Soluble Parasite Antigens Research Articles

OMNIGEN KIT ACCURACY FOR PLASMODIUM FALCIPARU

Malaria is a serious public health burden in Senegal. Accurate diagnosis is essential to avoided unnecessary presumptive treatment. Malaria diagnosis currently relies on identifying malaria parasite using microscopy and detecting soluble parasite antigens by Rapid Diagnostic Tests (RDT). These techniques do not detect low-level, sub-patent malaria infections and are inherently hazardous and invasive. To overcome these obstacles, alternatives diagnostics method were explored. In contrast to blood, saliva presents a reduced biohazard and can be painlessly collected in relatively large quantities by individuals with moderate training. The objective of this study was to use saliva collected in OmnIGEN Kits as an alternative sample for malaria detection. Methods: A total of 77 febril patients tested malaria positive by mRDT were enrolled in this study. From each patient, blood sample was collected for dried blood spot and blood smear; and saliva sample on OMNIGEN kit. Parasite density was determined from smear and Plasmodium falciparum DNA was extracted from both dried blood spot and saliva samples collected from the same patients. Extracted DNA was amplied by qPCR machine. Results: Malaria prevalence using qPCR was 98,7% and 60% respectively with blood and saliva. Compared to blood, saliva showed a sensitivity; specicity; positive predictive value of 60%; 100 %; and 100 %. The concordance between parasites detection from saliva and blood was (p = 0.45). In addition, no difference was found between these two methods and the microscopy counting. Conclusions:Saliva could be a non-invasive alternative method for P. falciparum detection and epidemiological surveillance in country with limited ressources.

Discovery of a recombinant Babesia canis supernatant antigen that protects dogs against virulent challenge infection

Soluble parasite antigens (SPA) in supernatants of in vitro cultures of Babesia canis can be used to vaccinate dogs against virulent B. canis infection. The moment that immunity becomes apparent coincides with the appearance of antibodies against SPA in the serum of the vaccinated animals. This so-called vaccination-challenge serum (VC-serum) was used to precipitate antigens from B. canis culture supernatants in agarose gels. This antigen preparation was then used to analyse the reactivity of sera from vaccinated dogs on western blots. Resultsshowed that the first appearance of antibody reactivity against a protein that migrated at the 39kDa position in SDS-PAGE gels was associated with the moment vaccinated dogs started to recover from a virulent challenge infection. In addition, pulse-chase experiments revealed that a 39–40kDa doublet was released into the supernatant of B. canis cultures starting 15min after the chase. This doublet was specifically precipitated by VC-serum, thus corroborating that the 39–40kDa doublet in SPA preparations was of parasite origin. Partial amino acid sequencing allowed the discovery of the gene that encoded the 39–40kDa doublet (canine Babesia antigen; CBA). The full-length gene was cloned and expressed in E. coli. The recombinant CBA protein (rCBA) was recognized by VC-serum, and antibodies against rCBA precipitated the 39kDa antigen of SPA preparations and of merozoites of B. canis. In addition, anti-rCBA serum reacted with the surface of B. canis merozoites (but not with B. rossi merozoites) in immunofluorescence. Vaccination of dogs with rCBA induced antibodies against rCBA, which recognized B. canis merozoites. Vaccinated dogs were protected against virulent challenge infection by limiting parasite proliferation. As a result, the development of clinical signs was prevented and the animals self-cured. In contrast, six out of seven non-vaccinated control dogs developed relatively high parasitaemia and serious clinical signs associated with poor tissue perfusion. This antigen can be used to replace the SPA antigen in the conventional B. canis vaccines, which eliminates the need for dog blood and serum for vaccine production.

Atypical Manifestations of Cutaneous Leishmaniasis in a Region Endemic for Leishmania braziliensis: Clinical, Immunological and Parasitological Aspects.

BackgroundAtypical cutaneous leishmaniasis (ACL) has become progressively more frequent in Corte de Pedra, Northeast Brazil. Herein we characterize clinical presentation, antimony response, cytokine production and parasite strains prevailing in ACL.Methodology/Principal FindingsBetween 2005 and 2012, 51 ACL (cases) and 51 temporally matched cutaneous leishmaniasis (CL) subjects (controls) were enrolled and followed over time in Corte de Pedra. Clinical and therapeutic data were recorded for all subjects. Cytokine secretion by patients’ peripheral blood mononuclear cells (PBMC) stimulated with soluble parasite antigen in vitro, and genotypes in a 600 base-pair locus in chromosome 28 (CHR28/425451) of the infecting L. (V.) braziliensis were compared between the two groups. ACL presented significantly more lesions in head and neck, and higher rate of antimony failure than CL. Cytosine–Adenine substitutions at CHR28/425451 positions 254 and 321 were highly associated with ACL (p<0.0001). In vitro stimulated ACL PBMCs produced lower levels of IFN-γ (p = 0.0002) and TNF (p <0.0001), and higher levels of IL-10 (p = 0.0006) and IL-17 (p = 0.0008) than CL PBMCs.Conclusions/SignificanceACL found in Northeast Brazil is caused by distinct genotypes of L. (V.) braziliensis and presents a cytokine profile that departs from that in classical CL patients. We think that differences in antigenic contents among parasites may be in part responsible for the variation in cytokine responses and possibly immunopathology between CL and ACL.

Linear B-cell epitope mapping of MAPK3 and MAPK4 from Leishmania braziliensis: implications for the serodiagnosis of human and canine leishmaniasis.

The correct and early identification of humans and dogs infected with Leishmania are key steps in the control of leishmaniasis. Additionally, a method with high sensitivity and specificity at low cost that allows the screening of a large number of samples would be extremely valuable. In this study, we analyzed the potential of mitogen-activated protein kinase 3 (MAPK3) and mitogen-activated protein kinase 4 (MAPK4) proteins from Leishmania braziliensis to serve as antigen candidates for the serodiagnosis of human visceral and tegumentary leishmaniasis, as well as canine visceral disease. Moreover, we mapped linear B-cell epitopes in these proteins and selected those epitopes with sequences that were divergent in the corresponding orthologs in Homo sapiens, in Canis familiaris, and in Trypanosoma cruzi. We compared the performance of these peptides with the recombinant protein using ELISA. Both MAPK3 and MAPK4 recombinant proteins showed better specificity in the immunodiagnosis of human and canine leishmaniasis than soluble parasite antigens and the EIE-leishmaniose-visceral-canina-bio-manguinhos (EIE-LVC) kit. Furthermore, the performance of this serodiagnosis assay was improved using synthetic peptides corresponding to B-cell epitopes derived from both proteins.

An in vitro model of antibody-enhanced killing of the intracellular parasite Leishmania amazonensis.

Footpad infection of C3HeB/FeJ mice with Leishmania amazonensis leads to chronic lesions accompanied by large parasite loads. Co-infecting these animals with L. major leads to induction of an effective Th1 immune response that can resolve these lesions. This cross-protection can be recapitulated in vitro by using immune cells from L. major-infected animals to effectively activate L. amazonensis-infected macrophages to kill the parasite. We have shown previously that the B cell population and their IgG2a antibodies are required for effective cross-protection. Here we demonstrate that, in contrast to L. major, killing L. amazonensis parasites is dependent upon FcRγ common-chain and NADPH oxidase-generated superoxide from infected macrophages. Superoxide production coincided with killing of L. amazonensis at five days post-activation, suggesting that opsonization of the parasites was not a likely mechanism of the antibody response. Therefore we tested the hypothesis that non-specific immune complexes could provide a mechanism of FcRγ common-chain/NADPH oxidase dependent parasite killing. Macrophage activation in response to soluble IgG2a immune complexes, IFN-γ and parasite antigen was effective in significantly reducing the percentage of macrophages infected with L. amazonensis. These results define a host protection mechanism effective during Leishmania infection and demonstrate for the first time a novel means by which IgG antibodies can enhance killing of an intracellular pathogen.

Soluble parasite antigens from Babesia canis do not directly activate the kallikrein system in dogs infected with Babesia canis

Soluble parasite antigens (SPA) from Babesia canis have been shown to induce protective immunity when used as vaccine. In order to explain the immune mechanisms of vaccination, the precise role of SPA in the pathogenesis of canine babesiosis is under investigation. Earlier studies suggested that the plasma kallikrein system is central in the pathogenesis of babesiosis, malaria and trypanosomosis, and significant plasma kallikrein activation during acute B. bovis and P. knowlesi infections has been described. In the studies presented here dogs were experimentally infected with B. canis to investigate whether the plasma kallikrein system is activated during babesiosis infection. Results showed that prekallikrein levels decreased during episodes of peak parasitaemia. No effect was found on the kallikrein levels. In order to determine whether B. canis SPA could activate plasma kallikrein, dogs were infused with variable amounts of B. canis SPA and plasma samples were taken for (pre-) kallikrein determination. The results indicated that B. canis SPA did not affect plasma (pre-) kallikrein levels. In addition, the effect of B. canis SPA on (pre-) kallikrein levels in normal dog plasma was determined in vitro. Again, no effect on (pre-) kallikrein levels was found. The results suggest that, although the kallikrein pathway may be involved in B. canis-associated pathology, the system is not directly activated by B. canis SPA. Furthermore, infusion of B. canis SPA as well as stroma of normal dog erythrocytes triggered the production of the acute phase reactant, C-reactive protein. This suggests that the inflammatory response that is triggered during B. canis infection could be in part due to the release and exposure of self molecules. The implications of these findings are discussed.

Do antenatal parasite infections devalue childhood vaccination?

On a global basis, both potent vaccine efficacy and high vaccine coverage are necessary to control and eliminate vaccine-preventable diseases. Emerging evidence from animal and human studies suggest that neglected tropical diseases (NTDs) significantly impair response to standard childhood immunizations. A review of efficacy and effectiveness studies of vaccination among individuals with chronic parasitic infections was conducted, using PUBMED database searches and analysis of data from the authors' published and unpublished studies. Both animal models and human studies suggest that chronic trematode, nematode, and protozoan infections can result in decreased vaccine efficacy. Among pregnant women, who in developing countries are often infected with multiple parasites, soluble parasite antigens have been shown to cross the placenta and prime or tolerize fetal immune responses. As a result, antenatal infections can have a significant impact on later vaccine responses. Acquired childhood parasitic infections, most commonly malaria, can also affect subsequent immune response to vaccination. Additional data suggest that antiparasite therapy can improve the effectiveness of several human vaccines. Emerging evidence demonstrates that both antenatal and childhood parasitic infections alter levels of protective immune response to routine vaccinations. Successful antiparasite treatment may prevent immunomodulation caused by parasitic antigens during pregnancy and early childhood and may improve vaccine efficacy. Future research should highlight the varied effects that different parasites (alone and in combination) can have on human vaccine-related immunity. To optimize vaccine effectiveness in developing countries, better control of chronic NTDs may prove imperative.

Comparison of Babesia rossi and Babesia canis isolates with emphasis on effects of vaccination with soluble parasite antigens : a review : review article

Babesia canis and B. rossi are large Babesia species that infect dogs and cause clinical disease. The spectrum of disease is highly diverse with either parasite, but upon evaluation of field cases it has been suggested that in general B. rossi is more virulent than B. canis. This difference was also found in experimental infections using B. canis and B. rossi isolates and appeared to be related to a difference in parasitaemia. Whether this difference reflects the essential difference between B. canis and B. rossi species in general, or merely reflects the variability in virulence of individual isolates cannot be discerned. Comparative in vitro and in vivo studies revealed a number of qualitative differences between the B. canis and B. rossi isolates studied; however, more research is required to determine any causal relationship between in vitro and in vivo characteristics. Vaccination with a bivalent vaccine (containing soluble parasite antigen [SPA] from supernatants of in vitro cultures of B. canis and B. rossi) induced protection against clinical babesiosis upon challenge infection with either parasite. The dynamics of parasitaemia upon challenge infection of vaccinated animals indicated a biological difference between the B. canis and B. rossi isolates studied. Vaccinated dogs that were challenged with B. rossi parasites (2 isolates tested) effectively controlled parasitaemia. By contrast, in vaccinated dogs that were challenged with B. canis isolates (2 isolates tested) there was little or no effect on parasitaemia but levels of SPA in plasma were reduced. Apparently the nature of vaccine-induced immunity differs with respect to the challenge species.

Regulation of immune responses in lymphatic filariasis.

The nature and intensity of immune reactions to filarial antigens appear to be controlled by two broad mechanisms: immunoregulation and immune tolerance. Parasite molecules of high molecular weight activate suppressor T lymphocytes; suppressive parasite products are present in sera from microfilaraemic patients. Prenatal or perinatal exposure to soluble parasite antigens may influence a person's future ability to react to filarial antigens.

Immunity against Babesia rossi infection in dogs vaccinated with antigens from culture supernatants

Soluble parasite antigens (SPA) from different Babesia species have been shown earlier to induce protective immunity when used as vaccine. However, initial attempts to produce such vaccine against Babesia rossi infection using SPA from B. rossi culture supernatants were not or only partially successful. Here we show that when dogs were vaccinated with a vaccine comprising SPA from B. rossi combined with SPA from Babesia canis protective immunity against experimental challenge infection was induced. Immunity was reflected in reduced clinical signs that resolved spontaneously, and reduction of parasitaemia and SPA in the blood. Not a single infected erythrocyte could be found in blood smears of dogs that had been repeatedly boosted (three vaccinations in total). In contrast, three out of four control dogs required chemotherapeutic treatment to prevent death. The fourth control dog showed a transient parasitaemia that resolved spontaneously. Vaccination did not prevent the development of a transient anaemia. It is concluded that a vaccine containing a mixture of SPA obtained from in vitro culture supernatants of B. rossi and B. canis induces protection in dogs against heterologous challenge infection with B. canis (as shown before) or B. rossi.

Onset and duration of immunity against Babesia canis infection in dogs vaccinated with antigens from culture supernatants

It has previously been shown that dogs can be vaccinated against heterologous Babesia canis infection using a vaccine containing soluble parasite antigens (SPA) from in vitro cultures of B. canis and B. rossi that are adjuvanted with saponin. In the present study the onset and duration of immunity of vaccinated dogs were studied. Results showed that 3–26 weeks after initial vaccination, dogs effectively limit the level of SPA in plasma upon challenge infection, which was reflected in limited duration and extent of clinical manifestations. There was no statistically significant effect of vaccination on the parasite load in the circulation, which was determined from blood smears. It was further shown that the level of immunity of primary vaccinated dogs (priming and booster vaccination with a 6-week interval) and that of repeatedly vaccinated dogs (a single additional vaccination 6 months after primary vaccination) is comparable. From this study it is concluded that vaccination with this preparation induces protective immunity against clinical babesiosis from 3 weeks after booster vaccination onwards, and remains effective for a period of at least another 6 months. A single booster vaccination is sufficient to maintain immunity for at least another 6 months.

Vaccination against canine babesiosis

It has been known for several decades that the soluble parasite antigen (SPA) of several Babesia species can be used as a vaccine against the clinical manifestations of babesiosis. Originally observed in the plasma of infected animals, SPA can also be recovered from the supernatants of in vitro cultures of these parasites. Variable success has been reported for vaccines against the bovine and canine Babesia parasites, which seems to be related to antigenic diversity within Babesia species. In this article, an overview is presented of the development of such vaccines for dogs, and additional research that has led to improvement of an SPA-based vaccine against Babesia canis in dogs.

Vaccination of dogs against heterologous Babesia canis infection using antigens from culture supernatants

Soluble parasite antigens (SPA) from European Babesia canis can be used to protect dogs against a homologous but not heterologous challenge infection. In this study it is shown that when dogs are vaccinated with a mixture of SPA from both, a European B. canis isolate and a South African Babesia rossi isolate, protective immunity against heterologous B. canis infection is induced. Three groups of five beagle dogs each were vaccinated twice with graded doses of SPA derived from in vitro cultures of B. canis and B. rossi, with a 3-week interval. Saponin was used as adjuvant. Three weeks after booster vaccination immunological responsiveness against heterologous B. canis antigen was measured by seroconversion against infected erythrocytes and lymphocyte transformation using SPA. Upon vaccination dogs produced antibodies against infected erythrocytes and lymphoblastogenic responses against SPA in a dose-dependent manner. Dogs were then challenged with heterologous B. canis parasites. Dogs appeared to be protected against challenge infection, which was reflected in less severe decrease of packed cell volume (PCV) and reduced clinical signs. The level of protection to clinical signs (but not excessive PCV drop) was related to the level of SPA in plasma and spleen size, and not related to peripheral parasitaemia. The results suggest that vaccination with this bivalent vaccine primes T-helper cells that recognise common epitopes on SPA from an antigenically distinct B. canis isolate. These cells provide the essential Th signal to mount an effective and timely antibody response against SPA and parasites or parasitised erythrocytes, which prevents the further development of clinical babesiosis.

Parasite localization and dissemination in the Babesia-infected host.

Babesia bovis infections in cattle and B. canis infections in dogs are characterized by non-haemolytic anaemia and low parasitaemia during the acute phase of the disease. In this phase of the disease, animals suffer from hypotension followed by disturbances of the coagulation system. This review discusses the hypothesis that may explain the process of parasite localization in the host, and the consequences of such localization. It is suggested that hypotension favours the interaction between infected erythrocytes and the endothelial lining, thus facilitating localization of the infection. In addition, activation of the coagulation system by a parasite-derived molecule (one associated with the surface of infected erythrocytes or a soluble antigen) might consolidate this situation by causing cellular plugs to form. The continued proliferation of parasites in such plugs may then result in the occurrence of capillaries that are particularly heavily parasitised. An explanation is also suggested for the protective effect of vaccines against clinical babesiosis, based on the soluble parasite antigens that are released into the medium in cultures of babesial parasites.

Parasite localization and dissemination in theBabesia-infected host

Babesia bovis infections in cattle and B. canis infections in dogs are characterized by non-haemolytic anaemia and low parasitaemia during the acute phase of the disease. In this phase of the disease, animals suffer from hypotension followed by disturbances of the coagulation system. This review discusses the hypothesis that may explain the process of parasite localization in the host, and the consequences of such localization. It is suggested that hypotension favours the interaction between infected erythrocytes and the endothelial lining, thus facilitating localization of the infection. In addition, activation of the coagulation system by a parasite-derived molecule (one associated with the surface of infected erythrocytes or a soluble antigen) might consolidate this situation by causing cellular plugs to form. The continued proliferation of parasites in such plugs may then result in the occurrence of capillaries that are particularly heavily parasitised.An explanation is also suggested for the protective effect of vaccines against clinical babesiosis, based on the soluble parasite antigens that are released into the medium in cultures of babesial parasites.

Vaccination of dogs against Babesia canis infection

This paper describes the clinico-pathological parameters measured in dogs that were vaccinated against Babesia canis using soluble parasite antigens (SPA) and then challenged. The packed cell volume (PCV) and the plasma creatinine value decreased immediately after challenge. Actual PCV values could be predicted in the first 5–6 days of the infection, assuming that creatinine values were modulated by increase of plasma volume. This association no longer existed after that time, and observations indicated splenic involvement in reduction of numbers of circulating erythrocytes. The anaemia due to B. canis infection appears to be the result of a multifactorial process including plasma volume increase, erythrocyte retention in the spleen and erythrocyte destruction, partly due to parasite proliferation. Vaccination limited the reduction of PCV values, and the development of splenomegaly. Differences in protection between vaccinated and control animals became apparent 6 days after infection, when a memory immune response becomes operative, and the onset of recovery of vaccinated animals correlated with the onset of antibody production against SPA.

Evidence for direct interaction between mast cells and Leishmania parasites

When stimulated through IgE-(or IgG-) immune complexes with parasite antigens, mast cells can release several cytokines, including IL-4, IL-6, IL-10, IL-12, Interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) that may influence the host response to Leishmania major in modulating lesion size and persistence during experimental infection in the mouse. Moreover, recent data demonstrated that mast cells are able to be antibody-independently activated by direct contact with bacteria, making them important elements in innate immunity. Given these data, we asked whether cell-parasite contact could directly induce mast cell mediator release and whether mast cells could be infected by L. major or L. infantum parasites. In this study, we showed that a pure homogeneous population of mouse bone marrow derived mast cells (BMMC) in contact with living L. major or L. infantum promastigotes, but not with attenuated parasites or soluble parasite antigens, released preformed mediators such as beta-hexosaminidase and the preformed pool of TNF-alpha within minutes. Furthermore, direct cell-parasite contact induced TNF-alpha synthesis by mast cells within hours. Moreover, we demonstrated by in vitro co-culture experiments that metacyclic L. major or L. infantum promastigotes are directly infective for a significant proportion of BMMC and are transformed into intracellular amastigotes. Taken together, these data suggest that mast cell can participate in the first line of defence, i.e. innate immunity, during local cutaneous infection with Leishmania parasites.

Cryptosporidium infection in major histocompatibility complex congeneic strains of mice: variation in susceptibility and the role of T-cell cytokine responses

Studies with murine infection models have shown that immunity to the protozoan parasite Cryptosporidium involves T-cells and interferon-gamma (IFN-gamma) activity. The present study was performed to compare the course of infection of Cryptosporidium muris in major histocompatibility complex (MHC) congeneic strains of mice and examine the relationship between susceptibility to infection and production of T-cell cytokines. In experiments with BALB mice, the BALB/c strain (H-2d) produced significantly fewer oocysts and recovered from infection sooner than the BALB/B (H-2b) or BALB/K (H-2k) strains. BALB/B X BALB/c F1 hybrid mice were found to express the more susceptible phenotype of the BALB/B parent strain, indicating that the gene(s) in the H-2 locus conferring increased susceptibility to C. muris infection was dominant. At different times during infection of the resistant BALB/c strain and the susceptible BALB/B strain, splenocytes were cultured with soluble parasite antigen and measurements were made of production of a number of T-cell cytokines. Similar patterns of increasing levels of IFN-gamma and interleukin 2 (IL-2) were observed in both the resistant and susceptible strains during the patent stage of infection, indicating that production of these type 1 T-helper-cell (TH1) cytokines (i.e. involved in cell-mediated responses) correlated with the development of immunity. This also suggested that the increased susceptibility of BALB/B mice was not associated with a defective TH1 cytokine response. In the study of TH2 cytokines (involved in induction of an antibody response), low levels of IL-10 were detected during infection of BALB/c and BALB/B mice. In contrast, although IL-4 was released by splenocytes of both strains, significantly larger amounts were obtained from cells of the susceptible BALB/B mice in the early stages of infection. Thus, the H-2-dependent variation in susceptibility to infection between these BALB strains correlated with a difference in the pattern of IL-4 secretion.

Not peripheral parasitaemia but the level of soluble parasite antigen in plasma correlates with vaccine efficacy against Babesia canis.

Groups of five dogs were vaccinated against Babesia canis using soluble parasite (SPA) antigens from in vitro cultures. Although vaccination did not significantly alter peripheral parasitaemia upon challenge, protected animals had lower levels of SPA in the plasma after a challenge infection. The severity of anaemia correlated with the SPA-load during the post-challenge period in that high levels of SPA were associated with low haematocrit values. In addition, it was found that recovery was associated with the production of antibodies against SPA. The results suggest that SPA induce anaemia during B. canis infection, and that vaccination with SPA results in antibody production that can neutralize the effects of SPA after a challenge infection.

Strain variation limits protective activity of vaccines based on soluble Babesia canis antigens

Groups of five dogs were immunized with vaccines containing soluble parasite antigens (SPA) derived from in vitro culture of Babesis canis parasites, either obtained commercially (Pirodog) or produced at laboratory scale. Both vaccines generated antibodies that reacted with parasitised erythrocytes (PE). Upon challenge infection with homologous parasites, protection was evident from less severe decreases of haematocrit values, and reduced morbidity. Vaccinated animals, however, were not protected against challenge infection with heterologous B. canis parasites. Recovery from challenge infection coincided with the production of antibodies against parasitized erythrocytes. The results suggest that SPA from B. canis carry strain-specific determinants that are crucial for inducing protection in dogs against challenge infection, and explain vaccination failures in the field.