Development of nested multiplex polymerase chain reaction (PCR) assay for the detection of Toxocara canis, Toxocara cati and Ascaris suum contamination in meat and organ meats

Ascarid Larva Migrans Syndrome (ascarid LMS) is a clinical syndrome in humans, caused by the migration of animal roundworm larvae such as Toxocara canis, Toxocara cati and Ascaris suum. Humans may acquire infection by ingesting embryonated eggs, or infective larvae of these parasites in contaminated meat and organ meats. To detect these pathogenic contaminations, a novel nested multiplex PCR system was developed. Our novel nested multiplex PCR assay showed specific amplification of T. canis, T. cati and Ascaris spp. Detection limit of the nested multiplex PCR was tested with serial dilution of T. canis, T. cati or A. suum genomic DNA (gDNA) from 100?pg to 100 ag and found to be 10?fg, 1?fg and 100?fg, respectively. When larvae were spiked into chicken liver tissue, DNA of T. canis and A. suum was detected from the liver spiked with a single larva, while the assay required at least 2 larvae of T. cati. Moreover, the ascarid DNA was detected from the liver of mice infected with 100 and 300 eggs of T. canis, T. cati or A. suum. This nested multiplex PCR assay could be useful for the detection of contamination with ascarid larvae in meat and organ meats.     

Longevity of Toxocara cati Larvae and Pathology in Tissues of Experimentally Infected Chickens

This study was conducted to determine the distribution patterns and duration of stay of Toxocara cati larvae in organs of chickens and to investigate chronic phase and potential zoonotic risk of toxocariasis in chickens. Chickens were orally infected with 1,000 embryonated T. cati eggs and necropsied 240 days post-infection. Organs of the chickens were examined at gross and microscopic levels; tissues were digested to recover larvae. Peribronchiolitis with infiltration of lymphocytes, and hyperplasia of bronchiolar associated lymphatic tissues (BALT) and goblet cells, were evident in the lungs of infected chickens. There were mild hemorrhages and infiltration of lymphocytes and a few eosinophils in the meninges. Larvae were recovered from 30% of the exposed chickens. Larvae recovery indicated that T. cati larvae stay alive for at least 240 days in the chicken brain. Therefore, chickens may potentially act as a paratenic host in nature and transfer T. cati larvae to other hosts.     

Modification of gDNA extraction from soil for PCR designed for the routine examination of soil samples contaminated with Toxocara spp. eggs

A modification of gDNA extraction was developed for the polymerase chain reaction (PCR) technique, intended for the detection and differentiation of Toxocara spp. eggs in soil or sediments. Sand samples from sandpits confirmed as being contaminated with Toxocara spp. eggs by the flotation technique were analysed by PCR. The use of proteinase K made it possible to obtain genomic DNA from the sample without needing to isolate eggs using flotation or to inactivate PCR inhibitors present in the sand. Specific primers in the PCR reaction allowed discrimination between T. canis and T. cati eggs. The modification simplified the procedure, thanks to eliminating the step of gDNA isolation from eggs, which is both laborious and difficult.     

A sensitive and specific PCR assay for the detection of Baylisascaris schroederi eggs in giant panda feces

Baylisascaris schroederi is one of the most common intestinal nematodes in giant pandas. It can cause severe baylisascariasis which is highly infectious in its natural hosts. A rapid and reliable diagnosis of parasite infections is crucial to protect giant pandas, as well as for environmental monitoring and disease surveillance. Here, we established a specific PCR assay for B. schroederi detection which was targeting a 331-bp long fragment of the mitochondrial cytochrome c oxidase subunit II (COII) gene. Fifty fresh fecal samples collected from captive giant pandas were tested by the established PCR assay and the traditional flotation technique. DNA extracted from a single B. schroederi egg could be successfully amplified, while no cross-reactivity was found with DNA from Ancylostoma caninum eggs. The detection rate of the PCR assay was 68%, which was higher than that of the traditional egg flotation (46%). Our findings demonstrated that the PCR assay is sensitive and specific for the detection and identification of B. schroederi eggs. Therefore, it could become a useful tool for the investigation of B. schroederi infections in giant pandas.     

PCR-Based Molecular Characterization of Toxocara spp. Using Feces of Stray Cats: A Study from Southwest Iran

Feces of stray cat are potential sources of gastrointestinal parasites and play a crucial role in spreading and transmitting parasite eggs, larvae, and oocysts through contamination of soil, food, or water. In this study, we investigated the prevalence of Toxocara spp. infection in stray cats in Ahvaz city, southwest Iran. Eggs of Toxocara spp. in feces of stray cats were detected by the sucrose flotation method, and identification was conducted by polymerase chain reaction (PCR) and DNA sequencing. Of the 140 fecal samples that were randomly collected from public environments during the months of January to May 2012, 45% were found to harbour Toxocara spp. eggs. The highest prevalence of Toxocara spp. eggs was found in the central area of Ahvaz city (28.6%). T. canis eggs were found in 4 (6.34%) of the 63 positive samples. Stray cats are found in parks, playgrounds, and other public places and may be a potential contamination risk. Identification of Toxocara spp. using molecular methods is sufficiently sensitive to detect low levels of parasites and identify the different Toxocara spp. in feces. The relatively high prevalence of Toxocara spp. infection may continue to increase due to lack of effective environmental hygiene control in Iran. Consequently, there is a need to plan adequate programs to detect, identify, and control this infection as well as stray cats in the region.     

Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4±0.09℃ and 85.9±0.08℃ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.     

Detection of Schistosoma mansoni eggs in feces through their interaction with paramagnetic beads in a magnetic field

Background

Diagnosis of intestinal schistosomiasis in low endemic areas is a problem because often control measures have reduced egg burdens in feces to below the detection limits of classical coproparasitological methods. Evaluation of molecular methods is hindered by the absence of an established standard with maximum sensitivity and specificity. One strategy to optimize method performance, where eggs are rare events, is to examine large amounts of feces. A novel diagnostic method for isolation of Schistosoma mansoni eggs in feces, and an initial evaluation of its performance is reported here.

Methodology/Principal Findings

Known amounts of S. mansoni eggs were seeded into 30 g of normal human feces and subjected to a sequence of spontaneous sedimentation, sieving, Ritchie method, incubation and isolation through interaction with paramagnetic beads. Preliminary tests demonstrated the efficacy of lectins as ligands, but they also indicated that the paramagnetic beads alone were sufficient to isolate the eggs under a magnetic field through an unknown mechanism. Eggs were identified by microscopic inspection, with a sensitivity of 100% at 1.3 eggs per gram of feces (epg). Sensitivity gradually decreased to 25% at a concentration of 0.1 epg. In a preliminary application of the new method to the investigation of a recently established focus in southern Brazil, approximately 3 times more eggs were detected than with the thick-smear Kato-Katz method.

Conclusions/Significance

The novel S. mansoni detection method may significantly improve diagnosis of infections with low burdens in areas of recent introduction of the parasite, areas under successful control of transmission, or in infected travelers. It may also improve the evaluation of new treatments and vaccines.     

Ancylostoma caninum: calibration and comparison of diagnostic accuracy of flotation in tube, McMaster and FLOTAC in faecal samples of dogs

We performed a calibration of flotation in tube, McMaster and FLOTAC to determine the optimal flotation solution (FS) and the influence of faecal preservation for the diagnosis of Ancylostoma caninum in dogs, and compared the accuracy of the three copromicroscopic techniques. Among nine different FS, sodium chloride and sodium nitrate performed best for detection and quantification of A. caninum eggs. Faecal samples, either fresh or preserved in formalin 5%, resulted in higher A. caninum egg counts, compared to frozen samples or preserved in formalin 10% or sodium acetate–acetic acid–formalin. FLOTAC consistently resulted in higher A. caninum eggs per gram of faeces (EPG) and lower coefficient of variation (CV) than McMaster and flotation in tube. The best results in terms of mean faecal egg counts (highest value, i.e. 117.0 EPG) and CV (lowest value, i.e. 4.8%) were obtained with FLOTAC using sodium chloride and faecal samples preserved in formalin 5%. Our findings suggest that the FLOTAC technique should be considered for the diagnosis of A. caninum in dogs.     

Improved Nematode Extraction from Carrot Disk Culture

Radopholus spp. were reared in carrot tissue culture via established procedures, with slight modification. Several plant tissue maceration enzymes and flotation media (salts and sucrose) were evaluated with regard to nematode toxicity and extraction efficiency. Best extraction of viable nematodes and eggs was attained when carrot tissue infested with Radopholus citrophilus or R. similis was macerated with a mixture of 0.50% driselase and 0.50% cellulysin, w/v each, with 2.5 ml of enzyme solution based for each gram of carrot tissue. Maceration slurries containing carrot tissue and nematodes were maintained in open flasks on a rotary shaker (175 rpm) at 26 C for 24 hours. Nematodes and eggs were extracted from resultant culture slurries by flotation with MgSO₄-7H₂0 (sp gr 1.1). A protocol is presented to extract large quantities of viable burrowing nematodes and their eggs from carrot disk cultures.     

Growth of the Fungus Duddingtonia flagrans in Soil Surrounding Feces Deposited by Cattle or Sheep Fed the Fungus to Control Nematode Parasites

The results reported in this paper represent work from two separate experiments, namely a plot trial using cattle feces conducted at Kungsãngen in Uppsala, Sweden and a plot trial using sheep feces undertaken at Tåstrup in Copenhagen, Denmark. In both trials, a technique was used to monitor the level of Duddingtonia flagrans propagules in soil surrounding feces. The feces were from animals fed or not fed D. flagrans fungal chlamydospores. Also presented are the numbers of soil nematodes in soil surrounding sheep feces. The results indicate that D. flagrans has little growth beyond the fecal environment into surrounding soil when chlamydospores are fed to either sheep or cattle. This is substantiated by the soil nematode data. No statistical differences in the number of nematode taxa identified, Shannon Weiner H′, proportion of various feeding groups, and B/B + F (B and F are the proportions of bacterial and fungal-feeding nematodes) were found when soil surrounding sheep feces containing chlamydospores and parasitic nematode eggs was compared to soil surrounding feces containing parasitic nematode eggs alone. It is unlikely that the application of D. flagrans as a biological control agent against the free-living stages of nematode parasites of these livestock will negatively affect populations of nontarget soil nematodes.     

Seasonal and biogeographical patterns of gastrointestinal parasites in large carnivores: wolves in a coastal archipelago

Parasites are increasingly recognized for their profound influences on individual, population and ecosystem health. We provide the first report of gastrointestinal parasites in gray wolves from the central and north coasts of British Columbia, Canada. Across 60 000 km(2), wolf feces were collected from 34 packs in 2005-2008. At a smaller spatial scale (3300 km(2)), 8 packs were sampled in spring and autumn. Parasite eggs, larvae, and cysts were identified using standard flotation techniques and morphology. A subset of samples was analysed by PCR and sequencing to identify tapeworm eggs (n=9) and Giardia cysts (n=14). We detected ≥14 parasite taxa in 1558 fecal samples. Sarcocystis sporocysts occurred most frequently in feces (43·7%), followed by taeniid eggs (23·9%), Diphyllobothrium eggs (9·1%), Giardia cysts (6·8%), Toxocara canis eggs (2·1%), and Cryptosporidium oocysts (1·7%). Other parasites occurred in ≤1% of feces. Genetic analyses revealed Echinococcus canadensis strains G8 and G10, Taenia ovis krabbei, Diphyllobothrium nehonkaiense, and Giardia duodenalis assemblages A and B. Parasite prevalence differed between seasons and island/mainland sites. Patterns in parasite prevalence reflect seasonal and spatial resource use by wolves and wolf-salmon associations. These data provide a unique, extensive and solid baseline for monitoring parasite community structure in relation to environmental change.     

High variability in the number of E. multilocularis eggs in cat feces collected in the field

Echinococcus multilocularis is the causative agent of alveolar echinococcosis that is considered as the most severe parasitic disease in Europe. The contribution of cat to environmental contamination by E. multilocularis is generally considered as extremely low based on results of experimental infections and worm burden estimations from natural infections. However, the recent collection of numerous cat feces from kitchen gardens in high endemic areas and the detection of E. multilocularis DNA in a significant number of these feces raise the question of the risk of human transmission from cats. This study aimed to provide a quantitative estimation of E. multilocularis eggs in feces from naturally infected cats. A field sampling conducted in 192 kitchen gardens during a joint study led to the collection and analysis of 597 cat feces, among them 7 (1.2%) yielded positive results for E. multilocularis real-time PCR. The entire pellets obtained after homogenization, filtration and centrifugation of a 5 g-sample for each of these 7 feces were examined under a stereoscopic microscope. After assessing their number, 20 taeniid eggs were individually isolated and specifically identified by real-time PCR. Morphologically mature E. multilocularis eggs were identified in 4 samples and the counting of 4 to 43 E. multilocularis eggs per gram in these samples, i.e. 62 to 2331 eggs per feces when the total mass of the feces is considered. The number of eggs counted in 2 feces suggests a biotic potential of some naturally infected cats that largely exceed the previous experimental estimations.     

Fasciola hepatica: Comparison of the sedimentation and FLOTAC techniques for the detection and quantification of faecal egg counts in rats

We compared the sedimentation and FLOTAC techniques for the detection and quantification of Fasciola hepatica eggs in faecal samples obtained from 120 experimentally-infected rats before intervention, and in 42 rats after drug administration. Additionally, the average time for a single test was determined. A single FLOTAC showed a higher sensitivity (92.6%) than 2, 4 and 8 sedimentation readings (63.0-85.2%) for detecting F. hepatica eggs in rat faeces post-treatment. On average, it took 21 min to prepare and examine a single FLOTAC, whereas 114 min were needed for the sedimentation method including the reading of 8 slides. In both treated and untreated rats, the sedimentation method resulted in higher mean faecal egg counts (FECs) than FLOTAC (P < 0.05). In view of the high sensitivity and efficiency, the FLOTAC technique holds promise for experimental work in the F. hepatica-rat model. Additional research is needed to determine the reasons for the observed differences in FECs.     

Sensitivity comparison between Mini-FLOTAC and conventional techniques for the detection of Echinococcus multilocularis eggs

Canines serve as the definitive host of Echinococcus multilocularis. This study evaluated the sensitivity of the Mini-FLOTAC technique (MF) for the detection of E. multilocularis eggs in definitive hosts. First, we investigated the effects of heat inactivation and preservative conditions on the detection rate of eggs obtained from experimentally infected dogs. The sensitivity of MF was compared with that of eight other techniques: the centrifugal flotation with sucrose or zinc sulfate, MGL, AMS III, and a combination of MF and flotation/sedimentation techniques. Finally, we compared the sensitivity of MF and the centrifugal flotation with sucrose for the feces of E. multilocularis-infected foxes. The detection rate reached a plateau level with a specific gravity (s.g.) 1.22 for fresh eggs, but the highest rates were obtained with s.g. greater than 1.32 for heat-inactivated eggs. There was no significant difference in the detection rate among the preservative conditions. MF showed significantly higher EPG than the other techniques. Moreover, it showed higher diagnostic sensitivity for the fox feces than the centrifugal flotation technique. These results suggest that heat inactivation may alter s.g. of E. multilocularis eggs and that MF with zinc sulfate (s.g. = 1.32) would be effective for detecting heat-inactivated E. multilocularis eggs.     

Identification of Japanese Lymantria species (Lepidoptera: Lymantriidae) based on PCR–RFLP analysis of mitochondrial DNA

A polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP)-based method for species identification was applied to seven Japanese Lymantria species, including four Asian gypsy moth (AGM) species. We sequenced the partial end of the cytochrome c oxidase I (COI) gene, tRNA leucine, COII gene, and partial end of the tRNA lysine in mitochondrial DNA (mtDNA) for one individual of each of the seven species. We analyzed the recognition sites of three restriction endonucleases and constructed a scheme for Lymantria species identification using PCR–RFLP. We then applied the scheme to 291 individuals from 45 populations of seven species. We found that all seven species were correctly identified using PCR–RFLP. These results suggest that PCR–RFLP is useful for identifying Japanese Lymantria species, which may be detected at Japanese ports.     

Genomic RFLP Analysis of Meloidogyne arenaria Race 2 Populations

Traditional morphological methods of Meloidogyne identification have been unsuccessful in distinguishing three South Carolina, USA Meloidogyne arenaria race 2 populations—Govan, Pelion, and Florence. These populations differ greatly in reproductive rate and aggressiveness on soybean hosts. Total genomic DNA from eggs of each population was digested with the restriction endonuclease Eco RI and Southern hybridization analyses were performed with single-copy and interspersed multi-copy cloned probes. Probes were isolated from a genomic library of Eco RI, M. arenaria DNA fragments cloned into pUC8. One probe, designated pE1.6A, when hybridized to Southern blots of M. arenaria genomic DNAs, displayed an interspersed repetitive pattern, and the RFLPs distinguished the Govan population from the Pelion and Florence populations. Another clone, pE6.0A, carrying moderately repeated sequences, distinguished the Pelion and Florence isolates. This communication demonstrates the utility of genomic RFLP analysis for distinguishing populations of the same race within the same species. To test the possible utility of these moderately repeated sequence probes for detecting the presence of nematode DNA in DNA samples from roots inoculated with varying numbers of nematodes, dot blot hybridization analyses were performed. It is possible to detect as few as 30 nematodes per root sample with these cloned probes.     

Molecular Differentiation of Schistosoma japonicum and Schistosoma mekongi by Real-Time PCR with High Resolution Melting Analysis

Human schistosomiasis caused by Schistosoma japonicum and Schistosoma mekongi is a chronic and debilitating helminthic disease still prevalent in several countries of Asia. Due to morphological similarities of cercariae and eggs of these 2 species, microscopic differentiation is difficult. High resolution melting (HRM) real-time PCR is developed as an alternative tool for the detection and differentiation of these 2 species. A primer pair was designed for targeting the 18S ribosomal RNA gene to generate PCR products of 156 base pairs for both species. The melting points of S. japonicum and S. mekongi PCR products were 84.5±0.07℃ and 85.7±0.07℃, respectively. The method permits amplification from a single cercaria or an egg. The HRM real-time PCR is a rapid and simple tool for differentiation of S. japonicum and S. mekongi in the intermediate and final hosts.     

Seeding experiments demonstrate poor performance of the hatching test for detecting small numbers of Schistosoma mansoni eggs in feces

Parasitological methods for the evaluation of schistosomiasis tend to be limited when parasitic burdens are low, which is a major characteristic of low intensity transmission areas. While the hatching test (HT) method has been considered to be “very sensitive”, reports of its capacity to detect low numbers of eggs remain scarce in the published literature. Our main hypothesis is that HT has limitations and cannot be recommended for diagnosing light infections or as a control of cure. Hence, this study aims to describe the performance of HT in detail, with respect to seeding experiments for egg numbers in the range of 4 to 24 eggs per gram (epg) of feces. Different numbers of eggs of Schistosoma mansoni were seeded in normal human feces. The first set of experiments evaluated the amount of feces (higher than 0.5 g prevented hatching), the proximity of the light source (50 cm was preferred), and the observation time required for the detection of miracidia (more than 3 h did not add to sensitivity). HT was subsequently performed with 12, 10, 8, 6, 4, and 2 eggs in 0.5 g of feces. The final set of experiments was performed to analyze the initial filtration step, in which surgical gauze versus a 500 μm nylon mesh was compared and demonstrated losses of eggs that occurred with washing and gauze (better with nylon) sieving steps. The proposed method was found to produce 100% positivity for up to 12 epg, with a sharp decrease to 33% for 8 epg and less. In conclusion, HT is not recommended for diagnosing intestinal schistosomiasis in populations with light infections, considering the complexity of the procedure and its lack of effectiveness with fecal amounts higher than 0.5 g even at optimized conditions.     

Prevalence,Associated Risk Factors,and Phylogenetic Analysis of Toxocara vitulorum Infection in Yaks on the Qinghai Tibetan Plateau,China

Toxocara vitulorum has been rarely reported in yaks at high altitudes and remote areas of Sichuan Province of Tibetan Plateau of China. The current study was designed to investigate the prevalence, associated risk factors, and phylogenetic characteristics of T. vitulorum in yak calves on the Qinghai Tibetan plateau. Fecal samples were collected from 891 yak calves and were examined for the presence of T. vitulorum eggs by the McMaster technique. A multivariable logistic regression model was employed to explore variables potentially associated with exposure to T. vitulorum infection. T. vitulorum specimens were collected from the feces of yaks in Hongyuan of Sichuan Province, China. DNA was extracted from ascaris. After PCR amplification, the sequencing of ND1 gene was carried out and phylogenetic analyses was performed by MEGA 6.0 software. The results showed that 64 (20.1%; 95% CI 15.8–24.9%), 75 (17.2; 13.8–21.1), 29 (40.9; 29.3–53.2), and 5 (7.6; 2.5–16.8) yak calves were detected out to excrete T. vitulorum eggs in yak calve feces in Qinghai, Tibet, Sichuan, and Gansu, respectively. The present study revealed that high infection and mortality by T. vitulorum is wildly spread on the Qinghai Tibetan plateau, China by fecal examination. Geographical origin, ages, and fecal consistencies are the risk factors associated with T. vitulorum prevalence by logistic regression analysis. Molecular detection and phylogenetic analysis of ND1 gene of T. vitulorum indicated that T. vitulorum in the yak calves on the Qinghai Tibetan plateau are homologous to preveiously studies reported.     

Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria

A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10⁶ to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10⁶ cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.