ABSTRACT
The aim of this study was to detect vector-borne pathogens (Anaplasmataceae family, Rickettsia genus, and Bartonella genus) in bats from Misiones (Argentina). Thirty-three specimens were captured over 8 days using mist nets. Twenty (60.6%) blood samples were positive (11/13 Artibeus lituratus, 4/10 Desmodus rotundus, 4/8 Carollia perspicillata, and 1/2 Myotis nigricans) by PCR for the gltA gene fragment of Bartonella. All samples were negative by PCR for the Anaplasmataceae family and Rickettsia genus. The phylogenetic analysis showed seven Bartonella genotypes. The three genotypes obtained from A. lituratus, 2 from C. perspicillata, and 1 from D. rotundus were related to Bartonella spp. from New World bats, while the sequence obtained from M. nigricans was related to Old World bats. We identified a considerable diversity of Bartonella genotypes in a small number of bats, thus further research is required to better understand the complex bat-pathogen interaction.
ABSTRACT
Bats are potential reservoirs of many vector-borne bacterial pathogens. The aim of the present study was to detect species of Anaplasma, Ehrlichia, Neorickettsia, Rickettsia, Borrelia and Bartonella in Brazilian free-tailed bats (Tadarida brasiliensis, Molossidae) from Buenos Aires city, Argentina. Between 2012 and 2013, 61 T. brasiliensis from urban areas of Buenos Aires city were studied. The samples were molecularly screened by PCR and sequencing. Five bats (8.2%) were positive to Neorickettsia risticii, one (1.6%) was positive to Rickettsia sp. and three bats (4.9%) to Bartonella sp. For molecular characterization, the positive samples were subjected to amplification and sequencing of a fragment of p51 gene for N. risticii, a fragment of citrate synthase gene (gltA) for Rickettsia genus and a fragment of gltA for Bartonella genus. Phylogenetic tree was constructed using the maximum-likelihood method. Phylogenetic analysis of N. risticii detect in our study revealed that it relates to findings in the USA West Coast; Rickettsia sp. detected is phylogenetically within R. bellii group, which also includes many other Rickettsia endosymbionts of insects; and Bartonella sp. found is related to various Bartonella spp. described in Vespertilionidae bats, which are phylogenetically related to Molossidae. Our results are in accordance to previous findings, which demonstrate that insectivorous bats could be infected with vector-borne bacteria representing a potential risk to public health. Future research is necessary to clarify the circulation of these pathogens in bats from Buenos Aires.
Subject(s)
Bartonella/isolation & purification , Chiroptera/microbiology , Disease Reservoirs , Neorickettsia risticii/isolation & purification , Rickettsia/isolation & purification , Anaplasmataceae Infections/epidemiology , Anaplasmataceae Infections/veterinary , Animals , Argentina/epidemiology , Bacterial Proteins/genetics , Bartonella/genetics , Bartonella Infections/epidemiology , Bartonella Infections/veterinary , Citrate (si)-Synthase/genetics , Neorickettsia risticii/genetics , Phylogeny , Polymerase Chain Reaction , Rickettsia/genetics , Rickettsia Infections/epidemiology , Rickettsia Infections/veterinary , Sequence Analysis, DNAABSTRACT
The bone morphogenetic proteins' (BMPs) pathway is one of the evolutionarily oldest used by animal embryos and is required for dorsal-ventral patterning of the early embryo of both vertebrates and invertebrates. Nevertheless, the role of this system in preimplantation embryo development has not been extensively studied yet. Taking into account that the preimplantation period is different among species though the BMP system is conserved, information regarding comparative embryo development and the role of BMPs in different mammalian models is revised and discussed in this chapter. BMP system is expressed by maternal tissues (the ovary, the oviduct, and the uterus) as well as by the embryo and extraembryonic tissues. The reviewed information demonstrates a very important role for BMP signaling system at different stages of embryo preimplantation development from acquisition of gamete competence to regulation of trophoblast development and differentiation in mice as well as in ungulates.
Subject(s)
Blastocyst/cytology , Bone Morphogenetic Proteins/metabolism , Cell Differentiation/physiology , Embryonic Development/physiology , Signal Transduction/physiology , Animals , Blastocyst/metabolism , Female , Humans , PregnancyABSTRACT
The import of exogenous DNA (eDNA) from the cytoplasm to the nucleus represents a key intracellular obstacle for efficient gene delivery in mammalian cells. In this study, cumulus cells or oolemma vesicles previously incubated with eDNA, and naked eDNA were injected into the cytoplasm of MII oocytes to evaluate their efficiency for eDNA expressing bovine embryo production. Our study evaluated the potential of short time co-incubation (5 min) of eDNA with; (1) cumulus cells, to be used as donor cells for SCNT and (2) oolemma vesicles (vesicles) to produce parthenogenic transgene expressing embryos. In addition, we included a group consisting of the injection of eDNA alone (plasmid) followed by parthenogenic activation. Two different pCX-EGFP plasmid concentrations (50 and 500 ng/µl) were employed. The results showed that embryos produced by SCNT and by vesicle injection assisted by chemical activation were able to express the eDNA in higher rates than embryos injected with plasmid alone. The lower plasmid concentration allowed the highest development rates in all groups. Using confocal microscopy, we analyzed the interaction of FITC- labeled eDNA with cumulus cells and vesicles as well as oocytes injected with labeled plasmid alone. Our images demonstrated that eDNA interacted with cumulus cells and vesicles, resulting an increase in its expression efficiency. In contrast, oocytes injected with DNA alone did not show signs of transgene accumulation, and their eDNA expression rates were lower. In a further experiment, we evaluated if transgene-expressing embryos could be produced by means of vesicle injection followed by IVF. The lower plasmid concentration (50 ng/µl) injected after IVF, produced the best results. Preliminary FISH analysis indicated detectable integration events in 1/5 of SCNT blastocysts treated. Our studies demonstrate for the first time that short term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT embryos and also that parthenogenic, eDNA- expressing embryos can be obtained by injection of vesicles or eDNA alone. Moreover, eDNA-expressing embryos can be also obtained by cytoplasmic injection vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection technique.
Subject(s)
Embryo Culture Techniques/methods , Fertilization in Vitro/methods , Gene Expression Profiling/methods , Gene Transfer Techniques , Parthenogenesis , Animals , Cattle , Culture Media/metabolism , Cumulus Cells/cytology , Cumulus Cells/metabolism , Cytoplasm/genetics , Cytoplasm/metabolism , DNA/genetics , DNA/metabolism , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Gene Expression , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , In Situ Hybridization, Fluorescence , Ionomycin/pharmacology , Microinjections , Microscopy, Confocal , Nuclear Transfer Techniques , Oocytes/cytology , Oocytes/drug effects , Oocytes/metabolism , Plasmids/genetics , Plasmids/metabolism , Time FactorsABSTRACT
BACKGROUND: BMP4 is a member of the transforming growth factor beta (TGFbeta) superfamily and Noggin is a potent BMP inhibitor that exerts its function by binding to BMPs preventing interactions with its receptors. The aim of this work was to investigate the role of BMP4 and Noggin, on oocytes in vitro maturation (m experiments) and embryos in vitro development (c experiments) of bovine. METHODS: For m experiments, COCs were collected from slaughterhouse ovaries and in vitro matured in TCM with 100 ng/ml of either BMP4 or Noggin. After 24 h, the nuclear stage of the oocytes was determined by staining with Hoechst 33342. In addition, RT-qPCR was performed on MII oocytes to study the relative concentration of ZAR1, GDF9, BAX, MATER and HSP70 transcripts. Treated oocytes were submitted to parthenogenic activation (PA) or in vitro fertilization (IVF) and cultured in CR2. For c experiments, non-treated matured oocytes were submitted to PA or IVF to generate embryos that were exposed to 100 ng/ml of BMP4 or Noggin in CR2 until day nine of culture. Cleavage, blastocyst and hatching rates, expression pattern of the transcription factor Oct-4 in blastocysts and embryo cell number at day two and nine post-activation or fertilization were evaluated. RESULTS: We found that Noggin, as BMP4, did not affect oocyte nuclear maturation. Noggin supplementation up-regulated the expression of HSP70 and MATER genes in matured oocytes. Moreover, BMP4 during maturation increased the proportion of Oct-4 positive cells in parthenogenic embryos. On the other hand, when Noggin was added to embryo culture medium, developmental rates of parthenogenic and in vitro fertilized embryos were reduced. However, BMP4 addition decreases the development only for in vitro fertilized embryos. BMP4 and Noggin during culture reduced the proportion of Oct-4-expressing cells. CONCLUSIONS: Our results show that BMP4 is implicated in bovine oocytes maturation and embryo development. Moreover, our findings demonstrate, for the first time, that a correct balance of BMP signaling is needed for proper pre-implantation development of bovine embryos.
Subject(s)
Bone Morphogenetic Protein 4/physiology , Carrier Proteins/physiology , Animals , Autoantigens/biosynthesis , Blastocyst/drug effects , Blastocyst/metabolism , Bone Morphogenetic Protein 4/antagonists & inhibitors , Cattle , Embryonic Development/drug effects , Fertilization in Vitro , HSP70 Heat-Shock Proteins/biosynthesis , Parthenogenesis/physiologyABSTRACT
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Subject(s)
Humans , Community Medicine/education , Family Practice/education , Rural Health , SpainABSTRACT
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