Comparison of oocyst shedding and the serum immune response to Cryptosporidium parvum in cattle and pigs

 A comparison was made between oocyst shedding and the presence of specific serum IgG antibodies to Cryptosporidium parvum in 108 bovines and 90 pigs. Oocysts were detected by a commercial immunofluorescence assay in feces from 26.8% of bovines and 34.4% of pigs, whereas positive titers as determined by an indirect fluorescent antibody method were found in sera from 12.9% and 48.9% of the respective animals. Infection was significantly most frequent in suckling calves (82.7%) and weaned piglets (87.5%). By contrast, the numbers of seropositives were highest in weaned calves (17.1%) and fattening pigs (76.6%). The results of coprological and serological analysis corresponded in 65.7% of bovines and 56.7% of pigs. When used to diagnose the shedding of cryptosporidial oocysts, the detection of specific IgG antibodies had a sensitivity ranging from 10.3% (cattle) to 58.1% (pigs) and a specificity of 86.1% (cattle) and 55.9% (pigs). Received: 07 December 1995 / Accepted: 16 January 1996     

CP2 gene as a useful viability marker for Cryptosporidium parvum

The validity of the CP2 gene of Cryptosporidium parvum as a viability marker was evaluated using absolute quantitative real-time polymerase chain reaction (qPCR) assays. Total ribonucleic acid (RNA) was isolated from live and heat-killed C. parvum oocysts, and complementary deoxyribonucleic acid was synthesized and used as a template. The most accurate number of viable C. parvum oocysts was predicted when the CP2 gene was used as a target gene. The lower detection limit of the CP2 gene was ten oocysts, which was the most sensitive among examined target genes. With heat shock induction, only hsp70 messenger RNA (mRNA) was induced, and the predicted viable oocyst number was increased by heat shock for this marker. The CP2, hsp70, Cryptosporidium oocyst wall protein, and β-tubulin mRNAs were not detected in heat-killed oocysts, but the 18S ribosomal ribonucleic acid (rRNA) showed heat stability until 48 h after heat killing. Although the 18S rRNA demonstrated the fastest response in crossing point (CP) value among the examined primer sets in qPCR, overestimation of viable oocysts was noted in the analysis with this gene. In conclusion, the CP2 gene was identified as the most sensitive, reliable, and accurate candidate of a viability marker of C. parvum by qPCR evaluation.     

Arginine-derived nitric oxide reduces fecal oocyst shedding in nude mice infected with Cryptosporidium parvum.

Dietary L-arginine (4%) significantly reduced fecal oocyst shedding in athymic nude mice chronically infected with Cryptosporidium parvum. This effect appeared to be due to an increase in host nitric oxide (NO) production as it was not observed in arginine-supplemented animals administered the NO synthase inhibitor, N-nitro-L-arginine methyl ester. N-Nitro-L-arginine methyl ester alone significantly increased fecal oocyst shedding in chronically infected animals. In in vitro assays, oocyst excystation and sporozoite viability were significantly reduced by the NO donors sodium nitroprusside and S-nitroso-L-acetyl penicillamine in a concentration-dependent manner. These data suggest that arginine-derived NO may reduce the parasite load in experimental cryptosporidiosis.     

The terminal sialic acid of glycoconjugates on the surface of intestinal epithelial cells activates excystation of Cryptosporidium parvum

The apicomplexan Cryptosporidium parvum reproduces in the intestinal epithelial cells of many mammalian species and is an agent of the important diarrheal disease cryptosporidiosis. Infection is transmitted fecal-orally by oocysts that pass through the stomach and excystation occurs in the intestine, releasing four invasive sporozoites. Some factors involved in inducing excystation have been identified, but the role of the enterocyte is not known. The present study showed that excystation was accelerated in the presence of the three enterocyte cell lines Caco2, HCT8, and CMT93. Epithelial cell lines derived from other organs, including the stomach, had no effect on excystation. No evidence was obtained that factors secreted from enterocytes induced excystation, but an enterocyte membrane preparation promoted sporozoite release. In addition, modification of the enterocyte surface by trypsin digestion or paraformaldehyde fixation abrogated the ability to enhance excystation. Importantly, the level of excystation in the presence of enterocytes decreased after treatment with either sialidase/neuraminidase to deplete surface terminal sialic acid or with lectins that specifically bind to sialic acid. Furthermore, the addition of sialic acid to oocysts in the absence of cells increased the level of excystation. These results suggest that sialic acid on the surface of enterocytes may provide an important local signal for the excystation of C. parvum sporozoites.     

Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae) oocyst and sporozoite antigens recognized by bovine colostral antibodies.

Colostral whey from seven hyperimmunized and two control cows (hyperimmune bovine colostrum) was examined by Western immunoblotting for the presence of antibody against oocysts and sporozoites of Cryptosporidium parvum, using rabbit anti-bovine immunoglobulin A (IgA), IgG1, IgG2, and IgM antibodies, followed by a horseradish peroxidase goat anti-rabbit polyvalent antibody. Although considerable variation was found in binding activity between cows on different immunization protocols, IgA and IgG1 in whey recognized a greater variety of C. parvum antigens than did IgG2 and IgM. A band at 9 to 10 kilodaltons appeared unique in that it was recognized only by IgA.     

Differential regulation of beta-defensin gene expression during Cryptosporidium parvum infection

Invasion of enterocytes by pathogenic microbes evokes both innate and adaptive immune responses, and microbial pathogens have developed strategies to overcome the initial host immune defense. beta-Defensins are potentially important endogenous antibiotic-like effectors of innate immunity expressed by intestinal epithelia. In this study, the interplay between the enteric protozoan parasite Cryptosporidium parvum and host epithelial beta-defensin expression was investigated. Using human and murine models of infection, we demonstrated that C. parvum infection differentially regulates beta-defensin gene expression. Downregulation of murine beta-defensin-1 mRNA and protein was observed in both in vitro and in vivo models of infection. Infection of the human colonic HT29 cell line with the parasite resulted in differential effects on various members of the defensin gene family. Partial reduction in human beta-defensin-1 (hBD-1), induction of hBD-2, and no effect on hBD-3 gene expression was observed. Recombinant hBD-1 and hBD-2 peptides exhibited significant antimicrobial activity against C. parvum sporozoites in vitro. These findings demonstrate that C. parvum infection of enterocytes may affect the expression of various defensins in different ways and suggest that the overall outcome of the effect of antimicrobial peptides on early survival of the parasite may be complex.     

Effect of three concentration techniques on viability of Cryptosporidium parvum oocysts recovered from bovine feces.

Bovine fecal samples (1 g) negative for Cryptosporidium sp. oocysts were seeded with 7 x 10(4) Cryptosporidium parvum oocysts and purified by either water-ether concentration, sucrose density flotation, or zinc sulfate flotation to evaluate oocyst recovery. The effect of these purification techniques on the viability of recovered oocysts was also evaluated. Significantly higher numbers of seeded oocysts were recovered by water-ether concentration (recovery rate, 46 to 75%) than by sucrose density (24 to 65%) or zinc sulfate (22 to 41%) flotation methods. In addition, water-ether concentration did not exert a significant effect on the viability of the population of oocysts recovered, whereas sucrose density flotation and zinc sulfate flotation selectively concentrated viable oocysts. The water-ether concentration procedure is recommended for use in epidemiological studies in which both oocyst enumeration and viability assessment are required.     

Characterization of the Cryptosporidium antigens from sporulated oocysts of Cryptosporidium parvum.

The antigenic constituents of sporulated Cryptosporidium parvum oocyst antigens were characterized with antisera from mice immunized against C. parvum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining defined the major proteins. Six of seven lectins used recognized as many as 15 bands. The lectins concanavalin A, Dolichos biflorus, and wheat germ agglutinin showed strong activity against the same eight bands with molecular weights ranging from 72,000 to greater than 100,000. An enzyme-linked immunosorbent assay was used to detect antibody to C. parvum. Antibody binding was significantly decreased by heat and enzymatic treatment with trypsin, protease, and mixed glycosidases. C. parvum antigens were further defined by the reactivity of immune sera with a C. parvum sonicate preparation separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose paper. Antisera from orally infected mice consistently recognized four antigens with molecular weights ranging from 72,000 to greater than 100,000. These antigens also bound concanavalin A. Treatment of the antigen preparation with mixed glycosidases reduced the reactivity of antisera with most antigens with molecular weights greater than 60,000. The data suggest that the antigenic composition of C. parvum is complex and that carbohydrates alone or in association with lipids or proteins may be important in the immune response to C. parvum.     

Using flow cytometry to determine the viability of Cryptosporidium parvum oocysts extracted from spiked environmental samples in chambers

Cryptosporidium parvum oocyst viability was determined by a dye permeability assay using a flow cytometric method. Oocysts were inoculated into small chambers with soil and biosolids. Oocysts extracted from soil and biosolids were then stained with propidium iodide (PI) and labeled with a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody. The oocyst population in each sample was determined using forward and side scatter plots, then further analyzed with fluorescence. A red and green fluorescence detector using gates established single populations of unstained, PI-stained, or FITC-labeled oocysts. No statistical difference was observed between viability of oocysts extracted from soil and biosolids as determined by either flow cytometry or microscopy. The location of excysted oocysts was changed in forward and side scatter plots. Results indicated that, although oocysts are not identified if they excyst, the flow cytometric method could be used to determine oocyst viability from spiked environmental samples.     

A Random Survey of the Cryptosporidium parvum Genome

Cryptosporidium parvum is an obligate intracellular pathogen responsible for widespread infections in humans and animals. The inability to obtain purified samples of this organism's various developmental stages has limited the understanding of the biochemical mechanisms important for C. parvum development or host-parasite interaction. To identify C. parvum genes independent of their developmental expression, a random sequence analysis of the 10.4-megabase genome of C. parvum was undertaken. Total genomic DNA was sheared by nebulization, and fragments between 800 and 1,500 bp were gel purified and cloned into a plasmid vector. A total of 442 clones were randomly selected and subjected to automated sequencing by using one or two primers flanking the cloning site. In this way, 654 genomic survey sequences (GSSs) were generated, corresponding to >320 kb of genomic sequence. These sequences were assembled into 408 contigs containing >250 kb of unique sequence, representing approximately 2.5% of the C. parvum genome. Comparison of the GSSs with sequences in the public DNA and protein databases revealed that 107 contigs (26%) displayed similarity to previously identified proteins and rRNA and tRNA genes. These included putative genes involved in the glycolytic pathway, DNA, RNA, and protein metabolism, and signal transduction pathways. The repetitive sequence elements identified included a telomere-like sequence containing hexamer repeats, 57 microsatellite-like elements composed of dinucleotide or trinucleotide repeats, and a direct repeat sequence. This study demonstrates that large-scale genomic sequencing is an efficient approach to analyze the organizational characteristics and information content of the C. parvum genome.     

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.     

Characterization of bovine cellular and serum antibody responses during infection by Cryptosporidium parvum.

Cellular and serum antibody responses of calves were monitored for 23 days after oral inoculation of the calves with oocysts of Cryptosporidium parvum. In vitro blastogenic responses of peripheral blood lymphocytes were assessed after stimulation with a C. parvum preparation. Specific lymphocyte blastogenic responses to the parasite were detected 2 days after inoculation. Parasite-specific antibody titers were demonstrable 7 days after inoculation with oocysts and achieved peak levels 9 days after inoculation, coinciding with oocyst shedding at 5 to 10 days after inoculation. Both lymphocyte and antibody responses remained elevated until the termination of the experiment. Immunoblotting the C. parvum preparation with serum from an infected calf revealed six major parasite antigens. Five of these antigens reacted on immunoblots from 7 to 14 days after inoculation with oocysts. A parasite antigen of approximately 11,000 molecular weight demonstrated intense reactivity on immunoblots from 7 to 23 days after inoculation. The 11,000-molecular-weight antigen also reacted on immunoblots with parenterally raised antioocyst and antisporozoite rabbit sera. These results indicate that cell-mediated as well as humoral immune responses are initiated by cryptosporidial infection in calves and that the 11,000-molecular-weight parasite antigen is immunodominant.     

Fine structure of the oocyst wall and excystation ofEimeria procyonis from the American raccoon (Procyon lotor)

Oocysts ofEimeria procyonis, from the American raccoon (Procyon lotor), were broken, added to a cell suspension, fixed in Karnovsky's fluid, and studied with the electron microscope. The oocyst wall has three layers: a thin electron-dense inner layer (8–15 nm), an electron-lucent middle layer (25–35 nm), and a thick outer layer (120–140 nm). The outer layer has an electron-dense inner portion and an electron-lucent outer portion that contains membrane-bound vesicles. When exposed to a trypsin-sodium taurocholate fluid, sporozoites excysted from most sporocysts which were 35–43 months old, but not from sporocysts that looked normal and were 106 months old. Excysted sporozoites measured 13–16×3–4 (mean 14.3×3.2) m, usually had two refractile bodies, and had a nucleus with a prominent nucleolus.     

应用巢式PCR方法诊断微小隐孢子虫感染的实验研究

目的应用巢式PCR扩增方法诊断微小隐孢子虫感染。方法用昆明种小鼠通过免疫抑制方法建立动物模型，通过不连续蔗糖密度梯度离心法分离、纯化隐孢子虫卵囊。抽提DNA后，用巢式PCR扩增隐孢子虫的18S核糖体DNA，电泳、割胶回收后测序。结果成功建立隐孢子虫的小鼠免疫抑制动物模型，纯化后的隐孢子虫卵囊形状均一，呈圆形或椭圆形，大小在3-6um左右。经巢式PCR扩增，在840bp左右有一条特异性条带。通过测序和生物信息学分析，证实该基因序列与微小隐孢子虫18S具有高度同源性。结论巢式PCR方法扩增18S基因是诊断微小隐孢子虫感染的敏感方法。