Enzyme-linked immunoassay for detection of Cryptosporidium antigens in fecal specimens.

Cryptosporidium sp. is a ubiquitous 4- to 6-micron protozoan parasite infecting the intestinal tract of humans. It causes mild to fulminant diarrhea in patients, especially immunocompromised persons, and it may be hard to detect by microscopic fecal examination. An indirect, double-antibody enzyme-linked immunosorbent assay (ELISA) was developed using specifically produced goat and rabbit antisera to detect Cryptosporidium antigens in human feces. Of 62 frozen stools from patients with cryptosporidiosis, as detected by at least two microscopic diagnostic techniques, 51 were positive by ELISA; all ELISA-negative specimens came from patients with fewer than five oocysts per 0.01 ml of concentrated fecal sample examined after modified acid-fast or fluorescent monoclonal antibody staining. A total of 182 specimens from persons without Cryptosporidium infection were negative by ELISA in 176 instances; 3 ELISA-positive specimens came from patients with cryptosporidiosis diagnosed earlier. The sensitivity of the assay was 82.3%, and specificity was 96.7%. The predictive value of a positive ELISA was 89.5%, and the predictive value of a negative ELISA was 94.2%. The ELISA was not affected by the presence of eight other intestinal parasites but was sometimes affected by repeated freezing and thawing of fecal specimens. All fecal specimens were heated to 100 degrees C for 2 min to reduce proteolytic enzyme activity, although the necessity of this step needs further evaluation. This first-generation ELISA is a simple, rapid, easily standardized test for Cryptosporidium antigens in stool samples which will be useful for diagnosis and for large-scale epidemiologic studies.     

Flow cytometric detection of Cryptosporidium oocysts in human stool samples.

Cryptosporidium parvum is an important pathogen that causes diarrhea in virtually all human populations. Improved diagnostic methods are needed to understand the risk factors, modes of transmission, and impact of cryptosporidiosis. In the present study, we fluorescently labeled and counted C. parvum oocysts by flow cytometry (FC) and developed a simple and efficient method of processing human stool samples for FC analysis. Formed stool (suspended in phosphate-buffered saline) from an asymptomatic, healthy individual was seeded with known concentrations of oocysts, and oocysts were labeled with a cell wall-specific monoclonal antibody and detected by FC. The method described herein resulted in a mean oocyst recovery rate of 45% +/- 16% (median, 42%), which consistently yielded a fourfold increase in sensitivity compared to direct fluorescent-antibody assay of seeded stool samples. However, in many instances, FC detected as few as 10(3) oocysts per ml. Thus, FC provides a reproducible and sensitive method for C. parvum oocyst detection.     

Development and Applications of a Bovine Coronavirus Antigen Detection Enzyme-Linked Immunosorbent Assay

We developed a monoclonal antibody-based, antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) for bovine coronavirus. We compared the ELISA with electron microscopy and the hemagglutination test and found a close correlation between them. The sensitivity of the ELISA was 10⁴ bovine coronavirus particles per ml of 10% fecal suspension. Compared with electron microscopy, bovine coronavirus ELISA had 96% specificity.     

Serological response to Cryptosporidium parvum in adult cattle from the Andean region of Colombia

Single faecal and serum samples were individually collected from 135 asymptomatic adult cows on seven farms in Cundinamarca (Colombian Andean region). Tests for the presence of oocysts of Cryptosporidium parvum (carbol fuchsin stain) and Eimeria spp (flotation in saturated saline solution) revealed that none of the animals had coccidia in their faeces. The IgG antibody levels to C. parvum were measured by an enzyme-linked immunosorbent assay (ELISA) technique and the reactivity to C. parvum antigens by a Western blotting procedure. Cryptosporidial antibodies were detected in cattle from all farms, with 53.3% (72 animals) being seropositive. Sera recognized 5-11 protein fractions with molecular masses ranging from 12 14 kDa to 97-100 kDa. Sera considered as positive by ELISA reacted intensely and more frequently with protein fractions of approximately 20-22, 42-48, 51-57 and 60-69 kDa, whereas only the 42-48 kDa antigen was strongly recognized by sera without IgG antibodies. The presence of IgG antibody against C. parvum in most animals, as well as the reactivity to major proteins of C. parvum, could be indicative of continuous exposure to this parasite.     

Threshold of detection of Cryptosporidium oocysts in human stool specimens: evidence for low sensitivity of current diagnostic methods.

To determine the minimum number of Cryptosporidium oocysts that can be detected in stool specimens by diagnostic procedures, stool samples seeded with known numbers of Cryptosporidium parvum oocysts were processed by the modified Formalin-ethyl acetate (FEA) stool concentration method. FEA concentrates were subsequently examined by both the modified cold Kinyoun acid-fast (AF) staining and fluorescein-tagged monoclonal antibody (immunofluorescence [IF]) techniques. Oocysts were more easily detected in watery diarrheal stool specimens than they were in formed stool specimens. For watery stool specimens, a 100% detection rate was accomplished at a concentration of 10,000 oocysts per g of stool by both the AF staining and IF techniques. In formed stool specimens, 100% of specimens seeded with 50,000 oocysts per gram of stool were detected by the IF technique, whereas 500,000 oocysts per g of stool were needed for a 100% detection rate by AF staining. Counting of all oocysts on IF slides indicated a mean oocyst loss ranging from 51.2 to 99.6%, depending on the stool consistency as determined by the FEA concentration procedure. Our findings suggest that the most commonly used coprodiagnostic techniques may fail to detect cryptosporidiosis in many immunocompromised and immunocompetent individuals.     

Enzyme-linked immunosorbent assay for shigella toxin.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of shigella toxin. For the assay, a mouse monoclonal antibody against the B subunit of the toxin and a rabbit polyclonal antibody against the holotoxin were employed. The monoclonal antibody was used to coat wells of a microtiter plate, and the polyclonal antibody preparation was used as the detecting antibody. The amount of bound polyclonal antibody was determined by using a goat anti-rabbit immunoglobulin G-alkaline phosphatase conjugate and substrate. The ELISA was able to detect as little as 12 pg (0.06 ng/ml) of shigella toxin. The assay was specific for shigella toxin, not detecting a variety of other bacterial enterotoxins and lethal toxins. The ELISA values correlated well with cytotoxin activity during toxin purification. Shigella toxin was detected by ELISA and by immunoblot analysis in human fecal specimens from persons with S. dysenteriae infections, demonstrating that this toxin is produced in vivo.     

Cross-reactivity of polyclonal serum antibodies generated against Cryptosporidium parvum oocysts.

Polyclonal antibodies raised against Cryptosporidium parvum oocysts were found to cross-react with Eimeria spp. oocyst antigens in an indirect immunofluorescence assay, and sera from Eimeria spp.-infected lambs reacted with some antigens from sonicated C. parvum oocysts (between 29 to 30 and 66 to 69 kDa) by Western blot (immunoblot). No cross-reaction was observed with cystozoites of Toxoplasma and Sarcocystis spp. These results show the existence of epitopes common to C. parvum and various Eimeria spp.     

Bovine antibody against Cryptosporidium parvum elicits a circumsporozoite precipitate-like reaction and has immunotherapeutic effect against persistent cryptosporidiosis in SCID mice.

Control of cryptosporidiosis is currently hampered by the absence of drugs or vaccines proven consistently effective against Cryptosporidium parvum. On the basis of observations that anti-C. parvum antibody has therapeutic effect against cryptosporidiosis, cows were immunized with C. parvum to produce hyperimmune colostral antibody. An antibody-rich fraction was prepared and differentiated from control (nonhyperimmune) antibody by enzyme-linked immunosorbent assay, immunofluorescence assay, immunoelectron microscopy, and in vitro neutralizing titer against DEAE-cellulose-isolated C. parvum sporozoites. Oocyst, purified sporozoite, and merozoite antigens recognized by hyperimmune antibody were defined by Western blot (immunoblot). Hyperimmune antibody recognized antigens common to oocysts, sporozoites, and merozoites, as well as stage-specific antigens. Upon incubation with hyperimmune antibody, sporozoites underwent distinct morphologic changes characterized by progressive formation and eventual release of membranous sporozoite surface antigen-antibody complexes, similar to the malaria circumsporozoite precipitate reaction. The infectivity of sporozoites having undergone this reaction was neutralized. The reaction was minimal or absent on sporozoites incubated with control antibody. To determine therapeutic effect in vivo, persistent C. parvum infection was established in adult severe combined immune-deficient (SCID) mice by oral inoculation with 10(7) oocysts. At 5 weeks postinfection, infected mice were treated for 10 days with hyperimmune or control antibody by inclusion in drinking water and daily gavage. Fecal oocyst shedding and infection scores in the gastrointestinal tract and gall bladder/common bile duct in hyperimmune antibody-treated mice were significantly lower than those in the control antibody-treated mice. Hyperimmune bovine antibody prepared against C. parvum may provide a first-generation therapy for control of cryptosporidiosis. Additionally, the defined antigens can be evaluated as subunit immunogens to produce better-characterized polyclonal antibody for control of cryptosporidiosis or as targets for monoclonal antibody-based immunotherapy.     

Development of a biotin-streptavidin-enhanced enzyme-linked immunosorbent assay which uses monoclonal antibodies for detection of group C rotaviruses.

A biotin-streptavidin-enhanced enzyme-linked immunosorbent assay (ELISA) which uses monoclonal antibodies (MAbs) for the detection of group C rotaviruses was developed. An assay in which plates were coated with three pooled MAbs and biotinylated polyclonal immunoglobulin G (IgG) (polyclonal antibody [PAb]) was used as the detector (MAb capture-PAb detector) was found to be the most sensitive and specific of the assays when it was compared with assays in which plates were coated with polyclonal antiserum and detection was done with either biotinylated polyclonal antiserum (PAb capture-PAb detector) or biotinylated pooled MAbs (PAb capture-MAb detector). The MAb capture-PAb detector ELISA detected 83% of samples confirmed to be positive for group C rotaviruses, whereas the PAb capture-PAb detector assay detected 63% of positive samples and the PAb capture-MAb detector assay detected 65% of positive samples. All three procedures detected both of the bovine and the two human group C rotaviruses, but none of the three procedures detected fecal samples containing group A and B rotaviruses or fecal samples negative for group C rotaviruses used in this study. The sensitivity of the MAb capture-PAb detector ELISA was determined by serially diluting fecal group C rotaviruses; antigens were detected in maximal positive dilution ranges of 1:1,000 to 1:3,000 for the samples tested. On the basis of the cell culture immunofluorescence assay infectivity titer of semipurified cell culture-passaged Cowden group C rotavirus, the sensitivity of the MAb capture-PAb detection ELISA for detection of homologous group C rotavirus was 53 fluorescent focus units per ml. Epitope mapping by use of the biotinylated MAbs in competition assay suggested that our MAbs may bind to three different but overlapping epitopes. These results suggest that the MAb capture-PAb detector ELISA can be used to study the epidemiology of group C rotaviruses in humans and animals.     

Detection of Cryptosporidium parvum DNA in formed human feces by a sensitive PCR-based assay including uracil-N-glycosylase inactivation.

We developed a PCR-based method that can be used to identify Cryptosporidium parvum in human feces. Fecal oocysts were concentrated by centrifugation on a sodium chloride gradient and filtration on a nitrocellulose filter prior to DNA extraction and PCR amplification of a 452-bp C. parvum-specific DNA sequence with a protocol including dUTP and uracil-N-glycosylase. All samples obtained from naturally infected humans (n = 10), calves (n = 4), and goats (n = 2) were positive. A 100% detection rate was achieved with both formed and solid stools (n = 10) seeded with 1,000 C. parvum oocysts per g. Procedures based on stool concentration by a modified Ritchie method and subsequent oocyst identification by immunofluorescent labeling or acid-fast staining require concentrations of 50,000 to 500,000 oocysts per g to achieve a 100% detection rate with formed stools. The described PCR-based assay thus has a 50- to 500-fold increase in sensitivity compared to those of the methods commonly used to analyze formed feces.     

Persistent cryptosporidiosis in horses with severe combined immunodeficiency.

Cryptosporidial infections were established in five young foals with severe combined immunodeficiency following oral administration of 10(8) Cryptosporidium parvum oocysts. All foals shed oocysts (average of 8 x 10(6) to 2 x 10(8)/g of feces) until death. Inflammation and C. parvum organisms were observed in the common bile duct, duodenum, jejunum, and ileum. Since foals with severe combined immunodeficiency lack functional T and B lymphocytes and are incapable of antigen-specific immune responses, they are well suited for evaluating the pathogenesis and treatment of persistent cryptosporidiosis.     

Comparative evaluation of several techniques for purification of Cryptosporidium parvum oocysts from rat feces.

Three methods of isolating Cryptosporidium parvum oocysts from rat feces were evaluated. Oocysts were initially isolated by sucrose density gradient centrifugation. They were further purified by passage through a glass bead column or a Percoll gradient or by dialysis. Although oocysts recovered from the glass bead column and by dialysis were relatively free of fecal debris, only oocysts recovered from the Percoll gradient were free of bacteria. Recovered oocysts retained their antigenicity and infectivity. The ability to effectively recover oocysts from rat feces suggests that the laboratory rat may be a convenient substitute for ruminants in the propagation and maintenance of C. parvum oocysts for in vitro and in vivo use.     

Resistance of calves to Cryptosporidium parvum: effects of age and previous exposure.

Cryptosporidium parvum is a coccidian parasite that causes diarrheal disease in many vertebrate species, including young (less than or equal to 1 month old) calves. Older calves and adult cattle are resistant to infection. In this study, newborn calves were raised in isolation from C. parvum for 1 week to 3 months before experimental challenge with the parasite. Calves orally challenged with C. parvum at 1 week of age shed oocysts in their feces and had diarrhea after challenge exposure. When these calves were rechallenged at 1 and 3 months of age, they neither shed oocysts nor had diarrhea. There was no significant increase in the mean anticryptosporidium enzyme-linked immunosorbent assay serum antibody titer in these calves following any of the challenge exposures. Calves orally inoculated with C. parvum for the first time at 1 month of age shed oocysts, had diarrhea after challenge exposure, and were resistant to rechallenge at 3 months of age. These calves had a twofold increase in serum antibody titer after the first challenge and no increase after the second challenge. Calves orally inoculated with C. parvum for the first time at 3 months of age shed oocysts, and two of seven animals had diarrhea. These calves had a 10-fold increase in serum antibody to C. parvum after exposure. This study demonstrates that calves raised in isolation from C. parvum remain susceptible to challenge until at least 3 months of age. Furthermore, within this time period, initial exposure and recovery renders calves resistant to further challenge with the parasite. The data also suggest that exposure of young calves to C. parvum may inhibit the development of a serum antibody response to the parasite.     

Evidence of Thymus-Independent Local and Systemic Antibody Responses to Cryptosporidium parvum Infection in Nude Mice

Differences in susceptibility to persistent cryptosporidial infection between two strains of adult athymic nude mice prompted us to investigate the immune mechanism(s) that may control resistance to infection in these T-cell-deficient mice. We studied fecal oocyst shedding, serum and fecal parasite-specific antibody responses, and fecal immunoglobulin levels in athymic C57BL/6J nude and athymic BALB/cJ nude mice following oral inoculation with Cryptosporidium parvum oocysts at 8 to 9 weeks of age. C57BL/6J nude mice had significantly higher fecal parasite-specific immunoglobulin A (IgA) (days 27, 31, 35, and 42 postinoculation) and IgM (days 10, 17, 24, 28, 31, 38, 42, and 48 postinoculation) levels than BALB/cJ nude mice (P < 0.05) and significantly higher serum parasite-specific IgA levels at 63 days postinoculation (P < 0.03). Moreover, C57BL/6J nude mice shed significantly fewer C. parvum oocysts than BALB/cJ nude mice from days 52 to 63 postinoculation (P < 0.05). In contrast, BALB/cJ nude mice had higher levels of non-parasite-specific IgA (days 38 to 63 postinoculation) and IgM (days 24, 35, 38, and 52 postinoculation) than C57BL/6J nude mice in feces (P < 0.05). These data suggest that parasite-specific fecal antibodies may be associated with resistance to C. parvum in C57BL/6J nude mice.     

Development of antigen detection assay for diagnosis of tuberculosis using sputum samples

The rising incidence of tuberculosis worldwide means an increasing burden on diagnostic facilities, so tests simpler than Ziehl-Neelsen staining are needed. Such tests should be objective, reproducible, and have at least as good a detection limit as 10(4) bacteria/ml. A capture enzyme-linked immunosorbent assay (ELISA) was developed for detection of lipoarabinomannan (LAM) in human sputum samples. As a capture antibody, we used a murine monoclonal antibody against LAM, with rabbit antiserum against Mycobacterium tuberculosis as a source of detector antibodies. The sensitivity of the capture ELISA was evaluated by using purified LAM and M. tuberculosis whole cells. We were able to detect 1 ng of purified LAM/ml and 10(4) M. tuberculosis whole cells/ml. LAM could also be detected in culture filtrate of a 3-week-old culture of M. tuberculosis. The culture filtrate contained approximately 100 microgram of LAM/ml. The detection limit in sputum pretreated with N-acetyl-L-cysteine and proteinase K was 10(4) M. tuberculosis whole cells per ml. Thirty-one (91%) of 34 sputum samples from 18 Vietnamese patients with tuberculosis (32 smear positive and 2 smear negative) were positive in the LAM detection assay. In contrast, none of the 25 sputum samples from 21 nontuberculous patients was positive. This specific and sensitive assay for the detection of LAM in sputum is potentially useful for the diagnosis of tuberculosis.     

Monoclonal anti-idiotypes for the rapid detection of human cytomegalovirus.

A novel enzyme-linked immunosorbent assay (ELISA) was developed for human cytomegalovirus (HCMV) utilizing a monoclonal anti-idiotype specific for CMVB1, an antibody to HCMV. Samples of HCMV were measured by their inhibition of the binding of CMVB1 to anti-idiotype. The ELISA detected HCMV in a concentration-dependent manner from 20 to 0.6 x 10(3) PFU/ml, with 50% inhibition at approx. 3 x 10(3) PFU/ml. These data demonstrate the potential of anti-idiotype antibodies as the basis of simple and rapid diagnostic tests for infectious agents.     

In vitro inhibition of Cryptosporidium parvum infection by human monoclonal antibodies.

Cryptosporidium parvum infection of the small epithelial intestine causes unremitting diarrhea and malabsorption that can lead to chronic and sometimes fatal illness in patients with AIDS. The illness may be ameliorated by passive oral immunoglobulin therapy. The objective of this study was to produce anti-Cryptosporidium human monoclonal antibodies for evaluation as potential therapy. All human monoclonal cell lines that produced C. parvum antibodies were originally generated from the peripheral blood lymphocytes of a human immunodeficiency virus-seronegative woman. She had recovered from C. parvum infection and had a high specific antibody titer. Hybridization of these lymphocytes with a tumor cell line was accomplished by hypo-osmolar electrofusion. Twelve clones were identified by enzyme-linked immunosorbent assay (ELISA) as secreting anti-Cryptosporidium antibodies after the initial hybridization. From the 12 positive clones, two high antibody-secreting clones, 17A and 17B, were maintained in long-term culture. A second hybridization produced two other human monoclonal cell lines, EC5 and BB2. Human monoclonal antibody from the first two cell lines bound to C. parvum sporozoites and oocysts by immunofluorescence. The ability of human monoclonal antibodies to inhibit C. parvum infection in vitro was assessed by using a human enterocyte cell line, HT29.74. The antibodies of the four different human hybridomas inhibited infection by 35 to 68% (P < 0.05) compared to a control irrelevant human monoclonal antibody derived in a similar fashion. Human monoclonal antibodies are candidate molecules for immunotherapy of C. parvum infection.     

Development of an immunomagnetic bead separation-coupled quantitative PCR method for rapid and sensitive detection of Cryptosporidium parvum oocysts in calf feces

Cattle feces are the environmental vehicle for the zoonotic Cryptosporidium oocysts, but there are drawbacks associated with reliability of the existing methods for the detection of oocysts in the feces. Quantification of the immunomagnetic bead separation (IMS) coupled with real-time TaqMan PCR (qPCR) was accomplished by comparing the fluorescence signals obtained from the calf fecal samples of Cryptosporidium parvum oocysts with those obtained from standard dilutions of C. parvum oocysts. TaqMan qPCR assays were developed for the detection of C. parvum based on 18S rDNA gene. This IMS-qPCR assay allowed a reliable quantification of C. parvum oocysts over seven orders of magnitude with a baseline sensitivity of 8.7 oocysts. The newly developed IMS-qPCR technique proved specific as confirmed by negative reactivity against a wide panel of non-parvum Cryptosporidium oocysts. As a field application, experimentally infected calves (15 infected and 9 non-infected) were screened for oocysts shedding on 16, 18, and 21 days postinfection. Acid-fast staining microscopy of infected calves revealed oocysts in the feces of 11, 7, and 4 calves, respectively, compared to 15, 15, and 12 in case of screening by IMS-qPCR. Taken together, the proposed IMS-qPCR method significantly improved the diagnostic capacity for C. parvum infection in calves, making the technique a useful, sensitive, reliable, and time-saving.     

Antibody response of healthy adults to recombinant thrombospondin-related adhesive protein of cryptosporidium 1 after experimental exposure to cryptosporidium oocysts

Thrombospondin-related adhesive protein of Cryptosporidium 1 (TRAP-C1) belongs to a group of proteins that are also found in Toxoplasma gondii, Eimeria tenella, and Plasmodium species. TRAP-related proteins are needed for gliding motility, host-cell attachment, and invasion. The objective of this study was to characterize the antibody response to recombinant TRAP-C1 (rTRAP-C1) in healthy volunteers exposed to C. parvum and their association with clinical illness. A total of 31 healthy adult volunteers participated. Seven volunteers received the C. parvum TAMU isolate (inocula, 10 to 300 oocysts), and 24 volunteers received the C. parvum UCP isolate (500 to 10(5) oocysts). The total antibody (immunoglobulin M [IgM], IgG, and IgA) response to rTRAP C-1 was measured by enzyme-linked immunosorbent assays prior to and after exposure to Cryptosporidium parvum (days 0 to 45). Results of this study showed that individuals who were uninfected demonstrated higher reactivity at baseline compared to those who became infected. After challenge, increases in antibody reactivity were seen on days 30 and 45 compared to the results seen on days 0 to 5. The increases in antibody reactivity were statistically significant in subjects with diarrhea and with or without detectable oocysts compared to the results seen with those who were uninfected and asymptomatic. These findings suggest that increases in antibody reactivity to rTRAP-C1 occur after recent exposure to C. parvum.     

The magnetic immuno polymerase chain reaction assay for direct detection of salmonellae in fecal samples.

Direct polymerase chain reaction (PCR)-based detection with fecal specimens is hampered by inhibitory compounds, such as bilirubin and bile salts. These fecal compounds showed significant inhibition of PCR at low concentrations (10 to 50 micrograms/ml). For direct PCR analysis, fecal samples must be diluted 500-fold to overcome inhibition. Therefore, the magnetic immuno PCR assay (MIPA), which combines immunomagnetic separation by using specific monoclonal antibodies and PCR, was used to directly detect salmonellae in feces from humans. Immunomagnetically extracted stool samples needed to be diluted only 10-fold when 1 microgram of T4 gene 32 protein was added to the PCR. The MIPA sensitivity obtained was 10(5) CFU/ml of feces. A panel of monoclonal antibodies specific for Salmonella serogroups A to E was used to extract salmonellae from clinical samples. MIPA detection of salmonellae occurred with 11 out of 14 stool samples stored at 4 degrees C for 2 months. MIPA detection of salmonellae in stool samples is a promising, fast method for detection and identification.