Identification of Echinococcus granulosus eggs

Diagnostic Microbiology and Infectious Disease 44 (2002) 29 –34 www.elsevier.com/locate/diagmicrobio Parasitology Identification of Echinococcus granulosus eggs Marta Cabrera, Sergio Canova, Mara Rosenzvit, Eduardo Guarnera* Departamento de Parasitologı́a. Instituto Nacional de Enfermedades Infecciosas “Dr. Carlos G. Malbrán,” Av. Vélez Sársfield 563. (1281) Buenos Aires, Argentina Received 15 February 2002; accepted 15 May 2002 Abstract The eggs from Echinococcus granulosus contaminate the environment spreading out the disease among the herbivorous. The differential diagnosis of the embriophores recovered from the soil is very difficult by morphologic and immunologic methods. In this paper we evaluate the EgO/DNA-IM1 for identification of E. granulosus oncosphere DNA and differentiation of eggs from other Taeniid. The positive result of the PCR technique shows an amplification fragment of the expected size (285 bp) corresponding to the partial sequence of the mitochondrial gene of the citochrome oxidase CO1 from E. granulosus (391 bp). The fragment is not present in the DNA from Echinococcus multilocularis, Taenia hydatigena, Taenia saginata, Diphyll-obothrium latum, and Hymenolepis nana. It could be useful to rule out Taenia taeniformis, Taenia solium, Taenia pisiformis, and Taenia crassiceps, which sequences do not belong to the primer. We concluded that the PCR amplification employing the EgO/DNA-IM1 primer set showed high sensitivity and specificity for the identification of Echinococcus granulosus eggs. © 2002 Elsevier Science Inc. All rights reserved. 1. Introduction The proglottids with embrionated eggs mature in the intestine of parasitized dogs with adult specimens of Echinococcus granulosus. At this moment, the eggs are released to the environment, contaminating the pasture ground, vegetables, fruits and soil. It is very difficult to recover eggs from these places and to identify the species, due to the facts that the relationship between the contaminating eggs and the environment is very low (Schultz & Kroegel, 1992) and the Taeniid eggs are similar (Euzeby, 1966; Craig, 1986; Aluja et al., 1987). Craig et al.(1988) proposed some methods to recover E. granulosus eggs as direct identification in fecal samples, from perianal scotch-tape swabs or in concentrated canine stool samples. However, these procedures are not speciesspecific and lack sensitivity. Eggs have been also obtained from soil and water (Craig et al., 1988; Guarnera, 2001), employing concentrating methods and washing with Tween 80 and sodium dodecyl sulfate (SDS). The coproparasitological analysis is the best test for Taeniid eggs finding, especially with the concentration method of Teleman (1908) by the formaldehyde/ether technique. * Corresponding author. Tel.: ⫹54-11-4301-7437; fax: ⫹54-11-43017437. E-mail address: [email protected] (E. Guarnera). Craig et al. (1988) advanced obtaining an species specific indirect immunofluorescence test using an anti-Echinococcus oncosphere monoclonal antibody (Eg0H6 – 4E5), applied to identify Echinococcus oncospheres released from Taeniid eggs collected in environmental soil and water samples. The specificity of the immunodetection from naturally infected dogs was 100% when compared to Taenia hydatigena infections, and a sensitivity of 73% was obtained in the detection of infected dogs using perianal scotch-tape swabs. The authors considered this test suitable for application to the problem of identification of Echinococcus eggs in the environment. Nevertheless this assay was not utilized in any control program. Bretagne et al. (1993) developed a method in order to identify Echinococcus multilocularis DNA in fox feces for epidemiologic purposes. The target sequence for amplification was the E. multilocularis U1 sRNA gene. The isolates of E. multilocularis showed the same pattern of 337 bp, but this band was not observed for other DNAs including E. granulosus. Until now, there is not any available assay for identification of E. granulosus eggs. The need of arrange an accurate method for the diagnosis at the species level is important in endemic areas where samples are collected from places with prevalence of more than one specie of Taeniid. It is also important to identify the eggs recovered from the environment because they are a sanitary indicator of the exposure level. 0732-8893/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 2 - 8 8 9 3 ( 0 2 ) 0 0 4 1 4 - 5 30 M. Cabrera et al. / Diagnostic Microbiology and Infectious Disease 44 (2002) 29 –34 The aim of this paper is to evaluate the EgO/DNA-IM1 (Echinococcus granulosus oncosphere/DNA-Malbran Institute 1) primer set for the specific diagnosis of Echinococcus granulosus eggs collected from the environment, with the purpose of understanding the risk level of transmission in endemic areas of Echinococcosis and the degree of local contamination. 2. Materials and methods 2.1. Sample collection Adult specimens of Echinococcus granulosus were obtained by necropsy of a dog from the village of Balsa Las Perlas, Neuquén, Argentina. Two adult samples of Taenia hydatigena and one of Dipylidium caninum were obtained by purge with arecoline bromhydrate in a deparasitation park in Buenos Aires, Argentina. Gravid proglottids from Taenia saginata and Hymenolepis nana eggs from human patients were also employed. DNA from E. multilocularis was kindly provided by Dr. Donald P. McManus. Adult specimens, proglottids and fecal samples were inactivated by freezing at ⫺70°C during 7 days in ethanol 70%. All samples were employed in the next 30 days from collection. Mature proglottids from each specimen were separated for egg recovery by mechanical disruption. The obtained material was filtered through 40 ␮m mesh to separate eggs from the remaining tissue and intestinal mucose. The egg suspension was washed three times with phosphate-buffered saline (PBS) pH 7.6 by centrifugation at 12,500⫻ g during 5 min. The fecal samples were filtered through double gauze in centrifuge tubes of 15 mL and were concentrated by centrifugation at 12,000⫻ g during 15 min. The eggs at the pellet were washed three times with PBS pH 7.4 by centrifugation at 1,000⫻ g during 15 min. The eggs obtained from proglottids and the extracted by concentration/centrifugation from fecal samples were adjusted at a high final quantity of about 12,000 eggs of each parasite and a low number of 240 eggs of each one. The eggs were counted by light microscopy according to the volume. The DNA was extracted from oncospheres from each final number of eggs. 2.2. DNA purification The procedure for DNA extraction was adapted from that employed for extraction of DNA from mycobacterium (Van Soolingen et al., 1992) with the following modifications. The eggs obtained by mechanic disruption of gravid proglottids and those obtained by concentration/centrifugation of fecal samples were adjusted to two final quantities arbitrary chosen, as starting point for DNA purification. The higher number was of 12,000 units and the lower ones were of 400 total eggs for T. saginata and 240 for the other cestode. The eggs count was carried out by light microscopy in all samples, and the adjustment of concentration was done in 400 ␮L of TE (100 mM Tris-ClH, 1 mM EDTA). The eggs were digested by the addition of 50 ␮L of lysozyme (10 mg/ml) and overnight incubation at 37°C. In order to favor the disruption of the keratin embryosphere, extremely resistant to chemical attack, the samples were frozen in liquid nitrogen during 3 min and thawed at 65°C during 5 min, for three times. The enzymatic lysis was continued by the addition of 75 ␮L of the mixture Proteinase K/SDS 10% (5 ␮L of proteinase K 20 mg/ml and 70 ␮L of SDS 10%) and incubation at 65°C during 10 min. Then, the lysis buffer (100 ␮L of 5 M ClNa and 100 ␮L of CTAB (N-cetyl-N,N,N,-trimethyl ammonium bromide, Merck)/ClNa solution) prewarmed at 65°C and shaking for 10 min and 65°C. The DNA extraction was done with 1 volume of chloroform:isoamyl alcohol (24:1), approximately 750 ␮L, vortexing 10 min and centrifuging at room temperature for 5 min at 12,000⫻ g. The DNA was precipitated in 0.6 volume of isopropanol (approximately 450 ␮L) overnight at 4°C. The pellet was concentrated by centrifugation at 12,000⫻ g at room temperature for 5 min, washed with 1 mL of cold ethanol 70% without mixing, and centrifuged for 5 min. The supernatant was carefully removed and the pellet was airdried. The obtained DNA was diluted in 50 ␮L of Milli Q water and quantified by 1% agarose gel electrophoresis and UV fluorescence in the presence of ethidium bromide and quantitation markers. The sample was conserved at 4°C until used. 2.3. Polymerase chain reaction (PCR) Alignment of the following already published mitochondrial cytochrome c oxidase subunit 1 (CO1) sequences was performed: E. granulosus G1, G2, G4, G5, G6 and G7 genotype, E. vogeli, E. oligarthrus, E. multilocularis, Taenia hydatigena and Taenia crassiceps. Echinococcus specific nucleotides were identified from the above alignment. The sequence of the forward primer was 5⬘-TCATATTTGTTTGAGKATYAGTKC-3⬘, the 3⬘ cytosine being only present in all the Echinococcus granulosus strains, E. vogeli and E. oligartrhus. The sequence of the reverse primer was 5⬘-GTAAATAAMACTATAAAAGAAAYMAC-3⬘, the 3⬘ cytosine being only present in the four Echinococcus species tested. With these primers, it was expected to obtain an amplification product 285 bp long, only with genomic DNA from E. granulosus, E. oligartrhus and E. vogeli (Fig. 1). The reaction mixture contained 5 ␮L (approximately 10 ng) of DNA, 1 U of Taq DNA polymerase, 10 mM Tris-HCl pH 9.6, 50 mM KCl, 1.5 mM magnesium chloride, 0.1% Triton, 25 mM each deoxynucleotide triphosphate, 0.2 ␮M each primer. The reaction conditions were: denaturation for M. Cabrera et al. / Diagnostic Microbiology and Infectious Disease 44 (2002) 29 –34 Fig. 1. Schematic representation of an amplified region of the mitochondrial CO1 gene for PCR. The sequences of the two primers are given in the Material and Methods section. 3 min at 94°C, 39 cycles of a denauration step at 94°C for 1 min, an annealing step at 50°C for 1 min and an elongation step at 72°C for 1 min, final elongation at 72°C for 3 min. The amplification products were run in 2% agarose gel electrophoresis using 100 bp ladder as molecular size marker to determine the fragment length. 2.4. DNA sequencing The nucleotide sequence of the amplification product (named IM1) was determined by automatic DNA sequencing employing the ABI PRISM Big DyeTM Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Applied Biosystems). Gel electrophoresis was carried out on an ABI 377 Automated DNA Sequencer (Perkin Elmer). 3. Results 3.1. PCR amplification From the samples of 12,000 eggs, 1.5 and 2.0 ng/␮L of DNA were recovered that were initially employed for secure the reaction performance. When the reaction was carried out with the sample of 240 eggs, an optimal signal was obtained by PCR, equivalent to 24 oncospheres. This sample was used to make dilutions of DNA until a concentration equivalent to 100 eggs in 50 ␮L of solution. In the PCR reaction, 2 ␮L were employed, equivalent to the detection of 4 eggs. The PCR reaction with the primer set EgO/DNA-IM1 31 Fig. 2. Agarose gel electrophoresis (ethidium bromide stain of PCRamplified products. Lane 1, reaction mixture (opened tube); lanes 2 to 5, DNA extracted from 240 eggs of Taenia saginata, H. nana, T. hydatigena and D. caninum, respectively; Lane 6, DNA from E. multilocularis; lanes 7 and 8, DNA purified from 240 and 12,000 eggs of E. granulosus, respectively; lane 9, reaction mixture (closed tube); lane 10, 100 bp ladder. with DNA samples from E. granulosus showed an amplification fragment of the expected size (285 bp), using as target the corresponding sequence of the mitochondrial gene of the citochrome oxidase CO1 of Echinococcus granulosus (size: 391 bp). The DNA from E. multilocularis, T. hydatigena, T. saginata, D. caninum and H. nana never showed this amplification band (Fig. 2). Simultaneously, other reactions were carried out to rule out the presence of inhibitors. This was demonstrated by spiking DNA of E. granulosus to the DNA of each sample in the reaction mixture. The samples of high (12,000 eggs) and low (240 eggs) quantities of each Taeniid produce the same results. 3.2. Sequence analysis The nucleotide sequence of the amplification product IM1 was compared with E. granulosus (sheep and camel strains), E. vogeli and E. oligarthrus mitochondrial cytochrome c oxidase subunit 1 (CO1) sequences deposited in the database (Fig. 3). The overall nucleotide identity varied from 89% to 99%, showing that the amplification product belongs to E. granulosus G6 genotype. This result is in concordance with a previous report describing the presence of E. granulosus G6 genotype in hidatid cyst from humans living in the same area where the sample employed in this work was obtained (Rozenzvit at al., 1999). 4. Discussion If we assume that the soil is the place where transmission of Echinococcosis to the intermediate hosts occur, the demonstra- 32 M. Cabrera et al. / Diagnostic Microbiology and Infectious Disease 44 (2002) 29 –34 Fig. 3. Comparison of the nucleotide sequence of the amplicon IM1 with four available sequences of the mitochondrial CO1 gene. Alignment between IM1 and CO1 sequences corresponding to Accesion numbers AF408687 (E. granulosus G6 genotype), U50464 (E. granulosus sheep strain), M84671 (E. oligarthrus) and M84670 (E. vogeli) showed identities of 99, 91, 91 and 89%, respectively. tion of the stage in the soil will be an unequivocal measure of the epidemiologic importance of each location. At present, there is not an species-specific diagnosis of Echinococcus granulosus eggs collected from the environment. In this paper, we performed an study with eggs just obtained from proglottids and stool samples to evaluate the behavior of the EgO/ DNA-IM1 primer set, designed on the basis of known sequences of cestode with sanitary importance. Although several authors (Rishi & McManus, 1987; Smyth & McManus, 1989; Bretagne et al., 1993) mentioned chemical means for the disruption of the keratin embryosphere, the physical treatment by freezing and thawing the M. Cabrera et al. / Diagnostic Microbiology and Infectious Disease 44 (2002) 29 –34 eggs followed by chemical method facilitate the extraction of the oncospheres. The sensitivity of the PCR with the EgO/DNA-IM1 was evaluated by the optimal signal obtained with the sample containing 240 eggs. In accordance with Rishi et al. (1987), it had been employed 1920 pg of DNA. Although this quantity seems to be very high compared with the raised by Bretagne et al. (1993) of 8 pg added to 4 g of fox feces free of parasites, to identify by PCR eggs of E. multilocularis, it seems more natural to analyze the sensitivity on the basis of the number of necessary eggs to give a signal than to evaluate pure DNA obtained by an indeterminated number of eggs. The quantity suggests that to obtain the specific band is necessary a half of the oncospheres present in a gravid proglottid. This is important because not all the eggs contained DNA of the same quality (Smyth & McManus, 1989). The comparison of the initial quantity of 240 eggs with the equivalence in DNA of the content of 24 eggs, which correspond to the signal value obtained by PCR, suggests that the yield of DNA extraction correspond to 10% of the content of the embryosphores. This indicates that the sensitivity measure in DNA was of 192 pg. However, when DNA dilutions where used, the sensitivity was 32 pg, equivalent to 4 eggs. The specificity of the PCR with the EgO/DNA-IM1 was evaluated with a variety of cestode of sanitary importance, whose eggs also contaminate the environment mixtured with those of E. granulosus. All the PCR reactions performed with DNA of this last parasite, employing high and low quantities of eggs, showed the same pattern, characterized by an expected band of 285 bp, which sequence analysis demonstrate that belongs to E. granulosus G6 genotype. It can be assumed that this set of primers is useful to rule out E. multilocularis, T. hydatigena, D. caninum, T. saginata, and H. nana, which did not produce the band with any number of eggs. It could be also useful to rule out T. taeniaeformis, T. solium, T. psiformis and T. crassiceps, which sequences were excluded from the primers. T. hydatigena and T. saginata, which sequences either integrate the primers, did not show any band by PCR. This demonstration suggests that the sequences of the parasites that were excluded from the primers, effectively do not produce amplification fragments by PCR. In this paper we evaluate DNA of eggs from known source and under this conditions, we did not detect discordant positive or negative results. Due to the fact that the primer set contain the cytosine in 3⬘ position shared by all Echinococcus species with the exception of E. multilocularis, it will only behave as species-specific where do not exist E. oligarthrus and E. vogeli. The geographic area of these species is the tropical forest of South and Central America. The other endemic regions in the world have only E. granulosus or share the habitat with E. multilocularis; under this situation the primer set behave as species-specific because it do not recognize E. multilocu- 33 laris. This characteristic turns it into a useful tool for epidemiologic surveillance and evaluation of the magnitude of environmental contamination. Acknowledgments We are grateful to Dr. Donald P. 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