Evaluation of Five Membrane Filtration Methods for Recovery of
            <i>Cryptosporidium</i>
            and
            <i>Giardia</i>
            Isolates from Water Samples

Wohlsen, T.; Bates, John; Gray, Bruce; Katouli, Mohammad

doi:10.1128/aem.70.4.2318-2322.2004

Cited by 54 publications

(50 citation statements)

References 33 publications

(39 reference statements)

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“…Minor modifications to the manufacturer's protocol were 133 made. Briefly, samples were subjected to five cycles of freeze-thaw (freezing each sample Using Envirochek filters (Pall Life Sciences, New South Wales, Australia), filtration of the 144 water samples was performed in accordance with the manufacturer's instructions, except 145 for some slight modifications as described by Wohlsen et al, (2004). The eluate was 146 subjected to the same genomic DNA extraction method detailed above.…”

Section: Genomic Dna Extraction 129mentioning

confidence: 99%

Longitudinal investigation of protozoan parasites in meat lamb farms in southern Western Australia

Sweeny

Ryan

Robertson

et al. 2011

Preventive Veterinary Medicine

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14In this study, 96 faecal samples were collected from pregnant Merino ewes, at two broad-15 acre, commercial sheep farms in southern Western Australia, on two separate occasions 16 (16 and 2 weeks prior to lambing). Following lambing, 111 (Farm A) and 124 (Farm B) 17 female crossbred lambs (2 -6 weeks old), were individually identified using ear tags (a 18 numbered tag and a radio-frequency tag). A total of 1,155 faecal samples were collected 19 only from these individually identified lambs on five separate sampling occasions. All 20 samples were screened using PCR to detect Cryptosporidium (18S rRNA and actin loci) 21and Giardia duodenalis (glutamate dehydrogenase and triosephosphate isomerise loci). 22The overall prevalences (lambs positive for a parasite on at least one of the five 23 samplings) at Farm A and B were 81.3% and 71.4%, respectively for Cryptosporidium and 24 similarly 67.3% and 60.5% for Giardia, respectively. Cryptosporidium and Giardia 25 prevalences at individual samplings ranged between 18.5 -42.6% in lambs and were 26 <10% in the ewes. Cryptosporidium xiaoi was the most prevalent species detected at all 27 five samplings and was also isolated from lamb dam water on Farm B. Cryptosporidium 28 ubiquitum was most commonly detected in younger lambs and C. parvum was detected in 29 lambs at all five samplings, typically in older lambs and as part of a mixed species infection 30 with C. xiaoi. A novel, possibly new genotype (sheep genotype I), was identified in six 31Cryptosporidium isolates from Farm B. Giardia duodenalis assemblage E was the most 32 common genotype detected at all five samplings, with greater proportions of assemblage A 33 and mixed assemblage A and E infections identified in older lambs. This longitudinal study 34 identified high overall prevalences of Cryptosporidium and Giardia in lambs grazed 35 extensively on pastures, whilst reinforcing that sampling a random selection of animals 36 from a flock/herd on one occasion (point prevalence), underestimates the overall 37 prevalence of these parasites in the flock/herd across an extended time period. Based on 38 these findings, grazing lambs were identified as a low risk source of zoonotic 39Page 3 of 36 Cryptosporidium and Giardia species/genotypes, with these protozoa detected at all five 40 samplings in some lambs, indicating that these individuals were either unable to clear the 41 naturally acquired protozoan infections or were repeatedly re-infected from their 42 environment or other flock members. 43

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Section: Genomic Dna Extraction 129mentioning

confidence: 99%

Longitudinal investigation of protozoan parasites in meat lamb farms in southern Western Australia

Sweeny

Ryan

Robertson

et al. 2011

Preventive Veterinary Medicine

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“…And so, different filter systems are applied for the filtration required in these methods. As it turned out, the efficiency of those methods, assessed in practice, shows different degrees of recovery of cysts and/or oocysts depending on the applied filter system (Wohlsen et al, 2004). These and many other studies show that these methods have got many restrictions.…”

Section: Development In the Methods Of Detectionmentioning

confidence: 92%

Methods for parasitic protozoans detection in the environmental samples

Skotarczak

2009

Parasite

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Summary :The environmental route of transmission of many parasitic protozoa and their potential for producing large numbers of transmissive stages constitute persistent threats to public and veterinary health. Conventional and new immunological and molecular methods enable to assess the occurrence, prevalence, levels and sources of waterborne protozoa. Concentration, purification, and detection are the three key steps in all methods that have been approved for routine monitoring of waterborne cysts and oocysts. These steps have been optimized to such an extent that low levels of naturally occurring (oo)cysts of protozoan can be efficiently recovered from water. Ten years have passed since the United States Environmental Protection Agency (USEPA) introduced the 1622 and 1623 methods and used them to concentrate and detect the oocysts of Cryptosporidium and cysts of Giardia in water samples. Nevertheless, the methods still need studies and improvements. Pre-PCR processing procedures have been developed and they are still improved to remove or reduce the effects of PCR inhibitors. The progress in molecular methods allows to more precise distinction of species or simultaneous detection of several parasites, however, they are still not routinely used and need standardization. Standardized methods are required to maximize public health surveillance. Résumé : MÉTHODES DE DÉTECTIONS DES PROTOZOAIRES PARASITES DANS L'ENVIRONNEMENT Les voies de transmissions de nombreux protozoaires parasites dans l'environnement et leur aptitude à y produire un nombre élevé de formes transmissibles constituent des menaces persistantes tant en santé humaine qu'animale. Des méthodes conventionnelles et de nouvelles méthodes moléculaires et immunologiques permettent d'évaluer la fréquence, la prévalence, les niveaux et les origines des protozoaires transmis par l'eau. La concentration, la purification et la détection sont les trois étapes clés de toutes les méthodes agréées pour la surveillance de routine des kystes et des oocystes transmis par l'eau. Ces étapes ont été optimisées à un niveau d'efficacité tel que les faibles concentrations naturelles d'(oo)cystes présents dans l'eau sont efficacement détectées. Dix ans se sont écoulés depuis l'introduction par l'United States

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“…However, IMS is not only expensive; its performance is affected by the type of commercial kit used, pH, and dissociation procedures (Ware et al 2003). Earlier studies have shown various degrees of recovery efficiency for Cryptosporidium (Hsu et al 2001;Simmons et al 2001;Wohlsen et al 2004). …”

Section: Discussionmentioning

confidence: 99%

“…The 1622 method detects and calculates only Cryptosporidium oocysts in water samples; the 1623 method -Cryptosporidium oocysts and Giardia cysts, and introduces some improvements. However, the recovery of oocysts from environmental water samples with the use of these both methods has been reported to be very variable (Simmons et al 2001;Kuhn and Oshima 2002;Wohlsen et al 2004;Krometis et al 2009;Karim et al 2010). With regard to this fact, matrix spikes are routinely analysed to determine the influence of environmental matrices on the effectiveness of the methods for detecting target organisms (Francy et al 2004).…”

mentioning

confidence: 99%

Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR

Adamska¹,

Leońska-Duniec²,

Sawczuk³

et al. 2012

Vet. Med.

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ABSTRACT:Cryptosporidium parvum is a common intestinal protozoan parasite infecting humans and a wide range of animals, whose diagnostics present considerable difficulties. These arise from the exceptionally robust nature of the oocyst's walls, which necessitates more stringent treatments for disruption and recovery of DNA for analysis using molecular methods. In the case of water, which is the major source of Cryptosporidium oocysts, investigations concern the detection of the presence of the oocysts. Their concentration in water is very low, and moreover, many substances that may have significance as inhibitors of DNA amplification, are present in environmental water and stool. We have carried out trials in order to assess the effectiveness of recovery of C. parvum oocysts, from spiked environmental and distilled water samples, filtrated and concentrated with the use of special laboratory equipment. Inactivation of inhibitors was carried out with use of bovine serum albumin (BSA) in PCR mixes at ten different concentrations. DNA extraction was carried out from stool samples spiked with C. parvum oocysts, concentrated using two methods, and unconcentrated. Nested PCR and a TaqMan nested real time PCR assay, targeting the 18S rRNA gene, was used to detect C. parvum DNA in spiked water and additionally in spiked stool samples. The obtained results showed that losses of C. parvum oocysts occur during the filtration and concentration of spiked water samples. The addition of small amounts of BSA (5-20 ng/µl) to PCR and TaqMan PCR mixes increases the sensitivity of both methods, but a high concentration of BSA (100 ng/µl and above) has an inhibiting effect on the polymerase reaction. The extraction of DNA from C. parvum oocysts from spiked stool samples preceded by concentration with PBS, ether and Percoll resulted in a higher copy number of the 18S rRNA gene.

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Evaluation of Five Membrane Filtration Methods for Recovery of Cryptosporidium and Giardia Isolates from Water Samples

Cited by 54 publications

References 33 publications

Longitudinal investigation of protozoan parasites in meat lamb farms in southern Western Australia

Longitudinal investigation of protozoan parasites in meat lamb farms in southern Western Australia

Methods for parasitic protozoans detection in the environmental samples

Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR

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