Immunoglobulin Y for Potential Diagnostic and Therapeutic Applications in Infectious Diseases.

Antiviral, antibacterial, and antiparasitic drugs and vaccines are essential to maintaining the health of humans and animals. Yet, their production can be slow and expensive, and efficacy lost once pathogens mount resistance. Chicken immunoglobulin Y (IgY) is a highly conserved homolog of human immunoglobulin G (IgG) that has shown benefits and a favorable safety profile, primarily in animal models of human infectious diseases. IgY is fast-acting, easy to produce, and low cost. IgY antibodies can readily be generated in large quantities with minimal environmental harm or infrastructure investment by using egg-laying hens. We summarize a variety of IgY uses, focusing on their potential for the detection, prevention, and treatment of human and animal infections.

Avid binding by B cells to the Plasmodium circumsporozoite protein repeat suppresses responses to protective subdominant epitopes.

Antibodies targeting the NANP/NVDP repeat domain of the Plasmodium falciparum circumsporozoite protein (CSPRepeat) can protect against malaria. However, it has also been suggested that the CSPRepeat is a decoy that prevents the immune system from mounting responses against other domains of CSP. Here, we show that, following parasite immunization, B cell responses to the CSPRepeat are immunodominant over responses to other CSP domains despite the presence of similar numbers of naive B cells able to bind these regions. We find that this immunodominance is driven by avid binding of the CSPRepeat to cognate B cells that are able to expand at the expense of B cells with other specificities. We further show that mice immunized with repeat-truncated CSP molecules develop responses to subdominant epitopes and are protected against malaria. These data demonstrate that the CSPRepeat functions as a decoy, but truncated CSP molecules may be an approach for malaria vaccination.

Protein/AS01B vaccination elicits stronger, more Th2-skewed antigen-specific human T follicular helper cell responses than heterologous viral vectors.

Interactions between B cells and CD4+ T follicular helper (Tfh) cells are key determinants of humoral responses. Using samples from clinical trials performed with the malaria vaccine candidate antigen Plasmodium falciparum merozoite protein (PfRH5), we compare the frequency, phenotype, and gene expression profiles of PfRH5-specific circulating Tfh (cTfh) cells elicited by two leading human vaccine delivery platforms: heterologous viral vector prime boost and protein with AS01B adjuvant. We demonstrate that the protein/AS01B platform induces a higher-magnitude antigen-specific cTfh cell response and that this correlates with peak anti-PfRH5 IgG concentrations, frequency of PfRH5-specific memory B cells, and antibody functionality. Furthermore, our data indicate a greater Th2/Tfh2 skew within the polyfunctional response elicited following vaccination with protein/AS01B as compared to a Th1/Tfh1 skew with viral vectors. These data highlight the impact of vaccine platform on the cTfh cell response driving humoral immunity, associating a high-magnitude, Th2-biased cTfh response with potent antibody production.

Comparative Immunological Response and Pathobiology of Mice Inoculated with Toxoplasma gondii Isolated from Different Hosts.

Toxoplasma gondii is an obligate intracellular parasite that has a worldwide distribution and can infect almost all warm-blood animals. Serological tests are the main detection methods for T. gondii infection in animals and humans. Little is known of biological behavior, antibody responses, and virulence of T. gondii strains in mice from China. Here we document antibody responses, tissue cyst burden, and mouse virulence of T. gondii strains isolated from different hosts in China. All T. gondii strains formed tissue cysts in the brains of mice and positively correlated with the T. gondii antibody titer (R2 = 0.3345). These results should aid in the diagnosis and characterization of T. gondii isolates.

Glycan moieties in Entamoeba histolytica ubiquitin are immunodominant.

The ubiquitin-proteasome system plays a central role performing several functions to maintain parasite homeostasis. We have reported the partial characterization of N-linked glycosylation profile in E. histolytica ubiquitin (EhUb). Here we examined the immunogenicity and antigenicity of carbohydrates in EhUbiquitin. Rabbits were immunized with purified EhUbiquitin or purified recombinant rUb expressed by E. coli. Using Western Blot, we explored the immunogenicity and antigenicity of protein portion and carbohydrates moiety. Interestingly, immunized rabbits produced antibodies to both Ub glycoprotein and rUb; but antibodies against carbohydrates were immunodominant, rather than antibodies to the protein moiety of EhUbiquitin. In addition, we observed that antibodies to protein moiety are not conserved in serum unless antigen is continually administrated. Conversely, anti-Ub glycoprotein antibodies are well maintained in circulation. In humans, infection with Entamoeba histolytica induces strong IgG anti-Ub response. The human antibodies recognize both, the protein moieties and the glycosylated structure. Entamoeba histolytica ubiquitin is immunogenic and antigenic. The glycan moieties are immunodominant and induces IgG. These data open the door to use carbohydrates as potential targets for diagnose tests, drugs and vaccine to prevent this parasitic disease.

Epitope Analysis and Efficacy Evaluation of Phosphatase 2C (PP2C) DNA Vaccine Against Toxoplasma gondii Infection.

Toxoplasma gondii infects almost all warm-blooded animals and negatively affects the health of a wide range of these animals, including humans. Protein phosphatase 2C (PP2C) is a T. gondii protein secreted by rhoptry organelles during host cell invasion. However, very little is known about whether this protein can induce protective immunity against T. gondii. In this study, bioinformatics analysis of PP2C revealed some useful information in the context of anti-toxoplasmosis treatments and vaccine research. In addition, the PP2C gene was amplified, and a eukaryotic expression vector (pEGFP-PP2C) was successfully constructed to express PP2C. Finally, the constructed pEGFP-PP2C was injected into mice to evaluate whether it could induce immunoprotection. Compared with the control groups, we found that immunizations with the pEGFP-PP2C plasmid could elicit specific IgG antibodies and cytokines against T. gondii infection. The survival of mice immunized with the pEGFP-PP2C plasmid was significantly prolonged compared with that of the control group mice. Based on the ability of pEGFP-PP2C to induce specific immune responses against T. gondii, we propose that PP2C merits consideration as a potential vaccine candidate against toxoplasmosis.

The Case for Exploiting Cross-Species Epitopes in Malaria Vaccine Design.

The infection dynamics between different species of Plasmodium that infect the same human host can both suppress and exacerbate disease. This could arise from inter-parasite interactions, such as competition, from immune regulation, or both. The occurrence of protective, cross-species (heterologous) immunity is an unlikely event, especially considering that strain-transcending immunity within a species is only partial despite lifelong exposure to that species. Here we review the literature in humans and animal models to identify the contexts where heterologous immunity can arise, and which antigens may be involved. From the perspective of vaccine design, understanding the mechanisms by which exposure to an antigen from one species can elicit a protective response to another species offers an alternative strategy to conventional approaches that focus on immunodominant antigens within a single species. The underlying hypothesis is that certain epitopes are conserved across evolution, in sequence or in structure, and shared in antigens from different species. Vaccines that focus on conserved epitopes may overcome the challenges posed by polymorphic immunodominant antigens; but to uncover these epitopes requires approaches that consider the evolutionary history of protein families across species. The key question for vaccinologists will be whether vaccines that express these epitopes can elicit immune responses that are functional and contribute to protection against Plasmodium parasites.

Fowlerstefin, a cysteine protease inhibitor of Naegleria fowleri, induces inflammatory responses in BV-2 microglial cells in vitro.

BACKGROUND: Naegleria fowleri is a free-living amoeba that causes an opportunistic fatal infection known as primary amoebic meningoencephalitis (PAM) in humans. Cysteine proteases produced by the amoeba may play critical roles in the pathogenesis of infection. In this study, a novel cysteine protease inhibitor of N. fowleri (fowlerstefin) was characterized to elucidate its biological function as an endogenous cysteine protease inhibitor of the parasite as well as a pathogenic molecule that induces immune responses in microglial cells. METHODS: Recombinant fowlerstefin was expressed in Escherichia coli. The inhibitory activity of fowlerstefin against several cysteine proteases, including human cathepsins B and L, papain and NfCPB-L, was analyzed. Fowlerstefin-induced pro-inflammatory response in BV-2 microglial cells was anayzed by cytokine array assay, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. RESULTS: Fowlerstefin is a cysteine protease inhibitor with a monomeric structure, and belongs to the stefin family. Recombinant fowlerstefin effectively inhibited diverse cysteine proteases including cathepsin B-like cysteine proteases of N. fowleri (NfCPB-L), human cathepsins B and L, and papain. Expression of fowlerstefin in the amoeba was optimal during the trophozoite stage and gradually decreased in cysts. Fowlerstefin induced an inflammatory response in BV-2 microglial cells. Fowlerstefin induced the expression of several pro-inflammatory cytokines and chemokines including IL-6 and TNF in BV-2 microglial cells. Fowlerstefin-induced expression of IL-6 and TNF in BV-2 microglial cells was regulated by mitogen-activated protein kinase (MAPKs). The inflammatory response induced by fowlerstefin in BV-2 microglial cells was downregulated via inhibition of NF-κB and AP-1. CONCLUSIONS: Fowlerstefin is a pathogenic molecule that stimulates BV-2 microglial cells to produce pro-inflammatory cytokines through NF-κB- and AP-1-dependent MAPK signaling pathways. Fowlerstefin-induced inflammatory cytokines exacerbate the inflammatory response in N. fowleri-infected areas and contribute to the pathogenesis of PAM.

Radiation effects on Toxoplasma antigens: different immune responses of irradiated intact tachyzoites or soluble antigens in experimental mice models.

Purpose: Purpose: Protein irradiation causes aggregation, chain breakage, and oxidation, enhancing its uptake by antigen-presenting cells. To evaluate if irradiated proteins participate on the protection, we studied the immune response induced in mice immunized with irradiated soluble extracts of T. gondii tachyzoites (STag) or irradiated intact T. gondii RH tachyzoites (RH0.25 kGy).Material and Methods: Soluble extracts of Toxoplasma gondii tachyzoites (STag) were irradiated at different dose by Cobalt-60 source. By polyacrylamide gel electrophoresis (SDS-Page) we evaluated the effects on primary structures of protein STags induced by irradiation. By Enzyme-linked Immunosorbent Assay (ELISA) we evaluated the difference between humoral immune response induced by irradiated STag or RH tachyzoites in immunized mice from the detection of specific immunoglobulin G (IgG) antibodies in the serum of immunized mice. From challenge with viable RH strain of T. gondii we evaluated the protection induced in the immunized animals. By cytometry we performed the phenotyping of T and B lymphocytes in the peripheral blood of the immunized animals.Results: Irradiation dose of 1.5 kGy induced minimal changes in most proteins, without affecting their antigenicity or immunogenicity. Immunization showed saturation at the dose of 10 µg/mice, with worst response at higher doses. STag irradiated at 1.5 kGy (STag1.5 kGy) induced higher survival and protection similar to T. gondii RH strain irradiated at 0.25 kGy (RH0.25 kGy), with higher serum levels of high affinity IgG compared to STag native. Blood immune memory cells of mice immunized with STag1.5 kGy had higher proportions of CD19+ (cluster of differentiation 19) and CD4+ (cluster of differentiation 14) cells, whereas mice RH0.25 kGy had high proportion of memory CD8+ (cluster of differentiation 8) cells.Conclusions: Our data suggest that major histocompatibility complex type I (MHCI) pathway, appears seem to be used by RH0.25 kGy to generate cytotoxic cells while STag1.5 kGy uses a major histocompatibility complex type II (MHCII) pathway for B-cell memory, but both induce sufficient immune response for protection in mice without any adjuvant. Irradiation of soluble protein extracts enhances their immune response, allowing similar protection against T. gondii in mice as compared to irradiated intact parasites.

An immunocompetent rat model of infection with Cryptosporidium hominis and Cryptosporidium parvum.

A major obstacle to developing vaccines against cryptosporidiosis, a serious diarrheal disease of children in developing countries, is the lack of rodent models essential to identify and screen protective immunogens. Rodent models commonly used for drug discovery are unsuitable for vaccine development because they either are purposefully immunodeficient or immunosuppressed. Here, we describe the development and optimization of an immunocompetent intratracheal (IT) rat model susceptible to infections with sporozoites of Cryptosporidium parvum and Cryptosporidium hominis - the primary causes of human cryptosporidiosis. A model suitable for screening of parasite immunogens is a prerequisite for immunogen screening and vaccine development.

Acquisition of IgG to ICAM-1-Binding DBLß Domains in the Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigen Family Varies between Groups A, B, and C.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important malaria virulence factor. The protein family can be divided into clinically relevant subfamilies. ICAM-1-binding group A PfEMP1 proteins also bind endothelial protein C receptor and have been associated with cerebral malaria in children. IgG to these PfEMP1 proteins is acquired later in life than that to group A PfEMP1 not binding ICAM-1. The kinetics of acquisition of IgG to group B and C PfEMP1 proteins binding ICAM-1 is unclear and was studied here. Gene sequences encoding group B and C PfEMP1 with DBLß domains known to bind ICAM-1 were used to identify additional binders. Levels of IgG specific for DBLß domains from group A, B, and C PfEMP1 binding or not binding ICAM-1 were measured in plasma from Ghanaian children with or without malaria. Seven new ICAM-1-binding DBLß domains from group B and C PfEMP1 were identified. Healthy children had higher levels of IgG specific for ICAM-1-binding DBLß domains from group A than from groups B and C. However, the opposite pattern was found in children with malaria, particularly among young patients. Acquisition of IgG specific for DBLß domains binding ICAM-1 differs between PfEMP1 groups.

Antibody-Dependent, Gamma Interferon-Independent Sterilizing Immunity Induced by a Subunit Malaria Vaccine.

The development of effective malaria vaccines is hampered by incomplete understanding of the immunological correlates of protective immunity. Recently, the moderate clinical efficacy of the Plasmodium falciparum circumsporozoite protein (CSP)-based RTS,S/AS01E vaccine in phase 3 studies highlighted the urgency to design and test more efficacious next-generation malaria vaccines. In this study, we report that immunization with recombinant CSP from Plasmodium yoelii (rPyCSP), when delivered in Montanide ISA 51, induced sterilizing immunity against sporozoite challenge in C57BL/6 and BALB/c strains of mice. This immunity was antibody dependent, as evidenced by the complete loss of immunity in B-cell-knockout (KO) mice and by the ability of immune sera to neutralize sporozoite infectivity in mice. Th2-type isotype IgG1 antibody levels were associated with protective immunity. The fact that immunized gamma interferon (IFN-γ)-KO mice and wild-type (WT) mice have similar levels of protective immunity and the absence of IFN-γ-producing CD4+ and CD8+ T cells in protected mice, as shown by flow cytometry, indicate that the immunity is IFN-γ independent. Protection against sporozoite challenge correlated with higher frequencies of CD4+ T cells that express interleukin-2 (IL-2), IL-4, and tumor necrosis factor alpha (TNF-α). In the RTS,S study, clinical immunity was associated with higher IgG levels and frequencies of IL-2- and TNF-α-producing CD4+ T cells. The other hallmarks of immunity in our study included an increased number of follicular B cells but a loss in follicular T helper cells. These results provide an excellent model system to evaluate the efficacy of novel adjuvants and vaccine dosage and determine the correlates of immunity in the search for superior malaria vaccine candidates.

Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli.

A transmission-blocking vaccine targeting the sexual stages of Plasmodium species could play a key role in eradicating malaria. Multiple studies have identified the P. falciparum proteins Pfs25 and Pfs48/45 as prime targets for transmission-blocking vaccines. Although significant advances have been made in recombinant expression of these antigens, they remain difficult to produce at large scale and lack strong immunogenicity as subunit antigens. We linked a self-assembling protein, granule lattice protein 1 (Grl1p), from the ciliated protozoan, Tetrahymena thermophila, to regions of the ectodomains of either Pfs25 or Pfs48/45. We found that resulting protein chimera could be produced in E. coli as nanoparticles that could be readily purified in soluble form. When produced in the E. coli SHuffle strain, fusion to Grl1p dramatically increased solubility of target antigens while at the same time directing the formation of particles with diameters centering on 38 and 25 nm depending on the antigen. In a number of instances, co-expression with chaperone proteins and induction at a lower temperature further increased expression and solubility. Based on Western blotting and ELISA analysis, Pfs25 and Pfs48/45 retained their transmission-blocking epitopes within E. coli-derived particles, and the particles themselves elicited strong antibody responses in rabbits when given with an aluminum-based adjuvant. Antibodies against Pfs25-containing nanoparticles blocked parasite transmission in standard membrane-feeding assays. In conclusion, fusion to Grl1p can act as a solubility enhancer for proteins with limited solubility while retaining correct folding, which may be useful for applications such as the production of vaccines and other biologics.

Immune Stimulation of RAP domain binding protein (rTgRA15) from Toxoplasma gondii.

Toxoplasmosis, a parasitic disease in human and animals, is caused by Toxoplasma gondii. Our previous study has led to the discovery of a novel RAP domain binding protein antigen (TgRA15), an apparent in-vivo induced antigen recognised by antibodies in acutely infected individuals. This study is aimed to evaluate the humoral response and cytokine release elicited by recombinant TgRA15 protein in C57BL/6 mice, demonstrating its potential as a candidate vaccine for Toxoplasma gondii infection. In this study, the recombinant TgRA15 protein was expressed in Escherichia coli, purified and refolded into soluble form. C57BL/6 mice were immunised intradermally with the antigen and CASAC (Combined Adjuvant for Synergistic Activation of Cellular immunity). Antigen-specific humoral and cell-mediated responses were evaluated using Western blot and ELISA. The total IgG, IgG1 and IgG2a antibodies specific to the antigen were significantly increased in treatment group compare to control group. A higher level of interferon gamma (IFN-γ) secretion was demonstrated in the mice group receiving booster doses of rTgRA15 protein, suggesting a potential Th1-mediated response. In conclusion, the rTgRA15 protein has the potential to generate specific antibody response and elicit cellular response, thus potentially serve as a vaccine candidate against T. gondii infection.

Correlating efficacy and immunogenicity in malaria vaccine trials.

The availability of an effective and appropriately implemented malaria vaccine would form a crucial cornerstone of public health efforts to fight this disease. Despite many decades of research, however, no malaria vaccine has yet shown satisfactory protective efficacy or been rolled-out. Validated immunological substitute endpoints have the potential to accelerate clinical vaccine development by reducing the required complexity, size, duration and cost of clinical trials. Besides facilitating clinical development of existing vaccine candidates, understanding immunological mechanisms of protection may drive the development of fundamentally new vaccination approaches. In this review we focus on correlates of protection in malaria vaccine development: Does immunogenicity predict malaria vaccine efficacy and why is this question particularly difficult? Have immunological correlates accelerated malaria vaccine development in the past and will they facilitate it in the future? Does Controlled Human Malaria Infection represent a valid model for identifying such immunological correlates, or a correlate of protection against naturally-acquired malaria in itself?

Propranolol efficacy as a novel adjuvant for immunization against Toxoplasma gondii tachyzoites.

Severe or lethal damages, caused by Toxoplasma gondii infection in congenital cases and immunocompromised patients implies the necessity for development of a vaccine and an appropriate adjuvant would be needed to elicit a protective Th1 biased-immune response. The adjuvant activity of propranolol was surveyed and compared with alum by immunization of BALB/c mice with protein components of T. gondii tachyzoites. Five groups of BALB/c mice were immunized with phosphate buffered saline (negative control), Toxoplasma lysate antigen (TLA), alum plus TLA, Propranolol plus TLA, and alum, propranolol and TLA. Immunization efficacy was evaluated by lymphocyte proliferation and DTH tests, challenge with live tachyzoites, IFN-γ production by spleen cells, serum TNF-α concentration and anti- Toxoplasma total IgG, IgG1 and IgG2a measurements. Mice of the PRP-TLA group induced significantly more IFN-γ and TNF-α production and lymphocyte proliferation than other groups. This group of mice also showed more anti-T. gondii IgG2a and DTH responses and showed a significantly increased survival time after challenge. These findings indicate that propranolol as an adjuvant in combination with TLA, may enhance cellular immunity against T. gondii.

Prophylactic treatment of L-Arg improves malaria outcomes by regulating host immune responses during Plasmodium yoelii 17XL infection.

L-arginine (L-Arg), the precursor of nitric oxide (NO), plays multiple, important roles in nutrient metabolism and immune regulation. Hypoargininemia is one of the distinctive features of malaria patients in endemic areas. To understand the immunoregulatory function of L-Arg in malaria, we investigated the effects of L-Arg, pre- or/and post-treatment, on the cellular/humoral immune response during Plasmodium yoelii 17XL (P.y17XL) infection in DBA/2 mice. Populations of splenic CD4+T-bet+IFN-γ+ T cells (Th1), F4/80+ macrophages, CD4+GATA-3+IL-4+ T cells (Th2), B220+CD138+ plasmacytes and antibody-producing cells (IgG+/IgG1+-plasma cells) were assessed by flow cytometry. Pro-inflammatory cytokines and antibodies (IgG and IgG1) were quantified by immunoassays. We found that treatment with L-Arg significantly decreased parasitemia and shortened disease duration. Prophylactic treatment with L-Arg promotes an enhanced Th1 cell response during the early stages of P.y17XL infection, and treatment with L-Arg in the course of infection facilitates the later humoral immune response. Our findings suggest that treatment with L-Arg may decrease parasite burden and control the host's susceptibility to parasite synchronously by regulating host immune responses against P.y17XL, producing better outcomes for malaria infection. This implies that the supplementation of L-Arg may be a promising adjunctive therapy to reduce malaria-associated mortality in endemic areas.

Sm-p80-based schistosomiasis vaccine: double-blind preclinical trial in baboons demonstrates comprehensive prophylactic and parasite transmission-blocking efficacy.

Schistosomiasis is of public health importance to an estimated one billion people in 79 countries. A vaccine is urgently needed. Here, we report the results of four independent, double-blind studies of an Sm-p80-based vaccine in baboons. The vaccine exhibited potent prophylactic efficacy against transmission of Schistosoma mansoni infection and was associated with significantly less egg-induced pathology, compared with unvaccinated control animals. Specifically, the vaccine resulted in a 93.45% reduction of pathology-producing female worms and significantly resolved the major clinical manifestations of hepatic/intestinal schistosomiasis by reducing the tissue egg-load by 89.95%. A 35-fold decrease in fecal egg excretion in vaccinated animals, combined with an 81.51% reduction in hatching of eggs into the snail-infective stage (miracidia), demonstrates the parasite transmission-blocking potential of the vaccine. Substantially higher Sm-p80 expression in female worms and Sm-p80-specific antibodies in vaccinated baboons appear to play an important role in vaccine-mediated protection. Preliminary analyses of RNA sequencing revealed distinct molecular signatures of vaccine-induced effects in baboon immune effector cells. This study provides comprehensive evidence for the effectiveness of an Sm-p80-based vaccine for schistosomiasis.

Inducible nitric oxide synthase controls experimental Strongyloides infection.

Infection with Strongyloides sp. induces a host immune response, predominantly the Th2 type, that is able to eliminate the parasite. However, little is known about the role of the nitric oxide (NO) mediator, induced by the enzyme nitric oxide synthase (NOS), in strongyloidiasis. Therefore, in this study, we investigated the immune response of mice genetically deficient in the enzyme inducible nitric oxide synthase (iNOS-/- ), infected with Strongyloides venezuelensis. C57BL/6 wild-type (WT) and iNOS-/- mice were individually inoculated by subcutaneous injection of 3000 S. venezuelensis L3 larvae. In the absence of iNOS, mice were more susceptible to the infection than WT animals, in which the parasite was completely eliminated. The overall production of cytokines and specific IgG, IgG1 or IgE antibodies against the parasite was significantly lowered in infected iNOS-/- mice. The expression of iNOS was observed in the intestine of WT hosts but mainly in the wall of the parasite, despite the presence of iNOS in mice. Altogether, we concluded that iNOS expression may play an important role in the control of S. venezuelensis infection.

Production of a monoclonal antibody against a mannose-binding protein of Acanthamoeba culbertsoni and its localization.

Amoebae from the genus Acanthamoeba are facultative pathogens of humans and other animals. In humans they most frequently infect the eye causing a sight threatening infection known as Acanthamoeba keratitis (AK), and also cause an often fatal encephalitis (GAE). A mannose-binding protein (MBP) has been identified as being important for Acanthamoeba infection especially in AK. This lectin has previously been characterized from Acanthamoeba castellanii as consisting of multiple 130 kDa subunits. MBP expression correlates with pathogenic potential and is expressed in a number of Acanthamoeba species. Here we report the purification of a similar lectin from Acanthamoeba culbertsoni and the production of a monoclonal antibody to it. The A. culbertsoni MBP was isolated by affinity chromatography using α-D-mannose agarose and has an apparent molecular weight of 83 kDa. The monoclonal antibody is an IgM that is useful in both western blots and immunofluorescence. We expect that this antibody will be useful in the study of the pathology of A. culbertsoni and in its identification in clinical samples.