Journal of Medical Entomology, Lanham, v. 45, n. 6, p ,

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1 Universidade de São Paulo Biblioteca Digital da Produção Intelectual - BDPI Departamento de Medicina Veterinária Prevenção e Saúde Animal Artigos e Materiais de Revistas Científicas - FMVZ/VPS - FMVZ/VPS 2008 Comparative susceptibility of larval stages of Amblyomma aureolatum, Amblyomma cajennense, and Rhipicephalus sanguineus to infection by Rickettsia rickettsii Journal of Medical Entomology, Lanham, v. 45, n. 6, p , Downloaded from: Biblioteca Digital da Produção Intelectual - BDPI, Universidade de São Paulo

2 Comparative Susceptibility of Larval Stages of Amblyomma aureolatum, Amblyomma cajennense, and Rhipicephalus sanguineus to Infection by Rickettsia rickettsii Author(s) :Marcelo B. Labruna, Maria Ogrzewalska, Thiago F. Martins, Adriano Pinter, and Maurício C. Horta Source: Journal of Medical Entomology, 45(6): Published By: Entomological Society of America DOI: / (2008)45[1156:CSOLSO]2.0.CO;2 URL: full/ / %282008%2945%5b1156%3acsolso%5d2.0.co %3B2 BioOne ( is a a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne s Terms of Use, available at terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

3 VECTOR/PATHOGEN/HOST INTERACTION, TRANSMISSION Comparative Susceptibility of Larval Stages of Amblyomma aureolatum, Amblyomma cajennense, and Rhipicephalus sanguineus to Infection by Rickettsia rickettsii MARCELO B. LABRUNA, 1,2 MARIA OGRZEWALSKA, 1 THIAGO F. MARTINS, 1 ADRIANO PINTER, 3 AND MAURÍCIO C. HORTA 1 J. Med. Entomol. 45(6): 1156Ð1159 (2008) ABSTRACT The current study compared the susceptibility of larval stages of Amblyomma cajennense (F.), Amblyomma aureolatum (Pallas), and Rhipicephalus sanguineus (Latreille) to infection by a Brazilian strain of Rickettsia rickettsii. Guinea pigs experimentally infected by R. rickettsii were simultaneously infested by larvae of the three tick species. Recovered engorged larvae were allowed to molt to nymphs and held in an incubator at 23 C and 85Ð90% RH. Subsequent ßat nymphs were tested for rickettsial infection by polymerase chain reaction (PCR). Concomitant infestations with sibling ticks on noninfected guinea pigs (control) were done. While 10Ð60% of the A. cajennense nymphs were shown to be infected by R. rickettsii, both A. aureolatum and R. sanguineus were highly susceptible to R. rickettsii, since 80Ð100% of their nymphs were shown to be infected in the corresponding trials. Most of the engorged larvae ( 70Ð95%), regardless of being infected or not, successfully molted to nymphs. Mortality rates for engorged larvae tended to be statistically similar (P 0.05) for ticks recovered from R. rickettsii-infected and noninfected guinea pigs, within each tick species. The only exceptions were the signiþcantly higher mortalities (P 0.05) for engorged A. cajennense larvae recovered from two infected guinea pigs. Therefore, A. cajennense was less susceptible to R. rickettsii infection than A. aureolatum and R. sanguineus, while feeding on rickettsemic guinea pigs. These two later species were similarly highly susceptible. KEY WORDS Amblyomma aureolatum, Amblyomma cajennense, Rhipicephalus sanguineus, Rickettsia rickettii, spotted fever The bacterium Rickettsia rickettsii is the etiological agent of Rocky Mountain spotted fever (RMSF), a disease also called Brazilian spotted fever (BSF) in Brazil. It is the most severe spotted fever group rickettsiosis in the world. In Brazil, the cayenne tick Amblyomma cajennense (F.) is the vector of BSF in most of the endemic areas, whereas the yellow dog tick Amblyomma aureolatum (Pallas) is the vector in a few areas of the São Paulo Metropolitan area (Pinter and Labruna 2006, Horta et al. 2007). The brown dog tick Rhipicephalus sanguineus (Latreille) is a recognized vector of R. rickettsii in the United States and Mexico and also a suspected vector in Brazil (Bustamante and Varela 1947, Demma et al. 2005, Moraes-Filho et al. 2008). 1 Faculty of Veterinary Medicine, University of São Paulo, São Paulo, SP, Brazil. 2 Corresponding author: 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 , Brazil ( labruna@usp.br). 3 Superintendência de Controle de Endemias, SUCEN, São Paulo, SP, Brazil. This study compared susceptibility of the larval stages of A. cajennense, A. aureolatum, and R. sanguineus to infection by strain Taiaçu, a Brazilian strain of R. rickettsii. Materials and Methods Strain Taiaçu was originally isolated from an A. aureolatum tick from Mogi das Cruzes (23 38 S, W), state of São Paulo, through the inoculation of guinea pigs with infected tick homogenate and subsequent adaptation of the strain to Vero cell culture (Pinter and Labruna 2006). Only the guinea pig lineage of strain Taiaçu (never cultured in vitro) was used in this study. For this purpose, guinea pig infected organs were thawed at room temperature and crushed in a mortar with brain-heart infusion (BHI), and the resultant homogenate was used to inoculate guinea pigs intraperitoneally. All guinea pigs used in this study were provided by a private laboratory that rears them under proper sanitary conditions. The study was divided into three experiments. In experiment 1, six guinea pigs were used: four inoculated by a homogenate of R. rickettsii infected guinea pig organs (brain or spleen from the Þrst guinea pig /08/1156Ð1159$04.00/ Entomological Society of America

4 November 2008 LABRUNA ET AL.: SUSCEPTIBILITY OF TICKS TO R. rickettsii 1157 passage) and two uninfected controls inoculated only with BHI. Each guinea pig had three feeding chambers glued on its shaved back, with each feeding chamber infested by 400 larvae of each of the tick species A. aureolatum, A. cajennense, and R. sanguineus. The A. aureolatum larvae were the F 1 progeny of engorged females collected in Atibaia (23 06 S, W), state of São Paulo. The A. cajennense larvae were the F 2 progeny of a laboratory colony derived from engorged females collected in Pirassununga (21 59 S, W), state of São Paulo. The R. sanguineus larvae were the F 5 progeny of a laboratory colony derived from engorged females collected in Cabreúva (23 18 S, W), state of São Paulo. In experiment 2, six guinea pigs were used: four inoculated by a homogenate of R. rickettsii infected guinea pig brain (second guinea pig passage) and two uninfected controls inoculated only with BHI. Each guinea pig had two feeding chambers glued on its shaved back, with each feeding chamber infested with 400 larvae of each of the tick species A. aureolatum and R. sanguineus. The A. aureolatum larvae were the F 1 progeny of engorged females collected in São Roque (23 31 S, W), state of São Paulo. The R. sanguineus larvae were the F 8 progeny of a laboratory colony derived from engorged females collected in Seropédica (22 44 S, W), state of Rio de Janeiro. In experiment 3, four guinea pigs were inoculated by a homogenate of R. rickettsii infected guinea pig organs (spleen or blood from the third guinea pig passage). Each guinea pig had two feeding chambers glued on its shaved back, and each feeding chamber was infested with 400 larvae of each of the tick species A. aureolatum and A. cajennense. The A. aureolatum larvae were the F 1 progeny of engorged females collected in São Paulo city (23 35 S, W), state of São Paulo. The A. cajennense larvae were the F 1 progeny of engorged females collected in Pedreira (22 47 S, W), state of São Paulo. All larvae used in this study were indirectly shown to be free of Rickettsia infection by testing the original progenitor female after oviposition by PCR targeting the rickettsial glta gene (same protocol described below). All infestations with A. aureolatum larvae were performed at the same day of guinea pig inoculation (day 0), whereas infestations by both A. cajennense and R. sanguineus were performed 2 d after inoculation (DAI). This procedure was adopted to synchronize the detachment peak of engorged larvae of the three species, because larval feeding period tends to be 2 d longer for A. aureolatum (mean feeding period 5d) than for A. cajennense or R. sanguineus (mean feeding period 3 d). Naturally detached engorged larvae were recovered daily inside the feeding chambers and held in an incubator at 23 C and 85Ð90% RH for molting to nymphs. Samples of 10Ð20 ßat nymphs 15Ð20 d old from each tick species, molted from engorged larvae recovered from each guinea pig were submitted to DNA extraction by the guanidine isothiocyanate-phenol technique, as previously described (Sangioni et al. 2005). For every 10 individual ticks, a blank tube was included in the DNA extraction. Samples were tested individually by PCR targeting a 401-bp fragment of the rickettsial gene glta as previously described (Labruna et al. 2004). In each set of reactions, Þve negative control tubes containing water were included, and also a positive control tube containing R. parkeri DNA. At the end of experiment 1, PCR product from at least one nymph from each infected guinea pig underwent DNA sequencing, and the resultant sequence was compared with GenBank data by Blast analysis. During experiment 1, the total number of dead engorged larvae and ßat nymphs were counted for each guinea pig to calculate the mortality of engorged larvae for each tick species. The proportions of ticks that successfully molted after exposure to infected and uninfected guinea pigs were analyzed using 2 tests. We followed the protocol that agrees with Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation (COBEA) and that was approved by the Faculdade de Medicina Veterinária e Zootecnia /USP-Ethical Committee for Animal Research. Permits and approvals are on Þle in the ofþce of M.B.L. Results and Discussion Results of experiments 1, 2, and 3 are given in Table 1. Although only 10Ð60% of the A. cajennense ticks were shown to be infected by R. rickettsii, both A. aureolatum and R. sanguineus were highly susceptible to R. rickettsii, because 80Ð100% of their nymphs were shown to be infected after feeding as larvae on infected guinea pigs. The only exceptions were guinea pig 2 in experiment 2, from which no infected tick was found, and guinea pig 3 in experiment 3, from which only 20 and 5% of the A. aureolatum and A. cajennense nymphs, respectively, were shown to be infected. In experiment 2, guinea pig 2 developed no fever (in contrast to guinea pigs 1, 3, and 4); thus, it seems that its infection by R. rickettsii was not successful, resulting in the absence of infected nymphs. In experiment 3, guinea pig 3 developed only 2 d of fever, with temperatures never reaching 40 C, and survived the infection, in contrast to the other infected ones that developed 3Ð5 d of fever with temperatures reaching 40 C (temperature data not shown), and died during the febrile period. Thus, it seems that guinea pig 3 developed a milder infection, resulting in fewer infected ticks, either A. aureolatum or A. cajennense. PCR products from one to two nymphs from each infected guinea pig in experiment 1 were DNA sequenced and shown to be 100% identical to R. rickettsii strain Taiaçu, available in GenBank (DQ115890). In experiment 1, most of the engorged larvae, regardless of being infected or not, successfully molted to nymphs. Mortality rates for engorged larvae tended to be statistically similar (P 0.05) for ticks recovered from R. rickettsii infected and noninfected guinea pigs, within each tick species (Table 2). The only exceptions were the signiþcantly higher mortalities

5 1158 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 45, no. 6 Table 1. Results of three experiments (1, 2, and 3) that tested the percent (%) flat nymphs of three tick species that were infected by R. rickettsii, after the larval stage fed on R. rickettsii infected guinea pigs No. Guinea pigs Inoculation source b Ticks used for larval infestation a Amblyomma aureolatum Amblyomma cajennense Rhipicephalus sanguineus Days of fever c Percent Percent Percent Colony infection d Colony infection d Colony infection d Experiment 1 e 1 Brain 3 f Atibaia 100 (15/15) Pirassununga 60 (6/10) Cabreúva 100 (12/12) 2 Brain 2 f Atibaia 100 (10/10) Pirassununga 50 (5/10) Cabreúva 100 (10/10) 3 Spleen 3 f Atibaia 100 (10/10) Pirassununga 30 (3/10) Cabreúva 90 (9/10) 4 Spleen 3 f Atibaia 100 (10/10) Pirassununga 0 (0/10) Cabreúva 90 (9/10) Experiment 2 e 1 Brain 4 f São Roque 80 (8/10) Ñ Ñ Seropédica 80 (8/10) 2 Brain 0 São Roque 0 (0/10) Ñ Ñ Seropédica 0 (0/10) 3 Brain 4 f São Roque 100 (10/10) Ñ Ñ Seropédica 100 (10/10) 4 Brain 5 f São Roque 100 (10/10) Ñ Ñ Seropédica 90 (9/10) Experiment 3 1 Spleen 3 f São Paulo 100 (20/20) Pedreira 15 (3/20) Ñ Ñ 2 Spleen 4 f São Paulo 100 (20/20) Pedreira 25 (5/20) Ñ Ñ 3 Blood 2 São Paulo 20 (4/20) Pedreira 5 (1/20) Ñ Ñ 4 Blood 5 f São Paulo 100 (20/20) Pedreira 10 (2/20) Ñ Ñ a Inoculated guinea pigs were infested by larvae (larvae of each tick species were infested separately within a feeding chamber glued to the guinea pig shaved dorsum). Engorged larvae were recovered and allowed to molt to nymphs. The percent infection was determined by testing the resultant nymphs by PCR targeting rickettsial DNA. b Guinea pigs were inoculated intraperinoneally with 1Ð2 ml of BHI containing a homogenate of an internal organ (brain, spleen, or blood) collected from a R. rickettsii infected guinea pig that was killed during the febrile period. c Number of days that guinea pigs developed fever (rectal temperature 39.5 C) during the larval feeding period. d Percent infected nymphs (no. PCR-positive nymphs/no. nymphs tested by PCR). e In this experiment, two additional guinea pigs were inoculated only with BHI (uninfected controls) and infested with larval ticks as done with the infected guinea pigs. None of the examined ßat nymphs (10 per guinea pig) originated from engorged larvae were PCR positive. f Guinea pig died during the acute febrile period. (P 0.05) for A. cajennenseðengorged larvae recovered from guinea pigs 2 and 3. The absence of signiþcantly different mortality between R. rickettsii infected and Ðnoninfected nymphs of either A. aureolatum or R. sanguineus indicates that R. rickettsii had no significant lethal effect on the engorged larvae of these two species. Experimental studies with Dermacentor andersoni Stiles (natural vector of R. rickettsii in western United States) showed that most of the engorged larvae successfully molted to nymphs after feeding on R. rickettsii infected guinea pigs, in contrast to high mortality of the subsequent nymphal and adult feeding (Niebylski et al. 1999). These authors argued that because of the highest tolerance of the D. andersoni larval stage to the R. rickettsii infection, this tick stage is a critical link for R. rickettsii to cycle between vertebrates. Table 2. Mortality of engorged larvae of A. aureolatum, A. cajennense, and R. sanguineus ticks fed on uninfected guinea pigs (5, 6) and guinea pigs infected by R. rickettsii (1, 2, 3, 4) Guinea pig No. dead engorged larvae/total no. engorged larvae (% mortality) A. aureolatum A. cajennense R. sanguineus 1 17/166 (10.2) a 5/119 (4.2) a 5/48 (10.4) a 2 12/173 (6.9) a 13/38 (34.2) b 6/55 (10.9) a 3 8/133 (6.0) a 4/21 (19.0) b 2/25 (8.0) a 4 10/207 (4.8) a 3/158 (1.9) a 13/99 (13.1) a 5 13/140 (9.3) a 5/245 (2.0) a 3/14 (21.4) a 6 11/110 (10.0) a 1/120 (0.8) a 11/61 (18.0) a Different letters in the same column mean statistically signiþcant differences (P 0.05). This study showed that A. cajennense is clearly less susceptible to R. rickettsii infection than A. aureolatum and R. sanguineus while feeding on rickettsemic guinea pigs. These two latter species were similarly highly susceptible. In this regard, the following statements should be noted: (1) detachment of engorged larvae from guinea pigs peaked at the Þfth DAI for the three tick species, and we tested only nymphs that molted from larvae detached on this single day; (2) engorged larvae of A. cajennense and A. aureolatum are of similar size, whereas the engorged larvae of R. sanguineus are visually smaller; thus, it is unlikely that lower infection rates of A. cajennense nymphs were caused by a smaller amount of ingested blood during the larval stage; (3) even though there was a signiþcantly higher mortality for R. rickettsii--exposed A. cajennense larvae after feeding on two guinea pigs in experiment 1, the majority of engorged larvae survived; thus, one cannot say that the reason for fewer R. rickettsii infected A. cajennense nymphs was because more infected larvae died in this species; (4) and Þnally, we obtained similar results for A. cajennense ticks from two different populations, one from Pedreira (a BSF-endemic area), representing an A. cajennense population that is incriminated in transmitting R. rickettsii to humans and animals under natural conditions (Sangioni et al. 2005). Thus, the reasons for lower susceptibility of A. cajennense to R. rickettsii remain to be studied. Amblyomma cajennense is the most frequent human-biting tick in Brazil (Guglielmone et al. 2006). Its high aggressiveness to humans is evident when, not

6 November 2008 LABRUNA ET AL.: SUSCEPTIBILITY OF TICKS TO R. rickettsii 1159 rarely, humans suffer mass attack by hundreds to thousands of larvae and/or dozens of nymphs and adults. We observed that A. cajennense is always present in high population levels in the BSF-endemic areas (Sangioni et al. 2005). In contrast, both A. aureolatum and R. sanguineus are less anthropophilic, because they do not attack humans frequently, and when they do, there is usually only a single adult specimen (Pinter et al. 2004, Dantas-Torres et al. 2006, Guglielmone et al. 2006). Moreover, A. aureolatum is usually present in low population levels in its speciþc BSF-endemic areas (Pinter et al. 2004). Interestingly, during the last 10 yr in the state of São Paulo, the number of BSF-conÞrmed cases per locality was similar among the endemic areas where either A. cajennense or A. aureolatum was the vector. In this regard, we compared ofþcial data available in the São Paulo State Health Secretary website ( from 1998 to 2007, the mean number of BSF-conÞrmed cases per endemic area was (range: 1Ð11) among seven A. aureolatum areas and (range: 1Ð38) among 28 A. cajennense areas (mean values statistically similar; StudentÕs t-test, P 0.616, df 23). In addition, a recent study (Sangioni et al. 2005) in three A. cajennense BSF-endemic areas in the state of São Paulo (where both A. aureolatum and R. sanguineus were absent) failed to detect any R. rickettsii infected A. cajennense ticks (810 adult ticks were tested by PCR), whereas another study (Pinter and Labruna 2006) in an A. aureolatum BSF-endemic area (where both A. cajennense and R. sanguineus were absent) found 6 (0.89%) of 669 A. aureolatum adult ticks to be infected by R. rickettsii. Based on the results of this study, it is likely that many fewer individual ticks are infected by R. rickettsii within A. cajennense populations than in the A. aureolatum populations in the BSF-endemic areas. These infection rate differences might account for the similar number of BSF cases among A. cajennense and A. aureolatum BSF-endemic areas, even though there are many more human infestations by A. cajennense than by A. aureolatum. Finally, our study showed that R. sanguineus larvae are as susceptible as are A. aureolatum larvae for R. rickettsii infection. Because R. sanguineus is a recognized vector of RMSF in the United States and Mexico and was recently found naturally infected by R. rickettsii in a BSF-endemic area in the state of São Paulo (Moraes-Filho et al. 2008), the role of this tick in the epidemiology of R. rickettsii in Brazil might have been possibly underestimated. Acknowledgments We thank Laboratório Biovet, Brazil, for providing naõ ve guinea pigs, and E. M. Piranda (Federal Rural University of Rio de Janeiro) for providing R. sanguineus larvae from Seropédica. This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientõ Þco e Tecnológico (CNPq). References Cited Bustamante, M. E., and G. Varela Distribuicion de las rickettsiasis en Mexico. Rev. Inst. Salub. Enf. Trops. 8: 3Ð14. Dantas-Torres, F., L. A. Figueiredo, and S. P. Brandão-Filho Rhipicephalus sanguineus (Acari: Ixodidae), the brown dog tick, parasitizing humans in Brazil. Rev. Soc. Bras. Med. Trop. 39: 64Ð67. Demma, L. J., M. Traeger, W. L. Nicholson, C. Paddock, D. Blau, M. Eremeeva, G. Dasch, M. Levin, J. Singleton, S. R. Zaki, J. Cheek, D. Swerdlow, and J. McQuiston Rocky Mountain spotted fever from an unexpected tick vector in Arizona. N. Engl. J. Med. 353: 587Ð594. Guglielmone, A. A., L. Beati, D. M. Barros-Battesti, M. B. Labruna, S. Nava, J. M. Venzal, A. J. Mangold, M.P.J. Szabó, J. R. Martins, D. Gonzalez-Acuna, and A. Estrada- Peña Ticks (Ixodidae) on humans in South America. Exp. Appl. Acarol. 40: 83Ð100. Horta, M. C., M. B. Labruna, A. Pinter, P. M. Linardi, and T.T.S. Schumaker Rickettsia infection in Þve areas of the state of São Paulo, Brazil. Mem. Inst. Oswaldo Cruz. 102: 793Ð801. Labruna, M. B., T. Whitworth, M. C. Horta, D. H. Bouyer, J. W. McBride, A. Pinter, V. Popov, S. M. Gennari, and D. H. Walker Rickettsia species infecting Amblyomma cooperi ticks from an area in the state of Sao Paulo, Brazil, where Brazilian spotted fever is endemic. J. Clin. Microbiol. 42: 90Ð98. Moraes-Filho, J., A. Pinter, R. C. Pacheco, T. B. Gutmann, S. O. Barbosa, M.A.R.M. Gonzáles, M. A. Muraro, S.R.M. Cecílio, and M. B. Labruna New epidemiological data on Brazilian spotted fever in an endemic area of the state of São Paulo, Brazil. Vectorborne Zoonotic Dis. (in press). Niebylski, M. L., M. G. Peacock, and T. G. Schwan Lethal effect of Rickettsia rickettsii on its tick vector (Dermacentor andersoni). Appl. Environ. Microbiol. 65: 773Ð 778. Pinter, A., and M. B. Labruna Isolation of Rickettsia rickettsii and Rickettsia bellii in cell culture from the tick Amblyomma aureolatum in Brazil. Ann. N.Y. Acad. Sci. 1078: 523Ð529. Pinter, A., R. A. Dias, S. M. Gennari, and M. B. Labruna Study of the seasonal dynamics, life cycle, and host speciþcity of Amblyomma aureolatum (Acari: Ixodidae). J. Med. Entomol. 41: 324Ð332. Sangioni, L. A., M. C. Horta, M.C.B. Vianna, S. M. Gennari, R. M. Soares, M.A.M. Galvão, T.T.S. Schumaker, F. Ferreira, O. Vidotto, and M. B. Labruna Rickettsial infection in animals and Brazilian spotted fever endemicity. Emerg. Infect. Dis. 11: 265Ð270. Received 14 June 2008; accepted 1 August 2008.