Exp Appl Acarol DOI 10.1007/s10493-016-0070-1
Ecology of a tick-borne spotted fever in southern Brazil Felipe S. Krawczak1 • Lina C. Binder1 • Caroline S. Oliveira2 • Francisco B. Costa1 • Jonas Moraes-Filho1,3 • Thiago F. Martins1 Jonas Sponchiado4 • Geruza L. Melo5 • Fa´bio Gregori1 • Gina Polo1 • Stefan V. Oliveira6 • Marcelo B. Labruna1
•
Received: 29 April 2016 / Accepted: 5 July 2016 Ó Springer International Publishing Switzerland 2016
Abstract Rio Grande do Sul is the southernmost state of Brazil, bordering Uruguay. Clinical cases of spotted fever group (SFG) rickettsiosis were recently reported in Rio Grande do Sul. None of these cases was lethal, and all were confirmed by seroconversion to R. rickettsii antigens. Because serological cross-reactions are well known to occur between different SFG agents, the SFG agent responsible for the clinical cases remains unknown in Rio Grande do Sul, where no rickettsial agent is known to infect ticks. During 2013–2014, ticks and blood sera samples were collected from domestic dogs and wild small mammals, and from the vegetation in a SFG-endemic area of Rio Grande do Sul. Dogs were infested by Amblyomma ovale adult ticks, whereas small mammals were infested by immature stages of A. ovale, Ixodes loricatus, and adults of I. loricatus. Ticks collected on vegetation were adults of A. ovale, and immature stages of A. ovale, Amblyomma dubitatum, and Amblyomma longirostre. Three Rickettsia species were detected: Rickettsia bellii in I. loricatus, Rickettsia amblyommii in A. longirostre, and a Rickettsia parkeri-like agent (Rickettsia sp. strain Atlantic rainforest) in A. ovale. Seroreactivity to SFG antigens were detected in 19.7 % (27/137) canine and 37.5 % (15/40) small mammal sera, with highest titers to R. parkeri. Results indicate that the R. parkeri-like agent, strain
& Marcelo B. Labruna
[email protected] 1
Departamento de Medicina Veterina´ria Preventiva e Sau´de Animal, Faculdade de Medicina Veterina´ria e Zootecnia, Universidade de Sa˜o Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universita´ria, Sa˜o Paulo, SP 05508-270, Brazil
2
Departamento de Medicina Veterina´ria Preventiva, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
3
Mestrado em Medicina Veterina´ria e Bem Estar Animal, Universidade de Santo Amaro, Sa˜o Paulo, SP, Brazil
4
Departamento de Biologia, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
5
Departamento de Ecologia, Universidade Federal do Mato Grosso do Sul, Campo Grande, MS, Brazil
6
Secretaria de Vigilaˆncia em Sau´de, Ministe´rio da Sau´de, Brası´lia, DF, Brazil
123
Exp Appl Acarol
Atlantic rainforest, is circulating between A. ovale ticks, dogs and small mammals in the study area, suggesting that this SFG pathogen could be one of the etiological agents of SFG clinical cases in Rio Grande do Sul. Keywords Rickettsia sp. strain Atlantic rainforest Rickettsia amblyommii Rickettsia bellii Amblyomma ovale Amblyomma longirostre Ixodes loricatus
Introduction Spotted fevers are diseases caused by bacterial agents belonging to the spotted fever group (SFG) of the genus Rickettsia. Different SFG rickettsial agents are transmitted to humans by different ixodid tick species in every continent, except for Antarctica (Parola et al. 2013). Until 2009, Brazilian spotted fever or Rocky Mountain spotted fever, caused by Rickettsia rickettsii, was the only known tick-borne SFG disease known to occur in Brazil, with laboratory-confirmed cases restricted to the southeastern area of the country (Labruna 2009). Two tick species, Amblyomma sculptum and Amblyomma aureolatum, act as major vectors of R. rickettsii to humans in Brazil (Labruna et al. 2014). In 2010, a novel SFG agent, designated as Rickettsia sp. strain Atlantic rainforest, was described from a patient in the state of Sa˜o Paulo, southeastern Brazil (Spolidorio et al. 2010). In 2011, a second clinical case caused by this novel agent was described from the state of Bahia, northeastern Brazil (Silva et al. 2011). Recently, a third clinical case was reported from the state of Santa Catarina, southern Brazil (Krawczak et al. 2016a). These three clinical cases were epidemiologically linked to the tick Amblyomma ovale (Szabo´ et al. 2013; Barbieri et al. 2014; Nieri-Bastos et al. 2016); which was shown to be a competent vector of Rickettsia sp. strain Atlantic rainforest (Krawczak et al. 2016b). In addition, these three cases presented mild symptoms with no lethality, contrasting to the highly lethal pattern of the classically severe Brazilian spotted fever that occurs in southeastern Brazil (Labruna et al. 2014). The state of Rio Grande do Sul is the southernmost state of Brazil, bordering Uruguay. From 2005 to 2015, there were 12 laboratory-confirmed clinical cases of SFG rickettiosis in Rio Grande do Sul. None of these cases was lethal, and they were all confirmed by seroconversion to R. rickettsii antigens through analyses of acute and convalescent serum samples (official data from the Brazilian Ministry of Health, available at: http:// portaldasaude.saude.gov.br). Because serological cross-reactions are well known to occur between different SFG agents (Parola et al. 2013), the SFG agent responsible for the clinical cases in Rio Grande do Sul remains unknown. This scenario is even more obscure because until now, no rickettsial agent is known to infect ticks in Rio Grande do Sul. Here, we investigated rickettsial infection in ticks, wild, and domestic animals in an area of Rio Grande do Sul from where human SFG clinical cases have been reported.
Materials and methods Study area This study was performed in the rural area of Cerro Largo municipality (28°080 4900 S, 54°440 1700 W, altitude 211 m), located in the center-western portion of Rio Grande do Sul, within the Atlantic forest biome. In 2010, Cerro Largo had a human population of 13,289
123
Exp Appl Acarol
habitants, from which 2718 (20.5 %) lived in the rural area (http://censo2010.ibge.gov.br). According to unpublished official data from the Brazilian Ministry of Health, four nonfatal clinical cases of spotted fever were confirmed in Cerro Largo from 2005 to 2015. For the present study, samples were collected on three field campaigns (July, October 2013 and January 2014). This study was previously approved by the Chico Mendes Institute for biodiversity (ICMBio Permit No. 38502-1) and the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Veterinary Medicine of the University of Sa˜o Paulo (protocol 2908/2013).
Dogs Dogs were sampled primarily to be tested for the presence of seroreactivity to SFG rickettsial antigens. For this purpose, the minimal number of sampled dogs was estimated to be 119, considering a canine population of 860 dogs for the rural area of Cerro Largo. This canine population was estimated according to Soto et al. (2006), who reported a 3.16:1 ratio (number of dogs: number of humans) for the rural area of another Brazilian municipality. Sample size calculation considered an expected seropositivity of 10, and 95 % sample confidence level, according to Arya et al. (2012). Blood samples for serological analysis were obtained from a total of 137 different dogs, 103 sampled in the first campaign, and 34 in the third campaign. Among these 137 dogs, 103, 32, and 52 were examined for tick infestations during the first, second, and third field campaigns, respectively, resulting in a total of 187 canine examinations. Each dog had its entire body examined for the presence ticks, which were collected in plastic tubes and sent to the laboratory. While immature ticks were preserved in absolute ethanol, adult ticks were sent alive to the laboratory.
Small mammals Attempts to capture wild small mammals were performed within forest patches surrounding the households in which dogs were sampled. For this purpose, a total of 80 livetraps (75 Sherman and five Tomahawk) baited with bacon, banana, apple and peanut butter were installed for four consecutive nights during each field campaign. Additionally, two pitfall station traps with five bucket of 42.5 cm diameter and 60 cm height in each station connected by a plastic fence (of at least 30 m long and 50 cm high) (Umetsu et al. 2011) were installed for the same period. The total sampling effort was 960 trap-nights for livetraps and 120 trap-nights for pitfall-traps. Trapped animals were then anaesthetized with ketamine and xylazine, and carefully examined for ticks, which were collected in plastic tubes and sent to the laboratory. Blood samples for serological analysis were collected from all trapped animals.
Host-seeking ticks Host-seeking ticks were collected from the vegetation in the same forest patches where small mammals were trapped. In each field campaign, ticks were collected by cloth dragging in trails within the forest, as described elsewhere (Terassini et al. 2010). Dragging was performed by one person through a 50–100 m-trail in each campaign.
123
Exp Appl Acarol
Tick identification Morphological identification of tick species were based on Barros-Battesti et al. (2006) for adult ticks, and Martins et al. (2010) for Amblyomma nymphs. Larval ticks were morphologically identified through direct comparisons with laboratory-reared larvae from engorged females of A. ovale (Martins et al. 2010) and Ixodes loricatus (Labruna et al. 2002), and following the morphological description of the larval stage of these two tick species (Marques et al. 2004; Barbieri et al. 2008). The morphological identification of these larvae was confirmed by molecular analysis of a few specimens, which had the DNA extracted by boiling (Horta et al. 2007) and tested by polymerase chain reaction (PCR) targeting a fragment of &460 nucleotides of the tick mitochondrial 16S rDNA gene (Mangold et al. 1998). PCR products were DNA sequenced in an automatic sequencer (Model ABI 3500 Genetic Analyzer; Applied Biosystems/Thermo Fisher Scientific, Foster City, CA, USA) according to manufacturer’s instructions. Generated sequences were submitted to BLAST analysis to determine the closest similarities available in GenBank.
Rickettsial infection in ticks Ticks that were still alive upon arrival in the laboratory were frozen in a -80 °C freezer. Attempts to isolate rickettsiae in Vero cell culture were performed with 4 ticks, which were defrosted and immediately processed by the shell vial technique, as previously described (Labruna et al. 2004). A rickettsial isolate was considered to be established in Vero cells after at least three passages at 28 °C with the prevalence of infected cells exceeding 90 %. A sample of 3rd passage-infected cells was submitted to DNA extraction by the DNeasy Tissue Kit (Qiagen, Chatsworth, CA, USA), and tested by two PCR protocols, one with primers CS-78 and CS-323, and another with primers CS-239 and CS-1069, which amplify two overlapping fragments (401 and 834-bp, respectively) of the rickettsial citrate synthase gene (gltA) (Labruna et al. 2004). These two fragments were DNA sequenced; generated sequences were aligned and the consensus submitted to BLAST analyses, as cited above. Body remnants from the ticks that were processed for inoculation of shell vials, as well as other frozen or alcohol-preserved adults were processed individually for DNA extraction by the guanidine isothiocyanate phenol technique (Sangioni et al. 2005). Larval and nymphs ticks were processed individually or in pools of up to 10 ticks by boiling (Horta et al. 2007). Tick DNA samples were tested by a taqman real-time PCR assay targeting a 147-bp fragment of the rickettsial gltA gene (Labruna et al. 2004; Guedes et al. 2005). Once a tick was demonstrated by real-time PCR to contain rickettsial DNA, amplification of a larger fragment of the gltA gene was attempted by two conventional PCR protocols. One used primers CS-78 and CS-323 for the gltA gene (Labruna et al. 2004), and the second protocol used primers Rr190.70F and Rr190.701R, which amplify a & 632 bp fragment of the 190 kDa outer membrane protein gene (ompA) of rickettsiae belonging to the spotted fever group (Roux et al. 1996). The PCR products were sequenced and submitted to BLAST analysis to determine their closest similarities to Rickettsia sequences available in GenBank. All tick DNA samples that were shown to contain no rickettsial DNA by the real-time PCR described above were tested by the conventional PCR targeting the tick mitochondrial 16S rDNA gene (Mangold et al. 1998), in order to validate the DNA extraction protocol. If the tick sample yielded no product by this PCR, it was considered that DNA extraction was not successful, and the individual tick was discarded from the study.
123
Exp Appl Acarol
Serology Canine, rodent and marsupial sera were tested by the indirect immunofluorescence assay (IFA) using crude antigens derived from 6 Rickettsia isolates from Brazil (R. rickettsii strain Taiac¸u, R. parkeri strain At24, R. amblyommii strain Ac37, R. rhipicephali strain HJ5, R. felis strain Pedreira, and R. bellii strain Mogi), as previously described (Labruna et al. 2007). Briefly, sera were diluted in twofold increments with phosphate-buffered saline (PBS), starting from the 1:64 dilution. Slides were incubated with fluorescein isothiocyanate-labelled rabbit anti-dog IgG (Sigma,St Louis, MO, USA), goat anti-mouse IgG (Sigma, St Louis, MO, USA) and sheep anti-opossum IgG (CCZ, Sa˜o Paulo, Brazil) for canine, rodent and marsupial sera, respectively. For each sample, the endpoint IgG titer reacting with each of the 6 Rickettsia antigens was determined. An endpoint titer at least fourfold higher for a Rickettsia species than that observed for any other Rickettsia species was considered probably homologous to the first Rickettsia species or to a very closely related species (Labruna et al. 2007; Szabo´ et al. 2013). In each slide, a serum previously shown to be non-reactive (negative control) and a known reactive serum (positive control), both from the studies of Szabo´ et al. (2013) and Krawczak et al. (2016b), were tested at the 1:64 dilution.
Results Because the three field campaigns encompassed a study period of 6 months (from July 2013 to January 2014), all data were pooled for presentation. From a total of 187 canine examinations, 23 tick specimens were collected, which were all identified as adults of A. ovale (11 males, 12 females). A total of 40 small mammals of four species (1 marsupial and 3 rodent) were captured (Table 1). A single marsupial species, Didelphis albiventris, was parasitized by adults of Ixodes loricatus (1 male, 12 females), and immature stages of Amblyomma spp. (1 nymph of A. ovale and 30 larvae of Amblyomma sp.). The most abundant rodent species, Akodon montensis, was parasitized by immature stages of both A. ovale (1 nymph, 9 larvae) and I. loricatus (14 larvae). Two rodent species (Oligoryzomys nigripes, Sooretamys angouya) were not infested by ticks (Table 1). A total of 119 hostseeking ticks were collected by dragging, and were identified as A. ovale (2 males, 4 females, 2 nymphs), A. dubitatum (2 nymphs), A. longirostre (1 nymph, 3 larvae), and Amblyomma sp. (105 larvae). Three A. longirostre larvae were molecularly identified by sequencing a fragment of their 16S rRNA gene, which were identical to each other and 100 % identical to a corresponding sequence of A. longirostre from GenBank (KM262205). In addition, 16S rRNA partial sequences were also generated for four A. ovale larvae that were collected from A. montensis, generating two haplotypes that differed by 3.9 %, and at the same time were 100 % identical to corresponding sequences of A. ovale from GenBank (KR605468 and KR605466, respectively). Finally, taxonomic identification of a I. loricatus larva from A. montensis was also confirmed by molecular analysis, generating a 16S rRNA partial sequence that was 99.5 % identical to I. loricatus from GenBank (AF549840). Although not foreseen, taxonomic identification of an A. dubitatum nymph was also confirmed by molecular analysis, generating a 16S rRNA partial sequence 99.7 % identical to A. dubitatum from GenBank (DQ858955). Attempts to isolate rickettsiae in Vero cell culture were performed individually with two I. loricatus and two A. ovale adult ticks. Rickettsiae were successfully established in Vero
123
Exp Appl Acarol Table 1 Infestation by ticks on domestic dogs and wild small mammals in Cerro Largo municipality, state of Rio Grande do Sul, Brazil, from July 2013 to January 2014 Animal species (N)
Tick species collected from the animals* Amblyomma ovale
Amblyomma sp.
Ixodes loricatus
Adults
Nymphs
Larvae
Larvae
Adults
Larvae
9 (4.8); 2.6
–
–
–
–
–
–
1 (25.0); 1
–
1 (25.0); 30
4 (100); 3.3
–
Akodon montensis (29)
–
1 (3.4); 1
4 (13.8); 2.3
–
5 (17.2); 2.8
Oligoryzomys nigripes (6)
–
–
–
–
–
–
Sooretamys angouya (1)
–
–
–
–
–
–
23
2
9
30
13
14
Domestic dogs (187) Marsupials Didelphis albiventris (4) Rodents
Total number of ticks
N number of examined individuals * Values presented as: number of infested animals (%); mean number of ticks per infested host
cell culture from one I. loricatus tick. Rickettsial DNA samples amplified by PCR from the isolate were sequenced and submitted to BLAST analyses. The isolate was identified as Rickettsia bellii, since its gltA sequence (1109 nucleotides, excluding primer sequences) was identical to each other and 100 % equal to corresponding sequence of R. bellii in GenBank (accession number DQ146481). This R. bellii isolate, designated as strain RBCL, has been cryopreserved and deposited at the Rickettsial Collection of the Faculty of Veterinary Medicine, University of Sa˜o Paulo, Sa˜o Paulo, Brazil, and at the Rickettsial Collection of the Rickettsial Zoonoses Branch of the Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA. Real-time PCR targeting rickettsial DNA was performed on 192 tick specimens, from which three A. ovale adults from dogs, one A. ovale adult and two A. longirostre larvae from vegetation, and one I. loricatus adult from D. albiventris were shown to contain rickettsial DNA (Table 2). This I. loricatus adult was shown to be infected by R. bellii, since this agent was isolated in Vero cells from this tick, as presented above. The remaining real-time PCR-positive ticks were further tested by conventional PCR assays, which amplified fragments of the rickettsial genes gltA and ompA. All ompA products were DNA sequenced, while the gltA product was sequenced from only one A. ovale specimen. By BLAST analysis, the ompA partial sequences of the four A. ovale ticks were identical to each other and 100 % (590/590-bp) identical to corresponding sequence of Rickettsia sp. strain Atlantic rainforest from GenBank (KJ855085). The gltA partial sequence of one of these ticks was 99.9 % (1077/1078-bp) identical to corresponding sequence of Rickettsia sp. strain Atlantic rainforest (GQ855235). The ompA partial sequence of the A. longirostre tick was 99.5 % identical to corresponding sequence of R. amblyommii (KF702333). Overall, 20.4 % (28/137) of the dogs were seroreactive (titer C 64) to at least one Rickettsia species. A total of 27 (19.7 %), 21 (15.3 %), 14 (10.2 %), 19 (13.9 %), 12 (8.8 %), and
123
Exp Appl Acarol Table 2 Rickettsial infection in ticks collected in Cerro Largo municipality, state of Rio Grande do Sul, Brazil, from July 2013 to January 2014 Number infected ticks/ number tested ticks (% infection)
Rickettsia species
Tick species
Stage
Source
Amblyomma ovale
Adults
Dogs
3/20 (15.0)
Strain Atlantic rainforest
Adults
Vegetation
1/6 (16.7)
Strain Atlantic rainforest
Nymphs
Vegetation
0/2 (0)
Nymph
Akodon montensis
0/1 (0)
Larvae
A. montensis
0/9 (0)
A. dubitatum
Nymph
Vegetation
0/1 (0)
A. longirostre
Larvae
Vegetation
2/3 (66.7)
Ixodes loricatus
Adults
Didelphis albiventris
1/13 (7.7)
Larvae
A. montensis
0/12 (0)
Amblyomma sp.
Larvae
D. albiventris
Amblyomma sp.
Larvae
Vegetation
Total
R. amblyommii R. bellii
0/20 (0) 0/105 (0) 7/192 (3.6)
6 (4.4 %) dogs were seroreactive to R. parkeri, R. rickettsii, R. amblyommii, R. rhipicephali, R. felis, and R. bellii, respectively (Table 3). Only one canine serum reacted with another Rickettsia species without reacting with R. parkeri. This single serum had an endpoint titer of 64 for R. bellii, and non-reactive for the remaining five antigens. A total of 16 (11.7 %) canine sera showed titers to R. parkeri at least fourfold higher than those to any of the other five antigens. The antibody titers in these 16 dogs were considered to have been stimulated by R. parkeri or a very closely related species. Canine IFA endpoint titers for R. parkeri varied from 64 to 8192. Among the 27 R. parkeri–seroreactive dogs, 15 animals (55.6 %) had titers C1024, and 9 animals (33.3 %) had titers C4096 to R. parkeri. Among the 40 small mammals, 13 (44.8 %) out of 29 A. montensis and 3 (75 %) out of 4 D. albiventris were seroreactive to at least one Rickettsia species (Table 3). The antibody titers in at least 2 seroreactive D. albiventris were considered to have been stimulated by R. parkeri or a very closely related species, as they showed titers to R. parkeri at least fourfold higher than those to any of the other five antigens. GenBank nucleotide sequence accession numbers for the partial sequences generated in the present study are KX137894-KX137898 for the 16S rRNA gene of A. longirostre, I. loricatus, A. dubitatum, A. ovale, and KX137902, KX137899 for the ompA and gltA genes of Rickettsia sp. strain Atlantic rainforest, KX137901 for the ompA gene of R. amblyommii, and KX137900 for the gltA gene of Rickettsia bellii. Voucher specimens of the tick species collected in the present study have been deposited in the tick collection ‘‘Colec¸a˜o Nacional de Carrapatos’’ of the University of Sa˜o Paulo, under accession numbers CNC 3293-3297.
Discussion The present study was carried out in an area of Rio Grande do Sul state, southern Brazil, where human cases of spotted fever have been reported, albeit with no fatalities. We found that &15 % of the A. ovale ticks collected from dogs and vegetation were infected by the human pathogen Rickettsia sp. strain Atlantic rainforest, a R. parkeri-like agent that has
123
123
3 (75.0)
12 (41.4)
0
0
A. montensis (29)
O. nigripes (6)
S. angouya (1)
0
0
13 (44.8)
2 (50.0)
21 (15.3)
R. rickettsii
0
0
8 (27.6)
1 (25.0)
14 (10.2)
R. amblyommii
0
0
9 (31.0)
1 (25.0)
19 (13.9)
R. rhipicephali
0
0
0
0
12 (8.8)
R. felis
0
0
5 (17.2)
1 (25.0)
6 (4.4)
R. bellii
–
–
–
2 (R. parkeri)
16 (R. parkeri)
No. animals with determined homologous reaction (PAIHR in parenthesis)*
* A homologous reaction was determined when an endpoint titer to a Rickettsia species was at least fourfold higher than those observed for the other Rickettsia species. In this case, the Rickettsia species (or a very closely related species) involved in the highest endpoint titer was considered the possible antigen involved in a homologous reaction (PAIHR)
N number of examined individuals
27 (19.7)
D. albiventris (4)
R. parkeri
No. seroreactive animals to each of the Rickettsia species (% seroreactivity for each animal species)
Dogs (137)
Animal species (N)
Table 3 Seroreactivity to six Rickettsia species of animals from Cerro Largo municipality, state of Rio Grande do Sul, Brazil, from July 2013 to January 2014
Exp Appl Acarol
Exp Appl Acarol
been associated with human spotted fever in several other areas of Brazil (Spolidorio et al. 2010; Silva et al. 2011; Krawczak et al. 2016a). In these other areas, A. ovale was epidemiologically assigned as vector of Rickettsia sp. strain Atlantic rainforest (Szabo´ et al. 2013; Barbieri et al. 2014; Nieri-Bastos et al. 2016), a condition recently demonstrated under laboratory conditions (Krawczak et al. 2016b). In the present study, &20 % of the dogs were seroreactive to spotted fever group rickettsiae, notably to R. parkeri, since most of the seroreactive dogs presented high endpoint titers to R. parkeri, and at least four-fold higher than the endpoint titers to the other rickettsial antigens that were tested. These serological results, coupled with the findings of Rickettsia sp. strain Atlantic rainforest infecting A. ovale ticks, provides evidence that dogs from the study area have been infected by strain Atlantic rainforest through the parasitism by A. ovale infected ticks. Because A. ovale is a recognized human-biting tick throughout South America (Guglielmone et al. 2006), we can infer that A. ovale is acting as a vector of strain Atlantic rainforest in Rio Grande do Sul. This assumption is reinforced by the fact that A. ovale was previously reported as the sole human-biting tick in the study area (Sangioni et al. 2011), and the clinical profile of the spotted fever cases of Rio Grande do Sul (mild course, no fatalities) are compatible with the disease caused by strain Atlantic rainforest (Spolidorio et al. 2010; Silva et al. 2011; Krawczak et al. 2016a). In a transmission area of strain Atlantic rainforest in southeastern Brazil, A. ovale was found to use dogs as hosts for adult ticks, and the Cricetidae rodent Euryoryzomys russatus and the marsupial Didelphis aurita as hosts for immature ticks (Szabo´ et al. 2013). In an area of Colombia, where A. ovale was also found infected by strain Atlantic rainforest, dogs were reported as main hosts for the tick adult stage, where immature stages of A. ovale were found on the rodents Proechimys semispinosus (Echimyidae) and Transandinomys talamancae (Cricetidae), and the marsupial Didelphis marsupialis (London˜o et al. 2014). In the present study, we found A. ovale adults on dogs, and immature stages on the Cricetidae rodent A. montensis and the marsupial D. albiventris. While these findings indicated that small mammals are main hosts for immature stages of A. ovale, it seems that different small mammal species might assume a host role at different ecological zones within the distribution area of A. ovale. It would be interesting to evaluate the role of these different small mammal species as amplifier hosts of Rickettsia sp. strain Atlantic rainforest to A. ovale ticks, since it could have a direct impact on the tick infection rate in nature. In fact, a laboratory study showed that Rickettsia sp. strain Atlantic rainforest is partially deleterious to A. ovale engorged females, suggesting that amplifier vertebrate hosts might be necessary for persistent perpetuation of Rickettsia sp. strain Atlantic rainforest in A. ovale under natural conditions (Krawczak et al. 2016b). Two other Rickettsia species are reported for the first time in Rio Grande do Sul state, namely R. bellii in I. loricatus and R. amblyommii in A. longirostre. While the pathogenicity role of these two agents for humans has not been confirmed, they have both been found infecting these respective tick species in different areas of Latin America (Horta et al. 2007; Ogrzewalska et al. 2015). The present study expand the distribution of the tick-borne pathogen Rickettsia sp. strain Atlantic rainforest to Rio Grande do Sul, the southernmost state of Brazil. This report also corresponds to the southernmost record of this tick-borne-pathogen, together with a previous report from another tick species, A. dubitatum, in northeastern Argentina (Monje et al. 2015). Once strain Atlantic rainforest has also been reported in A. ovale ticks from Colombia (London˜o et al. 2014), and as north as Belize (Lopes et al. 2016), it is likely that human illness have been unnoticed within many places within the distribution area of A. ovale, from Mexico to Argentina (Guglielmone et al. 2003).
123
Exp Appl Acarol Acknowledgments This work was financially supported by the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP process 2012/21915-4), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), and Coordenadoria de Apoio a Pesquisa e Desenvolvimento (CAPES), and has been authorized by the ‘‘Instituto Chico Mendes de Conservac¸a˜o da Biodiversidade’’ (authorization SISBIO 38502-1) and the Ethical Committee in Animal Research of the Faculty of Veterinary Medicine of the University of Sa˜o Paulo (Protocol 2908/2013).
References Arya R, Antonisamy B, KumarIndian S (2012) Sample size estimation in prevalence studies. J Pediatr 79:1482–1488 Barbieri FS, Brito LG, Labruna MB, Barros-Battesti DM, Camargo LMA, Famadas KM (2008) Description of the larva of Amblyomma ovale Koch, 1884 (Acari: Ixodidae) by light and scanning electron microscopy. Syst Appl Acarol 13:109–119 Barbieri AR, Filho JM, Nieri-Bastos FA, Souza JC Jr, Szabo´ MP, Labruna MB (2014) Epidemiology of Rickettsia sp. strain Atlantic rainforest in a spotted fever-endemic area of southern Brazil. Ticks Tickborne Dis 5:848–853 Barros-Battesti DM, Arzua M, Bechara GH (2006) Carrapatos de Importaˆncia Medico-Veterina´ria da Regia˜o Neotropical: um Guia Ilustrado para Identificac¸a˜o de Espe´cies. Vox/ICTTD-3/Butantan, Sa˜o Paulo, p 223 Guedes E, Leite RC, Prata MC, Pacheco RC, Walker DH, Labruna MB (2005) Detection of Rickettsia rickettsii in the tick Amblyomma cajennense in a new Brazilian spotted fever-endemic area in the state of Minas Gerais. Mem Inst Oswaldo Cruz 100:841–845 Guglielmone AA, Estrada-Pen˜a A, Mangold AJ, Barros-Battesti DM, Labruna MB, Martins JR, Venzal JM, Arzua M, Keirans JE (2003) Amblyomma aureolatum (Pallas, 1772) and Amblyomma ovale Koch, 1844 (Acari: Ixodidae): hosts, distribution and 16S rDNA sequences. Vet Parasitol 113:273–288 Guglielmone AA, Beati L, Barros-Battesti DM, Labruna MB, Nava S, Venzal JM, Mangold AJ, Szabo MP, Martins JR, Gonzalez-Acuna D (2006) Ticks (Ixodidae) on humans in South America. Exp Appl Acarol 40:83–100 Horta MC, Labruna MB, Pinter A, Linardi PM, Schumaker TT (2007) Rickettsia infection in five areas of the state of Sa˜o Paulo, Brazil. Mem Inst Oswaldo Cruz 102:793–801 Krawczak FS, Mun˜oz-Leal S, Guztazaky AC, Oliveira SV, Santos FCP, Angerami RN, Moraes-Filho J, Souza Jr JC, Labruna MB (2016a) Case Report: Rickettsia sp. strain Atlantic rainforest infection in a human patient from a spotted fever-endemic area in southern Brazil. Am J Trop Med Hyg (in press) Krawczak FS, Agostinho WC, Polo G, Moraes-Filho J, Labruna MB (2016b) Comparative evaluation of Amblyomma ovale ticks infected and noninfected by Rickettsia sp. strain Atlantic rainforest, the agent of an emerging rickettsiosis in Brazil. Ticks Tick-borne Dis 7:502–507 Labruna MB (2009) Ecology of Rickettsia in South America. Ann N Y Acad Sci 1166:156–166 Labruna MB, Marrelli MT, Heinemann JM, Fava AB, Cortez A, Soares RM, Sakamoto SM, Richtzenhain LJ, Marinotti O, Schumaker TT (2002) Taxonomic status of Ixodes didelphidis (Acari: Ixodidae). J Med Entomol 39:135–142 Labruna MB, Whitworth T, Horta MC, Bouyer DH, McBride J, Pinter A, Popov V, Gennari SM, Walker DH (2004) Rickettsia species infecting Amblyomma cooperi ticks from an area in the state of Sa˜o Paulo, Brazil, where Brazilian spotted fever is endemic. J Clin Microbiol 42:90–98 Labruna MB, Horta MC, Aguiar DM, Cavalcante GT, Pinter A, Gennari SM, Camargo LM (2007) Prevalence of Rickettsia infection in dogs from the urbanand rural areas of Monte Negro municipality, western Amazon, Brazil. Vector Borne Zoonotic Dis 7:249–255 Labruna MB, Santos FC, Ogrzewalska M, Nascimento EM, Colombo S, Marcili A, Angerami RN (2014) Genetic identification of rickettsial isolates from fatal cases of Brazilian spotted fever and comparison with Rickettsia rickettsii isolates from the American continents. J Clin Microbiol 52:3788–3791 London˜o AF, Dı´az FJ, Valbuena G, Gazi M, Labruna MB, Hidalgo M, Mattar S, Contreras V, Rodas JD (2014) Infection of Amblyomma ovale by Rickettsia sp. strain Atlantic rainforest, Colombia. Ticks Tick-borne Dis 5:672–675 Lopes MG, May J Jr, Foster RJ, Harmsen BJ, Sanchez E, Martins TF, Quigley H, Marcili A, Labruna MB (2016) Ticks and rickettsiae from wildlife in Belize, Central America. Parasit Vectors 9:62 Mangold AJ, Bargues MD, Mas-Coma S (1998) Mitochondrial 16S rDNAsequences and phylogenetic relationships of species of Rhipicephalus and othertick genera among Metastriata (Acari: Ixodidae). Parasitol Res 84:478–484
123
Exp Appl Acarol Marques S, Barros-Battesti DM, Onofrio VC, Famadas KM, Faccini JL, Keirans JE (2004) Redescription of larva, nymph and adults of Ixodes (I.) loricatus Neumann, 1899 (Acari: Ixodidae) based on light and scanning electron microscopy. Syst Parasitol 59:135–146 Martins TF, Onofrio VC, Barros-Battesti DM, Labruna MB (2010) Nymphs of thegenus Amblyomma (Acari: Ixodidae) of Brazil: descriptions, redescriptions, and identification key. Ticks Tick-borne Dis 1:75–99 Monje LD, Nava S, Eberhardt AT, Correa AI, Guglielmone AA, Beldomenico PM (2015) Molecular detection of the human pathogenic Rickettsia sp. strain Atlantic rainforest in Amblyomma dubitatum ticks from Argentina. Vector Borne Zoonotic Dis 15:167–169 Nieri-Bastos FA, Horta MC, Barros-Battesti DM, Moraes-Filho J, Ramirez DG, Martins TF, Labruna MB (2016) Isolation of the pathogen Rickettsia sp. strain Atlantic rainforest from its presumed tick vector, Amblyomma ovale, from two areas of Brazil. J Med Entomol (in press) ´ , Rodrı´guez BC, Prudencio C, Martins TF, Ogrzewalska M, Litera´k I, Capek M, Sychra O, Caldero´n VA Labruna MB (2015) Bacteria of the genus Rickettsia in ticks (Acari: Ixodidae) collected from birds in Costa Rica. Ticks Tick-borne Dis 6:478–482 Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, Abdad MY, Stenos J, Bitam I, Fournier PE, Raoult D (2013) Update on tick-borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev 26:657–702 Roux V, Fournier PE, 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 Sangioni LA, Horta MC, Vianna MC, Gennari SM, Soares RM, Galva˜o MA, Schumaker TT, Ferreira F, Vidotto O, Labruna MB (2005) Rickettsial infection in animals and Brazilian spotted fever endemicity. Emerg Infect Dis 11:265–270 Sangioni LA, Vogel FFS, Cadore GC, Hilger RB, Tonim R, Pacheco RC, Ogrzewalska M, Labruna MB (2011) Rickettsial infection in Cerro Largo, State of Rio Grande do Sul, Brazil. Arq Bras Med Vet Zootec 63:511–514 Silva N, Eremeeva ME, Rozental T, Ribeiro GS, Paddock CD, Ramos EAG, Favacho ARM, Reis MG, Dasch GA, de Lemos ERS, Ko AI (2011) Eschar-associated spotted fever Rickettsiosis, Bahia, Brazil. Emerg Infect Dis 17:275–278 Soto FRM, Ferreira F, Pinheiro SR, Nogari F, Risseto MR, Souza O, Amaku M (2006) Dinaˆmica populacional canina no municı´pio de Ibiu´na-SP: estudo retrospectivo. Braz J Vet Res Anim Sci 43:178–185 Spolidorio MG, Labruna MB, Mantovani E, Branda˜o P, Richtzenhain LJ, Yoshinari NH (2010) Novel spotted fever group rickettsioses, Brazil. Emerg Infect Dis 16:521–523 Szabo´ MP, Nieri-Bastos FA, Spolidorio MG, Martins TF, Barbieri AM, Labruna MB (2013) 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 140:719–728 Terassini FA, Barbieri FS, Albuquerque S, Szabo´ MP, Camargo LM, Labruna MB (2010) Comparison of two methods for collecting free-living ticks in the Amazonian forest. Ticks Tick-borne Dis 1:194–196 Umetsu F, Naxara L, Pardini R (2011) Evaluating the efficiency of pitfall traps for sampling small mammals in the Neotropics. J Mammal 87:757–765
123