AJTMH Transactions of the Royal Society of Tropical Medicine and Hygiene
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Am. J. Trop. Med. Hyg., 78(5), 2008, pp. 777-783
Copyright © 2008 by The American Society of Tropical Medicine and Hygiene

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Evaluation of Three PCR Assays for the Identification of the Sheep Strain (Genotype 1) of Echinococcus granulosus in Canid Feces and Parasite Tissues

Belgees S. Boufana*, Maiza Campos-Ponce, Ariel Naidich, Imad Buishi, Selma Lahmar, Eberhard Zeyhle, David J. Jenkins, Benoit Combes, Hao Wen, Ning Xiao, Minoru Nakao, Akira Ito, Jiamin Qiu, AND Philip S. Craig
Biomedical Sciences Research Institute, University of Salford, United Kingdom; Department of Infectious Diseases, Institute for Health Sciences, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands; Departamento de Parasitología, Instituto Nacional de Enfermedades Infecciosas, Capital Federal, Argentina; El-Fateh University, Tripoli, Libya; Service de Parasitologie, École Nationale de Médecine Vétérinaire, Tunisia; Hydatid Unit, African Medical and Research Foundation, Nairobi, Kenya; Australian Hydatid Control and Epidemiology Program, Fyshwick, Australia; Entente Interdepartementale de Lutte Contre la Rage et Autres Zoonoses, Malzeville, France; First Teaching Hospital, Xinjiang Medical University, Urumqi, Xinjiang, China; Department of Parasitology, Asahikawa Medical College, Asahikawa, Japan; Institute of Parasitic Diseases, Sichuan Centre for Disease Control and Prevention, Chengdu, Sichuan, China


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The performance of 3 PCR assays for the identification of the G1 sheep genotype of Echinococcus granulosus was evaluated using tissue and canid fecal samples. The "Dinkel" and "Stefanic" primers were the most sensitive in detecting E. granulosus DNA in feces of necropsied dogs (73.7% and 100%, respectively). The "Abbasi" primers detected 52.6% of E. granulosus infected dogs but were the most species-specific, cross-reacting only with Echinococcus shiquicus (tissue 90.9%; feces 75%). The Stefanic primers were the least specific (tissue, 27.3%; feces, 25%) for E. granulosus. The Dinkel primers also showed inter-species cross-reactivity (tissue, 63.6%; feces, 100%) but were found to be strain-specific for the E. granulosus G1 sheep genotype. Improvement of PCR tests for Echinococcus species and subspecific variants should rely on the use of less-conserved genes and development of protocols that improve the quality and quantity of DNA extracted from feces.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cystic echinococcosis (CE) is a zoonotic infection caused by the larval stage of the dog tapeworm Echinococcus granulosus. Humans become infected through the accidental ingestion of eggs that give rise to hydatid cysts mainly in the liver and lungs. The life cycle is perpetuated between carnivores (especially the domestic dog) and a wide range of ungulates (particularly herbivorous livestock) that serve as definitive and intermediate hosts, respectively. In view of the impact on human health and economic losses to livestock,1 there is a need for the development of reliable methods for the detection of infection in the dog definitive host.2 This is particularly important when assessing transmission dynamics and the potential risk of human hydatidosis as well as for surveillance of hydatid control programs. Gold standard detection of E. granulosus in dogs has always been at post-mortem (necropsy) which, although highly sensitive and specific, is evidently laborious, raises ethical issues, and is biohazardous.3 Co-proantigen detection-based laboratory tests (usually ELISA) for canine echinococcosis have also been described by several groups.4–8 Despite a lack of absolute species specificity, co-proantigen ELISA is a useful tool for large-scale screening of dog populations.3 There is a need for the development of more specific tests, particularly tests based on DNA detection in light of emerging genotypes and variants of E. granulosus, many of which are infective to humans.9,10

The first PCR method optimized for coprodiagnosis of E. granulosus relied on the detection of a tandem repeat unit within the genome of this cestode.11 In addition, 2 PCR assays to amplify regions of the 12S rRNA gene for the identification of E. granulosus have been successfully developed.12,13 Little information, however, is available on the specificity and sensitivity of these 3 PCR tests in routine laboratory practice. Also the genotypic specificity of PCR assays for isolates of E. granulosus has not been thoroughly assessed. The current study is an evaluation of the performance of these single-step PCR methods in detecting E. granulosus DNA from parasite tissue (hydatid cyst or adult tapeworm derived) including a number of E. granulosus strains/genotypes, and from fecal samples derived from naturally or experimentally infected canids.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fecal and parasite samples. Sensitivity of the coproPCR tests was determined using dog fecal samples from which E. granulosus worms were collected at necropsy and thus of known worm burden. These originated from China (n = 7), Libya (n = 7), Kenya (n = 4), and Jordan (n = 1). Fecal samples collected 21–37 days post-infection from experimentally infected Tunisian (n = 15) and Australian dogs (n = 10) were also included. PCR assay detection limit was evaluated using 1 g of a negative dog fecal sample spiked with 1, 10, 100, or 1000 E. granulosus eggs isolated from worms collected from dogs naturally infected with the sheep (G1) strain in China.

Parasite specificity of the E. granulosus PCR tests under evaluation was checked using tissue derived DNA from the following cestodes of canids (stage and place of origin): Dipylidium caninum (adult, Wales, U.K.), Taenia crassiceps (cysts, experimental mice, Belfast, U.K.), Taenia hydatigena (adult, Wales), Taenia multiceps (adult, Wales), Taenia ovis (adult, Wales), Taenia pisiformis (adult, Wales; cysts, Malham, U.K.), Echinococcus multilocularis (adult, China) and Echinococcus shiquicus (adult, Tibet, China). In addition, tapeworms of Taenia solium (adult and cysts, Peru), Taenia saginata (human derived adult, Tanzania), and Hymenolepis diminuta (adult, experimental rats, U.K.) were also used for DNA extraction.

Species specificity was also assessed using fecal samples from dogs after purgation (n = 14, China) and necropsied red foxes (Vulpes vulpes) (n = 6, France) from which E. multilocularis worms were collected. A fecal sample from 1 Tibetan fox (Vulpes ferrilata) from which the newly identified species E. shiquicus had been isolated was also available.14 Fecal samples from necropsied dogs naturally infected with T. crassiceps (n = 2, Libya) and fecal samples from dogs experimentally infected with T. multiceps (n = 2) were also included.

Strain specificity was tested using DNA extracted from protoscoleces or the germinal layer from hydatid cysts of various E. granulosus genotypes,15,16 namely, the sheep (G1, Libya, 4 isolates), horse (G4, UK, 3 isolates), cattle (G5, Switzerland, 2 isolates), camel (G6, Sudan), pig (G7, Slovak Republic, 3 isolates), cervid (G8, Minnesota), and Fennoscandian (G10, Finland) strains.

Fecal samples from 32 necropsied dogs (Libya, 28; Kenya, 4) and 17 necropsied red foxes from Franche-Comté, France, served as endemic-negative controls. Fecal samples (n = 17) from pet dogs in the Manchester area (U.K.) were used as "non-endemic" controls. DNA extracted from adults of the E. granulosus sheep strain (G1) (2 from Australia, 4 from China, 4 from Kazakhstan) as well as from protoscoleces (14 isolates from Libya) were used as positive DNA controls.

DNA extraction. Fecal DNA was retrieved from samples using the QIAmp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions with 1 minor adjustment: the fecal suspension was heated for 10 minutes at 85°C. In order to increase the amount of starting material and hence maximize the chance of detecting free Echinococcus DNA and/or DNA in eggs, an additional step was carried out prior to the use of the kit. One or two grams of fecal sample was emulsified in 0.01 M PBS and poured into a 100-µm cell strainer (BD Biosciences, Franklin Lakes, NJ) fitted onto a 50-mL tube containing 1.5 mL of Percoll (Sigma, U.K.) and 43 mL of 0.01 M PBS in order to remove light fecal debris (M. Campos-Ponce and P. S. Craig, unpublished). The tube was then centrifuged at 3600g for 30 minutes, and DNA extraction was performed using the pellet. DNA from parasite proglottids or metacestode tissue was extracted using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions.

Target DNA. The presence of DNA generic to all species and strains of E. granulosus used in this study was ascertained through the use of cestode-specific primers which amplified a fragment of the mitochondrial 12S rRNA gene.13,17,18 The presence of E. multilocularis DNA in Chinese dog purges and French fox fecal samples was verified through the use of a species-specific PCR.19 Detection of DNA of E. shiquicus extracted from feces of a Tibetan fox and generic cestode DNA derived from tissue and host fecal samples of a range of cestode species was achieved through the amplification of a fragment of the mitochondrial NADH dehydrogenase subunit 1.14,20

E. granulosus (G1) PCR assays. Three published PCR tests optimized for the detection of the G1 strain of E. granulosus were evaluated. These were the "Abbasi,"11 "Stefanic", 12 and "Dinkel"13 tests, respectively. PCR setups and cycling parameters were carried out as described by the respective authors with minor modifications as follows. The reagent concentrations quoted in the Abbasi protocol did not provide optimal results. Therefore, 250 µM (each) dNTPs, 1 µM of each primer, and 2% formamide were used instead. For the Stefanic test using Hotstart PCR, uracil DNA glycosylase and the internal control were not included and standard dTTP was used instead of the recommended dUTP. Finally the Dinkel test was used as specified by the authors with no modifications.

A Stratagene (La Jolla, CA) RoboCycler was used for all cycling profiles. The Hotstart procedure in the Stefanic test was carried out using a PCR Sprint Hybaid cycler (Thermo Electron Corporation, Waltham, MA). GoTaq (Promega U.K. Ltd., Southampton, U.K.) was used for DNA amplification of all protocols. Primers, as specified by the respective protocols were synthesized by MWG-Biotech AG (Ebersberg, Germany) and Invitrogen (Scotland, U.K.). PCR amplicons underwent electrophoresis in a 1.5% agarose TBE gel and were stained with ethidium bromide. A molecular weight marker (HyperLadder I, Bioline, London, England) was included on each gel for confirmation of amplicon sizes. Positive controls to monitor PCR success and negative controls to check for false-positive results that may have arisen from carry-over contamination were also included in all experiments. For all protocols, the same set of DNA samples within each group were systematically tested at least 3 times, and representative results are shown here. Gels were visualized under UV illumination using a Flowgen Alpha 1220 gel imaging system (Alpha Innotech Ltd., Staffordshire, U.K.).

Sequencing and analysis. DNA derived from both tissue and feces of all non-Echinococcus cestode species, E. shiquicus DNA from feces, and tissue DNA of E. multilocularis and E. granulosus genotypes G1 and G4 was verified by double-stranded sequencing (Cogenics, Norwich, U.K.). The sequence reports were analyzed using Chromas (http://www.technelysium.com.au/Chromas.html) and compared with the NCBI nucleotide database through the use of BLAST biosoftware (www.ncbi.nlm.nih.gov/BLAST/). Other genotypes of Echinococcus and parasite tissue of E. shiquicus had been molecularly typed by the individuals who kindly provided them.

Evaluation of reproducibility. The specificity of the Stefanic primers was further assessed by an independent laboratory (Departamento de Parasitología, Instituto Nacional de Enfermedades Infecciosas, Buenos Aires, Argentina). This was considered necessary in order to evaluate the reproducibility of results observed in the current work using the Stefanic assay as compared to the published data. This laboratory was not aware of our PCR data. Species specificity was assessed using a separate panel of tissue DNA derived from E. granulosus sensu stricto, E. multilocularis (cysts, Japan), E. vogeli (cysts, Venezuela), E. oligarthrus (cysts, Venezuela), and T. hydatigena (adult, Argentina). Strain specificity was also tested using DNA extracted from protoscoleces of E. granulosus genotypes (sheep G1, Tasmanian G2, horse G4, cattle G5, camel G6, and pig G7). Most E. granulosus samples tested were from Argentina; genotype G4 was from Spain. The uracil DNA glycosylase was not included, thus standard dTTP was used. An internal control was included for T. hydatigena test. DNA extraction was accomplished using the conventional phenol–chloroform method.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sensitivity of PCR tests. Sensitivity was used to compare the limits of detection of each PCR assay for the same set of samples and calculated as the percentage of E. granulosus samples correctly amplified. All 3 PCR tests performed well when DNA extracted from tissues of E. granulosus G1 adult worms or protoscoleces was tested, giving a 100% sensitivity with the amplification of the respective diagnostic products (data not shown).

All of the E. granulosus PCR protocols were capable of detecting DNA extracted from at least a single egg, yet test sensitivity for detection of E. granulosus in feces of naturally infected dogs ranged from 52.6% to 100% (Table 1Go). The Stefanic assay was the most sensitive with 100% copro-detection (worm burden from 4 to > 4000 worms/animal), although at times the PCR appeared to be performing suboptimally, as manifested by the detection of very faint target bands for feces from 7 necropsy-positive E. granulosus-infected dogs. The Abbasi test did not detect DNA in 4 dogs with E. granulosus (worm burdens < 130 worms/animal). Fecal controls from endemic and non-endemic areas gave negative PCR signals for all 3 sets of E. granulosus-specific primers. Surprisingly, all 3 PCR tests demonstrated the ability for amplification of E. granulosus DNA in feces of prepatent experimentally infected dogs. The Abbasi and Stefanic tests detected parasite DNA with 100% sensitivity in dog feces from 21 to 27 days post-infection (dpi) and from 30 to 37 dpi, respectively. The Dinkel test detected E. granulosus DNA by 30 dpi (Table 2Go).


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TABLE 1
Comparison of the sensitivity of 3 PCR tests for detection of Echinococcus granulosus DNA in egg-spiked fecal samples and from necropsied dogs with specific worm burdens from natural infections*
 

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TABLE 2
Sensitivity of 3 PCR assays in detecting Echinococcus granulosus prepatent infections of experimentally infected dogs*
 
Species specificity of PCR tests. Specificity is used here to designate the correct amplification of target species (E. granulosus DNA) and the absence of a PCR product from nontarget species-derived DNA. All 3 protocols were tested using closely related Echinococcus and Taenia species (Figure 1Go). The Abbasi test was the most species-specific (90.9%), yielding a diagnostic band only from non-E. granulosus DNA with the amplification of a strong 133-bp band for E. shiquicus DNA Figure 1AGo; lane 10). The lowest specificity (27.3%) was recorded for the Stefanic primers, which amplified 2 bands for tissue DNA of E. multilocularis, one of which was the diagnostic 255-bp band and the other was probably of host origin (Figure 1BGo; lane 9). Furthermore, Stefanic primers amplified DNA extracted from tissue of E. shiquicus, T. hydatigena, T. multiceps, T. ovis, T. pisiformis, D. caninum, and T. solium. A diagnostic band of 254 bp was observed 50% of the time when the Dinkel primers were used to amplify tissue-derived DNA from T. hydatigena, T. multiceps, T. ovis, and T. pisiformis, giving a specificity of 63.6% (Figure 1CGo).


Figure 1
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  		    FIGURE 1. PCR amplification of tissue and fecal DNA from Echinococcus granulosus and related species; (A) Abbasi, (B) Stefanic, and (C) Dinkel assays, respectively. Lane M, 100-bp molecular DNA ladder; lane 1, negative control; lane 2, positive control; lanes 3–13, tissue-derived DNA of Dipylidium caninum, Taenia crassiceps, Taenia hydatigena, Taenia multiceps, Taenia ovis, Taenia pisiformis, Echinococcus multilocularis, Echinococcus shiquicus, Taenia solium, Taenia saginata, and Hymenolepis diminuta; lanes 14–17, fecal-derived DNA of Echinococcus multilocularis, Echinococcus shiquicus, Taenia crassiceps, and Taenia multiceps.

 
Specificity was also tested using E. multilocularis DNA extracted from fecal samples recovered from naturally infected purged dogs and necropsied foxes. Furthermore, E. shiquicus DNA from feces of one infected Tibetan fox, and fecal extracted DNA of T. crassiceps and T. multiceps from necropsied and purged dogs was tested (Figure 1Go; Table 3Go). The Abbasi and Dinkel PCR tests showed 100% specificity in respect of no cross-reactions with fecal extracted DNA of E. multilocularis, T. crassiceps, or T. multiceps. The Stefanic test was positive for 8 of 14 E. multilocularis dog-purge–derived fecal DNA samples as well as for T. multiceps-infected feces but remained negative for E. multilocularis and T. crassiceps DNA from necropsied foxes and dogs, respectively (Table 3Go). Both the Abbasi and Stefanic primers gave positive signals for E. shiquicus DNA extracted from fox feces, whereas the Dinkel assay remained negative when tested with the same sample. The overall specificity of the coproPCR tests for DNA extracted from feces of these 4 species (E. multilocularis, E. shiquicus, T. crassiceps, T. multiceps) was 25%, 75%, and 100% for the Stefanic, Abbasi, and Dinkel primers, respectively.


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TABLE 3
Species specificity of 3 PCR assays using Echinococcus granulosus "specific" primers in detecting DNA in fecal samples from dogs or foxes with natural infections of Echinococcus multilocularis or other heterologous species*
 
E. granulosus genotype specificity. The 3 PCR assays were tested against a panel of 7 E. granulosus genotypes (G1 sheep, G4 horse, G5 cattle, G6 camel, G7 pig, G8 cervid, and G10 Finnish). The Dinkel test was found to be strain-specific to the E. granulosus G1 sheep genotype. In contrast, the Abbasi primers amplified DNA from G1, G4, G5, G7, and G10 genotypic isolates while the Stefanic PCR amplified DNA of all 7 E. granulosus genotypes tested (Figure 2Go).


Figure 2
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  		    FIGURE 2. PCR amplification of Echinococcus granulosus genotypes; (A) Abbasi, ( B) Stefanic, and (C) Dinkel assays, respectively. Lane M, 100-bp molecular DNA ladder; lane 1, negative control; lane 2, G1; lanes 3–7, G4–G8; lane 8, G10 genotypes.

 
Independent analysis. The results obtained by our collaborator (A.N.) with regard to the performance of the Stefanic primers were consistent with those observed in our laboratory. A 255-bp diagnostic product was amplified for E. granulosus G1 genotype, E. multilocularis, E. vogeli, E. oligarthrus, as well as for the G2, G4, G5, G6, and G7 genotypes of E. granulosus. In contrast, no such product was amplified using T. hydatigena DNA.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Human cystic echinococcosis (CE) is an important and widespread zoonotic disease. The causative cestode E. granulosus utilizes domestic dogs or wild canids as the main definitive host. CoproPCR testing is a potentially effective way of detecting species or even strain/genotype infections in living dogs and hence may provide an important confirmatory tool for epidemiologic studies and surveillance of hydatid-control programs. Species-specific detection of E. granulosus in the definitive host using coproPCR has to date been reported by 2 research groups.11,12 A third assay for the amplification of E. granulosus from tissue has also been documented.13 These 3 PCR tests, here referred to as the Abbasi,11 Stefanic,12 and Dinkel13 tests, were assessed in the current study against panels of DNA directly extracted from tissue of Echinococcus species (E. granulosus, E. multilocularis, and E. shiquicus), E. granulosus genotypes (G1, G4, G5, G6, G7, G8, and G10), as well as DNA from other cestode species (D. caninum, T. crassiceps, T. hydatigena, T. multiceps, T. ovis, T. pisiformis, T. solium, T. saginata, and Hymenolepis diminuta). In addition, a panel of fecal samples derived from canids with confirmed E. granulosus or Echinococcus species (E. multilocularis, E. shiquicus) or Taenia species (T. crassiceps, T. multiceps) was also used.

In terms of specificity, the Abbasi PCR test was the most species-specific for E. granulosus. Apart from E. granulosus, only DNA from either tissue or host feces of E. shiquicus14 was detected. This latter result appears to indicate that the tandem repetitive unit described by Abbasi and others11 is not unique to E. granulosus. The Stefanic E. granulosus primers were not species-specific in our hands as they cross-amplified DNA of E. multilocularis, E. shiquicus, and T. multiceps extracted from either tissue or feces; furthermore, DNA was amplified from 4 canid tapeworm species (T. hydatigena, T. ovis, T. pisiformis, D. caninum) and also from the human tapeworm T. solium. This lack of specificity with regard to E. multilocularis was confirmed by a second laboratory (A.N.). Additionally, E. vogeli DNA, which was not tested in the current study but was used to assess the specificity of the Stefanic protocol by the original authors, was positive in the hands of the collaborating researcher (A.N.). The Dinkel test primers also lacked specificity in our hands, amplifying tissue DNA from 4 canid Taenia species (T. hydatigena, T. ovis, T. pisiformis, T. multiceps) but not detecting DNA of T. multiceps from feces of infected dogs.

A 100% specificity for E. granulosus DNA was originally reported for the Stefanic and Dinkel tests,12,13 although the previous assessment of the former test was primarily based on purged fecal samples from dogs naturally infected with this cestode. The lack of specificity we obtained for the Stefanic and Dinkel PCR tests in the current study may be a reflection of the fact that both sets of primers were designed within a highly conserved gene (12S rRNA).12,13 In contrast, the high specificity we observed for the Abbasi primers for E. granulosus was only compromised by the new Echinococcus species (E. shiquicus), which was not available in the original study.11 It should be noted that DNA samples from the neotropical species of Echinococcus (E. vogeli, E. oligarthrus) were not available for testing in the present assessment and were only used by the collaborating researcher (A.N.) for evaluation of the Stefanic assay.

The Dinkel PCR was described essentially as a strain-typing PCR optimized to differentiate between the dominant G1 sheep–dog strain and the cattle (G5), camel (G6), and pig (G7) genotypes of E. granulosus.13 The current assessment has both confirmed this and further shown the Dinkel test primers to be strain-specific against other genotypes of E. granulosus not evaluated in their original study.13 Complete strain/genotype specificity, however, would need to include testing of the genotypes G2, G3, and G9 of E. granulosus, which were not evaluated in the present study. A rigorous molecular assessment of strain specificity was not included by the authors in the optimization of the Abbasi coproPCR assay; however, they reported a degree of cross-detection of the primers with DNA from other non-sheep (G1) strains of E. granulosus.11 The current study confirmed this lack of strain specificity for E. granulosus with the Abbasi PCR test. Similar results to those obtained here were also recently reported in Argentina, in which the G1, G2, G4, G5, G6, and G7 genotypes of E. granulosus gave positive diagnostic products with the Abbasi primers.21 This further emphasizes the probable occurrence of the tandem repeat units within the various strains or genotypes of E. granulosus. In fact, tandem-repeated sequences have also been described in the genome of the pig strain (G7) of E. granulosus.22 The Stefanic primers were reported to be optimized for specific detection of the G1 strain of E. granulosus against control DNA isolates of the G4, G5, G6, and G7 genotypes.12 The lack of E. granulosus genotype specificity we obtained for both the Abbasi and Stefanic PCR test primers may partly be due to the somewhat low annealing temperatures used in the respective protocols,11,12 which we speculate were probably not sufficient to eliminate the amplification of DNA from other E. granulosus genotypes.

Overall sensitivities for amplification of DNA from feces of dogs with prepatent experimental infections of E. granulosus were 92% for the Abbasi and Stefanic tests and 52% for the Dinkel assay. Positive DNA amplification from feces was obtained as early as 21 days post-infection in dogs experimentally infected with E. granulosus using either the Abbasi or Stefanic primers and by 30 days post-infection for the Dinkel assay. This indicates the ability of all 3 PCR assays to detect DNA in the absence of E. granulosus eggs because the pre-patent period for the G1 strain is known to be about 45 days.23 The ability of the Abbasi primers to detect prepatent E. granulosus from 25 to 33 days post-infection has also recently been reported in a separate study.21 Tapeworm DNA has also been reported to be detected by coproPCR in human feces prior to patency.24 These observations confirm that non-egg–derived DNA may also be present in feces of E. granulosus-infected canids and probably originates from proglottid breakdown, proglottid separation (apolysis), or tegumental turnover/disruption of tapeworms prior to patency. The ability of all 3 PCR protocols to detect prepatent E. granulosus infections is particularly relevant in follow-up of hydatid-control programs, where recently infected dogs could also be monitored as part of surveillance.

The overall sensitivities of the 3 PCR tests were 52.6% (Abbasi test), 73.7% (Dinkel test), and 100% (Stefanic test) for amplification of DNA from feces of naturally infected dogs with E. granulosus worm burdens ranging from 4 to > 4000. Only 2 of the PCR assays evaluated here were originally optimized to detect E. granulosus DNA in dog feces.11,12 The Abbasi test was reported to be capable of detecting at least 1 fg of DNA, which was much less than the 8 pg calculated for 1 taeniid egg.25 The high detection potential of the Abbasi test primers for E. granulosus DNA is derived from the amplification of a tandem repeat unit, with multiple targets for the primers to bind to.11 Despite this, however, the Abbasi test was least sensitive when tested with feces from necropsied dogs infected with E. granulosus. The 100% sensitivity of coproPCR obtained by the Stefanic primers for E. granulosus may in part be due to the Hotstart procedure used and because the same DNA samples were used for all PCR reactions, results obtained using the Abbasi and Dinkel primers may not have been caused by PCR inhibitors but are rather a reflection of poor template quality and its reduced quantity. This factor may have affected the reaction dynamics of the primers and their ability to interact with the Taq polymerase and target DNA. Indeed, studies have shown that mammal host fecal DNA is of low concentration and is usually degraded.26 This was clearly observed in this study because 100% sensitivity was recorded for all primers with DNA derived from E. granulosus (G1) tissue samples. Furthermore, some primers are affected more than others by impurities present in crude DNA samples.27

Sensitivity has also been noted to be affected by host and bacterial DNA.21 Host DNA has been detected in the current study, particularly when generic cestode primers were used to ascertain the presence of Echinococcus DNA in fecal samples (data not shown). Chromatograms of the sequencing data of E. granulosus and T. crassiceps DNA amplified from infected host feces (as compared to that amplified from parasite tissue) had low signals and high background, indicating low DNA quantity as well as the presence of contaminants as manifest by other sequences probably pertaining to DNA of other organisms (data not shown). High concentrations of nontarget DNA may compete with target DNA for the primers, resulting in weak diagnostic signals or complete inhibition.28 This may explain the occasional suboptimal performance of the Stefanic primers in detecting E. granulosus DNA in confirmed necropsy dogs. Primers are known to be affected by the presence of nontarget DNA which is known to be inhibitory to PCR.29 Inhibition of amplification was more closely associated with natural as opposed to the experimental E. granulosus infections because all 3 PCR tests were capable of detecting 1 egg from spiked fecal samples. This suggests that prolonged exposure of parasite DNA to fecal matter may hasten its degradation, which would result in reduction in both quantity and quality of available DNA. The amplification of coproDNA of parasite origin may therefore require the implementation of specific procedures to maximize its detection when present. The importance of the use of good extraction methods for recovery of DNA from feces and its affect on PCR sensitivity has been stressed by many workers.30,31

In conclusion, the results obtained in the present E. granulosus PCR study show that, although the Abbasi PCR primers were not E. granulosus G1 strain-specific, they were highly species specific and thus most useful in confirmation of an E. granulosus infection in dogs. Conversely, the Dinkel primers appear to have the potential of being strain-specific for the E. granulosus G1 sheep strain and were 100% specific for E. granulosus in dog feces, but they showed evidence of cross-reaction with tissue-derived DNA from other canid tapeworm species. The Stefanic primers were 100% sensitive in detecting coproDNA from confirmed E. granulosus infections in dogs but in our assessment did not appear to be species-specific even when the recommended annealing temperature (53°C) was increased to 57°C. It is therefore evident that, although the Stefanic and Dinkel PCR tests for E. granulosus were reportedly specific in the hands of their original developers, the current assessment found them poorly reproducible. Finally, improvement in PCR tests for detection of E. granulosus (species and subspecies levels) should be directed to the identification of alternative, less-conserved gene targets to eliminate interspecies cross-reactions and on development of protocols to maximize and improve the quality of DNA extracted from host feces.

Received November 13, 2007. Accepted for publication February 8, 2008.

Acknowledgments: The laboratory technical support of Mrs. Helen Bradshaw is gratefully acknowledged. Material used in this study was kindly provided by Kevin Shaddick (protoscoleces from horse hydatid G4, Bristol abattoir, Bristol, U.K.), A. Dinkel (E. granulosus G5 DNA, G6 and G7 protoscoleces), B. Gottstein (E. granulosus G5 DNA), P. Dubinsky (E. granulosus G7, protoscoleces and germinal layer), D. McManus (E. granulosus G8 protoscoleces), A. Lavikainen and A. Oksanen (E. granulosus G10 protoscoleces), S. Abdel-Hafez (for access to a dog necropsy isolated from Jordan), Paul Torgerson (feces from dogs experimentally infected with T. multiceps and adult E. granulosus from Kazakhstan), and H. Garcia (T. solium cysts and adult worms, Peru).

Financial support: The research described was supported by a grant (RO1 TW001565) from the Fogarty International Centre.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the Fogarty International Centre or the National Institutes of Health (Principal Investigator, Philip S. Craig).

* Address correspondence to Belgees S. Boufana, Biomedical Sciences Research Institute, University of Salford, United Kingdom. E-mail: b.boufana{at}salford.ac.uk Back

Authors’ addresses: Belgees S. Boufana and Philip S. Craig, Cestode Zoonoses Research Group, Biomedical Sciences Research Institute, University of Salford, United Kingdom, Tel: 44-0161-295 4299, Fax: 44-0161-295 5129, E-mail: B.Boufana{at}salford.ac.uk. Maiza Campos-Ponce, Department of Infectious Diseases, Institute for Health Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands. Ariel Naidich, Departamento de Parasitología, Instituto Nacional de Enfermedades Infecciosas, Av. Velez Sarsfield 563, CP 1281, Capital Federal, Argentina. Imad Buishi, El-Fateh University, PO Box 606, Tripoli, Libya. Selma Lahmar, Service de Parasitologie, Ecole Nationale de Médecine Vétérinaire, 2020 Sidi Thabet, Tunisia. Eberhard Zeyhle, Hydatid Unit, African Medical and Research Foundation, PO Box 30125, Nairobi, Kenya. David J. Jenkins, Australian Hydatid Control and Epidemiology Program, 12 Mildura Street, Fyshwick, ACT 2609, Australia. Benoit Combes, Entente Interdepartementale de Lutte Contre la Rage et Autres Zoonoses, Domaine de Pixérécourt, 54220 Malzeville, France. Hao Wen, First Teaching Hospital, Xinjiang Medical University, Urumqi, Xinjiang, China. Ning Xiao, Minoru Nakao, and Akira Ito, Department of Parasitology, Asahikawa Medical College, Asahikawa 078-8510, Japan. Jiamin Qiu, Institute of Parasitic Diseases, Sichuan Centre for Disease Control and Prevention, Chengdu, Sichuan 610041 China.

Reprint requests: Belgees S. Boufana, Biomedical Sciences Research Institute, University of Salford, United Kingdom, Tel: 44-0161-295 4299, Fax: 44-0161-295 5129, E-mail: B.Boufana{at}salford.ac.uk.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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