Cryptosporidium spp. in Wild,Laboratory, and Pet Rodents in China: Prevalence and Molecular Characterization

To understand the prevalence of Cryptosporidium infection in rodents in China and to assess the potential role of rodents as a source for human cryptosporidiosis, 723 specimens from 18 rodent species were collected from four provinces of China and examined between August 2007 and December 2008 by microscopy after using Sheather''s sugar flotation and modified acid-fast staining. Cryptosporidium oocysts were detected in 83 specimens, with an overall prevalence of 11.5%. Phodopus sungorus, Phodopus campbelli, and Rattus tanezumi were new reported hosts of Cryptosporidium. The genotypes and subtypes of Cryptosporidium strains in microscopy-positive specimens were further identified by PCR and sequence analysis of the small subunit rRNA and the 60-kDa glycoprotein (gp60) genes. In addition to Cryptosporidium parvum, C. muris, C. andersoni, C. wrairi, ferret genotype, and mouse genotype I, four new Cryptosporidium genotypes were identified, including the hamster genotype, chipmunk genotype III, and rat genotypes II and III. Mixed Cryptosporidium species/genotypes were found in 10.8% of Cryptosporidium-positive specimens. Sequence analysis of the gp60 gene showed that C. parvum strains in pet Siberian chipmunks and hamsters were all of the subtype IIdA15G1, which was found previously in a human isolate in The Netherlands and lambs in Spain. The gp60 sequences of C. wrairi and the Cryptosporidium ferret genotype and mouse genotype I were also obtained. These findings suggest that pet rodents may be potential reservoirs of zoonotic Cryptosporidium species and subtypes.Cryptosporidium spp. are protozoan parasites that infect a wide range of vertebrates, including humans. Cryptosporidiosis is acute and self-limiting in immunocompetent hosts but life threatening in immunocompromised individuals (48). Humans and animals can acquire Cryptosporidium infection through direct contact with infected individuals or contaminated fomites or by consumption of contaminated food or water (16, 47). Rodents, which are abundant and widespread, have been considered reservoirs of cryptosporidiosis in humans and farm animals. Previous studies based on oocyst morphology showed that many wild rodents might serve as hosts of Cryptosporidium parvum-like and C. muris-like parasites (4, 8, 42). The reported prevalence rates of Cryptosporidium in rodents ranged from 5.0% to 39.2% (11-13). Nearly 40 rodent species belonging to 11 families (Sciuridae, Muridae, Cricetidae, Castoridae, Geomyidae, Hystricidae, Erethizontidae, Myocastoridae, Caviidae, Hydrochoeridae, and Chinchillidae) have been reported as hosts of Cryptosporidium spp. (10, 12, 30, 53).Recently, PCR-based genotyping and subtyping tools have been used in assessing the human-infective potential of Cryptosporidium spp. in animals and the extent of cross-species transmission of cryptosporidiosis in animals (47, 49, 51). Five Cryptosporidium species and nearly 20 Cryptosporidium genotypes of uncertain species status have been identified in rodents worldwide in recent studies (3, 6, 12, 13, 18-20, 23, 26, 30, 31, 36, 39, 52, 53). Among them, C. parvum, C. meleagridis, cervine genotype, C. muris, C. andersoni, chipmunk genotype I, and skunk genotype have been associated with cryptosporidiosis in humans although the last four species and genotypes are each responsible for only one or a few cases (47). Subtyping based on sequence analysis of the 60-kDa glycoprotein (gp60) gene has been used in tracking the transmission of six Cryptosporidium species and genotypes, including C. hominis, C. parvum, C. meleagridis, C. fayeri, and the rabbit and horse genotypes (7, 37). There are at least 10 gp60 subtype families of C. parvum, two (IIa and IId) of which are involved in zoonotic transmission. In rodents, natural C. parvum infection is rare (11), and only one C. parvum subtype (IIaA15G2R1) has been reported in capybaras (Hydrochoerus hydrochaeris) in Brazil (30).Until recently there has been no genetic characterization of Cryptosporidium spp. in rodents in China. Worldwide, there are also hardly any genetic data on Cryptosporidium spp. from pet rodents. The purpose of this study was to determine the prevalence of Cryptosporidium in some wild, laboratory, and pet rodents in China and to assess the zoonotic potential of Cryptosporidium spp. from rodents.     

Development of a Two-Color Fluorescence In Situ Hybridization Technique for Species-Level Identification of Human-Infectious Cryptosporidium spp.

A two-color fluorescence in situ hybridization assay that allows for the simultaneous identification of Cryptosporidium parvum and C. hominis was developed. The assay is a simple, rapid, and cost-effective tool for the detection of the major Cryptosporidium species of concern to public health.Cryptosporidium (Apicomplexa) is a genus of protozoan parasites with species and genotypes that infect humans, domesticated livestock, companion animals, and wildlife worldwide (5, 6, 14, 15, 20, 23). The majority of cases of cryptosporidiosis in humans are caused by Cryptosporidium parvum or C. hominis (8, 10, 19, 24), although rare cases due to species such as C. meleagridis, C. felis, or C. canis have been reported (8, 9, 11-13, 17, 18, 22). The specific identification and characterization of Cryptosporidium species are central to the control of this disease in humans and a wide range of animals.One of the most widely adopted techniques for the identification of microorganisms in complex microbial communities is fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes (2-4). This method relies on the hybridization of synthetic oligonucleotide probes to specific regions within the rRNA of the organism. While FISH has been applied for the detection of Cryptosporidium oocysts in water samples (21), no FISH probes that successfully differentiate C. hominis from C. parvum have been reported.We have reported previously on the design of a species-specific probe, Cpar677, that detects C. parvum (1). In this study, we report on the design and validation of a C. hominis species-specific probe, Chom253. Together, the two probes were used here for the development of a two-color, microscopy-based FISH assay for the simultaneous detection of C. parvum and C. hominis.     

Effect of Daily Temperature Fluctuation during the Cool Season on the Infectivity of Cryptosporidium parvum

The present work calculated the rate of inactivation of Cryptosporidium parvum oocysts attributable to daily oscillations of low ambient temperatures. The relationship between air temperature and the internal temperature of bovine feces on commercial operations was measured, and three representative 24-h thermal regimens in the ∼15°C, ∼25°C, and ∼35°C ranges were chosen and emulated using a thermocycler. C. parvum oocysts suspended in deionized water were exposed to the temperature cycles, and their infectivity in mice was tested. Oral inoculation of 10³ treated oocysts per neonatal BALB/c mouse (∼14 times the 50% infective dose) resulted in time- and temperature-dependent reductions in the proportion of infected mice. Oocysts were completely noninfectious after 14 24-h cycles with the 30°C regimen and after 70 24-h cycles with the 20°C regimen. In contrast, oocysts remained infectious after 90 24-h cycles with the 10°C regimens. The estimated numbers of days needed for a 1-log₁₀ reduction in C. parvum oocyst infectivity were 4.9, 28.7, and 71.5 days for the 30, 20, and 10°C thermal regimens, respectively. The loss of infectivity of oocysts induced by these thermal regimens was due in part to partial or complete in vitro excystation.It is well recognized that the protozoan parasite Cryptosporidium parvum causes waterborne enteric disease and poses a significant threat to public health. Fecal contamination from infected hosts, such as humans and some species of livestock and wildlife (17), can lead to elevated concentrations of C. parvum oocysts in drinking, recreational, and irrigation water supplies (6, 8). Once excreted, C. parvum oocysts can be eluted from fresh fecal matrices during precipitation events that generate surface flow or runoff conditions (4, 5, 12, 21, 32). During cool moist conditions oocysts can persist for months in the environment (10, 11, 25, 30), but factors such as extremes of temperature, exposure to UV radiation, and desiccation can substantially reduce the number of infective oocysts prior to waterborne transport (2, 7, 9, 11, 19, 24, 25, 29, 30).To examine thermal stress, most studies have used constant thermal regimens to investigate the effect of temperature on the viability or infectivity of Cryptosporidium oocysts (11, 14, 20, 28, 30). To complement this work, we previously investigated the impact of large daily changes in the ambient temperature on C. parvum oocyst infectivity, using spring through autumn thermal regimens and temperatures measured inside bovine fecal pats that were exposed to solar radiation at cow-calf and dairy production facilities (23). Under California''s summer climatic conditions, internal fecal pat temperatures range from 45°C to 75°C during the day and decrease 10 to 60°C during the night. Exposing oocysts to these large thermal fluctuations results in >3.3-log₁₀ reductions in oocyst infectivity in each 24-h cycle (23). The present study was conducted in order to measure the effect of exposure to oocysts to cool-season daily temperatures (with peaks at temperatures greater than 10°C, 20°C, and 30°C) on the rate of inactivation of C. parvum oocysts. Determining the temperature-dependent rate of C. parvum oocyst inactivation for these lower temperatures would allow grazing management and source water assessment plans to more properly predict the amount of time needed for exclusion of cattle prior to the onset of winter precipitation in order to inactivate sufficient numbers of oocysts in critical watersheds.     

Evidence for Mucin-Like Glycoproteins That Tether Sporozoites of Cryptosporidium parvum to the Inner Surface of the Oocyst Wall

Cryptosporidium parvum oocysts, which are spread by the fecal-oral route, have a single, multilayered wall that surrounds four sporozoites, the invasive form. The C. parvum oocyst wall is labeled by the Maclura pomifera agglutinin (MPA), which binds GalNAc, and the C. parvum wall contains at least two unique proteins (Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) identified by monoclonal antibodies. C. parvum sporozoites have on their surface multiple mucin-like glycoproteins with Ser- and Thr-rich repeats (e.g., gp40 and gp900). Here we used ruthenium red staining and electron microscopy to demonstrate fibrils, which appear to attach or tether sporozoites to the inner surface of the C. parvum oocyst wall. When disconnected from the sporozoites, some of these fibrillar tethers appear to collapse into globules on the inner surface of oocyst walls. The most abundant proteins of purified oocyst walls, which are missing the tethers and outer veil, were COWP1, COWP6, and COWP8, while COWP2, COWP3, and COWP4 were present in trace amounts. In contrast, MPA affinity-purified glycoproteins from C. parvum oocysts, which are composed of walls and sporozoites, included previously identified mucin-like glycoproteins, a GalNAc-binding lectin, a Ser protease inhibitor, and several novel glycoproteins (C. parvum MPA affinity-purified glycoprotein 1 [CpMPA1] to CpMPA4). By immunoelectron microscopy (immuno-EM), we localized mucin-like glycoproteins (gp40 and gp900) to the ruthenium red-stained fibrils on the inner surface wall of oocysts, while antibodies to the O-linked GalNAc on glycoproteins were localized to the globules. These results suggest that mucin-like glycoproteins, which are associated with the sporozoite surface, may contribute to fibrils and/or globules that tether sporozoites to the inner surface of oocyst walls.Cryptosporidium parvum and the related species Cryptosporidium hominis are apicomplexan parasites, which are spread by the fecal-oral route in contaminated water and cause diarrhea, particularly in immunocompromised hosts (1, 12, 39, 47). The infectious and diagnostic form of C. parvum is the oocyst, which has a single, multilayered, spherical wall that surrounds four sporozoites, the invasive forms (14, 27, 31). The outermost layer of the C. parvum oocyst wall is most often absent from electron micrographs, as it is labile to bleach used to remove contaminating bacteria from C. parvum oocysts (27). We will refer to this layer as the outer veil, which is the term used for a structure with an identical appearance on the surface of the oocyst wall of another apicomplexan parasite, Toxoplasma gondii (10). At the center of the C. parvum oocyst wall is a protease-resistant and rigid bilayer that contains GalNAc (5, 23, 43). When excysting sporozoites break through the oocyst wall, the broken edges of this bilayer curl in, while the overall shape of the oocyst wall remains spherical.The inner, moderately electron-dense layer of the C. parvum oocyst wall is where the Cryptosporidium oocyst wall proteins (Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) have been localized with monoclonal antibodies (4, 20, 28, 32). COWPs, which have homologues in Toxoplasma, are a family of nine proteins that contain polymorphic Cys-rich and His-rich repeats (37, 46). Finally, on the inner surface of C. parvum oocyst walls are knob-like structures, which cross-react with an anti-oocyst monoclonal antibody (11).Like other apicomplexa (e.g., Toxoplasma and Plasmodium), sporozoites of C. parvum are slender, move by gliding motility, and release adhesins from apical organelles when they invade host epithelial cells (1, 8, 12, 39). Unlike other apicomplexa, C. parvum parasites are missing a chloroplast-derived organelle called the apicoplast (1, 47, 49). C. parvum sporozoites have on their surface unique mucin-like glycoproteins, which contain Ser- and Thr-rich repeats that are polymorphic and may be modified by O-linked GalNAc (4-7, 21, 25, 26, 30, 32, 34, 35, 43, 45). These C. parvum mucins, which are highly immunogenic and are potentially important vaccine candidates, include gp900 and gp40/gp15 (4, 6, 7, 25, 26). gp40/gp15 is cleaved by furin-like proteases into two peptides (gp40 and gp15), each of which is antigenic (42). gp900, gp40, and gp15 are shed from the surface of the C. parvum sporozoites during gliding motility (4, 7, 35).The studies presented here began with electron microscopic observations of C. parvum oocysts stained with ruthenium red (23), which revealed novel fibrils or tethers that extend radially from the inner surface of the oocyst wall to the outer surface of sporozoites. We hypothesized that at least some of these fibrillar tethers might be the antigenic mucins, which are abundant on the surface of C. parvum sporozoites. To test this hypothesis, we used mass spectroscopy to identify oocyst wall proteins and sporozoite glycoproteins and used deconvolving and immunoelectron microscopy (immuno-EM) with lectins and anti-C. parvum antibodies to directly label the tethers.     

Significance of Wall Structure,Macromolecular Composition,and Surface Polymers to the Survival and Transport of Cryptosporidium parvum Oocysts

The structure and composition of the oocyst wall are primary factors determining the survival and hydrologic transport of Cryptosporidium parvum oocysts outside the host. Microscopic and biochemical analyses of whole oocysts and purified oocyst walls were undertaken to better understand the inactivation kinetics and hydrologic transport of oocysts in terrestrial and aquatic environments. Results of microscopy showed an outer electron-dense layer, a translucent middle layer, two inner electron-dense layers, and a suture structure embedded in the inner electron-dense layers. Freeze-substitution showed an expanded glycocalyx layer external to the outer bilayer, and Alcian Blue staining confirmed its presence on some but not all oocysts. Biochemical analyses of purified oocyst walls revealed carbohydrate components, medium- and long-chain fatty acids, and aliphatic hydrocarbons. Purified walls contained 7.5% total protein (by the Lowry assay), with five major bands in SDS-PAGE gels. Staining of purified oocyst walls with magnesium anilinonaphthalene-8-sulfonic acid indicated the presence of hydrophobic proteins. These structural and biochemical analyses support a model of the oocyst wall that is variably impermeable and resistant to many environmental pressures. The strength and flexibility of oocyst walls appear to depend on an inner layer of glycoprotein. The temperature-dependent permeability of oocyst walls may be associated with waxy hydrocarbons in the electron-translucent layer. The complex chemistry of these layers may explain the known acid-fast staining properties of oocysts, as well as some of the survival characteristics of oocysts in terrestrial and aquatic environments. The outer glycocalyx surface layer provides immunogenicity and attachment possibilities, and its ephemeral nature may explain the variable surface properties noted in oocyst hydrologic transport studies.Previous studies of the survival of Cryptosporidium parvum under natural and laboratory conditions have shown that the oocyst phase is a durable stage in the life cycle of this apicomplexan parasite and is crucial for parasite transmission. A major public health problem is the resistance of oocysts to chlorine at normal concentrations used in water treatment systems. Oocysts have the reputation of being tough, durable structures; however, they can be inactivated by many physical and chemical disinfectants, including UV radiation, ozone, ammonia, high temperature, desiccation, freezing, and exposure to extreme alkaline or acidic conditions (10, 11, 12, 17, 18, 22, 35). Low temperatures above freezing extend oocyst viability and infectivity for very long times (12, 18, 19, 20, 35). Environmental temperature is a major factor controlling oocyst survival (23, 24, 32). While there have been many studies documenting the significance of temperature for oocyst survival and the influence of temperature on stored energy reserve utilization has been recognized (see references 10 and 32 for reviews), the effects of temperature on the key oocyst wall structures and macromolecules have not been well investigated.While oocyst wall structure and macromolecular chemistry have been investigated in some detail (10, 14, 31, 33, 34, 41) and survival and transport in natural environments have been studied (5, 6, 8, 10, 23, 24), neither the underlying mechanisms by which oocysts resist environmental pressures nor the surface properties that control environmental transport have been well characterized (23, 24).In this study, we investigated details of the ultrastructure and chemical composition of the C. parvum oocyst wall with the aim of understanding the key physical and chemical properties of the oocyst wall that may confer environmental resistance and affect environmental transport.     

Detection of Toxoplasma gondii Oocysts in Water Sample Concentrates by Real-Time PCR

PCR techniques in combination with conventional parasite concentration procedures have potential for the sensitive and specific detection of Toxoplasma gondii oocysts in water. Three real-time PCR assays based on the B1 gene and a 529-bp repetitive element were analyzed for the detection of T. gondii tachyzoites and oocysts. Lower sensitivity and specificity were obtained with the B1 gene-based PCR than with the 529-bp repeat-based PCR. New procedures for the real-time PCR detection of T. gondii oocysts in concentrates of surface water were developed and tested in conjunction with a method for the direct extraction of inhibitor-free DNA from water. This technique detected as few as one oocyst seeded to 0.5 ml of packed pellets from water samples concentrated by Envirocheck filters. Thus, this real-time PCR may provide a detection method alternative to the traditional mouse assay and microscopy.Toxoplasma gondii is a ubiquitous parasite found in all classes of warm-blooded vertebrates. Nearly one-third of humans have been exposed to this parasite (15). In immunocompetent adults, acute infection normally results in transient influenza-like symptoms, but in immunocompromised persons retinochoroiditis and encephalitis are more common. Infected individuals can retain the parasite as quiescent tissue cysts for long periods, but invasive infection can occur if the immune status of the infected person deteriorates (42). If women become infected during pregnancy, the parasite can cause abortion or seriously damage the fetus. The potential morbidity from the ingestion of oocysts of T. gondii and the organism''s low infectious dose are a great concern for public health. There are at least four reported waterborne outbreaks of toxoplasmosis (2, 3, 14, 44), and endemic toxoplasmosis in Brazil is associated with the consumption of water or ice contaminated with T. gondii oocysts (1, 23), demonstrating the potential for the waterborne transmission of this disease (15).There is no rapid detection method for T. gondii oocysts recovered from water or other environmental samples. Traditionally, the detection of protozoa in water required their concentration from large volumes of water by filtration or centrifugation, isolation from concentrated particulates by immunomagnetic separation (IMS) or other methods, and detection by immunofluorescence microscopy, the infection of cultured cells, biochemistry, animal infection tests, molecular techniques, or combinations of these (17, 58). For T. gondii oocysts there are no commercially available IMS techniques, no widely available immunofluorescent staining reagents, and no standardized cultivation protocols. The identification of oocysts from environmental samples has included differential floatation and mouse inoculation (27). Recently, IMS techniques have been developed for the isolation of T. gondii oocysts and sporocysts in water (16, 18). Both the oocyst and sporocyst IMS assays, however, had poor specificity, because antibodies cross-reacted with water debris and the sporocyst wall of Hammondia hammondi, Hammondia heydorni, and Neospora caninum (16).PCR is becoming a favored technique for the detection of T. gondii oocysts in water (32, 35, 36, 46, 49, 55) over the conventional mouse bioassay (27, 55), as it reduces the detection time from weeks to 1 to 2 days. Although they have been developed for the detection of T. gondii in clinical specimens (50), no real-time PCR assays have been adapted for the detection of oocysts in water samples, possibly because of expected high concentrations of PCR inhibitors and low numbers of T. gondii oocysts in environmental samples (55).There are several unresolved issues regarding the effectiveness of the PCR detection of T. gondii oocysts in water. The most readily available method for the isolation of T. gondii oocysts from water samples is flocculation or sucrose floatation prior to DNA extraction (35, 36, 49, 55). Because sucrose flotation and flocculation result in oocyst losses, the recovery rate of using these methods is poor. For DNA extraction, the phenol-chloroform method or QIAamp mini kit frequently is used (16, 35, 36, 46, 55). When oocysts are recovered from water either by the Environmental Protection Agency (EPA) information collection rule method (53) or EPA Method 1623 (54) without purification by IMS, neither the conventional phenol-chloroform DNA extraction nor the QIAamp mini kit is effective at removing PCR inhibitors (30, 55, 57).Recently, a method was used effectively in the analysis of Cryptosporidium oocysts in surface water, storm water, and wastewater samples (30). This method extracted DNA directly from water concentrates without pathogen IMS, differential flotation, or enrichment cultures, and it utilized a commercial DNA extraction kit, the FastDNA spin kit for soil, and a high concentration of nonacetylated bovine serum albumin in PCR. The FastDNA soil kit has a higher capacity for PCR inhibitor removal than several other commercial extraction kits designed for environmental samples. The use of nonacetylated bovine serum in the PCR neutralizes residual PCR inhibitors that are coextracted with the DNA (30).In the present study, the performance of two published LightCycler real-time PCR assays based on the multicopy B1 gene and 529-bp repetitive element (13, 45) and a newly developed LightCycler real-time PCR assay using a common primer set were analyzed for the detection of T. gondii, using pure DNA and DNA extracted by the aforementioned extraction method (30) from water sample concentrates seeded with known number of oocysts.     

Seasonal Retention and Release of Cryptosporidium parvum Oocysts by Environmental Biofilms in the Laboratory

Cryptosporidium is a genus of waterborne protozoan parasites that causes significant gastrointestinal disease in humans. These parasites can accumulate in environmental biofilms and be subsequently released to contaminate water supplies. Natural microbial assemblages were collected each season from an eastern Pennsylvania stream and used to grow biofilms in laboratory microcosms in which influx, efflux, and biofilm retention were determined from daily oocyst counts. For each seasonal biofilm, oocysts attached to the biofilm quickly during oocyst dosing. Upon termination of oocyst dosing, the percentage of oocysts retained within the biofilm decreased to a new steady state within 5 days. Seasonal differences in biofilm retention of oocysts were observed. The spring biofilm retained the greatest percentage of oocysts, followed (in decreasing order) by the winter, summer, and fall biofilms. There was no statistically significant correlation between the percentage of oocysts attached to the biofilm and (i) any measured stream water quality parameter (including temperature, pH, conductivity, and dissolved organic carbon concentration) or (ii) experimental temperature. Seasonal differences in oocyst retention persisted when biofilms were tested with stream water from a different season. These data suggest that seasonal variation in the microbial community and resulting biofilm architecture may be more important to oocyst transport in this stream site than water quality. The biofilm attachment and detachment dynamics of C. parvum oocysts have implications for public health, and the drinking water industry should recognize that the potential exists for pathogen-free water to become contaminated during the distribution process as a result of biofilm dynamics.Cryptosporidium is a genus of waterborne protozoan parasites that cause a gastrointestinal disease in humans (cryptosporidiosis) that can be prolonged and life-threatening for people with compromised immune systems. Recent advances in medical treatment for cryptosporidiosis exist but are not entirely effective for immunocompromised patients (1). In addition, conventional water treatment does not effectively target Cryptosporidium oocysts because the oocysts'' small size (4 to 8 μm) limits the ability of filters to remove them and, more importantly, oocysts are resistant to chlorine (25). Therefore, environmental control of Cryptosporidium is important to protect public health. To determine the risk of human exposure and infection, the fate and transport of Cryptosporidium oocysts in the environment, including biofilms, should be examined.Within the past two decades, biofilms have been recognized as ubiquitous habitats found on most surfaces exposed to water (20, 24). Environmental biofilms can rapidly accumulate pathogens at densities much higher than water column densities, and the potential for gradual or sudden pathogen loss from the biofilm exists long after entrapment (8, 22). Biofilm sloughing events are commonplace, occurring when a biofilm detaches from the substrate to be resuspended as large particles in the water column, and may result in the release of pathogen reservoirs from the biofilm into the water column (8).Biofilms have been identified as a possible contamination source for drinking water supplies, which may lead to infections for which the source cannot be identified (7, 9). An example of the impact of biofilm sloughing events on human health is seen in the cryptosporidiosis outbreak that occurred in Lancashire County, England, in March 2000 (10). After the outbreak, the oocyst source was identified as cattle feces from adjacent farmland that contaminated the drinking water after abnormally heavy rainfall. The water source was subsequently changed to two upland impounding reservoirs containing filtered surface water. However, oocysts persisted in the water distribution system for 19 days, with large peaks associated with major water main disturbance events, including the initial flushing of the system and a burst in the main supply pipe. This persistence of oocysts in the water supply was attributed to the release of oocysts trapped in biofilms on the interior surface of the distribution pipes and may have contributed to additional infections.Several studies have examined pathogen transport dynamics in biofilms using glass or latex beads of various sizes as surrogates for pathogens (5, 8, 16, 17). A few studies examined the attachment of C. parvum oocysts to biofilms but did not use natural microbial assemblages to make the biofilms (3, 23) or quantify how many oocysts attached or sloughed (9, 22). Rogers and Keevil (22) showed that oocysts attached to a biofilm composed of a natural microbial assemblage collected from a reservoir at a concentration of 1,400 oocysts/cm² after the addition of 10⁸ oocysts in 10 ml of sterile water. Dai and Hozalski (3) and Searcy et al. (23) used pure culture biofilms to demonstrate oocyst attachment; however, only Searcy et al. (23) accounted for sloughing, although no oocyst release from the biofilm was seen during the course of their experiments. Helmi et al. (9) noted attachment and detachment of oocysts from a natural biofilm but did not include a quantitative analysis to account for all oocysts in the flow system over time. None of these studies examined pathogen attachment seasonally over the course of a year. Seasonal changes in temperature, precipitation, and water quality (including nutrient availability) may have significant impacts on the microbial composition and functional structure of a biofilm (14). These changes include structural changes in the biofilm thickness and morphology, as well as changes in the water composition and suspended matter. In addition, seasonal changes in stream flow dynamics may alter biofilm composition and morphology, as well as oocyst attachment and release patterns.This study provides novel information about C. parvum oocyst attachment to biofilms grown in the laboratory from natural microbial assemblages collected seasonally (i.e., in January, April, July, and October) from Monocacy Creek in Bethlehem, PA. Previous work (26) showed that (i) a significant fraction of C. parvum oocysts adhered to the surface of experimental biofilms during a 3-day oocyst dosing period, (ii) a portion of the adhered oocysts immediately released from the biofilm, and (iii) a portion of the oocysts remained attached to the biofilm for a period of days after termination of oocyst dosing. Here, we test the hypotheses that (i) oocyst retention by biofilms varies seasonally and (ii) seasonal changes in water quality influence oocyst retention.     

Identification of Cryptosporidium Species and Genotypes in Scottish Raw and Drinking Waters during a One-Year Monitoring Period

We analyzed 1,042 Cryptosporidium oocyst-positive slides (456 from raw waters and 586 from drinking waters) of which 55.7% contained 1 or 2 oocysts, to determine species/genotypes present in Scottish waters. Two nested PCR-restriction fragment length polymorphism (RFLP) assays targeting different loci (1 and 2) of the hypervariable region of the 18S rRNA gene were used for species identification, and 62.4% of samples were amplified with at least one of the PCR assays. More samples (577 slides; 48.7% from raw water and 51.3% from drinking water) were amplified at locus 1 than at locus 2 (419 slides; 50.1% from raw water and 49.9% from drinking water). PCR at loci 1 and 2 amplified 45.4% and 31.7% of samples containing 1 or 2 oocysts, respectively. We detected both human-infectious and non-human-infectious species/genotype oocysts in Scottish raw and drinking waters. Cryptosporidium andersoni, Cryptosporidium parvum, and the Cryptosporidium cervine genotype (now Cryptosporidium ubiquitum) were most commonly detected in both raw and drinking waters, with C. ubiquitum being most common in drinking waters (12.5%) followed by C. parvum (4.2%) and C. andersoni (4.0%). Numerous samples (16.6% total; 18.9% from drinking water) contained mixtures of two or more species/genotypes, and we describe strategies for unraveling their identity. Repetitive analysis for discriminating mixtures proved useful, but both template concentration and PCR assay influenced outcomes. Five novel Cryptosporidium spp. (SW1 to SW5) were identified by RFLP/sequencing, and Cryptosporidium sp. SW1 was the fourth most common contaminant of Scottish drinking water (3%).The protozoan parasite Cryptosporidium has been implicated in numerous waterborne and food-borne outbreaks of cryptosporidiosis (3, 6, 16, 17, 18). Currently, there are 22 valid Cryptosporidium species: Cryptosporidium hominis, infecting mainly humans; C. parvum, in humans and numerous other mammals, including cattle; C. andersoni, C. bovis (previously bovine genotype B), and C. ryanae (previously deer-like genotype) in cattle; C. xiaoi (previously bovis-like genotype) in sheep; C. muris in mice; C. felis in cats; C. suis (previously pig genotype I) in pigs; C. wrairi in guinea pigs; C. canis in dogs; C. meleagridis and C. baileyi in birds; C. galli in finches and chickens; C. fayeri (previously marsupial genotype I) and C. macropodum (previously marsupial genotype II) in various species of marsupials; C. fragile in toads; C. varanii (previously C. saurophilum) in lizards and snakes; C. serpentis in snakes; C. scophthalmi and C. molnari in fish (20); and C. ubiquitum (previously Cryptosporidium cervine genotype) in a wide variety of host species, including white-tailed deer, sheep, cattle, goat, mouse, various species of rodents, and humans (4). In addition, there are over 60 Cryptosporidium genotypes, which differ significantly in their molecular sequences but, as yet, have not been ascribed species status (13, 29).Genetic analyses reveal that at least eight species (C. hominis, C. parvum, C. meleagridis, C. felis, C. canis, C. suis, C. muris, and C. ubiquitum) and seven Cryptosporidium genotypes (C. hominis monkey, C. andersoni-like, and Cryptosporidium chipmunk I, skunk, horse, rabbit, and pig genotype II) are associated with human disease (1, 9, 22), but C. parvum and C. hominis remain the most common species infecting humans. Environmental contamination with oocysts of Cryptosporidium species that are not infectious to susceptible human hosts contributes to the difficulties in assessing the risk to public health from waterborne oocysts.Oocysts occur at low densities in water (16, 17, 21), and molecular methods which can genotype small numbers of organisms reliably and reproducibly from water concentrates are required to determine which species occur, and with what frequency, in water. We used our standardized, maximized freezing and thawing method for DNA extraction (10) and our procedure for retrieving oocysts from Cryptosporidium water monitoring slides to maximize DNA extraction for PCR-restriction fragment length polymorphism (RFLP) analysis (11, 12, 19) in this study.We undertook a 1-year survey to identify the species and genotypes of Cryptosporidium oocysts detected in the Scottish Water (SW) Routine Cryptosporidium Monitoring Programme to gain information on the occurrence and diversity of Cryptosporidium oocysts in drinking water sources and drinking waters in order to determine predominant types in water catchment areas and monitor variations in oocyst population distribution over a 1-year period with a view to adding value to current assessments of risk to human health.     

Comparison of Single- and Multilocus Genetic Diversity in the Protozoan Parasites Cryptosporidium parvum and C. hominis

The genotyping of numerous isolates of Cryptosporidium parasites has led to the definition of new species and a better understanding of the epidemiology of cryptosporidiosis. A single-locus genotyping method based on the partial sequence of a polymorphic sporozoite surface glycoprotein gene (GP60) has been favored by many for surveying Cryptosporidium parvum and C. hominis populations. Since genetically distinct Cryptosporidium parasites recombine in nature, it is unclear whether single-locus classifications can adequately represent intraspecies diversity. To address this question, we investigated whether multilocus genotypes of C. parvum and C. hominis cluster according to the GP60 genotype. C. hominis multilocus genotypes did not segregate according to this marker, indicating that for this species the GP60 sequence is not a valid surrogate for multilocus typing methods. In contrast, in C. parvum the previously described “anthroponotic” genotype was confirmed as a genetically distinct subspecies cluster characterized by a diagnostic GP60 allele. However, as in C. hominis, several C. parvum GP60 alleles did not correlate with distinct subpopulations. Given the rarity of some C. parvum GP60 alleles in our sample, the existence of additional C. parvum subgroups with unique GP60 alleles cannot be ruled out. We conclude that with the exception of genotypically distinct C. parvum subgroups, multilocus genotyping methods are needed to characterize C. parvum and C. hominis populations. Unless parasite virulence is controlled at the GP60 locus, attempts to find associations within species or subspecies between GP60 and phenotype are unlikely to be successful.The lack of variable morphological traits to identify oocysts from different Cryptosporidium species has driven the development of numerous genotyping methods to survey the diversity in this genus. Genetic markers such as single-nucleotide polymorphisms (24), restriction fragment length polymorphisms (7, 34), random amplification methods (17, 20), conformational polymorphisms (11), simple sequence repeats (3, 10), and DNA sequence polymorphisms (6, 36) have been used to type Cryptosporidium oocysts excreted by humans and animals and oocysts recovered from the environment. This effort has led to a deeper understanding of the taxonomy of the genus Cryptosporidium and the epidemiology of cryptosporidiosis in humans and livestock. As a result of this work, two species responsible for a majority of human infections, Cryptosporidium parvum and C. hominis, were identified (21) and our understanding of the taxonomy of the genus was refined (35).The application of genetic markers to define species, i.e., reproductively isolated populations, is straightforward. At this taxonomic level, all genotypes cosegregate and the choice of marker will have little impact on the outcome, provided that the marker, or combination thereof, is sufficiently polymorphic. The classical example is the variable region of the small-subunit rRNA gene which has been used, as in other taxa, to define many Cryptosporidium species. For studying intraspecies polymorphism, the choice of genotyping methods needs to take into consideration the potential for genetic recombination. This is clearly the case for species such as those belonging to the genus Cryptosporidium, which are known to undergo an obligatory sexual cycle during which genetically dissimilar haplotypes can recombine (28).Among the many markers that have been applied in epidemiological surveys of C. parvum and C. hominis, a variable fragment of the gene encoding a sporozoite surface glycoprotein (8, 26) has been particularly popular. As a result of the widespread adoption of this marker, variously named GP60, cpgp40/15, or gp40, numerous alleles have been identified and deposited in GenBank. The analysis of this continuously growing collection of GP60 sequences has led to the identification of groups of related sequences (18, 27, 32, 33). In an attempt to simplify the comparison of GP60 genotypes among different laboratories, a GP60 nomenclature distinguishing the main groups of alleles has been created (26) and later refined (27).The desire to streamline the genotyping of large numbers of Cryptosporidium isolates collected during surveys has led to the widespread adoption of the GP60 genotype as the only marker for defining intraspecies groups. Since this approach is not compatible with the reassortment of unlinked loci, the classification of isolates on the basis of the GP60 genotype, or any other single marker, needs to be evaluated. Within a recombining population, no single genetic marker can a priori be expected to serve as a surrogate for other loci or multilocus genotypes (MLGs), and any apparent clustering of isolates is dependent on the marker. To investigate the validity of the GP60 genotyping method as commonly applied to the classification of C. parvum and C. hominis isolates, the GP60 genotype was added to a previously described 9-locus genotype (29) and a diversified collection of 10-locus genotypes was examined for intraspecies clusters. We show that, with the exception of some GP60 alleles apparently restricted to human C. parvum, neither C. parvum nor C. hominis GP60 alleles define subspecies genotypes. These results are discussed in the context of ongoing research to better understand the population structure of these parasites and identify genotypes associated with virulence traits.     

Concentrations,Viability, and Distribution of Cryptosporidium Genotypes in Lagoons of Swine Facilities in the Southern Piedmont and in Coastal Plain Watersheds of Georgia

Waste lagoons of swine operations are a source of Cryptosporidium oocysts. Few studies, however, have reported on oocyst concentrations in swine waste lagoons; none have reported on oocyst viability status, nor has there been a systematic assessment of species/genotype distributions across different types of swine facilities. Ten swine waste lagoons associated with farrowing, nursery, finishing, and gestation operations were each sampled once a month for a year. Oocysts were extracted from triplicate 900-ml effluent samples, enumerated by microscopy, and assessed for viability by dye exclusion/vital stain assay. DNA was extracted from processed samples, and 18S ribosomal DNA (rDNA) genes were amplified by PCR and sequenced for species and genotype identification. Oocysts were observed at each sampling time at each lagoon. Annual mean concentrations of total oocysts and viable oocysts ranged between 24 and 51 and between 0.6 and 12 oocysts ml⁻¹ effluent, respectively. The species and genotype distributions were dominated (95 to 100%) by Cryptosporidium suis and Cryptosporidium pig genotype II, the latter of which was found at eight of the lagoons. The lagoon at the gestation facility was dominated by Cryptosporidium muris (90%), and one farrowing facility showed a mix of pig genotypes, Cryptosporidium muris, and various genotypes of C. parvum. The zoonotic C. parvum bovine genotype was observed five times out of 407 18S rDNA sequences analyzed. Our results indicate that pigs can have mixed Cryptosporidium infections, but infection with C. suis is likely to be dominant.Over the last few decades, pork production in North America has undergone significant growth and centralization into large concentrated swine (Sus scrofa) operations with more animals on fewer farms (18). A consequence of the increase in numbers of swine per facility is a concomitant increased concentration of swine waste. Present housing facilities for swine are designed to collect feces and urine in wastewater lagoons, in which the waste undergoes anaerobic transformations. One of several public health concerns over swine lagoons is the potential presence of infectious bacteria, viruses, and protozoa (4). Because of the notoriety given to swine waste lagoon spills in the coastal flood plain of North Carolina that were associated with a series of hurricanes in 1998 and 1999 (21), large-scale swine operations have become a focus of environmental and public health concerns.The cause of the massive outbreak of cryptosporidiosis in Milwaukee, WI, in 1993 was afterwards determined to be Cryptosporidium hominis, the human genotype of C. parvum and an obligate parasite of humans (33, 44). At the time, however, it was thought to be caused by C. parvum (22). Because of this initial misidentification of the cryptosporidial source of the outbreak, the connection between C. parvum and large-scale confined livestock operations has become a focused area of research. Although manure-associated outbreaks of C. parvum have implicated bovine sources, a Canadian study found that the prevalence of Cryptosporidium in swine lagoons was greater than that in dairy liquid manure (9). Olson et al. (24) also reported the presence of Cryptosporidium oocysts of undetermined genotype at four of six hog operations in Canada. Atwill et al. (2) observed C. parvum oocysts in feces of feral pigs. Hutchison et al. (13) observed C. parvum oocysts of undetermined genotype in 5 and 13% of fresh and stored fecal samples, respectively, from pigs of undeclared age. Guselle et al. (10) followed the course of a naturally occurring C. parvum infection in 33 weaned pigs. Following the protocol of the genetic analysis of Morgan et al. (23), Guselle et al. (10) identified this C. parvum genotype as being adapted to pigs. At the time, the zoonotic potential of this C. parvum pig-adapted genotype was considered uncertain (23).Recently, two genotypes of Cryptosporidium have been recognized as host adapted to swine: Cryptosporidium suis (formerly Cryptosporidium pig genotype I) and Cryptosporidium pig genotype II (28, 29). Xiao et al. (37) reported on an immunocompromised person who was infected with a Cryptosporidium pig genotype and thus implicated Cryptosporidium from swine as potentially zoonotic and a public health concern. Before molecular methods were developed to differentiate pig genotypes of Cryptosporidium from other species, C. parvum was thought to infect 152 species of mammals and consist of several cryptic species (6). An extensive survey of swine effluent from swine finishing operations in Ireland indicated a prevalence of both C. suis and Cryptosporidium pig genotype II (39). Hamnes et al. (11) reported prevalence of both C. suis and Cryptosporidium pig genotype II in feces of suckling pigs across Norway and thus implicated farrowing operations as sources of this parasite.Other than the prevalence of Cryptosporidium in feces of young pigs and effluent lagoons of older pigs in finishing operations, little comprehensive data on oocyst concentrations, viability of oocysts, and distributions of Cryptosporidium species and genotypes have been reported. No systematic study of swine lagoon effluents from large-scale facilities has been reported for the four separate stages of swine development, (i) breeding and gestation, (ii) farrowing (parturition), (iii) nursery (in which weaned piglets are kept until 8 to 9 weeks of age), and (iv) finishing (in which 8- to 9-week-old pigs are kept to market weight). The objective of this investigation was to determine for 1 year the frequencies, concentrations, viability statuses, and distributions of Cryptosporidium species and genotypes in lagoons associated with the four types of swine operations in the Southern Piedmont and in coastal plain watersheds of Georgia.     

Molecular Characterization of Cryptosporidium molnari Reveals a Distinct Piscine Clade

Multilocus phylogenetic analysis of small-subunit (SSU) rRNA and actin from Cryptosporidium molnari clustered this species with the C. molnari-like genotype of an isolate from the guppy, although the two fish isolates seem to be distinct species. The analysis of available piscine genotypes provides some support for cladistic congruence of the genus Piscicryptosporidium, but additional piscine genotypes are needed.Recent reviews accept more than 20 valid cryptosporidium species (7, 20), and characterization of additional isolates is expanding this list rapidly (http://www.vetsci.usyd.edu.au/staff/JanSlapeta/icrypto/index.htm). In addition, numerous morphotypes or genotypes have been proposed whose taxonomic affiliation is unsettled due to incomplete characterization according to minimum consensus standards (5, 7, 24). Five species have been proposed for fish isolates (15), but only Cryptosporidium molnari and Cryptosporidium scophthalmi (2, 4) stand as valid species (20), although not without discussion (7). Fish cryptosporidia present some unique features, which have even led to the genus Piscicryptosporidium being proposed (13). However, lack of genetic support keeps this genus and several fish morphotypes as incertae sedis (12, 15, 24). Detailed biological data on C. molnari and C. scophthalmi have been previously presented (3, 18, 19), but no molecular characterization has yet been conducted, thus hampering species identification of other fish isolates (7, 24) and evaluation of their relationships within the genus (15). Ribosomal and actin gene data on an isolate from guppy fish (Poecilia reticulata) have been obtained, and preliminary analyses of these sequences indicated a basal position in the cryptosporidial tree (17). Although it was regarded as C. molnari-like, biological characterization of this isolate was limited. The purpose of this work was to provide the necessary C. molnari comparative genetic data and to clarify the relationship of available fish isolates in a phylogenetic context.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Propagation of Human Enteropathogens in Constructed Horizontal Wetlands Used for Tertiary Wastewater Treatment

Constructed subsurface flow (SSF) and free-surface flow (FSF) wetlands are being increasingly implemented worldwide into wastewater treatments in response to the growing need for microbiologically safe reclaimed waters, which is driven by an exponential increase in the human population and limited water resources. Wastewater samples from four SSF and FSF wetlands in northwestern Ireland were tested qualitatively and quantitatively for Cryptosporidium spp., Giardia duodenalis, and human-pathogenic microsporidia, with assessment of their viability. Overall, seven species of human enteropathogens were detected in wetland influents, vegetated areas, and effluents: Cryptosporidium parvum, C. hominis, C. meleagridis, C. muris, G. duodenalis, Encephalitozoon hellem, and Enterocytozoon bieneusi. SSF wetland had the highest pathogen removal rate (i.e., Cryptosporidium, 97.4%; G. duodenalis, 95.4%); however, most of these values for FSF were in the negative area (mean, −84.0%), meaning that more pathogens were discharged by FSF wetlands than were delivered to wetlands with incoming wastewater. We demonstrate here that (i) the composition of human enteropathogens in wastewater entering and leaving SSF and FSF wetlands is highly complex and dynamic, (ii) the removal and inactivation of human-pathogenic microorganisms were significantly higher at the SSF wetland, (iii) FSF wetlands may not always provide sufficient remediation for human enteropathogens, (iv) wildlife can contribute a substantial load of human zoonotic pathogens to wetlands, (v) most of the pathogens discharged by wetlands were viable, (vi) large volumes of wetland effluents can contribute to contamination of surface waters used for recreation and drinking water abstraction and therefore represent a serious public health threat, and (vii) even with the best pathogen removal rates achieved by SSF wetland, the reduction of pathogens was not enough for a safety reuse of the reclaimed water. To our knowledge, this is the first report of C. meleagridis from Ireland.Demand for high-quality drinking and recreational waters rises exponentially due to global demographic growth in the human population, reinforcing an urgent need for microbiologically safe reclaimed waters (12). Wastewater discharges are worldwide risk factors for the introduction of human pathogens into surface waters used as drinking and recreational resources. Cryptosporidium parvum, C. hominis, Giardia duodenalis, and human-virulent microsporidia (i.e., Encephalitozoon intestinalis, E. hellem, E. cuniculi, and Enterocytozoon bieneusi) are waterborne enteropathogens inflicting considerable morbidity in healthy people and mortality (e.g., Cryptosporidium and microspora) in immunodeficient individuals (34, 44). Their transmissive stages, i.e., oocysts, cysts, and spores, are resistant to environmental stressors and are therefore long-lasting and relatively ubiquitous in the environment (13, 27, 45). These pathogens are category B biodefense agents on the U.S. National Institutes of Health list, and microsporidian spores are on the Contaminant Candidate List of the U.S. Environmental Protection Agency (29) because spore identification, removal, and inactivation in drinking water are technologically challenging. Surface water is not routinely monitored for these pathogens, despite evidence demonstrating environmental contamination derived from wastewater discharges (12). Environmentally, all aforementioned pathogens (except C. hominis) have a broad zoonotic reservoir (13, 27, 34).Constructed wetlands of either vertical or horizontal flow are increasingly used worldwide for secondary or tertiary treatment of municipal wastewater due to minimum electric requirements and low maintenance costs (6, 32). The wetland concept has become an attractive wastewater treatment alternative to conventional tertiary treatment processes for (i) municipal wastewater, (ii) on-site domestic wastewater treatment, and (iii) concentrated animal feeding operations (24). In wetlands, human-pathogenic microorganisms are physically removed and biodegraded by sedimentation (2, 23), filtration and evapotranspiration-driven attachment to plant roots (10, 43), natural die-off (28), UV radiation, straining and sorption by biofilm (31), and protozoan predation (37). It is thought that the performance of subsurface flow (SSF) wetlands in removing human pathogens is superior to that of secondary wastewater treatment, i.e., conventional sewage sludge activation (40). Horizontal wetlands usually discharge to surface waters that are frequently used for recreation or drinking water production (6). It is commonly assumed that human pathogens identified in wetland effluents originate from the wastewater (39). However, this was never proven because studies of human pathogens in wetlands (10, 23, 28, 31, 32, 39, 40) did not utilize molecular epidemiology techniques to differentiate pathogen species or assess their viability.In general, wastewater can be injected under the wetland surface for plug flow hydraulics, i.e., SSF (43), or be delivered to the wetland surface for free-surface flow (FSF). Because the wastewater resides in wetlands, these areas can act as endemic sites supporting both propagation and transmission of human zoonotic pathogens (15). Sizing reed-bed systems for a residence time of 5 days has become a standard practice (6, 31, 39), leaving plenty of time for the propagation and spreading of wastewater-derived pathogens in wetland habitats via a wide variety of wildlife (12, 15). Any temporal or permanent malfunctioning caused by clogged inlet pipes can cause (i) hydraulic short circuits that bypass part of the filtration area in FSF wetlands or (ii) chance SSF wetland filtration dynamics to FSF dynamics (31, 40). This can additionally increase wastewater retention time in wetlands.The purposes of the present study were to (i) determine species of human protozoan and fungal enteropathogens entering, residing, and leaving constructed horizontal wetlands used for tertiary treatment of municipal wastewater; (ii) determine the efficacy for removal of Cryptosporidium oocysts, G. duodenalis cysts, and human-virulent microsporidian spore species by wetlands from wastewater subjected to secondary treatment; and (iii) compare pathogen removal efficacies between SSF and FSF wetlands. We used a multiplexed fluorescence in situ hybridization (FISH) assay with immunofluorescence antibody (IFA) to identify C. parvum and C. hominis oocysts and microsporidian spores and to assess their viability in a quantitative manner. Since multiplexed FISH specifically identifies C. parvum and C. hominis oocysts but does not differentiate between these species (36), we used PCR-restriction fragment length polymorphism (RFLP) to identify other potential oocyst species. In addition, we used PCR to confirm species of microsporidian spores identified by FISH.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Effect of Estuarine Wetland Degradation on Transport of Toxoplasma gondii Surrogates from Land to Sea

The flux of terrestrially derived pathogens to coastal waters presents a significant health risk to marine wildlife, as well as to humans who utilize the nearshore for recreation and seafood harvest. Anthropogenic changes in natural habitats may result in increased transmission of zoonotic pathogens to coastal waters. The objective of our work was to evaluate how human-caused alterations of coastal landscapes in California affect the transport of Toxoplasma gondii to estuarine waters. Toxoplasma gondii is a protozoan parasite that is excreted in the feces of infected felids and is thought to reach coastal waters in contaminated runoff. This zoonotic pathogen causes waterborne toxoplasmosis in humans and is a significant cause of death in threatened California sea otters. Surrogate particles that mimic the behavior of T. gondii oocysts in water were released in transport studies to evaluate if the loss of estuarine wetlands is contributing to an increased flux of oocysts into coastal waters. Compared to vegetated sites, more surrogates were recovered from unvegetated mudflat habitats, which represent degraded wetlands. Specifically, in Elkhorn Slough, where a large proportion of otters are infected with T. gondii, erosion of 36% of vegetated wetlands to mudflats may increase the flux of oocysts by more than 2 orders of magnitude. Total degradation of wetlands may result in increased Toxoplasma transport of 6 orders of magnitude or more. Destruction of wetland habitats along central coastal California may thus facilitate pathogen pollution in coastal waters with detrimental health impacts to wildlife and humans.Estuaries are recognized as being critically endangered worldwide. Pollution of estuarine waters is a significant threat to the health of aquatic life, as well as to humans who depend on coastal habitats (23). Contamination of nearshore waters with terrestrially derived, zoonotic pathogens has received little attention in the field of marine water pollution, which has primarily focused on chemical and nutrient pollutants (22, 42, 46, 55). Yet, studies have documented the presence of fecal pathogens from terrestrial animals in coastal waters and filter-feeding shellfish (7, 37, 48), as well as infections and deaths in aquatic wildlife and humans who become exposed through recreation activities or seafood (4, 18, 39). The zoonotic parasite Toxoplasma gondii is emerging as an important waterborne pathogen in both human and marine wildlife populations (2, 3, 6, 11, 15, 38). Consumption of raw oysters, clams, or mussels has recently been determined to be a risk factor for human exposure to T. gondii (24). Moreover, this parasite is an important cause of death in threatened Southern sea otters (Enhydra lutris nereis) (10, 29). Sea otter infection appears most likely to result from ingestion of environmentally resistant T. gondii oocysts that reach coastal waters in contaminated freshwater runoff (35, 36). These oocysts are shed in the feces of infected wild and domestic felids, with an individual cat capable of shedding up to 1 billion oocysts over several days postinfection (12).Elkhorn Slough, within Monterey Bay in California, is one of the high-risk sites for sea otter infection with T. gondii, with seroprevalence rates of 79% in otters sampled in this area (35). To date, the reasons for the high sea otter prevalence of infections with T. gondii at this site remain unknown. This estuarine habitat has been extensively altered by human activities and is listed as an impaired body of water by the State of California (9). Specifically, extensive degradation has been observed in the slough, with over one-third of vegetated wetlands converted to mudflats due to erosion (49). While the effect of this landscape alteration on the transport of waterborne pathogens is not currently known, such degradation may facilitate contamination of nearshore waters with T. gondii.Wetland habitats provide valuable ecosystem services, including improvement of effluent water quality characteristics through removal of a variety of pollutants (28, 50, 57). Artificially constructed wetlands are now used globally in water treatment facilities to remove nutrients, chemical pollutants, and fecal pathogens from contaminated waters before discharge into receiving water bodies (8, 17, 21, 26, 27). However, compared with freshwater and constructed wetlands, significantly less research has focused on the effects of natural, estuarine wetlands on water quality. In the few studies that investigated the impact of saltwater marshes on marine water quality, these habitats were shown to reduce concentrations of chemicals and nutrients that reach coastal waters in contaminated overland runoff (5, 51). In addition, the percentage of watershed-impervious surface coverage and reduction of natural coastal habitats due to anthropogenic changes has been associated with increased coastal water pollution (33, 34). Despite previous research suggesting a link between wetland degradation and coastal pathogen pollution (5, 33, 34, 51), the role estuarine wetlands play in the transport of terrestrial pathogens from land to sea has not been previously investigated.The overall goal of our research was to evaluate the effect of coastal wetland degradation on contamination of estuarine and coastal waters with terrestrially derived, zoonotic pathogens. Specifically, the objective of this study was to measure T. gondii oocyst transport through vegetated estuarine wetlands and nonvegetated mudflats to quantify the effect of vegetation loss on the flux of this zoonotic pathogen to coastal waters. Due to the biohazard risks associated with the release of environmentally resistant oocysts, experiments used previously validated surrogate microspheres and a specially designed flume that was deployed in vegetated and mudflat (nonvegetated) estuarine wetland habitats. The flume-in-field study design allowed for replication of experiments using specific hydrological parameters while conducting the study within a natural estuarine environment with in situ vegetation, substrate, and water. The two autofluorescent microspheres used in this study have similar physical and surface chemistry properties to T. gondii oocysts and have been previously evaluated as surrogate particles for this protozoan parasite (44). Our results provide novel insights into the consequences of changes in coastal habitat on the ecology of zoonotic infectious disease organisms in coastal marine ecosystems.     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.