The purpose of this study was to determine ultrastructural and cytoskeletal changes that result from vitrification of porcine germinal vesicle- (GV-) and meiosis II- (MII-) stage oocytes. To investigate the effects of vitrification on developmental competence, oocytes were divided into three groups: fresh GV-oocytes (control), vitrified GV-oocytes, and vitrified MII-oocytes. In both GV- and MII-oocytes, vitrification resulted in a high proportion with normal morphology (92.4 vs. 94.2%, P > 0.05), while vitrified GV-oocytes yielded a higher survival rate than did vitrified MII-oocytes (56.8 vs. 41.9%, P < 0.05). In vitrified GV-oocytes, 12 of 154 oocytes underwent cleavage after fertilization in vitro, and 6 of these developed to the 8-cell stage, 3 developed to the 16-cell stage, and 3 developed into morulae. No cleavage was obtained from vitrified MII-oocytes. For ultrastructural analysis of oocytes, fresh and vitrified-warmed GV- and MII-oocytes were randomly selected for transmission electron microscopy (TEM). Results showed that vitrification caused various degrees of cryodamage in GV-oocytes. Cumulus cells of some oocytes were separated from the cumulus-oocyte complex (COC), and the zona pellucida adjacent to cumulus cells was fractured. The gap junctions between cumulus cells were ruptured, and many microvilli were disrupted or disappeared. Only homogeneous lipid droplets were observed. After vitrification, cortical granules still lined the oolemma of MII-oocytes. Only morphologically irregular, nonhomogeneous lipid droplets surrounding large vacuoles were found. To examine cytoskeletal structures, fresh and vitrified-warmed MII-oocytes were analyzed by laser-scanning confocal microscopy (LSCM); vitrified-warmed GV-oocytes were cultured for 42-44 hr before LSCM. Of 58 control oocytes, 79.5% displayed normal spindles with chromosomes aligned along the equatorial plate. In vitrified oocytes the percentage with normal spindle organization was decreased significantly in both vitrified GV-oocytes and MII-oocytes (10.1 and 12.9%, respectively, P < 0.05). The proportion of oocytes with normal distribution of F-actin was lower for vitrified GV- and MII-oocytes than for controls (16.9 and 37.2% vs. 72.3%). Results of this experiment suggest that irreversible damage to the cytoskeleton of porcine GV- and MII-oocytes after vitrification could be an important factor affecting developmental competence.

The ibeA gene is located on a genomic island, GimA, which is involved in the pathogenesis of neonatal meningitis Escherichia coli (NMEC) and avian pathogenic E. coli (APEC). The prevalence of ibeA in the APEC collection in China was investigated, and 20 of 467 strains (4.3%) were positive. In addition, analysis of the association of the E. coli reference (ECOR) groups with positive strains revealed that ibeA was linked to group B2. The ibeA gene in DE205B was analyzed and compared to those of APEC and NMEC, which indicated that the specificity of ibeA was not consistent along pathotypes. The invasion of chicken embryo fibroblast DF-1 cells by APEC DE205B and RS218 was observed, which suggested that DF-1 cells could be a model to study the mechanism of APEC invasion. The inactivation of ibeA in APEC DE205B led to the reduced capacity to invade DF-1 cells, defective virulence in vivo, and decreased biofilm formation compared to the wild-type strain. In addition, strain AAEC189 expressing ibeA exhibited enhanced invasion capacity and biofilm formation. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) analysis and animal system infection experiments indicated that the loss of ibeA decreased the colonization and proliferation capacities of APEC in the brain during system infection.Escherichia coli typically colonizes the mammalian and avian gastrointestinal tract and other mucosal surfaces. While many of these strains are commensal, certain pathogenic strains can cause severe diseases (33). Extraintestinal pathogenic E. coli (ExPEC) is a group of strains that have been implicated in a large range of infections in humans and animals, such as neonatal meningitis, urinary tract infections (UTIs), pneumonia, osteomyelitis, and septicemia (14,16,30,35). Among these, typical infections caused by ExPEC in humans are UTIs and neonatal meningitis (5). Similarly, systemic infections caused by avian pathogenic E. coli (APEC) are economically devastating to poultry industries (14, 16). APEC enters and colonizes the avian respiratory tract by inhalation of fecal dust, leading to localized infections, such as airsacculitis and pneumonia. In certain cases, they spread into various internal organs, typically causing pericarditis, perihepatitis, peritonitis, salpingitis, and other extraintestinal diseases. Systemic infection of poultry is characterized in its acute form by septicemia, commonly resulting in sudden death (14,16,52).Previous studies showed that certain subsets of ExPEC strains isolated from different host organisms are highly similar (17,31,44,51), thus increasing the need to study their zoonotic potential. Virulence determinants common to uropathogenic E. coli (UPEC), APEC, neonatal meningitis Escherichia coli (NMEC), or septicemia-associated E. coli (SEPEC), such as the aerobactin iron transport system, the K1 capsule, and type 1 and P fimbriae, have been identified (2,14,19,21,43,47,59). Furthermore, the function of the K1 capsule was similar in virulence to those of APEC and NMEC. The K1...

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Avian pathogenic E. coli and human extraintestinal pathogenic E. coli serotypes O1, O2 and O18 strains isolated from different hosts are generally located in phylogroup B2 and ST complex 95, and they share similar genetic characteristics and pathogenicity, with no or minimal host specificity. They are popular objects for the study of ExPEC genetic characteristics and pathogenesis in recent years. Here, we investigated the evolution and genetic blueprint of APEC pathotype by performing phylogenetic and comparative genome analysis of avian pathogenic E. coli strain IMT5155 (O2:K1:H5; ST complex 95, ST140) with other E. coli pathotypes. Phylogeny analyses indicated that IMT5155 has closest evolutionary relationship with APEC O1, IHE3034, and UTI89. Comparative genomic analysis showed that IMT5155 and APEC O1 shared significant genetic overlap/similarities with human ExPEC dominant O18:K1 strains (IHE3034 and UTI89). Furthermore, the unique PAI I5155 (GI-12) was identified and found to be conserved in APEC O2 serotype isolates. GI-7 and GI-16 encoding two typical T6SSs in IMT5155 might be useful markers for the identification of ExPEC dominant serotypes (O1, O2, and O18) strains. IMT5155 contained a ColV plasmid p1ColV5155, which defined the APEC pathotype. The distribution analysis of 10 sequenced ExPEC pan-genome virulence factors among 47 sequenced E. coli strains provided meaningful information for B2 APEC/ExPEC-specific virulence factors, including several adhesins, invasins, toxins, iron acquisition systems, and so on. The pathogenicity tests of IMT5155 and other APEC O1:K1 and O2:K1 serotypes strains (isolated in China) through four animal models showed that they were highly virulent for avian colisepticemia and able to cause septicemia and meningitis in neonatal rats, suggesting zoonotic potential of these APEC O1:K1 and O2:K1 isolates.