Serum butyrylcholinesterase activity in healthy dogs with and without exposure to diazinon.

BACKGROUND: Cholinesterase is a biomarker for poisonings by anticholinesterase agents, but its reference values are scarce, and possible interaction with collars containing parasiticides has not been studied. OBJECTIVES: We aimed to evaluate the serum cholinesterase activity of healthy dogs without a history of contact with anticholinesterase agents and healthy animals exposed to commercial collars containing organophosphate. METHODS: Ninety-nine dogs were used and included healthy animals without recent exposure to anticholinesterase agents and healthy animals previously exposed to diazinon collars. Serum quantification of the enzyme butyrylcholinesterase (BuchE) through spectrophotometry was conducted on all samples. In experiment 1, BuchE activity was quantified at time 0 and 7 days after, a time when the samples were kept at -18°C. In experiment 2, sampling times were 0, 7, 14, 21, 28, and 56 days. RESULTS: Time 0 values were 4622.38 ± 1311.53 U/L. After 7 days, a significant decay was observed, with a mean of 3934.45 ± 1430.45 U/L. Spearman's test was performed, finding a weak correlation between ALT, creatinine, total plasma proteins, age, weight, red blood cells, platelets, leukocytes, and BuchE activities. In experiment 2, the mean at time 0 was 4753 ± 454.8 U/L. With exposure to the collar, there was a decay of up to 93% after 14 days. CONCLUSIONS: Normality values of serum BuchE in healthy dogs without a history of exposure to anticholinesterase agents were 4360.8-4883.96 U/L. Freezing serum caused a decrease in BuchE activity. Exposure to commercial collars containing diazinon also reduced BuchE activity without clinical signs, indicating that previously exposed animals should be evaluated carefully.

In Vivo Cardiotoxic Potential of Micrurus frontalis Venom.

In the present study, we investigated the cardiotoxic potential of Micrurus frontalis venom. Twelve guinea pigs (Cavia porcellus) were distributed in two groups (n = 6), named control and envenomed. Control groups received 0.2 ml of PBS/BSA, while envenomed group received 0.2 ml of the same solution containing 450 µg/kg of M. frontalis venom. Both were intramuscular injections. Electrocardiography, echocardiogram, blood count, and serum biochemistry were performed before and 2 h after inoculation. Necropsy was performed, and histological and ultrastructural analysis of the heart were conducted. First clinical signs were presented as early as 18 min after venom inoculation. All poisoned animals presented flaccid paralysis of both hind and forelimbs, followed by fasciculations and respiratory arrythmia. However, the animals did not die in the first 2 h of poisoning. ECG of the poisoned animals revealed severe ventricular arrythmias, corroborated by reduction of both ejection and shortening fractions, increase in CK, CK-MB, troponin, cardiomyocyte degeneration, fragmentation and mitochondrial damage. M. frontalis venom causes severe heart damage, eliciting both morphological and arrhythmogenic effects after only 2 h of envenomation.

Alginate-chitosan microcapsules improve vaccine potential of gamma-irradiated Listeria monocytogenes against listeriosis in murine model.

Listeria monocytogenes is a cause of infectious food-borne disease in humans, characterized by neurological manifestations, abortion, and neonatal septicemia. It is intracellular bacterium, which limits the development of protective inactivated vacines. Adjuvants capable of stimulating cellular immune response are important tools for developing novel vaccines against intracellular bacteria. The aim of this study was to evaluate the vaccine potential of L. monocytogenes inactivated by gamma irradiation (KLM-γ) encapsulated in alginate microcapsules associated or not with chitosan against listeriosis in the murine model. At the fourth day after challenge there was a reduction in bacterial recovery in mice vaccinated with KLM-γ encapsulated with alginate or alginate-chitosan, with lower bacterial loads in the spleen (10 fold) and liver (100 fold) when compared to non-vaccinated mice. In vitro stimulation of splenocytes from mice vaccinated with alginate-chitosan-encapsulated KLM-γ resulted in lymphocyte proliferation, increase of proportion of memory CD4+ and CD8+ T cell and production of IL-10 and IFN-γ. Interestingly, the group vaccinated with alginate-chitosan-encapsulated KLM-γ had increased survival to lethal infection with lower L. monocytogenes-induced hepatic inflammation and necrosis. Therefore, KLM-γ encapsulation with alginate-chitosan proved to have potential for development of novel and safe inactivated vaccine formulations against listeriosis.

Hemostatic evaluation of rabbits envenomed with Bothrops alternatus treated with anti-bothropic serum, desmopressin and tranexamin acid / Avaliação hemostática de coelhos envenenados com Bothrops alternatus e tratados com soro antibotrópico, desmopressina e ácido tranexâmico

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly composed of proteolytic enzymes. Although the antibothropic serum produced by the Brazilian Institutes is remarkably efficient, more studies are necessary, especially in veterinary medicine. The venom contain enzymes and non-enzymatic proteins that interfere with hemostasis leading to hemorrhage or even thrombosis. Possible treatment associations with known bothropic antivenom were the reason for the development of the present study. The aim of this study was to evaluate hemostasis alterations caused by Bothrops alternatus venom in rabbits followed by treatments with anti-bothropic serum, tranexamic acid and desmopressin. Twenty New Zealand rabbits were distributed into five groups (n=4) that were experimentally envenomed with 150mcg/kg of B. alternatus venom via intramuscular injection and treated as follow: Group 1 (G1) was the positive control and received venom and PBS/BSA; Group 2 (G2) was treated with tranexamic acid; Group 3 (G3) with desmopressin; Group 4 (G4) with tranexamic acid and anti-bothropic serum; and Group 5 (G5) with anti-bothropic serum and desmopressin. Blood samples were collected before venom administration, and one, four, eight and 12 hours after, for Partial activated partial thromboplastin time, Prothrombin Time, Thrombin Time and fibrinogen evaluation. Thrombin generation (TG) test was carried out with a pool of samples from final times (8 and 12h). At the end of 12h, all animals were euthanized and necropsy was conducted. Samples from muscle tissue, heart, lungs and kidney were analyzed. Classic coagulation tests showed no significant differences amongst groups and times. However, TG indicated that the venom causes a hypocoagulability state, which was not reversed by proposed treatments. Histology showed muscle inflammation, hemorrhage and necrosis, as well as hemorrhage in other tissues with no differences amongst groups. B. alternatus envenomation causes hypocoagulability detected by TG assay, but not through classical coagulation tests. The use of tranexamic acid and desmopressin for hemostasis stabilization after inoculation of the venom did not show advantage in coagulation restoration.(AU)

No Brasil, as serpentes do gênero Bothrops são responsáveis por milhares de acidentes, e seus venenos são compostos principalmente de enzimas proteolíticas. Embora o soro antiofídico produzido pelos institutos brasileiros seja notavelmente eficiente, mais estudos são necessários, especialmente na medicina veterinária. O veneno contem enzimas e proteínas não-enzimáticas que interferem com a hemostasia levando a hemorragias ou trombose. A associação de outros tratamentos ao soro antibotrópico foi a razão para o desenvolvimento do presente estudo. O objetivo deste estudo foi avaliar as alterações da hemostasia causadas pelo veneno de Bothrops alternatus em coelhos, após tratamento com soro antibotrópico, ácido tranexâmico e desmopressina. Vinte coelhos da Nova Zelândia foram distribuídos em cinco grupos (n = 4) que foram submetidos a experimentos com 150mcg/kg de veneno de B. alternatus por injeção intramuscular. O Grupo 1 (G1) foi o controle positivo e recebeu veneno e PBS / BSA, enquanto o Grupo 2 (G2) foi tratado com ácido tranexâmico, o Grupo 3 (G3) com desmopressina, o Grupo 4 (G4) com ácido tranexâmico e soro antibotrópico, e o Grupo 5 (G5) com soro antibotrópico e desmopressina. As amostras de sangue foram coletadas antes da administração do veneno, e uma, quatro, oito e 12 horas após os tratamentos para realização de tempo de tromboplastina parcial ativada parcial (TTPa), tempo de protrombina (TP), tempo de trombina (TT) e mensuração de fibrinogênio. Para o ensaio de geração de trombina (TG) foi realizado com um pool de amostras nos tempos finais (8 e 12h). Ao final das 12h, todos os animais foram sacrificados e a necropsia foi realizada. Amostras de tecido muscular, coração, pulmões e rins foram analisadas. Os testes TTPa, TP, TT e fibrinogênio não mostraram diferenças significativas entre os grupos e os tempos. No entanto, o TG indicou que o veneno causa um estado de hipocoagulabilidade, que não foi revertido pelos tratamentos propostos. Na histologia, foram observadas inflamação muscular, hemorragia e necrose, além de hemorragia em outros tecidos, sem diferenças entre os grupos. O envenenamento por B. alternatus causa hipocoagulabilidade detectada mais precocemente pelo teste de geração de trombina. O uso de ácido tranexâmico e desmopressina para estabilização da hemostasia após a inoculação do veneno não mostrou vantagem na restauração da coagulação.(AU)

Use of bone marrow for detection of toxic chemicals for the elucidation of poisoning in forensic veterinary medicine / Uso de medula óssea para detecção de produtos químicos tóxicos para a elucidação de intoxicação em patologia veterinária forense

In forensic toxicology, the detection of toxic chemicals from human bone marrow is often used in cases with an extended post mortem interval; however, in veterinary medicine, this practice is not used. Therefore, this study was performed to investigate the suitability of bone marrow for toxicological analysis in dogs and cats. Six animals with suspected poisoning were selected; the carcasses were sent for necropsy, and the organs were collected and preserved in buffered formalin and processed routinely for histological examination. In addition, bone marrow samples from the femur, humerus, and tibia were collected for toxicological analysis by liquid chromatography coupled to mass spectrometry detection (LC-MS). This analysis confirmed the presence of aldicarb, aldicarb sulfone, asulam, carbendazim, chlorpyrifos, dichlorvos, thifensulfuron methyl and trifloxysulfuron-sodium and associated with clinical symptoms and anatomo-histopathological alterations it was recognized the poisonings. It is expected that this study will promote the toxicological investigation of bone marrow and open avenues for the use of this tissue as an option for the detection of toxic chemicals in cases of forensic pathology.(AU)

Na toxicologia forense, a detecção de substâncias químicas tóxicas provenientes da medula óssea humana é frequentemente usada em casos com intervalo post mortem prolongado; no entanto, na medicina veterinária, essa prática não é utilizada. Portanto, este estudo foi realizado para investigar a utilização da medula óssea nas análises toxicológicas em cães e gatos. Seis animais com suspeita de intoxicação foram selecionados; as carcaças foram enviadas para necropsia e os órgãos foram coletados e preservados em formalina tamponada e processados rotineiramente para exame histológico. Amostras de medula óssea de fêmur, úmero e tíbia foram coletadas para análise toxicológica por cromatografia líquida acoplada à espectrometria de massa-massa (LC-MS). A análise por LC-MS confirmou a presença dos agrotóxicos aldicarbe, aldicarbe sulfona, asulam, carbendazim, clorpirifós, diclorvós, tifensulfuron metil e trifloxisulfuron-sódico, e em associação com sinais clínicos e achados anatomo-histopatológicos comprovou-se as intoxicações. Espera-se que este estudo promova a utilização da medula óssea como uma opção na investigação toxicológica para a detecção de produtos químicos tóxicos em casos de patologia forense.(AU)

Comparative Cardiotoxicity of Low Doses of Digoxin, Ouabain, and Oleandrin.

The aim of this study was to evaluate the comparative effects of CGs on heart physiology. Twenty-eight Wistar rats were distributed into four groups (n = 7), control group received NaCl 0.9% every 24 h for 21 days; treated groups received respectively 50 µg/kg of digoxin (DIG), ouabain (OUA) and oleandrin (OLE) every 24 h for 21 days. Serial ECGs were performed, as well as serum levels of creatinine kinase (CK), its MB fraction, troponin I (cTnI), calcium (Ca2+) and lactic dehydrogenase (LDH). Heart tissue was processed for histology, scanning electron microscopy and Western blot analysis for cTnI, brain natriuretic peptide (BNP), sodium potassium pump alpha-1 and alpha-2. Ventricle samples were also analyzed for thiobarbituric acid reactive substances and antioxidant enzymes (SOD, GPX, and CAT). ECGs showed decrease in QT and progressive shortening of QRS. No arrhythmias were observed. No significant differences were associated with CGs treatment and serum levels of CK, CK-MB, and cTnI. Only oleandrin increased LDH levels. Histological analysis showed degenerative changes and only oleandrin promoted moderate focal necrosis of cardiomyocytes. Scanning microscopy also confirmed the greatest effect of oleandrin, with rupture and shortening of cardiac fibers. The expression of troponin I and alpha-1 isoform were not altered, however, the protein levels of BNP and alpha-2 were higher in the groups that received oleandrin and ouabain in relation to the digoxin group. All GCs affected the production of ROS, without causing lipid peroxidation, through the activation of different antioxidant pathways. It is concluded that the administration of digoxin, ouabain, and oleandrin at 50 µg/kg for 21 days caused cardiovascular damage that represent an important limitation into its future use in heart failure and antineoplastic therapy.

Brucella ovis mutant in ABC transporter protects against Brucella canis infection in mice and it is safe for dogs.

BACKGROUND/OBJECTIVES: Vaccination is the most important tool for controlling brucellosis, but currently there is no vaccine available for canine brucellosis, which is a zoonotic disease of worldwide distribution caused by Brucella canis. This study aimed to evaluate protection and immune response induced by Brucella ovis ΔabcBA (BoΔabcBA) encapsulated with alginate against the challenge with Brucella canis in mice and to assess the safety of this strain for dogs. METHODS: Intracellular growth of the vaccine strain BoΔabcBA was assessed in canine and ovine macrophages. Protection induced by BoΔabcBA against virulent Brucella canis was evaluated in the mouse model. Safety of the vaccine strain BoΔabcBA was assessed in experimentally inoculated dogs. RESULTS: Wild type B. ovis and B. canis had similar internalization and intracellular multiplication profiles in both canine and ovine macrophages. The BoΔabcBA strain had an attenuated phenotype in both canine and ovine macrophages. Immunization of BALB/c mice with alginate-encapsulated BoΔabcBA (108 CFU) induced lymphocyte proliferation, production of IL-10 and IFN-γ, and protected against experimental challenge with B. canis. Dogs immunized with alginate-encapsulated BoΔabcBA (109 CFU) seroconverted, and had no hematologic, biochemical or clinical changes. Furthermore, BoΔabcBA was not detected by isolation or PCR performed using blood, semen, urine samples or vaginal swabs at any time point over the course of this study. BoΔabcBA was isolated from lymph nodes near to the site of inoculation in two dogs at 22 weeks post immunization. CONCLUSION: Encapsulated BoΔabcBA protected mice against experimental B. canis infection, and it is safe for dogs. Therefore, B. ovis ΔabcBA has potential as a vaccine candidate for canine brucellosis prevention.

Coenzyme Q10 Cardioprotective Effects Against Doxorubicin-Induced Cardiotoxicity in Wistar Rat.

In the present study, we investigated the cardioprotective effects of coenzyme Q10 (Q10) against doxorubicin (DOXO) induced cardiomyopathy. Twenty adult rats were distributed in four experimental groups: group 1 received NaCl 0.9% at 1 ml/day for 14 days; group 2 received Q10 at 1 mg/kg/day for 14 days; group 3 received initial 7 days of treatment with NaCl 0.9% followed by a single dose of doxorubicin (12.5 mg/kg IP) and another 7 days of NaCl; and group 4 received initial 7 days of Q10 1 mg/kg/day, followed by a single dose of doxorubicin (12.5 mg/kg IP) and another 7 days of Q10. At the end of 14 days, systolic, diastolic and mean blood pressure, electrocardiogram (ECG), complete blood count, and serum biochemical profile were evaluated. We also analyzed heart histological and ultrastructure analysis, and estimated heart's oxidative stress and lipid peroxidation. DOXO administration altered ECG, with increase heart rate, P-wave duration, PR interval duration, and T-wave amplitude. All the parameters were significantly reduced following Q10 treatment. DOXO also caused increase in CK, CK-MB, LDH, and urea levels, which were not mitigated by Q10 treatment. However, Q10 reduced oxidative stress by interfering with superoxide dismutase, significantly decreasing lipid peroxidation in heart tissue. DOXO administration also leads to several histological and ultrastructure alterations including cardiomyocyte degeneration and intense intracelullar autophagosomes, all minimized by Q10 treatment. Q10 treatment prevented the ECG changes, minimized oxidative stress, lipid peroxidation, and DOXO-induced heart tissue alterations. Our findings suggest that pre- and post-treatment with Q10 exerts potential cardioprotective effect against the DOX-induced cardiotoxicity.

Non-invasive ECG recording and QT interval correction assessment in anesthetized rats and mice / Eletrocardiograma não invasivo e avaliação da correção do intervalo QT em roedores anestesiados

Rats and mice are the most common species used in experimental cardiac electrophysiology studies. Electrocardiogram (ECG) recording shows paramount importance for monitoring arrhythmias and cardiac function in several disease models, including QT syndrome. However, the lack of standardized reference values and QT correction formula for different animal species and lineages represent a challenge for ECG interpretation. The aim of this study is to provide an improved method for ECG recording, establishing reference range values and determine the QT formulas with higher correlation to heart rate (HR). A total of 10 Wistar rats, 10 Swiss mice, 10 C57BL/6 mice and 10 FVB/NJ mice were used in the study. Animals were submitted to anesthesia with isoflurane and ECG recording was performed using a six-channel non-invasive electrocardiograph. QT was corrected using the following formulas: Bazzett, Fridericia, Mitchell, Hodges, Van der Water and Framingham. Normal range values for ECG parameters were established in all animals studied. Pearsons' correlation defined Hodges formula as the most suitable for QT correction. This study demonstrated an improved method of ECG recording with reference values for Swiss, FVB/NJ, C57BL/6 mice, and Wistar rats. Hodges' formula was the most effective formula for QT correction in rodents, whereas Bazett's and Friderica formulas were ineffective for such animals. The present work contributes to arrhythmias investigation in experimental cardiology and may reduce misinterpretations in rodents' ECG.(AU)

Ratos e camundongos são as espécies mais comumente utilizadas em estudos experimentais de eletrofisiologia cardíaca. O registro do eletrocardiograma (ECG) é de suma importância para o monitoramento de arritmias e função cardíaca em vários modelos de patologias. No entanto, a falta de valores de referência padronizados e a fórmula de correção do QT para diferentes espécies e linhagens animais representam um desafio para a interpretação do ECG. O objetivo deste estudo é fornecer um método melhorado para o registro de ECG, estabelecendo valores de referência e determinar as fórmulas QT com maior correlação com a freqüência cardíaca (FC). Um total de 10 ratos Wistar, 10 camundongos Swiss, 10 camundongos C57BL/6 e 10 camundongos FVB/NJ foram utilizados no estudo. Os animais foram submetidos à anestesia com isoflurano e o registro de ECG foi realizado com eletrocardiógrafo não invasivo de seis canais. O QT foi corrigido usando as seguintes fórmulas: Bazzett, Fridericia, Mitchell, Hodges, Van der Water e Framingham. Os valores da normalidade para os parâmetros do ECG foram estabelecidos em todos os animais estudados. A correlação de Pearson definiu a fórmula de Hodges como a mais adequada para a correção do QT. Este estudo demonstra um método melhorado de registro de ECG com valores de referência para camundongos Swiss, FVB/NJ, C57BL/6 e Wistar. A fórmula de Hodges foi a mais eficaz para correção de QT em roedores, enquanto as fórmulas de Bazett e Friderica apresentaram valores mais baixos de correlação. O presente trabalho contribui para a investigação de arritmias em cardiologia experimental e pode reduzir interpretações erradas no ECG de roedores.(AU)

Clinical biochemistry profile of American Quarter Horse broodmares fed Tifton-85 (Cynodon spp. ) hay and haylage / Perfil bioquímico clínico de matrizes Quarto de Milha alimentadas com feno e haylage Tifton 85 (Cynodon spp. )

The conservation of haylage (a pre-dried feed) can be challenging, since there is an increased risk of mould growth, which can contaminate this foodstuff with mycotoxins. However, when the hygienic quality is secured, haylage enhances grass palatability and provide enough supply of dry matter throughout the year. Due to the lack of information regarding its effect on blood parameters in horses fed exclusively with this foodstuff, the aim of this study is to provide information regarding its use in comparison to hay and ensure that it does not affect horses' biochemical profile. Twelve Quarter Horse broodmares were distributed into two groups, each fed with Tifton-85 (Cynodon spp.) hay or haylage for a period of 28 days, and the biochemical profile was done in five different times (T0 before the experiment started and, chronologically, seven days apart - T1, T2, T3 and T4), It was analyzed total protein (TP) and its fractioning; enzymes alanine aminotransferase, aspartate aminotransferase and γ-glutamyl-transferase; endogenous catabolism products urea and creatinine; and ions calcium and phosphorus. Mycotoxins in haylage were also investigated and remained below the legislation thresholds. Only TP was higher in the last sampling (T4) of the haylage group, which may be related to the foodstuff's higher protein digestibility. No differences were observed between serum enzymes, urea, creatinine and Ca/P from both experimental groups. Haylage has proven to be safe, when well prepared for horses, without causing impairing side effects, as shown by the normal serum biochemistry parameters presented in this study.(AU)

A conservação do haylage (alimento pré-seco) pode ser desafiadora, considerando o aumento do risco de crescimento de fungos, com consequente produção de micotoxinas. Entretanto, quando a qualidade da higiene e armazenamento é assegurada, o haylage aumenta a palatabilidade da forragem e fornece suplemento de matéria seca suficiente ao longo do ano. Devido à falta de informação relativa aos efeitos dessa alimentação nos parâmetros sanguíneos de equinos alimentados exclusivamente com essa dieta, o objetivo do presente estudo é avaliar o perfil bioquímico sanguíneo dos equinos após administração da haylage em comparação com feno. Doze matrizes Quarto de Milha foram distribuídas em dois grupos, cada um recebendo feno ou haylage de Tifton 85 (Cynodon spp.) por um período de 28 dias. O perfil bioquímico foi realizado em cinco tempos (T) diferentes (T0, antes do início do experimento e cronologicamente, a cada sete dias após o fornecimento das dietas - T1, T2, T3 e T4) para análise de proteína total (PT) e seu perfil fracionado, das enzimas alanina aminotransferase, aspartato aminotransferase, γ-glutamil-transferase, dos produtos de catabolismo creatinina e ureia e, dos íons cálcio e fósforo. Micotoxinas no haylage foram investigadas e mantiveram-se abaixo dos limites determinados pela legislação brasileira. O perfil bioquímico revelou, somente, elevação da PT em T4 no grupo que recebeu haylage, o que pode estar relacionado à sua maior digestibilidade proteica. Nenhuma diferença foi observada nos outros parâmetros estudados em ambos os grupos experimentais. Conclui-se que Haylage é comprovadamente seguro, quando bem preparado para equinos, sem causar efeitos na saúde geral, conforme demonstrado pelos exames bioquímicos no presente estudo.(AU)

Sequential administrations of trifluoroacetate induce tolerance to Palicourea maccgravii, a monofluoroacetate-containing plant, in calves.

Monofluoroacetate (MFA) is considered one of the most toxic substances known. It is found naturally in plants, and causes sudden death syndrome in ruminants. Due to hyperacute evolution of poisoning and the absence of effective treatment, induction of resistance in animals might be the best tool to control MFA poisoning in ruminants. The objective of this study was to promote resistance in cattle against the toxic effects of MFA through its degradation by the ruminal microbiota after the administration of sodium trifluoroacetate (TFA). Ten calves were distributed into two groups: control group (n = 3) and treated group (n = 7). The calves in the treated group received 0.1 mg/kg live weight of TFA, whereas, those in the control group received water; both for 28 consecutive days. The calves were subjected to daily clinical evaluation and weekly blood biochemical determination to identify any signs of poisoning. After 28 d of administration of TFA or water, 2.0 g/kg body weight of Palicourea marcgravii leaves (containing 0.15% MFA) were administered using a stomach tube to determine the occurrence of resistance. The administration of TFA did not induce any clinical or biochemical changes in blood. The administration of P. marcgravii induced clinical changes in the calves of control group, but there was no change in the calves of the treated group. In conclusion, the administration of TFA to cattle can induce effective resistance against MFA poisoning.

Liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (QTOF/MS) assay for quantification of protodioscin in Brachiaria grasses.

This work aimed to develop and validate a method to detect and quantify protodioscin in Brachiaria grasses using ultraperformance liquid chromatography (UPLC) coupled to high-resolution quadrupole time-of-flight mass spectrometry. Samples were extracted by acetonitrile-water 50:50 v/v mixture and ultrasonication. The mobile phase consisted of 5 mM ammonium acetate in water-methanol and acetonitrile containing 0.1% formic acid. The parameters used to validate the method for determining protodioscin comprised determination of the selectivity, ionization suppression/enhancement (matrix effect), linearity of the calibration curve, the limit of detection (LOD), the lower limit of quantitation (LLOQ), and the precision and accuracy of the method. The LLOQ of protodioscin was determined as 0.1 µg mL-1, and the LOD was 0.03 µg mL-1. The developed method was applied for determining protodioscin levels in B. decumbens collected from three pastures where sheep showed clinical signs of photosensitization. The obtained values ranged from 0.71% to 1.12%. Thus, the developed method for determining protodioscin in Brachiaria grasses by LC coupled to high-resolution quadrupole time-of-flight mass spectrometry showed high accuracy, precision, and sensitivity.

Conotoxin MVIIA improves cell viability and antioxidant system after spinal cord injury in rats.

This study evaluates whether intrathecal MVIIA injection after spinal cord injury (SCI) elicits neuroprotective effects. The test rats were randomly distributed into six groups- sham, placebo, MVIIA 2.5 µM, MVIIA 5 µM, MVIIA 10 µM, and MVIIA 20 µM-and were administered the treatment four hours after SCI. After the optimal MVIIA dose (MVIIA 10 µM) was defined, the best time for application, one or four hours, was analyzed. Locomotor hind limb function and side effects were assessed. Forty-eight hours after the injury and immediately after euthanasia, spinal cord segments were removed from the test rats. Cell viability, reactive oxygen species, lipid peroxidation, and glutamate release were investigated. To examine the MVIIA mechanism of action, the gene expressions of pro-apoptotic (Bax, nNOS, and caspase-3, -8, -9, -12) and anti-apoptotic (Bcl-xl) factors in the spinal cord tissue samples were determined by real-time PCR, and the activities of antioxidant enzymes were also investigated. Application of intrathecal MVIIA 10 µM four hours after SCI prompted a neuroprotective effect: neuronal death decreased (22.46%), oxidative stress diminished, pro-apoptotic factors (Bax, nNOS, and caspase-3, -8) were expressed to a lesser extent, and mitochondrial viability as well as anti-apoptotic factor (Bcl-xl) expression increased. These results suggested that MVIIA provided neuroprotection through antioxidant effects. Indeed, superoxide dismutase (188.41%), and glutathione peroxidase (199.96%), reductase (193.86%), and transferase (175.93%) expressions increased. Therefore, intrathecal MVIIA (MVIIA 10 µM, 4 h) application has neuroprotective potential, and the possible mechanisms are related to antioxidant agent modulation and to intrinsic and extrinsic apoptotic pathways.

Bem-estar em equinos do Jockey Club do Paraná: indicadores clínicos, etológicos e ritmo circadiano do cortisol / Welfare in horses of the Jockey Club Paraná: clinic ethologic indicators and circadian rhythm of cortisol

Com o objetivo de estudar parâmetros indicativos de bem-estar, 30 equinos de corrida foram avaliados quanto ao temperamento, comportamento, parâmetros clínicos (condição geral, escoriações, escore corporal, cólicas no último ano, frequências cardíaca e respiratória) e níveis de cortisol plasmático (matutino, vespertino e ritmo circadiano). Todos os animais apresentaram boas condições físicas gerais, parâmetros clínicos normais para a espécie e não tiveram cólica no ano anterior ao estudo, demonstrando a preocupação dos tratadores com a saúde física dos animais. Contudo, 36,7% dos animais estudados apresentavam estereotipias. Apesar da alta incidência de indivíduos com ritmo circadiano do cortisol (RCC) alterado dentro do grupo de animais com comportamentos anormais (63,64%), não foi encontrada diferença estatística (P>0,05) quando comparado com a incidência do grupo de animais sem estereotipia (31,58%). Quatro animais com estereotipia portavam dispositivos para restringir a expressão de anomalias. Desses, todos apresentaram RCC alterado, permitindo demonstrar que equinos restringidos fisicamente de realizar sua estereotipia apresentam 18 vezes mais chances de ter alteração no RCC do que um animal de comportamento normal. Conclui-se que os equinos do Jockey Club do Paraná, apesar de apresentarem condições físicas satisfatórias comparados a estudos nacionais anteriores, possuíam alterações comportamentais e de função adrenocortical compatíveis com estresse crônico. Os achados também corroboram noções prévias de que a estereotipia é uma forma de tentar aliviar o estresse crônico em equinos, e que o uso de meios físicos para restringir sua manifestação aumenta a chance de distúrbios no metabolismo do cortisol.(AU)

The objective was to study indicative parameters of welfare in 30 race horses evaluated for temperament, behavior, clinical parameters (general condition, abrasions, body score, colic in the last year, heart and respiratory rates) and plasma cortisol levels (morning, afternoon and circadian rhythm). All animals presented good general physical condition, normal clinical parameters for the species and no colic episodes in the year preceding the study. However, 36.7% of animals had stereotypies. Despite the high incidence of altered circadian cortisol rhythmicity (CCR) within the group of animals with abnormal behavior (63.64%), there was no statistical difference (P>0.05) when compared with the incidence in the group animals without stereotyping (31.58%). Four animals with stereotypic behavior carried devices to restrict the expression of anomalies. All these four individuals had altered CCR, showing that restricting the stereotypic behavior of a horse increases 18 times the chances of altered CCR when compared to an individual with normal behavior. The horses of the Jockey Club of Paraná, despite having good physical condition compared to previous national studies, had behavioral and adrenocortical function consonant with chronic stress changes. The results also corroborate previous concepts of stereotyping as stress relieving in horses, and that the use of its physical restriction increases the chances of disturbances in cortisol metabolism.(AU)

Improved method for diagnosis of Nerium oleander poisoning in necropsy tissues / Método aprimorado para diagnóstico da intoxicação por Nerium oleander em tecidos de necropsia

Nerium oleander is an ornamental cardiotoxic plant found in tropical and subtropical areas of the World. Its toxicity is related to the content of cardioactive glycosides, mainly oleandrin, found throughout the plant. The present study aimed to describe a new and improved method for oleandrin detection in tissue samples. The determination of oleandrin was made after extraction with a modified QuEChERS technique and measurement by UFLC-MS/MS. A total of 36 guinea pigs (Cavia porcellus) were distributed into 3 groups (n=12): control group that received only water orally (CON), and two treated groups that received hydroalcoholic oleander extract at doses of 150mg.kg-1 (OLE 150) and 300mg.kg-1 (OLE 300) in single oral dose. After three hours, fragments of heart, kidneys, liver and brain were collected for determination of oleandrin levels. The extraction and chromatographic procedures were effective for oleandrin detection and quantification in tissues, with retention time of 1.2 min and detection limit of 0.001μg g-1. The chromatographic analysis of treated guinea pigs indicated that oleandrin is distributed equally among the analyzed tissues. The developed methodology is a reliable, effective and rapid form of diagnosis of N. oleander poisoning based on necropsy tissue samples.(AU)

Nerium oleander é uma planta cardiotóxica ornamental encontrada em áreas tropicais e subtropicais do mundo. Sua toxicidade é relacionada á presença de glicosídeos cardioativos, principalmente a oleandrina, encontrada em toda a planta. O presente estudo objetiva descrever um novo e aprimorado método para detecção da oleandrina em amostras de tecido. A determinação da oleandrina foi feita após extração utilizando técnica modificada de QuEChERS e mensuração por UFLC-MS/MS. Um total de 36 cobaios (Cavia porcellus) foi distribuído em três grupos (n=12): grupo controle que recebeu apenas água por via oral (CON), e dois grupos tratados que receberam extrato hidroalcóolico de oleander nas doses de 150mg.kg-1 (OLE 150) e 300mg.kg-1 (OLE 300) em uma única dose oral. Após três horas, fragmentos do coração, rins, fígado e cérebro foram coletados para determinação dos níveis de oleandrina. A extração e procedimentos cromatográficos foram eficientes na detecção e quantificação da oleandrina nos tecidos, com tempo de retenção de 1,2min e limite de detecção de 0,001μg g-1. A análise cromatográfica dos animais tratados indicou que a oleandrina é distribuída de forma equalizada pelos tecidos analisados. A metodologia desenvolvida representa uma forma de diagnóstica segura, efetiva e rápida da intoxicação por N. oleander a partir de amostras de tecidos de necropsia.(AU)

Comparação dos efeitos das folhas de Cestrum axillare Vell. com as saponinas isoladas em caprinos / Comparison of the effects of Cestrum axillare leaves with isolated saponins in goats

Cestrum axillare Vell. (formerly Cestrum laevigatum Schltd.), family Solanaceae, is the most important hepatotoxic plant in Brazil that causes acute poisoning. It occurs in the Southeast and Center-West regions and in coastal areas of the Northeast Brazil. Spontaneous poisoning was described in cattle, goats and sheep, with clinical signs evidenced within 24 hours after ingestion of the leaves and death within 48 hours after signs onset. The clinical signs observed in acute poisoning are apathy, anorexia, ruminal arrest, arched back, constipation with feces in small spheres, sometimes covered with mucus and blood streaks, muscle tremors, staggering gait and sometimes sialorrhoea. Neurological signs may be observed, due to interference in the urea cycle due to hepatic insufficiency resulting in hyperammonemia (hepatic encephalopathy). The main pathological finding is centrilobular hepatic necrosis. The toxic principle present in C. axillare was not yet definitively proven, but some authors attribute the toxicity of the plant to the presence of saponins gitogenin and digitogenin. However, it has not been determined whether the saponins present in C. axillare are responsible for the hepatotoxic effect of the plant. Thus, the objective of this work is to determine if the saponins are the compounds responsible for the hepatotoxic effects produced by the ingestion of the leaves of C axillare, using goats as experimental model. For this, the effects of the administration of the leaves were compared with those produced by the saponins isolated from the leaves in goats. Six goats were randomly assigned to three experimental groups that received [1] dry leaves of C. axillare (animals A1 and A2), [2] saponins extract from leaves (animals S1 and S2) or [3] control group (animals C1 and C2). For goats receiving the dry leaves the administered dose of plant was 10g/kg for one animal (A1) and 5g/kg for the other one (A2). For animals receiving the saponins extract, administration was done at a dose equivalent to 20g/kg repeated after 24 hours. The dry leaves administered at a dose of 10g/kg to a goat produced toxic effects, with alterations in biochemistry (indicating hepatic lesion) and histopathology showing centrilobular hepatic necrosis. At the dose of 5 g/kg of dry leaves, clinical signs of poisoning were not observed, but hepatic necrosis was found; after 15 days after the last administration, the hepatic parenchyma of this animal was already normal, with only hemorrhagic areas, demonstrating full regeneration. The administration of extracts of saponins containing gitogenin and digitogenin to goats did not produce significant toxic effects, proving that these compounds are not responsible for intoxication. In addition, goats are a good experimental model for studies of this intoxication.(AU)

Cestrum axillare Vell. (anteriormente C. laevigatum Schltd.), família Solanaceae, é a mais importante planta hepatotóxica do Brasil que causa intoxicação aguda. Tem ocorrência nas regiões Sudeste e Centro-Oeste e em áreas litorâneas do Nordeste. A intoxicação natural foi descrita em bovinos, caprinos e ovinos, com sinais clínicos evidenciados em até 24 horas após a ingestão das folhas e morte em até 48 horas após o início da sintomatologia. Os sinais clínicos observados na intoxicação aguda são apatia, anorexia, parada ruminal, dorso arqueado, constipação com fezes em formas de pequenas esferas, por vezes recobertas com muco e com estrias de sangue, tremores musculares, andar cambaleante e, às vezes, sialorreia. Podem ser observados sinais neurológicos, devido à interferência no ciclo da ureia pela insuficiência hepática resultando em hiperamonemia (encefalopatia hepática). O principal achado patológico é a necrose hepática centrolobular. O princípio tóxico presente no C. axillare ainda não está definitivamente comprovado, mas alguns autores atribuem a toxicidade da planta à presença das saponinas gitogenina e digitogenina. No entanto, ainda não foi determinado se as saponinas presentes em C. axillare são as responsáveis pelo efeito hepatotóxico da planta. Assim, o objetivo deste trabalho é determinar se as saponinas são os compostos responsáveis pelos efeitos hepatotóxicos produzidos pela ingestão das folhas de C. axillare, usando caprinos como modelo experimental. Para isto, foram comparados os efeitos da administração das folhas com os produzidos pelas saponinas isoladas destas folhas em caprinos. Foram utilizados seis caprinos, distribuídos aleatoriamente em três grupos experimentais que receberam [1] folhas secas de C. axillare (Caprinos A1 e A2), [2] extrato de saponinas das folhas (Caprinos S1 e S2), e [3] grupo controle (Caprinos C1 e C2). Para os caprinos que receberam as folhas secas a dose administrada de planta foi de 10g/kg para um animal (A1) e de 5g/kg para outro (A2). Para os animais que receberam o extrato de saponinas, a administração foi feita na dose equivalente a 20g/kg, repetida após 24 horas. Foi verificado que as folhas secas, quando administradas na dose de 10g/kg a um caprino, produziram efeitos tóxicos, com alterações na bioquímica (indicando lesão hepática) e histopatológica apresentando necrose hepática centrolobular. Na dose de 5g/kg de folhas secas, não foi observado sintomatologia clínica da intoxicação, mas houve necrose hepática; 15 dias após a última administração, o parênquima hepático deste animal já se encontrava normal, apenas com áreas hemorrágicas, demonstrando plena regeneração. A administração do extrato de saponinas contendo gitogenina e digitogenina a caprinos não produziu efeitos tóxicos significantes, comprovando não serem estes compostos os responsáveis pela intoxicação. Além disto, a espécie caprina é um bom modelo experimental para estudos desta intoxicação.(AU)

In vitro study of gossypol's ovarian toxicity to rodents and goats.

Gossypol interferes with reproduction, causing damage to sperm, disrupting the estrous cycle and resulting in embryonic lethality. In females, gossypol administration promotes degeneration of ovarian follicles, but it is unknown whether this effect is direct or indirect. Thus, the aim of this study is to determine whether gossypol interferes with folliculogenesis in vitro in rats, mice and goats. Ovaries from rats and mice and fragments of goat ovaries were grown in cell culture for 24 h or 7 days. Four groups were tested: 0 (control), 5, 10 and 20 µg gossypol/ml. After incubation, the ovaries were fixed and processed for histological analysis. Follicles were classified according to their stage of development as either viable or atretic. It was found that the ovaries of rats, mice and goats cultured with gossypol showed an increase in the proportion of atretic follicles and a consequent reduction in the proportion of viable follicles at all stages of follicular development. Compared to the control group, the viability of all ovarian follicles in the rat, mouse and goat groups was reduced after cultivation for 24 h by 56.9%, 56.5% and 68.0%, respectively, with the highest concentration of gossypol (20 µg/mL), and after seven days, the respective reductions were 65.4%, 65.3% and 88.2%. Thus, it is possible that gossypol may directly affect follicular maturation, and consequently female fertility.

Simultaneous quantification of gatifloxacin, moxifloxacin, and besifloxacin concentrations in cornea and aqueous humor by LC-QTOF/MS after topical ocular dosing.

The fourth-generation fluoroquinolones are widely used as ophthalmic antimicrobials. This study aimed to validate a new analytical technique for simultaneous quantification of gatifloxacin, moxifloxacin, and besifloxacin concentrations in the cornea and aqueous humor by liquid chromatography (LC) coupled to quadrupole time-of-flight mass spectrometry (QTOF/MS) at 10min and 1h after instillation of topical ophthalmic antimicrobial suspensions. It was used twenty-two male dogs without ocular lesions verified by ophthalmic and histologic examinations. Methanol:water (4:1) was used for the extraction procedure for cornea and acetonitrile:water (4:1) was used for aqueous humor. The chromatographic separations were carried out on a C18 column with a linear gradient of water and methanol, both containing 0.1% formic acid. The total chromatographic run time was 4min. Mass spectrometry analyses were performed on a Xevo™ G2-S QTof tandem mass spectrometer, operated in a positive ion electrospray ionization (ESI+) mode. The retention times were approximately 1.42min for gatifloxacin, 1.87min for moxifloxacin, and 3.01min for besifloxacin. No interference peak was detected for the three tested antimicrobials in samples obtained from both cornea and aqueous humor, ensuring that the peak response was exclusive to the analyte of interest. The limit of detection for the three antimicrobials was 0.11µg/mL and the limit of quantification was 0.42µg/mL for both cornea and aqueous humor samples. At both time points post instillation of the three antimicrobials, moxifloxacin had the highest corneal concentration and besifloxacin demonstrated the highest concentration in the aqueous humor.

Computerized electrocardiography in healthy conscious guinea pigs (Cavia porcellus) / Eletrocardiografia computadorizada em cobaios (Cavia porcellus) saudáveis e conscientes

The purpose of this study is to evaluate healthy conscious guinea pigs as a model for electrophysiology assessment and to describe normal electrocardiographic patterns in controlled laboratory environment, establishing the best QT formula for this method. Electrocardiographic recordings of fifty adult conscious guinea pigs were obtained using a computerized electrocardiography. The electrocardiographic measurements of three different tracings were analyzed. The results obtained established normal mean and range values for the parameters: heart rate, waves and intervals of P-QRS-T deflections, as well as the mean cardiac axis. Groups were separated by body weight: group 1 gathered animals with 500-699g and group 2 with animals 700-900g. No differences were found when measurements were compared between groups, showing no significant difference between weight/body sizes to the electrocardiographic parameters (P<0.05). The mean corrected QT values (QTc) obtained using diverse formulae were significantly different (P<0.05), were the most consistent was Van der Water (QTcV). QTcV values were strongly correlated (r=98) and 95% confidence interval 185.7 to 195.2ms.Considering its simplicity and reliability, the QTcV was deemed the most appropriate to be used for the correction of QT interval in conscious guinea pigs.The results of this study also suggest that the values found can be used as reference for the species.(AU)

O objetivo desse estudo foi avaliar cobaios hígidos e conscientes como modelos para estudos de eletrofisiologia e descrever os padrões eletrocardiográficos normais em ambiente laboratorial controlado, estabelecendo a melhor fórmula QT para esse método. Gravações eletrocardiográficas de cinquenta cobaios adultos conscientes foram obtidas usando eletrocardiografia computadorizada. As medidas eletrocardiográficas de três trechos diferentes foram analisadas. Os resultados estabeleceram média e desvio padrão para os parâmetros: frequência cardíaca, ondas e intervalos P-QRS-T, assim como o eixo cardíaco médio. Grupos foram separados de acordo com o peso: grupo 1 incluiu animais com 500-699g e o grupo 2 animais de 700-900g. Nenhuma diferença foi encontrada quando as medidas foram comparadas entre os grupos, mostrando que não há diferença significativa entre peso/tamanho corporal com os parâmetros eletrocardiográficos (p<0.05). As médias corrigidas dos valores do QT (QTc) obtidas usando diferentes fórmulas foram significativamente diferentes (p<0.05), sendo a mais consistente a de Van der Water (QTcV). Valores de QTcV fortemente correlacionam com o QT (r=98), com intervalo de confiança a 95% de 185.7 a 195.2 ms. Considerando a simplicidade e confiabilidade, o QTcV foi considerado apropriado para correção do intervalo QT em cobaios conscientes. Os resultados do presente estudo também sugerem que os valores encontrados possam ser utilizados como referência para essa espécie.(AU)

Toxicity of crude and detoxified Tityus serrulatus venom in anti-venom-producing sheep.

Specific anti-venom used to treat scorpion envenomation is usually obtained from horses after hyperimmunization with crude scorpion venom. However, immunized animals often become ill because of the toxic effects of the immunogens used. This study was conducted to evaluate the toxic and immunogenic activities of crude and detoxified Tityus serrulatus (Ts) venom in sheep during the production of anti-scorpionic anti-venom. Sheep were categorized into three groups: G1, control, immunized with buffer only; G2, immunized with crude Ts venom; and G3, immunized with glutaraldehyde-detoxified Ts venom. All animals were subjected to clinical exams and supplementary tests. G2 sheep showed mild clinical changes, but the other groups tolerated the immunization program well. Specific antibodies generated in animals immunized with either Ts crude venom or glutaraldehyde-detoxified Ts venom recognized the crude Ts venom in both assays. To evaluate the lethality neutralization potential of the produced sera, individual serum samples were pre-incubated with Ts crude venom, then subcutaneously injected into mice. Efficient immune protection of 56.3% and 43.8% against Ts crude venom was observed in G2 and G3, respectively. Overall, the results of this study support the use of sheep and glutaraldehyde-detoxified Ts venom for alternative production of specific anti-venom.