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Simple Summary: As a global zoonotic parasitic disease, fasciolosis can cause serious economic losses to animal husbandry. The timely detection of Fasciola hepatica (F. hepatica)-infected animals and the adoption of epidemic prevention measures are of great significance. This manuscript provides a new technical method, a biotin–streptavidin signal amplification ELISA (SA-ELISA), based on anti-rFhGST monoclonal and polyclonal antibodies, which can detect circulating antigens in the serum of sheep infected with F. hepatica and achieve early detection. This study explores its application value in immune diagnosis, laying the foundation for the development of serum detection preparations for F. hepatica infection. Fasciolosis is a global zoonotic parasitic disease caused by F. hepatica infection that is particularly harmful to cattle and sheep. A biotin–streptavidin signal amplification ELISA (streptavidin-ELISA/SA-ELISA) based on circulating antigens can allow for the early detection of F. hepatica-infected animals and is suitable for batch detection. It is considered to be a better means of detecting F. hepatica infection than traditional detection methods. In this study, using the serum of sheep artificially infected with F. hepatica, the cDNA expression library of F. hepatica was screened, 17 immunodominant antigen genes of F. hepatica were obtained, and glutathione s-transferase (GST) was selected as the candidate detection antigen. Firstly, the GST cDNA sequence was amplified from F. hepatica, followed by the preparation of recombinant protein GST (rFhGST). Then, monoclonal and polyclonal antibodies against rFhGST were prepared using the GST protein. Afterward, the immunolocalization of the target protein in the worm was observed via confocal microscopy, and it was found that the GST protein was localized in the uterus, intestinal tract, and body surface of F. hepatica. Finally, a double-antibody sandwich SA-ELISA based on the detection of circulating antigens was established. There was no cross-reaction with positive sera infected with Dicrocoelium lanceatum (D. lanceatum), Haemonchus contortus (H. contortus), Neospora caninum (N. caninum), or Schistosoma japonicum (S. japonicum). Forty serum and fecal samples from the same batch of sheep in Nong'an County, Changchun City, Jilin Province, China were analyzed using the established detection method and fecal detection method. The positive rate of the SA-ELISA was 17.5%, and the positive rate of the fecal detection method was 15%. The detection results of this method were 100% consistent with commercial ELISA kits. A total of 152 sheep serum samples were tested in Nong'an County, Changchun City, Jilin Province, and the positive rate was 5.92%. This study laid the foundation for the development of serological detection preparations for F. hepatica infection based on the detection of circulating antigens. [ABSTRACT FROM AUTHOR]

Detection of Schistosoma eggs in stool or urine is known for its low sensitivity in diagnosing light infections. Alternative diagnostics with better sensitivity while remaining highly specific, such as real-time PCR and circulating antigen detection, are progressively used as complementary diagnostic procedures but have not yet replaced microscopy. This study evaluates these alternative methods for the detection of Schistosoma infections in the absence of microscopy. Schistosomiasis presence was determined retrospectively in 314 banked stool and urine samples, available from a previous survey on the prevalence of taeniasis in a community in the Democratic Republic of the Congo, using real-time PCR, the point-of-care circulating cathodic antigen (POC-CCA) test, as well as the up-converting particle lateral flow circulating anodic antigen (UCP-LF CAA) test. Schistosoma DNA was present in urine (3%) and stool (28%) samples, while CCA (28%) and CAA (69%) were detected in urine. Further analysis of the generated data indicated stool-based PCR and the POC-CCA test to be suitable diagnostics for screening of S. mansoni infections, even in the absence of microscopy. A substantial proportion (60%) of the 215 CAA-positive cases showed low antigen concentrations, suggesting that even PCR and POC-CCA underestimated the “true” number of schistosome positives.

Objective: Timely Schistosoma japonicum detection improves outcomes in schistosomiasis. Here, we established a double antibody sandwich ELISA to detect Schistosoma japonicum. Methods: Sj29 polyclonal and monoclonal antibodies were developed and identified. A Sj29 double antibody sandwich ELISA was evaluated. Results: Assay sensitivity for detecting Schistosoma japonicum circulating antigen Sj29 was 76.7% (23/30), 54.5% (18/33) and 50.0% (18/36) in patients with acute, chronic and advanced schistosomiasis. No false positives or cross-reactivity was observed in healthy controls or patients with clonorchiasis, paragonimiasis, or ancylostomiasis, respectively. By contrast, false positives (5.7%) and cross-reactivity (6.5%–10%) were detected using an AWA-ELISA. The circulating antigen positive rates decreased significantly faster than that of the antibody detection after 6 months treatment (22.2%, 4/18 and 88.9%, 16/18). Chi-Square Tests revealed that Sj29 sandwich ELISA had lower sensitivity than AWA indirect ELISA in the detection of S. japonicum infected patients (p

The human parasitic disease Schistosomiasis is caused by the Schistosoma trematode flatworm that infects freshwaters in tropical regions of the world, particularly in Sub-Saharan Africa, South America, and the Far-East. It has also been observed as an emerging disease in Europe, due to increased immigration. In addition to improved therapeutic strategies, it is imperative to develop novel, rapid, and sensitive diagnostic tests that can detect the Schistosoma parasite, allowing timely treatment. Present diagnosis is difficult and involves microscopy-based detection of Schistosoma eggs in the feces. In this context, we present the 3.22 Å resolution crystal structure of the circulating antigen Serine protease inhibitor from S. mansoni (SmSPI), and we describe it as a potential serodiagnostic marker. Moreover, we identify three potential immunoreactive epitopes using in silico-based epitope mapping methods. Here, we confirm effective immune sera reactivity of the recombinant antigen, suggesting the further investigation of the protein and/or its predicted epitopes as serodiagnostic Schistosomiasis biomarkers.

The inadequacy of current diagnostics for the detection of low worm burdens in humans means that schistosomiasis mansoni is more widespread than previously acknowledged. With the inception of mass drug treatment programmes aimed at disease elimination and the advent of human vaccine trials, the need for more sensitive diagnostics is evident. In this review, we evaluate the merits and limitations of the principal diagnostic methods, namely detection of eggs in faeces; anti-schistosome antibodies in serum; parasite-derived proteins and glycans in serum or urine; parasite DNA in blood, faeces or urine. Only in the baboon model, where actual worm burden is determined by portal perfusion, have faecal smear and circulating antigen methods been calibrated, and shown to have thresholds of detection of 10–19 worm pairs. There is scope for improvement in all the four methods of detection, e.g. the identification of single targets for host antibodies to improve the specificity of enzyme linked immunosorbent assay. Despite recent advances in the definition of the schistosome secretome, there have been no comprehensive biomarker investigations of parasite products in the urine of infected patients. Certainly, the admirable goal of eliminating schistosomiasis will not be achieved unless individuals with low worm burdens can be diagnosed. [ABSTRACT FROM AUTHOR]

Abstract Background Schistosomiasis remains a serious public health problem in affected countries, and routine, highly sensitive and cost-effective diagnostic methods are lacking. We evaluated two immunodiagnostic techniques for the detection of Schistosoma japonicum infections: circulating antibody and circulating antigen assays. Methods A total of 1864 individuals (between 6 and 72 years old) residing in five administrative villages in Hubei province were screened by serum examination with an indirect hemagglutination assay (IHA). The positive individuals (titer ≥20 in IHA) were reconfirmed by stool examination with the Kato-Katz method (three slides from a single stool specimen). Samples of good serum quality and a volume above 0.5 ml were selected for further testing with two immunodiagnostic antibody (DDIA and ELISA) and two antigen (ELISA) assays. Results The average antibody positive rate in the five villages was 12.7%, while the average parasitological prevalence was 1.50%; 25 of the 28 egg-positive samples were also circulating antigen-positive. Significant differences were observed between the prevalence according to the Kato-Katz method and all three immunodiagnostic antibody assays (P-value

Babesia canis is the predominant Babesia species in dogs in Europe and is responsible for a severe and fatal disease. An increase in global pet tourism and a widening of the geographic distribution of the tick vector has led to the emergence of infections in areas where previously only imported cases have been reported. Due to the potential for rapid and serious disease progression, direct parasite detection by stained blood smears and light microscopy or DNA-based methods have traditionally been used for the diagnosis of acute infections. This study describes the production of a murine monoclonal antibody (‘mAb BcFIII 7/1/2’) that reacts to a 65 kDa corpuscular epitope present in B. canis -infected erythrocytes and can be used in an ELISA to detect circulating Babesia antigen during acute infections. The sensitivity of the ELISA was 100% (95%CI: 84.5–100) as determined using blood lysate samples from 27 dogs with acute B. canis infections. Sensitivity was reduced to 53.8% in 13 patent Babesia vogeli infections (95%CI: 26.1–79.6) based on the current test design using convalescent serum from a B. canis -infected dog. The specificity was determined to be 86.4% (95%CI: 64–96.4) using 22 samples from healthy canine blood donors. In the course of acute B. canis infections, the ELISA showed a positive result at the same time as a positive PCR result was recorded. This was 24–48 h before parasites could be detected by light microscopy. Convalescent samples collected from 6 B. canis -infected dogs at least 14 days post treatment resulted in negative ELISA reactions. The hyper-acute to acute phase of a B. canis infection represents an emergency situation with high mortality. To increase the chances of survival, a fast and accurate diagnosis and immediate treatment is required. The current study demonstrates the opportunity of an early and specific detection of acute infections by an AgELISA that is potentially translatable to a rapid diagnostic test design. [ABSTRACT FROM AUTHOR]

Adult Schistosoma mansoni parasites have the capacity to degrade ingested host hemoglobin and other host plasma proteins by using a series of gut proteolytic enzymes, including cathepsin B; this enzyme is released to the host intravascular environment during regurgitations of adult worms. Cathepsin B becomes thus a circulating parasite component that has been shown to be specifically recognized as the Sm31 antigen by antibodies present in most S. mansoni infected patients. Taking advantage of this immunological property, we attempted here to immunocapture Sm31 from sera of infected patients using specific polyclonal rabbit antibodies raised against a highly enriched preparation of Sm31 and detect its intrinsic proteolytic activity using a previously described solid-phase procedure called Cysteine Protease Immuno Assay (CPIA). To produce highly specific anti-Sm31/cathepsin B antibodies, cathepsin B (Sm31 or SmCB) was enriched more than 3000-folds from an adult worm preparation using a series of conventional biochemical steps including ion exchange and affinity chromatography. Anti-cathepsin B antibodies were generated by immunizing rabbits with the enriched cathepsin B fraction; these antibodies recognized a band of Mr. ~ 31 kDa in Western-blot (WB) analysis of this fraction and were able to capture, in a modified CPIA procedure, Sm31/SmCB present in sera from infected Venezuelan patients living in low endemic areas for schistosomiasis . CPIA showed 100% sensitivity and 100% specificity; representing a new diagnostic tool to detect circulating Sm31 antigen in actual infections. [ABSTRACT FROM AUTHOR]

Seven patients with active neurocysticercosis (NCC) received an eight days treatment with albendazole and were followed up using computed tomography (CT-scan) and a monoclonal antibody based ELISA for the detection of circulating antigen (Ag-ELISA). Only three patients were cured as was shown by CT-scan and by the disappearance of circulating antigens one month after treatment. After a second course of albendazole therapy, two other patients became seronegative. CT-scan showed the disappearance of viable cysts in all persons who became seronegative whereas patients who were not cured remained seropositive. These preliminary results show that this Ag-ELISA is a promising technique for monitoring the success of treatment of NCC patients because of the excellent correlation between the presence of circulating antigens and of viable brain cysts.

Mouse infection models are described that demonstrate reduction of egg production in Schistosoma haematobium infections and both worm loss and reduced fecundity in S. bovis infections. Neither phenomenum could be shown in S. mansoni infected mice. The immunological basis for these anti-adult responses was inferred by comparison with infections in T-cell deprived mice and by the serum transfer of the ability to reduce a S. bovis worm burden into immunocompromised hosts. Vaccination with irradiation attenuated parasites was also shown to have consequences for the adults of a challenge infections of S. haematobium and S. bovis specifically. Prior vaccination resulted in an abrogation of the anti-fecundity and adult worm elimination that occurred in non-vaccinated similary infected mice. hese models are being used to define the targets and mechanisms involved in anti-adult attrition. A serological assay, quantitation of a circulating antigen (CAA) has been assessed for its ability to measure worm burdens of different species of schistosome in mice. This assay will be used to question whether anti-adult immunity contributes to the pattern of infection with S. mansoni and S. haematobium in man.

Highlights: [•] Improved lateral flow strip assay to diagnose active Schistosoma infection. [•] Assay materials can be stored and shipped worldwide, both at ambient temperature. [•] Assay materials shipped from the Netherlands, evaluated in Africa by local staff. [•] The lateral flow strip assay performed as least as good as the ELISA. [•] Validation of lightweight reader for analysis of the test strips. [Copyright &y& Elsevier]

Abstract: In this study, a sandwich ELISA based on IgY (egg yolk immunoglobulin) and IgM monoclonal antibody (mAb) against excretory-secretory (ES) antigens of Trichinella spiralis muscle larvae was developed for detection of circulating antigens (CAg) in serum from mice infected with T. spiralis. The IgY–IgM sandwich ELISA involved the use of chicken antibody IgY as a capture antibody and mouse IgM mAb 5A7G4 as a detecting antibody. This assay was able to detect as little as 1ng/ml of ES antigens added to normal mouse serum. Two groups of BALB/c mice infected with T. spiralis larvae were used: heavily infected mice (20 mice infected with 300 larvae) and lightly infected mice (20 mice infected with 100 larvae) and 10 normal mice as control. The CAg was detectable as early as 4days post infection (dpi) in the sera from both groups of infected mice, then increased rapidly, reached a peak with detection rate of 100% in heavily infected mice at 10dpi and 80% in lightly infected mice at 22dpi, respectively. The anti-Trichinella IgM antibodies was first detected in 40% of heavily infected mice and 20% of lightly infected mice at 8dpi, and reached a peak positive rate of 100% in heavily infected mice at 16dpi and in lightly infected mice at 26dpi, respectively. The novel assay appears to be sensitive for detection of antigens of T. spiralis and valuable to the early diagnosis of trichinellosis. [Copyright &y& Elsevier]

Abstract: Schistosomiasis is an important zoonosis and some livestock especially bovine and swine play a crucial role on the disease transmission in endemic areas. The gold standard for animal Schistosoma japonicum infection is fecal examination although indirect agglutination assay (IHA) is so far mostly used in field survey and laboratory examination. Lack of sensitivity, poor practicality and high false positivity limit the use of those methods for routine veterinary detection as well as human diagnosis. A novel immunomagnetic bead ELISA based on IgY (egg yolk immunoglobulin) was developed for detection of circulating schistosomal antigen (CSA) in sera of hosts infected with S. japonicum. To assess the application of this method for diagnosis of domestic animal schistosomiasis with urine sample, the immunomagnetic bead ELISA based on IgY (IgY-IMB-ELISA) was employed in the present study to detect CSA in urine of murine schistosomiasis with either light (10 S. japonicum cercariae infection per mouse) or heavy infection (30 S. japonicum cercariae infection per mouse). The results showed that the CSA levels in urine of heavily and lightly infected mice reached a peak in 8 and 10 weeks after infection, respectively, remaining at a constant plateau in both groups by the end of the experiment (14 weeks after infection). The CSA level in urine of heavily infected mice was much higher than that of lightly infected mice from 8 to 14 weeks after infection. The effect of praziquantel treatment on the CSA level in urine of heavily infected mice was also investigated. It was found that the CSA level in urine of heavily infected mice with treatment was much lower than that of untreated mice at 4 weeks post-treatment, although still higher than that of control mice, and then gradually descended to the background level by 8 weeks after treatment. Our findings suggested that the IgY-IMB-ELISA may be an efficient and practical tool in non-invasive diagnosis of schistosome infection based on antigen detection, and evaluation of the efficacy of chemotherapy as well. [Copyright &y& Elsevier]

Abstract: Cysticercosis results from tissue infection with the larval stage of the pig tapeworm Taenia solium. Infection of the brain may cause neurocysticercosis, the most frequent cause of acquired epilepsy in developing countries. Information on human cysticercosis in the Democratic Republic of Congo (DRC) is scarce and outdated. We believe this is the first reported study on human cysticercosis and epilepsy in a village community of DRC. The proportion of villagers seropositive by ELISA for T. solium circulating antigen was 21.6%, the highest figure reported to date. The adjusted prevalence of active epilepsy in the community was 12.7 in 1,000. [Copyright &y& Elsevier]

Abstract: Canine angiostrongylosis is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We developed a sandwich-ELISA based on a monoclonal antibody (mAb Av 56/1/2) and on polyclonal rabbit antibodies directed against Angiostrongylus vasorum adult excretory/secretory – antigen for the detection of circulating serum antigen of A. vasorum. The sensitivity of the test was 95.7% (78.1–99.9, 95% CI) as determined with sera of 23 dogs naturally infected with A. vasorum. The specificity was 94.0% (83.5–98.7, 95% CI) using 50 dog sera (control group) submitted for reasons other than parasitic infections. Potential cross-reactions were investigated with sera of a group of totally 61 dogs with proven infections with Dirofilaria immitis (n =23), Crenosoma vulpis (n =14), Ancylostoma caninum (n =4) or Toxocara canis (n =20). No significant difference was observed concerning the proportion of positive reactions between the control group and the group with proven helminth infections other than A. vasorum. In experimentally inoculated dogs with proven worm burdens of A. vasorum, the proportion of seropositive dogs increased over the first 3 months of infection, starting from 35 days post inoculation (dpi) which was before the onset of larval excretion. Ten weeks post inoculation, 98.6% of the dogs were seropositive, and circulating antigen persisted in two dogs with long-term follow-up over 286 and 356 days, respectively. In contrast, in dogs with a single treatment with imidacloprid/moxidectin at four or 32 dpi, no circulating antigen was observed, while in dogs treated at 88–92 dpi, OD values decreased within 13–34 days. The specific detection of circulating A. vasorum antigen by ELISA represents a valid alternative for reliable diagnosis and for follow-up investigations after anthelmintic treatment. Moreover, the test can be used for mass screening in large epidemiological investigations. [Copyright &y& Elsevier]

We have developed an ELISA that employs monoclonal anti-Toxoplasma SAG1 (p30) as the capture antibody to detect T. gondii circulating antigens in patients' serum samples. Using serum spiked with Toxoplasma soluble and with SAG1 recombinant proteins, the detection limits were 31.25 ng/mL and 62.50 ng/mL, respectively. We obtained positive results in 28% (21/75) and 11% (23/206) of probable active and chronic toxoplasmosis serum samples, respectively. Western blot analysis on pooled antigen-positive serum samples showed antigenic bands of molecular weights 25 and 75 kDa from sera of probable active infection and five antigenic bands ranging in size from 26 to 33 kDa from chronic infection sera. This assay would be useful as an initial serum selection step in developing a Toxoplasma antigen detection test and for characterization studies. [ABSTRACT FROM AUTHOR]

Schistosoma mansoni cathepsin B (Sm31) is a major antigen from adult worms that circulates in the blood of infected patients (Li et al., Parasitol Res 1996; 82: 14–18). An analysis of the Sm31 sequence (Klinkert et al., Mol Biochem Parasitol 1989; 33: 113–122) allowed the prediction of seven hydrophilic regions that were confirmed to be exposed on the surface of a 3D model of Sm31; the species specificity of these regions was checked using BLAST analysis. The corresponding peptides were chemically synthesized in polymerazible forms using the t-Boc technique. Rabbits developed a high humoral response against these peptides as tested by a multiple antigen blot assay; it recognized native Sm31 in crude S. mansoni extracts and as circulating antigen in sera of S. mansoni-infected patients by western blot. Relevant antigenic determinants were located at the N- and C-terminus sequences. Antibodies against these regions recognized the native enzyme in an ELISA-like assay called cysteine protease immuno assay in which the immunocaptured enzyme was revealed by the intrinsic cathepsin B hydrolytic activity of Sm31. The method successfully and specifically detected Sm31 in sera of infected individuals, most of them (83·3%) with light infections, offering a rationale for the development of parasite enzyme capture assays using anti-synthetic peptide antibodies for possible use in the diagnosis of schistoso,iasis. [ABSTRACT FROM AUTHOR]

Abstract: We developed a novel immunomagnetic bead ELISA based on IgY (egg yolk immunoglobulin) for detection of circulating antigen (CA) in sera of mice infected with Schistosoma japonicum. The assay involved the use of chicken polyclonal antibodies IgY against soluble egg antigens (SEA) of S. japonicum as a capture antibody and anti-SEA mouse monoclonal antibody NP28-5B labeled horseradish peroxidase (HRP) as a detecting antibody. Two groups of BALB/c mice infected with S. japonicum cercariae were used: lightly infected mice (infected with 10 S. japonicum cercariae) and heavily infected mice (infected with 30 S. japonicum cercariae). The CA was detectable as early as 4 and 5 weeks after infection in the sera of heavily and lightly infected mice, respectively. The CA levels rose rapidly and reached a peak in 8 weeks after infection and then remained a plateau for at least another 6 weeks in both groups. Moreover, the effect of praziquantel on the CA levels was also investigated. The heavily infected mice were treated with praziquantel and the CA levels in sera increased dramatically in the first week post-treatment and then decreased to the control level by 6 weeks after treatment. The novel assay appears to be sensitive for detection of schistosomal antigenemia and valuable to judge the efficacy of chemotherapy in murine schistosomiasis. [Copyright &y& Elsevier]

Abstract: An oral infection model with Taenia solium whole proglottids was used to study host–parasite relationships and the mechanisms underlying resistance to infection in pigs. In addition, an attempt was made to link the parasitological findings to serological data. Groups of six piglets aged 1, 3 and 5 months were infected and slaughtered 3 months p.i. Circulating antibody and antigen levels were monitored weekly. At autopsy total cyst counts were performed. Although the detailed carcass dissection at necropsy revealed a high variation in the number of cysts, the trend was that the number of viable cysts decreased with the age at which the animals were infected. The kinetics of the antigen levels throughout the course of the infection differed markedly between the three age groups of the experimental infection model. In the younger animals, a fast increase in titres of circulating antigen was observed in most animals, reaching a plateau as early as 2 weeks p.i. Besides its faster increase, antigen levels in pigs infected at younger ages also reached higher levels than in older animals and were associated with weaker antibody responses. Results also demonstrated that a relationship exists between the number of cysts and the titre of circulating antigen. This is promising in view of the development of an assay to quantify the progress of an active T. solium infection and would be a useful tool in epidemiological studies to assess the infection burden and the risk of transmission of the disease. The use of specific antibody-detection assays combined with circulating antigen detection could improve our understanding of this relationship. [Copyright &y& Elsevier]

Abstract: We developed a sandwich ELISA for the detection of circulating Toxoplasma gondii MIC10 antigens. In T. gondii culture supernatant, MIC10 was detected in a growth dependent manner. Mice were infected with a lethal dose of either a virulent RH strain, an avirulent Beverley strain or a sub-lethal dose of a PLK strain of T. gondii. MIC10 appeared 2 days after infection and increased gradually in the sera of RH-infected mice. A detectable but significantly lower amount of MIC10 was observed in the sera of mice infected intraperitoneally with Beverley tachyzoites. In contrast, the MIC10 antigen in mice sera following oral infection with Beverley cysts was below detectable levels during the course of the experiment. In sera of PLK-infected mice, MIC10 was predominantly observed between late acute and early chronic phase. Our data show that the kinetics of circulating MIC10 differs depending on the strain and route of infection. [Copyright &y& Elsevier]

To evaluate a modified rapid ELISA method for detecting CAg during Toxoplasma gondii infection, we analyzed the specificity and sensitivity of the ELISA method by using experimental Toxoplasma infection in rabbits and also tested this method in human samples including 5428 serum, 548 cerebrospinal fluid and two breast milk samples. We prepared PcAb, and used it for rapid one-step sandwich ELISA testing in which an incubation time in the regular ELISA procedure was omitted. This method detected CAg at the concentration of 31.2 ng/mL, and no cross-reaction was found with antigens of protozoa ( Cryptosporidium parvum, Plasmodium falciparum), trematode ( Schistosoma japonicum, Paragonimus sp.) and nematode ( Brugia malayi, Ancylostoma duodenale, Ascaris lumbricoides and Trichinella spiralis). CAg was detected in rabbit serum 3 days after infection, and optical density values reached a peak 9 ∼ 13 days after infection, then declined gradually. Among human serum samples, the positive rate of CAg was 2.11% in cerebral paralysis patients, whereas it was 0.22% or 0.71% in patients without neurological symptoms or in uncomplicated pregnant women. The difference among these three groups was statistically significant ( P < 0.05). The positive rate of cerebrospinal fluid samples from cerebral paralysis patients was 10.58%. There is a statistically significant difference between the positive rates of meat-packing workers and blood donors ( P < 0.01). In the retrospective analysis, CAg was detected in accordance with the onset of clinical symptoms, suggesting that CAg could reflect the clinical course in humans. Together with these results, CAg detected in the modified rapid sandwich ELISA could be a sensitive marker for acute and active infection of T. gondii. [ABSTRACT FROM AUTHOR]

Abstract: An antigen capture or sandwich ELISA (sELISA) was evaluated for the diagnosis of Hypoderma lineatum in cattle under field conditions in northwestern Spain. The kinetics of circulating hypodermin C (HyC) and specific antibodies during the course of natural infestation were determined in a group of 10 Frisian calves. In addition, oesophagi and blood samples were taken from 105 cows at a slaughterhouse in order to compare three methods for the diagnosis of H. lineatum: sandwich ELISA for the detection of the antigen HyC (sELISA), indirect ELISA for the detection of antibodies anti-HyC (iELISA) and the detection of first instars (L1) in the oesophagus. In naturally infested cattle, HyC was present in circulation at low levels during the early and late phases of the infestation. However, in the middle phase, coinciding with the presence of L1 in the oesophagus, two peaks of increased HyC concentration were observed. Specific antibodies increased progressively until the first appearance of larvae in warbles on the back. There was no correlation between antigen or antibody levels and the number of grubs in the back. Prevalence of first instars in the oesophagi of slaughtered cows was 21.9% (23/105). The percentage of cattle that were positive for circulating antigen was slightly higher (24.8%), suggesting the recent destruction of migrating larvae in some animals. However, there was no correlation between the number of L1 and HyC levels. With the iELISA, 79% of the animals were positive to Hypoderma, which means that a high percentage of those animals have been exposed to the parasite but they had no apparent current infestation. The sELISA is a good tool to follow larval development within the host; however, the episodic elevation of HyC levels limits the usefulness of this test for the early diagnosis of Hypoderma under field conditions. [Copyright &y& Elsevier]

Summary: Diethylcarbamazine/albendazole (DEC/ALB) therapy is widely used in mass drug administration (MDA) programmes aimed at eliminating lymphatic filariasis. We studied the effects of repeated annual treatments with DEC/ALB on Wuchereria bancrofti microfilaraemia, filarial antigenaemia and IgG4 antibodies to Bm14 antigen. Fifty-seven subjects with asymptomatic microfilaraemia were treated with one or seven daily doses of DEC/ALB at time zero. All subjects were re-treated with single-dose DEC/ALB 12, 24 and 36 months later. The two treatment groups had comparable pre-treatment microfilaria counts. Multidose treatment cleared microfilaraemia more effectively than single-dose treatment. Filarial antigen levels decreased equally in both treatment groups. Total antigen clearance was observed in 29.6%, 52.0%, 63.6% and 79.5% of subjects at 12, 24, 36 and 48 months. These clearance rates are much higher than those observed in prior treatment trials with DEC or ivermectin. Antibody levels increased 4 weeks after treatment and then slowly decreased in most subjects. Antibody tests turned negative in 20%, 35%, 39.4% and 52.5% of treated subjects at 12, 24, 36 and 48 months post treatment. These results show that the studied parameters decline at different rates and to differing degrees following DEC/ALB treatment. These findings have important implications regarding strategies for monitoring the effects of MDA in populations. [Copyright &y& Elsevier]

Abstract: Background: Diagnosis of extra-pulmonary tuberculosis is often difficult to establish using standard methods. Recently, a 55-kDa mycobacterial antigen was identified in sera of individuals with pulmonary TB using a simple and rapid dot-ELISA based on monoclonal antibody (TB-55 mAb). Here, we have evaluated the application of the dot-ELISA for the detection of target antigen in sera of individuals with extra-pulmonary TB. Methods: The Western blot and indirect immunoperoxidase staining was used to identify the target TB antigen using the TB-55 mAb. The dot-ELISA was used to detect the target antigen in serum samples. Results: The target antigen was identified at 55-kDa molecular weight in serum, ascitic fluid and CSF samples from individuals with extra-pulmonary TB. The purified antigen from these samples showed similar biochemical properties to the previously described antigen. The target antigen was localized in areas without caseous necrosis in lymph tissues. The dot-ELISA detected the target antigen in 90% sera of individuals with extra-pulmonary TB and in 87% sera of individuals with pulmonary TB with a specificity of 97% among control individuals. Conclusion: The detection of the 55-kDa antigen using dot-ELISA can be routinely employed to support clinical diagnosis of extra-pulmonary TB and pulmonary TB. [Copyright &y& Elsevier]

The antibody response and circulating antigen levels in bovine calves, infected experimentally with Fasciola gigantica, were monitored using enzyme-linked immunoelectrotransfer blot (EITB) and sandwich ELISA, respectively. By EITB, the infected calves’ sera recognized the polypeptides in the range of 54–58 kDa as early as 2 weeks post-infection. By 12th week post-infection, the lower two polypeptides of 12 and 8 kDa had disappeared. In sandwich ELISA, the circulating 54 kDa and whole worm antigen of F. gigantica were detected in the sera samples of infected calves as early as 2 weeks post-infection and persisted until the end of experiment (26th week PI). The 54 kDa antigen of F. gigantica appears to be specific and possesses promising immunodiagnostic potential for early prepatent diagnosis of bovine fasciolosis. [Copyright &y& Elsevier]

Three groups of four piglets were experimentally infected with different doses (103, 104 and 105) of Taenia solium eggs whereas a fourth group of two pigs received gravid proglottids. At autopsy 6 months post infection, the two latter pigs were heavily infected with more than 3000 living cysts per kg of muscle. Ten of the 12 other pigs harboured light infections, i.e. between 2 and 107 cysticerci, 42.4% of which were degenerated. The two remaining pigs had no detectable cysts at post mortem examination. Circulating antigens (CA) were detected in the sera of all pigs harbouring living cysticerci using a monoclonal antibody based ELISA. CA were first detected between 2 and 6 weeks post infection and remained present generally throughout the entire observation period even in pigs carrying only five to eight living cysts, although strong fluctuations of the level of CA were observed in some pigs. In animals without living cysts at post mortem CA were only detected for a short period and disappeared presumably when the cysticerci became degenerated. The minimum number of living cysts, which could be detected using this ELISA, was 1. [Copyright &y& Elsevier]

We developed a serodiagnostic test which is based on the agglutination of hybridoma cells. In the presence of specific antigen, agglutination of the fixed and stained cells occurs and can be visualized in analogy to traditional erythrocyte agglutination. The procedures were developed with a murine cell line producing a monoclonal antibody against a schistosome gut protein and sera of patients and mice infected with Schistosoma japonicum. This test is capable of detecting circulating antigen during pre-patency in mice infected with 50 cercariae. Its sensitivity was high with acute schistosomiasis japonica (97%, n = 32) and moderate with chronic cases (75%, n = 57). No positive reactions were obtained with healthy persons (n = 78) or patients infected with other parasites (Chlonorchis sinensis, n = 20; Paragonimus westermani, n = 20; Plasmodium vivax, n = 10) or suffering from lupus erythomatodus (n = 5) or mononucleosis (n = 10). [ABSTRACT FROM AUTHOR]

An antigen capture assay for the detection of circulating hypodermin C was developed for diagnosis of hypodermosis. A murine monoclonal antibody to recombinant hypodermin C was raised using rapid immunization and a one-step hybridization-cloning technique. A highly reactive, specific monoclonal antibody was tested using sera spiked with known quantities of purified, native hypodermin C or with recombinant hypodermin C. Sensitivity of 96.4% and specificity of 95.6% for the antigen capture assay was assessed using a panel of sera from animals unexposed to cattle grubs and from cattle with palpation proven cattle grub infestations. Data from this panel of sera was used to establish the cut-off OD for further testing. The kinetics of circulating hypodermin C was assessed using the assay in three groups of cattle artificially infested with 50, 100 or 200 first instar Hypoderma lineatum. Antigen was first detected approximately 6 weeks after infestation. The amount of antigen detected increased in each group of animals reaching peaks at different times in each group. Levels of antigen fell quickly following arrival of grubs at the back and completion of the molt to second instar. [Copyright &y& Elsevier]

We analysed specific antibody (Ab) and circulating antigen (CAg) profiles along experimental mouse infection using as control a group of mice immunized with intact but dead parasites. Results from this experiment showed an early major CAg peak followed by a larger Ab peak which partially overlaps with other minor CAg peaks. These results suggest that CAg may be a marker of early mouse infection. In order to study the relevance of these findings in humans we similarly analysed by ELISA 148 sera provided by retrospective post-surgical follow-up of 19 patients. Available records showed that 14 patients developed new cysts one to ten years after surgery while no new disease was observed in the other five. Some of the former patients showed CAg as early as two months after surgery while no CAg was observed in the other five patients at any time. In addition, a collection of 38 sera obtained before surgery were similarly tested and five of them showed only CAg, while 18 showed only Ab and 12 sera showed Ab&CAg. These results in humans are consistent with the findings in the mouse experimental model and suggest that CAg may be an early marker of hydatid infection, thus being relevant for post-surgical follow-up. [ABSTRACT FROM AUTHOR]

The effect of circulating antigen (human kappa light chains (LC)) on radioimmunoscintigraphy was examined in a normal rat model. Rats were implanted subcutaneously with kappa (test) and lambda (control) LC coupled to sepharose beads (500 μg LC/ml swollen gel). When I-labelled monoclonal antibody, K-1-21 was injected, clear images of specific antibody localisation to the kappa implant were seen 6 days later. The kappa implant: blood ratio (K:B) was 7.69±0.45, which represented an implant uptake of 2.52±0.20 percent injected dose per gram (%ID/g). When I-K-1-21 was preincubated with I-kappa LC at ratios of up to 1:10 antibody: antigen, (w/w) before injection; or if antigen was given daily by bolus injection or continuously infused via an osmotic pump, very clear specific images of antibody localisation to the kappa implant were still obtained. There was, however, a decline in the absolute uptake of label by the kappa implant. The greatest change was observed following continuous antigen infusion, when kappa implant uptake fell to 1.26±0.1 %ID/g. Nevertheless, the K:B ratio was still maintained at 6.8±0.2, due to increased clearance of antibody from the blood. Thus, in this case, where antigen and antibody might be expected to form small, soluble immune complexes, there was no major deleterious effect on radioimmunoscintigraphy. [ABSTRACT FROM AUTHOR]

Two circulating antigens were detected in the serum of ICR/Timco female mice infected intraperitonealy with tetrathyridia of the cestode Mesocestoides corti Hoeppli, 1925. One circulating antigen appeared by day 2 postinfection (p.i.) and remained in all mice until at least 90 days p.i. A second antigen appeared in the serum on day 14 p.i. and disappeared from all mice by day 28 p.i. Infected mouse serum also contained antibodies against one secretory/excretory antigen and two antigens in crude homogenate, as judged by double diffusion in two dimensions (Ouchterlony). Immune deposits were observed in the kidney tissue of Rockland mice by transmission electron microscopy, and their identity as products of tetrathyridia was confirmed by immunofluorescence. Further studies showed that the main antibody subclass associated with the mesangial immune deposits was 7Sγl, and that other subclasses of IgG and IgM were not involved. Antigen was found in the proximal renal tubules of infected mice, as demonstrated by fluorescein-labeled IgG fraction of rabbit antitetrathyridia secretory/excretory antigen antisera. The presence of tetrathyridia antigen in the urine of infected mice was confirmed using the Ouchterlony technique. [ABSTRACT FROM AUTHOR]

The applicability of ELISA detection of circulating Aspergillus spp. antigen (Ag) and systemic antibody (Ab) of IgG class, and the blood parameter values were evaluated for diagnosis of invasive aspergillosis in Aspergillus spp.-challenged Peking ducks (Anas platyrhynchos). The protective role of Aspergillus spp. IgG was evaluated in Cape shelducks (Tadorna cana) immunized with Aspergillus spp. Ag. Challenged but non-immunized A. platyrhynchos developed invasive aspergillosis on day 21 as demonstrated histopathologically by the presence of fungal microgranuloma in air sacs and lung tissue, with serum antigenemia fluctuating from 65 to 270 ng of 55-kD basic protein Ag per ml. Immunized A. platyrhynchos did not demonstrate Aspergillus spp. serum antigenemia but did show rare histopathological changes in some air sacs associated with fungal inflammation. Although the differences between immunized and non-immunized T. cana in blood evaluation parameters did not differ significantly, immunized birds mounted high Aspergillus spp.-specific IgG titer. There was no correlation between the blood parameter values and post-immunization timepoints in T. cana and in A. platyrhynchos. Intramuscular immunization with Aspergillus spp. mycelial phase cultures Ag provided protection against the pathogens. The lack of relations between blood parameter values and increasing Aspergillus spp. IgG titers (in T. cana and A. platyrhynchos) indicate low applicability of these parameters in evaluation of a bird Aspergillus spp. status. Detection of circulating 55-kDa Aspergillus spp. Ag has high early predictive values for invasive aspergillosis in birds. [ABSTRACT FROM AUTHOR]