Quantitative assessment of viable Cryptosporidium parvum load in commercial oysters (Crassostrea virginica) in the Chesapeake Bay

The epidemiological importance of increasing reports worldwide on Cryptosporidium contamination of oysters remains unknown in relation to foodborne cryptosporidiosis. Thirty market-size oysters (Crassostrea virginica), collected from each of 53 commercial harvesting sites in Chesapeake Bay, MD, were quantitatively tested in groups of six for Cryptosporidium sp. oocysts by immunofluorescent antibody (IFA). After IFA analysis, the samples were retrospectively retested for viable Cryptosporidium parvum oocysts by combined fluorescent in situ hybridization (FISH) and IFA. The mean cumulative numbers of Cryptosporidium sp. oocysts in six oysters (overall, 42.1±4.1) were significantly higher than in the numbers of viable C. parvum oocysts (overall, 28.0±2.9). Of 265 oyster groups, 221 (83.4%) contained viable C. parvum oocysts, and overall, from 10–32% (mean, 23%) of the total viable oocysts were identified in the hemolymph as distinct from gill washings. The amount of viable C. parvum oocysts was not related to oyster size or to the level of fecal coliforms at the sampling site. This study demonstrated that, although oysters are frequently contaminated with oocysts, the levels of viable oocysts may be too low to cause infection in healthy individuals. FISH assay for identification can be retrospectively applied to properly stored samples.     

Contamination of Atlantic coast commercial shellfish with Cryptosporidium

Shellfish (oysters and/or clams) were obtained from 37 commercial harvesting sites in 13 Atlantic coast states from Maine to Florida and one site in New Brunswick, Canada. Gill washings from each of 25 shellfish at each site were examined by immunofluorescence microscopy (IFA) for oocysts of Cryptosporidium. Gill washings from another 25 shellfish at each site were grouped into five pools of five shellfish each. DNA from each pool was utilized for PCR and genotyping. Oocysts were found in 3.7% of 925 oysters and clams examined by IFA in shellfish from New Brunswick and 11 of 13 states. Cryptosporidium DNA was detected by PCR in 35.2% of 185 pools. Cryptosporidium parvum genotypes 1 and 2, and Cryptosporidium meleagridis,all of which have been identified in infected humans, were identified at 37.8% of the sites. Gill washings from every site were tested for the presence of infectious oocysts by biological assay in neonatal BALB/c mice but no mice were found infected, suggesting that either the oocysts were no longer infectious or infections in mice were below the level of detection. Collectively, these findings indicate that Cryptosporidium species, indicative of pollution from human and animal feces and potentially infectious for humans, were found in commercial shellfish from 64.9% of sites examined along the Atlantic coast by either microscopy or molecular testing. Previous reports link periods of high rainfall with the elevated numbers of pathogen contaminated shellfish. Because shellfish in the present study were examined during a period of exceptionally low precipitation, the data are thought to underestimate the number of Cryptosporidium contaminated shellfish likely to be found during periods of normal or above normal precipitation.     

Cryptosporidium oocysts in Bent mussels (Ischadium recurvum) in the Chesapeake Bay

Filter-feeding molluscan shellfish can concentrate environmentally derived waterborne pathogens of humans, which can be utilized in the sanitary assessment of water quality. In the present study, oocysts of Cryptosporidium were detected in Bent mussels (Ischadium recurvum) at two Chesapeake Bay sites from which C. parvum-contaminated oysters had previously been collected. Spiking of Cryptosporidium-free blue mussel (Mytilus edulis) tissue with C. parvum oocysts showed a 51.1% recovery rate of oocysts, giving an oocyst detection limit of 19 oocysts/0.7 ml of mussel tissue homogenate. The results indicate that Bent mussels, which are common throughout the Chesapeake Bay region, may prove to be useful as biological indicators of water contamination with Cryptosporidium oocysts. Received: 24 November 1998 / Accepted: 20 January 1999     

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.     

Genetic diversity of<Emphasis Type="Italic"> Cryptosporidium</Emphasis> spp. in cattle in Michigan: implications for understanding the transmission dynamics

Epidemiological and molecular data on 248 bovine, 17 human, and 16 water samples of Cryptosporidium spp. collected from the lower peninsula of Michigan between 1997 and 2000 were analysed. Cryptosporidium parvum bovine genotype and Cryptosporidium andersoni were found in 56 and four cattle samples, respectively. A total of six C. parvum subgenotypes were found in 34 bovine samples, and five of the eight farms had two or three subgenotypes in cattle. Six water samples from these farms had C. andersoni, five had the C. parvum bovine genotype, and one had Cryptosporidium muris. In contrast, four PCR-positive human samples produced the C. parvum bovine genotype and two had the C. parvum human genotype. Among the C. parvum bovine genotype samples, two human samples and one water sample had subgenotypes identical to those found on cattle farms. The results of this study demonstrate the potential use of molecular methods in tracking the transmission of Cryptosporidium.     

Infectivity and neutralization of Cryptosporidium parvum sporozoites.

Cryptosporidiosis, a diarrheal disease of calves and humans caused by the coccidian parasite Cryptosporidium parvum, is terminated in hosts with normal immune systems. To assess the mechanisms of immunity in cryptosporidiosis, it is necessary to isolate and quantitate sporozoites, the infective stage of Cryptosporidium spp. Here we report the (i) separation of infective C. parvum oocysts from calf feces by ether extraction, sieving, and hypochlorite treatment; (ii) separation of viable C. parvum sporozoites from intact and excysted oocysts by anion-exchange chromatography; and (iii) quantitation of sporozoite infectivity in vivo by direct intraintestinal injection of isolated sporozoites in 7-day-old BALB/c mice. When isolated sporozoites were incubated with heat-inactivated immune bovine serum, 25 times the 50% infective dose for 7-day-old mice was completely neutralized. Sporozoites incubated with preimmune bovine serum were infectious for 7-day-old mice.     

Detection of cryptosporidium and identification to the species level by nested PCR and restriction fragment length polymorphism

Cryptosporidiosis is an emerging protozoan disease associated with large waterborne outbreaks. Diagnosis relies on microscopic examination of stools, but this method cannot identify the infecting species of Cryptosporidium. We have developed a test based on nested PCR and restriction fragment length polymorphism (RFLP) that offers simple identification of Cryptosporidium hominis, Cryptosporidium parvum, and most other human infective species in stool samples. Purified C. parvum oocysts were used for PCR development. Extracted DNA was amplified by nested PCR targeting a 214-bp fragment of the 18S RNA gene. Enzymatic restriction sites were identified by bioinformatic analysis of all published Cryptosporidium 18S rRNA sequences. Experiments with spiked stool samples gave an estimated PCR detection limit of one oocyst. Specificity was assessed by testing 68 stool samples from patients with microscopically proven cryptosporidiosis and 31 Cryptosporidium-negative stools. Sixty-seven (98.5%) of the 68 stool samples from patients with microscopically proven cryptosporidiosis and 2 of the other stool samples were positive by PCR and could be genotyped. RFLP analysis identified 36 C. hominis, 19 C. parvum, 8 Cryptosporidium meleagridis, and 6 Cryptosporidium felis or Cryptosporidium canis samples. Species determination in 26 PCR-positive cases was in full agreement with DNA sequencing of the 18S rRNA hypervariable region. The excellent sensitivity of PCR, coupled with the accuracy of RFLP for species identification, make this method a suitable tool for routine diagnosis and genotyping of Cryptosporidium in stools.     

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.     

Molecular characterization of Cryptosporidium isolates from human and bovine using 18s rRNA gene in Shahriar county of Tehran, Iran

Cryptosporidiosis is a widespread cause of diarrheal diseases of humans, young calves, and many mammals. Although in recent years molecular investigations on Cryptosporidium have increased, no data are available on Iran in this regard. Two species of Cryptosporidium are known to infect human beings; Cryptosporidium hominis which shows anthroponotic transmission among humans and Cryptosporidium parvum which shows zoonotic transmission between animals and humans. Cryptosporidium oocysts, morphologically identified as C. parvum, were isolated from 24 human and 35 bovine cases in Shahriar (suburb of Tehran, Iran), and genotyped by means of a Nested-polymerase chain reaction/restriction fragment length polymorphism analysis of the 18s rRNA gene. The isolates from bovine samples gave zoonotic or 2 genotype (C. parvum), and DNA profiles of human isolates gave two distinct genotypes, namely an anthroponotic or 1 (C. hominis) and zoonotic genotype or 2 (C. parvum).     

Molecular characterisation of Cryptosporidium isolates from humans in Slovenia

Twenty-nine faecal specimens from Slovenian patients in which Cryptosporidium oocysts had been identified were studied. A fragment of the Cryptosporidium 18S rRNA gene and a fragment of the Cryptosporidium COWP gene were amplified by PCR and sequenced. Cryptosporidium parvum was identified in 26 of the 29 specimens, Cryptosporidium hominis in two, and Cryptosporidium cervine genotype in one. The fact that C. parvum, which is associated traditionally with animals, was identified in the majority of human faecal specimens suggests that cryptosporidiosis may have primarily a zoonotic origin in Slovenia.     

Rapid microsphere assay for identification of cryptosporidium hominis and cryptosporidium parvum in stool and environmental samples

Cryptosporidium hominis and Cryptosporidium parvum are associated with massive disease outbreaks worldwide. Because these two species have different transmission cycles, identification of these parasites to the species level in clinical samples may provide laboratory data of crucial importance in epidemiologic investigations. To date, the most reliable way to differentiate C. hominis and C. parvum is based on DNA sequencing analysis of PCR amplicons. Although this approach is very effective for differentiation of Cryptosporidium species, it is labor-intensive and time-consuming compared with methods that do not require DNA sequencing analysis as an additional step and that have been successfully used for specific identification of a number of pathogens. In this study, we describe a novel Luminex-based assay that can differentiate C. hominis from C. parvum in a rapid and cost-effective manner. The assay was validated by testing a total of 143 DNA samples extracted from clinical specimens, environmental samples, or samples artificially spiked with Cryptosporidium oocysts. As few as 10 oocysts per 300 microl of stools could be detected with this assay. The assay format includes species-specific probes linked to carboxylated Luminex microspheres that hybridize to a Cryptosporidium microsatellite-2 region (ML-2) where C. hominis and C. parvum differ by one nucleotide substitution. The assay proved to be 100% specific when samples that had been characterized by direct fluorescent antibody test (DFA) and DNA sequencing analysis were tested. In addition, the assay was more sensitive than DFA and provided species identification, which is an advantage for epidemiologic studies.     

Cryptosporidium spp. in farms in the eastern region of Venezuela

Cryptosporidiosis is caused by the coccidian protozoa Cryptosporidium. It is the most worldwide prevalent pathogen associated with diarrhea in young cattle and usually is caused by C. parvum which is also responsible of infections in humans, specially in immunocompromised patients. Faecal samples from 75 young cattle aged between 2 to 20 weeks, were collected in four Western Dairy farms of Venezuela and screened for the presence of Cryptosporidium oocysts. Positive samples were detected in 22 animals (29.3%). Results of this current study demonstrate that Cryptosporidium spp. is frequently involved in the aetiology of calf neonatal diarrhea in this region, and adults carriers seem to play an important role as a source of infection.     

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.     

Comparison of tests for viable and infectious Cryptosporidium parvum oocysts

The purpose of this study was to compare different assays for viable Cryptosporidium parvum incubated in water at a temperature commonly found in the environment. C. parvum oocysts were stored in sterile water for 9 months at 15 degrees C. A sample was removed monthly and analyzed by five different assays to determine oocyst viability. Mouse infection and cell culture showed that C. parvum oocysts remained viable and infectious when stored for 7 months at this temperature. Fluorescence in situ hybridization (FISH) using probes directed to ribosomal RNA was also applied to these oocysts. The proportion of FISH-positive oocysts was 70-80% for the first 2 months of storage, decreased and remained nearly constant at 40-50% for 3-7 months, then decreased to 20% by 8 months, and to 0% by 9 months. Amylopectin content and mRNA for amyloglucosidase (CPAG), as measured by RT-PCR, decreased much more rapidly. By 3 months and for the remainder of the incubation period, amylopectin content was 20% of the original amount present in the oocysts. The CPAG RT-PCR signal at 3 months was 50% of that observed after 1 month storage, 20% at 4 months, and was not detected thereafter. Thus, results from cell culture and mouse infection assay exhibited the best agreement, the FISH assay showed modest agreement with these assays, and CPAG RT-PCR and the amylopectin assay displayed marginal agreement with the other three assays.     

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.     

Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis

Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum beta-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same beta-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts.     

Fecal antibodies to Cryptosporidium parvum in healthy volunteers

This study examined the intestinal antibody response in 26 healthy volunteers challenged with Cryptosporidium parvum oocysts. Fecal extracts were assayed for total secretory immunoglobulin A (IgA) and C. parvum-specific IgA reactivity. Specific IgA reactivity was standardized to IgA concentration and expressed as a reactivity index (RI). Anti-C. parvum fecal IgA (fIgA) increased significantly in 17 of 26 (65.4%) following oocyst ingestion. Of those with detectable responses, 59, 76.5, and 94.1% were positive by days 7, 14, and 30, respectively. Volunteers receiving high challenge doses (>1,000 and 300 to 500 oocysts) had higher RIs (RI = 5.57 [P = 0. 027] and RI = 1.68 [P = 0.039], respectively) than those ingesting low doses (30 to 100 oocysts; RI = 0.146). Subjects shedding oocysts and experiencing a diarrheal illness had the highest fIgA reactivity. When evaluated separately, oocyst excretion was associated with an increased fIgA response compared to nonshedders (RI = 1.679 versus 0. 024, respectively; P = 0.003). However, in subjects experiencing diarrhea with or without oocyst shedding, a trend toward a higher RI (P = 0.065) was seen. Extracts positive for fecal IgA were further examined for IgA subclass. The majority of stools contained both IgA1 and IgA2, and the relative proportions did not change following challenge. Also, no C. parvum-specific IgM or IgG was detected in fecal extracts. Thus, fecal IgA to C. parvum antigens was highly associated with infection in subjects who had no evidence of previous exposure and may provide a useful tool in detecting recent infections.     

Molecular characterization of Cryptosporidium isolates obtained from humans in France

Cryptosporidium parvum is usually considered the agent of human cryptosporidiosis. However, only in the last few years, molecular biology-based methods have allowed the identification of Cryptosporidium species and genotypes, and only a few data are available from France. In the present work, we collected samples of whole feces from 57 patients from France (11 immunocompetent patients, 35 human immunodeficiency virus [HIV]-infected patients, 11 immunocompromised but non-HIV-infected patients) in whom Cryptosporidium oocysts were recognized by clinical laboratories. A fragment of the Cryptosporidium 18S rRNA gene encompassing the hypervariable region was amplified by PCR and sequenced. The results revealed that the majority of the patients were infected with cattle (29 of 57) or human (18 of 57) genotypes of Cryptosporidium parvum. However, a number of immunocompromised patients were infected with C. meleagridis (3 of 57), C. felis (6 of 57), or a new genotype of C. muris (1 of 57). This is the first report of the last three species of Cryptosporidium in humans in France. These results indicate that immunocompromised individuals are susceptible to a wide range of Cryptosporidium species and genotypes.     

Viability and infectivity of oocysts recovered from clams, Ruditapes philippinarum, experimentally contaminated with Cryptosporidium parvum

This study confirms the important role of marine bivalve molluscs, destined for human consumption, as transmitters of cryptosporidiosis, zoonotic diarrhoeal disease caused by Cryptosporidium parvum. C. parvum oocysts recovered from seawater clams (Ruditapes philippinarum) were viable and infective in five of eight infected neonatal CD-1 Swiss mice. Oocysts were observed in clam gill and gastrointestinal tract tissue homogenates as well as in gill histological sections, by an immunofluorescent antibody technique. In vitro viability of recovered oocysts was also determined using fluorogenic vital dyes (75% viability).