Substance P receptor antagonist reverses intestinal pathophysiological alterations occurring in a novel ex-vivo model of Cryptosporidium parvum infection of intestinal tissues derived from SIV-infected macaques

BACKGROUND: Cryptosporidium infection leads to life-threatening diarrhea in AIDS patients. Pathogenesis of cryptosporidiosis is due to intestinal physiological alterations. We devised an ex-vivo model using ex-vivo Cryptosporidium parvum infection of jejunal tissues derived from SIV-infected macaques and studied the role of substance P (SP) in the pathogenesis of cryptosporidiosis. METHODS: We measured jejunal SP protein levels using ELISA, and electrophysiological alterations using the Ussing chamber technique in an ex vivo model of Cryptosporidium infection. Paraformaldehyde-fixed jejunum from SIV-infected macaques with and without naturally occurring cryptosporidiosis was studied for SP protein expression by immunohistochemistry and fluorescence deconvolution microscopy. RESULTS: Ex-vivo Cryptosporidium-infected tissues and tissues from SIV-infected macaques with naturally occurring cryptosporidiosis demonstrated elevated SP protein levels compared with tissues from SIV-infected animals without ex-vivo C. parvum infection or tissues from SIV-infected animals that have no evidence of cryptosporidiosis. In our ex-vivo model of Cryptosporidium infection, we demonstrated pathophysiological alterations that were blocked by SP-receptor antagonist treatment. CONCLUSIONS: These studies suggest that SP-receptor antagonists could prove useful for treatment of AIDS-related cryptosporidiosis.     

Cryptosporidiosis in children and adults: parasitological and clinico-epidemiological features

By using the elective diagnosis methodology for Cryptosporidium (modified Ziehl-Neelsen and Heine stainings), 481 faeces samples from patients--children and adults--presenting acute or prolonged gastroenteritis and also from asymptomatic ones, but with professional, immunological or pharmacological risk for infection, were studied. Twelve Cryptosporidium positive cases were identified, meaning a general prevalence of 2.48% Cryptosporidium infection was detected in all investigated epidemiological groups: children, animal handlers, immunodeficient patients, immunocompetent adults with acute gastroenteritis. The risk factors hierarchy elicited the role of children collectivities attending, for acquiring the parasite in infantile cryptosporidiosis, while the opportunistic and zoonotic feature of the disease was especially present in adults' cryptosporidiosis. The assessment of cryptosporidiosis evolution, according to patients' immune status, has not allowed an obvious clinical discrepancy, since the immunocompetent subjects with professional risk had presented a prolonged oocysts excretion. The different clinico-epidemiological and parasitological features of Cryptosporidium infection are commented and the importance of applying Cryptosporidium methodology in current diagnosis practice is emphasized.     

Cryptosporidium--biotechnological advances in the detection, diagnosis and analysis of genetic variation

Cryptosporidiosis is predominantly a gastrointestinal disease of humans and other animals, caused by various species of protozoan parasites representing the genus Cryptosporidium. This disease, transmitted mainly via the faecal-oral route (in water or food), is of major socioeconomic importance worldwide. The diagnosis and genetic characterization of the different species and population variants (usually recognised as "genotypes" or "subgenotypes") of Cryptosporidium is central to the prevention, surveillance and control of cryptosporidiosis, particularly given that there is presently no broadly applicable treatment regimen for this disease. Although traditional phenotypic techniques have had major limitations in the specific diagnosis of cryptosporidiosis, there have been major advances in the development of molecular analytical and diagnostic tools. This article provides a concise account of Cryptosporidium and cryptosporidiosis, and focuses mainly on recent advances in nucleic acid-based approaches for the diagnosis of cryptosporidiosis and analysis of genetic variation within and among species of Cryptosporidium. These advances represent a significant step toward an improved understanding of the epidemiology as well as the prevention and control of cryptosporidiosis.     

Environmental risk factors for Cryptosporidium infection in an island from Western Venezuela

Few investigations have been conducted on risk factors for Cryptosporidium infection in communities from developing countries. A study was conducted to determine the prevalence and risk factors for cryptosporidiosis in San Carlos island, Venezuela. A sample of 515 subjects (mean age +/- SD: 21.4 +/- 17.8 years) was surveyed. Single fecal specimens were collected and modified Ziehl-Neelsen carbolfuchsin staining of formalin-ether concentrate stools were examined for identification of the parasite. Infections with Cryptosporidium (67 of 515, 13%) were common. Prevalence of the parasite varied among sectors of the community; 34 of 67(50.7%) cases of cryptosporidiosis clustered in two sectors with extreme poverty. Variables strongly associated with a higher risk for the infection (p < 0.01) were residing in these sectors versus the remainder, living in a hut or small residence versus a brick or larger house, using an area of backyard rather than a toilet or latrine for defecation, and having contact with soil contaminated with human feces. Crowding was also a risk (p < 0.05). Contact with human feces contaminated-soil may be an important mode of transmission and poverty a predisposing factor for the infection.     

A review of the biology and epidemiology of cryptosporidiosis in humans and animals

The epidemiology of cryptosporidiosis, an infection caused by several genotypically and phenotypically diverse Cryptosporidium species, has been dynamically changing over the past decade from that of a rare, largely asymptomatic infection to an acute enteric disease of animals and humans. In this review, the current understanding of factors (biology and epidemiology) contributing to the emergence of cryptosporidiosis in animals, including parasite biology, genetic diversity, environmental spread, livestock production trends, presence of the parasite in livestock and companion animals, and potential risk of transmission from animals to humans is highlighted. Potential control measures and the role of veterinary and medical professionals in the prevention of cryptosporidiosis are also discussed.     

Substance P receptor antagonism for treatment of cryptosporidiosis in immunosuppressed mice

Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum, causes self-limited diarrhea in normal hosts but can cause life-threatening diarrhea for immunosuppressed patients. There is an urgent need for new drugs to treat this chronic disease. Cryptosporidium parvum infection is associated with intestinal structural and pathophysiologic changes, including villi blunting and glucose malabsorption. Substance P (SP), a neuropeptide and pain transmitter, is associated with the gastrointestinal tract and is elevated in humans and macaques after experimental C. parvum challenge. To examine the relevance of SP in the pathogenesis of cryptosporidiosis, and to determine if SP receptor antagonism can be employed for treatment of cryptosporidiosis in immunosuppressed hosts, we used an immunosuppressed murine model (dexamethasone-immunosuppressed mice) that is frequently utilized for examining chemotherapeutic potential of drugs. Quantitative ELISA was used to measure intestinal SP levels in immunosuppressed mice with, and without, C. parvum infection. Intestinal physiological alterations, as studied by the Ussing chamber technique, plus weight change, fecal oocyst shedding, and villi measurements, were compared in infected mice with, and without, SP receptor antagonist (aprepitant) treatment. Immunosuppressed mice infected with C. parvum demonstrated increased SP levels as well as physiological alterations (glucose malabsorption), weight loss, fecal oocyst shedding, and structural alterations (increased intestinal villi blunting) compared to uninfected mice. Each of these defects was significantly inhibited by aprepitant treatment. These studies demonstrate the potential of SP receptor antagonism for treatment of pathogenesis of cryptosporidiosis in immunosuppressed hosts.     

Cryptosporidiosis in ferrets

The diagnosis of cryptosporidiosis in two ferrets who died from unrelated causes prompted a survey to determine the prevalence and incidence of the infection in ferrets at our facility. The survey of the existing ferret population and all new arrivals indicated cryptosporidiosis occurred as a subclinical disease in a high percentage of young ferrets: 40% of the ferret population and 38 to 100% of the new arrivals had cryptosporidial oocysts in their feces. The infection was found to persist for several weeks in both immunocompetent and immunosuppressed ferrets. The interspecies transmission of Cryptosporidium implies that infected ferrets should be considered a potential source of infection for the general population.     

The role of humoral immunity in Cryptosporidium spp. infection. Studies with B cell-depleted mice

Cryptosporidiosis has become an important infection in man, particularly among very young or immunocompromised individuals. Here, the protective role of antibody responses to Cryptosporidium was examined using a well established murine model of cryptosporidiosis. Although normal neonatal BALB/c mice exhibited good IgM and IgG serum antibody responses, no correlation could be drawn between the intensity of these responses and the severity or duration of cryptosporidiosis. Moreover, B cell-deficient (anti-mu-treated) neonatal BALB/c mice did not differ from untreated or normal rabbit Ig-treated, age-matched controls in the onset, peak, or duration of cryptosporidiosis. The apparent absence of a role for antibody in these self resolving infections was supported by the lack of susceptibility of anti-mu-treated adult BALB/c to attempted infection with doses of Cryptosporidium 10 times the dose required to infect 100% of normal (Ig producing) neonates. The results suggest that the role of specific in vivo antibody responses in the resolution of murine infection with this coccidian parasite is minor and that the likelihood of success for cryptosporidial vaccines aimed solely at enhancing in vivo antibody production may be limited.     

Molecular epidemiology of human cryptosporidiosis

Species within the genus Cryptosporidium are protozoan parasites that infect a wide range of vertebrates, and represent a significant cause of morbidity and mortality in those animals. In humans, cryptosporidiosis is a common cause of diarrhoeal disease with a global distribution. Unravelling the epidemiology of human infection has proven to be difficult, due to the existence of multiple transmission routes (person-to-person, animal-to-person, waterborne, foodborne and airborne transmission), and to the difficulties in identifying the different species using conventional criteria, such as oocyst morphology. The advent of molecular techniques has had a remarkable impact on the way the epidemiology of cryptosporidiosis can be studied. Molecular investigations have shown that the vast majority of human cases are caused by C. hominis and C. parvum. Interestingly, differences in geographical and temporal distribution, disease presentations and risk factors for infection have been identified for both C. hominis and C. parvum. Further, molecular analyses have revealed that other species, including C. meleagridis, C. felis, C. canis, C. suis, C. muris and two Cryptosporidium genotypes, can infect humans and may be linked to clinical disease, not only in immunocompromised but also in immunocompetent individuals.     

Worldwide human zoonotic cryptosporidiosis caused by Cryptosporidium felis

Cryptosporidium is an important enteric pathogen worldwide distributed causing diarrhoeal illness in humans and animals. Identifying Cryptosporidium species using conventional criteria, such as oocyst morphology, is inadequate. The advent of molecular techniques has conducted to characterize different species and genotypes of Cryptosporidium infecting humans. The vast majority of human cases of cryptosporidiosis in the world are caused by both species, Cryptosporidium hominis and Cryptosporidium parvum. However other species including Cryptosporidium felis can infect humans too. In this review, we analyse 58 reported cases of human C. felis infection in different parts of the world. To date this emerging protozoan disease is present in humans around the world, except in Australia and Oceania. Adults and children are infected, more often when immunocompromised by HIV infection (83 % of reported cases). Apparently immunocompetent individuals are also infected by C. felis. In developing countries, inhabitants are more likely infected by C. felis probably through the oocyst contamination of drinking or recreational water. The public health importance of C. felis infection in tropical countries remains to be evaluated.     

Protection of calves against cryptosporiosis by oral inoculation with gamma-irradiated Cryptosporidium parvum oocysts

The purpose of this study was to determine whether gamma-irradiated Cryptosporidium parvum oocysts could elicit protective immunity against cryptosporidiosis in dairy calves. Cryptosporidium parvum Iowa strain oocysts (1 x 10(6) per inoculation) were exposed to various levels of gamma irradiation (350-500 Gy) and inoculated into 1-day-old dairy calves. The calves were examined daily for clinical signs of cryptosporidiosis, and fecal samples were processed for the presence of C. parvum oocysts. At 21 days of age, the calves were challenged by oral inoculation with 1 x 10(5) C. parvum oocysts and examined daily for oocyst shedding and clinical cryptosporidiosis. Calves that were inoculated with C. parvum oocysts exposed to 350-375 Gy shed C. parvum oocysts in feces. Higher irradiation doses (450 or 500 Gy) prevented oocyst development, but the calves remained susceptible to C. parvum challenge infection. Cryptosporidium parvum oocysts exposed to 400 Gy were incapable of any measurable development but retained the capacity to elicit a protective response against C. parvum challenge. These findings indicate that it may be possible to protect calves against cryptosporidiosis by inoculation with C. parvum oocysts exposed to 400-Gy gamma irradiation.     

Cryptosporidium andersoni is the predominant species in post-weaned and adult dairy cattle in China

Dairy industry plays an important role in the agricultural economy of China. To estimate the prevalence and public health significance of cryptosporidiosis in post-weaned and adult dairy cattle in China, during four consecutive years (from 2006 to 2009), a total of 1315 fecal samples from 22 dairy cattle farms in ten prefectures in Henan Province were examined for the presence of Cryptosporidium oocysts. The overall prevalence of Cryptosporidium was 7.9%, with the highest infection rate (11.3%) in 3 to 11-month-old calves and the lowest infection rate (1.0%) in >2-year-old cows (p<0.01). Cryptosporidium-positive samples (n=104) were analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis of the small subunit (SSU) rRNA gene, and 25 representative samples were further analyzed by DNA sequencing of the PCR products. Cryptosporidium bovis and Cryptosporidium andersoni were identified. C. andersoni (84/104) was the predominant species and was found in all age groups, whereas C. bovis (20/104) was only detected in 3 to 11-month-old calves. Thus, C. andersoni appears to be the dominant species in weaned dairy calves and heifers in China, in contrast with its common occurrence in adult cattle in other parts of the world.     

The role of breast milk in protecting urban Peruvian children against cryptosporidiosis.

To test the hypothesis that breast milk of nursing mothers may afford children protection against cryptosporidiosis, a prospective cohort study was carried out in the young peoples' community of San Juan de Miraflores near Lima, Peru. Mothers and newborn children were sorted into cohort groups based on the mothers' breast milk antibody response to Cryptosporidium sporozoites using an antibody-capture enzyme-linked immunosorbent assay to detect parasite-specific immunoglobulin A. Children were monitored for Cryptosporidium infection using an indirect immunofluorescence assay. Of 211 mothers enrolled in the study, 39 (18.5%) had high breast milk antibody titers, 107 (50.7%) had medium titers, and 65 (30.8%) had low titers. Sixty-one episodes of Cryptosporidium infection were detected in 50 children of these mothers. Eleven (22%) had mothers in the high antibody titer group, 20 (40%) had mothers in the medium titer group, and 19 (38%) had mothers in the low titer group. The prevalence of infection within children of each group was 0.17, 0.19 and 0.38 respectively. There was no significant difference in the prevalence or duration of infection among children of the different groups. The data does not support the notion that there is protection from Cryptosporidium infection afforded children whose mothers have demonstrable breast milk antibodies against the parasite.     

Prevalence of cryptosporidiosis among the villagers and domestic animals in several rural areas of Korea

The present study was undertaken to investigate the infection status of Cryptosporidium parvum in the villagers and the reservoir hosts in several rural areas in Korea. A total 5,262 fecal samples were collected from the inhabitants residing at Gangwon-do, Chungcheongbuk-do, Jeollanam-do, and Gyeongsangnam-do between the dates of September, 2001 to June, 2002. In addition, 1,453 fecal samples were collected from livestock reared in Gokseong-gun, Jeollanam-do and Chungju-si, Chungcheongbuk-do. All the fecal smears were prepared by formalin-ether sedimentation, and examined by light microscopy after modified acid-fast staining. The overall positive rate of human cryptosporidiosis was 3.3%. Gokseong-gun, Jeollanam-do showed a 8.2% positive rate and appeared as the highest endemic area among the surveyed areas. Haman-gun, Gyeongsangnam-do showed a 0.4% positive rate and was the lowest endemic area. The positive rate of livestock infection in Gokseong-gun, Jeollanam-do was 94%, which was more than ten times higher than that of Chungju-si, Chungcheongbuk-do (9.3%). From these results, it was revealed that cryptosporidiosis was an endemic disease in some rural areas of Korea, and the livestock could be an important source of human infection.     

Variation in Cryptosporidium: towards a taxonomic revision of the genus

Cryptosporidium is an important cause of enteric disease in humans and other animals. Limitations associated with conventional diagnostic methods for cryptosporidiosis based on morphological features, coupled with the difficulty of characterising parasites isolated in the laboratory, have restricted our ability to clearly identify species. The application of sensitive molecular approaches has obviated the necessity for laboratory amplification. Such studies have found considerable evidence of genetic heterogeneity among isolates of Cryptosporidium from different species of vertebrate, and there is now mounting evidence suggesting that a series of host-adapted genotypes/strains/species of the parasite exist. In this article, studies on the molecular characterisation of Cryptosporidium during the last 5 years are reviewed and put into perspective with the past and present taxonomy of the genus. The predictive value of achieving a sound taxonomy for the genus Cryptosporidium with respect to understanding its epidemiology and transmission and controlling outbreaks of the disease is also discussed.     

Multiple Cryptosporidium genotypes detected in wild black rats (Rattus rattus) from northern Australia

As part of a broader investigation into the potential role of black rats (Rattus rattus) as disease vectors into native small mammal populations of northern Australia, blood and faecal samples from wild black rats were screened by molecular methods, for piroplasms (Babesia and Theileria), trypanosomes and the enteric parasite Cryptosporidium. While piroplasms and trypanosomes were not detected in the blood of these animals, the overall prevalence of Cryptosporidium 18S rDNA in faecal samples was 8.2% (7/85). Co-occurrence of multiple genotypes was observed in 57.1% of the infected individuals (4/7); cloning and re-sequencing resulted in 14 sequences which broadly grouped with Cryptosporidium sp. rat-genotypes II and III. A novel rat-derived Cryptosporidium sp. genotype at the actin locus was also obtained from five animals. The relatively low infection rate detected, and the epidemiological data on cryptosporidiosis, do not conclusively support a current threat to native Australian mammals from black rats carrying Cryptosporidium. However, this observation is based on sampling limited isolates, in limited regions. Further studies, also including sampling of native mammals, are required on larger sample sizes and from wider geographic areas, to determine the significance of these findings, including the public health importance of Cryptosporidium spp. from rodents.     

Genetic characterization of Cryptosporidium species from humans in Spain

Several species of Cryptosporidium have been associated with infection. Cryptosporidium parvum and Cryptosporidium hominis are the main agents of cryptosporidiosis in humans. Stool samples from 108 Cryptosporidium-infected patients were submitted to PCR-RFLP analysis for a 553-bp fragment of Cryptosporidium oocyst wall protein (COWP) gene and an 826-864 bp fragment of the small-subunit ribosomal RNA (SSU-rRNA) gene. Ninety-two patients were immunocompetent children and 16 were HIV-infected adults. C. hominis was detected in 69 patients (59 immunocompetent and 10 HIV-infected); C. parvum, in 34 patients (28 immunocompetent and 6 HIV-infected); and C. meleagridis and C. felis in one patient each (both immunocompetent children). Three samples yielded negative results. C. parvum was significantly more frequent in children from rural areas than in those of urban residence (p=0.010). As far as we know, this is the first surveillance study about the molecular characterization of Cryptosporidium in humans performed in Spain. The finding of zoonotic species infecting humans calls for further research on this subject.     

Drug treatment and novel drug target against Cryptosporidium

Cryptosporidiosis emergence triggered the screening of many compounds for potential anti-cryptosporidial activity in which the majority were ineffective. The outbreak of cryptosporidiosis which occurred in Milwaukee in 1993 was not only the first significant emergence of Cryptosporidium spp. as a major human pathogen but also a huge waterborne outbreak thickening thousands of people from a major city in North America. Since then, outbreaks of cryptosporidiosis are regularly occurring throughout the world. New drugs against this parasite became consequently urgently needed. Among the most commonly used treatments against cryptosporidiosis are paromomycin, and azithromycin, which are partially effective. Nitazoxanide (NTZ)'s effectiveness was demonstrated in vitro, and in vivo using several animal models and finally in clinical trials. It significantly shortened the duration of diarrhea and decreased mortality in adults and in malnourished children. NTZ is not effective without an appropriate immune response. In AIDS patients, combination therapy restoring immunity along with antimicrobial treatment of Cryptosporidium infection is necessary. Recent investigations focused on the potential of molecular-based immunotherapy against this parasite. Others tested the effects of probiotic bacteria, but were unable to demonstrate eradication of C. parvum. New synthetic isoflavone derivatives demonstrated excellent activity against C. parvum in vitro and in a gerbil model of infection. Newly synthesized nitro- or non nitro- thiazolide compounds, derived from NTZ, have been recently shown to be at least as effective as NTZ against C. parvum in vitro development and are promising new therapeutic agents.     

Efficacy of S-adenosylhomocysteine hydrolase inhibitors, D-eritadenine and (S)-DHPA, against the growth of Cryptosporidium parvum in vitro

D-eritadenine and (S)-DHPA are aliphatic adenosine analogues known to target S-adenosylhomocysteine hydrolase (SAHH) and potent antiviral compounds. In the present study, we demonstrate that these two compounds also display efficacy against recombinant SAHH enzyme of the protozoan parasite Cryptosporidium parvum, as well as inhibition of parasite growth in vitro. Our data confirm that SAHH could serve as a rational drug target in cryptosporidial infection and antiviral adenosine analogues are potential candidates for drug development against cryptosporidiosis.