Performance of the ELISA test for swine cysticercosis using antigens of Taenia solium and Taenia crassiceps cysticerci

Studies were conducted to evaluate antigens of Taenia solium (Tso) and Taenia crassiceps (Tcra) cysticerci in the ELISA test for the diagnosis of swine cysticercosis. The samples analyzed were cysticercosis positive and negative control sera and heterologous sera. Four antigens were assayed: vesicular fluid (VF) and crude (T) Tcra and scolex (S) and crude (T) Tso. All antigens showed good performance, but VF-Tcra was the best followed by T-Tcra. Sensitivity rates of ELISA were respectively, in 2nd and 3rd standard deviation cut-offs, 96.0 and 80.0% for the VF antigen and specificity of 97.5 and 100.0%. Cross-reactivity was verified only for hidatidosis and ascaridiosis. Due to the high performance observed, the ELISA test using Tcra antigens should be recommended for the diagnosis of swine cysticercosis.     

Development of a serologic assay for cysticercosis, using an antigen isolated from Taenia spp cyst fluid

An ammonium sulfate-soluble fraction of Taenia hydatigena cyst fluid (ThFAS) was further evaluated for use in the immunodiagnosis of cysticercosis. Analysis of ThFAS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblot analysis confirmed earlier reports of a highly specific, low molecular weight antigen in this preparation; in contrast, other components of ThFAS were shown to react nonspecifically. Antibodies against the less than 12-kD diagnostic antigen were detected in sera from 10 cattle and 4 swine inoculated with metacestodes of T saginata and T solium, respectively, but not in animals inoculated with Fasciola hepatica, Trichinella spiralis, Brucella abortus, or Toxoplasma gondii, or in noninoculated controls. Isolation and immobilization of the less than 12-kD antigen on a hydrophobic transfer membrane resulted in development of an unambiguous dipstick assay capable of correctly identifying fully developed (10-week) experimentally induced infections in cattle and swine. In addition, the dipstick assay was highly specific for diagnosis of the disease in human beings, and offers the potential of distinguishing between human clinical cases of cysticercosis and taeniasis. A similar reactive antigen of diagnostic potential was also identified and isolated from T crassiceps and T taeniaeformis cyst fluids.     

Evaluation of an antigenic fraction of Taenia hydatigena metacestode cyst fluid for immunodiagnosis of bovine cysticercosis

An antigenic fraction (ThFAS) isolated from Taenia hydatigena metacestode cyst fluid was used in an ELISA to detect antibodies to T saginata in experimentally and naturally infected cattle. In 10 calves given 1,000 to 100,000 T saginata eggs (20% to 60% viability), IgG and IgM antibodies were detected in all the calves by post-inoculation week 3. Immunoglobulin G antibody values remained increased until calves were slaughtered at post-inoculation weeks 13 to 26. Six naturally infected calves (determined by postmortem examination) were considered positive, using the ELISA. Shared antigens were demonstrated between ThFAS and T saginata and T crassiceps; there were no shared antigens between ThFAS and Haemonchus contortus or Fasciola hepatica. Specific lectin binding to ThFAS indicated the presence of glycoconjugates. Immunoblot analysis indicated that a low molecular weight polypeptide (10,000 Mr) bears the immunodiagnostic antigen.     

Bovine cysticercosis in cattle slaughtered at Awassa municipal abattoir, Ethiopia: prevalence, cyst viability, distribution and its public health implication

A cross-sectional study was conducted from October 2005 to April 2006 on bovine cysticercosis in cattle slaughtered at Awassa municipal abattoir with the objective of determining the prevalence of Taenia saginata cysticercosis, cyst viability, distribution and its public health implication. Questionnaire survey involving 120 respondents was also conducted on human taeniasis. A total of 400 carcasses were examined during the study period, of which 105 (26.25%) were infected with T. saginata metacestodes. From a total of 3200 samples inspected, 500 cysticerci were detected in 141 samples, of which 221 (44.2%) were alive. The anatomical distribution of cysticerci were 65 (29.2%) heart, 56 (25.3%) shoulder muscle, 59 (26.7%) masseter, 23 (10.4%) tongue, 12 (5.4%) diaphragm, three (1.4%) liver, two (0.9%) lung and one (0.5%) kidney samples. The prevalence varied significantly between local and crossbred animals (OR = 3.15, P < 0.05), but not varied between sex, age groups and origin of the animals. T. saginata taeniasis was a widespread public health problem in the town with an overall prevalence of 64.2% (77 of 120). The potential risk factors for disease contraction were raw meat consumption, religion and occupational risks. In conclusion, the study revealed high prevalence of T. saginata metacestodes throughout the edible organs together with existence of deep-rooted tradition of raw meat consumption. This may magnify the public health hazards of T. saginata in the study area. As a result, the disease deserves due attention to safeguard the public health and further promote beef industry in the country.     

Specific and shared antigenic components of Taenia saginata oncospheres

Taenia saginata oncosphere components were analysed by double diffusion. Antigenic components in saline or detergent (Triton x-100) extracts of T saginata oncospheres were identified using a rabbit polyclonal serum directed against the oncosphere and compared with extracts prepared from the metacestodes and proglottids of T saginata and six other helminths commonly found in cattle. There were seven antigenic components found in the saline extract of the oncospheres, of which six were shared with the metacestodes and proglottids. None of these components was consistently different from those of the other six helminths. However, one of the four components in the detergent extract of the oncospheres was present in neither of the extracts of other stages of T saginata nor in extracts of other helminths. This may be of diagnostic significance.     

Evaluation of the results of intradermal tests in spontaneous cysticercosis in cattle

The diagnostic value of intradermal tests was verified on 126 bulls (out of this number 32 autopsically positive) subject to intravital diagnostics of bovine cysticercosis. As allergen were used homologous antigens from adult larvae of Taenia saginata and heterologous antigens from larvae of Taenia crassiceps, as well as hydatid fluid from Echinococcus granulosis. With the antigen T. saginata the sensitivity reached 53.1% and specificity 90.4%, with the antigen T. crassiceps 53.1% and 80.9% and with the antigen E. granulosus 16.7% and 89.5%.     

Serological diagnosis of cysticercosis by an enzyme-linked immunosorbent assay in experimentally infected cattle.

Taenia saginata infections were established in four groups of calves by administering doses of 10, 10(2), 10(3) and 10(4) infective eggs respectively by gavage. A fifth group remained as uninfected controls. Sera were collected from all calves over a period of 210 days. The sera were examined by an enzyme-linked immunosorbent assay (ELISA) with a fraction of larval Taenia hydatigena cyst fluid as antigen for the presence of anti-T. saginata IgG antibodies. At slaughter, the tongue, masseter, diaphragm and cardiac muscles and liver were examined for cysticerci. The higher dose rates of T. saginata eggs were reflected in higher numbers of cysticerci found in the calves at necropsy. There was also a correlation between higher levels of antibodies produced as measured by the ELISA and the numbers of eggs given. Sero-conversion was first detected about 25 days postinfection in heavy infections and later in the lighter infections. Maximal levels of antibody occurred between 40 and 60 days postinfection, followed by a gradual decrease in levels of antibody. A secondary increase in antibody occurred between 160 and 200 days postinfection which might have been due to release of antigen after death of the cysticerci. The low level of circulating antibodies in light infections may result in false positive or false negative diagnoses depending upon the selection of the cut-off point.     

Ag-ELISA and PCR for Monitoring the Vaccination of Cattle against Taenia saginata Cysticercosis Using an Oncospheral Adhesion Protein (HP6) with Surface and Secreted Localization

A Taenia saginata oncosphere-derived adhesion protein (HP6) with surface and secreted localization was used to successfully vaccinate calves against oral challenge with T. saginata eggs. In contrast, vaccination using a combination of T. saginata oncosphere-derived peptides, selected on the basis of their antigenic index, and including three derived from the HP6 molecule (HP6-1, HP6-2 and HP6-3), was unsuccessful. This either indicated that the wrong peptides were selected or, in the case of the HP6 protein, that the protective epitope is conformational in nature. The protection experiments were monitored using a parasite antigen detection ELISA (HP10 Ag-ELISA), which allowed the early determination of the success of the vaccination protocol, subsequently confirmed at autopsy. PCR assays were used for the first time to confirm the presence of T. saginata DNA in lesions recovered at autopsy and thus verify the parasite origin of the lesions.     

A sero-epidemiological study of bovine cysticercosis in Zambia.

A sero-epidemiological study of Taenia saginata cysticercosis was carried out in adult cattle in Zambia to determine the prevalence and study the influence of the farming system on the infection rate. Serum samples were examined for circulating parasite antigen by a monoclonal-based sandwich ELISA. Thirty-eight of 628 serum samples were found positive (prevalence 6.1%). Cysticercosis was significantly more prevalent in feedlots and in traditional farming systems than in dairy farms. It is suggested that the continuous man to animal contact and the use of casual workers in feedlots may be factors that are conductive to T. saginata transmission.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for detection of Taenia saginata cysticercosis in cattle.

Serum IgG response of cattle with cysticercosis caused by Taenia saginata was studied in an enzyme-linked immunosorbent assay (ELISA) where a T. saginata metacestode surface extract was used as antigen. In experimentally infected calves, a sharp rise in specific antibody levels was found 3-4 weeks after the infection followed by a logical level of detection corresponded to about 25 cysts. The ELISA was employed in cattle herds where cysticercosis outbreaks had occurred and also in supposedly uninfected herds. Significantly increased antibody levels were found in the herds with massive cysticercosis cases. The test was not adapted for individual diagnosis as some animals of the uninfected herds, especially within the older age groups, had elevated antibody values. The ELISA was, however, useful in the investigation of outbreaks to determine the extent and pattern of the infection in the herd. The rate of decline in antibody levels in these herds was studied by follow up sampling. The increased antibody levels in the infected herds were also reflected in colostrum-fed calves. This observation was employed to estimate the time of infection.     

Isolation of two lipoprotein antigens from the metacestodes of Taenia hydatigena (Pallas, 1766) and Taenia multiceps (Leske, 1780) and their evaluation in sero-diagnosis

Two identical host contaminant-free lipoprotein antigens were isolated from the metacestodes of T. hydatigena and T. multiceps, of which antigen 1 was more reactive than antigen 2. These antigens appear to be identical with antigens B and A, respectively, and antigen 2 with arc 5 of Echinococcus granulosus described by other workers. Attempts to use these antigens in serodiagnosis showed that only in the indirect haemagglutination (IHA) technique was there any increased sensitivity and specificity compared with the unpurified cyst fluid antigens. No correlation was found between the size of the infecting dose, the number of cysts found at necropsy, the IHA titres, or number of precipitation lines seen in immunodiffusion tests.     

Taenia saginata derived synthetic peptides with potential for the diagnosis of bovine cysticercosis

Immunity in Taeniids is predominantly antibody mediated and thus many serological immuno-determinants will have potential in both protection and diagnosis. The antigenicity of six peptides derived from four potentially protective molecules cloned from a Taenia saginata oncospheres cDNA library have been evaluated as targets for the specific diagnosis of bovine cysticercosis. The six peptides consist of: two peptides (HP6-2 and HP6-3) derived from the sequence of the 18 kDa surface/secreted oncospheral adhesion antigen identified by McAb-HP6, two peptides (Ts45W-1 and Ts45W-5) derived from the sequence of the T. saginata homologue of the T. ovis 45W protective gene family, one peptide (TS45S-10) derived from a T. saginata sequence with significant similarity to the T. ovis 45S protective antigen, and one peptide (TEG-1) derived from the sequence of the T. saginata homologue of Echinococcus spp. main surface protein. Longitudinal studies indicate that T. saginata infected cattle respond to all six peptides by 3-4 weeks post-infection and that the antibody levels remain high for at least 12 weeks post-infection. As protection against Taeniid parasites is predominantly antibody mediated, some of these six peptides may be of value as immuno-prophylactic tools and hence also in assays to determine resistance to infection with the parasite. For diagnosis, on the other hand, only three peptides (HP6-2, TEG-1 and Ts45S-10) performed with the necessary sensitivity and specificity to determine exposure to infection with T. saginata, and now merit an exhaustive evaluation prior to employment as routine diagnostic tools.     

Countercurrent immunoelectrophoresis in the diagnosis of Taenia hydatigena cysticercosis in goats

The procedure of countercurrent immunoelectrophoresis in the diagnosis of Taenia hydatigena cysticercosis in goats was carried out for antemortem diagnosis of T. hydatigena cysticercosis in experimentally and naturally infected goats. The antigens of cyst fluid, scolex and membrane of T. hydatigena metacestodes were purified and compared. The sensitivity of the test in experimentally and naturally infected goats was 57.1 and 52.5%, respectively, whereas its specificity using antisera raised against T. solium cysticercosis, hydatid cyst and Fasciola gigantica was 66.7 and 83.4% with partially purified and fractionated antigens, respectively. Of all three antigens, the cyst fluid antigen was found to be most reactive. The test could be employed for antemortem diagnosis of T. hydatigena cysticercosis using purified antigen.     

Cross-reactivity of anti-Taenia crassiceps cysticerci immune antibodies with Taenia solium antigens

A comparative study of antibody production was carried out using BALB/c mice immunized with 20 or 50microg vesicular fluid (VF)-Tcra (Taenia crassiceps) antigens, and gel of <30kD or eluate from <30kD peptides. Good IgM, IgA and IgG levels were detected by ELISA-Tcra and the antibodies presented reactivity with the <20kD peptides when tested by immunoblotting-Tcra. The antibodies from animals immunized with 20 and 50microg presented high anti-Tso cross-reactivity in ELISA (IgG>IgM and IgA). All groups presented IgG antibodies identifying the 12kD Tso-peptide.     

Isolation of a 14 kDa antigen from Taenia solium cyst fluid by HPLC and its evaluation in enzyme linked immunosorbent assay for diagnosis of porcine cysticercosis

A fraction with a major band of 14kDa was obtained from crude cyst fluid of Taenia solium cysticerci by 2-step chromatography. A first fraction isolated by gel filtration (Sephacryl S-300 high resolution) was purified using an anion exchange column (Mono Q HR 5/5) on high performance liquid chromatography. Evaluation of the analytic sensitivity of this fraction (F3) was carried out in an antibody enzyme linked immunosorbent assay (Ab-ELISA-F3) using serum samples from pigs experimentally infected with different doses of T. solium eggs. The cross-reactivity of F3 was evaluated with serum samples from pigs that were naturally or experimentally infected with Taenia hydatigena, Taenia saginata asiatica, Fasciola hepatica, Trichinella spiralis, Metastrongylus apri, Trypanosoma congolense and Sarcoptes scabiei, and with serum samples of rabbits hyper-immunised with metacestode cyst fluid of T. hydatigena and T. solium. Antibody titres of lightly or heavily infected pigs differed in their kinetics. However, the increase in F3-specific antibodies could not be related to the infection level. Analysis of the specificity of the F3 showed that serum samples of pigs infected with other parasites did not recognise this antigen. Cross-reaction with T. hydatigena occurred in ELISA using cyst fluid as antigen, but the F3 antigen fraction was not recognized by rabbit hyper-immune serum samples to T. hydatigena. Evaluation of the diagnostic sensitivity and specificity of the Ab-ELISA-F3 was done by a non-parametric receiver operating characteristic (ROC) analysis using 66 serum samples from Zambian village pigs. The total number of cysticerci of these pigs was determined by dissection (28 pigs harboured T. solium cysticerci and 38 were negative at dissection). In addition, 58 serum samples from Cameroonian pigs (28 pigs from cysticercosis-free farms and 30 pigs with cysticerci at tongue inspection) were used in a separate ROC analysis. The results from the ROC analysis yielded a low diagnostic value (area under ROC curve=0.48) with the sera from the Zambian pigs while a relatively high diagnostic value was obtained with the sera from Cameroonian pigs (area under ROC curve=0.78). The main factor contributing to a low diagnostic value based on the Zambian serum samples seemed to be the false-positive reactions that were likely caused by the occurrence of transient antibodies in the non-infected animals.     

Production and consumption of T cell growth factor by peripheral blood lymphocytes of cattle infected with Taenia saginata

Calves were infected with 25,000 or 50,000 viable eggs of Taenia saginata. Larval numbers ranged between 2077 and 6005. During infection the animals developed leucocytosis, which was mainly due to lymphocytosis. An apparently positive correlation was observed between the lymphocytosis and the in vitro proliferative response of the lymphocytes to antigen prepared from proglottids. Maximal in vitro blast transformation of the cells stimulated with antigen occurred on Days 12 and 32 post-infection (p.i.). Specific antibodies to T. saginata were demonstrated on Day 14 p.i. At that time, the proliferative response of the cells paralleled the formation of specific antibodies, particularly of the IgG class. The stimulated cells produced a lymphokine showing interleukin 2 (IL 2)-like activity, since the addition of supernatant of such cells to IL 2-dependent concanavalin A (Con A)-blast cells supported the in vitro growth of the cells. In addition, peripheral blood lymphocytes (PBL) specific for T. saginata could be maintained in long-term cultures when they were cultured in medium containing supernatants of MLA-144 cell lines. The data presented in this study indicate that cells specific for T. saginata produced and consumed T cell growth factor (TCGF).     

Preliminary observations on vaccination against bovine cysticercosis in the Sudan

Four Zebu calves, 1-1.5 years old, were vaccinated subcutaneously with hatched ova of Taenia saginata. The immunity elicited protected the animals from subsequent oral infections with this cestode as manifested by the early degeneration of the metacestodes and failure to attain maturity in three of four animals. Three viable cysts were found in the fourth calf compared to more than 300 specimens in non-vaccinated controls.     

Serum antibody levels to Taenia saginata in cattle grazed on Scottish pastures

The serum antibody levels to Taenia saginata of three groups of cattle were assessed by an enzyme-linked immunosorbent assay (ELISA). The first group of cattle were from four farms which had a confirmed T saginata cysticercosis outbreak, all of which had cattle classed as infected by ELISA. The second group were from four farms where sewage sludge had been applied to pasture subsequently grazed by the cattle. One of these farms had cattle classed as infected by ELISA. The control cattle, which were all classed as uninfected by ELISA, came from five farms whose pasture had not been treated with sewage sludge. In a wider survey, involving sera from 47 additional farms, the majority could not be distinguished from the control farms in the earlier survey. However, samples from three of the farms had a similar number of positives to two of the known infected farms in the initial survey. Since the ELISA assay may indicate infected herds, farms such as these warrant further investigation.     

An enzyme-linked immunosorbent assay for the detection of vesicular stomatitis virus antibodies

A liquid-phase enzyme-linked immunosorbent assay (ELISA) was developed for the detection of vesicular stomatitis virus (VSV) types New Jersey (VSV-NJ) and Indiana subtype Indiana I (VSV-IND₁) antibodies in the sera of naturally and experimentally infected cattle, horses and swine. Four different VSV preparations were compared for use as antigen in the ELISA: virus used in neutralization tests, complement-fixation antigen, immunodiffusion ager gel antigen and viral glycoprotein. Comparative antibody titration results of virus neutralization (VN) and ELISA showed no statistically significant difference between serum titers obtained with the four antigens to VSV-NJ (P=0.21) and VSV-IND₁ (P=0.14). The viral glycoprotein antigen was incorporated in the ELISA system because it is non-infectious and induces neutralizing antibodies. The reliability and variability of the ELISA was determined by testing 516 bovine, equine and swine sera which originated from areas free of vesicular stomatitis, and by testing 186 sera from areas where outbreaks occur. ELISA and VN results were correlated (P < 0.001 for both serotypes), and no statiscafically difference was found between replications of the tests. The ELISA allows the testing of a larger number of sera in a shorter time than is possible with the VN test and it can be used in diagnostic laboratories in VSV-free areas for the support of epidemiological surveillance programs.     

The highly antigenic 53/25 kDa Taenia solium protein fraction with cathepsin-L like activity is present in the oncosphere/cysticercus and induces non-protective IgG antibodies in pigs

Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection.