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Municipal wastewater treatment plants play a vital role in reducing the microbial load of sewage before the end-products are discharged to surface waters (final effluent) or local environments (biosolids). This study was to investigate the presence of human-virulent microsporidian spores (Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Encephalitozoon hellem) and enterococci during treatment processes at four Irish municipal secondary wastewater treatment plants (plants A-D). Microsporidian abundance was significantly related to seasonal increase in water temperature. Plant A had the least efficient removal of E. intestinalis spores (32%) in wastewater, with almost 100% removal at other plants both in April and July. Some negative removal efficiencies were obtained for E. bieneusi (at plants C and D, -100%) and for E. hellem (at plants A and D, -90% and -50%). In addition, a positive correlation was found between the levels of enterococci and E. bieneusi in July (r (s) = 0.72, P 0.05). In terms of the dewatered biosolids, a median concentration as high as 32,000 spores/Kg of E. hellem was observed at plant D in July. Plant C sewage sludge contained the lowest microsporidian loadings (E. bieneusi; 450 spores/L and 1,000 spores/L in April and July, respectively). This study highlights the seasonal variation in concentrations of microsporidian spores in the incoming sewage. Spores in final effluents and dewatered biosolids can be the source of human-virulent microsporidian contamination to the local environment. This emphasizes a considerably high public health risk when sewage-derived biosolids are spread during summer months. This study also suggested enterococci as a potential indicator of the presence of microsporidian spores in wastewater, especially for E. bieneusi.

During summer months, samples of marine beach water were tested weekly for human waterborne pathogens in association with high and low bather numbers during weekends and weekdays, respectively. The numbers of bathers on weekends were significantly higher than on weekdays (P < 0.001), and this was associated with a significant (P < 0.04) increase in water turbidity. The proportion of water samples containing Cryptosporidium parvum, Giardia duodenalis, and Enterocytozoon bieneusi was significantly higher (P < 0.03) on weekends than on weekdays, and significantly (P < 0.01) correlated with enterococci counts. The concentration of all three waterborne pathogens was significantly correlated with bather density (P < 0.01). The study demonstrated that: (a) human pathogens were present in beach water on days deemed acceptable for bathing according to fecal bacterial standards; (b) enterococci count was a good indicator for the presence of Cryptosporidium, Giardia, and microsporidian spores in recreational marine beach water; (c) water should be tested for enterococci during times when bather numbers are high; (d) re-suspension of bottom sediments by bathers caused elevated levels of enterococci and waterborne parasites, thus bathers themselves can create a non-point source for water contamination; and (e) exposure to recreational bathing waters can play a role in epidemiology of microsporidiosis. In order to protect public health, it is recommended to: (a) prevent diapered children from entering beach water; (b) introduce bather number limits to recreational areas; (c) advise people with gastroenteritis to avoid bathing; and (d) use showers prior to and after bathing.

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

Objectives. We assessed the prevalence of recreational activities in the waterways of Baltimore, MD, and the risk of exposure to Cryptosporidium among persons with HIV/AIDS. Methods. We studied patients at the Johns Hopkins Moore Outpatient AIDS Clinic. We conducted oral interviews with a convenience sample of 157 HIV/AIDS patients to ascertain the sites used for recreational water contact within Baltimore waters and assess risk behaviors. Results. Approximately 48% of respondents reported participating in recreational water activities (fishing, crabbing, boating, and swimming). Men and women were almost equally likely to engage in recreational water activities (53.3% versus 51.3%). Approximately 67% (105 of 157) ate their own catch or that of friends or family members, and a majority (61%, or 46 of 75) of respondents who reported recreational water contact reported consumption of their own catch. Conclusions. Baltimoreans with HIV/AIDS are engaging in recreational water activities in urban waters that may expose them to waterborne pathogens and recreational water illnesses. Susceptible persons, such as patients with HIV/AIDS, should be cautioned regarding potential microbial risks from recreational water contact with surface waters.

This study investigates the fate of Cryptosporidium parvum and C. hominis oocysts and Giardia duodenalis cysts at four Irish municipal wastewater treatment plants (i.e., Plant A, B, C, and D) that utilize sludge activation or biofilm-coated percolating filter systems for secondary wastewater treatment. The fate of these pathogens through the sewage treatment processes was determined based on their viable transmissive stages, i.e., oocysts for Cryptosporidium and cysts for Giardia. Analysis of final effluent indicated that over 97% of viable oocysts and cysts were eliminated, except at Plant C, which achieved only 64% of oocyst removal. A significant correlation between the removal of oocysts and cysts was found at Plants A, B, and D (R = 0.98, P < 0.05). All sewage sludge samples were positive for C. parvum and C. hominis, and G. duodenalis, with maximum concentrations of 20 oocysts and eight cysts per gram in primary sludge indicating the need for further sludge sanitization treatments. This study provides evidence that C. parvum and C. hominis oocysts and G. duodenalis cysts are present throughout the wastewater processes and in end-products, and can enter the aquatic environment with consequent negative implications for public health.

The zebra mussel (Dreissena polymorpha) is an abundant and invasive molluscan shellfish species which arrived in Ireland’s river basins in the early 1990’s. Inland and coastal surface waters can be contaminated by human waterborne zoonotic enteropathogens such as Cryptosporidium parvum, Giardia lamblia, Encephalitozoon intestinalis, E. hellem and Enterocytozoon bieneusi originating predominantly from wastewater treatment plant effluents and agricultural runoff. Bivalve species, i.e., the invasive zebra mussel, Mytilus edulis (blue mussel) and Anodonta anatina (duck mussel) were used as sentinels and also as biomonitors of the aforementioned waterborne pathogens at twelve sites located in three Irish river basin districts impacted by pollution related to various water quality pressures. A variety of advanced biomolecular techniques were utilized to assess the presence and concentration of these pathogens in molluscan shellfish. At least one pathogen species was detected in bivalves at each of the twelve sites. Cryptosporidium, implicated in several recent Irish gastrointestinal epidemics, was recorded at all sites subjected to agricultural runoff and at one treated wastewater discharge site, linking source-track directly to animal and human fecal wastes. Overall, the results demonstrated a long-term human enteropathogen contamination of Irish waters with consequent public health risk-factors for drinking water abstraction and water-based recreational activities. The study provided further solid evidence that zebra mussels can recover and concentrate environmentally derived human pathogens and therefore can be used for the sanitary assessment of surface water quality.

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.

The capacity of synanthropic flies belonging to the families Calliphoridae, Sarcophagidae, and Muscidae to serve as mechanical vectors of Cryptosporidium and Giardia among livestock and wildlife in a multispecies agricultural complex in northwest Georgia (USA) was studied using fluorescent in situ hybridization (FISH) and immunofluorescent antibody (IFA) techniques. Flies from all three families were carrying viable Cryptosporidium oocysts and Giardia cysts internally and externally, and deposited them on trap surfaces. Furthermore, the contaminated flies and trap surfaces occurred in association with each of the four domestic animal units (beef, dairy, equine, and sheep), and in wildlife areas dominated by deer (Odocoileus virginianus) and Canada Geese (Branta canadensis). Cryptosporidium was isolated from 55.56% of the fly samples, whereas Giardia was isolated from only 7.94%. The highest numbers of Cryptosporidium were associated with cattle and wildlife areas, while Giardia occurred in greatest numbers in the sheep area. Cryptosporidium was isolated comparably from exoskeletal elutants and fly homogenates, but Giardia was isolated primarily from homogenates, indicating primarily internal transport in the latter. Surfaces visited by the flies (i.e., traps) became contaminated by both Cryptosporidium (18.89%) and Giardia (1.11%). In conclusion, these flies can serve as mechanical vectors of both Cryptosporidium and Giardia in all of these settings, and undoubtedly play a role in movement of these pathogens among the various host species.

Numerous studies have documented the presence of Cryptosporidium parvum , an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters ( Crassostrea virginica ) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups ( P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.

A taste-enhancement process for cold-stored, raw shell-stock Crassostrea virginica oysters (i.e., application of table salt to shells) when externally contaminated with human enteropathogens intensified spillage of these enteropathogens to oyster' storage containers (77% compared to 27% for controls) but did not, however, cause contamination of edible oyster tissue.

Helminth infections are prevalent in malaria-endemic areas, yet the potential for helminths to alter malaria transmission has not been closely examined. We used the Echinostoma caproni-Plasmodium yoelii murine model of co-infection to assess the impact of helminth co-infection on malaria transmission. In four replicate experiments, Anopheles stephensi mosquitoes exposed to co-infected mice five days post-malaria infection had a higher rate of infectivity (80.1%, n = 241) than those exposed to malaria only-infected mice (72.0%, n = 232, P = 0.039). Intensity of malaria parasite transmission was also greater, with approximately two-fold more oocysts (geometric mean = 19.2 versus 10.5, P = 0.004) and an increase in sporozoite burden observed in mosquitoes exposed to co-infected mice. Malaria parasite prevalence and anemia were similar between co-infected and malaria only-infected mice, which suggested that enhanced malaria parasite transmission was due to helminth-induced modulation of host responses.

Although well-known methods for the detection of intestinal parasitic protozoans in water samples exist, they are insufficiently sensitive, expensive, of little practical value in the routine monitoring of waterborne pathogens and time- and labour-consuming. In the investigation reported here we have assessed Cryptosporidium oocyst detection using both the so-called Method 1623[recommended by the U.S. Environmental Protection Agency (USEPA)] and a direct method involving the determination of oocysts of Cryptosporidium in rotifers as detection tools of surface water contamination by dispersive stages of intestinal protozoans. Rotifers were sampled from three lakes located near the city of Poznan (Poland). To detect the oocysts of Cryptosporidium, we applied the fluorescent in situ hybridisation technique, an immunofluorescent assay and an enzyme immunoassay. Oocysts of Cryptosporidium were detected both in water collected from the lakes and in rotifers. The FISH technique applied to rotifers enabled the detection of biological contamination of surface water through an assessment of the dispersive stages of the parasite and was found to be more sensitive, less time-consuming and cheaper than the method recommended by the USEPA.

Human microsporidiosis, a serious disease of immunocompetent and immunosuppressed people, can be due to zoonotic and environmental transmission of microsporidian spores. A survey utilizing conventional and molecular techniques for examining feces from 570 free-ranging, captive, and livestock birds demonstrated that 21 animals shed microsporidian spores of species known to infect humans, including Encephalitozoon hellem (20 birds; 3.5%) and Encephalitozoon intestinalis (1 bird; 0.2%). Of 11 avian species that shed E. hellem and E. intestinalis , 8 were aquatic birds (i.e., common waterfowl). The prevalence of microsporidian infections in waterfowl (8.6%) was significantly higher than the prevalence of microsporidian infections in other birds (1.1%) ( P < 0.03); waterfowl fecal droppings contained significantly more spores (mean, 3.6 × 10 5 spores/g) than nonaquatic bird droppings contained (mean, 4.4 × 10 4 spores/g) ( P < 0.003); and the presence of microsporidian spores of species known to infect humans in fecal samples was statistically associated with the aquatic status of the avian host ( P < 0.001). We demonstrated that a single visit of a waterfowl flock can introduce into the surface water approximately 9.1 × 10 8 microsporidian spores of species known to infect humans. Our findings demonstrate that waterborne microsporidian spores of species that infect people can originate from common waterfowl, which usually occur in large numbers and have unlimited access to surface waters, including waters used for production of drinking water.

The introduction of nonnative oysters (i.e., Crassostrea ariakensis ) into the Chesapeake Bay has been proposed as necessary for the restoration of the oyster industry; however, nothing is known about the public health risks related to contamination of these oysters with human pathogens. Commercial market-size C. ariakensis triploids were maintained in large marine tanks with water of low (8-ppt), medium (12-ppt), and high (20-ppt) salinities spiked with 1.0 × 10 5 transmissive stages of the following human pathogens: Cryptosporidium parvum oocysts, Giardia lamblia cysts, and microsporidian spores (i.e., Encephalitozoon intestinalis , Encephalitozoon hellem , and Enterocytozoon bieneusi ). Viable oocysts and spores were still detected in oysters on day 33 post-water inoculation (pwi), and cysts were detected on day 14 pwi. The recovery, bioaccumulation, depuration, and inactivation rates of human waterborne pathogens by C. ariakensis triploids were driven by salinity and were optimal in medium- and high-salinity water. The concentration of human pathogens from ambient water by C. ariakensis and the retention of these pathogens without (or with minimal) inactivation and a very low depuration rate provide evidence that these oysters may present a public health threat upon entering the human food chain, if harvested from polluted water. This conclusion is reinforced by the concentration of waterborne pathogens used in the present study, which was representative of levels of infectious agents in surface waters, including the Chesapeake Bay. Aquacultures of nonnative oysters in the Chesapeake Bay will provide excellent ecological services in regard to efficient cleaning of human-infectious agents from the estuarine waters.

The effect of chronic intestinal trematode infection on malaria was examined in a murine model of co-infection using Echinostoma caproni and Plasmodium yoelii. BALB/c mice (n = 32) infected with a low dose of E. caproni (approximately 10 cysts) 25-35 days before malaria infection displayed significantly increased malaria parasitemia (P = 0.01), extended patency of malaria (P = 0.03), and increased fatality (47%; P0.001) compared to mice infected only with P. yoelii (17X nonlethal strain) (n = 18). Further analysis revealed that differences in malaria parasitemia between fatal co-infections and infections with P. yoelii only were highly significant (P0.0001), whereas nonfatal co-infections were not statistically different. Exacerbation of malaria was demonstrated to be reversible through clearance of E. caproni worms by praziquantel treatment administered 10 days before malaria infection. No deaths were observed during malaria infection in mice cleared of their E. caproni infection (n = 10), and parasitemia was significantly reduced from that of untreated co-infected mice (P = 0.03) and was not different from that of mice infected with P. yoelii only. Further studies examining parasite-parasite interactions and host immune response in the echinostome model are warranted to understand the mechanisms affecting the course and outcome of malaria infection during concomitant helminth infection.

Food-borne trematodiases still remain a public health problem world-wide, despite changes in eating habits, alterations in social and agricultural practices, health education, industrialization, environmental alteration, and broad-spectrum anthelmintics. Food-borne trematodiases usually occur focally, are still persistently endemic in some parts of the world, and are most prevalent in remote rural places among school-age children, low-wage earners, and women of child-bearing age. Intestinal fluke diseases are aggravated by socio-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. Control programs implemented for food-borne zoonoses and sustained in endemic areas are not fully successful for intestinal food-borne trematodiases because of centuries-old traditions of eating raw or insufficiently cooked food, widespread zoonotic reservoirs, promiscuous defecation, and the use of "night soil" (human excrement collected from latrines) as fertilizer. This review examines food-borne intestinal trematodiases associated with species in families of the Digenea: Brachylaimidae, Diplostomidae, Echinostomatidae, Fasciolidae, Gastrodiscidae, Gymnophallidae, Heterophyidae, Lecithodendriidae, Microphallidae, Nanophyetidae, Paramphistomatidae, Plagiorchiidae, and Strigeidae. Because most of the implicated species are in the Echinostomatidae and Heterophyidae, emphasis in the review is placed on species in these families.

Captive juvenile African black-footed penguins (Spheniscus demersus) housed in an outdoor enclosure at the Baltimore Zoo have an average 50% mortality from avian malarial (Plasmodium sp.) infection each year without intense monitoring for disease and chemotherapeutic intervention. During the 1996 malaria transmission season, the safety and efficacy of an anti-circumsporozoite (CSP) DNA vaccine encoding the Plasmodium gallinaceum CSP protein against P. relictum were studied. The goal was to reduce clinical disease and death without initiating sterile immunity after release into an area with stable, endemic avian malaria. The birds were monitored for adverse clinical signs associated with vaccination, the stimulation of an anti-CSP antibody response, and protection afforded by the vaccine. The presence of P. relictum in trapped culicine mosquitoes within the penguin enclosure was monitored to assess parasite pressure. Among the vaccinated penguins, the parasitemia rate dropped from approximately 50% to approximately 17% despite intense parasite pressure, as determined by mosquito infection rate. During the year of the vaccine trial, no mortalities due to malaria occurred and no undesirable vaccination side effects occurred. This is the first trial of an antimalarial vaccine in a captive penguin colony.

The effectiveness of malaria control measures depends not only on the potency of the control measures themselves but also upon the influence of variables associated with the environment. Environmental variables have the capacity either to enhance or to impair the desired outcome. An optimal outcome in the field, which is ultimately the real goal of vaccine research, will result from prior knowledge of both the potency of the control measures and the role of environmental variables. Here we describe both the potential effectiveness of control measures and the problems associated with testing in an area of endemicity. We placed canaries with different immunologic backgrounds (e.g., naïve to malaria infection, vaccinated naïve, and immune) directly into an area where avian malaria,Plasmodium relictum, is endemic. In our study setting, canaries that are naïve to malaria infection routinely suffer approximately 50% mortality during their first period of exposure to the disease. In comparison, birds vaccinated and boosted with a DNA vaccine plasmid encoding the circumsporozoite protein ofP. relictumexhibited a moderate degree of protection against natural infection (P< 0.01). In the second year we followed the fate of all surviving birds with no further manipulation. The vaccinated birds from the first year were no longer statistically distinguishable for protection against malaria from cages of naïve birds. During this period, 36% of vaccinated birds died of malaria. We postulate that the vaccine-induced protective immune responses prevented the acquisition of natural immunity similar to that concurrently acquired by birds in a neighboring cage. These results indicate that dominant environmental parameters associated with malaria deaths can be addressed before their application to a less malleable human system.

Wild-caught synanthropic flies were tested for the presence of Cryptosporidium parvum and Giardia lamblia on their exoskeletons and in their digestive tracks by fluorescent in situ hybridization and fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (MAb) against Cryptosporidium and Giardia cell wall epitopes. The levels of C. parvum were positively correlated with the levels of G. lamblia, indicating a common source of contamination. The majority of oocysts and cysts were potentially viable (C. parvum = 80% and G. lamblia = 69%). More G. lamblia cysts occurred on the exoskeleton of the flies than within the digestive tracts; the opposite relationship was observed for C. parvum. No genotype other than C. parvum G2 was found to be associated with flies. Because filth flies carry viable C. parvum oocysts and G. lamblia cysts acquired naturally from unhygienic sources, they can be involved in the epidemiology of cryptosporidiosis and giardiasis. Fluorescent oligonucleotide probes used together with FITC-conjugated MAb represent a convenient and cost-effective technique for rapid and specific identification of human-infectious species of Cryptosporidium and Giardia mechanically transported by flies, and for the assessment of the viability of these pathogens.

Little is known about engineered nanoparticles (NPs) exposures on oysters. As sessile filter feeders, oysters are likely to be exposed to NPs suspended in the water column with unknown effects of NP exposure on oyster functioning. Our study indicates that waterborne NPs alter oyster hemocyte phagocytosis dynamics, an indication of sub-lethal effects of NP exposures. Silver NPs, titanium dioxide (TiO2) NPs, and silver nitrate exposures reduced phagocytosis compared to the control. Increasing TiO2 NPs and silver nitrate concentrations reduced phagocytosis. Silver NPs, up to 120 ppb, increased phagocytosis, but higher concentrations reduced phagocytosis.

To facilitate ecotourism and research, free-ranging mountain gorillas of Uganda have been habituated to humans. Testing of fecal samples of gorillas (n = 100), people sharing gorilla habitats (n = 62). and local pre- and postweaned cattle (n = 50) having access to these habitats with fluorescein isothiocyanate-conjugated monoclonal antibodies revealed Giardia duodenalis cysts at prevalences of 2, 5, and 10%, respectively. The identification of G. duodenalis was confirmed by fluorescent in situ hybridization with 2 species-specific 18-bp oligonucleotide probes conjugated to hexachlorinated 6-carboxyfluorescein. The mean pathogen concentration was 2.5, 2.8, and 0.2 x 10(4) cysts/g of the gorilla, people, and cattle feces, respectively. All cyst isolates aligned with genotype (assemblage) A, as confirmed by polymerase chain reaction amplification and sequencing of a 130-bp region near the 5' end of the small subunit-ribosomal RNA gene. A single genotype (assemblage) A recovered from 3 genetically distant but geographically united host groups indicates anthropozoonotic transmission of G. duodenalis. A large percentage of the local community does not follow park regulations regarding the disposal of their fecal waste, as self-reported in a questionnaire. This genotype may have been introduced into gorilla populations through habituation activities and may have then been sustained in their habitats by anthropozoonotic transmission.

Microsporidian spores have been detected by Chromotrope 2R and calcofluor stains in fecal samples of three free-ranging human-habituated mountain gorillas in Uganda and in two people who share gorilla habitats. All spore isolates have been identified by PCR with species-specific primers and fluorescent in situ hybridization with a species-specific oligonucleotide probe to be Encephalitozoon intestinalis. Sequencing analyses of the full length SSUrRNA amplified from all spore isolates were identical with Enc. intestinalis SSUrRNA GenBank SIU09929. Sequences generated from a fragment containing the internal transcribed spacer of these isolates were identical to GenBank sequence Y11611, i.e., Enc. intestinalis of anthroponotic origin. A single pathogen genotype in two genetically distant but geographically united host groups indicates anthropozoonotic transmission of Enc. intestinalis. It is highly unlikely that these two identical Enc. intestinalis genotypes were acquired independently by gorillas and people; it is much more probable that one group initiated infection of the other.

A semidistributed watershed model is applied over the Mahantango Creek catchment in Pennsylvania to estimate future changes in direct runoff under 22 different climate scenarios. It is shown how different subcatchments of the watershed may respond to possible changes in the precipitation and temperature regimes. Subcatchments with the most unfavorable future runoff responses can be identified where possible changes in land use management practices may be suggested.

Microsporidial infections may be asymptomatic in immunocompetent hosts, but can be severe and disseminated in HIV/AIDS patients, children, the elderly, or in immunocompromised individuals, including those with primary or medically-induced immunodeficiencies. 209 faecal samples were collected from 80 clinical patients, with or without abdominal symptoms, and tested for the presence of the parasites. Microsporidia were found in 10 of the 80 patients (12.5%) using trichrom staining of faecal smears and/or PCR. Encephalitozoon intestinalis and 1 unidentified species were identified in 2 of the 32 children with primary immunodeficiencies (6%), presenting with diarrhoea, including one co-infection with Cryptosporidium meleagridis. In the group of patients with medically-induced immunosuppression (transplant recipients), 8 of the 48 patients (17%) were tested positive for microsporidia. Thus, these pathogens should be taken into account when the other etiological agents cannot be found in diarrheic patients with PIDs or undergoing immunosuppressive treatment before or after transplantation. This article presents the results of the first epidemiological study on the occurrence and prevalence of microsporidia in patients with primary and secondary immunodeficiency in Poland.

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.

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.

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.

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 nonhuman 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 landuse 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 landuse and alterations in global climate disrupt the natural ecosystem and can increase the risk of transmission of parasitic diseases to the human population. q 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

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-water-borne protozoan contaminant, Cryptosporidium parvum.

Filter-feeding molluscan shellfish can concentrate zoonotic and anthroponotic waterborne pathogens. Cysts of Giardia sp. were detected by immunofluorescent antibodies in tissues of the clams Macoma balthica and M. mitchelli from Rhode River, a Chesapeake Bay (Maryland) subestuary. Molecular tests identified the cysts as Giardia duodenalis Genotype A, the most common genotype recovered from humans. Macoma clams are burrowers in mud or sandy-mud substrata and preferentially feed on the surface sediment layer. Waterborne Giardia cysts settle rapidly to the bottom in slow-moving waters and contaminate the sediment. Macoma clams do not have economic value, but can serve as biologic indicators of sediment contamination with Giardia sp. cysts of public health importance. These clams can be used for sanitary assessment of water quality.

Oocysts of Cryptosporidium parvum, a zoonotic waterborne pathogen, can be removed by bivalve molluscs from contaminated water and retained on gills and in hemolymph. We identified oocysts of C. parvum in oysters from seven sites in the Chesapeake Bay area. These findings document the presence of C. parvum infectious for humans in oysters intended for human consumption.

Apicomplexan Cryptosporidium parasites infect a wide range of vertebrate hosts. While some species are limited to a single host group, such as Cryptosporidium baileyi , which infects chickens, other species of this genus, such as C. parvum , infect a wide range of mammalian species from mice to humans. During an investigation of Cryptosporidium infection in cattle on a farm in northern Poland, we identified an infection caused by C. felis , in addition to known infections with C. muris and C. parvum . This new infection was identified based on the size of the oocysts (mean size, 4.3 ± 0.4 μm; range, 3.5 to 5.0 μm), as well as by analysis of the molecular sequence of the variable region of the small-subunit rRNA. This finding demonstrates the complex host specificity and circulation in the environment of Cryptosporidium species.

The study demonstrated that the resuspension of bottom sediments caused by bathers and their direct microbial input resulted in elevated levels of Cryptosporidium parvum oocysts, Giardia lamblia cysts, and microsporidian spores, particularly Enterocytozoon bieneusi, in recreational beach water on days deemed acceptable for bathing by fecal bacterial standards.

Human echinostomiasis, endemic to southeast Asia and the Far East, is a food-borne, intestinal, zoonotic parasitosis attributed to at least 16 species of digenean trematodes transmitted by snails. Two separate life cycles of echinostomes, human and sylvatic, efficiently operate in endemic areas. Clinical symptoms of echinostomiasis include abdominal pain, violent watery diarrhea, and anorexia. The disease occurs focally and transmission is linked to fresh or brackish water habitats. Infections are associated with common sociocultural practices of eating raw or insufficiently cooked mollusks, fish, crustaceans, and amphibians, promiscuous defecation, and the use of night soil (human excrement collected from latrines) for fertilization of fish ponds. The prevalence of infection ranges from 44% in the Philippines to 5% in mainland China, and from 50% in northern Thailand to 9% in Korea. Although the patterns of other food-borne trematodiases have changed in Asia following changes in habits, cultural practices, health education, industrialization, and environmental alteration, human echinostomiasis remains a health problem. The disease is most prevalent in remote rural places among low-wage earners and in women of child bearing age. Echinostomiasis is aggravated by socioeconomic factors such as poverty, malnutrition, an explosively growing free-food market, a lack of supervised food inspection, poor or insufficient sanitation, other helminthiases, and declining economic conditions. Furthermore, World Health Organization control programs implemented for other food-borne helminthiases and sustained in endemic areas are not fully successful for echinostomiasis because these parasites display extremely broad specificity for the second intermediate host and are capable of completing the life cycle without involvement of the human host.

Oocysts of Cryptosporidium parvum placed in artificial seawater at salinities of 10, 20, and 30 ppt at 10°C and at 10 ppt at 20°C were infectious after 12 weeks. Those placed in seawater at 20 ppt and 30 ppt at 20°C were infectious for 8 and 4 weeks, respectively. These findings suggested that oocysts could survive in estuarine waters long enough to be removed by filter feeders such as oysters. Thereafter, 30 Eastern oysters, Crassostrea virginica , were collected with a dredge or with hand tongs at each of six sites within Maryland tributaries of the Chesapeake Bay in May and June and in August and September of 1997. Hemocytes and gill washings from all oysters were examined for the presence of Cryptosporidium oocysts and Giardia cysts by immunofluorescence microscopy utilizing a commercially available kit containing fluorescein isothiocyanate-conjugated monoclonal antibodies. Giardia was not detected by this method from any of the 360 oysters examined. Presumptive identification of Cryptosporidium oocysts was made in either hemocytes or gill washings of oysters from all six sites both times that surveys were conducted. In addition, during August and September, for each of the six sites, hemocytes from the 30 oysters were pooled and gill washings from the oysters were pooled. Each pool was delivered by gastric intubation to a litter of neonatal mice to produce a bioassay for oocyst infectivity. Intestinal tissue from two of three mice that received gill washings from oysters collected at a site near a large cattle farm and shoreline homes with septic tanks was positive for developmental stages of C. parvum . These findings demonstrate for the first time that oysters in natural waters harbor infectious C. parvum oocysts and can serve as mechanical vectors of this pathogen.

Asian freshwater clams, Corbicula fluminea , exposed for 24 h to 38 liters of water contaminated with infectious Cryptosporidium parvum oocysts (1.00 × 10 6 oocysts/liter; approximately 1.9 × 10 5 oocysts/clam) were examined (hemolymph, gills, gastrointestinal [GI] tract, and feces) on days 1, 2, 3, 7, and 14 postexposure (PE). No oocysts were detected in the water 24 h after the contamination event. The percentage of oocyst-containing clams varied from 20 to 100%, depending on the type of tissue examined and the technique used—acid-fast stain (AFS) or immunofluorescent antibody (IFA). The oocysts were found in clam tissues and feces on days 1 through 14 PE; the oocysts extracted from the tissues on day 7 PE were infectious for neonatal BALB/c mice. Overall, the highest number of positive samples was obtained when gills and GI tracts were processed with IFA (prevalence, 97.5%). A comparison of the relative oocyst numbers indicated that overall, 58.3% of the oocysts were found in clam tissues and 41.7% were found in feces when IFA was used; when AFS was used, the values were 51.9 and 48.1%, respectively. Clam-released oocysts were always surrounded by feces; no free oocysts or oocysts disassociated from fecal matter were observed. The results indicate that these benthic freshwater clams are capable of recovery and sedimentation of waterborne C. parvum oocysts. To optimize the detection of C. parvum oocysts in C. fluminea tissue, it is recommended that gill and GI tract samples be screened with IFA (such as that in the commercially available MER IFLUOR test kit).

Therapy based on the protective passive immunity of Hyperimmune Bovine Colostrum (HBC) (raised against Cryptosporidium parvum in dairy cows immunized during gestation) was tested for heterologous efficacy in subclinical and clinical infections of 12 captive snakes with C. serpentis. Six gastric HBC treatments of 1% snake weight at 1-week intervals each, have histologically cleared C. serpentis in three subclinically infected snakes, and regressed gastric histopathological changes in one of these snakes. In all snakes, each subsequent HBC treatment significantly decreased the number of oocysts recovered in gastric lavage eluants (P < 0.03). The treatments induced oocyst-negative gastric eluants and stools in all snakes, and improved clinical signs of infection. Clinically infected snakes displayed severe histopathological changes in the gastric region; however, the numbers of developmental stages of C. serpentis were moderate. Considering the severity of pathology, much lower than expected pathogen numbers were observed, and it is believed that clinically infected snakes did not have enough time to repair tissue damage that had occurred over the years of infection. As the HBC treatment was safe and highly efficacious, it is recommended to gastrically administer the HBC therapeutically to snakes that are clinically or subclinically infected with C. serpentis. Hyperimmune bovine colostrum can also be used in snake supportive therapy or prophylaxis.

The cellulose-acetate membrane (CAM)-filter dissolution method implemented into a Millipore Glass Microanalysis system was used for recovery of Cryptosporidium parvum oocysts seeded into 25 l of drinking water in polyethylene carboy aspirator bottles. CAM-entrapped oocysts were detected by immunofluorescence microscopy. From 65 to 94 oocysts/l (mean 75 oocysts/l), 34.7% overall of the inoculated oocysts, were unrecovered as determined after the water had been drained from the bottle, rinsed with 1 l of eluting fluid (EF), and CAM-filtered. Efficiency rates of oocyst recovery ranged from 24.0% to 64.0% (mean 44.1%), without the use of EF and from 72.1% to 82.3% (mean 78.8%) when EF was used. To ensure a high recovery efficiency of Cryptosporidium oocysts from sampled water by the CAM-filter dissolution method, it is recommended that 1 l of EF per 25 l of water be used.

The applicability of ELISA detection of circulating Aspergillus spp. antigen (Ag) and systemic antibody (Ab) of IgG class, and the blood parameter values were evaluated for diagnosis of invasive aspergillosis in Aspergillus spp.-challenged Peking ducks (Anas platyrhynchos). The protective role of Aspergillus spp. IgG was evaluated in Cape shelducks (Tadorna cana) immunized with Aspergillus spp. Ag. Challenged but non-immunized A. platyrhynchos developed invasive aspergillosis on day 21 as demonstrated histopathologically by the presence of fungal micro-granuloma in air sacs and lung tissue, with serum antigenemia fluctuating from 65 to 270 ng of 55-kD basic protein Ag per ml. Immunized A. platyrhynchos did not demonstrate Aspergillus spp. serum antigenemia but did show rare histopathological changes in some air sacs associated with fungal inflammation. Although the differences between immunized and non-immunized T. cana in blood evaluation parameters did not differ significantly, immunized birds mounted high Aspergillus spp.-specific IgG titer. There was no correlation between the blood parameter values and post-immunization timepoints in T. cana and in A. Platyrhynchos. Intramuscular immunization with Aspergillus spp. mycelial phase cultures Ag provided protection against the pathogens. The lack of relations between blood parameter values and increasing Aspergillus spp. IgG titers (in T. cana and A. platyrhynchos) indicate low applicability of these parameters in evaluation of a bird Aspergillus spp. status. Detection of circulating 55-kDa Aspergillus spp. Ag has high early predictive values for invasive aspergillosis in birds.

Microsporidia are intracellular parasites that cause opportunistic infections in humans of various immunological status. Only a few case reports exist on microsporidial infection in solid organ transplant recipients worldwide. The presented study demonstrates the first case in Poland of Enterocytozoon bieneusi infection in a liver transplant patient. Parasites were diagnosed in stool samples using both modified trichrome staining and PCR.

Microsporidia are single-celled intracellular parasites which occur in a number of animals, both vertebrates and invertebrates. Several species of microsporidia can cause disease in humans in both immunocompromised and immunocompetent individuals. However, the sources of human infection and the routes of transmission of microsporidia have not yet been fully determined, although more and more researchers are of the opinion that microsporidia in humans is zoonotic. The aim of the presented study was to identify the most common microsporidial species in the droppings of raised and wild pigeons in Poland. A total of 139 collective samples of droppings (33 samples of droppings from feral pigeons and 106 samples from raised pigeons) were examined using conventional staining and molecular techniques. Using chromotrope staining and multiplex FISH techniques, the microsporidial spores were found in 12 (8.6%) of the 139 samples of droppings. The spores of Encephalitozoon hellem were detected in five samples of pigeon droppings (3.6%), spores of E. intestinalis in four samples of droppings (2.9%), while spores of E. cuniculi and E. bieneusi were only detected in two samples (1.4%). Furthermore, a mixed infection of E. bieneusi and E. cuniculi was found in a single sample of droppings (0.7%). The presence of microsporidial spores in droppings collected from raised pigeons indicates a risk of infection to humans, mainly pigeon fanciers.

The applicability of stomach lavage and cloacal swab techniques for diagnosis of subclinical cryptosporidiosis were tested in eight captive snakes subclinically infected with Cryptosporidium serpentis. Two feeding regimes were employed. The snakes were first fed 7 days prior to stomach and cloaca sampling, and then 3 days prior to sampling, and the oocysts were detected by fluorescein labeled monoclonal antibody (mAb) and by acid-fast stained (AFS) direct wet smear (DWS). The overall sensitivity of AFS DWS was 95% for stomach samples and 57% for cloacal samples, with false-negativity of 5% and 43%, respectively. A significant relationship (P0.01) was found between stomach and cloacal samples when mAb were used for oocyst detection. Stomach sampling was diagnostically superior to cloacal sampling for identifying snake subclinical cryptosporidiosis. Based on gastric aspirates, cryptosporidial infection was diagnosed in all eight animals, and only in two or four snakes when cloacal swab material was processed by AFS or by mAb, respectively. Feeding snakes 3 days prior to sampling facilitated diagnosis based on stomach samples; however, it did not improve diagnosis when cloacal samples were used. The fraction of oocyst-positive stomach samples was significantly higher (P0.05) for snakes fed 3 days prior to gastric lavage when compared with the fraction of positive samples of snakes fed 7 days prior to lavage. If subclinical cryptosporidiosis is suspected in a non-eating snake patient, force-feeding and stomach lavage, 3 days after the meal, is recommended.

The sampling of 50 microliters of the yolk (average 1.7% of the total volume) from northern bobwhite quail (Colinus virginianus) eggs on day 0, 6, 12, and 18 of incubation resulted in significantly (P0.05) increased embryo mortality in the day 12 and day 18 egg groups. Performance parameters and reproductive indices of the non-sampled eggs and the eggs sampled on day 0, and 6 of incubation did not differ from other reports on C. virginianus. No differences was found in postnatal responsiveness among hatchlings originating from controls and from all the groups of sampled eggs. As demonstrated in this representative galliform species, the technique can be applied for the screening of unembryonated eggs or early-embryogenesis-eggs of wild birds. If female birds are not accessible but their eggs are, information on the reproductive flock can be accessed without stressing adult birds.

The therapeutic effect of halofuginone hydrobromide (Steronol) and spiramycin solubilized (Spirasol) applied to clinical Cryptosporidium serpentis infections in captive snakes was investigated. Pathological changes induced by C. serpentis were typical for snake cryptosporidiosis. Spiramycin induced no significant change in the pattern of shedding of fecal Cryptosporidium oocysts; biopsies and necropsies revealed cryptosporidiosis in gastric mucosa of all spiramycin-treated animals. In all, 8 of 21 (38%) halofuginone-treated snakes stopped shedding C. serpentis oocysts; examination of gastric tissue of 6 of these 8 animals revealed cryptosporidiosis in 2 snakes. Hepatotoxic and nephrotoxic pathological changes induced by halofuginone included focal or multifocal, severe, acute liver necrosis; severe liver hemosiderosis; and bilateral, severe, acute diffuse cortical and tubular necrosis and iron deposition. Postprandial regurgitation associated with midbody swelling, observed in 4 halofuginone-treated and 2 spiramycin-treated snakes at 15 and 21 weeks after drug withdrawal, did not coincide with oocyst-positive feces. Neither halofuginone nor spiramycin treatment produced a satisfactory therapeutic outcome when applied against clinical C. serpentis infections in snakes.

Anti-Aspergillus spp. antibody (Ab) levels of seven mating pairs of captive African penguins (Spheniscus demersus) and the maternal Ab levels of their embryonated and unembryonated eggs were determined by ELISA. The egg Ab levels were significantly higher than the Ab levels of their female or male parents. Maternal Ab of unembryonated eggs and Ab of the embryos were significantly correlated with the Ab levels of their female parents. Because eggs can be sampled where adult penguins are not accessible, this technique can be applied for prediction of Aspergillus spp. Ab titers of penguin females in wild populations.

The value profiles of 5 intracellular enzymes, 15 metabolites (with 2 associated ratios), and 3 electrolytes were monitored over time in 9 captive-reared African black-footed penguins (Spheniscus demersus) with different avian malaria clinical status: uninfected, subclinically infected, and clinically infected with fatal outcome. Fatal infections were caused by Plasmodium relictum. Numerous schizonts were visible in the lungs, liver, spleen, and interstitial tissue of the kidneys. The reference ranges of 23 serum clinical chemistry parameters and 2 ratios were established for S. demersus. The mean values obtained for 8 of 23 parameters of the infected penguins were significantly different from those recorded for the uninfected birds, indicating impaired renal function, hepatic dysfunction, and nonspecific tissue damage related to the infestation with exoerythrocytic schizonts. Analysis of sensitivity, specificity, and negative and positive predictive values (PPVs) showed that gamma-glutamyltranspeptidase (GGTP), alanine aminotransferase (ALT), and creatinine reached PPVs and a specificity over 57% for avian malaria infections in penguins. Creatinine, ALT, and GGTP values should be consulted in evaluation of the clinical malaria status of S. demersus.