Genotyping of Giardia lamblia isolates from humans in China and Korea using ribosomal DNA Sequences

Genetic characterization of a total of 15 Giardia lamblia isolates, 8 from Anhui Province, China (all from purified cysts) and 7 from Seoul, Korea (2 from axenic cultures and 5 from purified cysts), was performed by polymerase chain reaction amplification and sequencing of a 295-bp region near the 5' end of the small subunit ribosomal DNA (eukaryotic 16S rDNA). Phylogenetic analyses were subsequently conducted using sequence data obtained in this study, as well as sequences published from other Giardia isolates. The maximum parsimony method revealed that G. lamblia isolates from humans in China and Korea are divided into 2 major lineages, assemblages A and B. All 7 Korean isolates were grouped into assemblage A, whereas 4 Chinese isolates were grouped into assemblage A and 4 into assemblage B. Two Giardia microti isolates and 2 dog-derived Giardia isolates also grouped into assemblage B, whereas Giardia ardeae and Giardia muris were unique.     

Risk of human infection with Giardia duodenalis from cats in Japan and genotyping of the isolates to assess the route of infection in cats

The number of facilities in which customers make contact with cats before eating and drinking, called 'cat cafés', has recently increased in Tokyo, Japan. In a survey to clarify the possibility of zoonotic transmission in Giardia duodenalis, the infection rates of G. duodenalis in 321 stool samples of cats from 16 cat cafés, 31 pet shops, and the Animal Care and Consultation Center of Tokyo were 19·1% (22/115), 1·2% (1/85), and 2·5% (3/121), respectively. In the molecular analysis of 26 G. duodenalis isolates, 6 samples from 2 cat cafés belonged to the zoonotic genotype assemblage A I, and 20 other samples were of assemblage F. Moreover, phylogenetic analysis of glutamate dehydrogenase (GDH) and triosephosphate isomerase (TPI) genes of the 20 assemblage F isolates revealed 2 major lineages. The 6 assemblage A isolates belonged to the same cluster with regard to the GDH gene; however, 2 of the 6 isolates belonged to a different cluster from the other 4 isolates with regard to the TPI gene. Therefore, a risk of transmission from cats to humans is suggested because of the detection of zoonotic Giardia genotypes in cat cafés.     

Detection of Giardia intestinalis assemblages A, B and D in domestic cats from Warsaw, Poland

Giardia intestinalis is a complex species divided into 7 assemblages (A - G). Two of them (A and B) are infective for both humans and animals. In cats four assemblages can occur: A, B, D, and F Assemblages A and B infect either cats, dogs and humans, assemblage D infects cats and dogs and assemblage F only cats. The purpose of this study was to determine the prevalence and genotypes of G. intestinalis in cats from Warsaw. From November 2006 to March 2007 a hundred sixty samples of stool were collected and examined by light microscopy. G. intestinalis cysts were detected in 3.75% of samples. DNA extracted from positive samples was used as template for PCR-RFLP using Giardia specific primers and the amplicons were sequenced. A comparison of the obtained DNA sequences with the Giardia sequences in the GeneBank database revealed assemblage A in 1.25% of the investigated cats, assemblage B in 1.25% and D in 1.25%.     

Prevalence of Giardia cysts and Cryptosporidium oocysts and characterization of Giardia spp. isolated from drinking water in Canada.

This study was carried out to estimate the prevalence and potential for human infectivity of Giardia cysts in Canadian drinking water supplies. The presence of Cryptosporidium oocysts was also noted, but isolates were not collected for further study. A total of 1,760 raw water samples, treated water samples, and raw sewage samples were collected from 72 municipalities across Canada for analysis, 58 of which treat their water by chlorination alone. Giardia cysts were found in 73% of raw sewage samples, 21% of raw water samples, and 18.2% of treated water samples. There was a trend to higher concentration and more frequent incidence of Giardia cysts in the spring and fall, but positive samples were found in all seasons. Cryptosporidium oocysts were found in 6.1% of raw sewage samples, 4.5% of raw water samples, and 3.5% of treated water samples. Giardia cyst viability was assessed by infecting Mongolian gerbils (Meriones unguiculatus) and by use of a modified propidium iodide dye exclusion test, and the results were not always in agreement. No Cryptosporidium isolates were recovered from gerbils, but 8 of 276 (3%) water samples and 19 of 113 (17%) sewage samples resulted in positive Giardia infections. Most of the water samples contained a low number of cysts, and 12 Giardia isolates were successfully recovered from gerbils and cultured. Biotyping of these isolates by isoenzyme analysis and karyotyping by pulsed-field gel electrophoresis separated the isolates into the same three discrete groups. Karyotyping revealed four or five chromosomal bands ranging in size from 0.9 to 2 Mb, and four of the isolates had the same banding pattern as that of the WB strain. Analysis of the nucleotide sequences of the 16S DNA coding for rRNA divided the isolates into two distinct groups corresponding to the Polish and Belgian designations found by other investigators. The occurrence of these biotypes and karyotypes appeared to be random and was not related to geographic or other factors (e.g., different types were found in both drinking water and sewage from the same community). Biotyping and karyotyping showed that isolates from this study were genetically and biochemically similar to those found elsewhere, including well-described human source strains such as WB. We conclude that potentially human-infective Giardia cysts are commonly found in raw surface waters and sewage in Canada, although cyst viability is frequently low. Cryptosporidium oocysts are less common in Canada. An action level of three to five Giardia cysts per 100 liters in treated drinking water is proposed on the basis of the monitoring data from outbreak situations. This action level is lower than that proposed by Haas and Rose (C. N. Haas and J. B. Rose, J. Am. Water Works Assoc. 87(9):81-84, 1995) for Cryptosporidium spp. (10 to 30 oocysts per 100 liters).     

Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B

Giardia duodenalis is a widespread parasite of mammalian species, including humans. Due to its invariant morphology, investigations of aspects such as host specificity and transmission patterns require the direct genetic characterisation of parasites from faecal samples. We performed a sequence analysis of four genes (ssrRNA, β-giardin, glutamate dehydrogenase and triose phosphate isomerase) of 61 human isolates and 29 animal isolates. The results showed that multilocus genotypes (MLGs) can be readily defined for G. duodenalis isolates of assemblage A but not for assemblage B. Indeed, for assemblage A isolates, there was no evidence of intra-isolate sequence heterogeneity, and congruent genotyping results were obtained at the four genetic loci investigated. Sequence comparison and phylogenetic analysis showed that human-derived and animal-derived MLGs are different, and further indicated the presence of a new sub-assemblage (referred to as “AIII”), which was found exclusively in wild hoofed animals. On the other hand, there were variable levels of intra-isolate sequence heterogeneity (i.e., the presence of two overlapping nucleotide peaks at specific positions in the chromatograms, or “heterogeneous templates”) in assemblage B isolates from humans and animals, and this prevented the unambiguous identification of MLGs. Furthermore, in five human isolates and one non-human primate isolate, the assignment to assemblage B was problematic, given that one of the four markers supported an assignment to assemblage A. These findings raise concerns about the interpretation of genotyping data based on single markers, and indicate the need to understand the mechanisms that are responsible for the differences between G. duodenalis assemblages A and B.     

Novel and canine genotypes of Giardia duodenalis in harbor seals ( Phoca vitulina richardsi)

Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.     

Real-time PCR for quantification of Giardia and Cryptosporidium in environmental water samples and sewage

The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the beta-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.     

Application of genotyping during an extensive outbreak of waterborne giardiasis in Bergen, Norway, during autumn and winter 2004

During the autumn and winter of 2004 and 2005, an extensive outbreak of waterborne giardiasis occurred in Bergen, Norway. Over 1,500 patients were diagnosed with giardiasis. Analysis of water from the implicated source revealed low numbers of Giardia cysts, but the initial contamination event probably occurred up to 10 weeks previously. While sewage leakage from a residential area is now considered to be the probable source of contamination, during the episode waste from one particular septic tank was thought to be a possible source. Genotyping of cysts from the septic tank demonstrated that they were assemblage A cysts, although the sequences were not identical to any previously published sequences. For the beta-giardin gene, the closest published subgenotype was subgenotype A3; for the gdh gene, the closest published subgenotype was subgenotype A2. Genotyping of cysts from 21 patient samples revealed that they were assemblage B cysts; thus, the septic tank was unlikely to be the contamination source. Sequencing of the beta-giardin and gdh genes from patient samples and a comparison of the sequences gave complex results. For the beta-giardin gene, three isolates had sequences identical to subgenotype B3 sequences. However, other isolates had between one and four single-nucleotide polymorphisms (SNPs). For the gdh gene, none of the sequences were identical to the sequence published for subgenotype B3, and the sequences had between one and three SNPs. One isolate, which was identical to subgenotype B3 at the beta-giardin gene, was more similar to subgenotype B2 at the gdh gene. Grouping the isolates on the basis of SNPs resulted in different groups for the two genes. The results are discussed in relation to giardiasis in Norway and to other Giardia genotyping studies.     

Genotyping of Giardia duodenalis from humans and dogs from Mexico using a beta-giardin nested polymerase chain reaction assay

Cysts of Giardia duodenalis were collected in Mexico from symptomatic children (n = 9) and from pet dogs (n = 5), and they were directly characterized by nested polymerase chain reaction (PCR) amplification of the beta-giardin gene. Eight isolates of human origin established as in vitro cultures and 2 reference strains, representing assemblages A and B of G. duodenalis, were also analyzed. PCR-restriction fragment length polymorphism showed that all isolates belonged to assemblage A. Sequence analyses indicated that the large majority of isolates were of the A1 genotype; interestingly, 2 human isolates displayed the A3 genotype, which has been previously identified in human isolates from Italy. The presence of cysts of the A1 and A3 genotypes in isolates from pet dogs is consistent with their role as reservoirs for human infection, although further studies are needed to confirm the occurrence of zoonotic transmission. Remarkably, cysts of assemblage B have not been found in any of the Mexican isolates studied to date.     

Genotyping of Giardia duodenalis Isolates from Dogs in Guangdong,China Based on Multi-Locus Sequence

This study aimed to identify the assemblages (or subassemblages) of Giardia duodenalis by using normal or nested PCR based on 4 genetic loci: glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), β-giardin (bg), and small subunit ribosomal DNA (18S rRNA) genes. For this work, a total of 216 dogs'' fecal samples were collected in Guangdong, China. The phylogenetic trees were constructed with MEGA5.2 by using the neighbor-joining method. Results showed that 9.7% (21/216) samples were found to be positive; moreover, 10 samples were single infection (7 isolates assemblage A, 2 isolates assemblage C, and 1 isolate assemblage D) and 11 samples were mixed infections where assemblage A was predominant, which was potentially zoonotic. These findings showed that most of the dogs in Guangdong were infected or mixed-infected with assemblage A, and multi-locus sequence typing could be the best selection for the genotype analysis of dog-derived Giardia isolates.     

Giardia duodenalis cysts isolated from wild moose and reindeer in Norway: genetic characterization by PCR-rflp and sequence analysis at two genes

There are few genotyping studies of Giardia duodenalis isolates from cervid hosts, although a previous study suggested that cervids may be a source of infection for humans and cattle. Giardia duodenalis isolates collected from wild moose (Alces alces) and reindeer (Rangifer tarandus) in Norway during 2002 and 2003 were characterized by polymerase chain reaction-restriction fraction length polymorphism (PCR-RFLP) at the beta-giardin gene, and sequence analysis at both the beta-giardin and glutamate dehydrogenase (gdh) genes. All results suggested that these isolates (n=25) belonged to assemblage A. Three different restriction patterns were obtained with PCR-RFLP, one of which has previously been associated with assemblage A. At the beta-giardin gene, sequences from six reindeer isolates and one moose isolate were identical to a previously published assemblage A sequence from G. duodenalis cysts isolated from dairy calves. The other 10 moose isolates could be divided into five groups, with between two and 14 single nucleotide polymorphisms (SNPs) from the published genotype A2. At the gdh gene, three different sequences were obtained, differing from each other by between one and 15 SNPs and which have all been previously published as genotype A1, but with different specific hosts. Grouping of the isolates based on the sequences from both genes gave complex results; whereas all the G. duodenalis isolates from reindeer grouped together, two moose isolates, which had identical sequences at the beta-giardin gene, had sequences that differed from each other by 15 SNPs at the gdh gene. The results of these studies, together with the large Norwegian populations of these cervids and the amount of fecal matter they produce, indicate that moose and reindeer may be significant reservoirs of G. duodenalis infection in Norway, which may be of importance to veterinary and public health.     

Occurrence of Cryptosporidium oocysts and Giardia cysts in sewage in Norway

Samples of sewage influent from 40 sewage treatment works (STW) throughout Norway were examined for Cryptosporidium oocysts and Giardia duodenalis cysts. Both parasites were detected frequently (80% of STW were Cryptosporidium positive; 93% of STW were Giardia positive) and at maximum concentrations of > 20,000 parasites/liter. The data suggest giardiasis is more widespread, and/or occurs with greater infection intensity, than cryptosporidiosis in Norway. STW serving higher person equivalents were more likely to be positive and had higher parasite concentrations. Parasite concentrations were used to estimate the proportion of contributing populations that could be clinically infected. For Cryptosporidium, the highest estimates were up to 5 per 100,000 individuals for two populations in eastern Norway. For Giardia, the highest estimate was 40 infected per 100,000 persons (approximately five times the usual national annual average) contributing to an STW in western Norway. As this population experienced a large waterborne giardiasis outbreak 6 months after sampling, it can be speculated that regular challenge with Giardia may occur here. Most Giardia isolates in sewage influent were assemblage A, although some assemblage B isolates were detected. There was substantial heterogeneity, but most samples contained isolates similar to genotype A3. Removal efficiencies at two STW with secondary treatment processes were estimated to be approximately 50% for Cryptosporidium and > 80% for Giardia. An STW with minimal treatment had negligible removal of both parasites. Many STW in Norway have minimal treatment and discharge effluent into rivers and lakes, thus, risk of contamination of water courses by Cryptosporidium and Giardia is considerable.     

Occurrence of Giardia duodenalis assemblages in alpacas in the Andean region

In this study, 352 fecal samples were analyzed for G. duodenalis from alpaca mothers and crias from three different areas of highland in Peru. The triosephosphate isomerase (TPI) gene of Giardia was amplified using a nested PCR protocol. Forty-six G. duodenalis-PCR positive samples were sequenced. G. duodenalis assemblage A was the most frequent followed by assemblage E. The former was seen in 37 animals whereas the latter was seen in nine. Most of the assemblage A infections were caused by the A1 subtype of sub-assemblage AI, except for three, which were caused by the A2 subtype of sub-assemblage AI. Assemblage A was found in all three geographic regions, while assemblage E was detected in crias from two regions. Among the four alpaca mothers positive for Giardia, three had assemblage AI and one had assemblage AII. Results of this study indicate that possible zoonotic transmission human to alpacas.     

High prevalence Giardia duodenalis assemblage B and potentially zoonotic subtypes in sporadic human cases in Western Australia

Giardia duodenalis is a widespread parasite of mammalian species, including humans. Fecal samples from sporadic human clinical cases of giardiasis in Western Australia were analysed at two loci; 18S rRNA and glutamate dehydrogenase (gdh), and G. duodenalis assemblage B isolates were identified in 75% of isolates. Sequence analyses of 124 isolates at the 18S rRNA locus identified 93 isolates as assemblage B and 31 as assemblage A. Analyses of 109 isolates at the gdh locus identified 44 as B3, 38 as B4 and 27 were A2. Infection with Giardia was highest amongst children <5 years of age, with >56% of infections in this age group. The majority of the isolates were from rural areas (91/124) compared with urban areas (33/124). The assemblage A isolates were completely homogenous genetically at the gdh locus, while assemblage B isolates showed variability at the nucleotide but not at the amino acid level at this locus. Some of the assemblage B3 and B4 subtypes identified in humans were previously identified in marsupials in Australia and in a fox, indicating potential zoonotic transmission.     

Molecular characterization of Giardia duodenalis isolates from police and farm dogs in China

To assess the potential zoonotic transmission of giardiasis from dogs in China, a total of 205 fecal specimens from dogs were screened for Giardia duodenalis using PCR and sequence analysis of the triosephosphate isomerase gene. The prevalence of G. duodenalis in dogs was 13.2% (27/205). The potentially zoonotic assemblage A and the dog-specific assemblage C was identified in 25 (12.2%) and two (1.0%) dogs, respectively. All assemblage A isolates belonged to sub-assemblage AI, genotype AI-1. Likewise, one subtype was found in assemblage C. The high occurrence of potentially zoonotic G. duodenalis subtype AI-1 in dogs that are in close contact with humans is of public health concern.     

Occurrence and Molecular Identification of Giardia duodenalis from Stray Cats in Guangzhou,Southern China

The objective of this study was to genetically characterize isolates of Giardia duodenalis and to determine if zoonotic potential of G. duodenalis could be found in stray cats from urban and suburban environments in Guangzhou, China. Among 102 fresh fecal samples of stray cats, 30 samples were collected in Baiyun district (urban) and 72 in Conghua district (suburban). G. duodenalis specimens were examined using light microscopy, then the positive specimens were subjected to PCR amplification and subsequent sequencing at 4 loci such as glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), β-giardin (bg), and small subunit ribosomal RNA (18S rRNA) genes. The phylogenetic trees were constructed using obtained sequences by MEGA5.2 software. Results show that 9.8% (10/102) feline fecal samples were found to be positive by microscopy, 10% (3/30) in Baiyun district and 9.7% (7/72) in Conghua district. Among the 10 positive samples, 9 were single infection (8 isolates, assemblage A; 1 isolate, assemblage F) and 1 sample was mixed infection with assemblages A and C. Based on tpi, gdh, and bg genes, all sequences of assemblage A showed complete homology with AI except for 1 isolate (CHC83). These findings not only confirmed the occurrence of G. duodenalis in stray cats, but also showed that zoonotic assemblage A was found for the first time in stray cats living in urban and suburban environments in China.     

LNA probes in a real‐time TaqMan PCR assay for genotyping of Giardia duodenalis in wastewaters

Aims: This study describes an approach for genotyping Giardia cysts obtained from wastewater treatment plants (WTPs) in Spain using real‐time PCR (qPCR) in combination with immunomagnetic beads. Methods and Results: A 50‐cycle amplification of a 74‐bp fragment of the Giardia beta‐giardin gene was adopted from a previous qPCR method. Additionally, two locked nucleic acid (LNA) probes were designed (LNA P434 P1 for assemblage A and LNA P434 H3 for assemblage B). All 16 wastewater samples analysed were positive with the immunofluorescence assay (IFA). Assemblage A was detected in all WTP samples using primer–LNA probe P434 P1 set. Giardia duodenalis identification was confirmed by PCR–RFLP analysis and sequencing of the β‐giardin gene in the water samples found positive by IFA and qPCR. Among the 16 assemblage A isolates that were sequenced, two subtypes were identified; 11 corresponded to the A2 subgenotype, whereas three corresponded to the subgenotype A3. A mixture of subgenotypes was found in the remaining two isolates. Conclusions: The newly developed qPCR assays were able to discern G. duodenalis assemblages A and B in wastewater. Significance and Impact of the Study: The real‐time PCR assays provided a rapid method for detection and one‐step genotyping of G. duodenalis from wastewater samples, and its application would contribute to understanding the distribution and abundance of G. duodenalis assemblages A and B in wastewater.     

Intestinal parasites and genotyping of Giardia duodenalis in children: first report of genotype B in isolates from human clinical samples in Mexico

Giardia duodenalis is one of the most prevalent enteroparasites in children. This parasite produces several clinical manifestations. The aim of this study was to determine the prevalence of genotypes of G. duodenalis causing infection in a region of southeastern Mexico. G. duodenalis cysts were isolated (33/429) from stool samples of children and molecular genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, targeting the triosephosphate isomerase ( tpi ) and glutamate dehydrogenase ( gdh ) genes. The tpi gene was amplified in all of the cyst samples, either for assemblage A (27 samples) or assemblage B (6 samples). RFLP analysis classified the 27 tpi -A amplicons in assemblage A, subgenotype I. Samples classified as assemblage B were further analysed using PCR-RFLP of the gdh gene and identified as assemblage B, subgenotype III. To our knowledge, this is the first report of assemblage B of G. duodenalis in human clinical samples from Mexico.     

Detection of Norovirus and Sapovirus from diarrheic dogs and cats in Japan

Norovirus (NoV) and sapovirus (SaV) are important causes of human diarrhea. In this study, between 2007 and 2014 fecal samples were collected from 97 dogs and 83 cats with diarrhea and examined to determine the prevalence of NoV and SaV infections in Japan. To detect caliciviruses, approximately 300 bases targeting the polymerase gene were amplified using RT‐PCR and subjected to phylogenetic and homology analyses. Specific PCR products were obtained from four canine and nine feline samples: two canine and one feline isolate were classified as NoV, two canine isolates as SaV and the remaining eight feline isolates as vesivirus (VeV). The three NoV isolates were classified into the same clade as that of known canine and feline NoVs; their homologies (75.9–92.3%) were higher than those with human genogroup IV (GIV) NoVs (59.1–65.9%). The homology of the feline NoV isolate with previously reported feline NoV isolates was particularly high (91.7–92.3%). Regarding SaV, the two canine isolates were classified into the same clade as known canine SaVs and their homologies (72.5–86.5%) were higher than those with other mammal SaVs (20.7–58.0%). The eight feline VeV isolates were assumed to be feline calicivirus. The present study is the first report of the presence of NoV‐ and SaV‐infected dogs and cats in Japan. The findings suggest there are species‐specific circulations of NoV and SaV among dogs and cats, in Japan.     

Antibody-magnetite method for selective concentration of Giardia lamblia cysts from water samples.

An antibody-magnetite method was developed in order to selectively concentrate Giardia cysts from water samples. The indirect technique employed a mouse immunoglobulin G anti-Giardia antibody as a primary antibody and an anti-mouse immunoglobulin G antibody-coated magnetite particle as a secondary labeling reagent. The magnetically labeled cysts were then concentrated by high-gradient magnetic separation. Ninety percent of the seeded cysts were recovered from buffer when this method was employed. An average of 82% of the seeded cysts were recovered from water samples with various turbidities. Significantly higher cyst recoveries were obtained from water samples with turbidities below 600 nephelometric turbidity units.