Acanthopanax senticosus cultures fermented by Lactobacillus rhamnosus enhanced immune response through improvement of antioxidant activity and inflammation in crucian carp (Carassius auratus).

Lactic acid bacteria (LAB) have been recognized as safe microorganism that improve micro-flora disturbances and enhance immune response. A well-know traditional herbal medicine, Acanthopanax senticosus (As) was extensively utilized in aquaculture to improve growth performance and disease resistance. Particularly, the septicemia, skin wound and gastroenteritis caused by Aeromonas hydrophila threaten the health of aquatic animals and human. However, the effects of probiotic fermented with A. senticosus product on the immune regulation and pathogen prevention in fish remain unclear. Here, the aim of the present study was to elucidate whether the A. senticosus fermentation by Lactobacillus rhamnosus improve immune barrier function. The crucian carp were fed with basal diet supplemented with L. rhamnosus fermented A. senticosus cultures at 2 %, 4 %, 6 % and 8 % bacterial inoculum for 8 weeks. After trials, the weight gain rate (WGR), specific growth rate (SGR) were significantly increased, especially in LGG-6 group. The results confirmed that the level of the CAT, GSH-PX, SOD, lysozyme, and MDA was enhanced in fish received with probiotic fermented product. Moreover, the L. rhamnosus fermented A. senticosus cultures could trigger innate and adaptive immunity, including the up-regulation of the C3, C4, and IgM concentration. The results of qRT-PCR revealed that stronger mRNA transcription of IL-1ß, IL-10, IFN-γ, TNF-α, and MyD88 genes in the liver, spleen, kidney, intestine and gills tissues of fish treated with probiotic fermented with A. senticosus product. After infected with A. hydrophila, the survival rate of the LGG-2 (40 %), LGG-4 (50 %), LGG-6 (60 %), LGG-8 (50 %) groups was higher than the control group. Meanwhile, the pathological damage of the liver, spleen, head-kidney, and intestine tissues of probiotic fermentation-fed fish could be alleviated after pathogen infection. Therefore, the present work indicated that L. rhamnosus fermented A. senticosus could be regard as a potential intestine-target therapy strategy to protecting fish from pathogenic bacteria infection.

Infection of epithelioma papulosum cyprini (EPC) cells with spring viremia of carp virus (SVCV) induces autophagy and apoptosis through endoplasmic reticulum stress.

Spring viremia of carp virus (SVCV) is a lethal freshwater pathogen of cyprinid fish that has caused significant economic losses to aquaculture. To reduce the economic losses caused by SVCV, its pathogenic mechanism needs to be studied more thoroughly. Here, we report for the first time that SVCV infection of Epithelioma papulosum cyprini (EPC) cells can induce cellular autophagy and apoptosis through endoplasmic reticulum stress. The presence of autophagic vesicles in infected EPC cells was shown by transmission electron microscopy. Quantitative fluorescence PCR and Western blot results showed that p62 mRNA expression was decreased, and the expression of Beclin1 and LC3 mRNA was increased. The p62 protein was decreased, and the Beclin1 protein and LC3 were increased in the endoplasmic reticulum stress activation state. To further clarify the mode of death of SVCV-infected EPC cells, we examined caspase3, caspase9, BCL-2, and Bax mRNA, which showed that they were all increased. Apoptosis of SVCV-infected cells increased upon activation of endoplasmic reticulum stress. Our results suggest that endoplasmic reticulum stress can regulate SVCV infection-induced autophagy and apoptosis. The results of this study provide theoretical data for the pathogenesis of SVCV and lay the foundation for future drug development and vaccine construction.

Detection of changes in biological characteristics of Aeromonas veronii TH0426 after deletion of lsrB gene by homologous recombination.

Aeromonas veronii is a widespread pathogenic microorganism that can infect humans, animals, and a variety of aquatilia, at the same time, can cause diseases, mainly sepsis and ulcer syndrome. In this research, we first deleted the gene of lsrB's nucleotide sequences by homologous recombination. The results showed that the median lethal dose (LD50) of the mutant strain (ΔlsrB) for zebrafish was 1.28-times higher than that of the TH0426 strain. The toxicity of TH0426 to epithelioma papulosum cyprini (EPC) cells was 1.15-times and 1.64-times higher than that of ΔlsrB, 1 and 2 h after infection. The production ability of the biofilm of ΔlsrB decreased by 1.38-times, and the adhesion ability of ΔlsrB to EPC cells greatly decreased by 1.96-times than the TH0426. The result of motility detection pointed out that the swimming ability of ΔlsrB was down by 1.67-times. The results indicated that almost all of them lost their flagella after deleting the lsrB gene. In general, the virulence of TH0426 was reduced after deleting the lsrB gene. The final results point out that the lsrB gene of TH0426 is related to motility, biofilm formation, adhesion, and virulence.

Immune enhancement effects of inactivated vaccine against extracellular products of Aeromonas caviae AC-CY on crucian carp.

Aeromonas caviae is a zoonotic pathogen that can cause disease in aquatic organisms and mammals, including humans, and it is widespread in nature, especially in freshwater environments. Previous research has reported that extracellular products (ECPs) secreted by pathogens during growth are effective protective antigens that can induce the host immune response and protect the host from pathogens. However, little is known about how ECPs enhance immunity. Here, we prepared extracellular products by the cellophane plate method, determined the total protein concentration, and analysed the protein composition of the extracellular products by SDS-PAGE. Subsequently, their enzyme activity and pathogenicity were evaluated separately. Crucian carp were randomly divided into four groups to receive formalin-inactivated A. caviae vaccine (FKC), ECPs mixed with the same amount of Freund's complete adjuvant, the same amount of ECPs mixed with an equal volume of A. caviae inactivated vaccine (FKC + ECPs), sterile PBS alone via intraperitoneal injection. On Days 7, 14, 21, and 28 after immunization, the expression levels of IgM, SOD, and CAT and the lysozyme (LYS) activity in the serum were detected by ELISA, and the relative expression levels of the TNF-α, IFN-γ, IL-1ß, and IL-10 genes in the liver, kidney, spleen, intestine, and gills were measured by qPCR. The extracellular products generated five clearly visible protein bands and exhibited lipase, protease, amylase, DNase and lysozyme but no urease or lecithinase activities. In addition, the median lethal doses of A. caviae and ECPs to crucian carp were 411.64 µg/fish and 1.6 × 105 CFU/mL, respectively. Compared with those of the control group, the IgM, SOD, and CAT contents and serum LYS activity were significantly increased in the experimental groups, and the qRT-PCR results showed that the relative expression levels of TNF-α, IFN-γ, IL-1ß, and IL-10 genes in the liver, kidney, spleen, and intestine were significantly increased after injection immunization. In addition, the relative immune protection rates of the three experimental groups were 60%, 65%, and 45%, all of which were significantly higher than those of the control group. Collectively, our findings show that the extracellular products of A. caviae can be used as a vaccine to significantly improve the immune level of crucian carp and have obvious anti-infection ability. This may represent a promising approach to prevent and control infection by A. caviae and provides strong theoretical support for the development of new inactivated vaccines.

Oral vaccination with recombinant Lactobacillus casei expressing Aha1 fused with CTB as an adjuvant against Aeromonas veronii in common carp (Cyprinus carpio).

Aeromonas veronii (A. veronii) is a pathogenic that can infect human, animal and aquatic organisms, in which poses a huge threat to the health of many aquatic organisms such as Cyprinus carpio. In this study, Lactobacillus casei (L. casei) strain CC16 was used as antigen deliver carrier and fused with cholera toxin B subunit (CTB) as an adjuvant to construct the recombinant L. casei pPG-Aha1/Lc CC16(surface-displayed) and pPG-Aha1-CTB/Lc CC16(surface-displayed) expressing Aha1 protein of A. veronii, respectively. And the immune responses in Cyprinus carpio by oral route was explored. Our results demonstrated that the recombinant strains could stimulate high serum specific antibody immunoglobulin M (IgM) and induce a stronger acid phosphatase (ACP), alkaline phosphatase (AKP), C3, C4, lysozyme (LZM), Lectin and superoxide dismutase (SOD) activity in Cyprinus carpio compared with control groups. Meanwhile, the expression of Interleukin-10 (IL-10), Interleukin-1ß (IL-1ß), Tumor Necrosis Factor-α (TNF-α), immunoglobulin Z1 (IgZ1) and immunoglobulin Z2 (IgZ2) in the tissues were significantly upregulated compared with Lc-pPG or PBS groups, indicating that humoral and cell immune response were triggered. Additionally, recombinant L. casei could survive and colonize in fish intestine. Significantly, recombinant L. casei provides immune protection against A. veronii infection, which Cyprinus carpio received pPG-Aha1-CTB/Lc CC16 (64.29%) and pPG-Aha1/Lc CC16 (53.57%) had higher survival rates compared with the controls. Thus, we demonstrated that recombinant pPG-Aha1/Lc CC16 and pPG-Aha1-CTB/Lc CC16 may be the promising strategy for the development of an oral vaccine against A. veronii.

Functional analysis of fis in Aeromonas veronii TH0426 reveals a key role in the regulation of virulence.

Aeromonas veronii is a comorbid pathogen that can infect humans, and animals including various aquatic organisms. In recent years, an increasing number of cases of A. veronii infection has been reported, indicating serious risks. This bacterium not only threatens public health and safety but also causes considerable economic loss in the aquaculture industry. Currently, some understanding of the pathogenic mechanism of A. veronii has been obtained. In this study, we first constructed the A. veronii TH0426 fis gene deletion strain Δfis and the complementation strain C-fis through homologous recombination technology. The results showed that the adhesion and invasion ability of the Δfis strain towards Epithelioma papulosum cyprini (EPC) cells and the cytotoxicity were 3.8-fold and 1.38-fold lower, respectively, than those of the wild-type strain. In the zebrafish infection model, the lethality of the deleted strain is 3-fold that of the wild strain. In addition, the bacterial load of the deletion strain Δfis in crucian carp was significantly lower than the wild-type strain, and the load decreased with time. In summary, deletion of the fis gene led to a decrease in the virulence of A. veronii. Our research results showed that the deletion of the fis gene significantly reduces the virulence and adhesion ability of A. veronii TH0426. Therefore, the fis gene plays a vital role in the pathogenesis of A. veronii TH0426. This preliminary study of the function of the fis gene in A. veronii will help researchers further understand the pathogenic mechanism of A. veronii.

Functional analysis of hisJ in Aeromonas veronii reveals a key role in virulence.

Aeromonas veronii is an important aquatic zoonotic pathogen in humans and animals. In recent years, extracellular proteins from bacteria have been found to be the major pathogenic factors for aquatic animals. The aim of this study was to systematically analyze the extracellular proteins of nine sources of A. veronii and the effects of hisJ on virulence. We screened only the common proteins from nine different sources of A. veronii by liquid chromatography-tandem mass spectrometry and identified the gene hisJ. We then constructed ΔhisJ (deleted) and C-hisJ (complemented) variants of A. veronii TH0426 to assess the biological function of hisJ. While the ΔhisJ strain did not show altered growth (P > 0.05), we observed that it had reduced colony formation and biofilm formation and reduced adhesion to and invasion of epithelioma papulosum cyprini cells by 2.0-, 1.9-, and 10.8-fold, respectively. Additionally, infection experiments on zebrafish and mouse infection experiments showed that the virulence of the ΔhisJ strain was decreased by 865-fold (P < 0.001) compared with the wild-type strain; virulence of the complemented C-hisJ strain was reduced only 2.8-fold. Furthermore, in the context of hisJ deletion, flagella of A. veronii TH0426 were easily detached and the expression of virulence genes was downregulated. A persistence test (of bacterial colonies in crucian carp) showed that the number of bacteria in the immune organs of the ΔhisJ-infected group was lower than that in the wild-type-infected group. Overall, these results show that hisJ affects flagellar shedding, virulence, biofilm formation, adhesion, and invasion of A. veronii TH0426, and that hisJ is closely associated with virulence and plays a crucial role in its pathogenicity of A. veronii TH0426.

Effect of Bacillus velezensis on Aeromonas veronii-Induced Intestinal Mucosal Barrier Function Damage and Inflammation in Crucian Carp (Carassius auratus).

Aeromonas veronii is an emerging aquatic pathogen causing hemorrhagic septicemia in humans and animals. Probiotic is an effective strategy for controlling enteric infections through reducing intestinal colonization by pathogens. Here we report that the consumption of Bacillus velezensis regulated the intestinal innate immune response and decreased the degree of intestinal inflammation damage caused by the A. veronii in Crucian carp. In this study, we isolated four strains of B. velezensis, named C-11, S-22, L-17 and S-14 from apparently healthy Crucian carp, which exerted a broad-spectrum antimicrobial activity inhibiting both Gram-positive and Gram-negative bacteria especially the fish pathogens. B. velezensis isolates showed typical Bacillus characteristics by endospore staining, physiological and biochemical test, enzyme activity analysis (amylase, protease, and lipase), and molecular identification. Here, Bacillus-containing dietary was orally administrated to Crucian carp for 8 weeks before A. veronii challenge. Immunological parameters and the expression of immune-related genes were measured at 2, 4, 6, 8, and 10 weeks post-administration. The results showed that B. velezensis was found to promote the increase in the phagocytic activities of peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs), as well as the increase in interleukin 1ß (IL-1ß), IL-10 and tumor necrosis factor α (TNF-α) concentration of serum. Lysozyme levels (113.76 U/mL), ACP activity (25.32 U/mL), AKP activity (130.08 U/mL), and SOD activity (240.63 U/mL) were maximum (P < 0.05) in the B. velezensis C-11 treated group at 8 week. Our results showed that Crucian carp fed with the diet containing B. velezensis C-11 and S-22 developed a strong immune response with significantly higher (P < 0.05) levels of IgM in samples of serum, mucus of skin and intestine compared to B. velezensis L-17 and S-14 groups. Moreover, B. velezensis spores appeared to show no toxicity and damage in fish, which could inhabit the gut of Crucian carp. B. velezensis restrained up-regulation of pro-inflammation cytokines (IL-1ß, IFN-γ, and TNF-α) mRNA levels in the intestine and head kidney at final stage of administration, and the expression of IL-10 was increased throughout the 10-week trial. A. veronii infection increased the population of inflammatory cells in the intestinal villi in the controls. In contrast, numerous goblet cells and few inflammatory cells infiltrated the mucosa in the B. velezensis groups after challenge with A. veronii. Compared with A. veronii group, B. velezensis could safeguard the integrity of intestinal villi. The highest post-challenge survival rate (75.0%) was recorded in B. velezensis C-11 group. The present data suggest that probiotic B. velezensis act as a potential gut-targeted therapy regimens to protecting fish from pathogenic bacteria infection. IMPORTANCE: In this work, four Bacillus velezensis strains isolated from apparently healthy Crucian carp, which exhibited a broad-spectrum antibacterial activity especially the fish pathogens. Administration of B. velezensis induced the enhancement of the intestinal innate immune response through reducing intestinal colonization by pathogens. The isolation and characterization would help better understand probiotic can be recognized as an alternative of antimicrobial drugs protecting human and animal health.

Functional Analysis of preA in Aeromonas veronii TH0426 Reveals a Key Role in the Regulation of Virulence and Resistance to Oxidative Stress.

Aeromonas veronii is one of the main pathogens causing freshwater fish sepsis and ulcer syndrome. This bacterium has caused serious economic losses in the aquaculture industry worldwide, and it has become an important zoonotic and aquatic agent. However, little is known about the molecular mechanism of pathogenesis of A. veronii. In this study, we first constructed an unmarked mutant strain (ΔpreA) by generating an in-frame deletion of the preA gene, which encodes a periplasmic binding protein, to investigate its role in A. veronii TH0426. Our results showed that the motility and biofilm formation ability of ΔpreA were similar to those of the wild-type strain. However, the adhesion and invasion ability in epithelioma papulosum cyprini (EPC) cells were significantly enhanced (2.0-fold). Furthermore, the median lethal dose (LD50) of ΔpreA was 7.6-fold higher than that of the wild-type strain, which illustrates that the virulence of the mutant was significantly enhanced. This finding is also supported by the cytotoxicity test results, which showed that the toxicity of ΔpreA to EPC cells was enhanced 1.3-fold relative to the wild type. Conversely, tolerance test results showed that oxidative stress resistance of ΔpreA decreased 5.9-fold compared to with the wild-type strain. The results suggest that preA may negatively regulate the virulence of A. veronii TH0426 through the regulation of resistance to oxidative stress. These insights will help to further elucidate the function of preA and understand the pathogenesis of A. veronii.

OmpW expressed by recombinant Lactobacillus casei elicits protective immunity against Aeromonas veronii in common carp.

Aeromonas veronii is an opportunistic pathogen that is capable of infecting both aquatic livestock and mammals. Natural infection in fishes results in irreparable damage to the aquaculture industry. In this study, we sought to investigate whether recombinant Lactobacillus casei expressing the outer membrane protein W (OmpW) of A.veronii could elicit protective immunity against A.veronii infections. We generated two recombinant Lactobacillus casei (L.casei) strains expressing the OmpW of A.veronii (surface-displayed or secreted) and evaluated the effect on immune responses in a fish model. A 600-bp gene fragment was subcloned into the L.casei expression plasmids pPG-1 (surface-displayed) and pPG-2 (secreted). Expression of the recombinant OmpW protein was also confirmed by Western blot and immunofluorescence assays. Common carp immunized with Lc-pPG-1- OmpW and Lc-pPG-2- OmpW via oral administration elicited high serum specific antibody titers and high LZM, ACP, and SOD activities. High levels of the IL-10, IL-ß, IFN-γ, and TNF-α genes in different organs indicated that the inflammatory response and cell immune response were triggered. Additionally, when immunized fish were challenged with A.veronii, Lc-pPG1-OmpW and Lc-pPG2-OmpW demonstrated 40% and 50% protective efficacy. These data indicate that the combination of OmpW delivery and the lactic acid bacteria (LAB) approach may be a promising mucosal therapeutic strategy for treatment of A.veronii.

Immunogenicity of extracellular products from an inactivated vaccine against Aeromonas veronii TH0426 in koi, Cyprinus carpio.

Aeromonas veronii is a type of human-livestock-aquatic animal pathogen; it is widely found in nature and causes many deaths among aquatic animals. Extracellular products (ECPs) are secreted by the pathogen during growth and reproduction. These products are considered effective protective antigens that can induce the host to produce an immune response. In this study, the ECPs of A.veronii TH0426 were prepared by ultrafiltration, and then the pathogenicity and enzymatic activity of the ECPs were determined. All the groups were injected intraperitoneally, as follows: group one: ECP protein with an equal volume of Freund's adjuvant; group two: ECPs and formalin-killed cells (FKC) of A.veronii combined with an equal volume of Freund's adjuvant (FKC + ECPs); group three: formalin-killed cells (FKC) of A.veronii combined with an equal volume of Freund's adjuvant (FKC); and, group four: sterile PBS as the control group. The expression levels of IgM, IL-1ß, and TNF-α and the lysozyme activity in blood were examined at 7, 14, and 21 days after the immunizations. The results show that the ECPs can produce protease, lipase, amylase and hemolyase, and there was no lecithinase, urease, or gelatinase activity. The results indicate that the ECPs were clearly pathogenic to koi fish, and the LD50 dose was 391.6 µg/fish. Throughout this study, the RPS of the three experimental groups were 75%, 50%, and 70%. This study indicates that the ECPs of A.veronii can effectively enhance the ability of kio fish to resist bacterial invasion.

Mycobacterium vaccae induces a strong Th1 response that subsequently declines in C57BL/6 mice.

Mycobacterium (M.) vaccae is a fast-growing species of saprophytic bacteria that is widely distributed. To understand the host immune responses induced by M. vaccae isolated from bovine submaxillary lymph nodes, C57BL/6 mice were infected with reference strain M. bovis Bacillus Calmette-Guérin (BCG) and isolated M. vaccae using intraperitoneal injections. Comparison of the bacterial replication and organ pathology between M. vaccae and M. bovis BCG revealed that M. vaccae was more malignant than M. bovis in mice. We also demonstrated that serum from the M. vaccae-infected mice contained a higher expression level of gamma-interferon (IFN-γ), tumor necrosis factor alpha, monocyte chemoattractant protein-1, interleukin (IL)-4, IL-12, IL-10 and transforming growth factor beta than did the other groups, especially after week 4. Furthermore, when the numbers of CD3⁺CD4⁺IFN-γ⁺ and CD3⁺CD4⁺IL4⁺cells in the infected mice were observed by flow cytometry, we found that a powerful T helper 1 (Th1) response was induced by M. vaccae infection, which was associated with the emergence of CD3⁺CD4⁺IFN-γ⁺cells. However, the Th1 response declined over time, which was associated with appearance of the CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁺CD152⁺Treg cell reaction. In addition, a strong Th2 response was found. Finally, we found that M. vaccae infection increased the production of type I IFNs, which was associated with a reduced Th1 response.

Toxoplasma gondii, Dirofilaria immitis, feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV) infections in stray and pet cats (Felis catus) in northwest China: co-infections and risk factors.

This study was conducted to estimate the prevalence of Toxoplasma gondii, Dirofilaria immitis, feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV) infections among stray and pet cats in Lanzhou, northwest China, and to identify the influence of age, gender, and regions on seropositivity. T. gondii antibodies were examined in cat sera by the modified agglutination test (MAT). The circulating antigens of D. immitis and FeLV and specific antibodies to FIV were examined using kits commercially available. The overall prevalence of T. gondii, FIV, FeLV, and D. immitis was 19.34, 9.12, 11.33, and 3.04 %, respectively. For the genetic characterization of T. gondii genotypes in cats, genomic DNA was extracted from the seropositive cats and the T. gondii B1 gene was amplified using a semi-nested PCR. DNA samples giving positive B1 amplification were then genotyped using multilocus PCR-RFLP. Two T. gondii genotypes (ToxoDB#9 and ToxoDB#1) were identified. Results of the multivariate logistic regression analysis showed that older cats are more likely to be seropositive than juveniles for T. gondii, FIV, FeLV, and D. immitis. This is the first report of T. gondii genotypes in cats in northwest China. Moreover, the present study is the first study of retrovirus and D. immitis seroprevalence in cats in China. The results revealed that T. gondii, FIV, and FeLV infections are common in stray and pet cats in northwest China.

Mycobacterium avium Subspecies paratuberculosis and Bovine Leukemia Virus Seroprevalence and Associated Risk Factors in Commercial Dairy and Beef Cattle in Northern and Northeastern China.

Mycobacterium avium subspecies paratuberculosis (MAP) and bovine leukemia virus (BLV) are important pathogens, commonly responsible for economical loss to cattle farms all over the world, yet their epidemiology in commercial dairy and beef cattle in China is still unknown. Thus, from September 2013 to December 2014, a large-scale seroprevalence study was conducted to determine the seroprevalence and identify herd-level risk factors associated with MAP and BLV infection. The source sample was 3674 cattle from 113 herds in northern and northeastern China. Antibodies against MAP and BLV were detected using ELISA tests. At animal-level, the seroprevalence of antibodies against MAP and BLV was 11.79% (433/3674) and 18.29% (672/3674), respectively. At herd-level, the seroprevalence of antibodies against MAP and BLV was 20.35% and 21.24% (24/113), respectively. Herd size was identified to be associated with MAP infection while herd size and presence of cattle introduced from other farms were significantly associated with BLV infection. Further research is needed to confirm these findings and improve the knowledge of the epidemiology of these two pathogens in these regions and elsewhere in China.

Seroprevalence and risk factors of Mycobacterium avium subspecies paratuberculosis infection in domestic sika deer in China.

Paratuberculosis or Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic infectious granulomatous enteritis of ruminants and other animals, which has a worldwide occurrence, but little is known of MAP infection in domestic sika deer in Jilin Province, China. The objective of the present investigation was to examine seroprevalence and risk factors of MAP infection in Jilin Province. Serum samples collected from 1400 sika deer from 16 sika deer herds were collected in the 4 districts of the province between May 2013 and August 2014 and were tested independently for the presence of antibodies against MAP. A total of 247 (17.64 %) sika deer tested positive for MAP antibodies using a commercially available enzyme immunoassay kit. The management level of farm and collecting region of sika deer was the main risk factor associated with MAP infection. The present study revealed the seroprevalence of MAP infection in sika deer in Jilin Province, China, which provided the baseline data for taking comprehensive countermeasures and measures in effectively preventing and controlling MAP infection in sika deer.

Toxoplasma gondii infection in cancer patients: prevalence, risk factors, genotypes and association with clinical diagnosis.

Prevalence of human infection with Toxoplasma gondii has been increasing in China due to the increasing number of cats. However, little is known of the epidemiology of T. gondii infection in different cancer patient groups. Thus, a case-control study of 900 cancer patients and 900 controls was conducted to detect anti-T. gondii antibodies by ELISA in China. Genomic DNA was extracted from the diseased tissues of 510 patients and the T. gondii B1 gene was amplified using a semi-nested PCR. DNA samples giving positive B1 amplification were then genetically characterized using multi-locus PCR-RFLP. The prevalence of anti-T. gondii IgG in cancer patients (35.56%) was significantly higher than that in controls (17.44%). The highest T. gondii seroprevalence was detected in lung cancer patients (60.94%), followed by cervical cancer patients (50%), brain cancer patients (42.31%) and endometrial cancer patients (41.67%). Exposure with soil and consumption of raw/undercooked meat were significantly associated with T. gondii infection in cancer patients. Three T. gondii genotypes (ToxoDB#9, ToxoDB#10 and Type I variant) were identified. In conclusion, T. gondii infection is a severe problem in cancer patients and it is imperative that improved integrated measures should be conducted to prevent and control T. gondii infection in cancer patients.

Cepharanthine, an alkaloid from Stephania cepharantha Hayata, inhibits the inflammatory response in the RAW264.7 cell and mouse models.

The aim of this study was to investigate the protective effects of cepharanthine (CEP) on inflammation in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in vitro and a LPS-induced lung injury model in vivo. RAW264.7 cells were treated with various concentrations of CEP for 1 h followed by incubation with or without 1 µg/ml LPS for 18 h. TNF-α, IL-6, and IL-1ß in the supernatants were measured by ELISA. Nuclear factor-κB (NF-κB) and mitogen-activated protein kinase pathways were analyzed by Western blot. Mice were randomly divided into control group, LPS group, CEP + LPS group, and dexamethasone + LPS group. A male BALB/c mouse model of acute lung injury was induced by LPS. Bronchoalveolar lavage fluid was collected for inflammatory cell count and cytokine assays. Histopathologic examination was performed on mice that were not subjected to bronchoalveolar lavage fluid collection. CEP dose-dependently inhibited the release of TNF-α, IL-6, and IL-1ß in LPS-stimulated RAW264.7 cells. Significantly, CEP dose-dependently suppressed NF-κB activation, IκBα degradation, and phosphorylation of ERK, JNK, and p38 induced by LPS. In vivo, it was also observed that CEP attenuated lung histopathologic changes and down-regulated the level of pro-inflammatory cytokines, including TNF-α, IL-1ß, and IL-6, in the mouse acute lung injury model. These results suggest that CEP potentially decreases inflammation in vitro and in vivo and might be a therapeutic agent against inflammatory diseases.

Protective effect of glutathione S-transferase-fused mutant staphylococcal enterotoxin C against Staphylococcus aureus-induced bovine mastitis.

Recent studies have demonstrated that immunization with nontoxic mutant staphylococcal enterotoxin C (mSEC) provides protection against Staphylococcus aureus infection in mouse models. In the present study, we investigated whether vaccination with a glutathione S-transferase-fused SEC (GST-mSEC) can protect against S. aureus-induced bovine mastitis. Cows were immunized with the GST-mSEC plus alum adjuvant and then challenged with viable S. aureus by an intramammary route. The results showed that immunization with GST-mSEC-induced production of SEC-specific antibodies in sera and the high titers of antibodies could persist for over 12 weeks. Importantly, immunization with GST-mSEC also induced production of SEC-specific antibodies in milk. The somatic cell counts in the milk from S. aureus challenged quarters of vaccinated lactating cows were significantly lower than those of the non-vaccinated control animals. Furthermore, the sera from GST-mSEC-immunized cows significantly inhibited interferon-gamma and tumor necrosis factor-alpha production from mouse spleen cells induced by wild-type SEC. These results suggest that vaccination with GST-mSEC provides protection against S. aureus-induced bovine mastitis and that the protection might be mediated by SEC-neutralizing antibodies.

Immunization with glutathione S-transferase and mutant toxic shock syndrome toxin 1 fusion protein protects against Staphylococcus aureus infection.

To investigate whether immunization with glutathione S-transferase (GST) and mutant toxic shock syndrome toxin 1 (mTSST-1) fusion protein can protect against Staphylococcus aureus infection, we purified a non-toxic mutant GST-mTSST-1 fusion protein. Mice were immunized with the GST-mTSST-1 plus alum adjuvant and then challenged with viable S. aureus. The results showed that the survival rate of GST-mTSST-1-immunized group was higher and the bacteria counts in the organs were significantly lower than those of the non-immunized mice. Immunization with GST-mTSST-1 induced strongly the production of TSST-1 specific antibodies, especially immunoglobulin G1 and immunoglobulin G2b. Furthermore, the serum samples from GST-mTSST-1-immunized mice also significantly inhibited interferon-gamma and tumor necrosis factor-alpha production from murine spleen cells by TSST-1. These results suggest that vaccination with GST-mTSST-1 provides protection against S. aureus infection and that the protection might be mediated by TSST-1-neutralizing antibody.