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Influence of microhabitat use and behavior of Amblyomma sculptum and Amblyomma dubitatum nymphs (Acari: Ixodidae) on human risk for tick exposure, with notes on Rickettsia infection
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Original article
Influence of microhabitat use and behavior of Amblyomma sculptum and Amblyomma dubitatum nymphs (Acari: Ixodidae) on human risk for tick exposure, with notes on Rickettsia infection Adalberto Albuquerque Pajuaba Netoa, Vanessa do Nascimento Ramosb, Maria Marlene Martinsa, Carolina Fonseca Osavac, Jamile de Oliveira Pascoala, Adriane Suzina, Jonny Yokosawad, ⁎ Matias Pablo Juan Szabóa, a Laboratório de Ixodologia, Faculdade de Medicina Veterinária, Universidade Federal de Uberlândia, Av. Pará, 1720/Campus Umuarama-Bloco 2T, CEP 38400-902 Uberlândia, Minas Gerais, Brazil b Programa de Pós-doutorado, Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitária, São Paulo, SP CEP 05508-270, Brazil c Instituto Federal Goiano – Campus Urutaí, Rod. Geraldo S. Nascimento Km 2,5, CEP 75790-000 Urutaí, Goiás, Brazil d Laboratório de Virologia, Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Av. Pará 1720/Campus Umuarama – Bloco 2 B – CEP 38400-902 – CP 592, Uberlândia, Minas Gerais, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Amblyomma sculptum Amblyomma dubitatum Southeast Brazil Ecology behavior Tick-bite risk Rickettsia
Brazilian spotted fever (BSF) is a potentially lethal human disease caused by Rickettsia rickettsii transmitted by ticks, including Amblyomma sculptum. However, in Southeast Brazil, where most BSF cases occur, capybaras are key hosts for both A. sculptum and Amblyomma dubitatum. We therefore compared the risk of human exposure to these ticks at a non-endemic anthropogenic site in Southeast Brazil where both tick species are maintained by capybaras and occur at high abundance. Cloth dragging, human baits and CO2 traps were used to assess tick abundance and risk for human exposure. The two tick species displayed profound differences in behavior and microhabitat use. Notably, A. sculptum but not A. dubitatum quested for hosts openly from vegetation (ambush behavior) and infested human baits. Furthermore, A. dubitatum was more aggregated at a specific site whereas A. sculptum was more widespread along differing and drier microhabitats. Adults and nymphs of both species were infected with Rickettsia bellii. Overall, the results indicate that even though both species co-existed in the same area, A. sculptum posed a greater risk for biting humans and thus also for transmitting tick-borne pathogens.
1. Introduction The main human tick-borne disease in Brazil is spotted fever caused by Rickettsia rickettsii (Labruna, 2009). This potentially fatal disease is also known as Brazilian spotted fever (BSF). Two tick species, Amblyomma sculptum (referred to as Amblyomma cajennense before 2014) and Amblyomma aureolatum, are recognized as vectors of the pathogen to humans, although a potential role for other two species, Amblyomma dubitatum and Rhipicephalus sanguineus sensu lato, cannot be discounted (Szabó et al., 2013a). Most disease cases occur in areas where A. sculptum are abundant and feed on capybaras (Labruna, 2009; Szabó et al., 2013a). Nonetheless several features of BSF epidemiology are unknown and it is intriguing that in Southeast Brazil, where most BSF
cases occur, capybaras are key hosts for both A. sculptum and A. dubitatum (Queirogas et al., 2012; Souza et al., 2006). Furthermore, under experimental conditions both tick species were shown to transmit R. rickettsii to susceptible hosts (Sakai et al., 2014; Soares et al., 2012). Despite low efficiency of transovarial transmission, tick infection may result from feeding on infectious capybaras, serving as amplifier hosts, and subsequent transstadial passage (Labruna, 2009). Such R. rickettsii tick infection rate amplification was experimentally proven for A. sculptum (Souza et al., 2009). One possible explanation for A. sculptum, and not A. dubitatum, serving as a primary vector for R. rickettsii to humans is the high aggressiveness of the former species. In fact, A. cajennense (designated as A. sculptum after 2014 in southeast and central Brazil) is considered a
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corresponding author. E-mail addresses: [email protected] (A.A. Pajuaba Neto), [email protected] (V.d.N. Ramos), [email protected] (M.M. Martins), [email protected] (C.F. Osava), [email protected] (J.d.O. Pascoal), [email protected] (A. Suzin), [email protected] (J. Yokosawa), [email protected] (M.P.J. Szabó). http://dx.doi.org/10.1016/j.ttbdis.2017.10.007 Received 18 June 2017; Received in revised form 8 October 2017; Accepted 10 October 2017 1877-959X/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Pajuaba Neto, A.A., Ticks and Tick-borne Diseases (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.10.007
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major human biting tick species in Brazil (Guglielmone et al., 2006) but the lack of species identification keys for immatures (a key for nymphs in Brazil was published only in 2010 by Martins and colleagues) may have caused evaluation bias. To date A. dubitatum human bites have been recorded but the extent of such parasitism remains unclear (Labruna et al., 2007). Thus, considering that both tick species coexist throughout many capybara territories in southeast Brazil where BSF occurs, the potential for human exposure to both tick species should be examined in greater detail to enhance our knowledge of the risks of tick-borne diseases, particularly of BSF. Therefore, we herein compare risk of human exposure to A. sculptum and A. dubitatum, and identify their associated Rickettsia, at an anthropogenic area that is non-endemic for BSF and where populations of both tick species are maintained by capybaras.
2. Materials and methods 2.1. Study location The study was conducted on the grounds of a social and sports club (Praia Clube −18°55′49”S; 48°17′ 33”W) in Uberlândia city, Minas Gerais State, Southeast Brazil. Uberlândia is a major town in Southeast Brazil (circa 700,000 inhabitants) and is not endemic for Brazilian spotted fever. The town is within the “Cerrado” biome, a savannah, and has a tropical climate with two distinct seasons; a hot and rainy summer and a cool and dry winter. The club is located along, approximately, 1100 m and on both margins of the Uberabinha River. This river flows over several kilometers within the city boundaries and most of its margins are covered by vegetation from a few to several meters wide. Vegetation is heterogeneous, from grassy to several patches of a riparian forest. Several capybara (Hydrochoerus hydrochaeris) groups inhabit river margins within the city (Queirogas et al., 2012).
Fig. 1. Locations of tick sampling. (A) River side with several high and dense bamboo tussocks: (1) 1.5 m from the fence and (2) immediately besides the fence; (B) Club side with 4 m wide grass area from the fence to a blue concrete path, and additional 14 m wide grass area until a jogging path: (3) the ivy on the fence, (4) beside the fence, (5) 4 m from the fence and (6) 6 m from the fence after the concrete walking path. Direction of cloth dragging is indicated by yellow arrows. Black arrows show the direction of CO2 traps. Human bait locations are indicated by stars. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2.2. Site and time of tick sampling Tick sampling occurred twice, a preliminary one, in March 2015, and the other in August 2015. The preliminary one, in summer, evaluated infestation sites along the riverside within the club and determined the main tick species. Adult stages of Amblyomma prevail in this season in Brazil (Labruna et al., 2009) and therefore species identification is easier. This sampling occurred along the full extension of the club on both sides of the fence that separates the club from the river. The second sampling focused on evaluating human tick-bite risk and was conducted on a single morning in August 2015. This sampling occurred at the most heavily infested site of the Uberabinha River as determined by the preliminary sampling. August is in the middle of winter in Brazil, when nymphs of A. sculptum are found in high numbers. This tick species and life stage commonly infest humans (Ramos et al., 2014) and is associated with spotted-fever occurrence (Labruna, 2009). This second sampling, occurred parallel to the river, on both sides of a 1.3 m high and ivy-covered fence that separates the club from the river margin and along, approximately, 130 m (Fig. 1). Along the studied path, the river margin was high precluding flooding in the wet season, 6–10 m wide until the fence, and had several high and dense bamboo tussocks that provided a constant shadow precluding the growth of other vegetation. Thus, the ground was mostly devoid of vegetation and partially covered with bamboo leaves. A group of capybaras frequently used this shadowed area to rest and was restricted to the river side by the fence. The other side of the fence (the club side) had a four-meter-wide grass area from the fence until a concrete path (two meter-wide), and then an additional 14 m-wide grass area until a jogging path and a sports field. This whole area by the club side was fully exposed to the sun light and capybaras could not access it.
2.3. Tick sampling methods Three sampling methods, cloth dragging, human bait and CO2 traps were used. Cloth dragging is used to sample ticks questing on the tips of vegetation and trying to maximize contact with hosts that are passing by. For our sampling, a one-meter-wide and two-meter-long white cotton flannel was pulled over the ground vegetation or over the ivycovered fence and examined every 10–20 m of dragging to collect ticks. The second technique, human bait, was used to evaluate attraction of ticks specifically to this host when stationary in one spot. Such human behavior is common during sunbathing, fishing, hunting, picnicking and others. Human baits consisted of five of the authors each sitting on a white flannel (50 cm × 100 cm) for approximately 30 min, and collecting ticks crawling towards them on the flannel. The last technique used, CO2 trapping, was used to provide the actual tick population size since both targeted tick species are much attracted by such a trap (Szabó et al., 2007; Szabó et al., 2009). CO2 traps attracts both hunter ticks that are hiding but also those questing from the vegetation. Briefly, each CO2 trap consisted of a piece of white square flannel (50 × 50 cm) with 200 g of dry ice in the middle allowed to sublimate for 90 min. Ticks were collected with fine forceps and stored in ethyl alcohol-containing vials until identification in laboratory. 2.4. Preliminary sampling in March 36 CO2 traps were set up along both sides of the fence that separates 2
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the river margin from the club and on both margins of the river to determine the most tick infested location.
Table 1 Tick species, life stages and numbers collected per sampling method and location within a sports club, Uberlândia, Minas Gerais, Brazil, August 2015.
2.5. Evaluation of human tick-biting risk in August (second sampling)
Sampling method/ location
Human risk was evaluated in the location with the highest infestation, and the three tick sampling techniques described above were used in orderly fashion on a single morning in a four hours’ frame; initially cloth dragging, followed by human baits and finally CO2 traps. Cloth dragging intended to evaluate potential exposure of humans to ticks seeking hosts from the vegetation (ambush behavior). Cloth dragging was done along six 130 m-long paths, parallel to each other and to the river and fence (Fig. 1A an B). The first two paths were on the river side of the fence and the latter four on the club side and positioned as follows: 1st – river side 1.5 m from the fence, 2nd – river side immediately beside the fence, 3rd – the ivy on the fence (vertical dragging), 4th – beside the fence (club side), 5th – four meters from the fence on the club side and 6th – six meters from the fence (on the club side after the concrete walking path). To evaluate human tick attraction, five baits were equidistantly distributed along 130 m by the fence solely on the club side (Fig. 1B) and ticks collected in two time frames; 0–15 min and 15–30 min. For the last sampling, five parallel lines of four CO2 traps each, and perpendicular to the river were distributed equidistantly along the 130-m path; the first trap of each line was set up by the fence on the river margin (Fig. 1A), the second by the fence on the club side and coinciding with the previous human bait, the third trap four meters away and the fourth trap six meters away from the fence (after the concrete walking path-Fig. 1B). CO2 traps were used to assess tick populations and therefore provide baseline information for comparisons with results from the other techniques.
Dragging river side by the fence on the river side on the fence by the fence on the club side club side 4 m from the fence club side 6 m from the fence Human baits by the fence on the club side CO2 traps by the fence on the river side by the fence on the club side club side 4 m from the fence club side 6 m from the fence
Amblyomma spp.
A. dubitatum
A. sculptum
larvae
nymphs
adults
nymphs
adults
33 4
1 42
1 1
50 135
0 0
15 14
0 1
0 0
385 44
2 0
0
0
0
12
0
0
0
0
0
0
29
3
1
124
5
88
211
4
548
2
253
598
30
749
2
7
0
1
61
0
0
1
0
8
0
Amblyomma spp. The number of ticks found on each side of the fence was similar, with 110 ticks collected on the river side and 108 on the club side. However, the distribution of ticks was unequal within the club and 79.8% (n = 174) were found in one location, along the 130-mlong path that was later used to assess human tick-bite risk.
2.6. Tick identification Adults and nymphs were identified according to Onofrio et al. (2006), Nava et al. (2014), and Martins et al. (2010, 2016). Since nymphs were the targeted life stage, and currently there is no taxonomic key for Brazilian Amblyomma larvae, larvae were identified only to Genus level. Voucher specimens (646–649) were deposited in the Coleção de Carrapatos da Universidade Federal de Uberlândia.
3.2. Human tick-bite risk assessment 3.2.1. General aspects Tick species, life stages and numbers per sampling method are presented in Table 1. Overall, 3465 ticks were collected in August 2015, mainly by CO2 traps (74.0%) followed by dragging (21.4%) and human baits (4.7%). Amblyomma sculptum was the dominant tick species (70.4% of all nymphs and adults). Nymphs accounted for 85.8% of all ticks, and A. sculptum nymphs on their own made up 61.1% of all ticks. The spatial distribution of tick species and the results of sampling with the three methods differed profoundly.
2.7. Detection of Rickettsia in ticks A sample of collected ticks were submitted for DNA extraction using the guanidine isothiocyanate phenol technique (Sangioni et al., 2005) and tested for the presence of Rickettsia DNA by three different PCR protocols. Firstly, all DNA samples were tested by the amplification reaction with primers CS-78 and CS- 323, which target a conserved sequence of the citrate synthase gene (gltA) that occurs in all Rickettsia species (Labruna et al., 2004). Samples yielding a visible amplicon of the expected size were then tested by a second PCR protocol with primers Rr190.70F and Rr190.701R, which target a 632-bp segment of the 190-kDa outer membrane protein gene (ompA) (Roux et al., 1996) that is present only in Rickettsia species belonging to the Spotted Fever Group. Samples that yielded no product to the second PCR protocol were further submitted to a third one using primers specific for amplification of a 338-bp segment of the R. bellii gltA gene (Szabó et al., 2013b).
3.2.2. Dragging Out of the 740 ticks collected by dragging, 66 were Amblyomma spp. larvae. Of the remaining ticks, 46 (6.1%) were A. dubitatum whereas 628 (84.9%) were A. sculptum, overwhelmingly nymphs (Table 1). Most ticks were found on the fence itself (54.3%) and all of these were A. sculptum. Approximately 95.5% of all A. dubitatum nymphs were found by the fence on the river side and 2.3% by the fence on the club side, while the frequencies were 21.6% and 7.0%, respectively, for A. sculptum nymphs. Not a single tick and only 12 A. sculptum nymphs were collected, respectively, six and four meters from the fence on the club side. On the river side but away from the fence, one nymph and one A. dubitatum adult as well as 50 A. sculptum nymphs (8% of all A. sculptum nymphs) were found.
3. Results 3.1. Preliminary sampling
3.2.3. Human baits 162 Ticks were collected (Table 1), 25.3% in the first 15 min and 74.7% between 15 and 30 min. Most ticks (76.5%) attracted to humans were A. sculptum nymphs whereas only 2.5% of ticks were A. dubitatum. Tick numbers varied considerably from one human bait location to the
Overall, 218 ticks of two species, A. dubitatum and A. sculptum, were collected in March 2015. Of these ticks, 63.7% were A. dubitatum (54 adults and 85 nymphs) and 34.4% were A. sculptum (70 adults and 5 nymphs). An additional four larvae (1.8%) were identified as 3
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To evaluate the risk for human exposure to ticks, three methods were used during the second sampling. Non-nidicolous ticks, living in open environments as opposed to nidicolous species that live in shelters used by their hosts, exhibit two main host finding strategies; ambush or hunting (Sonenshine and Roe, 2014). Ambush species crawl up vegetation where they wait for a passing host whereas hunter ticks are attracted by CO2, ammonia and other odorants and move from their hiding places towards their host (Sonenshine and Roe, 2014). Thus, dragging rather samples ambush ticks seeking hosts from the vegetation, while CO2 attracts hunter ticks and human baits tend to attract hunter ticks that are attracted by exhaled CO2 and human odorants. Human behavior will correspondently influence exposure; ambush ticks will rather grab hosts passing by and that touch infested vegetation whereas hunter ticks will crawl toward humans that are stationary for a while. The second sampling occurred in August 2015, winter time in Brazil. As expected for this season (Labruna et al., 2009; Szabó et al., 2007) most collected ticks were nymphs. CO2 traps yielded the greatest tick numbers. However, tick collection by this method was used as the last technique to avoid depleting tick numbers before dragging and human bait collections. Higher efficacy of CO2 traps reinforced the results reported in other studies that both tick species collected are hunter ticks (Brites-Neto et al., 2013; Szabó et al., 2009). Further, this tick sampling method showed that A. sculptum was more widespread in the environment than A. dubitatum. In fact, the latter species was almost exclusively restricted to traps by the fence on the club side. The explanation for such restriction is not clear but it can be speculated that A. dubitatum was hiding in microhabitat with higher humidity since it is a tick species associated with marsh areas or those prone to flooding (Szabó et al., 2007; Queirogas et al., 2012) and the river bank at the sampling site was high precluding water pooling. The ground by the fence on the club side was covered by grass and close/below the ivycovered fence. This environment must have retained higher humidity in relation to, respectively, the shaded river side with the ground devoid of vegetation and the grass exposed to the sun on the club side. Amblyomma sculptum, on the other hand, was found in similar numbers on both sides of the fence and its numbers decreased exponentially, to four and six meters from tick source, i. e. fence, on the club side. This last observation shows that horizontal spreading of nymphs of this tick species is of, at least, six meters but with numbers decreasing sharply with each meter in the sun-exposed environment. In this context, a role for A. sculptum being spread by other animals known to use the area (small mammals or cats) cannot be disregarded but none has been ever shown to be important hosts for A. sculptum. Dragging showed that ticks seeking hosts from the vegetation (ambush behavior) were concentrated on the river side and, overwhelmingly, on the fence itself, with numbers decreasing sharply with increasing distance from the fence on the club side, similarly to those observed with CO2 traps. Furthermore, dragging collected almost exclusively A. sculptum whereas this technique yielded only 5% of all A. dubitatum. Altogether, these observations reinforce that ticks disseminate only a few meters from the infestation source and that, under the described environmental conditions, A. dubitatum ticks do not seek hosts from vegetation as ambush ticks would do, at least not in the same proportion as A. sculptum. On the other hand, high A. sculptum tick numbers on the fence clearly indicate that this tick species exhibits ambush behavior as well. Human baits were set up by the fence, which concentrated ticks on the club side and where human exposure would be more likely. At this site, humans attracted almost exclusively A. sculptum ticks, overwhelmingly nymphs, and mostly after 15 min. CO2 traps set up immediately afterwards and on the very same locations proved that A. sculptum and A. dubitatum could be attracted in similar numbers. There is no definitive explanation for the discrepancies in tick attraction between these two methods and several factors may have influenced this. For example, A. dubitatum ticks may react slower and had more time to
others, with one of them attracting close to half (41.9%) whereas another had only 4.9% of all ticks collected with this technique. 3.2.4. CO2 traps Overall, 2563 ticks were collected by CO2 traps (Table 1). Of these, 33.3% were collected by the fence on the river side, 63.7% by the fence on the club side. Most ticks were A. sculptum nymphs (53.3%) followed by A. dubitatum nymphs (31.6%). However, the spatial distributions of the two species were not homogenous; most A. dubitatum ticks (74.3% of all A. dubitatum ticks) were collected from traps by the fence on the club side, whereas A. sculptum ticks were distributed similarly on traps by both sides of the fence (40.1% and 54.8% of all A. sculptum on the river and club side, respectively). The latter species was also found, albeit in lower numbers, four and six meters from the fence on the club side (2.7% and 0.4% of all A. sculptum ticks, respectively). In the case of A. dubitatum, only one adult and one nymph were found, respectively, four and six meters from the fence on the club side. CO2 trapping was the most effective sampling method for A. dubitatum and A. sculptum and yielded, respectively, 94.5% and 64.6% of all nymphs of each tick species. The other two collection methods were less effective, and inefficient in the case of A. dubitatum nymphs. Of all collected A. dubitatum and A. sculptum nymphs, dragging yielded, respectively 5.1% and 29.6% whereas human baits 0.4% and 5.9% of each tick species. 3.2.5. Other observations Amblyomma spp. larvae represented 12.7% (N = 443) of all ticks and were collected as individuals because distinct larval clusters were not found, but rather scattered individuals. Most of these were collected from CO2 traps by the fence on the club side (57.1% of all larvae) followed by CO2 traps by the fence on the riverside (19.9% of all larvae). Adult ticks were the least numerous stage (1.4% of all ticks) and the majority was A. dubitatum collected from CO2 traps by the fence on the club side (61.2% from all adult ticks). 3.3. Rickettsia in ticks 244 nymphs (204 A. sculptum and 40 A.dubitatum) divided into 25 pools with, approximately 10 nymphs each, as well as four A. sculptum and three A. dubitatum adults were examined for Rickettsia. Four A. sculptum nymphal pools (40 nymphs) and one adult yielded visible amplicons on PCR targeting both Rickettsia’s gltA gene sequence and on the third PCR specific for the R. bellii gltA gene sequence. Similar products were obtained from one A. dubitatum nymph pool (10 nymphs) and one adult. No product was obtained from any PCR targeting the ompA gene sequence. The product of a PCR targeting the citrate synthase gene in one pool of A. dubitatum nymphs that also yielded a visible product in the third PCR (R. bellii specific) was sequenced and submitted for BLAST analysis (www.ncbi.nlm.nih.gov/Blast), to determine its similarities to the relevant sequences from identified Rickettsia (Altschul et al., 1990). The nucleotide sequence of the PCR product of this sample was 99% identical to R. bellii from South Brazil (KX137900) and Argentina (KX009410). This sequence was deposited at GenBank and assigned nucleotide accession number MF187460. 4. Discussion The preliminary sampling in March 2015 confirmed known features of tick ecology in southeast Brazil, where Praia Clube is located; capybaras at anthropogenic sites are associated with high environmental infestations of both A. sculptum and A. dubitatum and adult ticks prevail over other stages in summer (Brites-Neto et al., 2013; Queirogas et al., 2012; Souza et al., 2006). This sampling also showed the unevenness of infestations within the club, with one location harboring most of the ticks. Such intense infestations in one location may be attributed to the presence of capybaras that used it as resting place as well as adequate micro-environmental conditions for both tick species. 4
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Entomol. 26, 139–151. Sonenshine, D.E., Roe, R.M., 2014. Overview: ticks, people, and animals. In: second edition. In: Sonenshine, D.E., Roe, R.M. (Eds.), Biology of Ticks Vol. I. Oxford University Press, New York USA, pp. 3–15. Souza, S.S.A.L., Souza, C.E., Neto, E.J.R., Prado, A.P., 2006. Dinâmica sazonal de carrapatos (Acari Ixodidae) na mata ciliar de uma região endêmica para febre maculosa na região de Campinas, São Paulo, Brasil. Ciênc. Rural 36, 887–891. Souza, C.E., Moraes-Filho, J., Ogrzewalska, M., Uchoa, F.A., Horta, M.C., Souza, S.S.L., Borba, R.C.M., Labruna, M.B., 2009. Experimental infection of capybaras Hydrochaeris hydrochaeris by Rickettsia rickettsii and evaluation of the transmission of the infection to ticks Amblyomma cajennense. Vet. Parasitol. 161, 116–121. Szabó, M.P.J., Castro, M.B., Ramos, H.G.C., Garcia, M.V., Castagnolli, K.C., Pinter, A., Veronez, V.A., Magalhães, G.M., Duarte, J.M.B., Labruna, M.B., 2007. Species diversity and seasonality of free-living ticks (Acari: Ixodidae) in the natural habitat of wild Marsh deer (Blastocerus dichotomus) in Southeastern Brazil. Vet. Parasitol. 143, 147–154. Szabó, M.P.J., Labruna, M.B., Garcia, M.V., Pinter, A., Castagnolli, K.C., Pacheco, R.C., Castro, M.B., Veronez, V.A., Magalhães, G.M., Vogliotti, A., Duarte, J.M.B., 2009. Ecological aspects of free-living ticks (Acari: Ixodidae) on animal trails in an Atlantic rainforest of Southeastern Brazil. Ann. Trop. Med. Parasitol. 103, 57–72. Szabó, M.P.J., Pinter, A., Labruna, M.B., 2013a. Ecology, biology and distribution of spotted-fever tick vectors in Brazil. Front. Cell. Infect. Microbiol. 3, 27. Szabó, M.P.J., Nieri-Bastos, F.A., Spolidorio, M.G., Martins, T.F., Barbieri, A.M., Labruna, M.B., 2013b. In vitro isolation from Amblyomma ovale (Acari: Ixodidae) and ecological aspects of the Atlantic rainforest Rickettsia, the causative agent of a novel spotted fever rickettsiosis in Brazil. Parasitology 140.
reach CO2 traps that were maintained three times longer than human baits. In fact, in human baits, three of four A. dubitatum ticks were collected after 15 min, but again this number is too small for speculation. It is also possible that humans expel less CO2 then the traps and are less attractive for A. dubitatum. Whatever the case, results indicate that humans in stationary behavior are more likely to attract A. sculptum over A. dubitatum, at least during the time length of the sampling methods. Overall, differing aspects of behavior and microhabitat use of A. sculptum and A. dubitatum were observed that determined, respectively, increase and decrease, of human tick-bite risk. A. sculptum exhibited both hunting and ambush behavior as observed before (Ramos et al., 2017) whereas an ambush behavior was not apparent for A. dubitatum. This means that humans walking by infested areas will be rather exposed to A. sculptum over A. dubitatum. Furthermore, at drier areas (not flooded or marsh) A. sculptum is more widespread and the chance of human exposure is increased Considering the hunting strategy of ticks, stationary humans attract much more A. sculptum than A. dubitatum, at least within 30 min. Thus, human behaviors such as picnicking, fishing, meditating and so on will rather result in A. sculptum infestation in areas where both species are present. In this study, a high R. bellii infection prevalence was found for both A. dubitatum and A. sculptum. Rickettsia bellii is common in various hard tick species from Brazil, considered non-pathogenic (Labruna, 2009) and is highly associated with A. dubitatum tick populations where it might attain an infection prevalence of 100% (Pacheco et al., 2009; Sakai et al., 2014). This bacterium may have a role in the R. rickettsii ecology, since, at high tick infection rates it might prevent the establishment of R. rickettsii infection in tick populations by an interference mechanism (Labruna, 2009; Sakai et al., 2014), as previously described for other Rickettsia species (Macaluso et al., 2002). Thus, the outcome of A. sculptum, A. dubitatum and R. bellii coexistence on R. rickettsii transmission should be investigated further. Overall, our results highlight the potential for A. sculptum to infest humans. Thus, in BSF endemic areas with both tick species infected with Rickettsia, transmission will rather occur by exposure to A. sculptum, but occasional human encounters with A. dubitatum cannot be discounted. Acknowledgments Authors acknowledge Praia Clube, Uberlândia, Minas Gerais, for logistic and financial support. This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (Academic Career Research Fellowship to M.P.J. Szabó). Ticks were collected as authorized by Brazilian Environmental Ministry (SISBIO no. 10762-1). Permits are on files of senior researcher M P J Szabó. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Brites-Neto, J., Nieri-Bastos, F.A., Brasil, J., Duarte, K.M., Martins, T.F., Veríssimo, C.J., Barbieri, A.R., Labruna, M.B., 2013. Environmental infestation and rickettsial infection in ticks in an area endemic for Brazilian spotted fever. Rev. Bras. Parasitol. Vet. 22, 367–372. Guglielmone, A.A., Beati, L., Barros-Battesti, D.M., Labruna, M.B., Nava, S., Venzal, J.M., Mangold, A.J., Szabó, M.P.J., Martins, J.R., González-Acuña, D., Estrada-Peña, A., 2006. Ticks (Ixodidae) on humans in south america. Exp. Appl. Acarol. 40, 83–100. Labruna, M.B., Whitworth, T., Bouyer, D.H., McBride, J., Camargo, L.M.A., Camargo, E.P., Popov, V., Walker, D.H., 2004. Rickettsia bellii and Rickettsia amblyommii in Amblyomma ticks from the state of Rondônia, Western Amazon, Brazil. J. Med. Entomol. 41, 1073–1081. Labruna, M.B., Pacheco, R.C., Ataliba, A.C., Szabó, M.P.J., 2007. Human parasitism by
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Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Influence of microhabitat use and behavior of Amblyomma sculptum and Amblyomma dubitatum nymphs (Acari: Ixodidae) on human risk for tick exposure, with notes on Rickettsia infection Adalberto Albuquerque Pajuaba Netoa, Vanessa do Nascimento Ramosb, Maria Marlene Martinsa, Carolina Fonseca Osavac, Jamile de Oliveira Pascoala, Adriane Suzina, Jonny Yokosawad, ⁎ Matias Pablo Juan Szabóa, a Laboratório de Ixodologia, Faculdade de Medicina Veterinária, Universidade Federal de Uberlândia, Av. Pará, 1720/Campus Umuarama-Bloco 2T, CEP 38400-902 Uberlândia, Minas Gerais, Brazil b Programa de Pós-doutorado, Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitária, São Paulo, SP CEP 05508-270, Brazil c Instituto Federal Goiano – Campus Urutaí, Rod. Geraldo S. Nascimento Km 2,5, CEP 75790-000 Urutaí, Goiás, Brazil d Laboratório de Virologia, Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Av. Pará 1720/Campus Umuarama – Bloco 2 B – CEP 38400-902 – CP 592, Uberlândia, Minas Gerais, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Amblyomma sculptum Amblyomma dubitatum Southeast Brazil Ecology behavior Tick-bite risk Rickettsia
Brazilian spotted fever (BSF) is a potentially lethal human disease caused by Rickettsia rickettsii transmitted by ticks, including Amblyomma sculptum. However, in Southeast Brazil, where most BSF cases occur, capybaras are key hosts for both A. sculptum and Amblyomma dubitatum. We therefore compared the risk of human exposure to these ticks at a non-endemic anthropogenic site in Southeast Brazil where both tick species are maintained by capybaras and occur at high abundance. Cloth dragging, human baits and CO2 traps were used to assess tick abundance and risk for human exposure. The two tick species displayed profound differences in behavior and microhabitat use. Notably, A. sculptum but not A. dubitatum quested for hosts openly from vegetation (ambush behavior) and infested human baits. Furthermore, A. dubitatum was more aggregated at a specific site whereas A. sculptum was more widespread along differing and drier microhabitats. Adults and nymphs of both species were infected with Rickettsia bellii. Overall, the results indicate that even though both species co-existed in the same area, A. sculptum posed a greater risk for biting humans and thus also for transmitting tick-borne pathogens.
1. Introduction The main human tick-borne disease in Brazil is spotted fever caused by Rickettsia rickettsii (Labruna, 2009). This potentially fatal disease is also known as Brazilian spotted fever (BSF). Two tick species, Amblyomma sculptum (referred to as Amblyomma cajennense before 2014) and Amblyomma aureolatum, are recognized as vectors of the pathogen to humans, although a potential role for other two species, Amblyomma dubitatum and Rhipicephalus sanguineus sensu lato, cannot be discounted (Szabó et al., 2013a). Most disease cases occur in areas where A. sculptum are abundant and feed on capybaras (Labruna, 2009; Szabó et al., 2013a). Nonetheless several features of BSF epidemiology are unknown and it is intriguing that in Southeast Brazil, where most BSF
cases occur, capybaras are key hosts for both A. sculptum and A. dubitatum (Queirogas et al., 2012; Souza et al., 2006). Furthermore, under experimental conditions both tick species were shown to transmit R. rickettsii to susceptible hosts (Sakai et al., 2014; Soares et al., 2012). Despite low efficiency of transovarial transmission, tick infection may result from feeding on infectious capybaras, serving as amplifier hosts, and subsequent transstadial passage (Labruna, 2009). Such R. rickettsii tick infection rate amplification was experimentally proven for A. sculptum (Souza et al., 2009). One possible explanation for A. sculptum, and not A. dubitatum, serving as a primary vector for R. rickettsii to humans is the high aggressiveness of the former species. In fact, A. cajennense (designated as A. sculptum after 2014 in southeast and central Brazil) is considered a
⁎
corresponding author. E-mail addresses: [email protected] (A.A. Pajuaba Neto), [email protected] (V.d.N. Ramos), [email protected] (M.M. Martins), [email protected] (C.F. Osava), [email protected] (J.d.O. Pascoal), [email protected] (A. Suzin), [email protected] (J. Yokosawa), [email protected] (M.P.J. Szabó). http://dx.doi.org/10.1016/j.ttbdis.2017.10.007 Received 18 June 2017; Received in revised form 8 October 2017; Accepted 10 October 2017 1877-959X/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Pajuaba Neto, A.A., Ticks and Tick-borne Diseases (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.10.007
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major human biting tick species in Brazil (Guglielmone et al., 2006) but the lack of species identification keys for immatures (a key for nymphs in Brazil was published only in 2010 by Martins and colleagues) may have caused evaluation bias. To date A. dubitatum human bites have been recorded but the extent of such parasitism remains unclear (Labruna et al., 2007). Thus, considering that both tick species coexist throughout many capybara territories in southeast Brazil where BSF occurs, the potential for human exposure to both tick species should be examined in greater detail to enhance our knowledge of the risks of tick-borne diseases, particularly of BSF. Therefore, we herein compare risk of human exposure to A. sculptum and A. dubitatum, and identify their associated Rickettsia, at an anthropogenic area that is non-endemic for BSF and where populations of both tick species are maintained by capybaras.
2. Materials and methods 2.1. Study location The study was conducted on the grounds of a social and sports club (Praia Clube −18°55′49”S; 48°17′ 33”W) in Uberlândia city, Minas Gerais State, Southeast Brazil. Uberlândia is a major town in Southeast Brazil (circa 700,000 inhabitants) and is not endemic for Brazilian spotted fever. The town is within the “Cerrado” biome, a savannah, and has a tropical climate with two distinct seasons; a hot and rainy summer and a cool and dry winter. The club is located along, approximately, 1100 m and on both margins of the Uberabinha River. This river flows over several kilometers within the city boundaries and most of its margins are covered by vegetation from a few to several meters wide. Vegetation is heterogeneous, from grassy to several patches of a riparian forest. Several capybara (Hydrochoerus hydrochaeris) groups inhabit river margins within the city (Queirogas et al., 2012).
Fig. 1. Locations of tick sampling. (A) River side with several high and dense bamboo tussocks: (1) 1.5 m from the fence and (2) immediately besides the fence; (B) Club side with 4 m wide grass area from the fence to a blue concrete path, and additional 14 m wide grass area until a jogging path: (3) the ivy on the fence, (4) beside the fence, (5) 4 m from the fence and (6) 6 m from the fence after the concrete walking path. Direction of cloth dragging is indicated by yellow arrows. Black arrows show the direction of CO2 traps. Human bait locations are indicated by stars. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2.2. Site and time of tick sampling Tick sampling occurred twice, a preliminary one, in March 2015, and the other in August 2015. The preliminary one, in summer, evaluated infestation sites along the riverside within the club and determined the main tick species. Adult stages of Amblyomma prevail in this season in Brazil (Labruna et al., 2009) and therefore species identification is easier. This sampling occurred along the full extension of the club on both sides of the fence that separates the club from the river. The second sampling focused on evaluating human tick-bite risk and was conducted on a single morning in August 2015. This sampling occurred at the most heavily infested site of the Uberabinha River as determined by the preliminary sampling. August is in the middle of winter in Brazil, when nymphs of A. sculptum are found in high numbers. This tick species and life stage commonly infest humans (Ramos et al., 2014) and is associated with spotted-fever occurrence (Labruna, 2009). This second sampling, occurred parallel to the river, on both sides of a 1.3 m high and ivy-covered fence that separates the club from the river margin and along, approximately, 130 m (Fig. 1). Along the studied path, the river margin was high precluding flooding in the wet season, 6–10 m wide until the fence, and had several high and dense bamboo tussocks that provided a constant shadow precluding the growth of other vegetation. Thus, the ground was mostly devoid of vegetation and partially covered with bamboo leaves. A group of capybaras frequently used this shadowed area to rest and was restricted to the river side by the fence. The other side of the fence (the club side) had a four-meter-wide grass area from the fence until a concrete path (two meter-wide), and then an additional 14 m-wide grass area until a jogging path and a sports field. This whole area by the club side was fully exposed to the sun light and capybaras could not access it.
2.3. Tick sampling methods Three sampling methods, cloth dragging, human bait and CO2 traps were used. Cloth dragging is used to sample ticks questing on the tips of vegetation and trying to maximize contact with hosts that are passing by. For our sampling, a one-meter-wide and two-meter-long white cotton flannel was pulled over the ground vegetation or over the ivycovered fence and examined every 10–20 m of dragging to collect ticks. The second technique, human bait, was used to evaluate attraction of ticks specifically to this host when stationary in one spot. Such human behavior is common during sunbathing, fishing, hunting, picnicking and others. Human baits consisted of five of the authors each sitting on a white flannel (50 cm × 100 cm) for approximately 30 min, and collecting ticks crawling towards them on the flannel. The last technique used, CO2 trapping, was used to provide the actual tick population size since both targeted tick species are much attracted by such a trap (Szabó et al., 2007; Szabó et al., 2009). CO2 traps attracts both hunter ticks that are hiding but also those questing from the vegetation. Briefly, each CO2 trap consisted of a piece of white square flannel (50 × 50 cm) with 200 g of dry ice in the middle allowed to sublimate for 90 min. Ticks were collected with fine forceps and stored in ethyl alcohol-containing vials until identification in laboratory. 2.4. Preliminary sampling in March 36 CO2 traps were set up along both sides of the fence that separates 2
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the river margin from the club and on both margins of the river to determine the most tick infested location.
Table 1 Tick species, life stages and numbers collected per sampling method and location within a sports club, Uberlândia, Minas Gerais, Brazil, August 2015.
2.5. Evaluation of human tick-biting risk in August (second sampling)
Sampling method/ location
Human risk was evaluated in the location with the highest infestation, and the three tick sampling techniques described above were used in orderly fashion on a single morning in a four hours’ frame; initially cloth dragging, followed by human baits and finally CO2 traps. Cloth dragging intended to evaluate potential exposure of humans to ticks seeking hosts from the vegetation (ambush behavior). Cloth dragging was done along six 130 m-long paths, parallel to each other and to the river and fence (Fig. 1A an B). The first two paths were on the river side of the fence and the latter four on the club side and positioned as follows: 1st – river side 1.5 m from the fence, 2nd – river side immediately beside the fence, 3rd – the ivy on the fence (vertical dragging), 4th – beside the fence (club side), 5th – four meters from the fence on the club side and 6th – six meters from the fence (on the club side after the concrete walking path). To evaluate human tick attraction, five baits were equidistantly distributed along 130 m by the fence solely on the club side (Fig. 1B) and ticks collected in two time frames; 0–15 min and 15–30 min. For the last sampling, five parallel lines of four CO2 traps each, and perpendicular to the river were distributed equidistantly along the 130-m path; the first trap of each line was set up by the fence on the river margin (Fig. 1A), the second by the fence on the club side and coinciding with the previous human bait, the third trap four meters away and the fourth trap six meters away from the fence (after the concrete walking path-Fig. 1B). CO2 traps were used to assess tick populations and therefore provide baseline information for comparisons with results from the other techniques.
Dragging river side by the fence on the river side on the fence by the fence on the club side club side 4 m from the fence club side 6 m from the fence Human baits by the fence on the club side CO2 traps by the fence on the river side by the fence on the club side club side 4 m from the fence club side 6 m from the fence
Amblyomma spp.
A. dubitatum
A. sculptum
larvae
nymphs
adults
nymphs
adults
33 4
1 42
1 1
50 135
0 0
15 14
0 1
0 0
385 44
2 0
0
0
0
12
0
0
0
0
0
0
29
3
1
124
5
88
211
4
548
2
253
598
30
749
2
7
0
1
61
0
0
1
0
8
0
Amblyomma spp. The number of ticks found on each side of the fence was similar, with 110 ticks collected on the river side and 108 on the club side. However, the distribution of ticks was unequal within the club and 79.8% (n = 174) were found in one location, along the 130-mlong path that was later used to assess human tick-bite risk.
2.6. Tick identification Adults and nymphs were identified according to Onofrio et al. (2006), Nava et al. (2014), and Martins et al. (2010, 2016). Since nymphs were the targeted life stage, and currently there is no taxonomic key for Brazilian Amblyomma larvae, larvae were identified only to Genus level. Voucher specimens (646–649) were deposited in the Coleção de Carrapatos da Universidade Federal de Uberlândia.
3.2. Human tick-bite risk assessment 3.2.1. General aspects Tick species, life stages and numbers per sampling method are presented in Table 1. Overall, 3465 ticks were collected in August 2015, mainly by CO2 traps (74.0%) followed by dragging (21.4%) and human baits (4.7%). Amblyomma sculptum was the dominant tick species (70.4% of all nymphs and adults). Nymphs accounted for 85.8% of all ticks, and A. sculptum nymphs on their own made up 61.1% of all ticks. The spatial distribution of tick species and the results of sampling with the three methods differed profoundly.
2.7. Detection of Rickettsia in ticks A sample of collected ticks were submitted for DNA extraction using the guanidine isothiocyanate phenol technique (Sangioni et al., 2005) and tested for the presence of Rickettsia DNA by three different PCR protocols. Firstly, all DNA samples were tested by the amplification reaction with primers CS-78 and CS- 323, which target a conserved sequence of the citrate synthase gene (gltA) that occurs in all Rickettsia species (Labruna et al., 2004). Samples yielding a visible amplicon of the expected size were then tested by a second PCR protocol with primers Rr190.70F and Rr190.701R, which target a 632-bp segment of the 190-kDa outer membrane protein gene (ompA) (Roux et al., 1996) that is present only in Rickettsia species belonging to the Spotted Fever Group. Samples that yielded no product to the second PCR protocol were further submitted to a third one using primers specific for amplification of a 338-bp segment of the R. bellii gltA gene (Szabó et al., 2013b).
3.2.2. Dragging Out of the 740 ticks collected by dragging, 66 were Amblyomma spp. larvae. Of the remaining ticks, 46 (6.1%) were A. dubitatum whereas 628 (84.9%) were A. sculptum, overwhelmingly nymphs (Table 1). Most ticks were found on the fence itself (54.3%) and all of these were A. sculptum. Approximately 95.5% of all A. dubitatum nymphs were found by the fence on the river side and 2.3% by the fence on the club side, while the frequencies were 21.6% and 7.0%, respectively, for A. sculptum nymphs. Not a single tick and only 12 A. sculptum nymphs were collected, respectively, six and four meters from the fence on the club side. On the river side but away from the fence, one nymph and one A. dubitatum adult as well as 50 A. sculptum nymphs (8% of all A. sculptum nymphs) were found.
3. Results 3.1. Preliminary sampling
3.2.3. Human baits 162 Ticks were collected (Table 1), 25.3% in the first 15 min and 74.7% between 15 and 30 min. Most ticks (76.5%) attracted to humans were A. sculptum nymphs whereas only 2.5% of ticks were A. dubitatum. Tick numbers varied considerably from one human bait location to the
Overall, 218 ticks of two species, A. dubitatum and A. sculptum, were collected in March 2015. Of these ticks, 63.7% were A. dubitatum (54 adults and 85 nymphs) and 34.4% were A. sculptum (70 adults and 5 nymphs). An additional four larvae (1.8%) were identified as 3
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To evaluate the risk for human exposure to ticks, three methods were used during the second sampling. Non-nidicolous ticks, living in open environments as opposed to nidicolous species that live in shelters used by their hosts, exhibit two main host finding strategies; ambush or hunting (Sonenshine and Roe, 2014). Ambush species crawl up vegetation where they wait for a passing host whereas hunter ticks are attracted by CO2, ammonia and other odorants and move from their hiding places towards their host (Sonenshine and Roe, 2014). Thus, dragging rather samples ambush ticks seeking hosts from the vegetation, while CO2 attracts hunter ticks and human baits tend to attract hunter ticks that are attracted by exhaled CO2 and human odorants. Human behavior will correspondently influence exposure; ambush ticks will rather grab hosts passing by and that touch infested vegetation whereas hunter ticks will crawl toward humans that are stationary for a while. The second sampling occurred in August 2015, winter time in Brazil. As expected for this season (Labruna et al., 2009; Szabó et al., 2007) most collected ticks were nymphs. CO2 traps yielded the greatest tick numbers. However, tick collection by this method was used as the last technique to avoid depleting tick numbers before dragging and human bait collections. Higher efficacy of CO2 traps reinforced the results reported in other studies that both tick species collected are hunter ticks (Brites-Neto et al., 2013; Szabó et al., 2009). Further, this tick sampling method showed that A. sculptum was more widespread in the environment than A. dubitatum. In fact, the latter species was almost exclusively restricted to traps by the fence on the club side. The explanation for such restriction is not clear but it can be speculated that A. dubitatum was hiding in microhabitat with higher humidity since it is a tick species associated with marsh areas or those prone to flooding (Szabó et al., 2007; Queirogas et al., 2012) and the river bank at the sampling site was high precluding water pooling. The ground by the fence on the club side was covered by grass and close/below the ivycovered fence. This environment must have retained higher humidity in relation to, respectively, the shaded river side with the ground devoid of vegetation and the grass exposed to the sun on the club side. Amblyomma sculptum, on the other hand, was found in similar numbers on both sides of the fence and its numbers decreased exponentially, to four and six meters from tick source, i. e. fence, on the club side. This last observation shows that horizontal spreading of nymphs of this tick species is of, at least, six meters but with numbers decreasing sharply with each meter in the sun-exposed environment. In this context, a role for A. sculptum being spread by other animals known to use the area (small mammals or cats) cannot be disregarded but none has been ever shown to be important hosts for A. sculptum. Dragging showed that ticks seeking hosts from the vegetation (ambush behavior) were concentrated on the river side and, overwhelmingly, on the fence itself, with numbers decreasing sharply with increasing distance from the fence on the club side, similarly to those observed with CO2 traps. Furthermore, dragging collected almost exclusively A. sculptum whereas this technique yielded only 5% of all A. dubitatum. Altogether, these observations reinforce that ticks disseminate only a few meters from the infestation source and that, under the described environmental conditions, A. dubitatum ticks do not seek hosts from vegetation as ambush ticks would do, at least not in the same proportion as A. sculptum. On the other hand, high A. sculptum tick numbers on the fence clearly indicate that this tick species exhibits ambush behavior as well. Human baits were set up by the fence, which concentrated ticks on the club side and where human exposure would be more likely. At this site, humans attracted almost exclusively A. sculptum ticks, overwhelmingly nymphs, and mostly after 15 min. CO2 traps set up immediately afterwards and on the very same locations proved that A. sculptum and A. dubitatum could be attracted in similar numbers. There is no definitive explanation for the discrepancies in tick attraction between these two methods and several factors may have influenced this. For example, A. dubitatum ticks may react slower and had more time to
others, with one of them attracting close to half (41.9%) whereas another had only 4.9% of all ticks collected with this technique. 3.2.4. CO2 traps Overall, 2563 ticks were collected by CO2 traps (Table 1). Of these, 33.3% were collected by the fence on the river side, 63.7% by the fence on the club side. Most ticks were A. sculptum nymphs (53.3%) followed by A. dubitatum nymphs (31.6%). However, the spatial distributions of the two species were not homogenous; most A. dubitatum ticks (74.3% of all A. dubitatum ticks) were collected from traps by the fence on the club side, whereas A. sculptum ticks were distributed similarly on traps by both sides of the fence (40.1% and 54.8% of all A. sculptum on the river and club side, respectively). The latter species was also found, albeit in lower numbers, four and six meters from the fence on the club side (2.7% and 0.4% of all A. sculptum ticks, respectively). In the case of A. dubitatum, only one adult and one nymph were found, respectively, four and six meters from the fence on the club side. CO2 trapping was the most effective sampling method for A. dubitatum and A. sculptum and yielded, respectively, 94.5% and 64.6% of all nymphs of each tick species. The other two collection methods were less effective, and inefficient in the case of A. dubitatum nymphs. Of all collected A. dubitatum and A. sculptum nymphs, dragging yielded, respectively 5.1% and 29.6% whereas human baits 0.4% and 5.9% of each tick species. 3.2.5. Other observations Amblyomma spp. larvae represented 12.7% (N = 443) of all ticks and were collected as individuals because distinct larval clusters were not found, but rather scattered individuals. Most of these were collected from CO2 traps by the fence on the club side (57.1% of all larvae) followed by CO2 traps by the fence on the riverside (19.9% of all larvae). Adult ticks were the least numerous stage (1.4% of all ticks) and the majority was A. dubitatum collected from CO2 traps by the fence on the club side (61.2% from all adult ticks). 3.3. Rickettsia in ticks 244 nymphs (204 A. sculptum and 40 A.dubitatum) divided into 25 pools with, approximately 10 nymphs each, as well as four A. sculptum and three A. dubitatum adults were examined for Rickettsia. Four A. sculptum nymphal pools (40 nymphs) and one adult yielded visible amplicons on PCR targeting both Rickettsia’s gltA gene sequence and on the third PCR specific for the R. bellii gltA gene sequence. Similar products were obtained from one A. dubitatum nymph pool (10 nymphs) and one adult. No product was obtained from any PCR targeting the ompA gene sequence. The product of a PCR targeting the citrate synthase gene in one pool of A. dubitatum nymphs that also yielded a visible product in the third PCR (R. bellii specific) was sequenced and submitted for BLAST analysis (www.ncbi.nlm.nih.gov/Blast), to determine its similarities to the relevant sequences from identified Rickettsia (Altschul et al., 1990). The nucleotide sequence of the PCR product of this sample was 99% identical to R. bellii from South Brazil (KX137900) and Argentina (KX009410). This sequence was deposited at GenBank and assigned nucleotide accession number MF187460. 4. Discussion The preliminary sampling in March 2015 confirmed known features of tick ecology in southeast Brazil, where Praia Clube is located; capybaras at anthropogenic sites are associated with high environmental infestations of both A. sculptum and A. dubitatum and adult ticks prevail over other stages in summer (Brites-Neto et al., 2013; Queirogas et al., 2012; Souza et al., 2006). This sampling also showed the unevenness of infestations within the club, with one location harboring most of the ticks. Such intense infestations in one location may be attributed to the presence of capybaras that used it as resting place as well as adequate micro-environmental conditions for both tick species. 4
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the capybara tick, Amblyomma dubitatum (Acari Ixodidae) in Brazil. Entomol. News 118, 77–80. Labruna, M.B., Terassini, F.A., Camargo, L.M.A., 2009. Notes on population dynamics of Amblyomma ticks (Acari: Ixodidae) in Brazil. J. Parasitol. 95, 1016–1018. Labruna, M.B., 2009. Ecology of Rickettsia in South America. Ann. N. Y. Acad. Sci. 1166, 156–166. Macaluso, K.R., Sonenshine, D.E., Ceraul, S.M., Azad, A.F., 2002. Rickettsial infection in Dermacentor variabilis (Acari: Ixodidae) inhibits transovarial transmission of a second Rickettsia. J. Med. Entomol. 39, 808–813. Martins, T.F., Onofrio, V.C., Barros-Battesti, D.M., Labruna, M.B., 2010. Nymphs of the genus Amblyomma (Acari Ixodidae) of Brazil: descriptions, redescriptions, and identification key. Ticks Tick Borne Dis. 1, 75–99. Martins, T.F., Barbieri, A.R., Costa, F.B., Terassini, F.A., Camargo, L.M., Peterka, C.R., Pacheco, R.C., 2016. Geographical distribution of Amblyomma cajennense (sensu lato) ticks (Parasitiformes: Ixodidae) in Brazil, with description of the nymph of A. cajennense (sensu stricto). Parasites Vectors 9, 186. Nava, S., Beati, L., Labruna, M.B., Cáceres, A.G., Mangold, A.J., Guglielmone, A.A., 2014. Reassessment of the taxonomic status of Amblyomma cajennense (Fabricius, 1787) with the description of three new species, Amblyomma tonelliae n. sp., Amblyomma interandinum n. sp. and Amblyomma patinoi n. sp., and reinstatement of Amblyomma mixtum Koch, 1844 and Amblyomma sculptum Berlese, 1888 (Ixodida: ixodidae). Ticks Tick Borne Dis. 5, 252–276. Onofrio, V.C., Labruna, M.B., Pinter, A., Giacomin, F.G., Barros-Battesti, D.M., 2006. Comentários e chaves para as espécies do gênero. In: Barros-Battesti, D., Arzua, M., Bechara, G.H. (Eds.), Carrapatos de importância medico veterinária da região neotropical. Um guia ilustrado para identificação de espécies. Butantan, São Paulo, pp. 53–113. Pacheco, R.C., Horta, M.C., Pinter, A., Moraes-Filho, J., Martins, T.F., Nardi, M.S., Souza, S.S.A.L., Souza, C.E., Szabó, M.P.J., Richtzenhain, L.J., Labruna, M.B., 2009. Pesquisa de Rickettsia spp em carrapatos Amblyomma cajennense e Amblyomma dubitatum no Estado de Sóo Paulo. Rev. Soc. Bras. Med. Trop. 42, 351–353. Queirogas, V.L., Del Claro, K., Nascimento, A.R.T., Szabó, M.P.J., 2012. Capybaras and ticks in the urban areas of Uberlândia Minas Gerais, Brazil: ecological aspects for the epidemiology of tick-borne diseases. Exp. Appl. Acarol. 57, 75–82. Ramos, V.N., Osava, C.F., Piovezan, U., Szabo, M.P.J., 2014. Ticks on humans in the Pantanal wetlands, Brazil. Ticks Tick Borne Dis. 5, 497–499. Ramos, V.N., Osava, C.F., Piovezan, U., Szabó, M.P.J., 2017. Ambush behavior of the tick Amblyomma sculptum (Amblyomma cajennense complex) (Acari: Ixodidae) in the Brazilian Pantanal. Ticks Tick Borne Dis. 8, 506–510. Roux, V., Fournier, P.E., Raoult, D., 1996. Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCR-amplifed DNA of the gene encoding the protein rOmpA. J. Clin. Microbiol. 34, 2058–2065. Sakai, R.K., Costa, F.B., Ueno, T., Ramirez, D.G., Soares, J.F., Fonseca, A.H., Labruna, M.B., Barros-Battesti, D.M., 2014. Experimental infection with Rickettsia rickettsii in an Amblyomma dubitatum tick colony, naturally infected by Rickettsia bellii. Ticks Tick Borne Dis. 5, 917–923. Sangioni, L.A., Horta, M.C., Vianna, M.C.B., Gennari, S.M., Soares, R.M., Galvão, M.A.M., Schumaker, T.T.S., Ferreira, F., Vidotto, O., Labruna, M.B., 2005. Rickettsial infection in animals and Brazilian Spotted Fever endemicity. Emerg. Infect. Dis. 11, 265–270. Soares, J.F., Soares, H.S., Barbieri, A.M., Labruna, M.B., 2012. Experimental infection of the tick Amblyomma cajennense, Cayenne tick, with Rickettsia rickettsii, the agent of Rocky Mountain spotted fever. Med. Vet. Entomol. 26, 139–151. Sonenshine, D.E., Roe, R.M., 2014. Overview: ticks, people, and animals. In: second edition. In: Sonenshine, D.E., Roe, R.M. (Eds.), Biology of Ticks Vol. I. Oxford University Press, New York USA, pp. 3–15. Souza, S.S.A.L., Souza, C.E., Neto, E.J.R., Prado, A.P., 2006. Dinâmica sazonal de carrapatos (Acari Ixodidae) na mata ciliar de uma região endêmica para febre maculosa na região de Campinas, São Paulo, Brasil. Ciênc. Rural 36, 887–891. Souza, C.E., Moraes-Filho, J., Ogrzewalska, M., Uchoa, F.A., Horta, M.C., Souza, S.S.L., Borba, R.C.M., Labruna, M.B., 2009. Experimental infection of capybaras Hydrochaeris hydrochaeris by Rickettsia rickettsii and evaluation of the transmission of the infection to ticks Amblyomma cajennense. Vet. Parasitol. 161, 116–121. Szabó, M.P.J., Castro, M.B., Ramos, H.G.C., Garcia, M.V., Castagnolli, K.C., Pinter, A., Veronez, V.A., Magalhães, G.M., Duarte, J.M.B., Labruna, M.B., 2007. Species diversity and seasonality of free-living ticks (Acari: Ixodidae) in the natural habitat of wild Marsh deer (Blastocerus dichotomus) in Southeastern Brazil. Vet. Parasitol. 143, 147–154. Szabó, M.P.J., Labruna, M.B., Garcia, M.V., Pinter, A., Castagnolli, K.C., Pacheco, R.C., Castro, M.B., Veronez, V.A., Magalhães, G.M., Vogliotti, A., Duarte, J.M.B., 2009. Ecological aspects of free-living ticks (Acari: Ixodidae) on animal trails in an Atlantic rainforest of Southeastern Brazil. Ann. Trop. Med. Parasitol. 103, 57–72. Szabó, M.P.J., Pinter, A., Labruna, M.B., 2013a. Ecology, biology and distribution of spotted-fever tick vectors in Brazil. Front. Cell. Infect. Microbiol. 3, 27. Szabó, M.P.J., Nieri-Bastos, F.A., Spolidorio, M.G., Martins, T.F., Barbieri, A.M., Labruna, M.B., 2013b. In vitro isolation from Amblyomma ovale (Acari: Ixodidae) and ecological aspects of the Atlantic rainforest Rickettsia, the causative agent of a novel spotted fever rickettsiosis in Brazil. 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reach CO2 traps that were maintained three times longer than human baits. In fact, in human baits, three of four A. dubitatum ticks were collected after 15 min, but again this number is too small for speculation. It is also possible that humans expel less CO2 then the traps and are less attractive for A. dubitatum. Whatever the case, results indicate that humans in stationary behavior are more likely to attract A. sculptum over A. dubitatum, at least during the time length of the sampling methods. Overall, differing aspects of behavior and microhabitat use of A. sculptum and A. dubitatum were observed that determined, respectively, increase and decrease, of human tick-bite risk. A. sculptum exhibited both hunting and ambush behavior as observed before (Ramos et al., 2017) whereas an ambush behavior was not apparent for A. dubitatum. This means that humans walking by infested areas will be rather exposed to A. sculptum over A. dubitatum. Furthermore, at drier areas (not flooded or marsh) A. sculptum is more widespread and the chance of human exposure is increased Considering the hunting strategy of ticks, stationary humans attract much more A. sculptum than A. dubitatum, at least within 30 min. Thus, human behaviors such as picnicking, fishing, meditating and so on will rather result in A. sculptum infestation in areas where both species are present. In this study, a high R. bellii infection prevalence was found for both A. dubitatum and A. sculptum. Rickettsia bellii is common in various hard tick species from Brazil, considered non-pathogenic (Labruna, 2009) and is highly associated with A. dubitatum tick populations where it might attain an infection prevalence of 100% (Pacheco et al., 2009; Sakai et al., 2014). This bacterium may have a role in the R. rickettsii ecology, since, at high tick infection rates it might prevent the establishment of R. rickettsii infection in tick populations by an interference mechanism (Labruna, 2009; Sakai et al., 2014), as previously described for other Rickettsia species (Macaluso et al., 2002). Thus, the outcome of A. sculptum, A. dubitatum and R. bellii coexistence on R. rickettsii transmission should be investigated further. Overall, our results highlight the potential for A. sculptum to infest humans. Thus, in BSF endemic areas with both tick species infected with Rickettsia, transmission will rather occur by exposure to A. sculptum, but occasional human encounters with A. dubitatum cannot be discounted. Acknowledgments Authors acknowledge Praia Clube, Uberlândia, Minas Gerais, for logistic and financial support. This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (Academic Career Research Fellowship to M.P.J. Szabó). Ticks were collected as authorized by Brazilian Environmental Ministry (SISBIO no. 10762-1). Permits are on files of senior researcher M P J Szabó. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Brites-Neto, J., Nieri-Bastos, F.A., Brasil, J., Duarte, K.M., Martins, T.F., Veríssimo, C.J., Barbieri, A.R., Labruna, M.B., 2013. Environmental infestation and rickettsial infection in ticks in an area endemic for Brazilian spotted fever. Rev. Bras. Parasitol. Vet. 22, 367–372. Guglielmone, A.A., Beati, L., Barros-Battesti, D.M., Labruna, M.B., Nava, S., Venzal, J.M., Mangold, A.J., Szabó, M.P.J., Martins, J.R., González-Acuña, D., Estrada-Peña, A., 2006. Ticks (Ixodidae) on humans in south america. Exp. Appl. Acarol. 40, 83–100. Labruna, M.B., Whitworth, T., Bouyer, D.H., McBride, J., Camargo, L.M.A., Camargo, E.P., Popov, V., Walker, D.H., 2004. Rickettsia bellii and Rickettsia amblyommii in Amblyomma ticks from the state of Rondônia, Western Amazon, Brazil. J. Med. Entomol. 41, 1073–1081. Labruna, M.B., Pacheco, R.C., Ataliba, A.C., Szabó, M.P.J., 2007. Human parasitism by
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