Humoral immune responses during experimental infection with Fascioloides magna and Fasciola hepatica in goats and comparison of their excretory/secretory products.

This study investigated the humoral immune responses of goats experimentally infected with Fascioloides magna and Fasciola hepatica to F. magna excretory/secretory products (FmESP) or F. hepatica excretory/secretory products (FhESP), respectively. An enzyme-linked immunosorbent assay (ELISA) was used to determine serum antibody responses and for possible discrimination of F. magna and F. hepatica infections in goats. Comparison of ESPs of both flukes and evaluation of ESP antigenicity was also studied applying immunoblotting techniques. In all infected goats, antibody level was significantly increased (against negative control) since 2 weeks post infection (WPI). However, the dynamics of antibodies varied between F. magna and F. hepatica groups during the course of the infection. The cross-reaction of antibodies developed against F. magna and F. hepatica with ESP proteins was recorded by ELISA. The species-specific proteins 40, 120 kDa from FmESP and 80, 160 kDa from FhESP (with no antibody cross-reaction) were detected by two dimensional electrophoresis and immunoblot as the potential immunodiagnostic markers. Our results suggest that F. magna and F. hepatica infection could be distinguished by common immunological techniques based on species-specific antigen-antibodies interaction.

Identification of mouth part antigens of Fasciola gigantica and Toxocara vitulorum and its molecular targets recognized by homologous and heterologous adult anti-sera against adult.

Sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE) fractionation of whole-worm and mouth part antigens of F. gigantica and T. vitulorum showed obvious qualitative and quantitative differences. Three different anti-sera, raised in rabbits against adult extracts of F. gigantica, T. vitulorum and Monieza expansa, were utilized in immunoblotting for identification of mouth part antigens that cross-react with adult worm of the same species or of different species. 7 and 8 poly peptides were recognized in F. gigantica and T. vitulorum mouth parts respectively by their homologous rabbits anti-adult anti-sera. The cross reactive antigens in F. gigantica which recognized by T. vitulorum anti-sera was 234 KD, while two components of M.wt. 113 and 93 KD were detected by M. expansa serum in the same extract. Furthermore, T. vitulorum antigens which cross-reacted with F. gigantica anti-serum 150 and 65 KD and with M. expansa were 65 and 49 KD.

Enzyme-linked immunoelectrotransfer blot analysis of excretory-secretory proteins of Fascioloides magna and Fasciola hepatica.

Fasciola hepatica is a parasite of cattle (Bos taurus), but not of white-tailed deer (Odocoileus virginianus), while Fascioloides magna is a parasite of white-tailed deer which also infects cattle as dead-end host. Adult parasites were collected from naturally infected white-tailed deer or cattle. Excretory-secretory proteins (ESP) were obtained from each parasite. Protein banding patterns were analysed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed using sera from experimentally infected deer of cattle using enzyme-linked immunoelectrotransfer blot (EITB, also known as Western blot) analysis. Protein banding patterns of the two species were different. EITB analysis of Fascioloides magna ESP using sera from Fascioloides magna infected deer or cattle identified three bands of approximately 17, 22 and 27 kDa of which the 27 kDa antigen cross-reacted with sera from Fasciola hepatica infected cattle. EITB analysis of Fasciola hepatica ESP probed with sera from Fasciola hepatica infected cattle identified three bands of approximately 15, 26 and 46 kDa. The 46 and 26 kDa ESP cross-reacted with sera from Fascioloides magna infected cattle, but not with sera from Fascioloides magna infected deer. The band at 15 kDa which reacted specifically for Fasciola hepatica infected cattle sera consisted of two protein bands close to each other as seen on the SDS-PAGE gel. The EITB reaction at approximately 17 kDa and 22 kDa of Fascioloides magna ESP, and at approximately 15 kDa of Fasciola hepatica ESP can be used for species specific diagnosis.

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.