The MF6p/FhHDM-1 major antigen secreted by the trematode parasite Fasciola hepatica is a heme-binding protein.

Blood-feeding parasites have developed biochemical mechanisms to control heme intake and detoxification. Here we show that a major antigen secreted by Fasciola hepatica, previously reported as MF6p, of unknown function (gb|CCA61804.1), and as FhHDM-1, considered to be a helminth defense molecule belonging to the family of cathelicidin-like proteins (gb|ADZ24001.1), is in fact a heme-binding protein. The heme-binding nature of the MF6p/FhHDM-1 protein was revealed in two independent experiments: (i) immunopurification of the secreted protein·heme complexes with mAb MF6 and subsequent analysis by C8 reversed-phase HPLC and MS/MS spectrometry and (ii) analysis of the binding ability of the synthetic protein to hemin in vitro. By immunohistochemistry analysis, we have observed that MF6p/FhHDM-1 is produced by parenchymal cells and transported to other tissues (e.g. vitellaria and testis). Interestingly, MF6p/FhHDM-1 is absent both in the intestinal cells and in the lumen of cecum, but it can be released through the tegumental surface to the external medium, where it binds to free heme molecules regurgitated by the parasite after hemoglobin digestion. Proteins that are close analogs of the Fasciola MF6p/FhHDM-1 are present in other trematodes, including Clonorchis, Opistorchis, Paragonimus, Schistosoma, and Dicrocoelium. Using UV-visible spectroscopy and immunoprecipitation techniques, we observed that synthetic MF6p/FhHDM-1 binds to hemin with 1:1 stoichiometry and an apparent Kd of 1.14 × 10(-6) M(-1). We also demonstrated that formation of synthetic MF6p/FhHDM-1·hemin complexes inhibited hemin degradation by hydrogen peroxide and hemin peroxidase-like activity in vitro. Our results suggest that MF6p/FhHDM-1 may be involved in heme homeostasis in trematodes.

[Immunochemical properties of the excretory-secretory antigen of Trichinella spiralis].

In vitro cultivation of Trichinella spiralis provided data on the structure of somatic and excretory-secretory antigens of T. spiralis larvae, their immunochemical properties were studied. The findings suggest that work should be continued to produce monoclonal antibodies and to develop highly sensitive and specific ELISA test systems for the diagnosis of human and animal trichinosis.

Targeted glycoproteomic analysis reveals that kappa-5 is a major, uniquely glycosylated component of Schistosoma mansoni egg antigens.

Glycans present on glycoproteins from the eggs of the parasite Schistosoma mansoni are mediators of various immune responses of the human host, including T-cell modulation and granuloma formation, and they are the target of glycan-specific antibodies. Here we have analyzed the glycosylation of kappa-5, a major glycoprotein antigen from S. mansoni eggs using a targeted approach of lectin purification followed by mass spectrometry of glycopeptides as well as released glycans. We demonstrate that kappa-5 has four fully occupied N-glycosylation sites carrying unique triantennary glycans composed of a difucosylated and xylosylated core region, and immunogenic GalNAcß1-4GlcNAc (LDN) termini. Furthermore, we show that the kappa-5 specific IgE antibodies in sera of S. mansoni-infected individuals are directed against the core region of the kappa-5 glycans. Whereas two previously analyzed immunomodulatory egg glycoproteins, IPSE/alpha-1 and omega-1, both express diantennary N-glycans with a difucosylated core and one or two Galß1-4(Fucα1-3)GlcNAc (Lewis X) antennae, the kappa-5 glycosylation appears unique among the major soluble egg antigens of S. mansoni. The distinct structural and antigenic properties of kappa-5 glycans suggest a specific role for kappa-5 in schistosome egg immunogenicity.

Quality of milk of cows in the first lactation vs. presence of anti-Ostertagia antibodies in their milk.

Invasions of gastrointestinal nematodes in dairy cows may affect animals productivity. The most frequently detected internal parasite of dairy cattle is Ostertagia ostertagi. The objective of this study was to determine O. ostertagi invasion extensiveness in selected herds of dairy cattle, with special consideration to cows being in the first lactation, and to analyze the milk yield and contents of basic constituents of milk originating from sero-positive cows. Five herds of dairy cattle (403), with different populations of cows, were selected for the study. Invasion extensiveness in particular herds was determined and ranged from 11.9% to 27.27%. Cows being in the first lactation, the udder milk of which was shown to contain anti-O. ostertagi antibodies, were producing on average 470 kg of milk annually less than cows being in the same lactation period. The analysis of results did not confirm the statistical significance of this difference, likewise it did not demonstrate any statistically significant differences in contents of fat, protein and dry matter. Despite a lack of the statistical significance a producer suffers great economic losses. The conducted study proves that the occurrence of O. ostertagi invasion in herds of dairy cattle is a global problem and that it affects cost-effectiveness of milk production.

Molecular identification and characterization of leucine aminopeptidase 2, an excretory-secretory product of Clonorchis sinensis.

Aminopeptidases serve vital roles in metabolism of hormones, neurotransmission, turnover of proteins and immunological regulations. Leucine aminopeptidases catalyze the hydrolysis of amino-acid residues from the N-terminus of proteins and peptides. In the present study, leucine aminopeptidase 2 (LAP2) gene of Clonorchis sinensis (C. sinensis) was isolated and identified from an adult cDNA library of C. sinensis. Recombinant CsLAP2 was expressed and purified in Escherichia coli BL21. The open reading frame of LAP2 contains 1,560 bp equivalent to 519 amino acids, a similarity analysis showed a relatively low homology with Homo sapiens (19.0 %), Trypanosoma cruzi (18.0 %), Mus musculus (19.3 %), and relatively high homology with Schistosoma mansoni (65.6 %). The optimum condition of rCsLAP2 enzyme activity was investigated using a fluorescent substrate of Leu-MCA at 37 °C and pH 7.5. The K (m) and V (max) values of rCsLAP2 were 18.2 µM and 10.7 µM/min, respectively. CsLAP2 gene expression can be detected at the stages of the adult worm, metacercaria, excysted metacercaria and egg of C. sinensis using real-time PCR, no difference was observed at the stages of the adult worm, metacercaria and egg. However, CsLAP2 showed a higher expression level at the stage of excysted metacercaria than the adult worm (3.90-fold), metacercaria (4.60-fold) and egg (4.59-fold). Histochemistry analysis showed that CsLAP2 was located at the tegument and excretory vesicle of metacercaria, and the tegument and intestine of adult worm. The immune response specific to rCsLAP2 was characterized by a mixed response patterns of Th1 and Th2, indicating a compounded humoral and cellular immune response. The combined results from the present study indicate that CsLAP2 was an important antigen exposed to host immune system, and probably implicated as potential role in interaction with host cells in clonorchiasis.

Production and characterization of antibody against Opisthorchis viverrini via phage display and molecular simulation.

In this study, a key issue to be addressed is the safe disposal of hybridoma instability. Hybridoma technology was used to produce anti-O. viverrini monoclonal antibody. Previous studies have shown that antibody production via antibody phage display can sustain the hybridoma technique. This paper presents the utility of antibody phage display technology for producing the phage displayed KKU505 Fab fragment and using experiments in concomitant with molecular simulation for characterization. The phage displayed KKU505 Fab fragment and characterization were successfully carried out. The KKU505 hybridoma cell line producing anti-O. viverrini antibody predicted to bind to myosin was used to synthesize cDNA so as to amplify the heavy chain and the light chain sequences. The KKU505 displayed phage was constructed and characterized by a molecular modeling in which the KKU505 Fab fragment and -O. viverrini myosin head were docked computationally and it is assumed that the Fab fragment was specific to -O. viverrini on the basis of mass spectrometry and Western blot. This complex interaction was confirmed by molecular simulation. Furthermore, the KKU505 displayed phage was validated using indirect enzyme-linked immunosorbent assays (ELISA) and immunohistochemistry. It is worthy to note that ELISA and immunohistochemistry results confirmed that the Fab fragment was specific to the -O. viverrini antigen. Results indicated that the approach presented herein can generate anti-O. viverrini antibody via the phage display technology. This study integrates the use of phage display technology together with molecular simulation for further development of monoclonal antibody production. Furthermore, the presented work has profound implications for antibody production, particularly by solving the problem of hybridoma stability issues.

Associations of IL13 gene polymorphisms and immune factors with Schistosoma haematobium infection in schoolchildren in four schistosomiasis-endemic communities in Ghana.

BACKGROUND: Schistosomiasis remains a major public health issue with over 90% of the prevalence rates recorded in Sub-Saharan Africa. In this study, the relationships between different interleukin gene polymorphisms (IL-13-591A/G, IL-13-1055C/T, IL-13-1258A/G) and Schistosoma haematobium infection levels were evaluated; as well as the host plasma antibodies and cytokine profiles associated with schistosomiasis infection. METHODOLOGY: A total of 469 school children aged 6 to 19 years from four schistosomiasis-endemic communities in Ghana were involved. Single urine and stool samples were obtained from each pupil, processed via sedimentation and Kato-Katz, and examined via microscopy for Schistosoma and soil-transmitted helminth (STH) eggs. Next, venous blood samples were drawn from 350 healthy pupils, and used to measure antibody and plasma cytokine levels by ELISA. Single nucleotide polymorphisms in the IL-13 gene were genotyped on 71 selected blood samples using the Mass Array technique. PRINCIPAL FINDINGS AND CONCLUSION: The overall prevalence of urinary schistosomiasis was 21.11%. Community-level prevalences were 17.12%, 32.11%, 20.80%, and 15.32% for Asempaneye, Barikumah, Eyan Akotoguah, and Apewosika respectively. Generally, higher S. haematobium infection prevalence and intensity were recorded for participants with genotypes bearing the IL13-1055C allele, the IL13-591A, and the IL13-1258A alleles. Also, higher S. haematobium infection prevalence was observed among participants in the 12-14-year age group with the IL13-1055C, IL13-591A, and IL13-1258A alleles. Interestingly, higher STH prevalence was also observed among participants with the IL13-1055C, IL13-591A, and IL13-1258A alleles. Furthermore, the age-associated trends of measured antibodies and cytokines of S. haematobium-infected school-children depicted a more pro-inflammatory immune profile for pupils aged up to 1l years, and an increasingly anti-inflammatory profile for pupils aged 12 years and above. This work provides insight into the influence of IL-13 gene polymorphisms on S. haematobium, and STH infections, in school-aged children (SAC).

Broad specificity of immune helminth scFv library to identify monoclonal antibodies targeting Strongyloides.

Antibodies have different chemical properties capable of targeting a diverse nature of antigens. Traditionally, immune antibody libraries are perceived to be disease-specific with a skewed repertoire. The complexity during the generation of a combinatorial antibody library allows for a skewed but diverse repertoire to be generated. Strongyloides stercoralis is a parasite that causes strongyloidiasis, a potentially life-threatening disease with a complex diagnosis that impedes effective control and treatment of the disease. This study describes the isolation of monoclonal antibodies against S. stercoralis NIE recombinant protein using an immune antibody phage display library derived from lymphatic filaria-infected individuals. The isolated antibody clones showed both lambda and kappa light chains gene usage, with diverse amino acid distributions. Structural analysis showed that electropositivity and the interface area could determine the binding affinity of the clones with NIE. The successful identification of S. stercoralis antibodies from the filarial immune library highlights the breadth of antibody gene diversification in an immune antibody library that can be applied for closely related infections.

Case Report: First Molecular Diagnosis of Liver Abscesses Due to Fasciola hepatica Acute Infection Imported from Vietnam.

We report a case of Fasciola hepatica liver abscesses in a 67-year-old female returning from a trip to Vietnam. She has been suffering from a fever, right abdominal pain for 4 days, and major eosinophilia. Radiologic investigations showed multiple hypodense confluent abscesses in the right lobe of the liver, complicated by occlusive thrombosis of the right branch of the portal vein. The serological investigation of helminth-elicited eosinophilia showed only a positive serology for F. hepatica. Despite repeated negative stool examinations for any intestinal pathogen, the diagnosis was established by the detection of F. hepatica DNA in stool and pus aspirate samples.

In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA.

Recombinant proteins expressed in E. coli are frequently purified by immobilized metal affinity chromatography (IMAC). By means of this technique, tagged proteins containing a polyhistidine sequence can be obtained up to 95% pure in a single step, but some host proteins also bind with great affinity to metal ions and contaminate the sample. A way to overcome this problem is to include a second tag that is recognized by a preexistent monoclonal antibody (mAb) in the gene encoding the target protein, allowing further purification. With this strategy, the recombinant protein can be directly used as target in capture ELISA using plates sensitized with the corresponding mAb. As a proof of concept, in this study we engineered a Trichinella-derived tag (MTFSVPIS, recognized by mAb US9) into a His-tagged recombinant Fasciola antigen (rFhLAP) to make a new chimeric recombinant protein (rUS9-FhLAP), and tested its specificity in capture and indirect ELISAs with sera from sheep and cattle. FhLAP was selected since it was previously reported to be immunogenic in ruminants and is expressed in soluble form in E. coli, which anticipates a higher contamination by host proteins than proteins expressed in inclusion bodies. Our results showed that a large number of sera from non-infected ruminants (mainly cattle) reacted in indirect ELISA with rUS9-FhLAP after single-step purification by IMAC, but that this reactivity disappeared testing the same antigen in capture ELISA with mAb US9. These results demonstrate that the 6XHis and US9 tags can be combined when double purification of recombinant proteins is required.

Development and Validation of a Copro-Enzyme-Linked Immunosorbent Assay Sandwich for Detection of Echinococcus granulosus-Soluble Membrane Antigens in Dogs.

Cystic echinococcosis (CE) is a parasitic zoonosis caused by the larval stage of the tapeworm Echinococcus granulosus. Detection of the adult stage in the canine definitive host is essential for estimating infection rates, surveillance and monitoring of CE control programs. This study sought to develop and validate a coproantigen sandwich enzyme-linked immunosorbent assay (copro-ELISA), based on antibodies against E. granulosus-soluble membrane antigens (EGMA), that is capable of distinguishing infected and noninfected dogs. Anti-E. granulosus polyclonal immunoglobulin G antibodies were obtained from rabbit antiserum against EGMA. Optimization of the test was performed with 51 positive and 56 negative stool samples of canine echinococcosis. Specificity, sensitivity, cross-reactivity, intra- and inter-assay precision, and over time detection were evaluated. According to the receiver operating characteristic analysis, the diagnostic sensitivity and specificity were 96.1% (CI: 85.9-99.6) and 98.2% (CI: 89.5-100), respectively. Negative and positive predictive values were 96.5% (CI: 91.7-100) and 98% (CI: 94.1-100), respectively. No cross-reactivity with Taenia hydatigena, Dipylidium caninum, or Toxocara canis was observed. Intra- and inter-assay repeatability showed values of less than 15% of the variation coefficient. The over time detection was from 20 to 27 days postinfection with E. granulosus. The copro-ELISA based on EGMA detection offers a simplified in-house development of diagnostic testing. This assay showed high specificity and sensitivity and had no cross-reactivity with other parasites. Further studies and development of this test in a kit format may be useful for the detection of active infection in dogs living in CE endemic regions.

Use of a saliva-based diagnostic test to identify tapeworm infection in horses in the UK.

BACKGROUND: Anthelmintic resistance combined with limited chemotherapeutic options has prompted a change in approaches to control of equine helminth infections. Targeted selective treatment strategies use diagnostics to reduce anthelmintic use by treating individuals with worm burdens or egg shedding levels above a set threshold. While faecal egg count analysis has limitations for informing tapeworm treatment, a commercially available saliva-based diagnostic test accurately diagnoses horses with tapeworm infection. OBJECTIVES: Evaluation of a saliva-based diagnostic test to identify horses naturally infected with tapeworm and assess the impact of using the test to inform anthelmintic administration. STUDY DESIGN: Retrospective longitudinal study. METHODS: Saliva was collected from horses (n = 237) at a UK welfare charity from autumn 2015 to autumn 2016. Horses diagnosed as positive for tapeworm infection using the EquiSal® Tapeworm test were anthelmintic treated according to weight. The number of horses that received anthelmintic treatment based on the test result was compared with an all-group treatment approach and the reduction in anthelmintic usage calculated. Incoming horses were also tested (n = 143) and the information was used to inform quarantine treatments. RESULTS: In autumn 2015, 85% of 237 horses tested received no anthelmintic and the majority (71%) of these remained below the treatment threshold throughout the study. Of the 69 horses that received treatment, seven required treatment following three subsequent tests, while >50% of horses administered with anthelmintic fell below the treatment threshold at the following test. No increase in tapeworm prevalence within the 237 horses was observed during the study despite a substantial reduction in the application of antitapeworm treatments. A total of 41% of incoming horses required anticestode treatment. MAIN LIMITATIONS: Other management practices were not included in the analysis. CONCLUSIONS: Compared with an all-group treatment strategy, the diagnostic-led approach used here considerably reduced application of anticestode anthelmintics. This could reduce selection pressure for anthelmintic resistance.

Carrier-specificity of a phosphorylcholine-binding antibody requires the presence of the constant domains and is not dependent on the unique VH49 glycine or VH30 threonine residues.

Bacterially-produced antibody fragments, such as single-chain Fv (scFv) which comprises the variable regions of the light (VL) and heavy (VH) chains joined together by a short flexible linker, are useful as diagnostic and therapeutic agents. We previously constructed a scFv fragment from a hybridoma antibody (Mab2) but it unexpectedly lacked the unique carrier specificity of the native antibody. Thus, it bound indiscriminately to various phosphorylcholine (PC)-associated antigens, whereas the hybridoma antibody recognized the PC epitope only in the context of the immunizing antigen. Here, we investigated whether the problem was linker-related by changing the linker composition or by deleting it, but these attempts proved futile. Instead, we have constructed a recombinant Fab fragment of the antibody in bacteria that was carrier-specific. This suggests that constant regions are required for the carrier specificity, which presumably helps to mould the fine structure of the antibody combining site or in stabilizing such a structure. Consistent with this global effect is the finding that replacing specific residues in VH with germ-line residues, namely, VH49 glycine and VH30 threonine, both thought previously to be important for the carrier specificity, had no effect on the carrier specificity of the recombinant Fab.

Fasciola spp: Mapping of the MF6 epitope and antigenic analysis of the MF6p/HDM family of heme-binding proteins.

MF6p/FhHDM-1 is a small cationic heme-binding protein which is recognized by the monoclonal antibody (mAb) MF6, and abundantly present in parenchymal cells and secreted antigens of Fasciola hepatica. Orthologs of this protein (MF6p/HDMs) also exist in other causal agents of important foodborne trematodiasis, such as Clonorchis sinensis, Opisthorchis viverrini and Paragonimus westermani. Considering that MF6p/FhHDM-1 is relevant for heme homeostasis in Fasciola and was reported to have immunomodulatory properties, this protein is expected to be a useful target for vaccination. Thus, in this study we mapped the epitope recognized by mAb MF6 and evaluated its antigenicity in sheep. The sequence of the MF6p/FhHDM-1 ortholog from F. gigantica (MF6p/FgHDM-1) was also reported. By means of ELISA inhibitions with overlapping synthetic peptides, we determined that the epitope recognized by mAb MF6 is located within the C-terminal moiety of MF6p/FhHDM-1, which is the most conserved region of MF6p/HDMs. By immunoblotting analysis of parasite extracts and ELISA inhibitions with synthetic peptides we also determined that mAb MF6 reacted with the same intensity with F. hepatica and F. gigantica, and in decreasing order of intensity with C. sinensis, O.viverrini and P. westermani orthologs. On the contrary, mAb MF6 showed no reactivity against Dicrocoelium dendriticum and Schistosoma mansoni. The study of the recognition of peptides covering different regions of MF6p/FhHDM-1 by sera from immunized sheep revealed that the C-terminal moiety is the most antigenic, thus being of potential interest for vaccination. We also demonstrated that the production of antibodies to MF6p/FhHDM-1 in sheep infected by F. hepatica occurs relatively early and follows the same pattern as those produced against L-cathepsins.

Cross-Reactivity Pattern of Asian and American Human Gnathostomiasis in Western Blot Assays Using Crude Antigens Prepared from Gnathostoma spinigerum and Gnathostoma binucleatum Third-Stage Larvae.

Gnathostomiasis is a zoonotic parasitosis endemic in many Asian and some Latin American countries. Most human infections are caused by Gnathostoma spinigerum in Asia and Gnathostoma binucleatum in the Americas, and recently, imported cases have been increasing among travelers returning from endemic regions. Confirmation of the clinical diagnosis relies largely on serologic tests, with a G. spinigerum-antigen-based immunoblot currently being the diagnostic method of choice. However, we repeatedly experienced that sera from patients with clinically suspected American gnathostomiasis gave negative results in this assay. Therefore, we used homologous methods to prepare G. spinigerum- and G. binucleatum-antigen-based immunoblot assays, and evaluated the cross-reactivity of the two assays. The results show incomplete cross-reactivity between the two assays: the G. spinigerum-antigen-based immunoblot apparently only detects Asian gnathostomiasis caused by G. spinigerum, whereas the G. binucleatum-antigen-based immunoblot is apparently capable of detecting American as well as Asian gnathostomiasis.

Comparative diagnostic potentiality of ELISA and dot-ELISA in prepatent diagnosis of experimental Fasciola gigantica infection in cattle.

A glycoprotein (27 kDa) was isolated from crude somatic antigen of Fasciola gigantica by two steps affinity chromatography and was used in early detection of experimental fasciolosis in cattle by indirect ELISA and in dot-ELISA formats. Although, anti-27 kDa antibodies could be detected after 3 weeks post infection (WPI) by dot - ELISA which was one week later than indirect ELISA. The test, dot-ELISA, was more convenient in field application. By the test (dot-ELISA) the infection could be equally detected in animals infected with 100, 200 and 300 metacercariae of F. gigantica with high sensitivity. Further, the antigen (27 kDa) was not found to react with goat sera infected with Paramphistomum epiclitum, which are giving strong reaction to homologous immature and mature fluke antigens of P. epiclitum.

Identification of 38kDa Brugia malayi microfilarial protease as a vaccine candidate for lymphatic filariasis.

A FPLC purified 38kDa protease (Bm mf S-7) isolated from B. malayi microfilarial soluble antigen was identified. It showed pronounced reactivity with sera collected from 'putatively immune' asymptomatic and amicrofilaraemic individuals residing in an endemic area for bancroftian filariasis. Further the immune protective activity of Bm mf S-7 antigen was evaluated in susceptible hosts, jirds (Meriones unguiculatus) against B. malayi filarial infection. The antigen showed 89% cytotoxicity against mf and 87-89% against infective (L3) larvae in in vitro antibody dependent cellular cytotoxicity Assay (ADCC) and in situ micropore chamber methods. Bm mf S-7 immunized jirds after challenge infection showed 81.5% reduction in the adult worm burden. The present study has shown that, the 38kDa microfilarial proteases (Bm mf S-7) could stimulate a strong protective immune response against microfilariae and infective larvae in jird model to block the transmission of filariasis. Analysis of IgG subclasses against Bm mf S-7 revealed a significant increase in IgG2 and IgG3 antibodies in endemic normals. Lymphocyte proliferation to Bm mf S-7 was significantly high in endemic normal group as compared to that in clinical and microfilarial carriers. Significantly enhanced levels of IFN-gamma in the culture supernatant of PBMC of endemic normals followed by stimulation with Bm mf S-7 suggest that the cellular response in this group is skewed towards Th 1 type.

Applying a kinetic method to an indirect ELISA measuring Ostertagia ostertagi antibodies in milk.

Indirect enzyme-linked immunosorbent assays (ELISAs) are frequently run as endpoint ELISAs (e-ELISAs). However, kinetic ELISAs (k-ELISAs) have certain advantages over e-ELISAs. The objective of this study was to understand the relationship between e-ELISA and k-ELISA results. Specifically, to determine whether it was possible to run both k-ELISA and e-ELISA on the same plate and establish an appropriate time interval for k-ELISA measurements. A normalization method for k-ELISA slopes (slope ratio) is proposed. Using an indirect e-ELISA test measuring antibodies against Ostertagia ostertagi in milk from dairy cattle, we found that running a k-ELISA had no effect on optical density ratio results of an e-ELISA on the same plate, and that agreement was very strong at 10, 15, and 28 min, allowing for a reduction in the total processing time for ELISA tests.

Les épreuves immunoenzymatiques indirectes (ELISA) sont fréquemment effectuées comme ELISA avec un point limite (e-ELISA). Toutefois, une épreuve ELISA cinétique (k-ELISA) a certains avantages par rapport à une e-ELISA. L'objectif de la présente étude était de comprendre la relation entre les résultats d'une e-ELISA et ceux d'une k-ELISA. Spécifiquement, déterminer s'il est possible de réaliser simultanément sur la même plaque une épreuve k-ELISA et une épreuve e-ELISA et d'établir un intervalle de temps approprié pour les mesures de k-ELISA. Une normalisation de la méthode pour les pentes des k-ELISA (rapport de pente) est proposée. En utilisant une épreuve e-ELISA indirecte pour mesurer les anticorps dirigés contre Ostertagia ostertagi dans le lait de bovins laitiers, nous avons trouvé que d'effectuer une k-ELISA n'avait aucun effet sur les résultats des ratios de densité optique d'une e-ELISA effectuée sur la même plaque, et que l'accord était très élevé à 10, 15, et 28 min, permettant ainsi une réduction du temps de traitement total pour les épreuves ELISA.(Traduit par Docteur Serge Messier).

Prevalence and seasonality of bulk milk antibodies against Dictyocaulus viviparus and Ostertagia ostertagi in Irish pasture-based dairy herds.

Infections with Dictyocaulus viviparus and Ostertagia ostertagi nematode parasites are of importance to bovine health and production in temperate areas across the world. Losses due to these parasites in dairy herds can be considerable due to decreased milk productivity and fertility. However, information on current epidemiological patterns in Irish dairy herds is limited. Bulk milk samples were collected from a total of 319 dairy farms across the Republic of Ireland. The D. viviparus samples were tested with an ELISA based on recombinant major sperm protein, while the O. ostertagi samples were tested with an ELISA based on crude saline extract, whole worm O. ostertagi antigen. Management data were collected from the farms using a questionnaire. Logistic regression was used to find significant associations between the presence of antibodies against D. viviparus and O. ostertagi and management factors. The overall prevalence of D. viviparus infection was 62.8%, while over 98% of herds had antibodies to O. ostertagi at the specified cut-off. Both D. viviparus and O. ostertagi antibodies were highest in November, which could be explained by the accumulated uptake of larvae through the grazing season. In herds of farmers that dosed their in-calf heifers with anthelmintics were significantly more likely to be positive for antibodies against D. viviparus infection. This study highlights that both D. viviparus and O. ostertagi infections are widespread in dairy herds in Ireland throughout the grazing season.

Production and characterization of a monoclonal antibody against Schistosoma japonicum glutathione S-transferase.

Schistosoma japonicum glutathione S-transferase (GST), expressed from a pGEX plasmid, was isolated from Escherichia coli cells and used to immunize mice in order to generate specific anti-GST monoclonal antibodies. Using a modified immunization and fusion procedure, one stable hybridoma clone secreting an anti-GST antibody (alpha GST-1) was obtained. Milligram quantities of this antibody were produced in vitro in a miniPERM bioreactor and subsequently purified by protein G affinity chromatography. The characteristics of this antibody were investigated by enzyme-linked immunosorbent assays and immunoblotting experiments. The alpha GST-1 antibody was found to react specifically with GST and GST fusion proteins and demonstrated no reactivity with normal E. coli proteins. This monoclonal antibody should be a valuable reagent for tracing the production of GST fusion proteins and possibly for affinity purification of GST fusion proteins.