Molecular diversity of <i>Sarcocystis</i> spp. in opossums (<i>Didelphis</i> spp.) from Southeastern and Midwestern Brazil

De Santi, Mariele; André, Marcos Rogério; Werther, Karin; Gonçalves, Luiz Ricardo; Soares, Rodrigo Martins; Herrera, Heitor Miraglia; Santos, Filipe Martins; Sano, Nayara Yoshie; Assis, William Oliveira de; Rucco, Andreza Castro; Machado, Rosangela Zacarias

doi:10.1590/S1984-29612023008

Mariele De Santi

Departamento de Patologia, Reprodução e Saúde Única, Faculdade Ciências Agrárias e Veterinárias - FCAV, Universidade Estadual Paulista - UNESP, Jaboticabal, SP, Brasil

Marcos Rogério André

Departamento de Patologia, Reprodução e Saúde Única, Faculdade Ciências Agrárias e Veterinárias - FCAV, Universidade Estadual Paulista - UNESP, Jaboticabal, SP, Brasil

Karin Werther

Departamento de Patologia, Reprodução e Saúde Única, Faculdade Ciências Agrárias e Veterinárias - FCAV, Universidade Estadual Paulista - UNESP, Jaboticabal, SP, Brasil

Luiz Ricardo Gonçalves

Imunodot Diagnósticos, Jaboticabal, SP, Brasil

Rodrigo Martins Soares

Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo - USP, Pirassununga, SP, Brasil

Heitor Miraglia Herrera

Laboratório de Biologia Parasitária, Universidade Católica Dom Bosco - UCDB, Campo Grande, MS, Brasil

Filipe Martins Santos

Laboratório de Biologia Parasitária, Universidade Católica Dom Bosco - UCDB, Campo Grande, MS, Brasil

Nayara Yoshie Sano

Programa de Pós-graduação em Ecologia e Conservação, Universidade Federal do Mato Grosso do Sul - UFMS, Campo Grande, MS, Brasil

William Oliveira de Assis

Laboratório de Biologia Parasitária, Universidade Católica Dom Bosco - UCDB, Campo Grande, MS, Brasil

Andreza Castro Rucco

Laboratório de Biologia Parasitária, Universidade Católica Dom Bosco - UCDB, Campo Grande, MS, Brasil

Rosangela Zacarias Machado ^**Corresponding author: Rosangela Zacarias Machado. E-mail: rzacariasmachado@gmail.com

Departamento de Patologia, Reprodução e Saúde Única, Faculdade Ciências Agrárias e Veterinárias - FCAV, Universidade Estadual Paulista - UNESP, Jaboticabal, SP, Brasil

http://orcid.org/0000-0002-5176-2940

*Corresponding author: Rosangela Zacarias Machado. E-mail: rzacariasmachado@gmail.com

Ethics declaration

The present study was approved by the Ethics Committees in the Use of Animals of the (hidden) (protocol number 013444/2019) and (hidden) (1558030821), and by Institute Chico Mendes for Conservation of Biodiversity (ICMBio numbers 49662-8 and 76295-1).

Conflict of interest

We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.

Figures (3)
Tables (2)

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Figure 1
Phylogenetic relationships within the Sarcocystis genus based on 965 bp fragment of ITS-1. The tree was inferred by using the maximum likelihood (ML) method with the evolutionary model Kimura 2-parameter with gamma distributed rates. The numbers at the nodes correspond to bootstrap values higher than 50% accessed with 1000 replicates. A sequence from Sarcocystis lindsayi was used as an outgroup. The sequences from the present study are marked in bold.

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Figure 2
Phylogenetic relationships within the Sarcocystis genus based on 916 pb fragment of cox1. The tree was inferred by using the maximum likelihood (ML) method with the evolutionary model Tamura 3-parameter with gamma distributed rates. The numbers at the nodes correspond to bootstrap values higher than 50% accessed with 1000 replicates. Sequences from Toxoplasma gondii, Neospora caninum and Hammondia heydorni were used as an outgroup (hidden). The sequences from the present study are marked in bold.

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Figure 3
Phylogenetic relationships within the Sarcocystis genus based on SAG2, SAG3 and SAG4. The trees were inferred by using the maximum likelihood (ML) method with the evolutionary models Jukes-Cantor (SAG2 and SAG4) and Kimura 2-parameter (SAG3) with uniform rates. The numbers at the nodes correspond to bootstrap values higher than 50% accessed with 1000 replicates. The sequences from the present study are marked in bold.

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Table 1
Polymorphism and genetic diversity of Sarcocystis spp. detected in wild-caught opossums (Didelphis albiventris and Didelphis aurita) captured in Campo Grande state of Mato Grosso do Sul, and São Paulo, state of São Paulo, Midwestern, and Southeastern Brazil, respectively.

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Table 2
Sarcocystis spp. detected in wild-caught opossums Didelphis albiventris and Didelphis aurita captured in Campo Grande state of Mato Grosso do Sul, and São Paulo, state of São Paulo, Midwestern, and Southeastern Brazil, respectively. Numbers represent the alleles identified at ITS-1 (27), cox1 (14), SAG2 (4), SAG3 (13), and SAG4 (7) at genetic diversity assessment. Superscribed letters refer to each of the clones sequenced.

Molecular marker	Size of the fragment analyzed ^* * The number of nucleotides in all the sequences was standardized based on the length of the shorter sequence.	Number of sequences analyzed	Number of variable sites (VS)	Number of Alleles (a)	GC content (%)	Allele Diversity (mean ± SD)	Nucleotide Diversity per site = π (mean ± SD)	Average Number of Nucleotide Differences between All Sequences (K)
ITS-1	1075	29	54	27	46.3	0.99 ± 0.01	0.006 ± 0.00	7.32
cox1	972	39	53	14	37.2	0.81 ± 0.04	0.013 ± 0.00	13.01
SAG2	676	9	5	4	54.9	0.80 ± 0.08	0.003 ± 0.00	2.27
SAG3	1106	13	159	13	51.8	1.00 ± 0.03	0.055 ± 0.00	55.4
SAG4	280	15	22	7	58.9	0.87 ± 0.05	0.034 ± 0.00	9.71

Samples		Species of opossum	Molecular Markers
ID	Origin of DNA	Species of opossum	ITS-1	*cox1*	SAG2	SAG3	SAG4
Dal1-CG	Sporocyst	Didelphis albiventris	N	N	N	NS	NS
Dal3-CG	Sporocyst	Didelphis albiventris	NS	N	NS	1	1
Dal5-CG	Sporocyst	Didelphis albiventris	N	N	NS	NS	2
Dal6-CG	Sporocyst	Didelphis albiventris	1^a, 2^b	1^a, 2^b, 3^c	2	2	3
Dal9-CG	Sporocyst	Didelphis albiventris	3^a, 4^b, 5^c	2^a,b	1	NS	4
Dal11-CG	Sporocyst	Didelphis albiventris	6^a, 7^b, 8^c	2^a,c, 4^b	2	3	1
Dal12-CG	Merozoite	Didelphis albiventris	9^a, 10^b, 11^c	5^a,b,c	2	4	2
Dal13-CG	Sporocyst	Didelphis albiventris	N	NS	NS	N	N
Dal16-CG	Sporocyst	Didelphis albiventris	NS	2^a, 6^b, 7^c	N	N	N
Dal18-CG	Merozoite	Didelphis albiventris	12^a, 13^b	5^a,b,c	2	5	2
Dal23-CG	Merozoite	Didelphis albiventris	12^a	2^a,b,c	3	6	1
Dal26-CG	Sporocyst	Didelphis albiventris	N	8^a 9^b,c	N	7	5
Dal27-CG	Merozoite	Didelphis albiventris	14^a, 15^b, 16^c	2^a,b,c	3	8	1
Dau28-SP	Sporocyst	Didelphis aurita	17^a, 18^b	10^b, 2^c, 5^a	N	9	4
Dau29-SP	Sporocyst	Didelphis aurita	19^a, 20^b	2^b, 11^a,	1	10	4
Dau30-SP	Sporocyst	Didelphis aurita	17^b, 21^a	12^a, 13^b	NS	11	6
Dau36-SP	Merozoite	Didelphis aurita	22^a, 23^b, 24^c	9^a,b,c	NS	12	7
Dau37-SP	Merozoite	Didelphis aurita	25^a, 26^b, 27^c	14^a, 5^b,c	4	13	6

[1] *Corresponding author: Rosangela Zacarias Machado. E-mail: rzacariasmachado@gmail.com

Brasil

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Molecular diversity of Sarcocystis spp. in opossums (Didelphis spp.) from Southeastern and Midwestern Brazil

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Abstract