Vaccination with antioxidant enzymes confers protective immunity against challenge infection with Schistosoma mansoni

Schistosoma mansoni, an intravascular parasite, lives in a hostile environment in close contact with host humoral and cellular cytotoxic factors. To establish itself in the host, the parasite has evolved a number of immune evasion mechanisms, such as antioxidant enzymes. Our laboratory has demonstrated that the expression of antioxidant enzymes is developmentally regulated, with the highest levels present in the adult worm, the stage least susceptible to immune elimination, and the lowest levels in the larval stages, the most susceptible to immune elimination. Vaccination of mice with naked DNA constructs containing Cu/Zn cytosolic superoxide dismutase (CT-SOD), signal-peptide containing SOD or glutathione peroxidase (GPX) showed significant levels of protection compared to a control group. We have further shown that vaccination with SmCT-SOD but not SmGPX results in elimination of adult worms. Anti-oxidant enzyme vaccine candidates offer an advance over existing vaccine strategies that all seem to target the larval developmental stages in that they target adult worms and thus may have therapeutic as well as prophylactic value. To eliminate the potential for cross-reactivity of SmCT-SOD with human superoxide dismutase, we identified parasite-specific epitope-containing peptides. Our results serve as a basis for developing a subunit vaccine against schistosomiasis.

From genomes to vaccines via the proteome

An effective vaccine against schistosomiasis mansoni would be a valuable control tool and the high levels of protection elicited in rodents and primates by radiation-attenuated cercariae provide proof of principle. A major obstacle to vaccine development is the difficulty of identifying the antigens that mediate protection, not least because of the size of the genome at 280Mb DNA encoding 14,000 to 20,000 genes. The technologies collectively called proteomics, including 2D electrophoresis, liquid chromatography and mass spectrometry, now permit any protein to be identified provided there is extensive DNA data, and preferably a genome sequence. Applied to soluble (cytosolic) proteins from schistosomes, proteomics reveals the great similarity in composition between life cycle stages, with several WHO vaccine candidates amongst the most abundant constituents. The proteomic approach has been successfully applied to identify the secretions used by cercaria to penetrate host skin, the gut secretions of adult worms and the proteins exposed on the tegument surface. Soluble proteins can also be separated by 2D electrophoresis before western blotting to identify the full range of antigenic targets present in a parasite preparation. The next step is to discover which target proteins represent the weak points in the worm's defences.

Pathology and first occurrence of the kidney trematode Paratanaisia bragai (Santos, 1934) Freitas, 1959 (Digenea: Eucotylidae) in Phasianus colchicus L., 1758, from Brazil

The kidney trematode Paratanaisia bragai is reported for the first time parasitizing the ring-necked pheasant (Phasianus colchicus L., 1758) and the pathological alterations associated to the parasitism are referred on the basis of 50 specimens of this bird from backyard flocks in 11 counties of the state of Rio de Janeiro, Brazil after clinical examination, necropsies, and histopathological analysis. The counting of the kidney flukes was based on worms recovered from one of the kidneys, since the other was fixed in 10% formalin and then routinely processed for histopathological procedures. The prevalence of P. bragai was of 22%, with a mean intensity of 44.3, mean abundance of 9.7, and range of infection of 3-153. Parasitized birds did not present with clinical signs and kidney gross lesions. Microscopic lesions were mild and characterized by dilatation of the renal medullary collecting ducts, occasional flattening of the lining epithelium of the ducts and inflammatory reaction of variable intensity with granulocytes around the ureter branches and medullary collecting ducts. The severity and pattern of the microscopic lesions seem not to be associated to the size of the worm burden and could be related to the mechanic action of the parasites, without traumatism, in despite of the presence of the tegumentar spines in specimens of P. bragai.

Angiostrongylus costaricensis: complete redescription of the migratory pathways based on experimental Sigmodon hispidus infection

Angiostrongylus costaricensis lives in the cecal and mesenteric arteries of its vertebrate hosts, and causes an inflammatory disease in humans. To investigate unknown aspects of the abdominal angiostrogyliasis pathogenesis, infected Sigmodon hispidus were sequentially studied in different times of infection. The study revealed that L3 goes alternatively through two migratory courses during its development into an adult worm: lymphatic/venous-arterial and venous portal pathways. The former is considered the principal one, because it is used by most of the larvae. Like other metastrongylides, A. costaricensis passes over the pulmonary circulation to migrate from the lymphatic system to the arterial circulation, where they circulate during some days before reaching their definitive habitat. The oviposition by mature females began on 15th day. Eggs and L1 were detected mainly in the intestine and stomach, surrounded by inflammatory reaction constituted by macrophages, monocytes, and eosinophils. They were also spread to the lungs, mesenteric lymph nodes, pancreas, spleen, and kidneys. The larvae (L1) exhibited the centripetal capacity to invade the lymphatic and venous vessels of the intestine and mesentery. Adult worms that developed in the venous intrahepatic pathway migrated downstream to reach the mesenteric veins and laid eggs that embolized in the portal hepatic vessels.

Resistance to reinfection in rats induced by irradiated metacercariae of Clonorchis sinensis

A study was made to observe the association between the resistance to reinfection induced by irradiated metacercariae (MC) of Clonorchis sinensis and antigen specific Th1- and Th2-type cytokine productions in rats. Rats were infected with 20 MC of C. sinensis, previously exposed to a single dose of gamma irradiation, which varied from 0 to 100 Gy. All of them, single dose of 12 Gy showed higher IgG antibody titer with lowest worm recovery. Thus, 50 MC were used to challenge infection in rats previously infected with 20 MC irradiated at 12 Gy and the highest resistance to challenge infection was observed. The results of lymphocyte proliferation with specific antigen, ES Ag were shown no difference of proliferative responses as compared with primary and challenge infection at 12 Gy irradiation dose. In the case of cytokines production were observed that interferon (IFN-gamma) and interlukin (IL-2) were significantly enhanced, while IL-4 and IL-10 was almost unchanged to make comparison between primary and secondary infection at 12 Gy irradiation dose. In conclusion, the single dose of 12 Gy could be adopted for induction of the highest resistance to challenge infection. Up-regulation of Th1 type cytokines, IFN-gamma and IL-2 may be affected to develop vaccine by irradiated MC.

Efficacy of Citrus reticulata and Mirazid in treatment of Schistosoma mansoni

This work has been carried out to investigate the effect of Schistosoma mansoni infection on mice livers after treatment with the ethanolic extract of Citrus reticulata root or the oleo-resin extract from Myrrh of Commiphora molmol tree (Mirazid), as a new antishistosomal drug. Marker enzymes for different cell organelles were measured; succinate dehydrogenase (SDH); lactate dehydrogenase (LDH) and its isoenzymes; glucose-6-phosphatase (G-6-Pase); acid phosphatase (AP) and 5'- nucleotidase. Liver function enzymes; aspartate aminotransferase (AST); alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were also estimated. Parasitological studies through ova count and worm burden will also be taken into consideration. The results showed a marked reduction in SDH, LDH, AST, and ALT enzyme activities and a significant increase in G-6-Pase, AP, 5'- nucleotidase, and ALP after S. mansoni infection. A noticeable alteration in LDH subunits were also noticed. Treatment with C. reticulata or Mirazid improved all the previous enzyme activities with a noticeable reduction in ova count and worm burden.

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5% and 47.6% less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.

Localization of Brugia malayi (sub-periodic) adults in different organs of Mastomys coucha and its influence on microfilaraemia and host antibody response

Lymphatic filariasis caused by nematode parasites Wuchereria bancrofti or Brugia malayi is a spectral disease and produces wide range of immune responses and varying levels ofmicrofilaraemia in infected individuals. The relationship between the immune response of host and the developmental stage of the parasite as well as the microfilariae (mf) density and specific location of the adult worms is yet to be understood. As an experimental model, B. malayi adapted in the experimental animal Mastomys coucha has been used widely for various studies in filariasis. The present study was to assess microfilaraemia as well as the humoral immune response of M. coucha during various stages of B. malayi development and their localization in different organs. The result showed that the density of mf in the circulating blood of the experimental animal depended upon the number of female worms as well as the location and co-existence of male and female worms. The mf density in the blood increased with the increase in the number of females. The clearance of inoculated infective stage (L3) or single sex infection or segregation of male and female to different organs of infected host resulted in amicrofilaraemic condition. With respect to antibody response, those animals cleared L3 after inoculation and those with adult worm as well as mf showed low antibody levels. But those with developmental fourth stage and/or adult worms without mf showed significantly higher antibody levels.

Proteomic analysis of the shistosome tegument and its surface membranes

The tegument surface of the adult schistosome, bounded by a normal plasma membrane overlain by a secreted membranocalyx, holds the key to understanding how schistosomes evade host immune responses. Recent advances in mass spectrometry (MS), and the sequencing of the Schistosoma mansoni transcriptome/genome, have facilitated schistosome proteomics. We detached the tegument from the worm body and enriched its surface membranes by differential extraction, before subjecting the preparation to liquid chromatography-based proteomics to identify its constituents. The most exposed proteins on live worms were labelled with impearmeant biotinylation reagents, and we also developed methods to isolate the membranocalyx for analysis. We identified transporters for sugars, amino acids, inorganic ions and water, which confirm the importance of the tegument plasma membrane in nutrient acquisition and solute balance. Enzymes, including phosphohydrolases, esterases and carbonic anhydrase were located with their catalytic domains external to the plasma membrane, while five tetraspanins, annexin and dysferlin were implicated in membrane architecture. In contrast, few parasite proteins could be assigned to the membranocalyx but mouse immune response proteins, including three immunoglobulins and two complement factors, were detected, plus host membrane proteins such as CD44, integrin and a complement regulatory protein, testifying to the acquisitive properties of the secreted bilayer.

The effects of drugs, ions, and poly-l-lysine on the excretory system of Schistosoma mansoni

We have been able to label the excretory system of cercariae and all forms of schistosomula, immature and adult worms with the highly fluorescent dye resorufin. We have shown that the accumulation of the resorufin into the excretory tubules and collecting ducts of the male adult worm depends on the presence of extracellular calcium and phosphate ions. In the adult male worms, praziquantel (PZQ) prevents this accumulation in RPMI medium and disperses resorufin from tubules which have been prelabelled. Female worms and all other developmental stages are much less affected either by the presence of calcium and phosphate ions, or the disruption caused by PZQ. The male can inhibit the excretory system in paired female. Fluorescent PZQ localises in the posterior gut (intestine) region of the male adult worm, but not in the excretory system, except for the anionic carboxy fluorescein derivative of PZQ, which may be excreted by this route. All stages of the parasite can recover from damage by PZQ treatment in vitro. The excretory system is highly sensitive to damage to the surface membrane and may be involved in vesicle movement and damage repair processes. In vivo the adult parasite does not recover from PZQ treatment, but what is inhibiting recovery is unknown, but likely to be related to immune effector molecules.

Schistosome vaccine testing: lessons from the baboon model

The high level of protection elicited in rodents and primates by the radiation-attenuated schistosome vaccine gives hope that a human vaccine relying on equivalent mechanisms is feasible. In humans, a vaccine would be undoubtedly administered to previously or currently infected individuals. We have therefore used the olive baboon to investigate whether vaccine-induced immunity is compromised by a schistosome infection. We showed that neither a preceding infection, terminated by chemotherapy, nor an ongoing chronic infection affected the level of protection. Whilst IgM responses to vaccination or infection were short-lived, IgG responses rose with each successive exposure to the vaccine. Such a rise was obscured by responses to egg deposition in already-infected animals. In human trials it would be necessary to use indirect estimates of infection intensity to determine vaccine efficacy. Using worm burden as the definitive criterion, we demonstrated that the surrogate measures, fecal eggs, and circulating antigens, consistently overestimated protection. Regression analysis of the surrogate parameters on worm burden revealed that the principal reason for overestimation was the threshold sensitivity of the assays. If we extrapolate our findings to human schistosomiasis mansoni, it is clear that more sensitive indirect measures of infection intensity are required for future vaccine trials.

Low susceptibility of Achatina fulica from Brazil to infection with Angiostrongylus costaricensis and A. cantonensis

Introduction of Achatina fulica in Brazil has led to serious concerns about its role as vector for metaIylid worms: AngioIylus costaricensis and A. cantonensis. Experimental infection with both parasites was performed to evaluate the potential risk for their transmission by the giant African snail. Groups of 5 animals, both wild and bred at captivity were exposed at different inocula: 1, 5, and 10 ´ 10³ L1 of A. costaricensis and A. cantonensis. In all groups, few snails got infected and parasitic burden was low. Two different ways of infection were tested: ingestion produced higher numbers of L3 than the inoculation through an artificial hole in the shell. We also report the parasitological examination of 6 batches of wild A. fulica from Florianópolis, state of Santa Catarina, Brazil: only 1 out of 244 animals were infected with metaIylid larvae. Taken together these data indicate that the giant African snail occurring in Southern Brazil is not a permissive host for both AngioIylus species and does not represent a significant risk for transmission of these parasites.

Treatment of human acute schistosomiasis with oxamniquine induces an increase in interferon-gamma response to Schistosoma mansoni antigens

Patients with acute schistosomiasis were studied before and after oxamniquine treatment. They had been exposed to cercariae 5 to 9 weeks before, and presented compatible clinical manifestations, eosinophilia, and high levels of total IgE. Interferon-gamma (IFN-gamma) and interleukin-4 were measured by ELISA in whole blood samples under soluble egg antigen or soluble adult worm preparation stimulation. After treatment, the reduction of leukocytosis and eosinophilia were not significant, but total IgE levels decreased significantly, in contrast to IFN-gamma levels that were significantly increased. The oxamniquine treatment of acute schistosomiasis patients is followed by an improvement of a Th1 response in vitro. If this response has a protective aspect is unknown, and some investigations need to be realized.

First record in South America of Didymosulcus palati and Didymosulcus philobranchiarca (Digenea, Didymozoidae) with new hosts records and pathological alterations

Two species of Didymozoidae, Didymosulcus palati (Yamaguti 1970) and Didymosulcus philobranchiarca (Yamaguti 1970) were reported for the first time in South America, Atlantic Ocean, parasitizing three different tuna species from the coast of Rio de Janeiro, Brazil: Thunnus atlanticus (Lesson), Thunnus albacares (Bonnaterre) and Thunnus obesus (Lowe). Pairs of D. philobranchiarca were found on gill arches of T. albacares and T. obesus, in longitudinal rows of yellow cysts located inside grooves in the hard denticle palate (new site) of the three hosts species studied, and as disperse groups of cysts in the operculum (new site) and gill arches of T. atlanticus (new host record). D. palati occurred as disperse groups of encysted worm pairs in the gill arches of T. albacares and T. obesus and in gill arches and operculum of T. atlanticus (new host record). The pathological alterations induced by D. philobranchiarca in the palate of T. atlanticus are described for the first time. Original measurements and figures are presented.

Capillariid nematodes in Brazilian turkeys, Meleagris gallopavo (Galliformes, Phasianidae): pathology induced by Baruscapillaria obsignata and Eucoleus annulatus (Trichinelloidea, Capillariidae)

The pathology induced in turkeys (Meleagris gallopavo) by two capillariid nematodes, Baruscapillaria obsignata and Eucoleus annulatus is described together with data on prevalences, mean infection and range of worm burdens. B. obsignata occurred with a prevalence of 72.5% in the 40 examined hosts in a range of 2-461 nematodes and a mean intensity of 68.6, whereas E. annulatus was present in 2.5% of the animals, with a total amount of five recovered parasites. Gross lesions were not observed in the parasitized birds. Lesions due to B. obsignata mainly consisted of the thickening of intestinal villi with a mild mixed inflammatory infiltrate with the presence of mononuclear cells and heterophils. The lesions induced by E. annulatus were represented by foci of inflammatory infiltrate with heterophils in the crop epithelium and esophagus of a single infected female. These are the first pathological findings related to the presence of capillariid worms in turkeys to be reported in Brazil so far. Capillaria anatis, although present, was not pathogenic to the investigated turkeys.