Detection of papillomavirus DNA in formalin-fixed paraffin-embedded equine aural plaque samples / Detecção do DNA de papilomavírus em amostras de placa aural equina fixadas em formalina e embebidas em parafina

A placa aural é uma dermatopatia associada à quatro Equus caballus papillomavirus (EcPVs). Até o momento, o DNA de EcPVs não foi identificado em amostras de placa aural fixadas em formalina e embebidas em parafina (FFPE). O objetivo deste estudo foi otimizar um método para a detecção dos quatro tipos de EcPVs em 21 amostras FFPE usando a PCR. O DNA dos EcPVs foram detectados em 11 amostras (52.4%). O DNA do EcPV4 foi detectado em 38.1% (8/21) e do EcPV3 em 4.8% (1/21) das amostras. Coinfecção foi identificada em duas amostras (9.5%); EcPV4 e 5 foram detectados simultaneamente em uma amostra, enquanto o DNA dos EcPV4 e 6 foi detectado em outra. A especificidade do DNA dos papilomavírus equinos foi avaliada por sequenciamento gênico direto, que confirmou a especificidade dos produtos. A metodologia de PCR proposta possibilita o diagnóstico dos EcPV3, 4, 5 e 6 em amostras FFPE de placa aural equina.(AU)

Effects of different extracts of the mushroom Agaricus blazei Murill on the hematologic profile of mice with Ehrlich tumor / Efeitos de diferentes extratos do cogumelo Agaricus blazei Murill sobre o perfil hematológico de camundongos com tumor de Ehrlich

The aim of this study was to investigate the effect of the mushroom Agaricus blazeii Murril (ABM) extracts on the hematological profile of Swiss mice bearing an Ehrlich solid tumor. Three fractions (total extract, polysaccharides, and supernatant) of ABM extracts obtained by four methods (ultrasonic or water bath, at pH 4 or pH 7) were administered to mice over 21 days. Polysaccharide solutions were analyzed by gas and liquid chromatography that showed both mannose and glucose concentrations. The method of extraction influenced the degree of glucose polymerization and the mannose/glucose relationship. The treatment with ABM supernatant at pH 7 and water bath was associated with reduced concentrations of leukocytes and lymphocytes and altered the percentage of CD4+ and CD8+ lymphocytes in Ehrlich tumor-bearing mice. The treatment with the ABM extract in water bath and ultrasound at pH 4 resulted in lower lymphocyte counts, regardless of tumor presence, and greater granulocyte values in mice with Ehrlich tumor than in controls. We concluded that different fractions and methods of extraction of A. blazei produced differing blood profiles in mice inoculated with the Ehrlich tumor.

O objetivo deste estudo foi investigar o efeito de diferentes extratos do cogumelo Agaricus blazeii Murril (ABM) sobre o perfil hematológico de camundongos Swiss portadores de tumor de Ehrlich sólido. Três frações (extrato total, polissacarídeos e sobrenadante) dos extratos de ABM foram obtidas por quatro métodos (sonificador, banho-maria, em pH 4 ou pH 7) e administradas para camundongos durante 21 dias. Soluções de polissacarídeos foram analisadas por cromatografia gasosa e líquida, que mostraram concentrações de glucose e manose. O método de extração influenciou o grau de polimerização da glicose e a relação manose/glucose. O tratamento com o sobrenadante de ABM (em pH 7 e banho-maria) estava associado com reduzidas concentrações de leucócitos e linfócitos, além de alterar a porcentagem de linfócitos CD4+ e CD8+ em camundongos portadores de tumor sólido de Ehrlich. O tratamento com extratos de ABM, obtidos tanto em banho-maria como no sonificador em pH 4, resultou nas mais baixas contagens de linfócitos, independentemente da presença do tumor, e nos maiores valores de granulócitos em camundongos com tumor de Ehrlich. Conclui-se que os diferentes métodos de extração com as respectivas frações de A. blazei são capazes de intereferir no perfil hematológico de camundongos com tumor sólido de Ehrlich.

A critical evaluation of automated blood gas measurements in comparative respiratory physiology.

Precise measurements of blood gases and pH are of pivotal importance to respiratory physiology. However, the traditional electrodes that could be calibrated and maintained at the same temperature as the experimental animal are increasingly being replaced by new automated blood gas analyzers. These are typically designed for clinical use and automatically heat the blood sample to 37°C for measurements. While most blood gas analyzers allow for temperature corrections of the measurements, the underlying algorithms are based on temperature-effects for human blood, and any discrepancies in the temperature dependency between the blood sample from a given species and human samples will bias measurements. In this study we review the effects of temperature on blood gases and pH and evaluate the performance of an automated blood gas analyzer (GEM Premier 3500). Whole blood obtained from pythons and freshwater turtles was equilibrated in rotating Eschweiler tonometers to a variety of known P(O2)'s and P(CO2)'s in gas mixtures prepared by Wösthoff gas mixing pumps and blood samples were measured immediately on the GEM Premier 3500. The pH measurements were compared to measurements using a Radiometer BMS glass capillary pH electrode kept and calibrated at the experimental temperature. We show that while the blood gas analyzer provides reliable temperature-corrections for P(CO2) and pH, P(O2) measurements were substantially biased. This was in agreement with the theoretical considerations and emphasizes the need for critical calibrations/corrections when using automated blood gas analyzers.

The disposition of oxytetracycline to feathers after poultry treatment.

In the combat against bacterial resistance, there is a clear need to check the use of antibiotics in animal husbandry, including poultry breeding. The use of chicken feathers as a tool for the detection of use of antibiotics was investigated. An extraction method for the analysis of oxytetracycline (OTC) from feathers was developed and was tested by using incurred feathers obtained from a controlled animal treatment study. The use of McIlvain-ethylenediaminetetraacetic acid buffer only in combination with acetone gave the highest extraction yield, indicating the need of an organic solvent for feather extraction. By using the developed method, it was found that after a withdrawal time, the OTC concentration in feathers is in the mg kg⁻¹ range, far higher than that in muscle and liver tissue. Based on the analysis of individual segments of feathers from OTC-treated chicken, evidence was found supporting the hypothesis of secretion of antibiotics through the uropygial gland and external spread over feathers by grooming behaviour. It was also found that part of the administered OTC is built into the feather rachis. Finally, we provide the first evidence that the analysis of individual segments of the rachis can be used as a tool to discriminate among different treatment strategies, for example, therapeutic versus subtherapeutic. As a result, we concluded that the analysis of feathers is an extremely valuable tool in residue analysis of antibiotics.

Simultaneous and confirmative detection of multi-residues of ß2-agonists and ß-blockers in urine using LC-MS/MS/MS coupled with ß-receptor molecular imprinted polymer SPE clean-up.

A liquid chromatography-linear ion-trap spectrometry (LC-MS³) method using ß-receptor molecular-imprinted polymer (MIP) solid-phase extraction (SPE) as clean-up was developed to determine simultaneously and confirmatively residues of 25 ß2-agonists and 21 ß-blockers in urine samples. Urine samples were subjected to enzymatic hydrolysis by ß-glucoronidase/arylsulphatase, and then extracted with perchloric acid. Sample clean-up was performed using ß-receptor MIP SPE. A Supelco Ascentis® express Rp-Amide column was used to separate the analytes, and MS³ detection used an electrospray ionisation source in positive-ion mode. Recovery studies were carried out using blank urine samples fortified with the 46 analytes at the levels of 0.5, 1.0 and 2.0 µg l⁻¹. Recoveries were obtained ranging from 60.1% to 109.9% with relative standard deviations (RSDs, n = 7) from 0.5% to 19.4%. The limits of detection (LODs) and limits of quantitation (LOQs) of the 46 analytes in urine were 0.02-0.18 and 0.05-0.60 µg l⁻¹, respectively. As a result of the selective clean-up by MIP SPE and MS³ detection of the target drugs, the sensitivity and accuracy of the present method was high enough for monitoring ß2-agonist and ß-blocker residues in urine samples. Satisfactory results were obtained in the process of the determination of positive urine samples.

Chromatographic methods for biogenic amines determination in foods of animal origin / Métodos cromatográficos para determinar aminas biogênicasem alimentos de origem animal

Biogenic amines (BAs) are formed as a result of specific free amino acid decarboxylation. Analysis of these metabolitesmay be of great importance to determine food quality and for monitoring the levels of biogenic amines such as histamineand tyramine related to intoxication episodes in humans. Chromatography is a chemistry separation technique usedto characterize biogenic amines in foods. Variations of this technique (liquid, thin layer and gas chromatography)have been widely applied; however, the food matrix complex requires that changes in the methodology of extraction,derivatization and detection must be performed according to each group of foods. High-performance liquidchromatography is the most widely used chromatographic method applied for biogenic amines in foods. However, dueto the current importance of biogenic amines in quality control and consumer safety, researchers try to develop newmethods for a fast, reliable analysis of foods in the market. This review presents some chromatographic techniquesapplied to monitoring BAs in different foods of animal origin...

Aminas biogênicas são formadas como resultado da descarboxilação de aminoácidos livres específicos. A análise dessesmetabólitos é de grande importância na determinação da qualidade e monitoramento de biogênicas como histamina etiramina relacionadas com episódios de intoxicação em humanos. A cromatografia é uma técnica de separação químicausada para caracterizar aminas biogênicas. Variações da técnica (cromatografia líquida, em camada delgada e gasosa)têm sido amplamente usadas, porém a complexidade da matriz alimentar faz com que sejam realizadas mudanças nosprocessos de extração, derivatização e detecção em concordância com cada grupo de alimento. A cromatografia líquidade alta eficiência (CLAE) é o método mais utilizado na determinação de aminas biogênicas em alimentos. Contudo,devido à importância das aminas biogênicas no controle da qualidade e a segurança do consumidor, os pesquisadorestentam desenvolver novos métodos com o intuito de uma análise mais rápida e precisa para o controle de alimentos nomercado. O objetivo da revisão é apresentar algumas técnicas cromatográficas aplicadas no monitoramento de aminasbiogênicas em produtos de origem animal...

Natural occurrence of aflatoxins (B1 and M1) in feed, plasma and raw milk of lactating dairy cows in Beja, Tunisia, using ELISA.

Beja is an agricultural area in northwest Tunisia. It contributes to national needs by offering cereals and milk to the market for human and animal consumption. A small number of studies on mycotoxin occurrence in feedstuffs and raw milk from lactating dairy cows in this region are available. Therefore, 226 samples were collected from farms and local markets during November 2008 until April 2010. Samples consisted of 112 raw cow milk, 56 blood from lactating cows and 58 feed destined for dairy cows. Plasma and feed were analysed for aflatoxin B1 (AFB1). Milk samples were analysed for aflatoxin M1 (AFM1). All samples were treated using a simultaneous methanolic-aqueous extraction, followed by immunoaffinity column clean-ups and were investigated by competitive enzyme-linked immunoabsorbent assay (ELISA). Recoveries were 80%-95% and 81%-92% for AFB1 and AFM1, respectively, while the limit of detection (LOD) was 0.01 µg/kg or µg/l for both mycotoxins. Results revealed the presence of AFB1 in 84.4% of the feed samples (mean 18.7 ± 1.4 µg/kg), and 39.2% of the plasma-examined samples (median 7.1 ± 1.0 µg/l) were found to be contaminated at levels higher than the Tunisian and the European Union (EU) limit for dairy animals, which are 20 and 5 µg/kg in animal feed, respectively. AFM1 was detected in 60.7% of the cow raw milk samples examined (median 13.6 ± 1.4 µg/l). Contaminated levels were higher than the EU limit of 0.05 µg/l. It was concluded that more precaution should be taken on hygiene controls in order to prevent fungal contamination.