Biological Characteristics of Listeria monocytogenes Following Deletion of TatD-like Protein Gene.

TatD is the subunit of the twin-arginine translocation (Tat) pathway. Members of TatD family are multifunctional, conserved and widely presented proteins in most prokaryotes. It has been reported that Tat can affect bacterial motility in some bacteria. This study was conducted to determine the contribution of the TatD protein (herein named LmTatD) to the regulation of flagella in Listeria monocytogenes. We constructed an LmTatD gene mutant in L. monocytogenes strain 10403 s and evaluated its biological characteristics. The results showed no difference in growth or morphology between the wild-type strain and the ΔLmTatD mutant. Intriguingly, the ΔLmTatD mutant showed impaired swimming motility and flagella structure but increased biofilm formation. Comparative proteomic analysis using tandem mass tag (TMT) combined with liquid chromatography-tandem mass spectrometry (LC‒MS/MS) was performed to determine differentially expressed proteins (DEPs). The results revealed that 134 proteins out of 2228 total proteins identified were differentially expressed, among which 18 proteins were upregulated and 116 proteins were downregulated in the ΔLmTatD mutant. Analysis of DEPs indicated that the reduced expression levels of the proteins related to flagellar assembly in the ΔLmTatD mutant correlate with its characteristics. Compared to the wild-type strain, the most downregulated proteins in the ΔLmTatD mutant included FlaA, FliD, FliR, FlgD, FlgL, and FlgG. Collectively, our data suggest that although LmTatD is not required for growth in L. monocytogenes, loss of LmTatD reduces flagellar production and motility by regulating flagellar assembly-related protein expression.

Effects of Salmonella enterica serovar typhimurium sseK1 on macrophage inflammation-related cytokines and glycolysis.

Salmonella enterica serovar Typhimurium (S. Typhimurium), an important virulent intracellular pathogen, causes inflammatory gastroenteritis or typhoid. Macrophages play a key role in innate immunity against Salmonella. Salmonella secreted effector K1 (SseK1) encoded by SPI2 has been identified a novel translocated protein. To investigate the role of Salmonella enterica serovar Typhimurium sseK1 about the inflammation and glycolysis in macrophages, the levels of IL-1ß, IL-2, IL-4, IL-6, IFN-γ and Nitric Oxide in macrophages infected by S. Typhimurium SL1344 wild-type (WT) group, ΔsseK1 mutant group and sseK1-complemented group were measured. And the glycolysis level was determined in RAW 264.7 cells infected with these different Salmonella strains. The results showed that groups infected by wild-type strain, sseK1 mutant and sseK1-complemented strain upregulated the production of IL-1ß, IL-2, IL-4, IL-6, IFN-γ and NO at 3 h, 6 h and 12 h, respectively. The production of IL-1ß, IL-2, IL-4, IL-6, IFN-γ and NO in wild-type strain group were significantly decreased compared with the ΔsseK1 mutant group, which suggested that sseK1 down-regulated the production of related inflammatory factors. Moreover, hexokinase, lactic acid and pyruvic acid levels significantly decreased by infection with sseK1 mutant compared to the wild-type strain. The ATP level of ΔsseK1 mutant group was remarkably increased than WT group and sseK1-complemented group. These indicated that the sseK1 enhanced the level of glycolysis of macrophages infected by S. Typhimurium. In summary, the results demonstrated that sseK1 can down-regulate the inflammation-related cytokines and enhance the glycolysis level in macrophages infected by S. Typhimurium, which may be beneficial for S. typhimurium survival in macrophages.

Salmonella enterica serovar Typhimurium sseK3 induces apoptosis and enhances glycolysis in macrophages.

BACKGROUND: Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important infectious disease pathogen that can survive and replicate in macrophages. Glycolysis is essential for immune responses against S. Typhimurium infection in macrophages, and is also associated with apoptosis. S. Typhimurium secreted effector K3 (SseK3) was recently identified as a novel translated and secreted protein. However, there is no study about the role of sseK3 in the relationship between apoptosis and glycolysis in cells infected with S. Typhimurium. It is unclear whether this protein exerts a significant role in the progress of apoptosis and glycolysis in S. Typhimurium-infected macrophages. RESULTS: Macrophages were infected with S. Typhimurium SL1344 wild-type (WT), ΔsseK3 mutant or sseK3-complemented strain, and the effects of sseK3 on apoptosis and glycolysis were determined. The adherence and invasion in the ΔsseK3 mutant group were similar to that in the WT and sseK3-complemented groups, indicating that SseK3 was not essential for the adherence and invasion of S. Typhimurium in macrophages. However, the percentage of apoptosis in the ΔsseK3 mutant group was much lower than that in the WT and sseK3-complemented groups. Caspase-3, caspase-8, and caspase-9 enzyme activity in the ΔsseK3 mutant group were significantly lower than in the WT group and sseK3-complemented groups, indicating that sseK3 could improve the caspase-3, caspase-8, and caspase-9 enzyme activity. We also found that there were no significant differences in pyruvic acid levels between the three groups, but the lactic acid level in the ΔsseK3 mutant group was much lower than that in the WT and sseK3-complemented groups. The ATP levels in the ΔsseK3 mutant group were remarkably higher than those in the WT and sseK3-complemented groups. These indicated that the sseK3 enhanced the level of glycolysis in macrophages infected by S. Typhimurium. CONCLUSIONS: S. Typhimurium sseK3 is likely involved in promoting macrophage apoptosis and modulating glycolysis in macrophages. Our results could improve our understanding of the relationship between apoptosis and glycolysis in macrophages induced by S. Typhimurium sseK3.

Effect of gga-miR-155 on cell proliferation, apoptosis and invasion of Marek's disease virus (MDV) transformed cell line MSB1 by targeting RORA.

BACKGROUND: Marek's disease (MD) is caused by the oncogenic Marek's disease virus (MDV), and is a highly contagious avian infection with a complex underlying pathology that involves lymphoproliferative neoplasm formation. MicroRNAs (miRNAs) act as oncogenes or tumor suppressors in most cancers. The gga-miR-155 is downregulated in the MDV-infected chicken tissues or lymphocyte lines, although its exact role in tumorigenesis remains unclear. The aim of this study was to analyze the effects of gga-miR-155 on the proliferation, apoptosis and invasiveness of an MDV-transformed lymphocyte line MSB1 and elucidate the underlying mechanisms. RESULTS: The expression level of gga-miR-155 was manipulated in MSB1 cells using specific mimics and inhibitors. While overexpression of gga-miR-155 increased proliferation, decreased the proportion of G1 phase cells relative to that in S and G2 phases, reduced apoptosis rates and increased invasiveness. However, its downregulation had the opposite effects. Furthermore, gga-miR-155 directly targeted the RORA gene and downregulated its expression in the MSB1 cells. CONCLUSION: The gga-miR-155 promotes the proliferation and invasiveness of the MDV-transformed lymphocyte line MSB1 and inhibits apoptosis by targeting the RORA gene.

Identification and Characterization of the Nuclease Activity of the Extracellular Proteins from Salmonella enterica Serovar Typhimurium.

Pathogens have evolved an array of strategies to establish a productive infection. The extracellular proteins secreted by pathogens are one of unique mechanisms to evade the host innate immune response. Many secretory proteins transported by the bacterial secretion systems have been widely investigated in Salmonella. Certain extracellular nucleases are essential for bacterial pathogenesis. However, there is no current data available for the enzymatic properties of the proteins secreted by Salmonella. Therefore, in the present study we have identified and characterized the nuclease activity of the extracellular proteins from Salmonella enterica serovar Typhimurium. It was demonstrated that the extracellular proteins from S. Typhimurium exhibited the deoxyribonucleases activity against λDNA by agarose gel electrophoresis and agar plate diffusion method. The activity was observed at 16 °C, 37 °C and 42 °C, and found to be highest at 42 °C and inhibited at temperatures over 60 °C. The nuclease activity was stable under alkaline conditions (pH 7-10) and the optimum pH was 9.0. The nuclease activity was promoted at high ionic strength of Ba2+, Ca2+, Mg2+, and Ni2+. Nuclease zymography analysis revealed that there were four activity bands in the extracellular proteins; followed by LC-ESI/MS/MS analysis seven proteins were identified. As demonstrated by nuclease zymography, the recombinant 5'-nucleotidase protein expressed in the prokaryotic expression system displayed the DNase activity. To our knowledge, the present findings represent the first direct and unambiguous demonstration of the nuclease activity of the extracellular proteins from S. Typhimurium, and it provides an important fundamental for further investigation of the role of the extracellular proteins in pathogenicity and immune evasion.

The impact of sseK2 deletion on Salmonella enterica serovar typhimurium virulence in vivo and in vitro.

BACKGROUND: Salmonella enterica is regarded as a major public health threat worldwide. Salmonella secretes the novel translocated effector protein K2 (SseK2), but it is unclear whether this protein plays a significant role in Salmonella enterica Typhimurium virulence. RESULTS: A ΔsseK2 mutant of S. Typhimurium exhibited similar growth curves, adhesion and invasive ability compared with wild-type (WT) bacteria. However, deletion of sseK2 rendered Salmonella deficient in biofilm formation and the early proliferative capacity of the ΔsseK2 mutant was significantly lower than that of the WT strain. In vivo, the LD50 (median lethal dose) of the ΔsseK2 mutant strain was increased 1.62 × 103-fold compared with the WT strain. In addition, vaccinating mice with the ΔsseK2 mutant protected them against challenge with a lethal dose of the WT strain. The ability of the ΔsseK2 mutant strain to induce systemic infection was highly attenuated compared with the WT strain, and the bacterial load in the animals' internal organs was lower when they were infected with the ΔsseK2 mutant strain than when they were infected with the WT strain. CONCLUSIONS: We conclude that sseK2 is a virulence-associated gene that plays a vital role in Salmonella virulence.

cAMP Receptor Protein of Salmonella enterica Serovar Typhimurium Modulate Glycolysis in Macrophages to Induce Cell Apoptosis.

We studied the role of glycolysis in the mechanism of cAMP receptor protein-induced macrophage cell death of Salmonella enterica serovar Typhimurium (S. Typhimurium). Cell apoptosis, caspase-3, -8, -9 enzyme activity, and pyruvic acid, lactic acid, ATP, and hexokinase (HK) contents were determined after infection of macrophages with S. Typhimurium SL1344 wild-type and a cAMP receptor protein mutant strain. While cell apoptosis, caspase-3, -8, -9 enzyme activity, lactic acid, hexokinase, and ATP levels significantly changed by infection with crp mutants compared to the wild-type strain (P < 0.05). Our data suggest that the cAMP receptor protein of S. Typhimurium can modulate macrophage death by effecting glycolysis levels. This finding may help to elucidate the mechanisms of S. Typhimurium pathogenesis.

Role of the sseK1 gene in the pathogenicity of Salmonella enterica serovar enteritidis in vitro and in vivo.

Salmonella enteritidis is a common food-borne pathogen associated with consumption of contaminated poultry meat and eggs, which frequently causes gastroenteritis in humans. Salmonella secreted effector K1 (SseK1), as a translocated and secreted protein has been identified to be essential for the virulence of Salmonella typhimurium in host cells. However, the role of the sseK1 gene in the pathogenicity of S. enteritidis remain unclear. In this study, a sseK1 deletion mutant of S. enteritidis was constructed and its biological characteristics were examined. It was found that the sseK1 deletion mutant did not affect the growth, adherence and invasion of Salmonella enteritidis when compared to the wild-type S. enteritidis. However, the mutant showed decreased formation of biofilm and significantly reduced intracellular survival of bacteria in activated mouse peritoneal macrophages, as well as showed reduced pathogenicity to a murine model by increasing the lethal dose 50% (LD50) value and decreasing the proliferation ratio of bacteria in vivo. Taken together, this study determined an important role for SseK1 in the pathogenicity of S. enteritidis in vitro and in vivo.

Isolation, molecular characterization and an artificial infection model for a variant porcine epidemic diarrhea virus strain from Jiangsu Province, China.

Porcine epidemic diarrhea virus (PEDV) is a causative agent of porcine intestinal disease, which causes vomiting, diarrhea, and dehydration in piglets. PEDV is associated with the most severe pathogenesis in one-week-old piglets, with mortality rates reaching 100%. A PEDV strain was isolated from the intestinal tract of diarrheic piglets from a pig farm in Jiangsu Province in March 2016, termed the JS201603 isolate. The isolated virus was confirmed to be PEDV via RT-PCR, electron microscopy, a cytopathic effect assay and sequence analysis. The S and ORF3 genes of the JS201603 isolate were sequenced, revealing that the S gene was associated with a 15-base insertion at 167 nt, 176 - 186 nt, and 427 - 429 nt, as well as a six-base deletion in 487 - 492 nt, indicating that it was a current epidemic variant compared with the classical strain, CV777. No deletion occurred between 245 - 293 nt of the ORF3 gene in the JS201603 isolate compared with the vaccine isolates YY2013 and SQ2014. An experimental infection model indicated that the piglets in the challenge group successively developed diarrhea, exhibiting yellow-colored loose stools with a foul odor. The piglets in the JS201603 isolate challenge group displayed reduced food consumption, lost weight, and in severe cases even died. No abnormalities were observed in the control group. The JS201603 variant isolated in this study contributes to the evolutionary analysis of diarrhea virus. The experimental infection model has established a foundation for further studies on vaccine development.

Attenuation of cellular toxicity by calpain inhibitor induced by bacterial endotoxin: a mechanistic study using muscle precursor cells as a model system.

This investigation was under taken to explore probable mechanisms and signal pathways involved in cytotoxicity induced by bacterial endotoxin lipopolysaccharide (LPS). Herein, we selected muscle precursor C2C12 myoblasts as representative cells to test effect of calpain inhibitor 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) on LPS induced inflammation and apoptosis. In order to rule out the toxicity of endotoxin, mouse myoblasts were exposed to various concentrations of LPS and viability of cells and morphology were assessed using CCK-8 assay and simple microscopy respectively. Apoptotic cell death was examined by fluorescence microscope at regular time intervals. Additionally, LPS induced apoptosis in C2C12 cells were determined by mRNA expression of µ-calpain, caspase-3 and tumor necrosis factor alpha (TNF-α) and were quantified by qRT-PCR. Our results point out that LPS stimulation produced dose dependent toxicity in muscle precursor cells. Pre-treatment with a calpain inhibitor can significantly attenuate LPS-induced inflammation/apoptosis. Results of present research determined that mRNA expression of aforesaid genes was amplified (p<0.05) in LPS stimulated C2C12 cells, whereas a noticeable drop off in mRNA expression of these genes was observed when pre-exposed with calpain inhibitor PD150606. Our study has outlined the current understanding regarding the connection between µ-calpain and caspase-3 in skeletal muscle wasting and as a result provides suitable choice for designing promising chemotherapeutic system for muscle illness and atrophy.

Deletion of Invasion Protein B in Salmonella enterica Serovar Typhimurium Influences Bacterial Invasion and Virulence.

Salmonella enterica serovar Typhimurium (S. Typhimurium) has a wide host range and causes infections ranging from severe gastroenteritis to systemic infections in human, as well as causing typhoid-like disease in murine models of infection. S. Typhimurium translocates its effector proteins through the Salmonella pathogenicity island-I (SPI-I)-encoded T3SS-I needle complex. This study focuses on invasion protein B (SipB) of S. Typhimurium, which plays an active role in SPI-I invasion efficiency. To test our hypothesis, a sipB deletion mutant was constructed through double-crossover allelic using the suicide vector pRE112ΔsipB, and its biological characteristics were analyzed. The results showed that the SipB does not affect the growth of Salmonella, but the adherence, invasion, and virulence of the mutant were significantly decreased compared with wild-type S. Typhimurium (SL1344). This research indicates that SipB is an important virulence factor in the pathogenicity of S. Typhimurium.

[Construction of host-vector balanced lethal system of Salmonella typhimurium SL1344Δcya mutant and immune protection test of chickling].

OBJECTIVE: To develop a host-vector balanced lethal system of Salmonella typhimurium adenylate cyclase mutant, and detect its biological characteristics. METHODS: We constructed SL1344ΔcyaΔasd mutant strain by recombinant suicide plasmid (pREAasd), and screened by two-step method, transformed pYA3493 plasmid containing the asd gene without resistance electric into the lack of SL1344 AcyaΔasd, then the recombinant strains SL1344 ΔcyaΔasd (pYA3493) was constructed successfully. RESULTS: The biochemical characteristics and growth rate of the mutant were different from that of the wild strain SL1344, but almost the same as that of the parent strain SL1344Δcya. The mutant strain could neither ferment maltose, lactose, and sorbitol, nor decompose H2S, galactose and rat lee sugar, but still retained the ability to use glucose. The one-day chicken lethal test showed that SL1344ΔcyaΔasd (pYA3493) mutant was at least 104 times lower than SL1344 strain. The protection rate induced by the SL1344ΔcyaΔasd (pYA3493) mutant was 62. 5%. CONCLUSION: The SL1344ΔcyaΔasd (pYA3493) mutant was successfully constructed, and had good immune protection, it laid a foundation for developing potential oral vaccines.

Bursin-like peptide (BLP) enhances H9N2 influenza vaccine induced humoral and cell mediated immune responses.

Vaccination with H9N2 avian influenza whole-inactivated virus (WIV) has been shown to be ineffective at eliciting sufficient humoral and cellular immunity against H9N2 avian influenza virus. This study assessed the effects of a synthetic Bursin-like epitope peptide (BLP) as adjuvant for H9N2 WIV in mice. Titers HI and avian influenza virus neutralizing antibodies, subtypes of HA antibodies, T helper (Th) cytokine levels, cytotoxic T-lymphocyte activities and changes in spleen T-cell subsets and natural killer cells were determined. We found that BLP induced a balance between IgG1 and IgG2a secretion levels. WIV antigen alone induced mainly Th1 cytokines secretion, whereas BLP showed increased secretion of Th1 and Th2 cytokines, including interleukin (IL)-2, interferon-γ (IFN-γ) and IL-4, but not IL-10, and may be resembles a Th0 like response. BLP significantly promoted growth and expansion of natural killer cells and of CD4(+) and CD8(+) T-cell subsets in the spleen. Meanwhile, BLP induced a better cytotoxic T-lymphocyte response to H9N2 virus. Furthermore, virus challenge experiments confirmed that BLP contributed to inhibition replication of the virus from mouse lungs. Taken together, these findings suggest that BLP may be an effective adjuvant for H9N2 avian influenza vaccine.

Biofilm formation, host-cell adherence, and virulence genes regulation of Streptococcus suis in response to autoinducer-2 signaling.

Autoinducer-2 (AI-2) is a universal signal molecule mediating intra- and interspecies communication among bacteria. AI-2 is a byproduct of the LuxS enzyme during the catabolism of S-adenosylhomocysteine and plays critical roles in regulating various behaviors of bacteria. In our previous study, the function of LuxS in AI-2 production was verified in Streptococcus suis (SS). Decreased levels of SS biofilm formation and host-cell adherence as well as the inability to produce AI-2 were observed in SS having a luxS mutant gene. In this study, exogenous addition of a low concentration of AI-2 synthesized in vitro was found to promote biofilm formation and host-cell adherence. However, higher concentrations of AI-2 inhibited SS biofilm formation and host-cell adherence. Real-time PCR results showed that the mRNA level of virulence factors of SS biofilm, gdh, cps2, sly, and mrp increased and ef, fbps, and gapdh decreased with increasing AI-2 concentrations. These findings demonstrated that AI-2 supplemented exogenously acted as a concentration-dependent signaling molecule to regulate SS biofilm formation, host-cell adherence, and transcription levels of many virulence genes.

Overexpression of luxS cannot increase autoinducer-2 production, only affect the growth and biofilm formation in Streptococcus suis.

LuxS/AI-2 quorum sensing (QS) system involves the production of cell signaling molecules via luxS-based autoinducer-2 (AI-2). LuxS has been reported to plays critical roles in regulating various behaviors of bacteria. AI-2 is a byproduct of the catabolism of S-adenosylhomocysteine (SAH) performed by the LuxS and Pfs enzymes. In our previous study, the function of LuxS in AI-2 production was verified in Streptococcus suis (SS). Decreased levels of SS biofilm formation and host-cell adherence as well as an inability to produce AI-2 were observed in bacteria having a luxS mutant gene. In this study, the level of AI-2 activity exhibits a growth-phase dependence with a maximum in late exponential culture in SS. An SS strain that overexpressed luxS was constructed to comprehensively understand the function of AI-2. Overexpressed luxS was not able to increase the level of pfs expression and produce additional AI-2, and the bacteria were slower growing and produced only slightly more biofilm than the wild type. Thus, AI-2 production is not correlated with luxS transcription. luxS expression is constitutive, but the transcription of pfs is perhaps correlated with AI-2 production in SS.

Imidapril inhibits right ventricular remodeling induced by low ambient temperature in broiler chickens.

This study explored the effect of imidapril on the right ventricular remodeling induced by low ambient temperature in broiler chickens. Twenty-four broiler chickens were randomly divided into 3 groups (n = 8), including the control group, low temperature group, and imidapril group. Chickens in the control group were raised at normal temperature, whereas chickens in the low temperature group and imidapril group were exposed to low ambient temperature (12 to 18°C) from 14 d of age until 45 d of age. At the same time, chickens in the imidapril group were gavaged with imidapril at 3 mg/kg once daily for 30 d. The thickness of the right ventricular wall was observed with echocardiography. The BW and wet lung weight as well as weight of right and left ventricles and ventricular septum were measured. Both wet lung weight index and right ventricular hypertrophy index were calculated. Pulmonary arterial systolic pressure was assessed according to echocardiography. The expression of ACE and ACE2 mRNA in the right ventricular myocardial tissue was quantified by real-time PCR. Proliferating cell nuclear antigen-positive cells were detected by immunohistostaining. The concentration of angiotensin (Ang) II and Ang (1-7) in the right ventricular myocardial tissue was measured with ELISA. The results showed that right ventricular hypertrophy index, wet lung weight index, pulmonary arterial systolic pressure, expression of ACE mRNA in the right ventricular tissue, Ang II concentration, and the thickness of the right ventricular wall in the low temperature group increased significantly compared with those in the control group and imidapril group. The ACE2 mRNA expression increased 36%, whereas Ang (1-7) concentration decreased significantly in the low temperature group compared with that in the control group and imidapril group. In conclusion, imidapril inhibits right ventricular remodeling induced by low ambient temperature in broiler chickens.

Effects of IFN-γ on IL-18 Expression in Pregnant Rats and Pregnancy Outcomes.

The present study focused on establishing the effects of interferon-gamma (IFN-γ) on interleukin-18 (IL-18) expression patterns and pregnancy outcomes in pregnant rats. Pregnant rats at the post-implantation stage were randomized into control, low IFN-γ (L-IFN-γ) and high IFN-γ groups (H-IFN-γ) that received normal saline, 100 IU/g of IFN-γ and 500 IU/g of IFN-γ vaginal muscular injection, respectively. The effects of IFN-γ on IL-18 expression and pregnancy outcomes were assessed systematically using several methods, including immunohistochemistry streptavidin-perosidase (SP), image pattern analysis, enzyme-linked immune-sorbent assay (ELISA), whole blood count (WBC) count, microscopy and visual observation. IL-18 was detected in the uteri of all pregnant rats, and mainly distributed in the endometrium, decidual cells, vascular endothelium and myometrium. Immunohistochemistry and image pattern analyses revealed significantly lower IL-18 expression in the H-IFN-γ group compared to the L-IFN-γ and control groups (p<0.01), indicating that high doses of IFN-γ induce downregulation of IL-18 in the uterus of pregnant rats. ELISA results disclosed that IL-18 expression in peripheral blood of the H-IFN-γ group was lower than that of the L-IFN-γ group (p<0.05), and significantly reduced compared to the control group (p<0.01). Moreover, the number of peripheral leukocytes in the H-IFN-γ group was significantly higher than those in the control and L-IFN-γ groups (p<0.01). Morphology analysis showed no evident differences between the L-IFN-γ and control groups. However, for the H-IFN-γ group, uterine mucosa bleeding, necrosis and excoriation were observed using microscopy. Visual observation revealed marroon, swelling, crassitude and no embryo in the uterus, which are obvious indicators of abortion. These results indicate that IFN-γ plays a regulatory role in IL-18 expression in the uterus and peripheral blood of pregnant rats at the post-implantation stage. Moreover, high levels (500 IU/g) of IFN-γ influence normal pregnancy at the early stages in rats by downregulating IL-18 expression in the uterus and peripheral blood and increasing the number of peripheral leukocytes, consequently triggering termination of pregnancy.

Antimicrobial susceptibility of Bordetella bronchiseptica isolates from pigs with respiratory diseases on farms in China.

One hundred and 63 Bordetella bronchiseptica isolates from pigs, consisting of 65 isolated in 2003 and 98 isolated in 2007, were tested for antimicrobial susceptibility to 17 antimicrobial agents by the disk diffusion method. All 163 B. bronchiseptica isolates were sensitive to polymyxin B and cefoperazone/sulbactam; the majority of the strains were sensitive to amikacin (149/163; 91.4%), gentamicin (132/163; 81.0%), ampicillin/sulbactam (127/163; 77.9%) and ciprofloxacin (115/163; 70.6%). A high level of resistance was found for furazolidone (100%), ampicillin (90.2%), cefazolin (89.0%), streptomycin (87.7%), amoxicillin/clavulanic acid (74.2%) and tetracycline (64.4%). Comparison of the data revealed that isolates with multiresistance to at least six or eight of the 17 antimicrobials used became more frequent, with the proportions increasing from 32.3% or 16.9% in 2003 to 90.8% or 41.8% in 2007.

[Expression of bursin receptor on membranes of hybridoma cell and identification of binding peptide of bursin from 12-merrandom phage display peptide library].

OBJECTIVE: Bursin (BS) could greatly promote the production of monoclonal antibody in hybridoma cell. We studied the interaction between BS and hybridoma cell. METHODS: Fluorescence microscopy, Laser Scanning Confocal Microscope (LSCM) and fluorescence activated cell sorter (FACS) were used. RESULTS: Fluorescence microscopy revealed specific binding of FITC-BS to hybridoma cell. FACS analysis demonstrated that the binding was specific, saturable and reversibility. BS was used as target protein to screen its binding peptides from 12-mer random phage display peptide library. After four rounds of biopanning, 20 phage clones were randomly selected and identified. ELISA and competitive inhibition test results indicated that 2 phage clones were identified as positive clones. The amino acid sequences analysis shown that the sequences were ACTKHLCLLQPL or MSCNDTLCLLPN, which sharing a conservative sequence LCLL. In vitro experiments suggested that the two binding peptides can inhibit BS specific binding to hybridoma cell. CONCLUSION: These results confirmed that existence of BS receptor in the membrane of hybridoma cell, which involved in hybridoma cell secreting monoclonal antibody signal pathway.

[Immunogenicity and protective efficacy of pertactin recombinants against Bordetella bronchiseptica challenge].

OBJECTIVE: In this study we showed the immunogenicity and protective efficacy of five pertactin recombinants against Bordetella bronchiseptica (Bb) challenge. METHODS AND RESULTS: The complete coding sequence (2040 bp) of the prn gene (PRN) and its fragments,5'-terminal 1173 bp fragment (PN),3'-terminal 867 bp fragment (PC), two copies of region I (654 bp; PR I) in PN, and 2 copies of region II (678 bp; PR II) in PC, were separately cloned into the prokaryotic expression vector pGEX-KG, and expressed in the Eschierichia coli BL21 (DE3) using induction by isopropyl-beta-D-thiogalactopyranoside. The recombinant proteins were named GST-PRN, GST-PN, GST-PC, GST-2PR I and GST-2PR II. All five recombinant proteins showed immunological reactivity in the Western-blot analysis. Mice, immunized subcutaneously with two doses of the purified proteins mixed with an equal volume of Freund's adjuvant,produced robust PRN-specific IgG antibody levels. When challenged, 6 of 9 mice in GST-2PR I group and all 9 mice in the other groups survived intranasal challenge with three times the 50% lethal dose (LD50) of virulent Bb HH0809. After challenge with 10 LD50 7/9,3/9,6/9,1/10 and 6/10 of the mice survived. Furthermore, complete protection against intraperitoneal (i.p.) challenge with 10 LD50 of HH0809 was observed in mice that were injected i.p. with 0.5 ml rabbit anti-GST-PRN, GST-PN,GST-PC or GST-2PR II serum. Only 1 of 10 mice survived in the group of mice that received anti-GST-2PR I, and no survivors were noted in the group of mice that received PRN-absorbed rabbit antiserum (0/5). CONCLUSION: In this study,we showed that all of five pertactin recombinants had differential immunogenicity and protective efficacy against Bb challenge. Mice immunized with GST-PC had better survival against fatal Bb challenge than did those immunized with GST-PN. In addition, GST-2PR II and GST-2PR I provided the similar results These data may have implications for the development of safe and efficacious subunit vaccines for the prevention of bordetellosis on the basis of these five pertactin recombinants.