Precipitin responses of infected mice to exoantigens and cellular antigens of Trypanosoma musculi.

Plasma were collected from mice which had been immunosuppressed with 650 R from a cobalt-60 gamma radiation source and infected with Trypanosoma musculi. Trypanosomes were also collected from immuno-suppressed mice and from nonirradiated, infected animals. Rabbit antiserum was prepared against trypanosomes fron nonirradiated mice and employed in immunodiffusion analyses to detect trypanosome exoantigens (ExAg) in plasma of irradiated, infected mice and cellular antigens (CAg) in extracts of parasites which had been collected from immunosuppressed and nonirradiated hosts. The rabbit antiserum formed at least 3 precipitin lines with plasma from irradiated, infected mice and 8–9 precipitin lines with extracts of parasites which were obtained from immunosuppressed and untreated mice. Two of the precipitin reactions were against mouse plasma antigens (PAg). Lower levels of PAg appeared to be present in extracts of trypanosomes which were isolated from the irradiated mice than in those from nonirradiated animals.Mice synthesized antibodies against 1 ExAg which was demonstrable in immunodiffusion tests by 14 days after T. musculi infection. A single precipitin reaction was also seen after 21 days. One to 2 precipitin lines were formed with ExAg after 42 days of infection. Two to 3 precipitin lines formed between the ExAg and mouse antisera collected 98, 175 and 341 days after injection of the T. musculi.Similar immunodiffusion reactions were detected with CAg present in both the extracts of T. musculi which had been isolated from irradiated and those from nonirradiated mice and the mouse antisera. One to 2 precipitin lines were found between CAg and antisera from mice which had been infected for 14 days. Two precipitating antigen-antibody systems were seen with antisera collected after 21, 42 and 98 days and 2–3 precipitin reactions were formed between CAg and antisera collected from mice 175 and 341 days after infection.Absorption and immunodiffusion analyses conducted with rabbit and mouse antisera indicated parasite ExAg in plasma of irradiated, T. musculi infected mice were also present in preparations of CAg of the trypanosomes. The persistence of antibody and the increase in the numbers of antigen-antibody systems detected by immunodiffusion during the course of the infection may in part be related to the presence of parasites in capillaries of the kidneys long after they cannot be demonstrated in the peripheral blood of the host.     

Dipetalonema viteae: primary, secondary and tertiary infections in hamsters.

Hamsters were given primary infections of 100, 200, and 300 D. viteae larvae and groups killed at various intervals after infection. In addition, hamsters were sequentially infected with 100, 200, and 300 larvae and groups killed at 100 or 75 days after the secondary and tertiary infection, respectively. Blood microfilariae were detected on Day 60 following a primary infection, reached a maximum on Day 75, declined to low levels by Day 105, and were negative on Day 120. No microfilariae reappeared in the blood of hamsters given secondary or tertiary infections.Between 20–30% of the infecting larval dose had reached the adult stage by Days 75 or 100 postinfection in hamsters given primary, secondary, or tertiary infections. There was no evidence of arrested larval development in hamsters receiving a second or third challenge infection. Almost half of the tertiary infection hamsters developed subcutaneous nodules and their numbers varied greatly among individual animals. The nodules variously contained living worms, pus, and fragmented worms, or pus only. Hamsters given primary infections of 100, 200, or 300 larvae and killed 375 days after infection had no subcutaneous nodules; however, hamsters given the 200 and 300 larval infections were seen to have dead worms in the subcutaneous tissues. No stunting of adult worms was noted and all female worms had uteri packed with microfilariae.     

Nippostrongylus brasiliensis: effects of immunity on the pre-intestinal and intestinal larval stages of the parasite

Nippostrongylus brasiliensis: effects of immunity on the pre-intestinal and intestinal larval stages of the parasite. International journal for Parasitology4: 183–191. Migration of the pre-intestinal larval stages of N. brasiliensis was studied in rats undergoing either primary or challenge infections. In rats undergoing a primary infection, more than 67 percent of larvae successfully migrated from the skin to the oesophagus by 70 h after infection, and subsequently over 90 per cent of these larvae became established in the small intestine as sexually mature adults. In immune rats undergoing a second infection, 46 per cent of larvae completed migration to the oesophagus by 70 h and of these, only 1·6 per cent became established in the intestine to produce eggs. These inhibitory effects on the pre-intestinal and intestinal larval stages were even more pronounced in immune rats undergoing a third or fourth infection and in addition, there was a prolonged sojourn and substantial retention of larvae in their lungs. There was no evidence that the immune response had an adverse effect on oesophageal fourth stags larvae as these organisms (obtained from immune donors) were able to establish and develop to maturity when transferred per os to normal animals.Syngeneic transfer of immune mesenteric lymph node cells to normal recipients, caused expulsion of parasites from the intestine but failed to effect migration of pre-intestinal larval stages. The implications of these findings are discussed in the context of current knowledge of the mechanisms of immunity to helminths.     

Uniformity of protective antigens among isolates of the cattle tick, Boophilus microplus

Abstract. Gut membrane antigens were extracted from ten isolates of the cattle tick Boophilus microplus; the antigen extracts were probed with bovine antisera and three murine monoclonal antibodies (mAbs) in Western blots and dot-ELISA. The antisera had been obtained from cattle which were vaccinated with larval and gut extracts of B.microplus , and which were subsequently protected (84% and 94% respectively) against challenge with B.microplus. One of the mAbs (QU13) has been demonstrated to precipitate protective antigens from the midgut of B.microplus. Gut antigens from all ten isolates displayed similar reactivity profiles against bovine antisera and also against mAbs in Western blots. The end-point titres of antigens in dot-ELISA showed four-fold variation between isolates against bovine antisera, and also against mAb QUI 3. Larval membrane antigen extracted from N-strain B.microplus reacted with QU13 in dot-ELISA, indicating that protective antigens are common to both larval and adult stages of B.microplus. It was concluded that protective antigens recognized by QUI3 and antigens recognized by sera from protected cattle were conserved between the ten isolates examined, and between life-cycle stages.     

Pupal and larval cuticle proteins of Drosophila melanogaster

Proteins, soluble in 7 M urea, were extracted from third-instar larval and pupal cuticles of Drosophila melanogaster. Both extracts contain a limited number of polypeptides resolved by one- or two-dimensional electrophoresis. The five major larval proteins have low molecular weights (less than 20000) and are not glycosylated. The major pupal cuticle proteins fall into two size classes: two with apparent molecular weights of 56K and 82K and four with molecular weights between 15K and 25K. The proteins with high apparent molecular weights are glycosylated. In nondenaturing gels, no components of the larval and pupal cuticle extracts comigrate. One-dimensional "fingerprints" indicate that cuticle proteins from these two stages have unique primary structures. Immunological results indicate that the major low molecular weight larval and pupal cuticle proteins are comprised of two families of proteins that share antigenic determinants. The high molecular weight pupal cuticle proteins are immunologically unrelated to the low molecular weight components. We conclude that the pupal and larval proteins are encoded in part by multigene families that have arisen by gene duplication and evolutionary divergence.     

Correlation between gut hypersensitivity and resistance to Trichostrongylus colubriformis infection of outbred and inbred lines of guinea pigs

The relationship between gut sensitivity and immunity to challenge infection was examined in outbred and inbred guinea pig lines. Primary infections terminated at 3,6,9 or 13 days and multiple infections of 3 days' duration confirmed the importance of direct gut stimulation and the period of exposure in the induction of immunity and gut hypersensitivity. The studies with the multiple 3-day infections confirmed that the third-stage larvae alone are capable of inducing strong protective immunity and showed that this is accompanied by pronounced gut sensitivity to parasite extracts and secretions. Finally, two inbred guinea pig lines selected for enhanced resistance or susceptibility to T. colubriformis infection displayed corresponding high or low capacities to mount hypersensitivity reactions following a single truncated 3-day primary infection.     

Studies on the specific immunodiagnosis of larval cestode infections of cattle and sheep using antigens purified by affinity chromatography in an enzyme-linked immunosorbent assay (Elisa)

Craig P.S. and Rickard M.D. 1981. Studies on the specific immunodiagnosis of larval cestode infections of cattle and sheep using antigens purified by affinity chromatography in an enzyme-linked immunosorbent assay (ELISA). International Journal for Parasitology11: 441–449. Crude somatic or cyst fluid extracts prepared from Taenia saginata, T. hydatigena or Echinococcus granulosus were partially purified by absorption against homologous and heterologous bovine or ovine antisera on immunoabsorbent affinity columns. Antigens in parasite extracts which were eluted after binding to the homologous anti-parasite antisera (bovine or ovine) coupled to CNBr-activated Sepharose were then passed sequentially through affinity columns containing heterologous anti-parasite Ig and the ‘run-through’ antigens collected. The level of cross reactions to these absorbed antigens, in an enzyme-linked immunosorbent assay (ELISA) using sera from cattle or sheep given heterologous parasite infections (including Fasciola hepatica), were significantly decreased. Absolute specificity was not achieved, and some loss in sensitivity occurred. The absorption of cross-reactive antigen(s) using affinity Chromatographie techniques may be a useful first step in the production of species-specific immunodiagnostic antigens for larval cestode infections.     

Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation ofManduca sexta

Summary During the final larval instar the epidermis of the tobacco hornworm,Manduca sexta, synthesizes the larval cuticular proteins and the pigment insecticyanin. Then at the onset of metamorphosis the cells first become pupally-committed, then later produce the pupal cuticle. The changes in the pattern of epidermal protein synthesis during this period were followed by incubating the integument in vitro with either³H-leucine or³⁵S-methionine, then analyzing the proteins by 2-dimensional gel electrophoresis. Precipitation by larval and pupal cuticular antisera and by insecticyanin antibody identified these proteins. Three distinct changes in epidermal protein synthesis were noted: 1) Stage-specific proteins, some of which are larval cuticular proteins, appear just before and during the change of commitment on day 3. (2) By late the following day (wandering stage), synthesis of these and many other proteins including all the identified larval cuticular proteins and insecticyanin was undetectable. Several noncuticular proteins were transiently synthesized by this pupally committed cell during wandering and sometimes the following day. (3) During the production of pupal cuticle a new set of pupal-specific cuticular proteins as well as some common cuticular proteins (precipitated by both antisera) were synthesized. Some of the latter were also synthesized during the period between pupal commitment and pupal cuticle deposition.In spite of an apparent absence of methionine in both larval and pupal cuticle, many cuticular proteins incorporated³⁵S-methionine. Thus they may be synthesized as proproteins.Insecticyanin was shown to have two forms differing in isoelectric point, the cellular form being more acidic than the hemolymph form. Synthesis of the cellular form ceased before that of the hemolymph form.     

Echinococcus multilocularis: identification of proteins inducing antibody formation in metacestode infection

An approach to the identification of parasite proteins which are immunogenic in natural infections is described, using the infection with the larval stage of Echinococcus multilocularis as a parasite model. Metacestode proteins were separated by SDS-polyacrylamide gel electrophoresis, and transferred electrophoretically to nitrocellulose sheets (Western blotting). Subsequently, immune recognition of the proteins was performed with various host sera and antigen-antibody complexes were detected enzymatically. Using homologous antisera, different patterns of immunogenic bands were revealed by sera of different host species. Cross-reactions with sera from individuals infected with unrelated helminths were analysed. Four proteins of E. multilocularis which failed to show any cross-reaction were identified.     

Characterization of Protective Immune Responses Induced by Pneumococcal Surface Protein A in Fusion with Pneumolysin Derivatives

Pneumococcal surface protein A (PspA) and Pneumolysin derivatives (Pds) are important vaccine candidates, which can confer protection in different models of pneumococcal infection. Furthermore, the combination of these two proteins was able to increase protection against pneumococcal sepsis in mice. The present study investigated the potential of hybrid proteins generated by genetic fusion of PspA fragments to Pds to increase cross-protection against fatal pneumococcal infection. Pneumolisoids were fused to the N-terminus of clade 1 or clade 2 pspA gene fragments. Mouse immunization with the fusion proteins induced high levels of antibodies against PspA and Pds, able to bind to intact pneumococci expressing a homologous PspA with the same intensity as antibodies to rPspA alone or the co-administered proteins. However, when antibody binding to pneumococci with heterologous PspAs was examined, antisera to the PspA-Pds fusion molecules showed stronger antibody binding and C3 deposition than antisera to co-administered proteins. In agreement with these results, antisera against the hybrid proteins were more effective in promoting the phagocytosis of bacteria bearing heterologous PspAs in vitro, leading to a significant reduction in the number of bacteria when compared to co-administered proteins. The respective antisera were also capable of neutralizing the lytic activity of Pneumolysin on sheep red blood cells. Finally, mice immunized with fusion proteins were protected against fatal challenge with pneumococcal strains expressing heterologous PspAs. Taken together, the results suggest that PspA-Pd fusion proteins comprise a promising vaccine strategy, able to increase the immune response mediated by cross-reactive antibodies and complement deposition to heterologous strains, and to confer protection against fatal challenge.     

Immunization of sheep against infection with larvae of the blowfly Lucilia cuprina

Sheep were repeatedly exposed to a second stage excretory-secretory antigen preparation of Lucilia cuprina by intradermal injection (S group) or intranasal aerosol (I group) in an attempt to induce immunity to the larvae. Hypersensitivity responses to the injections were monitored and correlated with larval numbers at subsequent challenge. Intradermal injections showed that the immediate or IgE-mediated and the intermediate or Arthus response were the major skin hypersensitivity reactions to the larval antigens. At challenge there was no significant reduction in larval numbers between the S and the control group, however the Arthus reaction did show some correlation with larval recoveries in the S group. There was a significant reduction in larval numbers (P < 0.005 Mann-Whitney U Test) between the I and the control group. In addition, the animals which showed respiratory hypersensitivity to the aerosol during the immunization period had the lowest larval recoveries. The results of a second challenge in the I group did not show continued protection. It is suggested that the protective response was suppressed by exposure to antigens at the first challenge infection. Exudate samples recovered during the infection were analysed for protein amount and the results were correlated with larval survival. They suggested that two separate mechanisms of resistance are operating in this experiment. The first occurs early in the infection, is probably associated with immediate hypersensitivity, and may control the initiation of protein leakage. The second occurs later in the infection and may result in the leakage of proteins able to control larval survival.     

Trypanosoma lewisi: effects of immunosuppression on the serological reactivities of exoantigens and cellular antigens of bloodstream parasites.

Groups of rats were immunosuppressed with antithymocyte serum (ATS) and infected with Trypanosoma lewisi. Immunodiffusion studies were performed which demonstrated that trypanosome exoantigens, present in the plasma of these animals, were precipitated by antibodies in the sera of rats undergoing a typical primary T. lewisi infection; extracts of trypanosomes which had been collected from ATS-treated rats contained antigens which also were precipitated by antibodies in these sera. These precipitating antibodies could not be detected using either the plasma of untreated infected rats or extracts of trypanosomes which had been collected from untreated rats. With the exoantigens, precipitating antibodies were detected in serum samples collected from rats 14 to 250 days after infection. With the extract, precipitating antibodies were found as early as 5 days after infection and could be detected as late as 90 days after infection. Antigens of trypanosome extracts partially blocked the precipitin reactions between antisera and exoantigens, suggesting the presence of common antigens in the two preparations. Intact trypanosomes were serologically more reactive when collected from immunosuppressed rats. Trypanosomes collected from ATS-treated rats were agglutinated by antisera at titers fourfold higher than trypanosomes collected from untreated hosts. Absorption with exoantigens from immunosuppressed infected rats blocked trypanosome agglutination, indicating that these antigens are of cell surface origin. The experiments suggest that a likely result of immunosuppressing the host is a trypanosome antigen preparation that is a more reactive serodiagnostic reagent.     

Midgut amylase,lysozyme, aminopeptidase,and trehalase from larvae and adults of Musca domestica

Based on polyacrylamide gel electrophoresis, density-gradient ultracentrifugation and thermal inactivation, there is only one major molecular species of each of the following larval enzymes (soluble in water or solubilized in Triton X-100): membrane-bound aminopeptidase (pH optimum 8.5; K_m 0.21 mM L-leucine p-nitroanilide; M_r 322,000), amylase (pH optimum 6.5; K_m 0.14% starch; M_r 66,000), lysozyme (pH optimum 3.5; K_m 0.3 mg/ml; Mr 24,000); and membrane-bound trehalase (pH optimum 5.0; K_m 1.09 mM trehalose; M_r 94,000). Except for lysozyme, the properties of adult digestive enzymes are different from those described for larval enzymes. Larval aminopeptidase and trehalase were purified by electrophoresis and larval lysozyme (contaminated with amylase) by density-gradient ultracentrifugation, and were used to raise antibodies in a rabbit. Antibodies raised against larval aminopeptidase, trehalase, and amylase did not recognize the imaginal enzymes, whereas those against larval lysozyme recognize imaginal lysozyme. The data suggest that the genes coding for digestive enzymes (except for lysozyme) are different in larvae and imagoes.     

The cuticular proteins of Tenebrio molitor: I. Electrophoretic banding patterns during postembryonic development

Buffer-soluble cuticular proteins of the abdomen of the yellow mealworm, Tenebrio molitor, were analyzed by SDS-polyacrylamide gel electrophoresis. Since the abdominal epidermis of Tenebrio persists throughout the insect's life, these cuticular proteins reflect the secretory history of a continuous line of cells during its entire metamorphic developmental program. Twenty-two to thirty-eight bands were detected in extracts of larval cuticle, 11 to 35 in pupal cuticular extracts, and 30 to 41 in extracts from adults. No population polymorphism was apparent, nor was there any sexual dimorphism, in these cuticular proteins. At each metamorphic stage, the cuticular proteins formed a unique banding pattern. Bands unique to the larval and to the adult exocuticular extracts were observed. Extracts from cuticles of freshly ecdysed animals (exocuticle) differed from extracts from animals in which sclerotization and postecdysial (endocuticle) deposition had occurred, both in number of hands and in their molecular weight distributions. Some proteins became less soluble during sclerotization. The majority of the exocuticular bands from all three stages had molecular weights below 25,000; higher-molecular-weight proteins were extracted from postecdysial animals of each stage.     

Immunocytochemical and biochemical characterization of guanine nucleotide-binding regulatory proteins in mammalian spermatozoa

Polyclonal antisera directed against conserved and subtype-specific peptide sequences of the alpha-subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to characterize the nature of mammalian sperm G proteins and to determine whether their localization was consistent with their proposed roles in mediating ZP3-induced acrosomal exocytosis. Mouse and guinea pig sperm exhibit positive immunofluorescence in the acrosomal region using an antiserum directed against a peptide region common to all alpha-subunits of G proteins (G alpha). The immunofluorescence disappears after sperm have undergone the acrosome reaction, suggesting that the immunoreactive material is associated with the plasma membrane/outer acrosomal membrane region overlying the acrosome. The presence of G proteins in this region is confirmed by the presence of a Mr 41,000 substrate for pertussis toxin (PT)-catalyzed [32P]ADP-ribosylation in purified plasma membrane/outer acrosomal membrane hybrid vesicles obtained from acrosome-reacted guinea pig sperm. Immunoprecipitation and polyacrylamide gel electrophoresis of PT-catalyzed [32P]ADP-ribosylated protein(s) using anti-peptide antisera generated against sequences unique to Gi alpha 1, Gi alpha 2, and Gi alpha 3 confirm the existence of all three Gi subtypes in mouse sperm extracts. Indirect immunofluorescence using an antiserum directed against a peptide region present in Gz alpha, a PT-insensitive G protein, demonstrates positive immunoreactivity in the postacrosomal/lateral face region of the mouse sperm head. This immunoreactivity is retained during acrosomal exocytosis in response to solubilized ZP and then disappears subsequent to this exocytotic event. These data demonstrate that Gi protein alpha-subunits are present in the acrosomal region of mammalian sperm, consistent with their postulated role in regulating ZP3-mediated acrosomal exocytosis, and that PT-insensitive Gz alpha is found in a region of the sperm head distinct from that of the Gi alpha subunits.     

Serological comparison of polyhedron protein and virions from four nuclear polyhedrosis viruses of plusiine larvae (Lepidoptera: Noctuidae)

Purified polyhedron proteins and purified, ultrasonicated virions of four nuclear polyhedrosis viruses (NPVs), separable into two morphologic groups of singly and multiply embedded virion types (SEVs and MEVs), were investigated by immunodiffusion and immunoelectrophoresis. The four viruses were Pseudoplusia includens SEV, Trichoplusia ni SEV, T. ni MEV, and Autographa californica MEV. In immunodiffusion, SEV polyhedron proteins formed two precipitin bands with antiserum to SEV polyhedron proteins, while MEV polyhedron proteins formed only one. All four proteins formed one precipitin band with antiserum to MEV polyhedron protein, with a spur between SEV and MEV proteins. In immunoelectrophoresis, mobilities of SEV proteins were significantly different from those of MEVs. Precipitin arc patterns were similar to those in immunodiffusion when electrophoresis was carried out at 4 C; at room temperature, a single arc of precipitation formed with all four proteins. SEV virions formed five possibly identical precipitin bands in immunodiffusion with antiserum to SEV virions. MEV virions formed three possibly identical precipitin bands when reacted with antiserum to MEV virions. Little or no cross-reactions were observed between SEV and MEV virions or between virions and polyhedron proteins. In immunoelectrophoresis, SEV virions formed three precipitin arcs in reactions with SEV antisera and none with MEV antisera; MEV virions formed two arcs with MEV antisera and none with SEV antisera. When antisera were subjected to electrophoresis, five arcs were formed by SEVs and three by MEVs in homologous systems, and none were formed in heterologous systems.     

The use of multivariable standard curves in the radioimmunoassay of testosterone and 5α-dihydrotestosterone

Multivariable calibration curves have been used to enable testosterone and 5α-dihydrotestosterone to be assayed directly in plasma extracts without further pre-purification of the sample. Two antisera were used, both with relatively high, but different affinities for the substances measured, and with relatively low affinity towards all other substances tested. The antisera were obtained from rabbits immunized against testosterone-3-BSA and 5α-dihydrotestosterone-3-BSA. The technique was of adequate precision, accuracy and specificity. The last was examined by comparison of values obtained by the present method and those obtained following prepurification by thin layer chromatography.     

Taenia taeniaeformis: immunoprecipitation analysis of the protein antigens of oncospheres and larvae

Biosynthetically or exogenously labeled proteins and immunoprecipitated protein antigens of established 28-day-old larvae of Taenia taeniaeformis were compared with proteins and antigens of infective oncospheres using single and two-dimensional gel electrophoresis. Immunoprecipitation was carried out using sera from infected mice and mouse antisera raised to larvae or oncospheres, and emphasis was placed on identifying antigens common to both oncospheres and larvae. Two major larval antigens of Mr 40,000 and 200,000, designated Tt40 and Tt200, are common to somatic larval preparations and oncospheres. Additionally, two major oncosphere antigens of Mr 55,000 and 60,000, designated Tt55 and Tt60, are also present in larval excretory and secretory (i.e., ES or exoantigen) products. Information obtained from these immunoprecipitation analyses will facilitate isolation and production of common as well as stage-specific protein antigens in the development of defined-antigen vaccines in this model system of cysticercosis.     

Isolation of Fasciola hepatica genus-specific antigens

Santiago de Weil N., Hillyer G. V. and Pacheco E. 1984. Isolation of Fasciola hepatica genus-specific antigens. International Journal for Parasitology14: 197–206. The Fasciola hepatica antigens which induce antibody formation in acute fascioliasis were isolated by acid elution after reacting an F. hepatica tegument antigen extract with a CNBr-Sepharose 4B column coupled with IgG obtained from the serum of rabbits infected with fascioliasis for 6–10 weeks. These isolated antigens were further separated by gel filtration using a column packed with Sephacryl S-200. In this manner three major peaks were obtained. The best serologic antigens were found in peak 2 which had a mol. wt range of 14,000–43,000. This peak contains genus-specific F. hepatica antigens which are highly reactive with fascioliasis serum. These antigens do not cross-react with either Schistosoma mansoni or with bovine serum albumin by gel diffusion. Monitoring by ELISA and gel diffusion with heterologous and homologous antisera showed that as purification by antibody affinity chromatography proceeded, cross reactivity with S. mansoni was eliminated. The rabbit antiserum obtained against peak 2, when tested by immunoelectrophoresis with a crude F. hepatica extract shows one main band identical to the main band observed with serum from acutely infected rabbits. Up to two other minor bands can be detected using concentrated homologous antisera. Fractions obtained from preparative iso-electric focusing of the F. hepatica tegument extract were reacted with sera from rabbits with acute fascioliasis. Two main bands were observed in immunodiffusion with antigens eluting in a pH range of 7.4–8.7. When these fractions were monitored with anti peak 2 antisera, two precipitin bands appeared with antigens eluting in a pH range of 7.4–7.9. The F. hepatica genus-specific antigen pool was applied to ELISA to evaluate its ability to detect antibody in a primary F. hepatica infection in rabbits. A rise in absorbance values could be detected by 2 weeks of infection, reached high levels by 6 weeks and remained high through 28 weeks of infection.