Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C. parvum oocysts

The purpose of this study was to characterize the viral symbiont (CPV) of Cryptosporidium parvum sporozoites and evaluate the CPV capsid protein (CPV40) as a target for sensitive detection of the parasite. Recombinant CPV40 was produced in Escherichia coli, purified by affinity chromatography, and used to prepare polyclonal rabbit sera specific for the viral capsid protein. Anti-rCPV40 recognized a 40 kDa and a 30 kDa protein in C. parvum oocysts and appeared to localize to the apical end of the parasite. Anti-rCPV40 serum was capable of detecting as few as 1 C. parvum oocyst in a dot blot assay, the sensitivity being at least 1000-fold greater than sera reactive with total native C. parvum oocyst protein or specific for the 41 kDa oocyst surface antigen. Water samples were seeded with C. parvum oocysts and incubated at 4, 20, or 25 degrees C for greater than 3 months to determine if CPV levels were correlated with oocyst infectivity. Samples were removed monthly and subjected to mouse and cell culture infectivity, as well as PCR analysis for infectivity and viral particle presence. While sporozoite infectivity declined by more than 75% after 1 month at 25 degrees C, the CPV signal was similar to that of control samples at 4 degrees C. By 3 months at 20 degrees C, the C. parvum oocysts were found to be non-infectious, but retained a high CPV signal. This study indicates that CPV is an excellent target for sensitive detection of C. parvum oocysts in water, but may persist for an indefinite time after oocysts become non-infectious.     

Detection of cryptosporidium antibodies in sera and oral fluids using multiplex bead assay

For the first time, a multiplex bead assay (MBA) was used to assay oral fluid and serum specimens for immunoglobulin G (IgG) antibodies to specific Cryptosporidium parvum antigens that were coupled to polystyrene beads. Recombinant C. parvum 17- and 27-kDa antigens (r17 and r27, respectively) both linked with glutathione-S-transferase (GST) fusion proteins, native 17-kDa antigen, and GST alone were each coupled to microspheres that could be differentiated based on variable amounts of internally incorporated red fluorescent dye. Initial and follow-up serum and oral fluid specimens from a 1997 cryptosporidiosis outbreak in Spokane, Washington, were incubated with the coupled beads. Antibodies bound to the coupled beads were detected using biotinylated monoclonal anti-human IgG antibody and streptavidin-labeled r-phycoerythrin. Fluorescence intensity was measured by flow cytometry. For the 3 C. parvum antigens, the median of the mean fluorescence intensity (MFI) was significantly higher (P < 0.03) in the initial specimens than in the follow-up specimens. No significant change in IgG responses to GST in oral fluids or serum specimens was observed. For all Cryptosporidium antigens, the MFI in the initial serum specimens correlated with the MFI in the initial oral fluid specimens (P < 0.001, r > 0.673). For the recombinant antigens used in the MBA, the MFI correlated with the response as measured by an enzyme-linked immunosorbent assay that used r17 and r27 expressed without the GST fusion partner (P < 0.001, r > 0.854). MBA using sera or more conveniently collected oral fluids, especially from children, may be an option for immunodiagnosis of C. parvum infection and for prospective epidemiological studies designed to monitor infection risk.     

Antibody responses to Cryptosporidium antigen in HIV-positive patients in the Republic of Korea

The diagnosis of cryptosporidiosis has been carried out using coprologic techniques in the Republic of Korea. However, antibody responses to Cryptosporidium have rarely been studied. Serum antibodies from HIV-positive/oocyst-positive Korean patients recognized significantly 31 and 27 kDa antigens, and HIV-negative/oocyst-positive individuals clearly reacted to 15/17 kDa antigens. Compared with oocyst-positive cases, 18.7% and 75.8% of sera from HIV-positive patients reacted to 31 and 27 kDa antigens. Only 11.1% of HIV-negative individuals reacted to 15/17 kDa. Based on these findings, serum antibody responses were different between HIV-positive and HIV-negative individuals infected with Cryptosporidium, and it is suggested that HIV-positive patients are more frequently exposed to C. parvum compared to HIV-negative individuals.     

Trichinella spiralis: strong antibody response to a 49 kDa newborn larva antigen in infected rats

In this work, we analyzed the kinetics of anti-Trichinella spiralis newborn larva (NBL) antibodies (Ab) and the antigenic recognition pattern of NBL proteins and its dose effects. Wistar rats were infected with 0, 700, 2000, 4000 and 8000 muscle larvae (ML) and bled at different time intervals up to day 31 post infection (p.i.). Ab production was higher with 2000 ML dose and decreased with 8000, 4000 and 700 ML. Abs were not detected until day 10, peaked on day 14 for the 2000 ML dose and on day 19 for the other doses and thereafter declined slowly from 19 to 31 days p.i. In contrast, Abs to ML increased from day 10, peaked on day 19 and remained high until the end of the study. Abs bound strongly at least to three NBL components of 188, 205 and 49 kDa. NBL antigen of 188 and 205 kDa were recognized 10-26 days p.i. and that of 49 kDa from day 10 to day 31 p.i. A weak recognition towards antigens of 52, 54, 62 and 83 kDa was also observed during the infection. An early recognition of 31, 43, 45, 55, 68 and 85 kDa ML antigens was observed whereas the response to those of 43, 45, 48, 60, 64 and 97 kDa (described previously as TSL-1 antigens) occurred late in the infection. A follow-up of antigen recognition up to day 61 with the optimal immunization dose (2000 ML) evidenced a decline of Ab production to the 49 kDa NBL antigen 42 days p.i., which suggested antigenic differences with the previously reported 43 kDa ML antigen strongly recognized late in the infection. To analyze the stage-specificity of the 49 kDa NBL antigen, polyclonal antibodies (PoAb) were obtained in rats immunized with 49 kDa NBL antigen. PoAb reacted strongly with the 49 kDa NBL component in NBL total soluble extract but no reactivity was observed with soluble antigen of the other T. spiralis stages. Albeit with less intensity, the 49 kDa component was also recognized by PoAb together with other antigens of 53, 97 and 107 kDa, in NBL excretory-secretory products (NBL-ESP). Thus, our results reveal differences in the kinetics of anti-NBL and ML Ab responses. While anti-NBL Abs declined slowly from day 19 until the end of the experiment, Abs to ML antigen remained high in the same period. It is remarkable the optimal Ab response to NBL antigens with 2000 ML infective dose and the reduced number of NBL antigens identified throughout the experimental T. spiralis infection, standing out the immunodominant 49 kDa antigen. Interestingly, this antigen, which was prominently expressed in NBL somatic proteins, was also detected in NBL-ESP.     

Oral dosing of neonatal mice with sucrose reduces infection with Cryptosporidium parvum

Cryptosporidium parvum is a significant cause of diarrheal disease in humans and economically important livestock species. There is no effective treatment available for this protozoan parasite. Mechanisms of intestinal colonization by C. parvum are not well understood, but it has been suggested that the parasite may utilize a lectin-like receptor. We used an infant mouse model to test whether high sugar concentrations in the intestine would affect in vivo colonization with C. parvum. We found that a single oral dose of sucrose, administered to mice at the time of, or 24 hr before, challenge with C. parvum significantly reduced infection. Significant reduction of infection was also seen in mice given isomaltose. Histologic examination of intestinal sections of mice treated with sucrose or isomaltose, but not other sugars, showed marked vacuolation of the small intestinal epithelium 1 day after treatment. Three days after treatment, tissue appeared normal. Thus, sucrose and, to a lesser extent, isomaltose reduced in vivo colonization with C. parvum and altered epithelial cell morphology in intestines of mice.     

Stepwise phosphorylation of the NH2-terminal region of the simian virus 40 large T antigen

The simian virus 40 (SV40) large T antigen was immunoprecipitated from extracts of infected monkey cells and cleaved with trypsin under conditions of mild proteolysis. The digestion generated fragments from the NH2-terminal region of T antigen which were released from the immunoprecipitates. Pulse-chase experiments showed that most of the newly made T antigen (form A) generated an NH2-terminal fragment of 17 kDa in size, whereas most of the T antigen that had aged in the cell (form C) generated a fragment of 20 kDa. An intermediate form of T antigen (form B), which generated an 18.5- kDa NH2-terminal fragment, was produced in part from form A and was converted to form C during the chase. Phosphate-labeling experiments showed that form C was the species of T antigen that incorporated the most 32P radioactivity at the NH2-terminal region, although some label was also incorporated into forms A and B. In vitro dephosphorylation of gel-purified 18.5- and 20-kDa fragments labeled with [35S]methionine increased the electrophoretic mobility of the fragments to that of 17 kDa. This signified that phosphorylation of the NH2-terminal fragments was directly responsible for their aberrant behavior in acrylamide gels. Although peptide maps of the methionine-labeled tryptic peptides of the 17-, 18.5-, and 20-kDa fragments were very similar to one another, maps of the 32P-labeled tryptic Pronase E peptides of these fragments contained qualitative and quantitative differences. Analysis of the labeled phosphoamino acids of various peptides from these fragments indicated that the 20-kDa fragment was highly phosphorylated at Ser 123 and Thr 124, whereas the 17- and 18.5-kDa fragments were mostly unphosphorylated at these sites. These experiments indicated that T antigen is phosphorylated at the NH2-terminal region in a specific stepwise process and, therefore, that this post-translational modification of T antigen is tightly regulated.     

Antigen profiles of the fish pathogen Moritella viscosa and protection in fish

Aims: Moritella viscosa is a Gram‐negative bacterium that causes winter ulcer disease in salmonid fish cultured in sea water below 8°C. The aim of this study was to study the antigen profiles of these bacteria and to reveal the protection which the antigens induce in fish. Methods and Results: Lipooligosaccharides (LOS) and an approximately 17–19 kDa outer membrane antigen were shown to be the major specific antigens of M. viscosa. The size of the wall antigen differed between strain groups and even between strains reacting positively in the same sera. Four different serotypes of M. viscosa were determined by producing polyclonal sera. Western blot analysis revealed that sera from vaccinated fish groups that had good or fair protection reacted against the LOS and the 17/19 kDa antigen, while no antibody response was observed with sera from groups that showed no efficacy. Conclusions: The study provides evidence that LOS and an approximately 17–19 kDa outer membrane antigen are the major specific protective antigens of M. viscosa, and that the M. viscosa species consists of many different serotypes. Significance and Impact of the Study: The results are important for the production of vaccines against winter ulcers and should also lead to better methods of verifying the bacteria and monitoring winter ulcers.     

Gastric parietal cell antigens of 60-90, 92, and 100-120 kDa associated with autoimmune gastritis and pernicious anemia. Role of N-glycans in the structure and antigenicity of the 60-90-kDa component

Thirty-four human sera containing parietal cell autoantibodies (PCA) specifically immunoprecipitated two antigens, with apparent molecular masses of 60-90 kDa and 100-120 kDa under nonreducing conditions and 60-90 kDa and 120-150 kDa under reducing conditions, from porcine gastric membrane extracts. A third antigen of 92 kDa was only observed in immunoprecipitates analyzed under reducing conditions. By immunoblotting, 24 of the 34 PCA-positive sera reacted with only the 60-90-kDa antigen, three reacted with a broad 60-120-kDa smear, one reacted only with a 92-kDa antigen and six did not react. Reactivity with the 60-90-kDa antigen was observed with gastric membranes from dog, pig, rat, and rabbit. Twenty PCA-negative sera did not react with these components by immunoprecipitation or immunoblotting. PCA reactivity with the 60-90-kDa antigen was abolished when the gastric membranes were (a) digested with Pronase, (b) reduced with 100 mM dithiothreitol, (c) treated with sodium periodate, or (d) digested with N-glycanase. The 60-90-kDa and 100-120-kDa components were insensitive to neuraminidase treatment. N-glycanase digestion of 125I-labeled antigens purified by immunoprecipitation and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis collapsed the 60-90-kDa antigen to a sharp 34-kDa band; the 100-120-kDa component was unaffected. These observations suggest that (i) parietal cell antigens comprise three components of 60-90, 92, and 100-120 kDa; (ii) the epitopes differ in conformational sensitivity; (iii) the 60-90-kDa antigen is a conserved molecule comprising a 34-kDa core protein extensively glycosylated with N-linked oligosaccharides; (iv) sialic acid residues are not present in the 60-90- and 100-120-kDa molecules, and (v) the carbohydrate and protein moieties of the 60-90-kDa molecule are required for antibody binding.     

Metagonimus yokogawai: a 100-kDa Somatic Antigen Commonly Reacting with Other Trematodes

This study was undertaken to characterize the properties of a 100 kDa somatic antigen from Metagonimus yokogawai. Monoclonal antibodies (mAbs) were produced against this 100 kDa antigen, and their immunoreactivity was assessed by western blot analysis with patients'' sera. The mAbs against the 100 kDa antigen commonly reacted with various kinds of trematode antigens, including intestinal (Gymnophalloides seoi), lung (Paragonimus westermani), and liver flukes (Clonorchis sinensis and Fasciola hepatica). However, this mAb showed no cross-reactions with other helminth parasites, including nematodes and cestodes. To determine the topographic distribution of the 100 kDa antigen in worm sections, indirect immunoperoxidase staining was performed. A strong positive reaction was observed in the tegumental and subtegumental layers of adult M. yokogawai and C. sinensis. The results showed that the 100 kDa somatic protein of M. yokogawai is a common antigen which recognizes a target epitope present over the tegumental layer of different trematode species.     

Antigen Binding to Secretory Immunoglobulin A Results in Decreased Sensitivity to Intestinal Proteases and Increased Binding to Cellular Fc Receptors

In intestinal secretions, secretory IgA (SIgA) plays an important sentinel and protective role in the recognition and clearance of enteric pathogens. In addition to serving as a first line of defense, SIgA and SIgA·antigen immune complexes are selectively transported across Peyer''s patches to underlying dendritic cells in the mucosa-associated lymphoid tissue, contributing to immune surveillance and immunomodulation. To explain the unexpected transport of immune complexes in face of the large excess of free SIgA in secretions, we postulated that SIgA experiences structural modifications upon antigen binding. To address this issue, we associated specific polymeric IgA and SIgA with antigens of various sizes and complexity (protein toxin, virus, bacterium). Compared with free antibody, we found modified sensitivity of the three antigens assayed after exposure to proteases from intestinal washes. Antigen binding further impacted on the immunoreactivity toward polyclonal antisera specific for the heavy and light chains of the antibody, as a function of the antigen size. These conformational changes promoted binding of the SIgA-based immune complex compared with the free antibody to cellular receptors (FcαRI and polymeric immunoglobulin receptor) expressed on the surface of premyelocytic and epithelial cell lines. These data reveal that antigen recognition by SIgA triggers structural changes that confer to the antibody enhanced receptor binding properties. This identifies immune complexes as particular structural entities integrating the presence of bound antigens and adds to the known function of immune exclusion and mucus anchoring by SIgA.     

Cloning and Analysis of a Cryptosporidium parvum Gene Encoding a Protein with Homology to Cytoplasmic Form Hsp70

ABSTRACT. An intronless gene encoding a protein of 674 amino acid residues with a molecular mass of 73,403 Da showing homology to the cytoplasmic form of the 70 kDa heat shock proteins has been cloned and sequenced from the intestinal pathogen Cryptosporidium parvum . Monospecific polyclonal antibodies obtained to recombinant protein recognized a single band with an approximate molecular mass of 70 kDa on a Western blot of C. parvum proteins, as well as the 70 kDa heat shock protein from bovine brain. Southern blot analysis suggested the gene was single copy in the C. parvum genome. Eleven perfect repeats of the sequence GGMP were found in the predicted protein near the carboxyl terminus.     

Electron microscopic observation of cytoskeletal frame structures and detection of tubulin on the apical region of Cryptosporidium parvum sporozoites

Cryptosporidium parvum is an intracellular protozoan parasite belonging to the phylum Apicomplexa, and a major cause of waterborne gastroenteritis throughout the world. Invasive zoites of apicomplexan parasites, including C. parvum, are thought to have characteristic organelles on the apical apex; however, compared with other parasites, the cytoskeletal ultrastructure of C. parvum zoites is poorly understood. Thus, in the present study, we ultrastructurally examined C. parvum sporozoites using electron microscopy to clarify the framework of invasive stages. Consequently, at the apical end of sporozoites, 3 apical rings and an electron-dense collar were seen. Two thick central microtubules were seen further inside sporozoites and extended to the posterior region. Using anti-alpha and -beta tubulin antibodies generated from sea urchin and rat brain, both antibodies cross-reacted at the apical region of sporozoites in immunofluorescent morphology. The molecular mass of C. parvum alpha tubulin antigen was 50 kDa by Western blotting and the observed apical cytoskeletal structures were shown to be composed of alpha tubulin by immunoelectron microscopy. These results suggested that C. parvum sporozoites were clearly different in their cytoskeletal structure from those of other apicomplexan parasites.     

Uterine specific antigens in the ewe

Uterine flushings from ewes on days 0, 3, 6, 9, 12 and 15 of the estrous cycle were analyzed for total protein content. Flushings from days 9, 12 and 15 had greater (P<.01) amounts of protein than those from 0, 3 and 6. Antisera to uterine fluids from ewes at day 10 to 12 or day 14 to 15 of pregnancy detected two uterine-specific antigens in uterine flushings at day 7, 11 and 15 but not at days 0 and 3 of the cycle. A third uterine antigen was also detected in kidney tissue extracts. All three antigens were present in endometrial extracts at each stage examined. Progesterone, or estrogen plus progesterone, administration to ovariectomized ewes induced the appearance of the two uterine-specific antigens. The third antigen was detectable even in ovariectomized ewes. No pregnancy-specific antigens were detected in flushings from days 7, 11 or 15 of gestation. The effect of pregnancy on endometrial protein synthesis was examined in vitro . No differences were seen in the incorporation of (3)H-leucine in day 11 pregnant or nonpregnant or in day 14 pregnant or nonpregnant endometrium. No differences in total uterine lumenal protein were observed. Endometrial secretions, obtained by conditioning media with endometrial explant cultures, were evaluated to assess their effect on protein synthesis in day 11 embryos cultured in vitro . No significant effects of endometrial secretions or serum were observed.     

Role of murine Peyer's patch lymphocytes against primary and challenge infections with Cryptosporidium parvum

In order to determine the role of Peyeros patch lymphocytes (PPL) in self-clearing of Cryptosporidium parvum infection in murine models, changes in PPL subsets, their cytokine expression, and in vitro IgG1 and IgA secretions by PPL were observed in primary- and challenge-infected C57BL/6 mice. In primary-infected mice, the percentages of CD4+ T cells, CD8+ T cells, sIgA+ B cells, IL-2+ T cells, and IFN-gamma+ T cells among the PPL, increased significantly (P < 0.05) on day 10 post-infection (PI). Secretion of IgG1 and IgA in vitro by PPL also increased on day 10 PI. However, all these responses, with the exception of IgG1 and IgA secretions, decreased in challenge-infected mice on day 7 post-challenge (= day 13 PI); their IgG1 and IgA levels were higher (P > 0.05) than those in primaryinfected mice. The results suggest that murine PPL play an important role in self-clearing of primary C. parvum infections through proliferation of CD4+, CD8+, IL-2+, and IFN-gamma+ T cells, and IgG1 and IgA-secreting B cells. In challenge infections, the role of T cells is reduced whereas that of B cells secreting IgA appeared to be continuously important.     

Response of T lymphocytes from previously infected calves to recombinant Cryptosporidium parvum P23 vaccine antigen

We had previously demonstrated that a Type-1-like immune response involving interferon-gamma expression in lamina propria lymphocytes accompanied by IgG2 subclass fecal antibodies to Cryptosporidium parvum p23 emerged in gut mucosa of calves recovering from cryptosporidiosis. Because a recombinant p23 had been shown to protect calves from cryptosporidiosis when administered as a vaccine antigen to late gestation cattle, this study was undertaken to determine if the same vaccine antigen could induce a Type-1-like, in vitro response by T cells from calves that had recovered from C. parvum infection. We isolated peripheral blood mononuclear cells from calves that had been previously infected with C. parvum oocysts and incubated them in the presence or absence of the recombinant C. parvum p23 vaccine antigen. We used flow cytometry to simultaneously detect cells in cell cycle and identify the T cell subset containing cycling cells. We also used flow cytometry to identify interferon-gamma positive cells and 2-dimensional gel electrophoresis to profile proteins made by PBMC stimulated with the recombinant p23 vaccine antigen. The results demonstrated that CD4+ T lymphocytes proliferated and that interferon-gamma was synthesized by a subset of stimulated cells. Two-dimensional gel electrophoresis revealed the presence of several cytoplasmic proteins in a size range of approximately 25-80 kDa that were detected in p23-stimulated, but not in unstimulated, cytoplasmic samples. Together, the results show that the recombinant p23 vaccine antigen can stimulate a Type-1-like immune response by T cells from calves that have recovered from C. parvum infection.     

Dirofilaria immitis: identification and characterization of circulating parasite antigens

A method for the identification of circulating parasite antigens in filarial nematode infections was developed using canine infections with Dirofilaria immitis as a model. Filarial antigens ranging in molecular weight from 211 to 13 kDa were extracted from the sera of microfilaremic dogs by a solid phase immunobinding procedure and identified by immunostaining of Western blots. A major antigen of 104 kDa was selected for further characterization. The 104 kDa circulating antigen showed antigenic and biochemical identity with 104 kDa peptides found in extracts of adult male and microfilarial stages of the parasite. The 104 kDa peptide was antigenically stable under a variety of storage conditions. Its potential as a diagnostic target is discussed.     

Trichinella spiralis: murine strain variation in response to monoclonally defined, protective, nonstage-specific antigens

Nine hybridoma cell lines secreting monoclonal antibodies (mAbs) against Trichinella spiralis muscle larvae (ML) excretory/secretory antigens (ESA) were developed. Two mAbs, 6-D8-E3 (6D8) and 6-B1-G10 (6B1), were studied in detail. Western blot analysis using ML ESA showed that 6D8 recognized 35- and 40-kDa constituents whereas 6B1 identified a doublet of 33 kDa. However, Western blots of SDS-PAGE of crude ML homogenate showed that 6D8 identified proteins of approximately 35 and 43-60 kDa, whereas 6B1 recognized bands of 42-50 kDa. These results indicated substantial apparent MW differences between secreted and nonsecreted proteins recognized by both mAbs. Neither 6D8 nor 6B1 reacted with adult worm ESA, but both recognized antigens in aqueous extracts of homogenates of whole adult worms. Competitive inhibition experiments using ML ESA as a target demonstrated that the antigen epitopes recognized by monoclonals 6D8, 6B1, a rat mAb, 9D4, and a 37-kDa antigen previously defined were noncross-reactive. MAbs 6D8, 6B1, and 9D4 were used to isolate proteins possessing target determinants by affinity chromatography from crude ML homogenates. Each mAb isolated distinct protein species as determined by SDS-PAGE (6B1, approximately 42 kDa; 6D8, approximately 28, 37, and 61 kDa; 9D4, approximately 29, 33, 38-57, 80, and 86 kDa). NFS mice responded in a dose-dependent manner to affinity-purified antigens and were 25-fold more effective (by weight of antigen) than either C3Heb/Fe(C3H) or B10.BR mice. Immunization of mice with 6D8, 6B1, or 9D4 antigens induced strong protection against a subsequent challenge infection in NFS mice as indicated by accelerated intestinal adult worm expulsion, reduced fecundity of the female worms, and reduction of ML burden. Affinity-isolated antigens stimulated in vitro proliferation of spleen and MLN cells from immune mice; however, the mitogenic response to these antigens barely varied among NFS, C3H, and B10.BR strains.     

A comparative study on the biology of Cryptosporidium sp. from guinea pigs and Cryptosporidium parvum (Apicomplexa).

Cryptosporidium sp. from guinea pigs and C. parvum were compared morphologically, electrophoretically, and for the ability to infect suckling mice. Oocysts from guinea pigs measured 5.4 x 4.6 (4.8-5.6 x 4.0-5.0) microns and had a shape index (length/width) of 1.17 (1.04-1.33). Oocysts of C. parvum were similar and measured 5.2 x 4.6 (4.8-5.6 x 4.2-4.8) microns with a shape index of 1.16 (1.04-1.33). All suckling mice inoculated with oocysts of C. parvum became infected, whereas most, but not all, mice fed oocysts of the guinea pig isolate also became infected. However, mice inoculated with oocysts from guinea pigs produced on average 100-fold fewer oocysts by day 7 postinoculation than did mice infected with C. parvum, and the resulting infections were sparse and patchy along the ileum. Electrophoretic profiles were similar, but 125I surface labeling of outer oocyst wall proteins revealed striking differences between the two isolates. Cryptosporidium parvum had a wide molecular size range of 125I-labeled bands, whereas C. sp. from guinea pigs had a banding pattern clustered between 39 and 66 kDa, with a smaller number of bands greater than 100 kDa.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence