Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection

Cathepsin L cysteine proteinase from Fasciola gigantica was evaluated for its potential in the early prepatent detection of this helminth infection in bovine calves. Five cross-bred bovine calves were experimentally infected with 400 metacercariae/calf and evaluated for anti-cathepsin L antibody response. F. gigantica infection in these calves could be detected 4 weeks post-infection using an ELISA, dipstick ELISA and Western blotting with 100% sensitivity. The antigen was also used to detect F. gigantica field infection in cattle, by screening 256 sera of these animals by an ELISA, which demonstrated an overall infection rate of 26.95%. Preliminary studies showed that F. gigantica cathepsin L cysteine proteinase does not cross-react with Paramphistomum epiclitum, Gigantocotyle explanatum and hydatid cyst antigens. However, extensive studies on the cross-reactivity of this antigen with related helminth parasites of cattle and buffaloes are required, before this antigen can be considered suitable for immuno-diagnosis of fasciolosis in these ruminants.     

Evaluation of cercarial antigen for the serodiagnosis of fasciolosis in experimentally and naturally infected sheep

The value of cercarial antigen for diagnosis of experimental and natural sheep fasciolosis was studied by enzyme linked immunosorbent assay (ELISA) and enzyme linked immunotransfer blot (EITB). In ELISA, the antibody levels of experimentally infected sheep with Fasciola gigantica appeared at 2 weeks post infection (PI), gradually increased till 7 weeks PI and nearly remained at the same level from 7 to 13 weeks PI (the end of experiment). Also, the sensitivity and specificity of cercarial antigen for diagnosis of naturally sheep fasciolosis were 100 and 90%, respectively.In EITB, in the sheep experimentally infected with F. gigantica, the band of 32.5kDa molecular weight polypeptide appeared at 2 weeks PI and continued till the end of experiment. Also, the cercarial antigen recognized 32.5kDa molecular weight band with all sera from naturally infected sheep with fasciolosis (n = 25). This band did not cross-react when tested with sera from infected sheep with Cysticercus tenuicollus (n = 20). This study suggests that, the 32.5kDa molecular weight polypeptide could be used as sensitive and specific epitope for the serodiagnosis of sheep fasciolosis.     

28 kDa Fasciola gigantica cysteine proteinase in the diagnosis of prepatent ovine fasciolosis

Coprological confirmation of ovine fasciolosis in the field, prior to out breaks of the disease and/or strategic antifluke medication, seem to be of little consequence. Efforts are, therefore, being made to evolve a putative antigen specific to serodiagnostic test for early diagnosis during prepatency. In the present investigation, 28 kDa cysteine proteinase was used in ELI SA and Western blot to detect Fasciola gigantica antibodies and further Dipstick-ELISA was developed for field application, using known positive monospecific sera from experimentally infected sheep with 100 F. gigantica metacercariae. Isolation of 28 kDa cysteine proteinase was achieved from bubalian origin flukes. The specific antigen, recognised homologous antifluke antibodies by Western blot as early as 2nd week post-infection (wpi) with 100% sensitivity, in sera samples of sheep harbouring 38 flukes and by 10th wpi in sheep harbouring 3-8 flukes. All sheep were found positive for the infection when ELISA and/or Dipstick-ELISA was applied from 4th wpi. In pooled sera of infected sheep, these were positive during 4th wpi.     

27 kDa Fasciola gigantica Glycoprotein for the Diagnosis of Prepatent Fasciolosis in Cattle

An indirect enzyme-linked immunosorbent assay (ELISA) using 27 kDa glycoprotein of Fasciola gigantica has been evaluated for its potential use in the diagnosis of bovine fasciolosis. Following experimental infection of rabbits, F. gigantica infection-induced antibodies were isolated and later used as ligands in affinity chromatography for isolation of infection-induced antibody-specific proteins. Among the five infection-specific proteins isolated, a glycoprotein of 27 kDa was later isolated by second-step purification using concanavalin A matrix. In crossbred cattle receiving different doses of infection (100, 200 and 400 metacercariae), the anti-27 kDa antibodies were detected as early as the 2nd week post infection. No direct correlation between initial dose, antibody response and fluke establishment was recorded. No cross-reaction was noted with the sera of goats experimentally infected with Paramphistomum epiclitum. ELISA with the 27 kDa glycoprotein could be a feasible diagnostic tool for the early detection of bovine fasciolosis.     

Immunoglobulin E recognition of Dirofilaria immitis antigens is more specific than immunoglobulin G.

The chronological development of the serum IgE and IgG response to microfilaria, third and fourth stage larvae, and male and female adult Dirofilaria immitis was examined by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunoelectrotransfer blot (EITB). Dirofilaria immitis-specific IgE and IgG levels peaked 16-18 weeks post-infection after increasing in response to the fourth larval molt. Specific IgG levels plateaued after patency, while IgE continued to decline. The use of ammonium sulfate cut sera showed there was no quenching or blocking of IgE binding by IgG in the ELISA and EITB methods used in this study. IgE-specific EITB showed 30-49 bands for the five respective extracts that were identified by M(r) or relative mobility. Eighty-five to 100 bands were visualized by IgG-specific EITB for the same five extracts. The isotype-specific ELISA and EITB were shown to be closely related by significant correlations (P < 0.0001) between S/N ratios and the number of bands found on blots. The isotype-specific EITB bands non-specifically recognized were greater in size than 21 kDa for IgG and 45 kDa for IgE. Recognition of bands changed over time with some bands being recognized only by prepatent sera. Ten antigen bands of seven M(r) were consistently and specifically recognized by IgE in the five-stage extracts by sera from prepatent and patent infections; only one such M(r) at 13.9 kDa, was described for IgG. A potentially diagnostic 31.9 kDa antigen band was identified on the IgE-specific EITB of D. immitis female extract and was shown to be recognized by IgE in sera from all infected dogs at all time points examined from 2 weeks until 1 year post-inoculation. Overall, IgE reactivity was more specific for D. immitis infections than IgG reactivity.     

Preparation and evaluation of the specificity of Parafilaria bovicola antigen for detection of specific antibodies by ELISA

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in bovine sera against Parafilaria bovicola nematodes was developed and its sensitivity was compared with the immunodiffusion (ID) method. An exoantigen of P. bovicola which was shown to contain four major polypeptides was used in these procedures. The serological reactivity of the antigen polypeptides was defined by using the enzyme-linked immunoelectrotransfer blot technique (EITB) and whole-worm extract proteins. It identified only four serologically reactive polypeptides with sera from one experimentally infected calf and a verified field case. These two positive sera reacted mainly with four major antigens which coincided in molecular weights of the polypeptides of the exoantigenic preparation, namely, 43, 39, 28 and 25 KDa. Calves experimentally infected with P. bovicola showed a positive reaction with ELISA at 4 months after inoculation, and after this period a rapid increase in serum antibody response occurred. In these cases the ID reaction was observed for the first time at 7 months after inoculation. The specificity of an ELISA method using crude exoantigen preparation of P. bovicola was tested for the diagnosis of bovine parafilariasis. No cross-reactivity was detected when the P. bovicola exoantigen preparation was tested against sera from calves experimentally infected with Onchocerca lienalis, as well as against the sera from cattle naturally infected with Dictyocaulus viviparus or from cattle chronically infected with Ostertagia ostertagi. In addition, testing of 740 field sera from cattle in areas non-endemic and endemic for P. bovicola indicated a specificity of the antigen preparation used. Forty sera from laboratory-confirmed field cases of P. bovicola infection were tested by ELISA and immunodiffusion. All of these sera were ELISA positive, whereas only 70% of these were positive in the ID test. Seven (2.1%) of 328 sera from 21 herds from non-endemic P. bovicola areas were ELISA positive, as opposed to none in the ID test. Of the 94 sera from six herds in areas endemic for P. bovicola infection, 51 (54%) were ELISA positive whereas only 24 (26%) were positive in the ID test. When 56 slaughtered cattle, with varying degrees of meat condemnations due to parafilariasis, were tested for P. bovicola specific antibody, 91% of the serum samples were positive by ELISA. These results suggest that the exoantigen of P. bovicola can be used in a sensitive and reliable serological detection of parafilariasis by ELISA.     

Antibody response of calves to a single infection of Fasciola gigantica determined by an indirect fluorescent antibody technique.

The antibody response of six calves infected with 500 metacercariae of Fasciola gigantica each was monitored throughout 30 weeks of infection using an indirect fluorescent antibody technique (IFA). In vitro excysted F gigantica were employed as the test antigen. All animals showed high antibody titres from two to six weeks post-infection. Thereafter the antibody titres diminished gradually. Although all the experimentally infected animals harboured fluke burdens at autopsy, most gave negative IFA tests from 22 weeks post infection onwards. Specific immunofluorescent staining occurred on the surface glycocalyx of the newly excysted flukes. It is likely that this glycocalyx provides one of the earliest antigenic stimuli for the host's immune reactions to fascioliasis.     

Detection of stable diagnostic antigen from bile and feces of Fasciola hepatica infected cattle.

Diagnostic antigens in bile and feces from Fasciola hepatica infected cattle were detected and characterized by enzyme-linked immunotransfer blot (EITB) techniques. As sources of antigen, samples of bile, intestinal contents and feces were collected from five uninfected calves and from 10 calves with known Fasciola hepatica burdens. A band detected by EITB using a densitometer in the area corresponding to 26 kDa reacted with rabbit anti-fresh fluke antigen and infected cattle sera but not with fluke-negative rabbit sera, rabbit anti-Fasciola hepatica egg sera, Fascioloides magna positive or negative cattle sera. This band was not detected by Coomassie blue in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels or by Ponceau-S stained nitrocellulose strips. Band groups located at 104-66, 66-42, 42-26 and 25-16 kDa reacted inconsistently with the above sera. Sera from mice hyperimmunized with Fasciola hepatica excretory-secretory (ES) products detected only the 26 kDa band by EITB, without cross-reactivity with bands in the other molecular weight (MW) ranges. The results suggest that the 26 kDa antigen may consist of a stable component of ES products and/or tegument-related worm antigen. Diagnosis of Fasciola hepatica through detection of specific, stable antigens in feces of infected animals offers potential advantages over serum-based tests of better sample accessibility, discrimination between previous and current infections, and possible semi-quantitation of fluke burdens.     

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.     

Humoral and cellular immune responses to Fasciola gigantica experimental infection in buffaloes

Humoral and cellular immune responses to Fasciola gigantica experimental infection in buffaloes were studied. The results showed that 33.4+/-9.1% of the infection dose was recovered as adult flukes from infected animals at necropsy. Significant differences of weight gain between infected and non-infected buffaloes was observed at 4 MPI (months post-infection). Anti F. gigantica excretory-secretory products (FgESP)-IgG levels increased significantly from 3 WPI (weeks post-infection) and displayed a peak at 13 WPI. Western blot indicated that in FgESP six major bands of 11.5, 19.0, 23.4, 29.8, 47.5 and 53.2kDa were recognized by F. gigantica-infected buffaloes sera after 0 WPI. Eosinophil numbers increased significantly from 3 WPI in F. gigantica-infected buffaloes and displayed a peak at 8 WPI. Peripheral blood mononuclear cells (PBMC) proliferation induced by FgESP increased from 2 WPI with a peak at 5 WPI. IFNgamma secretion by FgESP-stimulated PBMC appeared early from 1 WPI with three peaks at 2, 5 and 8 WPI, respectively. IL-10 production was observed from 2 WPI with two peaks at 4 and 9 WPI, respectively. Our results suggested that buffaloes were highly susceptible to F. gigantica infection, and this susceptibility could be associated with the late and weak cellular immune response in the early phase of infection and the Th0-like response throughout the infection.     

Interferon-gamma and interleukin-4 expression during Fasciola gigantica primary infection in crossbred bovine calves as determined by real -time PCR

Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) expression in crossbred (Bos taurusxBos indicus) bovine calves during primary infection with Fasciola gigantica was measured. Ten crossbred calves of 1-year age were divided into two groups of five calves each, group I uninfected control and group II calves orally infected with a dose of 1000 metacercariae of F. gigantica. The two cytokines were measured 10, 30 and 75 days post-infection (PI) by real-time polymerase chain reaction (RT-PCR) with the double stranded DNA-binding dye SYBR Green. IL-4 was present in detectable levels in peripheral blood mononuclear cells (PBMCs) of infected animals at 10, 30 and 75 days PI but no IFN-gamma was detected in PBMCs of infected animals at 10 and 30 days PI. However, at 75 days PI, IFN-gamma in two infected animals was present in detectable level. Eosinophil count increased from 2nd fortnight after infection and the increased level persisted till the termination of experiment. Present study indicated that T-cell response during F. gigantica infection was Th2-type during earlier phase of infection, which may be polarized in chronic infection to that of a Th0-type.     

Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fa(g)) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fa(g) and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.     

Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica.

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fag) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fag and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.     

囊虫病猪血清CA效价与虫负荷的相关性及CA与Ab的动态观察

应用ELISA双抗体夹心法检测囊虫病猪血清中的循环抗原(CA),对CA效价与虫负荷的相关性作了初步观察,发现两者密切相关.轻度感染猪血清CA效价≤000,中度为1000～10000,重度为10000～100000.人工感染病猪血清一般在感染后1周检出CA,于感染后4～5周达高峰;抗体一般于感染后2周检出.这种方法可检出的最小虫负荷约为20个/猪     

伊氏锥虫变异表面糖蛋白抗原双抗体夹心ELISA检测方法的建立及初步应用

A double antibody sandwich ELISA for detection of Trypanosoma evansi variant surface glucoprotein (VSG) antigen was established by using two monoclonal antibodies against Trypanosoma evansi VSG antigen.The result of sensitivity showed when positive serum was diluted to 3200 doubles, its D_{450 nm} value was still greater than the critical value of positive and negative,and specificity test result showed that there was no cross reaction with the antigens of Theileria,Toxoplasma gondii,Chlamydia and Fasciola gigantica.82 clinical sera were detected by this double antibody sandwich ELISA and its positive rate was 9.76%.The experiment results demonstrated that this double antibody sandwich ELISA for detection of Trypanosoma evansi VSG antigen had good detecting sensitivity and specificity,and could be used as an effective method for clinical detecting buffalo Trypanosoma evansi disease.     

Development of an antibody-detection ELISA for Fasciola hepatica and its evaluation against a commercially available test

An ELISA was developed for the detection of Fasciola hepatica antibody in serum of cattle. The assay was applied to sera from 258 naturally infected cattle, 256 non-infected cattle and six calves experimentally infected with F. hepatica. The diagnostic sensitivity and specificity of the ELISA test was 98% (95% confidence intervals, 96-100%) and 96% (95% confidence intervals, 93-98%) respectively at a cut-off value of 15% positivity. The results using sera from the experimentally infected calves showed that antibodies were first detected 2-4 weeks after infection. The ELISA test was also compared to the commercially available Bio-X bovine F. hepatica ELISA kit. A subset of 39 positive sera and 47 negative sera were selected from the samples used to evaluate the in-house test. The results indicated that the agreement between the two tests was almost perfect (k statistic=0.82).     

Immune response and antigen recognition in non-pregnant ewes experimentally infected with Neospora caninum tachyzoites

The cellular and humoral responses as well as the antigen recognition during the acute stage of a Neospora caninum (NC) infection were investigated in non-pregnant ewes. The experimentally infected ewes developed specific lymphoproliferative and humoral responses within 2 weeks post-infection (PI). The magnitude of the cellular response showed large variations between animals. A significant decrease in the proliferative response to Con A mitogen and N. caninum, Toxoplasma gondii (TG) antigens was recorded on day 21 post-infection (PI). The humoral response and the pattern of antigen recognition were similar among infected ewes. Proteins of 44, 42, 40, 39 and 28 kDa were intensively recognized by the infected animals during the experiment. The 42 and 28 kDa antigens should be considered as useful for the diagnostic of N. caninum infection, as the intensity of recognition infection of the other antigens had decreased markedly 8 weeks post-infection. For some antigens a sequential recognition was recorded. The 59, 54 and 38-37 kDa proteins were frequently recognized by infected sera during the first weeks of the infection, but recognition of these antigens was absent or rare at the end of the experiment. These antigens could be related to the acute stage of the infection.     

Characterization of Brucella canis protein antigens and polypeptide antibody responses of infected dogs

The cytoplasmic protein antigens (CPAg) of Brucella canis were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analysis of 35S-labeled polypeptides. Approximate molecular weights of the immunoreactive polypeptides were determined by migration patterns of the immunoprecipitated polypeptides after SDS-PAGE or Western immunoblotting of sera collected at various times after experimental infection of dogs. Polypeptides were specifically precipitated by sera of infected dogs, but not from the sera of normal or false-positive (seropositive, non-infected) animals. During the initial month after infection, proteins with molecular weight masses (MW) of approximately 18, 22, 31, 42 and 54 kDa were commonly recognized. A 20-kDa polypeptide was first recognized at 8-10 weeks after infection, but it was detected inconsistently after 6 months. Additional polypeptides detected from 2 to 12 months post-infection had MW of 22, 66-68 and, less regularly, 42, 60, 82, 100 and greater than 200 kDa. The polypeptides most consistently recognized in sera from B. canis-infected dogs had MW of 18, 22 and 68 kDa.     

Primary experimental infection of riverine buffaloes with Fasciola gigantica.

The clinical course of the primary experimental Fasciola gigantica infection was investigated in riverine buffalo calves of the Murrah breed. Nine male calves aged 12-15 months were randomly assigned to two groups of five (Group I) and four (Group II) animals. Each animal in Group I, was orally infected with 1000 metacercariae (mc) of F. gigantica, whereas Group II animals did not receive any infection dose and served as uninfected controls. No clinical signs of fasciolosis were observed until the sixth week post-infection (PI). Group I animals, however, developed recognised symptoms of acute fasciolosis, comprising apyrexic inappetance, anemia, poor weight gain, diarrhoea and sub-mandibular and facial oedema, respectively, from 5, 6, 8, 16 and 17 weeks PI. The signs were intermittent in nature and of variable duration. The prepatent period was of 92-97 days (mean 95.2 +/- 3.1). One of the five infected animals died on Day 147 PI. At necropsy, 36.8 +/- 11.0% of the infection dose was recovered as adult fluke population. The gross lesions were primarily biliary in nature. Group II, the uninfected controls, throughout the study period of 165 days PI, did not show any symptom and were negative for F. gigantica. The study demonstrated that the onset of adverse effects of F. gigantica on the growth and health of the infected host was mainly noted during late prepatency much before coprological prediction and diagnosis. The significance of preventive therapy against fasciolosis during prepatency has been stressed in endemic areas.     

IgG response against infective larvae of Dirofilaria immitis in experimentally infected cats

Somatic antigens from third stage larvae of Dirofilaria immitis (SL3) were used to detect IgG response against heartworm infection in 8 experimentally infected cats. A moderate specific anti-SL3 IgG response was found one month post-infection. Afterwards, antibodies decreased reaching a basal level 4 months post-infection and remained at this level until the end of the study. 6 months post-infection. Western blot analysis showed specific recognition of polypeptides of 79, 73, 60, 52, 40 and 39 kDa by sera from infected cats 1 month post-infection, but not by sera taken prior to the infection. The low antigenicity of the SL3 antigen in the cat should allow the parasite to escape the host's immune response.