[show abstract][hide abstract] ABSTRACT: A cross-sectional study was conducted to investigate risk factors for sporadic Cryptosporidium infection in a paediatric population in Nigeria. Of 692 children, 134 (19·4%) were infected with Cryptosporidium oocysts. Cryptosporidium spp. were identified in 49 positive samples using PCR-restriction fragment length polymorphism and direct sequencing of the glycoprotein60 (GP60) gene. Generalized linear mixed-effects models were used to identify risk factors for all Cryptosporidium infections, as well as for C. hominis and C. parvum both together and separately. Risk factors identified for all Cryptosporidium infections included malaria infection and a lack of Ascaris infection. For C. hominis infections, stunting and younger age were highlighted as risk factors, while stunting and malaria infection were identified as risk factors for C. parvum infection.
Epidemiology and Infection 06/2011; 139(6):946-54. · 2.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: We analyzed 1,042 Cryptosporidium oocyst-positive slides (456 from raw waters and 586 from drinking waters) of which 55.7% contained 1 or 2 oocysts, to determine species/genotypes present in Scottish waters. Two nested PCR-restriction fragment length polymorphism (RFLP) assays targeting different loci (1 and 2) of the hypervariable region of the 18S rRNA gene were used for species identification, and 62.4% of samples were amplified with at least one of the PCR assays. More samples (577 slides; 48.7% from raw water and 51.3% from drinking water) were amplified at locus 1 than at locus 2 (419 slides; 50.1% from raw water and 49.9% from drinking water). PCR at loci 1 and 2 amplified 45.4% and 31.7% of samples containing 1 or 2 oocysts, respectively. We detected both human-infectious and non-human-infectious species/genotype oocysts in Scottish raw and drinking waters. Cryptosporidium andersoni, Cryptosporidium parvum, and the Cryptosporidium cervine genotype (now Cryptosporidium ubiquitum) were most commonly detected in both raw and drinking waters, with C. ubiquitum being most common in drinking waters (12.5%) followed by C. parvum (4.2%) and C. andersoni (4.0%). Numerous samples (16.6% total; 18.9% from drinking water) contained mixtures of two or more species/genotypes, and we describe strategies for unraveling their identity. Repetitive analysis for discriminating mixtures proved useful, but both template concentration and PCR assay influenced outcomes. Five novel Cryptosporidium spp. (SW1 to SW5) were identified by RFLP/sequencing, and Cryptosporidium sp. SW1 was the fourth most common contaminant of Scottish drinking water (3%).
Applied and environmental microbiology 09/2010; 76(17):5977-86. · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Samples of fresh vegetables and soft fruit were collected from farmers' markets in the Lublin Area of Poland during 2006-2007; the produce was grown in areas of high to moderate livestock production. Cryptosporidium sp. oocysts were eluted from food surfaces, separated from residual food materials by IMS and identified by immunofluorescence and Nomarski differential interference contrast microscopy. Cryptosporidium sp. oocysts were detected in 6 of 128 vegetable samples (range 1-47 oocysts), but not in any of 35 fruit samples. Both empty and intact oocysts were detected. Species identity of oocyst-positive samples was performed by molecular analysis at four genetic loci. One of two 18S rRNA loci amplified DNA from 5 of the 6 oocyst-positive samples, but insufficient DNA for RFLP or sequencing analysis was available from 4 of these samples. An oocyst-positive celery sample generated an RFLP pattern consistent with C. parvum at two loci, but insufficient DNA was available for subtyping (GP60 sequencing) this isolate. Oocyst-contaminated foods originated from districts with the highest numbers of homesteads possessing cattle herds and no contaminated produce was detected from districts containing lower numbers of cattle-owning homesteads, strengthening the assumption that the origin of the contamination was livestock. The results of this study strengthen the evidence for the potential for zoonotic foodborne transmission of Cryptosporidium.
International journal of food microbiology 01/2010; 139(1-2):96-101. · 3.01 Impact Factor
[show abstract][hide abstract] ABSTRACT: Of 1346 faecal samples from the Chikwawa and Thyolo districts of Malawi, analysed for the presence of Cryptosporidium oocysts between October 2001 and May 2003, 61.3% were from cattle (29.8% of these were from calves <6 months old). Cryptosporidium oocysts were detected during all three seasons studied in Chikwawa and Thyolo. In Chikwawa, 13.6% of adult cattle and 11.7% of calves were infected, compared to 28.9% of adult cattle and 36.7% of calves in Thyolo. Dependent on season, between 7.8% and 37.7% (Chikwawa) and 16.7% and 39.3% (Thyolo) of cattle samples contained oocysts. In Chikwawa, the highest percentage of infections occurred in the cool season, whereas in Thyolo, the highest percentage of infections occurred in the dry season. Faecal samples from goats [n=225], pigs [n=92], sheep [n=6]), rabbits, guinea pigs, chickens, ducks, turkeys, doves and guinea fowls were also analysed. Up to 5.6% of goat samples contained oocysts in Chikwawa, compared to between 16.7% and 39.3% in Thyolo. Again, in Chikwawa, the highest percentage of infections occurred in the cool season and the lowest in the rainy season, whereas, in Thyolo, the highest percentage of infections occurred in the dry season and the lowest in the cool season. In pigs, more infections were detected in the dry season in Chikwawa, but infections in the cool season were similar (17.7%), whereas in Thyolo, infections occurred in all three seasons (17.9% in the rainy season, 25% in the cool season and 60% in the dry season). Of ten diarrhoeic, oocyst positive cattle faecal samples collected from Chikwawa and subjected to PCR-RFLP, four oocyst positive samples (two from heifers, one from a cow and one unknown) were amplified at an 18S rRNA and Cryptosporidium oocyst wall protein (COWP) loci. RFLP of the 18S rRNA locus indicated that Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium bovis and/or Cryptosporidium ryanae DNA, or a mixture of them was present. Cryptosporidium parvum DNA was identified in one sample that amplified at the COWP locus, indicating the presence of the major zoonotic Cryptosporidium species in Malawi.
The Onderstepoort journal of veterinary research 12/2009; 76(4):363-75. · 0.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: The objective of this cross-sectional study was to determine the prevalence and intensity of soil-transmitted helminths (STHs) in children aged 0-25 months and to identify the associated risk factors for Ascaris lumbricoides infections. The study was conducted in three villages outside Ile-Ife, Osun state, Nigeria in May/June 2005. Stool samples (369) were processed by formol-ether concentration. Ascaris lumbricoides (12.2%) was the dominant infection. Age, father's occupation and dog ownership were identified as the significant risk factors in the minimal adequate model for A. lumbricoides. The odds of being infected with A. lumbricoides increased as the children got older. Children aged 12-17 months and 18-25 months were 8.8 and 12.4 times, respectively, more likely to harbour Ascaris than those aged 7-11 months. The odds of harbouring Ascaris for children whose families owned a dog were 3.5 times that of children whose families did not own a dog. Children whose fathers were businessmen were 0.4 times less likely to be infected with Ascaris than those whose fathers were farmers. The findings from this study suggest that many of these young children, who are at a critical stage of development, are infected with Ascaris and that the prevalence of infection with this parasite increases with age. This study has highlighted the need to incorporate preschool children into deworming programmes in endemic regions and to investigate innovative ways of delivering cost-effective deworming treatment to this high-risk age group.
Journal of Helminthology 05/2009; 83(3):261-6. · 1.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: We determined the incidence of cryptosporidiosis in children aged <5 years presenting with diarrhoea in an urban and rural hospital-based setting in Malawi. Stools were collected over a 22-month period during both rainy and dry seasons. A range of microscopic methods were used to determine the presence of Cryptosporidium spp. oocysts. Species determination was by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of oocyst-extracted DNA using 18S rRNA and COWP gene loci. Cryptosporidium spp. oocysts were seen in 5.9% (50/848) of samples, of which 43 amplified by PCR-RFLP indicated the following species: C. hominis, C. parvum, C. hominis/C. parvum, C. meleagridis and C. andersoni. Seven samples could not be amplified by PCR. Wider species diversity was found in the rural setting, and may be a result of increased malnutrition and zoonotic exposure in this area. Improvements in water, sanitation, household hygiene and animal control are required to reduce the incidence of infection in this population.
Epidemiology and Infection 12/2007; 135(8):1307-15. · 2.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: We report a method for detecting Giardia duodenalis cysts on lettuce, which we subsequently use to examine salad products for the presence of Giardia cysts and Cryptosporidium oocysts. The method is based on four basic steps: extraction of cysts from the foodstuffs, concentration of the extract and separation of the cysts from food materials, staining of the cysts to allow their visualization, and identification of cysts by microscopy. The concentration and separation steps are performed by centrifugation, followed by immunomagnetic separation using proprietary kits. Cyst staining is also performed using proprietary reagents. The method recovered 46.0% +/- 19.0% (n = 30) of artificially contaminating cysts in 30 g of lettuce. We tested the method on a variety of commercially available natural foods, which we also seeded with a commercially available internal control, immediately prior to concentration of the extract. Recoveries of the Texas Red-stained Giardia cyst and Cryptosporidium oocyst internal controls were 36.5% +/- 14.3% and 36.2% +/- 19.7% (n = 20), respectively. One natural food sample of organic watercress, spinach, and rocket salad contained one Giardia cyst 50 g(-1) of sample as an indigenous surface contaminant.
Applied and Environmental Microbiology 12/2007; 73(22):7388-91. · 3.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cryptosporidium and Giardia are major causes of diarrhoeal disease in humans, worldwide and are major causes of protozoan waterborne diseases. Both Cryptosporidium and Giardia have life cycles which are suited to waterborne and foodborne transmission. There are 16 'valid'Cryptosporidium species and a further 33+ genotypes described. Parasites which infect humans belong to the Giardia duodenalis "type", and at least seven G. duodenalis assemblages are recognised. Cryptosporidium parvum is the major zoonotic Cryptosporidium species, while G. duodenalis assemblages A and B have been found in humans and most mammalian orders. In depth studies to determine the role of non-human hosts in the transmission of Cryptosporidium and Giardia to humans are required. The use of harmonised methodology and standardised and validated molecular markers, together with sampling strategies that provide sufficient information about all contributors to the environmental (oo)cyst pool that cause contamination of food and water, are recommended. Standardised methods for detecting (oo)cysts in water are available, as are optimised, validated methods for detecting Cryptosporidium in soft fruit and salad vegetables. These provide valuable data on (oo)cyst occurrence, and can be used for species and subspecies typing using appropriate molecular tools. Given the zoonotic potential of these organisms, epidemiological, source and disease tracking investigations involve multidisciplinary teams. Here, the role of the veterinarian is paramount, particularly in understanding the requirement for adopting comprehensive sampling strategies for analysing both sporadic and outbreak samples from all potential non-human contributors. Comprehensive sampling strategies increase our understanding of parasite population biology and structure and this knowledge can be used to determine what level of discrimination is required between isolates. Genetic exchange is frequent in C. parvum populations, leading to recombination between alleles at different loci, the generation of a very large number of different genotypes and a high level of resolution between isolates. In contrast, genetic exchange appears rare in Cryptosporidium hominis and populations are essentially clonal with far fewer combinations of alleles at different loci, resulting in a much lower resolution between isolates with many being of the same genotype. Clearly, more markers provide more resolution and high throughput sequencing of a variety of genes, as in multilocus sequence typing, is a way forward. Sub-genotyping tools offer increased discrimination, specificity and sensitivity, which can be exploited for investigating the epidemiology of disease, the role of asymptomatic carriers and contaminated fomites and for source and disease tracking for food and water contaminated with small numbers of (oo)cysts.
[show abstract][hide abstract] ABSTRACT: The occurrence of Giardia cysts in Scottish raw and potable waters was investigated. Giardia cysts were detected in 49% of raw waters, with concentrations of up to 1.05 cysts per litre, and in 19% of final waters, with concentrations of up to 1.67 cysts per litre. In some of the positive water samples the viability of the cysts was assessed by viewing the cyst morphology and inclusion/exclusion of propidium iodide. Viable cysts were detected in a proportion of both raw waters and positive final waters studied. Further investigations at 21 water-treatment plants revealed cysts in 9 (43%) of the raw waters, and in 4 (19%) of the final waters. Cysts were only detected in the final waters of plants in which cysts were also detected in the raw water. These data indicate that viable Giardia cysts may be ingested with potable water in Scotland.
Water and Environment Journal 07/2007; 7(6):632 - 635. · 0.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ x cm-2 doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4',6'-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ x cm-2. With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ x cm-2 of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ x cm-2 of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.
Applied and Environmental Microbiology 03/2007; 73(3):947-55. · 3.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: We used the fluorescent dye monochlorobimane (MCB) which binds glutathione (GSH) to localize between 2 and 6 distinctly labelled nuclear and cytoplasmic GSH foci in recently excreted and aged, intact Cryptosporidium parvum oocysts and sporozoites. Buthionine sulfoximine (BSO), a potent and specific inhibitor of GSH, was used to determine whether GSH is synthesized in BSO-treated C. parvum oocysts, by labelling treated oocysts with MCB. Both visual and electronic quantifications were performed. At 5 mM BSO, a significant inhibition of MCB fluorescence, reflecting reduced MCB uptake, was observed in GSH-depleted oocysts (mean +/- S.D. 35 +/- 3.7) compared with controls (3.3 +/- 1.2, P = 0). This clear reduction occurred only in viable oocysts. 1 mM BSO-treated oocysts exhibited weak or no MCB fluorescence, although they were viable (excluded propidium iodide, PI)), and intact and contained sporozoites by differential interference contrast microscopy (DIC). MCB was used in conjunction with PI to determine C. parvum oocyst viability. Oocysts labelled with MCB/PI or 4'6-diamidino-2-phenyl indole (DAPI)/PI produced comparable labelling patterns. Viable oocysts were labelled with MCB or DAPI whereas dead oocysts were labelled with PI only. The localization of GSH in viable, intact oocysts and excysted sporozoites and UV light-irradiated oocysts and sporozoites revealed no changes in MCB uptake at levels up to 40 mJ.cm(-2) irradiation. Although GSH can be detected following MCB localization in both the nucleus and cytoplasm of sporozoites, and can be specifically depleted by BSO treatment, MCB is unlikely to be useful as a surrogate for detecting UV damage in UV-treated Cryptosporidium oocysts.
[show abstract][hide abstract] ABSTRACT: No standard method is available for detecting protozoan parasites on foods such as soft fruit and salad vegetables. We report on optimizing methods for detecting Cryptosporidium parvum on lettuce and raspberries. These methods are based on four basic stages: extraction of oocysts from the foodstuffs, concentration of the extract and separation of the oocysts from food materials, staining of the oocysts to allow their visualization, and identification of oocysts by microscopy. The concentration and separation steps are performed by centrifugation, followed by immunomagnetic separation using proprietary kits. Oocyst staining is also performed using proprietary reagents. The performance parameters of the extraction steps were extensively optimized, using artificially contaminated samples. The fully developed methods were tested several times to determine their reliability. The method to detect C. parvum on lettuce recovered 59.0+/-12.0% (n=30) of artificially contaminated oocysts. The method to detect C. parvum on raspberries recovered 41.0+/-13.0% (n=30) of artificially contaminated oocysts.
International Journal of Food Microbiology 07/2006; 109(3):215-21. · 3.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: We report the results of interlaboratory collaborative trials of methods to detect oocysts of the protozoan parasite Cryptosporidium parvum on lettuce and raspberries. The trials involved eight expert laboratories in the United Kingdom. Samples comprised 30 g lettuce, and 60 g raspberries. Lettuce samples were artificially contaminated at three levels: low (8.5-14.2 oocysts), medium (53.5-62.6 oocysts), and high (111.3-135.0 oocysts). Non-contaminated lettuce samples were also tested. The method had an overall sensitivity (correct identification of all artificially contaminated lettuce samples) of 89.6%, and a specificity (correct identification of non-contaminated samples) of 85.4%. The total median percentage recovery (from all artificially contaminated samples) produced by the method was 30.4%. The method was just as reproducible between laboratories, as repeatable within a laboratory. Raspberry samples were artificially contaminated at three levels: low (8.5-26.8 oocysts), medium (29.7-65.7 oocysts), and high (53.9-131.3 oocysts). Non-contaminated raspberry samples were also tested. The method had an overall sensitivity (correct identification of all artificially contaminated raspberry samples) of 95.8%, and a specificity (correct identification of non-contaminated samples) of 83.3%. The total median percentage recovery (from all artificially contaminated samples) produced by the method was 44.3%. The method was just as reproducible between laboratories, as repeatable within a laboratory. The results of the collaborative trial indicate that these assays can be used effectively in analytical microbiological laboratories.
International Journal of Food Microbiology 07/2006; 109(3):222-8. · 3.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: Electrorotation is a non-invasive technique that is capable of detecting changes in the morphology and physicochemical properties of microorganisms. The first detailed electrorotation study of the egg (ovum) of a parasitic nematode, namely Ascaris suum is described to show that electrorotation can rapidly differentiate between fertilized and non-fertilized eggs. Support for this conclusion is by optical microscopy of egg morphology, and also from modelling of the electrorotational response. Modelling was used to determine differences in the dielectric properties of the unfertilized and fertilized eggs, and also to investigate specific differences in the spectra of fertilized eggs only, potentially reflecting embryogenesis. The potential of electrorotation as an investigative tool is shown, as undamaged eggs can be subjected to further non-destructive and destructive techniques, which could provide further insight into parasite biology and epidemiology.
Journal of Helminthology 04/2006; 80(1):25-31. · 1.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: The application of the AC electrokinetic technique of electrorotation for studying eukaryotic parasite transmission stages is reviewed. Electrorotation is a noninvasive technique that utilizes electrically energized microelectrode structures within micro-fluidic chambers to probe the physiological structure of micro-organisms. Application of the technique to the transmission life cycle stages of three separate genera of protozoan parasites, Cryptosporidium, Giardia and Cyclospora, and one nematode genus Ascaris, each of significant public health importance, is described.
Standard electrorotation apparatus, consisting of micro-fabricated electrodes in a fluidic chip, quadrature sinusoidal signal generator, microscope and image capture system, was used to study each organism. Spectra of cellular rotation rate were recorded as a function of applied electric field frequency and compared with standardized biological tests, where appropriate, to illustrate the effectiveness and versatility of the electrorotation technique.
Electrorotational determination of the viability of individual G. intestinalis cysts, Cryptosporidium parvum and Cyclospora cayetanensis oocysts has been achieved. The sporulation state of Cyclospora cayetanensis oocysts was also readily determined, as was the fertilization state of A. suum ova.
Electrorotation is a simple, noninvasive and versatile analytical technique suited to a wide range of particle types and capable of incorporation into integrated Lab-on-a-chip devices.
Journal of Applied Microbiology 02/2004; 96(1):24-32. · 2.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: We describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities of Cryptosporidium spp. oocysts in natural mineral waters and drinking waters. Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA. The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65 degrees C), followed by proteinase K digestion. Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels. DNA extracted from Cryptosporidium parvum oocysts, C. muris (RN 66), C. baileyi (Belgium strain, LB 19), human-derived C. meleagridis, C. felis (DNA from oocysts isolated from a cat), and C. andersoni was used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymes DraI and VspI. Discrimination between C. andersoni and C. muris isolates was confirmed by a separate, subsequent digestion with DdeI. Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR. The Cryptosporidium species detected in these finished water samples was C. parvum genotype 1. This method consistently and routinely detected >5 oocysts per sample.
Applied and Environmental Microbiology 07/2003; 69(7):4183-9. · 3.68 Impact Factor