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Molecular identification of Echinococcus granulosus genotypes (G1 and G7) isolated from pigs in Mexico
Veterinary Parasitology 147 (2007) 185–189 www.elsevier.com/locate/vetpar
Short communication
Molecular identification of Echinococcus granulosus genotypes (G1 and G7) isolated from pigs in Mexico N. Villalobos a, L.M. Gonza´lez a, J. Morales b, A.S. de Aluja b, M.I. Jime´nez a, M.A. Blanco a, L.J.S. Harrison c, R.M.E. Parkhouse d, T. Ga´rate a,* a
Servicio de Parasitologı´a, Centro Nacional de Microbiologı´a, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain b Facultad de Medicina Veterinaria y Zootecnia de la Universidad Nacional Auto´noma de Me´xico, Me´xico 04510, Mexico c Division of Veterinary Clinical Sciences, Incorporating Centre for Tropical Veterinary Medicine, Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easter Bush Veterinary Centre, Easter Bush, Roslin, Midlothian, Scotland EH25 9RG, UK d Instituto Gulbenkian de Ciencia, R. Quinta Grande 6, Apartado 14, P-2781 Oeiras Codex, Portugal Received 31 January 2007; received in revised form 14 March 2007; accepted 15 March 2007
Abstract With the aim of genotyping Echinococcus granulosus cysts found in Mexican livestock, we collected hydatid cysts from the livers and lungs of pigs in slaughterhouses in the state of Morelos, Central Region of Mexico. DNA was extracted from the parasites and examined by polymerase chain reaction (PCR) of rDNA internal transcribed spacer 1 (ITS1-PCR), Eg9-PCR, Eg16-PCR, and PCR-restriction fragment length polymorphism (PCR-RFLP). In addition, fragments of the genes coding for mitochondrial cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase 1 (ND1) were sequenced. Two different genotypes of E. granulosus were unequivocally identified, the common sheep genotype, G1, and the common pig genotype, G7. The G1 genotype of E. granulosus has not been previously demonstrated in Mexico. Because of its recognized infectivity in humans, G1 genotype is a direct threat to human health and its presence in Mexico is consequently of immediate public health importance and epidemiological relevance. # 2007 Elsevier B.V. All rights reserved. Keywords: E. granulosus; Genotyping characterization; PCR
1. Introduction The cestode parasite Echinococcus granulosus and its genetic variants are the agents of cystic hydatidosis (McManus and Thompson, 2003), a zoonotic disease of worldwide importance that is widespread in Europe, Asia, Africa, South America, Canada and Australia (Moro and Schantz, 2006). To date studies indicate that there are nine distinct genetic types (Genotypes G1-G9) within E. granulosus which vary in their infectivity to
* Corresponding author. Tel.: +34 918223675; fax: +34 915097034. E-mail address: [email protected] (T. Ga´rate). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.03.021
humans and livestock (Eckert and Thompson, 1997; McManus, 2002). The lifecycle of E. granulosus includes dogs and other canids as the definitive hosts of the adult parasite (Moro and Schantz, 2006), whilst sheep and numerous ungulates (goats, swine, cattle) are intermediate hosts, harbouring the hydatid cyst (Euzeby, 1991; Garippa et al., 2004). Although pigs can be infected by different genotypes of E. granulosus (Bowles and McManus, 1993a,b; Eckert et al., 1993), in Mexico there is limited information on this infection: (i) human disease is practically absent (Barroeta et al., 1962; Sarinana Natera et al., 1976; Herrera Merino et al., 1991; Villarreal et al., 1994; Sua´rez et al., 1995; Mene´ndez-Arzac et al., 2002; Palacios-Ruiz et al.,
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2003), with only one report demonstrating the existence of the G5 genotype in an infected human patient (Maravilla et al., 2004); (ii) the frequency of cystic echinococcosis in pigs and cattle is 0.27–6.5% and 0.1%, respectively (Martı´nez et al., 1994; Vargas-Rivera et al., 1995); (iii) the prevalence of dog echinococcosis is estimated to be 0.49% (Ferna´ndez and Canto´, 2002). In the present work, using various molecular protocols, we have identified not only the common pig genotype (G7) but, for the first time, the sheep genotype (G1) in pigs of Mexico.
conserved sequences of 18S and 5.8S rDNA subunits, were employed following the working conditions described elsewhere (Bowles and McManus, 1993b). (ii) Eg9-PCR and Eg16-PCR: PEg9F1/PEg9R1 primers (50 -ATGGCATGGGTAGCACGGAGAG-30 / 50 -GGT TTGGGAATGGCGATGTTGA-30 ) and PEg16F1/PEg16R1 primers (50 -CGCGCTATCGACCCACCAACAG-30 /50 -ACTCCGTCCCGCTACTCCACTCC-30 ) were used according to previous protocols (Gonza´lez et al., 2002).
2. Material and methods
All the primers were synthesized by Isogen Life Science. Amplifications were carried out in a GeneAmp TM PCR System 2400 Thermocycler (Applied Biosystems) and then analysed by electrophoresis on 2% and 3% (w/v) agarose gels, followed by ethidium bromide staining and photography (Sambrook and Russell, 2001).
2.1. Parasite material The parasite samples were obtained in December 2004 and December 2005 in the slaughterhouse of Zacatepec, state of Morelos, Mexico. The carcasses and viscera of 350 pigs were examined, and 21 animals among them showed cysts in different organs. Fifteen pigs showed Taenia cyst (Gonza´lez et al., 2006), and six pigs had fertile E. granulosus hydatid cysts, one per each individual animal. Five parasites were recovered from the livers of five animals (#1Lv, #2Lv, #3Lv, #4Lv, #5Lv) and one from the lung of the sixth pig (6#Ln). Each cyst was removed, washed in PBS and then conserved in 70% ethanol until use. 2.2. Extraction of DNA Total genomic DNA (gDNA) was extracted from each cyst sample using standard procedures (Sambrook and Russell, 2001). The cyst was washed with PBS, frozen in liquid nitrogen, ground to power and then digested for 12 h at 37 8C with 100 ml of proteinase K (10 mg/ml, Sigma) in 3.8 ml of extraction buffer (50 mM Tris–HCl pH 8, 50 mM EDTA, 100 mM NaCl) and 200 ml of 10% SDS, extracted with phenol– chloroform and precipitated with ethanol. The pellet was resuspended in Milli-Q sterile water (Millipore), concentration of DNA was determinated by spectrophotometry and the samples were stored at 20 8C. 2.3. Amplification of DNA The gDNAs (50 ng/sample) were amplified in a final reaction volume of 25 ml as described below: (i) ITS1-PCR: BD1 and 4S primers (50 -GTCGTAACAAGGTTTCCGTA-30 /50 -TCTAGATGCGTTCGAA(G/A)TGTCGATG-30 ), derived from the
2.4. Restriction fragment length polymorphismPCR (RFLP-PCR) The ITS1-PCR amplification products (16 ml) were digested with 10 units of the restriction endonucleases Msp I, Rsa I and Cfo l (Roche), in a final volume of 40 ml overnight at 37 8C. The Eg9-PCR amplification products (12.5 ml) were digested with the restriction endonucleases Rsa I and Cfo I (10 units) (Roche), in a final volume of 30 ml, overnight at 37 8C. Later, the products were fractionated in 3% agarose gels, visualized under UV light, after ethidium bromide staining, and photographed (Sambrook and Russell, 2001). 2.5. Sequencing of mitochondrial DNA Fragments of the ND1 and CO1 mitochondrial genes were PCR-amplified as described by Bowles and McManus (1993a) and Bowles et al. (1992), respectively, with ND1 JB11/JB12 primers (50 -AGATTCGTAAGGGGCCTAATA-3 0 /5 0 -ACCACTAACTAATTCACTTTC-30 ) and COI JB3/JB4.5 primers (50 TTTTTTGGGCATCCTGAGGTTTAT-3 0 /5 0 -TAAAGAAAGAACATAATGAAAATG-30 ). gDNA (100 ng) from E. granulosus isolates were used in both reactions. Amplicons were cut from the gel under UV exposure and the samples were purified by a QIAquick Gel Extraction Kit (Qiagen, Diagen, Hilden, Germany). The CO1 and ND1 sequences were automatically obtained using a 377 ABI PRISM system (Applied Biosystems). Nucleotide sequence analysis was undertaken by
N. Villalobos et al. / Veterinary Parasitology 147 (2007) 185–189
BLAST algorithms and databases from the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov). Multiple sequence alignments were made with the ClustalW method, with Bioedit software and compared with sequences retrieved from GenBank. 3. Results and discussion Conclusive identification of Echinococcus genotypes requires a combination of approaches for DNA characterization (Bowles and McManus, 1993a,b; Bowles et al., 1992; Gonza´lez et al., 2002). In the present work we used ITS1-PCR, ITS1-PCR-RFLP, Eg9-PCR-RFLP, Eg16-PCR and ND1/CO1 sequencing to characterize E. granulosus DNA isolated from cysts recovered from pigs, specifically five livers and one lung, in the State of Morelos, Mexico. The ITS1-PCR with BD1 and 4S primers yielded two amplification products of 1.0 and 1.1 kb, with the #1Lv, #2Lv, #3Lv, #4Lv, and #5Lv samples, characteristic of the G7 genotype, and two bands of 0.9 and 1.0 kb, characteristics of the G1 genotype, with the #6Ln sample (Fig. 1A). In order to confirm these results, we compared the PCR-RFLP patterns produced after digestion of the ITS1 fragments with the four-base recognizing restriction endonucleases Cfo I, Rsa I and Msp I (Fig. 1B). The samples from livers (#1Lv, #2Lv, #3Lv, #4Lv, #5Lv) yielded patterns of the G7 genotype and the sample from the lung (#6Ln) exhibited the G1 genotype digestion profile; Fig. 1B shows the digestion patterns obtained from samples Lv#1, Lv#2 and Ln#6, the other liver samples yielded identical results to the Lv#1 and Lv#2 ones (data not shown). Similar conclusions were drawn using the Eg9-PCR-RFLP and Eg16-PCR. The RFLP patterns from livers and lung
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samples after digestion of the Eg9-PCR amplicons by Rsa I enzyme, identified E. granulosus G7 genotype (two bands, 500 and 450 bp) and G1 genotype (two bands, 400 and 100 bp), respectively (Fig. 1C). Moreover, analysis with Cfo I digestion confirmed the E. granulosus genotypes identified above, because E. multilocularis DNA would have been digested by the enzyme, as did the Eg16-PCR. In the latter, the isolates of E. granulosus obtained from livers (#1Lv, #2Lv) yielded three bands between 500 and 450 bp (G7 genotype) and the samples from the lung (#6Ln) exhibited a pattern of three weak bands, 500, 450 and 400 bp (G1 genotype) (Fig. 1D). Both Eg9-PCR-RFLP and Eg16-PCR were done with the five cysts liver, with identical profiles (data not shown). Finally, partial sequences of the mitochondrial genes ND1 (471 bp) and CO1 (391 bp) of E. granulosus isolates were obtained and compared with those published for genotypes G1 and G7 (Bowles et al., 1992). Thus the sequences obtained of the five liver parasite samples (#1Lv, #2Lv, #3Lv, #4Lv, #5Lv) corresponded to the G7 genotype (pig) and the sequence of the lung parasite sample (#6Ln) was of the G1 genotype (sheep). In general, the sequences were highly homologous with few differences, corresponding to punctual base substitution (data not shown). To summarise our findings with ITS1-PCR, ITS1 PCR-RFLP, Eg-9 PCR-RFPL. Eg16-PCR, ND1 and CO1, all five samples extracted from pig livers were identified unequivocally by all the methods as genotype G7, while the one lung sample was unequivocally identified by all the methods as the sheep genotype G1. However, although the lung parasite was of the G1 genotype and the five liver parasites of the genotype G7, there is insufficient data to suggest any relationship between organ tropism and parasite genotype in E.
Fig. 1. Identification of G1 and G7 genotypes of E. granulosus recovered from Mexican pigs. (A) ITS1-PCR amplification patterns of the gDNA from #1Lv, #2Lv, #3Lv, #4Lv, #5Lv, #6Ln pig isolates from E. granulosus. (B) ITS1-PCR-RFLP (Msp I, Cfo I, Rsa I) profiles from samples #1Lv, #2Lv and #6Ln. (C) Eg9-PCR-RFLP (Cfo I and Rsa I) profiles from samples #1Lv, #2Lv and #6Ln. (D) Eg16-PCR amplification patterns from samples #1Lv, #2Lv and #6Ln. The PCR and PCR-RFLPs products were resolved by 2% (w/v) and 3% (w/v) agarose gel electrophoresis, respectively, and visualized by ethidium bromide staining. The numbers on the left indicate the sizes (in base pairs, bp) of the molecular weight markers. All the E. granulosus six pig isolates liver (Lv) and lung (Ln), were processed by PCRs and PCR-RFLPs described in the present work.
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granulosus infection in Mexico. More studies are being designed to try to explain this observation. There is only limited information on hydatid infection in humans and animals from Mexico, and indeed the extent of its prevalence is generally unknown. Nevertheless, a recent report described the presence of two genotypes in Mexico: the G7 genotype found in a hydatid cyst isolated from a pig liver and the G5 genotype in a hepatic cyst from an autochthonous human case (Maravilla et al., 2004). The present work now describes the presence of the G1 genotype of E. granulosus populations in Mexico. Interestingly, E. granulosus recovered from the infected pigs of Morelos included both the G7 (pig genotype in five pigs) and G1 (sheep genotype in one pig). These results are consistent with data shown previously by other authors using different molecular approaches (Gonza´lez et al., 2002; Varcasia et al., 2006). Moreover, the infectivity of the G1 genotype (sheep) in pigs has been demonstrated in different parts of Europe (Eckert et al., 1993; Kedra et al., 1999; Scott et al., 1997; Sˇna´bel et al., 2000), but not in Mexico. This result could have important epidemiological consequences and a direct impact on Mexican Public Health, as the pig isolates could infect humans. It is well known that the majority of human isolates of E. granulosus have been shown to be the common sheep G1 genotype, although the G7 genotype (Kedra et al., 1999) and others (McManus and Thompson, 2003) are also known to be infective to humans. Therefore, it is important to define the role of these pig parasite genotypes and their epidemiological implications for E. granulosus zoonotic control, for instance, in areas that lack adequate veterinary services and meat inspection with the possibility of the transmission of E. granulosus to humans and the consequent repercussion on public health. In conclusion, the molecular protocols used in this study distinguish two genetic groups in the E. granulosus isolates of the pigs examined: G1 and G7 genotypes. As far as we know, the G1 genotype has not been previously reported in Mexico. Acknowledgments We are very grateful to Roberto Oregon Morales MVZ, Conrrado Canovas Olmos, Elias Salinas Contreras, Cesar Medina Rojas, Ubaldo Leal Ponciano, for all their support during the slaughterhouse work. The present study was supported by grants from FIS (grants 97/0141 and 00/407) and Intramural ISCIII projects. N. Villalobos was sponsored by fellowships of FMV-
UNAM, CONACyT, Mexico and LM Gonza´lez by postdoctoral contracts of the ISCIII and RICET-FIS (RD06/ 0021/0019). References Barroeta, F., Biagi, F., Danchez de la Barquera, R., 1962. 1st case of autochthonous pulmonary hydatidosis in Me´xico. Neumol. Cir. Torax 23, 279–285. Bowles, J., McManus, D.P., 1993a. NADH dehydrogenase 1 gene sequences compared for species and strains of the genus Echinococcus. Int. J. Parasitol. 23, 969–972. Bowles, J., McManus, D.P., 1993b. Rapid discrimination of Echinococcus species and strains using a polymerase chain reactionbased RFLP method. Mol. Biochem. Parasitol. 57, 231–240. Bowles, J., Blair, D., McManus, D.P., 1992. Genetic variants within the genus Echinococcus identified by mitochondrial sequencing. Mol. Biochem. Parasitol. 54, 165–174. Eckert, J., Thompson, R.C.A., 1997. Intraspecific variation of Echinococcus granulosus and related species with emphasis on their infectivity to humans. Acta Trop. 64, 209–258. Eckert, J., Thompson, R.C.A., Lymbery, A.J., Pawlowski, Z.S., Gottstein, B., Morgan, U.M., 1993. Further evidence for the occurrence of a distinct strain of Echinococcus granulosus in European pigs. Parasitol. Res. 79, 42–48. Euzeby, J., 1991. The epidemiology of Hydatidosis with special reference to the Mediterranean area. Parassitologia 33, 25–39. Ferna´ndez, C.F., Canto´, A.J.F., 2002. Frecuencia de helmintos en intestinos de perros sin duen˜o sacrificados en la ciudad de Quere´taro, Quere´taro, Me´xico. Vet. Mex. 33, 247–253. Garippa, G., Varcasia, A., Scala, A., 2004. Cystic echinococcosis in Italy from the 1950s to today. Parasitologia 46, 387–391. Gonza´lez, L.M., Daniel-Mwambete, K., Montero, E., Rosenzvit, M.C., McManus, D.P., Ga´rate, T., Cuesta-Bandera, C., 2002. Further molecular discrimination of Spanish strains of Echinococcus granulosus. Exp. Parasitol. 102, 46–56. Gonza´lez, L.M., Villalobos, N., Montero, E., Morales, J., Alamo Sans, R., Muro, A., Harrison, L.J.S., Parkhouse, R.M.E., Ga´rate, T., 2006. Differential molecular identification of Taeniid spp. and Sarcocystis spp. cyst isolated from infected pigs and cattle. Vet. Parasitol. 142, 95–101. Herrera Merino, N., Abascal Morte, J., Dı´as del Rı´o Botas, M., et al., 1991. Quiste hidatı´dico de bazo. Aportacio´n de diecise´is casos. Rev. Esp. Digest. 79, 254–258. Kedra, A.H., Swiderski, Z., Tkach, V.V., Dubinsky, P., Pawlowski, Z., Stefaniak, J., Pawlowski, J., 1999. Genetic analysis of Echinococcus granulosus from human and pigs in Poland, Slovakia and Ukraine. A multicentre study. Acta Parasitol. 44, 248–254. Maravilla, P., Thompson, R.C.A., Palacios-Ruiz, J.A., Estcourt, A., Ramı´rez-Solis, E., Mondrago´n-de-la-Pen˜a, C., Moreno-Moller, M., Ca´rdenas-Mejia, M.T., Mata-Miranda, P., Aguirre-Alcantara, M.T., Bonilla-Rodrı´guez, C., Flisser, A., 2004. Echinococcus granulosus cattle strain identification in an autochthonous case of cysts echinococcosis in central Mexico. Acta Trop. 92, 231– 236. Martı´nez, M.J.J., Zu´n˜iga, A.I., Jaramillo, A.C.J., Ca´rdenas, L.J., Navarro, F.R., 1994. Caracterizacio´n de la equinococosis/hidatidosis en Zacatecas, Me´xico. Vet. Mex. 25, 231–237. McManus, D.P., 2002. The molecular epidemiology of Echinococcus granulosus and cystic hydatid disease. Trans. Roy. Soc. Trop. Med. Hyg. 96 (Supp. 1), S151–S157.
N. Villalobos et al. / Veterinary Parasitology 147 (2007) 185–189 McManus, D.P., Thompson, R.C., 2003. Molecular epidemiology of cystic echinococcosis. Parasitology 127, S37–S51. Mene´ndez-Arzac, R., Sanjua´n, A., Rebolledo, G., Ma´rquez, J.C., Recinos, E.G., Cue, A., Blas, C., Flisser, A., 2002. Quiste hidatı´dico esple´nico. Reporte de un caso en una mujer embarazada. Rev. Gastroenterol. Mex. 67, 195–198. Moro, P., Schantz, P.M., 2006. Cystic echinococcosis in the Americas. Parasitol. Int. 55, S181–S186. Palacios-Ruiz, J.A., Ramı´rez-Solis, E., Moreno-Moller, M., Ca´rdenasMejia, M.T., Aguirre-Alcantara, M.T., Bonilla-Rodrı´guez, C., Mata-Miranda, P., Maravilla, P., Flisser, A., 2003. Identificacio´n y manejo integral de un caso auto´ctono de hidatidosis en Me´xico. Rev. Gastroenterol. Mex. 68, 41–45. Sambrook, J., Russell, D.W.T., 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Sarinana Natera, C., Lara Aguilera, R., Vergara Rodrı´guez, J., Del Campo, N.M., 1976. Liver hydatidosis in an eight-year-old child. Bol. Med. Hosp. Infant Mex. 33, 555–565.
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Scott, J.C., Stefaniak, J., Pawlowski, Z.S., McManus, D.P., 1997. Molecular genetic analysis of human cystic hydatid cases from Poland: identification of a new genotypic group (G9) of Echinococcus granulosus. Parasitology 114, 37–43. Sˇna´bel, V., Da´melio, S., Mathiopoulos, K., Turcekova´, L., Dubinsky´, P., 2000. Molecular evidence for the presence of a G7 genotype of Echinococcus granulosus in Slovakia. J. Helminthol. 74, 177–181. Sua´rez, J.P., Rivera, E., Rico, F.G., Ramı´rez, P., Lo´pez del Lara, A., 1995. Paquipleuritis granulomatosa de origen parasitario. Rev. Inst. Nal. Enf. Resp. Me´x. 8, 54. Varcasia, A., Canu, S., Lightowlers, M.W., Scala, A., Garippa, G., 2006. Molecular characterization of Echinococcus granulosus strains in Sardinia. Parasitol. Res. 98, 273–277. Vargas-Rivera, I., Martı´nez-Maya, J.J., Jaramillo-Arango, C.J., 1995. Caracterizacio´n de la hidatidosis porcina en el rastro frigorı´fico Los Reyes La Paz, Estado de Me´xico, Me´xico. Vet. Mex. 26, 365–368. Villarreal, A., Padua, G., Lezama, C., Galindo, M.A., Argu¨ero, R., 1994. Quiste hidatı´dico pulmonar con ruptura mixta. Informe de un caso. Rev. Med. IMSS 33, 47.
Short communication
Molecular identification of Echinococcus granulosus genotypes (G1 and G7) isolated from pigs in Mexico N. Villalobos a, L.M. Gonza´lez a, J. Morales b, A.S. de Aluja b, M.I. Jime´nez a, M.A. Blanco a, L.J.S. Harrison c, R.M.E. Parkhouse d, T. Ga´rate a,* a
Servicio de Parasitologı´a, Centro Nacional de Microbiologı´a, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain b Facultad de Medicina Veterinaria y Zootecnia de la Universidad Nacional Auto´noma de Me´xico, Me´xico 04510, Mexico c Division of Veterinary Clinical Sciences, Incorporating Centre for Tropical Veterinary Medicine, Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easter Bush Veterinary Centre, Easter Bush, Roslin, Midlothian, Scotland EH25 9RG, UK d Instituto Gulbenkian de Ciencia, R. Quinta Grande 6, Apartado 14, P-2781 Oeiras Codex, Portugal Received 31 January 2007; received in revised form 14 March 2007; accepted 15 March 2007
Abstract With the aim of genotyping Echinococcus granulosus cysts found in Mexican livestock, we collected hydatid cysts from the livers and lungs of pigs in slaughterhouses in the state of Morelos, Central Region of Mexico. DNA was extracted from the parasites and examined by polymerase chain reaction (PCR) of rDNA internal transcribed spacer 1 (ITS1-PCR), Eg9-PCR, Eg16-PCR, and PCR-restriction fragment length polymorphism (PCR-RFLP). In addition, fragments of the genes coding for mitochondrial cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase 1 (ND1) were sequenced. Two different genotypes of E. granulosus were unequivocally identified, the common sheep genotype, G1, and the common pig genotype, G7. The G1 genotype of E. granulosus has not been previously demonstrated in Mexico. Because of its recognized infectivity in humans, G1 genotype is a direct threat to human health and its presence in Mexico is consequently of immediate public health importance and epidemiological relevance. # 2007 Elsevier B.V. All rights reserved. Keywords: E. granulosus; Genotyping characterization; PCR
1. Introduction The cestode parasite Echinococcus granulosus and its genetic variants are the agents of cystic hydatidosis (McManus and Thompson, 2003), a zoonotic disease of worldwide importance that is widespread in Europe, Asia, Africa, South America, Canada and Australia (Moro and Schantz, 2006). To date studies indicate that there are nine distinct genetic types (Genotypes G1-G9) within E. granulosus which vary in their infectivity to
* Corresponding author. Tel.: +34 918223675; fax: +34 915097034. E-mail address: [email protected] (T. Ga´rate). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.03.021
humans and livestock (Eckert and Thompson, 1997; McManus, 2002). The lifecycle of E. granulosus includes dogs and other canids as the definitive hosts of the adult parasite (Moro and Schantz, 2006), whilst sheep and numerous ungulates (goats, swine, cattle) are intermediate hosts, harbouring the hydatid cyst (Euzeby, 1991; Garippa et al., 2004). Although pigs can be infected by different genotypes of E. granulosus (Bowles and McManus, 1993a,b; Eckert et al., 1993), in Mexico there is limited information on this infection: (i) human disease is practically absent (Barroeta et al., 1962; Sarinana Natera et al., 1976; Herrera Merino et al., 1991; Villarreal et al., 1994; Sua´rez et al., 1995; Mene´ndez-Arzac et al., 2002; Palacios-Ruiz et al.,
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2003), with only one report demonstrating the existence of the G5 genotype in an infected human patient (Maravilla et al., 2004); (ii) the frequency of cystic echinococcosis in pigs and cattle is 0.27–6.5% and 0.1%, respectively (Martı´nez et al., 1994; Vargas-Rivera et al., 1995); (iii) the prevalence of dog echinococcosis is estimated to be 0.49% (Ferna´ndez and Canto´, 2002). In the present work, using various molecular protocols, we have identified not only the common pig genotype (G7) but, for the first time, the sheep genotype (G1) in pigs of Mexico.
conserved sequences of 18S and 5.8S rDNA subunits, were employed following the working conditions described elsewhere (Bowles and McManus, 1993b). (ii) Eg9-PCR and Eg16-PCR: PEg9F1/PEg9R1 primers (50 -ATGGCATGGGTAGCACGGAGAG-30 / 50 -GGT TTGGGAATGGCGATGTTGA-30 ) and PEg16F1/PEg16R1 primers (50 -CGCGCTATCGACCCACCAACAG-30 /50 -ACTCCGTCCCGCTACTCCACTCC-30 ) were used according to previous protocols (Gonza´lez et al., 2002).
2. Material and methods
All the primers were synthesized by Isogen Life Science. Amplifications were carried out in a GeneAmp TM PCR System 2400 Thermocycler (Applied Biosystems) and then analysed by electrophoresis on 2% and 3% (w/v) agarose gels, followed by ethidium bromide staining and photography (Sambrook and Russell, 2001).
2.1. Parasite material The parasite samples were obtained in December 2004 and December 2005 in the slaughterhouse of Zacatepec, state of Morelos, Mexico. The carcasses and viscera of 350 pigs were examined, and 21 animals among them showed cysts in different organs. Fifteen pigs showed Taenia cyst (Gonza´lez et al., 2006), and six pigs had fertile E. granulosus hydatid cysts, one per each individual animal. Five parasites were recovered from the livers of five animals (#1Lv, #2Lv, #3Lv, #4Lv, #5Lv) and one from the lung of the sixth pig (6#Ln). Each cyst was removed, washed in PBS and then conserved in 70% ethanol until use. 2.2. Extraction of DNA Total genomic DNA (gDNA) was extracted from each cyst sample using standard procedures (Sambrook and Russell, 2001). The cyst was washed with PBS, frozen in liquid nitrogen, ground to power and then digested for 12 h at 37 8C with 100 ml of proteinase K (10 mg/ml, Sigma) in 3.8 ml of extraction buffer (50 mM Tris–HCl pH 8, 50 mM EDTA, 100 mM NaCl) and 200 ml of 10% SDS, extracted with phenol– chloroform and precipitated with ethanol. The pellet was resuspended in Milli-Q sterile water (Millipore), concentration of DNA was determinated by spectrophotometry and the samples were stored at 20 8C. 2.3. Amplification of DNA The gDNAs (50 ng/sample) were amplified in a final reaction volume of 25 ml as described below: (i) ITS1-PCR: BD1 and 4S primers (50 -GTCGTAACAAGGTTTCCGTA-30 /50 -TCTAGATGCGTTCGAA(G/A)TGTCGATG-30 ), derived from the
2.4. Restriction fragment length polymorphismPCR (RFLP-PCR) The ITS1-PCR amplification products (16 ml) were digested with 10 units of the restriction endonucleases Msp I, Rsa I and Cfo l (Roche), in a final volume of 40 ml overnight at 37 8C. The Eg9-PCR amplification products (12.5 ml) were digested with the restriction endonucleases Rsa I and Cfo I (10 units) (Roche), in a final volume of 30 ml, overnight at 37 8C. Later, the products were fractionated in 3% agarose gels, visualized under UV light, after ethidium bromide staining, and photographed (Sambrook and Russell, 2001). 2.5. Sequencing of mitochondrial DNA Fragments of the ND1 and CO1 mitochondrial genes were PCR-amplified as described by Bowles and McManus (1993a) and Bowles et al. (1992), respectively, with ND1 JB11/JB12 primers (50 -AGATTCGTAAGGGGCCTAATA-3 0 /5 0 -ACCACTAACTAATTCACTTTC-30 ) and COI JB3/JB4.5 primers (50 TTTTTTGGGCATCCTGAGGTTTAT-3 0 /5 0 -TAAAGAAAGAACATAATGAAAATG-30 ). gDNA (100 ng) from E. granulosus isolates were used in both reactions. Amplicons were cut from the gel under UV exposure and the samples were purified by a QIAquick Gel Extraction Kit (Qiagen, Diagen, Hilden, Germany). The CO1 and ND1 sequences were automatically obtained using a 377 ABI PRISM system (Applied Biosystems). Nucleotide sequence analysis was undertaken by
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BLAST algorithms and databases from the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov). Multiple sequence alignments were made with the ClustalW method, with Bioedit software and compared with sequences retrieved from GenBank. 3. Results and discussion Conclusive identification of Echinococcus genotypes requires a combination of approaches for DNA characterization (Bowles and McManus, 1993a,b; Bowles et al., 1992; Gonza´lez et al., 2002). In the present work we used ITS1-PCR, ITS1-PCR-RFLP, Eg9-PCR-RFLP, Eg16-PCR and ND1/CO1 sequencing to characterize E. granulosus DNA isolated from cysts recovered from pigs, specifically five livers and one lung, in the State of Morelos, Mexico. The ITS1-PCR with BD1 and 4S primers yielded two amplification products of 1.0 and 1.1 kb, with the #1Lv, #2Lv, #3Lv, #4Lv, and #5Lv samples, characteristic of the G7 genotype, and two bands of 0.9 and 1.0 kb, characteristics of the G1 genotype, with the #6Ln sample (Fig. 1A). In order to confirm these results, we compared the PCR-RFLP patterns produced after digestion of the ITS1 fragments with the four-base recognizing restriction endonucleases Cfo I, Rsa I and Msp I (Fig. 1B). The samples from livers (#1Lv, #2Lv, #3Lv, #4Lv, #5Lv) yielded patterns of the G7 genotype and the sample from the lung (#6Ln) exhibited the G1 genotype digestion profile; Fig. 1B shows the digestion patterns obtained from samples Lv#1, Lv#2 and Ln#6, the other liver samples yielded identical results to the Lv#1 and Lv#2 ones (data not shown). Similar conclusions were drawn using the Eg9-PCR-RFLP and Eg16-PCR. The RFLP patterns from livers and lung
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samples after digestion of the Eg9-PCR amplicons by Rsa I enzyme, identified E. granulosus G7 genotype (two bands, 500 and 450 bp) and G1 genotype (two bands, 400 and 100 bp), respectively (Fig. 1C). Moreover, analysis with Cfo I digestion confirmed the E. granulosus genotypes identified above, because E. multilocularis DNA would have been digested by the enzyme, as did the Eg16-PCR. In the latter, the isolates of E. granulosus obtained from livers (#1Lv, #2Lv) yielded three bands between 500 and 450 bp (G7 genotype) and the samples from the lung (#6Ln) exhibited a pattern of three weak bands, 500, 450 and 400 bp (G1 genotype) (Fig. 1D). Both Eg9-PCR-RFLP and Eg16-PCR were done with the five cysts liver, with identical profiles (data not shown). Finally, partial sequences of the mitochondrial genes ND1 (471 bp) and CO1 (391 bp) of E. granulosus isolates were obtained and compared with those published for genotypes G1 and G7 (Bowles et al., 1992). Thus the sequences obtained of the five liver parasite samples (#1Lv, #2Lv, #3Lv, #4Lv, #5Lv) corresponded to the G7 genotype (pig) and the sequence of the lung parasite sample (#6Ln) was of the G1 genotype (sheep). In general, the sequences were highly homologous with few differences, corresponding to punctual base substitution (data not shown). To summarise our findings with ITS1-PCR, ITS1 PCR-RFLP, Eg-9 PCR-RFPL. Eg16-PCR, ND1 and CO1, all five samples extracted from pig livers were identified unequivocally by all the methods as genotype G7, while the one lung sample was unequivocally identified by all the methods as the sheep genotype G1. However, although the lung parasite was of the G1 genotype and the five liver parasites of the genotype G7, there is insufficient data to suggest any relationship between organ tropism and parasite genotype in E.
Fig. 1. Identification of G1 and G7 genotypes of E. granulosus recovered from Mexican pigs. (A) ITS1-PCR amplification patterns of the gDNA from #1Lv, #2Lv, #3Lv, #4Lv, #5Lv, #6Ln pig isolates from E. granulosus. (B) ITS1-PCR-RFLP (Msp I, Cfo I, Rsa I) profiles from samples #1Lv, #2Lv and #6Ln. (C) Eg9-PCR-RFLP (Cfo I and Rsa I) profiles from samples #1Lv, #2Lv and #6Ln. (D) Eg16-PCR amplification patterns from samples #1Lv, #2Lv and #6Ln. The PCR and PCR-RFLPs products were resolved by 2% (w/v) and 3% (w/v) agarose gel electrophoresis, respectively, and visualized by ethidium bromide staining. The numbers on the left indicate the sizes (in base pairs, bp) of the molecular weight markers. All the E. granulosus six pig isolates liver (Lv) and lung (Ln), were processed by PCRs and PCR-RFLPs described in the present work.
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granulosus infection in Mexico. More studies are being designed to try to explain this observation. There is only limited information on hydatid infection in humans and animals from Mexico, and indeed the extent of its prevalence is generally unknown. Nevertheless, a recent report described the presence of two genotypes in Mexico: the G7 genotype found in a hydatid cyst isolated from a pig liver and the G5 genotype in a hepatic cyst from an autochthonous human case (Maravilla et al., 2004). The present work now describes the presence of the G1 genotype of E. granulosus populations in Mexico. Interestingly, E. granulosus recovered from the infected pigs of Morelos included both the G7 (pig genotype in five pigs) and G1 (sheep genotype in one pig). These results are consistent with data shown previously by other authors using different molecular approaches (Gonza´lez et al., 2002; Varcasia et al., 2006). Moreover, the infectivity of the G1 genotype (sheep) in pigs has been demonstrated in different parts of Europe (Eckert et al., 1993; Kedra et al., 1999; Scott et al., 1997; Sˇna´bel et al., 2000), but not in Mexico. This result could have important epidemiological consequences and a direct impact on Mexican Public Health, as the pig isolates could infect humans. It is well known that the majority of human isolates of E. granulosus have been shown to be the common sheep G1 genotype, although the G7 genotype (Kedra et al., 1999) and others (McManus and Thompson, 2003) are also known to be infective to humans. Therefore, it is important to define the role of these pig parasite genotypes and their epidemiological implications for E. granulosus zoonotic control, for instance, in areas that lack adequate veterinary services and meat inspection with the possibility of the transmission of E. granulosus to humans and the consequent repercussion on public health. In conclusion, the molecular protocols used in this study distinguish two genetic groups in the E. granulosus isolates of the pigs examined: G1 and G7 genotypes. As far as we know, the G1 genotype has not been previously reported in Mexico. Acknowledgments We are very grateful to Roberto Oregon Morales MVZ, Conrrado Canovas Olmos, Elias Salinas Contreras, Cesar Medina Rojas, Ubaldo Leal Ponciano, for all their support during the slaughterhouse work. The present study was supported by grants from FIS (grants 97/0141 and 00/407) and Intramural ISCIII projects. N. Villalobos was sponsored by fellowships of FMV-
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