51 mg/kg降低到0.03 mg/kg,超标率由38.10%降到2.03%;甲拌磷的最高检出浓度由6.45 mg/kg降低到0.04 mg/kg,超标率由23.81%降到1.35%;甲基对硫磷的最高检出浓度由6.62 mg/kg降低到0.13 mg/kg,超标率由25.40%降到2.70%;毒死蜱的最高检出浓度由3.95 mg/kg降低到0.47 mg/kg,超标率由15.87%降到4.73%;对硫磷的最高检出浓度由22.69mg/kg降低到0.03 mg/kg,超标率由15.08%降到1.35%。【结论】上述农药的最高残留检出浓度和超标率大幅降低,但是韭菜中仍能检出少量残留。应加强安全综合防治技术的研发,加强安全用药的宣传和指导,保障韭菜产品安全。     

Veterinary medicinal product usage among food animal producers and its health implications in Central Ethiopia

Background

Antimicrobials and anthelmintics are the most commonly used veterinary drugs to control animal diseases. However, widespread use of these drugs could contribute to the emergence of drug resistance. Information on the practice of antimicrobial usage among food animal raising communities in Central Ethiopia is scarce. We used a standardised questionnaire survey to assess knowledge, awareness, and practices related to drug use and resistance in food animals among the farmers in and around Bishoftu town.

Results

Of the total of 220 livestock owners interviewed, around 80% of the respondents were not able to define what antimicrobials are and for what purposes they are used. Only 14.1% (n?=?31) of the respondents had awareness about antimicrobial resistance (AMR) and its consequences; and 35.5% (n?=?11/31) and 9.7% (n?=?3/31) of them agreed that the irrational use of antimicrobials in animals could lead to AMR in animals and humans. Oxytetracycline was the most commonly available antibiotic in veterinary drug shops/pharmacies and the most widely used drug in the area. However, 43.3% of the respondents did not see clinical improvements after using antibiotics. Similarly, the respondents explained that no response was observed in 73.3, 70.8 and 52.5% of the cases after medication with anthelmintics, antiprotozoal and acaricides, respectively. About 56.7% of the respondents considered traditional medicines equally important to modern medicines. It was also noted that there were illegal drug vendors, dispensing medicines under unfavourable conditions which include a direct exposure to sunlight, which practice violates the drug handling and storage recommendations given by WHO.

Conclusion

The study revealed that there is a general lack of awareness among food animal owners about the correct use of antibiotics and anthelmintics. The widespread misuse and improper drug dispensing and handling practices observed in this study can affect the drug quality and can also contribute to the development of drug resistance in central Ethiopia.

     

时间分辨荧光免疫分析在兽药残留检测中的应用

近年来,兽药残留引起食物中毒的报道日益增多,兽药残留检测的意义重大。传统的气相色谱法、液相色谱法存在前处理复杂、仪器成本昂贵等缺陷,酶联免疫吸附分析(enzyme-linked immunosorbent assay,ELISA)灵敏度也不高,而时间分辨荧光免疫分析(time-resolved fluoroimmunoassay,TRFIA)操作简便、灵敏度高,已在兽药残留检测领域引起重视。介绍了TRFIA的原理和优势,综述了其在促生长繁殖类、瘦肉增产类和杀菌驱虫类兽药残留检测中的应用,并与传统方法进行了对比,TRFIA有望取代传统的检测方法成为兽药残留检测的常规方法。     

Quantitative determination of dexamethasone in bovine milk by liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry

Dexamethasone (DXM) is a synthetic glucocorticoid that is authorized for therapeutic use in veterinary medicine. The European Community (EC) fixed a maximum residue limit (MRL) at 2ng/g for liver, 0.75ng/g for muscle and kidney tissues, and 0.3ng/ml for milk, while its use as growth-promoter is completely banned. The purpose of this study was to develop and validate a simple and reliable method to determine DXM residues in bovine milk. Milk proteins were removed by the addition of concentrated trichloroacetic acid and paper filtration. Solid-phase extraction clean-up on a C18 reversed phase column was performed to obtain an extract suitable for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatographic separation of DXM and the internal standard desoximetasone, was achieved on a PLRP-S polymeric reversed phase column, using a mixture of 0.1% (v/v) acetic acid in water (mobile phase A) and acetonitrile (mobile phase B) as the mobile phases. They were identified using the MS/MS detection technique, and were subsequently quantified. The method has been validated according to the requirements of the EC at 0.15, 0.30 and 0.60ng/ml (being half the MRL, the MRL and double the MRL levels fixed by the EC). Calibration graphs were prepared in the 0.15-5ng/ml range and good linearity was achieved (r>or=0.99 and goodness of fit 相似文献   

Ivermectin 20 years on: maturation of a wonder drug

Ivermectin has been on the veterinary market for almost a quarter of a century and has been approved for human use for 18 years. Its use has revolutionized the treatment of nematode and arthropod parasites in animals and has provided hope for the control or even eradication of filariases in humans. Although much remains to be learned about how the drug works and how resistance to it will develop, it has earned the title of "wonder drug".     

Impact of antimicrobial residues on gut communities: are the new regulations effective?

Veterinary medicines are subject to a rigorous evaluation with regard to safety, efficacy and quality before they are licensed. For drugs used in food producing animals, it is necessary to establish what is referred to as the acceptable daily intake (ADI), this is defined as an estimate of the amount of a substance, expressed on a body weight basis, that can be ingested daily over a lifetime without appreciable risk to human health. It is necessary to determine a toxicological, pharmacological and microbiological ADI. This article describes a recently harmonized guideline that outlines the process for determining the need for a microbiological ADI and discusses the test systems that take into account the complexity of the human intestinal flora. The described process is used to address the effects of antimicrobial drug residues on human intestinal flora for regulatory purposes. The guideline does not recommend any one particular system for use in regulatory decision making but provides recommendations for a harmonized approach to establish a microbiological ADI and offers test options rather than specifying a testing regimen. The process and the challenges of this new guideline are discussed.     

Veterinary use and antibiotic resistance

Globally, an estimated 50% of all antimicrobials serve veterinary purposes. Bacteria that inevitably develop antibiotic resistance in animals comprise food-borne pathogens, opportunistic pathogens and commensal bacteria. The same antibiotic resistance genes and gene transfer mechanisms can be found in the microfloras of animals and humans. Direct contact, food and water link animal and human habitats. The accumulation of resistant bacteria by the use of antibiotics in agriculture and veterinary medicine and the spread of such bacteria via agriculture and direct contamination are documented.     

Light-chain shuffling from an antigen-biased phage pool allows 185-fold improvement of an anti-halofuginone single-chain variable fragment

Halofuginone is an antiprotozoal drug used in the treatment of coccidiosis in poultry, a contagious enteric disease caused by parasites of the Eimeria spp. To ensure that food is free from any halofuginone residues and safe for human consumption, a rapid method to detect these residues below the maximum residue limits (MRLs) in a variety of matrices is necessary. To address this need, we constructed an immune single-chain variable fragment (scFv) library from the RNA of a halofuginone-immunized chicken and selected halofuginone-specific scFv by phage display. The best clone isolated from the library had a limit of detection of 30 ng/ml as determined by enzyme-linked immunosorbent assay (ELISA). However, the minimum MRL for halofuginone in certain foodstuffs can be as low as 1 ng/ml, well below the sensitivity of the selected antibody. The selected antibody was then affinity maturated by light-chain shuffling to further improve the antibody’s assay performance. The halofuginone-specific heavy-chain pool of the biopanned library was assembled with the light-chain repertoire amplified from the original prepanned library. This resulted in a heavy-chain-biased library from which an scFv with the potential to detect halofuginone residues as low as 80 pg/ml was isolated, a 185-fold improvement over the original scFv. This new chain-shuffled scFv was incorporated into a validated ELISA (according to Commission Regulation 2002/657/EC) for the sensitive detection of halofuginone in spiked processed egg samples.     

天然水蛭素冻干粉实验结果的分析

目的研究天然水蛭素冻干粉的生物化学成分及其毒理学的安全性。方法采用《中华人民共和国药典》的抗凝血酶活性测定法、电感耦合等离子体质谱法、《中华人民共和国国家标准》中的食品中水分、微生物、重金属检测、检验法等。结果天然水蛭素具有较高的抗凝血酶活性(809 ATU/g),无致病菌,微生物及重金属不超标,对动物和人体无毒副作用。结论天然水蛭素有潜在的药物开发价值。     

Current methodologies for the analysis of aminoglycosides

The aminoglycosides are a large and diverse class of antibiotics that characteristically contain two or more aminosugars linked by glycosidic bonds to an aminocyclitol component. Structures are presented for over 30 of the most important members of this family of compounds. The use of aminoglycosides in clinical and veterinary medicine and in agriculture is described. Qualitative methods for aminoglycoside analysis include X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). The major part of this article comprises a comprehensive review of quantitative methods for the determination of aminoglycosides. These are microbiological assay, radiochemical assay, radioimmunoassay, enzyme immunoassay, fluoroimmunoassay and other immunoassays, spectrophotometric and other non-separative methods, gas chromatography (GC), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE). Simple spectrophotometric methods may be adequate for the assay of bulk pharmaceuticals and their formulations. Microbiological assays make useful semi-quantitative screening tests for the analysis of veterinary drug residues in food, but rapid enzyme immunoassays are more suitable for accurate measurements of aminoglycosides in complex matrices. Automated immunoassays are the most appropriate methods for serum aminoglycoside determinations during therapeutic drug monitoring. HPLC techniques provide the specificity and sensitivity required for pharmacokinetic and other research studies, while HPLC–MS is employed for the confirmation of veterinary drug residues. The potential for further development of chromatographic and CE methods for the analysis of biological samples is outlined.     

Binding of the human "electron transferring flavoprotein" (ETF) to the medium chain acyl-CoA dehydrogenase (MCAD) involves an arginine and histidine residue

The interaction between the "electron transferring flavoprotein" (ETF) and medium chain acyl-CoA dehydrogenase (MCAD) enables successful flavin to flavin electron transfer, crucial for the beta-oxidation of fatty acids. The exact biochemical determinants for ETF binding to MCAD are unknown. Here we show that binding of human ETF, to MCAD, was inhibited by 2,3-butanedione and diethylpyrocarbonate (DEPC) and reversed by incubation with free arginine and hydroxylamine respectively. Spectral analyses of native ETF vs modified ETF suggested that flavin binding was not affected and that the loss of ETF activity with MCAD involved modification of one ETF arginine residue and one ETF histidine residue respectively. MCAD and octanoyl-CoA protected ETF against inactivation by both 2,3-butanedione and DEPC indicating that the arginine and histidine residues are present in or around the MCAD binding site. Comparison of exposed arginine and histidine residues among different ETF species, however, indicates that arginine residues are highly conserved but that histidine residues are not. These results lead us to conclude that this single arginine residue is essential for the binding of ETF to MCAD, but that the single histidine residue, although involved, is not.     

Prediction and confirmation of a switch-like region within the N-terminal domain of hSIRT1

Many proteins display conformational changes resulting from allosteric regulation. Often only a few residues are crucial in conveying these structural and functional allosteric changes. These regions that undergo a significant change in structure upon receiving an input signal, such as molecular recognition, are defined as switch-like regions. Identifying these key residues within switch-like regions can help elucidate the mechanism of allosteric regulation and provide guidance for synthetic regulation. In this study, we combine a novel computational workflow with biochemical methods to identify a switch-like region in the N-terminal domain of human SIRT1 (hSIRT1), a lysine deacetylase that plays important roles in regulating cellular pathways. Based on primary sequence, computational methods predicted a region between residues 186–193 in hSIRT1 to exhibit switch-like behavior. Mutations were then introduced in this region and the resulting mutants were tested for allosteric reactions to resveratrol, a known hSIRT1 allosteric regulator. After fine-tuning the mutations based on comparison of known secondary structures, we were able to pinpoint M193 as the residue essential for allosteric regulation, likely by communicating the allosteric signal. Mutation of this residue maintained enzyme activity but abolished allosteric regulation by resveratrol. Our findings suggest a method to predict switch-like regions in allosterically regulated enzymes based on the primary sequence. If further validated, this could be an efficient way to identify key residues in enzymes for therapeutic drug targeting and other applications.     

Development and application of indirect competitive enzyme immunoassay for detection of neomycin in milk

As a result of immunization of rabbits with neomycin B (NM) conjugated to sodium periodate-oxidized (SP) transferrin, polyclonal antibodies were generated and used to develop an indirect competitive enzyme-linked immunosorbent assay (ELISA) of NM. Several heterologous conjugates, namely, glutaraldehyde (GA)-polymerized NM, gelatin-ribostamycin (sp), and gelatin-NM (ga) were used as coating antigens in different ELISA variants for quantification of NM in milk. These variants were characterized by different dynamic ranges and detection limits of 1.0, 0.1, and 0.01 ng/ml, respectively. Maximum residue level (MRL) of this antibiotic in milk accepted in the EU can be detected without any special pretreatment at a 100-fold sample dilution in the least sensitive assay variant. The mean recovery rate from NM-spiked milk containing 1.5–10% fat was 111.7% and ranged from 84 to 125.2%. We found that 57 of 106 tested milk samples contained NM at concentrations higher than 100 ng/ml. In ten percent of cases (11/106), the residual level of this antibiotic was greater than 500 ng/ml. In one case, the MRL was exceeded (1690 ng/ml). The assay developed in this study is specific shows no cross-reactivity with other veterinary aminoglycosides, has a good sensitivity reserve, and can serve as an effective tool to monitor the NM content in milk stuff.     

Antibiotic residues in food animals: Public health concern

Antibiotics are used to treat disease and improve animal production. These antibiotics might result in deposition of residues in meat, milk and eggs which are not permitted in food intended for human consumption. This review report some health hazards of antibiotic residues in food. There are many factors influencing the occurrence of residues in animal products such as drug’s properties and their pharmacokinetic characteristics, physicochemical or biological processes of animals and their products. The use of antibiotics is necessary in the prevention and treatment of animal diseases. Moreover, these antibiotics also improve the performance of growth and feed efficiency, synchronizing the reproductive cycle and breeding performance. These may also lead to harmful residual effects. For this to be minimized, withdrawal periods must be observed. This withholding periods makes the residues to be negligible or no longer detected in foods. However, withdrawal period is established to safeguard human from exposure of antibiotics added food. Failure to respect this period, could result in one which produces potential threat to direct toxicity in human. Moreover, low levels of antibiotic exposure would result in alteration of microflora, causing disease and the possible development of resistant strains causing failure of antibiotic therapy. The regulation of these residues in food of animal origin is necessary to prevent the health of humans.     

Methicillin-resistant Staphylococcus aureus in animals

Methicillin-resistant Staphylococcus aureus (MRSA) is a critically important human pathogen that is also an emerging concern in veterinary medicine and animal agriculture. It is present in a wide range of animal species, including dogs, cats, rabbits, horses, cattle, pigs, poultry, and exotic species, both as a cause of infection and in healthy carriers. Identification of MRSA in various species and in food has led to concerns about the roles of animals, both pets and livestock, in the epidemiology of MRSA infection and colonization in humans. There is evidence of the role of food animals in human MRSA infections in some countries and of pets as a possible source of human infection. Some groups of individuals who work closely with animals, such as veterinarians, have high MRSA colonization rates. This article includes discussions of MRSA in human medicine, animals, and food, as well as its interspecies transmission, colonization, infection, strains, and affected populations. However, clear answers are lacking in many of these areas and limited studies may lead to premature conclusions. It is certain that animals are a source of human MRSA infection in some circumstances--but humans may also serve as sources of infection in animals. Changes in the epidemiology of MRSA in one species may be reflected in changes in other species. The true scope of MRSA in animals and its impact on human health are still only superficially understood, but it is clear that MRSA is a potentially important veterinary and public health concern that requires a great deal more study to enhance understanding and effective response.     

Phenotypic characterization of the binding of tetracycline to human serum albumin

Because of the widely usage of the veterinary drug tetracycline (TC), its residue exist extensively in the environment (e.g., animal food, soils, surface water, and groundwater) and can enter human body, being potential harmful. Human serum albumin (HSA) is a major transporter for endogenous and exogenous compounds in vivo. The aim of this study was to examine the interaction of HSA with TC through spectroscopic and molecular modeling methods. The inner filter effect was eliminated to get accurate binding parameters. The site marker competition experiments revealed that TC binds to site II (subdomain IIIA) of HSA mainly through electrostatic interaction, illustrated by the calculated negative ΔH° and ΔS°. Furthermore, molecular docking was applied to define the specific binding sites, the results of which show that TC mainly interacts with the positively charged amino acid residues Arg 410 and Lys 414 predominately through electrostatic force, in accordance with the conclusion of thermodynamic analysis. The binding of TC can cause conformational and some microenvironmental changes of HSA, revealed by UV-visible absorption, synchronous fluorescence, and circular dichroism (CD) results. The accurate and full basic data in the work is beneficial to clarifying the binding mechanism of TC with HSA in vivo and understanding its effect on protein function during the blood transportation process.     

Fluoroquinolone resistance of Escherichia coli and Salmonella from healthy livestock and poultry in the EU

The potential for transmission of antibiotic-resistant enteric zoonotic bacteria from animals to humans has been a public health concern for several decades. Bacteria carrying antibiotic resistance genes found in the intestinal tract of food animals can contaminate carcasses and may lead to food-borne disease in humans that may not respond to antibiotic treatment. It is consequently important to monitor changes in antimicrobial susceptibility of zoonotic and commensal organism; in this context, there are a number of veterinary monitoring programmes that collect bacteria in food-producing animals at slaughter and determine their susceptibility against antibiotics relevant for human medicine. The data generated are part of the risk analysis for potential food-borne transmission of resistance. There has been much debate about the use of fluoroquinolones in veterinary medicine, and so, this review will consider the fluoroquinolone data from two surveys and compare them to national surveillance programmes. At the outset, it must be pointed out that there is, however, a lack of agreement between several programmes on what is meant by the term 'fluoroquinolone resistance' through use of different definitions of resistance and different resistance breakpoints. An additional aim of this paper is to clarify some of those definitions. Despite the debate about the contribution of antibiotic use in veterinary medicine to the overall resistance development in human pathogens, the data suggest that clinical resistance to fluoroquinolones in Escherichia coli and nontyphoidal Salmonella is generally uncommon, except for a few countries. Ongoing surveillance will continue to monitor the situation and identify whether this situation changes within the respective animal populations. For the benefit of both the epidemiologist and the clinician, it would be strongly advantageous that national monitoring surveys report both percentages of clinical resistance and decreased susceptibility.     

动物性食品中兽药残留酶联免疫分析研究进展

介绍了动物性食品中兽药残留酶联免疫分析技术的原理、类型、技术环节和国内外的应用现状,并对其存在的问题和应用前景进行了讨论。