Exacerbation of allergic asthma by somatic antigen of Echinococcus granulosus in allergic airway inflammation in BALB/c mice

Background There is ample evidence demonstrating a reverse relationship between helminth infection and immune-mediated diseases. Accordingly, several studies have shown that Echinococcus granulosus infection and hydatid cyst compounds are able to suppress immune responses in allergic airway inflammation. Previous studies have documented the ability of hydatid cysts to suppress aberrant Th2 immune response in a mouse model of allergic asthma. However, there is a paucity of research on the effects of protoscoleces on allergic asthma. Thus, this study was designed to evaluate the effects of somatic antigens of protoscoleces in a murine model of allergic airway inflammation. Methods Ovalbumin (OVA)/aluminum hydroxide (alum) was injected intraperitoneally to sensitize BALB/c mice over a period of 0 to 7 days, followed by challenge with 1% OVA. The treatment group received somatic antigens of protoscoleces emulsified with PBS on these days in each sensitization before being challenged with 1% OVA on days 14, 15, and 16. The effects of somatic antigens of protoscoleces on allergic airway inflammation were evaluated by examining histopathological changes, the recruitment of inflammatory cells in the bronchoalveolar lavage, cytokine production in the homogenized lung tissue (IL-4, IL-5, IL-10, IL-17, and IFN-γ), and total antioxidant capacity in serum. Results Overall, administration of somatic antigens of protoscoleces exacerbated allergic airway inflammation via increased Th2 cytokine levels in the lung homogenate, recruitment of eosinophils into bronchoalveolar lavage fluid, and pathological changes. In addition, total antioxidant capacity and IFN-γ levels declined following the administration of somatic antigens. Conclusions The results revealed that the co-administration of somatic products of protoscoleces with OVA/alum contributed to the exacerbation of allergic airway inflammation in BALB/c mice. Currently, the main cause of allergic-type inflammation exacerbation is unknown, and further research is needed to understand the mechanism of these interactions. Graphical Abstract

Dysregulation of Ovine Toll-Like Receptors 2 and 4 Expression by Hydatid Cyst-Derived Antigens

Background: Cystic echinococcosis (CE) is a zoonotic disease caused by infection with Echinococcus granulosus. Toll-like receptors (TLRs) as the first line of defense against various parasites play a critical role in sensing and triggering anti-parasite responses. Methods: The study was conducted at the Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Iran in 2019. Ovine peripheral blood mononuclear cells (PBMCs) were stimulated with hydatid cyst-derived antigens including hydatid cyst fluid (HCF), germinal layer antigens (GL), somatic and excretory/secretory (ES) products of protoscoleces (PSC). To investigate whether the expression of TLR2 and TLR4 was altered during exposure to these antigens, PBMCs were stimulated with two different concentrations at different time points. Results: After exposure of PBMCs to ES and somatic antigens of protoscoleces (PSC) the expression of TLR2 and TLR4 was down-regulated in comparison with control group. Similarly, HCF markedly down-regulated TLR2 and TLR4 transcripts independent of dose and time. GL antigens significantly down-regulated TLR2, while TLR4 expression was up-regulated as compared with control group. Conclusion: Hydatid cyst-derived antigens could dysregulate the expression of TLR2 and TLR4 in ovine PBMCs, suggesting a possible mechanism to suppress host immunity to establish chronic infection.

Proteomic Analysis of Eluted Antigens from Crude Somatic Antigens of Trichinella Spiralis Muscle Larva by Igg-ELISA, Two-Dimensional Gel Electrophoresis, and Mass Spectrometry

Background: Trichinellosis is caused by Trichinella spiralis muscle larvae (ML), which in swine is the main source of transmission. Trichinellosis is detected by enzyme-linked immunosorbent assay (ELISA) using excretory–secretory antigens (ESAg). Preparation of ESAg is difficult, and it produces a low content. Furthermore, the sensitivity and specificity of the test depends on the quality of the antigen that is produced in each batch. Crude somatic antigens (CSAg) of T. spiralis ML at molecular weights (MWs) of 101, 79, and 43 kDa were previously positive for swine trichinellosis sera, with no cross-reaction observed for normal sera, except that 79 and 43 kDa each reacted with one of the coccidiosis and with mixed infections containing trichuriasis and coccidiosis. Therefore, the current study aimed to obtain eluted antigens of T. spiralis ML CSAg, which were analyzed by IgG-ELISA for detecting swine trichinellosis and identification by two-dimensional polyacrylamide gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC MS-MS).Methods: In this study, T. spiralis larvae CSAg at 101, 79, and 43 kDa (TsCSAg-101, TsCSAg-79, and TsCSAg-43, respectively) were eluted from sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The eluted antigens were analyzed by IgG-ELISA for sensitivities and specificities. In addition, three specific antigens were identified by 2-DE and LC MS-MS.Results: Sensitivity of IgG-ELISA using three eluted antigens was persistent at 100%. Specificities were 90.63%, 95.54%, and 97.77% following TsCSAg-101, TsCSAg-79, and TsCSAg-43, respectively. The LC MS-MS results showed that 18/20 spots of the antigens were identified for 11 different proteins. One protein has several isoforms; for example, a serine proteinase and the phosphoenolpyruvate carboxykinase protein.Conclusions: TsCSAg-43 showed the highest specificity compared to the other two eluted antigens, which indicated that these specific proteins (a 45k antigen–trichina [fragment], a DNA topoisomerase 2-alpha, an antigen targeted by protective antibodies, and a conserved hypothetical protein [gi339234223]) should be developed and produced in large volumes in further study.

Identification and Immunological Characterization of Somatic Proteins from Adults of Toxocara cati by Proteomics Technique

Background: Toxocara cati is considered as one of the main etiological agents of toxocariasis with global and regional importance. As there is no information on proteomics of T. cati, herein, we reported the results obtained by proteomic analysis of somatic proteins extract, using a mass spectrometry (LC–MS/MS) approach. Methods: Somatic extract fractions were separated by two-dimensional SDSPAGE and were electro blotted on to PVDF membranes for immunoblot analysis, then collected the immunogenic spots which response of antibodies of the paratenic hosts (mice) to the antigens ( Mashhad, 2017), and analyzed by LC–MS/MS. The LC-MS/MS data were analyzed by Mascot database, Taxonomy Toxocara, and common contaminants, in Omics Center, Biotechnology Medical University of Graz (Austria, 2018). Result: The protein spots were isolated between 15–140 kDa ranges using 3–10 non-linear IPG strips and Brilliant Blue Coomassie. Ten proteins were characterized as immunogenic proteins, seven of them were identified and three of them were unknown proteins. Conclusion: This study provided additional information about the somatic antigens of T. cati, which can lead to the development of new strategies for novel immuno-modulators, drug targets, subunit vaccines and immunodiagnostic kits for toxocariasis.

Development of an interferon-γ release assay (IGRA) for detection of Brucella abortus and clinical diagnosis of brucellosis

Introduction: Brucellosis, caused by Brucella abortus (B. abortus), is an important zoonosis posing a great risk to both livestock and humans. Currently, most assays for clinical diagnosis of brucellosis have been developed based on serological principles; however, these assays have a number of limitations and disadvantages. Methodology: To address this concern, the aim of this study was to develop a gamma interferon (IFN-γ) release assay (IGRA) for the diagnosis of brucellosis. Towards this end, the stimulating effect induced by different somatic antigens of B. abortus on the secretion of IFN-γ was evaluated. Results: The best antigen candidate, B. abortus strain 2308, able to induce high levels of IFN-γ expression in peripheral blood (PB) cells from cattle, was used for the development of the IGRA. The optimal concentration for stimulation was determined as 1.0×107 CFU/mL. This study demonstrated that IFN-γ was detectable on day 5 post infection (p.i.) and peaked on day 14 p.i.. Finally, the IGRA developed was used for detection of B. abortus in clinical samples, and a higher level of IFN-γ was detected in Brucella-infected samples compared to vaccination samples and negative controls. Conclusions: The optimal somatic antigen for B. abortus was identified and used to establish a robust IGRA. The IGRA developed is suitable for clinical diagnosis of brucellosis, especially in the early stages of infection.

Peptides isolation from crude somatic antigens of Haemonchus contortus through SDS- PAGE

Haemonchus (H.) contortus is a blood feeding gastrointestinal nematode, and is considered one of the major threats for goat health and production globally. Crude somatic antigens (CSA) have been reported as sources of immunogens and provide better level of immunity against gastrointestinal nematodes. The present study aimed to quantify CSA of H. contortus worms (n=25) of both sexes followed by isolation of peptides through Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and their characterization. The triturate contained proteins having concentration of 1.3mg/ml as measured through Bradford assay. Seven different bands were appeared on gel ranging from 35KDa to 170KDa. The future prospects may include identification of the immunogenic peptides which can be used as vaccine candidates against H. contortus.

Prevalence of Shiga toxin-producing and enteropathogenic Escherichia coli marker genes in diarrhoeic stools in a New Zealand catchment area

BackgroundShiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli are gastrointestinal pathogens causing diarrhoeal and extraintestinal disease. Due to lack of EPEC screening and use of Sorbitol-MacConkey (SMAC) agar in faecal screening, the true prevalence of EPEC and non-O157 STEC in New Zealand diarrhoeal cases is unknown.MethodsDiarrhoeic stools sourced from Dunedin hospital were pre-enriched, DNA extracted with Chelex-100 resin and screened using a multiplex TaqMan quantitative PCR assay amplifying stx1, sxt2 and EPEC (eae) gene markers.ResultsOf the 522 diarrhoeic samples surveyed, 8 (1.53%) were PCR positive for stx1/stx2 and 23 (4.41%) were positive for eae. Six (75%) of the stx+ samples were uncommon non-O157 serotypes, and the remainder were found to be positive for both O103 and O157 STEC somatic antigens.ConclusionsResults revealed shortcomings in current screening protocols for pathogenic E. coli; SMAC is not sufficiently discriminatory to detect emergent STEC serotypes and EPEC likely has an unappreciated role in cases of diarrhoea in New Zealand.