- Email: [email protected]
Genotyping of potentially zoonotic Giardia duodenalis from exotic and wild animals kept in captivity in Brazil
Veterinary Parasitology 180 (2011) 344–348
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Genotyping of potentially zoonotic Giardia duodenalis from exotic and wild animals kept in captivity in Brazil Rodrigo Martins Soares ∗ , Sílvio Luís Pereira de Souza, Luciane Holsback Silveira, Mikaela Renata Funada, Leonardo José Richtzenhain, Solange M. Gennari Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508-270 São Paulo, SP, Brazil
a r t i c l e
i n f o
Article history: Received 21 April 2010 Received in revised form 8 February 2011 Accepted 29 March 2011 Keywords: Giardia duodenalis Wild animals Glutamate dehydrogenase Molecular identification Sequencing Brazil
a b s t r a c t We have studied the variability of glutamate dehydrogenase (gdh) and small subunit ribosomal (SSU) rRNA coding genes of Giardia species in fecal samples isolated from wild and exotic animals in Brazil, and compared with homologous sequences of isolates from human and domestic animals characterized in previous studies. Cysts of Giardia duodenalis were obtained from feces of naturally infected monkeys (Alouatta fusca) (n = 20), chinchillas (Chinchilla lanigera) (n = 3), ostriches (Struthio camelus) (n = 2) and jaguar (Panthera onca) (n = 1). Assemblage AI was assigned to the unique isolate of jaguar. All the samples from monkeys, chinchillas, and ostriches were assigned to Assemblage B. There was little evolutionary divergence between the referred isolates and isolates described elsewhere. The Assemblage B isolates identified in this study were closely related to Assemblage BIV isolated from humans. The molecular identification of Assemblages A and B of G. duodenalis isolates from exotic and wild animals demonstrates that such hosts may be a potential reservoir for zoonotic transmission of G. duodenalis. © 2011 Elsevier B.V. All rights reserved.
1. Short communication Giardia duodenalis (syn: Giardia lamblia, Giardia intestinalis) is a ubiquitous enteric protozoan that infects humans, domestic animals and wildlife worldwide (Thompson, 2000). Although G. duodenalis isolates from different host species are morphologically indistinguishable, they can be differentiated by PCR-based procedures in conjunction with analysis of housekeeping genes including the genes coding for glutamate dehydrogenase (gdh), elongation factor-␣ and triose phosphate isomerase (Yee and Dennis, 1992; Monis et al., 1996; Cacció et al., 2002; Sulaiman et al., 2003). Molecular genetic studies using these markers have demonstrated that G. duodenalis is a species complex that comprises at least seven assemblages, identified from A to G (Monis et al., 1999, 2003).
∗ Corresponding author. Tel.: +55 11 3091 7653; fax: +55 11 3091 7653. E-mail address: [email protected] (R.M. Soares). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.03.049
Primates of the genus Allouata are characteristic of the New World. This genus has eight species distributed from Mexico to Argentina. One of the species occurring in Brazil, from the state of Bahia to the state of Rio Grande do Sul is the Alouatta fusca (howler monkey). Monkeys of the Allouata genus may harbor the two zoonotic assemblages of G. duodenalis (Assemblages A and B). Both zoonotic assemblages were found in stool samples of wild black howler monkeys (Alouatta pigra), in Belize (Vitazkova and Wade, 2006). Screening of isolates of G. duodenalis from asymptomatic southern brown howler monkeys (Alouatta clamitans) kept in captivity in South Brazil has shown the sub-assemblage AI (Volotão et al., 2008). Little is known about diseases that affect Ostriches (Struthio camelus) in Brazil, in particular those caused by protozoa. Reports of hexamitiasis in ratites concern ostriches and rheas, and are described as Giardia sp. (Clipsham, 1995; Tully and Shane, 1996; Huchzermeyer, 1999) and Hexamita-like flagellates (Tully and Shane, 1996; Huchzermeyer, 1999). Several protozoan species
R.M. Soares et al. / Veterinary Parasitology 180 (2011) 344–348
of the gastrointestinal tract have already been found in ostriches, but most of them needed to be confirmed as ratite specific parasites. Chinchilla lanigera is a rodent native to Chile which is bred for commercial purposes. Parasitic diseases, particularly giardiasis, may cause clinical and sanitary problems and lead to production and economic losses. A survey in a commercial breeding facility in southern Brazil revealed 36.4% (80/220) of animals shedding Giardia spp. cysts (Fialho et al., 2008). Like the ostriches, C. lanigera is also bred for commercial purposes in Brazil. The jaguar (Panthera onca) is the largest feline of the Americas. This felid is currently found from Mexico (in the coastal plains) to northern Argentina, and inhabits ecosystems such as the Atlantic Forest, Cerrado, Pantanal and Amazon. Recently, a fecal sample from a young panther was found to be positive for Giardia cysts. The diagnosis was performed in the Laboratory of Parasitic Diseases (Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo). The animal approximately two months old was being held captive and had diarrhea (unpublished data). To date, no genotyping studies using stocks from any of the above species, except monkeys, have been carried out in Brazil. The present study was aimed to compare, in terms of nucleotide diversity, the genetic sequences of gdh coding sequences of such isolates with homologous sequences of isolates from Brazil and other parts of the world. Cysts of G. duodenalis were obtained from feces of naturally infected howler monkeys (A. fusca) (n = 20), chinchillas (C. lanigera) (n = 3), ostriches (S. camelus) (n = 2) and jaguar (P. onca) (n = 1). The monkeys were sampled in a Center for the Study and Management of Wild Animals. The animals had been kept in captivity. Fecal samples from chinchilla and ostriches were collected from animals bred for commercial purposes. The feline was obtained from a state park for the management and recovery of wild animals. All animals in the samples were symptomatic. Except for the sample taken from the two-month-old young jaguar, all other samples were taken from adult animals of unknown age. The feces were examined for cysts by a conventional flotation method, using zinc sulfate solution. Floated material was transferred to a slide and examined by light microscopy. DNA extraction of the cysts, PCR amplification of a segment of 1190 bp within the gdh gene, and sequencing reactions of PCR products were performed exactly as described previously (Souza et al., 2007). PCR amplification of a segment of 292 bp within the small subunit rDNA (SSU-rRNA) coding sequences, and sequencing reactions of PCR products were also performed (Hopkins et al., 1997). The genetic sequences were deposited in GenBank (accession HM134192–HM134217). Multiple alignments from gdh nucleotide sequences and SSUrDNA were made using homologous sequences retrieved from Genbank. Genetic sequences of Assemblage A from Brazilian origin were compared with gdh sequences of the subassemblages and genotypes described by Cacció et al. (2008). These gdh genotypes were named AI-1, AI-5, AII-2, AII-3, AII-4, AII-5 and AIII-6.
345
Molecular characterization of the genetic sequences of Assemblage B was performed with phylogenetic reconstructions using the gdh segment from the nucleotide positions 258–650 (from the initial gene positions). Eight sequences of G. duodenalis of monkeys were not included in this analysis because they were shorter than the rest (Afu01 to Afu10). In order to demonstrate the phylogenetic relationships of parasite isolates of Assemblage B, the dataset gdh was analyzed by SplitsTree4 (Huson, 1998; Huson and Bryant, 2006). The result is shown as a reticulated network, which is an alternative to the traditional bifurcating phylogenetic tree in describing complex relationships in population biology (Morrison, 2005). Assemblage AI was assigned to the unique isolate of P. onca (Pon01). Novel substitutions in positions were detected, which occurred in the last third of the gdh coding sequences. The novel substitutions were observed at positions 993 and 1011. Three isolates from Brazil, Pon01, B4, and F11 (from P. onca, bovine and cat, respectively) share the same nucleotide at position 993, whereas three isolates from cats of Brazil (F5, F12, and F14) share the same nucleotide at position 1011. These isolates may constitute new genotypes but these could be confirmed only after other constitutive genes are sequenced. Isolates Pon01and B4 are identical and share the same character at position 621 (Suppl. data S1). At this site, these isolates differ from all the other isolates of sub-assemblage AI and share the same character with isolates from sub-assemblage AII. These results show that different mammals such as felines and cattle may become infected with the same genotype, suggesting that the genotype AI-1 has low hostspecificity. The SSU-rRNA coding sequences of the isolate Pon01 had 100% identity with the Assemblage A isolates Cat2BAC2, Portland and BAH40c11 (AF199445, M54878 and AF199446, respectively). With respect to Assemblage B, there was little evolutionary divergence between isolates from animals in this study and other isolates described elsewhere. All the samples from monkeys (n = 20), chinchillas (n = 3), and ostriches (n = 2) were assigned to Assemblage B, being closely related to isolates from humans which were previously identified as sub-assemblage BIV (Suppl. data S2). The isolates from monkeys, chinchillas and ostriches are named Afu, Cla and Sca, respectively. Such samples were also assigned to Assemblage B by SSU-rRNA analysis. All Assemblage B sequences had 100% identity with the Assemblage B isolates AMC-4, CM and BAH12c14 (U09491, U09492 and AF199447, respectively). It is worth mentioning that no mixed Assemblage A and B infections were reported among the animals in this study. A previous study from Belgium (Levecke et al., 2009) has shown a high prevalence of mixed Assemblage A and B infections in monkeys while using assemblage specific primers. In our survey assemblage specific primers were not used. However the combined results of SSU-rRNA and gdh analysis suggest that mixed infections were not present in our samples. Assemblage B isolates are usually subdivided into two sub-assemblages named BIII and BIV. Sequences of gdh gene of G. duodenalis were grouped into sub-assemblages according to the positions of their single nucleotide
346
R.M. Soares et al. / Veterinary Parasitology 180 (2011) 344–348
Fig. 1. Panel (A) NeighborNet phylogenetic network of Giardia duodenalis Assemblage B isolates from Brazil and from other parts of the world. Note that Giardia duodenalis Assemblage AI was not represented (this taxon is located in the terminal node of the interrupted branch in the bottom of the figure). The whole network is shown in panel (B). (BIV) Assemblage BIV; (BIV-like) Assemblage BIV-like; (B-central) Assemblage B-central; (BIII) Assemblage BIII; and (BIII-like) Assemblage BIII-like. There were a total of 393 positions in the final dataset.
R.M. Soares et al. / Veterinary Parasitology 180 (2011) 344–348
polymorphisms in relation to previously characterized reference isolates (Wielinga and Thompson, 2007). In the referred study, the authors divided gdh haplotypes into BIII, BIII-like, B-central, BIV and BIV-like. Gdh phylogenies cannot be used to reconstruct the evolutionary history of this assemblage because different authors sequenced distinct parts of the molecule. Gdh sequences BIII and BIII-like have characters available only between positions 258 and 650, while most of the sequences BIV and BIV-like have available sites upstream and downstream this fragment. A visual inspection of the alignment of sequences of gdh gene of Assemblage B isolates shows that isolates BIII and BIII-like are divergent from the other sub-assemblages (B-central, BIV and BIV-like) (Suppl. data S2). The sub-assemblages originally described within Assemblage B (BIII and BIV) were reproduced with the traditional bifurcating phylogenetic tree, but with low statistical support for the internal nodes (not shown). With the use of a reticulated network we were able to demonstrate sub-structuring within this assemblage (Fig. 1). All the sequences closely related to BIII and BIII-like are exclusively of human origin, whereas those related to BIV and BIV-like are of human and animal origin. Plot of similarity between fragments of gdh gene of Assemblage B reveals an extensive polymorphism at the segment downstream the nucleotide position 650 (Suppl. data S2). Thus, it will be possible to infer more robust phylogenetic reconstructions when this segment of gdh of BIII and BIII-like is sequenced. It has been demonstrated that non-human primates harbor the two zoonotic assemblages of G. duodenalis, but Assemblage B appears to be the most prevalent in European countries (Levecke et al., 2009). In a previous study in brown howler monkeys in Brazil, only sub-assemblage AI was found (Volotão et al., 2008). Although sequence data of the two studies with monkeys in Brazil are rather limited, it seems that both zoonotic assemblages are equally prevalent in howler monkeys kept in captivity. Exotic animals raised in a farm for food or other purposes were found to be susceptible to Assemblage B. Hosts as distant as chinchillas and ostriches may harbor the same sub-assemblage, which shows the low host specificity of this assemblage. Thus, the risk posed by this sub-assemblage for these animals has to be assessed in future studies. The finding of G. duodenalis Assemblage B in ostriches is interesting as birds are commonly infected with other Giardia species. However, trophozoytes of Giardia were not observed although the animals had diarrhea. Thus the actual importance of G. duodenalis for this host still has to be issued. Although three genotypic variants have been found among isolates from monkeys, they differ only marginally from the archetypes BIV (one to three nucleotide substitutions). A slight difference (only three nucleotide substitutions) between sequences of ostriches and archetypal sequences of BIV could also be observed. Gdh coding sequences of G. duodenalis of chinchillas were identical to BIV alleles of human origin. The sequence analysis of isolates of exotic and wild animals performed in the present study revealed none of
347
heterogeneous nucleotide positions within the fragment of the gdh gene. Heterogeneous templates can originate both from amplification of DNA from cysts from a mixed infection and from allelic sequence heterozygosity within single cysts (Cacció et al., 2008). Thus, homogeneous templates can only be obtained with assemblage, sub-assemblage or genotype specific primers. Herein, we employed generic primers, PCR amplification and direct sequencing of DNA from cysts, which makes the distinction between the two aforementioned conditions impossible. High prevalence of heterogeneous sequences is a commonly described phenomenon. We could not explain why the analyzed templates did not yield heterogeneous chromatograms. Acknowledgments Rodrigo M. Soares Leonardo J. Richtzenhain and Solange M. Gennari is in receipt of a productivity fellowship from CNPq. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vetpar.2011.03.049. References Cacció, S.M., De Giacomo, M., Pozio, E., 2002. Sequence analysis of the beta-giardin gene and development of a polymerase chain reactionrestriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int. J. Parasitol. 32, 1023–1030. Cacció, S.M., Beck, R., Lalle, M., Marinculic, A., Pozio, E., 2008. Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B. Int. J. Parasitol. 38, 1523–1531. Clipsham, R., 1995. Avian pathogenic flagellated enteric protozoa. Sem. Avian Exotic Pet Med. 4, 112–125. Fialho, C.G., Oliveira, R.G., Teixeira, M.C., Marques, S.M.T., Oliveira, R.G., Oliveira, R.G., Araujo, F.A.P., 2008. Comparac¸ão da infecc¸ão por protozoários em chinchila (Chinchilla lanigera) de uma criac¸ão comercial do município de Viamão-RS, Brasil, e de chinchilas no seu habitat natural, Chile. Parasitol. Latinoam. 63, 85–87. Hopkins, R.M., Meloni, B.P., Groth, D.M., Wetherall, J.D., Reynoldson, J.A., Thompson, R.C., 1997. Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality. J Parasitol. 83, 44–51. Huchzermeyer, F.W., 1999. PatologIa de avestruces y otras ratites. Ediciones Mundi-Prensa, Madrid, p. 284. Huson, D.H., 1998. SplitsTree: a program for analyzing and visualizing evolutionary data. Bioinformatics 14, 68–73. Huson, D.H., Bryant, D., 2006. Application of phylogenetic networks in evolutionary studies. Mol. Biol. Evol. 23 (2), 254–267. Levecke, B., Geldholf, P., Claerebout, E., Dorny, P., Vercammen, F., Cacció, S.M., Vercruysse, J., Geurden, T., 2009. Molecular characterization of Giardia duodenalis in captive non-human primates reveals mixed assemblage A and B infections and novel polymorphisms. Int. J. Parasitol. 39, 1595–1601. Monis, P.T., Mayrhofer, G., Andrews, R.H., Homan, W.L., Limper, L., Ey, P.L., 1996. Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus. Parasitology 112, 1–12. Monis, P.T., Andrews, R.H., Mayrhofer, G., Ey, P.L., 1999. Molecular systematics of the parasitic protozoan Giardia intestinalis. Mol. Biol. Evol. 16, 1135–1144. Monis, P.T., Andrews, R.H., Mayrhofer, G., Ey, P.L., 2003. Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin. Infect. Genet. Evol. 3, 29–38. Morrison, D.A., 2005. Networks in phylogenetic analysis: new tools for population biology. Int. J. Parasitol. 35, 567–582.
348
R.M. Soares et al. / Veterinary Parasitology 180 (2011) 344–348
Souza, S.L.P., Gennari, S.M., Richtzenhain, Pena, H.F.J., Funada, M.R., Cortez, A.D., Gregori, F., Soares, R.M., 2007. Molecular identification of Giardia duodenalis isolates from humans, dogs, cats and cattle from the state of São Paulo, Brazil, by sequence analysis of fragments of glutamate dehydrogenase (gdh) coding gene. Vet. Parasitol. 149, 258–264. Sulaiman, I.M., Fayer, R., Bern, C., Gilman, R.H., Trout, J.M., Schantz, P.M., Das, P., Lal, A.A., Xiao, L., 2003. Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg. Infect. Dis. 9, 1152–1444. Thompson, R.C.A., 2000. Giardiasis as a re-emerging infectious disease and its zoonotic potential. Int. J. Parasitol. 30, 1259–1267. Tully, T.N., Shane, S.M., 1996. Husbandry practices as related to infectious and parasitic diseases of farmed ratites. Rev. Sci. Tech. Off. Int. Epiz. 15, 73–89.
Vitazkova, S.K., Wade, S.E., 2006. Parasites of free-ranging Black Howler Monkeys (Alouatta pigra) from Belize and Mexico. Am. J. Primatol. 68, 1089–1097. Volotão, A.C.C., Souza Júnior, J.C., Grassini, C., Peralta, J.M., Fernandes, O., 2008. Genotyping of Giardia duodenalis from Southern Brown Howler Monkeys (Alouatta clamitans) from Brazil. Vet. Parasitol. 158, 133–137. Wielinga, C.M., Thompson, R.C.A., 2007. Comparative evaluation of Giardia duodenalis sequence data. Parasitology 134, 1795–1821. Yee, J., Dennis, P.P., 1992. Isolation and characterization of a NADP dependent glutamate dehydrogenase gene from the primitive eukaryote Giardia lamblia. J. Biol. Chem. 267, 7539–7544.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Genotyping of potentially zoonotic Giardia duodenalis from exotic and wild animals kept in captivity in Brazil Rodrigo Martins Soares ∗ , Sílvio Luís Pereira de Souza, Luciane Holsback Silveira, Mikaela Renata Funada, Leonardo José Richtzenhain, Solange M. Gennari Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508-270 São Paulo, SP, Brazil
a r t i c l e
i n f o
Article history: Received 21 April 2010 Received in revised form 8 February 2011 Accepted 29 March 2011 Keywords: Giardia duodenalis Wild animals Glutamate dehydrogenase Molecular identification Sequencing Brazil
a b s t r a c t We have studied the variability of glutamate dehydrogenase (gdh) and small subunit ribosomal (SSU) rRNA coding genes of Giardia species in fecal samples isolated from wild and exotic animals in Brazil, and compared with homologous sequences of isolates from human and domestic animals characterized in previous studies. Cysts of Giardia duodenalis were obtained from feces of naturally infected monkeys (Alouatta fusca) (n = 20), chinchillas (Chinchilla lanigera) (n = 3), ostriches (Struthio camelus) (n = 2) and jaguar (Panthera onca) (n = 1). Assemblage AI was assigned to the unique isolate of jaguar. All the samples from monkeys, chinchillas, and ostriches were assigned to Assemblage B. There was little evolutionary divergence between the referred isolates and isolates described elsewhere. The Assemblage B isolates identified in this study were closely related to Assemblage BIV isolated from humans. The molecular identification of Assemblages A and B of G. duodenalis isolates from exotic and wild animals demonstrates that such hosts may be a potential reservoir for zoonotic transmission of G. duodenalis. © 2011 Elsevier B.V. All rights reserved.
1. Short communication Giardia duodenalis (syn: Giardia lamblia, Giardia intestinalis) is a ubiquitous enteric protozoan that infects humans, domestic animals and wildlife worldwide (Thompson, 2000). Although G. duodenalis isolates from different host species are morphologically indistinguishable, they can be differentiated by PCR-based procedures in conjunction with analysis of housekeeping genes including the genes coding for glutamate dehydrogenase (gdh), elongation factor-␣ and triose phosphate isomerase (Yee and Dennis, 1992; Monis et al., 1996; Cacció et al., 2002; Sulaiman et al., 2003). Molecular genetic studies using these markers have demonstrated that G. duodenalis is a species complex that comprises at least seven assemblages, identified from A to G (Monis et al., 1999, 2003).
∗ Corresponding author. Tel.: +55 11 3091 7653; fax: +55 11 3091 7653. E-mail address: [email protected] (R.M. Soares). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.03.049
Primates of the genus Allouata are characteristic of the New World. This genus has eight species distributed from Mexico to Argentina. One of the species occurring in Brazil, from the state of Bahia to the state of Rio Grande do Sul is the Alouatta fusca (howler monkey). Monkeys of the Allouata genus may harbor the two zoonotic assemblages of G. duodenalis (Assemblages A and B). Both zoonotic assemblages were found in stool samples of wild black howler monkeys (Alouatta pigra), in Belize (Vitazkova and Wade, 2006). Screening of isolates of G. duodenalis from asymptomatic southern brown howler monkeys (Alouatta clamitans) kept in captivity in South Brazil has shown the sub-assemblage AI (Volotão et al., 2008). Little is known about diseases that affect Ostriches (Struthio camelus) in Brazil, in particular those caused by protozoa. Reports of hexamitiasis in ratites concern ostriches and rheas, and are described as Giardia sp. (Clipsham, 1995; Tully and Shane, 1996; Huchzermeyer, 1999) and Hexamita-like flagellates (Tully and Shane, 1996; Huchzermeyer, 1999). Several protozoan species
R.M. Soares et al. / Veterinary Parasitology 180 (2011) 344–348
of the gastrointestinal tract have already been found in ostriches, but most of them needed to be confirmed as ratite specific parasites. Chinchilla lanigera is a rodent native to Chile which is bred for commercial purposes. Parasitic diseases, particularly giardiasis, may cause clinical and sanitary problems and lead to production and economic losses. A survey in a commercial breeding facility in southern Brazil revealed 36.4% (80/220) of animals shedding Giardia spp. cysts (Fialho et al., 2008). Like the ostriches, C. lanigera is also bred for commercial purposes in Brazil. The jaguar (Panthera onca) is the largest feline of the Americas. This felid is currently found from Mexico (in the coastal plains) to northern Argentina, and inhabits ecosystems such as the Atlantic Forest, Cerrado, Pantanal and Amazon. Recently, a fecal sample from a young panther was found to be positive for Giardia cysts. The diagnosis was performed in the Laboratory of Parasitic Diseases (Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo). The animal approximately two months old was being held captive and had diarrhea (unpublished data). To date, no genotyping studies using stocks from any of the above species, except monkeys, have been carried out in Brazil. The present study was aimed to compare, in terms of nucleotide diversity, the genetic sequences of gdh coding sequences of such isolates with homologous sequences of isolates from Brazil and other parts of the world. Cysts of G. duodenalis were obtained from feces of naturally infected howler monkeys (A. fusca) (n = 20), chinchillas (C. lanigera) (n = 3), ostriches (S. camelus) (n = 2) and jaguar (P. onca) (n = 1). The monkeys were sampled in a Center for the Study and Management of Wild Animals. The animals had been kept in captivity. Fecal samples from chinchilla and ostriches were collected from animals bred for commercial purposes. The feline was obtained from a state park for the management and recovery of wild animals. All animals in the samples were symptomatic. Except for the sample taken from the two-month-old young jaguar, all other samples were taken from adult animals of unknown age. The feces were examined for cysts by a conventional flotation method, using zinc sulfate solution. Floated material was transferred to a slide and examined by light microscopy. DNA extraction of the cysts, PCR amplification of a segment of 1190 bp within the gdh gene, and sequencing reactions of PCR products were performed exactly as described previously (Souza et al., 2007). PCR amplification of a segment of 292 bp within the small subunit rDNA (SSU-rRNA) coding sequences, and sequencing reactions of PCR products were also performed (Hopkins et al., 1997). The genetic sequences were deposited in GenBank (accession HM134192–HM134217). Multiple alignments from gdh nucleotide sequences and SSUrDNA were made using homologous sequences retrieved from Genbank. Genetic sequences of Assemblage A from Brazilian origin were compared with gdh sequences of the subassemblages and genotypes described by Cacció et al. (2008). These gdh genotypes were named AI-1, AI-5, AII-2, AII-3, AII-4, AII-5 and AIII-6.
345
Molecular characterization of the genetic sequences of Assemblage B was performed with phylogenetic reconstructions using the gdh segment from the nucleotide positions 258–650 (from the initial gene positions). Eight sequences of G. duodenalis of monkeys were not included in this analysis because they were shorter than the rest (Afu01 to Afu10). In order to demonstrate the phylogenetic relationships of parasite isolates of Assemblage B, the dataset gdh was analyzed by SplitsTree4 (Huson, 1998; Huson and Bryant, 2006). The result is shown as a reticulated network, which is an alternative to the traditional bifurcating phylogenetic tree in describing complex relationships in population biology (Morrison, 2005). Assemblage AI was assigned to the unique isolate of P. onca (Pon01). Novel substitutions in positions were detected, which occurred in the last third of the gdh coding sequences. The novel substitutions were observed at positions 993 and 1011. Three isolates from Brazil, Pon01, B4, and F11 (from P. onca, bovine and cat, respectively) share the same nucleotide at position 993, whereas three isolates from cats of Brazil (F5, F12, and F14) share the same nucleotide at position 1011. These isolates may constitute new genotypes but these could be confirmed only after other constitutive genes are sequenced. Isolates Pon01and B4 are identical and share the same character at position 621 (Suppl. data S1). At this site, these isolates differ from all the other isolates of sub-assemblage AI and share the same character with isolates from sub-assemblage AII. These results show that different mammals such as felines and cattle may become infected with the same genotype, suggesting that the genotype AI-1 has low hostspecificity. The SSU-rRNA coding sequences of the isolate Pon01 had 100% identity with the Assemblage A isolates Cat2BAC2, Portland and BAH40c11 (AF199445, M54878 and AF199446, respectively). With respect to Assemblage B, there was little evolutionary divergence between isolates from animals in this study and other isolates described elsewhere. All the samples from monkeys (n = 20), chinchillas (n = 3), and ostriches (n = 2) were assigned to Assemblage B, being closely related to isolates from humans which were previously identified as sub-assemblage BIV (Suppl. data S2). The isolates from monkeys, chinchillas and ostriches are named Afu, Cla and Sca, respectively. Such samples were also assigned to Assemblage B by SSU-rRNA analysis. All Assemblage B sequences had 100% identity with the Assemblage B isolates AMC-4, CM and BAH12c14 (U09491, U09492 and AF199447, respectively). It is worth mentioning that no mixed Assemblage A and B infections were reported among the animals in this study. A previous study from Belgium (Levecke et al., 2009) has shown a high prevalence of mixed Assemblage A and B infections in monkeys while using assemblage specific primers. In our survey assemblage specific primers were not used. However the combined results of SSU-rRNA and gdh analysis suggest that mixed infections were not present in our samples. Assemblage B isolates are usually subdivided into two sub-assemblages named BIII and BIV. Sequences of gdh gene of G. duodenalis were grouped into sub-assemblages according to the positions of their single nucleotide
346
R.M. Soares et al. / Veterinary Parasitology 180 (2011) 344–348
Fig. 1. Panel (A) NeighborNet phylogenetic network of Giardia duodenalis Assemblage B isolates from Brazil and from other parts of the world. Note that Giardia duodenalis Assemblage AI was not represented (this taxon is located in the terminal node of the interrupted branch in the bottom of the figure). The whole network is shown in panel (B). (BIV) Assemblage BIV; (BIV-like) Assemblage BIV-like; (B-central) Assemblage B-central; (BIII) Assemblage BIII; and (BIII-like) Assemblage BIII-like. There were a total of 393 positions in the final dataset.
R.M. Soares et al. / Veterinary Parasitology 180 (2011) 344–348
polymorphisms in relation to previously characterized reference isolates (Wielinga and Thompson, 2007). In the referred study, the authors divided gdh haplotypes into BIII, BIII-like, B-central, BIV and BIV-like. Gdh phylogenies cannot be used to reconstruct the evolutionary history of this assemblage because different authors sequenced distinct parts of the molecule. Gdh sequences BIII and BIII-like have characters available only between positions 258 and 650, while most of the sequences BIV and BIV-like have available sites upstream and downstream this fragment. A visual inspection of the alignment of sequences of gdh gene of Assemblage B isolates shows that isolates BIII and BIII-like are divergent from the other sub-assemblages (B-central, BIV and BIV-like) (Suppl. data S2). The sub-assemblages originally described within Assemblage B (BIII and BIV) were reproduced with the traditional bifurcating phylogenetic tree, but with low statistical support for the internal nodes (not shown). With the use of a reticulated network we were able to demonstrate sub-structuring within this assemblage (Fig. 1). All the sequences closely related to BIII and BIII-like are exclusively of human origin, whereas those related to BIV and BIV-like are of human and animal origin. Plot of similarity between fragments of gdh gene of Assemblage B reveals an extensive polymorphism at the segment downstream the nucleotide position 650 (Suppl. data S2). Thus, it will be possible to infer more robust phylogenetic reconstructions when this segment of gdh of BIII and BIII-like is sequenced. It has been demonstrated that non-human primates harbor the two zoonotic assemblages of G. duodenalis, but Assemblage B appears to be the most prevalent in European countries (Levecke et al., 2009). In a previous study in brown howler monkeys in Brazil, only sub-assemblage AI was found (Volotão et al., 2008). Although sequence data of the two studies with monkeys in Brazil are rather limited, it seems that both zoonotic assemblages are equally prevalent in howler monkeys kept in captivity. Exotic animals raised in a farm for food or other purposes were found to be susceptible to Assemblage B. Hosts as distant as chinchillas and ostriches may harbor the same sub-assemblage, which shows the low host specificity of this assemblage. Thus, the risk posed by this sub-assemblage for these animals has to be assessed in future studies. The finding of G. duodenalis Assemblage B in ostriches is interesting as birds are commonly infected with other Giardia species. However, trophozoytes of Giardia were not observed although the animals had diarrhea. Thus the actual importance of G. duodenalis for this host still has to be issued. Although three genotypic variants have been found among isolates from monkeys, they differ only marginally from the archetypes BIV (one to three nucleotide substitutions). A slight difference (only three nucleotide substitutions) between sequences of ostriches and archetypal sequences of BIV could also be observed. Gdh coding sequences of G. duodenalis of chinchillas were identical to BIV alleles of human origin. The sequence analysis of isolates of exotic and wild animals performed in the present study revealed none of
347
heterogeneous nucleotide positions within the fragment of the gdh gene. Heterogeneous templates can originate both from amplification of DNA from cysts from a mixed infection and from allelic sequence heterozygosity within single cysts (Cacció et al., 2008). Thus, homogeneous templates can only be obtained with assemblage, sub-assemblage or genotype specific primers. Herein, we employed generic primers, PCR amplification and direct sequencing of DNA from cysts, which makes the distinction between the two aforementioned conditions impossible. High prevalence of heterogeneous sequences is a commonly described phenomenon. We could not explain why the analyzed templates did not yield heterogeneous chromatograms. Acknowledgments Rodrigo M. Soares Leonardo J. Richtzenhain and Solange M. Gennari is in receipt of a productivity fellowship from CNPq. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vetpar.2011.03.049. References Cacció, S.M., De Giacomo, M., Pozio, E., 2002. Sequence analysis of the beta-giardin gene and development of a polymerase chain reactionrestriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int. J. Parasitol. 32, 1023–1030. Cacció, S.M., Beck, R., Lalle, M., Marinculic, A., Pozio, E., 2008. Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B. Int. J. Parasitol. 38, 1523–1531. Clipsham, R., 1995. Avian pathogenic flagellated enteric protozoa. Sem. Avian Exotic Pet Med. 4, 112–125. Fialho, C.G., Oliveira, R.G., Teixeira, M.C., Marques, S.M.T., Oliveira, R.G., Oliveira, R.G., Araujo, F.A.P., 2008. Comparac¸ão da infecc¸ão por protozoários em chinchila (Chinchilla lanigera) de uma criac¸ão comercial do município de Viamão-RS, Brasil, e de chinchilas no seu habitat natural, Chile. Parasitol. Latinoam. 63, 85–87. Hopkins, R.M., Meloni, B.P., Groth, D.M., Wetherall, J.D., Reynoldson, J.A., Thompson, R.C., 1997. Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality. J Parasitol. 83, 44–51. Huchzermeyer, F.W., 1999. PatologIa de avestruces y otras ratites. Ediciones Mundi-Prensa, Madrid, p. 284. Huson, D.H., 1998. SplitsTree: a program for analyzing and visualizing evolutionary data. Bioinformatics 14, 68–73. Huson, D.H., Bryant, D., 2006. Application of phylogenetic networks in evolutionary studies. Mol. Biol. Evol. 23 (2), 254–267. Levecke, B., Geldholf, P., Claerebout, E., Dorny, P., Vercammen, F., Cacció, S.M., Vercruysse, J., Geurden, T., 2009. Molecular characterization of Giardia duodenalis in captive non-human primates reveals mixed assemblage A and B infections and novel polymorphisms. Int. J. Parasitol. 39, 1595–1601. Monis, P.T., Mayrhofer, G., Andrews, R.H., Homan, W.L., Limper, L., Ey, P.L., 1996. Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus. Parasitology 112, 1–12. Monis, P.T., Andrews, R.H., Mayrhofer, G., Ey, P.L., 1999. Molecular systematics of the parasitic protozoan Giardia intestinalis. Mol. Biol. Evol. 16, 1135–1144. Monis, P.T., Andrews, R.H., Mayrhofer, G., Ey, P.L., 2003. Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin. Infect. Genet. Evol. 3, 29–38. Morrison, D.A., 2005. Networks in phylogenetic analysis: new tools for population biology. Int. J. Parasitol. 35, 567–582.
348
R.M. Soares et al. / Veterinary Parasitology 180 (2011) 344–348
Souza, S.L.P., Gennari, S.M., Richtzenhain, Pena, H.F.J., Funada, M.R., Cortez, A.D., Gregori, F., Soares, R.M., 2007. Molecular identification of Giardia duodenalis isolates from humans, dogs, cats and cattle from the state of São Paulo, Brazil, by sequence analysis of fragments of glutamate dehydrogenase (gdh) coding gene. Vet. Parasitol. 149, 258–264. Sulaiman, I.M., Fayer, R., Bern, C., Gilman, R.H., Trout, J.M., Schantz, P.M., Das, P., Lal, A.A., Xiao, L., 2003. Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg. Infect. Dis. 9, 1152–1444. Thompson, R.C.A., 2000. Giardiasis as a re-emerging infectious disease and its zoonotic potential. Int. J. Parasitol. 30, 1259–1267. Tully, T.N., Shane, S.M., 1996. Husbandry practices as related to infectious and parasitic diseases of farmed ratites. Rev. Sci. Tech. Off. Int. Epiz. 15, 73–89.
Vitazkova, S.K., Wade, S.E., 2006. Parasites of free-ranging Black Howler Monkeys (Alouatta pigra) from Belize and Mexico. Am. J. Primatol. 68, 1089–1097. Volotão, A.C.C., Souza Júnior, J.C., Grassini, C., Peralta, J.M., Fernandes, O., 2008. Genotyping of Giardia duodenalis from Southern Brown Howler Monkeys (Alouatta clamitans) from Brazil. Vet. Parasitol. 158, 133–137. Wielinga, C.M., Thompson, R.C.A., 2007. Comparative evaluation of Giardia duodenalis sequence data. Parasitology 134, 1795–1821. Yee, J., Dennis, P.P., 1992. Isolation and characterization of a NADP dependent glutamate dehydrogenase gene from the primitive eukaryote Giardia lamblia. J. Biol. Chem. 267, 7539–7544.