1 Army Health Branch Research Unit, Medical Corps, Israel Defence Force, Military Post 02149, Israel.
2 Epidemiology and Health Services Evaluation Department, Soroka University Medical Center, Beer-Sheva, Israel.
3 Daniel Abraham International Center for Health and Nutrition, Soroka University Medical Center, Beer-Sheva, Israel.
4 Pediatric Infectious Disease Unit, Soroka University Medical Center and Ben-Gurion University of the Negev, Beer-Sheva, Israel.
5 Division of Environmental Health Services, School of Public Health, Columbia University, New York, NY.
6 Department of Pediatrics, Columbia University, New York, NY.
7 Institute of Human Nutrition, Columbia University, New York, NY.
8 Department of Epidemiology and Preventive Medicine, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Israel.
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ABSTRACT |
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antibodies; Cryptosporidium; enzyme-linked immunosorbent assay; infant; infection
Abbreviations: IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M.
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INTRODUCTION |
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Cryptosporidiosis has a worldwide distribution. Prevalence varies from 1.3 percent in Scandinavian countries (11) to 16.7 percent in Haiti (12
), and cryptosporidiosis spreads by person-to-person transmission as indicated by the occurrence of day-care center outbreaks (13
, 14
), multiple family infections (15
), and sequential infections in hospitalized patients and personnel (16
). Other ways of transmission include exposure to water (17
, 18
), animal contacts (19
, 20
), and travel (21
).
Immunity to Cryptosporidium infection is believed to be conferred by cellular mechanisms that are at least in part antibody dependent (1, 19
, 22
, 23
). The infection elevates specific antibody responses, and serology tests are potentially useful for the assessment of the extent of cryptosporidial infection in different populations. Infection has been described in all six continents, and specific anti-Cryptosporidium antibodies were found in 49.5 percent of examined children in endemic areas, such as three rural communities of Anhui (24
), or in more than 60 percent of the sera obtained from subjects in two low socioeconomic populations in Peru and Venezuela (3
). In general, the prevalence of Cryptosporidium infection was reported to be higher among children than among adults (24
, 25
).
A prospective study has been carried out in a cohort of Bedouin infants, during the first 2 years of life, to describe the natural history of infection with Cryptosporidium and to examine the association between Cryptosporidium infection and host and environmental risk factors (26). The Bedouin society, which is a society in transition from a nomadic to a settled semiurban lifestyle, can serve as a model for the study of parasitic infections in the high-risk groups of newly urbanized populations. Both frequent stool analyses (monthly) and serology were used to assess the extent of infection with Cryptosporidium among these infants in the first 2 years of life. We describe herein the seroconversion rates and kinetics of the Cryptosporidium serum antibodies among the Bedoiun infants at 6, 12, and 23 months after birth and among their mothers immediately following delivery.
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MATERIALS AND METHODS |
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Intake and follow-up procedures
During the period from November 1989 to December 1992, 234 healthy infants from the Bedouin town were recruited after obtaining informed consent to the study. Fifty-two of the infants, born in 1992, and their mothers were included in the serologic study. Maternal blood samples were obtained from routine postdelivery blood samples. Cord bloods from consenting mothers were retained. A pediatric examination was performed at the clinic at birth and at the ages of 6, 12, and 23 months, at which consecutive blood samples were drawn, separated into plasma, and frozen until tested. Blood samples were available from all the 52 mothers at the time of delivery. A total of 52, 48, 49, and 40 serum samples were obtained at birth and at 6, 12, and 23 months, respectively, from the 52 infants included in the serologic follow-up. From November 1989 to December 1992, surveillance for diarrheal disease was carried out routinely on arrival of the infants at the primary health clinic, at the emergency room of the hospital, or at the Maternal and Child Health clinics where study nurses were stationed. This morbidity surveillance system was supplemented by maternal reports at monthly home visits. From January 1993 to the end of the study (July 1994), morbidity was ascertained weekly by interviewers trained to inquire about diarrhea by a home visit (85 percent of homes) or by telephone interview (15 percent of homes) and by information obtained at the monthly home visit. Nurses and pediatricians examined the infants, and diarrheal episode forms were completed. Stool samples were obtained monthly as well as at all new episodes of diarrhea and 7- to 10-day intervals thereafter until the episode ended. Of the samples for collection during routine surveillance (at monthly home visits), 93.2 percent were obtained. During diarrheal episodes, 73.0 percent of the samples that should have been obtained were collected. The stool samples were examined routinely for the following parasites and bacterial enteropathogens: C. parvum, Giardia lamblia, Salmonella spp., Shigella spp., Campylobacter spp., enteroaggregative Escherichia coli, diffuse adherent E. coli, localized adherent enteropathogenic E. coli, enterotoxigenic E. coli expressing heat-stable toxin, enterotoxigenic E. coli expressing heat-labile toxin, enterotoxigenic E. coli expressing both toxins, enterohemorrhagic E. coli, enteroinvasive E. coli, and Rotavirus.
Stools were collected from diapers or directly into a disposable cup, transferred to tubes containing phenol-alcohol-formaldehyde (27), and transported on ice to the Parasitology Laboratory at Soroka University Medical Center. Cryptosporidium was detected by the modified Ziehl-Neelsen method and confirmed by indirect immunofluorescence assay using the Merifluor Cryptosporidium indirect immunofluorescence detection procedure (Meridian Diagnostics, Inc., Cincinnati, Ohio).
Serologic methods
An enzyme-linked immunosorbent assay was performed in polystyrene microtiter plates (model 3590; Costar, Cambridge, Massachusetts) according to the method of Ungar et al. (28) with some modifications. Briefly, 100 µl of coating buffer (0.05 m carbonate buffer, pH 9.6) containing lysate of 104 calf oocysts after 20 freeze-thaw cycles were added to each of 96 wells, and the plates were incubated for 1 hour at 37°C. After removal of the coating solution, the plates were incubated for 1 hour at 37°C with 0.05 M phosphate-buffered saline supplemented with casein and bovine serum albumin (both at 5 g/liter). The plates were then washed twice in phosphate-buffered saline-Tween 20 (Pierce Chemical Company, Rockford, Illinois) washing solution. Sera were diluted in blocking buffer (1:25 for immunoglobulin G (IgG) and immunoglobulin M (IgM) and 1:5 for immunoglobulin A (IgA)) and added to the first line of wells in the microtiter plates. The sera were then double diluted seven times in blocking buffer and incubated overnight at room temperature. After four additional washings, goat anti-human immuno-globulin G, A, or M conjugated to alkaline phosphatase (Kirkegaard and Perry Laboratories, Gaithersburg, Maryland) was added to the wells. The plates were incubated overnight at room temperature and washed, and an enzyme-linked immunosorbent assay was completed by the addition of the enzyme-substrate solution containing para-nitrophenylphosphate (1 mg/ml) in diethanolamine buffer at pH 9.8. The reactions were stopped with 3 M NaOH, and absorbance was read at 405 nm with an automatic enzyme-linked immunosorbent assay biokinetics EL340 reader (Bio-Tek Instruments, Winooski, Vermont). The consecutive serum samples of each subject were tested within the same assay, and positive and negative control sera were included in every microtitration plate in each of the assays. The adjusted absorbances derived from a linear regression analysis of eight doubling dilutions were expressed as endpoint titers (at absorbance = 0.3), and geometric mean titers were calculated.
The specificity of the enzyme-linked immunosorbent assay system used in the study was documented in two different ways.
Lack of cross-reactivity in paired sera of subjects exposed to bacterial enteropathogens. The same criterion for significant antibody response to Cryptosporidium (equal to or greater than a fourfold rise in titer) was used to detect a potential nonspecific rise in serum Cryptosporidium antibodies in pairs of sera (n = 10) obtained 14 days apart from healthy adult subjects vaccinated with an investigational enterotoxigenic E. coli vaccine and in pairs of sera obtained from cases of laboratory-proven Shigella sonnei (n = 5), Shigella flexneri (n = 5), Salmonella typhimurium (n = 5), and enterotoxigenic E. coli (n = 5) infections. The anti-Cryptosporidium titers in these paired sera were extremely stable. The ratios of the antibody titers between post- and preenterotoxigenic E. coli vaccination or post- and pre-Shigella, Salmonella, and enterotoxigenic E. coli infections ranged between 0.8 and 1.5.
Absorption study. One hundred µl of a 1:400 dilution of each of five sera with high IgG Cryptosporidium antibody levels were mixed with 100 µl of 5 x 105 oocysts and incubated for 2 hours, rotating at 37°C. The mixtures were then centrifuged at 13,000 rpm for 10 minutes. Eight twofold dilutions of the absorbed and unabsorbed sera were tested in the same enzyme-linked immunosorbent assay microplate for the presence of Cryptosporidium antibodies. A significant reduction of 4762 percent of the IgG Cryptosporidium geometric mean titer was measured in the absorbed as compared with the unabsorbed corresponding sera.
Definitions and data analysis
An episode of diarrhea was defined as the passing of loose stools at least four times in a 24-hour period for infants less than 1 month old and at least three times in a 24-hour period for older children. A new episode of diarrhea was preceded by three diarrhea-free days. Seroconversion was defined as an antibody response equal to or greater than a fourfold rise in titer compared with that of day 0, while the second serum sample had to reach an antibody titer higher than a threshold value. The threshold titers were defined in order to screen out very small rises that still meet the fourfold criterion. The values chosen were based on the mean baseline titer plus 2 standard deviations (baseline titer was that of age 0 for IgA and IgM and that of age 6 months for IgG). The threshold titers were 333, 17, and 17 for IgG, IgM, and IgA, respectively. The statistical significance for comparison of geometric mean titers at various times was calculated using Duncan's multiple range tests. The statistical significance for comparison of seroconversion rates was calculated using chi-square or Fisher's exact tests.
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RESULTS |
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The experience with diarrheal episodes was similar among the 52 infants included in the serologic study and the rest of the cohort. There were no significant differences in the total number of the diarrheal episodes, their distribution during the 2 years of follow-up, the length of the episodes, and the infants' age at which the first episode occurred. Almost all of the 52 infants who were followed up from birth to the age of 2 years experienced at least one episode of diarrhea during this period. Twenty percent of them had seven or more episodes, and 40 percent had suffered from 36 events.
C. parvum was identified in 25 (48 percent) of the 52 infants during the 23-month follow-up period. Eleven of the infants with Cryptosporidium were symptomatic, and 14 were asymptomatic when the parasite was detected. The timing of Cryptosporidium detection in the 25 infants was evenly distributed throughout the 23 months of follow-up: six infants were positive in the first 6 months of life, another six, between 7 and 12 months, and the rest of 13 infants harbored Cryptosporidium between the age of 13 and 23 months. Five of the 25 infants shed C. parvum in two or three consecutive stool samples for at least 917 days. These were the only subjects with multiple Cryptosporidium infections. Cryptosporidium was detected as a single pathogen among 17 of the 25 infants with positive stool samples, while the other Cryptosporidium-infected infants were coinfected with the following enteropathogens: G. lamblia (three infants), G. lamblia and enterotoxigenic E. coli expressing heat-stable toxin (one infant), Rotavirus and enteroaggregative E. coli (one infant), diffuse adherent E. coli (one infant), enterotoxigenic E. coli expressing heat-labile toxin (one infant), and Campylobacter spp. (one infant). The serologic tests carried out on the consecutive serum samples obtained from the 52 infants revealed an IgG anti-Cryptosporidium geometric mean titer of 339 (95 percent confidence interval: 290, 413) at birth, most probably of maternal origin (figure 1). The geometric mean titer of the mothers of the same infants using the postdelivery samples was 1,295 (95 percent confidence interval: 1,123, 1,486). The level of IgG antibodies dropped significantly by the age of 6 months and then rose continuously to the level of 481 (95 percent confidence interval: 385, 598) at the age of 23 months, when it reached less than half the titer measured in mothers.
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DISCUSSION |
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The specificity of the enzyme-linked immunosorbent assay system used in this study was documented by the lack of cross-reactivity with Cryptosporidium antigens of paired sera obtained from subjects exposed to various bacterial enteropathogens and by the significant reduction of the IgG Cryptosporidium geometric mean titer after absorption with Cryptosporidium oocysts. The specificity of the enzyme-linked immunosorbent assay using Cryptosporidium oocysts as antigens was previously addressed by others (1, 24
). It has been shown that IgG binding to solid-phase adsorbed Cryptosporidium oocysts was preferentially inhibited by preabsorption with Cryptosporidium antigens but not with any of the following formalin-fixed organisms: G. lamblia, Trichomonas vaginalis, Campylobacter jejuni, enterotoxigenic E. coli, and Candida albicans (1
). In a manner similar to that of our study, two other studies (1
, 24
), after one cycle of absorption with Cryptosporidium antigens, reported that an approximate 50 percent inhibition of specific antibody binding was achieved. A possible explanation for the limited inhibition is that more epitopes are exposed when the crude Cryptosporidium antigens are adsorbed to a solid phase because of partial denaturation as compared with the similar antigens in a suspension. In another study (29
), however, it was shown that the sera of lambs or rabbits that were experimentally immunized with Cryptosporidium oocytes cross-reacted in indirect immunofluorescence assay and Western blot with various ovine Eimeria spp. In that study the investigators did not detect cross-reactivity with Toxoplasma gondii or Sarcocystis gigantea (29
).
No significant association was found between the rate or magnitude of the immune response to Cryptosporidium during the first 2 years of life and detection of Cryptosporidium in stools from diarrhea- or nondiarrhea-associated samples. These findings suggest that the immune system of the infants immediately after birth is sensitive to even a very low level of Cryptosporidium antigenic stimuli and promptly reacts with a specific serum response. It has been shown that a very low infectious dose can cause symptomatic Cryptosporidium infection (30, 31
). It is conceivable that a subclinical immune response may be induced by exposure to even lower doses of Cryptosporidium oocysts.
The much higher titers of antibodies among Bedouin mothers, as compared with the Bedouin infants at the age of 2 years, are most probably a result of cumulative natural boosters following repeated exposure to Cryptosporidium in the community of Bedouins living in a township in the northern Negev. At birth, the infants had about a third of the level of anti-Cryptosporidium IgG measured in the sera of their mothers, suggesting that maternal IgG anti-Cryptosporidium passes through the placenta. The geometric mean titer of IgG Cryptosporidium antibodies decreased threefold from the levels measured at birth to those measured 6 months later, reflecting most probably the decay of antibodies of maternal origin. The continuous increase in the levels of serum IgM and IgA Cryptosporidium antibodies during the first 6 months of life suggests that the presence of maternal IgG did not interfere with the recognition of Cryptosporidium antigens by the infants' immune system.
Most of the serologic data related to Cryptosporidium infections have been reported in prevalence studies (1, 3
, 6
, 8
, 28
, 32
34
) that showed, in general, a higher prevalence rate of Cryptosporidium antibodies in populations living in developing as compared with developed countries. Ungar et al. (28
) showed that 20 percent of Ecuadorian children with diarrhea were seropositive for Cryptosporidium-specific IgM and IgG. Another study revealed that C. parvum-specific IgG was present in more than 60 percent of Peruvian children older than 2 years of age and in more than 50 percent of Venezuelan children, including those in the first year of life (3
). A serologic study carried out among institutionalized Thai children aged 260 months, living under crowded conditions with steady contact among ages, showed elevated levels of Cryptosporidium antibodies in virtually all the 35 subjects examined (1
). Similar to our data, this study did not reveal any difference in the Cryptosporidium antibody levels between subjects harboring or not harboring the parasite. Another seroprevalence study carried out among 803 children in Oklahoma showed that the seroprevalence of C. parvum antibodies was positively associated with the age of the children and negatively associated with their socioeconomic status (6). The seroprevalence rates in this population were 13 percent, 38 percent, and 58 percent for the age groups <5 years, 513 years, and 1421 years, respectively (6
). Seroprevalence studies carried out in adult populations showed the presence of detectable Cryptosporidium IgG antibodies in 32 percent of Peace Corps volunteers before they traveled to developing countries (8
). Another study reported a Cryptosporidium IgG seroprevalence rate of 36 percent among adults in a farming community in Wisconsin (33
). These studies defined seropositivity as an antibody level above a cutoff point derived from the measurement of detectable specific antibodies in "negative controls" considered as healthy subjects without any documented previous exposure to Cryptosporidium. Because the negative controls were different in the various studies, the comparisons drawn between the rates of seroprevalence reported are of limited value. Our study, in which the infants were followed up serologically from birth till the age of 2 years, clearly reveals that very early after birth infants naturally exposed to Cryptosporidium elicit significant rises in the specific Cryptosporidium antibody titers. The increase in Cryptosporidium antibody levels during the follow-up period reflects most probably cumulative exposures and repeated antigenic stimuli. Similarly, it has been recently shown that the IgG seroconversion rate increased among volunteers exposed to consecutive challenges, 1 year apart, with Cryptosporidium (30
, 35
, 36
). On the second challenge, the 50 percent infective dose for volunteers with preexisting IgG antibodies (by enzyme-linked immunosorbent assay) was found to be 20-fold higher than that for antibody-negative volunteers, indicating that anti-C. parvum IgG correlates with protection to exposures to low numbers of the parasite (36
). In our study, although without reaching statistical significance, a trend of an excess of parasitologically proven Cryptosporidium infections occurred during the 2-year follow-up among infants with low specific IgG titers measured at birth. It is not clear yet what will be the level of specific serum antibodies that could be associated with naturally acquired immunity to Cryptosporidium and whether the serum antibodies play an active role in mechanisms of immunity or are just a surrogate marker of another effector. It is believed that mechanisms of cell-mediated immunity are of importance in protection against cryptosporidiosis (22
, 23
, 34
), but it was also shown that the specific Cryptosporidium serum antibodies could be also involved in protection against recurrent disease, possibly as part of an antibody-dependent cell-mediated cytotoxic effect (22
, 23
, 34
).
In summary, the findings of the study indicate that Bedouin infants are naturally exposed to Cryptosporidium soon after birth. Serology was more sensitive than frequent prospective assessment of stool samples in detecting Cryptosporidium infections. The higher titers of antibodies among Bedouin mothers as compared with the young children at the age of 2 years may be a result of more natural boosters following repeated exposure to Cryptosporidium. It may also be possible that this difference reflects at least in part the immaturity of the infants' immune system.
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ACKNOWLEDGMENTS |
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The authors thank Dr. Cynthia Chappell for critical reading of the manuscript and useful discussions.
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NOTES |
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REFERENCES |
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