Marcia Helena Braga Catroxo

Isolation of chicken astrovirus from specific pathogen-free chicken embryonated eggs

Astroviruses have been associated with enteric disorders in many animal species, including chickens. Here, we describe the isolation, propagation, and pathological characteristics of chicken astrovirus (CAstV) in specific pathogen free (SPF) chicken embryonated eggs (CEE) from chickens with diarrhea and runting-stunting syndrome. The CEE were inoculated via the yolk sac route. Viral confirmation was carried out using PCR techniques and transmission electron microscopy negative staining with ammonium molybdate. The intestinal contents were screened for CAstV, and differential diagnostic testing was performed for avian nephritis virus, avian rotavirus, avian reovirus, chicken parvovirus, infectious bronchitis virus, and fowl adenovirus Group I to detect co-infection with other infectious agents. Seven- or 14-day-old CEEs presented with hemorrhages, edema, a gelatinous aspect, deformities, and dwarfism. The supporting membranes did not show any alterations. Here, we have described the isolation of CAstV and its pathological characteristics in SPF CEE.

Isolation of avian nephritis virus from chickens showing enteric disorders

Abstract Runting-stunting syndrome (RSS) is one of the diseases associated with many detected viruses. In Brazil, there were reports of several enteric disease outbreaks in chickens in which avian nephritis virus (ANV) was detected; however, the role of ANV in the outbreaks and whether the virus was a causative agent of these cases of enteric diseases were not determined. The aim of this study was to isolate ANV in specific pathogen-free (SPF) chicken embryonated eggs (CEE) from the enteric contents of chickens showing signs of RSS. For this purpose, 22 samples of chicken enteric contents that were positive only for ANV were inoculated into 7 and 14-day-old SPF-CEE via the yolk sac route and incubated for 5 d, with a total of 3 passages. Virus isolation was confirmed by the presence of embryo injuries, detection of viral RNA by RT-PCR, and visualization of viral particles using electron microscopy. Therefore, the 7-day-old inoculated embryos showed dwarfism, gelatinous consistency, hemorrhage, and edema in the embryos, whereas the 14-day-old did not show any alteration. Viral RNA was detected in the embryos of both ages of inoculation, and the same viral particles were visualized. The embryos from the mock group showed no alteration and were negative for all the tests. The viral cDNA was sequenced, and the molecular and phylogenetic analyses showed that the Brazilian isolates are more related with the ANV-1 serotype group; the sequences of these isolates showed a high percentage of nucleotide (86.4 to 94.9%) and amino acid (92.3 to 98.7%) similarity with other sequences from China, Japan, Australia, and the United States that belong to this serotype previously classified group. In this study, we isolated 8 samples of ANV in SPF-CEE from enteric content samples from chickens with RSS. In doing so, we showed the pathological injuries to the embryo caused by the virus and the molecular characterization of a part of the ORF 1b gene of the virus.

PVALB diminishes [Ca2+] and alters mitochondrial features in follicular thyroid carcinoma cells through AKT/GSK3β pathway

We have identified previously a panel of markers (C1orf24, ITM1 and PVALB) that can help to discriminate benign from malignant thyroid lesions. C1orf24 and ITM1 are specifically helpful for detecting a wide range of thyroid carcinomas, and PVALB is particularly valuable for detecting the benign Hürthle cell adenoma. Although these markers may ultimately help patient care, the current understanding of their biological functions remains largely unknown. In this article, we investigated whether PVALB is critical for the acquisition of Hürthle cell features and explored the molecular mechanism underlying the phenotypic changes. Through ectopic expression of PVALB in thyroid carcinoma cell lines (FTC-133 and WRO), we demonstrated that PVALB sequesters free cytoplasmic Ca(2+), which ultimately lowers calcium levels and precludes endoplasmic reticulum (ER) Ca(2+) refilling. These results were accompanied by induced expression of PERK, an ER stress marker. Additionally, forced expression of PVALB reduces Ca(2+) inflow in the mitochondria, which can in turn cause changes in mitochondria morphology, increase mitochondria number and alter subcellular localization. These findings share striking similarity to those observed in Hürthle cell tumors. Moreover, PVALB inhibits cell growth and induces cell death, most likely through the AKT/GSK-3β. Finally, PVALB expression coincides with Ca(2+) deposits in HCA tissues. Our data support the hypothesis that the loss of PVALB plays a role in the pathogenesis of thyroid tumors.

Alterações na morfologia e na viabilidade no desenvolvimento de embriões bovinos fecundados in vitro com sêmen contaminado experimentalmente à Escherichia coli produtora da toxina Shiga stx2

The objective of this study was to evaluate by optical microscopy and transmission electron, changes in morphology and viability of the development of bovine embryos, fertilized with semen experimentally contaminated (STEC). Oocytes were aspirated from ovaries of slaughtered cows and the intact zona pellucida were selected and matured. After 20-24 hours of maturation, the oocytes were divided into 2 groups. The first, control group (n = 4l8),fertilized with semen tested and without any type of contaminant and the second, the infected group (n = 415), fertilized with sperm exposed to STEC. Both semen were treated by the technique of discontinuous Percoll gradient. After the period of fertilization, embryos were evaluated for their morphology and viability by optical and electron microscopy. In morphologic evaluation, the oocytes fertilized with contaminated semen showed cytoplasmic shrinkage, gaps in the division, asymmetry of blastomeres, ooplasm grainy, dark brown color, vacuoles formation, degeneration and zona pellucid disruption. These changes were not observed in the control group. The cleavage rate was 70.3 and 52.8%, respectively, for control and infected groups, significant differences (p = 0.0001). After the 5th day of embryonic development, where it was observed 44.7% of morula in the control group, and 22.4% in the contaminated group, showing a significant difference (p = 0.0001). The presence of STEC interferes with the cleavage rate of embryos and also prevents and causes a decline in embryonic development to the morula stage and cause morphological changes during this development.