Determining potential indicators of Cryptosporidium oocysts throughout the wastewater treatment process.

Most research on wastewater treatment efficiency compliance focuses on physicochemical and microbial indicators; however, very little emphasis has been placed so far on determining suitable indicator organisms to predict the discharge level of pathogens from treatment plants. In this study, raw wastewater, activated sludge, and the resulting final effluents and biosolids in four municipal wastewater treatment plants (WWTPs A, B, C and D) were seasonally investigated for human-virulent water-borne pathogens Cryptosporidium parvum/hominis and Giardia duodenalis, and microsporidia (e.g. Encephalitozoon hellem, E. intestinalis, and Enterocytozoon bieneusi) between 2008 and 2009. A suite of potential microbial indicators for human-virulent protozoa and microsporidia was also determined. A combination of multiple fluorescent in situ hybridization and immunofluorescent antibody assays were applied to detect Cryptosporidium oocysts, Giardia cysts, and microsporidian spores. Escherichia coli, enterococci and Clostridium perfringens spores were cultivated in selective media. Positive correlations were found between the abundance of enterococci and E. coli and abundance of Cryptosporidium oocysts (r(s) > 0.47, p < 0.01) and Giardia cysts (r(s) > 0.44, p < 0.01) at WWTPs A-D. Cryptosporidium perfringens spores were positively correlated to Cryptosporidium oocysts (r(s) = 0.40, p < 0.01) and Giardia cysts (r(s) = 0.46, p < 0.01). There was a strong positive correlation between abundance of Giardia cysts and that of Cryptosporidium oocysts (r(s) > 0.89, p < 0.01). To sum up, a suite of faecal indicator bacteria can be used as indicators for the presence of Cryptosporidium oocysts and Giardia cysts in these activated-sludge systems (WWTPs A, B and C). Overall, Giardia duodenalis was noted to be the best Cryptosporidium indicator for human health in the community-based influent wastewater and throughout the treatment process.

Co-localized Crassostrea virginica and Crassostrea ariakensis oysters differ in bioaccumulation, retention and depuration of microbial indicators and human enteropathogens.

AIMS: To evaluate the bioaccumulation, retention and depuration rates of nine pathogens and surrogates when two oyster species were co-localized in tanks of seawater. METHODS AND RESULTS: Crassostrea ariakensis (n = 52) and Crassostrea virginica (n = 52) were exposed to five virus types, two protozoan and two microsporidian species for 24 h. Oysters were then placed in depuration tanks, and subsets were removed and analysed for micro-organisms at weekly intervals. The odds of C. ariakensis oysters harbouring mouse norovirus-1 (MNV-1), human norovirus (NoV) or haepatitis A virus (HAV) were significantly greater than the odds of C. virginica oysters harbouring the same viruses (MNV-1 OR = 5.05, P = 0.03; NoV OR = 6.97, P = 0.01; HAV OR = 7.40, P < 0.001). Additionally, compared to C. virginica, C. ariakensis retained significantly higher numbers of transmissive stages of all protozoan and microsporidian species (P < 0.01). Crassostrea ariakensis oysters are also capable of retaining multiple human pathogens for at least 1 month. CONCLUSIONS: Crassostrea ariakensis oysters were statistically more likely to harbour enteropathogens and microbial indicators, compared to C. virginica. Individual C. ariakensis were also statistically more likely to retain multiple viruses, protozoa and microsporidia than C. virginica, highlighting the role the species may play in the transmission of multiple diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: Nonnative Crassostrea ariakensis oysters are under review for their introduction into the Chesapeake Bay. The results of this study suggest that nonnative C. ariakensis oysters may present a serious public health threat to people consuming the oysters raw from contaminated sites.

Molecular markers and sentinel organisms for environmental monitoring.

Molecular methods are useful for both to monitor anthropogenic viral, bacterial, and protozoan enteropathogens, and to track pathogen specific markers in a complex environment in order to reveal sources of these pathogens. Molecular genetic markers for fecal viruses, bacteria, and protozoans hold promise for monitoring environmental pollution and water quality. The demand for microbiologically safe waters grows exponentially due to the global demographic rise of the human population. Economically important shellfish, such as oysters, which are harvested commercially and preferentially consumed raw can be of public health importance if contaminated with human waterborne pathogens. However, feral molluscan shellfish which do not have an apparent economic value serve as indicators in monitoring aquatic environments for pollution with human waterborne pathogens and for sanitary assessment of water quality. Current technology allows for multiplexed species-specific identification, genotyping, enumeration, viability assessment, and source-tracking of human enteropathogens which considerably enhances the pathogen source-tracking efforts.

Accumulation of human waterborne parasites by zebra mussels (Dreissena polymorpha) and Asian freshwater clams (Corbicula fluminea).

Zebra mussels (Dreissena polymorpha) and Asian freshwater clams (Corbicula fluminea) are nonindigenous invasive bivalves present in North American fresh waters that are frequently contaminated with human enteric parasites, Cryptosporidium parvum and Giardia lamblia. Six-week laboratory exposure of D. polymorpha and Corbicula fluminea to both parasites seeded daily at concentrations reported from surface waters demonstrated efficient removal of Cryptosporidium parvum oocysts and G. lamblia cysts by both bivalve species. The number of parasites in mollusk tissue progressively increased in relation to the concentration of waterborne contamination, and decreased after cessation of the contamination. Oocysts outnumbered cysts in the tissue of both bivalves, and more parasites were identified in D. polymorpha than in Corbicula fluminea; overall 35.0% and 16.3% of the parasites seeded, respectively. Because C. fluminea and D. polymorpha can accumulate human waterborne parasites in proportion to ambient concentrations, these species of bivalves can be effective bioindicators of contamination of freshwater habitats with Cryptosporidium and Giardia.

Temporal variability of Cryptosporidium in the Chesapeake Bay.

Although Cryptosporidium has been found worldwide in molluscan shellfish from waters contaminated with human and animal feces, little or no related environmental data have been obtained. In the present study, oysters ( Crassostrea virginica) were collected eight times over 3 years from seven sites in the Chesapeake Bay or its tributaries, with accompanying data on water temperature, salinity, rainfall, and streamflow. Oyster gill washings were examined by immunofluorescence microscopy for Cryptosporidium oocysts. Of 1,590 oysters collected, 19.6% had detectable oocysts. Of 53 collections, oocysts were detected 81% of the time. The time when the greatest percentage of oysters at most sites had detectable oocysts coincided with the time of greatest weekly and monthly rainfall, greatest streamflow into the Bay, and lowest water temperatures. In 28% of 53 collections, C. parvum genotypes 1 and 2 and C. baileyi were identified by PCR and gene sequencing. Oocyst infectivity was confirmed from 37.5% of 40 collections by initiating C. parvum genotype 2 infections in mice.

Cattle near the Bwindi Impenetrable National Park, Uganda, as a reservoir of Cryptosporidium parvum and Giardia duodenalis for local community and free-ranging gorillas.

The prevalence and intensity of shedding of Cryptosporidium parvum oocysts and Giardia duodenalis cysts was investigated in cattle grazing in the vicinity of the Bwindi Impenetrable National Park, Uganda. The prevalence of cryptosporidiosis and giardiosis was 38% and 12%, respectively, with 10% concomitant infections. Shedding intensity varied from 130 to 450 oocysts/g (mean of 215 oocysts/g) and from 110 to 270 cysts/g (mean of 156 cysts/g). Significantly more pre-weaned than post-weaned cattle were infected with either parasite, and the pre-weaned cattle shed significantly higher numbers of either parasite than the post-weaned cattle. Mathematical modeling indicated that the maximum prevalence of asymptomatic infections can reach approximately 80% for cryptosporidiosis and 35% for giardiosis in the sampled cattle. Because C. parvum and G. duodenalis recovered from cattle can infect people and gorillas, cattle that graze within the Bwindi Park should be considered as a significant reservoir of these anthropozoonotic parasites.

Cryptosporidium parvum genotype 2 infections in free-ranging mountain gorillas (Gorilla gorilla beringei) of the Bwindi Impenetrable National Park, Uganda.

For behavioral research and due to growing ecotourism, some populations of free-ranging mountain gorillas (Gorilla gorilla beringei) have become habituated to humans. Molecular analysis of two Cryptosporidium sp. oocyst isolates originating from two human-habituated gorilla groups and two oocyst isolates from non-habituated gorillas yielded positive identification of C. parvum Genotype 2 (G2; i.e., "cattle", "animal-adapted", or "zoonotic"). As G2 is cross-transmissible between humans and animals, C. parvum infections can be propagated in the habitats of human-habituated, free-ranging gorillas through both zoonotic and anthroponotic transmission cycles.

The role of non-biting flies in the epidemiology of human infectious diseases.

The feeding and reproductive habits of non-biting synanthropic flies make them important mechanical vectors of human pathogens. Synanthropic flies are major epidemiologic factors responsible for the spread of acute gastroenteritis and trachoma among infants and young children in (predominantly) developing countries. House flies are involved in mechanical transmission of nosocomial infections with multiple antibiotic-resistant bacteria in hospital environments.

Campylobacteriosis, salmonellosis, and shigellosis in free-ranging human-habituated mountain gorillas of Uganda.

For conservation purposes and due to growing ecotourism, free-ranging mountain gorillas (Gorilla gorilla beringei) have been habituated to humans. Fecal specimens (n = 62) collected in January 1999 from mountain gorillas of the Bwindi and Mgahinga National Parks, Uganda, were tested for Campylobacter spp., Salmonella spp., and Shigella spp., and the overall prevalence of infection was 19%, 13%, and 6%, respectively. The prevalence of positive specimens was not related to the year of habituation of a gorilla group to humans. Campylobacter spp., Salmonella, and Shigella spp. infections were not distributed equally among the age classes of gorillas; most of the enteropathogens (80%), and all Shigella spp. organisms, S. sonnei, S. boydii, and S. flexneri, were isolated from subadults and adult gorillas with ages ranging from 6.0 to 11.9 yr. The prevalence of Campylobacter spp. and Salmonella spp. infections among human-habituated gorillas has doubled during the last 4 yr, and isolation of Shigella spp. for the first time from mountain gorillas, may indicate enhanced anthropozoonotic transmission of these enteropathogens.

Cryptosporidium parvum oocysts in zebra mussels (Dreissena polymorpha): evidence from the St Lawrence River.

Molluscan shellfish can recover and concentrate environmentally derived waterborne pathogens and can be used for the sanitary assessment of water quality. Oocysts of Cryptosporidium parvum (genotype 1) were identified in zebra mussels (Dreissena polymorpha) from the St. Lawrence River, Quebec. Approximately 67 oocysts/ml of hemolymph and 129 oocysts/g of soft tissue were recovered. The adjusted concentration of oocysts per gram of tissue was 2.2 x 10(2), and approximately 4.4 x 10(2) oocysts were recovered from a single mussel. Zebra mussels can serve as biological indicators of waterborne contamination with Cryptosporidium.

Molecular characterization of cryptosporidium oocysts in samples of raw surface water and wastewater.

Recent molecular characterizations of Cryptosporidium parasites make it possible to differentiate the human-pathogenic Cryptosporidium parasites from those that do not infect humans and to track the source of Cryptosporidium oocyst contamination in the environment. In this study, we used a small-subunit rRNA-based PCR-restriction fragment length polymorphism (RFLP) technique to detect and characterize Cryptosporidium oocysts in 55 samples of raw surface water collected from several areas in the United States and 49 samples of raw wastewater collected from Milwaukee, Wis. Cryptosporidium parasites were detected in 25 surface water samples and 12 raw wastewater samples. C. parvum human and bovine genotypes were the dominant Cryptosporidium parasites in the surface water samples from sites where there was potential contamination by humans and cattle, whereas C. andersoni was the most common parasite in wastewater. There may be geographic differences in the distribution of Cryptosporidium genotypes in surface water. The PCR-RFLP technique can be a useful alternative method for detection and differentiation of Cryptosporidium parasites in water.

Fasciolopsiasis: is it a controllable food-borne disease?

Fasciolopsiasis, endemic to the Orient and Southeast Asia, is a snail-transmitted, intestinal, food-borne parasitic zoonosis caused by a trematode, Fasciolopsis buski, which also infects farm pigs. Fasciolopsiasis remains a public health problem despite changes in eating habits, alterations in social and agricultural practices, health education, industrialization, and environmental alterations. The disease occurs focally and is most prevalent in school-age children. In foci of parasite transmission, the prevalence of infection in children ranges from 57% in mainland China to 25% in Taiwan and from 50% in Bangladesh and 60% in India to 10% in Thailand. Control programs implemented for food-borne zoonoses are not fully successful for fasciolopsiasis because of century-old traditions of eating raw aquatic plants and using untreated water. Fasciolopsiasis is aggravated by social and economic factors such as poverty, malnutrition, an explosively growing free-food market, a lack of sufficient food inspection and sanitation, other helminthiases, and declining economic conditions.

Hyperkeratotic mange caused by Sarcoptes scabiei (Acariformes: Sarcoptidae) in juvenile human-habituated mountain gorillas (Gorilla gorilla beringei).

To facilitate ecotourism and behavioral research, free-ranging mountain gorillas (Gorilla gorilla beringei) have been habituated to humans. During routine health monitoring, five juvenile gorillas were observed with active crusted dermatitis and alopecia. Papular and vesicular lesions and crusts with papular eruption and oozing were numerous and disseminated over the body of one gorilla with a confirmed infestation of scabies. In this gorilla, the hyperkeratotic crusts were loose and thick with a flaky and scaly appearance. Histologically, the epidermis was thickened, displayed hyperkeratosis and was infiltrated with lymphocytes and neutrophils. Examination of skin scraping yielded a positive identification of adults and eggs of Sarcoptes scabiei mites. The gorillas were treated with ivermectin, 200 mg kg(-1). As S. scabiei mites can cross-infect various mammalian species causing self-limiting dermatitis, these ectoparasites can be propagated in the habitats shared by gorillas, people, and livestock, and therefore they represent an anthropozoonotic threat.

Effects of environmental change on emerging parasitic diseases.

Ecological disturbances exert an influence on the emergence and proliferation of malaria and zoonotic parasitic diseases, including, Leishmaniasis, cryptosporidiosis, giardiasis, trypanosomiasis, schistosomiasis, filariasis, onchocerciasis, and loiasis. Each environmental change, whether occurring as a natural phenomenon or through human intervention, changes the ecological balance and context within which disease hosts or vectors and parasites breed, develop, and transmit disease. Each species occupies a particular ecological niche and vector species sub-populations are distinct behaviourally and genetically as they adapt to man-made environments. Most zoonotic parasites display three distinct life cycles: sylvatic, zoonotic, and anthroponotic. In adapting to changed environmental conditions, including reduced non-human population and increased human population, some vectors display conversion from a primarily zoophyllic to primarily anthrophyllic orientation. Deforestation and ensuing changes in landuse, human settlement, commercial development, road construction, water control systems (dams, canals, irrigation systems, reservoirs), and climate, singly, and in combination have been accompanied by global increases in morbidity and mortality from emergent parasitic disease. The replacement of forests with crop farming, ranching, and raising small animals can create supportive habitats for parasites and their host vectors. When the land use of deforested areas changes, the pattern of human settlement is altered and habitat fragmentation may provide opportunities for exchange and transmission of parasites to the heretofore uninfected humans. Construction of water control projects can lead to shifts in such vector populations as snails and mosquitoes and their parasites. Construction of roads in previously inaccessible forested areas can lead to erosion, and stagnant ponds by blocking the flow of streams when the water rises during the rainy season. The combined effects of environmentally detrimental changes in local land use and alterations in global climate disrupt the natural ecosystem and can increase the risk of transmission of parasitic diseases to the human population.

Prevalence of Cryptosporidium, Giardia and Eimeria infections in post-weaned and adult cattle on three Maryland farms.

The prevalence of Cryptosporidium, Giardia and Eimeria, in healthy, asymptomatic, post-weaned and mature cattle was investigated on three Maryland farms. One farm, a dairy research facility, had 150 multiparous Holstein milking cows; 24 were examined and Cryptosporidium andersoni was detected in three (12.5%) but neither Giardia nor Eimeria was detected. The second farm, a commercial dairy, had 57 multiparous Holstein milking cows and an equal number of heifers. Of 19 cows examined, C. parvum, Giardia duodenalis, and Eimeria bovis and/or E. ellipsoidalis were detected in two (10.5%), two (10.5%) and one (5.26%) cow, respectively. Of 23 heifers examined, C. parvum, Giardia, and E. bovis and E. ellipsoidalis, was detected in two (8.7%), four (17.4%), and five (21.7%), heifers, respectively. The third farm, a beef cattle breeding and genetics research facility, had 180 7- to 9-month old purebred black Angus. Of 118 examined for C. parvum and Giardia, 34 (28.8%) and 44 (37.3%) were positive, respectively, of 97 examined for E. bovis and/or E. ellipsoidalis 32 (33.0%) were positive. These findings, based on a method with a minimum detection level of 100 oocysts of C. parvum/g of feces, which underestimates the number of infected cattle, clearly demonstrate the presence of low level, asymptomatic infections in post-weaned and adult cattle in the United States and indicate the potential role of such cattle as reservoirs of infectious parasites.

Foodborne infections vectored by molluscan shellfish.

Foodborne diseases cause approximately 76 million illnesses, 325,000 hospitalizations, and 5000 deaths each year in the United States. The authors present and analyze information derived from epidemiologic investigations and surveillance systems on foodborne infections caused by consumption of molluscan shellfish. This review focuses on the bias in reporting of shellfish-vectored illness, prevention and control of such infections, the origin of recognized viral and bacterial etiologic agents, and a new potential public health threat of a food-and-waterborne protozoan contaminant, Cryptosporidium parvum.

Successful hyperimmune bovine colostrum treatment of Savanna monitors (Varanus exanthematicus) infected with Cryptosporidium sp.

Therapy based on the protective passive immunity of hyperimmune bovine colostrum (HBC) (raised against Cryptosporidium parvum in cows) was applied to 4 Savanna monitors (Varanus exanthematicus) with gastric Cryptosporidium sp. infections. All lizards were moderately emaciated, and their fecal and gastric lavage samples contained moderate numbers of Cryptosporidium sp. oocysts. The first 3 of 7 gastric HBC treatments at 1-wk interval each decreased the numbers of oocysts in the fecal and gastric samples to undetectable levels. Neither feces nor lavages of the HBC-treated lizards contained Cryptosporidium sp. oocysts after the HBC therapy, whereas such samples of a single control lizard remained positive for oocysts. Two of the HBC-treated lizards died spontaneously due to metastasized carcinoma and septicemia of unknown etiology, respectively, and 2 lizards treated and killed during the experiment were histologically negative for developmental stages of Cryptosporidium sp. The control lizard died spontaneously of septicemia of unknown etiology and contained developmental stages of Cryptosporidium sp. in the gastric region. The HBC therapy was efficacious in V. exanthematicus and is recommended for lizards with gastric cryptosporidiosis.

Cryptosporidium serpentis oocysts and microsporidian spores in feces of captive snakes.

Fecal smears of 90 snakes, 29 lizards, and 8 turtles and tortoises were tested for Cryptosporidium spp. oocysts and microsporidian spores. Microsporidian spores measured mean = 3.7 microm in length and mean = 2.3 microm in width and were present in feces of 19 snakes and 1 lizard (16%); 13 of these snakes also shed Cryptosporidium serpentis oocysts. The oocysts were numerous in all positive samples, whereas microsporidian spores were always sparse, irrespective if whether fecal samples contained the oocysts. Retrospective examination of reptile clinical records revealed that all animals shedding microsporidian spores died naturally due to diseases, pathologic conditions, and clinical problems or were killed due to severe cryptosporidiosis. The present study indicates that microsporidian infections in reptiles have the features of an opportunistic infection.

Development of Fasciolopsis buski (Trematoda : Fasciolidae) in Hippeutis umbilicalis and Segmentina trochoideus (Gastropoda : Pulmonata).

Sequential development and histopathologic effects of Fasciolopsis buski larvae were examined in two species of pulmonate snail, Hippeutis umbilicalis and Segmentina trochoideus. Miracidial attachment and penetration began 5 min post-exposure (PE). On day 2 PE, the sporocysts rapidly increased in size and over days 3-7 PE, they transformed into mother rediae which migrated to the ovotestis on day 10 PE. In both species of snail, daughter rediae were initially observed on day 17 PE, the first cercariae emerged on day 21 PE, and from that day onward daughter rediae were the dominant larval form occupying the ovotestis. Histopathologic changes were confined exclusively to the ovotestis and included lytic lesions due to mechanical damage, and disappearance of all cellular elements. No inflammatory responses were observed in the ovotestis, and no degenerated larvae of any kind were observed in the snails. Infected snails did not produce egg masses and lived no longer than 35 days. The lack of differences in miracidial recognition and penetration, and in the temporal, sequential, and spatial development of F. buski larvae in H. umbilicalis and S. trochoideus indicate that both these snail species are equally susceptible to F. buski.

Environmental and geographical factors contributing to watershed contamination with Cryptosporidium parvum oocysts.

Cryptosporidium parvum is a waterborne parasite which infects cattle and produces life-threatening zoonosis in people with impaired immune systems. Digital maps of 100-year floodplain boundaries, land use/cover, and livestock operations were used to select and characterize cattle farms in the floodplain area in Lancaster County, Pennsylvania, U.S.A. Over 21% of the cattle farms were located within 100-year floodplain boundaries. On average, a single farm comprised 12.8 ha of pasture (including buildings and farmyard) at risk of inundation. In all farms cattle had unlimited access to the creek. Manure samples collected from closed-in calf pens, cow/heifer yard runoff, and cattle paths through the creek were tested for C. parvum. On 64% of the farms (n=50) at least one sample was positive for C. parvum, and 44% of the farms had oocysts in all manure samples. Concentration varied from 90 to 371 oocysts/g and was significantly higher (P<0.02) in calf samples than in manure from cow and cow/heifer.