Immunization of zebu calves against Fasciola gigantica, using irradiated metacercariae

The pathogenesis of unirradiated, 3 krad-irradiated and 20 krad-irradiated metacercarial infections was compared in zebu calves studied over a 10-week period. Calves exposed to 1000 unirradiated metacercariae (mc) became hypoalbuminaemic, and showed elevated serum concentrations of liver enzymes, whereas neither of the other groups was significantly affected. At slaughter, a mean of 332 flukes was recovered from the 0 krad group, while only 23% and 12% of this number were recovered from the 3 krad and and 20 krad groups, respectively. All the worms recovered from the 20 krad group were stunted, and found in biliary ductules, but a mean of 13% of the flukes recovered from the 3 krad group were large, and dwelling in main bile-ducts. Liver lesions typical of acute fascioliasis were present in the 0 krad group, but lesions in the other groups, and particularly the 20 krad group, were far less severe. Judged on clinico-pathological criteria, a single vaccination of calves with 1000 3 krad-irradiated mc induced partial resistance to a challenge with 1000 normal mc eight weeks later, but the reduction in worm recovery was not statistically significant. There was less evidence of protection when two vaccinating doses of 3 krad mc were given within four weeks, with challenge at week 8, and a single vaccination was ineffective against a challenge four weeks later. However, when the irradiation dose was increased to 20 krad, a hgh level of resistance (69% worm reduction) was induced by a single vaccination, given eight weeks before challenge, and liver pathology was strikingly reduced in the vaccinated animals.     

Studies on the viability of metacercariae of Fasciola gigantica.

The viability of metacercariae of Fasciola gigantica was tested by in vitro and in vivo methods. In vitro testing was based upon the motility of juvenile flukes within the inner cyst as examined under the light microscope. In vivo testing was undertaken through experimental infections of rabbits (two groups) and natural definitive hosts, lambs (one group). In the first group, out of six rabbits each given 25 metacercariae, worm establishment only took place in one rabbit with a single fluke recovery on 60 days post infection. In the second group of six rabbits each given 200 metacercariae, five were infected, with two or three flukes per host. All the lambs given 250 metacercariae became infected showing prevalences of 7.2-40% in comparison with rabbits in which low prevalences (0-4%) were recorded. The results indicated that even viable metacercariae which were already tested in vitro could not readily establish in rabbits. Such variability in worm establishment suggests that immunological and chemotherapeutic studies in rabbits infected with F. gigantica are likely to be unreliable.     

On the viability of Fasciola gigantica metacercariae ingested by Lymnaea auricularia

The effect of ingestion by Lymnaea auricularia on the viability and infectivity of Fasciola gigantica metacercariae was studied. The cyst wall was unaffected by the snail's digestive processes. Two rabbits, each infected with 50 ingested metacercariae, died at 83 and 87 days post-infection. Eight and 10 immature flukes were recovered from the livers, indicating that the metacercariae had remained infective after passing through the intestine of the snail.     

Comparison of humoral response in sheep to Fasciola hepatica and Fasciola gigantica experimental infection

Humoral response of sheep to F. gigantica was compared with the well known humoral response to F. hepatica, in order to explain the difference of susceptibility of sheep to these two parasites. In this work, a lesser susceptibility of sheep to F. gigantica than to F. hepatica infection was confirmed. Humoral response to F. hepatica infection is similar to that previously described by several authors. IgG level of F. gigantica infected sheep increased from week 2 post-infection (2WPI) and displayed a peak at 13WPI. F. gigantica excretory-secretory products (FgESP) analyzed by SDS-PAGE showed at least 31 bands from 12.0 to 127.6 kDa in FgESP. Western blot indicated that F. gigantica infected sheep sera recognized, in FgESP, at least 30 antigens from 7.8 to 119.2 kDa of which 12 major bands recognized after OWPI. In FhESP and FgESP, F. hepatica infected sheep serum reacted only with the lower molecular mass antigens, while F. gigantica infected sheep serum reacted with the lower and the higher molecular mass antigens. These differences of antigenic recognition might be associated with the difference of susceptibility of sheep. Further investigation must be done to study the mechanism of resistance between the sheep infected with F. hepatica or F. gigantica.     

The fate of Fasciola hepatica metacercariae following challenge infection of immune rats

Groups of rats, infected 7 weeks previously with Fasciola hepatica, together with appropriate control groups, were challenged either orally or intraperitoneally with 30 metacercariae. The mean worm recovery from the previously infected, orally challenged rats was significantly lower than from their respective controls (2.2 +/- 1.1 worms as opposed to 9.0 +/- 2.6). There was no significant difference in mean worm recovery from the previously infected, intraperitoneally challenged rats and their respective controls (5.3 +/- 3.2 worms as opposed to 6.2 +/- 1.9). Livers of the orally challenged group appeared to be largely free from secondary damage but considerable damage was evident in rats which received an intraperitioneal challenge. This evidence supports the view that the gut acts as an important barrier to metacercariae of a challenge infection. In a further experiment, young flukes were recovered from the gut, abdominal cavity and liver of immune and control rats 9, 18, 27, 36 and 45 h after oral challenge. It was found that fewer flukes successfully penetrated the guts of immune rats (3%) than those of uninfected controls (13%), again pointing to the gut as a barrier to metacercariae of a challenge infection. Protective mechanisms that may operate at the level of the gut are discussed.     

Fasciola hepatica and Fasciola gigantica: comparison of cellular response to experimental infection in sheep

Cellular responses to Fasciola gigantica and to Fasciola hepatica infection in sheep were compared. Eosinophil numbers increased more quickly and strongly in F. gigantica-infected sheep than in F. hepatica-infected sheep. In both groups, peripheral blood mononuclear cell (PBMC) proliferation in response to the parasitic excretory-secretory products (ESP) showed similar kinetics. Interferon-gamma (IFN-gamma) production by ESP-stimulated PBMC was early and showed similar kinetics in both groups. Interleukin-10 (IL-10) production by FhESP-stimulated PBMC was very high throughout infection even at 0 weeks post-infection (WPI) in F. hepatica-infected sheep, while in F. gigantica-infected sheep, IL-10 production by FgESP-stimulated PBMC increased between 1 and 4 WPI. IL-10 production in F. gigantica-infected sheep was significantly lower than in F. hepatica-infected sheep during infection. The lower susceptibility to F. gigantica infection in sheep could be explained by the more intense cellular response induced by the parasite and the weaker capacity of F. gigantica to evade the immune response.     

Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-β, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.     

Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis

Sheep fasciolosis is a devastating burden for the livestock industry. We herein report on immunodiagnosis of fasciolosis, and significant protection of sheep against challenge infection with Fasciola gigantica following immunization with a peptide based on the H-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH (Fas14p) sequence of F. gigantica cathepsin L-cysteine proteinase. This sequence was synthesized in three different forms: as N(alpha) acetylated (Ac-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAc14p), bearing at the amino-terminus an N(alpha) acetylated cystein (Ac-Cys-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAcCys14p), and conjugated to sequential oligopeptide carrier Ac-[Lys-Aib-Gly](4)-OH (Ac-SOC(4)) through an amide bond formed between Val(123) carboxylic group of the epitope and the lysine N(epsilon) groups of the carrier (Ac-[Lys(Fas14p)-Aib-Gly](4)-OH). Ac-[Lys(Fas14p)-Aib-Gly](4)-OH failed to readily discriminate between na?ve and infected sheep. In contrast, the free peptides reproducibly differentiated between parasite-free sheep, sheep infected with parasites other than Fasciola, and experimentally Fasciola-infected sheep. The data together indicated that the peptides might be of considerable use for discriminating between early and late, and low and high burden, sheep infection with F. gigantica. FasAc14p was chosen to determine whether a peptide based on a critical enzymatic site of cathepsin L proteinase may induce protection against challenge infection. Sheep immunization with FasAc14p peptide induced significant expression of interleukin-4 mRNA, and humoral antibodies that bound to molecule(s) on the intact surface membrane of newly excysted juvenile worms, and mediated their attrition. The immune responses were associated with significant (P < 0.02) decrease of 23.1% in worm recovery, but with no decrease in the size or maturation of worms recovered.     

Evaluation of Caenorhabditis elegans glycoproteins as protective immunogens against Haemonchus contortus challenge in sheep

High levels of protection can be attained against Haemonchus contortus challenge infection in sheep using native antigens isolated from the gut of the adult parasite. However, vaccination with recombinant forms of these antigens, or components thereof, has disappointingly failed to generate similar levels of protection, suggesting that appropriate nematode glycosylation may be a prerequisite for protection. The free-living nematode, Caenorhabditis elegans is closely related to H. contortus and has been shown to share similar glycan moieties. In order to investigate the potentially protective role of these glycan moieties, a complex set of glycoproteins was isolated from C. elegans using ConA-lectin chromatography and their efficacy as immunogens against H. contortus challenge infection evaluated in sheep. Despite the generation of a high titre systemic IgG antibody response to the C. elegans glycoproteins and the ability of these antibodies to bind to the microvillar surface of the gut of H. contortus, no protection against challenge infection was observed. Serum antibodies to the C. elegans glycoproteins cross-reacted with the H. contortus host-protective antigen, H-gal-GP, by ELISA, although the level of cross-reactivity was not of a magnitude considered protective. Qualitative differences were also determined between the glycan epitopes of the C. elegans ConA-binding proteins and those of H-gal-GP, suggesting the presence of H. contortus-specific patterns of glycosylation.     

Fasciola gigantica: evaluation of the effect of phenyl vinyl sulfone in vitro

Phenyl vinyl sulfone is a synthetic inhibitor of cysteine protease and has antihelminthic and antiprotozoal properties. Phenyl vinyl sulfone was assayed in vitro for antifasciola activity against adult Fasciola gigantica worms using a well-established culture medium. Worms were treated with phenyl vinyl sulfone for incubation periods ranging from 0 to 12h and its activity was assessed in terms of viability, motility and death of worms. Phenyl vinyl sulfone exhibited a minimum effective concentration of 50 ppm after 12h. Three hundred parts per million concentrations were most potent causing immediate death of adult flukes in vitro. Histopathological studies showed that there was tegumental flattening, rupture of vesicles, and spine loss. Marked reduction in size and number of ova and sperms in the convoluted tubules of the reproductive organs was observed in comparison to the untreated control group. In conclusion, phenyl vinyl sulfone shows potent activity against F. gigantica in vitro, and the authors recommend carrying out more studies to detect its efficacy in vivo.     

Babesia major: protection of intact calves against homologous challenge by the injection of irradiated piroplasms.

Blood from a splenectomized calf infected with Babesia major was divided into 20 ml aliquots which were γ-irradiated at doses of 0, 23.3, 27.3, 31.4, 35.4 and 39.5 krad and then inoculated into groups of three intact calves. Animals receiving non-irradiated blood had typical mild B. major reactions, but those receiving blood irradiated at 23.3, 27.3 and 31.4 krad and 2 of 3 receiving blood irradiated at 35.4 krad had minimal reactions. The remaining 4 animals had no detectable parasitaemic reactions. When the calves were challenged with a similar number (6.0 × 10⁹) of homologous parasites, they were all immune with the exception of the 4 animals which had not reacted initially. The immune status of individual cattle was reflected accurately in the results of the micro-ELISA test, which detected a significant rise in serum antibody titre of the 4 susceptible animals 7 days after challenge.     

The combined recombinant cathepsin L1H and cathepsin B3 vaccine against Fasciola gigantica infection

Protections against Fasciola gigantica infection in mice immunized with the individual and combined cathepsin L1H and cathepsin B3 vaccines were assessed. The vaccines comprised recombinant (r) pro-proteins of cathepsin L1H and B3 (rproFgCatL1H and rproFgCatB3) and combined proteins which were expressed in Pichia pastoris. The experimental trials were performed in ICR mice (n = 10 per group) by subcutaneous injection with 50 μg of the recombinant proteins combined with Alum or Freund's adjuvants. At two weeks after the third immunization, mice were infected with 15 F. gigantica metacercariae per mouse by oral route. The percents of protection of rproFgCatL1H, rproFgCatB3 and combined vaccines against F. gigantica were approximately 58.8 to 75.0% when compared with adjuvant-infected control. These protective effects were similar among groups receiving vaccines with Alum or Freund's adjuvants. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th1 and Th2 immune responses, it was found that both Th1 and Th2 humoral immune responses were significantly increased in vaccinated groups compared with the control groups, with higher levels of IgG1 (Th2) than IgG2a (Th1). Mice in vaccinated groups showed reduction in liver pathological lesions when compared with control groups. This study indicates that the combined rproFgCatB3 and rproFgCatL1H vaccine had a high protective potential than a single a vaccine, with Alum and Freund's adjuvants showing similar level of protection. These results can serve as guidelines for the testing of this F. gigantica vaccine in larger economic animals.     

Larval productivity of Fasciola gigantica in two lymnaeid snails

Two groups of Galba truncatula and two groups of Lymnaea natalensis were experimentally infected with Fasciola gigantica to determine if snail species had an influence on the redial burden and cercarial shedding of this trematode when snails of both species were infected with the same isolate of miracidia. In the two groups used for the study of redial burden, the total number of free rediae was significantly higher at day 49 post-exposure in L. natalensis than in G. truncatula. In the groups used for cercarial shedding, the life-span of cercaria-shedding snails and those of infected snails which died without cercarial emission, and the duration of the prepatent period were significantly longer in L. natalensis than those noted in G. truncatula. However, the mean numbers of shed cercariae did not significantly differ and showed no differences in their daily distribution throughout the shedding period. These results demonstrate that G. truncatula might be the principal intermediate host of F. gigantica in Egypt, at least in the areas where this lymnaeid species lives.     

Molecular analyses confirm the coexistence of Fasciola gigantica and parthenogenetic Fasciola in the Philippines

Fasciola flukes collected from domestic buffalos and cattle in the Philippines were confirmed as Fasciola gigantica and parthenogenetic Fasciola based on DNA analyses of nuclear pepck and pold genes, and the mitochondrial ND1 gene. This study is the first to elucidate that F. gigantica and parthenogenetic Fasciola coexist in the Philippines with prevalences of 90.6% and 9.4%, respectively. The F. gigantica population showed a high genetic diversity with 25 ND1 haplotypes, suggesting that F. gigantica has existed in the Philippines for a long time. In contrast, parthenogenetic Fasciola flukes showed a single ND1 haplotype (Fsp-ND1-P1), which was identical to the founder haplotype, Fg-C2 of parthenogenetic Fasciola in China. These results indicate that parthenogenetic Fasciola in the Philippines is a recently introduced population from a neighboring continent.     

The distribution of Fasciola hepatica and Fasciola gigantica within southern Tanzania--constraints associated with the intermediate host

In East Africa, Fasciola gigantica is generally the causative agent of fasciolosis but there have been reports of F. hepatica in cattle from highland regions of Kenya, Ethiopia, Uganda and Zaire. The topography of the Southern Highlands of Tanzania provides an environment where the climatic conditions exist for the sustenance of lymnaeid species capable of supporting both Fasciola hepatica and F. gigantica. Theoretically this would allow interaction between fasciolid species and the possible creation of hybrids. In this report we present molecular data confirming the existence of the snail, Lymnaea truncatula, at high altitude on the Kitulo Plateau of the Southern Highlands, Tanzania, along with morphometric and molecular data confirming the presence of F. hepatica in the corresponding area. At lower altitudes, where climatic conditions were unfavourable for the existence of L. truncatula, the presence of its sister species L. natalensis was confirmed by molecular data along with its preferred fasciolid parasite, F. gigantica. Analysis based on a 618 bp sequence of the 28S rRNA gene did not reveal the presence of hybrid fasciolids in our fluke samples.     

Studies on histoplasmosis. V protective effect of HIF-coated Histoplasma against challenge

Summary Results of testing a total of 145 hamsters in 2 separate trials (a total of 90 challenged animals) suggest the potentiality of HIF-coatedHistoplasma yeast cells as a vaccine. Animals challenged with 5×10⁵ Histoplasma yeast cells in saline 3 weeks after initial inoculation with 5 × 10⁴ Histoplasma yest cells in 1) saline or 2) crude HIF in kidney homogenate or 3) the active 280 mµ fraction thereof were protected by the 280 mµ fraction > the crude HIF > saline suspensions ofHistoplasma, based on analysis of tissue sections and cultures.It is suggested that since HIF has also been found in experimental blasomycosis it should be sought in other mycoses, perhaps in all infectious diseases, and its potential use in a vaccine preparation should be more extensively tested.Supported in part by Public Health Service Grant No. 1-SO-IFR-05359-01 to the senior author at The George Washington University.