Giardia cysts and Cryptosporidium oocysts detection in the intake and rapid filter system in a water purification plant

Water samples, taken from the intake and rapid filter system of a water purification plant, were analyzed using an immunofluorescence antibody method for detecting the presence of Giardia cysts and Cryptosporidium oocysts. Giardia cysts and Cryptosporidium oocysts were found in the intake water from zero to 38.7 cysts/100 l and 1.7–50.5 oocysts/100 l with averages of 9.6 cysts/100 l and 19.4 oocysts/100 l. Giardia cysts and Cryptosporidium oocysts were also detected in the samples taken from the rapid filtration unit with mean concentrations of zero to 2.3 cysts/100 l and 0–2.5 oocysts/100 l, respectively. The efficacy of the rapid filter in suspended material and (oo)cyst removal was significant. The removal late was 56–97% for suspended material and 69–100% for the (oo)cysts.     

Occurrence of Cryptosporidium and Giardia in Wild Ducks along the Rio Grande River Valley in Southern New Mexico

Fecal samples were taken from wild ducks on the lower Rio Grande River around Las Cruces, N. Mex., from September 2000 to January 2001. Giardia cysts and Cryptosporidium oocysts were purified from 69 samples by sucrose enrichment followed by cesium chloride (CsCl) gradient centrifugation and were viewed via fluorescent-antibody (FA) staining. For some samples, recovered cysts and oocysts were further screened via PCR to determine the presence of Giardia lamblia and Crytosporidium parvum. The results of this study indicate that 49% of the ducks were carriers of Cryptosporidium, and the Cryptosporidium oocyst concentrations ranged from 0 to 2,182 oocysts per g of feces (mean ± standard deviation, 47.53 ± 270.3 oocysts per g); also, 28% of the ducks were positive for Giardia, and the Giardia cyst concentrations ranged from 0 to 29,293 cysts per g of feces (mean ± standard deviation, 436 ± 3,525.4 cysts per g). Of the 69 samples, only 14 had (oo)cyst concentrations that were above the PCR detection limit. Samples did test positive for Cryptosporidium sp. However, C. parvum and G. lamblia were not detected in any of the 14 samples tested by PCR. Ducks on their southern migration through southern New Mexico were positive for Cryptosporidium and Giardia as determined by FA staining, but C. parvum and G. lamblia were not detected.     

Effects of the Norwegian Winter Environment on <Emphasis Type="Italic">Giardia</Emphasis> Cysts and <Emphasis Type="Italic">Cryptosporidium</Emphasis> Oocysts

The structural integrity of Cryptosporidium oocysts and Giardia cysts in the Norwegian winter environment was investigated. During winter 2001/2002, Cryptosporidium oocysts and Giardia cysts were placed in the upper layers of soil in different matrices contained in chambers and exposed to the Norwegian climate. Morphological characteristics and inclusion/exclusion of vital dyes were monitored and compared to refrigerated controls. Reduction in parasite numbers was recorded for all parasites, geographical locations, and matrices. Shear forces generated during freeze–thaw cycles are postulated to have disintegrated the parasites exposed to the Norwegian winter and retrospective laboratory studies support this theory. Increased dye inclusion, possibly indicative of viability loss, was also noted. The refrigerated control parasites exhibited no decline in numbers, and alteration in dye inclusion characteristics for refrigerated parasites was slower. Cryptosporidium oocysts were apparently more robust than Giardia cysts; differences between isolates were also noted. These results suggest Cryptosporidium oocysts and Giardia cysts do not persist in the Norwegian terrestrial environment over winter, and when detected, will have been excreted since the previous winter. Differences in the morphological characteristics, matrix effects, and the possible relationship of the dye data to parasite survival are discussed in relation to further studies.     

Prevalence and molecular identification of Cryptosporidium isolates from pet lizards and snakes in Italy

In order to acquire prevalence and genetic data on Cryptosporidium infections in captive lizards and snakes kept as pets, a survey was conducted on 150 individual reptiles from southern Italy. Fecal samples were preserved in 5% formalin and analyzed using a commercial immunofluorescence assay (IFA) for the detection of Cryptosporidium oocysts and Giardia cysts. IFA revealed the presence of Cryptosporidium oocysts in nine of the 150 samples examined (6.0%), precisely in 6/125 snakes (4.8%) and in 3/25 lizards (12.0%); all fecal samples tested negative for the presence of Giardia cysts. Molecular characterization based on nested PCR amplification and sequencing of the SSU-rRNA gene, revealed the presence of Cryptosporidium serpentis in three samples from snakes (Boa constrictor constrictor, Elapheguttata guttata guttata and Python molurus).     

Removal of wastewater pathogen indicators in a constructed wetland in Leon, Spain

This paper studies the seasonal presence and removal of the pathogenous micro-organisms Escherichia coli, total coliforms (TC), Clostridium perfringens (Cp), faecal streptococci (FS), Giardia cysts, Cryptosporidium oocysts and helminth eggs, in a constructed wetland treatment system. The removal efficiency of this system with respect to the indicator micro-organisms achieved maximum values in spring and autumn at 99.9% for E. coli and TC, respectively, in winter at 97.0% for FS, in summer at 100% for Clostridium and throughout the year, also at 100%, in the case of Giardia cysts, Cryptosporidium oocysts and helminth eggs. In general, very low protozoan and helminth egg counts were found, and the system demonstrated efficient reduction of the wastewater indicator pathogens.     

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies ¹². The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used^1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocystsand Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously^{1-3,5-8,10,11}. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters⁴. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices^9,13 .This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.     

Mechanisms for Parasites Removal in a Waste Stabilisation Pond

A waste stabilisation pond (WSP) system formed by two anaerobic ponds, a facultative pond and a maturation pond was studied from December 2003 to September 2004 in north-western Spain in order to evaluate its efficiency in the removal of faecal indicator bacteria (total coliforms, Escherichia coli, faecal streptococci), coliphages, helminth eggs and protozoan (oo)cysts (Cryptosporidium and Giardia). Furthermore, sediment samples were collected from the bottom of the ponds to assess the settling rates and thus determine the main pathogen removal mechanisms in the WSPs system. The overall removal ranged from 1.4 log units for coliphages in the cold period to 5.0 log units for E. coli in the hot period. Cryptosporidium oocysts were reduced by an average of 96%, Giardia cysts by 98% and helminth eggs by 100%. The anaerobic ponds showed significantly higher surface removal rates (4.6, 5.2 and 3.7 log (oo)cysts/eggs removed m⁻² day⁻¹, respectively) than facultative and maturation ponds. Sunlight and water physicochemical conditions were the main factors influencing C. parvum oocysts removal both in the anaerobic and maturation ponds, whereas other factors like predation or natural mortality were more important in the facultative pond. Sedimentation, the most commonly proposed mechanism for cyst removal had, therefore, a negligible influence in the studied ponds.     

Enterocytozoon bieneusi,Giardia, and Cryptosporidium Infecting White‐tailed Deer

Despite a white‐tailed deer (WTD) population in the United States of approximately 32 million animals extremely little is known of the prevalence and species of the protists that infect these animals. This study was undertaken to determine the presence of potential human protist pathogens in culled WTD in central Maryland. Feces from fawns to adults were examined by molecular methods. The prevalence of Enterocytozoon bieneusi, Cryptosporidium, and Giardia was determined by PCR. All PCR‐positive specimens were sequenced to determine the species and genotype(s). Of specimens from 80 WTD, 26 (32.5%) contained 17 genotypes of E. bieneusi. Four genotypes were previously reported (I, J, WL4, LW1) and 13 novel genotypes were identified and named DeerEb1‐DeerEb13. Genotypes I, J, and LW1 are known to infect humans. Ten (12.5%) specimens contained the Cryptosporidium deer genotype, and one (1.25%) contained Giardia duodenalis Assemblage A. The identification zoonotic G. duodenalis Assemblage A as well as four E. bieneusi genotypes previously identified in humans suggest that WTD could play a role in the transmission of those parasites to humans.     

Detection of <Emphasis Type="Italic">Giardia,Entamoeba,</Emphasis> and <Emphasis Type="Italic">Cryptosporidium</Emphasis> in unprocessed food items from northern India

This study was conducted to evaluate the load of three food- and water-borne parasites, namely, Giardia, Entamoeba, and Cryptosporidium on food items that are consumed either raw or in an unprocessed state in the northern parts of India. A two-step diagnostic method was employed to assess the presence of Giardia and Cryptosporidium, the immunofluorescence assay (IFA) combined with the polymerase chain reactiion (PCR assay), whereas a Tech lab diagnostic kit in combination with PCR assay was used for accurate detection of Entamoeba histolytica in the samples. The methods for isolation and enrichment of cysts/oocysts from the various food items were tested and discussed here. Our results showed that the overall spectrum of incidence of the three parasites on food items in decreasing order were Giardia > Entamoeba > Cryptosporidium. When data were subjected to the chi-square test, the prevalence of all three parasites was found to be independent of the food items. To determine whether the presence of two types of parasites in a food item is uniform, a Poisson distribution test was conducted. On comparing the intensity of occurrence of the different parasites in various food items, it was observed that the occurrence of Giardia and Entamoeba was not of the same order at 5% level of significance only in case of samples of raw meat and milk. This confirmed that a high number of raw or unpasteurized milk and meat samples are more likely to be contaminated with Giardia than with Entamoeba. Therefore, our observations point to the unhygienic practices of food handlers being a major contributor in the transmission of parasites to unprocessed food products.     

Assessment of Giardia and Cryptosporidium spp. as a Microbial Source Tracking Tool for Surface Water: Application in a Mixed-Use Watershed

Knowledge of host specificity, combined with genomic sequencing of Giardia and Cryptosporidium spp., has demonstrated a microbial source tracking (MST) utility for these common waterborne microbes. To explore the source attribution potential of these pathogens, water samples were collected in a mixed rural-urban watershed in the Township of Langley, in southwestern British Columbia (BC), Canada, over a 2-year period. Cryptosporidium was detected in 63% of surface water samples at concentrations ranging from no positive detection (NPD) to 20,600 oocysts per 100 liters. Giardia was detected in 86% of surface water samples at concentrations ranging from NPD to 3,800 cysts per 100 liters of water. Sequencing at the 18S rRNA locus revealed that 50% of Cryptosporidium samples and 98% of Giardia samples contained species/genotypes (Cryptosporidium) or assemblages (Giardia) that are capable of infecting humans, based on current knowledge of host specificity and taxonomy. Cryptosporidium genotyping data were more promising for source tracking potential, due to the greater number of host-adapted (i.e., narrow-host-range) species/genotypes compared to Giardia, since 98% of Giardia isolates were zoonotic and the potential host could not be predicted. This report highlights the benefits of parasite genomic sequencing to complement Method 1623 (U.S. Environmental Protection Agency) and shows that Cryptosporidium subtyping for MST purposes is superior to the use of Giardia subtyping, based on better detection limits for Cryptosporidium-positive samples than for Giardia-positive samples and on greater host specificity among Cryptosporidium species. These additional tools could be used for risk assessment in public health and watershed management decisions.     

DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer''s protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit''s protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ≈ 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.     

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.     

Detection of Helicobacter pylori in stool samples of young children using real‐time polymerase chain reaction

Background

The aims of this study were to develop and validate a multiplex real‐time polymerase chain reaction (q‐PCR) assay of Helicobacter pylori in stool samples of healthy children. Additionally, we determined the prevalence of clarithromycin resistance and cagA gene in H. pylori‐positive samples.

Materials and methods

Archived stool samples from 188 children aged 6‐9 years and 272 samples of 92 infants aged 2‐18 months were tested for H. pylori antigens using enzyme immunoassay (EIA). A multiplex q‐PCR assay was designed to detect H. pylori 16S rRNA and urease and the human RNase P gene as an internal control. Kappa coefficient was calculated to assess the agreement between q‐PCR and EIA.

Results

Laboratory validation of the q‐PCR assay using quantitated H. pylori ATCC 43504 extracted DNA showed S‐shaped amplification curves for all genes; the limit of detection was 1 CFU/reaction. No cross‐reactivity with other bacterial pathogens was noted. Applying the multiplex q‐PCR to DNA extracted from fecal samples showed clear amplification curves for urease gene, but not for 16S rRNA. The prevalence of H. pylori infection was 50% (95% CI 43%‐57%) by q‐PCR (urease cycle threshold <44) vs 59% (95% CI 52%‐66%) by EIA. Kappa coefficient was .80 (P < .001) and .44 (P < .001) for children aged 6‐9 years and 2‐18 months, respectively. Sixteen samples were positive for cagA and three were positive for clarithromycin resistance mutation (A2143G) as confirmed by sequencing.

Conclusions

The developed q‐PCR can be used as a cotechnique to enhance the accuracy of H. pylori detection in epidemiological studies and in clinical settings.     

Phenotypic and genotypic profile of clinical and animal multidrug‐resistant Salmonella enterica isolates from Mexico

Aims

The objective of this study was to obtain a phenotypic and genotypic profile of Salmonella enterica including multidrug‐resistant (MDR) isolates from food‐producing animals and clinical isolates, as well as their genetic relatedness in two different States of Mexico (Jalisco and State of Mexico).

Methods and Results

A total of 243 isolates were evaluated in terms of antimicrobial resistance (AMR) and related genes through a disk diffusion method and PCR respectively; we found 16 MDR isolates, all of them harbouring the bla_CMY gene but not qnr genes, these isolates represent less than 10% of the collection. The pulsed‐field gel electrophoresis revealed a higher genotypic similitude within isolates of State of Mexico than Jalisco.

Conclusions

A low percentage of Salmonella isolates were resistant to relevant antibiotics in human health, nevertheless, the AMR and involved genes were similar despite the different serovars and origin of the isolates.

Significance and Impact of the Study

This investigation provided an insight of the current status of AMR of Salmonella isolates in two States of Mexico and pinpoint the genes involved in AMR and their epidemiological relationship, the information could help to determine an adequate therapy in human and veterinary medicine.     

Species‐specific detection of Candida tropicalis using evolutionary conserved intein DNA sequences

Inteins (internal proteins) are self‐splicing transportable genetic elements present in conserved regions of housekeeping genes. The study highlights the importance of intein as a potential diagnostic marker for species‐specific identification of Candida tropicalis, a rapidly emerging opportunistic human pathogen. Initial steps of primer validation, sequence alignment, phylogenetic tree analysis, gel electrophoresis and real‐time polymerase chain reaction (PCR) assays were performed to confirm the specificity of the designed primers. The primers were selective for C. tropicalis with 100% inclusivity and showed no cross‐species or cross‐genera matches. The established technique is a prototype for developing multifaceted PCR assays and for point‐of‐care testing in near future.

Significance and Impact of the Study

Development of molecular markers for specific detection of microbial pathogens using real‐time polymerase chain reaction (PCR) is an appealing and challenging technique. A real‐time PCR is an emerging technology frequently used to detect the aetiologic agents. In recent times, designing species‐specific primers for pathogen detection is gaining momentum. The method offers rapid, accurate and cost‐effective strategy to identify the target, thus providing sufficient time to instigate appropriate chemotherapy. The study highlights the use of intein DNA sequence as molecular markers for species‐specific identification of Candida tropicalis. The study also offers a prototype model for developing multifaceted PCR assays using intein DNA sequences, and provides a developmental starting point for point‐of‐care testing in near future.     

Validity of the Indicator Organism Paradigm for Pathogen Reduction in Reclaimed Water and Public Health Protection

The validity of using indicator organisms (total and fecal coliforms, enterococci, Clostridium perfringens, and F-specific coliphages) to predict the presence or absence of pathogens (infectious enteric viruses, Cryptosporidium, and Giardia) was tested at six wastewater reclamation facilities. Multiple samplings conducted at each facility over a 1-year period. Larger sample volumes for indicators (0.2 to 0.4 liters) and pathogens (30 to 100 liters) resulted in more sensitive detection limits than are typical of routine monitoring. Microorganisms were detected in disinfected effluent samples at the following frequencies: total coliforms, 63%; fecal coliforms, 27%; enterococci, 27%; C. perfringens, 61%; F-specific coliphages, ~40%; and enteric viruses, 31%. Cryptosporidium oocysts and Giardia cysts were detected in 70% and 80%, respectively, of reclaimed water samples. Viable Cryptosporidium, based on cell culture infectivity assays, was detected in 20% of the reclaimed water samples. No strong correlation was found for any indicator-pathogen combination. When data for all indicators were tested using discriminant analysis, the presence/absence patterns for Giardia cysts, Cryptosporidium oocysts, infectious Cryptosporidium, and infectious enteric viruses were predicted for over 71% of disinfected effluents. The failure of measurements of single indicator organism to correlate with pathogens suggests that public health is not adequately protected by simple monitoring schemes based on detection of a single indicator, particularly at the detection limits routinely employed. Monitoring a suite of indicator organisms in reclaimed effluent is more likely to be predictive of the presence of certain pathogens, and a need for additional pathogen monitoring in reclaimed water in order to protect public health is suggested by this study.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Presence of Cryptosporidium spp. and Giardia duodenalis in Drinking Water Samples in the North of Portugal

Cryptosporidium and Giardia are 2 protozoan parasites responsible for waterborne diseases outbreaks worldwide. In order to assess the prevalence of these protozoans in drinking water samples in the northern part of Portugal and the risk of human infection, we have established a long term program aiming at pinpointing the sources of surface water, drinking water, and environmental contamination, working with the water-supply industry. Total 43 sources of drinking water samples were selected, and a total of 167 samples were analyzed using the Method 1623. Sensitivity assays regarding the genetic characterization by PCR and sequencing of the genes, 18S SSU rRNA, for Cryptosporidium spp. and β,-giardin for G. duodenalis were set in the laboratory. According to the defined criteria, molecular analysis was performed over 4 samples. Environmental stages of the protozoa were detected in 25.7% (43 out of 167) of the water samples, 8.4% (14 out of 167) with cysts of Giardia, 10.2% (17 out of 167) with oocysts of Cryptosporidium and 7.2% (12 out of 167) for both species. The mean concentrations were 0.1-12.7 oocysts of Cryptosporidium spp. per 10 L and 0.1-108.3 cysts of Giardia duodenalis per 10 L. Our results suggest that the efficiency in drinking water plants must be ameliorated in their efficiency in reducing the levels of contamination. We suggest the implementation of systematic monitoring programs for both protozoa. To authors'' knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in drinking water samples in the northern part of Portugal.     

Cryptosporidium cuniculus and Giardia duodenalis in rabbits: genetic diversity and possible zoonotic transmission

Background

Cryptosporidium and Giardia are the two important zoonotic pathogens causing diarrhea of humans and animals worldwide. Considering the human cryptosporidiosis outbreak and sporadic cases caused by C. cuniculus, the important public health significance of G. duodenalis and little obtained information regarding rabbit infected with Cryptosporidium and Giardia in China, the aim of this study is to determine the prevalence and molecularly characterize Cryptosporidium and Giardia in rabbits in Heilongjiang Province, China.

Methodology/Principal Findings

378 fecal samples were obtained from rabbits in Heilongjiang Province. Cryptosporidium oocysts and Giardia cysts were detected using Sheather''s sugar flotation technique and Lugol''s iodine stain method, respectively. The infection rates of Cryptosporidium and Giardia were 2.38% (9/378) and 7.41% (28/378), respectively. Genotyping of Cryptosporidium spp. was done by DNA sequencing of the small subunit rRNA (SSU rRNA) gene and all the nine isolates were identified as Cryptosporidium cuniculus. The nine isolates were further subtyped using the 60-kDa glycoprotein (gp60) gene and two subtypes were detected, including VbA32 (n = 3) and a new subtype VbA21 (n = 6). G. duodenalis genotypes and subtypes were identified by sequence analysis of the triosephosphate isomerase (TPI) gene. The assemblage B (belonging to eight different subtypes B-I to B-VIII) was found in 28 G. duodenalis-positive samples.

Conclusions/Significance

The rabbits have been infected with Cryptosporidium and Giardia in Heilongjiang Province. The results show that the rabbits pose a threat to human health in the studied areas. Genotypes and subgenotypes of C. cuniculus and G. duodenalis in this study might present the endemic genetic characterization of population structure of the two parasites.