Chantal Boulard

Humoral and cellular immune responses to Fasciola hepatica experimental primary and secondary infection in sheep

Blood leukocyte changes, serum hepatic enzyme levels, lymphocyte proliferation in response to Concanavalin A (ConA) and to parasitic excretory-secretory products (FhESP), and antibody (IgG and IgM) responses (ELISA and Western blot) were studied in sheep, the natural susceptible host of F. hepatica, during the first 3 months of an experimental primary or secondary infection. The proportion of flukes established was similar in once- and twice-infected groups, but the flukes originating from the secondary infection migrated more rapidly to the bile ducts. Primary infection induced a marked peripheral eosinophilia from 3 to 13 weeks post-primary infection (PPIW). FhESP-specific IgM were produced from PPIW 2 with peaks in PPIW 3 and 9-10; FhESP-specific IgG increased from PPIW 2 to 6 and became stable afterwards. Western blotting revealed 12 major antigenic fractions in FhESP from 12, 15, 20, 24, 27, 28.5, 30, 41, 51, 56, 69 and 156 kDa; some non-specific ones have been characterized. A sequential recognition of higher then lower molecular weight antigens was observed. FhESP-specific lymphocyte proliferation was marked from PPIW 2 to 5. In contrast, ConA stimulation of lymphocytes was decreased. After secondary infection in PPIW 6, immune responses were modified. The ConA-induced lymphocyte proliferation was transitorily increased. In contrast, the humoral response, in particular against the early recognized antigens, and the level and the duration of the FhESP-specific lymphocyte proliferative response, were reduced.

Comparison of three ELISA tests for seroepidemiology of bovine fascioliosis.

The aim of the present study was to compare the sensitivity, specificity and usefulness of the DIG-ELISA, DOT-ELISA and Indirect ELISA tests for determining the seroprevalence of fasciolosis in cattle under tropical conditions in Mexico. To standardize the tests, positive and negative sera to F. hepatica from 88 Holstein Freisian adult cows located in an enzootic area of fascioliosis and 88 crossbred adult cattle from a fluke-free area were used. For the epidemiological study, 85 crossbred cattle between 1 to 7 years of age were used. Animals were bled every two months, from March 1995 to September 1996 and the sera obtained were stored at -70 degrees C, until used. Indirect ELISA showed a sensitivity of 96.5% and a specificity of 98.8%, DIG-ELISA 97.5% and 80.0% and DOT-ELISA 93.1% and 95.4%, respectively. During 1995, Indirect ELISA yielded the highest levels of IgG anti-F. hepatica antibodies. However, in 1996, after animal treatment with triclabendazole, DIG-ELISA tended to show higher percentages of antibody-positive animals, but it was not significantly different (p>0.05) from the other tests. Comparisons made in parallel to the faecal sedimentation test demonstrated that all serological tests detected higher percentages of positive animals. Only one serum out of ten (10%) of Paramphistomum spp. cross-reacted with the DOT-ELISA test, but no cross-reaction was observed with sera from animals with other parasites. All ELISA tests were highly sensitive and specific; they may be recommended for use in seroepidemiological surveys for F. hepatica.

Incidence of a subclinical fascioliasis on antipyrine clearance and metabolite excretion in sheep

1. The pharmacokinetics of i.v. antipyrine (25 mg/kg) used as a model compound, were determined in young male sheep, before and each month after an oral infestation by 150 metacercariae of Fasciola hepatica, and 8 weeks following a flukicidal treatment. 2. The parasitic pathology was ascertained by the clinical observation of animals and the increase in plasma antibodies directed against liver flukes. 3. A significant decrease in the total plasma clearance of the drug occurred by week 4 to 16, and a 1.7 fold increase in mean residence time occurred by week 12 post-infection. 4. Urinary excretion of antipyrine metabolites was determined before and 8 weeks following the infestation. 4-Hydroxyantipyrine was the major urinary metabolite and its excretion was decreased by 30% in infected sheep, whereas there was no change in the excretion of norantipyrine, 3-hydroxymethylantipyrine or unmetabolized drug. 5. It is concluded that the impairment of antipyrine clearance in the course of fascioliasis could be related to the decrease in liver microsomal cytochrome P-450-dependent mono-oxygenases observed in sheep with a similar parasitic burden.

Humoral and cellular immune responses in rats during a primary infestation with Fasciola hepatica

The antibody and lymphocyte responses to Fasciola hepatica were studied in rats. Infested rats were shown to produce antibodies against excretory-secretory (ES) products of adult flukes as early as the first week after infestation. Immunoblotting revealed fractions of ES products of adult flukes to which antibodies were progressively produced during the course of the infestation. Proliferation of peripheral blood lymphocytes, splenocytes and thymocytes when incubated with different mitogens (Concanavalin A (ConA) or Pokeweed mitogen (PWM) or different liver fluke antigens (metacercariae antigen (EM) or ES products of adult flukes) have been studied. In response to these mitogens or antigens, splenocytes were stimulated on the second and fourth weeks after infestation. Thymocytes were significantly activated by PWM on the second week but peripheral blood lymphocytes did not show any statistically significant response. Results obtained in antibody production, immunoblotting and lymphocyte proliferation suggested sequential releases of F. hepatica substances and the existence of common proteins between adult and juvenile parasite stages. Cellular and humoral responses observed in this work did not seem to confer a complete resistance to liver fluke primary infestation on the rat.

Kinetic responses of parasite-specific antibody isotypes, blood leucocyte pattern and lymphocyte subsets in rats during primary infestation with Fasciola hepatica.

Antibody responses, blood leucocyte and splenic lymphocyte subset patterns were studied during a primary infection with Fasciola hepatica in the rat. The infection induced parasite-specific IgM by 2 weeks after infection. High levels of IgM antibodies were maintained for many weeks. The IgE response was biphasic with peaks at 5 and 9 weeks after infection which were correlated with different phases in the development of F. hepatica in the rat. Both IgG2a and IgG1 antibodies were detected but the titre of IgG2a augmented slightly and rose more slowly than did that of IgG1. There was a rise in neutrophil and eosinophil numbers. Neutrophils did not increase before the fourth week but eosinophil numbers were raised by the second week after infection and remained high during the whole migratory phase of the parasite. In the spleen, the percentage of B lymphocytes increased and there was a decrease in the percentages of CD4+ and CD(8+)-like T lymphocytes. These results suggested that, in the rat infested with F. hepatica, TH2-like lymphocytes could be preferentially stimulated, as has been reported in murine schistosomiasis.

Characterization of a collagenolytic enzyme from larvae of Hypoderma lineatum (Insecta: Diptera, Oestriform)

Abstract 1. 1. An enzyme with a collagenolytic activity has been isolated by chromatography on SE Sephadex from the first instar larvae of Hypoderma lineatum. 2. 2. The purified collagenase of molecular weight around 16,000 and of isoelectric point pH 3.5, cleaves the collagen molecule in two fragments of 3 4 1 4 at neutral pH. 3. 3. This enzyme is slightly inhibited by diisopropylfluorophosphate but specific trypsin or chymotrypsin inhibitors have no effects, neither do EDTA, cyanide, or p-chloromercuribenzoate.

Use of pooled serum or milk samples for the epidemiological surveillance of bovine hypodermosis.

An enzyme-linked immunosorbent assay (ELISA) was used on pooled serum and milk samples to determine whether hypodermosis could be detected where a larger sero-epidemiological survey was required. This study was undertaken to assess the potential of this assay for testing sera on milk samples, pooled from 10 cows, and determining the period of the year when detection was optimal. The sensitivity of the assay was determined by increasingly diluting a positive serum with pooled negative sera, from 1:10 to 1:100. The diagnostic lower limit of the assay requires at least two serological reactors within a herd of 100. The kinetic development and depletion of anti-Hypoderma antibody of individual and pooled sera or milk from 30 cows was evaluated from November to July. Anti-Hypoderma antibody levels of two groups of 8 calves, one control and one teated with ivermectin (Ivomec), were tested from October to June. These preliminary results indicate that an ELISA assay on serum or milk samples pooled from 10 cows can be used between February and April to evaluate the prevalence of hypodermosis within cattle herds in France, demonstrating the feasibility of using pooled serum already collected for bovine leucosis testing.

Sequencing and gene expression of hypodermins A, B, C in larval stages of Hypoderma lineatum

The cDNAs of hypodermins, enzymes secreted by the larvae of the parasitic fly Hypoderma lineatum, were sequenced. Four cDNA clones were isolated, one encoding hypodermin A (HA), one encoding hypodermin C (HC), and the two others encoding proteins related to hypodermin B (HB). The amino acid sequences deduced from the nucleotide sequences confirmed that these enzymes are serine proteases. HA and one of the HB proteins had potential N-linked glycosylation sites. Analysis of hypodermin protein, RNA and DNA at different larval stages indicated that protein overexpression is regulated transcriptionally for HA and HB, and by transcriptional and DNA amplification for HC.

Early hepatic cytokine mRNA expression in experimental rat fasciolosis

We studied the development of the cellular response, particularly with respect to Th1 and Th2 cytokine mRNA levels, in rat liver during the first 14 days of experimental infection with Fasciola hepatica. We analysed the panel of cytokines involved in initiation of the inflammatory and immune response. The levels of various mRNAs, particularly those primarily associated with the acute inflammatory response, and those commonly associated with T-cell proliferation and differentiation, were assessed by reverse transcription-polymerase chain reaction (RT-PCR) in liver samples. We also investigated the immune and inflammatory mediators balance in the liver, draining lymph node and spleen, by RT-competitive PCR quantification of mRNA levels for IL-4, IL-10 and IFN-gamma. Our data provide the first evidence that, in the early phase of infection, the inflammatory response in the liver of infected animals is transiently depressed or delayed. A Th0 profile was initially observed in the liver and hepatic lymph node, which developed into a Th2 profile 2 weeks after infection in the liver only. In the spleen, cytokine down-regulation was initiated and maintained during this period, suggesting that the parasite acts differently locally and in the periphery.

Immunological responses of rabbits artificially infested with the cattle grubs Hypoderma bovis (L.) and H. Lineatum (De Vill.) (Diptera: Oestridae).

Abstract Boulard Chantal and Weintraub J. 1973. Immunological responses of rabbits artificially infested with the cattle grubs Hypoderma bovis (L.) and H. lineatum (De Vill.) (Diptera : Oestridae). International Journal for Parasitology3: 379–386. Immunological responses against first-instar larvae of Hypoderma bovis (L.) and H. lineatum (De Vill.) were traced in artificially infested rabbits in which larvae grew normally in the first instar but did not survive to the second instar. Passive haemagglutination demonstrated a rapid rise in antibody titres during the first 2 months to maximum levels that persisted until about 200 days of the infestation. Immunoelectrophoretic analyses of the sera demonstrated that larval metabolic products were more important than breakdown products of dead larvae in stimulating the production of antibodies. Larval collagenase, which seemed to induce the most significant immune response, was inhibited by homologous antibodies in the serum of a hyperimmunized rabbit. The implications of these results for infestations in the normal bovine host were discussed, with emphasis on the need to identify antigenic fractions other than collagenase, which by itself had not produced total control of larvae in previous vaccination tests.