Marta Fonseca Martins Guimarães

Toxigenic status of Staphylococcus aureus isolated from bovine raw milk and minas frescal cheese in Brazil.

A group of 291 Staphylococcus aureus isolates from mastitic cows milk (n = 125), bulk tank milk (n = 96), and Minas frescal cheese (n = 70) were screened for staphylococcal enterotoxin (SE) genes (sea, seb, sec, sed, see, seg, seh, sei, selj, and sell) and for the tst-1 gene encoding staphylococcal toxic shock syndrome toxin 1 by PCR assay. A total of 109 (37.5%) of the isolates were positive for at least one of these 11 genes, and 23 distinct genotypes of toxin genes were observed. Of the S. aureus isolates bearing SE genes, 17 (13.6%) were from mastitic cows milk, 41 (41.7%) were from bulk tank milk, and 51 (72.9%) were from Minas frescal cheese. The occurrence of exclusively more recently described SE genes (seg through sell) was considerably higher (87 of 109 PCR-positive strains) than that of classical SE genes (sea through see, 15 strains). The SE genes most commonly detected were seg and sei; they were found alone or in different combinations with other toxin genes, but in 60.8% of the cases they were codetected. No strain possessed see. The tst-1 gene was found in eight isolates but none from mastitic cows milk. Macrorestriction analysis of chromosomal DNA from 89 S. aureus isolates positive for SE gene(s) was conducted with the enzyme SmaI. Fifty-five distinct pulsed-field gel electrophoresis patterns were found, demonstrating a lack of predominance of any specific clone. A second enzyme, Apa I, used for some isolates was less discriminating than Sma I. The high genotype diversity of potential toxigenic S. aureus strains found in this study, especially from Minas frescal cheese, suggests various sources of contamination. Efforts from the entire production chain are required to improve consumer safety.

Association of MYF5 gene allelic variants with production traits in pigs

We studied the phenotypic effects of polymorphisms at the MYF5 gene in a divergent F2 swine population and found that one polymorphism was due to an insertion and another to a deletion. The genotypes of 359 F2 animals were obtained and the Normal/Normal (NN) and Normal/Insertion (NI) genotypes analyzed to determine associations with phenotypic data for performance, carcass and meat quality traits. Significant differences were observed (p < 0.05) between NN and NI animals for drip (NN = 3.14 ± 1.56; NI = 3.69 ± 2.78%), cooking (NN = 32.26 ± 2.41; NI = 33.21 ± 2.31%) and total loss (NN = 34.16 ± 2.63 and NI = 34.97 ± 2.08%). The Deletion marker was not statistically tested. The results indicate that the allelic variant Insertion is associated with a deleterious effect on meat quality traits and should be monitored in marker assisted selection programs.

Expression profile of genes associated with mastitis in dairy cattle

In order to characterize the expression of genes associated with immune response mechanisms to mastitis, we quantified the relative expression of the IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ and TNF- α genes in milk cells of healthy cows and cows with clinical mastitis. Total RNA was extracted from milk cells of six Black and White Holstein (BW) cows and six Gyr cows, including three animals with and three without mastitis per breed. Gene expression was analyzed by real-time PCR. IL-10 gene expression was higher in the group of BW and Gyr cows with mastitis compared to animals free of infection from both breeds (p < 0.05). It was also higher in BW Holstein animals with clinical mastitis (p < 0.001), but it was not significant when Gyr cows with and without mastitis were compared (0.05 < p < 0.10). Among healthy cows, BW Holstein animals tended to present a higher expression of all genes studied, with a significant difference for the IL-2 and IFN- γ genes (p < 0.001). For animals with mastitis no significant difference in gene expression was observed between the two breeds. These findings suggest that animals with mastitis develop a preferentially cell-mediated immune response. Further studies including larger samples are necessary to better characterize the gene expression profile in cows with mastitis.

Association of the bovine major histocompatibility complex (BoLA) BoLA-DRB3 gene with fat and protein production and somatic cell score in Brazilian Gyr dairy cattle (Bos indicus)

The effect of the bovine major histocompatibility complex (BoLA) locus on animal health may be due to a direct action of its alleles on immune functions, whereas its indirect effect on production traits might be explained by the better general health conditions of more productive animals. In the present study, the BoLA-DRB3 gene was investigated in 1058 cows belonging to seven Brazilian Gyr Dairy herds (Bos indicus, Zebu cattle). A total of 37 alleles were identified, 15 of them described for the first time in a Zebu breed. A highly significant association (p < 0.02) was observed between allele *54 and a decrease (-26.1 kg) in milk protein yield and there was a significant association (p < 0.05) between this allele and lower (-26.07 kg) milk fat yield. There was also a significant association (p < 0.05) between allele *6 and decreased (-12.47 kg) milk protein and allele *7 and increased (12.72 kg) milk protein. There were also indications of association (p < 0.10) between somatic cell score (SCS) and alleles *3 (SCS increased by 0.54 units) and *31 (SCS increased by 0.46 units). The highly significant association of allele *54 with lower protein yield suggests the possible use of this allele in marker-assisted selection programs.

Detection and quantification of Roundup Ready® soybean residues in sausage samples by conventional and real-time PCR

The increasing presence of products derived from genetically modified (GM) plants in human and animal diets has led to the development of detection methods to distinguish biotechnology-derived foods from conventional ones. The conventional and real-time PCR have been used, respectively, to detect and quantify GM residues in highly processed foods. DNA extraction is a critical step during the analysis process. Some factors such as DNA degradation, matrix effects, and the presence of PCR inhibitors imply that a detection or quantification limit, established for a given method, is restricted to a matrix used during validation and cannot be projected to any other matrix outside the scope of the method. In Brazil, sausage samples were the main class of processed products in which Roundup Ready® (RR) soybean residues were detected. Thus, the validation of methodologies for the detection and quantification of those residues is absolutely necessary. Sausage samples were submitted to two different methods of DNA extraction: modified Wizard and the CTAB method. The yield and quality were compared for both methods. DNA samples were analyzed by conventional and real-time PCR for the detection and quantification of Roundup Ready® soybean in the samples. At least 200 ng of total sausage DNA was necessary for a reliable quantification. Reactions containing DNA amounts below this value led to large variations on the expected GM percentage value. In conventional PCR, the detection limit varied from 1.0 to 500 ng, depending on the GM soybean content in the sample. The precision, performance, and linearity were relatively high indicating that the method used for analysis was satisfactory.

Differential expression of genes in follicular cells of swines.

The main purpose of the present study was to identify for candidate genes related to ovulation in swines. To do so, it was investigated in ovarian follicular cells through quantitative real-time PCR the differential expression of the following genes: steroidogenic acute regulator (STAR), GATA-binding protein 4 (GATA), prostaglandin F2α (PGF2α), progesterone receptor (P4R), follicle-stimulating hormone receptor (FSHR), and cytochrome P450 aromatase (CYP19). These genes encode hormone receptors (FSHR and P4R), hormone (PGF2α), steroidogenic proteins (STAR and CYP19) and transcription factor (GATA). Folicular cells were collected from sows with high and low number of piglets/litters during the follicular phase of the estrus cycle. There was difference in transcript abundance among low and high prolific sows for the STAR, GATA, PGF2α, P4R and CYP19 genes. For the FSHR gene, the fold change was not considered to be significantly different. Because in the present study only the transcript level of the above mentioned genes was analyzed, no inference can be made regarded to protein translation or activity. Therefore, gene sequence trials and other functional studies will be necessary to complement the present results, allowing a better understanding on biological complexity of these genes and their use as markers for prolificity in swines.

Análise de diversidade genética do gene da osteopontina em bovinos da raça girolando

The objective was to obtain the indices of genetic diversity for the SNP (single nucleotide polymorphism) of the 4 intron osteopontin gene (OPN) for 434 animals (87 bulls and 347 cows) participants in the Teste de Progenie da raca girolando (Girolando Progeny Test) in Brazil. For amplification, primers used were described for the Holstein breed, and differentiation of alleles C/T SNP that was obtained by PCR-RFLP. Genotype frequencies of TT (52.53%), CT (38.71%) and CC (8.76%) and allele frequencies of T (71.9%) and C (28.1%) indicate that the population is in Hardy-Weinberg principle (HWP). Although the OPN gene locus is in HWP, the higher frequency of allele T of SNP in these animals may suggest a setting-trend of allele T in the race. No difference was observed between the group of bulls and cows (FST = -0.018), supporting the estimate of population balance. Considering the values estimated by the FIS (0.043), it is likely that high numbers of individuals homozygous for the T allele observed in the population occur because of possible inheritance of this allele coming from the zebu breed, rather than inbreeding. Thus, to better characterize the OPN gene polymorphism, assessments in a larger number of animals must be performed, since only animals that participated in the Progeny Test were assessed.