Enterococcus faecium inhibits NF-κB/NLRP3/Caspase-1 signaling pathway to antagonize enterotoxigenic Escherichia coli-mediated inflammatory response.

Enterotoxigenic Escherichia coli (ETEC) can cause intestinal inflammation and diarrhea in yaks, which has a negative impact on their economic value. In recent years, probiotics have gained increasing attention as a pure, natural, nontoxic, harmless, and residue-free additive. However, the underlying mechanisms by which probiotics safeguard against ETEC are not completely elucidated. This study aimed to investigate the protective effect of Enterococcus faecium (E. faecium) against ETEC infection in mice through oral gavage. Morphological changes were examined through light microscopy. The expressions of inflammatory cytokines (IL-1ß, IL-6, TNF-α, IL-10, NF-κB, and NLRP3), tight junction protein (ZO-1, Claudin-1), and pyroptosis (Caspase-1, Caspase-4, and gasdermin D (GSDMD)) were detected using immunohistochemistry and quantitative real-time PCR. The results indicate that ETEC infection triggers the activation of inflammation-related pathways (NF-κB) and NLRP3 inflammasome, leading to the expression of a large number of inflammatory cytokines. Additionally, the activation of NLRP3 leads to the release of GSDMD activation through Caspase-1, ultimately resulting in inflammatory injury and pyroptosis. Feeding mice E. faecium early resulted in an increase in the expression of tight junction protein, a reduction in inflammatory cytokines, and alleviation of inflammatory injury and pyroptosis in intestinal tissues. Our research indicates that E. faecium has the ability to antagonize ETEC and provide protection to the gastrointestinal mucosa in mice.

Extraction of Polysaccharides from Root of Pseudostellaria heterophylla (Miq.) Pax. and the Effects of Ultrasound Treatment on Its Properties and Antioxidant and Immune Activities.

The hydrophilic polysaccharides (PS) were isolated and purified from the tuberous roots of Pseudostellaria heterophylla. The extraction process of PS from Pesudostellariae radix was optimized by single-factor experiments and orthogonal design. The extract was purified by DEAE cellulose column to obtain the pure polysaccharide PHP. Then PHP was treated with different intensities of sonication to study the effect of sonication on PHP's characteristics and its biological activity in vitro and in vivo. The results of this study revealed that ultrasound treatment did not significantly change the properties of PHP. Further, with the increase of ultrasound intensity, PHP enhanced the proliferation and phagocytosis of macrophage RAW264.7. Meanwhile, it could also significantly improve the body's antioxidant activity and immune function. The results of this study demonstrated that PHP has the potential as a food additive with enhanced antioxidant and immune functions, and its biological activities could be enhanced by sonication.

HABP4 overexpression promotes apoptosis in goat turbinate bone cells.

Hyaluronic acid-binding protein (HABP4) plays important roles in regulating cell cycle and apoptosis. However, its functions in regulating cell apoptosis remain unclear. To reveal the effects of HABP4 on cell proliferation, cell cycle and apoptosis, the HABP4 sequence was cloned, and we investigated the gain and loss functions of HABP4 in goat turbinate bone cells. Our results showed that a 1,496-bp HABP4 sequence was cloned successfully. The interference effect of siRNA1 on HABP4 was the strongest, reducing its mRNA expression level by 83%, decreasing the cells in the G0/G1 and S phases of the cell cycle and inhibiting cell growth and apoptosis. The overexpression of HABP4 produced contrasting results. Furthermore, an HABP4 knockdown caused the up-regulated expression of genes associated with apoptosis, including Bcl-2 and BCL2L11, but the down-regulation of Caspase3, Caspase7, Bax, PARP1, SOCS2 and P53 mRNA levels. Additionally, HABP4 overexpression significantly up-regulated the expression levels of Bax, Caspase3, Caspase7, BCL2L11, P53, SOCS2 and PARP1. However, the expression of Bcl-2 was down-regulated. These data provide an important foundation for further in-depth studies of HABP4 functions.

Enterococcus faecium inhibits NF-κB/NLRP3/IL-1ß signaling pathway and antagonizes Salmonella-mediated inflammatory response.

Aim: This study explored the protective effect of Enterococcus faecium as a probiotic against Salmonella typhimurium infection. Materials & methods: The protective role of E. faecium against tissue damage by S. typhimurium infection and the expression of inflammatory cytokines and tight junction proteins were detected by histological observation, real-time quantitative PCR and immunohistochemical methods. Results: E. faecium demonstrated a regulatory function that affected the expression of Claudin-1 and enhanced tight junctions, suppressed the NF-κB/NLRP3/IL-1ß signaling pathway and reduced the release of IL-6, TNF-α, IFN-γ, TLR4 and MYD88 and inflammatory damage to tissues by S. typhimurium in the duodenum, cecum and colon of mice. Conclusion: E. faecium antagonized S. Typhimurium alleviating inflammatory injury in mice through the NF-κB/NLRP3/IL-1ß signaling pathway.

Screening, Identification, and Probiotic Properties of Bacillus Pumilus From Yak.

The yak has a unique physiological structure suited to life in anoxic and cold environments at high altitudes. The aim of this study was to isolate Bacillus species with good probiotic properties from yak feces. A series of tests were performed on the isolated Bacillus: 16S rRNA identification, antibacterial activity, tolerance to gastroenteric fluid, hydrophobicity, auto-aggregation, antibiotic sensitivity, growth performance, antioxidants, and immune indexes. A safe and harmless Bacillus pumilus DX24 strain with good survival rate, hydrophobicity, auto-aggregation, and antibacterial activity was identified in the yak feces. Feeding mice with Bacillus pumilus DX24 increased their daily weight gain, jejunal villus length, villi/Crypt ratio, blood IgG levels, and jejunum sIgA levels. This study confirmed the probiotic effects of Bacillus pumilus isolated from yak feces and provides the theoretical basis for the clinical application and development of new feed additives.

Luteolin enhanced antioxidant capability and induced pyroptosis through NF-κB/NLRP3/Caspase-1 in splenic lymphocytes exposure to ammonia.

During feeding process in intensive chicken farms, the prolonged exposure of chickens to elevated level of ammonia leads to substantial economic losses within poultry farming industry. Luteolin (Lut), known as its anti-inflammatory and antioxidant properties, possesses the ability to eliminate free radicals and enhance the activities of antioxidant enzymes, thus rendering it highly esteemed in production. The objective of this study was to examine the effects of Lut on antioxidant and anti-inflammatory responses of chicken splenic lymphocytes exposed to ammonia. In order to achieve this, we have replicated a protective model involving Lut against ammonia exposure in chicken splenic lymphocytes. The findings of the study indicated that Lut mitigated the elevation of lactate dehydrogenase (LDH), malondialdehyde (MDA), and reactive oxygen species (ROS) induced by ammonia poisoning. Additionally, Lut demonstrated an increase in the expression of antioxidant enzymes, namely superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Furthermore, Lut exhibited a protective effect on cell morphology and ultrastructure following exposure to ammonia. Moreover, Lut exhibited a reduction in the expression of heat shock proteins (HSPs) and inflammatory cytokines, which were found to be highly expressed in splenic lymphocytes after ammonia exposure. Additionally, Lut demonstrated the ability to inhibit the overexpression of pyroptosis-related genes and proteins (NLRP3 and Caspase-1) in splenic lymphocytes following ammonia exposure. Lut exerted an antioxidant effect on lymphocytes, counteracting elevated levels of oxidative stress following exposure to ammonia. Additionally, Lut had the potential to modulate the expression of HSPs, suppressed the inflammatory response subsequent to ammonia exposure, and influenced the expression of NLRP3 and Caspase-1, thereby mitigating pyroptosis induced by ammonia exposure. The exploration of this subject matter can elucidate the protective properties of Lut against NH4Cl-induced damage in chicken splenic lymphocytes, while also offer insights and experimental groundwork for the utilization of natural therapeutics in animal husbandry to prevent and treat ammonia-related conditions.

Microbiome and metabolome analyses of milk and feces from dairy cows with healthy, subclinical, and clinical mastitis.

Mastitis is commonly recognized as a localized inflammatory udder disease induced by the infiltration of exogenous pathogens. In the present study, our objective was to discern fecal and milk variations in both microbiota composition and metabolite profiles among three distinct groups of cows: healthy cows, cows with subclinical mastitis and cows with clinical mastitis. The fecal microbial community of cows with clinical mastitis was significantly less rich and diverse than the one harbored by healthy cows. In parallel, mastitis caused a strong disturbance in milk microbiota. Metabolomic profiles showed that eleven and twenty-eight molecules exhibited significant differences among the three groups in feces and milk, respectively. Similarly, to microbiota profile, milk metabolome was affected by mastitis more extensively than fecal metabolome, with particular reference to amino acids and sugars. Pathway analysis revealed that amino acids metabolism and energy metabolism could be considered as the main pathways altered by mastitis. These findings underscore the notable distinctions of fecal and milk samples among groups, from microbiome and metabolomic points of view. This observation stands to enhance our comprehension of mastitis in dairy cows.

Golgi apparatus regulated pyroptosis through the miR-32-5p/Golga7/NLRP3 axis in chicken splenic lymphocytes exposure to ammonia.

Ammonia, a common toxic gas, posed a hazard to both human and chickens. The Golgi apparatus, an essential organelle, helped maintain the internal environment of the organism and supported the protein foundation for the endoplasmic reticulum to be involved in pyroptosis. Thus, the Golgi apparatus has garnered significant attention. The purpose of our research was to explore the mechanisms of Golgin A7 (Golga7) involved in pyroptosis after chicken exposure to ammonia. To reach our goal, we first created an in vitro ammonia model to study the effect of ammonia on chicken splenic lymphocyte pyroptosis. Then, leveraging this model, we established Golga7 and miR-32-5p knockdown and overexpression models to investigate their roles in ammonia-induced pyroptosis. We found the ultrastructural changes in the nucleus, Golgi apparatus, and mitochondria of chicken splenic lymphocytes exposure to ammonia. The damage of mitochondria increased the level of Reactive Oxygen Species (ROS), which caused the down-regulation of miR-32-5p. The miR-32-5p inhibitor increased the expression of Golga7 and pyroptosis-related genes (NOD-like receptor protein 3 (NLRP3), Cysteine aspartase-1 (Caspase-1), Golgin A3 (Golga3), Nuclear Factor-kappa B (NF-κB), and Tumor Necrosis Factor-alpha (TNF-α)), which induced the pyroptosis, but when miR-32-5p mimic/si-Golga7 (Golga7 inhibitor) was utilized, these effects were reduced. Our research demonstrated that miR-32-5p/Golga7 regulated NLRP3 involving in the pyroptosis of chicken splenic cells exposed to ammonia. Our study provided a valuable foundation for the prevention and treatment chickens ammonia poisoning in the livestock production.

Effects of Probiotic Enterococcus faecium from Yak on the Intestinal Microflora and Metabolomics of Mice with Salmonella Infection.

Salmonella spp. are pathogenic bacteria that cause diarrhea, abortion, and death in yak and severely harm livestock breeding. Therefore, it is vital to identify a probiotic that effectively antagonizes Salmonella. To the best of our knowledge, few prior studies have investigated the efficacy of Enterococcus faecium against Salmonella. Here, we evaluated the enteroprotective mechanism of E. faecium in a mouse Salmonella infection model using hematoxylin-eosin (H&E) staining, quantitative real-time polymerase chain reaction (Q-PCR) technology, microbial diversity sequencing, and metabonomics. Enterococcus faecium inhibited the proinflammatory cytokines IL-1ß, IL-6, TNF-α, and IFN-γ and promoted the anti-inflammatory cytokine IL-10. The Firmicutes/Bacteroidota (F/B) ratio and the abundances of Firmicutes and Akkermansia were significantly higher in the E. faecium than in the Salmonella group. Metabonomics and microbial diversity sequencing disclosed five different metabolites with variable importance in the projection (VIP) > 3 that were characteristic of both the Salmonella and E. faecium groups. Combined omics revealed that Lactobacillus and Bacteroides were negatively and positively correlated, respectively, with cholic acid, while Desulfovibrio was positively correlated with lipids in both the control and Salmonella groups. Desulfovibrio was also positively correlated with lipids in both the Salmonella and E. faecium groups. Enterococcus faecium antagonizes Salmonella by normalizing the abundance of the intestinal microorganisms and modulating their metabolic pathways. Hence, it may efficaciously protect the host intestine against Salmonella infection.

The protective effect of Luteolin on chicken spleen lymphocytes from ammonia poisoning through mitochondria and balancing energy metabolism disorders.

Ammonia poses a significant challenge in the contemporary intensive breeding industry, resulting in substantial economic losses. Despite this, there is a dearth of research investigating efficacious strategies to prevent ammonia poisoning in poultry. Consequently, the objective of this study was to investigate the molecular mechanisms through which Luteolin (Lut) safeguards mitochondria and restores equilibrium to energy metabolism disorders, thereby shielding chicken spleen lymphocytes from the detrimental effects of ammonia poisoning. Chicken spleen lymphocytes were categorized into 3 distinct groups: the control group, the ammonia group (with the addition of 1 mmol/L of ammonium chloride), and the Lut group (with the treatment of 0.5 µg/mL of Lut for 12 h followed by the addition of 1 mmol/L of ammonium chloride). These groups were then cultured for a duration of 24 h. To investigate the potential protective effect of Lut on lymphocytes exposed to ammonia, various techniques were employed, including CCK-8 analysis, ultrastructural observation, reagent kit methodology, fluorescence microscopy, and quantitative real-time PCR (qRT-PCR). The findings indicate that Lut has the potential to mitigate the morphological damage of mitochondria caused by ammonia poisoning. Additionally, it can counteract the decline in mitochondrial membrane potential, ATP content, and ATPase activities (specifically Na+/K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, and Ca/Mg2+-ATPase) following exposure to ammonia in lymphocytes. Lut also has the ability to regulate the expression of genes involved in mitochondrial fusion (Opa1, Mfn1, and Mfn2) and division (Drp1 and Mff) in spleen lymphocytes after ammonia exposure. This regulation leads to a balanced energy metabolism (HK1, HK2, LDHA, LDHB, PFK, PK, SDHB, and ACO2) and provides protection against ammonia poisoning.

Metabolomic Analysis of Multiple Biological Specimens (Feces, Serum, and Urine) by 1H-NMR Spectroscopy from Dairy Cows with Clinical Mastitis.

Due to huge economic losses to the dairy industry worldwide, mastitis can be considered as one of the most common diseases in dairy cows. This work aimed to study this disease by comparing multiple biological specimens (feces, serum, and urine) from individuals with or without clinical mastitis. This was performed by a single analytical platform, namely 1H-NMR, through a multi-matrix strategy. Thanks to the high reproducibility of 1H-NMR, we could characterize 120 molecules across dairy cow feces, serum, and urine. Among them, 23 molecules were in common across the three biofluids. By integrating the results of multi-matrix metabolomics, several pathways pertaining to energy metabolism and amino acid metabolism appeared to be affected by clinical mastitis. The present work wished to deepen the understanding of dairy cow mastitis in its clinical form. Simultaneous analysis of metabolome changes across several key biofluids could facilitate knowledge discovery and the reliable identification of potential biomarkers, which could be, in turn, used to shed light on the early diagnosis of dairy cow mastitis in its subclinical form.

The orf virus 129 protein can inhibit immune responses by interacting with host complement C1q binding protein in goat turbinate bone cells.

OBJECTIVE: Contagious ecthyma is a severe and highly contagious disease caused by an orf virus (ORFV). The virus is responsible for substantial economic losses in the goat industry and threatens humans. We previously determined the role of ORFV129 protein, one of the five ankyrin-repeat proteins coded by the orf genome, in suppressing the transcription of pro-inflammatory cytokines IL-6, IL-1ß and IFN-γ. In the present study, we identified 14 cellular proteins (complement C1q binding protein [C1QBP], MCM7, EIF5A, PKM, SLC6A, TSPAN6, ATP6AP2, GPS1, MMADHC, HSPB6, SLC35B1, MTF1, P3H4, and IL15RA) that interact with ORFV129 using a yeast two-hybrid system in goat turbinate bone cells (GFTCs). The interaction between ORFV129 and (C1QBP), an immune-related protein, was confirmed using immunofluorescence co-localization and co-immunoprecipitation assays. C1QBP overexpression inhibited ORFV replication, whereas the knockdown of C1QBP promoted ORFV replication in GFTCs. Furthermore, ORFV or ORFV129 increased C1QBP expression in GFTCs, indicated that ORFV129-C1QBP interaction might contribute to the ORFV-induced host immune process. In addition, our research showed that ORFV increased the expression of ORFV129, cytokine IL-6, IL-1ß and IFN-γ. C1QBP overexpression induced IFN-γ production and reduced IL-6 and IL-1ß production. Conversely, C1QBP knockdown induced IL-1ß production and reduced IFN-γ and IL-1ß production. Moreover, augmentation of ORFV129 expression enhanced the inhibition of the secretion of cytokines IL-6, IL-1ß, and IFN-γ induced by the altered expression of C1QBP. These findings suggest different downstream pathways might be involved in regulating different cytokines induced by ORFV129 expression in GFTCs.

An experimental study of the pathogenicity of a duck hepatitis A virus genotype C isolate in specific pathogen free ducklings.

Duck hepatitis A virus genotype C (DHAV-C), recognized recently, is one of the pathogens causing fatal duck viral hepatitis in ducklings, especially in Asia. To demonstrate the pathogenesis of the DHAV-C isolate, 3-day-old specific pathogen free ducklings were inoculated subcutaneously with a DHAV-C isolate and the clinical signs were observed. Virus distribution, histological and apoptotic morphological changes of various tissues were examined at different times post inoculation. The serial, characteristic changes included haemorrhage and swelling of the liver. Apoptotic cells and virus antigen staining were found in all of the tissues examined. Where more virus antigen staining was detected, there were more severe histopathological and apoptotic changes. The amount of virus antigen and the histological and apoptotic morphological changes agreed with each other and became increasingly severe with length of time after infection. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages and monocytes in immune organs such as the bursa of Fabricius, thymus and spleen, and in liver, kidney and cerebral cells. Necrosis was also observed within 72 h post inoculation in all organs examined, except the cerebrum, and was characterized by cell swelling and collapsed plasma membrane. These results suggest that the recent outbreak of disease caused by DHAV-C virus is pantropic, causing apoptosis and necrosis of different organs. The apoptosis and necrosis caused by the DHAV-C field strain in this study is associated with pathogenesis and DHAV-C-induced lesions.

First detection and molecular characteristics of bopivirus from goats in China.

A metavirome analysis was performed and detected bopivirus in the diarrhoeal fecal samples of goats in China. A total of 136 fecal samples were collected from yeanlings between the dates of June 2021 and January 2022 in Sichuan province, China. Moreover, "Bopivirus B" strains were detected by a specific RT-PCR targeting the 3D gene of the virus. The results showed that the overall detection rate of "Bopivirus B" was 19.12% (26/136). Additionally, there was a higher detection rate (24.05%, 19/79) in the fecal samples collected from yeanlings with diarrhea compared to those from asymptomatic animals (12.28%, 7/57). In these samples, no other common diarrhea-causing pathogens were detected except for three enteric viruses, namely caprine enterovirus, caprine kobuvirus and caprine hunnivirus (with detection rates of 13.97, 13.97, and 8.82%, respectively). Subsequently, full-length VP4, VP2, VP3, and VP1 genes from "Bopivirus B"-positive samples were amplified, cloned, sequenced, and analyzed. The phylogenetic analysis performed on the VP1 genes revealed that the identified bopivirus belonged to genotype B1 (seven strains) and B2 (three strains) and presented a high genetic diversity. Furthermore, a complete genome sequence of a "Bopivirus B" strain (SWUN/B1/2022) was obtained using PCR from fecal sample of a diarrhoeal yeanling. The complete genome was 7,309 nucleotides in length with a standard picornavirus genome organization, and shares 93.10% and 91.10% nucleotide similarity with bopivirus B1 genotype strain ovine/TB14/2010-HUN and bopivirus B2 genotype strain goat/AGK16/2020-HUN, respectively. According to the species classification criteria put forward by the International Committee on Taxonomy of Viruses and VP1 genotype, the strain SWUN/B1/2022 belongs to the bopivirus B1. This strain has unique amino acid substitutions in the VP4, VP2, VP3, and VP1 genes. Moreover, genomic recombination analysis revealed that this strain may be a minor parental strain of bopivirus B1 ovine/TB14/2010-HUN. Evolutionary analysis based on the 2C and 3CD genes revealed that the new bopivirus B1 strain SWUN/B1/2022 presents a unique evolutionary pattern. This study provided evidence to suggest that "Bopivirus B" is circulating with substantial genetic diversity in goats in China at present, and the mixed infection of "Bopivirus B" with other enteric viruses should be considered to be a composite factor in the occurrence of viral diarrhea in goats.

Development and validation of an insulated isothermal PCR assay for the rapid detection of Mannheimia haemolytica.

We developed a rapid insulated isothermal PCR (iiPCR) assay for on-site detection of Mannheimia haemolytica using a primer and probe set targeting the superoxide dismutase (sodA) gene. Our iiPCR assay detected M. haemolytica clinical isolates successfully and produced negative results on other bovine or ovine respiratory pathogens, including Histophilus somni, Bibersteinia trehalosi, Trueperella pyogenes, Streptococcus suis, and Mycoplasma spp., indicating that the PCR reactions were specific. Additionally, our iiPCR assay detected as few as 21 copies of genomic DNA and 17.2 cfu/mL of bacterial culture, which was 10 and 100 times more sensitive than conventional PCR, respectively. Our iiPCR assay can be performed on a portable device in a total of 58 min and may be a useful tool for the detection of M. haemolytica in bovine and ovine respiratory disease in the field.

Genome sequence of Mycoplasma ovipneumoniae strain SC01.

Mycoplasma ovipneumoniae is associated with chronic nonprogressive pneumonia in both sheep and goats. Studies concerning its molecular pathogenesis, genetic analysis, and vaccine development have been hindered due to limited genomic information. Here, we announce the first complete genome sequence of this organism.

Complete Genome Sequence of a Parabacteroides distasonis Strain (CavFT hAR46) Isolated from a Gut Wall-Cavitating Microlesion in a Patient with Severe Crohn's Disease.

Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) of the digestive tract in humans. There is evidence that Parabacteroides distasonis could contribute to IBD. Here, we present the complete genome sequence of a strain designated CavFT-hAR46, which was isolated from a gut intramural cavernous fistulous tract (CavFT) microlesion in a CD patient.

[Quantification of class 1 integrase gene expression of Pseudomonas aeruginosa in biofilm].

OBJECTIVE: To study the molecular mechanism of integron related gene transfer in biofilm and aqueous culture of P. aeruginosa by investigating the expression level of intI1 mRNA in class 1 integron positive strains. METHODS: A competitive reverse transcription-PCR (cRT-PCR) method was designed to quantify class 1 integrase mRNA production in two clinical P. aeruginosa strains called PA10 and PA39 when they were grown in biofilms and planktonic culture. In brief, competitive DNA (cDNA) was obtained via PCR from the genomic DNA of class 1 integron positive strains. Then the competitive RNA (cRNA) was amplified from the cDNA. Finally the serials diluted cRNA were mixed separately with the total RNA extracted from the biofilm and planktonic culture of P. aeruginosa and the competitive RT-PCR were conducted. After electrophoresis, the expression level of intI1 mRNA was quantified by comparing with the amount of the cDNA. RESULTS: The PA10 and PA39 strains produced intI1 mRNA both in their biofilms and planktonic cells. Furthermore the expression levels of intI1 mRNA from the two strains were of approximation in their biofilm and plankton stage respectitively, while the quantities of intI1 mRNA expression in their biofilm stage were about 15 times higher than those in their planktonic stage. CONCLUSION: The integrase gene is up-regulated at mRNA level in P. aeruginosa biofilm, which may be one of the reseans for the spread of antibiotic resistance and the formation of multidrug resistance.

[Establishing a real-time PCR assay for anatid herpesvirus 1].

OBJECTIVES: To develop a real-time quantitative PCR assay to detect duck plague virus (DPV) for the rapid diagnosis of DPV infection, the investigation of its nosogenesis and the screening of effective antiviral drugs. METHODS: The primer and probe were designed according to the gene sequence of DPV DNA polymerase gene. To establish a standard curve, a plasmid containing 125 bp PCR product was constructed and severed as a positive control. The TaqMan based real-time PCR method was adopted and compared with the traditional PCR approach in sensitivity, reliability and specificity. RESULTS: A good linear correlation was demonstrated in the standard curve for the real-time PCR assay within the range from 2.3 x 10 to 2.3 x 10(5) copies. A minimum of 23 positive plasmids could be detected, indicating a good sensitivity of the assay. The coefficients of variance (CVs) were 1.22-6.69 and 2.09-8.84 for the intra-assay and inter-assay tests respectively, indicating a good reliability. No amplification products were found for DNA from other pathogens, indicating a good specificity. The comparative study proved that the TaqMan technology was much more sensitive than traditional PCR assay. CONCLUSION: The real-time quantitative PCR assay for DPV DNA has good sensitivity, specificity and reliability.

[The cloning and expression of rhlR, a quorum sensing gene in Pseudomonas aeruginosa PAO1, and a study of its effect on bacterial clearance rate in mouse lung].

OBJECTIVE: To inquire about the molecular characteristics of rhlR, a Quorum Sensing gene in Pseudomonas aeruginosa (P. aeruginosa) PAO1, and to explore the immunogenicity of RhlR protein in mouse. METHODS: The rhlR gene of PAO1 was amplified by PCR and cloned into pGEX4T-1 plasmid. The recombination was expressed in E. coli BL21 (DE3) and analyzed by SDS-PAGE and Western blotting. The fusion protein (GST-RhlR) was purified by GST purification Kit and the purified protein was used to immunize mice. P. aeruginosa PA0315 was injected into mouse lung to explore the immuno-protection of the protein. RESULTS: The 726 bp DNA fragment of rhlR was amplified from PAO1 general DNA. The restriction enzyme map showed that the inserted part of rhlR-pGEX4T-1 was successfully constructed and the gene was 100% homologous to rhlR in GenBank. The recombinant plasmid expressed a 54 kDa fusion protein (rhlR-GST) in E. coli BL21 (DE3) after induction by IPTG. The fusion protein could be recognized by mouse polyvalent antiserum against P. aeruginosa. The results showed that the bacterial clearance rate in mouse lung was 86. 92% in rhlR groups and 49.44% in the control group. CONCLUSION: A 54 kDa protein (RhlR-GST) has been successfully expressed in E. coli BL21 (DE3). The RhlR could increase the bacterial clearance rate in mouse lung and may serve as immunoprotective antigen to develop the genetic engineering vaccine.