Oo Cysts Research Articles

Protozoa as the “Underdogs” for Microbiological Quality Evaluation of Fresh Vegetables

The monitoring of the microbial quality of fresh products in the industrial environment has mainly focused on bacterial indicators. Protozoa, such as Giardia duodenalis, Cryptosporidium spp., Toxoplasma gondii, and Cyclospora cayetanensis, are routinely excluded from detection and surveillance systems, despite guidelines and regulations that support the need for tracking and monitoring these pathogens in fresh food products. Previous studies performed by our laboratory, within the scope of the SafeConsume project, clearly indicated that consumption of fresh produce may be a source of T. gondii, thus posing a risk for the contraction of toxoplasmosis for susceptible consumers. Therefore, preliminary work was performed in order to assess the microbiological quality of vegetables, highlighting not only bacteria (Escherichia. coli, Listeria monocytogenes, and Salmonella spp.), but also the zoonotic protozoa G. duodenalis and Cryptosporidium spp. Although all samples were found to be acceptable based on bacteriological parameters, cysts of G. duodenalis and oocysts of Cryptosporidium spp. were observed in vegetables. Moreover, it was possible to genetically characterize G. duodenalis positive samples as assemblage A, a genotype that poses risks to human health. Although these are preliminary results, they highlight the need to include protozoa in the microbiological criteria for foodstuffs, as required by EU Law No. 1441/2007, and to improve inactivation and removal procedures of (oo)cysts in fresh produce and water.

Removal of Giardia spp. cysts and Cryptosporididum spp. oocysts from anaerobic effluent by dissolved air flotation

ABSTRACT Lab-scale studies were carried out to investigate the efficiency of dissolved air flotation (DAF) for the removal of Giardia spp. cysts and Cryptosporidium spp. oocysts from anaerobic effluent from the pilot UASB reactor. Raw wastewater, UASB and DAF effluent samples were collected weekly and protozoan (oo)cysts were concentrated using IMS followed by protozoa detection using immunofluorescense assay (IFA). The number of Cryptosporidium spp. oocysts in the raw wastewater was always lower than that of Giardia spp. cysts with 28–33 oocysts L−1 and 3177–4267 cysts L−1, respectively. Log10 removal of Giardia cysts utilising polyaluminium chloride (PACl) was higher than that with FeCl3, but no statistically significant difference between the two coagulants was observed. Cryptosporidium was absent in most of the treated effluent samples. The results indicate that DAF reached more than 2 log of cyst removal. In addition, the results demonstrated that these parasites are prevalent in the study area and E. coli and total coliforms were not good indicator microorganisms in terms of cyst and oocysts numbers.

Humoral and cytokine response elicited during immunisation with recombinant Immune Mapped protein-1 (EtIMP-1) and oocysts of Eimeria tenella

Eimeria tenella, the causative agent of caecal coccidiosis, is a pathogenic gut dwelling protozoan which can cause severe morbidity and mortality in farmed chickens. Immune mapped protein-1 (IMP-1) has been identified as an anticoccidial vaccine candidate; in the present study allelic polymorphism was assessed across the IMP-1 coding sequence in E. tenella isolates from four countries and compared with the UK reference Houghton strain. Nucleotide diversity was low, limited to expansion/contraction of a CAG triplet repeat and five substitutions, three of which were non-synonymous. The EtIMP-1 coding sequence from a cloned Indian E. tenella isolate was expressed in E. coli and purified as a His-tagged thioredoxin fusion protein. An in-vivo vaccination and challenge trial was conducted to test the vaccine potential of recombinant EtIMP-1 (rEtIMP-1) and to compare post-vaccination immune responses of chickens to those stimulated by live oocyst infection. Following challenge, parasite replication measured using quantitative PCR was significantly reduced in chickens that had been vaccinated with rEtIMP-1 (rIC group; 67% reduction compared to UC or unimmunised controls; 79% reduction compared to rTC group or recombinant thioredoxin mock-immunised controls, p<0.05), or the birds vaccinated by infection with oocysts (OC group, 90% compared to unimmunised controls). Chickens vaccinated with oocysts (OC) had significantly higher levels of interferon gamma in their serum post-challenge, compared to rEtIMP-1 vaccinated birds (rIC). Conversely rEtIMP-1 (rIC) vaccinated birds had significantly higher antigen specific serum IgY responses, correlating with higher serum IL-4 (both p<0.05).

Immunization of Wistar female rats with 255-Gy-irradiated Toxoplasma gondii: Tissue parasitic load and lactogenic quantification

Toxoplasma gondii is one of the most significant parasite, due its importance in veterinary medicine and in public health, considered a food-borne pathogens, there is no available drug treatments to eliminate it from animal tissue, this reinforce the search for a vaccine against this parasite. This study was aimed to evaluate the dynamic of the distribution of T. gondii in tissues of female Wistar rats and their milk, after the immunization by oral rote with irradiated tachyzoites. One week after pregnancy confirmation, rats was challenged by gavage with T. gondii bradyzoites, oocysts or tachyzoites of T. gondii. Forty-eight pregnant rats were grouped as follows: immunized and challenged with bradyzoites (BZ*); non-immunized and challenged with bradyzoites (BZ); immunized and challenged with oocysts (OC*); non-immunized and challenged with oocysts (OC); immunized and challenged with tachyzoites (TZ*); non-immunized and challenged with tachyzoites (TZ); only immunized (I); control group (C). After parturition, milk samples were collected for 3 weeks and then rats were sacrificed and the tissues and milk samples were researched for T. gondii parasite load determined by the quantitative PCR (qPCR). It was verified that the immunization with irradiated tachyzoites of T. gondii induced the reduction of parasitic load in muscle samples in rats challenged by bradyzoites and oocysts, although not enabled the development of sterile immunity. The detection of parasite DNA in milk was found throughout the lactation period, from immunized and non-immunized rats, however no differences were found in the parasite load caused by immunization.

Immunization of Wistar female rats with 255-Gy-irradiated Toxoplasma gondii: Preventing parasite load and maternofoetal transmission

Toxoplasmosis, caused by an obligate intracellular protozoan parasite, Toxoplasma gondii, is an worldwide parasitic disease, with significant importance for animal production and considerable impact to the public health. This study was aimed to evaluate the dynamic of the distribution of T.gondii in tissues of female Wistar rats and their puppies tissues, after the immunization by oral rote with irradiated tachyzoites. One week after pregnancy confirmation, rats was challenged by gavage with T. gondii bradyzoites, oocysts or tachyzoites of T. gondii. Forty-eight pregnant rats were grouped as follow: immunized and challenged with bradyzoites (BZ∗); non-immunized and challenged with bradyzoites (BZ); immunized and challenged with oocysts (OC∗); non-immunized and challenged with oocysts (OC); immunized and challenged with tachyzoites (TZ∗); non-immunized and challenged with tachyzoites (TZ); only immunized (I); control group (C). After parturition the rats were sacrificed and the tissues were researched for the DNA of T. gondii by polymerase chain reaction (PCR) and the parasite load determined by the quantitative PCR (qPCR). It was verified that the immunization with irradiated tachyzoites of T. gondii induced the reduction of parasitic load in most organs analyzed, although not prevent the establishment of infection with the parasite. And also, the immunization showed a favorable effect on the birth rate and litter size.

Recovery of Cryptosporidium and Giardia organisms from surface water by counter-flow refining microfiltration

As waterborne parasitic cryptosporidiosis and giardiasis outbreaks continue globally, monitoring of Cryptosporidium parvum and Giardia lamblia in surface water continues to be challenging. Lack of non-clogging and high-efficiency methods for recovery of C. parvum oocysts and G. lamblia cysts in environmental water strongly limits the sensitivity of detection methods for these protozoan organisms. In this work, the Counter-Flow Micro-Refinery (CFMR) system was developed by employing the novel counter-flow microfiltration principle to enrich (oo)cysts for subsequent analytical purposes. The CFMR system was constructed with multiple counter-flow concentration units that were arranged into two refining levels. By use of different numbers of units, the CFMR offered an adjustable concentration ratio allowing the concentration of 10 L and 100 L to hundreds of mL with no recirculation processing. With spiked samples, recovery of 81.3% oocysts and 86.2% cysts at a variance of<7% was achieved for concentrations as low as 0.5–100 organisms L−1. The recovery efficiency showed consistent for a wide range of water turbidities as well as different sample volumes. No significant clogging has been observed in the experiments. Moreover, the refining filter was able to enrich and separate oocysts and cysts in water, simultaneously. This work verifies a feasible solution for recovering C. parvum oocysts and G. lamblia cysts in large-volume surface waters. The refining system has potential to be a high-efficiency monitoring tool when combined with proper analytical detection methods.

Detecting Cryptosporidium parvum and Giardia lamblia by coagulation concentration and real-time PCR quantification

Rapid and sensitive pathogen detection methods are essential for the effective functioning of the water treatment industry, yet for many pathogens, effective detection and removal methods are still lacking. Cryptosporidium parvum oocysts and Giardia lamblia cysts are two of the most common waterborne pathogens currently infecting the water supply. In this study, a new method was developed for the detection of Cryptosporidium parvum oocysts and Giardia lamblia cysts. The method includes multi-steps as coagulation concentration of (oo)cysts in water, the dissolution of the resulting flocs into a small volume using acid, filtration of the (oo)cysts solution, and DNA extraction, purification, and examination using real-time PCR. The method was tested using spiked tap water and reservoir water as references, and the mean recovery ranged from 19.6% to 97.6% for oocysts, and from 51.4% to 98.7% for cysts. The method is economical and convenient, and is especially suitable for relatively high turbidity surface water.

Enumeration of Cryptosporidium sp. and Giardia sp. (oo)cysts in a tropical eutrophic lake

The biodynamic of Cryptosporidium sp. oocysts and Giadia sp. cysts have been assessed in the municipal lake of Yaounde, which is an artificial eutrophic lake that has been constructed on the Mingoa stream of the Mfoundi River Basin of Cameroon. The oocysts were identified by the Ziehl-Neelsen method while the cysts were identified by the Lugol iodine coloration. The values obtained suggest an increase in (oo)cysts density from the surface to the bottom of the lentic ecosystem. The oocysts varied from 56 oocysts/L in the upper layer to 2640 oocysts/L of water in the lowest layer, while the cysts population dynamics ranged from 24 cysts/L at the surface layer of the lake to 1713 cysts/L at the lowest layers of the lake. There was a considerable reduction in the resistant forms of these emerging pathogenic protozoa between the surface water of the Mingoa stream entering the Lake and that coming out from the Lake. The highest value of Cyptosporidium oocysts determined at the entrance of the lake is 1480 oocysts/L while at the outlet the highest value of oocysts identified is 620 oocysts/L. The highest value of Giardia cysts determined at the entrance of the lake is 352 cysts/L while at the outlet the highest value recorded is 294 cysts/L.

Evaluation of cyst loss in standard procedural steps for detecting ofGiardia lamblia andCryptosporidium parvum in water

The standard procedure outlined by the United States Environmental Protection Agency (US EPA) in Method 1623 for analyzingGiardia lamblia cysts andCryptosporidium parvum oocysts in water samples consists of filtration, elution, centrifugal concentration, immunomagnetic separation (IMS), and immunofluorescence assay (IFA) followed by microscopic examination. In this study, the extent of (oo)cyst loss in each step of this procedure was evaluated by comparing recovery yields in segmented analyses: (i) IMS+IFA, (ii) concentration +IMS+IFA, and (iii) filtration/elution + concentration +IMS+IFA. The complete (oo)cyst recovery by the full procedure was 52–57%. The (oo)cyst loss in the IMS step was only 0–6%, implying that IMS is a fairly reliable method for (oo)cyst purification. Centrifugal concentration of the eluted sample and pellet collection before IMS resulted in a loss of 8–14% of the (oo)cysts. The largest (oo)cyst loss occurred in the elution step, with 68–71% of the total loss. The permeated loss of (oo)cysts was negligible during filtration of the water sample with a 1.0-μm pore polyethersulfone (PES) capsule. These results demonstrated that the largest fraction of (oo)cyst loss in this procedure occurred due to poor elution from the filter matrix. Improvements in the elution methodology are therefore required to enhance the overall recovery yield and the reliability of the detection of these parasitic protozoa.

A Practical Assessment of the Use of Particle Counters for Monitoring the Risk of Cryptosporidium and Giardia

AbstractThe paper describes a project which was carried out to establish whether particle counts can be used to predict the risk of Cryptosporidium and Giardia breakthrough at a large water‐treatment works. The project was based at a direct river‐abstraction works which treats up to 120 MI/d. On‐line particle counters were installed following three of the rapid‐gravity filters, and a fourth counter was used to measure particles in spot samples of raw and clarified water.There was a clear relationship between particle counts and the occasional low‐level (oo)cyst (crypto oocysts and giardia cysts) occurrence in the filter samples, and no (oo)cysts were found in the final water. There appear to be complex inter‐relationships between particle counts and other parameters such as raw‐water conditions, environmental factors and treatment‐plant variables.

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.

Recovery and viability ofCryptosporidium parvumoocysts andGiardia intestinaliscysts using the membrane dissolution procedure

Previously, the cellulose acetate membrane filter dissolution method was reported to yield Cryptosporidium parvum oocyst recoveries of 70.5%, with recovered oocysts retaining their infectivity. In contrast, high spike doses (approximately 1 × 105Cryptosporidium parvum oocysts and Giardia intestinalis cysts) yielded recoveries ranging from 0.4% to 83.9%, and 3.2% to 90.3%, respectively, in this study. Recoveries with low spike doses (approximately 100 (oo)cysts) continued to demonstrate high variability also. Efforts to optimize the method included increased centrifugation speeds, suspension of the final concentrate in deionized water for organism detection on well slides, and analysis of the entire concentrate. A comparison of two monoclonal antibodies was also conducted to identify potential differences between antibodies in detection of organisms. Archived source and finished water samples were spiked, yielding variable recoveries of C. parvum oocysts (11.8% to 71.4%) and G. intestinalis cysts (7.4% to 42.3%). Effects of organic solvents used in the membrane dissolution procedure on the viability of recovered (oo)cysts was determined using a fluorogenic vital dyes assay in conjunction with (oo)cyst morphology, which indicated &gt;99% inactivation. These data indicate that the membrane dissolution procedure yields poor and highly variable (oo)cyst recoveries, and also renders the majority of recovered organisms non-viable.Key words: Giardia, Cryptosporidium, membrane dissolution, viability.

Recovery and viability of &lt;i&gt;Cryptosporidium parvum&lt;/i&gt; oocysts and &lt;i&gt;Giardia intestinalis&lt;/i&gt; cysts using the membrane dissolution procedure

Previously, the cellulose acetate membrane filter dissolution method was reported to yield Cryptosporidium parvum oocyst recoveries of 70.5%, with recovered oocysts retaining their infectivity. In contrast, high spike doses (approximately 1 x 10(5) Cryptosporidium parvum oocysts and Giardia intestinalis cysts) yielded recoveries ranging from 0.4% to 83.9%, and 3.2% to 90.3%, respectively, in this study. Recoveries with low spike doses (approximately 100 (oo)cysts) continued to demonstrate high variability also. Efforts to optimize the method included increased centrifugation speeds, suspension of the final concentrate in deionized water for organism detection on well slides, and analysis of the entire concentrate. A comparison of two monoclonal antibodies was also conducted to identify potential differences between antibodies in detection of organisms. Archived source and finished water samples were spiked, yielding variable recoveries of C. parvum oocysts (11.8% to 71.4%) and G. intestinalis cysts (7.4% to 42.3%). Effects of organic solvents used in the membrane dissolution procedure on the viability of recovered (oo)cysts was determined using a fluorogenic vital dyes assay in conjunction with (oo)cyst morphology, which indicated > 99% inactivation. These data indicate that the membrane dissolution procedure yields poor and highly variable (oo)cyst recoveries, and also renders the majority of recovered organisms non-viable.

Improved detection and vital staining of Cryptosporidium and Giardia with flow cytometry

Flow cytometry with fluorescence activated cell sorting (FACS) was used to purify Cryptosporidium (oo)cysts and Giardia cysts from water. With this purification step Cryptosporidium and Giardia were found in a higher percentage of the samples and significantly higher Giardia concentrations were detected in these positive samples. Because FACS removed most of the debris from the concentrated water sample, the microscopic preparations could be examined more rapidly for the presence of (oo)cysts and the morphological characteristics of the (oo)cysts could be interpreted more unambiguously than with the conventional immunofluorescence microscopy method. The use of FACS made it possible to apply PI-staining on environmental samples to determine the fraction of dead (oo)cysts. Sample processing did not appear to influence the PI-staining characteristics of the Cryptosporidium (oo)cysts, but did increase the percentage of PI-positive Giardia cysts. This suggests that this protocol can be used for determining the percentage of dead Cryptosporidium oocysts in environmental samples. Analysis of environmental samples suggests that reservoir storage increases the percentage of PI-positive (dead) oocysts.

Occurrence of Cryptosporidium spp oocysts and Giardia spp cysts in sewage influents and effluents from treatment plants in England

Cryptosporidium parvum and Giardia duodenalis can cause severe diarrhoea in infected individuals and their transmissive stages, oocysts and cysts, are voided in large numbers with the faeces of infected hosts. Contaminated sewage effluents are recognised as a potential source of waterborne (oo)cysts. In this investigation methods optimised for the recovery of both from a range of wastewaters were used to determine the occurrence of these organisms in influents, effluents and sludges from seven sewage treatment works in England. The data indicated the presence of small numbers of oocysts both in sewage influent and effluent samples whereas cysts were detected more frequently and at higher concentrations in both influents and effluents. Whilst sludge samples from 1/5 sites contained oocysts, cysts were detected from all five sites. These investigations indicate that discharge of sewage effluents into a watercourse, which may be used for potable water abstraction, can contaminate that watercourse with potentially infectious oocysts. In addition, the application of sludge to land can be responsible for contaminating watercourses with (oo)cysts following run-off or leaching.