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Patent and pre-patent detection of Echinococcus granulosus genotypes in the definitive host
Molecular and Cellular Probes 20 (2006) 5–10 www.elsevier.com/locate/ymcpr
Patent and pre-patent detection of Echinococcus granulosus genotypes in the definitive host Ariel Naidich a,*, Donald P. McManus b, Sergio G. Canova a, Ariana M. Gutierrez a, Wenbao Zhang b, Eduardo A. Guarnera a, Mara C. Rosenzvit a a
Departamentode Parasitologı´a, Instituto Nacional de Enfermedades Infecciosas, ANLIS Dr Carlos G. Malbra´n, Av. Velez Sarsfield 563, 1281 Ciudad de Buenos Aires, Argentina b Division of Infectious Diseases and Immunology, Molecular Parasitology Laboratory, Queensland Institute of Medical Research, Royal Brisbane Hospital Post Office, Herston, Qld. 4006, Australia Received 16 June 2005; accepted for publication 22 August 2005 Available online 14 October 2005
Abstract The detection of Echinococcus granulosus in dogs is important for epidemiological surveillance and evaluation of cystic hydatic disease control programs. We report the efficacy of two PCR-based methods to detect patent and pre-patent infection in dogs experimentally infected with E. granulosus. The detection is based on amplification of a fragment of a mitochondrial gene (Mit-PCR) and a DNA repetitive element (Rep-PCR) of E. granulosus. We tested the ability of both methods to detect several genotypes of the parasite. Both PCR methods could detect E. granulosus in pre-patent and patent periods, even when microscopical observation of eggs resulted negative in fecal samples. The Mit-PCR produced the same amplification pattern for all the parasite genotypes tested while the amplification patterns with the Rep-PCR differed among groups of strains. Fecal samples collected from dogs of an endemic area were diagnosed with more sensitivity than arecoline hydrobromide purgation. These molecular methods could be applied in the confirmation of coproantigen-positive fecal samples and to verify the success of control programs. q 2005 Elsevier Ltd. All rights reserved. Keywords: Echinococcus granulosus; PCR; Fecal; Genotypes; Experimental infection; Patency
1. Introduction Echinococcus granulosus is the causative agent of cystic hydatid disease (CHD), a zoonosis that affects the public health and economy in many areas worldwide. Domestic livestock, wild life animals and humans infected with the larval stage of the parasite develop fluid-filled cysts, most often in the liver and lungs. Dogs are the usual definitive hosts and the parasite eggs shed in their feces become the source of infection for sheep, pigs, cows and other intermediate hosts including humans. Detecting E. granulosus infection in dogs is important for epidemiological surveillance and evaluation of control programs. Traditionally, the methods used for detecting dog infection were dog necropsy and further intestinal examination, and arecoline hydrobromide purgation [1]. The latter method is
* Corresponding author. Tel.: C54 11 4301 7437; fax: C54 11 4303 2382. E-mail address: [email protected] (A. Naidich).
0890-8508/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2005.08.001
inconvenient due to environmental contamination, low sensitivity and reproducibility; and detrimental effects on animals [2]. Assays for detection of parasite antigens in fecal samples from definitive hosts have been developed [3], but this procedure is not species-specific [4,5] and its sensitivity appears to be affected by parasite burdens [4,6,7]. Molecular methods, especially those based on PCR should provide the required specificity and sensitivity for E. granulosus detection in dogs. Although a PCR-based method for detecting a patent infection in the definitive host has been recently developed [8], it was not reported whether the assay could detect a pre-patent infection, i.e before eggs are released in the feces. Ten distinct genotypes (G1–G10) of E. granulosus, differing in biological characters such as intermediate host specificity and developmental rates have been described [9–14]. Although the sheep–dog strain (G1 genotype), is the predominant strain infective to humans, there is increasing evidence that other strains can also cause significant human hydatid disease. In Argentina, for example, Tasmanian sheep (G2 genotype), cattle (G5 genotype) and camel (G6 genotype) strains produced 42% of cases of human CHD [15]. Also, the camel strain was
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reported to cause CHD in humans from Iran [16] and East Africa [17]. This genetic variation makes it necessary to assess the performance of a PCR-based assay to detect a range of E. granulosus strains for reliable diagnosis in definitive hosts. Cabrera et al. [18] showed that a primer pair designed for amplification of a fragment of the mitochondrial cytochrome oxidase subunit 1 (coxI) gene was able to specifically identify E. granulosus DNA extracted from oncospheres. In addition, a primer set and a copro-PCR method were described for the detection of E. granulosus infection in dogs with high sensitivity and specificity [19]. The amplification target is a repetitive DNA element present in the genome of the sheep strain of E. granulosus but the efficacy of this latter method for detection of other E. granulosus strains was not documented. In the present study, the performances of both copro-PCR methods have been evaluated for the detection of distinct strains of E. granulosus and for diagnosis of pre-patent infections. 2. Materials and methods 2.1. DNA extraction and purification Total E. granulosus genomic DNA was prepared from fresh, frozen in liquid nitrogen, or 70% ethanol preserved protoscoleces or germinal layer from individual E. granulosus cysts by conventional techniques [20]. In brief, protoscoleces from an individual cyst or adult worm were washed three times in phosphate-buffered saline, pH 7.2, crushed in liquid nitrogen, and digested in 200 mg proteinase K/ml and 0.5% SDS during 3 h at 56 8C. After proteinase K treatment, phenol/chloroform extractions were made and DNA was precipitated with ethanol [20]. The germinal layers were processed as described by Kamenetzky et al. [21]. E. granulosus genotypes were determined by sequencing a fragment of the mitochondrial coxI gene as described [10]. All faecal samples from dogs were examined microscopically in triplicate to determine the presence of taenid eggs. DNA extraction and purification, from 200 mg of each fecal sample, was performed using the QIAmp DNA Stool Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. Eluates of purified DNA samples were strored at K20 8C for 24–48 h after extraction until their use in PCR reactions. Ten microliters of eluate were employed as template in PCRs. 2.2. Echinococcus granulosus eggs Isolated E. granulosus eggs were obtained from a gravid proglottid of an adult worm (previously genotyped as the G1 genotype) that was mechanically disrupted. The released eggs were washed three times with phosphate saline buffer and counted under the microscope. 2.3. Experimental infection E. granulosus protoscoleces were aspirated from sheep liver hydatid cysts collected from a slaughterhouse in Urumqi,
Xinjiang, PR China. Five dogs, previously treated with 10 mg/body weight of albendazole to remove all tapeworms, were each fed 0.8 ml packed and sedimented protoscoleces (approximately 100,000) together with a small amount of cyst fluid. Animals were maintained under humane conditions, according to the Guide for the Care and Use of Laboratory Animals [22]. Stool samples were collected at 25, 33, 35, 39, 43, 46 and 50 days post-infection (p.i.). In addition, two samples were collected at 55 days p.i. from two dogs. All samples were maintained in 70% ethanol at K70 8C for at least two weeks to inactivate oncospheres. After the collection of the last sample, all dogs were humanely killed and autopsied to determine parasite burdens. 2.4. Samples from endemic areas Stool samples were collected from nine domestic dogs from Carmen de Patagones, Buenos Aires province and Ingeniero Jacobacci, Rı´o Negro province, Argentina, after arecoline hydrobromide purging. The samples were kept in 70% ethanol at room temperature until their shipment to the laboratory, where they were stored at K70 8C for at least two weeks to inactivate oncospheres. 2.5. Polymerase chain reactions (PCR) Three PCR reactions were performed: Mit-PCR for mitochondrial DNA amplificaction, Rep-PCR for repetitive DNA amplification and one control Cnt-PCR. For Mit-PCR, the primer set EgO/DNA-IM1 (forward primer 5 0 -TCATATTTGTTTGAGKATYAGTKC-3 0 , and reverse primer 5 0 GTAAATAAMACTATAAAAGAAAYMAC-3 0 ), designed to amplify a fragment of the coxI gene specific for E. granulosus [18], was used. The thermal profile used with this set of primers involved 15 min at 95 8C, followed by 40 cycles of 1 min at 94 8C, 1 min at 50 8C and 1 min at 72 8C followed by a final elongation step of 10 min at 72 8C. This PCR is specific for E. granulosus, as was demonstrated by Cabrera et al. [18]. For Rep-PCR the primer set used was Eg1121a/22a (forward primer Eg1121a 5 0 -GAATGCAAGCAGCAGATG3 0 and reverse primer Eg1122a 5 0 -GAGATGAGTGAGAAGGAGTG-3 0 ) that amplifies a DNA repetitive element from E. granulosus genome [19]. The thermal profile involved 15 min at 94 8C, followed by 35 cycles, each of 1 min at 95 8C, 1 min at 55 8C, 1 min at 72 8C, and a final elongation step for 10 min at 72 8C. This PCR method has proved to be specific for E. granulosus [19]. As bacterial proteases and nucleases in feces, as well as cell debris, bile acids, and other factors, may prevent amplification by physicochemical and enzymic effects [23], a control was required to be sure that no false negatives by defective extraction and/or PCR inhibition occurred. The control used involved a polymerase chain reaction (Cnt-PCR) performed with eluates of each sample analyzed to detect DNA from bacteria that are always present in the intestine of dogs. The primer set used, DG74 5 0 -AGGAGGTGATCCAACCGCA-3 0 and RW01 5 0 -AACTGGAGGAAGGTGGGGAT-3 0 , amplifies
A. Naidich et al. / Molecular and Cellular Probes 20 (2006) 5–10
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a 16S rRNA bacterial gene, conserved among divergent groups of eubacteria [24]. The thermal profile involved 15 min at 95 8C, followed by 30 cycles, each of 30 s at 94 8C, 30 s at 55 8C, 30 s at 72 8C, and a final elongation step of 10 min at 72 8C. Ten microliliters of eluate from stool samples were used as templates for the three PCR reactions. The concentrations of reagents for the three PCR reactions were: 0.5 mM each primer, 1U HotStart Taq (QIAGEN, Germany); 0.4 mM dNTPs; 1X PCR buffer, 1.5 mM MgCl2 and BSA (0.1 mg/ml). The lengths of PCR products were determined by comparison with a 100 bp Molecular Ruler DNA Size Standard (Bio-Rad, USA) following electrophoresis in 1.5% agarose gels and ethidium bromhyde staining. The software UNSCAN-IT gel (Silk Scientific Inc, Utah, USA) was used for an accurate comparison of PCR fragment lengths. 3. Results
Fig. 2. PCR with DNA from E. granulosus G1, G2, G4, G5, G6 and G7 genotypes: (a) Mit-PCR with 10 ng of DNA as template. (b) Rep-PCR with 1 ng of DNA as template. Lanes 1–6: DNA from G1, G2, G4, G5, G6 and G7 genotypes respectively. Lane 7: negative control.
3.1. Sensitivity of Mit-PCR and Rep-PCR The sensitivity of Mit-PCR was evaluated using different amounts of purified DNA. As little as 1 fg per reaction could be detected using Mit-PCR (data not shown), a value also reported by Abassi et al. [19] for Rep-PCR. However, the presence of host and bacterial DNA and other contaminating substances including PCR inhibitors in feces, may affect the sensitivity. Sensitivity was thus determined for both Mit-PCR and RepPCR using fecal samples spiked with 1, 10, 100 and 1000 E. granulosus egg (Fig. 1). The detection limits of Mit-PCR and Rep-PCR were 100 eggs and 1 egg, respectively. In an attempt to increase the sensitivity of Mit-PCR, an additional round of PCR with the same primers was performed, but no improvement in sensitivity was achieved (data not shown). 3.2. Detection of different E. granulosus genotypes In order to determine if different E. granulosus genotypes could be detected, equal amounts of genomic DNA from
the G1, G2, G4, G5, G6 and G7 genotypes were used as templates in both PCRs. The Mit-PCR amplification showed a single band of 285 bp for all genotypes analyzed while Rep-PCR produced different patterns for some of the genotypes (Fig. 2). The primers used for Rep-PCR for the G1 genotype produced several amplification products sized 940, 671, 600, 402, 300 and 133 bp [19]. As can be seen in Fig. 2b, G2 genotype amplification produced a similar banding pattern to the G1 genotype, sharing the three slower running bands with similar intensities. In our hands, the G1 and G2 genotype DNAs produced a smeared pattern unless minimal DNA quantities (100 pg or less) were used. The G4 genotype showed a distinct pattern of three bands at 671, 402 and 133 bp, while the G5, G6 and G7 genotypes showed a similar banding pattern, with two intense bands of 671 and less than 133 bp in length, and a weaker band of 402 bp in length. It is important to note that with the G5, G6 and G7 genotypes the intensity of the 671 bp fragment was very pronounced compared with the others fragments. The pattern of the G5/G6/G7 genotypes thus differed from the G1/G2 pattern and the G4 pattern, especially with respect to the relative intensities of the major bands. 3.3. E. granulosus detection at different times post-infection
Fig. 1. Sensitivity of (a) Mit-PCR and (b) Rep-PCR. Lanes 1–4: negative fecal samples spiked with 1, 10, 100 and 1000 eggs, respectively.
The samples obtained from experimentally infected dogs were analyzed by Mit-PCR and Rep-PCR. Results show that both PCR methods could detect E. granulosus in pre-patent and patent periods (Table 1). The parasite burdens of the experimentally infected dogs ranged from 30 to 32,000 adult worms per dog. The Cnt-PCR positive results that were observed for all samples confirmed that the DNA extraction procedure was successful and no PCR inhibitors were present after DNA purification. Fig. 3 shows the results for one of the dogs (dog number 3).
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A. Naidich et al. / Molecular and Cellular Probes 20 (2006) 5–10
Table 1 Mit-PCR, Rep-PCR and Cnt-PCR of faecal samples from dogs experimentally infected with E. granulosus collected at different times post-infection Days postinfection 25 33 35 39 43 46 50 55
Dog No.1
Dog No.2
Dog No.3
Dog No.4
Dog No.5
Mit
Rep
Cnt
Mit
Rep
Cnt
Mit
Rep
Cnt
Mit
Rep
Cnt
Mit
Rep
Cnt
K C C C C C C C
C C C C C C C C
C C C C C C C C
C C C C C C Ca NC
C C C C C C Ca NC
C C C C C C Ca NC
C C C C C C Ca NC
C C C C C C Ca NC
C C C C C C Ca NC
C C C C C C Ca Ca
C C C C C C Ca Ca
C C C C C C Ca Ca
K C C C C C C NC
C C C C C C C NC
C C C C C C C NC
NC, not collected. a Samples where E. granulosus eggs could be microscopically detected.
3.4. Natural infections Samples from dogs from endemic areas were tested by MitPCR and Rep-PCR and compared with arecoline hydrobromide purgation. The results are summarized in Table 2. For six samples (66.7%) there was coincidence with the three methods. Both copro-PCR methods were able to detect DNA from E. granulosus in two cases where arecoline hydrobromide purgation was negative. The remaining sample was positive for arecoline hydrobromide purgation and Rep-PCR, but negative for Mit-PCR. 4. Discussion It is very important to detect E. granulosus infection in dogs, since current CHD control programs are based mainly on definitive host treatment as a strategy for interrupting the parasite life cycle [25]. The utility of PCR to diagnose naturally infected dogs during the patent period has already been described [8], but to date, no study has used a PCR detection method for diagnosis during prepatency. Coproantigen methods have shown some promise for detecting pre-patent infections, but with low specificity and sensitivity depending on the parasite burden [26].
The present study shows the ability of two PCR methods to detect E. granulosus infection from 25 or 33 days p.i. As the pre-patent period of the sheep strain (G1 genotype) is 45 days [27,28], PCR detection was possible before patency. In fact, we could observe parasite eggs only on day 50 p.i. in three out of five infected dogs. Probably, the release of fragments of worm tissue can account for the positive PCR results. It is very important to note that the sensitivity of these PCR-based methods of detection does not depend on parasite burden, at least within the range assayed in the present experiment (30–32,000 adult worms) in contrast with coproantigen methods where sensitivity depends on parasite burden [7]. There is increasing evidence of the infectivity to humans by strains of E. granulosus other than the sheep–dog strain [15–17]. Thus, it is also important to determine the usefulness of the PCR methods to detect different strains. The primers used in the Mit-PCR were designed to amplify DNAs from E. granulosus G1–G7 genotypes. Here, we confirmed that these primers were able to amplify DNA from these genotypes, producing the same pattern for all of them. We also showed the capacity of the Rep primers to amplify E. granulosus DNA irrespective of the strain. Although we have analyzed a small number of samples, Rep-PCR seemed to discriminate among genotypes G1-G2, G4 and the G5-G6-G7 group. This is a valuable feature, because a single test could identify an
Fig. 3. Detection in pre-patent period: (a) Mit-PCR (b) Rep-PCR corresponding to dog No.3 at different days post-infection (PI), and a representative example of Cnt-PCR. Lanes 1–7: 25, 33, 35, 39, 43, 46 and 50 days PI, respectively; lane 8: Cnt-PCR of a representative sample.
A. Naidich et al. / Molecular and Cellular Probes 20 (2006) 5–10 Table 2 Samples from dogs natuarlly infected with E. granulosus analyzed by Mit-PCR and Rep-PCR, compared with results obtained by arecoline hydrobromide purgation Sample
CP10 CP11 IJ1 IJ2 IJ3 IJ4 IJ6 IJ7 IJ8
Arecoline hydrobromide purge
Mit-PCR
C C K K K K C C K
K C C C K K C C K
Rep-PCR
C C C C K K C C K
Cnt-PCR
C C C C C C C C C
E. granulosus infection and provide preliminary information on the infecting genotype using a small quantity of DNA. Previously described genotyping methods are more laborious and require higher quantities of DNA [10,29–31]. The results obtained with Rep-PCR are in agreement with previous studies with mitochondrial or nuclear markers used for E. granulosus strain identification [15,32,33] which also grouped the G1/G2 and G6/G7 genotypes as two separate clusters, with the G5 genotype closer to the G6/G7 group and the G4 genotype quite distinct from the other genotypes. Although the Rep-PCR proved to be far more sensitive than the Mit-PCR, a disadvantage of this method is that the banding pattern appeared to differ with the quantity of DNA used for PCR amplification, unlike the Mit-PCR which always showed a single 285 bp band. In conclusion, the results obtained in the present study suggest that the Mit-PCR and Rep-PCR methods are reliable for detecting infections in dogs, even during the pre-patency period with high sensitivity and specificity for at least six of the recognized E. granulosus genotypes. In addition, both PCRbased assays can be used to detect natural E. granulosus infections in dogs without causing undue stress to animals and avoiding environment contamination of highly infective eggs following arecoline purging. Deplazes et al. [34] suggested that copro-PCR methods could be used for confirmation of copro-antigen-positive faecal samples from canines infected with E. multilocularis. In our opinion, the approach is equally suitable for detection of E. granulosus in dogs. In areas where control programs and surveillance for canine echinococcosis are undertaken, the copro-PCR could be used to verify that dogs are no longer infected with E. granulosus due to its high sensitivity and specificity. Acknowledgements We would like to thank Dr Edmundo Larrieu and Dr Karen L. Haag for providing samples from naturally infected dogs and G4 genotype E. granulosus DNA, respectively. Research supported by Instituto Nacional de Enfermedades
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Infecciosas, ANLIS Dr Carlos G. Malbra´n, CABBIO and CONICET. References [1] Eckert J, Deplazes P, Craig PS, Gemmel MA, Gottstein B, Heath D, et al. In: World Health Organization, editor. WHO/OIE Manual on echinococcosis in humans and animals, a public health problem of global concern, 2001, p. 72–7 [Geneva]. [2] Ligthowlers MW, Gottstein B. Echinococcosis/Hydatidosis: antigens, immunological and molecular diagnostics. In: Thompson RCA, Lymbery J, editors. Echinococcosis and hydatid disease. Wallingford, UK: CAB International; 1995. p. 389–91. [3] Allan JC, Craig PS, Garcia Noval J, Mencos F, Liu D, Wang Y, et al. Coproantigen detection for immunodiagnosis of echinococcosis and taeniasis in dogs and humans. Parasitology 1992;104:347–55. [4] El-Shehabi FS, Kamhawi SA, Schantz PM, Craig PS, Abdel-Hafez SK. Diagnosis of canine echinococcosis: comparison of coproantigen detection with necropsy in stray dogs and red foxes from Norhern Jordan. Parasite 2000;7:83–90. [5] Christofi G, Deplazes P, Christofi N, Tan˜er I, Economides P, Eckert J. Screening of dogs for Echinococcus granulosus coproantigen in a low endemic situation in Cyprus. Vet Parasitol 2002;104:299–306. [6] Craig PS, Passer RB, Parada L, Cabrera P, Parietti S, Borgues C, et al. Diagnosis of canine echinococcocosis: comparison of coproantigen and serum antibody tests with arecoline purgation in Uruguay. Vet Parasitol 1995;56:293–301. [7] Zhang W, Li J, McManus DP. Concepts in immunology and diagnosis of hydatid disease. Clin Microbiol Rev 2003;16:18–36. [8] Sˆtefanic´ S, Block SS, Deplazes P, Dinkel A, Torgerson PR, Mathis A. Polimerase chain reaction for detection of patent infections of Echinococcus granulosus (“sheep strain”) in naturally infected dogs. Parasitol Res 2004;92:347–51. [9] Smyth JD, Davies Z. Occurrence of physiological strains of Echinococcus granulosus demonstrated by in vitro culture of protoscoleces from sheep and horse hydatid cysts. Int J Parasitol 1974;4:443–5. [10] Bowles J, Blair D, McManus DP. Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing. Mol Biochem Parasitol 1992;54:165–74. [11] Scott JC, McManus DP. The random amplification of polymorphic DNA can discriminate species and strains of Echinococcus. Trop Med Parasitol 1994;45:1–4. [12] Scott JC, Stefaniak J, Pawlowski ZS, McManus DP. Molecular genetic analysis of human cystic hydatid cases from Poland: identification of a new genotypic group (G9) of Echinococcus granulosus. Parasitology 1997;114:37–43. [13] Lavikainen A, Lehtinen MJ, Meri T, Hirvela-Koski V, Meri S. Molecular genetic characterization of the Fennoscandian cervid strain, a new genotypic group (G10) of Echinococcus granulosus. Parasitology 2003; 127:207–15. [14] Eckert J, Deplazes P. Biological, epidemiological, and clinical aspects of Echinococcosis, a zoonosis of increasing concern. Clin Microbiol Rev 2004;17:107–35. [15] Kamenetzky L, Gutierrez AM, Canova SG, Haag KL, Guarnera EA, Parra A, et al. Several strains of Echinococcus granulosus infect livestock and humans in Argentina. Infect Genet Evol 2002;2:129–36. [16] Harandi MF, Hobbs RP, Adams PJ, Mobedi I, Morgan-Ryan UM, Thompson RC. Molecular and morphological characterization of Echinococcus granulosus of human and animal origin in Iran. Parasitology 2002;125:367–73. [17] Dinkel A, Njoroge EM, Zimmermann A, Wa¨lz M, Zeyhle E, Elmahdi IE, et al. A PCR system for detection of species and genotypes of the Echinococcus granulosus-complex, with reference to the epidemiological situation in eastern Africa. Int J Parasitol 2004;34:645–53. [18] Cabrera M, Canova S, Rosenzvit M, Guarnera E. Identification of Echinococcus granulosus eggs. Diagn Microbiol Infect Dis 2002;44: 29–34.
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[19] Abbasi I, Branzburg A, Campos-Ponce M, Abdel Hafez SK, Raoul F, Craig PS, et al. Copro-diagnosis Of Echinococcus granulosus infection in dogs by amplification of a newly identified repeated DNA sequence. Am J Trop Med Hyg 2003;69:324–30. [20] Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989. [21] Kamenetzky L, Canova SG, Guarnera EA, Rosenzvit MC. Echinococcus granulosus: DNA extraction from germinal layers allows strain determination in fertile and nonfertile hydatid cysts. Exp Parasitol 2000;95:122–7. [22] Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. Washington, DC: National Academy Press; 1996. [23] Wilson I. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 1997;63:3741–51. [24] Greisen K, Loeffelholz M, Purohit A, Leong D. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clin Microbiol 1994;32:335–51. [25] Gemmell MA, Roberts MG, Beard TC, Campano Diaz S, Lawson JR, Nonnemaker JM. In: World Health Organization, editor. WHO/OIE Manual on echinococcosis in humans and animals, a public health problem of global concern; 2001, 2001. p. 195–237 [Geneva]. [26] Jenkins DJ, Fraser A, Bradshaw H, Craig PS. Detection of Echinococcus granulosus coproantigens in Australian canids with natural and experimental infection. J Parasitol 2000;86:140–5.
[27] Kumaratilake LM, Thompson RCA, Dunsmore JD. Comparative strobilar development of Echinococcus granulosus of sheep origin from different geographical areas of Australia in vivo and in vitro. Int J Parasitol 1983; 13:151–6. [28] Thompson RCA, Kumaratilake LM, Eckert J. Observation on Echinococcus granulosus of cattle origin in Switzerland. Int J Parasitol 1984;14: 283–91. [29] Bowles J, McManus DP. Rapid discrimination of Echinococcus species and strains using a polymerase chain reaction-based RFLP method. Mol Biochem Parasitol 1993;57:231–40. [30] Bowles J, McManus DP. NADH deshidrogenase 1 gene sequences compared for species and strains of the genus Echinococcus. Int J Parasitol 1993;23:969–72. [31] Gasser RB, Zhu X, McManus DP. Display of sequence variation in PCR-amplified mitochondrial DNA regions of Echinococcus by single-strand conformation polymorphism. Acta Trop 1998;71: 107–15. [32] Rosenzvit MC, Canova SG, Kamenetzky L, Guarnera EA. Echinococcus granulosus: intraespecific genetic variation assessed by a DNA repetitive element. Parasitology 2001;123:381–8. [33] Bowles J, Blair D, McManus DP. A molecular phylogeny of the genus Echinococcus. Parasitology 1995;110:317–28. [34] Deplazes P, Dinkel A, Mathis A. Molecular tools for studies on the transmission biology of Echinococcus multilocularis. Parasitology 2003; 127(Suppl.):S53–S61.
Patent and pre-patent detection of Echinococcus granulosus genotypes in the definitive host Ariel Naidich a,*, Donald P. McManus b, Sergio G. Canova a, Ariana M. Gutierrez a, Wenbao Zhang b, Eduardo A. Guarnera a, Mara C. Rosenzvit a a
Departamentode Parasitologı´a, Instituto Nacional de Enfermedades Infecciosas, ANLIS Dr Carlos G. Malbra´n, Av. Velez Sarsfield 563, 1281 Ciudad de Buenos Aires, Argentina b Division of Infectious Diseases and Immunology, Molecular Parasitology Laboratory, Queensland Institute of Medical Research, Royal Brisbane Hospital Post Office, Herston, Qld. 4006, Australia Received 16 June 2005; accepted for publication 22 August 2005 Available online 14 October 2005
Abstract The detection of Echinococcus granulosus in dogs is important for epidemiological surveillance and evaluation of cystic hydatic disease control programs. We report the efficacy of two PCR-based methods to detect patent and pre-patent infection in dogs experimentally infected with E. granulosus. The detection is based on amplification of a fragment of a mitochondrial gene (Mit-PCR) and a DNA repetitive element (Rep-PCR) of E. granulosus. We tested the ability of both methods to detect several genotypes of the parasite. Both PCR methods could detect E. granulosus in pre-patent and patent periods, even when microscopical observation of eggs resulted negative in fecal samples. The Mit-PCR produced the same amplification pattern for all the parasite genotypes tested while the amplification patterns with the Rep-PCR differed among groups of strains. Fecal samples collected from dogs of an endemic area were diagnosed with more sensitivity than arecoline hydrobromide purgation. These molecular methods could be applied in the confirmation of coproantigen-positive fecal samples and to verify the success of control programs. q 2005 Elsevier Ltd. All rights reserved. Keywords: Echinococcus granulosus; PCR; Fecal; Genotypes; Experimental infection; Patency
1. Introduction Echinococcus granulosus is the causative agent of cystic hydatid disease (CHD), a zoonosis that affects the public health and economy in many areas worldwide. Domestic livestock, wild life animals and humans infected with the larval stage of the parasite develop fluid-filled cysts, most often in the liver and lungs. Dogs are the usual definitive hosts and the parasite eggs shed in their feces become the source of infection for sheep, pigs, cows and other intermediate hosts including humans. Detecting E. granulosus infection in dogs is important for epidemiological surveillance and evaluation of control programs. Traditionally, the methods used for detecting dog infection were dog necropsy and further intestinal examination, and arecoline hydrobromide purgation [1]. The latter method is
* Corresponding author. Tel.: C54 11 4301 7437; fax: C54 11 4303 2382. E-mail address: [email protected] (A. Naidich).
0890-8508/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2005.08.001
inconvenient due to environmental contamination, low sensitivity and reproducibility; and detrimental effects on animals [2]. Assays for detection of parasite antigens in fecal samples from definitive hosts have been developed [3], but this procedure is not species-specific [4,5] and its sensitivity appears to be affected by parasite burdens [4,6,7]. Molecular methods, especially those based on PCR should provide the required specificity and sensitivity for E. granulosus detection in dogs. Although a PCR-based method for detecting a patent infection in the definitive host has been recently developed [8], it was not reported whether the assay could detect a pre-patent infection, i.e before eggs are released in the feces. Ten distinct genotypes (G1–G10) of E. granulosus, differing in biological characters such as intermediate host specificity and developmental rates have been described [9–14]. Although the sheep–dog strain (G1 genotype), is the predominant strain infective to humans, there is increasing evidence that other strains can also cause significant human hydatid disease. In Argentina, for example, Tasmanian sheep (G2 genotype), cattle (G5 genotype) and camel (G6 genotype) strains produced 42% of cases of human CHD [15]. Also, the camel strain was
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A. Naidich et al. / Molecular and Cellular Probes 20 (2006) 5–10
reported to cause CHD in humans from Iran [16] and East Africa [17]. This genetic variation makes it necessary to assess the performance of a PCR-based assay to detect a range of E. granulosus strains for reliable diagnosis in definitive hosts. Cabrera et al. [18] showed that a primer pair designed for amplification of a fragment of the mitochondrial cytochrome oxidase subunit 1 (coxI) gene was able to specifically identify E. granulosus DNA extracted from oncospheres. In addition, a primer set and a copro-PCR method were described for the detection of E. granulosus infection in dogs with high sensitivity and specificity [19]. The amplification target is a repetitive DNA element present in the genome of the sheep strain of E. granulosus but the efficacy of this latter method for detection of other E. granulosus strains was not documented. In the present study, the performances of both copro-PCR methods have been evaluated for the detection of distinct strains of E. granulosus and for diagnosis of pre-patent infections. 2. Materials and methods 2.1. DNA extraction and purification Total E. granulosus genomic DNA was prepared from fresh, frozen in liquid nitrogen, or 70% ethanol preserved protoscoleces or germinal layer from individual E. granulosus cysts by conventional techniques [20]. In brief, protoscoleces from an individual cyst or adult worm were washed three times in phosphate-buffered saline, pH 7.2, crushed in liquid nitrogen, and digested in 200 mg proteinase K/ml and 0.5% SDS during 3 h at 56 8C. After proteinase K treatment, phenol/chloroform extractions were made and DNA was precipitated with ethanol [20]. The germinal layers were processed as described by Kamenetzky et al. [21]. E. granulosus genotypes were determined by sequencing a fragment of the mitochondrial coxI gene as described [10]. All faecal samples from dogs were examined microscopically in triplicate to determine the presence of taenid eggs. DNA extraction and purification, from 200 mg of each fecal sample, was performed using the QIAmp DNA Stool Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. Eluates of purified DNA samples were strored at K20 8C for 24–48 h after extraction until their use in PCR reactions. Ten microliters of eluate were employed as template in PCRs. 2.2. Echinococcus granulosus eggs Isolated E. granulosus eggs were obtained from a gravid proglottid of an adult worm (previously genotyped as the G1 genotype) that was mechanically disrupted. The released eggs were washed three times with phosphate saline buffer and counted under the microscope. 2.3. Experimental infection E. granulosus protoscoleces were aspirated from sheep liver hydatid cysts collected from a slaughterhouse in Urumqi,
Xinjiang, PR China. Five dogs, previously treated with 10 mg/body weight of albendazole to remove all tapeworms, were each fed 0.8 ml packed and sedimented protoscoleces (approximately 100,000) together with a small amount of cyst fluid. Animals were maintained under humane conditions, according to the Guide for the Care and Use of Laboratory Animals [22]. Stool samples were collected at 25, 33, 35, 39, 43, 46 and 50 days post-infection (p.i.). In addition, two samples were collected at 55 days p.i. from two dogs. All samples were maintained in 70% ethanol at K70 8C for at least two weeks to inactivate oncospheres. After the collection of the last sample, all dogs were humanely killed and autopsied to determine parasite burdens. 2.4. Samples from endemic areas Stool samples were collected from nine domestic dogs from Carmen de Patagones, Buenos Aires province and Ingeniero Jacobacci, Rı´o Negro province, Argentina, after arecoline hydrobromide purging. The samples were kept in 70% ethanol at room temperature until their shipment to the laboratory, where they were stored at K70 8C for at least two weeks to inactivate oncospheres. 2.5. Polymerase chain reactions (PCR) Three PCR reactions were performed: Mit-PCR for mitochondrial DNA amplificaction, Rep-PCR for repetitive DNA amplification and one control Cnt-PCR. For Mit-PCR, the primer set EgO/DNA-IM1 (forward primer 5 0 -TCATATTTGTTTGAGKATYAGTKC-3 0 , and reverse primer 5 0 GTAAATAAMACTATAAAAGAAAYMAC-3 0 ), designed to amplify a fragment of the coxI gene specific for E. granulosus [18], was used. The thermal profile used with this set of primers involved 15 min at 95 8C, followed by 40 cycles of 1 min at 94 8C, 1 min at 50 8C and 1 min at 72 8C followed by a final elongation step of 10 min at 72 8C. This PCR is specific for E. granulosus, as was demonstrated by Cabrera et al. [18]. For Rep-PCR the primer set used was Eg1121a/22a (forward primer Eg1121a 5 0 -GAATGCAAGCAGCAGATG3 0 and reverse primer Eg1122a 5 0 -GAGATGAGTGAGAAGGAGTG-3 0 ) that amplifies a DNA repetitive element from E. granulosus genome [19]. The thermal profile involved 15 min at 94 8C, followed by 35 cycles, each of 1 min at 95 8C, 1 min at 55 8C, 1 min at 72 8C, and a final elongation step for 10 min at 72 8C. This PCR method has proved to be specific for E. granulosus [19]. As bacterial proteases and nucleases in feces, as well as cell debris, bile acids, and other factors, may prevent amplification by physicochemical and enzymic effects [23], a control was required to be sure that no false negatives by defective extraction and/or PCR inhibition occurred. The control used involved a polymerase chain reaction (Cnt-PCR) performed with eluates of each sample analyzed to detect DNA from bacteria that are always present in the intestine of dogs. The primer set used, DG74 5 0 -AGGAGGTGATCCAACCGCA-3 0 and RW01 5 0 -AACTGGAGGAAGGTGGGGAT-3 0 , amplifies
A. Naidich et al. / Molecular and Cellular Probes 20 (2006) 5–10
7
a 16S rRNA bacterial gene, conserved among divergent groups of eubacteria [24]. The thermal profile involved 15 min at 95 8C, followed by 30 cycles, each of 30 s at 94 8C, 30 s at 55 8C, 30 s at 72 8C, and a final elongation step of 10 min at 72 8C. Ten microliliters of eluate from stool samples were used as templates for the three PCR reactions. The concentrations of reagents for the three PCR reactions were: 0.5 mM each primer, 1U HotStart Taq (QIAGEN, Germany); 0.4 mM dNTPs; 1X PCR buffer, 1.5 mM MgCl2 and BSA (0.1 mg/ml). The lengths of PCR products were determined by comparison with a 100 bp Molecular Ruler DNA Size Standard (Bio-Rad, USA) following electrophoresis in 1.5% agarose gels and ethidium bromhyde staining. The software UNSCAN-IT gel (Silk Scientific Inc, Utah, USA) was used for an accurate comparison of PCR fragment lengths. 3. Results
Fig. 2. PCR with DNA from E. granulosus G1, G2, G4, G5, G6 and G7 genotypes: (a) Mit-PCR with 10 ng of DNA as template. (b) Rep-PCR with 1 ng of DNA as template. Lanes 1–6: DNA from G1, G2, G4, G5, G6 and G7 genotypes respectively. Lane 7: negative control.
3.1. Sensitivity of Mit-PCR and Rep-PCR The sensitivity of Mit-PCR was evaluated using different amounts of purified DNA. As little as 1 fg per reaction could be detected using Mit-PCR (data not shown), a value also reported by Abassi et al. [19] for Rep-PCR. However, the presence of host and bacterial DNA and other contaminating substances including PCR inhibitors in feces, may affect the sensitivity. Sensitivity was thus determined for both Mit-PCR and RepPCR using fecal samples spiked with 1, 10, 100 and 1000 E. granulosus egg (Fig. 1). The detection limits of Mit-PCR and Rep-PCR were 100 eggs and 1 egg, respectively. In an attempt to increase the sensitivity of Mit-PCR, an additional round of PCR with the same primers was performed, but no improvement in sensitivity was achieved (data not shown). 3.2. Detection of different E. granulosus genotypes In order to determine if different E. granulosus genotypes could be detected, equal amounts of genomic DNA from
the G1, G2, G4, G5, G6 and G7 genotypes were used as templates in both PCRs. The Mit-PCR amplification showed a single band of 285 bp for all genotypes analyzed while Rep-PCR produced different patterns for some of the genotypes (Fig. 2). The primers used for Rep-PCR for the G1 genotype produced several amplification products sized 940, 671, 600, 402, 300 and 133 bp [19]. As can be seen in Fig. 2b, G2 genotype amplification produced a similar banding pattern to the G1 genotype, sharing the three slower running bands with similar intensities. In our hands, the G1 and G2 genotype DNAs produced a smeared pattern unless minimal DNA quantities (100 pg or less) were used. The G4 genotype showed a distinct pattern of three bands at 671, 402 and 133 bp, while the G5, G6 and G7 genotypes showed a similar banding pattern, with two intense bands of 671 and less than 133 bp in length, and a weaker band of 402 bp in length. It is important to note that with the G5, G6 and G7 genotypes the intensity of the 671 bp fragment was very pronounced compared with the others fragments. The pattern of the G5/G6/G7 genotypes thus differed from the G1/G2 pattern and the G4 pattern, especially with respect to the relative intensities of the major bands. 3.3. E. granulosus detection at different times post-infection
Fig. 1. Sensitivity of (a) Mit-PCR and (b) Rep-PCR. Lanes 1–4: negative fecal samples spiked with 1, 10, 100 and 1000 eggs, respectively.
The samples obtained from experimentally infected dogs were analyzed by Mit-PCR and Rep-PCR. Results show that both PCR methods could detect E. granulosus in pre-patent and patent periods (Table 1). The parasite burdens of the experimentally infected dogs ranged from 30 to 32,000 adult worms per dog. The Cnt-PCR positive results that were observed for all samples confirmed that the DNA extraction procedure was successful and no PCR inhibitors were present after DNA purification. Fig. 3 shows the results for one of the dogs (dog number 3).
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A. Naidich et al. / Molecular and Cellular Probes 20 (2006) 5–10
Table 1 Mit-PCR, Rep-PCR and Cnt-PCR of faecal samples from dogs experimentally infected with E. granulosus collected at different times post-infection Days postinfection 25 33 35 39 43 46 50 55
Dog No.1
Dog No.2
Dog No.3
Dog No.4
Dog No.5
Mit
Rep
Cnt
Mit
Rep
Cnt
Mit
Rep
Cnt
Mit
Rep
Cnt
Mit
Rep
Cnt
K C C C C C C C
C C C C C C C C
C C C C C C C C
C C C C C C Ca NC
C C C C C C Ca NC
C C C C C C Ca NC
C C C C C C Ca NC
C C C C C C Ca NC
C C C C C C Ca NC
C C C C C C Ca Ca
C C C C C C Ca Ca
C C C C C C Ca Ca
K C C C C C C NC
C C C C C C C NC
C C C C C C C NC
NC, not collected. a Samples where E. granulosus eggs could be microscopically detected.
3.4. Natural infections Samples from dogs from endemic areas were tested by MitPCR and Rep-PCR and compared with arecoline hydrobromide purgation. The results are summarized in Table 2. For six samples (66.7%) there was coincidence with the three methods. Both copro-PCR methods were able to detect DNA from E. granulosus in two cases where arecoline hydrobromide purgation was negative. The remaining sample was positive for arecoline hydrobromide purgation and Rep-PCR, but negative for Mit-PCR. 4. Discussion It is very important to detect E. granulosus infection in dogs, since current CHD control programs are based mainly on definitive host treatment as a strategy for interrupting the parasite life cycle [25]. The utility of PCR to diagnose naturally infected dogs during the patent period has already been described [8], but to date, no study has used a PCR detection method for diagnosis during prepatency. Coproantigen methods have shown some promise for detecting pre-patent infections, but with low specificity and sensitivity depending on the parasite burden [26].
The present study shows the ability of two PCR methods to detect E. granulosus infection from 25 or 33 days p.i. As the pre-patent period of the sheep strain (G1 genotype) is 45 days [27,28], PCR detection was possible before patency. In fact, we could observe parasite eggs only on day 50 p.i. in three out of five infected dogs. Probably, the release of fragments of worm tissue can account for the positive PCR results. It is very important to note that the sensitivity of these PCR-based methods of detection does not depend on parasite burden, at least within the range assayed in the present experiment (30–32,000 adult worms) in contrast with coproantigen methods where sensitivity depends on parasite burden [7]. There is increasing evidence of the infectivity to humans by strains of E. granulosus other than the sheep–dog strain [15–17]. Thus, it is also important to determine the usefulness of the PCR methods to detect different strains. The primers used in the Mit-PCR were designed to amplify DNAs from E. granulosus G1–G7 genotypes. Here, we confirmed that these primers were able to amplify DNA from these genotypes, producing the same pattern for all of them. We also showed the capacity of the Rep primers to amplify E. granulosus DNA irrespective of the strain. Although we have analyzed a small number of samples, Rep-PCR seemed to discriminate among genotypes G1-G2, G4 and the G5-G6-G7 group. This is a valuable feature, because a single test could identify an
Fig. 3. Detection in pre-patent period: (a) Mit-PCR (b) Rep-PCR corresponding to dog No.3 at different days post-infection (PI), and a representative example of Cnt-PCR. Lanes 1–7: 25, 33, 35, 39, 43, 46 and 50 days PI, respectively; lane 8: Cnt-PCR of a representative sample.
A. Naidich et al. / Molecular and Cellular Probes 20 (2006) 5–10 Table 2 Samples from dogs natuarlly infected with E. granulosus analyzed by Mit-PCR and Rep-PCR, compared with results obtained by arecoline hydrobromide purgation Sample
CP10 CP11 IJ1 IJ2 IJ3 IJ4 IJ6 IJ7 IJ8
Arecoline hydrobromide purge
Mit-PCR
C C K K K K C C K
K C C C K K C C K
Rep-PCR
C C C C K K C C K
Cnt-PCR
C C C C C C C C C
E. granulosus infection and provide preliminary information on the infecting genotype using a small quantity of DNA. Previously described genotyping methods are more laborious and require higher quantities of DNA [10,29–31]. The results obtained with Rep-PCR are in agreement with previous studies with mitochondrial or nuclear markers used for E. granulosus strain identification [15,32,33] which also grouped the G1/G2 and G6/G7 genotypes as two separate clusters, with the G5 genotype closer to the G6/G7 group and the G4 genotype quite distinct from the other genotypes. Although the Rep-PCR proved to be far more sensitive than the Mit-PCR, a disadvantage of this method is that the banding pattern appeared to differ with the quantity of DNA used for PCR amplification, unlike the Mit-PCR which always showed a single 285 bp band. In conclusion, the results obtained in the present study suggest that the Mit-PCR and Rep-PCR methods are reliable for detecting infections in dogs, even during the pre-patency period with high sensitivity and specificity for at least six of the recognized E. granulosus genotypes. In addition, both PCRbased assays can be used to detect natural E. granulosus infections in dogs without causing undue stress to animals and avoiding environment contamination of highly infective eggs following arecoline purging. Deplazes et al. [34] suggested that copro-PCR methods could be used for confirmation of copro-antigen-positive faecal samples from canines infected with E. multilocularis. In our opinion, the approach is equally suitable for detection of E. granulosus in dogs. In areas where control programs and surveillance for canine echinococcosis are undertaken, the copro-PCR could be used to verify that dogs are no longer infected with E. granulosus due to its high sensitivity and specificity. Acknowledgements We would like to thank Dr Edmundo Larrieu and Dr Karen L. Haag for providing samples from naturally infected dogs and G4 genotype E. granulosus DNA, respectively. Research supported by Instituto Nacional de Enfermedades
9
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