Detection of Cryptosporidium oocysts in wild mammals of mainland Britain

This paper combines the results from a preliminary survey of occurrence of Cryptosporidium species in faecal samples from a range of wild mammal species inhabiting mainland Britain with a tabulated literature review of world-wide reports of the parasite in those British mammals. In the literature, C. parvum was reported from 11 wild mammals found in Britain and elsewhere, mainly in rodents but also in insectivores, lagomorphs and ungulates. C. muris has been reported only in wild rodents. The sample survey detected C. parvum in seven additional British species, including carnivores. Overall, 12% of 184 faecal samples tested with a genus-specific monoclonal antibody contained oocysts of C. parvum. The results further emphasise the widespread distribution of Cryptosporidium amongst wild mammals in Britain, highlight the potential for transmission between host species and warn of the possibility of direct exposure for anybody using the countryside for professional or recreational purposes (e.g. farmers and ramblers) to previously unregarded sources of infection. It seems increasingly likely that most, if not all, mammalian species can be infected with C. parvum.     

Detection of vancomycin-resistant enterococci in fecal samples by PCR

Surveillance cultures for vancomycin-resistant enterococci (VRE) are time-consuming and expensive for the laboratory to perform. Therefore, we investigated the use of PCR as an alternative method of detecting and identifying VRE directly in fecal samples. PCR primers directed to vanA, vanB, vanC1, vanC2, and enterococcal ligase genes were used to detect and identify VRE in fecal material obtained by rectal or perirectal swabbing. Although PCR-inhibitory substances were present in DNA prepared directly from the swabs, the inhibitory substances could be reduced by processing the nucleic acid with two commercially available DNA preparation columns. Fecal material from 333 swabs was cultured on several selective agar media before and after broth enrichment. DNA was extracted from the fecal material and was analyzed by PCR. By using all four primer sets, only 59 (67.8%) of the samples were positive for vanA. However, after retesting the negative samples with only the vanA primer set, 77 (88.5%) of 87 specimens that were culture positive for Enterococcus faecium containing vanA were positive by PCR. One specimen was PCR positive for the vanA gene but culture negative for enterococci. The specificity of the vanA assay was 99.6%. PCR analysis of enrichment broth samples with all four primers sets after 15 to 18 h of incubation detected 74 (85.1%) of the 87 culture-positive specimens. The specificity of the vanA assay after the enrichment step was 100%. No vanB-containing enterococci were recovered by culture. Since 16 samples can be tested by PCR in 4 h (including electrophoresis), identification of VRE is possible within 8 h of specimen submission at a cost of approximately $10.12/assay. Thus, PCR may be a cost-effective alternative to culture for surveillance of VRE in some hospitals.     

Immunomagnetic capture PCR to detect viable Cryptosporidium parvum oocysts from environmental samples

A method to detect viable Cryptosporidium parvum oocysts was developed. Polyclonal immunoglobulin G against C. parvum oocyst and sporozoite surface antigens was purified from rabbit immune serum, biotinylated, and bound to streptoavidin-coated magnetic particles. C. parvum oocysts were captured by a specific antigen-antibody reaction and magnetic separation. The oocysts were then induced to excyst, and DNA was extracted by heating at 95 degrees C for 10 min. A 452-bp fragment of C. parvum DNA was amplified by using a pair of C. parvum-specific primers in PCR. The method detected as few as 10 oocysts in purified preparations and from 30 to 100 oocysts inoculated in fecal samples. The immunomagnetic capture PCR (IC-PCR) product was identified and characterized by a nested PCR that amplified a 210-bp fragment, followed by restriction endonuclease digestion of the IC-PCR and nested-PCR products at the StyI site and a nonradioactive hybridization using an internal oligonucleotide probe labeled with biotin. PCR specificity was also tested, by using DNAs from other organisms as templates. In the control experiments, inactivated oocysts were undetectable, indicating the ability of this method to differentiate between viable and nonviable oocysts. Thus, this system can be used to specifically detect viable C. parvum oocysts in environmental samples with great sensitivity, providing an efficient way to monitor the environment for C. parvum contamination.     

Detection of Cryptosporidium parvum in horses: thresholds of acid-fast stain, immunofluorescence assay, and flow cytometry

Feces collected from three asymptomatic horses and seeded with Cryptosporidium parvum oocysts (10(1) to 10(6)/g of feces) were evaluated by acid-fast staining (AF), an immunofluorescent antibody (IFA) technique, and flow cytometry. The thresholds of detection were 5 x 10(5) oocysts/g of feces for the IFA and AF techniques and 5 x 10(4) oocysts/g for flow cytometry.     

Method detection limits of PCR and immunofluorescence assay for Cryptosporidium parvum in soil

We determined and compared the method detection limits (MDLalpha) of a PCR and an immunofluorescence assay (IFA) for detection of Cryptosporidium parvum oocysts in soils. Based on the MDLalpha and the quantitative nature and stability of the IFA, PCR analysis is not a useful screening step for soil studies of oocyst transport.     

Infectivity of Cryptosporidium parvum oocysts stored in water at environmental temperatures

We showed that the efficacy of the new 2'-deoxycytidine (2'-dCyd) analogue antimetabolite 2'-deoxy-2'-methylidenecytidine (DMDC) correlates well with tumor levels of cytidine (Cyd) deaminase in human cancer xenograft models. DMDC was highly effective in tumors with higher levels of Cyd deaminase, whereas lower levels yielded only slight activity. In contrast, gemcitabine (2',2'-difluorodeoxycytidine), which has action mechanisms similar to those of DMDC, is only slightly active in tumors with higher levels of the enzyme. In the present study, we investigated the roles of Cyd deaminase in the antitumor activity of the two 2'-dCyd antimetabolites in 13 human cancer cell lines. Tetrahydrouridine, an inhibitor of Cyd deaminase, reduced the antiproliferative activity of DMDC (P = 0.0015). Furthermore, tumor cells transfected with the gene of human Cyd deaminase become more susceptible to DMDC both in vitro and in vivo. These results indicate that Cyd deaminase is indeed essential for the activity of DMDC. In contrast, the antiproliferative activity of gemcitabine was increased to some extent by tetrahydrouridine (P = 0.0277), particularly in tumor cell lines with higher levels of Cyd deaminase. This suggests that higher levels of Cyd deaminase may inactivate gemcitabine. Among nucleosides and deoxynucleosides tested, only dCyd, a natural substrate of both Cyd deaminase and dCyd kinase, suppressed the antiproliferative activity of DMDC by up to 150-fold. Because the Vmax/Km of DMDC for dCyd kinase was 8-fold lower than that for dCyd, the activation of DMDC to DMDC monophosphate (DMDCMP) by dCyd kinase might be competitively inhibited by dCyd. In addition, the dCyd concentrations in human cancer xenografts were inversely correlated with levels of Cyd deaminase activity. It is therefore suggested that higher levels of Cyd deaminase reduce the intrinsic cellular concentrations of dCyd in tumors, resulting in efficient activation of DMDC to DMDCMP by dCyd kinase. These results indicate that the efficacy of DMDC may be predicted by measuring the activity of Cyd deaminase in tumor tissues before treatment starts and that DMDC may be exploited in a new treatment modality: tumor enzyme-driven cancer chemotherapy.     

Fuchsin fluorescence and autofluorescence in Cryptosporidium, Isospora and Cyclospora oocysts

Cryptosporidium parvum and Isospora belli oocysts stained with carbol-fuchsin, as in a modified Ziehl Neelsen technique, fluoresce bright red under green light (546nm). Cryptosporidium oocysts tend to fluoresce more brightly the less intensely stained they appear under transmitted light; this is not the case with Isospora. Fuchsin-stained Cyclospora cayetanensis oocysts fluoresce rather dimly, but those not taking the dye retain their typical autofluorescence. Cryptosporidium and Isospora oocysts are also autofluorescent, appearing violet under u.v. light (365 nm), and green under violet (405 nm) and blue-violet light (436 nm). Their autofluorescence does not survive the staining procedure.     

Effect of pasteurization on infectivity of Cryptosporidium parvum oocysts in water and milk

Cryptosporidium parvum is a major cause of diarrheal disease in humans and has been identified in 78 other species of mammals. The oocyst stage, excreted in feces of infected humans and animals, has been responsible for recent waterborne outbreaks of human cryptosporidiosis. High temperature and long exposure time have been shown to render oocysts (suspended in water) noninfectious, but for practical purposes, it is important to know if high-temperature--short-time conditions (71.7 degrees C for 15 s) used in commercial pasteurization are sufficient to destroy infectivity of oocysts. In this study, oocysts were suspended in either water or whole milk and heated to 71.7 degrees C for 15, 10, or 5 s in a laboratory-scale pasteurizer. Pasteurized and nonpasteurized (control) oocysts were then tested for the ability to infect infant mice. No mice (0 of 177) given 10(5) oocysts pasteurized for 15, 10, or 5 s in either water or milk were found to be infected with C. parvum on the basis of histologic examination of the terminal ileum. In contrast, all (80 of 80) control mice given nonpasteurized oocysts were heavily infected. These data indicate that high-temperature--short-time pasteurization is sufficient to destroy the infectivity of C. parvum oocysts in water and milk.     

Evaluation of a direct immunofluorescence cytospin assay for the detection of herpes simplex virus in clinical samples

A comparison between a direct immunofluorescence assay (DFA) and the shell-vial culture (SVC) was conducted to evaluate their efficacies according to the quality and origin of the sample and the type of herpes simplex (HSV) responsible for the infection. The SVC detected all 58 HSV-infected samples, while the DFA detected only 49 (84.5%) positive samples. The DFA detected HSV type 1 in 22 of 89 samples (24.7%) and HSV type 2 in 27 of 96 samples (28.1%). Compared with the SVC, the DFA had a sensitivity of 75.8% for HSV type 1 and 93.1% for HSV type 2. The sensitivity of the DFA depends on the quality of the sample. Thus, while DFA is recommendable as a screening method, the SVC remains the method of choice for obtaining the maximum diagnostic yield from the sample.     

Detection of Cryptosporidium oocysts and Giardia cysts in the tissues of eastern oysters (Crassostrea virginica) carrying principal oyster infectious diseases

The potential cross-reactivity of the combined Cryptosporidium/Giardia direct immunofluorescence antibodies (IFA) of MERIFLUOR and HYDROFLUOR-COMBO tests was examined against tissues containing known developmental stages of 12 pathogens causing the principal infectious diseases in oysters. Spores of Haplosporidium nelsoni and Haplosporidium costale produced positive acid-fast stain (AFS) reactions similar in intensity to Cryptosporidium parvum oocysts. Hexamia nelsoni trophozoites produced positive IFA reactions in both IFA tests; however, the intensity of fluorescence was considerably lower and the fluorescein-staining pattern different than those of Giardia cysts. The applicability of AFS for screening oysters for Cryptosporidium oocysts is low, and positive identification of Cryptosporidium oocysts cannot be accomplished based on the AFS. Presumptive IFA identification of the Cryptosporidium oocysts or Giardia cysts in the oyster tissue should fulfill 3 criteria, i.e., bright-green fluorescence of the same intensity as C. parvum oocysts and Giardia cysts in the positive control, correct size and shape of the fluorescein-stained objects, and oocyst or cyst shell clearly visible.     

Detection of Helicobacter pylori in fecal samples of gnotobiotic mice infected with H. pylori by an immunomagnetic-bead separation technique

By an immunomagnetic-bead (IMB) separation technique, isolation of Helicobacter pylori from gastrointestinal and fecal samples of gnotobiotic mice infected with the microorganism was tried. The isolation rate of H. pylori from stomach samples after IMB separation was not higher than that of direct culture of the samples. After IMB separation of feces, H. pylori was detectable by PCR, although H. pylori was not culturable.     

Antagonistic effect of human alpha-1-antitrypsin on excystation of Cryptosporidium parvum oocysts

This study evaluated the effects of the human serine protease inhibitor alpha-1-antitrypsin (AAT) on in vitro excystation and infectivity of Cryptosporidium parvum. Excystation was monitored at 37 C in RPMI medium in the presence of 0, 100, 500, or 1,000 micrograms/ml AAT. AAT significantly inhibited (P < 0.05) excystation of bleach-decontaminated oocysts in a concentration-dependent manner at incubation intervals from 15 to 90 min but did not alter the excystation dynamics of unbleached oocysts. Bleach-treated oocysts, suspended in RPMI containing 0, 1, 10, 100, 500, or 1,000 micrograms/ml AAT, were used to inoculate bovine fallopian tube epithelial (BFTE) cell monolayers. Alternately, sporozoites, excysted at 37 degrees C and collected by filtration, were used to inoculate BFTE cells under the same conditions. The mean number of parasites counted in AAT-treated, oocyst-inoculated cells was significantly less (P < 0.01) than control mean values at 24 and 48 hr post-inoculation (PI); longer PI intervals (72-96 hr) exhibited a decreased inhibitory effect. AAT did not inhibit parasite infection when cultures were inoculated with C. parvum sporozoites. The findings of this study show that the anticryptosporidial potential of AAT is primarily associated with an antagonistic effect on oocyst excystation.     

The occurrence of Prototheca in fecal samples of cattle

Prototheca spp. were detected in 146 (48.7%) of culturally examined fecal samples of cattle, using the selective medium, developed by Pore (1973). 114 fecal samples (78.1%) contained a monoculture of Prototheca (P.) zopfii, while 10 fecal samples (6.8%) contained a monoculture of P. moriformis. In further 22 fecal samples (15.1%) a mixed culture of P. zopfii and P. moriformis was detected. The results of this study permit to conclude that cattle can harbour and shed Prototheca spp. in variable frequency.