Evidence of heterozygosity and recombinant alleles in single cysts of <i>Giardia duodenalis</i>

Giardia duodenalis is divided into eight assemblages (named A to H). Isolates of assemblage A are divided into four sub-assemblages (AI, AII, AIII and AIV). While isolates of sub-assemblage AII are almost exclusively detected in human hosts, isolates of assemblage B are encountered in a multitude of animal hosts and humans. Here, we isolated single cysts of G. duodenalis from a human stool sample and found that one of them had overlaps of assemblage AII and B alleles and an unexpectedly high number of variants of the beta-giardin (Bg) and GLORF-C4 (OrfC4) alleles. In addition, one of the Bg alleles of that cyst had a fragment of sub-assemblage AII interspersed with fragments of assemblage B, thus indicating that this allele may be a recombinant between sequences A and B. Our results are unprecedented and put a check on the statement that different assemblages of G. duodenalis represent species with different host specificities.

Keywords:
Giardia duodenalis; micromanipulation; heterozygosity; genetic recombination; cysts

Authorship

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

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (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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Evidence of heterozygosity and recombinant alleles in single cysts of <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