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.

Challenges in the rabbit haemorrhagic disease 2 (RHDV2) molecular diagnosis of vaccinated rabbits

Molecular methods are fundamental tools for the diagnosis of viral infections. While interpretation of results is straightforward for unvaccinated animals, where positivity represents ongoing or past infections, the presence of vaccine virus in the tissues of recently vaccinated animals may mislead diagnosis. In this study, we investigated the interference of RHDV2 vaccination in the results of a RT-qPCR for RHDV2 detection, and possible associations between mean Cq values of five animal groups differing in age, vaccination status and origin (domestic/wild). Viral sequences from vaccinated rabbits that died of RHDV2 infection (n = 14) were compared with the sequences from the commercial vaccines used in those animals. Group Cq means were compared through Independent t-test and One-way ANOVA. We proved that RHDV2 vaccine-RNA is not detected by the RT-qPCR as early as 15 days post- vaccination, an important fact in assisting results interpretation for diagnosis. Cq values of vaccinated and non-vaccinated infected domestic adults showed a statistically significant difference (p < 0.05), demonstrating that vaccination-induced immunity reduces viral loads and delays disease progression. Contrarily, in vaccinated young rabbits higher viral loads were registered compared to non-vaccinated kittens. No significant variation (p = 0.3824) was observed between viral loads of non- vaccinated domestic and wild RHDV2-victimised rabbits. Although the reduced number of vaccinated young animals analysed hampered a robust statistical analysis, this occurrence suggests that passively acquired maternal antibodies may inhibit the active immune response to vaccination, delaying protection and favouring disease progression. Our finding emphasises the importance of adapting kitten RHDV2 vaccination schedules to circumvent this interference phenomenon.