First report of Cryptosporidium spp. oocysts in oysters (Crassostrea rhizophorae) and cockles (Tivela mactroides) in Brazil

The consumption of oysters and cockles, which are usually eaten raw or lightly-cooked, can cause outbreaks of human diseases, especially if these shellfish are harvested from polluted areas. In Brazil data about the occurrence of pathogens, like hepatitis A virus, in shellfish have been reported but research on natural contamination for pathogenic protozoa is still non-existent. Cryptosporidium oocyst contamination of oysters (Crassostrea rhizophorae) and cockles (Tivela mactroides) was evaluated during two different periods in a coastal area from S?o Paulo, Brazil. From June to November 2005, and from July to December 2006, 180 mollusks were harvested for tissue examination. The gills and gastrointestinal tract (n = 36 pools) were carefully extracted from the animals and homogenized in a tissue homogenizer by adding surfactant Tween 80 (0.1%). Immunofluorescence assays were performed and Cryptosporidium oocysts were detected in 50.0% of gill pools of cockles and 10.0% of gill pools of oysters. In order to evaluate seawater quality in shellfish growing areas, total levels of thermotolerant coliforms, Escherichia coli and enterococci were determined. This is the first time that Cryptosporidium oocysts were found in shellfish from the coastal region of Brazil, and to the best of our knowledge it is also the first report in Latin America and the case might be of public health importance, reflecting the extension of the contamination on seafood, requiring a need for quality control standards.     

First investigations into the prevalence of Cryptosporidium and Giardia spp. in Hungarian drinking water

Safe drinking water is a top priority in preventing disease outbreaks and is of general concern to everyone. This study examines the occurrence of Cryptosporidium and Giardia in Hungarian drinking water supplies for the first time. A total of 76 raw and drinking water samples were examined using the U.S. EPA Method 1623. From these 15 of 34 (48.4%) raw water samples tested positive for Giardia and 7 (26.6%) for Cryptosporidium. Twelve of 45 (26.7%) drinking water samples were positive for Giardia and 6 (13.3%) for Cryptosporidium. Overall, Giardia cysts and/or Cryptosporidium oocysts were detected in 48% of the raw water samples and 35% of the drinking water samples. The highest levels in drinking water were found to be 3 oocysts/100 litres of Cryptosporidium and 63.6 cysts/100 litres for Giardia, enough to cause giardiasis. The highest levels in raw water were 1,030 cysts/100 litres for Giardia and 50 oocysts/100 litres for Cryptosporidium and higher oocyst densities were associated with source water receiving effluents from sewage treatment plants or originating from a forest environment. In addition to this monitoring, riverbank filtrated water and raw water from the River Danube in Budapest were monitored in order to ascertain protozoan removal efficiency of riverbank filtration (RBF). A total of 157 samples, including 87 samples from the River Danube and 70 samples post RBF, were examined. Cryptosporidium and Giardia were detected regularly in the river water but never in riverbank filtered water suggesting the effectiveness of RBF as a purification method.     

Comparison of two methodologies for detection of Giardia spp. cysts and Cryptosporidium spp. oocysts in activated sludge samples from a sewage treatment plant in the city of Campinas, Sao Paulo State, Brazil

Giardia spp. and Cryptosporidium spp. are recognized worldwide as highly infectious protozoan parasites that can cause severe gastrointestinal disease in humans and animals. The detection of these pathogens in activated sludge samples becomes interesting since there is an increasing trend for the use of sewage sludge (biosolids) in agriculture. A total of 22 samples were collected and evaluated by means of Centrifugal – Concentration, followed or not followed by a purification process (ether clarification and sucrose flotation). Student t tests for comparison of the two procedures indicated a higher recovery rate of Giardia cysts with Centrifugal – Concentration; with regard to Cryptosporidium oocysts, no significant differences were found between the two methods, as only two samples were positive. The Centrifugal – Concentration procedure was shown to be the simplest and cheapest to perform, as emphasized by the efficiency recovery results.     

Relationship between the contamination of gulls (Larus dominicanus) and oysters (Crassostrea gigas) with Salmonella serovar Typhimurium by PCR-RFLP

Shellfish can bioaccumulate in their tissues pathogenic contaminants present in water and they have been related with several outbreaks of food-borne diseases worldwide. With their increased population in urban areas, gulls have been reported as an important source of water environment contamination. During a 10-month period, water, gulls feces and oyster samples were collected in a shellfish harvesting site and analyzed for total and fecal coliform counts (water) and Salmonella presence (gull feces and oyster meat). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to differentiate Salmonella species detected in gulls and oysters. Salmonella presence was detected in 3/10 of oyster samples and in 6/10 of gull feces samples by PCR. There was a relationship between Salmonella presence in oysters and fecal contamination in water. Restriction profiles of both gulls and oyster samples were similar to Salmonella Typhimurium profile by RFLP. These findings indicate strong evidence that gulls can contribute to Salmonella contamination of harvested oysters.     

Occurrence of Cryptosporidium oocysts and Giardia cysts in Norwegian groundwater wells in bedrock

The occurrence of Cryptosporidium oocysts and Giardia cysts in Norwegian groundwater wells in bedrock has been investigated for the first time. Wells close to risk areas such as farming and septic tanks were chosen. In all, 20 water samples from 20 different waterworks were collected. The samples were analysed for Cryptosporidium and Giardia, using US EPA Method 1623. Turbidity was also measured. Water samples from 10 of the waterworks were also analysed for Clostridium perfringens by membrane filtration. Cryptosporidium was detected in the groundwater samples from 3 of the waterworks. Giardia and Clostridium perfringens were not detected. Too few samples were analysed to verify whether Giardia is indeed absent from bedrock wells, and further studies are recommended to give more reliable data.     

The prevalence of infection of Giardia spp. and Cryptosporidium spp. in wild animals on farmland,southeastern North Island,New Zealand

Giardia spp. cysts were detected in the faeces of 23.6% of 76 possums ( Trichosurus vulpecula ), 42.1% of 19 ship rats ( Rattus rattus ), 30.5% of 46 house mice ( Mus musculus ), 33.3% of 6 hedgehogs ( Erinaceus europaeus ), 33.3% of 3 ferrets ( Mustela furo ), 20% of 5 rabbits ( Oryctolagus cuniculus ), 35% of 20 blackbirds ( Turdus merula ), 50% of 14 thrushes ( Turdus philomelos ), 15.4% of 104 sparrows ( Passer domesticus ), 60% of 10 chaffinches ( Fringilla coelebs ), and 14.3% of 14 hedge sparrows ( Prunella modularis ) trapped on two farms in the Dannevirke area, southeastern North Island, New Zealand. Cryptosporidium cysts were detected in 12.8% of 39 possums, 37.5% of 8 ship rats, 11.8% of 17 house mice, 8.2% of 61 house sparrows, 21.4% of 14 thrushes and 1 of 2 starlings ( Sturnus vulgaris ) from the same area. Although one of the two farms had a history of human Giardia infection, no significant differences were found between the prevalence of Giardia in wild animals on the farms. There was a significantly lower prevalence of Cryptosporidium than Giardia in the wild animals tested. The role of wild animals in maintaining and spreading Giardia and Cryptosporidium in New Zealand is discussed.     

Occurrence of Cryptosporidium oocysts and Giardia cysts in the Nakdong River and their removal during water treatment

This study was conducted in preparation of a pending Cryptosporidium regulation in Korea. The study had two main objectives: 1) to examine the occurrence of Cryptosporidium oocysts in the Nakdong River; and 2) to evaluate their removal during water treatment. Occurrence of Giardia cysts was also examined. Average (arithmetic mean) numbers of Cryptosporidium oocysts and Giardia cysts at the treatment intake site were 2.6 l^-1 and 4.8 l^-1, respectively. Generally, the number of Giardia cysts was higher than that of Cryptosporidium oocysts at more sites, but the difference was minimal. Comparison of tributaries indicated that livestock wastes were more serious pollutants than sewage in terms of protozoa contamination. In general, fewer oocysts and cysts were detected during winter. No correlation was found for such water quality parameters as T-N, T-P, TOC, DO, pH and temperature with the numbers of oocysts and cysts except for suspended solids, which showed the highest correlation (R²=0.55). Removal of Cryptosporidium oocysts was evaluated using a Cryptosporidium tracer, which has similar characteristics to Cryptosporidium oocysts. The tracer removal depended on turbidity removal. Coagulation followed by sedimentation resulted in 1.2–1.5 log removal of the tracer under optimal conditions. Filtration resulted in 1.3–1.5 log removal of the tracer. These treatability experiments showed that traditional water treatment processes could achieve 2.5–3.0 log removal of the oocysts.     

First report of zoonotic Cryptosporidium spp., Giardia intestinalis and Enterocytozoon bieneusi in golden takins (Budorcas taxicolor bedfordi)

Genetic study of Cryptosporidium spp., Giardia intestinalis and Enterocytozoon bieneusi at species/assemblage/genotype/subtype level facilitates understanding their mechanical transmissions and underpins their control. A total of 191 fresh faecal samples were collected from golden takins in China and examined using multilocus sequence typing (MLST). Cryptosporidium spp. was detected in 15 faecal samples (7.9%), including Cryptosporidium parvum (2/15) and Cryptosporidium andersoni (13/15). MLST tool identified C. andersoni subtypes (A1, A4, A4, A1) and (A4, A4, A4, A1), and C. parvum gp60 gene subtype IId A19G1. The prevalence of G. intestinalis infection was 8.9% (17/191) and assemblage analysis identified 14 assemblage E and three assemblage B. Intra-variations were observed at triose phosphate isomerase (tpi), beta giardin (bg) and glutamate dehydrogenase (gdh) loci within the assemblage E, showing seven, three and three new subtypes in respective locus. Ten and one multilocus genotypes (MLGs) were present in assemblages E and B, respectively. E. bieneusi infection was positive in 14.7% (28/191) of the examined specimens, with three genotypes known (BEB6, D and I) and four novel internal transcribed spacer (ITS) genotypes (TEB1–TEB4). The present study revealed, for the first time, the presence of zoonotic C. parvum IId A19G1, G. intestinalis assemblage B and E. bieneusi genotype D and four novel genotypes in golden takins in China. These findings expand the host range of three zoonotic pathogens and have important implications for controlling cryptosporidiosis, giardiasis and microsporidiosis in humans and animals.     

Pathogenic bacteria associated with oysters (Crassostrea brasiliana) and estuarine water along the south coast of Brazil

Oysters and estuarine water samples were collected monthly, from June 1998 to March 1999, in the Cananéia estuary, on the south coast of São Paulo, Brazil, and analyzed for bacterial hazards with and without depuration in filtered estuarine water. Aeromonas spp., Plesiomonas shigelloides, Vibrio cholerae O1, Vibrio parahaemolyticus, and Vibrio vulnificus were counted in oyster samples using the most probable number (MPN) and their presence verified in the surrounding estuarine water samples. The presence of Salmonella, Shigella, Escherichia coli O157:H7, and fecal coliforms counts were determined in oysters and in water samples too. Sixty percent of water samples contained fecal coliforms ranging from <1 to >200 CFU/100 ml and 100%, 30%, 20% and 10% were positive for V. parahaemolyticus, Salmonella, Aeromonas, and V. vulnificus in 5 l of water samples, respectively. In oyster samples, the fecal coliforms concentration ranged from <3.0 to ≥2.4 × 10³ MPN/g in 40% of untreated and from <3.0 to 1.1 × 10³ MPN/g in 40% of treated samples. Vibrio parahaemolyticus Kanagawa-negative was detected in all oyster samples and their concentration varied from 3.6 to ≥2.4 × 10³ MPN/g. For the untreated oyster samples 80%, 70%, and 10% were positive for V. vulnificus (<3 – 11.0 MPN/g), Aeromonas (<3 – 15 MPN/g), and Salmonella (presence in 25 g), respectively. However, for treated oyster samples 60%, 30%, and 0% of them contained the same bacteria, respectively. Escherichia coli O157:H7, Shigella spp., P. shigelloides, and V. cholerae O1 were not detected in any of the samples. Fecal indicators did not correlate with Vibrio presence (p>0.05), although the isolation of Aeromonas species had a positive correlation (p = 0.017). The results showed no correlation between temperature, salinity, and bacteria (p > 0.05). The comparison between bacterial concentration in treated and untreated oyster samples, showed that only Aeromonas was higher in untreated oyster samples (p = 0.039). This study contributes toward creating a more global understanding of food-borne bacterial pathogens. The presence and concentration of viable bacterial hazards in oysters and water surrounding areas was determined for the first time on the south coast of São Paulo and it helps to define better the true microbial hazard in the aquatic environment and oysters.     

Detection of Cryptosporidium oocysts and Giardia cysts in swimming pool filter backwash water concentrates by flocculation and immunomagnetic separation

The purpose of the present study was to evaluate techniques for detection of Giardia cysts and Cryptosporidium oocysts in swimming pool filter backwash water. Calcium carbonate flocculation was used for water samples concentration of 1 l filter backwash water samples. Immunomagnetic separation (IMS) was used for separation of cysts and oocysts from detrital material. Cysts and oocysts were then detected using direct immunofluorescence. ColorSeed C&G was used as an internal standard. Recoveries were examined at several processing points. Highest recoveries (23% Cryptosporidium, 18% Giardia) were obtained using lower filter backwash volumes, greater IMS volumes, and addition of Tween20 to backwash samples prior to processing. The combination of CaCO3 flocculation, IMS and direct immunofluorescence was found to be an effective tool for the detection and quantification of Giardia spp. and Cryptosporidium spp. in filter backwash water samples.     

Hemotropic Mycoplasma spp. in Aquatic Mammals,Amazon Basin,Brazil

Hemotropic Mycoplasma spp. (hemoplasmas) are uncultivable bacteria that infect mammals, including humans. We detected a potentially novel hemoplasma species in blood samples from wild river dolphins in the Amazon River Basin, Brazil. Further investigation could determine pathogenicity and zoonotic potential of the detected hemoplasma.     

Prevalence of Cryptosporidium spp. and Giardia intestinalis in swimming pools, Atlanta, Georgia

Cryptosporidium spp. and Giardia intestinalis have been found in swimming pool filter backwash during outbreaks. To determine baseline prevalence, we sampled pools not associated with outbreaks and found that of 160 sampled pools, 13 (8.1%) were positive for 1 or both parasites; 10 (6.2%) for Giardia sp., 2 (1.2%) for Cryptosporidium spp., and 1 (0.6%) for both.     

Heavy cryptosporidial infections in children in northeast Brazil: comparison of Cryptosporidium hominis and Cryptosporidium parvum

Cryptosporidium is an important cause of infectious diarrhoea worldwide, but little is known about the course of illness when infected with different species. Over a period of 5 years, Cryptosporidium was identified in the stools of 58 of 157 children prospectively followed from birth in an urban slum (favela) in northeast Brazil. Forty isolates were available for quantification and 42 for speciation (24 Cryptosporidium hominis and 18 C. parvum). Children with C. hominis shed significantly more oocysts/ml of stool (3.5 x 10(6) vs. 1.7 x 10(6)perml; P=0.001), and oocyst counts were higher among symptomatic children (P=0.002). Heavier C. parvum shedding was significantly associated with symptoms (P=0.004), and symptomatic C. parvum-infected children were significantly more likely than asymptomatic children to be lactoferrin-positive (P=0.004). Height-for-age (HAZ) Z-scores showed significant declines within 3 months of infection for children infected with either C. hominis (P=0.028) or C. parvum (P=0.001). However, in the 3-6 month period following infection, only C. hominis-infected children continued to demonstrate declining HAZ score and asymptomatic children showed even greater decline (P=0.01). Cryptosporidium hominis is more common than C. parvum in favela children and is associated with heavier infections and greater growth shortfalls, even in the absence of symptoms.     

First investigations into the prevalence of Cryptosporidium and Giardia spp. in Hungarian drinking water

Safe drinking water is a top priority in preventing disease outbreaks and is of general concern to everyone. This study examines the occurrence of Cryptosporidium and Giardia in Hungarian drinking water supplies for the first time. A total of 76 raw and drinking water samples were examined using the U.S. EPA Method 1623. From these 15 of 34 (48.4%) raw water samples tested positive for Giardia and 7 (26.6%) for Cryptosporidium. Twelve of 45 (26.7%) drinking water samples were positive for Giardia and 6 (13.3%) for Cryptosporidium. Overall, Giardia cysts and/or Cryptosporidium oocysts were detected in 48% of the raw water samples and 35% of the drinking water samples. The highest levels in drinking water were found to be 3 oocysts/100 litres of Cryptosporidium and 63.6 cysts/100 litres for Giardia, enough to cause giardiasis. The highest levels in raw water were 1,030 cysts/100 litres for Giardia and 50 oocysts/100 litres for Cryptosporidium and higher oocyst densities were associated with source water receiving effluents from sewage treatment plants or originating from a forest environment. In addition to this monitoring, riverbank filtrated water and raw water from the River Danube in Budapest were monitored in order to ascertain protozoan removal efficiency of riverbank filtration (RBF). A total of 157 samples, including 87 samples from the River Danube and 70 samples post RBF, were examined. Cryptosporidium and Giardia were detected regularly in the river water but never in riverbank filtered water suggesting the effectiveness of RBF as a purification method. The occurrence of Cryptosporidium oocysts and Giardia cysts in the investigated water supplies may require the water utilities and water authorities in Hungary to apply additional monitoring and treatment and/or watershed controls.     

Effects of salinity on biomarker responses in Crassostrea rhizophorae (Mollusca, Bivalvia) exposed to diesel oil

Crassostrea rhizophorae is a euryhaline oyster that inhabits mangrove areas, which are widely distributed along the Brazilian coast. The aim of this study was to investigate the effects of salinity (9, 15, 25, and 35ppt) on the activities of glutathione S-transferase (GST), glucose 6-phosphate dehydrogenase (G6PDH), catalase (CAT), and acetylcholinesterase (AChE) in the digestive gland of this species after exposure to diesel oil for 7 days at nominal concentrations of 0.01, 0.1, and 1mlL(-1) and after depuration for 24h and 7 days. GST activity increased in a diesel oil concentration-dependent manner at salinities 25 and 15ppt and remained slightly elevated even after depuration periods of 24h and 7 days. No changes were observed in the activities of G6PDH, CAT, and AChE in the oysters exposed to diesel and depurated. Based on these results, GST activity in the digestive gland of C. rhizophorae might be used as a biomarker of exposure to diesel oil in sites where the salinity is between 15 and 25ppt, values usually observed in mangrove ecosystems.     

First report of diffuse cutaneous leishmaniasis and Leishmania amazonensis infection in Rio de Janeiro State, Brazil

Diffuse cutaneous leishmaniasis (DCL) is characterised by multiple and progressive cutaneous lesions, resistance to chemotherapy and Leishmania-specific T-cell anergy. We report the first autochthonous DCL case and the first human infection with Leishmania amazonensis in Rio de Janeiro State, Brazil, where only L. braziliensis is considered to be the causative agent of cutaneous leishmaniasis. Leishmania amazonensis was identified by multilocus enzyme electrophoresis and PCR-RFLP. Our case was diagnosed as DCL according to clinical, parasitological, histopathological and immunological criteria. These observations indicate that L. amazonensis is increasing its geographical distribution in Brazil, accounting for unusual clinical presentations in new transmission areas.     

Uptake of cadmium from Pacific oysters (Crassostrea gigas) in British Columbia oyster growers

Background

Pacific oysters along the North American coast from Washington to Alaska contain concentrations of cadmium (Cd) that are high by comparison with Atlantic oysters, frequently exceeding 2 μg/g wet weight, but it is unclear whether this Cd is absorbed by consumers.

Objectives

To determine the effect of oyster consumption on Cd in blood and urine among a group with high oyster consumption.

Methods

Sixty-one non-smoking oyster growers and family members with a mean age of 47.3±7.6 years (range 33-64) were interviewed by telephone to assess their oyster consumption and other sources of Cd exposure at present and 5 years prior to the start of oyster farming. Their blood and urine Cd concentrations were measured.

Results

The geometric mean Cd concentration in blood was 0.83 μg/L and in urine was 0.76 μg/g creatinine. Thirty-six percent of participants had urinary Cd levels above 1 μg/g creatinine and 5% were above 2 μg/g creatinine. Recent (last 12 months) and long-term oyster consumptions were positive predictors of blood Cd but did not directly predict urinary Cd. The optimal model for predicting the variance in blood Cd included recent intake of oyster-derived Cd, serum iron concentration and recent ketchup consumption (R²=0.34, p=0.00004), with the latter two variables showing a protective effect. The factors found to predict urinary Cd were blood Cd concentration and duration of oyster farming. A rise in blood Cd was observed after 12 years of farming oysters, likely caused by higher consumption of oysters during this period.

Conclusions

Oyster-derived Cd is bioavailable and affects body stores of the metal.     

Destabilized lysosomes and elimination of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in eastern oysters (Crassostrea virginica)

Contaminated oysters from near the Galveston Bay Ship Channel ([GBSC], TX, USA) were transplanted into clean aquariums in order to investigate the recovery of their lysosomal health. Destabilized lysosomes in the oyster hemocytes recovered from 81% to 34% after 23 d of elimination. Chemical analyses showed that transplanted oysters eliminated organic chemicals they had accumulated in their tissue. Enhanced lysosomal health (i.e., decreased lysosomal destabilization) was correlated positively with lower tissue concentrations of chemicals. This study indicates that lysosomal destabilization is a transient biomarker, which indicates only the current status (e.g., within one month) of chemical exposure and potential adverse biological effects. Analytical measurements revealed that polycyclic aromatic hydrocarbons (PAHs) generally were eliminated faster than polychlorinated biphenyls (PCBs) by a factor of 5 to 7. Body burdens of PCBs decreased exponentially, while the elimination pattern of PAHs were biphasic, representing a faster elimination during the early phase of elimination and, subsequently, a much slower elimination. This indicates that elimination of PAHs in the oysters is more complex and may include additional mechanisms compared to the uptake and elimination of PCBs.     

First report of Cryptosporidium canis in foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) and identification of several novel subtype families for Cryptosporidium mink genotype in minks (Mustela vison) in China

Despite the rapid and extensive advances in molecular epidemiology of Cryptosporidium in humans and a variety of animals, the prevalence and genetic traits of the parasite in wildlife bred in captivity and the role of the neglected hosts in zoonotic transmission of human cryptosporidiosis are rarely understood. This study investigated the prevalence, species/genotype, and subtype of Cryptosporidium in farmed fur animals in China and assessed the possibility of zoonotic transmission. Three of 191 (1.6%) foxes (Vulpes vulpes), 17 of 162 (10.5%) raccoon dogs (Nyctereutes procyonoides), and 48 of 162 (29.6%) minks (Mustela vison) were positive for Cryptosporidium by nested PCRs targeting the small subunit rRNA gene. Sequence analysis indicated the presence of only Cryptosporidium canis in foxes and raccoon dogs. There is no significant difference in prevalence between young and adult foxes (or raccoon dogs). Three Cryptosporidium species or genotype including C. canis, Cryptosporidium meleagridis, and mink genotype were determined in minks aged five to six months. Subtyping based on nucleotide and amino acid sequence polymorphisms of the 60 kDa glycoprotein facilitated identification of three novel subtype families named as Xb to Xd for Cryptosporidium mink genotype. The presence of zoonotic C. canis, C. meleagridis, and Cryptosporidium mink genotype in captive-bred fur animals is of public health concerns. The findings expanded the host ranges of C. canis and C. meleagridis and confirmed genetic diversity at the subtype level in Cryptosporidium mink genotype. This is the first study reporting Cryptosporidium infections in foxes and raccoon dogs in China.