Parvum Infection Research Articles

Comparison of viability and infectivity of Cryptosporidium parvum oocysts stored in potassium dichromate solution and chlorinated tap water

The present study was undertaken to compare the viability and infectivity of Cryptosporidium parvum oocysts that had been stored for 1, 4, 7, 10, 13, 16, 20, 25 and 30 months at 4 °C in 2.5% potassium dichromate (Cr) or chlorinated tap water, respectively. An excystation protocol was performed in vitro to evaluate viability. One hundred and eighty female BABL/c mice were used to evaluate the infectivity of oocysts by investigating the prepatent period of C. parvum infection, the quantity of oocysts excreted, and the number of parasites that colonized the villi of the ileum. The results showed that C. parvum oocysts preserved in Cr for 1–16 months or in water for 1–13 months were capable of excystation in vitro and infection of mice. The excystation rates of oocysts and the prepatent periods in mice infected by oocysts stored in Cr and water were not significantly different ( p > 0.05), and there was a strong correlation between prepatent period and duration of oocyst storage (Cr: R 2 = 0.92 ; water: R 2 = 0.98). There were no significant differences in oocyst shedding from feces or parasitism of the terminal ilea of mice by Cryptosporidia between the two storage media ( p > 0.05). In conclusion, C. parvum oocysts may be stored at 4 °C in water instead of Cr for the purposes of laboratory research. However, the presence of viable C. parvum oocysts in water is a severe challenge to the drinking water treatment industry.

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

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. 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. 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. These studies suggest that SP-receptor antagonists could prove useful for treatment of AIDS-related cryptosporidiosis.

Artificial UV-B and Solar Radiation Reduce in Vitro Infectivity of the Human Pathogen Cryptosporidium parvum

The potential for solar ultraviolet (UV) radiation to act as a significant abiotic control of Cryptosporidium parvum oocysts in nature is unknown. Infectivity of C. parvum following exposure to artificial UV-B and natural solar radiation, with and without UV wavelengths, was tested under controlled pH and temperature conditions. Percent infectivity of exposed oocysts was determined by in vitro cell culture. Artificial UV-B exposures of 32 and 66 kJ/m2 significantly decreased oocyst infectivity by an average of 58 and 98%, respectively. Exposure of oocysts to approximately half and full intensity of full solar spectrum (all wavelengths) for a period of less than 1 day (10 h) in mid-summer reduced mean infectivity by an average of 67% and >99.99%, respectively. Exposure of the C. parvum oocysts to UV-shielded solar radiation (>404 nm) in early autumn reduced mean infectivity by 52%, while full spectrum solar radiation (exposure at all wavelengths) reduced mean infectivity by 97%. The data provide strong evidence that exposure to natural solar radiation can significantly reduce C. parvum infectivity. Direct effects of solar radiation on oocysts in nature will depend on the depth distribution of the oocysts, water transparency, mixing conditions, and perhaps other environmental factors such as temperature, pH, and stress.

Cryptosporidium parvum infection in cattle: are current perceptions accurate?

The perception that cattle are major reservoirs for Cryptosporidium parvum infections in humans and that C. parvum is a major cause of diarrhea and production loss in cattle might not reflect the whole situation. Numerous management factors influence the epidemiological and clinical picture associated with C. parvum infections in cattle. Whereas C. parvum is highly prevalent in young dairy calves and confined beef calves, it occurs rarely in calves on range and in adult cattle. In well-managed herds, clinical disease due to C. parvum is also rare. Therefore, C. parvum infections in cattle might not be as important as current perceptions would indicate.

A Cellular Micro-RNA, let-7i, Regulates Toll-like Receptor 4 Expression and Contributes to Cholangiocyte Immune Responses against Cryptosporidium parvum Infection

Toll-like receptors (TLRs) are important pathogen recognition molecules and are key to epithelial immune responses to microbial infection. However, the molecular mechanisms that regulate TLR expression in epithelia are obscure. Micro-RNAs play important roles in a wide range of biological events through post-transcriptional suppression of target mRNAs. Here we report that human biliary epithelial cells (cholangiocytes) express let-7 family members, micro-RNAs with complementarity to TLR4 mRNA. We found that let-7 regulates TLR4 expression via post-transcriptional suppression in cultured human cholangiocytes. Infection of cultured human cholangiocytes with Cryptosporidium parvum, a parasite that causes intestinal and biliary disease, results in decreased expression of primary let-7i and mature let-7 in a MyD88/NF-kappaB-dependent manner. The decreased let-7 expression is associated with C. parvum-induced up-regulation of TLR4 in infected cells. Moreover, experimentally induced suppression or forced expression of let-7i causes reciprocal alterations in C. parvum-induced TLR4 protein expression, and consequently, infection dynamics of C. parvum in vitro. These results indicate that let-7i regulates TLR4 expression in cholangiocytes and contributes to epithelial immune responses against C. parvum infection. Furthermore, the data raise the possibility that micro-RNA-mediated post-transcriptional pathways may be critical to host-cell regulatory responses to microbial infection in general.

Cryptosporidium parvum infection in orphan lambs on a farm open to the public

A longitudinal survey was undertaken on an open farm to investigate the occurrence of Cryptosporidium species infection in orphan lambs obtained from three local flocks. During an initial pilot study,...

CHARACTERIZATION OF SUBTILASE PROTEASE IN CRYPTOSPORIDIUM PARVUM AND C. HOMINIS

Cryptosporidium spp., enteropathogens of humans and other animals, are members of the Apicomplexa. In parasites belonging to this phylum, proteases have been shown to play a key role in the invasion of host cells, organelle biogenesis, and intracellular survival. The subtilases constitute a family of serine proteases present in prokaryotes, eukaryotes, and viruses. The C. parvum subtilase gene, CpSUB1, encodes a transcript of 3972 base pairs (bp) and 1324 amino acids. Using homologous polymerase chain reaction primers, a similar gene, ChSUB1, which has 98% (4007 bp/4050 bp) identity to CpSUB1, was found in C. hominis. The alignment of the CpSUB1 and ChSUB1 nucleotide sequences identified primarily silent substitutions, consistent with the absence of diversifying selection. The catalytic domain of CpSUB1 is very similar to that of other Apicomplexa (> 38% amino acid identity and >57% similarity) and to the bacterial subtilisin BPN from B. subtilis (36 and 47%). Transcriptional upregulation during merozoite development was observed in cell culture, and a predicted 76-bp intron located near the 3' end of the open reading frame was confirmed experimentally. Cryptosporidium parvum infection in cell culture was significantly inhibited by subtilisin inhibitor III and other serine protease inhibitors, emphasizing the importance of the parasite's subtilase for intracellular development and the enzyme's potential as a drug target.

(2 R,3 S)-(+)- and (2 S,3 R)-(−)-Halofuginone lactate: Synthesis, absolute configuration, and activity against Cryptosporidium parvum

The trans-enantiomers of the commercially important anti-protozoal compound Halofuginone have been prepared and characterized, and the absolute configuration was assigned by X-ray crystallography. The activity of both enantiomers against Cryptosporidium parvum was determined in vitro and related to acute toxicity in vivo. It was shown that both the activity and the toxicity are properties of the (2 R,3 S)-enantiomer. We conclude that with respect to broadening the therapeutic window there is no advantage in application of one enantiomer over the application of the racemic mixture in the treatment of C. parvum infections.

Induction of murine immune responses by DNA encoding a 23-kDa antigen of Cryptosporidium parvum

Cp23 has been identified as one of the immunodominant antigens involved in the immune response to Cryptosporidium parvum infection. Thus, in this study, Cp23 antigen was investigated as a vaccine candidate using the DNA vaccine model in adult interleukin-12 (IL-12) knockout (KO) mice, which are susceptible to C. parvum infection. Our data show that subcutaneous immunization in the ear with DNA encoding Cp23 (Cp23-DNA) cloned into the pUMVCb4 vector induced a significant anti-Cp23 immunoglobulin G1 (IgG1) and IgG2a antibody response and specific in vitro spleen cell proliferation to recombinant Cp23 as compared to control mice. Long-term memory responses were also detected after administration of the Cp23-DNA vaccine. Furthermore, Cp23-DNA vaccination induced a 50-60% reduction in oocysts shedding, indicating a partial protection against C. parvum infection in IL-12 KO mice. However, it is possible that this protective response was nonspecific because mice immunized with vector only also exhibited lower oocyst shedding than the naive controls. These results suggest that DNA encoding for immunodominant C. parvum antigens may provide an effective means of eliciting humoral and cellular responses and possibly in generating protective immunity against C. parvum infections in mammals.

Natural infection with zoonotic subtype of Cryptosporidium parvum in Capybara ( Hydrochoerus hydrochaeris) from Brazil

A total of 145 capybara ( Hydrochoerus hydrochaeris) fecal samples from the state of São Paulo, Brazil, were screened for Cryptosporidium spp. oocysts using the malachite green method. Eight samples (5.52%) showed positive results and were further submitted to nested PCR reaction for amplification of fragments of 18S rRNA gene and 60-kDa glycoprotein gene for determination of species, alleles and subtypes of Cryptosporidium. Sequencing of the PCR products of the 18S rRNA gene fragments and 60-kDa glycoprotein gene fragments showed that for both genes all Cryptosporidium isolates from capybara were respectively 100% genetically similar to a bovine isolate of C. parvum and to C. parvum subtype IIaA15G2R1. To the best of our knowledge this is the first report of Cryptosporidium infection in this rodent. The finding of zoonotic C. parvum infection in a semi-aquatic mammal that inhabits anthroponotic habitats raises the concern that human water supplies may be contaminated with zoonotic Cryptosporidium oocysts from wildlife.

Symptomatic and Asymptomatic Cryptosporidiosis in Young Children in Iran

The present study was conducted during the period May 2003 to October 2003 in 616 children less than three years of age with and without diarrheal disease presenting at the pediatric clinic of teaching hospitals in Kermanshah, Iran. Single stool specimens were collected from 515 diarrheic and 99 non-diarrheic children. Two smears were made from each stool samples and were stained by a modified Ziehl Neelsen technique. Cryptosporidium parvum (C. parvum) were detected in 10.4% of children. The C. parvum infection rate was significantly higher in diarrheic children (11.6%) than in non-diarrheic children (4.0%). C. parvum was observed more frequently in stool samples of children who lived in rural areas (15.2%) than those who lived in urban areas (7.2%). In regard to the presence of animals, the infection rate was 18.5% among children who lived in association with animals in comparison with 8.2% among those who lived in compounds with no animals. The majority of C. parvum cases occurred in children between the ages of 0-12 months (11.9%), followed by in children between the ages of 13-24 months (9.2%) and in children between the ages of 25-36 months (3.0%). The data suggest that C. parvum is relatively endemic in young children and that Cryptosporidium may be an important pathogen associated with diarrhea in young children.

PREVALENCE OF ENTERIC PROTOZOA IN HUMAN IMMUNODEFICIENCY VIRUS (HIV)–POSITIVE AND HIV-NEGATIVE MEN WHO HAVE SEX WITH MEN FROM SYDNEY, AUSTRALIA

A prospective, comparative study of the prevalence of enteric protozoa was determined among human immunodeficiency virus (HIV)- positive and HIV-negative men who have sex with men (MSM) in Sydney, Australia. A total of 1,868 patients submitted stool specimens; 1,246 were from MSM (628 HIV positive and 618 HIV positive) and 622 from non-MSM were examined over a 36-month period. A total of 651 (52.2%) stool specimens from MSM were positive for protozoa compared with 85 (13%) from non-MSM. There was a significant difference in the prevalence of Blastocystis hominis, Endolimax nana, Entamoeba histolytica/dispar complex, Entamoeba hartmanni, Iodamoeba butschlii, and Enteromonas hominis detected between MSM and non-MSM (P<0.001). The only notable difference between HIV-negative and HIV-positive MSM was that HIV-infected MSM were found to more likely have a Cryptosporidium parvum infection. Entamoeba histolytica was found in 3 patients, E. dispar in 25, and E. moshkovskii in 17, all of whom were MSM. When compared with a control group, MSM were significantly more likely to harbor intestinal protozoa and have multiple parasites present. The results of this study show high rates of enteric parasites persist in MSM and highlight the importance of testing for intestinal parasites in MSM. This is the first report of E. moshkovskii from MSM.

Involvement of intestinal epithelial cells in dendritic cell recruitment during C. parvum infection

Dendritic cells (DCs) play a key role in activating and orientating immune responses. Little is currently known about DC recruitment during Cryptosporidium parvum infection. In the intestine, epithelial cells act as sensors, providing the first signals in response to infection by enteric pathogens. We analyzed the contribution of these cells to the recruitment of DCs during cryptosporidiosis. We found that intestinal epithelial cells produced a broad range of DC-attracting chemokines in vitro in response to C. parvum infection. The supernatant of the infected cells induced the migration of both bone marrow-derived DCs (BMDC) and the SRDC lymphoid dendritic cell line. Chemokine neutralization abolished DC migration in these assays. We next analyzed chemokine mRNA expression in the mucosa of C. parvum-infected neonatal mice and recruitment of the various subsets of DCs. Myeloid (CD11c+ CD11b+) and double-negative DCs (CD11c+ CD11b− CD8α−) were the main subsets recruited in the ileum during C. parvum infection, via a mechanism involving IFNγ. DCs were also recruited and activated in the draining lymph nodes during C. parvum infection, as shown by the upregulation of expression of MHC II and of the costimulation molecules CD40 and CD86.

Comparative genome analysis of Ureaplasma parvum clinical isolates

Ureaplasma parvum colonizes human mucosal surfaces, primarily in the respiratory and urogenital tracts, causing a wide spectrum of diseases, from non-gonococcal urethritis to pneumonitis in immunocompromised hosts. Although the basis for these diverse clinical outcomes is not yet understood, more severe disease may be associated with strains harboring a certain set of strain-specific genes. To investigate this, whole genome DNA macroarrays were constructed and used to assess genomic diversity in 10 U. parvum clinical strains. We found that 7.6% of U. parvum genes were dispersed into one or more strains, thus defining a minimal functional core of 538 U. parvum genes. Most of the strain-specific genes (79%) were of unknown function and were unique to U. parvum. Four hypervariable plasticity regions were identified in the genome containing 93% of the variability in the gene pool (UU32–UU33, UU145–UU170, UU440–UU447 and UU527–UU529). We hypothesized that one of them (UU145–UU170) was a pathogenicity island in U. parvum and we characterized it. Thus, we propose that the clinical outcome of U. parvum infection is probably associated with this newly identified pathogenicity island.

Giardia and Cryptosporidium in water buffaloes (Bubalus bubalis)

A cross-sectional survey of Giardia duodenalis and Cryptosporidium parvum infection in the water buffalo (Bubalus bubalis) was carried out in central Italy. A geographical information system (GIS) was constructed utilizing as data-layers the topographic base map and the digital aerial photographs of the study area, as well as the geo-referenced points of all the buffalo farms. The survey was conducted on a sample of 90 farms, selected using a grid approach followed by proportional allocation. For this purpose, a grid representing quadrants of 5 x 5 km was overlaid on the study area within the GIS. As a result, the study area was divided in equal quadrants, and the number of farms sampled in each quadrant was proportional to the total number of study population in that quadrant. On each farm, faecal samples were collected per rectum from three to five asymptomatic buffalo calves, aged from 1 to 9 weeks. The total number of faecal samples collected was 347. Each faecal sample was tested for the presence of copro-antigens of G. duodenalis and of C. parvum using two commercially available enzyme-linked immunosorbent assays. Out of the 90 farms, 27 (30.0%) resulted positive for G. duodenalis and 22 (24.4%) for C. parvum. Co-infection was found in ten (11.1%) farms. With respect to animals, out of the 347 faecal samples, 63 (18.1%) were found to have antigens of G. duodenalis and 51 (14.7%) of C. parvum. Co-infection was found in ten buffalo calves (2.9%). The results of the logistic regression models showed a positive association between the positivity to G. duodenalis and the presence of sheep on farm and between the positivity to C. parvum and the high number of buffaloes on farms. No significant co-infection between the two protozoa was found. In conclusion, the findings of the present study, derived from a systematic territorial survey planned with GIS, are noteworthy because they provided additional data on C. parvum and the first evidence of G. duodenalis infection in water buffaloes.

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

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

An updating on Cryptosporidium parvum in the water buffalo

A cross-sectional survey of Cryptosporidium parvum infection in the water buffalo was carried out in central Italy. The survey was carried out on a sample of 90 farms, selected using a grid approach within a Geographical Information System, followed by proportional allocation. On each farm, faecal samples were collected from three to five asymptomatic buffalo calves, aged from 1 to 9 weeks (total number = 347). Each sample was tested for the presence of copro-antigens of C. parvum using a commercially available ELISA. Out of the 90 farms, 22 (24.4%) resulted positive. With respect to animals, out of the 347 faecal samples, 51 (14.7%) were found to have antigens of C. parvum. The results of the logistic regression model showed a positive association between the positivity to C. parvum and the high number of buffaloes on farms.

QUANTITATIVE TRACKING OF CRYPTOSPORIDIUM INFECTION IN CELL CULTURE WITH CFSE

Immunofluorescence-based assays have been developed to detect and quantitate Cryptosporidium parvum infection in cell culture. Here, we describe a method that tracks and quantifies the early phase of attachment and invasion of C. parvum sporozoites using a fluorescent dye. Newly excysted sporozoites were labeled with the amine-reactive fluorescein probe carboxyfluorescein diacetate succinimidyl esters (CFSE) using an optimized protocol. The initial invasion of cells by labeled parasites was detected with fluorescent or confocal microscopy. The infection of cells was quantified by flow cytometry. Comparative analysis of infection of cells with CFSE-labeled and unlabeled sporozoites showed that the infectivity of C. parvum was not affected by CFSE labeling. Quantitative analysis showed that C. parvum Iowa and MD isolates were considerably more invasive than Cryptosporidium hominis isolate TU502. Unlike immunofluorescent assays, CFSE labeling permitted the tracking of the initial invasion of C. parvum. Such an assay may be useful for studying the dynamics of host cell-parasite interaction and possibly for drug screening.

Molecular evidence of Ureaplasma urealyticum and Ureaplasma parvum colonization in preterm infants during respiratory distress syndrome.

BackgroundUreaplasma urealyticum and U. parvum have been associated with respiratory diseases in premature newborns, but their role in the pathogenesis of the respiratory distress syndrome (RDS) is unclear. The aim of this study was to detect, using molecular techniques, the role of Mycoplasma spp. and Ureaplasma spp. in respiratory secretion and blood specimens of preterm newborns with or without RDS and to evaluate the prevalence of perinatal U. urealyticum or U. parvum infection. The influence of chemotherapy on the clinical course was also evaluated.MethodsTracheal aspirate or nasopharingeal fluid samples from 50 preterm babies with (24) or without RDS (26) were analysed for detection of U. urealyticum and U. parvum by culture identification assay and PCR. Sequencing analysis of amplicons allowed us to verify the specificity of methods. Clarithromycin (10 mg kg-1 twice a day) was administered in ureaplasma-positive patients who presented clinical signs of RDS.Results15/24 neonates with RDS (p < 0.001) and 4/26 without RDS were found PCR-positive for U. urealyticum or U. parvum. Culture identification assay was positive in 5/50 newborns, three of which with RDS. Sequencing analyses confirmed the specificity of these methods. Association of patent ductus arteriosus with ureaplasma colonization was more statistically significant (p = 0.0004) in patients with RDS than in those without RDS.ConclusionColonization of the lower respiratory tract by Ureaplasma spp. and particularly by U. parvum in preterm newborns was related to RDS. The routine use of molecular methods could be useful to screen candidate babies for etiologic therapy.

The suppressive effect of Mekabu fucoidan on an attachment of Cryptosporidium parvum oocysts to the intestinal epithelial cells in neonatal mice

The present study was done to investigate the effects of fucoidan and de-sulfated fucoidan isolated from the sporophyll of Undaria pinnatifida on the C. parvum adhesion to the cultured human intestinal cells and on the C. parvum infection in neonatal mice. The C. parvum adhesion to human Intestinal 407 cells was significantly suppressed by a low dose (1 μg/ml) of Mekabu fucoidan (1 μg/ml) (approx. 20.5 oocysts, p < 0.0001), but not by de-sulfated fucoidan (approx. 138.2 oocysts), as compared with that (approx. 121.0 oocysts) of phosphate-buffered saline (PBS). The in vivo experiments presented here revealed that C. parvum oocysts in the fucoidan-treated mice was reduced nearly one fifth (approx. 5.4 × 10 4 oocysts, p < 0.02) of the total number of oocysts (approx. 3.0 × 10 5) in mice treated with PBS, but no significant effect of de-sulfated fucoidan was observed. These results show that (i) fucoidan effectively inhibits the growth of C. parvum in mice; and (ii) the ester sulfate of fucoidan is an active site to prevent the adhesion of C. parvum to the intestinal epithelial cells. Finally, we concluded that fucoidan might inhibit cryptosporidiosis through the direct binding of fucoidan to the C. parvum-derived functional mediators in the intestinal epithelial cells in neonatal mice.