Molecular Detection of Rickettsia spp. in Free Living Ticks in Military Instruction Areas in Southeast Brazil

Prado, Rubens Fabiano Soares; Araújo, Izabela Mesquita; Ferreira, Gilliard; Baêta, Bruna de Azevedo; Freitas, Aline Nascimento Furtado de; Guterres, Alexandro; Fonseca, Adivaldo Henrique da; Cordeiro, Matheus Dias

doi:10.1590/1678-4324-2023230052

Rubens Fabiano Soares Prado

Universidade Federal Rural do Rio de Janeiro - UFRRJ, Programa de Pós-graduação em Ciências Veterinárias, Seropédica, Rio de Janeiro, Brasil

https://orcid.org/0000-0002-6431-3677

Izabela Mesquita Araújo

Universidade Federal Rural do Rio de Janeiro - UFRRJ, Programa de Pós-graduação em Ciências Veterinárias, Seropédica, Rio de Janeiro, Brasil

https://orcid.org/0000-0002-5471-2404

Gilliard Ferreira

Universidade Federal Rural do Rio de Janeiro - UFRRJ, Programa de Pós-graduação em Ciências Veterinárias, Seropédica, Rio de Janeiro, Brasil

https://orcid.org/0000-0002-8622-849X

Bruna de Azevedo Baêta

Universidade Federal Rural do Rio de Janeiro - UFRRJ, Programa de Pós-graduação em Ciências Veterinárias, Seropédica, Rio de Janeiro, Brasil

https://orcid.org/0000-0002-0172-556X

Aline Nascimento Furtado de Freitas

Universidade Federal Rural do Rio de Janeiro - UFRRJ, Programa de Pós-graduação em Ciências Veterinárias, Seropédica, Rio de Janeiro, Brasil

https://orcid.org/0000-0002-7037-6076

Alexandro Guterres

Instituto Oswaldo Cruz - FIOCRUZ, Laboratório de Hantaviroses e Rickettsioses, Rio de Janeiro, Brasil

https://orcid.org/0000-0001-8323-1477

Adivaldo Henrique da Fonseca

Universidade Federal Rural do Rio de Janeiro - UFRRJ, Programa de Pós-graduação em Ciências Veterinárias, Seropédica, Rio de Janeiro, Brasil

https://orcid.org/0000-0002-5834-141X

Matheus Dias Cordeiro ^**Correspondence: mathcordeiro@hotmail.com, +55 21986191846 (M.D.C.)

Universidade Federal Rural do Rio de Janeiro - UFRRJ, Programa de Pós-graduação em Prática em Desenvolvimento Sustentável, Seropédica, Rio de Janeiro, Brasil

https://orcid.org/0000-0003-2045-8716

*Correspondence: mathcordeiro@hotmail.com, +55 21986191846 (M.D.C.)

Editor-in-Chief:

Paulo Vitor Farago

Associate Editor:

Marcelo Ricardo Vicari

Conflicts of interest:

The authors declare no competing or conflicting interests.

Figures (4)
Tables (3)

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Figure 1
Study area maps: Brazil, with emphasis on the state of São Paulo, Minas Gerais e Rio de Janeiro in the southerneast region; State of São Paulo, highlighting the municipality of Campinas; State of Minas Gerais, highlighting the municipality of Três Corações and Juiz de Fora. State of Rio de Janeiro, highlighting the municipality of Resende and Paracambi.

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Figure 2
Phylogenetic tree of the Rickettsia species identified in this study (in bold), based on the sequences of the gltA gene (511nt) of the genus Rickettsia, using the Maximum Likelihood method. Numbers (>70%) above branches indicate bootstrap values. Scale bars indicate an evolutionary distance of 0.04 substitutions per sequence position, and branch labels include GenBank accession numbers. The Tamura 3-parameter model with Gamma-distributed heterogeneity (T92+G) was selected as the best-fit evolutionary model.

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Figure 3
Phylogenetic tree of the Rickettsia species identified in this study (in bold), based on the sequences of the 17kDa gene of the genus Rickettsia (590 bp), using the Maximum Likelihood method. Numbers (>70%) above branches indicate bootstrap values. Scale bars indicate an evolutionary distance of 0.007 substitutions per sequence position, and branch labels include GenBank accession numbers. The Tamura 3-parameter model assuming that a certain fraction of the sites are evolutionarily invariant (T92+I) was selected as the best-fit evolutionary model.

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Figure 4
Phylogenetic tree of the Rickettsia species identified in this study (in bold), based on the sequences of the ompA gene (Rr190.70p/Rr190.701 primers) of the genus Rickettsia (590 bp), using the Maximum Likelihood method. Numbers (>70%) above branches indicate bootstrap values. Scale bars indicate an evolutionary distance of 0.007 substitutions per sequence position, and branch labels include GenBank accession numbers. The Tamura 3-parameter model assuming that a certain fraction of the sites are evolutionarily invariant (T92+I) was selected as the best-fit evolutionary model.

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Table 1
Number of tick, by stages of development and species, from MO in the Southeast region of Brazil used for molecular research of Rickettsia spp. in this study.

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Table 2
Sequences of the primers used, together with the respective target genes and the size of the amplified fragment.

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Table 3
Tick samples collected from the environment in military training areas in southeastern Brazil, positive for molecular detection of the genus Rickettsia spp., by species, stage, MO and area where it was collected.

Species/Stage	Number of tick samples tested by MO
Species/Stage	ESA	CIJF	DCMun	AMAN	11ª Bda Inf L/EsPCEx	Total
Amblyomma sp. larvae	32	43	0	44	35	154
Amblyomma sp. larvae pool	0	0	4	13	24	41
Amblyomma sculptum - nymph	238	200	299	374	378	1489
Amblyomma sculptum - adult	7	0	2	8	20	37
Amblyomma dubitatum - nymph	3	28	0	3	1	35
Amblyomma dubitatum - adult	0	1	0	3	9	13
Amblyomma brasiliense - nymph	0	0	0	42	0	42
Amblyomma brasiliense - adult	0	0	0	1	0	1
Amblyomma longirostre - nymph	0	0	0	2	0	2
Amblyomma aureolatum - nymph	2	0	0	0	0	2
Dermacentor nitens - larvae	0	0	0	7	0	7
Dermacentor nitens - larvae pool	0	0	0	26	0	26
Rhipicephalus sp. - larvae	18	0	0	0	0	18
Ixodes sp. - larvae	0	5	0	0	0	5
Haemaphysalis sp. - larvae	0	13	0	0	0	13
Total	300	290	305	523	467	1885

Gene/ Primers	Specificity	Aim	Molecular assay	*Sequence of primers* (5’-3’)**	Amplified fragment	Reference
16SrRNA	Family Ixodidae
16S+		Quality of the extracted DNA	cPCR	CCGGTCTGAACTCAGATCAAGT	460 bp	[²⁴24 Mangold AJ, Bargues MD, Mas-Coma S. Mitochondrial 16S rDNA sequences and phylogenetic relationships of species of Rhipicephalus and other tick genera among Metastriata (Acari: Ixodidae). Parasitol Res. 1998;84:478-84.]
16S-		Quality of the extracted DNA	cPCR	GCTCAATGATTTTTTAAATTGCTGT	460 bp
gltA	Rickettsia spp.
CS239 F		Screening	cPCR	GCTCTTCTCATCCTATGGCTATTAT	834 bp	[⁶²62 Labruna MB, Whitworth T, Horta MC, Bouyer DH, McBride JW, Pinter A, Popov V, Gennari SM, Walker DH. Rickettsia species infecting Amblyomma cooperi ticks from an area in the state of São Paulo, Brazil, where spotted fever is endemic. J Clin Microbiol, 2004; 42(1): 90-8.]
CS1069 R		Screening	cPCR	CAGGGTCTTCGTGCATTTCTT	834 bp
CSF1		Characterization	nPCR	CATCCTATGGCTATTATGCTTGC	885 bp	[⁶³63 Blanco CM, Teixeira BR, Da Silva AG, De Oliveira R, Strecht CL, Ogrzewalska M, De Lemos ERS. Microorganisms in ticks (Acari: Ixodidae) collected on marsupials and rodents from Santa Catarina, Paraná, and Mato Grosso do Sul states, Brazil. Ticks and tick-borne diseases, 2017; 8(1): 90-98. https://doi.org/10.1016/j.ttbdis.2016.10.003. https://doi.org/10.1016/j.ttbdis.2016.10... ]
CSR1				TATACTCTCTATGWACRTRACC	885 bp
CSF2				CTTACCGCTATTAGAATGATTGC	572 bp
CSR2				GAGCGAKAGCTTCAAGYTCTAT	572 bp
17kDa	Rickettsia spp.
17k-5		Characterization	cPCR	GCTTTACAAAATTCTAAAAACCATATA	549 bp	[⁶²62 Labruna MB, Whitworth T, Horta MC, Bouyer DH, McBride JW, Pinter A, Popov V, Gennari SM, Walker DH. Rickettsia species infecting Amblyomma cooperi ticks from an area in the state of São Paulo, Brazil, where spotted fever is endemic. J Clin Microbiol, 2004; 42(1): 90-8.]
17k-3		Characterization	cPCR	TGTCTATCAATTCACAACTTGCC	549 bp
ompA	RSFG
ompA F1		Characterization	nPCR	GTCAGGCYCTGAAGYTAAACTT	777 bp	[⁶³63 Blanco CM, Teixeira BR, Da Silva AG, De Oliveira R, Strecht CL, Ogrzewalska M, De Lemos ERS. Microorganisms in ticks (Acari: Ixodidae) collected on marsupials and rodents from Santa Catarina, Paraná, and Mato Grosso do Sul states, Brazil. Ticks and tick-borne diseases, 2017; 8(1): 90-98. https://doi.org/10.1016/j.ttbdis.2016.10.003. https://doi.org/10.1016/j.ttbdis.2016.10... ]
ompA R1				CCATTAACTYAAGCGATTTC	777 bp
ompA F2				TGTTGTAGGRACTGCGGYTAAT	460 bp
ompA R2				ATTAGTAGCAGTAACAACAASTGCT	460 bp
Rr190.70p		Characterization	cPCR	ATGGCGAATATTTCTCCAAAA	632 bp	[⁶²62 Labruna MB, Whitworth T, Horta MC, Bouyer DH, McBride JW, Pinter A, Popov V, Gennari SM, Walker DH. Rickettsia species infecting Amblyomma cooperi ticks from an area in the state of São Paulo, Brazil, where spotted fever is endemic. J Clin Microbiol, 2004; 42(1): 90-8.]
Rr190-701		Characterization	cPCR	GTTCCGTTAATGGCAGCATCT	632 bp

Ticks					Genes
Specie	Stage	Nº	MO/ Nr Area	Positives (%)	Rickettsia spp.	RSFG
A. dubitatum	nymph	3	CIJF/ Area 1	2 (66,67)	2
A. dubitatum	nymph	1	CIJF/ Area 2	1 (100)	1
A. dubitatum	nymph	19	CIJF/ Area 3	6 (31,58)	4 2	2
A. sculptum	nymph	120	CIJF/ Area 3	1 (0,08)	1
A. sculptum	nymph	58	ESA/Area 1	1 (1,72)	1

[1] *Correspondence: mathcordeiro@hotmail.com, +55 21986191846 (M.D.C.)

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Molecular Detection of Rickettsia spp. in Free Living Ticks in Military Instruction Areas in Southeast Brazil

Abstract

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