Fasciola gigantica fatty acid binding protein (FABP) as a prophylactic agent against Schistosoma mansoni infection in CD1 mice

Although schistosomicidal drugs and other control measures exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. In this study, native fatty acid binding protein (FABP) from Fasciola gigantica was purified from the adult worm's crude extract by saturation with ammonium sulphate followed by separation on DEAE-Sephadex A-50 anion exchange chromatography and gel filtration using Sephacryl HR-100, respectively. CD1 mice were immunized with the purified, native F. gigantica FABP in Freund's adjuvant and challenged subcutaneously with 120 Schistosoma mansoni cercariae. Immunization of CD1 mice with F. gigantica FABP has induced heterologous protection against S. mansoni, evidenced by the significant reduction in mean worm burden (72.3%), liver and intestinal egg counts (81.3% and 80.8%, respectively), and hepatic granuloma counts (42%). Also, it elicited mixed IgG(1)/IgG(2b) immune responses with predominant IgG1 isotype, suggesting that native F. gigantica FABP is mediated by a mixed Th1/Th2 response. However, it failed to induce any significant differences in the oogram pattern or in the mean granuloma diameter. This indicated that native F. gigantica FABP could be a promising vaccine candidate against S. mansoni infection.     

The rational simplification of a recombinant cocktail vaccine to control the parasitic nematode Teladorsagia circumcincta

Using data from five independent vaccine trials, which employed a subunit cocktail vaccine containing eight recombinant proteins to protect sheep against Teladorsagia circumcincta, a strategy was developed to simplify antigen complexity of the vaccine. A meta-analysis of data from these five trials demonstrated statistically significant reductions in cumulative faecal egg count and worm burden in vaccinated sheep when compared with those which had received adjuvant only (P = 0.009 and P < 0.0001, respectively). Relationships between antigen-specific antibody levels, antibody avidity and parasitological parameters of efficacy were analysed for each of the eight proteins in these trials. Of these, the strongest correlations between percentage reduction in cumulative faecal egg count and avidity were obtained for the vaccine antigen T. circumcincta apyrase-1 (Tci-APY-1) in relation to either total antigen-specific IgG or IgG1 in sera (P = 0.019 and P = 0.030, respectively). In addition, IgG and IgA within the serum and abomasal mucus of control (parasite challenged) lambs strongly recognised Tci-APY-1 and T. circumcincta metalloproteinase-1 (Tci-MEP-1) but only weakly bound the other six antigens, indicating Tci-APY-1 and Tci-MEP-1 are most effectively recognised by the parasite-induced antibody response. On the basis of these findings, a two-protein vaccine comprising Tci-APY-1 and Tci-MEP-1 was tested in a direct comparison with the original eight-component vaccine. A further group was immunised with Tci-MEP-1 in combination with a mutated form of Tci-APY-1 (mTci-APY-1), which had no enzymatic activity. Across the trial, the mean faecal egg count levels of the eight-antigen recipients were lower than those of the adjuvant only control group (P = 0.013) and the mean FEC of the mTci-APY-1 and Tci-MEP-1 recipients was lower, although not statistically significantly, than that of the adjuvant-only control group (P = 0.093). Mean cumulative faecal egg count levels were reduced by 43% in lambs immunised with mTci-APY-1 plus Tci-MEP-1 compared with the controls (P = 0.079).     

Glutathione S-transferase. Novel vaccine against Fasciola hepatica infection in sheep

The potential of GST as a vaccine candidate against liver fluke infection in ruminants was studied by vaccinating sheep (n = 9) with GST purified from adult worms of Fasciola hepatica and challenging with 500 F. hepatica metacercariae. The immunization induced a high antibody response to GST in contrast to the poor or undetectable response to this Ag observed in naturally infected sheep. Throughout the trial, the progress of the fluke infection was monitored by measuring RBC hemoglobin levels, the extent of liver damage and the fecal egg output in the sheep. This analysis indicated that a subpopulation (n = 4) of the GST vaccinated animals exhibited no anemia, reduced liver damage and a lower mean fecal egg count relative to the infected control group suggesting a lower fluke burden in these animals. Worm burdens in the livers of the GST vaccine group (107 +/- 22) were 57% lower than in the infected control group (250 +/- 25). The subpopulation of the GST vaccine group demonstrated a 78% reduction in mean worm burdens relative to the control group. These results show that GST of adult F. hepatica is a novel Ag that can significantly protect sheep against liver fluke infection. The results suggest that the immune response to GST is directed to the juvenile worm reducing the number of worms that can establish in the liver of the vaccinated animals.     

Schistosoma Japonicum UDP-Glucose 4-Epimerase Protein Is Located on the Tegument and Induces Moderate Protection against Challenge Infection

Schistosomiasis is an important global public health problem, as millions of people are at risk of acquiring this infection. An ideal method for sustainable control of schistosomiasis is using a vaccine alone or in combination with drugs. In the present study, we cloned the SjGALE gene and generated the expression product in E. coli. The expression level of SjGALE during different developmental stages of S. japonicum was evaluated by real-time RT-PCR and western blotting. Immunolocalization indicated that the protein was mainly located on the tegument of the parasite. Infection of rSjGALE-immunized mice demonstrated a 34% and 49% reduction of the mean worm burden and liver egg burden, respectively, in two independent experiments, indicating immune protection. The liver egg count from each female adult worm was significantly reduced by 63% in the two trials. The cytokine profile and IgG isotype analysis demonstrated the induction of a Th1 immune profile in response to immunization with this protein, further suggesting protection against infection. In conclusion, these findings indicated that SjGALE is a potential vaccine against S. japonicum.     

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.     

Vaccination with cathepsin L phage-exposed mimotopes,single or in combination,reduce size,fluke burden,egg production and viability in sheep experimentally infected with Fasciola hepatica

Fascioliasis is a worldwide emergent zoonotic disease that significantly constrains the productivity of livestock. In this study, fluke burdens, liver fluke size and biomass, faecal eggs counts, serum levels of hepatic enzymes and immune response were assessed in sheep vaccinated with peptide mimotopes of cathepsin L and infected with metacercariae. A total of 25 sheep were allocated randomly into five groups of five animals each, and experimental groups were immunised with 1 × 10¹³ filamentous phage particles of cathepsin L1 (CL1) (TPWKDKQ), CL2 (YGSCFLR) and mixtures of CL1 + CL2 mimotopes, in combination with Quil A adjuvant, and wild-type M13KE phage in a two-vaccination scheme on weeks 0 and 4. The control group received phosphate-buffered saline. All groups were challenged with 300 metacercariae two weeks after the last immunisation and euthanised 16 weeks later. The CL1 vaccine was estimated to provide 57.58% protection compared with the control group; no effect was observed in animals immunised with CL2 and CL1 + CL2 (33.14% and 11.63%, respectively). However, animals receiving CL2 had a significant reduction in parasite egg output. Vaccinated animals showed a significant reduction in fluke length and width and wet weights. In the CL1 group, there was a significant reduction in the total biomass of parasites recovered. Egg development was divided into seven stages: dead, empty, unembryonated, cell division, eyespot, hatched and hatching. The highest percentage of developmental stages was detected for vaccinated sheep administered CL1 + CL2 with cell division, and the lowest percentage was observed in the hatching stage. Furthermore, a significant difference in all developmental stages was observed between vaccinated animals and the control group (P < 0.01). The levels of anti-phage total IgG in immune sera increased significantly at four weeks after immunisation and were always significantly higher for cathepsin L vaccine group than in the challenged control group. Total IgG was inversely and significantly correlated with worm burden in the CL1 group.     

Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum

Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42–44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (pcDNA/SjGST), IL-12 expressing-plasmid (pIL-12), and rSjGST. Co-vaccination with pcDNA/SjGST, pIL-12, and rSjGST led to a reduction in worm burden, hepatic egg burden, and the size of liver tissue granulomas than that in the untreated infection controls. In addition, we detected high levels of specific IgG, IgG1, and IgG2a against the rSjGST antigen in infected mice vaccinated with this combination of pcDNA/SjGST, pIL-12, and rSjGST. Moreover, high expression levels of Th2 cytokines, including IL-4 and IL-10, were also detected in this group, without diminished levels of IL-12, INF-γ, and TNF-α cytokines that are related to parasite killing. In conclusion, we have developed a new vaccination regimen against S. japonicum infection and shown that co-immunization with pcDNA/SjGST vaccine, pIL-12, and rSjGST has significant anti-parasite, anti-hepatic egg and anti-pathology effects in mice. The efficacy of this vaccination method should be further validated in large animals such as water buffalo. This method may help to reduce the transmission of zoonotic schistosomiasis japonica.     

Immune responses in Indonesian thin tail and Merino sheep during a primary infection with Fasciola gigantica: lack of a specific IgG2 antibody response is associated with increased resistance to infection in Indonesian sheep.

After a primary infection with Fasciola gigantica, the immune responses in a resistant (Indonesian thin tail) and a susceptible (Merino) breed of sheep were analysed. The number of adult flukes recovered from the livers of the Indonesian thin tail sheep were significantly lower than those found in the Merino animals. On days 8, 14 and 25 p.i., Indonesian thin tail sheep exhibited a significantly higher eosinophilia than Merino sheep, whereas neutrophilia was significantly elevated in the Indonesian thin tail sheep on days 36 and 48 p.i. Serum from both sheep breeds demonstrated IgM, IgG1 and IgE responses to F. gigantica. In contrast, the Indonesian thin tail sheep produced significantly lower levels of IgG2 antibodies relative to the high level detected in Merino sheep. The IgE response was biphasic in both sheep breeds with the first response detected by day 14 and the second response developing from days 30 to 60 p.i. Western blotting showed that a similar profile of adult fluke antigens was recognised by IgG1 and IgE antibodies in both the Indonesian thin tail and Merino sheep. The IgE response was directed to a major antigen at about 92 kDa. We postulate that IgG2 could act as a blocking antibody for protective effector responses against F. gigantica in sheep and that the Indonesian thin tail sheep, by downregulating IgG2 responses, have an enhanced capacity for killing F. gigantica in vivo.     

A human case of Stellantchasmus falcatus infection in Korea

In an attempt to find the worm producing unidentified egg, one minute fluke was collect from a Korean patient after praziquantel administration. The fluke was identified to be Stellantchasmus falcatus by the expulsor. Brackish water fish was suggested to be a probable source of the infection.     

Concurrent infections of Fasciola, Schistosoma and Amphistomum spp. in cattle from Kafue and Zambezi river basins of Zambia

This study investigated interactions among Fasciola gigantica, Schistosoma spp. and Amphistomum spp. concurrent natural infections in Zambian cattle, based on egg and worm counts. In the abattoir 315 cattle were screened for worms of F. gigantica in the liver, Schistosoma spp. in mesenteric veins and/or Amphistomum spp. in the rumen. One hundred and thirty-three (42.2%) of the abattoir-examined cattle harboured one, two or all three trematodes. Of 133 cattle, 50 were randomly selected for worm and egg counts. The mean numbers (+/- SD) of Amphistomum, Schistosoma and Fasciola were 622.08 (+/- 97.87), 33.68 (+/- 7.44) and 19.46 (+/- 4.58), respectively. A total of 32% harboured all the three trematodes, 66% had F. gigantica and Amphistomum spp. infections, 52% had Schistosoma spp. and Amphistomum spp. infections while 32% had F. gigantica and Schistosoma infections. A positive correlation (P = 0.014) was found between F. gigantica and Amphistomum worm burdens. There were no correlations between Amphistomum and Schistosoma worm burdens and between F. gigantica and Schistosoma worm burdens. It may be concluded that there is no significant cross-protection among these trematodes in cattle in endemic areas.     

Experimental concurrent infection of Afar breed goats with Oestrus ovis (L1) and Haemonchus contortus (L3): interaction between parasite populations, changes in parasitological and basic haematological parameters

The study was conducted to examine the occurrence and interaction between Oestrus ovis and Haemonchus contortus in experimentally infected Ethiopian Afar breed of goats. Twenty goats were divided into four groups (O, OH, H, and C) of five animals each. Each animal of groups O and OH received weekly infections for 5 weeks with 66 first instar larvae (L₁) of O. ovis. Then animals of groups OH and H were infected with a single dose of 5000 third stage larvae (L₃) of H. contortus. Goats of group C were kept free of any infection as non-infected control. Faecal egg count (FEC), blood cell count, total serum protein level and body weight were recorded weekly throughout the study period. At necropsy worm burden, female worm length, fecundity and larval burden of O. ovis in the nasal-sinus cavities of infected animals were assessed. The results showed that the presence of H. contortus in the abomasum of goats of group OH had no influence on the development of O. ovis. On the contrary, a significant reduction (P < 0.05) in FEC, worm burden, fecundity and female worm length was revealed in group OH animals compared to the mono-infected animals (group H). This was associated with eosinophilia and reduced packed cell volume.     

Counterregulation of Th2 immunity by interleukin 12 reduces host defenses against Strongyloides venezuelensis infection

The aim of this study was to investigate the role of interleukin 12 (IL-12) during Strongyloides venezuelensis infection. IL-12^−/− and wild-type C57BL/6 mice were subcutaneously infected with 1500 larvae of S. venezuelensis. On days 7, 14, and 21 post-infection, we determined eosinophil and mononuclear cell numbers in the blood and broncoalveolar lavage fluid (BALF), Th2 cytokine secretion in the lung parenchyma, and serum antibody levels. The numbers of eggs in the feces and worm parasites in the duodena were also quantified. The eosinophil and mononuclear cell counts and the concentrations of IL-3, IL-5, IL-10, IL-13, and IgG1 and IgE antibodies increased significantly in infected IL-12^−/− and wild-type mice as compared with uninfected controls. However, the number of eosinophils and mononuclear cells in the blood and BALF and the Th2 cytokine levels in the lungs of infected IL-12^−/− mice were greater than in infected wild-type C57BL/6 mice. In addition, serum IgE and IgG1 levels were also significantly enhanced in the infected mice lacking IL-12. Meanwhile, parasite burden and fecal egg counts were significantly decreased in infected IL-12^−/− mice. Together, our results showed that the absence of IL-12 upregulates the Th2 immune response, which is important for control of S. venezuelensis infection.     

Co-administration of plasmid expressing IL-12 with 14-kDa Schistosoma mansoni fatty acid-binding protein cDNA alters immune response profiles and fails to enhance protection induced by Sm14 DNA vaccine alone

Schistosomiasis is an endemic disease that affects 200 million people worldwide. DNA-based vaccine is a promising strategy to induce protective immunity against schistosomiasis, since both humoral and cellular immune responses are involved in parasite elimination. In this study, we evaluated the ability of Sm14 cDNA alone or in association with a plasmid expressing murine interleukin (IL)-12 to induce protection against challenge infection. Mice were immunized with four doses of the DNA vaccine and the levels of protection were determined by worm burden recovery after challenge infection. Specific antibody production to rSm14 was determined by ELISA, and cytokine production was measured in splenocyte culture supernatants stimulated with rSm14 and in bronchoalveolar lavage of vaccinated mice after challenge infection. DNA immunization with pCI/Sm14 alone induced 40.5% of worm reduction. However, the use of pCI/IL-12 as adjuvant to pCI/Sm14 immunization failed to enhance protection against challenge infection. Protection induced by pCI/Sm14 immunization correlates with specific IgG antibody production against Sm14, Th1 type of immune response with high levels of interferon (IFN)-gamma and low levels of IL-4 in splenocyte culture supernatants and in bronchoalveolar lavage after challenge infection. IL-12 co-administration with pCI/Sm14 induced a significant production of nitric oxide in splenocyte culture supernatants and also lymphocyte suppression, with reduced percentage of T cells producing IFN-gamma and tumor necrosis factor-alpha.     

Cysteine Peptidases as Schistosomiasis Vaccines with Inbuilt Adjuvanticity

Schistosomiasis is caused by several worm species of the genus Schistosoma and afflicts up to 600 million people in 74 tropical and sub-tropical countries in the developing world. Present disease control depends on treatment with the only available drug praziquantel. No vaccine exists despite the intense search for molecular candidates and adjuvant formulations over the last three decades. Cysteine peptidases such as papain and Der p 1 are well known environmental allergens that sensitize the immune system driving potent Th2-responses. Recently, we showed that the administration of active papain to mice induced significant protection (P<0.02, 50%) against an experimental challenge infection with Schistosoma mansoni. Since schistosomes express and secrete papain-like cysteine peptidases we reasoned that these could be employed as vaccines with inbuilt adjuvanticity to protect against these parasites. Here we demonstrate that sub-cutaneous injection of functionally active S. mansoni cathepsin B1 (SmCB1), or a cathepsin L from a related parasite Fasciola hepatica (FhCL1), elicits highly significant (P<0.0001) protection (up to 73%) against an experimental challenge worm infection. Protection and reduction in worm egg burden were further increased (up to 83%) when the cysteine peptidases were combined with other S. mansoni vaccine candidates, glyceraldehyde 3-phosphate dehydrogenase (SG3PDH) and peroxiredoxin (PRX-MAP), without the need to add chemical adjuvants. These studies demonstrate the capacity of helminth cysteine peptidases to behave simultaneously as immunogens and adjuvants, and offer an innovative approach towards developing schistosomiasis vaccines     

Immunomodulatory effects of IL-12 released from poly-N-acetyl glucosamine gel matrix during schistosomiasis infection

We have reported recently that Interleukin-12 (IL-12) released from poly-N-acetyl glucosamine gel matrix (F2 gel/IL-12) is more effective than free IL-12 to enhance vaccination of mice with Schistosoma soluble worm antigen preparation. The aim of this study is to evaluate the effect of F2 gel/IL-12 on the inflammatory responses in mice undergoing schistosomiasis infection in absence of vaccination. To achieve this, mice undergoing Schistosoma mansoni infection or cured from this infection, after treatment with praziquantil (PZQ), were treated with subcutaneous injection of IL-12 for 3 consecutive days or once with F2 gel loaded with IL-12 (F2 gel/IL-12). The treatment was started on day 35 days after infection. For infection, mice were infected with 100 cercariae of S. mansoni using tail immersion method. We found that treatment with F2 gel/IL-12 induced significant decreases in the egg burden with a moderate reduction in the size of granuloma and decrease in the cellular granulomatous reaction in the lung as compared to infected mice treated with IL-12. These effects of F2 gel/IL-12 were more pronounced in infected mice previously treated with the anti-schistosomal drug PZQ. The total numbers of white blood cells in all treated mice showed similar profile. Treatment with IL-12 or F2 gel/IL-12, however, showed significant reduction in the number of mononuclear cells when compared with non-treated infected mice. In conclusion, this study showed the ability of IL-12 released from F2 gel to lower the inflammatory response to Schistosoma infection even in absence of vaccination.     

Transforming growth factor-β and Th17 responses in resistance to primary murine schistosomiasis mansoni

Discovery of the T-helper (Th) 17 cell lineage and functions in immune responses of mouse and man prompted us to investigate the role of transforming growth factor-beta (TGF-β) and interleukin (IL)-17 in innate resistance to murine schistosomiasis mansoni. Schistosoma mansoni-infected BALB/c and C57BL/6 mice were administered with recombinant TGF-β or mouse monoclonal antibody to TGF-β to evaluate the impact of this cytokine on host immune responses against lung-stage schistosomula, and subsequent effects on adult worm parameters. Developing schistosomula elicited increase in peripheral blood mononuclear cells (PBMC) mRNA expression and/or plasma levels of IL-4, IL-17, and interferon-gamma (IFN-γ), cytokines known to antagonize each other, resulting in impaired Th1/Th2, and Th17 immune responses and parasite evasion. Mice treated with TGF-β showed elevated PBMC mRNA expression of IL-6, IL-17, TGF-β, and TNF-α mRNA and increased IL-23 and IL-17 or TGF-β plasma levels, associated with significantly (P < 0.02–<0.0001) lower S. mansoni adult worm burden compared to controls in both mouse strains, thus suggesting that TGF-β led to heightened Th17 responses that mediated resistance to the infection. Mice treated with antibody to TGF-β showed increase in PBMC mRNA expression and plasma levels of IL-4, IL-12p70, and IFN-γ, and significantly (P < 0.02 and <0.0001) reduced worm burden and liver worm egg counts than untreated mice, indicating that Th1/Th2 immune responses were potentiated, resulting in significant innate resistance to schistosomiasis. The implications of these observations for schistosome immune evasion and vaccination were discussed.     

Ecological parasitology of polymorphic Arctic charr, Salvelinus alpinus (L.), in Thingvallavatn, Iceland

The helminth endoparasite fauna in four Arctic charr morphs, Salvelinus alpinus (L.), small benthivorous (SB), large benthivorous (LB), planktivorous (PL) and piscivorous (PI) charr, from Thingvallavatn, Iceland consisted of: Crepidostomum farionis (Trematoda: Allocreadiidae); Diplosttomum sp. (Trematoda: Diplostomatidae); Eubothrium salvelini; Diphyllobothrium dendriticum; D. ditremum (Cestoda: Pseudophyllidae); Proteocephalus longicollis (Cestoda: Proteocepha-lidae): and Philonema oncorhynchi (Nematoda: Filariidae). The morphs exhibited distinctive patterns in prevalences and parasite burdens (mean intensity and mean relative density of parasites). SB charr had high prevalence and parasite burden of the eye fluke Diplostomum sp. and none to very light infections of the other parasite species. LB charr had relatively high prevalence and parasite burden of the intestinal fluke C. farionis , whereas infections of the remaining parasite species were light to moderate. PL and PI charr had high prevalences and worm burdens of Diphyllobothrium spp. and P. longicollis . PL charr differed from PI charr in higher worm burden off P. longicollis and lighter burden of £. salvelini . Prevalences of P. oncorhynchi were high in PL and PI charr. Association of parasite intensities and age and length offish were investigated. The different infection patterns among the morphs agree well with their partitioning in food and habitat utilization, and confirm that there is a high degree of ecological segregation between the morphs. The results demonstrate the importance of ecological factors influencing transmission efficiency of parasites to the fish host.