Identification of Somatic Antigens of Adult Fasciola gigantica Isolated from Bali Cattle

In most tropical countries, such as Indonesia, fasciolosis is generally caused by Fasciola gigantica known as tropical liver fluke. However, most fasciolosis serodiagnostic tests have been developed solely for diagnosing fasciolosis caused by Fasciola hepatica (non-tropical liver fluke), and very few have been specifically designed for F. gigantica. The aim of this study was to determine the profile of antigenic proteins from the somatic extract of F. gigantica isolated from Bali cattle (Bos javanicus). The liver flukes were collected from a slaughtering house in Mataram, Indonesia. The somatic extracts were prepared by homogenizing in buffers containing 0.05 M NaCl, 0.02 M PMSF, and 0.05% Triton X-100. The characterization of the somatic extract proteins was performed using one-dimension gel electrophoresis and followed by Western blotting to determine the profile of its antigenic proteins. There were 14 bands of the somatic extracts with an estimated molecular weight ranging from 8 to105 8 kDa shown on the gel electrophoresis. The results of the Western blot show that there were five prominent protein bands. Three out of five prominent antigenic proteins with molecular weights of 8, 27, and 33 kDa are promising to enrich the existence of antigens that have immunodiagnostic value for fasciolosis. Therefore, further studies are required to examine more deeply the potency of those three antigenic somatic proteins of F. gigantica.

Immunoregulatory Effects of Somatic Extract of Toxocara canis on Airway Inflammations in Murine Model

Background: The immunomodulatory role of many parasites is well-documented. The current study designed to assess the immunoregulatory effects of the somatic extract (SE) of Toxocara canis on murine model of airway inflammations. Methods: The experiment was performed in department of parasitology of Tarbiat Modares University, Tehran, Iran from November 2018 to May 2019. Totally 30 female BALB/c mice divided into one control group and two experimental groups (10 mice in each group). The ovalbumin (OVA) group was sensitized with OVA in alum, while the SE group was administered with SE and OVA in alum intraperitoneally. The control group was injected with PBS in alum. Then, SE and OVA groups were intranasally challenged with OVA for three consecutive days and the control group encountered with PBS at the same time. One day after the last challenge, real-time PCR and histopathology survey were conducted on isolated lung tissues. Results: The gene expression of IL-25, IL-33, TNF-α and TLR-4 in SE group was significantly lower than OVA group (P<0.05). The level of IL-10, TGF-β and IFN-γ were considerably higher than the OVA group (P<0.05). The inflammation was reduced in SE group, as the total cell number of bronchoalveolar lavage fluid was less than OVA group. Based on the histopathology findings the inflammation was decreased in SE group compared to the OVA group. Conclusion: Although, an inhibitory effect of SE of T. canis on airway inflammations was detected, there is still a long way ahead regarding the indication of the precise mechanisms.

Metabolomes and Lipidomes of the Infective Stages of the Gastrointestinal nematodes, Nippostrongylus brasiliensis and Trichuris muris

Soil-transmitted helminths, including hookworms and whipworms, infect billions of people worldwide. Their capacity to penetrate and migrate through their hosts’ tissues is influenced by the suite of molecules produced by the infective developmental stages. To facilitate a better understanding of the immunobiology and pathogenicity of human hookworms and whipworms, we investigated the metabolomes of the infective stage of Nippostrongylus brasiliensis third-stage larvae (L3) which penetrate the skin and Trichuris muris eggs which are orally ingested, using untargeted liquid chromatography-mass spectrometry (LC-MS). We identified 55 polar metabolites through Metabolomics Standard Initiative level-1 (MSI-I) identification from N. brasiliensis and T. muris infective stages, out of which seven were unique to excretory/secretory products (ESPs) of N. brasiliensis L3. Amino acids were a principal constituent (33 amino acids). Additionally, we identified 350 putative lipids, out of which 28 (all known lipids) were unique to N. brasiliensis L3 somatic extract and four to T. muris embryonated egg somatic extract. Glycerophospholipids and glycerolipids were the major lipid groups. The catalogue of metabolites identified in this study shed light on the biology, and possible therapeutic and diagnostic targets for the treatment of these critical infectious pathogens. Moreover, with the growing body of literature on the therapeutic utility of helminth ESPs for treating inflammatory diseases, a role for metabolites is likely but has received little attention thus far.

ANTIPROLIFERATIVE EFFECT OF ANISAKIS SIMPLEX RUDOLPHI, 1809 (DUJARDIN, 1845) L3 EXTRACT ON VERO LINE CELLS IN VITRO

The effect of a sterile crude extract from the frozen Anisakis simplex Rudolphi, 1809 (Dujardin, 1845) third stage larvae on the transplantable culture of mammalian cells is described. At the first time fibroblasts cells line Vero (from African green monkey, kidney) obtained from the «BioloT», St Petersburg, Russia, on a nutrient media EMEM were used. In the in vitro conditions a significant antiproliferative action of crude somatic extract expressed by complete destruction of the monolayer of fibroblast-like cells after 48 hours of cultivation is proven. Therefore, our results can be regard as a manifestation of a pronounced inhibitory effect of the studied extract on the proliferation of Vero culture, which is of considerable interest in the field of cell technology and Parasitology and may be use as the basis for subsequent experiments to study the pathogenesis and prevention of anisakiasis in humans and animals.

Protein Detection of Excretory-Secretory Products and Somatic Extracts from Fasciola hepatica and F. gigantica Using Two-Dimensional Electrophoresis

Background: The aim of this research was to compare excretory-secretory and somatic extract materials of Fasciola hepatica and F. gigantica to detect protein maps of two species. Methods: Twenty infected livers were collected from sheep in industrial slaughterhouse in Tehran, 2017-2019. Worms were detached from bile ducts, then recognized according to morphologic and morphometric criteria. After three times washing, worms were incubated in RPMI culture media and excretory-secretory products were collected. Worms were crushed and homogenized for preparation of somatic extract. Two Dimensional Electrophoresis gels were accomplished for both excretory-secretory material and somatic extracts. Gels were scanned with densitometer and analyzed with Same Spots software and protein spots were identified with Expasy database. Results: For both excretory-secretory products and somatic extract, protein spots were appeared with two-dimensional electrophoresis technique. Quantitative analysis showed 40 and 28 protein spots for excretory-secretory of F. hepatica and F. gigantica respectively. For somatic extract 19 and 12 protein spots were recognized for F. hepatica and F. gigantica in that order. Conclusion: The rate of expression of some proteins were more in F. hepatica while expression of other proteins was high in F. gigantica. The expression of protease enzyme was higher in F. gigantica than F. hepatica. These data could be considered for biochemical differentiation of Fasciola species and subsequently to design and prepare of antigens for diagnosis/vaccine development.

PROTECTIVE EFFICACY OF A COMPLEX BIOPREPARATION BASED ON A SOMATIC EXTRACT OF TRICHINELLA AND IMUNOFAN IN EXPERIMENTAL TRICHINOSIS IN WHITE MICE

The purpose of this placebo-controlled randomized laboratory study was to evaluate the effectiveness of a complex biological product based on the Trichinella somatic antigen (SET) at a dose of 40 μg / mouse and an immunostimulant immunofan at a dose of 1 mg / mouse for T. spiralis larvae with experimental infection with them 40 days after the last injection of drugs. The preparations were administered twice, intramuscularly in 0.2 ml of sterile saline with an interval of 48 hours to 20 animals from 2 therapeutic groups. 10 mice of the control group received a placebo. Mice were experimentally infected with T. spiralis larvae at a dose of 30 ± 5 larvae / mouse orally. After 40-45 days, a control assessment was performed to detect T. spiralislarvae. The number of larvae of the parasite in animals treated with the complex drug was reduced by 61.98%, and in mice treated with an immunostimulant - by 31.42% (calculated from the geometric mean). The largest number of Trichinella larvae was found in the control group that received placebo.According to the results of the study, a conclusion was drawn about the effectiveness of double administration of a complex immunopreparation based on SET-40 and Imunofan, as well as on the prospects and feasibility of continuing further research in search of the most promising immunoprophylactic agents, their doses and frequency of use for trichinosis.

Analysis of excretory–secretory and somatic antigens ofGastrothylax crumenifer

The excretory/secretory (ES) metabolic products released byGastrothylax crumenifer(Trematoda: Digenea) duringin vitroincubations and the somatic extract of the adult parasite were analysed using polyacrylamide gel electrophoresis (PAGE). Immunogenicity of ES and somatic extracts were evaluated by immunoblotting and ELISA using sera raised against ES and somatic antigens in rabbits. The electropherograms of ES and somatic extracts have been resolved into 38 and 41 polypeptides, respectively. The apparent molecular weights of these polypeptides range from < 29 to > 205 kDa. A total of 14 polypeptides were found to be common to both of the samples. The analysis of immunoblot results revealed 22 and 27 antigenic polypeptides in ES and somatic extracts respectively. Only 11 and 13 antigenic polypeptides were found specific to ES and tissue extract respectively. The molecular weights of these specific polypeptides were calculated to be < 14.4, 16, 20, 25, 33, 42, 119, 125 and > 205 kDa for metabolic products and < 14.4, 25, 30, 35, 78, 84 and > 205 kDa for the tissue extracts, respectively. Analysis of ELISA results revealed that a dilution of up to 1:3200 of the test sera could react with the ES product. Further, when the ES antigens were allowed to react with antisomatic extracts in hyperimmune sera the titre of IgG increased up to a dilution of 1:12800. The potential importance of these antigens in the immunodiagnosis of amphistomiasis is discussed.