Comparative ex vivo infection with Trypanosoma cruzi and Toxoplasma gondii of human, canine and ovine placenta: Analysis of tissue damage and infection efficiency.

Trypanosoma cruzi, the causative agent of Chagas disease, and Toxoplasma gondii, which is responsible for Toxoplasmosis, are two parasites that cause significant protozoan zoonoses and consequently important economic losses in human, companion animals and livestock. For the congenital transmission to occur, both parasites must cross the barrier present in the mammalian placenta, which differs between species. Particularly, hemochorial, endotheliochorial and epitheliochorial placental barriers are present, respectively, in human, dog and sheep. The type of placental barrier has been associated with the probability of transmission of pathogens. In this study, we used experimental placental ex vivo infection models of T. cruzi and T. gondii in the above-mentioned mammals in order to study tissue alterations and to compare infection efficiency. Here, we infected placental term explants from human, dog and sheep and analyzed tissue damage by standard histological and histochemical methods. Comparative infection efficiency was determined by quantitative PCR. Both parasites are able to infect the different placental explants; however, more T. gondii parasites were detected, and T. gondii causes a more severe tissue damage in human and canine explants than T. cruzi. The histopathological changes observed in ovine placenta explants were similar in presence of both parasites. We conclude that the infection efficiency of T. gondii is higher, compared to T. cruzi, during the ex vivo infection of human, canine and ovine placental explants. In addition, the ex vivo infection of mammalian placental explants constitutes an interesting experimental approach to study part of the infection mechanisms as well as host responses during congenital infection of both parasites.

Use of Fecal Indices as a Non-Invasive Tool for Nutritional Evaluation in Extensive-Grazing Sheep.

The aim of the present study was to assess the reliability of fecal indices as predictors of nutrient intake in sheep under extensive grazing conditions. Fecal concentrations of 2,6-diaminopimelic acid (DAPAf), nitrogen (FN) and phosphorous (FP) were determined in four sheep kept in an extensive grazing system on annual Mediterranean grassland in the vegetative, reproductive and dry phenological stages. Metabolizable energy (MEI), crude protein (CPI) and phosphorus (PI) intake were calculated using the botanical composition, metabolizable energy, crude protein and phosphorus concentrations in each vegetal species making up the animal's diet. Significant differences were observed in the nutrient intake for each phenological stage (p < 0.0001). The highest MEI, CPI and PI were observed during the vegetative stage (p < 0.0001). FN and FP were different in each phenological stage (p < 0.0001), with significant correlations observed between these variables (r = 0.916; p < 0.0001). Regressions among nutrient intake and fecal indices were significant, except in the cases of DAPAf and MEI, and DAPAf and CPI. Based on these results, fecal indices could be used to estimate nutrient intake in sheep under extensive grazing on annual Mediterranean grassland.

Myogenic Differentiation Potential of Mesenchymal Stem Cells Derived from Fetal Bovine Bone Marrow.

The myogenic potential of bovine fetal MSC (bfMSC) derived from bone marrow (BM) remains unknown; despite its potential application for the study of myogenesis and its implications for livestock production. In the present study, three protocols for in vitro myogenic differentiation of bfMSC based on the use of DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza), myoblast-secreted factor Galectin-1 (Gal-1), and myoblast culture medium SkGM-2 BulletKit were used. Plastic-adherent bfMSC were isolated from fetal BM collected from abattoir-derived fetuses. Post-thaw viability analyses detected 85.6% bfMSC negative for propidium iodine (PI). Levels of muscle regulatory factors (MRF) MYF5, MYF6, MYOD, and DES mRNA were higher (P < 0.05) in bfMSC cultured under 100 µM of 5-Aza compared to 1 and 10 µM. Treatment of bfMSC with 10 µM of 5-Aza resulted in down-regulation of MYOD mRNA (Days 7 to 21) and up-regulation of MYF6 (Day 7), MYF5, and DES mRNA (Day 21). Gal-1 and SkGM-2 BulletKit induced sequential down-regulation of early MRF (MYF5) and up-regulation of intermediate (MYOD) and late MRF (DES) mRNA. Moreover, DES and MYF5 were immunodetected in differentiated bfMSC. In conclusion, protocols evaluated in bfMSC induced progress into myogenic differentiation until certain extent evidenced by changes in MRF gene expression.

Expression of growth differentiation factor 9 (GDF-9) during in vitro maturation in canine oocytes.

The aim of this study was to characterize in canine oocytes and cumulus cells the dynamic expression of growth differentiation factor 9 (GDF-9) in relation to meiotic development and cumulus expansion throughout in vitro maturation (IVM). Cumulus oocytes complexes (COCs) from ovaries of adult bitches were cultured intact for IVM during 0, 48, 72, and 96 hours. At 0 hours or after IVM, COCs were divided into two groups: one group remained with their cumulus cells and in the other group the cumulus cells were extracted. The expression levels of GDF-9 were determined in both groups using indirect immunofluorescence and Western blot analysis. For immunofluorescence assay, in vivo-matured oocytes collected from oviducts were also used as a positive control. The nuclear stage was analyzed in parallel with 4'-6-diamidino-2-phenylindole staining in denuded oocytes from all maturing groups. The intensity of fluorescence, indicative of GDF-9 expression level, decreased with time (P < 0.05). High expression was observed only in germinal vesicle nonmature oocytes; in contrast, second metaphase oocytes showed only low expression. Western blot analysis showed bands of approximately 56 kd and a split band of approximately 20 kd representing the proprotein and possibly two mature protein forms of GDF-9, respectively. The proprotein was detected in all samples, and it was highly expressed before IVM and in a lesser degree, during the first 48 hours, declining thereafter in coincidence with the expansion of the cumulus cell (P < 0.05). There was a negative correlation (r = -0.97; P < 0.05) between the expression level of GDF-9 and mucification. Mature forms were evident only in COCs, before culture and up to 48 hours of IVM. It was concluded that GDF-9 is expressed in canine oocytes and cumulus cells, mainly in the early developmental states, with low levels in mature oocytes in vitro and in vivo, representing the first approach of GDF-9 dynamic in dog oocyte maturation.

Perinatal neuroendocrine regulation. Development of the circadian time-keeping system.

During gestation, the perinatal neuroendocrine axis keeps clock time. In primates, the suprachiasmatic nucleus (biological clock in mammals), shows oscillatory function by midgestation. There is evidence in rodents that the mother, during pregnancy, entrains the fetal suprachiasmatic nucleus (SCN) and newborn circadian rhythms. We are investigating the role of maternal melatonin as an entraining signal for the newborn circadian time-keeping system in the Cebus apella (New World non-human primate). Twenty-four hour rhythms of temperature and cortisol are present in the 4 days old C. apella newborn. Preliminary data suggests that inhibition of maternal melatonin by exposing pregnant females to constant light alters these rhythms. We have found binding sites for melatonin and expression of mRNA for Mel 1A receptor in hypothalamus, kidney and testis. These preliminary results suggest that maternal melatonin may play a role in relating the perinatal circadian time-keeping system to environmental signals.