Excretory-secretory Antigens Research Articles

Evaluation of Toxocara canis Glycosylated TES Produced in Pichia pastoris for Immunodiagnosis of Human Toxocariasis

Recombinant proteins are a suggested alternative for the diagnosis of toxocariasis. The current Escherichia coli recombinant protein overexpression system usually produces insoluble products. As an alternative, yeast such as Pichia pastoris have secretory mechanisms, which could diminish the cost and time for production. This study aimed to produce recombinant proteins in Pichia pastoris and verify their sensibility and specificity in an indirect ELISA assay. Two sequences (rTES-30 and rTES-120) of Toxocara canis excretory-secretory antigens were cloned in a pPICZαB vector and expressed in P. pastoris KM71H. Sera samples collected from human adults infected by Toxocara spp. were tested by indirect ELISA using rTES-30 and rTES-120 as antigens. Recombinant proteins were detected at 72 hours after induction, in the supernatant, as pure bands between 60~70 kDa with hyperglycosylation. Regarding diagnosis potential, recombinant antigens had high specificity (95.6%); however, sensitivity was 55.6% for rTES-30 and 68.9% for rTES-120. Further deglycosylation of the P. pastoris antigens did not seem to affect ELISA performance (p>0.05). The low sensitivity in the serodiagnosis diminished any advantage that P. pastoris expression could have. Therefore, we do not recommend P. pastoris recombinant TES production as an alternative for the diagnosis of toxocariasis.

Neurotoxocariasis.

Human toxocariasis, caused by larvae of the Toxocara spp., is one of the most prevalent zoonosis with a worldwide distribution. Toxocara larvae can cross the blood-brain barrier, invading the central nervous system (CNS), leading to neurotoxocariasis. The clinical presentation consists of a wide spectrum of neurological manifestations such as meningitis, encephalitis, myelitis and cerebral vasculitis, but asymptomatic CNS infection is common. Despite the high seroprevalence, neurotoxocariasis is thought to be rare, even if in many animal models larvae usually migrate to the brain. Indeed, diagnosis of neurotoxocariasis is rarely considered, and the exact prevalence of CNS involvement is still unknown. Diagnosis of neurotoxocariasis is generally based on the detection of specific antibodies to Toxocara excretory-secretory antigens by ELISA in CSF or serum, and clinical and radiological improvement after anthelmintic therapy, but definitive diagnosis is given by histological confirmation, which is rarely available. Treatment is based on anti-helminthic drugs, commonly albendazole, generally administered with concomitant corticosteroids coverage. At the population level, serological studies suggest an association between Toxocara infections and epilepsy, as well as with other neurodegenerative and psychiatric disorders. Nonetheless the possible role of Toxocara spp. in the pathogenesis of these disorders is still matter of debate.

Development of point-of-care testing tool using immunochromatography for rapid diagnosis of human paragonimiasis

Paragonimiasis, an important food-borne zoonosis, is caused by lung flukes of the genus Paragonimus. Several million people are actually infected or at risk. Paragonimiasis is a re-emerging disease in developing countries. Diagnosis of pulmonary paragonimiasis is made by finding eggs in sputa and/or fecal samples. Eggs are typically not found in ectopic paragonimiasis cases, so diagnosis depends on supportive information, such as a history of eating fresh water crabs or crayfishes, radiographic findings and immunological tests. Here, a new point-of-care-testing (POCT) tool is presented. It uses immunochromatography for serodiagnosis of human paragonimiasis using excretory-secretory antigen from Paragonimus heterotremus. It proved effective in diagnosing infections due to P. heterotremus, and was also successfully diagnosed with sera from infections with P. westermani and P. miyazakii. The diagnostic sensitivity, specificity, positive and negative predictive values and accuracy were 97.9%, 87.6%, 78%, 98.9%, and 90.8%, respectively. The developed POCT tool is rapid and simple to use not only for clinical diagnosis of paragonimiasis at the bedside or at well-equipped laboratories, but also at local and remote hospitals with limited facilities. Moreover, the POCT tool could be applied for epidemiological surveys of paragonimaisis in Asia where P. heterotremus, P. westermani and P. miyazakii are endemic.

Seroprevalence and associated risk factors of toxocariasis among nomads in Boyer-Ahmad County, southwest Iran.

Human toxocariasis is caused by the larval stage of Toxocara canis and Toxocara cati. This cross-sectional study was conducted to evaluate the seroprevalence and the risk factors associated with Toxocara infection in nomads of Boyer-Ahmad County in the southwest of Iran. A total of 968 blood samples were collected from nomads. A structured questionnaire, containing sociodemographic data, was completed for each participant. Sera were evaluated for anti-Toxocara antibodies, using the T. canis excretory-secretory antigens prepared from the second stage larvae, in an ELISA system. Among the 968 participants, 220 (22.7%) were males and 748 (77.3%) were females; the mean age was 40.71y. Anti-Toxocara IgG antibodies were detected in 14 (1.4%) of the 968 nomads. The association between Toxocara infection and gender, age, contact with dogs, residential area and level of education were not statistically significant (p>0.05). The findings of the study revealed a low prevalence rate of toxocariasis in nomadic communities in southwest Iran. Geographic location and climatic conditions as well as the lifestyle and sociodemographic features of the participants may contribute to the low prevalence rate of Toxocara infection.

High-Throughput Measurement of Microneme Secretion in Toxoplasma gondii.

Micronemes are specialized secretory organelles present in all motile forms of apicomplexan parasites. Microneme vesicles hold adhesins and other proteins that are secreted to facilitate parasite attachment, invasion of host cells, and egress following replication-all processes indispensable for cell-to-cell transmission of these obligate intracellular parasites. Defining the signaling pathways that lead to microneme secretion is an important part of understanding the infectious cycle of apicomplexan parasites. However, the classical method of measuring microneme secretion by immunoblotting for microneme proteins in parasite excreted/secreted antigen (ESA) preparations is low-throughput and only semiquantitative. We recently reported a new luciferase-based method for measuring microneme secretion in a 96-well format with high sensitivity in the model apicomplexan Toxoplasma gondii. Here, we aim to elaborate on this detection method and review current practices for stimulating microneme secretion in vitro.

The Neurotropic Parasite Toxoplasma gondii Induces Astrocyte Polarization Through NFκB Pathway.

Background: Toxoplasma gondii is a protozoan parasite that chronically infects nearly one-third of the world's human population. In immunosuppressed individuals and fetus, infection with T. gondii contributes to a series of devastating conditions, including toxoplasmic encephalitis (TE), which is characterized by neuron damage in the central nervous system (CNS). Astrocyte polarization is currently found in some neurodegenerative diseases, and A1 subtype of astrocyte leads to neuron apoptosis. However, little information has been available on the role of astrocyte polarization in TE.Methods: In the present study, we established a mouse model to study TE and detected A1 astrocyte in the brains of mice with TE. Expression level of A1 astrocyte-specific marker C3 was evaluated using indirect fluorescent assay (IFA) and Western blotting. Primary mouse astrocytes were incubated with different concentrations of T. gondii excreted-secreted antigens (TgESAs) in vitro. Expression level of C3 and A1 astrocyte-specific transcription levels were assessed using Western blotting and qRT-PCR, respectively. Bay11-7082 was used to study nuclear factor (NF) κB pathway in TgESA-induced astrocyte polarization.Results: In mice with TE, the proportion of A1 astrocyte (GFAP+C3+) increased significantly. The results of in vitro study showed that TgESAs induced astrocyte polarization to A1 subtype. Blocking of NFκB pathway by Bay11-7082 inhibited TgESA-induced astrocyte polarization.Conclusions: Our preliminary study showed the involvement of A1 astrocyte in the process of TE in mice, and TgESAs could trigger astrocyte to polarize to A1 subtype. These findings suggest a new mechanism underlying the neuropathogenesis induced by T. gondii infection.

Establishment of a serodiagnosis system for the detection of Toxocara spp. and Ascaris suum infection in chickens

Chickens are considered to act as paratenic hosts for agents, Toxocara canis, T. cati and Ascaris suum; which cause ascarid larva migrans syndrome (ascarid LMS) in humans. In addition, they are the definitive host for Ascaridia galli, considered not to be infective for humans. All ascarid parasites can have a high homology of antigenicity, leading to cross-reactivity in serodiagnostic assays. This study was conducted to establish a procedure for the serological detection of those roundworm infections in chickens.Twenty-five male Julia chickens were divided into five groups (n = 5); T. canis-, T. cati-, Ascaris suum- and Ascaridia galli-infected, and an uninfected control group. In Ascaris suum-soluble worm antigen preparation (As-SWAP) ELISA, all infected groups showed an elevation of anti-ascarid antibodies, indicating the usefulness of As-SWAP as a screening antigen for the detection of ascarid infections. For infecting species identification, T. canis-excretory/secretory (Tc-ES) and Ascaris suum-ES (As-ES) antigen ELISA were conducted by serial dilution sera. Toxocara spp.-infected sera showed stronger binding to Tc-ES than As-ES, while Ascaris suum and Ascaridia galli-infected sera bound to As-ES more strongly than Tc-ES. To discriminate between Ascaris suum and Ascaridia galli infection, sera were pre-incubated with Ascaridia galli-SWAP antigen and applied to Tc-ES and As-ES ELISAs. In this pre-adsorbed ES antigen ELISAs, only the Ascaris suum infected group showed positive binding to As-ES, resulting from the adsorption of cross-reactive antibodies in Ascaridia galli-infected sera. Finally, anti-Toxocara specific antibodies were confirmed by Tc-ES western blot (WB). Toxocara spp.-infected sera showed toxocariasis-specific band pattern in Tc-ES WB, while no specific band appeared on any strip incubated with Ascaris suum, Ascaridia galli-infected and uninfected sera.In conclusion, the serodiagnostic assays evaluated in this study are useful for the detection of ascarid infections in chickens.

Development of an Immunochromatographic Point-of-Care Test for Serodiagnosis of Opisthorchiasis and Clonorchiasis.

Chronic infections with the food-borne liver flukes, Opisthorchis viverrini or Clonorchis sinensis, associate with cholangiocarcinoma, bile duct cancer, which generally has a poor prognosis. We have produced a rapid and simple immunochromatographic test (ICT) kit for the diagnosis of opisthorchiasis and clonorchiasis by the detection of IgG antibodies in human infection sera. Sera from volunteers with proven opisthorchiasis and several other parasitic diseases and from healthy controls were evaluated for the presence of liver fluke infection-specific antibodies using a preparation of excretory-secretory antigen from adult stage O. viverrini absorbed onto ICT strips. Diagnostic values were compared with an ELISA. The diagnostic sensitivity, specificity, and positive and negative predictive values of the ELISA were 100%, 98.3%, 97.9%, and 100%, whereas those for the ICT were 94.6%, 91.2%, 89.7%, and 95.4%, respectively. There was 91.7% concordance between the ICT with ELISA, and differences in performance between the tests were not statistically significant (P > 0.05). Twenty-seven of 30 (90%) of the clonorchiasis sera also were positive by ICT. This new ICT provides a facile, rapid test for point-of-care testing tool, which can be used at the bedside without the need for sophisticated equipment. Moreover, the ICT can be anticipated to supplement stool examination as a screening tool in the clinic for the diagnosis of opisthorchiasis and clonorchiasis, and in addition, it may be useful in screens of populations at risk of liver fluke infection-associated cholangiocarcinoma.

Electrochemical microfluidic immunosensor based on TES-AuNPs@Fe3O4 and CMK-8 for IgG anti-Toxocara canis determination

We report a microfluidic immunosensor for the electrochemical determination of IgG antibodies anti-Toxocara canis (IgG anti-T. canis). In order to improve the selectivity and sensitivity of the sensor, core-shell gold-ferric oxide nanoparticles (AuNPs@Fe3O4), and ordered mesoporous carbon (CMK-8) in chitosan (CH) were used. IgG anti-T. canis antibodies detection was carried out using a non-competitive immunoassay, in which excretory secretory antigens from T. canis second-stage larvae (TES) were covalently immobilized on AuNPs@Fe3O4. CMK-8-CH and AuNPs@Fe3O4 were characterized by transmission electron microscopy, scanning electron microscopy, energy dispersive spectrometry, cyclic voltammetry, electrochemical impedance spectroscopy, and N2 adsorption-desorption isotherms.Antibodies present in serum samples immunologically reacted with TES, and then were quantified by using a second antibody labeled with horseradish peroxidase (HRP-anti-IgG). HRP catalyzes the reduction from H2O2 to H2O with the subsequent oxidation of catechol (H2Q) to p-benzoquinone (Q). The enzymatic product was detected electrochemically at _100 mV on a modified sputtered gold electrode. The detection limit was 0.10 ng mL−1, and the coefficients of intra- and inter-assay variation were less than 6%, with a total assay time of 20 min. As can be seen, the electrochemical immunosensor is a useful tool for in situ IgG antibodies anti-T. canis determination.

Seroprevalence of Larval Toxocarosis in the Czech Republic.

Larval toxocarosis (LT), a zoonotic disease transmitted by dogs, cats, and other carnivores, is caused by roundworms of the genus Toxocara. Humans become infected by ingesting embryonated eggs of this parasite. In this study, we present data on the seroprevalence of LT in the Czech Republic collected by the National Reference Laboratory for Tissue Helminthoses in 2012-2016. Using enzyme-linked immunosorbent assay, a total of 4428 adults and children with or without clinical symptoms were examined for the presence of IgG antibodies against Toxocara canis excretory-secretory antigens. Of all the persons examined, specific Toxocara antibodies were detected in 160 (3.6%) individuals. There were, however, significant differences between various regions, with seropositivity rates ranging from 1.4 to 7.5%. In comparison to studies from 1998 and 2004, our results suggest a decrease in overall Toxocara seroprevalence in the Czech population, whereby the rates are similar to or even lower than rates in some other Central European countries.

Inhibition of Toxoplasma gondii excretory - secretory antigens on growth of murine Lewis lung carcinoma

To investigate the effect of Toxoplasma gondii excretory-secretory antigens (ESA) on CD4+ CD25+ Foxp3+ T (Treg) cells in mice carrying Lewis lung carcinoma, and examine the inhibitory effect of T. gondii ESA on tumor growth. C57BL/6 mice were randomly assigned into the PBS group (n = 14) and the Lewis group (n = 34). Mice in the Lewis group were subcutaneously injected with 2 × 105 Lewis lung carcinoma cells in the right axilla, while animals in the PBS group were injected with the same volume of sterile PBS. On day 7 post-injection (D7), mice in the PBS group were further divided into the PBS2 group and the PBS2 + ESA group, of 7 mice in each group, and mice in the Lewis group were further divided into the Lewis2 group and the Lewis2 + ESA group, of 17 mice in each group. Then, mice in the PBS2 + ESA group and the Lewis2 + ESA group were intraperitoneally injected with 100 μL of ESA. The mouse spleen coefficient was calculated in each group 7 days post-injection with ESA, and the changes of Treg cell counts and the long-term tumor growth were measured in tumor-bearing mice. The spleen coefficient was significantly greater in the PBS2 + ESA group and the Lewis2 + ESA group than in the PBS2 (0.66% ± 0.09% vs. 0.30% ± 0.02%, P < 0.05) and Lewis2 groups (0.69% ± 0.07% vs. 0.33% ± 0.03%, P < 0.05) 7 days post-treatment with ESA, respectively, and the percentage of splenic Treg cells in splenocytes was significantly lower in the PBS2 + ESA group and the Lewis2 + ESA group than in the PBS2 (1.28% ± 0.14% vs. 2.06% ± 0.07%, P < 0.05) and Lewis2 groups (1.58% ± 0.14% vs. 2.44% ± 0.23%, P < 0.05), respectively. T. gondii ESA treatment caused a delay in tumor growth, and the tumor size was significantly smaller in the Lewis2 + ESA group than in the Lewis2 group (P < 0.05). T. gondii ESA may reduce the proportion of splenic Treg cells in splenocytes and inhibit tumor growth in mice carrying Lewis lung carcinoma.

Resistance against Trichinella spiralis infection in pups delivered by T. spiralis-infected dam

Maternal antibody transmission via placenta and breastmilk are known to confer protection in infants. In this study, we investigated the maternal immunity transmission in pups delivered by rats infected with Trichinella spiralis and assessed the resulting resistance against subsequent parasitic infection. Our results revealed that parasite-specific IgG, IgG1 and IgG2a antibodies were present in pups prior to breastmilk ingestion (pre-milk), in which IgG and IgG1 antibodies persisted until week 8 after birth while parasite-specific IgG2a antibodies only lasted until week 4. After weaning on week 3, pups delivered by T. spiralis-infected dam and subsequently challenge-infected (immune-challenge) were found to possess higher mucosal IgG antibodies than control groups, whereas mucosal IgA levels were not significantly different across all groups. T. spiralis excretory-secretory antigen was discovered to react with pup sera until week 8, correlating with the resistance against parasitic infection which is represented by lessened worm burden. Upon T. spiralis infection at weeks 3 and 8, lower levels of eosinophil responses were detected in immune-challenge pups compared to naïve-challenge pups, indicating correlates of resistances in which ADCC may be involved. Findings from the present study demonstrate that resistances against T. spiralis infection in pups can be acquired by maternally-derived IgG, IgG1 and IgG2a antibody transmission through the placenta and breastmilk from T. spiralis-infected dam, which lasts until week 8.

Lack of evidence for Toxocara infection in Italian myelitis patients.

Acute myelitis is a common neurological manifestation due to different causes, but in about 15-30% of cases its etiology remains unknown (idiopathic myelitis). Myelitis represents the most common manifestation of neurotoxocariasis, the infection of the human nervous system by larvae of the nematode Toxocara spp.; however, despite the high seroprevalence worldwide, its contribution to the burden of disease has not been assessed. We evaluated the presence of antibodies against Toxocara spp. in cerebrospinal fluid (CSF) from a sample of 28 patients with a diagnosis of idiopathic myelitis (N = 20) or encephalomyelitis (N = 8) who attended the Neurological Unit of the University Hospital of Catania, Sicily. Antibodies against Toxocara spp. were measured using a multiplex bead-based assay and Toxocara immunoblot using Toxocara canis excretory secretory antigens. All samples tested negative for the presence of anti-T. canis IgG antibodies. In this series, we found no evidence of a contribution of neurotoxocariasis to the burden of myelitis.

Identification of novel antigens for serum IgG diagnosis of human toxoplasmosis

In the present study, we attempted to identify antigens with high sensitivity and specificity for the serological diagnosis of human toxoplasmosis. We investigated soluble proteins from the tachyzoites of the RH strain of Toxoplasma gondii (T. gondii) and excreted/secreted antigens (ESAs) from the peritoneal protein of T. gondii-infected mice. One-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis revealed that in both soluble tachyzoite antigens and ESAs, the antigens located between 25 and 35 kDa had high diagnostic sensitivity. Further analysis of antigenic specificity revealed that the antigens located between 25 and 35 kDa were specifically recognized by the sera of toxoplasmosis patients, but other parasitic diseases were not. The protein spots between 25 and 35 kDa were selected after two-dimensional electrophoresis of both soluble tachyzoite antigens and ESAs. GRA2, GRA7, and triosephosphate isomerase (TPI) were successfully characterized from the protein spots using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy analysis. We expressed, purified, and evaluated proteins GRA2, GRA7, and TPI. TPI is a novel antigen with potential for the serological diagnosis of toxoplasmosis, and composite recombinant proteins (TPI, GRA2, and GRA7) have great sera diagnostic value for the detection of the disorder.

Clinico-Seroepidemiological Evaluation of Toxocariasis in Asthmatic Egyptian Children

Background: Toxocariasis was recorded as one of the most commonly zoonotic helminthic infections in the world and still a poorly diagnosed disease hence largely unknown either to health professionals and/or the general of Egyptian population. The excretory-secretory antigens of T. canis larvae (TES) are widely used for both the diagnosis and seroepidemiological studies. Aim of the work: The aim of the present study was to assess the relationship between toxocariasis and bronchial asthma in asthmatic children through means of a case-control study in Cairo, Egypt and to determine its relation to epidemiological risk factors, laboratory tests and clinical signs. Patients and Methods: The study included two groups (Group1): asthmatic group included 72 children with confirmed asthmatic bronchitis and (Group 2): non-asthmatic group included 72 children selected randomly as a control group. The sociodemographic data was assessed as risk factors for toxocariasis based on a questionnaire collected from the children’s parents or guardians. The selected cases were subjected to stool analysis to exclude other parasitic infection, CBC for eosinophilia and immunoblot assay for detection of Toxocara canis IgG. Results: Out of 72 asthmatic patients toxocariasis IgG was positive in 16 cases (22.2%), negative in 56 cases (78.6%) compared to 5 positive cases (6.9%), and 67 negative cases (93.1%) in control group. There was a significant relation between positive antiToxocara IgG and asthmatic bronchitis. This study confirmed a significant correlation between the seroprevalence of Toxocara and possible socioepidemiological factors as contact with pets, geophagia and residence. Conclusion: Rising knowledge of toxocariasis will enable pediatricians to consider it as a cause of asthmatic bronchitis in their patients, initiate appropriate treatment, and educate patients and parents on how to avoid becoming infected

Clinico-Seroepidemiological Evaluation of Toxocariasis in Asthmatic Egyptian Children

Background: Toxocariasis was recorded as one of the most commonly zoonotic helminthic infections in the world and still a poorly diagnosed disease hence largely unknown either to health professionals and/or the general of Egyptian population. The excretory-secretory antigens of T. canis larvae (TES) are widely used for both the diagnosis and seroepidemiological studies. Aim of the work: The aim of the present study was to assess the relationship between toxocariasis and bronchial asthma in asthmatic children through means of a case-control study in Cairo, Egypt and to determine its relation to epidemiological risk factors, laboratory tests and clinical signs. Patients and Methods: The study included two groups (Group1): asthmatic group included 72 children with confirmed asthmatic bronchitis and (Group 2): non-asthmatic group included 72 children selected randomly as a control group. The sociodemographic data was assessed as risk factors for toxocariasis based on a questionnaire collected from the children’s parents or guardians. The selected cases were subjected to stool analysis to exclude other parasitic infection, CBC for eosinophilia and immunoblot assay for detection of Toxocara canis IgG. Results: Out of 72 asthmatic patients toxocariasis IgG was positive in 16 cases (22.2%), negative in 56 cases (78.6%) compared to 5 positive cases (6.9%), and 67 negative cases (93.1%) in control group. There was a significant relation between positive antiToxocara IgG and asthmatic bronchitis. This study confirmed a significant correlation between the seroprevalence of Toxocara and possible socioepidemiological factors as contact with pets, geophagia and residence. Conclusion: Rising knowledge of toxocariasis will enable pediatricians to consider it as a cause of asthmatic bronchitis in their patients, initiate appropriate treatment, and educate patients and parents on how to avoid becoming infected

Kinetics Evaluation of IgM and IgG Levels in the Mice Infect-ed with Trichinella spiralis Experimentally Using ES Antigens from Different Developmental Stages of the Parasite

To assay the Trichinella-specific IgM and IgG antibody responses during the early stage of infection, serum was collected from mice infected with the muscle larvae (ML) of T. spiralis (ISS534) at different dpi (days post infection) up to 60 days. The levels of IgM and IgG antibodies in serum were measured by ES antigens from different stage of T. spiralis using the ELISA method in Shanghai, China in 2017. The anti-Trichinella IgM and IgG could be detected by ES antigens from the adult three days worm (Ad3) as early as 5 dpi and 8 dpi, respectively. ES antigens from the mixture of adult six days worm & new born larvae (Ad6+NBL) was similar to Ad3. When antibodies were detected by these two antigens, the levels of IgM peaked at 14 dpi and then declined from 15 dpi to 60 dpi; the IgG peaked at 20 dpi, and gradually declined, however, higher detection levels were maintained until 60 dpi. Ad3 ES antigens showed more antigenicity than Ad6+NBL ES on titer detection of IgM and IgG antibodies, and the production of Ad3 ES is easier. In terms of early diagnosis, these two antigens are better than the ML ES antigens of T. spiralis, which antibodies could not be detected before 20dpi. Ad3 ES antigens might be good candidate for the early diagnosis of trichinellosis or the mixture of Ad3 and Ad6+NBL ES might be used.

Canine Brugia malayi microfilarial excretory/secretory protein-based antibody assay for the diagnosis of brugian filariasis in dogs.

Brugian filariasis is reported in dogs in Kerala, India. Antibody detection kits are not available worldwide, for detection of Brugian filariasis in dogs. A study was carried out to develop Indirect plate ELISA using excretory secretory antigen isolated from canine brugian microfilariae and compare the sensitivity and specificity with that of blood smear examination. Identification of microfilariae was done by acid phosphatase staining using Naphthol AS-TR method and Polymerase Chain Reaction for Hha 1 repeat sequence. The microfilariae were identified as Brugia malayi. Isolation of brugian microfilariae from canine blood was done by gradient centrifugation method. The isolated microfilariae were maintained in RPMI-1640 media. The pooled media was then concentrated to obtain excretory secretory protein (ESP). This ESP was used to develop Indirect ELISA. The sensitivity and specificity of the plate ELISA developed was 84 and 100 per cent respectively when compared with blood smear examination. This is the first report of successful isolation of ESP from Brugia malayi microfilariae from dogs and standardization of plate ELISA using the antigen.

Toxoplasma gondii excreted-secreted antigens suppress Foxp3 via PI3K-AKT-mTOR signaling pathway.

Toxoplasma gondii excreted-secreted antigens (ESA) cause spontaneous abortion or fetal teratogenesis during the pregnancy in mice, especially in the early stage. Those adverse pregnancy outcomes are due to the deficit in regulatory T cells (Tregs). Forkhead box P3 (Foxp3), a critical transcription factor, modulates Tregs differentiation and its function. Besides, phosphatidylinositol 3-kinase-protein kinase B-mammalian target of rapamycin (PI3K-AKT-mTOR) signaling network is implicated in interfering with Foxp3 induction. We previously demonstrated that ESA diminished the number of Tregs and inhibited its function. And ESA suppressed Foxp3 expression via the attenuation of transforming growth factor β RII/Smad2/Smad3/Smad4 pathway. The current study aimed to investigate whether the PI3K-AKT-mTOR signaling network is involved in Foxp3 downregulation induced by ESA. We found that ESA upregulated PI3K, P-AKT, mTOR, and P-mTOR. Knockdown of PI3K cooperated with ESA to restore Foxp3 expression mediated by ESA. This suppressive role of ESA on Foxp3 expression was abrogated by AKT inhibitor. In addition, neutralization of Toll-like receptor 4 could restore the expression of Foxp3, PI3K, and its downstream effectors induced by ESA. Collectively, the findings indicated that ESA inhibited Foxp3 expression via the upregulation of PI3K-AKT-mTOR signaling pathway.

A Survey on the Adjuvant Role of Naloxone Alone or Combined with Alum in Vaccination Against Fasciolosis in BALB/c Mice.

Fasciolosis is a zoonotic parasitic diseaseimposinga heavy load of livestock losses worldwide. We aimed to evaluate immune-stimulatory effects of naloxone (NLX), an opioid receptor antagonist, in combination with alum in mice vaccinated with excretory-secretory antigens (E/S) of Fasciola hepatica. 8-week-old female BALB/c mice were subcutaneously vaccinated using E/S antigens of F. hepatica. Experimental groups (14 mice per group) included: vaccine (E/S antigen), alum vaccine (E/S antigen plus alum), NLX vaccine (E/S antigen plus NLX), and alum-NLX vaccine (E/S antigen plus a mixture of alum-NLX). The control group was infused with PBS. Lymphocyte proliferation and the levels of IFN-γ, IL-4, IgG2a, IgG1, and total IgG were measured. Mice vaccinated with NLX or alum-NLX adjuvants showed significantly higher rates of lymphocyte proliferation, IFN-γ, total IgG, and IgG2a levels. The mice that were injected with alum showed a significantly higher concentration of IL-4. Ratios of IFN-γ/Il-4 and IgG2a/IgG1 were significantly higher in the NLX and alum-NLX groups in comparison with the groups vaccinated either with alum or without any adjuvant. A significantly higher protection rate (62.5%) was seen in mice vaccinated with the alum-NLX adjuvant compared to the other groups. NLX can be effective in conferring cellular immunity and protection against F. hepatica. It is recommended to consider this agent as a potential adjuvant in vaccines against fasciolosis.