Field detection of Schistosoma japonicum cercariae in environmental water samples by quantitative PCR.
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ABSTRACT:
SUBMITTER: Worrell C
PROVIDER: S-EPMC3067307 | biostudies-literature | 2011 Mar
REPOSITORIES: biostudies-literature
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Field detection of Schistosoma japonicum cercariae in environmental water samples by quantitative PCR.
Worrell Caitlin C Xiao Ning N Vidal Jorge E JE Chen Lin L Zhong Bo B Remais Justin J
Applied and environmental microbiology 20110128 6
A species-specific quantitative PCR (qPCR) assay was combined with two novel water-sampling methods and compared with the mouse bioassay for the quantitative detection of S. japonicum in surface waters. The novel methods were capable of capturing cercariae and, with subsequent analysis through qPCR, detecting the presence of a minimum of 1 cercaria. ...[more]
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Project description:In China alone, an estimated 30 million people are at risk of schistosomiasis, caused by the Schistosoma japonicum parasite. Disease has re-emerged in several regions that had previously attained transmission control, reinforcing the need for active surveillance. The environmental stage of the parasite is known to exhibit high spatial and temporal variability, and current detection techniques rely on a sentinel mouse method which has serious limitations in obtaining data in both time and space. Here we describe a real-time PCR assay to quantitatively detect S. japonicum cercariae in laboratory samples and in natural water that has been spiked with known numbers of S. japonicum. Multiple primers were designed and assessed, and the best performing set, along with a TaqMan probe, was used to quantify S. japonicum. The resulting assay was selective, with no amplification detected for Schistosoma mansoni, Schistosoma haematobium, avian schistosomes nor organisms present in non-endemic surface water samples. Repeated samples containing various concentrations of S. japonicum cercariae showed that the real-time PCR method had a strong linear correlation (R(2) = 0.921) with light microscopy counts, and the detection limit was below the DNA equivalent of half of one cercaria. Various cercarial concentrations spiked in 1 liter of natural water followed by a filtration process produced positive detection from 93% of samples analyzed. The real-time PCR method performed well quantifying the relative concentrations of various spiked samples, although the absolute concentration estimates exhibited high variance across replicated samples. Overall, the method has the potential to be applied to environmental water samples to produce a rapid, reliable assay for cercarial location in endemic areas.
| S-EPMC2580822 | biostudies-literature
Project description:As a unique feature among otherwise hermaphroditic trematodes, Schistosoma species are gonochoric parasites whose sex is genetically determined (ZZ for males and ZW for females). However, schistosome larvae are morphologically identical, and sex can only be discriminated by molecular methods. Here, we integrated published Schistosoma. japonicum transcriptome and genome data to identify W chromosome-specific genes as sex biomarkers. Three W chromosome-specific genes of S. japonicum were identified as sex biomarkers from a panel of 12 genes expressed only in females. An efficient duplex real-time PCR (qPCR) method for sexing cercariae was developed which could identify the sex of cercariae within 2 h without DNA extraction. Moreover, this method can be used to identify not only single-sex but also mixed-sex schistosome-infected snails. We observed a nearly equal proportion of single-male, single-female, and mixed-sex schistosome infections in artificially infected snails. Sex-known schistosome-infected snail models can be efficiently constructed with the aid of duplex qPCR. A field study revealed that single-sex schistosome infections were predominant among naturally infected snails. Finally, a schistosomiasis mouse model based on sex-known cercariae infection was shown to be more reliable than a model based on sex-unknown cercariae infection. The developed duplex qPCR method for sexing S. japonicum cercariae can be widely used for schistosomiasis modeling, genetic experiments, and field-based molecular epidemiological studies.
| S-EPMC7467314 | biostudies-literature
Project description:The gender of cercarial larvae can only be determined using molecular methods. End point PCR methods that amplify repetitive markers on the W chromosome of the female (ZW) parasites have been developed, but sometimes results are ambiguous or incorrect. To more effectively distinguish sexes, and to determine why end point PCR can be incorrect, we quantified the W6 repeat sequence and a specific Z chromosome gene using real-time PCR. The ratio between copy number of W6 and a Z chromosome marker unambiguously identifies gender: females have higher ratios (421-4371) than males (0-21). However, some males have low numbers of W6 elements in their genome, and qPCR demonstrated significantly higher W6/Z marker ratios for male genotypes giving ambiguous end point PCR results compared with males giving clear end point results. The quantitative PCR sexing method developed will be particularly useful where reliable sexing of cercariae is critical, for example when staging genetic crosses.
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Project description:BackgroundSchistosomiasis is a parasitic disease that is transmitted by skin contact with waterborne schistosome cercariae. Mass drug administration with praziquantel is an effective control method, but it cannot prevent reinfection if contact with cercariae infested water continues. Providing safe water for contact activities such as laundry and bathing can help to reduce transmission. In this study we examine the direct effect of UV light on Schistosoma mansoni cercariae using ultraviolet light-emitting diodes (UV LEDs) and a low-pressure (LP) mercury arc discharge lamp.MethodologyS. mansoni cercariae were exposed to UV light at four peak wavelengths: 255 nm, 265 nm, 285 nm (UV LEDs), and 253.7 nm (LP lamp) using bench scale collimated beam apparatus. The UV fluence ranged from 0-300 mJ/cm2 at each wavelength. Cercariae were studied under a stereo-microscope at 0, 60, and 180 minutes post-exposure and the viability of cercariae was determined by assessing their motility and morphology.ConclusionVery high UV fluences were required to kill S. mansoni cercariae, when compared to most other waterborne pathogens. At 265 nm a fluence of 247 mJ/cm2 (95% confidence interval (CI): 234-261 mJ/cm2) was required to achieve a 1-log10 reduction at 0 minutes post-exposure. Cercariae were visibly damaged at lower fluences, and the log reduction increased with time post-exposure at all wavelengths. Fluences of 127 mJ/cm2 (95% CI: 111-146 mJ/cm2) and 99 mJ/cm2 (95% CI: 85-113 mJ/cm2) were required to achieve a 1-log10 reduction at 60 and 180 minutes post-exposure at 265 nm. At 0 minutes post-exposure 285 nm was slightly less effective, but there was no statistical difference between 265 nm and 285 nm after 60 minutes. The least effective wavelengths were 255 nm and 253.7 nm. Due to the high fluences required, UV disinfection is unlikely to be an energy- or cost-efficient water treatment method against schistosome cercariae when compared to other methods such as chlorination, unless it can be demonstrated that UV-damaged cercariae are non-infective using alternative assay methods or there are improvements in UV LED technology.
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Project description:BackgroundSchistosomiasis japonica is a common zoonosis. Domestic animals are the primary source of infection and play an important role in disease transmission. The prevalence and infectivity of this disease in domestic animals in China have significantly decreased and, for this reason, diagnostics with a higher sensitivity have become increasingly necessary. It was reported that polymerase chain reaction (PCR)-based methods could be used to detect schistosome infection in humans and animals and presented a high sensitivity and specificity. The present study aimed to develop a PCR-based method for detection of Schistosoma japonicum infection in domestic animals.MethodsA specific nested-PCR assay was developed to detect S. japonicum infection in domestic animals via amplification of a 231-bp DNA fragment of retrotransposon SjR2. The developed assay was first used in sera and dry blood filter paper (DBFP) from goats and buffaloes at different time points of infection. Then, 78 DBFPs from 39 artificially-infected bovines at 14 and 28 days post-infection and 42 DBFPs from schistosome-negative bovines from the city of Huangshan in the Anhui province were used to evaluate the diagnostic validity. Furthermore, this assay was used to detect S. japonicum infection in domestic animals in Dongzhi and Wangjiang counties.ResultsThe expected PCR product was detected in eggs and adult worms of S. japonicum and blood samples from S. japonicum-infected goats and water buffaloes, but not from Fasciola and Haemonchus contortus worms. The nested-PCR assay could detect the target S. japonicum DNA in DBFPs from goats and buffaloes after day 3 post-infection. The sensitivity in buffaloes at 14 and 28 days post-infection was 92.30% (36/39) and 100% (39/39), respectively. The specificity was 97.60% (41/42). The positivity rates in Dongzhi and Wangjiang counties were 6.00% and 8.00% in bovines and 22.00% and 16.67% in goats, respectively. The positivity rates in goats in both counties were higher than those in bovines with a significant difference in Dongzhi County but not in Wangjiang County (P < 0.05 and P = 0.23, respectively).ConclusionsOur results suggest that the developed nested-PCR assay may be used for the diagnosis of S. japonicum infection in domestic animals, and the control of S. japonicum infection in goats should be paid more attention.
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Project description:Contamination of hospital water systems with legionellae is a well-known cause of nosocomial legionellosis. We describe a new real-time LightCycler PCR assay for quantitative determination of legionellae in potable water samples. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from Legionella spp. and a specifically cloned fragment of the phage lambda, added to each sample as an internal inhibitor control, were used. The amplified products were detected by use of a dual-color hybridization probe assay design and quantified with external standards composed of Legionella pneumophila genomic DNA. The PCR assay had a sensitivity of 1 fg of Legionella DNA (i.e., less than one Legionella organism) per assay and detected 44 Legionella species and serogroups. Seventy-seven water samples from three hospitals were investigated by PCR and culture. The rates of detection of legionellae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae calculated from the PCR results were associated with the CFU detected by culture (r = 0.57; P < 0.001), but PCR results were mostly higher than the culture results. Since L. pneumophila is the main cause of legionellosis, we further developed a quantitative L. pneumophila-specific PCR assay targeting the macrophage infectivity potentiator (mip) gene, which codes for an immunophilin of the FK506 binding protein family. All but one of the 16S rRNA gene PCR-positive water samples were also positive in the mip gene PCR, and the results of the two PCR assays were correlated. In conclusion, the newly developed Legionella genus-specific and L. pneumophila species-specific PCR assays proved to be valuable tools for investigation of Legionella contamination in potable water systems.
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Project description:Arcobacter spp. are emerging pathogens associated with gastroenteritis in humans. The objective of this study was to develop a highly sensitive and broadly reactive quantitative PCR (qPCR) assay for Arcobacter spp. and to apply the developed assay to different water sources in the Kathmandu Valley, Nepal. Fifteen samples to be analyzed by next-generation sequencing were collected from 13 shallow dug wells, a deep tube well, and a river in the Kathmandu Valley in August 2015. Among the 86 potential pathogenic bacterial genera identified, Acinetobacter, Pseudomonas, Flavobacterium, and Arcobacter were detected with relatively high abundance in 15, 14, 12, and 8 samples, respectively. A primer pair was designed with maximal nucleotide homologies among Arcobacter spp. by comparing the sequences of 16S rRNA genes. These primers were highly specific to most of the known species of Arcobacter and quantified between 1.0×101 and 6.4×106 copies reaction-1 and sometimes detected as few as 3 copies reaction-1. The qPCR assay was used to quantify Arcobacter spp. in bacterial DNA in not only the above 15 water samples, but also in 33 other samples collected from 15 shallow dug wells, 6 shallow tube wells, 5 stone spouts, 4 deep tube wells, and 3 springs. Thirteen (27%) out of 48 samples tested were positive for Arcobacter spp., with concentrations of 5.3-9.1 log copies 100 mL-1. This qPCR assay represents a powerful new tool to assess the prevalence of Arcobacter spp. in environmental water samples.
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Project description:BackgroundSchistosomiasis is a parasitic disease which is spread through skin contact with water containing Schistosoma cercariae. Drug treatment has been the main control method, but it does not prevent reinfection. The use of soap can be a complementary measure to reduce transmission. Therefore, this study investigates the quantitative effect of different soaps on the mortality of Schistosoma mansoni cercariae.MethodologyFour soaps including two powder soaps (Kleesoft and Omo) and two bar soaps (B29 and Rungu) which are used in a schistosomiasis-endemic Tanzanian village were studied. S. mansoni cercariae were exposed to powder soaps of 0 (control), 10, 50, 75, 100 and 1000 mg/L and to bar soaps of 0 (control), 100, 500 and 1000 mg/L. The highest concentration of 1000 mg/L was selected based on the laboratory-estimated average soap concentration during handwashing. Cercariae were observed under a microscope after 0, 5, 15, 30, 45 and 60 minutes of exposure to determine their survival.ConclusionsAll four soaps can kill S. mansoni cercariae and this lethal effect was related to soap concentration and exposure time. At the highest concentration of 1000 mg/L, all cercariae were dead at 5 minutes post-exposure with two powder soaps and Rungu, while 100% cercarial death was achieved between 5 minutes to 15 minutes for B29. Almost all cercariae survived after being exposed to 10 mg/L powder soaps and 100 mg/L bar soaps for 60 minutes. Powder soaps were more lethal than bar soaps. Considering the widely varying concentrations of soap during real-world hygiene activities and the necessity for a very high soap concentration to eliminate all cercariae in a short 5-minute exposure, providing the efficacy of soap in preventing schistosomiasis becomes challenging. Future studies should investigate whether soap can influence alternative mechanisms such as making cercariae unable to penetrate the skin, thereby providing protection.
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