Waterborne Protozoan Parasites Research Articles

Lower micromolar activity of the antifungal imidazoles on the bacterial-type bifunctional aldehyde/alcohol dehydrogenase (AdhE) in Cryptosporidium parvum and in vitro efficacy against the zoonotic parasite

Cryptosporidium parvum is a waterborne and foodborne zoonotic protozoan parasite, a causative agent of moderate to severe diarrheal diseases in humans and animals. However, fully effective treatments are unavailable for medical and veterinary uses. There is a need to explore new drug targets for potential development of new therapeutics. Because C. parvum relies on anaerobic metabolism to produce ATP, fermentative enzymes in this parasite are attractive targets for exploration. In this study, we investigated the ethanol-fermentation in the parasite and characterized the basic biochemical features of a bacterial-type bifunctional aldehyde/alcohol dehydrogenase, namely CpAdhE. We also screened 3892 chemical entries from three libraries and identified 14 compounds showing >50% inhibition on the enzyme activity of CpAdhE. Intriguingly, antifungal imidazoles and unsaturated fatty acids are the two major chemical groups among the top hits. We further characterized the inhibitory kinetics of selected imidazoles and unsaturated fatty acids on CpAdhE. These compounds displayed lower micromolar activities on CpAdhE (i.e., IC50 values ranging from 0.88 to 11.02 μM for imidazoles and 8.93 to 35.33 μM for unsaturated fatty acids). Finally, we evaluated the in vitro anti-cryptosporidial efficacies and cytotoxicity of three imidazoles (i.e., tioconazole, miconazole and isoconazole). The three antifungal imidazoles exhibited lower micromolar efficacies against the growth of C. parvum in vitro (EC50 values ranging from 4.85 to 10.41 μM and selectivity indices ranging from 5.19 to 10.95). The results provide a proof-of-concept data to support that imidazoles are worth being further investigated for potential development of anti-cryptosporidial therapeutics.

A cloth-based hybridization array system for rapid detection of the food- and waterborne protozoan parasites Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii

Protozoan parasites in food or water samples are generally detected using microscopy or PCR followed by Sanger sequencing. However, microscopy is subjective, requires a high degree of expertise and has limited sensitivity, while DNA sequencing requires expensive and specialized equipment and facilities. This study describes a cloth-based hybridization array system (CHAS) that is an alternative to Sanger sequencing to confirm PCR-positive samples. CHAS is an inexpensive, rapid and reliable method for the simultaneous detection of multiple protozoan parasite species based on the colorimetric detection of PCR amplicons on a polyester cloth. PCR primers and CHAS hybridization probes were developed to detect the protozoan parasites Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii. In addition, CHAS probes were designed for the differentiation of G. duodenalis Assemblages A and B. In artificially contaminated fresh produce (lettuce, parsley) and water samples (river water, wastewater), this CHAS assay allowed for the successful detection of G. duodenalis, Cryptosporidium spp., and T. gondii. The present study demonstrates that the CHAS detection method is a simple and inexpensive alternative to DNA sequencing for the confirmation of PCR-positive results in laboratories testing for parasites in food or water samples. This assay may also be beneficial in developing countries, where DNA sequencing facilities may not be readily available.

Wastewater-based epidemiology-surveillance and early detection of waterborne pathogens with a focus on SARS-CoV-2, Cryptosporidium and Giardia.

Waterborne diseases are a major global problem, resulting in high morbidity and mortality, and massive economic costs. The ability to rapidly and reliably detect and monitor the spread of waterborne diseases is vital for early intervention and preventing more widespread disease outbreaks. Pathogens are, however, difficult to detect in water and are not practicably detectable at acceptable concentrations that need to be achieved in treated drinking water (which are of the order one per million litre). Furthermore, current clinical-based surveillance methods have many limitations such as the invasive nature of the testing and the challenges in testing large numbers of people. Wastewater-based epidemiology (WBE), which is based on the analysis of wastewater to monitor the emergence and spread of infectious disease at a population level, has received renewed attention in light of the current coronavirus disease 2019 (COVID-19) pandemic. The present review will focus on the application of WBE for the detection and surveillance of pathogens with a focus on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the waterborne protozoan parasites Cryptosporidium and Giardia. The review highlights the benefits and challenges of WBE and the future of this tool for community-wide infectious disease surveillance.

Cryptosporidium spp. and Giardia duodenalis emissions from humans and animals in the Three Gorges Reservoir in Chongqing, China.

Cryptosporidium spp. and Giardia duodenalis are two waterborne protozoan parasites that can cause diarrhea. Human and animal feces in surface water are a major source of these pathogens. This paper presents a GloWPa-TGR-Crypto model that estimates Cryptosporidium and G. duodenalis emissions from human and animal feces in the Three Gorges Reservoir (TGR), and uses scenario analysis to predict the effects of sanitation, urbanization, and population growth on oocyst and cyst emissions for 2050. Our model estimated annual emissions of 1.6 × 1015 oocysts and 2.1 × 1015 cysts from human and animal feces, respectively. Humans were the largest contributors of oocysts and cysts, followed by pigs and poultry. Cities were hot-spots for human emissions, while districts with high livestock populations accounted for the highest animal emissions. Our model was the most sensitive to oocyst excretion rates. The results indicated that 74% and 87% of total emissions came from urban areas and humans, respectively, and 86% of total human emissions were produced by the urban population. The scenario analysis showed a potential decrease in oocyst and cyst emissions with improvements in urbanization, sanitation, wastewater treatment, and manure management, regardless of population increase. Our model can further contribute to the understanding of environmental pathways, the risk assessment of Cryptosporidium and Giardia pollution, and effective prevention and control strategies that can reduce the outbreak of waterborne diseases in the TGR and other similar watersheds.

Food and waterborne protozoan parasites: The African perspective

Parasitic food-borne diseases, particularly those caused by the protozoan parasites Cryptosporidium, Giardia, Cyclospora cayetanensis and Entamoeba are increasingly becoming common and have received considerable attention in the last two decades. The ability of the transmission stages of the parasites to survive in the environment for prolonged periods, globalization of the food industry and changes in eating habits have contributed to the numbers of human infections. This systematic scoping review highlights these important water- and foodborne parasites in the African context, detailing the burden in African water sources, wastewater/effluents and fresh produce. A scoping review search targeting African countries was conducted in Medline, Web of science and African journals online as well as back referencing from included studies covering the period 1990 to January 2020. Out of 1134 studies, 68 were included in the review. The articles covered 17 out of 54 African countries. There were 39/68 studies reporting on water sources while the rest reported on fresh produce. Cryptosporidium prevalence ranged from 6 to 100% in surface water, 4 to 100% in tap water and up to 100% in wastewater and sludge. In fresh produce, Cryptosporidium was reported from five countries with prevalence of 0.8–75%. Giardia was reported in 47 out of 68 articles; prevalence ranged from 2.4% in surface water; 1% to over 70% in tap water; 28–100% in wastewater and 2% - 99% in fresh produce. Prevalence of Cyclospora cayetanensis was lower. Prevalence of Entamoeba was 78% in surface water; 100% in wastewater and up to 99% in fresh produce. This study finds that Africa is no exception to the risk presented by the subject parasites from water and/or food sources. Routine screening for these parasites particularly at household level and provision of adequate and safe drinking water would help to control the parasites.

Molecular characterization of <i>Cryptosporidium</i> spp. from migratory ducks around Tokachi subprefecture, Hokkaido, Japan

Cryptosporidium, a waterborne protozoan parasite, has a substantial veterinary and medical impact worldwide. This parasite is more often recognized during waterborne outbreaks because of its resistance to chlorine disinfection, small size making it difficult to inactivate/eliminate through filtration, and presence in many animal species including humans. Migratory waterfowl, in addition to acting as mechanical carriers of Cryptosporidium oocysts, can also serve as natural reservoirs of infection by host-specific Cryptosporidium species. For better understanding of the extent of genetic diversity and inter-relationships among avian isolates of Cryptosporidium, 200 fecal samples of migratory ducks from the Tokachi subprefecture, Hokkaido, Japan were collected and analyzed by nested PCR (N-PCR) at the 18S rRNA gene. N-PCR revealed that 11.5% (23/200) were positive for Cryptosporidium spp. Among all samples, sequence analysis identified that 10% (20/200) were 98–100% identical to Cryptosporidium avian genotype III. On the other hand, 1.5% (3/200) were 99–100% identical to C. baileyi. This is the first molecular study reporting the prevalence of Cryptosporidium in migratory ducks in Japan. Genetic diversity among Cryptosporidium isolates from humans and birds has been reported worldwide. Nevertheless, further studies are important to assess genetic variety and to elucidate the transmission dynamics of Cryptosporidium parasites.

Development of Molecular Diagnosis Using Multiplex Real-Time PCR and T4 Phage Internal Control to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from Human Stool Samples.

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler’s diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAM™, HEX™, Cy5™, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×103 copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler’s diarrhea.

Use of ultrasound irradiation to inactivate Cryptosporidium parvum oocysts in effluents from municipal wastewater treatment plants

Water reuse is currently considered an innovative way to addressing water shortage that can provide significant economic, social and environmental benefits, particularly -but not exclusively- in water deficient areas. The potential transmission of infectious diseases is the most common concern in relation to water reclamation. Cryptosporidium is an important genus of protozoan enteropathogens that infect a wide range of vertebrate hosts, including humans. The infective form (oocyst) is highly resistant to the environmental conditions and disinfection treatments. Consequently, Cryptosporidium is the most common etiological agent identified in waterborne outbreaks attributed to parasitic protozoa worldwide. The present study evaluates the efficacy of ultrasound disinfection, at three power levels (60, 80 and 100 W), pulsed at 50% or in continuous mode, for inactivating the waterborne protozoan parasite Cryptosporidium parvum in simulated and real effluents from municipal wastewater treatment plants (MWTPs). Overall interpretation of the results shows that the application of ultrasound irradiation at 80 W power in continuous mode for an exposure time of 10 min drastically reduced the viability of C. parvum. Thus, oocyst viabilities of 4.16 ± 1.93%; 1.29 ± 0.86%; 3.16 ± 0.69%; and 3.15 ± 0.87% were obtained in distilled water, simulated, real and filtered MWTP effluents, respectively (vs 98.57 ± 0.01%, initial oocyst viability), as determined using inclusion/exclusion of the fluorogenic vital dye propidium iodide, an indicator of the integrity of the oocyst wall. Independently of the mode used (pulsed/continuous) and at 80 W power, higher level of oocyst inactivation was detected in MWTP effluents than in distilled water used as a control solution, may be due to the differences in the chemical composition of the samples. Comparison of the results obtained in both modes showed that use of the continuous mode yielded significantly lower oocyst viability. However, when the Dose parameter was considered (energy per volume unit), no statistically significant differences in oocyst viability were observed in relation to the type of mode used. The results demonstrate that ultrasound technology represents a promising alternative to the disinfection methods (ultraviolet irradiation and chlorine products) currently used in water reclamation as it drastically reduces the survival of Cryptosporidium oocysts, without changing the chemical composition of the water or producing toxic by-products.

Comparison of in vitro viability methods for Cryptosporidium oocysts

The water-borne protozoan parasite Cryptosporidium parvum forms oocysts that can persist for long periods of time in the environment, even though the sporozoites inside the oocysts may no longer be viable, making it difficult to assess the associated risk of infection. In this study, we compared the ability of various in vitro methods to discriminate viable from non-viable oocysts, including excystation, DAPI/PI staining, RNA FISH, PMA-qPCR and a novel polymer slide adhesion method. With the notable exception of our in vitro excystation protocol, all methods were found to be useful for identifying viable oocysts.

Multiple identification of most important waterborne protozoa in surface water used for irrigation purposes by 18S rRNA amplicon-based metagenomics.

Understanding waterborne protozoan parasites (WPPs) diversity has important implications in public health. In this study, we evaluated a NGS-based method as a detection approach to identify simultaneously most important WPPs using 18S rRNA high-throughput sequencing. A set of primers to target the V4 18S rRNA region of WPPs such as Cryptosporidium spp., Giardia sp., Blastocystis sp., Entamoeba spp, Toxoplasma sp. and free-living amoebae (FLA) was designed. In order to optimize PCR conditions before sequencing, both a mock community with a defined composition of representative WPPs and a real water sample inoculated with specific WPPs DNA were prepared. Using the method proposed in this study, we have detected the presence of Giardia intestinalis, Acanthamoeba castellanii, Toxoplasma gondii, Entamoeba histolytica and Blastocystis sp. at species level in real irrigation water samples. Our results showed that untreated surface irrigation water in open fields can provide an important source of WPPs. Therefore, the methodology proposed in this study can establish a basis for an accurate and effective diagnostic of WPPs to provide a better understanding of the risk associated to irrigation water.

Intestinal parasitic infections: Current prevalence and risk factors among schoolchildren in capital area of the Republic of Marshall Islands

Intestinal parasitic infections (IPIs) among schoolchildren in Republic of Marshall Islands (RMI) largely remains unknown, thus investigation on IPIs status to establish the baseline data is urgently needed. This cross-sectional study intended to investigate the current IPIs status and associated risk factors among schoolchildren at capital of RMI. Single stool sample from 400 schoolchildren (207 boys and 193 girls) aged 9.73±2.50 yrs old was examined by employing merthiolate-iodine-formaldehyde concentration method. Demographic characteristics, uncomfortable symptoms and risk factors were obtained by questionnaires investigation. The overall prevalence of IPIs in schoolchildren was 22.8% (91/400), of them 24.2% harbored at least 2 different parasites. Notably, the majority was infected by waterborne protozoan parasites (82.4%, 75/91). Nine different intestinal parasites have been identified, of which six were pathogenic including Hook worm, Trichuris trichiura, Enterobius vermicularis, Entamoeba histolytica/dispar, Giardia intestinalis and Blastocystis hominis. Schoolchildren who ever complained dizziness or headache showed a significant higher prevalence of pathogenic IPIs than those who did not (p<0.05). Schoolchildren who lived in urban area than rural area had higher chance to acquire pathogenic IPIs (p=0.03). However, none of risk factors were identified to be associated with pathogenic IPIs.

Photocatalytic inactivation of the waterborne protozoan parasite Cryptosporidium parvum using TiO2/H2O2 under simulated and natural solar conditions

This is the first study that evaluates the efficacy of the photocatalytic disinfection against the waterborne protozoan parasite Cryptosporidium using a combination of TiO2 and H2O2 under simulated and natural solar conditions. Samples of distilled water containing 100mg/L of TiO2 and/or 50mg/L of H2O2 were spiked with purified Cryptosporidium parvum oocysts and exposed to solar radiation for 5h. The oocyst global viability was determined by inclusion/exclusion of the fluorogenic vital dye propidium iodide. A strong decrease in the oocyst global viability was observed in samples containing TiO2 and TiO2/H2O2 under simulated solar conditions (4.16±2.35% and 3.82±4.26%, respectively, vs 90.44±5.87%, initial global viability). Similarly, a drastic reduction in the oocyst global viability was observed under real sunlight (2.29±1.99% and 0.92±0.71% in samples containing TiO2 and TiO2/H2O2, respectively, vs 99.45±0.95%, initial global viability). These results prove the efficacy of the TiO2 photocatalytic disinfection against C. parvum, decreasing the time needed to reach the oocyst inactivation in comparison with exclusive solar disinfection. However, the addition of H2O2 at low concentrations (50mg/L) did not enhance the TiO2 photocatalytic process against Cryptosporidium.

Static mode microfluidic cantilevers for detection of waterborne pathogens

This paper reports on the first demonstration of polymeric microfluidic cantilever sensors. Microcantilever sensors, magnetic beads, and microfluidic technology have been combined to create a polymer based biosensor. Using cheap materials like polyimide, a simple fabrication method has been developed to produce cantilevers with an embedded microfluidic channel. The advantage of this approach is that the addition of a microfluidic channel enables the analysis of smaller volumes and increases the capture efficiency in applications detecting rare analytes. As a proof of principle the system has been applied for the detection of the waterborne protozoan parasite Cryptosporidium, achieving sensitivity comparable to QCM, whereas a previous set-up without the microfluidic channel was unable to detect the parasite.

Occurrence of Giardia in Different Water Sources in District Bannu

Background: After air, water is one of the greatest significant essentials for life, which is considered as one of the nutrients. Giardia lamblia (G. lamblia) is one of the most common waterborne protozoan parasites, causing diarrheal disease in human beings and animal diseases throughout the world. Material and Methods: A total of 150 containing 1.5 L from each water samples were collected from different water sources of district Bannu from 1 May, 2012 to 30 April, 2013 and for further Original Research Article Khan et al.; BMRJ, 6(2): 119-125, 2015; Article no.BMRJ.2015.064 120 process the samples were brought to the Department of Zoology Kohat University of Science and Technology, Kohat within 24 hours. Water samples containing different water sources (Tap water, Bore water, Stream water and Pond water) in seven different areas of District Bannu (Pakistan). The water was filtered through Whattman filter paper No. 42 having 2.5μm pore size and the residue was subjected to Microscopy, DNA extraction and PCR was conducted for detection of G. lamblia. To increase the sensitivity of the test a small region (125-bp) of the SSU rRNA was targeted for the PCR amplification. Results: The overall prevalence of G. lamblia in drinking water of district Bannu was 20% microscopically, including 28.33% in Stream water, 12.5% in Tap water, 20% in Tap water and was absent in Bore water. While that of PCR based study the overall prevalence of parasite (G. lamblia) was 24%, including Stream 28.33%, Tap water, 20%, Pond water 26.66% and Bore water 15 %. The highest prevalence of G. lamblia was 25% recorded in Tap water of Basia Khel through microscopic study & that of PCR based study, the highest prevalence was recorded in the Stream water of Bannu City which was 37.5% and P<.05 was considered significant. Conclusion: It was revealed from the current study that G. lamblia is present in water sources in some areas in district Bannu, which may be due to flooding and improper management of water scheme. The study recommended that a proper treatment of water for human consumption is required, especially in Bannu City and Basia Khel in district Bannu.

Human waterborne protozoan parasites in freshwater bivalves (Anodonta anatina and Unio tumidus) as potential indicators of fecal pollution in urban reservoir

The presence of environmentally robust dispersive stages of intestinal protozoan parasites in waters represents an important public health threat since these pathogens have caused numerous outbreaks related to either drinking or recreational waters. The purpose of this study was to evaluate the presence of Cryptosporidium oocysts, Giardia and Blastocystis cysts, and microsporidian spores in mussels collected from municipal reservoir, Lake Malta (Poland, Europe). Two species of freshwater bivalves ( Anodonta anatina and Unio tumidus ) were tested for the enteropathogens. A direct wet smear and smears stained with chromotrope 2R, Ziehl–Neelsen and iron hematoxylin made from each pellet of the hemolymph, gills and gastrointestinal homogenates of mussels were examined microscopically. In the study the immunofluorescence antibody test kit MERIFLUOR Cryptosporidium/Giardia was also used for all bivalve samples. None of investigated parasites were found in U. tumidus . In A. anatina , Cryptosporidium oocysts and Blastocystis cysts were detected in 15.4 and 5.1% of mussel samples, respectively. The present results indicate contamination of Lake Malta with Cryptosporidium and Blastocystis , which is important from the point of view of public health threats because of different human uses of studied reservoir.

Giardia duodenalis genotype A and B are responsible for post infectious visceral hypersensitivity in an immunocompetent suckling rat model: Is commensal bacterial translocation one of the mechanisms leading to PI‐IBS?

Enteric pathogens such as Giardia duodenalis, common waterborne protozoan parasite, have been linked to Post‐infectious Irritable Bowel Syndrome (PI‐IBS) a functional intestinal disorder, in which visceral hypersensitivity is a key factor. We aimed to develop a model characterizing PI‐IBS triggered by Giardia infection and, reveal mechanisms responsible for the PI‐IBS‐like symptoms, such as the role of the commensal microbiota. Five day‐old Sprague‐Dawley rats gavaged with 104 trophozoites (genotypes A or B) were assessed for visceral hypersensitivity 50 days post‐infection (PI) via jejunal/rectal balloon distension. Changes in blood pressure were recorded as measurements of visceral hypersensitivity. Caco‐2 monolayers were used to assess if Giardia facilitates the translocation of a non‐invasive E. coli at various time points. On day 50 PI, jejunal and rectal hypersensitivity was observed with both genotypes. G. duodenalis also facilitated translocation of non‐invasive E. coli through Caco‐2 monolayers 3h PI. Hypersensitivity was detected in the jejunum, as well as in the rectum, a site beyond the infection area, indicating that the effect is not limited to the presence of the pathogen. Giardia facilitates the passage of non‐invasive bacteria through the intestinal epithelium, mechanism which could cause PI‐IBS. This study describe a new model for the study of protozoa‐induced PI‐IBS.fundings: NSERC Create, FFCR (France‐Canada Research Fund), CCFC

Dysfunctions at human intestinal barrier by water-borne protozoan parasites: lessons from cultured human fully differentiated colon cancer cell lines

Some water-borne protozoan parasites induce diseases through their membrane-associated functional structures and virulence factors that hijack the host cellular molecules and signalling pathways leading to structural and functional lesions in the intestinal barrier. In this Microreview we analyse the insights on the mechanisms of pathogenesis of Entamoeba intestinalis, Giardia and Cryptosporidium observed in the human colon carcinoma fully differentiated colon cancer cell lines, cell subpopulations and clones expressing the structural and functional characteristics of highly specialized fully differentiated epithelial cells lining the intestinal epithelium and mimicking structurally and functionally an intestinal barrier.

PCR Detection of &lt;i&gt;Entamoeba histolytica&lt;/i&gt; in Microscopically Positive Stool Samples of Hospital Patients in Soroti, Eastern Uganda

Amoebiasis is an infection caused by water borne protozoan parasite Entamoeba histolytica. In Uganda where sanitation infrastructure and health education was not adequate, amoebiasis was thought to be still an important health problem. However there was little or no data on prevalence of this very important protozoan infection. In addition, microscopy remained the main method for the diagnosis of amoebiasis but could not differentiate between Entamoeba dispar/moshkovskii and Entamoeba histolytica infections. This made determination of true prevalence of Entamoeba histolytica infections difficult. It was against this background that this study was designed to carry out species specific diagnosis of Entamoeba histolytica and Entamoeba dispar/moshkovskii in Uganda where these species had been reported to be endemic. This study used microscopy and polymerase chain reaction amplification of Serine-rich Entamoeba histolytica (SREHP) gene. It was shown that 36.7% (n=22) of the samples initially diagnosed as positive by microscopy were positive by PCR. The true prevalence of E. histolytica and E.dispar/ moshkovskii was found to be 7.31% and 12.6% respectively. It was concluded that Entamoeba infection in Soroti, Eastern Uganda is more frequently due to E. dispar /moshkovskii (13.3%) the non-pathogenic forms than to E. histolytica, the pathogen (7.31%).Key words: Entamoeba histolytica, Microscopy, Polymerase chain reaction, Prevalence.

Comparison of immobilization strategies for Cryptosporidium parvum immunosensors

Cryptosporidium is a waterborne protozoan parasite, which is problematic for the water industry due to widespread environmental presence, low infectious dose and resistance to chlorine disinfection. To replace the existing, slow regulatory monitoring procedure, immunosensors have been proposed. The performance of such sensors is often limited by the antibody immobilization. The aim of this article was to determine the immobilization protocol offering the highest oocyst capture efficiency. Four methods were tested both under static and convective conditions: physisorption, cysteamine–glutaraldehyde linkage, 3-aminopropyl-triethoxysilane functionalization and protein G linkage. The protein G protocol was shown to present the highest recovery rates in both conditions. For the protein G protocol, different antibody concentrations were tested and it was concluded that there is little difference in the recovery rates when the concentration of IgG antibody is higher than 20μg/mL. Additionally, operation under convective rather than static conditions increased the recovery for every protocol. These results can be useful to inform the design of future biosensor systems, using antibodies as the detection element, for Cryptosporidium.

Biofilm roughness determines Cryptosporidium parvum retention in environmental biofilms.

The genus Cryptosporidium is a group of waterborne protozoan parasites that have been implicated in significant outbreaks of gastrointestinal infections throughout the world. Biofilms trap these pathogens and can contaminate water supplies through subsequent release. Biofilm microbial assemblages were collected seasonally from three streams in eastern Pennsylvania and used to grow biofilms in laboratory microcosms. Daily oocyst counts in the influx and efflux flow allowed the calculation of daily oocyst retention in the biofilm. Following the removal of oocysts from the influx water, oocyst attachment to the biofilm declined to an equilibrium state within 5 days that was sustained for at least 25 days. Varying the oocyst loading rate for the system showed that biofilm retention could be saturated, suggesting that discrete binding sites determined the maximum number of oocysts retained. Oocyst retention varied seasonally but was consistent across all three sites; however, seasonal oocyst retention was not consistent across years at the same site. No correlation between oocyst attachment and any measured water quality parameter was found. However, oocyst retention was strongly correlated with biofilm surface roughness and roughness varied among seasons and across years. We hypothesize that biofilm roughness and oocyst retention are dependent on environmentally driven changes in the biofilm community rather than directly on water quality conditions. It is important to understand oocyst transport dynamics to reduce risks of human infection. Better understanding of factors controlling biofilm retention of oocysts should improve our understanding of oocyst transport at different scales.