The aim of this study was to develop and validate different innovative DNA extraction methods to detect Mycobacterium avium subsp. paratuberculosis (MAP) DNA from bovine and buffalo colostrum. Paratuberculosis is a chronic inflammatory infection of domestic and wild animals, especially ruminants, caused by MAP. The primary route of disease transmission is feces, but MAP can also be excreted in milk and colostrum. In 2015, the Italian Ministry of Health has issued a voluntary control plan of MAP in order to allow risk‐based certification of bovine and buffaloes farms. In addition to the annual diagnostic screening and to the clinical surveillance of animals the plan includes the adoption of biosecurity and management measures to progressively mitigate the incidence of MAP. To achieve this goal it is crucial to ensure the accuracy of the methods used to detect the presence of MAP in bovine and buffaloes milk and colostrum, in order to: (1) support a "safe colostrum farm‐bank" set‐up and thus prevent the main within‐farm MAP transmission route and (2) to allow the MAP‐free certification of milk products for export purposes. To achieve these goals, seven different DNA extraction protocols were identified from bibliography, out of which three methods were finally selected after the adoption of an evaluation procedure aimed at assessing the efficiency of extraction of DNA, the purity of DNA and the adaptability of the DNA amplification: NucleoSpin® Food Kit (Macherey‐Nagel), NucleoSpin® Food Kit (Macherey‐Nagel) combined with the magnetic beads, and QIAamp Cador Pathogen Mini kit (QIAGEN). In particular, the NucleoSpin® Food Kit (Macherey‐Nagel) and the QIAamp Cador Pathogen Mini kit (QIAGEN) were tested on bovine and buffalo colostrum, showing a LOD between 4 × 104 (2.6 × 106 cfu/ml) and 4.08 (26.7 cfu/ml) IS900 target copies and a LOD between 5.3 × 105 (4.1 × 106 cfu/ml) and 53 (4.1 × 103 cfu/ml) IS900 target copies, respectively.
Aethina tumida (Coleoptera: Nitidulidae - Small Hive Beetle - SHB), is a parasite of honey bee colonies that causes the notifiable disease called aethinosis. In 2014, SHB was detected in Southern Italy, where it is still present (Calabria region). As part of surveillance activities, official diagnosis of the disease is performed by veterinary services via visual inspection of single hives in the apiaries. New outbreaks can be eradicated and the spread of SHBs limited by early detection of new introductions. We report an alternative protocol for the diagnosis of SHB through swabs and hive debris analysis by PCR. This was tested in three apiaries through the evaluation of different SHB infestation levels with a hive inspection method. This approach for sampling, followed by biomolecular methods, was fast and useful in unfavorable conditions (bad weather, aggressiveness, robbing), could be integrated in the official diagnosis procedures and may act as pre-clinic indicator.
Acarapis woodi is a pathogen affecting honey bees health worldwide. Its prevalence may be underestimated due to the time-consuming traditional method for its diagnosis and the attitude in focusing the attention only onto Varroa destructor. New PCR techniques have allowed for the verification of the presence of A. woodi in 44 samples of honey bees and 11 samples of hive debris collected from 17 apiaries by the veterinary services of the Latium region (Central Italy). Overall, 9.1% of adult honey bee samples (all belonging to one apiary) and 6.3% of hive debris samples (belonging to 6 apiaries) were positive in an end point PCR and presence of the pathogen was confirmed through Sanger sequencing. Results demonstrated the potential underestimation of A. woodi occurrence in Italian apiaries and reported the first detection of A. woodi in hive debris samples.
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