Establishment of antibody panels and histochemical techniques in routine tumor diagnosis in Veterinary Pathology

In Portugal, Veterinary Pathology is developing rapidly, and in recent years we assist to the emergence of private laboratories and the restructuring of universities,polytechnics and public laboratories.The Portuguese Society of Animal Pathology,through its actions and its associates has been keeping the discussion among its peers in order to standardizethe criteria of description,classification and evaluation of cases which are the subject of our daily work.One of the last challenges is associated with the use of routine histochemical techniques and immunohistochemistry, in an effort to establish standardized panels for tumour diagnosis, which could eventually reduce each analysis cost.For this purpose a simple survey was built, in which all collaborators answered questions about the markers used for carcinoma, sarcoma and round cell tumour diagnosis, as well as general questions related with the subject. We obtained twenty-one answered to the questions, from public and private laboratories.In general, in most cases immunohistochemical and histochemical methods are used for diagnosis.The wide spectrum cytokeratins are universally used to confirm carcinoma, and vimentin for sarcoma. The CD3 marker is used by all laboratories to identify T lymphocytes. For the diagnosis of B-cell lymphoma, the marker used is not consensual. In each laboratory there are different markers for more specific situations and only two labs perform PCR techniques for diagnosis. These data will be presented to promote extended discussion,namely to reach a consensus when different markers are used.

Veterinary allergy diagnosis: past, present and future perspectives

Fleas, several aeroallergens as well as many food allergens are the most common allergenic sources for animals and frequent cause of allergic reactions with different target organs such as skin, eyes, and respiratory or digestive systems. Allergy diagnosis needs to follow well-established guidelines under clinical and laboratory approaches. Since 1980 with the Hanifin & Rajka’s criteria for the diagnosis of atopic dermatitis (AD) in humans, successive proposals have been developed to identify atopic dermatitis in dogs. A consensual plan was first proposed by Willemse in 1986 undergoing several modifications in 1994. Prélaud and colleagues made important changes to the plan in 1998 and it was further adjusted by Favrot in 2009. In 2010, this plan was approved by the International Task Force on Canine Atopic Dermatitis (CAD). It was subjected in 2015 to minor updates with regard to therapeutic options. To improve diagnostic accuracy by integrating the basic knowledge on sensitization development and allergen nature and diversity, allergen sources and implicated molecular allergens for animals should be clearly identified. As well as in human medicine, this molecular epidemiology concept is essential for the veterinary allergy diagnosis in the near future, standing as the basis of a component-resolved diagnosis (CRD). Besides current pharma- cotherapy, it will be highly relevant to increase the efficiency of the avoidance measures and specific immunotherapy. Clinical guidelines will lead to at least 80 % of positive diagnosis of atopy, but newer laboratory methods in veterinary medicine aiming to a more precise diagnosis and a better integration of the clinical/laboratory diagnostic course are needed. Allergoms identification for animals, from different allergen sources proteoms should become a priority in veterinary allergology, in order to allow the intended CRD, which is essential to understand the cross-reaction phenomena, allowing a more precise and possibly effective component-resolved immunotherapy (CRIT). Further research has been carried out for a better understanding of the interaction between allergic clinical condition and immune pathophysiology. As well as in human medicine, a deeper knowledge of the molecular immunological mechanisms in veterinary allergy — with their specific allergen triggers — will also provide the veterinary allergist with the necessary information to act more efficiently in the future.

“Biomarkers in a One Health perspective: current knowledge of their ability to merge human and animal health

Biomarkers are nowadays essential tools to be one step ahead for fighting disease, enabling an enhanced focus on disease prevention and on the probability of its occurrence. Research in a multidisciplinary approach has been an important step towards the repeated discovery of new biomarkers. Biomarkers are defined as biochemical measurable indicators of the presence of disease or as indicators for monitoring disease progression. Currently, biomarkers have been used in several domains such as oncology, neurology, cardiovascular, inflammatory and respiratory disease, and several endocrinopathies. Bridging biomarkers in a One Health perspective has been proven useful in almost all of these domains. In oncology, humans and animals are found to be subject to the same environmental and genetic predisposing factors: examples include the existence of mutations in BR-CA1 gene predisposing to breast cancer, both in human and dogs, with increased prevalence in certain dog breeds and human ethnic groups. Also, breast feeding frequency and duration has been related to a decreased risk of breast cancer in women and bitches. When it comes to infectious diseases, this parallelism is prone to be even more important, for as much as 75% of all emerging diseases are believed to be zoonotic. Examples of successful use of biomarkers have been found in several zoonotic diseases such as Ebola, dengue, leptospirosis or West Nile virus infections. Acute Phase Proteins (APPs) have been used for quite some time as biomarkers of inflammatory conditions. These have been used in human health but also in the veterinary field such as in mastitis evaluation and PRRS (porcine respiratory and reproductive syndrome) diagnosis. Advantages rely on the fact that these biomarkers can be much easier to assess than other conventional disease diagnostic approaches (example: measured in easy to collect saliva samples). Another domain in which biomarkers have been essential is food safety: the possibility to measure exposure to chemical contaminants or other biohazards present in the food chain, which are sometimes analytical challenges due to their low bioavailability in body fluids, is nowadays a major breakthrough. Finally, biomarkers are considered the key to provide more personalized therapies, with more efficient outcomes and fewer side effects. This approach is expected to be the correct path to follow also in veterinary medicine, in the near future.

Role of organic acids and phytonutrients as natural alternatives to antibiotics in supporting intestinal functionality

The gastrointestinal tract (GIT) represents the major portion of the body that interfaces with the external environment, with the double function of food processing and line of defense of the body. Numerous components support and regulate the barrier function of the GIT, such as tight junctions (TJs), cytokines, commensal and pathogenic microorganisms, and other systems of the organism, as the endocannabinoid system (ECS). The ECS can control several gastrointestinal functions, as well as the regulation of intestinal inflammation. Failure of the intestinal barrier function triggers an increase of the concentration of pro-inflammatory cytokines and leads to a reduction in intestinal functionality. This thesis aimed to explore the potential of natural compounds as a new alternative approach to antibiotics not only as antimicrobial, but also supporting intestinal maturation and integrity, and as immune-boosting agents. Different experiments were performed to evaluate the potential of nature-identical compounds (NICs), organic acids (OAs), and essential oils (EOs) to support and fight various stressful stimuli. In vitro, a well characterized blend of NICs and OAs were able to improve TJs and transepithelial electrical resistance (TEER) in an intestinal cell line, exerting an anti-inflammatory potential. EOs enhanced TEER and TJs mRNA levels, with a reduction of paracellular permeability, showing antioxidant and antimicrobial properties. In vivo, thymol modulates the gene expression of ECS and gut chemosensing in the GIT of piglets, where the precise localization of the cannabinoid receptors was immunohistochemically confirmed, suggesting an anti-inflammatory potential. In conclusion, natural alternative molecules represent an effective alternative to support or replace the classical pharmacological prophylaxis. These alternative molecules act not only as antimicrobial agents, but also exerted a crucial role in supporting the intestinal barrier function, preventing oxidative stress, and reducing inflammation. Moreover, thymol seems able to modulate the ECS, representing a novel frontier to support animal health and productivity.

Extensão máxima de retalho pediculado de omento maior através de túnel subcutâneo para ossos longos em cães

Há tempo sabe-se que o omento humano pode promover atividade angiogênica em estruturas adjacentes nas quais ele é aplicado. Na medicina veterinária, são poucas as pesquisas com retalho pediculado de omento maior como indutor angiogênico e imunogênico, porém suas propriedades de adesão e drenagem são bem conhecidas. Os objetivos deste estudo foram criar um retalho pediculado de omento maior, mensurar seu comprimento durante as etapas de criação e avaliar a possibilidade de alcance para ossos longos (fêmur, tíbia, úmero, rádio e ulna) através de túnel subcutâneo, visando a utilizá-lo futuramente como indutor angiogênico em focos de fratura, para aceleração da osteogênese e controle de infecções ósseas. Foram utilizados 30 cadáveres frescos de cães de todas as raças, com exceção dos condrodistróficos. Os resultados foram conclusivos e confirmaram a possibilidade de alcance do retalho de omento para ossos longos de cadáveres de cães em que todos os retalhos alcançaram a metáfise distal dos ossos avaliados. A média de comprimento do omento, em camada dupla, dos 30 animais avaliados foi de 30,87cm; da camada simples foi de 54,37cm e do retalho em L foi de 92,7cm. Com a extensão máxima do omento, foi possível alcançar as metáfises distais de todos os ossos propostos, com comprimento médio excedente de 29,87cm para fêmur, 20,73cm para tíbia/fíbula, 25,13cm para úmero e 16,27cm para rádio/ulna. As variáveis peso e retalho em L avaliadas estatisticamente de cada indivíduo apresentaram correlação positiva moderada. Concluiu-se que, em cadáveres de cães, é possível levar o retalho pediculado de omento maior através de túnel subcutâneo para metáfise distal de ossos longos e que, quanto maior o peso do animal, maior o comprimento do retalho em L.

Fluorokinolonit eläinlääkinnässä : kirjallisuuskatsaus

Summary: Fluoroquinolones in veterinary medicine : an overview

Eläinlääkinnässä käytettävät solusalpaajat : kirjallisuuskatsaus

Summary: Sytostatic substances in veterinary medicine - literature overview

Therapeutic applications of viscous and injectable poly(ortho esters).

Poly(ortho esters) (POE) are hydrophobic and bioerodible polymers that have been investigated for pharmaceutical use since the early 1970s. Among the four described generations of POE, the third (POE III) and fourth (POE IV) are promising viscous and injectable materials which have been investigated in numerous biomedical applications. POE III has been extensively studied for ophthalmic drug delivery, it presents an excellent biocompatibility and is currently being investigated as a vehicle for sustained drug delivery to treat diseases of the posterior segment of the eye. POE IV is distinguishable by a highly reproducible and controlled synthesis, a higher hydrophobicity, and an excellent biocompatibility. It is currently under development for a variety of applications, such as ocular delivery, periodontal disease treatment and applications in veterinary medicine. This review will also focus on new perspectives for this promising family of polymers, such as guided tissue regeneration, treatment of osteoarthritis, as well as peptide and protein delivery.

Les conflits d’intérêts en médecine : un exemple vétérinaire

Étude de cas / Case study

Métrologie de la douleur animale : validation sur des modèles de douleur viscérale bovine et articulaires canins

La douleur est une expérience multidimensionnelle comportant des aspects sensoriels, émotionnels et cognitifs. Théoriquement, des méthodes de mesures comportementales, physiologiques, neurophysiologiques et sensorielles peuvent quantifier la douleur. Peu d’études ont étudié la validation des mesures utilisées en médecine vétérinaire. La recherche combine les travaux de Maîtrise et de Doctorat, traite en partie de la validité de méthodes. Dans cet objectif, nos travaux de recherche étudiaient la validité de méthodes comportementales, physiologiques et neurophysiologiques usuelles pour la mesure de la douleur en comparant les expressions de douleur (vache et chien) chez des animaux contrôle par comparaison à des animaux sous analgésie préventive ou sous traitement curatif suivant une douleur induite par chirurgie (modèles de douleur viscérale bovine ou orthopédique canine) ou causée par une maladie naturelle (arthrose canine). Une première étude comparait les mesures de la douleur entre les vaches du groupe placebo et celles sous analgésie postopératoire sur une durée de 21 jours suivant l’induction d’une douleur viscérale chronique. Les vaches du groupe placebo ont présenté une plus forte sensibilité à la douleur et une diminution de la noradrénaline et de la transthyrétine mesurées dans le liquide cérébro-spinal, une diminution de l’activité motrice (AM) (moindre que dans les groupes avec analgésie), de l’agitation enregistrée par vidéo-analyse et une augmentation du stress selon la mesure de l’activité électrodermique (AED). Les méthodes d’intérêt identifiées étaient les marqueurs spinaux, la mesure de la sensibilisation, de comportements par vidéo-analyse et de l’AM par bio-télémétrie. En utilisant des méthodes semblables à celles précédemment décrites, deux études expérimentales de douleur orthopédique ont été réalisées afin de comparer les réponses à la douleur entre des chiens traités avec une analgésie préventive (opioïdes et anti-inflammatoires, étude #2) ou un biphosphonate (tiludronate, étude #3) par comparaison à des chiens contrôles. Seules les échelles de douleur étaient différentes entre les études de recherche. Pour l’étude #2, les ii chiens sous analgésie ont présenté de plus faibles scores de douleur mesurés avec l’échelle de douleur nommée 4A-VET et ceci simultanément à une faible réponse de l’AED une heure après la chirurgie de trochléoplastie. La fréquence du comportement spontané de ‘la marche avec plein appui de la patte opérée’ mesurée à l’aide de la vidéo-analyse augmentait chez les chiens sous analgésie préventive 24 heures après la chirurgie. L’étude #3 démontrait surtout l’apparition de sensibilisation centrale (à la fois par l’évaluation sensorielle quantitative et les marqueurs spinaux) chez les chiens contrôle, 56 jours après l’induction d’arthrose chirurgicale. Ainsi, les chiens traités avec le tiludronate ont présenté une différence sur la substance P et la transthyrétine cérébro-spinale, une diminution de la sensibilisation périphérique, plus d’appui de la patte opérée lors de la marche selon la mesure du pic de force verticale (PFV), une augmentation de la fréquence de ‘la marche avec plein appui de la patte opérée’. La sensibilisation centrale était associée à la diminution de PFV, et une augmentation de l’AED et du comportement spontané de ‘la marche avec plein appui de la patte opérée’. Pour l’étude #4, la validité et la sensibilité des méthodes ont été évaluées dans une condition d’arthrose naturelle chez des chiens traités avec une diète enrichie en moule verte, un produit ayant des effets anti-inflammatoires et chondroprotecteurs attendus. Les chiens traités présentaient une diminution des scores de douleur via l’échelle nommée CSOM, une augmentation de PFV et une augmentation de l’AM. Dans l’ensemble, les résultats confirment que la vidéo-analyse évaluait la douleur de façon objective et pour des modèles différents de douleur et les marqueurs spinaux sont prometteurs. Le PFV était spécifique de la douleur orthopédique. La sensibilisation était présente lors de douleur pathologique. L’AED n’est pas valide pour la mesure de la douleur. La baisse d’AM suggèrerait un comportement de douleur. Les études étaient exploratoires pour les échelles de douleur en raison de leur niveau (débutant) de développement et du manque d’informations sur les qualités métrologiques de ces mesures.

Etude d'un anticoagulant oral (le rivaroxaban) sur les paramètres hémostatiques de chiens en santé

Chez le chien, les thromboses représentent une complication majeure de nombreuses conditions qui sont revues dans ce manuscrit. L’arsenal thérapeutique actuel présente certaines limites: des effets anticoagulants variables d’un patient à l’autre, des hémorragies et une administration par voie sous-cutanée pour l’héparine. Le rivaroxaban est un nouvel anticoagulant oral approuvé pour la prévention et le traitement des thromboses chez l’humain. C’est un inhibiteur direct du facteur Xa. La présente étude a pour objectif d’évaluer les effets hémostatiques du rivaroxaban chez des chiens en santé, en utilisant les tests de coagulation suivants: temps de prothrombine (PT), temps partiel de thromboplastine (aPTT), activité anti-facteur X, génération de thrombine (GT) et thromboélastographie (TEG®). Tout d’abord, l’effet anticoagulant du rivaroxaban a été évalué in vitro : le plasma citraté pauvre en plaquettes provenant de 20 Beagle en santé a été aliquoté et enrichi avec des solutions de rivaroxaban à des concentrations de 0 à 1000 mg/L d’anticoagulant. Une prolongation concentration-dépendante de tous les tests de coagulation a été notée. Les concentrations de 0.024 et 0.053 mg/L diminuent respectivement de 50% la vitesse de propagation de la GT et la densité optique de l’activité anti-facteur X. Ces derniers tests sont les plus sensibles et précis pour détecter l’effet anticoagulant du rivaroxaban. Ensuite, 24 Beagle en santé ont été répartis aléatoirement en 3 groupes (n=8). Chaque groupe a reçu par voie orale un placebo, ou 20 mg de rivaroxaban une ou deux fois à 8h d’intervalle. Quinze échantillons sanguins ont été prélevés pour chaque chien sur 30 heures. Pour tous les tests de coagulation excepté la TEG®, une différence significative a été notée dans les résultats entre les groupes traités et le groupe placebo (p<0.0001). La durée de l’effet anticoagulant du rivaroxaban était de 7.9-18.7h dans le groupe traité une fois; et de 17.5-26.8h dans le groupe traité deux fois. Le pic d’action de l’effet anticoagulant était d’environ 2h. Seul le paramètre R de la TEG® était significativement affecté dans les groupes traités. En conclusion, le rivaroxaban exerce un effet anticoagulant chez le chien à la dose de 2 mg/kg. Une administration biquotidienne semble appropriée pour un effet de 24h.

Contribuição para o estudo das transfusões sanguíneas no cão e no gato : situação em Portugal e estudo de 61 transfusões

Os avanços na área da Medicina transfucional, nomeadamente a descoberta de nova informação sobre os grupos sanguíneos de várias espécies, a introdução rotineira da tipificação sanguínea e das provas de compatibilidade eritrocitária, o estudo das reacções transfusionais adversas, o despiste de doenças infeciosas no dador e a aplicação da terapia por componentes, têm contribuído para aumentar a segurança da transfusão sanguínea em Medicina Veterinária e, por consequência, a sua utilização é cada vez mais frequente. O presente trabalho é constituído por três objectivos: perspectivar a medicina transfusional em Portugal através da análise dos resultados de um inquérito, dirigido aos CAMV, nomeadamente sobre o uso da terapia por componentes, as principais indicações de transfusão e a ocorrência de reacções transfusionais; caracterizar a população de gatos e cães receptores de transfusões sanguíneas, com enfoque na prevalência dos diferentes grupos sanguíneos, na indicação para a realização da transfusão e tipo de produto administrado; determinar a ocorrência de reacções transfusionais através da monotorização do doente antes, durante e após a administração das transfusões. No presente estudo, 86% dos Centros de Atendimento Médico Veterinário (CAMV) inquiridos recorrem à transfusão sanguínea como terapia complementar. Destes, 54.7% utiliza sangue total e produtos do sangue, 41.3% apenas sangue total e 4% apenas produtos do sangue. Os produtos do sangue mais utilizados são o concentrado de glóbulos vermelhos e o plasma fresco congelado (34.2% e 31.6% respectivamente). A anemia constitui o principal motivo para a realização de transfusões sanguíneas e a hipertermia a reacção transfusional mais frequente. Relativamente à caracterização da população de cães e gatos que receberam transfusão sanguínea conclui-se que 77.8% dos cães pertenciam ao grupo DEA 1.1 negativo e 22.2% ao grupo DEA 1.1 positivo. Todos os gatos incluídos neste estudo pertenciam ao grupo sanguíneo A. A anemia por hemorragia foi a indicação predominante para a administração de sangue nos cães (54.3%). Nos gatos a anemia por não produção de eritrócitos prevalece (60%). No que respeita às reacções transfusionais, das 61 transfusões realizadas apenas se registou uma dispneia num gato.

Investigation of whole blood of SJL/J mice using neutron activation analysis

The Br (0.0022 +/- A 0.0006 gL(-1)), Ca (0.113 +/- A 0.012 gL(-1)), Cl (3.07 +/- A 0.36 gL(-1)), K (2.63 +/- A 0.14 gL(-1)), Mg (0.045 +/- A 0.002 gL(-1)) and Na (2.09 +/- A 0.10 gL(-1)) concentrations were determined in whole blood of SJL/J mice using the Neutron Activation Analysis (NAA) technique. Eleven whole blood samples were analyzed in the IEA-R1 nuclear reactor at IPEN (So Paulo, Brazil). These data contribute for applications in veterinary medicine related to biochemistry analyses using whole blood. Moreover, the correlation with human blood estimation allows to checking the similarities for studying muscular dystrophy using this model animal.

Isolation, Characterization, and Differentiation Potential of Canine Adipose-Derived Stem Cells

Adipose tissue may represent a potential source of adult stem cells for tissue engineering applications in veterinary medicine. It can be obtained in large quantities, under local anesthesia, and with minimal discomfort. In this study, canine adipose tissue was obtained by biopsy from subcutaneous adipose tissue or by suction-assisted lipectomy (i.e., liposuction). Adipose tissue was processed to obtain a fibroblast-like population of cells similar to human adipose-derived stem cells (hASCs). These canine adipose-derived stem cells (cASCs) can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of cASCs are of mesodermal or mesenchymal origin. cASCs are able to differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirate, canine adipose tissue may also contain multipotent cells and represent an important stem cell source both for veterinary cell therapy as well as preclinical studies.

Physical-chemical analysis of the cerebrospinal fluid of healthy dogs submitted to different storage periods and temperatures

Disfunções envolvendo o sistema nervoso são de grande importância na Medicina Veterinária, pois tratam-se de enfermidades de elevada incidência e com poucos subsídios auxiliares no seu diagnóstico, prognóstico e na avaliação de terapias empregadas. Ainda hoje, o diagnóstico baseia-se, em grande parte, no histórico e no exame clínico neurológico. Dessa forma, a análise dos constituintes do fluido cefalorraquidiano torna-se uma das poucas alternativas de acesso clínico ao sistema nervoso central (SNC). Mesmo com a grande utilidade do exame físico-químico e citoscópico do liquor na neurologia veterinária, poucos são os estudos sobre a estabilidade dos seus constituintes sob estocagem. Dessa forma, o presente trabalho teve como finalidade verificar a influência da temperatura e do tempo de conservação nas características físico-químicas do liquor de cães hígidos. Para tanto, foram coletadas amostras de LCR, através da punção da cisterna cerebelo-medular de cães clinicamente sadios, as quais foram submetidas à análise da densidade específica, do pH, da glicorraquia, das proteínas totais e das atividades das enzimas creatina quinase (CK) e aspartato aminotransferase (AST), após conservá-las em diferentes temperaturas (25°C, 4°C e -4°C) e por diferentes períodos de tempo (logo após a colheita, 24 horas, 48 horas, uma semana e um mês). Dentre os resultados obtidos, foi possível verificar, principalmente, que houve estabilidade dos parâmetros estudados por até um mês de estocagem nas amostras mantidas sob a temperaturas de congelamento de -4°C.