SHORT COMMUNICATION
New Records and Human Parasitism by Ornithodoros mimon (Acari: Argasidae) in Brazil MARCELO B. LABRUNA,1,2 ARLEI MARCILI,1 MARIA OGRZEWALSKA,1 DARCI M. BARROS-BATTESTI,3 FILIPE DANTAS-TORRES,4,5 ANDRE´ A. FERNANDES,6 ROMARIO C. LEITE,6 AND JOSE M. VENZAL7
J. Med. Entomol. 51(1): 283Ð287 (2014); DOI: http://dx.doi.org/10.1603/ME13062
ABSTRACT The bat tick Ornithodoros mimon Kohls, Clifford & Jones is currently known by only few reports in Bolivia, Uruguay, Argentina, and the state of Sa˜o Paulo in southeastern Brazil. Here, we expand the distribution of O. mimon in Brazil to the states of Minas Gerais (southeastern region), Goia´s (central-western), Pernambuco, and Rio Grande do Norte (northeastern). Ticks were collected on human dwellings, where there had been repeated complains of tick bites on persons during the night. Tick bites were generally followed by intense inßammatory reactions that lasted for several weeks at the bite site. Bats and opossums were reported to inhabit the attic of the infested houses. In addition, a free-ranging opossum (Didelphis albiventris Lund) trapped in Rio Grande do Norte was found infested by argasid larvae. Based on morphological and/or molecular analysis, all ticks were identiÞed as O. mimon. From one of the sites (Tiradentes, state of Minas Gerais), 20 Þeld-collected nymphs were tested by a battery of polymerase chain reaction protocols targeting tick-borne microorganisms of the genera Babesia, Hepatozoon, Rickettsia, Borrelia, Anaplasma, Ehrlichia, and Coxiella; no tick specimen was found infected by any of these microorganism genera. The current study expands northwards the distribution of O. mimon, which has been shown to be very harmful to humans because of the intense inßammatory response that usually occurs after tick bites. KEY WORDS Ornithodoros mimon, human infestation, bat, opossum, Brazil
The argasid tick Ornithodoros mimon Kohls, Clifford & Jones was originally described from larvae collected on bats from Bolivia and Uruguay (Kohls et al. 1969). Decades later, O. mimon larvae were reported in Argentinean and Brazilian bats (Venzal et al. 2004, BarrosÐBattesti et al. 2006). More recently, Barros-Battesti et al. (2011) described the adult stages, and Landulfo et al. (2012) evaluated the life cycle of O. mimon based on specimens collected on the ceiling and walls of a household in the state of Sa˜o Paulo, southeastern Brazil, where this tick was reported to be aggressive to humans in the house. To our knowledge, there have been no additional reports of O. mimon. Here, we provide new records of O. mimon, expanding 1 Department of Preventive Veterinary Medicine and Animal Health, Faculty of Veterinary Medicine, University of Sa˜o Paulo, Sa˜o Paulo, SP, Brazil. 2 Corresponding author, e-mail:
[email protected]. 3 Laboratory of Parasitology, Butantan Institute, Sa ˜o Paulo, SP, Brazil. 4 Department of Immunology, Centro de Pesquisas Aggeu Magalha˜es-Fiocruz, Recife, PE, Brazil. 5 Universita ` degli Studi di Bari, Valenzano, Italy. 6 Department of Preventive Veterinary Medicine, School of Veterinary, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. 7 Departamento de Parasitologõ´a Veterinaria, Facultad de Veterinaria, Universidad de la Repu´ blica, Regional Norte, Salto, Uruguay.
its distribution to four additional Brazilian states. In addition, we describe Þve households where this tick was found biting humans. Materials and Methods Ticks were collected in six localities (municipalities), encompassing four states: Minas Gerais in southeastern Brazil; Goia´s in centralÐwestern Brazil; Pernambuco, and Rio Grande do Norte in northeastern Brazil (Fig. 1). In Þve of these localities, ticks were collected on human dwellings, internal walls, beds, ceilings, and attics of the houses (Fig. 2). In these Þve houses, there had been repeated complains of tick bites on persons while sleeping during the night. Tick bites were generally followed by intense inßammatory reactions that lasted for several weeks at the tick bite site (Fig. 3). In all houses, bats were reported to inhabit the attic; in two houses (in Touros and Sõ´tio dÕAbadia) there were also opossums (Didelphis sp.) living in the attic. Finally, ticks (11 larvae) were collected on a free-ranging opossum (Didelphis albiventris Lund) that was trapped in an Atlantic forest Reserve (Parque da Cidade) in Natal, RN. Ticks collected on human dwellings comprised postlarval stages (nymphs and/or adults) of Ornithodoros sp. Similarly, all human complains of tick
0022-2585/14/0283Ð0287$04.00/0 䉷 2014 Entomological Society of America
284
JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 51, no. 1
Fig. 1. Map of Brazil indicating the localities where O. mimon have been found in Brazil. The phylogenetic tree inferred from the 16S rDNA mitochondrion gene includes localities from where 16S rDNA sequences of O. mimon were generated in the current study as well as the sequence of O. mimon from Araraquara (GU198362) retrieved from GenBank. Sequences of other tick species (GenBank accession numbers in brackets) were used to support the tree. No sequence of O. mimon from Vespasiano (locality indicated by a black triangle) was available for the current study. Numbers on tree nodes indicate bootstrap support. The sequences of O. mimon were segregated into two genogroups, one marked in the map by black circles and the other marked by black squares. SP, state of Sa˜o Paulo; MG, state of Minas Gerais; GO, state of Goia´s; PE, state of Pernambuco; RN, state of Rio Grande do Norte.
bites were conÞrmed to be caused by ticks because specimens found attached to skin during the night were saved and also conÞrmed to be postlarval stages of Ornithodoros sp. Because taxonomic identiÞcation to the species level of New World Ornithodoros generally relies on morphometry and chaetotaxy of the larval stage (Jones and Clifford 1972), some of these ticks were taken alive to the laboratory where they were fed on rabbits. The resultant engorged females were left in an incubator at 25⬚C and 85% relative humidity (RH) to obtain larvae. Hatched larvae (10to 20-d-old) were mounted in HoyerÕs medium to make semipermanent slides and examined by light microscopy for morphological analyses. In all localities (except for Vespasiano), ticks were also submitted for molecular analysis for taxonomic identiÞcation. For this purpose, 1Ð2 specimens of each locality were individually submitted for DNA extraction by the Wizard Genome DNA puriÞcation kit (Promega, Madison, WI), and processed by polymer-
ase chain reaction (PCR) using primers targeting a ⬇460 bp fragment of the 16S rDNA mitochondrial gene, as previously described (Mangold et al. 1998). PCR products were sequenced in an ABI automated sequencer (Applied Biosystems/Perkin Elmer, model ABI Prism 310 Genetic, Foster City, CA), with the same primers used for PCR. Generated sequences were submitted for basic local alignment search tool (BLAST) analysis (Altschul et al. 1990) to determine closest similarities to available corresponding 16S rDNA sequences of ticks. Partial 16S rDNA sequences generated in the current study were aligned by ClustalX (Thompson et al. 1997) and manually reÞned using Genedoc (Nicholas et al. 1997) with some Argasidae sequences available in GenBank. The phylogenetic trees were constructed by the parsimony method using Paup *4.0b10 software (Swofford 2002). ConÞdence values for individual branches of the resulting trees were determined by bootstrap analysis with 1,000 replicates.
January 2014
LABRUNA ET AL.: NEW RECORDS AND HUMAN PARASITISM BY O. mimon
285
rDNA PCR protocol cited above, to verify the suitability of the extracted DNA. Results
Fig. 2. Households infested by O. mimon. (A) Researcher (arrow) collecting ticks on the walls of a house at Vespasiano, state of Minas Gerais. (B) Nymphal and adult ticks on the wall in the Vespasiano house. (C) Researcher collecting ticks in the attic of a house at Tiradentes, state of Minas Gerais. (D) Ticks found over the ceiling in the Tiradentes house. (Online Þgure in color.)
Among the ticks collected in the house of Tiradentes, DNA was also extracted individually from 20 nymphs and subsequently tested by a battery of PCR protocols targeting tick-borne microorganisms of the genera Babesia, Hepatozoon, Rickettsia, Borrelia, Anaplasma, Ehrlichia, and Coxiella, as previously described (Almeida et al. 2012). In each reaction, appropriate positive control DNA samples (the same used by Almeida et al. 2012) were used. Furthermore, DNA from the 20 nymphs was tested by the same 16S
In three municipalities (Vespasiano, Ipojuca, and Natal), larvae were identiÞed morphologically as O. mimon according to Kohls et al. (1969) and Jones and Clifford (1972). Taxonomic identiÞcation through molecular analysis was applied on ticks from Þve localities (Table 1). In all cases, the generated DNA sequences were 99% identical to the corresponding sequence of O. mimon from the state of Sa˜o Paulo, Brazil, available in GenBank (GenBank GU198362). Overall, the DNA sequences (⬇400-bp long, without primers) of O. mimon reported in the current study, generated from four Brazilian states, differed from each other by only 0 Ð 4 nucleotide substitutions. These sequences have been deposited in GenBank under GenBank KC677675ÐKC677680. Voucher specimens (larvae, nymphs, and adults) of the ticks collected in this study have been deposited in the tick collections “Colec¸ a˜o Nacional de carrapatos” under CNC-971 and 2,292Ð2,297, “Colec¸ a˜o de A´ caros do Instituto Butantan” under IBSP-10,569, and “Coleccio´n del Departamento de Parasitologõ´a Veterinaria,” Faculty of Veterinary, Universidad de la Repu´ blica, Salto, Uruguay under DPVURU-826. Phylogenetic analysis revealed two distinct branches of O. mimon in Brazil according to biogeographic origin (Fig. 1): one composed by ticks from northern parts of the country (Touros, Natal, Ipojuca, and Sitio dÕAbadia), and the other composed by ticks from southern localities (Minas Gerais, Sa˜o Paulo). The O. mimon clade presented considerable variability (1.2% divergence of sequences) between northern and southern branches. Regarding the 20 nymphs from Tiradentes that were tested for tick-borne microorganisms, no tick yielded PCR amplicons in the PCR protocols, whereas the respective positive control DNA sample validated the reactions by providing robust bands of the expected size in ethidium bromide-stained agarose gels. In addition, these 20 nymphs yielded amplicons of the expected size through the mitochondrial 16S rDNA PCR protocol, indicating that the extracted DNA was suitable. Discussion
Fig. 3. Tick bite lesions caused by O. mimon on the wrist (A) and on the ankle (B) of residents of the Tiradentesinfested house in the state of Minas Gerais. (Online Þgure in color.)
Among the six new records of O. mimon in the current study, Þve were on human dwellings where tick presence was associated with bats colonizing the attic of the house. All previous host records of O. mimon were on insectivorous bats of the genera Mimon, Eptesicus, and Histiotus (Kohls et al. 1969; Venzal et al. 2003, 2004). These bats are known to roost in tree hollows, holes, caves, and also human dwellings (Emmons and Feer 1997, DÕAuria et al. 2010). Although owners of the tick-infested houses of the current study reported the presence of bats in the attic, no bat specimen was available for taxonomic identiÞcation. Interestingly, the only report from the wild environ-
Ñ 1 nymph, 1 larva 2 nymphs 2 nymphs 2 nymphs 2 larvae Yesb Yesb Ñ Ñ Ñ Yes Nymphs, adults Nymphs Adults Nymphs, adultsd Nymphs, adults Larvae
c
b
a
Number of ticks that had a portion of their mitochondrial 16S rDNA sequenced. Larvae were obtained from a laboratory colony that was established from ticks collected in the house. Ticks were also found infesting dogs sleeping in the house. d Ticks were also collected on an opossum (Didelphis sp.) that was resting in the attic.
Yes Yes Yes Yes Yes No Bats in the attic Bats in the attic Bats, opossums in the atticc Bats, opossums in the attic Bats in the attic Opossum (trapped) Sep. 2006 Jan. 2009 Dec. 2010 Aug. 2011 March 2012 Oct. 2012
Vespasiano Ipojuca Touros SDÕAbadia Tirandentes Natal
MG PE RN GO MG RN
19o 41⬘ S; 43⬚ 54⬘ W 08o 30⬘ S; 35⬚ 00⬘ W 05o 11⬘ S; 35⬚ 27⬘ W 14o 48⬘ S; 46⬚ 15⬘ W 21o 06⬘ S; 44⬚ 10⬘ W 05o 47⬘ S; 35⬚ 15⬘ W
House indoor (wall, bed) House indoor (wall) House indoor (wall, roof) House indoor (walls, roof, bed) House indoor (wall, roof) Atlantic forest
Moleculara Morphology Municipality
State
Coordinates Locality Date
Table 1.
Data for the O. mimon ticks collected in the present study
Type
Tick habitat
Associated fauna
Complains of human infestations
Stage collected
IdentiÞcation method
JOURNAL OF MEDICAL ENTOMOLOGY
O. mimon ticks
286
Vol. 51, no. 1
ment in the current study was from an opossum trapped in an Atlantic forest Reserve in northeastern Brazil. Wild opossums typically rest during the day in tree hollows (Emmons and Feer 1997), where they could possible share the space with bats and, consequently, could become infested with O. mimon. This seemed to be the case of Touros and Sõ´tio dÕAbadia, where opossums inhabited the attic with bats. In one of these houses, an opossum was found infested by O. mimon (Table 1); therefore, it is not known if the ticks were introduced in these houses via bats or opossums. Indeed, the role of opossums in the ecology of O. mimon should be further investigated. Decades ago, Carvalho (1942) reported infestations by Ornithodoros talaje (Gue´ rin-Meneville) in farmhouses in the state of Minas Gerais, where tick bites caused severe inßammatory response in persons. This report was based on morphological examination of adult and nymphal ticks collected inside dwellings that harbored large numbers of Histiotus velatus (I. Geoffroy) (Chiroptera: Vespertilionidae) bats. At that time, the species O. mimon was not known, and because the nymphal and adult stages of O. talaje and O. mimon cannot be separated through morphologic analysis (Jones and Clifford 1972, Barros-Battesti et al. 2011), it is quite possible that the ticks reported by Carvalho (1942) were actually O. mimon. More recently, Schumaker and Barros (1995) evaluated biological and morphological aspects of an O. talaje colony started with specimens collected from the attic of a house in the state of Minas Gerais. An optical microscopy picture of a larva presented by the authors is compatible with O. mimon rather than with O. talaje. In fact, a recent study proposed that O. talaje is not established in Brazil, where previous reports of this species were possibly misidentiÞcation with closely related species that were further described during the last decades (Venzal et al. 2008). The current study expands the distribution of O. mimon northward because all previous records of these ticks were from Bolivia, Uruguay, Argentina, and the state of Sa˜o Paulo in southeastern Brazil. Indeed, the distribution area of this tick species should encompass much more localities than the few currently known because its primary host species (bats) have high vagility. Moreover, phylogenetic analysis revealed a distinct genogroup of O. mimon northwards. Here, we tested a relatively small sample of ticks from one O. mimon population, from which no tick-borne pathogen was found. Although this preliminary result cannot discard the possibility that some O. mimon populations are infected by tick-borne pathogens, the simple presence of this tick in close contact with humans has shown to be very harmful because of the intense inßammatory response that usually occurs after tick bites. Acknowledgments This work was supported by the Fundac¸ a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientõ´Þco e Tecnolo´ gico
January 2014
LABRUNA ET AL.: NEW RECORDS AND HUMAN PARASITISM BY O. mimon
(CNPq), and Coordenac¸ a˜o de Aperfeic¸ oamento de Pessoal de Nõ´vel Superior (CAPES).
References Cited Almeida, A. P., A. Marcili, R. C. Leite, F. A. Nieri-Bastos, L. N. Domingues, J. R. Martins, and M. B. Labruna. 2012. Coxiella symbiont in the tick Ornithodoros rostratus (Acari: Argasidae). Ticks Tick Borne Dis. 3: 203Ð206. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403Ð 410. Barros-Battesti, D. M., M. Arzua, and G. H. Bechara. 2006. Carrapatos de importaˆncia me´ dico-veterina´ria da Regia˜o Neotropical: Um guia ilustrado para identiÞcac¸ a˜o de espe´ cies. Vox/International Consortium on Ticks and TickBorne Diseases (ICTTD-3)/Butantan, Sa˜o Paulo, Brazil. Barros-Battesti, D. M., G. A. Landulfo, V. C. Onofrio, J.L.H. Faccini, A. Marcili, F. A. Nieri-Bastos, J. M. Venzal, and M. B. Labruna. 2011. Carios mimon (Acari: Argasidae): description of adults and redescription of larva. Exp. Appl. Acarol. 54: 93Ð104. Carvalho, J.C.M. 1942. Note on the association of the tick Ornithodoros talaje (Gue´ rin-Meneville), with infestations in human dwellings in Brazil. J. Parasitol. 28: 165. D’Auria, S. R., M. C. Camargo, R. C. Pacheco, E. S. Savani, M. A. Dias, A. R. da Rosa, M. F. de Almeida, and M. B. Labruna. 2010. Serologic survey for rickettsiosis in bats from Sa˜o Paulo city, Brazil. Vector Borne Zoonotic Dis. 10: 459 Ð 463. Emmons, H., and F. Feer. 1997. Neotropical rainforest mammals, a Þeld guide, 2nd ed. University of Chicago Press, Chicago. Jones, E. K., and C. M. Clifford. 1972. The systematics of the subfamily Ornithodorinae (Acarina: Argasidae).V. A revised key to larval Argasidae of the western hemisphere and description of seven new species of Ornithodoros. Ann. Entomol. Soc. Am. 65: 730 Ð740. Kohls, G. M., C. M. Clifford, and E. K. Jones. 1969. The systematics of the subfamily Ornithodorinae (Acarina: Argasidae). IV. Eight new species of Ornithodoros from the Western Hemisphere. Ann. Entomol. Soc. Am. 62: 1035Ð1043.
287
Landulfo, G. A., L. V. Pevidor, J. Dos Santos Sampaio, H. R. Luz, V. C. Onofrio, J.L.H. Faccini, and D. M. BarrosBattesti. 2012. Life cycle of Ornithodoros mimon (Acari: Argasidae) under laboratory conditions. Exp. Appl. Acarol. 58: 69 Ð 80. Mangold, A. J., M. D. Bargues, and S. Mas-Coma. 1998. Mitochondrial 16S rDNA sequences and phylogenetic relationships of species of Rhipicephalus and other tick genera among Metastriata (Acari: Ixodidae). Parasitol. Res. 84: 478 Ð 484. Nicholas, K. B., H. B. Nicholas, and D. W. Deerfield. 1997. GeneDoc: analysis and visualization of genetic variation. Embnet News 4: 14. Schumaker, T.T.S., and D. M. Barros. 1995. Life cycle of Ornithodoros (Alectorobius) talaje (Acari: Argasidae) in laboratory. J. Med. Entomol. 32: 249 Ð254. Swofford, D. L. 2002. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Sinauer, Sunderland, MA. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX windows interface: ßexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876 Ð 4882. Venzal, J. M., E. M. Gonza´ lez, D. Capellino, A. Estrada-Pen˜ a, and A. A. Guglielmone. 2003. First record of Amblyomma triste Koch, 1844 (Acari: Ixodidae) and new records of Ornithodoros mimon Kohls, Clifford & Jones, 1969 (Acari: Argasidae) from neotropical bats. Syst. Appl. Acarol. 8: 93Ð96. Venzal, J. M., A. G. Autino, S. Nava, and A A. Guglielmone. 2004. Ornithodoros mimon Kohls, Clifford & Jones, 1969 (Acari: Argasidae) on Argentinean bats, and new records from Uruguay. Syst. Appl. Acarol. 9: 37Ð39. Venzal, J. M., A. Estrada-Pen˜ a, A. J. Mangold, D. Gonza´ lezAcun˜ a, and A. A. Guglielmone. 2008. The Ornithodoros (Alectorobius) talaje species group (Acari: Ixodida: Argasidae): description of Ornithodoros (Alectorobius) rioplatensis n. sp. from southern South America. J. Med. Entomol. 45: 832Ð 840. Received 26 March 2013; accepted 25 September 2013.
