Detection of K88 Fimbriae Gene of Escherichia coli from Mink

[Objective] This paper aimed to study the mechanism of diarrhea of mink caused by Escherichia coil [Method] Through the detection of K88 fimbriae gene of E. coli, cloning of gene fragments and identification, then PCR amplification was used to detect adhesion factor K88 gene, which was connected to T-vector and transformed into competent cells, and positive clones were selected. [ Results] E. coli 078, 029 and 038 were isolated from organs and feces of mink died of diarrhea in 3 mink farms, respectively, the 3 serotypes of E. coliwere detected in carrying K88 fimbriae gene and 3 positive clones were screened, respectively. [ Conclusion] The E. coli causing mink diarrhea carry K88 fimbriae gene.

Adhesion of <i>Gallibacterium anatis</i>to Chicken Oropharyngeal Epithelial Cells and the Identification of Putative Fimbriae

Microbial infections are typically initiated by the colonization of tissues by a specific mechanism that promotes adherence to host cells or tissues. In this work, we characterized the ability of Gallibacterium anatis F149T to express fimbriae that may be involved in mucosal attachment. Using transmission electron microscopy, the fimbriae-like structures could be observed on the surface of negatively stained G. anatis F149T, and these structures were further visualized after being released by physical shaking. When the fimbriae-like structures were separated by SDS-PAGE, the proteins comprising them were isolated and sized at 13 and 25 kDa. G. anatis F149T was able to adhere to chicken oropharyngeal epithelial cells. Adhesion could be completely inhibited by pretreatment of the bacterial cells with trypsin, whereas 25% inhibition was attained after pretreatment with an antiserum against the 13 kDa protein. We demonstrated by immuno-gold electron microscopy that the antibodies from the antiserum were specifically associated with the fimbria-like structures on G. anatis. These results indicated that G. anatis F149T expresses fimbriae that contribute to its adhesion to chicken oropharyngeal epithelial cells and may be important for colonization of the upper respiratory tract.

CS3 fimbriae of enterotoxigenic Escherichia coli as chimeric expression carriers of heterologous antigenic determinants

CS3 fimbriae, which are the strong immunogen, are produced by human enterotoxigenic Escherichia coli (ETEC). The possibility of using CS3 as a carrier of foreign antigenic determinants was investigated. A SacⅡ site sequence was inserted into the structural gene of CS3 subunit by site-specific mutagenesis based on analyzing and predicting the properties of the proteins. A recombinant strain expressing CS3/CTP3 hybrid fimbriae was constructed by inserting the sequence encoding CTP3 into the SacII site created by directed mutagenesis in the structural gene of CS3 subunit and transforming the recombinant plasmid into the host of DH5α. The result of SDS-PAGE showed that the hybrid CS3/CTP3 molecules were the fusion proteins with molecular masses of about 18.5 ku. Inoculating mice orally and intraperitoneally showed that both antibodies against CS3 and CTP3 were elicited. These results indicate that CS3 pili could be an exposure vector for heterologous antigenic determinants and become a powerful tool for the development of oral vaccines directed against mucosal pathogens.

Humoral Immune Response of Immunized Sows with Recombinant Proteins of Enterotoxigenic <i>Escherichia coli</i>

Enteric disorders in pigs are related to the fimbriae F4 (K88), F5 (K99), F6 (987P), F41 and F18 of enterotoxigenic Escherichia coli (ETEC). Immunization of sows with adhesins is important to stimulate the production of antibodies and the consequent transfer of these to the piglets via colostrum to prevent diarrhea during the neonate period and after weaning. The objective of this study was to evaluate the immune response of the sows immunized with recombinant ETEC proteins (F4, F5, F6, F18 and F41). The immune response of the sows immunized with the recombinant proteins was compared with a commercial vaccine containing ETEC bacterins. The study was performed on a commercial farm and included nine pregnant sows divided into three groups: G1 was vaccinated with recombinant proteins (n = 3);G2 was vaccinated with the commercial vaccine (n = 3);and G3 was vaccinated with sterile buffered saline (PBS) (n = 3). All the sows were fed a balanced diet without antibiotics and water ad libitum. The recombinant fimbriae stimulated the specific humoral immune response of the immunized sows. There was a statistically significant increase in the levels of antibodies to the fimbriae F4 (K88), F5 (K99), F6 (987P) and F18 in the sows vaccinated with the recombinant proteins compared with the control group. The colostrum IgG titers for all fimbriae in all the immunized sows were significantly increased compared to the control group. Additionally, all the piglets exhibited significantly increased antibody levels relative to all fimbriae when compared with those in the unimmunized control group, demonstrating successful antibody transfer via colostrum of the sows to the piglets.

Regulation of fim genes in uropathogenic Escherichia coli

Uropathogenic Escherichia coli(UPEC)is the leading cause of urinary tract infections in women,causing significant morbidity and mortality in this population.Adherence to host epithelial cells is a pivotal step in the pathogenesis of UPEC.One of the most important virulence factors involved in mediating this attachment is the type 1 pilus(type 1 fimbria)encoded by a set of fim genes arranged in an operon.The expression of type 1 pili is controlled by a phenomenon known as phase variation,which reversibly switches between the expression of type 1 pili(Phase-ON)and loss of expression(Phase-OFF).Phase-ON cells have the promoter for the fimA structural gene on an invertible DNA element called fimS,which lines up to allow transcription,whereas transcription of the structural gene is silenced in Phase-OFF cells.The orientation of the fimS invertible element is controlled by two site-specific recombinases,FimB and FimE.Environmental conditions cause transcriptional and post-transcriptional changes in UPEC cells that affect the level of regulatory proteins,which in turn play vital roles in modulating this phase switching ability.The role of fim gene regulation in UPEC pathogenesis will be discussed.

Intermittent vs Continuous Administration of Nerve Growth Factor to Injured Medial Septal Cholinergic Neurons in Rat Basal Forebrain

Many medial septal neurons of the basal forebrain are dependent on nerve growth factor (NGF) from the hippocampus for survival and maintenance of a cholinergic phenotype. When deprived of their source of NGF by axotomy, medial septal neuronal cell bodies atrophy and lose their cholinergic markers. This is similar to what is observed in the basal forebrain during Alzheimer’s disease (AD). In the present study, medial septal neurons were axotomized in female rats by way of a fimbria/fornix lesion. For fourteen days following axotomy, varying NGF doses (1 - 250 μg/ml) were administered to the lateral cerebral ventricle with either mini-osmotic infusion or daily injection. The responsiveness of medial septal neurons was evaluated with choline acetyltransferase immunohistochemistry. Within the mini-osmotic pumps, NGF activity diminished greatly during the first five days of implantation, but increased dramatically in the CSF after five days of infusion. The responsiveness of medial septal neurons to NGF was dose dependent and the ED50 for NGF injection was determined to be 14.08 μg/ml compared to 27.60 μg/ml for NGF infusion. Intermittent injections at varying intervals were evaluated with 30 μg/ml NGF over a fourteen-day time period (2, 3, 6, or 12 injections). No differences occurred in the number of choline acetyltransferase neurons from rats that received weekly injections to those that received daily injections of NGF. NGF administration has been suggested as a therapy for AD. The results of these studies continue to highlight the need for NGF stability within the delivery system and AD patient CSF, the choice of delivery system, frequency of administration, and the NGF dose for maintaining basal forebrain cholinergic neurons during AD.

Prophylactic Herbal Therapy Prevents Experimental Ascending Urinary Tract Infection in Mice

Objective： To study the preventive effect of herbal formulation on experimental murine urinary tract infection （UTI） induced by Dr Escherichia coil 11128. Methods： E. coil 11128 carrying Dr fimbriae was isolated from patients with chronic pyelonephritis. The minimal inhibitory concentration （MIC） value of herbal solution for E. coil 11128 was determined for further studies. Forty C3H/HeJ mice were divided into the herb-treated group （n=20, given Chinese herbs by gavage at an average dose of 20 g/kg body weight daily 3 days before inoculation）, and control group （n=20, given the same amount of distilled water by gavage）. Three and 6 days after infection, bacteria were counted in the urine and the kidneys of the mice. Kidney histopathologic changes were evaluated. Neutrophils infiltration and accumulation were detected. Results： The MIC value of herbal solution was 0.1 g/mL for the E. cofi 11128. In herb-treated mice, there was a significant reduction in bacterial counts in urine and colonization densities of kidneys. Microscopic studies revealed signs of inflammation in kidneys. In herb-treated mice, herbal administration resulted in significantly reduced neutrophilic infiltrates （P〈0.05）. The semi-quantitative scores for renal lesions were significantly lower （P〈0.05）. Conclusion： Prophylactic administration of herbal formulation potentiated the effect in partially preventing experimental murine ascending UTI.