Standardization of a SYBR Green Based Real-Time PCR System for Detection and Molecular Quantification of Babesia bovis and B. bigemina in Water Buffaloes (Bubalus bubalis)
Pages 44-52
Dasiel Obregón, Marcio D. Rabelo, Rodrigo Giglioti, Thalita B. Bilhassi, Thalita A. Néo, Belkis Corona, Pastor Alfonso, Rosangela Z. Machado and Marcia C.S. Oliveira

DOI: http://dx.doi.org/10.6000/1927-520X.2016.05.02.4

Published: 30 November 2016

Abstract: Water buffalo (Bubalus bubalis) is a potential reservoir for Babesia bovis and B. bigemina in tropical regions, but the epidemiological evidence of their reservoir competence is limited, especially due to the lack of diagnostic tests capable of detecting and quantifying the low-level parasitemia present in the carrier animals. In this paper we present the standardization process of a SYBR Green based real-time PCR system (qPCR), consisting of two single qPCR assays, for the detection and quantification of B. bovis and/or B. bigemina. Both assays were optimized in similar protocols, including reagent concentrations and thermocycling parameters, so it is possible its use as a multiple qPCR in a single run. Both single assays showed a suitable analytical performance, especially by allowing detection of a greater number of carrier animals when compared with nested PCR assays (nPCR) against a reference panel of 60 DNA samples extracted from blood of both, infected- and non-infected buffaloes. Furthermore, a mathematical algorithm to convert the qPCR outcomes in percent of infected red blood cell was used, and was found that the estimated parasitemia in carrier buffaloes within the reference sample panels were close to those described in carrier cattle. This method could be a useful tool for epidemiological studies on the participation of the bubaline specie in the epidemic process of bovine babesiosis.

Selection of Biomarkers from Differentially Expressed Genes in Leukocytes of Buffalos Treated with Recombinant Bovine Somatotropin: The Importance of Sample Size for Reliable Discriminating Systems
Pages 1-13
Lorenzo Castigliego, Filippo Jodi Carrieri, Andrea Armani, Marco Mazzi, Carlo Boselli, Goffredo Grifoni, Daniela Gianfaldoni and Alessandra Guidi

DOI: http://dx.doi.org/10.6000/1927-520X.2016.05.01.1

Published: 06 April 2016

Abstract: The research on biomarkers to detect livestock treated with recombinant bovine somatotropin (rbST) is still an open issue. In fact, beyond undertaking confirmation methods, there is the need to develop simple and inexpensive screening tests. In this direction, some proposals have been forwarded, mostly involving the measurement of circulating molecules, whereas the possibility of using biomarkers related to gene expression is a field under investigation. The present study was carried out on sixteen buffalos, eight of which treated with rbST. Blood samples were collected six times during the treatment to investigate on the presence of differentially expressed genes in leukocytes. Analysis with the microarray technique was performed on two sampling moments, in order to obtain a first selection of genes. Further analysis was carried out by real time RT-PCR, in order to create a discriminating linear system. A study on the variation of the error related to the number of samples included in statistics was also performed. Results showed that, including an increasing number of samples to build the discriminating algorithm, the b-error grows and tends to stabilize on 6.5%. This study clearly shows the paramount importance of including a proper number of samples to obtain reliable algorithms..