Cirurgia e clínica de espécies pecuárias

Neste relatório são, primeiramente, descritas as atividades desenvolvidas durante o Estágio Curricular do Mestrado Integrado em Medicina Veterinária da Universidade de Évora, no âmbito de clínica e cirurgia de espécies pecuárias. Pretendeu-se, depois, determinar se a suplementação de bovinos de aptidão leiteira com β-caroteno injetável contribui para reduzir o número de inseminações artificiais necessárias para uma gestação. Por outro lado avaliou-se a influência que podem ter algumas variáveis, como sejam a estação do ano ou o número de partos da vaca. Procurou-se, ainda, estudar a evolução dos teores séricos de β-caroteno nos animais suplementados e não suplementados com Dalmafertyl®, utilizando a técnica de cromatografia líquida de alta eficácia ultravioleta. A suplementação parenteral de β-caroteno, nomeadamente com Dalmafertyl®, na Primavera, mostra resultados benéficos na reprodução de vacas e novilhas de aptidão leiteira, aumentando a produtividade e diminuindo custos; ABSTRACT: LARGE ANIMAL SURGERY AND CLINICS On the following report are described the activities that took place during the Curricular Externship as part of the Veterinary Medicine Integrated Masters from University of Évora, in large animal surgery and clinics. Also, it was intended to determine if injectable β-carotene supplementation on dairy cattle can be a decreasing factor on the artificial insemination number needed to result in pregnancy. On the other hand, it was assessed its influence on some variables that can affect pregnancy, such as seasons and calving number. It has been studied the evolution of β-carotene serum levels on Dalmafertyl® supplemented and non-supplemented animals using the high-performance liquid chromatography ultraviolet detection method, as well. β-carotene parenteral supplementation using Dalmafertyl® seems to have benefic results on dairy cattle reproduction when it comes to Spring, which leads to productivity increase and cost decrease

A rapid staining-assisted wood sampling method for PCR-based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR-based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining-assisted wood sampling method aiding PCR-based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5-mg wood cross-sections, of 0.5 · 0.5 · 0.015 cm dimension, that were cut from a wood sample of 0.5 · 0.5 · 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5-mg wood cross-sections (that adjoined the three 5-mg wood cross-sections, mentioned above) were cut. Nematode-staining-spots (NSSs) in each of the two stained sections were counted under a microscope at 100· magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus – specific amplicon of 403 bp (DQ855275) was generated by PCR assay from 100.00% of 5-mg wood cross-sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx-detection methods such as PCR assay.

Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus , is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the Msp I satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus , up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation.