Crude Antigen Research Articles

Molecular identification and characterization of Trichinella spiralis proteasome subunit beta type-7

BackgroundPrevious study showed that Trichinella spiralis proteasome subunit beta type-7 (Tspst) gene is an up-regulated gene in intestinal infective larvae (IIL) compared to muscle larvae (ML), which was screened by using suppression subtractive hybridization (SSH) and confirmed by real-time PCR. Tspst may be related to the larval invasion of intestinal epithelial cells (IECs). The aim of this study was to identify Tspst and to investigate its immune protection against intestinal T. spiralis infection.MethodsThe Tspst gene encoding a 29 kDa protein from T. spiralis infective larvae was cloned, and recombinant Tspst protein (rTspst) was produced in an Escherichia coli expression system. The rTspst was used to immunize BALB/c mice. Anti-rTspst antibodies were used to determine the immunolocolization of Tspst in the parasite. Transcription and expression of Tspst at T. spiralis different developmental stages were observed by RT-PCR and immunofluorescence test (IFT). The in vitro or in vivo immune protection of anti-rTspst serum or rTspst against intestinal T. spiralis infection in BALB/c mice was evaluated.ResultsAnti-rTspst serum recognized the native Tspst protein with 29 kDa in ML crude antigens. Transcription and expression of gene was observed at all T. spiralis different developmental stages (IIL, adult worms, newborn larvae, and ML). An immunolocalization analysis identified Tspst in the cuticle and internal organs of the parasite. An in vitro invasion assay showed that, when anti-rTspst serum, serum of mice infected with T. spiralis or normal mouse serum were added to the medium, the invasion rate of the infective larvae in an IEC monolayer was 25.2%, 11.4%, and 79%, respectively (P < 0.05), indicating that anti-rTspst serum partially prevented the larval invasion of IECs. After a challenge infection with T. spiralis muscle larvae, mice immunized with rTspst conferred a 45.7% reduction in adult worm burden in intestines.ConclusionsIn the present study, Tspst was first identified and characterized. Tspst is an invasion-related protein of T. spiralis IIL and could be considered as a potential vaccine candidate antigen against intestinal T. spiralis infection that merits further study.

Serological Diagnosis and Follow-Up of Human Cystic Echinococcosis: A New Hope for the Future?

Cystic echinococcosis (CE) is an important helminthic zoonotic disease caused by the Echinococcus granulosus complex. In humans, CE is a chronic disease driven by the growth of echinococcal cysts in different organs. Prognosis of this disease depends on multiple factors, including location, number, size, and stage of the cysts, making CE a disease of complex management. CE is usually asymptomatic for years and attracts limited attention from funding organizations and health authorities. For this reason, only experts' recommendations are available but no evidence-based conclusions have been drawn for CE clinical management. One of those pitfalls refers to the lack of evidence to support the use of serological tools for the diagnosis and follow-up of CE patients. In this respect, crude antigens are used to detect specific antibodies in patients, giving rise to false positive results. The advent of molecular techniques allowing the production of recombinant proteins has provided a number of candidate antigens that could overcome the problems associated with the use of crude parasite extracts in the serological assays. In this review, we present the last advances in this field, proposing the use of serology to support cyst stage-specific diagnosis and follow-up.

Indian Major CarpCirrhinus mrigala(Hamilton, 1822) Inoculated with LiveAeromonas hydrophilaShows Dynamic Changes in Specific Immune-Cellular Activities

The Indian major carp, mrigal (Cirrhinus mrigala Ham.), sensitised with live Aeromonas hydrophila, were used to evaluate in vitro cellular immune-effector activities against A. hydrophila antigens. Fishes were maintained in the laboratory in four groups, namely, two control C1 (n = 10) and C2 (n = 10) and two test (treated) groups namely T1 (n = 10) and T2 (n = 10). Test fishes (T1 and T2) were inoculated (100 μl) intra-peritoneally (I/P) with live A. hydrophila at 2 × 107 cfu/ml for sensitisation. At 7th, 15th and 21st day post inoculation (DPI), fish (n = 2) from each tank were sampled, mononuclear cells from spleen were isolated and plated at 2 × 105 cells per well. The stimulation indices (SI) of leucocytes stimulated by crude somatic antigens (SAs) were observed to be much higher in the sensitised fish than in control of all DPI. However, stimulation was observed to be peak at 15 DPI. The in vitro percent cytotoxicity of sensitised leucocytes was observed to be 65.8% at 7 DPI, 38.1% at 15 DPI and 13.21% at 21 DPI. The results showed high specific effector activities by immune cells of Indian major carp mrigal in the initial 15 DPI. The study reveals for the first time the dynamic changes in specific immune-cellular activities of mrigal fish in initial infective phases, experimentally inoculated with live A. hydrophila.

Antigenic profile of heat-killed versus thimerosal-treated Leishmania major using sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Background:Leishmania is a parasitic protozoan of trypanosomatidae family which causes a wide spectrum of diseases ranging from self-healing cutaneous lesions to deadly visceral forms. In endemic areas, field trials of different preparations of Leishmania total antigen were tested as leishmaniasis vaccine. Two preparations of killed Leishmania major were produced In Iran, which were heat-killed vaccine called autoclaved L. major (ALM) and thimerosal-treated freeze-thawed vaccine called killed L. major (KLM). In this study, the protein content of both ALM and KLM were compared with that of freshly harvested intact L. major promastigotes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Materials and Methods:L. major (MRHO/IR/75/ER) from pre-infected Balb/c mice was isolated with modified Novy-MacNeal-Nicolle (NNN) medium and then subcultured in liquid RPMI 1640 medium supplemented with fetal calf serum (FCS) 20% for mass production. Two preparations of KLM and ALM were produced by Razi Vaccine and Serum Research Institute, Iran, under WHO/TDR supervision. Electrophoresis was performed by SDS-PAGE method and the gel was stained by Coomassie brilliant blue dye. The resultant unit bands were compared using standard molecular proteins.Results:Electrophoresis of the two preparations produced many bands from 10 kDa to 100 kDa. KLM bands were much like those of freshly harvested intact L. major.Conclusion:It is concluded that although there are similar bands in the three forms of Leishmania antigens, there are some variations which might be considered for identification and purification of protective immunogens in a total crude antigen, and detection of their stability is essential for the production and marketing of a putative vaccine.

Loiasis in a Japanese Traveler Returning from Central Africa

We encountered a probable case of loiasis in a returned traveler from Central Africa. A 52-year-old Japanese woman presented to our hospital complaining of discomfort in her eyes and skin. She reported having frequently visited Central Africa over many years and having been extensively exposed to the rainforest climate and ecosystem. Although no microfilariae were found in her blood, there was an elevated level of IgG antibodies against the crude antigens of Brugia pahangi, which have cross-reactivity with Loa loa. She was treated with albendazole for 21 days, after which the antigen-specific IgG level decreased and no relapse occurred.

Cross-reactivity of Toxocariasis with Crude Antigen of Toxascaris leonina Larvae by ELISA.

Roundworms of Toxocara canis and Toxascaris leonina are common gastrointestinal helminths of canids over the world. Humans are infected with T. canis larvae through ingestion of infective eggs in contaminated environments or larvae by consumption of raw or uncooked meat or livers. Recently, patients of clinically diagnosed toxocariasis are increasing and require correct diagnosis in Korea. The present study investigated serological cross-reactivity between crude antigens of T. canis (TCLA) and T. leonina (TLLA) larvae. We collected serum specimens from 177 toxocariasis patients who were clinically suspected in the Seoul National University Hospital and 115 healthy controls. An ELISA method for toxocariasis was used to evaluate diagnostic efficacy of TLLA for serodiagnosis of human toxocariasis. The IgG ELISA using TLLA gave 14 (14.3%) positives of 98 TCLA positive specimens among 177 suspected toxocariasis patients. Most of them showed high absorbances with TCLA. In conclusion, there is a partial cross reaction between serum specimens of toxocariasis and TLLA.

Serological diagnosis of canine visceral leishmaniasis in Brazil: systematic review and meta-analysis.

To evaluate the quality and accuracy of serological diagnosis of canine visceral leishmaniasis in the Americas. A systematic review found original studies in the databases MEDLINE, EMBASE and LILACS up to November 2012 and in complementary sources up to February 2013. Studies were evaluated in accordance with QUADAS 2 and STARD parameters and recommended in accordance with GRADE parameters. Meta-analysis was carried out with Meta-DiSc software, using the random-effect model. Two hundred and eighty-four studies were identified, of which 25 met the inclusion criteria, comprising the final synthesis. All but one was conducted in Brazil, and only two were judged to be of good quality. 15 studies involving immuno-enzymatic tests with crude antigens (cELISA), 11 studies on indirect immunofluorescence tests (IFAT) and three on the immunochromatographic dual-path platform (DPP) test were meta-analysed. The combined results for sensitivity and specificity were cELISA: 0.89 (CI 95% 0.87-0.91) and 0.87 (CI 95% 0.86-0.88); IFAT: 0.88 (CI 95% 0.85-0.91) and 0.63 (CI 95% 0.61-0.65); and DPP: 0.83 (CI 95% 0.78-0.88) and 0.73 (CI 95% 0.70-0.75). Enzyme-linked immunosorbent assay with crude antigens and DPP tests have moderate accuracy for the diagnosis of canine visceral leishmaniasis, and the quality of the design, implementation and analysis of validation studies on diagnostic tests for this disease urgently require improvement. The recommendation for use of the evaluated tests is based on evidence that is scarce and restricted to Brazil.

Molecular identification of Trichinella spiralis nudix hydrolase and its induced protective immunity against trichinellosis in BALB/c mice.

BackgroundNudix hydrolases (Nd) is a widespread superfamily, which is found in all classes of organism, hydrolyse a wide range of organic pyrophosphates and has a ‘housecleaning’ function. The previous study showed that Trichinella spiralis Nd (TsNd) bound to intestinal epithelial cells (IECs), and the vaccination of mice with T7 phage-displayed TsNd polypeptides produced protective immunity. The aim of this study was to clone, express and identify the full-length TsNd and to investigate its immune protection against T. spiralis infection.MethodsThe full-length cDNA sequence of TsNd gene encoding a 46 kDa protein from T. spiralis intestinal infective larvae (IIL) was cloned and identified. The antigenicity of rTsNd was analyzed by Western blot. Transcription and expression of TsNd at T. spiralis different stages were observed by RT-PCR and IFT. The levels of the specific total IgG, IgG1 and IgG2a antibodies to rTsNd were determined by ELISA. The immune protection of rTsNd against T. spiralis infection was investigated.ResultsSequence and phylogenetic analysis revealed that TsNd had a nudix motif located at 226-244aa, which had high homology and the closest evolutionary status with T. pseudospiralis. The rTsNd was obtained after expression and purification. Western blot analysis showed that anti-rTsNd serum recognized the native TsNd protein in crude antigens of muscle larvae (ML), IIL, adult worms (AW) and newborn larvae (NBL), and ES antigens of ML. Transcription and expression of TsNd gene was observed in all developmental stages of T. spiralis (ML, IIL, AW and NBL), with high level expression in IIL. An immunolocalization analysis identified TsNd in the cuticle, stichocytes and reproductive organs of the parasite. Following immunization, anti-rTsNd IgG levels were increased, and the levels of IgG1 were more significantly higher than that of IgG2a. After a challenge infection with T. spiralis, mice immunized with the rTsNd displayed a 57.7% reduction in adult worms and a 56.9% reduction in muscle larval burden.ConclusionsTsNd induced a partial protective immunity in mice and could be considered as a novel candidate vaccine antigen against trichinellosis.

Immunoreactive proteins of Saccharopolyspora rectivirgula for farmer's lung serodiagnosis

Saccharopolyspora rectivirgula is the principal cause of farmer's lung disease (FLD). Serodiagnosis is based on immunoprecipitation techniques or enzyme immunoassays with homemade crude antigens and is not standardized. We aimed to produce specific recombinant antigens for the development of a standardized ELISA. We recruited 41 patients and 43 healthy exposed controls from five university hospital pneumology departments in France and Switzerland. S. rectivirgula proteins were extracted, separated by 2D electrophoresis, and subjected to Western blotting, with sera from FLD patients or controls. FLD-specific proteins were identified by MS and were produced as recombinant antigens. The diagnostic performance of ELISA tests using the recombinant antigens was assessed with all the sera from FLD patients and controls. We identified 25 FLD-specific proteins, some of which play important roles in transport, nutrition, or virulence. We produced 17 of these proteins as recombinant antigens and assessed their suitability for inclusion in the ELISA test. A combination of three of these proteins (SR1FA, SR17, and SR22) proved remarkably effective at discriminating between patients and controls, with a sensitivity of 83% and a specificity of 77%. The recombinant antigens produced in this study constitute a major step toward the improvement of diagnostic performance and the standardization of FLD serodiagnosis.

Characterization of a Trichinella spiralis 31 kDa protein and its potential application for the serodiagnosis of trichinellosis

The Trichinella spiralis 31kDa protein (Ts31) was screened from the excretory-secretory (ES) proteins of muscle larvae (ML) by immunoproteomics using serum from mice infected with T. spiralis at 18 days post infection (dpi). The aim of this study was to characterize the Ts31 protein and to evaluate the potential of the recombinant Ts31 protein (rTs31) for serodiagnosis of human trichinellosis. Ts31 gene was cloned and rTs31 was produced in an E. coli expression system. An anti-rTs31serum recognized the native protein migrating in a 25–55kDa range by Western blotting of ML crude or ES antigens. Expression of Ts31 gene was observed at all developmental stages of T. spiralis (adult worms, newborn larvae, pre-encapsulated larvae and ML). An immunolocalization analysis identified Ts31 in the cuticle and stichocytes of the parasite. The sensitivity of rTs31-ELISA and ES antigen ELISA for detecting anti-Trichinella IgG antibodies in sera of patients with trichinellosis was 97.83% (45/46) and 86.78% (39/46), respectively (P>0.05); The specificity of rTs31-ELISA was 99.13% (114/115), which was significantly higher than 85.22% (98/115) of ES antigen ELISA (P<0.01). The rTs31 protein of T. spiralis could be considered as a potential diagnostic antigen for trichinellosis.

Application of crude and recombinant ELISAs and immunochromatographic test for serodiagnosis of animal trypanosomosis in the Umkhanyakude district of KwaZulu-Natal province, South Africa.

A total of 231 serum samples were collected from sheep (n=9), goats (n=99) and cattle (n=123) in northeastern KwaZulu-Natal, South Africa. Trypanosome infection was detected using Trypanosoma brucei brucei crude antigen (TbbCA) and T. congolense crude antigen (TcoCA) ELISA assays. Recombinant antigen (T. evansi GM6 which consisted of 4 repeat domains, TeGM6-4r) ELISA and immunochromatographic test (ICT) were also used. Crude antigen ELISA, TeGM6-4r-ELISA and ICT detected 27.3%, 29% and 19.9% of trypanosome seropositive samples, respectively. Trypanosome infection prevalence in cattle and goats was 35.8–46.3% and 0–9.1%, respectively. Out of 9 sheep serum samples, 2–4 sera (22.2–44.4%) were positive. The detection performance of crude and recombinant antigen ELISAs was relatively similar (K=0.6–0.7); both are recommended for reference diagnosis and large scale epidemiological surveys. There is potential application for ICT in on-site diagnosis, but its sensitivity should be improved.

Immune response assessment of inactivated Newcastle disease virus liposomal-based vaccine

Liposomes were evaluated as an alternative carrier to deliver inactivated Newcastle disease virus (NDV). The ability of a liposomal-based NDV vaccine to activate both cell-mediated immunity (CMI) and humoral immunity against NDV was assessed and compared with conventional NDV vaccines. Birds were assigned to 4 groups and received either no vaccine (control), NDV entrapped into liposomes (LN-NDV), NDV with oil adjuvant (oil-NDV), or live attenuated NDV (live-NDV). All birds were stimulated after 40 days of vaccination i&lt;em&gt;n vivo&lt;/em&gt; by an intravenous injection of inactivated NDV crude antigen, which is considered an &lt;em&gt;in vivo&lt;/em&gt; NDV-specific stimulation of the chicken immune system. After vaccination and&lt;em&gt; in vivo&lt;/em&gt; stimulation, serum samples were collected for NDV-specific antibody response evaluation by a hemagglutination inhibition test (HI) and an enzyme-linked immune sorbent assay (ELISA). The CMI and humoral immunity were evaluated by a measurement of the chicken interferon gamma and specific antibody response in the serum, respectively. Conventional NDV vaccines were able to stimulate a strong humoral and CMI response. Although the newly tested vaccine induced a weak NDV-specific antibody response after vaccination, the response was highly up-regulated, several folds above the protective level, after in vivo stimulation. All NDV vaccine formulas were able to induce a CMI response after vaccination at variable time points. This study revealed that a liposomal NDV-based vaccination in this experimental model tends to induce CMI and can only be beneficial in priming vaccinated birds to promote a strong anti-body response to later NDV exposure.

Characterisation of a plancitoxin-1-like DNase II gene in Trichinella spiralis.

BackgroundDeoxyribonuclease II (DNase II) is a well-known acidic endonuclease that catalyses the degradation of DNA into oligonucleotides. Only one or a few genes encoding DNase II have been observed in the genomes of many species. 125 DNase II-like protein family genes were predicted in the Trichinella spiralis (T. spiralis) genome; however, none have been confirmed. DNase II is a monomeric nuclease that contains two copies of a variant HKD motif in the N- and C-termini. Of these 125 genes, only plancitoxin-1 (1095 bp, GenBank accession no. XM_003370715.1) contains the HKD motif in its C-terminus domain.Methodology/Principal FindingsIn this study, we cloned and characterised the plancitoxin-1 gene. However, the sequences of plancitoxin-1 cloned from T. spiralis were shorter than the predicted sequences in GenBank. Intriguingly, there were two HKD motifs in the N- and C-termini in the cloned sequences. Therefore, the gene with shorter sequences was named after plancitoxin-1-like (Ts-Pt, 885 bp) and has been deposited in GenBank under accession number KF984291. The recombinant protein (rTs-Pt) was expressed in a prokaryotic expression system and purified by nickel affinity chromatography. Western blot analysis showed that rTs-Pt was recognised by serum from T. spiralis-infected mice; the anti-rTs-Pt serum recognised crude antigens but not ES antigens. The Ts-Pt gene was examined at all T. spiralis developmental stages by real-time quantitative PCR. Immunolocalisation analysis showed that Ts-Pt was distributed throughout newborn larvae (NBL), the tegument of adults (Ad) and muscle larvae (ML). As demonstrated by DNase zymography, the expressed proteins displayed cation-independent DNase activity. rTs-Pt had a narrow optimum pH range in slightly acidic conditions (pH 4 and pH 5), and its optimum temperature was 25°C, 30°C, and 37°C.ConclusionsThis study indicated that Ts-Pt was classified as a somatic protein in different T. spiralis developmental stages, and demonstrated for the first time that an expressed DNase II protein from T. spiralis had nuclease activity.

Molecular characterization of a Spirometra mansoni antigenic polypeptide gene encoding a 28.7 kDa protein.

The Spirometra mansoni antigenic polypeptide (SmAP) gene was expressed in Escherichia coli, and its characteristics and value as an antigen for the serodiagnosis of sparganosis were investigated. The recombinant SmAP protein (rSmAP) has the molecular weight of 28.7 kDa. On Western blotting analysis, the rSmAP strongly reacted with the sera of mice infected with spargana, but not with normal sera; the anti-rSmAP serum obviously recognized the 28.7-kDa band in the crude antigens and excretory-secretory (ES) antigens of spargana. The immunofluorescence test (IFT) results showed that the positive staining was observed at different stages of spargana from the infected frogs and mice, but not adult worm of S. mansoni. An immunolocalization analysis identified SmAP in the teguments and parenchymal tissues of spargana. ELISA with rSmAP antigen or sparganum ES antigens were evaluated for the serodiagnosis of sparganosis. The results showed that the sensitivity of rSmAP-ELISA and ES-ELISA was 83.3% (25/30) and 100% (30/30), respectively, for the detection of anti-sparganum IgG antibodies in sera of the experimentally infected mice (P > 0.05), the specificities of both ELISA were 100% (67/67). It is suggested that the rSmAP might be a potential candidate antigen for serodiagnosis of sparganosis.

Phage-displayed peptides as capture antigens in an innovative assay for Taenia saginata-infected cattle.

Bovine cysticercosis is detected during the routine post mortem examination of carcasses by visual inspection (knife and eye method). However, the sensitivity of this procedure is several times lower than immunoassays, even when it is performed by qualified professionals. In the present study, a new generation capture antigens were screened from a phage display peptide library using antibodies from Taenia saginata-infected animals. Eight phage clones were selected, and one, Tsag 3 (VHTSIRPRCQPRAITPR), produced similar results to the T. saginata metacestode crude antigen (TsCa) when used as a capture antigen in an ELISA. The phage-displayed peptides competed with TsCa for binding sites, reducing the reactivity by approximately 30 %. Alanine scanning indicated that proline, arginine, and serine are important residues for antibody binding. Tsag 1 (HFYQITWLPNTFPAR), the most frequent affinity-selected clone, and Tsag 6 (YRWPSTPSASRQATL) shared similarity with highly conserved proteins from the Taeniidae family with known immunogenicity. Due to their epitopic or mimotopic properties, these affinity-selected phages could contribute to the rational design of an ante mortem immunodiagnosis method for bovine cysticercosis, as well as an epitope-based vaccine to interrupt the taeniosis/cysticercosis complex.

Autoantibodies to iron-binding proteins in pigs infested with Sarcoptes scabiei

Despite the availability of effective treatments, Sarcoptes scabiei remains a major health problem in the pig industry. Unsuccessful control of the disease is often due to the lack of reliable detection methods, with current tests relying on skin scrapings and crude antigen ELISAs. A previous analysis of antigens in pig skin scrapings reported that anti-transferrin antibodies were present in S. scabiei infected animals and that this finding might be considered as a useful diagnostic tool. This paper confirms IgG autoantibodies against transferrin, including the first report of IgM autoantibodies, in both naturally and experimentally infected pigs using ELISA and dot blot assays. Autoantibodies were also detected in pigs to ferritin and to a lesser extent lactoferrin. Immunoblotting confirmed the presence of IgG and IgM autoantibodies in mange positive pigs, as well as IgM antibodies to transferrin and albumin in mange negative pigs. These findings suggest the presence of natural autoantibodies to transferrin and albumin in pigs. The development of the IgG autoimmune response may either be a host mechanism for limiting iron to the mite via antibody mediated clearance, the result of host exposure to mite iron-binding homologues or because of a mite-induced antigenic change to host transferrin. Further investigation into the formation of these autoantibodies may provide insights into the importance of iron in scabies infections and the development and perseverance of S. scabiei infections in pigs. The specificity and sensitivity of the anti-transferrin response reinforces its potential in the diagnosis of scabies in pigs.

Elevated antibody reactivity to measles virus NCORE protein among patients with multiple sclerosis and their healthy siblings with intrathecal oligoclonal immunoglobulin G production

BackgroundPatients with multiple sclerosis (MS) and their healthy siblings with the MS oligoclonal bands (OCB) trait, (a hyperimmune condition in form of two or more CSF enriched OCBs) harbor in cerebrospinal fluid (CSF) and serum elevated immunoglobulin G (IgG) titers against measles crude whole-cell antigen. The underlying mechanism resulting in the increased IgG antibody reactivity to measles remains unclear. The response may represent specific IgG reactivity to measles antigens or unspecific auto-antibodies targeting cellular components in the crude whole virus antigens commonly used in detection assays. ObjectiveTo determine the specificity of the measles IgG antibody reactivity by using a purified single nucleoprotein as antigen, thereby minimizing the contribution from auto-antibodies. Study designThe conserved N-terminal portion of the measles nucleocapsid protein (NCORE) was expressed as a specific antigen devoid of human or primate components. Serological analyses were performed on CSF and sera from MS patients, their clinically healthy siblings and healthy unrelated controls. ResultsMS patients demonstrated higher IgG reactivity compared to healthy controls in both CSF (P<0.001) and serum (P<0.001), and compared to siblings in CSF (P<0.001) and serum (P=0.2). Siblings with MS OCB trait showed higher IgG reactivity than healthy controls in CSF (P=0.002) and serum (P=0.01). Comparison between siblings with MS OCB trait and siblings without MS OCB trait showed (P=0.04) for CSF and (P=0.08) for serum. ConclusionThese findings suggest a measles-specific component in the antibody reactivity demonstrated by MS patients and their siblings with the MS OCB trait.

Cytotoxic effects of crude soluble antigen of Eimeria tenella on cultured cells

Coccidiosis is a common intestinal parasitic disease of poultry caused by species of the genus Eimeria. It causes severe economic losses and welfare issues. In this study we investigated the possible cytotoxic and genotoxic effects of the crude soluble antigen (CSA) of Eimeria tenella oocysts on cultured African green monkey cell line (Vero) cells. CSA was prepared from E. tenella oocysts and incubated with cultured cells for two hours. Cytotoxic and genotoxic damages were then quantified using a trypan blue exclusion assay and the comet assay, respectively. The results indicated that exposure to CSA of E. tenella oocysts induces a significant degree of cytotoxicity with tentative evidence of genotoxicity in cultured Vero cells.

Cytokine responses to the anti-schistosome vaccine candidate antigen glutathione-S-transferase vary with host age and are boosted by praziquantel treatment.

BackgroundImproved helminth control is required to alleviate the global burden of schistosomiasis and schistosome-associated pathologies. Current control efforts rely on the anti-helminthic drug praziquantel (PZQ), which enhances immune responses to crude schistosome antigens but does not prevent re-infection. An anti-schistosome vaccine based on Schistosoma haematobium glutathione-S-transferase (GST) is currently in Phase III clinical trials, but little is known about the immune responses directed against this antigen in humans naturally exposed to schistosomes or how these responses change following PZQ treatment.MethodologyBlood samples from inhabitants of a Schistosoma haematobium-endemic area were incubated for 48 hours with or without GST before (n = 195) and six weeks after PZQ treatment (n = 107). Concentrations of cytokines associated with innate inflammatory (TNFα, IL-6, IL-8), type 1 (Th1; IFNγ, IL-2, IL-12p70), type 2 (IL-4, IL-5, IL-13), type 17 (IL-17A, IL-21, IL-23p19) and regulatory (IL-10) responses were quantified in culture supernatants via enzyme-linked immunosorbent assay (ELISA). Factor analysis and multidimensional scaling were used to analyse multiple cytokines simultaneously.Principal FindingsA combination of GST-specific type 2 (IL-5 and IL-13) and regulatory (IL-10) cytokines was significantly lower in 10–12 year olds, the age group at which S. haematobium infection intensity and prevalence peak, than in 4–9 or 13+ year olds. Following PZQ treatment there was an increase in the number of participants producing detectable levels of GST-specific cytokines (TNFα, IL-6, IL-8, IFNγ, IL-12p70, IL-13 and IL-23p19) and also a shift in the GST-specific cytokine response towards a more pro-inflammatory phenotype than that observed before treatment. Participant age and pre-treatment infection status significantly influenced post-treatment cytokine profiles.Conclusions/SignificanceIn areas where schistosomiasis is endemic host age, schistosome infection status and PZQ treatment affect the cellular cytokine response to GST. Thus the efficacy of a GST-based vaccine may also be shaped by the demographic and epidemiological characteristics of targeted populations.

An international collaborative study to determine the prevalence of contagious caprine pleuropneumonia by monoclonal antibody-based cELISA

BackgroundFew serological tests are available for detecting antibodies against Mycoplasma capricolum subsp. capripneumoniae, the causal agent of contagious caprine pleuropneumonia (CCPP). The complement fixation test, the test prescribed for international trade purposes, uses a crude antigen that cross-reacts with all the other mycoplasma species of the “mycoides cluster” frequently infecting goat herds. The lack of a more specific test has been a real obstacle to the evaluation of the prevalence and economic impact of CCPP worldwide. A new competitive ELISA kit for CCPP, based on a previous blocking ELISA, was formatted at CIRAD and used to evaluate the prevalence of CCPP in some regions of Kenya, Ethiopia, Mauritius, Tajikistan and Pakistan in an international collaborative study.ResultsThe strict specificity of the test was confirmed in CCPP-free goat herds exposed to other mycoplasma species of the “mycoides cluster”. Prevalence studies were performed across the enzootic range of the disease in Africa and Asia. Seroprevalence was estimated at 14.6% in the Afar region of Ethiopia, whereas all the herds presented for CCPP vaccination in Kenya tested positive (individual seroprevalence varied from 6 to 90% within each herd). In Mauritius, where CCPP emerged in 2009, nine of 62 herds tested positive. In Central Asia, where the disease was confirmed only recently, no positive animals were detected in the Wakhan District of Afghanistan or across the border in neighboring areas of Tajikistan, whereas seroprevalence varied between 2.7% and 44.2% in the other districts investigated and in northern Pakistan. The test was also used to monitor seroconversion in vaccinated animals.ConclusionsThis newly formatted CCPP cELISA kit has retained the high specificity of the original kit. It can therefore be used to evaluate the prevalence of CCPP in countries or regions without vaccination programs. It could also be used to monitor the efficacy of vaccination campaigns as high-quality vaccines induce high rates of seroconversion.