Parallel detection of multiple zoonotic parasites using a real-time fluorogenic loop-mediated isothermal amplification-based quadruple-sample microfluidic chip.

Zoonotic parasites pose significant health risks globally. In the present study, we combined a microfluidic chip with loop-mediated isothermal amplification (on-chip LAMP) to detect five zoonotic parasites: Toxoplasma gondii, Cryptosporidium parvum, Cryptosporidium hominis, Clonorchis sinensis, and Taenia solium. This method enabled the simultaneous parallel analysis of five genetic markers from a maximum of four samples per chip. The on-chip LAMP assay was conducted in a highly automated format via the addition (by pipetting) of each sample in a single operation. The reaction was performed in volumes as low as 5 µL at a temperature of 65°C for 60 min, achieving limits of detection ranging from 10-2 to 10-3 pg./µL of recombinant plasmid DNA. All the time-to-positive values were less than 40 min, and almost all the coefficients of variation were less than 10%, even when using limit of detection concentrations for multiple pathogens, indicating robust reproducibility among replicates. The clinical sensitivity and specificity for detecting 135 field samples were 98.08 and 97.59%, respectively, compared with traditional biological methods, indicating good applicability in the detection of field samples. This on-chip LAMP assay allows for low reagent consumption, ease of operation, and multiple analyses of samples and genetic targets, and is applicable for on-site detection and the routine monitoring of multiple zoonotic parasites.

Annexin A2-Mediated Internalization of Staphylococcus aureus into Bovine Mammary Epithelial Cells Requires Its Interaction with Clumping Factor B.

BACKGROUND: Staphylococcus aureus is a leading cause of contagious mastitis in dairy cattle. Internalization of S. aureus by bovine mammary gland epithelial cells is thought to be responsible for persistent and chronic intramammary infection, but the underlying mechanisms are not fully understood. METHODS: In the present study, we evaluated the role of Annexin A2 (AnxA2), a membrane-binding protein, in S. aureus invasion into bovine mammary epithelial cell line (MAC-T). In vitro binding assays were performed to co-immunoprecipitate the binding proteins of AnxA2 in the lysates of S. aureus. RESULTS: AnxA2 mediated the internalization but not adherence of S. aureus. Engagement of AnxA2 stimulated an integrin-linked protein kinase (ILK)/p38 MAPK cascade to induce S. aureus invasion. One of the AnxA2-precipitated proteins was identified as S. aureus clumping factor B (ClfB) through use of mass spectrometry. Direct binding of ClfB to AnxA2 was further confirmed by using a pull-down assay. Pre-incubation with recombinant ClfB protein enhanced S. aureus internalization, an effect that was specially blocked by anti-AnxA2 antibody. CONCLUSION: Our results demonstrate that binding of ClfB to AnxA2 has a function in promoting S. aureus internalization. Targeting the interaction of ClfB and AnxA2 may confer protection against S. aureus mastitis.

Hc-hrg-2, a glutathione transferase gene, regulates heme homeostasis in the blood-feeding parasitic nematode Haemonchus contortus.

BACKGROUND: Haemonchus contortus, a blood-feeding parasite, is constantly surrounded by large quantities of heme released from the catabolism of host red blood cells. To cope with the toxicity of free heme, H. contortus needs to uptake and detoxify the heme, a process believed to be paramount for parasite survival. METHODS: A heme-responsive gene Hc-hrg-2 was identified which is the homologue of Ce-hrg-2. The transcriptional levels in all developmental stages and heme-responsive ability of Hc-hrg-2 were analyzed by qRT-PCR. Immunofluorescence analysis and cell transfections were performed to analyze the expression pattern of Hc-HGR-2. Statistical analyses were performed with GraghPad Prism 6.0 using Student's t-test. RESULTS: To investigate the heme homeostasis of H. contortus, we first identified a heme-responsive gene Hc-hrg-2, a homolog of Ce-hrg-2 that is involved in heme transport in the hypodermis of Caenorhabditis elegans. Using qRT-PCR, we showed that Hc-hrg-2 mRNA was expressed throughout all life-cycle stages of H. contortus with the highest level in the third-stage larvae (L3s). Notably, transcription of Hc-hrg-2 in the exsheathed L3s was significantly upregulated in the presence of high concentration of heme. We found that Hc-HRG-2 protein was mainly located in the hypodermal tissues of adult H. contortus in vivo and the endoplasmic reticulum in the transfected mammalian cells. Our in vitro assay demonstrated that Hc-HRG-2 is a heme-binding protein with glutathione S-transferase activity and heme had a significant effect on its enzymatic activity when a model substrate 1-chloro-2, 4-dinitrobenzene (CDNB) was used. CONCLUSIONS: Hc-hrg-2 is a heme-responsive gene and engaged in heme homeostasis regulation in hypodermal tissues during the free-living stages of H. contortus.

Evaluation of potential anti-toxoplasmosis efficiency of combined traditional herbs in a mouse model.

Toxoplasma gondii is a worldwide spread protozoan and is able to infect almost all warm-blood animals. No effective drugs are available clinically on toxoplasmosis. Chinese traditional herbal medicines have provided remedies for many health problems. There exists a possibility that Chinese herbs may provide protection against T. gondii. This work aims to assess the protective efficacy of combined Chinese herbs against T. gondii. We screened five herbal medicines that have different pharmacological effects and combined them into a prescription according to the traditional Chinese medicine compatibility principle. The drug potential and protective efficacy were evaluated through a mouse model by determining the survival time, the parasite load in blood and tissues, the change of cell proportions in blood and histological detection. The results showed that the survival time of mice in the 500 mg Chinese herbs group and sulfadiazine group was significantly longer than that of the PBS control group. Also the parasite load in blood and tissues of 500 mg Chinese herbs and sulfadiazine groups was significantly lower than that of PBS group at 7 days post infection (dpi), which was in accordance with the result of histological detection. Monocyte and neutrophil of infected mice were remarkably increased while lymphocyte was dramatically decreased compared to that of blank group at 7 dpi. The results demonstrated that the 500 mg dosage of our Chinese herbs could slow down the replication of T. gondii and prolong the survival time of mice and could be considered as possible candidate drug against toxoplasmosis.

Profiling of differentially expressed genes in sheep T lymphocytes response to an artificial primary Haemonchus contortus infection.

BACKGROUND: Haemonchus contortus is a common bloodsucking nematode causing widespread economic loss in agriculture. Upon H. contortus infection, a series of host responses is elicited, especially those related to T lymphocyte immunity. Existing studies mainly focus on the general immune responses of sheep T lymphocyte to H. contortus, lacking investigations at the molecular level. The objective of this study was to obtain a systematic transcriptional profiling of the T lymphocytes in H. contortus primary-infected sheep. METHODS: Nematode-free sheep were orally infected once with H. contortus L3s. T lymphocyte samples were collected from the peripheral blood of 0, 3, 30 and 60 days post infection (dpi) infected sheep. Microarrays were used to compare gene transcription levels between samples. Quantitative RT-PCR was employed to validate the microarray data. Gene Ontology and KEGG pathway analysis were utilized for the annotation of differentially expressed genes. RESULTS: Our microarray data was consistent with qPCR results. From microarrays, 853, 242 and 42 differentially expressed genes were obtained in the 3d vs. 0d, 30d vs. 0d and 60d vs. 0d comparison groups, respectively. Gene Ontology and KEGG pathway analysis indicated that these genes were involved in metabolism, signaling, cell growth and immune system processes. Functional analysis of significant differentially expressed genes, such as SLC9A3R2, ABCB9, COMMD4, SUGT1, FCER1G, GSK3A, PAK4 and FCER2, revealed a crucial association with cellular homeostasis maintenance and immune response. Our data suggested that maintaining both effective immunological response and natural cellular activity are important for T lymphocytes in fighting against H. contortus infection. CONCLUSIONS: Our results provide a substantial list of candidate genes in sheep T lymphocytes response to H. contortus infection, and contribute novel insights into a general immune response upon infection.

Expression of Caenorhabditis elegans-expressed Trans-HPS, partial aminopeptidase H11 from Haemonchus contortus.

Aminopeptidase H11 present in the surface of intestine microvilli in Haemonchus contortus was identified as the most effective antigen candidate. However, its recombinant forms produced in Escherichiacoli, insect cells and yeast could not provide promising protection against H. contortus challenge, probably due to the inappropriate glycosylation and/or conformational folding. Herein, partial H11 containing the potential zinc-binding domain and two predicted glycosylation sites (nt 1 bp-1710 bp, Trans-HPS) was subcloned downstream of 5' flanking region of Caenorhabditis elegans cpr-1 gene in pPD95.77 vector, with the deletion of GFP gene. The recombinant was expressed in C. elegans and verified by blotting with anti-H11 and anti-Trans-HPS rabbit polyclonal antibodies and anti-His monoclonal antibody. Stably inherited Trans-HPS in worm descendants was achieved by integration using UV irradiation. Immunization with the crude Trans-HPS extracted from transgenic worms resulted in 37.71% reduction in faecal egg counts (FEC) (P<0.05) and 24.91% reduction in worm burden, but an upward curve with moderate rate of daily FEC in goats. These results suggested an apparent delay against H. contortus egg-laying in goats, which differed from that with bacteria-origin form of partial H11 (nt 670 bp-1710 bp, HPS) (26.04% reduction in FEC and 18.46% reduction in worm burden). These findings indicate the feasibility of sufficient C. elegans-expressed H11 for the immunological research and vaccine development.

Anticoccidial activity of traditional Chinese herbal Dichroa febrifuga Lour. extract against Eimeria tenella infection in chickens.

The study was conducted on broiler birds to evaluate the anticoccidial efficacy of an extract of Chinese traditional herb Dichroa febrifuga Lour. One hundred broiler birds were assigned to five equal groups. All birds in groups 1-4 were orally infected with 1.5 × 10(4) Eimeira tenella sporulated oocysts and birds in groups 1, 2 and 3 were medicated with 20, 40 mg extract/kg feed and 2 mg diclazuril/kg feed, respectively. The bloody diarrhea, oocyst counts, intestinal lesion scores, and the body weight were recorded to evaluate the anticoccidial efficacy. The results showed that D. febrifuga extract was effective against Eimeria infection; especially 20 mg D. febrifuga extract/kg feed can significantly increase body weight gains and reduce bloody diarrhea, lesion score, and oocyst excretion in comparison to infected-unmedicated control group.

Anticoccidial effect of halofuginone hydrobromide against Eimeria tenella with associated histology.

Halofuginone (stenorol) has been used as an effective anticoccidial reagent for decades but very little is known about its mode of action. In this study, chickens were inoculated with Eimeria tenella oocysts on 14-day-old and medicated with halofuginone at days 0, 1, 2, 3, 4, 5 and 6 post inoculations (groups 0, 1, 2, 3, 4, 5 and 6, respectively). Chickens in group 7 were taken as challenge-unmedicated control and in group 8 unchallenged-unmedicated control. The survival rate, body weight gains (BWG), oocysts production, cecal scores, bloody diarrhea and histological examinations were analyzed to evaluate the anticoccidial efficacy of halofuginone and to initially elucidate its mechanisms. Results showed that halofuginone which acted as a coccidiostatic can significantly enhance the BWG, and decrease both the oocyst shedding and cecal destruction caused by E. tenella infection. The histological slide examination noted that halofuginone was effective when provided 0-2 days post inoculation but only partially effective when applied 3-7 days post infection. The second-generation schizonts treated with halofuginone appeared vacuolated and degenerated. It is concluded that halofuginone can inhibit the parasite's invasion of host cecal hypothetical cell at the early stages of life cycle and later disturb the parasite's development by vacuolation of the schizonts. The resulting abnormal schizonts could not divide into schizoites and were eventually eliminated by the host's immune response.

Adjuvant effect of ginsenoside-based nanoparticles (ginsomes) on the recombinant vaccine against Eimeria tenella in chickens.

An experiment was conducted to study the adjuvant effect of ginsomes on the recombinant profilin in coccidian-infected breeding birds. Three-day-old chickens were vaccinated with Eimeria tenella recombinant profilin antigen (10, 50, and 100 µg per chicken) with or without 50 µg ginsomes per chicken. The boost vaccination was carried out 14 days later. Two weeks after the booster, the chickens were challenged with 1.5 × 10(4) homologous sporulated oocysts. The specific antibody response, lymphocyte proliferation, and IL-1 release from lymphocyte were measured at 1-42 days after boost vaccination. Seven days post-challenge, the rate of survival, body weight gains (BWG) were examined then all chickens were sacrificed and lesion scores and oocysts per gram were monitored to evaluate the protective effects of the vaccination after challenge. Compared with the group of vaccinating with profilin only, groups of 50 and 100 µg antigen plus ginsomes significantly enhanced lymphocyte proliferation and IL-1 secretion. The profilin specific antibody level in the four vaccinated groups was significantly higher than in the control group and in groups vaccinated with profilin containing ginsomes than profilin only. In the groups vaccinated with profilin plus ginsomes, the BWG was significantly higher than that of group of profilin only, but there was no significant difference between profilin plus adjuvant ginsomes, diclazuril medicated and uninfected-unmedicated-unvaccinated control groups. The lesion scores in groups immunized with profilin plus ginsomes was significantly lower than that both of groups unimmunized-challenged-unmedicated control and group vaccinated with profilin only. Oocyst excretion in groups vaccinated with 50 or 100 µg profilin plus ginsomes was lower than that of groups vaccinated with profilin only. These results demonstrate that the adjuvant ginsomes can promote subunit vaccine to induce a strong immune response and protective effects.

Seroprevalence of Toxoplasma gondii infection in pigs, in Zhejiang Province, China.

Seroprevalence of Toxoplasma gondii infection in pigs in 10 regions of Zhejiang Province, China, was obtained by enzyme-linked immunosorbent assay (ELISA). Anti- T. gondii antibodies were found in 53.4% (434/813) of pigs. Results were analyzed by a chi-square (χ(2)) test. Differences were observed according to farm size, animal age, and sampling regions. Seroprevalences in pigs raised on small farms (71.4%) were significantly higher than that (42.7%) on large farms (P < 0.05), and seroprevalence increased progressively with age. The seroprevalence ranged from 28.1% to 66.0% in different regions, with Jiaxing having the lowest level (28.1%), followed by Hangzhou (36.0%) and Taizhou (42.0%). This is the first study on seroprevalence of T. gondii infection in pigs in Zhejiang Province.

Ginsenoside Rg1 enhances immune response induced by recombinant Toxoplasma gondii SAG1 antigen.

Ginsenoside, the most important component isolated from Panax ginseng, exhibits a variety of biological activities. Particularly, ginsenoside Rg1 is known to have immune-modulating activities such as increase of immune activity of T helper (Th) cells. In the present study, we evaluated the immunomodulatory potentials of the Rg1 at three dose levels on the cellular and humoral immune responses of ICR mice against T. gondii recombinant surface antigen 1 (rSAG1). ICR mice were immunized subcutaneously with 50 µg Rg1 alone, 100 µg rSAG1 alone or with 100 µg rSAG1 dissolved in saline containing ginsenoside Rg1 (10 µg, 50 µg or 100 µg). After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that the groups immunized with rSAG1 and Rg1 (50 µg, 100 µg) developed a high level of specific antibody responses against T. gondii rSAG1, a strong lymphoproliferative response, and significant levels of cytokine production, compared with the other groups. After lethal challenge, the mice immunized with the rSAG1 and Rg1 (50 µg, 100 µg) showed a significantly increased survival time compared with control mice which died within 6 days of challenge. Our data demonstrate that by addition of ginsenoside Rg1, the rSAG1 triggered a stronger humoral and cellular response against T. gondii, and that Rg1 is a promising vaccine adjuvant against toxoplasmosis, worth further development.

The gene structure and promoter region of the vaccine target aminopeptidase H11 from the blood-sucking nematode parasite of ruminants, Haemonchus contortus.

Aminopeptidase H11, an integral membrane glycoprotein present only in the gut of Haemonchus contortus, could provide substantial protection as shown by 90% reduction in fecal egg counts, while its recombinant version expressed in E. coli induced little. To investigate the characteristics further, we amplified mRNA of H11 gene via reverse transcriptase polymerase chain reaction, followed by isolation of its 1,517-bp 5'-flanking region and determination of its genomic organization. The H11 gene contained 25 exons separated by 24 introns and spans 14,959 bp of genomic DNA. Analysis of the 1,517 bp 5'-flanking region of the H11 gene revealed a putative "TATA-less" promoter. Partial sequences of the last exon and its 3'-UTR of H11 isoform H11-4 were also identified upstream to the H11 gene with the same transcription orientation. The 1,517-bp 5'-flanking region and part of the first exon of the H11 gene were subcloned into the vector upstream of green fluorescence protein reporter gene and microinjected into the gonads of Caenorhabditis elegans. The transformed animals exhibited fluorescence in the distal intestine in the L4 larvae stage and adult worms. This study characterized gene structure of aminopeptidase H11, demonstrated different transcriptional pattern of its promoter region between free-living and blood-sucking nematode species, and highlights the utility of C. elegans as a heterologous system to study the biology roles of H11 isoforms.