Molecular characterization of Eurytrema coelomaticum in cattle from Paraná, Brazil

Caracterização molecular de Eurytrema coelomaticum em bovinos do estado do Paraná, Brasil

Gustavo Freire Figueira Victor Henrique Silva de Oliveira Alessandra Taroda Amauri Alcindo Alfieri Selwyn Arlington Headley About the authors

Abstracts

This study investigated the occurrence of Eurytremaspp. in cattle by analysis of the partial 18S rRNA gene sequence. Trematodes from 44 bovine pancreas were collected and classified based on typical morphological features. PCR assay and sequence analyses of amplified products confirmed that the trematodes classified as Eurytrema coelomaticum were phylogenetically distinct from those identified as E. pancreaticum. The results of this study represent the first molecular characterization of E. coelomaticum within the Americas, and provide an efficient method to differentiate digenean trematodes of domestic animals.

Bovine trematode; Eurytrema spp.; PCR, phylogeny


Este estudo avaliou a ocorrência de Eurytremaspp. em bovinos por meio da análise da sequência parcial do gene 18S rRNA. Trematódeos procedentes de 44 pâncreas bovinos foram coletados e classificados com base em aspectos morfológicos típicos. A técnica de PCR e a análise da sequência de nucleotídeos dos produtos amplificados confirmaram que os trematódeos classificados como Eurytrema coelomaticum eram filogeneticamente distintos daqueles identificados como E. pancreaticum. Os resultados deste estudo representam a primeira caracterização molecular de E. coelomaticum nas Américas e disponibiliza um método eficiente para diferenciar trematódeos digenéticos em animais domésticos.

Trematoda bovino; Eurytrema spp.; PCR; filogenia


Introduction

Eurytrema spp. is a fluke that parasitizes the pancreatic ducts and rarely the bile ducts of cattle, buffaloes, camels, sheep, and goats (BASCH, 1966Basch PF. Patterns of transmission of the trematode Eurytrema pancreaticum in Malaysia. Zahnarztl Prax 1966; 17(2): 234-240. PMid:5222978.; SOULSBY, 1982Soulsby EJL. Helminths, arthropods and protozoa of domesticated animals. 7th ed. London: Baillière Tindall;1982.; ISHII et al., 1983Ishii Y, Koga M, Fujino T, Higo H, Ishibashi J, Oka K, et al. Human infection with the pancreas fluke, Eurytrema pancreaticum. Am J Trop Med Hyg 1983; 32(5): 1019-1022. PMid:6625056.; MATTOS JUNIOR; VIANNA, 1987). Parasitism frequently results in epithelial hyperplasia, hypertrophy of pancreatic ducts, and periductal fibrosis that lead to eurytrematosis (TANG, 1950Tang CC. Studies on the life history of Eurytrema pancreaticum Janson, 1889. J Parasitol 1950; 36(6): 559-573. PMid:14804225. http://dx.doi.org/10.2307/3273289
http://dx.doi.org/10.2307/3273289...
; MATTOS JUNIOR; VIANNA, 1987; HEADLEY et al., 2009Headley SA, Saut JPE, Bassani CA, Sangioni LA, Birgel EH Jr, Yamamura MH. Histopathologic patterns of pancreatic lesions induced by Eurytrema coelomaticum in cattle from the central-west region of the State of Paraná, Southern Brazil. Braz J Vet Pathol 2009; 2(1): 3-7.). Although bovine eurytrematosis is more frequently associated with Eurytrema coelomaticum in Brazil (TRAVASSOS et al., 1969Travassos L, Freitas JFT, Kohn A. Trematódeos do Brasil. Mem Inst Oswaldo Cruz 1969; 67: 1-886. PMid:5397756.), E. pancreaticumhas been described in studies that have not examined the morphological characteristics of the trematode (CAMPOS et al., 1974Campos MS, Ragusa AL, Miguel O, Ishizuka MM. Correlação entre número de parasitas e peso de pâncreas em bovinos naturalmente infestados por Eurytrema pacreaticum, Janson, 1889 (Trematoda-Dicrocoeliidae). Rev Fac Med Vet Univ S Paulo 1974; 11: 295-299. http://dx.doi.org/10.11606/issn.2318-3659.v11i1p295-299
http://dx.doi.org/10.11606/issn.2318-365...
; BUSETTI et al., 1983Busetti ET, Paske A, Ruis MCE, Thomaz V, Golinelli A. Helmintos parasitos de Bubalus bubalis no estado do Paraná Brasil. Arq Bras Med Vet Zootec 1983; 35(3): 399-404.).

Most investigations relative to Eurytrema spp. in Brazil have been based on morphological characterization (TRAVASSOS et al., 1969Travassos L, Freitas JFT, Kohn A. Trematódeos do Brasil. Mem Inst Oswaldo Cruz 1969; 67: 1-886. PMid:5397756.; YAMAMURA et al., 1995Yamamura MH, Horner MR, Lopes CWG. Avaliação patológica da euritrematose em bovinos naturalmente infectados na região de Londrina, Paraná. Semina: Cienc Agrar 1995; 16(1): 89-99.; PINHEIRO et al., 2012Pinheiro J, Franco-Acuña DO, Oliveira-Menezes A, Brandolini SV, DaMatta RA, De Souza W. New insight into the morphology of Eurytrema coelomaticum (Trematoda, Dicrocoeliidae) cercariae by light, scanning, and transmission electron microscopies. Parasitol Res 2012; 111(4): 1437-1445. PMid:22763703. http://dx.doi.org/10.1007/s00436-012-2977-8
http://dx.doi.org/10.1007/s00436-012-297...
), biological cycle (BRANDOLINI; AMATO, 2001Brandolini SVPB, Amato SB. Desenvolvimento de Eurytrema coelomaticum (Giard & Billet) (Digenea, Dicrocoeliidae) em Bradybaena similaris (Férussac) (Gastropoda, Xanthonychidae). Rev Bras Zool 2001; 18(2): 499-510. http://dx.doi.org/10.1590/S0101-81752001000200021
http://dx.doi.org/10.1590/S0101-81752001...
), epidemiological trends (AZEVEDO et al., 2004Azevedo JR, Mannigel RC, Agulhon AZ, Borba TR, Barbiéri AW, Oliveira DCLD, et al. Prevalence and geographical distribution of bovine eurytrematosis in cattle slaughtered in northern Paraná, Brazil. Pesq Vet Bras 2004; 24(1): 23-26. http://dx.doi.org/10.1590/S0100-736X2004000100006
http://dx.doi.org/10.1590/S0100-736X2004...
; BASSANI et al., 2006Bassani CA, Sangioni LA, Saut JP, Yamamura MH, Headley SA. Epidemiology of eurytrematosis (Eurytrema spp. Trematoda: Dicrocoeliidae) in slaughtered beef cattle from the central-west region of the State of Paraná, Brazil. Vet Parasitol 2006; 141(3-4): 356-361. PMid:16860482. http://dx.doi.org/10.1016/j.vetpar.2006.06.003
http://dx.doi.org/10.1016/j.vetpar.2006....
), and pathological manifestations (YAMAMURA et al., 1995Yamamura MH, Horner MR, Lopes CWG. Avaliação patológica da euritrematose em bovinos naturalmente infectados na região de Londrina, Paraná. Semina: Cienc Agrar 1995; 16(1): 89-99.; HEADLEY, 2000Headley SA. Bovine eurytrematosis: life cycle, pathologic manifestations and public health considerations. Iniciac Cient CESUMAR 2000; 2(2): 59-62.; HEADLEY et al., 2009Headley SA, Saut JPE, Bassani CA, Sangioni LA, Birgel EH Jr, Yamamura MH. Histopathologic patterns of pancreatic lesions induced by Eurytrema coelomaticum in cattle from the central-west region of the State of Paraná, Southern Brazil. Braz J Vet Pathol 2009; 2(1): 3-7.). Additionally, there are also descriptions of Eurytrema spp. associated with chronic wasting disease (RACHID et al., 2011Rachid MA, Aquino Neto HM, Facury-Filho EJ, Carvalho AU, Valle GR, Vasconcelos AC. Chronic interstitial pancreatitis and chronic wasting disease caused by Eurytrema coelomaticum in Nelore cow. Arq Bras Med Vet Zootec 2011; 63(3): 741-743. http://dx.doi.org/10.1590/S0102-09352011000300028
http://dx.doi.org/10.1590/S0102-09352011...
) and simultaneous intoxication due to chronic seneciosis (HEADLEY et al., 2004Headley SA, Saut JPE, Gomes DA, Silva DRM, Almeida IA, Sangioni LA. Simultaneous chronic seneciosis and eurytrematosis in a cow. Semina: Cienc Agrar 2004; 25(2): 131-138.) in cattle. However, there are no studies relative to the molecular characterization of Eurytremaspp. from Brazil.

Recent studies have demonstrated that molecular analysis is an important tool for the identification (OTRANTO et al., 2007Otranto D, Rehbein S, Weigl S, Cantacessi C, Parisi A, Lia RP, et al. Morphological and molecular differentiation between Dicrocoelium dendriticum (Rudolphi, 1819) and Dicrocoelium chinensis (Sudarikov and Ryjikov, 1951) Tang and Tang, 1978 (Platyhelminthes: Digenea). Acta Trop 2007; 104(2-3): 91-98. PMid:17803950. http://dx.doi.org/10.1016/j.actatropica.2007.07.008
http://dx.doi.org/10.1016/j.actatropica....
) and phylogenetic analyses (CAI et al., 2012Cai Z, Zhang Y, Ye X. Phylogenetic relationships of the genus Eurytrema from domestic and wild animal based on 18S rRNA sequences. Parasitol Res 2012; 111(4): 1637-1644. PMid:22760238. http://dx.doi.org/10.1007/s00436-012-3002-y
http://dx.doi.org/10.1007/s00436-012-300...
) of trematodes. Moreover, partial fragments of 18S rRNA gene were successfully used to design phylogenetic trees and study the different orders of digenean trematodes (FERNANDEZ et al., 1998; ZHENG et al., 2007Zheng Y, Luo X, Jing Z, Hu Z, Cai X. Comparison of 18S ribosomal RNA gene sequences of Eurytrema coelmaticum and Eurytrema pancreaticum. Parasitol Res 2007; 100(3): 645-646. PMid:17004097. http://dx.doi.org/10.1007/s00436-006-0281-1
http://dx.doi.org/10.1007/s00436-006-028...
). This study determined the species of Eurytrema present in cattle from northern Paraná, Brazil by the molecular characterization of the partial fragment of the 18S rRNA gene of the digenean trematode.

Materials and Methods

Study location, trematodes sampling, collection, and morphological identification

All trematodes were collected from individual cows submitted for slaughter at a municipal abattoir located in northern Paraná, southern Brazil. All cattle were of different breeds, sex, and ages, and originated from different geographical regions of the state of Paraná, including Campo do Tenente (25° 58′ 41″ S; 49° 40′ 58″ W), Borrazópolis (23° 56′ 28″ S; 51° 35′ 15″ W), and Jaboti (23° 44′ 36″ S; 50° 04′ 33″ W), and Jandaia do Sul (23° 36′ 11″ S; 51° 38′ 36″ W).

All pancreas were evaluated for the presence of trematodes, after which pools of two trematodes were used for DNA extraction and subsequent PCR analysis. The number of parasites used for molecular investigation was determined by the sample weight required for DNA extraction. From each pool of parasites, an equal number of trematodes were prepared for identification based on typical morphological characteristics (TRAVASSOS et al., 1969Travassos L, Freitas JFT, Kohn A. Trematódeos do Brasil. Mem Inst Oswaldo Cruz 1969; 67: 1-886. PMid:5397756.; YAMAMURA et al., 1995Yamamura MH, Horner MR, Lopes CWG. Avaliação patológica da euritrematose em bovinos naturalmente infectados na região de Londrina, Paraná. Semina: Cienc Agrar 1995; 16(1): 89-99.).

DNA extraction, PCR, sequencing, and phylogenetic analyses

Trematode DNA was extracted by using the Easy-DNA Kit-Life (Invitrogen Corp. Carlsbad, CA, USA), and then used in a PCR assay designed to amplify the partial 18S rRNA gene of Eurytrema spp. (ZHENG et al., 2007Zheng Y, Luo X, Jing Z, Hu Z, Cai X. Comparison of 18S ribosomal RNA gene sequences of Eurytrema coelmaticum and Eurytrema pancreaticum. Parasitol Res 2007; 100(3): 645-646. PMid:17004097. http://dx.doi.org/10.1007/s00436-006-0281-1
http://dx.doi.org/10.1007/s00436-006-028...
); positive controls were included in all PCR assays. Nuclease free water (Invitrogen Corp. Carlsbad, CA, USA) was used as the negative control. The PCR assay was performed as described (ZHENG et al., 2007Zheng Y, Luo X, Jing Z, Hu Z, Cai X. Comparison of 18S ribosomal RNA gene sequences of Eurytrema coelmaticum and Eurytrema pancreaticum. Parasitol Res 2007; 100(3): 645-646. PMid:17004097. http://dx.doi.org/10.1007/s00436-006-0281-1
http://dx.doi.org/10.1007/s00436-006-028...
), with slight adaptation. Briefly, the PCR protocol consisted of an initial denaturing cycle of 94°C for 5 min, 35 cycles of 45 s at 94°C, 45 s at 55°C, 2 min at 72°C followed by 72°C for 10 min. All PCR products were separated by electrophoresis in 2% agarose gels, stained with ethidium bromide, and examined under ultraviolet light.

The amplified PCR products were then purified (illustra GFX PCR DNA and Gel Band Purification Kit, GE Healthcare, Little Chalfont, Buckinghamshire, UK) and submitted for direct sequencing using the forward and reverse primers. The obtained sequences were examined for quality analysis of chromatogram readings by using the PHRED software (http://asparagin.cenargen.embrapa.br/phph); sequences were only accepted if base quality was equal to or greater than 20. Consensus sequences were then generated by the CAP3 program (http://asparagin.cenargen.embrapa.br/cgi-bin/phph/cap3.pl). The partial nucleotide sequences obtained were initially compared by the BLAST (http://www.ncbi.nlm.nih.gov/BLAST) program with similar sequences deposited in GenBank. Phylogenetic tree and sequence alignments based on the 18S rRNA gene of the digenean trematodes family were then created by using MEGA 5.2 (TAMURA et al., 2011Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA 5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 2011; 28(10): 2731-2739. PMid:21546353 PMCid:PMC3203626. http://dx.doi.org/10.1093/molbev/msr121
http://dx.doi.org/10.1093/molbev/msr121...
), constructed by the neighbor-joining method, based on 1,000 bootstrapped data sets; distance values were calculated by using the Kimura 2 parameter model. Fasciola gigantica was used as the out-group to provide stability to the generated tree. The sequence identity was generated by using the software BioEdit (HALL, 1999Hall TA. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp 1999; 41: 95-98.).

Results

Trematodes and morphological classification

The pancreas of 44 cows from the cities of Campo do Tenente (n=4), Jandaia do Sul (n=7), Borrazópolis (n=15), and Jaboti (n=18) contained trematodes, resulting in 13 samples that were used for molecular analyses. All trematodes collected were classified as E. coelomaticum based on typical morphological features (TRAVASSOS et al., 1969Travassos L, Freitas JFT, Kohn A. Trematódeos do Brasil. Mem Inst Oswaldo Cruz 1969; 67: 1-886. PMid:5397756.; YAMAMURA et al., 1995Yamamura MH, Horner MR, Lopes CWG. Avaliação patológica da euritrematose em bovinos naturalmente infectados na região de Londrina, Paraná. Semina: Cienc Agrar 1995; 16(1): 89-99.).

Molecular characterization, phylogenetic analysis, and sequence identities

The PCR assays amplified the 528 bp fragment of the 18S rRNA gene of E. coelomaticum from all pools of trematodes; thee of the 13 samples were sequenced and used in phylogenetic evaluation. Initial BLAST analyses revealed that these sequences (GenBank accession number KJ010808, KJ010809, and KJ010810) demonstrated 100% similarity with sequences deposited in GenBank. The phylogenetic analyses revealed that the isolates from this study formed a cluster that contained E. coelomaticum, E. pancreaticum (DQ401034), and Lyperosomum collurionis (AY222143). However, these isolates were more closely related to E. coelomaticum(DQ401035) and E. pancreaticum (DQ401034). The nucleotide sequences used for phylogenetic analyses during this study are given in Figure 1. Additionally, the generated matrix identity (data not show) revealed that the isolates from this study demonstrated 99.8% similarity with E. coelomaticum but 99.4% similarity with E. pancreaticum.

Figure 1.
Phylogenetic tree based on the 18S rRNA gene sequences of trematodes generated by MEGA 5.2. Fasciola gigantica was used as the out-group. The sequences derived from this study are highlighted (star).

Discussion

This study confirmed at the molecular level that all trematodes previously identified by morphological characteristics are E. coelomaticum; similar studies have been done in other geographical locations (ZHENG et al., 2007Zheng Y, Luo X, Jing Z, Hu Z, Cai X. Comparison of 18S ribosomal RNA gene sequences of Eurytrema coelmaticum and Eurytrema pancreaticum. Parasitol Res 2007; 100(3): 645-646. PMid:17004097. http://dx.doi.org/10.1007/s00436-006-0281-1
http://dx.doi.org/10.1007/s00436-006-028...
). Additionally, these findings represent the first characterization of Eurytrema spp. in the Americas by molecular techniques. The methods used during this investigation are more efficient to characterize trematodes, can demonstrate genetic variation between different species originated from different geographical regions, and reduce errors that could occur when morphological characteristics are being identified.

When the data identify matrix derived from this study was analyzed, it was shown that the sequences derived from this study were 99.8% and 99.4% similar to those of E. coelomaticum and E. pancreaticum, respectively, observed in China (ZHENG et al., 2007Zheng Y, Luo X, Jing Z, Hu Z, Cai X. Comparison of 18S ribosomal RNA gene sequences of Eurytrema coelmaticum and Eurytrema pancreaticum. Parasitol Res 2007; 100(3): 645-646. PMid:17004097. http://dx.doi.org/10.1007/s00436-006-0281-1
http://dx.doi.org/10.1007/s00436-006-028...
). Moreover, these sequences clustered with other closely related members of the Dicrocoeliidae family, but were distant from Dicrocoelium dendriticum and D. orientalis, while these trematodes formed a cluster that was phylogenetically distant from F. gigantica; similar results were described (ZHENG et al., 2007Zheng Y, Luo X, Jing Z, Hu Z, Cai X. Comparison of 18S ribosomal RNA gene sequences of Eurytrema coelmaticum and Eurytrema pancreaticum. Parasitol Res 2007; 100(3): 645-646. PMid:17004097. http://dx.doi.org/10.1007/s00436-006-0281-1
http://dx.doi.org/10.1007/s00436-006-028...
). Unfortunately, the sequences of a recent phylogenetic investigation that analyzed the differences of Eurytrema spp. (CAI et al., 2012Cai Z, Zhang Y, Ye X. Phylogenetic relationships of the genus Eurytrema from domestic and wild animal based on 18S rRNA sequences. Parasitol Res 2012; 111(4): 1637-1644. PMid:22760238. http://dx.doi.org/10.1007/s00436-012-3002-y
http://dx.doi.org/10.1007/s00436-012-300...
) by the 18S rRNA gene were not located in GenBank, and consequently not included in this study.

In addition, the results of the polygenetic analysis suggest that the trematode E. coelomaticum from distinct geographical regions might be closely related, considering that the isolates from this study and that from China (ZHENG et al., 2007Zheng Y, Luo X, Jing Z, Hu Z, Cai X. Comparison of 18S ribosomal RNA gene sequences of Eurytrema coelmaticum and Eurytrema pancreaticum. Parasitol Res 2007; 100(3): 645-646. PMid:17004097. http://dx.doi.org/10.1007/s00436-006-0281-1
http://dx.doi.org/10.1007/s00436-006-028...
) clustered together. Alternatively, these results suggest that there is phylogenetic difference between E. coelomaticum and E. pancreaticum, since these trematodes were grouped in different branches, indicating that these are two different parasites; similar results were described when trematodes from different host were analyzed phylogenetically (CAI et al., 2012Cai Z, Zhang Y, Ye X. Phylogenetic relationships of the genus Eurytrema from domestic and wild animal based on 18S rRNA sequences. Parasitol Res 2012; 111(4): 1637-1644. PMid:22760238. http://dx.doi.org/10.1007/s00436-012-3002-y
http://dx.doi.org/10.1007/s00436-012-300...
). However, there seems to be phylogenetic differences between the same species of Eurytrema spp. from the different hosts and geographical locations (CAI et al., 2012Cai Z, Zhang Y, Ye X. Phylogenetic relationships of the genus Eurytrema from domestic and wild animal based on 18S rRNA sequences. Parasitol Res 2012; 111(4): 1637-1644. PMid:22760238. http://dx.doi.org/10.1007/s00436-012-3002-y
http://dx.doi.org/10.1007/s00436-012-300...
).

Since bovine eurytrematosis is endemic in the state of Paraná (AZEVEDO et al., 2004Azevedo JR, Mannigel RC, Agulhon AZ, Borba TR, Barbiéri AW, Oliveira DCLD, et al. Prevalence and geographical distribution of bovine eurytrematosis in cattle slaughtered in northern Paraná, Brazil. Pesq Vet Bras 2004; 24(1): 23-26. http://dx.doi.org/10.1590/S0100-736X2004000100006
http://dx.doi.org/10.1590/S0100-736X2004...
; BASSANI et al., 2006Bassani CA, Sangioni LA, Saut JP, Yamamura MH, Headley SA. Epidemiology of eurytrematosis (Eurytrema spp. Trematoda: Dicrocoeliidae) in slaughtered beef cattle from the central-west region of the State of Paraná, Brazil. Vet Parasitol 2006; 141(3-4): 356-361. PMid:16860482. http://dx.doi.org/10.1016/j.vetpar.2006.06.003
http://dx.doi.org/10.1016/j.vetpar.2006....
), and has been described in the states of Minas Gerais (RACHID et al., 2011Rachid MA, Aquino Neto HM, Facury-Filho EJ, Carvalho AU, Valle GR, Vasconcelos AC. Chronic interstitial pancreatitis and chronic wasting disease caused by Eurytrema coelomaticum in Nelore cow. Arq Bras Med Vet Zootec 2011; 63(3): 741-743. http://dx.doi.org/10.1590/S0102-09352011000300028
http://dx.doi.org/10.1590/S0102-09352011...
), Rio de Janeiro (MATTOS JUNIOR; VIANNA, 1987), Mato Grosso do Sul (YAMAMURA et al., 1995Yamamura MH, Horner MR, Lopes CWG. Avaliação patológica da euritrematose em bovinos naturalmente infectados na região de Londrina, Paraná. Semina: Cienc Agrar 1995; 16(1): 89-99.), efforts are being made to obtain examples of pancreatic trematodes of ruminants from these regions to effectively characterize the species of Eurytrema existent in Brazil.

Conclusion

The results from this study confirmed that trematodes of cattle from southern Brazil were morphologically and phylogenetically consistent with E. coelomaticum.

Acknowledgements

Amauri A. Alfieri and Selwyn A. Headley are recipients of the National Council for Scientific and Technological Development (CNPq; Brazil) fellowships.

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Publication Dates

  • Publication in this collection
    Jul-Sep 2014

History

  • Received
    16 Jan 2014
  • Accepted
    26 Feb 2014
Colégio Brasileiro de Parasitologia Veterinária FCAV/UNESP - Departamento de Patologia Veterinária, Via de acesso Prof. Paulo Donato Castellane s/n, Zona Rural, , 14884-900 Jaboticabal - SP, Brasil, Fone: (16) 3209-7100 RAMAL 7934 - Jaboticabal - SP - Brazil
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