Anthropozoonotic Giardia duodenalis genotype (assemblage) a infections in habitats of free-ranging human-habituated gorillas,Uganda

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.     

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.     

An assay to measure the affinity of proteins for microtubules by quantitative fluorescent microscopy

We report a fluorescence-based assay for measuring the affinity of microtubule binding proteins for microtubules. The affinity of any fluorescently tagged protein for taxol-stabilized microtubules can be measured with this assay. We describe the assay and provide a detailed protocol. Using this assay, we found that the affinity of the Dam1 complex for microtubules is decreased by the presence of free unpolymerized tubulin and is sensitive to the salt concentration in the binding buffer. These effects may account for the previous differences in binding affinities reported.     

Urban feral pigeons (Columba livia) as a source for air- and waterborne contamination with Enterocytozoon bieneusi spores

This study demonstrated that a person with 30 min of occupational or nonoccupational exposure to urban feral pigeons, such as exposure through the cleaning of surfaces contaminated with pigeon excrement, could inhale approximately 3.5 x 10(3) Enterocytozoon bieneusi spores and that 1.3 x 10(3) spores could be inhaled by a nearby person.     

Maximizing recovery and detection of Cryptosporidium parvum oocysts from spiked eastern oyster (Crassostrea virginica) tissue samples

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.     

Quantitative evaluation of the impact of bather density on levels of human-virulent microsporidian spores in recreational water

Microsporidial gastroenteritis, a serious disease of immunocompromised people, can have a waterborne etiology. During summer months, samples of recreational bathing waters were tested weekly for human-virulent microsporidian spores and water quality parameters in association with high and low bather numbers during weekends and weekdays, respectively. Enterocytozoon bieneusi spores were detected in 59% of weekend (n = 27) and 30% of weekday (n = 33) samples, and Encephalitozoon intestinalis spores were concomitant in a single weekend sample; the overall prevalence was 43%. The numbers of bathers, water turbidity levels, prevalences of spore-positive samples, and concentrations of spores were significantly higher for weekend than for weekday samples; P values were <0.001, <0.04, <0.03, and <0.04, respectively. Water turbidity and the concentration of waterborne spores were significantly correlated with bather density, with P values of <0.001 and <0.01, respectively. As all water samples were collected on days deemed acceptable for bathing by fecal bacterial standards, this study reinforces the scientific doubt about the reliability of bacterial indicators in predicting human waterborne pathogens. The study provides evidence that bathing in public waters can result in exposure to potentially viable microsporidian spores and that body contact recreation in potable water can play a role in the epidemiology of microsporidiosis. The study indicates that resuspension of bottom sediments by bathers resulted in elevated turbidity values and implies that the microbial load from both sediments and bathers can act as nonpoint sources for the contamination of recreational waters with Enterocytozoon bieneusi spores. Both these mechanisms can be considered for implementation in predictive models for contamination with microsporidian spores.     

Quantitative assessment of contamination of fresh food produce of various retail types by human-virulent microsporidian spores

This study demonstrated that fresh food produce, such as berries, sprouts, and green-leafed vegetables, sold at the retail level can contain potentially viable microsporidian spores of human-virulent species, such as Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Encephalitozoon cuniculi, at quantities representing a threat of food-borne infection.     

Novel malonamide derivatives as potent kappa opioid receptor agonists

A novel series of malonamide derivatives was synthesized. These amides were shown to be potent and selective kappa opioid receptor agonists.