Simple modification to improve reliability of copro-DNA examinations for diagnosing Echinococcus multilocularis infections in red foxes

2020 ◽  
Vol 94 ◽  
Author(s):  
T. Irie ◽  
T. Ito ◽  
H. Kouguchi ◽  
K. Uraguchi
Keyword(s):  
Dna Extraction ◽  
Echinococcus Multilocularis ◽  
Epidemiological Studies ◽  
Sequencing Analysis ◽  
Red Foxes ◽  
Simple Modification ◽  
Pcr Inhibitors ◽  
Examination Technique ◽  
Modified Method ◽  
Infection Pressure

Abstract Epidemiological studies of Echinococcus multilocularis infections in definitive hosts require a reliable and economic diagnostic method. In this study, the current copro-DNA examination technique was modified by increasing the faecal amounts tested and adding a step to neutralize the faeces before DNA extraction. Reliability of the modified method was evaluated using rectal faecal samples from red foxes and comparing them with intestinal worms detected using the sedimentation and counting technique (SCT) following necropsy. The modified copro-DNA examination method demonstrated 93.9% sensitivity (138/147) on the SCT. Its detectability increased depending on the worm burden, and the sensitivity was 100% in cases harbouring over 1000 worms. From 111 SCT-negative cases, six (5.4%) were copro-DNA-positive, and all were confirmed as E. multilocularis via sequencing analysis. Five of the remaining 105 SCT-negative cases (4.8%) retained polymerase chain reaction (PCR) inhibitors in the extracted solution, suggesting that approximately 5% of the red fox faeces retained these inhibitors after treatment with the present copro-DNA extraction method. Although further evaluation is needed for faeces deposited in the wild, the present copro-DNA examination technique will help monitor the E. multilocularis prevalence in definitive hosts. When used for detailed evaluations of endemicity (e.g. changes in infection pressure or spread in non-endemic areas), the absence of PCR inhibitors should be confirmed, and multiple trials on faecal subsamples are recommended.

scholarly journals Monitoring of Echinococcus multilocularis infection in red foxes in Shiretoko, Japan, by coproantigen detection

Parasitology ◽  
1998 ◽  
Vol 117 (2) ◽  
pp. 193-200 ◽  
Author(s):  
N. NONAKA ◽  
H. TSUKADA ◽  
N. ABE ◽  
Y. OKU ◽  
M. KAMIYA
Keyword(s):  
Echinococcus Multilocularis ◽  
National Park ◽  
Close Contact ◽  
The Other ◽  
Management Tool ◽  
Red Foxes ◽  
Monitoring Method ◽  
Early Autumn ◽  
Detection Assay ◽  
Infection Pressure

The study was conducted to evaluate a mAb-based coproantigen detection assay for monitoring fox prevalence of Echinococcus multilocularis infections in the Shiretoko National Park in Hokkaido, Japan. Eight fox families, each consisting of 2–4 adults and their pups, were selected because their territories have been determined. Faeces of each family (total n=537) were collected monthly from April to October, 1994. Detection of coproantigen and taeniid eggs was recognized in 58 and 27 faeces, respectively, but mostly restricted in 1 family, in which coproantigen ELISA OD values had dual peaks, one in June and the other in October, whereas taeniid eggs were detected only from April to July. Fox pup faeces (n=51) collected around the dens used by the positive family were all coproantigen and taeniid egg negative except for 1 faeces. These results suggest that the prevalence and infection pressure in the study area were low and the infected family terminated the infection in the summer and acquired a new one in the early autumn, 1994. This monitoring method for fox infection will be a practical management tool for E. multilocularis infection, especially at the tourist spots in Hokkaido where close contact of fox with humans is frequently observed.

scholarly journals Comparison of Two DNA Extraction Methods and Two PCRs for Detection of Echinococcus multilocularis in the Stool Samples of Naturally Infected Red Foxes

Animals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2381
Author(s):  
Katarzyna Skrzypek ◽  
Jacek Karamon ◽  
Małgorzata Samorek-Pieróg ◽  
Joanna Dąbrowska ◽  
Maciej Kochanowski ◽  
...  
Keyword(s):  
Positive Result ◽  
Multiplex Pcr ◽  
Dna Extraction ◽  
Echinococcus Multilocularis ◽  
Nested Pcr ◽  
Genetic Material ◽  
Extraction Methods ◽  
Red Foxes ◽  
Stool Samples ◽  
Positive Results

(1) Background: Due to the increasing distribution of Echinococcus multilocularis infections in final hosts, epidemiological investigations are important for recognizing the spreading pattern of this parasite and also to estimate risk infection for humans. (2) Methods: Investigations were conducted with two commercial kits dedicated for DNA extraction from feces: ZR Fecal DNA Mini Prep (Zymo Research, Freiburg, Germany) and QIAamp FAST DNA Stool Mini Kit (Qiagen, Hilden, Germany) (marked as Z and Q), together with two common PCR protocols (nested PCR and multiplex PCR). The goal was to compare their efficiency in detecting the genetic material of E. multilocularis in the samples of feces. Stool samples from red foxes were collected in a highly endemic area in Poland. Sedimentation and counting technique (SCT) was used as a reference method. (3) Results: From 48 samples, 35 were positive in SCT. Further investigations showed that 40.0% of samples (from those with SCT positive result) after Z-DNA extraction and 45.7% after Q-DNA extraction gave positive results in nested PCR. In multiplex PCR, positive results were obtained in 54.3% of samples after Z isolation and 48.6% of samples after Q. Additionally, one sample that resulted in being negative in SCT gave a positive result in PCR. The number of worms detected in the intestines had no influence on PCR results. (4) Conclusions: Both of the extraction methods showed similar efficiency in DNA isolation and dealing with inhibitors; however, they showed relatively low sensitivity. This was probably caused by degradation of genetic material in the field-collected samples.

Echinococcus multilocularis in carnivores from the Klatovy district of the Czech Republic

2001 ◽  
Vol 75 (1) ◽  
pp. 61-66 ◽  
Author(s):  
K. Martínek ◽  
L. Kolárová ◽  
J. Cervený
Keyword(s):  
Czech Republic ◽  
Echinococcus Multilocularis ◽  
Alveolar Echinococcosis ◽  
Meles Meles ◽  
Epidemiological Studies ◽  
Human Case ◽  
Red Foxes ◽  
The Czech Republic ◽  
Source Of Infection ◽  
Faecal Samples

A unique human case of alveolar echinococcosis was described in 1979 from the Klatovy district of the Czech Republic. However, there were no previous epidemiological studies in this area focusing on detection of the source of infection –Echinococcus multilocularisadults producing eggs. During the period June 1997 to April 1999, 29 out of a total of 46 (63.3%) red foxes (Vulpes vulpes) in the Klatovy district and one of four foxes (25.0%) in the Pilsen South district were found to be infected with adult worms ofE. multilocularis. NoE. multilocularisadults were found in other animals from the Klatovy district (i.e. three specimens ofMartes martes, twoMartes foina, oneMustela erminea, twoMeles melesand oneFelis catusf.domestica). An examination of faecal samples from 55 dogs (Canis familiaris) from the Klatovy district resulted in the detection ofE. multilocularisDNA in one (1.8%) sample. The present results support the possibility that human alveolar echinococcosis previously described in the Czech Republic had the character of an autochthonous infection. There are also indications of a potential risk of infection to humans.

Potential remedy against Echinococcus multilocularis in wild red foxes using baits with anthelmintic distributed around fox breeding dens in Hokkaido, Japan

Parasitology ◽  
2002 ◽  
Vol 125 (2) ◽  
pp. 119-129 ◽  
Author(s):  
H. TSUKADA ◽  
K. HAMAZAKI ◽  
S. GANZORIG ◽  
T. IWAKI ◽  
K. KONNO ◽  
...  
Keyword(s):  
Echinococcus Multilocularis ◽  
Red Fox ◽  
Red Foxes ◽  
Clethrionomys Rufocanus ◽  
Efficient Control ◽  
Positive Rate ◽  
Potential Remedy ◽  
Patent Infection

The effect of bait-delivered anthelmintic to reduce the prevalence of Echinococcus multilocularis in wild red foxes was evaluated in Koshimizu, in the eastern part of Hokkaido, Japan. The study area (200 km2) was divided into baited and non-baited sections. The anthelmintic baits were distributed around fox den sites in the baited section every month for 13 months. After 1 year of the anthelmintic bait distribution, the prevalence of E. multilocularis in foxes, evaluated either by the parasite egg examination (from 27.1 to 5.6%) or coproantigen ELISA (from 59.6 to 29.7%), decreased in the baited section contrasting to that in the non-baited section (parasite egg: from 18.8 to 24.2%; ELISA: from 41.9 to 45.8%). The prevalence of E. multilocularis in grey red-backed vole Clethrionomys rufocanus, caught around fox dens, born after bait distribution also decreased and was significantly lower than that in non-baited section. However, within the study periods, the coproantigen-positive rate in fox faeces sporadically increased, while egg-positive rate constantly decreased. Since coproantigen ELISA can detect pre-patent infection, this observation indicates that reinfection pressure in the baited section was still high even after the 13 months of anthelmintic bait distribution. Therefore, the bait distribution longer than our study period is required for the efficient control of E. multilocularis in wild red fox population.

Reduced egg production of Echinococcus multilocularis in experimentally infected and re-infected red foxes (Vulpes vulpes)

2008 ◽  
Vol 155 (1-2) ◽  
pp. 59-66 ◽  
Author(s):  
Mohammad Nafi Solaiman Al-Sabi ◽  
Christian M.O. Kapel ◽  
Pia Webster ◽  
Peter Deplazes
Keyword(s):  
Echinococcus Multilocularis ◽  
Vulpes Vulpes ◽  
Egg Production ◽  
Red Foxes

scholarly journals Evaluation of DNA extraction methods for quantifying Helicobacter pylori in water with PCR inhibitors

2021 ◽  
Author(s):  
Eun-Sook Lee ◽  
So-Yang Cha ◽  
Jong-Soon Jung
Keyword(s):  
Helicobacter Pylori ◽  
Real Time ◽  
Dna Extraction ◽  
Water Samples ◽  
Real Time Pcr ◽  
Heavy Rain ◽  
Extraction Methods ◽  
Pcr Inhibitors ◽  
H Pylori ◽  
Dna Extraction Methods

Abstract DNA extraction methods were evaluated to reduce PCR inhibitors and quantify Helicobacter pylori directly from water samples using real-time PCR. Three nucleic acid extraction methods were evaluated for different types of water samples. While the QIAamp DNA mini kit for tissue was suitable for DNA extraction from treated water, the QIAamp DNA stool mini kit was still efficient in analyzing samples from river water after heavy rain and with high concentration of PCR inhibitors. The FastDNA SPIN Kit for Soil could extract DNA effectively from microbes in river and stream waters without heavy rain. Immunomagnetic separation (IMS) was used prior to DNA extraction and was a useful tool for reducing PCR inhibitors in influent and stream samples. H. pylori in various waters could be quantified directly by real-time PCR while minimizing the effect of PCR inhibitors by an appropriate method through the evaluation of DNA extraction methods considering the characteristics of the matrix water. The findings of the present study suggest that the types or characteristics of water sample by source and precipitation are an important factor in detecting H. pylori and they can be applied when detecting and monitoring of other pathogens in water.

scholarly journals Occurence of Echinococcus multilocularis in red foxes (Vulpes vulpes) in southern Poland

2008 ◽  
Vol 45 (1) ◽  
pp. 24-27 ◽  
Author(s):  
A. Borecka ◽  
J. Gawor ◽  
M. Malczewska ◽  
A. Malczewski
Keyword(s):  
Echinococcus Multilocularis ◽  
Vulpes Vulpes ◽  
Early Recognition ◽  
Risk Groups ◽  
Post Mortem ◽  
Red Foxes ◽  
Southern Poland ◽  
Regular Screening ◽  
High Prevalence ◽  
Epidemiological Situation

AbstractThe prevalence of Echinococcus multilocularis in 214 red foxes (Vulpes vulpes) in the southern part of Poland (Małopolskie voivodship) was evaluated post mortem in 2005. Infected foxes were found in 8 districts within 17 examined, with the prevalence from 10.0 % to 63.9 % (a mean 20.1 % in the whole study area). The highest prevalence (41.2 % and 63.9 %) was revealed in two districts, in the south and the east of the province, respectively. The high prevalence of E. multilocularis in red foxes in the region previously recognised as that of low parasite endemicity provides evidence for the need of regular screening of the current epidemiological situation in foxes as well as the monitoring of humans from risk groups for early recognition of possible AE cases.

scholarly journals Genetic Structure and Gene Flow in Red Foxes (Vulpes vulpes) in Scandinavia: Implications for the Potential Future Spread of Echinococcus multilocularis Tapeworm

2019 ◽  
Vol 9 (24) ◽  
pp. 5289
Author(s):  
Mari Hagenlund ◽  
Arne Linløkken ◽  
Kjartan Østbye ◽  
Zea Walton ◽  
Morten Odden ◽  
...  
Keyword(s):  
Gene Flow ◽  
Population Ecology ◽  
Wildlife Management ◽  
Echinococcus Multilocularis ◽  
Vulpes Vulpes ◽  
Red Fox ◽  
Emerging Diseases ◽  
Red Foxes ◽  
Genetic Structuring ◽  
High Level

Knowledge about the dispersal and gene flow patterns in wild animals are important for our understanding of population ecology and the connectedness of populations. It is also important for management relating to disease control and the transmission of new and emerging diseases. Our study aimed to evaluate the genetic structuring among comparative samples of red foxes in a small part of Scandinavia and to estimate the gene flow and potential directionality in the movements of foxes using an optimized set of microsatellite markers. We compared genetic samples of red foxes (Vulpes vulpes) from two areas in Sweden and two areas in Norway, including red fox samples from areas where the occurrence of the cyclophyllic tapeworm Echinococcus multilocularis has been documented, and areas without known occurrence of the parasite. Our results show a high level of gene flow over considerable distances and substantiates migration from areas affected with E. multilocularis into Norway where the parasite is not yet detected. The results allow us to better understand the gene flow and directionality in the movement patterns of red foxes, which is important for wildlife management authorities regarding the spread of E. multilocularis.

scholarly journals RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors

BioTechniques ◽  
2020 ◽  
Vol 69 (4) ◽  
pp. 270-280 ◽  
Author(s):  
Mustafa Ahmad Munawar ◽  
Frank Martin ◽  
Anna Toljamo ◽  
Harri Kokko ◽  
Elina Oksanen
Keyword(s):  
Reaction Kinetics ◽  
Dna Extraction ◽  
Pcr Primers ◽  
Sybr Green ◽  
Recombinase Polymerase Amplification ◽  
Pcr Inhibitors ◽  
Plant Dna ◽  
Pcr Assays ◽  
Flanking Regions

DNA extraction can be lengthy and sometimes ends up with amplification inhibitors. We present the potential of recombinase polymerase amplification (RPA) to replace plant DNA extraction. In our rapid ‘RPA-PCR couple’ concept, RPA is tuned to slower reaction kinetics to promote amplification of long targets. RPA primers amplify target and some flanking regions directly from simple plant macerates. Then PCR primers exponentially amplify the target directly from the RPA reaction. We present the coupling of RPA with conventional, TaqMan and SYBR Green PCR assays. We applied the concept to strawberry Phytophthora pathogens and the Phytophthora identification marker atp9-nad9. We found RPA-PCR couple specific, sensitive and reliable. The approach may also benefit other difficult samples such as food, feces and ancient samples.

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