Development of PCR/dot blot assay for specific detection and differentiation of taeniid cestode eggs in canids

We report the development of a colourimetric PCR/dot blot assay targeting the mitochondrial gene NADH dehydrogenase subunit 1 (nad1) for differential diagnosis of taeniid eggs. Partial sequences of the cestode nad1 gene were aligned and new primers were designed based on conserved regions. Species-specific oligonucleotide probes (S-SONP) for canine taeniid cestodes were then designed manually based on the variable region between the conserved primers. Specifically, S-SONP were designed for the Taenia crassiceps, T. hydatigena, T. multiceps, T. ovis, T. taeniaeformis, Echinococcus granulosus (genotype 1), E. multilocularis and E. vogeli. Each probe showed high specificity as no cross-hybridisation with any amplified nad1 fragment was observed. We evaluated the assay using 49 taeniid egg-positive samples collected from dogs in Zambia. DNA from 5 to 10 eggs was extracted in each sample. Using the PCR/dot blot assay, the probes successfully detected PCR products from T. hydatigena in 42 samples, T. multiceps in 3 samples, and both species (mixed infection) in the remaining 4 samples. The results indicate that the PCR/dot blot assay is a reliable alternative for differential diagnosis of taeniid eggs in faecal samples.     

Echinococcus multilocularis: purification and characterization of glycoprotein antigens with serodiagnostic potential for canine infection

We show that a conventionally purified glycoprotein component of Echinococcus multilocularis protoscolex, designated as Emgp-89, may be useful as a serodiagnostic antigen for detecting E. multilocularis infection in dogs domesticated in endemic areas. Emgp-89 was obtained from the parasite material by a simple procedure using Con A-agarose and subsequent gel filtration chromatography. The purified fraction showed a molecular weight of >4000 kDa upon gel filtration and reacted with a series of lectins that specifically bind to mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Subsequently, serodiagnostic performance of Emgp-89 was evaluated through enzyme-linked immunosorbent assays (ELISAs) by using sera from normal, domestic dogs and dogs infected with other helminths. Emgp-89 positively reacted with all 16 serum samples from E. multilocularis-infected dogs, thus showing that this antigen is highly sensitive. On the other hand, the specificity of Emgp-89-based ELISA, determined using 41 serum samples from dogs infected with other helminths, was relatively low (83%). As an attempt to improve the specificity of Emgp-89-based ELISA, we pretreated Emgp-89 with proteinase K or sodium periodate, expecting that these treatments would enable discrimination of true positives from false positives. The ELISA value increased after treatment with sodium periodate in most false-positive samples, whereas significant decreases were observed in sera from all dogs infected with E. multilocularis. Further evaluation of this antigen should be performed using sera from dogs infected with closely-related parasites, including taeniid cestodes, which are expected to prove that this serodiagnostic system is sufficiently specific for clinical and field applications.     

Intestinal cestodes of stray dogs in Jordan

Five species of cestodes namely Echinococcus granulosus, Taenia hydatigena, Taenia pisiformis, Taenia ovis and Dipylidium caninum were recovered post mortem from 120 out of 173 stray dogs collected from the 5 governorates of Jordan during the period June 1979 to November 1980. Twenty-five of the examined dogs (14%) were found to be infected with E. granulosus, 79 (46%) with T. hydatigena, 14 (8%) with T. pisiformis and 5 (3%) with T. ovis. Dipylidium caninum was encountered in 33 (19%) of the examined dogs and infection with this parasite was significantly higher in males than in females. The parasites, except for D. caninum which was encountered in the ileum, were almost exclusively recovered from the duodenum and the jejunum. Single, double and triple infections with those cestodes were recorded.     

Molecular and biological characterization of human monoclonal antibodies binding to the spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus

Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.     

The use of excretory and secretory antigens of the scolex of Taenia ovis for the serodiagnosis of infection in dogs

The excretory and secretory antigens from the evaginated scoleces of Taenia ovis were collected for 3 days in vitro, and used in an ELISA test to detect antibodies to T. ovis in the serum of dogs. When tested on sequentially collected sera, diagnostic ELISA values could be detected in many dogs 4 wk after infection, and remained for an average of a further 4 wk after worms were removed from dogs with an anthelmintic. Using an ELISA discriminant value that eliminated all false positives from 70 uninfected laboratory dog sera and from 57 uninfected farm dog sera, 54/62 true positives were found in sera from dogs infected with various numbers of T. ovis for various intervals. Sera from dogs infected with T. hydatigena gave 11/15 false positive reactions, whereas sera from 15 dogs infected with Echinococcus granulosus or 7 dogs infected with T. pisiformis were all negative. For T. ovis the test had a high repeatability, was not greatly influenced by the number of worms carried by the dog and higher titres were correlated with long-standing infections. Approximately 1,000 scoleces could be recovered from each experimentally infected sheep. Using the ELISA test with undiluted antigen and serum diluted 1:40, approximately 10 sera could be tested in duplicate with the excretions and secretions from each T. ovis scolex.     

Antibody responses against natural Taenia hydatigena infection in dogs in Kenya

Antibody responses (IgG) against Taenia hydatigena infection in dogs in Kenya were analysed in ELISA using excretory/secretory products of T. hydatigena scoleces derived from goat cysticercus cysts. Helminth infections of individual dogs were confirmed at autopsy. T. hydatigena worms were found in 89.5% of 143 dogs, and positive anti-T. hydatigena antibody levels were detected in 58.7% of infected dogs. Positive antiscolex antibody levels were detected in 40.0% of Turkana dogs uninfected with T. hydatigena, suggesting previous infection. Antibody was not detected in 34.4% of infected dogs. There was no relationship between individual T. hydatigena worm burdens and absorbance values for sera in ELISA. It was not possible to distinguish between sera from T. hydatigena-infected and uninfected dogs.     

Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface.

We studied the reactivity of 66 anti-Escherichia coli B/r porin monoclonal antibodies (MAbs) with several E. coli and Salmonella typhimurium strains. Western immunoblots showed complete immunological cross-reactivity between E. coli B/r and K-12; among 34 MAbs which recognized porin in immunoblots of denatured outer membranes of E. coli B/r, all reacted with OmpF in denatured outer membranes of E. coli K-12. Extensive reactivity, although less than that for strain B/r (31 of 34 MAbs), occurred for porin from a wild-type isolate, E. coli O8:K27. Only one of the MAbs reacted with porin in denatured outer membranes of S. typhimurium. Even with immunochemical amplification of the Western immunoblot technique, only six MAbs recognized S. typhimurium porin (OmpD), demonstrating that there is significant immunological divergence between the porins of these species. Antibody binding to the bacterial surface, which was analyzed by cytofluorimetry, was strongly influenced by lipopolysaccharide (LPS) structure. An intact O antigen, as in E. coli O8:K27, blocked adsorption of all 20 MAbs in the test panel. rfa+ E. coli K-12, without an O antigen but with an intact LPS core, bound seven MAbs. When assayed against a series of rfa E. coli K-12 mutants, the number of MAbs that recognized porin surface epitopes increased sequentially as the LPS core became shorter. A total of 17 MAbs bound porin in a deep rough rfaD strain. Similar results were obtained with S. typhimurium. None of the anti-E. coli B/r porin MAbs adsorbed to a smooth strain, but three antibodies recognized porin on deep rough (rfaF, rfaE) mutants. These data define six distinct porin surface epitopes that are shielded by LPS from reaction with antibodies.     

Preliminary study of the presence of antibodies against excretory-secretory antigens from protoscoleces of Echinococcus granulosus in dogs with intestinal echinococcosis

The aim of the present study was to analyze the antibody response against excretory-secretory antigens (ES-Ag) from Echinococcus granulosus protoscoleces, using sera from dogs infected with E. granulosus and other helminths. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. Immunochemical characterization was performed by immunoblotting with sera from dogs naturally infected with E. granulosus (n = 12), sera from dogs infected with helminths other than E. granulosus (n = 30), and helminth-free dog sera (n = 20). These findings were compared to those obtained from a somatic extract of protoscoleces (S-Ag). ES-Ag only showed four cross-reacting proteins of 65, 61, 54, and 45-46 kDa. Antigens with apparent masses of 89 and 50 kDa in ES-Ag and of 130 and 67 kDa in S-Ag were identified by sera of dogs infected with E. granulosus only, whereas a protein of 41-43 kDa was recognised by the majority of the sera from dogs with non-echinococcal infection. Employing ELISA to study the same sera, S-Ag revealed higher immunoreactivity than ES-Ag, but also showed higher cross-reactivity levels when sera from dogs with non-echinococcal infection were assayed in immunoblotting.     

Characterization of monoclonal antibodies generated against Norwalk virus GII capsid protein expressed in Escherichia coli

The Norwalk virus (NV) causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. The capsid protein of NV36 (genogroup II, Mexico virus type) was expressed in an Escherichia coli system and ten monoclonal antibodies (MAbs) were generated against it. The reactivity of these MAbs was characterized using enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analysis towards 20 overlapping fragments of the NV36 capsid protein expressed in E. coli. All of the MAbs recognized sequential (continuous) epitopes on the three antigenic regions. Six of the 10 MAbs recognized fragment 2 (equivalent residues 31-70), three MAbs recognized fragment 13 (residues 361-403) and one MAb recognized fragment 7 (residues 181-220), suggesting that the N-terminal domain (residues 1-220) may contain more antigenic epitopes than the C-terminal domain (residues 210-548). Furthermore, two MAbs (1B4 and 1F6) reacted in WB with three purified NV strains (genogroup II) derived from patients' stool samples. It was also found that genogroup I recombinant NV96-908 (genogroup I, KY89 type) could be detected as sensitively as recombinant NV36 (genogroup II) by ELISA with a set of the MAbs produced here.     

Intraspecific variability among NADH dehydrogenase subunit 1 sequences of Taenia hydatigena.

This paper describes intraspecific variability of the partial sequences of the mitochondrial ND1 gene among isolates of Taenia hydatigena from pigs in Poland, Ukraine and Wales. The differences between studied isolates ranged from 0.4 to 5.5%, which exceeds the variability within the same fragment between the different genetic variants of Echinococcus multilocularis and is comparable with the variability between the most closely related strains (G5/G6/G7) of E. granulosus. The biggest difference (5.5%) was found between the geographically most distant Ukrainian and Welsh samples of T. hydatigena while the samples collected from the neighbouring locations in Poland, were most similar to each other.     

Giardia lamblia: RNA translation products

The in vitro translation products of two different human isolates of Giardia lamblia, WB 2x and GS/E, were compared in order to determine common protein constituents and to identify proteins recognized by the infected host. Multiple polypeptides ranging from 20 to 185 kDa were synthesized using a rabbit reticulocyte cell-free translation system and although most were identical some differences were noted. GS/E compared to WB 2x showed different polypeptides of 23.5, 24.5, 26.5, 27.5, 32.5, 33.5, and 41 kDa. Some of these polypeptides were antigenic and were immunoprecipitated with anti-isolate antiserum from experimentally infected humans and gerbils. The sera of humans experimentally infected with isolate GS/M recognized a 24-kDa polypeptide from WB 2x and 23.5- and 24.5-kDa polypeptides from GS/E in vitro translation products. Sera from WB 2x- and GS/E-infected gerbils recognized 74- and 24-kDa polypeptides present in WB 2x translation products and 23.5-, 24.5-, 32.5-, 33.5-, and 74-kDa polypeptides when GS/E in vitro translation products were used. These studies identified both unique and common antigens in two different Giardia isolates and they may be of use in the serologic diagnosis of giardiasis and characterization of Giardia isolates.     

Diagnosis of canine echinococcosis: comparison of coproantigen detection with necropsy in stray dogs and red foxes from northern Jordan

The sandwich enzyme linked immunosorbent assay (ELISA) was used as a diagnostic test for Echinococcus granulosus infection by detecting coproantigens in 94 stray dogs Canis familiaris and eight red foxes (Vulpes vulpes) from northern Jordan. The results were analyzed in relation to actual helminth infection as revealed by necropsy. The infection rate of dogs with E. granulosus was 13.8% with a worm load ranging between 3-> 10,000 per infected dog. In contrast, eight of 13 E. granulosus infected dogs were coproantigen positive (overall sensitivity 61.5%). The sensitivity increased to 87.5% and 100% in dogs harboring > 20 and > 100 worms/dog, respectively. The specificity of coproantigen-ELISA was 91%. The greatest cross-reactivity was found in dogs infected with Dipylidium caninum. The positive and negative predictive values for the coproantigen-ELISA test were 50% and 94.2%, respectively. Thus, a coproantigen negative dog is most probably truly negative for E. granulosus. In contrast, a coproantigen positive dog may not be truly positive for E. granulosus, except if it has a high worm burden of > 100 worms/animal.     

Immunoelectron microscopy of Chlamydia psittaci with monoclonal antibodies.

An immunoelectron microscopic study was performed to determine the distribution of antigenic components on particles of Chlamydia psittaci and infected cells using a number of monoclonal antibodies (MAbs). Of three anti-lipopolysaccharide (LPS) antibodies (4D5, A2 and 4G5), two antibodies (4D5 and A2) reacted with the surface of reticulate bodies (RBs) but not with that of elementary bodies (EBs). The other antibody (4G5) reacted with both EBs and RBs. Examination of infected cells in thin sections revealed that 4D5 and A2 combined with the membranes of both EBs and RBs. These results indicate that each LPS epitope localized at a different position in the chlamydial membrane. Most MAbs directed to protein antigens reacted on the surface of both EBs and RBs though 3E9 specific for the 90 kDa and 50 kDa protein components combined with RBs only.     

Antibody responses of experimentally infected lambs to antigens collected during in vitro maintenance of the adult, metacestode or oncosphere stages of Taenia hydatigena and Taenia ovis with further observations on anti-oncospheral antibodies

The antigenicity and specificity of crude antigens collected during the in vitro maintenance of Taenia hydatigena and T. ovis, excretory/secretory (ES) antigens, were assessed in a peroxidase microenzyme-linked immunosorbent assay (ELISA), using sera from lambs given experimental monospecific infections with T. hydatigena, T. ovis, Echinococcus granulosus or Fasciola hepatica. ES antigens of larval cysts of T. ovis and T. hydatigena were less reactive than those of adult or oncosphere stages. Strong interspecific cros-reactions occurred between all antigen preparations, and these antigens offered no better specificity than crude somatic extracts. IgG1 was the major immunoglobulin detected in sera from lambs experimentally infected with T. ovis or T. hydatigena using antigens prepared from sonicated oncospheres. Discrete peaks of anti-oncospheral antibodies were detected following initial and challenge infections with eggs (whether the homologous or heterologous species), when sera were assayed with a PBS sonicate or an ES antigen from oncospheres. However, when oncospheres solubilised with sodium deoxycholate were used, the antibody response was prolonged and resembled that reported previously when somatic extracts of adult and metacestode stages were used as antigen. The results showed that oncospheres share antigens in common with other life-cycle stages, but also support the notion that they may possess some unique stage-specific antigenic determinants.     

Echinococcus multilocularis coproantigen detection by enzyme-linked immunosorbent assay in fox, dog, and cat populations

A sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Echinococcus multilocularis coproantigens (EM-ELISA) was developed with polyclonal rabbit (solid phase) and chicken egg (catching) antibodies that were directed against E. multilocularis coproantigens and somatic worm antigens, respectively. In experimentally infected dogs and cats, coproantigens were first detectable 6-17 days postinfection (PI) in samples of 8 dogs (worm burdens at necropsy: 6,330-43,200) and from 11 days PI onward in samples of 5 cats infected with 20-6,833 worms. After anthelmintic treatment of 4 dogs and 5 cats at day 20 PI, coproantigen excretion disappeared within 3-5 days. The sensitivity of the ELISA was 83.6% in 55 foxes infected with 4-60,000 E. multilocularis, but reached 93.3% in the 45 foxes harboring more than 20 worms. The EM-ELISA was used in surveys of "normal" dog and cat populations in Switzerland. Among 660 dogs and 263 cats, 5 dogs and 2 cats exhibited a positive reaction. In 2 of these dogs (0.30%) and 1 cat (0.38%), intestinal E. multilocularis infections were confirmed by necropsy, polymerase chain reaction PCR, or both. The specificites of the ELISA in these groups were found to be 99.5% and 99.6%, respectively, if positive ELISA results that could not be confirmed by other methods were classified as "false positive" reactions.     

Monoclonal antibodies of IgA isotype specific for lipopolysaccharide of Salmonella enteritidis: production, purification, characterization and application as serotyping reagents

Hybridomas secreting immunoglobulin A (IgA) monoclonal antibodies (MAbs) against Salmonella enteritidis lipopolysaccharide (LPS) were generated after mucosal immunization of BALB/c mice with heat killed bacteria. Antigen binding properties and specificity of the produced MAbs were studied in ELISA and immunoblotting with purified LPS. Two IgA MAbs agglutinated all Salmonella OD1 strains and all S. enteritidis clinical isolates. MAb 178H11 recognized O:9 antigen of subserogroup OD1 LPS. MAb 177E6/A9 reacted also with OD3 LPS antigen and agglutinated OD3 strains. These data suggest the existence of different O:9 antigen subspecificities, one presented in subgroup OD1 and the other common for OD1 and OD3. Thus the produced IgA MAbs prove to be useful reagents, which could differentiate OD1 and OD3 from OD2 strains.     

Shared and non-shared antigens from three different extracts of the metacestode of Echinococcus granulosus

Hydatid cyst fluid (HCF), somatic antigens (S-Ag) and excretory-secretory products (ES-Ag) of Echinococcus granulosus protoscoleces are used as the main antigenic sources for immunodiagnosis of human and dog echinococcosis. In order to determine their non-shared as well as their shared antigenic components, these extracts were studied by ELISA-inhibition and immunoblot-inhibition. Assays were carried out using homologous rabbit polyclonal antisera, human sera from individuals with surgically confirmed hydatidosis, and sera from dogs naturally infected with E. granulosus. High levels of cross-reactivity were observed for all antigenic extracts, but especially for ES-Ag and S-Ag. Canine antibodies evidenced lesser avidity for their specific antigens than antibodies from human origin. The major antigenic components shared by HCF, S-Ag, and ES-Ag have apparent molecular masses of 4-6, 20-24, 52, 80, and 100-104 kDa, including doublets of 41/45, 54/57, and 65/68 kDa. Non-shared polypeptides of each antigenic extract of E. granulosus were identified, having apparent masses of 108 and 78 kDa for HCF, of 124, 94, 83, and 75 kDa for S-Ag, and of 89, 66, 42, 39, 37, and 35 kDa for ES-Ag.     

Production and characterization of two monoclonal antibodies specific for Plasmopara halstedii

Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races of P. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.     

Humoral response to oligomeric human immunodeficiency virus type 1 envelope protein.

The humoral immune response to human immunodeficiency virus type 1 (HIV-1) is often studied by using monomeric or denatured envelope proteins (Env). However, native HIV-1 Env complexes that maintain quaternary structure elicit immune responses that are qualitatively distinct from those seen with monomeric or denatured Env. To more accurately assess the levels and types of antibodies elicited by HIV-1 infection, we developed an antigen capture enzyme-linked immunosorbent assay using a soluble, oligomeric form of HIV-1IIIB Env (gp140) that contains gp120 and the gp41 ectodomain. The gp140, captured by various monoclonal antibodies (MAbs), retained its native oligomeric structure: it bound CD4 and was recognized by MAbs to conformational epitopes in gp120 and gp41, including oligomer-specific epitopes in gp41. We compared the reactivities of clade B and clade E serum samples to captured Env preparations and found that while both reacted equally well with oligomeric gp140, clade B seras reacted more strongly with monomeric gp120 than did clade E samples. However, these differences were minimized when gp120 was captured by a V3 loop MAb, which may lead to increased exposure of the CD4 binding site. We also measured the ability of serum samples to block binding of MAbs to epitopes in gp120 and gp41. Clade B serum samples consistently blocked binding of oligomer-dependent MAbs to gp41 and, to a slightly lesser extent, MAbs to the CD4 binding site in gp120. Clade E serum samples showed equivalent or greater blocking of oligomer-dependent gp41 antibodies and considerably less blocking of CD4-binding-site MAbs. Finally, we found that < 5% of the antibodies in clade B sera bound to epitopes present only in monomeric gp120, 30% bound to epitopes present in both monomeric gp120 and oligomeric gp140, and 70% bound to epitopes present in oligomeric gp140, which includes gp41. Thus, captured oligomeric Env closely reflects the antigenic characteristics of Env protein on the surface of virions and infected cells, retains highly conserved epitopes that are recognized by antibodies raised against different clades, and makes it possible to detect a much greater fraction of total anti-HIV-1 Env activity in sera than does native monomeric gp120.     

Echinococcus multilocularis: Two-dimensional Western blotting method for the identification and expression analysis of immunogenic proteins in infected dogs

Domesticated dogs are an important potential source of Echinococcus multilocularis infection in humans; therefore, new molecular approaches for the prevention of the parasite infection in dogs need to be developed. Here, we identified and characterized an immunogenic protein of the parasite by using a proteome-based approach. The total protein extracted from protoscoleces was subjected to two-dimensional Western blotting with sera from dogs experimentally infected with E. multilocularis. Two protein spots showed major reactivity to the sera from infected dogs. The N-terminal amino acid sequences of these spots were identical to the deduced amino acid sequence of the product of the putative hsp20 gene. RT-PCR and Western blot analyses revealed that the putative hsp20 gene and its products were expressed in almost all stages of the parasite life cycle. Furthermore, recombinant hsp20 showed specific reactivity to the sera from infected dogs, suggesting that this molecule may facilitate the development of a practical vaccine.