Comparative evaluation of an indirect ELISA test for diagnosis of swine cysticercosis employing antigen from Taenia solium and Taenia crassiceps metacestodes

Taenia solium cysticercosis is still a serious public health problem in several countries where poverty and lack of hygiene favor transmission. Because pigs are the primary intermediate hosts, prevalence of porcine cysticercosis is a reliable indicator of active transmission zones. Serological diagnostic methods are important tools for epidemiological studies since they can be applied to living animals on a large scale. Four antigen preparations (cyst fluid and crude) from T. solium and T. crassiceps metacestodes were compared for swine cysticercosis diagnosis by indirect ELISA (IE). Twenty-eight serum samples from swine naturally and experimentally infected by cysticerci of T. solium and 56 serum samples from swine reared in commercial herds were tested. Best results of overall sensitivity were obtained by the use of cyst fluid and crude antigen of T. crassiceps metacestode (100 and 96.4%, respectively). Using homologous antigen preparations we have observed higher specificity percentage (98.2% for cyst fluid and 96.4% for crude metacestode T. solium antigen). We concluded that sensitivity is of far more importance than specificity for identification of endemic areas in order to prevent transmission to man. We conclude, therefore, that IE performed with cyst fluid antigen of T. crassiceps metacestode is a better tool for that purpose. (C) 2000 Elsevier B.V. B.V. All rights reserved.

Development and validation of an ELISA for detecting antibodies to Eimeria tenella in chickens

The aim of this study was to develop and validate an ELISA for detecting chicken antibodies to Eimeria tenella. An initial comparison of merozoite and sporozoite antigen preparations revealed few differences in their ability to monitor the onset, kinetics and magnitude of the antibody response suggesting that both antigens would be equally useful for development of an ELISA. Furthermore the cross-reactivity of these antigens with sera from birds infected with chicken Eimeria species was similar. The merozoite antigen was selected for further evaluation because it was easier to prepare. Discrimination between sera from birds experimentally infected with E. tenella and birds maintained in an Eimeria-free isolation facility was excellent. In sera collected from free-range layers and commercial broilers there also appeared to be clear discrimination between infected and uninfected birds. The ELISA should prove useful for monitoring infectivity in vaccination programmes in layer and breeder flocks and for assessing the effectiveness of biosecurity measures in broiler flocks.

Characterization of the antibody response in birds following infection with wild-type and attenuated strains of Eimeria tenella and Eimeria necatrix.

Live vaccines containing attenuated parasite strains are increasingly used to control chicken coccidiosis. In this paper antibody responses elicited by infections with wild-type and attenuated strains of Eimeria tenella and E.necatrix were characterized by immunoblotting and ELISA with homologous and heterologous antisera. Few differences between antisera from birds infected with wild and attenuated strains of E. tenella were evident in immunoblots conducted with merozoite antigen preparations from both E. tenella strains, however the reactivity of sera raised in birds infected with the wild-type strain was noticeably more intense. In ELISAs conducted with merozoite antigen preparations, antisera from birds infected with the wild-type strains of E. tenella and E. necatrix consistently produced a significantly higher (P < 0.05) antibody response than antisera from birds infected with the attenuated strains. Likewise, avidity ELISAs conducted with the E. tenella strains demonstrated that antibodies in birds infected with the wild-type strain were of significantly higher avidity (P < 0.05) than antibodies in birds infected with the attenuated strain. The differences in the antibody responses are probably due to changes in the attenuated strain as a result of selection for precocious development and the less severe tissue damage and inflammation of the intestine resulting from infection with the attenuated strain.

Evaluation of serological tests for the diagnosis of Neospora caninum infection in dogs: Optimization of cut off titers and inhibition studies of cross-reactivity with Toxoplasma gondii

Diagnosis of Neospora caninum infection in dogs is based on serological assays such as the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assays (ELISA). This study evaluated two serological tests (IFAT and ELISA) for the detection of IgG antibodies to N. caninum in 300 serum samples of dogs through the optimization of cut off titers by using the two-graph receiveroperating characteristic (TG-ROC) curve. In addition, the identification of major cross-reactive antigens with Toxoplasma gondii was investigated by inhibition ELISA and immunoblotting (IB) assays. IFAT and ELISA results showed 74% agreement, with a good negative concordance (P-neg=0.83), but a poor positive concordance (P-pos=0.42). The great majority (86%) of sera with positive concordant results (IFAT+/ELISA+) recognized at least two out of three N. caninum immunodominant antigens, particularly the 29-32 and 35-37 kDa bands. Optimization of cut off titers in IFAT and ELISA was performed considering the reactivity to at least two out of three N. caninum immunodominant antigens as infection markers, obtaining a titer of 50 for IFAT and 200 for ELISA. Seropositivity to N. caninuin was significantly associated with T gondii-seropositive samples, particularly in ELISA (55.4%). Inhibition ELISA curves for N. caninum showed a partial heterologous inhibition, indicating some degree of cross-reactivity between N. caninum and T gondii antigens. Inhibition IB assays showed a moderate heterologous inhibition for N. caninum antigens above 45-50 kDa. These results indicate that ELISA should be used critically when crude tachyzoite antigen preparations are employed, due to possible cross-reactivity with other related parasites as T gondii. Also, the cut off dilution of 1:50 in IFAT showed to be the most appropriated for N. caninum serology in dogs. Therefore, we suggest that N. caninum immunodominant antigens, specially the 17 and 29-32 kDa proteins, should be selected markers in serological assays for canine neosporosis. (c) 2006 Elsevier B.V. All rights reserved.

Cryptic species of Paracoccidioides brasiliensis: impact on paracoccidioidomycosis immunodiagnosis

We aimed to evaluate whether the occurrence of cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii, has implications in the immunodiagnosis of paracoccidioidomycosis (PCM). Small quantities of the antigen gp43 were found in culture filtrates of P. lutzii strains and this molecule appeared to be more variable within P. lutzii because the synonymous-nonsynonymous mutation rate was lower, indicating an evolutionary process different from that of the remaining genotypes. The production of gp43 also varied between isolates belonging to the same species, indicating that speciation events are important, but not sufficient to fully explain the diversity in the production of this antigen. The culture filtrate antigen AgEpm83, which was obtained from a PS3 isolate, showed large quantities of gp43 and reactivity by immunodiffusion assays, similar to the standard antigen (AgB-339) from an S1 isolate. Furthermore, AgEpm83 was capable of serologically differentiating five serum samples from patients from the Botucatu and Jundiaí regions. These patients had confirmed PCM but, were non-reactive to the standard antigen, thus demonstrating an alternative for serological diagnosis in regions in which S1 and PS2 occur. We also emphasise that it is not advisable to use a single antigen preparation to diagnose PCM, a disease that is caused by highly diverse pathogens.

Serological Diagnosis of Paracoccidioidomycosis: High Rate of Inter-laboratorial Variability among Medical Mycology Reference Centers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of the double agar gel immunodiffusion test and of the enzyme-linked immunosorbent assay in the diagnosis and follow-up of patients with chronic pulmonary aspergillosis

Coordenação de Aperfeiçoamento do Pessoal de Nível Superior (CAPES)

A collaborative study on larval excretory/secretory antigens of Toxocara canis for the immunodiagnosis of human toxocariasis with ELISA.

Two batches of excretory/secretory (E/S) antigens from second stage larvae of Toxocara canis maintained in vitro were prepared independently in two different laboratories (Zürich and Basel) and analysed in order to obtain information for future efforts to standardize the enzyme-linked immunosorbent assay (ELISA) used for the serodiagnosis of human toxocariasis. SDS-PAGE and "Western-blotting" revealed at least 10 different antigenic components common to the two antigen preparations. However, distinct qualitative and quantitative differences among the two E/S-antigens were observed, since one antigen had a more complex composition than the other. Despite these differences, an accordance of serodiagnosis was obtained in 80% of 25 sera from patients with suspected Toxocara infection tested independently in two different ELISA systems (Basel and Zürich) with the corresponding E/S-antigens. The specificity was 93% as determined (BS-antigen, BS-ELISA) by testing 46 out of 3396 sera from patients with parasitologically proven extra-intestinal helminthic infections. Cross-reactions occurred mainly with sera from patients infected with filariae (5 from 13 cases) exhibiting very high extinction values in their homologous ELISA-system. The reproducibility (intra- and inter-test variations) of two ELISA systems using the corresponding E/S-antigens varied from 5-15%. The results demonstrate that T. canis E/S-antigens may well be applicable for standardization of the ELISA used for the serodiagnosis of human toxocariasis.

Micromethods for the characterization of lipid A-core and O-antigen lipopolysaccharide

Methods for rapid and simple analysis of lipopolysaccharide (LPS) from bacterial whole-cell lysates or membrane preparations have contributed to advancing our knowledge of the genetics of the LPS biogenesis. LPS, a major constituent of the outer membranes in Gram-negative bacteria, has a complex mechanism of synthesis and assembly that requires the coordinated participation of many genes and gene products. This chapter describes a collection of methods routinely used in our laboratory for the characterization of LPS in Escherichia coli and other bacteria.

Association of a transglutaminase-related antigen with intermediate filaments.

A mouse monoclonal antibody, G92.1.2, raised against guinea pig liver transglutaminase (TGase) recognizes an antigen present in primary mouse dermal fibroblasts. A filamentous pattern, bearing remarkable similarity to the vimentin intermediate filament (IF) network, is seen when these cells are fixed and processed for indirect immunofluorescence with the antibody. Double-label immunofluorescence reveals that the antigen reacting with the antibody colocalizes precisely with vimentin IF and that this colocalization is retained after the treatment of fibroblasts with colchicine, which induces a redistribution of the majority of IFs into perinuclear aggregates. These morphological observations are further supported by the finding that the protein reacting with G92.1.2 is retained in IF-enriched cytoskeletal preparations made by using nonionic detergent-containing high ionic strength solutions. Western blots of the IF fraction show that G92.1.2 recognizes a major band of approximately 280 kDa and does not cross react with vimentin. Furthermore, when the antibody is microinjected into live dermal fibroblasts, it causes a collapse of the vimentin IF network in the majority of injected cells. The results suggest that a form of TGase, or a TGase-related antigen, is closely associated with the vimentin IF network of primary cultures of mouse dermal fibroblasts.

Novel formulations for antigen delivery using biodegradable polymers: new approaches for the use of new and established adjuvants

The use of immunological adjuvants has been established since 1924 and ever since many candidates have been extensively researched in vaccine development. The controlled release of vaccine is another area of biotechnology research, which is advancing rapidly with great potential and success. Encapsulation of peptide and protein drugs within biodegradable microspheres has been amongst the most successful of approaches within the past decade. The present studies have focused on combining the advantages of microsphere delivery systems composed of biodegradable polylactide (PLLA) and polylactide-co-glycolide (PLGA) polymers with that of safe and effective adjuvants. The research efforts were directed to the development of single-dose delivery vehicles which, can be manufactured easily, safely, under mild and favourable conditions to the encapsulated antigens. In pursuing this objective non ionic block copolymers (NIBCs) (Pluronics@ LI01 and L121) were incorporated within poly-dl-lactide (PDLA) micorospheres prepared with emulsification-diffusion method. LI0I and L121 served both as adjuvants and stabilising agents within these vaccine delivery vehicles. These formulations encapsulating the model antigens lysozyme, ovalbumin (OVA) and diphtheria toxoid (DT) resulted in high entrapment efficiency (99%), yield (96.7%) and elicited high and sustained immune response (IgG titres up to 9427) after one single administration over nine months. The structural integrity of the antigens was preserved within these formulations. In evaluating new approaches for the use of well-established adjuvants such as alum, these particles were incorporated within PLLA and PLGA microspheres at much lesser quantities (5-10 times lower) than those contained within conventional alum-adsorbed vaccines. These studies focused on the incorporation of the clinically relevant tetanus toxoid (TT) antigen within biodegradable microspheres. The encapsulation of both alum particles and TT antigen within these micropheres resulted in preparations with high encapsulation efficiency (95%) and yield (91.2%). The immune response to these particles was also investigated to evaluate the secretion of serum IgG, IgG1, IgG2a and IgG2b after a single administration of these vaccines. The Splenic cells proliferation was also investigated as an indication for the induction of cell mediated immunity. These particles resulted in high and sustained immune response over a period of 14 months. The stability of TT within particles was also investigated under dry storage over a period of several months. NIBC microspheres were also investigated as potential DNA vaccine delivery systems using hepatitis B plasmid. These particles resulted in micro spheres of 3-5 μm diameter and were shown to preserve the integrity of the encapsulated (27.7% entrapment efficiency) hepatitis B plasmid.

Radiolabelling of antigen and liposomes for vaccine biodistribution studies

A relatively simple and effective method to follow the movement of pharmaceutical preparations such as vaccines in biodistribution studies is to radiolabel the components. Whilst single radiolabelling is common practice, in vaccine systems containing adjuvants the ability to follow both the adjuvant and the antigen is favourable. To this end, we have devised a dual-radiolabelling method whereby the adjuvant (liposomes) is labelled with 3H and the antigen (a subunit protein) with 125I. This model is stable and reproducible; we have shown release of the radiolabels to be negligible over periods of up to 1 week in foetal calf serum at 37° C. In this paper we describe the techniques which enable the radiolabelling of various components, assessing stability and processing of samples which all for their application in biodistribution studies. Furthermore we provide examples derived from our studies using this model in tuberculosis vaccine biodistribution studies. © 2010 by the authors.

Lysosome-associated membrane glycoprotein (LAMP)--preliminary study on a hidden antigen target for vaccination against schistosomiasis

Our previously reported gene atlasing of schistosome tissues revealed transcripts that were highly enriched in the digestive tract of Schistosoma mansoni. From these, we selected two candidates, Sm-LAMP and Sm-NPC2 for testing as vaccine targets. The two molecules were selected on the basis of relatively high expression in the gastrodermis, their potentially important biological function, divergence from homologous molecules of the host and possible apical membrane expression in the gastrodermis. Bacterially expressed recombinant peptides corresponding to regions excluding trans-membrane domains of the selected vaccine targets were used in blinded vaccine trials in CBA mice using alum-CpG as adjuvant. Vaccine trials using the recombinant insoluble Sm-LAMP protein showed 16-25% significant reduction in total worm burden. Faecal egg count reduction was 52% and 60% in two trials, respectively, with similar results for the solubly expressed protein. Liver egg burden was reduced significantly (20% and 38%) with an insoluble recombinant Sm-LAMP in two trials, but not with the soluble recombinant form. Parasite fecundity was not affected by either Sm-LAMP protein preparations in the trials. It is concluded that Sm-LAMP may provide limited protection towards S. mansoni infections but could be used in combination with other vaccine candidates, to provide more comprehensive protection.