Bovine T cell responses to recombinant thioredoxin of Fasciola hepatica

Fasciolosis is an economically significant disease of ruminants, caused by infection with the digenetic trematodes, Fasciola hepatica and F. gigantica. Some vaccination trials using irradiated metacercariae or isolated proteins have been shown to afford significant protection. However, the mechanisms of specific immunity against this pathogen have not been elucidated. We have identified thioredoxin, a tegument antigen of F. hepatica, among several proteins that are common to both the juvenile and adult fluke within the mammalian host and have undertaken studies to characterize bovine T cell responses to recombinant thioredoxin protein (FH 2020). Peripheral blood mononuclear cells from immune cattle proliferated specifically to crude F. hepatica antigenic extract but not to FH 2020. However, after repeated stimulation of lymphocytes by alternating crude extract and FH 2020, FH 2020-specific proliferation by T cell lines was observed. T cell clones were subsequently generated and found to respond specifically but weakly to both crude antigen and FH 2020. Thioredoxin appears to be only weakly antigenic for bovine T cells and is, therefore, an unpromising candidate for inducing resistance to F. hepatica.     

Fasciola hepatica: studies on vaccination of rats and mice with a surface antigen prepared from fluke homogenate by means of a monoclonal antibody

T1 tegumental antigen was isolated from a homogenate of eight- to 10-week-old Fasciola hepatica using a T1-specific monoclonal antibody bound to sepharose in an antibody-affinity column. Rats and mice were vaccinated with T1 antigen in Freund's complete adjuvant, and control groups received equivalent amounts of non-T1 antigen (eluted from the antibody-affinity column) or ovalbumin. On completion of the immunisation programme, serum samples were collected for ELISA and IFA testing. The animals were challenged by oral infection with F hepatica metacercariae or, for several vaccinated rats, by intraperitoneal transplantation of live adult flukes. At autopsy, worm-burden and liver damage was assessed for each animal and the condition of transplanted flukes was examined. Comparison of test and control groups of animals showed that neither T1 nor non-T1 antigens provided significant protection against challenge, although specific antibody responses against the appropriate sensitising antigen were engendered. Flukes transplanted to the peritoneal cavity of immunised rats survived without damage, although they became encased in hollow fibrous capsules of host origin. The results lend support to the pre-existing concept that glycocalyx turnover by discharge of T1 secretory bodies at the apical surface of migrating flukes provides an efficient means of protection for the parasite against host immunity.     

Serum antibody isotype responses of Fasciola-infected sheep and cattle to excretory and secretory products of Fasciola species

This study investigated the immunoglobulin isotype responses of sheep and cattle chronically infected with Fasciola hepatica and Fasciola gigantica to adult F. hepatica excretory/secretory products (Fh-ES) or F. gigantica excretory/secretory products (Fg-ES), respectively. An antibody enzyme-linked immunosorbent assay (Ab-ELISA) was used to determine serum antibody (total Ig, IgG(1), IgM, IgG(2) and IgA) responses. At necropsy, the mean number of flukes recovered was lower in cattle than in sheep. All F. hepatica and F. gigantica infected sheep and cattle showed an increased total Ig levels from 3 to 4 weeks post-infection (wpi). Among isotypes IgG(1) was most dominant while IgM was the earliest (2 wpi) to be detected in both sheep and cattle infected with both F. hepatica and F. gigantica animals. IgG(2) response was early (2 wpi) in sheep infected by F. hepatica but there was no response in sheep infected with F. gigantica. There was a late and strong IgG(2) response in cattle infected with both flukes. The IgA isotype showed an early and a clear biphasic response in sheep with F. hepatica but was less pronounced in F. gigantica infected sheep. While IgA response to Fh-ES was noticed 5 wpi in F. hepatica infected cattle, it appeared much later (21 wpi) in those infected with F. gigantica. The dominance of IgG(1) isotype in infected sheep and cattle suggest an associated Th2 response. This early response to adult Fasciola spp. ES antigen suggests an early exposure to the antigen presumably through the cross-reacting ES products of juvenile flukes. There is clearly difference in IgG(2) isotype response in cattle (resistant) compared to sheep (susceptible). The late IgG(2) response in cattle may suggest late Th1 involvement in bovine cellular responses to adult Fh-ES/Fg-ES.     

The effect of Taenia hydatigena infection on existing and concurrent infections of Fasciola hepatica in sheep

Sheep given a primary infection of Fasciola hepatica were challenged 18 weeks later with Taenia hydatigena or F hepatica, or both parasites together, or were not challenged. At the same time, control sheep were infected with T hydatigena and/or F hepatica separately or concurrently. All sheep were killed seven weeks after challenge and the number of cysts and flukes counted. Challenge infection with T hydatigena did not affect the numbers of flukes recovered from either primary or challenge F hepatica infections. On the other hand, the numbers of cysticerci were reduced in sheep previously infected with F hepatica but not in those given T hydatigena and F hepatica concurrently.     

肝片吸虫免疫显性抗原基因的筛选及鉴定

为筛选出特异的肝片吸虫诊断候选抗原,拓展诊断靶标,利用肝片吸虫阳性血清筛选肝片吸虫cDNA表达文库,获得肝片吸虫特异性抗原基因,采用RT-PCR技术扩增目的基因,连接表达载体pET-32a(+),构建重组质粒,转化入感受态细胞BL21(DE3)中,利用IPTG对重组蛋白进行诱导表达,通过SDS-PAGE及Western blot技术对蛋白表达情况进行鉴定。结果显示:经筛选获得了17个肝片吸虫免疫显性抗原基因,其中假定蛋白Fh010935为肝片吸虫特异性抗原基因;扩增得到的Fh010935核苷酸序列大小为144 bp,编码48个氨基酸,A+T含量为55.56%;Fh010935蛋白由1个α-螺旋,2个β-折叠和3个β-转角构成,具有3个抗原表位,推测该蛋白可能具有较好的抗原性;重组蛋白主要以包涵体形式表达,分子量大小约24 ku,可以被肝片吸虫感染阳性血清特异性识别,具有较好的反应原性。提示:假定蛋白Fh010935可作为肝片吸虫病诊断候选抗原,为疾病诊断制剂的开发提供前期基础。     

Effect of parasite burden on the detection of Fasciola hepatica antigens in sera and feces of experimentally infected sheep

The effect of Fasciola hepatica parasite burden on the detection of excretory/secretory (E/S) antigens in sera and feces of experimentally infected sheep was evaluated using a double antibody-based capture enzyme-linked immunosorbent assay (ELISA). Four groups of five sheep each were used. The first three groups were infected with 50, 100 and 200 metacercariae of F. hepatica, and the fourth group remained as non-infected control. On the day of infection and weekly thereafter, serum and fecal samples were taken. ELISA detected F. hepatica E/S antigen levels in serum from the first week post-infection (wpi) and in fecal supernatant from the fourth wpi, which were significantly (p<0.05) higher than controls. F. hepatica eggs were not detected until after the eighth wpi. The correlation between absorbance of E/S antigens in serum with the fluke burden was 0.77 (p<0.0001) and in feces 0.76 (p<0.0001) at 12th wpi. The sensitivity of the assay to detect E/S antigens in serum was 86.6% and in feces 93.3%. It is concluded that the ELISA technique used in this study offers a diagnostic alternative for detecting early infections of F. hepatica in sheep.     

肝片吸虫免疫显性抗原基因的筛选及鉴定

为筛选出特异的肝片吸虫诊断候选抗原,拓展诊断靶标,利用肝片吸虫阳性血清筛选肝片吸虫cDNA表达文库,获得肝片吸虫特异性抗原基因,采用RT-PCR技术扩增目的基因,连接表达载体pET-32a(+),构建重组质粒,转化入感受态细胞BL21(DE3)中,利用IPTG对重组蛋白进行诱导表达,通过SDS-PAGE及Western blot技术对蛋白表达情况进行鉴定。结果显示:经筛选获得了17个肝片吸虫免疫显性抗原基因,其中假定蛋白Fh010935为肝片吸虫特异性抗原基因;扩增得到的Fh010935核苷酸序列大小为144 bp,编码48个氨基酸,A+T含量为55.56%;Fh010935蛋白由1个α-螺旋,2个β-折叠和3个β-转角构成,具有3个抗原表位,推测该蛋白可能具有较好的抗原性;重组蛋白主要以包涵体形式表达,分子量大小约24 ku,可以被肝片吸虫感染阳性血清特异性识别,具有较好的反应原性。提示:假定蛋白Fh010935可作为肝片吸虫病诊断候选抗原,为疾病诊断制剂的开发提供前期基础。     

Vaccine potential of inclusion bodies containing cysteine proteinase of Fasciola hepatica in calves and lambs experimentally challenged with metacercariae of the fluke

Despite intensive research efforts, progress in the development of effective anti-Fasciola hepatica vaccine has not been satisfactory. However, it has been found that cysteine proteinases of F. hepatica are very important candidates for a vaccine antigen because of their role in fluke biology and in the host-parasite relationship. In our previous experiments we found that recombinant cysteine proteinase which we have cloned from adult F. hepatica (CPFhW) can protect rats against the liver fluke infection when administered intramuscularly or when given intranasally in the form of cDNA. In the present experiments we aimed to evaluate the protectivity of the mucosal vaccination in calves and lambs with inclusion bodies containing recombinant CPFhW using different vaccination doses and various sites of antigen delivery. Female calves vaccinated intranasally with two doses of 300 microg of the recombinant CPFhW showed 54.2% protection against the subsequent challenge of 400 metacercariae (mc). Flukes which developed in vaccinated calves showed a reduction of reproductive potential. Male Corriedale lambs vaccinated at the age of 4 months demanded three doses of the antigen to gain 56.5% of protection to a challenge with 250 mc of F. hepatica. Vaccinated animals showed significantly lower blood eosinophil counts. No correlation was found between serum and mucosal IgG or IgA reacting with F. hepatica ES antigens and the protection level.     

Cysteine proteinases Fas1 and Fas2 are diagnostic markers for Fasciola hepatica infection in alpacas (Lama pacos)

Circulating antibody against Fasciola hepatica antigens was determined by enzyme-linked immunosorbent assay (ELISA) and immunoelectrophoresis in alpacas naturally exposed to F. hepatica. Serological assay parameters were established by using sera from eight infected animals and seven controls with no record of this parasitic infection. Excretory--secretory (ES-) products, Fas1- and Fas2-ELISA were used to survey 307 alpacas from a F. hepatica endemic area in the Peruvian Andes. Seroprevalence of F. hepatica infection varied from 56.7, 64.8 and 66.8% measured by Fas1-, Fas2- and ES-ELISA, respectively. The sensitivity for ES-ELISA was 95%, corresponding Fas1- and Fas2-ELISA sensitivity values were 90 and 95%. In this population, 7% of animals were positive for F. hepatica eggs in faeces, other parasites detected were Trichuris sp. (40%), Nematodirus sp. (34.6%), Lamanema sp. (12.8%) and Eimeria sp. (11.8%). The results show that F. hepatica infected animals elicit circulating antibodies against ES, Fas1 and Fas2. Fas2-ELISA may be proposed as a sensitive assay for the immunodiagnosis of fasciolosis in alpacas.     

Immunization of rats against Fasciola hepatica using crude antigens conjugated with Freund's adjuvant or oligodeoxynucleotides

Chronic Fasciola hepatica infection is correlated with the development of a T helper (Th2)-predominant immune response. To determine whether immunostimulatory CpG-containing oligodeoxynucleotides (CpG-ODN) or Freund's complete adjuvant (FCA), known to promote a Th1 (T helper 1) immune responses, could provide protection from F. hepatica infection, total homogenate (TH) of F. hepatica mixed with CpG-ODN or FCA were injected subcutaneously (s.c.) into Wistar rats. A F. hepatica-specific Th1-predominant immune response was induced with CpG-ODN or FCA in lymph nodes of immunized animals. Lymph node cells from TH-CpG-ODN or TH-FCA immunized rats showed increased antigen-specific proliferation with high levels of INFgamma, compared to lymphocytes from rats injected with TH alone. In contrast, these two groups of immunized animals did not modify IL-4 release by draining lymph node cells, when they were subsequently stimulated with TH in vitro. However, a significant reduction in the burden of flukes (76.7%) was only observed in rats immunized with TH-FCA. Conversely, immunization of rats with TH-CpG-ODN did not promote protection against the parasite. Therefore, even though CpG-ODNs and FCA induced Th1 type responses, only FCA provided a significant protection to rats infected with F. hepatica.     

Progress in the development of Fasciola hepatica vaccine using recombinant fatty acid binding protein with the adjuvant adaptation system ADAD

Fatty acid binding proteins (FABP) have been designed as a potential vaccine against fasciolosis. In this work, the immunoprophylaxis of the recombinant Fh15 FABP from F. hepatica (Fh15) in adjuvant/immunomodulator ADAD system was evaluated using mice and sheep challenged with F. hepatica. The ADAD system combines the Fh15 antigen with an immunomodulator (hydroalcoholic extract of Polypodium leucotomos; PAL) and/or an adjuvant (saponins of Quillaja saponaria; Qs) in a water/oil emulsion (30/70) with a non-mineral oil (Montanide). All the infected control mice died by 41-48 days post-infection. The mice vaccinated with ADAD only with PAL+Fh15 present a survival rate of 40-50% and those vaccinated with ADAD containing PAL+Qs+Fh15 had a survival rate of 50-62.5%. IgG1 antibodies were lower in surviving mice in comparison with non-surviving mice. The sheep vaccinated with ADAD PAL+Qs+Fh15 showed lower fluke recovery (43%), less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Thus, the ADAD system using recombinant fatty acid binding proteins from F. hepatica could be a good option to develop vaccines against F. hepatica.     

Immunity of schistosomes using heterologous trematode antigens--a review

This review summarizes the field of cross-protection in schistosomiasis due to other parasitic infections and with subcellular fractions of the parasite trematodes. Regarding parasitic infections, the clearest evidence of cross-protection to Schistosoma mansoni was found with the trematodes, particularly with Fasciola hepatica. Evidence was also presented which demonstrated that the protective F. hepatica worm antigens were those which bound to antibodies to S. mansoni, and that as antigen purification proceeded, smaller amounts were required to obtain significantly high levels of protection. These 2 factors, cross-reactivity and improved protection with increasing antigen purity (and possibly improved immunogenicity), are both supportive of an immunological basis for protection against S. mansoni. A Fasciola/Schistosoma-defined immunity cross-reactive antigen from F. hepatica worms was isolated and designated as FhSmIII(M). An antiserum to this antigen was developed and used as a probe to detect the presence of this antigen (or its determinants) in different extracts of parasitic trematodes. In this manner, it was possible to demonstrate that FhSmIII(M) (or at least some of its determinants) were found on (or in) S. mansoni, S. bovis and Paragonimus westermani. Since mice immunized with P. westermani worm extracts acquire resistance to challenge with S. mansoni cercariae, a common link of cross-protection to the parasitic trematodes is suggested; i.e., FhSmIII(M). Although immunity to schistosomes is undoubtedly multifactorial, the demonstration of a common protective antigen (or determinant) will provide a handle for the evaluation of additional candidate protective antigens.     

PCR diagnosis of Fasciola hepatica in field-collected Lymnaea columella and Lymnaea viatrix snails

Fasciolosis, caused by the trematode Fasciola hepatica, is a zoonosis of economic importance in livestock that is emerging as a chronic disease in humans. The intermediate hosts are lymnaeid snails, in which diagnosis of infection is traditionally based on cercarial shedding, tissue sectioning and crushing. We developed a PCR assay for the sensitive and specific detection of F. hepatica in field-collected Lymnaea sp. snails. A primer pair was designed to amplify a 405 bp fragment of the cytochrome c oxidase subunit 1 gene of F. hepatica. The PCR assay showed a limit of detection of 10 pg of genomic F. hepatica DNA. No cross-reactions were observed with samples from other related trematode species or from the snail hosts Lymnaea columella and Lymnaea viatrix. DNA sequencing of the amplicon showed 100% homology with F. hepatica, and 75-89% homology with other trematodes on regions that did not include the entire set of primers. Two samples from Argentina were analysed. For snails in sample 1 (n = 240), identified as L. columella, the infection rate was 17.5 and 51.3% by direct examination and PCR, respectively. For snails in sample 2 (n = 34), identified as L. viatrix, the infection rate was 2.9 and 61.8% by direct examination and PCR, respectively. Differences in infection rates between these diagnosis methods were significant for both samples. Our PCR technique showed to be effective for detecting specific F. hepatica infections of low intensity in the intermediate host, and hence it could be used to study the epidemiological situation in a given area, as well as to assess host suitability for the parasite.     

Serological responses of cattle after treatment and during natural re-infection with Fasciola hepatica, as measured with a dot-ELISA system

The dot-ELISA reaction was used to study the dynamics of IgG titers in cattle naturally infected with Fasciola hepatica after anthelmintic treatment and during reinfection. Excretion/secretion products (ES) of the parasite were used as antigens for the dot-ELISA. IgG antibodies were no longer detectable by dot-ELISA, 4-6 months after nine animals received the first of three weekly doses of triclabendazole (15 mg kg(-1)) and were then maintained on a pasture free of F. hepatica metacercariae. Six fluke-free cattle began shedding F. hepatica eggs 3-6 months after grazing a pasture contaminated with metacercariae of the parasite. A detectable increase in dot-ELISA IgG antibody levels was observed 2-4 weeks after natural reinfection by grazing a similar pasture contaminated with F. hepatica metacercariae. The usefulness of the dot-ELISA system to diagnose chronic infection by serology is complicated by previous treatment against the parasite. It is concluded that the ES antigens can be useful to detect early infection of cattle with F. hepatica in a dot-ELISA system     

Worm recovery, haemagglutinating antibodies and IgE-levels after immunisation against Fasciola hepatica in rats

Female inbred Hooded Lister (HL) rats were each infected with 20 metacercariae (Mc) of Fasciola hepatica. Remarkable variations between the number of flukes established in the bile ducts suggest the presence of individual, perhaps genetically controlled, differences in immune responsiveness of HL rats to F. hepatica. Serum (4 ml) from HL rats infected with 20 Mc 6 weeks prior to transfer partially protected rats against a F. hepatica challenge infection. However, 1 X 10(6) lymphoid cells originating from rats of the same age and stage of infection did not show the same protective qualities. Furthermore, attempts to immunise HL rats i.p. with either juvenile or adult excretory/secretory (ES) products, or somatic tissue antigens and AlOH3-gel as adjuvant failed. When compared to other investigations, the present results further suggest that both the adjuvant and the route of administration are crucial for the stimulation of a protective immunity to F. hepatica. Low titers and low anamnestic responses of haemagglutinating antibodies after prior immunisation with juvenile ES antigens or both juvenile ES and somatic tissue antigen suggest the occurrence of an immunosuppressive effect caused by juvenile ES products. The total serum IgE-levels in immunised groups were generally lower when compared to the challenge control group, whereas the F. hepatica ES-specific IgE-levels rose after challenge, but immediately decreased again when compared to challenge controls. These findings support the hypothesis of an immunomodulatory effect caused by the vaccination scheme.     

Histopathological and immunohistochemical study of lambs experimentally infected with Fasciola hepatica and Schistosoma bovis

The aim of this study was to investigate the cross-resistance between Fasciola hepatica and Schistosoma bovis in lambs assessing parasitologic, gross pathologic, histopathologic and immunohistochemical changes in liver and small intestine. Thirty Castellana breed lambs were divided into five comparable groups and exposed to F. hepatical S. bovis (group F/S), S. bovis/F. hepatica (group S/F), S. bovis (group S) or F. hepatica (group F) and six unexposed lambs were used as non-infected controls (group C). Primary patent infection with F. hepatica induced a lower number of schistosome eggs and a higher number of lymphocytes in intestinal and liver schistosome egg-induced granulomas in group F/S than in the groups S/F and S, liver damage being mainly attributed to F. hepatica. S. bovis infection followed by challenge with F. hepatica particularly increased the severity of the most significant liver alterations (cholangiohepatitis by F. hepatica and mesoendophlebitis by S. bovis) and F. hepatica seemed not to have an influence on established S. bovis infection. In addition, immunohistochemical results suggested that the predominant local immune response in both double-infected groups was different, being mainly a cell-mediated immune response in group F/S and a mucosal response in group S/F.     

Isolation and purification of Fasciola hepatica eggs

The present egg isolation method is both a rapid and simple technique for recovering large numbers of Fasciola hepatica eggs (1 X 10(7) eggs/gradient) from bovine bile. Bile from infected cattle was first passed through a 45 micron screen sieve. The F. hepatica eggs were collected from the surface of the screen by backwashing with a jet of distilled water. The resultant egg suspension was layered on a 60% to 100% (v/v) linear Percoll gradient prepared in distilled water. Centrifugation at 450 g for 20 min resulted in the formation of 2 visible bands and a pellet. The top band (density of 1.075 g ml-1) contained viscous debris and crystallized bile pigments. The second visible band (density of 1.093-1.099 g ml-1) consisted of a relatively pure population of F. hepatica eggs (greater than 93%) while the pellet contained only F. hepatica egg shells.     

Evaluation of the flukicide treatment policy for dairy cattle in Galicia (NW Spain)

Fasciola hepatica infection is an important cause of lost productivity in livestock worldwide. Effective control of fasciolosis is difficult, especially in milking cows, which can only be treated during dry periods, a control strategy that has not been yet evaluated. In this cross-sectional study, we investigated the effect of the type of flukicide treatment on the prevalence and intensity of infection in dairy cattle from Galicia, an area where fasciolosis is endemic and which is also the main milk-producing region in Spain. Faecal samples were taken from 5188 dairy cows on 275 randomly selected farms for measurement of the concentration of F. hepatica coproantigens by a monoclonal antibody based immunoassay (MM3-COPRO ELISA). On the same day as the sampling, each farm owner/manager was questioned about the types of treatment used on the farm. Three groups of farms were considered according to the fasciolicide treatment: (A) flukicides were not used, (B) an anthelmintic effective against mature stages of flukes was used (albendazole or netobimin) and (C) a fasciolicide effective against immature and mature stages was used (triclabendazole: TCBZ). Results indicated that 16.0% (832/5188) cows from 61.1% (168/275) herds were infected by F. hepatica. The mean coproantigen concentration in infected herds was 13.0ng/ml (range 0.9-112.6ng/ml). The highest individual concentration recorded was 496.6ng/ml. Herd and within-herd prevalences of F. hepatica were similar in all three groups, but surprisingly, individual prevalence and antigen concentration were higher in Group C (p<0.05). The percentage of farms with within-herd prevalences >25% was very high in all three groups, and no significant differences were observed. In contrast, the percentage of herds with mean antigen concentrations >20ng/ml was significantly lower (p<0.05) in Groups A and B (14.4% and 14.9%, respectively) than in Group C (50.0%). The proportion of herds that exceeded both limits (25% for prevalence and/or 20ng/ml for coproantigen concentration) was also significantly higher (p<0.05) in Group C than in untreated animals (Group A). The survey showed that most dairy farmers are unaware of the existence of F. hepatica infection on their farms, and treatments, when given, are administered without prior diagnosis. Treatment with TCBZ administered only at drying off did not show advantages over other measures including no treatment, or treatment with other benzimidazoles. Consequently, TCBZ should only be used to treat individual animals after correct diagnosis of the infection, and correct management measures taken to control re-infection.     

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.     

Partial purification of somatic and excretory-secretory products of adult Fasciola hepatica and their application for the serodiagnosis of experimental and natural fascioliasis using an ELISA

Six partially purified antigen fractions from adult Fasciola hepatica (three somatic tissues [Fhs] and three excretory products [Fhm]) were used in a micro-ELISA to monitor the serum antibody levels of an experimental rabbit F hepatica infection. Fhs 1 detected infection after 19 to 26 days and the titre remained significantly higher than that of the controls until day 103 of infection (end of experiment). Using Fhm 1 and Fhm 2, antibodies were detected between 12 and 19 days after infection. Fhm 2 distinguished infected from uninfected rabbits during the entire experimental period, whereas Fhm 1 did not. Excretory-secretory products of a low molecular weight were also antigenic and could differentiate between infected animals and controls. One hundred and nine sera from naturally infected cattle and uninfected controls were tested with the same antigens. Although antibody was detected, the results were inconsistent and further purification of the antigens may eventually improve the sensitivity of the method.