Cryptosporidium oocysts and Giardia cysts-A practical and sensitive method for counting and manipulating small numbers.

A rapid and inexpensive method is described for accurate and reproducible counting and manipulating small numbers of Cryptosporidium oocysts and Giardia cysts. From a suspension of oocysts or cysts at concentration from 1000 to 5000/mL (1-5/µL), replicate 5µL droplets are micro pipetted onto the edge of a microscope slide. Unstained oocysts or cysts in each droplet can be counted in a few minutes and replicated for statistical strength. The concentration of the suspension can then be verified by pipetting desired volumes containing approximately desired numbers onto confined 13mm membranes for IFA staining and counting with replication as desired. Requiring only a micropipette, analytical balance, and a microscope, this provides a useful tool accessible to virtually any microbiology laboratory.

Investigating source water Cryptosporidium concentration, species and infectivity rates during rainfall-runoff in a multi-use catchment.

Protozoan pathogens present a significant human health concern, and prevention of contamination into potable networks remains a key focus for drinking water providers. Here, we monitored the change in Cryptosporidium concentration in source water during high flow events in a multi-use catchment. Furthermore, we investigated the diversity of Cryptosporidium species/genotypes present in the source water, and delivered an oocyst infectivity fraction. There was a positive and significant correlation between Cryptosporidium concentration and flow (ρ = 0.756) and turbidity (ρ = 0.631) for all rainfall-runoff events, despite variable source water pathogen concentrations. Cell culture assays measured oocyst infectivity and suggested an overall source water infectious fraction of 3.1%. No infectious Cryptosporidium parvum or Cryptosporidium hominis were detected, although molecular testing detected C. parvum in 7% of the samples analysed using PCR-based molecular techniques. Twelve Cryptosporidium species/genotypes were identified using molecular techniques, and were reflective of the host animals typically found in remnant vegetation and agricultural areas. The inclusion of molecular approaches to identify Cryptosporidium species and genotypes highlighted the diversity of pathogens in water, which originated from various sources across the catchment. We suggest this mixing of runoff water from a range of landuses containing diverse Cryptosporidium hosts is a key explanation for the often-cited difficulty forming strong pathogen-indicator relationships.

wFlu: characterization and evaluation of a native Wolbachia from the mosquito Aedes fluviatilis as a potential vector control agent.

There is currently considerable interest and practical progress in using the endosymbiotic bacteria Wolbachia as a vector control agent for human vector-borne diseases. Such vector control strategies may require the introduction of multiple, different Wolbachia strains into target vector populations, necessitating the identification and characterization of appropriate endosymbiont variants. Here, we report preliminary characterization of wFlu, a native Wolbachia from the neotropical mosquito Aedes fluviatilis, and evaluate its potential as a vector control agent by confirming its ability to cause cytoplasmic incompatibility, and measuring its effect on three parameters determining host fitness (survival, fecundity and fertility), as well as vector competence (susceptibility) for pathogen infection. Using an aposymbiotic strain of Ae. fluviatilis cured of its native Wolbachia by antibiotic treatment, we show that in its natural host wFlu causes incomplete, but high levels of, unidirectional cytoplasmic incompatibility, has high rates of maternal transmission, and no detectable fitness costs, indicating a high capacity to rapidly spread through host populations. However, wFlu does not inhibit, and even enhances, oocyst infection with the avian malaria parasite Plasmodium gallinaceum. The stage- and sex-specific density of wFlu was relatively low, and with limited tissue distribution, consistent with the lack of virulence and pathogen interference/symbiont-mediated protection observed. Unexpectedly, the density of wFlu was also shown to be specifically-reduced in the ovaries after bloodfeeding Ae. fluviatilis. Overall, our observations indicate that the Wolbachia strain wFlu has the potential to be used as a vector control agent, and suggests that appreciable mutualistic coevolution has occurred between this endosymbiont and its natural host. Future work will be needed to determine whether wFlu has virulent host effects and/or exhibits pathogen interference when artificially-transfected to the novel mosquito hosts that are the vectors of human pathogens.

A high-flux isopore micro-fabricated membrane for effective concentration and recovering of waterborne pathogens.

A high-flux metallic micro/nano-filtration membrane has been fabricated and validated for isolation of waterborne pathogens from drinking water. Obtained membrane with smooth surface and perfectly ordered pores was achieved by a high yield and cost effective multilevel lithography and electroplating technique. The micro-fabricated membrane was also strengthened with an integrated back-support, which can withstand a high pressure during filtration. The results of microfiltration tests with model particles revealed the superior performance of the micro-fabricated filter than current commercial filters in sample throughput, recovery ratio, and reusability. This study highlighted the potential application of micro-fabricated filer in rapid filtration and recovery of C. parvum oocysts for downstream analysis.

Prevalence of protozoa, viruses, coliphages and indicator bacteria in groundwater and river water in the Kathmandu Valley, Nepal.

Limited information is available on the prevalence of waterborne pathogens in aquatic environments in developing countries. In this study, water samples were collected from nine shallow wells and a river in the Kathmandu Valley, Nepal, during the rainy season in 2009 and were subjected to detection of waterborne protozoa, viruses and coliphages using a recently developed method for simultaneous concentration of protozoa and viruses in water. Escherichia coli and total coliforms were also tested as indicator bacteria. At least one type of the five pathogens tested (Cryptosporidium, Giardia, human adenoviruses, and noroviruses of genogroups I and II) was detected in five groundwater samples (56%) (1000 ml each) from shallow wells. Compared with groundwater samples, the pathogens were more abundant in the river water sample (100ml); the concentrations of Cryptosporidium and Giardia were 140 oocysts/l and 8500 cysts/l, respectively, and the mean threshold cycle (Ct) values in real-time RT-PCR were 34.3, 36.8 and 34.0 for human adenoviruses and noroviruses of genogroups I and II, respectively. Genotyping of F-RNA coliphages by real-time RT-PCR was successfully used to differentiate human and animal faecal contamination in the samples. Moreover, for the groundwater samples, protozoa and viruses were detected only in E. coli-positive samples, suggesting that E. coli may be an appropriate indicator of pathogen contamination of valley groundwater.

The identification of the Cryptosporidium ubiquitum in pre-weaned Ovines from Aba Tibetan and Qiang autonomous prefecture in China.

OBJECTIVE: Cryptosporidium spp. are prevalent globally and sheep are an important zoonotic reservoir. Little data regarding the rates of Cryptosporidium infections in ovines in China are available. This study assessed the prevalence of Cryptosporidium spp. in pre-weaned ovines from Aba Tibetan and Qiang Autonomous Prefecture in the Sichuan province of China. METHODS: A total of 213 fecal samples were collected from pre-weaned ovines and were examined microscopically (following modified acid fast staining). In addition, 18S rRNA genetic sequences were amplified from fecal samples by nested PCR and phylogenetically analyzed. RESULTS: The prevalence of Cryptosporidium in the collected samples was at 14.6% (31/213) and four isolates identified by PCR belonged to the Cryptosporidium cervine genotype (Cryptosporidium ubiquitum) demonstrating that this species was the primary sheep species found in sheep in China. CONCLUSION: The present study suggested that the high incidence of Cryptosporidium in sheep poses a significant public health threat and that surveillance practices must be established to prevent zoonotic disease of humans.

Interaction of Escherichia coli K1 and K5 with Acanthamoeba castellanii trophozoites and cysts.

The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission.

Interaction of Escherichia coli K1 and K5 with Acanthamoeba castellanii Trophozoites and Cysts

The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission.