Prepatent detection of Fasciola gigantica infection in bovine calves using metacercarial antigen

Metacercarial antigen of Fasciola gigantica was evaluated for early immunodiagnosis of experimental bovine fasciolosis using ELISA and Western blot. In ELISA, the experimental F. gigantica infection was detected as early as 2 weeks post-infection (WPI). The gradual increasing trend of antibody level was observed from 2 to 7 WPI, followed by a plateau, which was maintained up to 14 WPI. In Western blot, sera from experimentally infected calves recognized one distinct polypeptide of 21 kDa in fractionated metacercarial antigen as early as 10th day post infection. From 2 WPI, more polypeptide bands were reacting. Recognition of these protein bands persisted till the end of the experiment (14 WPI). Cattle sera collected from the field showed 34.5% seroprevalence of fasciolosis by ELISA using MAg. Comparative immunoblot studies of metacercarial antigen with anti-Gigantocotyle explanatum and anti-Paramphistomum epiclitum sera revealed that 21 and 25 kDa polypeptides of metacercarial antigen did not cross-react with any of these sera and appear to be unique to F. gigantica and having the desirable qualities of early and specific immunodiagnosis.     

Analysis of thioredoxin peroxidase as a promising antigen for diagnosis of Fasciola gigantica infection: a preliminary study

Buffalo fasciolosis induced by Fasciola gigantica causes important economic losses in tropical areas of Asia. Detection of prepatent infection is essential to control this disease. Classical tools such as coprology, necroscopy or ELISA based on crude extracts from F. gigantica are poorly sensitive or specific. Purified antigens could be used to increase these parameters. Western blot analysis and mass spectrometry of a fraction of F. gigantica excretory-secretory products obtained by gel filtration showed that thioredoxin peroxidase could be a potential antigen for serodiagnosis: it was recognized from the 2nd week after infection, by all buffalo experimentally or naturally infected with F. gigantica but not by healthy animals.     

Detection of antibodies to Streptobacillus moniliformis in rats by an immunoblot procedure

An immunoblot (IB) technique for detecting antibodies to Streptobacillus moniliformis in rat sera was evaluated. Immune sera to three S. moniliformis strains showed a similar reactivity pattern with both autologous and homologous antigens in the 18-87 kDa range. Using a rat S. moniliformis strain as the antigen, a similar reactivity pattern was found with sera from rats infected experimentally with S. moniliformis and sentinels. Two to five proteins were detected in the 32-55 kDa range. Over a period of 2.5 years, 27/133 rat serum panels submitted for routine monitoring yielded one or more S. moniliformis enzyme-linked immunosorbent assay (ELISA)-positive samples. In one of these 27 panels, sera showed an IB reactivity pattern resembling that observed with immune sera and with sera from infected and exposed rats. S. moniliformis was confirmed in the colony by both culture and polymerase chain reaction (PCR). Sera from the remaining 26 ELISA-positive serum panels frequently showed activity to a 57 kDa antigen but not more than one antigen was detected in the 32-55 kDa range. We conclude that the IB can be used as a confirmatory test for the detection of S. moniliformis infection in ELISA-positive rats.     

Fasciola gigantica: Immunodiagnosis of fasciolosis by detection of circulating 28.5 kDa tegumental antigen

A monoclonal antibody (MoAb)-based sandwich ELISA was developed for the detection of circulating 28.5 kDa tegumental antigen (28.5 kDa TA) in the sera from mice experimentally infected with Fasciola gigantica. The MoAb was immobilized on a microtiter plate, and the antigen in the serum was captured and detected with biotinylated polyclonal rabbit anti TA antibody. The test could detect 28.5 kDa in the extracts of tegument (TA), whole body (WB) and excretory-secretory (ES) fractions at the concentrations of these crude antigens as low as 600 pg/ml, 16 and 60 ng/ml, respectively. This sandwich ELISA assay could detect the infection from day 1 to 35 post infection and showed that circulating level of 28.5 kDa TA peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using sera from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as healthy mice and hamsters. The sandwich ELISA exhibited a sensitivity and specificity at 94.55% and 100%, respectively, and with a positive predictive value of 100%, a negative predictive value of 97.39%, false positive rate of 0%, false negative rate of 5.50% and an accuracy of 98.2%. Thus, this detection method exhibited high specificity and sensitivity as well as could be used for early diagnosis of fasciolosis by F. gigantica.     

A dot-ELISA mimicry western blot test for the detection of swine trichinellosis

A dot enzyme-linked immunosorbent assay (dot-ELISA) using antigens purified by monoclonal antibody affinity chromatography was developed for detecting Trichinella spiralis infection in swine. The test was as sensitive as an ELISA using excretory-secretory products as antigen and western blot analysis, and nearly as specific as the western blot. The dot-ELISA detected all of 20 low infections (0.08-4.74 larvae per gram of diaphragm), most of them by 5-6 wk postinfection. Sera from 1,960 farm-reared swine were tested by conventional ELISA, dot-ELISA, and western blot. Of the 1,960 sera, 262 (13.4%) were considered positive on conventional ELISA, 16 (0.82%) by dot-ELISA, and 15 (0.77%) by western blot. The improved specificity was achieved by employing species-specific denatured antigens. More importantly, the dot-ELISA was much simpler to perform than western blot analysis. The principles employed in this test can be adapted to other infectious diseases, such as AIDS.     

Development and application of a fecal antigen diagnostic sandwich ELISA for estimating prevalence of Fasciola gigantica in cattle in central Java, Indonesia

The purpose of this study was to compare the sensitivity and specificity of an ELISA test to detect Fasciola gigantica antigens (coproantigens) in bovine feces, with fecal egg counting and an ELISA for detecting anti-F. gigantica antibodies in serum. Monoclonal antibodies to cathepsin L were generated and used to capture this antigen in feces of infected cattle. Blood, feces, and livers were collected from 150 cattle at an abattoir in Jakarta, Indonesia, for anti-Fasciola antibodies, coproantigen detection, and F. gigantica egg and worm counts. Fluke recovery varied from 1 to 426 per host, with a mean of 32 flukes. The results showed that the sensitivity and specificity of coproantigen detecting ELISA (95 and 91%, respectively) was better than the anti-F. gigantica antibody ELISA (91 and 88%, respectively) and to fecal egg counting (87 and 100%, respectively). The coproantigen ELISA was able to detect 100% of the cattle with >15 flukes. A survey of 305 cattle in central Java over a 10-mo period validated this test in the field, demonstrating a high prevalence of fascioliasis and establishing the test as a useful diagnostic method to determine patent F. gigantica infections in cattle.     

Comparison of humoral response in sheep to Fasciola hepatica and Fasciola gigantica experimental infection

Humoral response of sheep to F. gigantica was compared with the well known humoral response to F. hepatica, in order to explain the difference of susceptibility of sheep to these two parasites. In this work, a lesser susceptibility of sheep to F. gigantica than to F. hepatica infection was confirmed. Humoral response to F. hepatica infection is similar to that previously described by several authors. IgG level of F. gigantica infected sheep increased from week 2 post-infection (2WPI) and displayed a peak at 13WPI. F. gigantica excretory-secretory products (FgESP) analyzed by SDS-PAGE showed at least 31 bands from 12.0 to 127.6 kDa in FgESP. Western blot indicated that F. gigantica infected sheep sera recognized, in FgESP, at least 30 antigens from 7.8 to 119.2 kDa of which 12 major bands recognized after OWPI. In FhESP and FgESP, F. hepatica infected sheep serum reacted only with the lower molecular mass antigens, while F. gigantica infected sheep serum reacted with the lower and the higher molecular mass antigens. These differences of antigenic recognition might be associated with the difference of susceptibility of sheep. Further investigation must be done to study the mechanism of resistance between the sheep infected with F. hepatica or F. gigantica.     

An evaluation of the dot-ELISA procedure as a diagnostic test in an area with a high prevalence of human Toxocara canis infection

The aim of this work was to evaluate a dot-enzyme-linked immunosorbent assay (dot-ELISA) using excretory-secretory antigens from the larval stages of Toxocara canis for the diagnosis of toxocariasis. A secondary aim was to establish the optimal conditions for its use in an area with a high prevalence of human T. canis infection. The dot-ELISA test was standardised using different concentrations of the antigen fixed on nitrocellulose paper strips and increasing dilutions of the serum and conjugate. Both the dot-ELISA and standard ELISA methods were tested in parallel with the same batch of sera from controls and from individuals living in the problem area. The best results were obtained with 1.33 μg/mL of antigen, dilutions of 1/80 for the samples and controls and a dilution of 1/5,000 for the anti-human IgG-peroxidase conjugate. All steps of the procedure were performed at room temperature. The coincidence between ELISA and dot-ELISA was 85% and the kappa index was 0.72. The dot-ELISA test described here is rapid, easy to perform and does not require expensive equipment. Thus, this test is suitable for the serological diagnosis of human T. canis infection in field surveys and in the primary health care centres of endemic regions.     

严重急性呼吸综合征患者血清特异性抗体消涨规律的长期追踪研究

目的:追踪检测SARS冠状病毒(SARS-CoV)抗体在严重急性呼吸综合征(SARS)患者血清中的产生及其转归规律,为SARS诊断及防治提供依据。方法:对41例临床诊断SARS患者的血清进行了连续3年的检测,分别应用间接免疫荧光(IFA)检测患者血清特异性IgG抗体平均滴度,应用双抗原夹心ELISA法检测患者血清核衣壳蛋白(N蛋白)抗体的平均滴度,绘制消涨曲线,得出消涨规律。结果:应用IFA检测患者血清特异性IgG抗体与应用双抗原夹心ELISA法检测N蛋白抗体所得到的消涨规律不同,前者测得康复者血清IgG抗体滴度维持在较低水平,但后者检测35例康复者血清N蛋白抗体仍维持在较高水平。结论:SARS-CoV的N蛋白是免疫原性较强的抗原,感染3年后仍存在高滴度抗体;抗原夹心ELISA检测SARS-CoV N蛋白抗体的灵敏度较IFA方法高。     

Paramphistomum cervi: antigenic profile of adults as recognized by infected cattle sera

An antigenic profile of adult Paramphistomum cervi was revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from cattle naturally infected with P. cervi, Fasciola gigantica and strongylids. SDS-PAGE of whole worm extracts exhibited 26 distinct protein bands. Immunoblotting analysis of these proteins showed five major antigenic bands which were recognized by serum of individual cattle naturally infected with P. cervi. These antigenic proteins had molecular weights ranging from 23 to 116kDa. One antigenic protein with a molecular weight of 52kDa exhibited a consistent reaction with sera from all infected cattle. It's diagnostic sensitivity, specificity and accuracy using this test were 100%, 98% and 98.9%, respectively. The positive and negative predictive values were 97.6% and 100%, respectively. This finding suggests that the 52kDa protein may be a diagnostic antigen for paramphistomosis.     

Suppression of responses to cryptococcal antigen in murine cryptococcosis

Subpopulations of spleen cells responsible for responsiveness and unresponsiveness to cryptococcal antigen in vitro were identified. Lymphocytes which responded in lymphocyte transformation (LT) assays were nylon wool nonadherent and theta antigen positive. These lymphocytes required the presence of an accessory cell which could be supplied by normal peritoneal exudate cells. Spleen cells taken from mice which had been infected for 3 to 15 days were tested to determine their ability to respond to cryptococcal antigen in LT assays. A minimal response was detected at the ninth day of infection. The response of infected spleen cells was attributed to a nonadherent lymphocyte. Nonadherent spleen cells of infected animals had enhanced responses after removal of adherent cells and addition of normal peritoneal exudate cells. Suppressor cells were detected in the spleens of infected mice by the 12th day of infection and thereafter. A nonadherent suppressor cell was identified, but indirect evidence suggested that an adherent cell could also be present in infected spleens.     

Comparative evaluation of recombinant BP26 protein for serological diagnosis of Brucella melitensis infection in goats

An enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of caprine brucellosis with purified recombinant BP26 (outer membrane protein 28) of Brucella melitensis 16M produced in Escherichia coli. Brucella protein named CP28, BP26, or Omp28 has been identified as an immunodominant antigen in infected cattle, sheep, goats, and humans. The recombinant BP26 (rBP6) ELISA performed with serum samples (n = 1738) taken from organized farms and field goats from Northern India and tested in two different batches of 778 and 960 animals for brucellosis. Positive results were compared with the traditional serum agglutination test (SAT), complement fixation test (CFT) and dot-ELISA. The diagnostic sensitivity of rBP26 ELISA, SAT, CFT and dot-ELISA was 87.5%, 56.25%, 62.5% and 75% respectively. The specificity of the rBP26 ELISA, SAT, CFT and dot-ELISA was 90%, 75%, 80%, and 85% respectively. The results of rBP26 ELISA positive animals were further compared and evaluated by tissue PCR using B. melitensis BP26 gene as target. This resulted in 100% positive correlation between rBP26 ELISA and BP26 PCR. Thus, these results confirmed the importance of BP26 as a suitable antigen and rBP26 ELISA, if well standardized, proved to be a good screening test for the serological diagnosis of caprine brucellosis.     

Profiling serum antibodies to Mycobacterium tuberculosis proteins in rhesus monkeys with nontuberculous Mycobacteria

Recent evidence indicates that the prevalence of diseases caused by nontuberculous mycobacteria (NTM) has been increasing in both human and animals. In this study, antibody profiles of NTM in rhesus monkeys (Macaca mulatta) were determined and compared with those of monkeys infected with Mycobacterium tuberculosis complex (MTBC). Antibodies against 10 M. tuberculosis proteins, purified protein derivative (PPD), and mammalian old tuberculin (MOT) were detected in 14 monkeys naturally infected with NTM by indirect ELISA. Sera from 10 monkeys infected with MTBC and 10 healthy monkeys were set as controls. All antigens showed high serological reactivities to MTBC infections and low reactivities in healthy monkeys. NTM infections showed strong antibody responses to MOT and PPD; moderate antibody responses to 16kDa, U1, MPT64L, 14kDa, and TB16.3; and low antibody responses to 38kDa, Ag85b, CFP10, ESAT-6, and CFP10-ESAT-6. According to the criteria of MTBC, only CFP10, ESAT-6, and CFP10-ESAT-6 showed negative antibody responses in all NTM infections. Taken together, these results suggest that positive results of a PPD/MOT-based ELISA in combination with results of antibodies to M. tuberculosis-specific antigens, such as CFP10 and ESAT-6, could discriminate NTM and MTBC infections. Two positive results indicate an MTBC infection, and a negative result for an M. tuberculosis-specific antigen may preliminarily predict an NTM infection.     

Serological diagnostic potential of recombinant outer membrane protein (Omp31) from Brucella melitensis in goat and sheep brucellosis

Outer membrane proteins of Brucella have been classified as group 1 (94 or 88 kDa), group 2 (36–38 kDa), and group 3 (31–34 and 25–27 kDa). Two proteins of 25 and 31 kDa with only 34% of identity are included in group 3 and they are coded for by the omp25 and omp31 genes. Proposed study planned to detect antibodies to Brucella melitensis Omp31 in farm goats having history of B. melitensis induced abortions, in B. melitensis-infected goats and sheep. By enzyme-linked immunosorbent assay (ELISA), using recombinant Omp31 as antigen, of 872 farm goats antibodies to Omp31 were detected in 112 (12.8%) cases. Out of 14 naturally infected goats infected with B. melitensis 12 (85.7%) showed anti Omp31 antibodies. Out of 10 naturally infected sheep with Brucella ovis, antibodies to Omp31 were detected only in 6 (60%) cases and in 18 (81.8%) out of 22 cases infected with B. melitensis. Obtained results were also compared with the rose Bengal plate test (RBPT). In controlled experiments, sensitivity and specificity of recombinant Omp31 (rOmp31) ELISA and RBPT were also evaluated and it was found that former test is 100% specific though RBPT has slightly higher sensitivity. In this study, we found a significant difference between the two groups (B. melitensis and B. ovis infected) in terms of the percentage of positive reactions or signal level by an ELISA. The reactivity of the positive sera against the purified rOmp31 was also tested by Western blotting. Sera from B. melitensis-infected animals showed a strong reactivity in comparison to sera from B. ovis-infected animals. The potential diagnostic usefulness of this antigen in combination with other recombinant proteins from B. melitensis would be of great importance in future in eradication of brucellosis.     

Detection of Colletotrichum falcatum causing red rot of sugarcane by enzyme-linked immunosorbent assay

Red rot disease of sugarcane caused by Colletotrichum falcatum Went is one of the most destructive diseases of sugarcane (Saccharum officinarum L.) worldwide. The pathogen spreads primarily through infected sugarcane setts and hence the use of disease-free setts is essential to prevent the disease. In order to develop immunological method for detection of C. falcatum, two proteins with molecular weights of 27 kDa and 45 kDa were purified from the mycelium of C. falcatum race Cf 05 and used as antigen source to raise polyclonal antibodies in NewZealand white rabbit. The developed polyclonal antibodies were tested for detection of C. falcatum by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. The polyclonal antibodies specifically detected C. falcatum in extracts from infected plants, both in immunoblot and ELISA. The ELISA results showed that the developed polyclonal antibodies were highly specific to C.falcatum. The developed antibodies were very sensitive and could detect C.falcatum proteins even at a dilution of 1:50,000. Higher ELISA absorbance values were recorded even at an antigen dilution of 1:500. In western blot analysis, protein bands with molecular weights of 27 kDa and 45 kDa reacting to antisera raised against 27 kDa and 45 kDa mycelial proteins of C. falcatum, respectively, were detected in protein samples from red rot infected canes. The high specific reactivity and sensitivity of the antisera indicate its potential suitability for ELISA-based detection of C. falcatum.     

Comparative evaluation of antibody positive titer by ELISA and IFA in Theileria annulata vaccinated cattle in Iran

An enzyme linked immunosorbent assay (ELISA) was used to evaluate antibody positive titer in vaccinated and non-vaccinated cattle using schizont infected myeloid cells as an antigen. The result was compared with indirect fluorescent antibody level in the same animals. For this study 116 milking cows, 95 vaccinated and 21 non-vaccinated, were bleeded in order to prepare sera. They were tested with both ELISA and IFA tests. 94 sera had positive antibody titer and 22 sera were negative through ELISA test but, with IFA test, only 89 sera showed positive antibody titer and 27 were negative. Thereby, it was concluded that the sensitivity and specificity of ELISA test in comparison with IFA test was 95.5% and 66.6% respectively. This study generally indicated that ELISA could be an effective test for sero-epidemiological investigations of bovine tropical theileriosis, and it is considered to be valid as an additional test to distinguish the vaccinated from the non vaccinated cattle in order to schedule vaccination programs.     

Antibody reactivities of Mycobacterium paratuberculosis infected sheep as analyzed by enzyme-linked immunosorbent assay and Western blotting

Antibody reactivities in sera from Mycobacterium paratuberculosis (M. ptb) infected and vaccinated sheep were analyzed by enzyme-linked immunosorbent assay (ELISA) and Western (immuno)blotting using a sonicate antigen from M. ptb. Both methods allowed good differentiation between infected/vaccinated animals and noninfected controls. Removal of nonspecific crossreactive antibodies by absorption with a M. phlei sonicate antigen coupled to Sepharose reduced ELISA reactivities of positive sera by 50% and those of noninfected serum by 85%. Immunoblotting analysis revealed that reduction by M. phlei absorption was due to lower reactivities of M. ptb antigens in the range of 30 to 45 kDa. However, one protein with a molecular mass of approx. 27 kDa seemed to be specific for M. ptb since it reacted similarly with nonabsorbed and absorbed serum but not with antibodies which were eluted from M. phlei-Sepharose after absorption. Our findings indicate that M. ptb and M. phlei share a number of common antigens of potential pathogenic importance and that only a smaller part of proteins (i.e. the 27 kDa protein) might be specific for M. ptb.     

Immune responses in Indonesian thin tail and Merino sheep during a primary infection with Fasciola gigantica: lack of a specific IgG2 antibody response is associated with increased resistance to infection in Indonesian sheep.

After a primary infection with Fasciola gigantica, the immune responses in a resistant (Indonesian thin tail) and a susceptible (Merino) breed of sheep were analysed. The number of adult flukes recovered from the livers of the Indonesian thin tail sheep were significantly lower than those found in the Merino animals. On days 8, 14 and 25 p.i., Indonesian thin tail sheep exhibited a significantly higher eosinophilia than Merino sheep, whereas neutrophilia was significantly elevated in the Indonesian thin tail sheep on days 36 and 48 p.i. Serum from both sheep breeds demonstrated IgM, IgG1 and IgE responses to F. gigantica. In contrast, the Indonesian thin tail sheep produced significantly lower levels of IgG2 antibodies relative to the high level detected in Merino sheep. The IgE response was biphasic in both sheep breeds with the first response detected by day 14 and the second response developing from days 30 to 60 p.i. Western blotting showed that a similar profile of adult fluke antigens was recognised by IgG1 and IgE antibodies in both the Indonesian thin tail and Merino sheep. The IgE response was directed to a major antigen at about 92 kDa. We postulate that IgG2 could act as a blocking antibody for protective effector responses against F. gigantica in sheep and that the Indonesian thin tail sheep, by downregulating IgG2 responses, have an enhanced capacity for killing F. gigantica in vivo.     

Immunological diagnosis of cytomegalovirus infections. Application to blood transfusion centers

The Blood Transfusion Centers (B.T.C.) are mainly concerned with the selection of CMV infection free blood donors, the screening of the anti CMV antibody high titre plasma donors and the evaluation of specific anti CMV Immunoglobulin preparations. Various serological methods could be used but they are of different value depending the purposes of the B.T.C. The neutralization test (Nt), with the addition of complement is specific and detects the protecting AB against the glycoproteins of the viral envelope. The complement fixation test (CF) using extracts of CMV infected cells as antigen largely varies in its sensitivity according to the quality of the antigen. In any case, the CF test is not sensitive enough to detect a latent CMV infection in a certain percentage of the non immunosuppressed adults, but could be used for the selection of anti CMV antibody high titres carriers. Three sensitive methods: passive haemagglutination, indirect immunofluorescence and indirect ELISA tests, might be used for the detection of latent CMV infections. They detect various AB against various internal and external components of the CMV. They are submitted to various sources of errors. The sensitivity of the indirect IF test is mainly restricted by the quality of the antigen preparation, its specificity by the presence of anti cells antibodies in the sera, the Fc receptors in the antigens and the specificity of the conjugates. The indirect ELISA which is submitted to the same causes of errors is a highly sensitive test, easy to perform, reagents are available, and automatic processors have been developed. When compared with the previous techniques, the ELISA test is suitable for the screening of CMV free donors, when it is performed with an highly sensitive antigen. It could be also used for the screening of high antibody titre carriers: its correlation with the CF test is quite good (r = 0,82). When comparatively applied to the titration of Immunoglobulins preparations, made from plasmas or placentas, for either IM or IV administration, the Elisa test gives AB titres different from those obtained with Nt and indirect IF. The calibration of a standard Immunoglobulin reagent is urgently needed and double blind clinical assays of the protective effect of various preparations of specific anti CMV Immunoglobulins have to be promptly designed.