Comparative evaluation of an indirect ELISA test for diagnosis of swine cysticercosis employing antigen from Taenia solium and Taenia crassiceps metacestodes

Taenia solium cysticercosis is still a serious public health problem in several countries where poverty and lack of hygiene favor transmission. Because pigs are the primary intermediate hosts, prevalence of porcine cysticercosis is a reliable indicator of active transmission zones. Serological diagnostic methods are important tools for epidemiological studies since they can be applied to living animals on a large scale. Four antigen preparations (cyst fluid and crude) from T. solium and T. crassiceps metacestodes were compared for swine cysticercosis diagnosis by indirect ELISA (IE). Twenty-eight serum samples from swine naturally and experimentally infected by cysticerci of T. solium and 56 serum samples from swine reared in commercial herds were tested. Best results of overall sensitivity were obtained by the use of cyst fluid and crude antigen of T. crassiceps metacestode (100 and 96.4%, respectively). Using homologous antigen preparations we have observed higher specificity percentage (98.2% for cyst fluid and 96. 4% for crude metacestode T. solium antigen). We concluded that sensitivity is of far more importance than specificity for identification of endemic areas in order to prevent transmission to man. We conclude, therefore, that IE performed with cyst fluid antigen of T. crassiceps metacestode is a better tool for that purpose.     

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.     

Experimental infection model for Taenia solium cysticercosis in swine. Cysticercosis Working Group in Peru

A novel method for infecting pigs with Taenia solium using an intramuscular innoculum of oncospheres was investigated in a series of five experiments in 18 animals. The model is simple to perform, requires a minimal number of oncospheres, permits multiple infections per animal, and decreases the variation inherent in oral infection models. This intramuscular oncosphere assay (IMOA) may provide a valuable tool to evaluate therapeutic agents or potential vaccines for cysticercosis.     

A serodiagnostic ELISA using recombinant antigen of swine transmissible gastroenteritis virus nucleoprotein.

A serodiagnostic ELISA utilizing the recombinant nucleoprotein (rN protein) of transmissible gastroenteritis virus (TGEV) was developed, and evaluated by examining a panel of 141 virus neutralization (VN) positive and 101 negative sera. The rN protein-based ELISA (rnELISA) appeared to be highly sensitive and specific (98.6% and 98.0%, respectively) when it was compared to the VN test. The result was similar to that of an ELISA based on purified viral antigens with showing good correlation (R=0.829). No cross-reaction was detected with antisera against porcine epidemic diarrhea virus, hog cholera virus, type A rotavirus, pseudorabies virus and swine vesicular disease virus in this ELISA. The rnELISA can be an alternative for the diagnosis of TGE with a great advantage in antigen preparation.     

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.     

Evaluation of recombinant Bhlp29.7 as an ELISA antigen for detecting pig herds with swine dysentery

Swine dysentery (SD) results from infection of the porcine large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Diagnosis of SD traditionally has relied on detecting the spirochaete in the faeces of acutely affected pigs. To date simple and reliable serological assays that can be applied as a diagnostic tool at the herd level have not been available. In the current study a recombinant histidine tagged 29.7 kDa lipoprotein of B. hyodysenteriae (His6-Bhlp29.7) was used as an ELISA plate-coating antigen. Sera (n=1121) from slaughter-aged pigs on 19 farms were tested in this ELISA. Following optimization of the ELISA conditions using hyperimmune control sera, a set of 464 sera from slaughter-aged pigs from five herds where SD did not occur was tested. From these results a suitable cut-off value for herd negativity was defined as the mean optical density reading plus three standard deviations. Testing of 337 pig sera from six farms with SD then showed that the sensitivity of the test at the herd level was 100%, with all six farms having one or more serum samples exceeding the cut-off value for negativity. Finally, 320 sera from eight herds suspected of having SD were examined. Four of these herds were shown to have pigs with titres consistent with SD. The true health status of the other four herds that were serologically negative could not be confirmed. In conclusion, when used on sets of 40 sera from slaughter-aged pigs the His6-Bhlp29.7 ELISA as established proved to be a useful adjunct to the diagnosis of SD at the herd level.     

猪带绦虫TS11抗原基因接种小鼠的免疫应答

本实验以猪带绦虫 TS11抗原基因的真核表达型质粒 VTS11肌肉免疫注射 BAL B/ c小鼠 ,四甲基偶氮唑蓝 (MTT)比色法检测小鼠脾淋巴细胞刀豆蛋白 A(Con A)刺激的增殖反应及 IL- 2的诱生活性 ;用 EL ISA方法检测免疫小鼠 Ig G总量和特异性抗体水平 ,常规法检测外周血免疫细胞数量的动态变化。结果显示 VTS11免疫小鼠血清的特异性抗体滴度和 Ig G含量显著高于空白对照组小鼠 ;免疫小鼠脾脏淋巴细胞 Con A刺激增殖反应和 IL - 2诱生活性均比对照组小鼠显著增强 ;VTS11免疫小鼠的淋巴细胞和巨噬细胞等免疫细胞的数量也显著超过对照组。这表明 VTS11免疫小鼠 ,可诱导其产生特异性的细胞和体液免疫反应 ,VTS11具有很强的免疫激活作用 ,可望成为预防猪囊虫病的一种新型疫苗。     

Prevalence of Taenia solium cysticercosis in swine from a community-based study in 21 villages of the Eastern Cape Province, South Africa

The pork tapeworm, Taenia solium, causative organism of porcine cysticercosis and human neurocysticercosis is known to occur in areas of South Africa including Eastern Cape Province but, despite increasing reports of its occurrence throughout the subregion, the prevalence is yet to be clearly established. The parasite presents a potentially serious agricultural problem and public health risk in endemic areas. The human populations considered to be at highest risk of infection with this zoonotic helminth are people living in rural areas most of whom earn their livelihood wholly or partially through livestock rearing. Here we report on initial results of a community-based study of pigs owned by resource-poor, emerging pig producers from 21 villages in the Eastern Cape Province. Lingual examination (tongue palpation) in live pigs, two enzyme-linked immunosorbent assays (ELISAs), which detect parasite antigen (B158/B60 Ag-ELISA and HP10 Ag-ELISA) and an enzyme immunotransfer blot (EITB) assay, which detects antiparasite antibody, were used to verify endemicity and estimate apparent prevalence. In the absence of a gold standard true prevalence was obtained, using a Bayesian approach, with a model that uses both available data and prior information. Results indicate that the parasite is indeed present in the study villages and that true prevalence was 64.6%. The apparent prevalences as measured by each of the four tests were: 11.9% for lingual examination, 54.8% for B158/B60 Ag-ELISA, 40.6% for HP10 Ag-ELISA and 33.3% for EITB. This base-line knowledge of the prevalence of T. solium in pigs provides information essential to the design and monitoring of sustainable and appropriate interventions for cysticercosis prevention and control.     

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.     

Assessment of an enzyme-linked immunosorbent assay using a Taenia hydatigena fraction antigen in the diagnosis of cysticercosis in cattle.

Enzyme linked immunosorbent assay (ELISA) using a fraction of larval Taenia hydatigena cyst fluid antigen was carried out on 469 bovine sera collected at slaughter from feedlot cattle for the presence of anticysticercosis antibodies. Cysticerci, in low numbers, were found in the heart, tongue and/or masseter muscles of 84 of the 469 cattle at postmortem inspection. Only nine sera gave positive ELISA reactions and in only one of these nine animals were cysticerci found. Within the limitations of this study, the high rate of false negative and false positive reactions suggests that the ELISA with the antigen used is not a satisfactory procedure to diagnose cysticercosis in cattle, at least in animals with light infections.     

Prevalence of Taenia solium porcine cysticercosis in the Eastern, Southern and Western provinces of Zambia

Tongue examination and detection of circulating antigen (Ag-ELISA) were used to establish the prevalence of Taenia solium porcine cysticercosis in free-range pigs in selected districts of Eastern, Southern and Western provinces of Zambia, and to determine if prevalence of porcine cysticercosis was associated with age, breed and sex. Households with pigs were identified using the snowballing technique. A total of 1691 pigs were examined out of which 183 (10.8%) were positive on tongue examination. Ag-ELISA gave a sero-prevalence of 23.3%. When considering the factors in a logistic regression analysis, only breed type was significantly associated with porcine cysticercosis (OR=0.72; 95%CI=0.63-0.81). The crossbred pigs were 72% more likely to have had cysticercosis than the Nsenga (dwarf local) breed as determined by Ag-ELISA. The result that crossbred pigs had a higher prevalence of T. solium cysticercosis suggests that pig breeds may display different susceptibility to cysticercosis. The limited use of latrines in these areas implies that people use the nearby bush for defecation, resulting in pigs having access to human faeces. Therefore, investigation of taeniosis and cysticercosis in humans is warranted to better comprehend the local epidemiology and transmission risks. This should then be followed by extension programs to communities so that the control plans that could be instituted are more sustainable.     

Comparison of bovine cysticercosis prevalence detected by antigen ELISA and visual inspection in the North East of Spain

A cross-sectional serological study on cattle less than 2 years old, using an antigen ELISA for the detection of bovine cysticercosis was carried out between November 2009 and February 2010 in 10 slaughterhouses from the Catalonia region (North-Eastern Spain). Circulating antigen was detected in 23 of 2073 animals, i.e. a sero-prevalence of 1.11% (CI95%: 0.76-1.75%). The determined sero-prevalence was about 50 times higher than the prevalence obtained by visual inspection within the same period: 19 positive animals of 90,891 slaughtered animals (0.02%) in the same slaughterhouses. None of the animals with positive result in the Ag-ELISA was detected by meat inspection.     

Preliminary evaluation of Community-Led Total Sanitation for the control of Taenia solium cysticercosis in Katete District of Zambia

Taenia solium taeniasis/cysticercosis is a zoonotic disease endemic in sub-Saharan Africa. It is associated with poor sanitary practices, free-range pig husbandry and lack of disease awareness in endemic communities. A comparative research was conducted with pre and post-intervention assessments in nine villages to evaluate Community-Led Total Sanitation (CLTS) as an intervention measure for the control of porcine cysticercosis in Katete District in the Eastern Province of Zambia. Blood samples were collected from pigs for circulating antigen detection and a questionnaire focused on the household was administered to a total of 153 respondents whose pigs were examined (64 pre-intervention, 89 post-intervention), in order to obtain information on general demographic characteristics, pig husbandry practices, sanitation practices and associated knowledge and awareness of T. solium infections. The first sampling was conducted prior to the implementation of the CLTS and second sampling eight months after triggering of CLTS in the selected villages. A total of 379 pig serum samples were examined using the B158/B60 Ag-ELISA to detect T. solium cysticercosis, 104 pre-intervention and 275 post-intervention, of which 14 (13.5%) and 45 (16.4%) were positive, respectively. Wald test p-values were computed to assess significant differences in the variables of interest mentioned above for the pre and post CLTS. The research revealed that CLTS as a control measure did not significantly improve T. solium infections in pigs. The research also revealed that the sanitation practices and awareness of cysticercosis did not change. It is recommended that a longer term evaluation be undertaken when the villages have been declared open defaecation free. In addition, the research recommends that health education, mass drug treatment and pig vaccination be incorporated, as an essential component of prevention and control programmes for T. solium infections.     

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.     

Taenia solium porcine cysticercosis: viability of cysticerci and persistency of antibodies and cysticercal antigens after treatment with oxfendazole

The aim of this study was to assess the effect of treating Taenia solium infected pigs with oxfendazole (OFZ) on viability and clearance of cysticerci and the corresponding persistence of specific antibody isotypes (IgG(total), IgG1, IgG2 and IgA) and circulating cysticercal antigen (CCA). Antibody isotypes and CCA responses were measured by antibody-ELISA (Ab-ELISA) and antigen ELISA (Ag-ELISA), respectively. Correlations were made between antibodies, CCA and the total number of cysticerci enumerated at necropsy. Forty pigs with cysticercosis were randomly allocated into two groups: Treatment group (n=20) was treated with OFZ at 30 mg/kg orally while the treatment control group (n=20) was not treated. Five uninfected pigs served as negative controls. Pigs were killed at 1, 4, 8 and 26 weeks post-treatment (wkpt). Overall, the mean total cyst count in treated pigs was 2904+/-5397 (mean+/-S.D.) while in the controls it was 6235+/-6705. Mean cyst viability was 5+/-11% (mean+/-S.D.) and 97+/-4% in treated and control pigs, respectively. Results showed that OFZ killed muscular cysticerci over a period of 4 weeks but failed to kill cerebral cysticerci. Antibodies, CCA responses and clearance of dead cysts from the meat, depended on the cyst intensity of individual pigs at time of treatment since both antibody and CCA correlated with intensity of cysticerci at necropsy (r=0.441, P=0.005; r=0.654, P<0.001), respectively. IgG1 responses were the best indicator of treatment efficacy because they were predominant in both infected treated and control pigs and disappeared early after treatment. Both Ab/Ag-ELISA failed to detect cysts in the brain. Though dead cysticerci took some time (26 wkpt) to clear from the meat, treatment of porcine cysticercosis with OFZ should, in combination with other intervention measures be considered as an important, cost-effective measure in the control of taeniosis/cysticercosis.     

Evaluation of the ELISA for the serological diagnosis of trichinosis in Canadian swine

An ELISA using a Trichinella spiralis spiralis excretory-secretory antigen was evaluated as a procedure for the diagnosis of trichinosis in swine in Canada. Field and experimental trials were carried out using both indirect serological (ELISA) and direct parasitological (pepsin-digestion) methods concurrently on serum and musculature, respectively, from each animal. The ELISA is a sensitive and specific test for the detection of Trichinella antibodies in porcine sera when present. The development of Trichinella antibodies appears to be dependent on the magnitude of the infection established, age of the infection when the animal is tested and the immunocompetence or response to infection of individual animals. False negative reactions were recorded in both field and experimental trials. In the field study, five of the 1009 swine examined were parasitologically positive with light infections ranging from 0.01 to 0.046 larvae per gram (la/g) of musculature yet all were serologically negative. Experimentally it was shown that Trichinella antibodies develop slowly, at least two to three months postinfection, in pigs with very light infections. Even in pigs which developed infections of 33 to 55 la/g of musculature, seroconversion occurred greater than 23 and less than 30 days postinfection. The immunocompetence or response to infection of individual pigs was variable as illustrated by one pig inoculated with 3000 infective larvae which had consistently lower titers compared to others in the same group despite the establishment of a muscle infection of 8.5 la/g of musculature. One false positive reaction was recorded in the experimental trial in an animal which had received 100 larvae and seroconverted at about three months postinfection.(ABSTRACT TRUNCATED AT 250 WORDS)     

rNP-ELISA检测抗猪流感病毒抗体工作条件的优化

猪流感是生猪养殖的国家普遍都会遇到的猪的呼吸道病[1]。虽然HI检测已经被作为可信赖的抗流感病毒抗体的检测方法[2]。但是,应用ELISA方法检测抗体具有明显的优势,对于待检血清不需要进行任何处理,而且也不必要对应用的抗原进行更换与选择[3]。试验采用的包被抗原为重组NP蛋白     

Purification of an antigen from the Taenia crassiceps metacestode vesicular fluid highly sensitive in detecting IgG-antibodies (ELISA) in calves with experimental cysticercosis

This study reports on the purification of an antigen predominant within the vesicular fluid (VF) of T. crassiceps metacestodes and shown to share identity with the major vesicular fluid protein of the T. saginata larval stage. Purification was achieved by gel filtration of the VF on Sephacryl S-300 superfine, followed by ion exchange HPLC. The antigen represents a single polypeptide chain (Mr appr. 37,000) with carbohydrate moieties without affinity to ConA. Isoelectric focusing of the electrophoretical and immunological homogeneous antigen resulted in five bands focusing at pH 4.0, 4.2, 4.4, 4.6 and 4.8, respectively. In ELISA, the purified antigen detected serum IgG-antibodies in all 21-35 weeks old calves (n = 10) with experimental cysticercosis (70 to 6,000 viable larvae recovered). When compared to T. saginata metacestode VF the antigen was the better reagent for discriminating between infected and non-infected animals. As shown by immunodiffusion and ELISA the antigen is also common to the T. saginata adult stage and obviously to other taeniid metacestodes where it is accumulated in the VF or hydatid fluid.     

Efficacy of the immunoblot assay for cysticercosis in pigs and modulated expression of distinct IgM/IgG activities to Taenia solium antigens in experimental infections

A recently invented immunoblot assay for human cysticercosis was evaluated for efficacy in pigs. The test population consists of 45 pigs with parasitologically confirmed cysticercosis, 47 with heterologous infections, 45 SPF or concrete raised control animals. With this group of 137 animals the test performance was 100% sensitive and 100% specific. The antigen-specific responses of immunoglobulin A (IgA), IgG and IgM in four pigs infected with Taenia solium eggs derived from a human were quantified by immunoblot. Antigen-specific activities were observed as early as 1 week postinfection. The first antigen-specific isotypic response was IgM antibodies directed against a glycoprotein at 97 KD (GP97). This activity generally disappeared between the sixth and ninth week postinfection. Between Weeks 5 and 8, IgG activity rose as IgM activity fell. The IgG activity, however, was directed mostly towards GP50 and GP42 antigens. If the same response occurs in people with cysticercosis, identifying specific isotype activity may help to distinguish new infection from old.