Distinct Contributions of CD4+ and CD8+ T Cells to Pathogenesis of Trypanosoma brucei Infection in the Context of Gamma Interferon and Interleukin-10.

Although gamma interferon (IFN-γ) and interleukin-10 (IL-10) have been shown to be critically involved in the pathogenesis of African trypanosomiasis, the contributions to this disease of CD4(+) and CD8(+) T cells, the major potential producers of the two cytokines, are incompletely understood. Here we show that, in contrast to previous findings, IFN-γ was produced by CD4(+), but not CD8(+), T cells in mice infected with Trypanosoma brucei. Without any impairment in the secretion of IFN-γ, infected CD8(-/-) mice survived significantly longer than infected wild-type mice, suggesting that CD8(+) T cells mediated mortality in an IFN-γ-independent manner. The increased survival of infected CD8(-/-) mice was significantly reduced in the absence of IL-10 signaling. Interestingly, IL-10 was also secreted mainly by CD4(+) T cells. Strikingly, depletion of CD4(+) T cells abrogated the prolonged survival of infected CD8(-/-) mice, demonstrating that CD4(+) T cells mediated protection. Infected wild-type mice and CD8(-/-) mice depleted of CD4(+) T cells had equal survival times, suggesting that the protection mediated by CD4(+) T cells was counteracted by the detrimental effects of CD8(+) T cells in infected wild-type mice. Interestingly, CD4(+) T cells also mediated the mortality of infected mice in the absence of IL-10 signaling, probably via excessive secretion of IFN-γ. Finally, CD4(+), but not CD8(+), T cells were critically involved in the synthesis of IgG antibodies during T. brucei infections. Collectively, these results highlight distinct roles of CD4(+) and CD8(+) T cells in the context of IFN-γ and IL-10 during T. brucei infections.

[Antigenic determinants analysis and detection of virulence factors in F18 fimbriae Escherichia coli strains isolated from pigs].

OBJECTIVE: We determined the present distribution of serogroups and virulence factors among F18 Escherichia coli isolates from pigs with diarrhea and/or edema disease during 1998 to 2006. Epitope of F18 fimbriae was also analyzed. METHODS: A total of 75 E. coli isolates harbored fedA gene coding the major subunit of F18 fimbriae were identified by biochemical procedures and serological techniques. PCR was used to detect the virulence-related genes. An indirect ELISA was also used to analyze the antigen patterns of 33 F18 bearing E. coli strains. RESULTS: Among these 75 isolates, 62 were determined to be placed in total 8 serogroups, and 0107 and 0139 were main serogroups (61.3%) of fedA-positive isolates. The percentage of the detection of the estI, estII, elt, stx-2e, astA, orfA, irp2, fyuA, ler and eaeA among 75 strains was 64.0%, 46.7%, 28.0%, 62.7%, 26.7%, 9.3%, 9.3%, 9.3%, 1.3% and 1.3%, respectively. Of the 75 strains 19 were positive for stx-2e gene only, and 20 for estI/estII/stx-2e. The 11 MAbs against F18 "a", "b" and "c" had been used in indirect ELISA to detect F18 pili antigen, 44.0% (33/75) of these isolates were F18+ when cultured in TSB cultures. Among 33 isolates expressed F18 fimbriae, 21 isolates were identified as the "ac" variant, 2 were identified as the "ab" variant and 10 reacted only with F18 "a" specific MAbs while not with F18 "b", "c" specific MAbs. The results also revealed that the 11 MAbs recognized at least 6 epitopes including at least three common typespecific antigenic determinants "a" and two "b" antigen determinants and one "c" antigen determinant. CONCLUSION: The antigenic variants of F18 fimbriae are biologically distinct. F18ab fimbriae are expressed poorly in vitro, while F18ac are more efficiently expressed in vitro and most often are linked with enterotoxin (STa and/or STb) production and serogroups 0107. Some F18 fimbrial antigens experienced some variations comparing with those of F18ab and F18ac strains.

The transfer-messenger RNA-small protein B system plays a role in avian pathogenic Escherichia coli pathogenicity.

Extraintestinal pathogenic Escherichia coli (ExPEC) is capable of colonizing outside of the intestinal tract and evolving into a systemic infection. Avian pathogenic E. coli (APEC) is a member of the ExPEC group and causes avian colibacillosis. Transfer-mRNA-small protein B (tmRNA-SmpB)-mediated trans-translation is a bacterial translational control system that directs the modification and degradation of proteins, the biosynthesis of which has stalled or has been interrupted, facilitating the rescue of ribosomes stalled at the 3' ends of defective mRNAs that lack a stop codon. We found that disruption of one, or both, of the smpB or ssrA genes significantly decreased the virulence of the APEC strain E058, as assessed by chicken infection assays. Furthermore, the mutants were obviously attenuated in colonization and persistence assays. The results of quantitative real-time reverse transcription-PCR analysis indicated that the transcription levels of the transcriptional regulation gene rfaH and the virulence genes kpsM, chuA, and iss were significantly decreased compared to those of the wild-type strain. Macrophage infection assays showed that the mutant strains reduced the replication and/or survival ability in the macrophage HD11 cell line compared to that of the parent strain, E058. However, no significant differences were observed in ingestion by macrophages and in chicken serum resistance between the mutant and the wild-type strains. These data indicate that the tmRNA-SmpB system is important in the pathogenesis of APEC O2 strain E058.

Prolactin promotes crop epithelial proliferation of domestic pigeons (Columba livia) through the Hippo signaling pathway.

The purpose of this study was to investigate whether prolactin (PRL) regulates the proliferation of pigeon crop epithelium through the Hippo signaling pathway during the breeding cycle. Twenty-four pairs of adult pigeons were allotted to four groups by different breeding stages, and their crops and serum were sampled. Eighteen pairs of young pigeons were selected and divided into three groups for the injection experiments. The results showed that the serum PRL content and crop epithelial thickness of pigeons increased significantly at day 17 of incubation (I17) and day 1 of chick-rearing (R1). In males, the mRNA levels of yes-associated transcriptional regulator (YAP) and snail family transcriptional repressor 2 (SNAI2) were peaked at I17, and the gene levels of large tumor suppressor kinase 1 (LATS1), serine/threonine kinase 3 (STK3), TEA domain transcription factor 3 (TEAD3), connective tissue growth factor (CTGF), MYC proto-oncogene (c-Myc) and SRY-box transcription factor 2 (SOX2) reached the maximum value at R1. In females, the gene expression of YAP, STK3, TEAD3, and SOX2 reached the greatest level at I17, the expression profile of SAV1, CTGF, and c-Myc were maximized at R1. In males, the protein levels of LATS1 and YAP were maximized at R1 and the CTGF expression was upregulated at I17. In females, LATS1, YAP, and CTGF reached a maximum value at I17, and the expression level of phosphorylated YAP was minimized at I17 in males and females. Subcutaneous injection of prolactin (injected for 6 d, 10 µg per kg body weight every day) on the left crop of pigeons can promote the proliferation of crop epithelium by increasing the CTGF level and reducing the phosphorylation level of YAP. YAP-TEAD inhibitor verteporfin (injection for 6 d, 2.5 mg per kg body weight every day) can inhibit the proliferation of crop epithelium induced by prolactin by inhibiting YAP and CTGF expression. In conclusion, PRL can participate in crop cell proliferation of pigeons by promoting the expression of YAP and CTGF in Hippo pathway.

This study evaluated whether prolactin (PRL) regulates the proliferation of pigeon crops through Hippo signaling pathway during the breeding cycle. Twenty-four pairs of adult pigeons were allotted to four groups by different breeding stages, and their crops and serum were sampled. Eighteen pairs of young pigeons were selected and divided into three groups for the injection experiments. The crop epithelial thickness and serum PRL content of pigeons increased significantly at day 17 of incubation (I17) and day 1 of chick-rearing (R1). In males and females, the mRNA and protein levels of yes-associated transcriptional regulator (YAP) reached the maximum value at R1 and I17, respectively, and phosphorylation level of YAP were minimized at I17. Subcutaneous injection of prolactin on pigeon crops can promote the proliferation of crop epithelium by increasing the connective tissue growth factor (CTGF) level and reducing the phosphorylation level of YAP. YAP-TEAD inhibitor verteporfin can inhibit the proliferation of crop epithelium induced by prolactin by inhibiting YAP and CTGF expression. In conclusion, PRL can participate in crop cell proliferation of pigeons by promoting the expression of YAP and CTGF in Hippo pathway.

A DNA microarray assay for authenticating five important marine mammal species in food and feed.

Material identification in processed and unprocessed food and feed is crucial for ensuring the safety and hygiene of food and feed products. Therefore, to identify possible marine mammal components in feed, we study developed a DNA microarray with species-specific oligonucleotide probes that enable the rapid identification of five important marine mammal species (dolphins, seals, sea lions, white whales, and finless porpoises). The assay was tested using five target marine mammal species, and the probe patterns were compared with those of three fish meals (for feed) to see if they contained traces of marine mammals. All five marine mammal species could be distinguished by the microarray, and no marine mammal-derived ingredients were detected in the three fish meals. This study indicates that DNA microarray-based detection is relatively easy and effective for identification of non-compliant marine mammal ingredients in seafood or feed.

[Construction and pathogenic identification of aes-31 gene mutant of avian pathogenic Escherichia coli strain E058].

OBJECTIVE: To find the primary function of aes-31 fragment through construction of defined mutation of Avian Pathogenic Escherichia coli strain E058 and animal experiments. METHODS: The fragment of aes-31 was generated by PCR and cloned into pGEM-T-easy vector. A resultant suicide vector containing the aes-31 fragment named pMEG375-aes-31 was constructed and transformed to a receptor strain SM10. Then recombinant strain SM10 was hybridized with E058 strain in solid state. Mutant derivatives of strain E058 were generated by homologous recombination and were named E058 (delta aes-31). The 50% lethal dose (LD50) of E058 and E058 (delta aes-31) in commercial day-old chickens experimentally inoculated via intratrachea were 10(4.3) CFU and 10(3.5) CFU, respectively. The same way was used to inoculate with 10(8) CFU to obtain the pathogenic ability of E058 and E058 (delta aes-31) in 35-days-old SPF chickens. In the chicken challenge model,the mutant was tested to determine the individual function for virulence and persistence in 2-week-old SPF chicks. RESULTS: The pathogenicity test for E058 strain and E058 (delta aes-31) strain showed that the mutant had a higher mortality (75%) to 35-day-old specific pathogen-free (SPF) chicks than that of E058 (62.5%). In the chicken challenge model,there was no obviously CFUs difference in blood and lung in chicks of E058 group and E058 (delta aes-31) group 6 hours after inoculation. After 24 hours there was obvious CFUs difference in heart, liver, spleen, lung and blood in chicks of E058 group and E058 (delta aes-31) group. After 48 hours, there was also obvious CFUs difference in heart, liver and spleen in chicks of E058 group and E058 (delta aes-31) group E058 (delta aes-31) had a trend of increasing virulence in chicks. CONCLUSION: Aes-31 might be associated with negative regulatory gene for E058 virulence and its actual function needed further study.

Unexpected transcriptome pompT' contributes to the increased pathogenicity of a pompT mutant of avian pathogenic Escherichia coli.

The lambda red recombination system makes it suitable for screening virulence gene utility in avian pathogenic Escherichia coli (APEC) on account of its wide applicability, simplicity and high efficiency. In APEC E058 (O2 serogroup), there are two copies of the outer membrane protease (ompT) gene, compT encoding cOmpT that is located on the chromosome and pompT encoding pOmpT that is located on a ColV plasmid. However, the relationship between pathogenesis and pompT expression in APEC E058 has yet to be elucidated. Here, we successfully constructed two pompT gene mutants: E058ΔpompT containing a chloramphenicol (cat) resistance gene and E058ΔpompT' without the cat gene. By RT-PCR and sequencing analysis, an unexpected transcriptome pompT' was detected in mutant strain E058ΔpompT' after deletion of the cat gene induced by the lambda red recombination system. Surprisingly, the pathogenicity of mutant E058ΔpompT was significantly attenuated compared to its parental strain in the chicken infection model and HD11 cell model then the pompT gene was knocked out, while the pathogenicity of the other mutant strain E058ΔpompT' had no difference. Furthermore, the presence of unexpected transcriptome pompT' influenced the bactericidal activity of SPF chicken serum and decreased the transcription level of TLR2 in the heart tissue of chickens. Our study identifies the pompT gene plays an important role in the virulence of APEC E058, and the unexpected transcriptome pompT' contributes to the increased pathogenicity of APEC E058 mutants following deletion of the cat gene induced by the lambda red recombination system, which suggests that this system still has some limitations for construction of mutant strains particularly where these are used in development of live vaccine.

[Differential expression of virulence and potential virulence genes of avian pathogenic Escherichia coli in vitro with DNA microarray analysis].

We constructed avian pathogenic Escherichia coli (APEC) specific DNA microarray to analyze the transcriptomes of APEC highly pathogenic strain E058 and low pathogenic strain E526 (both belonging to O2 serotype). For cultures in Luria-Bertani (LB) broth medium, 16 distinctly expressed genes were observed in strain E526, and all of them were down-regulated. For cultures in the serum of chickens, 15 distinctly expressed genes were screened in strain E526, and all of them were also down-regulated. The results suggest that DNA microarray could be used to screen distinctly expressed genes among virulence- and potential virulence-associated genes of APEC. The expression of 11 common virulence- or potential virulence-associated genes were down-regulated for strain E526 compared to those of strain E058 cultured both in LB broth and chicken serum. Meanwhile, 4 potential virulence-associated genes, aes-3, aes-10, aes-13 and aes-15 were down-regulated for strain E526 but not for strain E058, when they grew in the chicken serum. Besides known virulence factors, we observed 10 new potential virulence-related genes including aes-1, aes-2, aes-3, aes-4, aes-6, aes-8, aes-10, aes-13, aes-15 and aes-31.

Comparison of virulence-associated traits between a UPEC strain HEC4 and a APEC strain E058.

Since avian pathogenic Escherichia coli (APEC) and human uropathogenic Escherichia coli (UPEC) may encounter similar challenges when establishing infection in the extra-intestinal locations of the hosts, they may share a similar content of virulence genes and capacity to cause disease. One APEC and one UPEC isolates were compared by their content of virulence genes and other traits. The two strains showed overlap in terms of their virulence genotypes, including their possession of certain genes associated with a large transmissible plasmid of APEC, and also shared some biochemical activities. Study of the pathogenicity of UPEC in chicks showed the similar symptoms and lesions compare to those caused by APEC. Based on these results, the potential whether APEC might serve as a reservoir of plasmid-linked and virulence genes for UPEC should be considered.

[Characterization of multidrug-resistant Salmonella serovars isolated from meats and human samples in some regions of Jiangsu].

A total of 117 Salmonella strains isolated from retail meats and human in some regions of Jiangsu were assayed for their antimicrobial susceptibility, the presence of integrons and antimicrobial resistance genes, and the ability of horizontal transfer of the characterized antimicrobial resistance determinants via conjugation. 111 (94.9%) were resistant to at least two antimicrobial agents. Resistance against streptomycin 92.3% (108/117) was the most common. Whereas 59 (50.4%) were resistant to at least five antimicrobials. Twelve isolates showed resistance to six antimicrobials: ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, and kanamycin (R type ACSSuTK). A total of 15 different antimicrobial resistance genes were identified in 36 multidrug-resistant Salmonella isolates. While 30 (83%) of these isolates tested carried integrons ranging in size from 0.3 to 1.6 kb. The most common integron was 1.6 kb which carried aadA5 and dfrl7 gene cassettes. Conjugation studies demonstrated that there was plasmid-mediated transfer of antimicrobial resistance genes. These data showed that the Salmonella isolates recovered from meats and human in some regions of Jiangsu were commonly resistant to multiple antimicrobials, and genes conferring antimicrobial resistance in Salmonella were often carried on integrons and plasmids and could be transmitted through conjugation. The mobile DNA elements might played very important role in transmission and dissemination of antimicrobial resistance determinants among Salmonella strains.

Isolation, identification, and pathogenicity of O142 avian pathogenic Escherichia coli causing black proventriculus and septicemia in broiler breeders.

Avian colibacillosis, characterized by black proventriculus and caused by avian pathogenic Escherichia coli (APEC) with an uncommon O142 serogroup, was diagnosed in young broiler breeders. Colonization and persistence assays performed in 7-day-old broilers showed that the bacterial load of the APEC 4d/9-1 O142 proventricular isolate in the lung was about 10-fold higher than that of the APEC 4d/9-1 O142 heart blood isolate (P<0.01), and about 100-fold higher in the heart blood, livers, spleens, kidneys, and proventriculi of inoculated broilers (P<0.001). When 32 common virulence genes of APEC were tested, the two isolates had nearly identical profiles, except that only the APEC 4d/9-1 O142 proventricular isolate carried the feoB gene. Furthermore, 100% mortality was observed in both 1-day-old Arbor Acres (AA) broilers and 1-day-old specific-pathogen-free (SPF) chickens inoculated with 10(6) colony-forming units of the APEC 4d/9-1 O142 proventricular isolate. However, black proventriculus was only observed in the dead AA broilers, consistent with the clinical occurrence of the disease. This implies that the black proventriculi seen in the dead birds, caused by the APEC 4d/9-1 O142 proventricular isolate, was breed-specific. Both the APEC 4d/9-1 O142 proventricular and heart blood isolates belong to phylogroup B2. However, the former was assigned to ST131 and the latter to ST2704 with multilocus sequence typing, demonstrating the genetic heterogeneity of these two bacterial isolates, although they were derived from the same dead broiler. These results suggest that the O142 APEC isolate was the main pathogenic agent for black proventriculi in 7-day-old broiler breeders.

Comparison of virulence factors and expression of specific genes between uropathogenic Escherichia coli and avian pathogenic E. coli in a murine urinary tract infection model and a chicken challenge model.

Avian pathogenic Escherichia coli (APEC) and uropathogenic E. coli (UPEC) establish infections in extraintestinal habitats of different hosts. As the diversity, epidemiological sources and evolutionary origins of extraintestinal pathogenic E. coli (ExPEC) are so far only partially defined, in the present study,100 APEC isolates and 202 UPEC isolates were compared by their content of virulence genes and phylogenetic groups. The two groups showed substantial overlap in terms of their serogroups, phylogenetic groups and virulence genotypes, including their possession of certain genes associated with large transmissible plasmids of APEC. In a chicken challenge model, both UPEC U17 and APEC E058 had similar LD(50), demonstrating that UPEC U17 had the potential to cause significant disease in poultry. To gain further information about the similarities between UPEC and APEC, the in vivo expression of 152 specific genes of UPEC U17 and APEC E058 in both a murine urinary tract infection (UTI) model and a chicken challenge model was compared with that of these strains grown statically to exponential phase in rich medium. It was found that in the same model (murine UTI or chicken challenge), various genes of UPEC U17 and APEC E058 showed a similar tendency of expression. Several iron-related genes were upregulated in the UTI model and/or chicken challenge model, indicating that iron acquisition is important for E. coli to survive in blood or the urinary tract. Based on these results, the potential for APEC to act as human UPEC or as a reservoir of virulence genes for UPEC should be considered. Further, this study compared the transcriptional profile of virulence genes among APEC and UPEC in vivo.