Evidência de heterozigose e alelos recombinantes em cistos individualizados de <i>Giardia duodenalis</i>

A espécie Giardia duodenalis é dividida em oito grupos (nomeados de A a H). Isolados do grupo A são divididos em quatro subgrupos (AI, AII, AIII and AIV). Enquanto isolados do subgrupo AII são detectados quase exclusivamente em hospedeiros humanos, isolados do subgrupo B são encontrados em uma grande variedade de hospedeiros entre animais e humanos. Neste trabalho, foi constatado que, dentre diversos cistos individualizados de G. duodenalis provenientes de fezes de origem humana, um cisto continha os alelos AII e B e um número inesperado de variantes de alelos codificadores de beta giardina e GLORF-C4. Ainda, um dos alelos beta giardina desse cisto possuía fragmentos AII intercalando um fragmento B, indicando que esse alelo pode ser um recombinante entre alelos AII e B. Os resultados aqui apresentados são inéditos e colocam em dúvida o conceito atual de que os diferentes grupos de G. duodenalis representam espécies distintas com diferentes graus de especificidade por hospedeiros.

Palavras-chave:
Giardia duodenalis; micromanipulação; heterozigose; recombinação gênica; cistos

Autoria

Juliana Martins Aguiar

Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo – USP, São Paulo, SP, BrasilUniversidade de São PauloBrasilSão Paulo, SP, BrasilDepartamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo – USP, São Paulo, SP, Brasil

Sheila Oliveira Silva

Valdir Azevedo dos Santos

Laboratório Clínico do Hospital Universitário, Departamento de Farmácia, Universidade de São Paulo – USP, São Paulo, SP, BrasilUniversidade de São PauloBrasilSão Paulo, SP, BrasilLaboratório Clínico do Hospital Universitário, Departamento de Farmácia, Universidade de São Paulo – USP, São Paulo, SP, Brasil

Sueli Akemi Taniwaki

Tricia Maria Ferreira de Sousa Oliveira

Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo – USP, São Paulo, SP, BrasilUniversidade de São PauloBrasilSão Paulo, SP, BrasilDepartamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo – USP, São Paulo, SP, Brasil

Helena Lage Ferreira

Lara Borges Keid

Fábio Gregori

Rodrigo Martins Soares ^**Corresponding author: Rodrigo Martins Soares. Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo – USP, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508-270, São Paulo, SP, Brasil. e-mail: rosoares@usp.br

*Corresponding author: Rodrigo Martins Soares. Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo – USP, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508-270, São Paulo, SP, Brasil. e-mail: rosoares@usp.br

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Figuras

Figuras (4)

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Figure 1
Chromatograms obtained after sequencing PCR products from single cysts. Sequences are unedited raw data and the nucleotide numbers on chromatograms do not follow the nucleotide numbering of genes. In each panel, sequences at the top were derived from the forward primer and sequences at the bottom were derived from the reverse primer. (A) Gdh sequences from cyst C3; (B) Gdh sequences from cyst C2; (C) Bg sequences from cyst C2.

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Figure 2
Alignment of PCR-derived sequences from Bg. The numbers on the ruler do not follow the nucleotide numbering of the gene. Sequences starting with C2 are alleles cloned from cyst C2. Sequence starting with C1 are alleles sequenced from cyst C1. Data from GenBank: isolate GS is assigned to Giardia duodenalis assemblage B (FJ560593); and isolates JH and WB are assigned to Giardia duodenalis assemblage A (FJ560592 and XM001705373, respectively). Assemblage A sequences differed from assemblage B sequences at several sites (fixed mutations). Between sites 104 and 188, the C2_BG_CLONE8 sequence shares mutations with assemblage A. Between sites 1 and 104 and between sites 188 and 365, the C2_BG_CLONE8 sequence shares mutations with assemblage B. Dots (.) represent identity.

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Figure 3
Evolutionary relationships of Bg sequences. Sequences starting with C2 are alleles cloned from cyst C2. Sequences starting with C1 are alleles sequenced from cyst C1. The evolutionary history was inferred using the neighbor-joining method. The optimal tree with the sum of branch length = 0.90220161 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances that were used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site. The analysis involved 18 nucleotide sequences. The codon positions included were 1^st + 2^nd + 3^rd + noncoding. All positions containing gaps and missing data were eliminated. There were a total of 343 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

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Figure 4
Evolutionary relationships of OrfC4 sequences. Sequences starting with C2 are alleles cloned from cyst C2. Sequences starting with C1 are alleles sequenced from cyst C1. The evolutionary history was inferred using the neighbor-joining method. The optimal tree with the sum of branch length = 0.90220161 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances that were used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site. The analysis involved 14 nucleotide sequences. The codon positions included were 1^st + 2^nd + 3^rd + noncoding. All positions containing gaps and missing data were eliminated. There were a total of 393 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

Figure 1 Chromatograms obtained after sequencing PCR products from single cysts. Sequences are unedited raw data and the nucleotide numbers on chromatograms do not follow the nucleotide numbering of genes. In each panel, sequences at the top were derived from the forward primer and sequences at the bottom were derived from the reverse primer. (A) Gdh sequences from cyst C3; (B) Gdh sequences from cyst C2; (C) Bg sequences from cyst C2.

Figure 2 Alignment of PCR-derived sequences from Bg. The numbers on the ruler do not follow the nucleotide numbering of the gene. Sequences starting with C2 are alleles cloned from cyst C2. Sequence starting with C1 are alleles sequenced from cyst C1. Data from GenBank: isolate GS is assigned to Giardia duodenalis assemblage B (FJ560593); and isolates JH and WB are assigned to Giardia duodenalis assemblage A (FJ560592 and XM001705373, respectively). Assemblage A sequences differed from assemblage B sequences at several sites (fixed mutations). Between sites 104 and 188, the C2_BG_CLONE8 sequence shares mutations with assemblage A. Between sites 1 and 104 and between sites 188 and 365, the C2_BG_CLONE8 sequence shares mutations with assemblage B. Dots (.) represent identity.

Figure 3 Evolutionary relationships of Bg sequences. Sequences starting with C2 are alleles cloned from cyst C2. Sequences starting with C1 are alleles sequenced from cyst C1. The evolutionary history was inferred using the neighbor-joining method. The optimal tree with the sum of branch length = 0.90220161 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances that were used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site. The analysis involved 18 nucleotide sequences. The codon positions included were 1^st + 2^nd + 3^rd + noncoding. All positions containing gaps and missing data were eliminated. There were a total of 343 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

Figure 4 Evolutionary relationships of OrfC4 sequences. Sequences starting with C2 are alleles cloned from cyst C2. Sequences starting with C1 are alleles sequenced from cyst C1. The evolutionary history was inferred using the neighbor-joining method. The optimal tree with the sum of branch length = 0.90220161 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances that were used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site. The analysis involved 14 nucleotide sequences. The codon positions included were 1^st + 2^nd + 3^rd + noncoding. All positions containing gaps and missing data were eliminated. There were a total of 393 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

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Evidência de heterozigose e alelos recombinantes em cistos individualizados de <i>Giardia duodenalis</i>

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[1] *Corresponding author: Rodrigo Martins Soares. Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo – USP, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508-270, São Paulo, SP, Brasil. e-mail: rosoares@usp.br