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    The southern portion of the state of Rio Grande do Sul, Brazil, indicating 33 localities where dogs were sampled for rickettsial infection.

  • 1

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    Guedes E, Leite RC, Prata MCA, 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.

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    Nascimento EM, Gehrke FS, Maldonado RA, Colombo S, Silva LJ, Schumaker TT, 2005. Detection of Brazilian spotted fever infection by polymerase chain reaction in a patient from the state of São Paulo. Mem Inst Oswaldo Cruz 100 :277–279.

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    Pinter A, Labruna MB, 2006. Isolation of Rickettsia rickettsii and Rickettsia bellii in cell culture from the tick Amblyomma aureolatum in Brazil. Ann N Y Acad Sci 1078 :523–529.

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    Evans DE, Martins JR, Guglielmone AA, 2000. A review of the ticks (Acari: Ixodidae) of Brazil, their hosts and geographic distribution 1. The state of Rio Grande do Sul, Southern Brazil. Mem Inst Oswaldo Cruz 95 :453–470.

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    La Scola B, Raoult D, 1997. Laboratory diagnosis of rickettsioses: current approaches to diagnosis of old and new rickettsial diseases. J Clin Microbiol 35 :2715–2727.

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  • 11

    Labruna MB, Horta MC, Aguiar DM, Cavalcante GT, Pinter A, Gennari SM, Camargo LMA, 2007. Prevalence of Rickettsia infection in dogs from the urban and rural areas of Monte Negro Municipality, western Amazon, Brazil. Vector Borne Zoonotic Dis 7 :249–255.

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    Breitschwerdt EB, Walker DH, Levy MG, Burgdorfer W, Corbett WT, Hurlbert SA, Stebbins ME, Curtis BC, Allen DA, 1988. Clinical, hematologic, and humoral immune response in female dogs inoculated with Rickettsia rickettsii and Rickettsia montana. Am J Vet Res 49 :70–76.

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    Santibáñez S, Ibarra V, Portillo A, Blanco JR, Martínez de Artola V, Guerrero A, Oteo JA, 2006. Evaluation of IgG antibody response against Rickettsia conorii and Rickettsia slovaca in patients with DEBONEL/TIBOLA. Ann NY Acad Sci 1078 :570–572.

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  • 14

    Nascimento EM, Schumaker TTS, 2004. Isolamento e identificação de riquétsias no Brasil. Rev Bras Parasitol Vet 13 :193–196.

  • 15

    Labruna MB, Whitworth T, Horta MC, Bouyer DH, McBride JW, Pinter A, Popov V, Gennari SM, Walker DH, 2004. Rickettsia species infecting Amblyomma cooperi ticks from an area in the State of São Paulo, Brazil, where Brazilian spotted fever is endemic. J Clin Microbiol 42 :90–98.

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  • 16

    Silveira I, Pacheco RC, Szabó MPJ, Ramos HGC, Labruna MB, 2007. First report of Rickettsia parkeri in Brazil. Emerg Infect Dis 13 :1111–1113.

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  • 17

    Labruna MB, Whitworth T, Bouyer DH, McBride JW, Camargo LMA, Camargo EP, Popov V, Walker DH, 2004. Rickettsia bellii and Rickettsia amblyommii in Amblyomma ticks from the State of Rondônia, Western Amazon, Brazil. J Med Entomol 41 :1073–1081.

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  • 18

    Labruna MB, Pacheco RC, Richtzenhain LJ, Szabo MP, 2007. Isolation of Rickettsia rhipicephali and Rickettsia bellii from ticks Haemaphysalis juxtakochi in the state of Sao Paulo, Brazil. Appl Environ Microbiol 73 :869–873.

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  • 19

    Horta MC, Labruna MB, Pinter A, Linardi PM, Schumaker TTS, 2007. Rickettsia infection in five areas of the State of São Paulo, Brazil. Mem Inst Oswaldo Cruz 102 :793–801.

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  • 20

    Paddock CD, Sumner JW, Comer JA, Zaki SR, Goldsmith CS, Goddard J, McLellan SL, Tamminga CL, Ohl CA, 2004. Rickettsia parkeri: a newly recognized cause of spotted fever rick-ettsiosis in the United States. Clin Infect Dis 38 :805–811.

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  • 21

    Aguiar DM, Cavalcante GT, Pinter A, Gennari SM, Camargo LMA, Labruna MB, 2007. Prevalence of Ehrlichia canis (Rick-ettsiales: Anaplasmataceae) in dogs and Rhipicephalus sanguineus (Acari: Ixodidae) ticks from Brazil. J Med Entomol 44 :126–132.

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  • 22

    Horta MC, Labruna MB, Sangioni LA, Vianna MCB, Gennari SM, Galvão MAM, Mafra CL, Vidotto O, Schumaker TTS, Walker DH, 2004. Prevalence of antibodies to spotted fever group Rickettsiae in humans and domestic animals in a Brazilian spotted fever-endemic area in the State of São Paulo, Brazil: serologic evidence for infection by Rickettsia rickettsii and another spotted fever group Rickettsia. Am J Trop Med Hyg 71 :93–97.

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  • 23

    Aguiar DM, Saito TB, Hagiwara MK, Machado RZ, Labruna MB, 2007. Diagnóstico sorológico de erliquiose canina com antígeno brasileiro de Ehrlichia canis. Cien Rural Santa Maria 37 :796–802.

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  • 24

    McBride JW, Corstvet RE, Breitschwerdt EB, Walker DH, 2001. Immunodiagnosis of Ehrlichia canis infection with recombinant proteins. J Clin Microbiol 39 :315–322.

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    SPSS for Windows, 1999. SPSS Base 9.0.1. Users Guide. Chicago: SPSS.

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    Paddock CD, 2005. Rickettsia parkeri as a paradigm for multiple causes of tick-borne spotted fever in the western hemisphere. Ann NY Acad Sci 1063 :315–326.

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  • 27

    Venzal JM, Portillo A, Estrada-Peña A, Castro O, Cabrera PA, Oteo JA, 2004. Rickettsia parkeri in Amblyomma triste from Uruguay. Emerg Infect Dis 10 :1493–1495.

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    Chapman AS, Bakken JS, Folk SM, Paddock CD, Bloch KC, Krusell A, Sexton DJ, Buckingham SC, Marshall GS, Storch GA, Dasch GA, McQuiston JH, Swerdlow DL, Dumler SJ, Nicholson WL, Walker DH, Eremeeva ME, Ohl CA, Tick-borne Rickettsial Diseases Working Group CDC, 2006. Diagnosis and management of tickborne rickettsial diseases: Rocky Mountain spotted fever, ehrlichioses, and anaplasmosis-United States: a practical guide for physicians and other health-care and public health professionals. MMWR Recomm Rep 55 :1–27.

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    Pinter A, Horta MC, Pacheco RC, Moraes-Filho J, Labruna MB, 2008. Serosurvey for Rickettsia spp. in dogs and humans from a Brazilian spotted fever endemic area in the state of São Paulo. Cad Saude Publica 24:.

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    Sangioni LA, Horta MC, Vianna MCB, Gennari SM, Soares RM, Galvão MAM, Schumaker TTS, Ferreira F, Vidotto O, Labruna MB, 2005. Rickettsial infection in animals and Brazilian spotted fever endemicity. Emerg Infect Dis 11 :265–270.

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  • 31

    Labruna MB, Pereira MC, 2001. Carrapato em cães no Brasil. Rev Clin Vet 6 :24–32.

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    Labarthe N, Campos Pereira M, Balbarini O, Mckee W, Coimbra CA, Hoskins J, 2003. Serologic prevalence of Dirofilaria immintis, Ehrlichia canis and Borrelia burgdorferi infection in Brazil. Vet Ther 4 :67–75.

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    Venzal JM, Castro O, Cabrera PA, De Souza CG, Guglielmone AA, 2003. Garrapatas de importancia médica y veterinaria en Uruguay. Entomol. Vect 10 :635–650.

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    Pacheco RC, Venzal JM, Richtzenhain LJ, Labruna MB, 2006. Rickettsia parkeri in Uruguay. Emerg Infec Dis 12 :1804–1805.

 

 

 

 

 

Canine Infection by Rickettsiae and Ehrlichiae in Southern Brazil

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  • 1 Faculty of Veterinary Medicine, University of São Paulo, São Paulo, SP, Brazil; Institute of Biology, Federal University of Pelotas, Pelotas, RS, Brazil; Faculty of Veterinary Medicine, Federal University of Pelotas, Pelotas, RS, Brazil

This study evaluated the infection caused by Rickettsia and Ehrlichia agents among dogs in southern Brazil. A total of 389 dogs were tested by the indirect immunofluorescence assay (IFA) for Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommii, Rickettsia rhipicephali, Rickettsia bellii, and Ehrlichia canis. Overall, 42.4% (165/389) of the dogs were seroreactive to at least one Rickettsia species, but only 11 canine sera reacted with another Rickettsia species without reacting with R. parkeri. A total of 100 (25.7%) canine sera showed titers to R. parkeri at least 4-fold higher than those to any of the other rickettsial antigens, allowing us to consider that these dogs were infected by R. parkeri. Dogs that had direct contact with pasture or forest areas were > 2 times more likely to be seroreactive to Rickettsia than dogs with no such direct contact. Only 19 (4.8%) of the 389 dogs were seroreactive to E. canis.

INTRODUCTION

The genus Rickettsia is comprised of obligate intracellular bacteria distributed worldwide, with some species maintained in nature and transmitted to vertebrate hosts by arthropod vectors.1 Most of the pathogenic Rickettsia species are vectored by ticks and are classified into the spotted fever group (SFG).1 Until the beginning of this century, the SFG agent Rickettsia rickettsii was the only tick-borne Rickettsia known to occur in South America. R. rickettsii is the most pathogenic SFG species, causing a severe clinical disease called Brazilian spotted fever (BSF) in Brazil and Rocky Mountain spotted fever (RMSF) in the United States. The lethality rate of BSF before the antibiotic era was up to 80%, and nowadays it is still ~40% in southeastern Brazil.24 BSF has been classically reported to occur in the southeastern region of Brazil, which includes the states of Minas Gerais, São Paulo, Rio de Janeiro, and Espírito Santo. In these states, the occurrence of the disease has been always accompanied by severe clinical cases that culminate in death, because of the absence of or delayed treatment with effective antirickettsial therapy, such as tetracyclines and chloramphenicol. Isolation and specific identification of R. rickettsii has been done in different occasions in Minas Gerais and São Paulo.36 In southeastern Brazil, BSF is transmitted mostly by the tick Amblyomma cajennense, and also by Amblyomma aureolatum in a few areas of the state of São Paulo.4,6

Recently, mild febrile illness diagnosed as BSF by serology using R. rickettsii antigens was reported in areas where the classic severe and lethal BSF was never reported. These reports were mostly in the states of Santa Catarina and Rio Grande do Sul, in the southern region of Brazil7 (Brazilian Ministry of Health, unpublished data). Interestingly, several of these cases resolved without antibiotic therapy, showing none of the clinical complications usually observed for the BSF in the southeastern region, such as jaundice and central nervous system dysfunction. In contrast, many of these southern cases showed lymphadenopathy, a clinical sign not reported for the classic BSF cases in the southeastern region.2,7 Finally, the most important vector (A. cajennense) of BSF in southeastern Brazil is not established in Santa Catarina and Rio Grande do Sul (southern Brazil) because of extreme winter temperatures.8,9

Cross-reactivity of rickettsial antigens results in antibody responses that are typically group specific but not necessarily species specific. For example, serologic tests that detect antibodies reactive with a given SFG species might have resulted from previous infections with another SFG species. Testing a clinical serum against the possible Rickettsia species known to occur in a given area is ideal because homologous antibody titers tend to be higher than heterologous antibody titers. In some cases, the differences in titers may be higher enough to differentiate between the rickettsial species,1012 whereas in other cases, both homologous and heterologous titers are similar.11,13

Clinical cases of BSF in southern Brazil have been laboratory confirmed solely by serology, using R. rickettsii antigens in the indirect immunofluorescence assay (IFA).7,14 Because cross-reaction antibodies between Rickettsia species are often observed, especially between SFG species, a serologic assay using a single rickettsial antigen is not sufficient to determine the Rickettsia species that elicited the serologic response.10 For this reason, the SFG Rickettsia species involved in the BSF cases reported in southern Brazil remains unknown.

Recently, a number of Rickettsia species have been reported infecting ticks in Brazil for the first time. These include the following SFG species: Rickettsia parkeri infecting Amblyomma ticks in the state of São Paulo,15,16 Rickettsia amblyommii infecting Amblyomma ticks in northern Brazil,17 and Rickettsia rhipicephali infecting Haemaphysalis juxtakochi in the state of São Paulo.18 In addition, Rickettsia bellii, a species classified outside the SFG, has been reported infecting a number of tick species belonging to the genera Amblyomma, Haemaphysalis, and Ixodes in northern and southeastern Brazil.6,15,1719 The pathogenicity of these Rickettsia species for humans or animals remains unknown, except for R. parkeri, which was first shown to cause spotted fever in humans in the United States in 2004, 65 years after this rickettsia was first isolated from the tick Amblyomma maculatum in that country.20

The ehrlichial agent Ehrlichia canis is currently the most important canine tick-borne pathogen of Latin America, where it is transmitted by the tick Rhipicephalus sanguineus. E. canis is the etiologic agent of canine monocytic ehrlichiosis (CME), recognized as the most prevalent tick-borne disease affecting dogs in Brazil, especially in the urban and suburban areas, where R. sanguineus is highly prevalent.21 This study evaluated infection caused by Rickettsia and Ehrlichia agents among dogs in the state of Rio Grande do Sul, southern Brazil, where there are only a few reports of canine ehrlichial infection, and Rickettsia was never reported infecting animals.

MATERIALS AND METHODS

This study sampled dogs living in 33 localities (i.e., beef and milk farms in case of rural area, households in case of urban area) in 10 municipalities located in the southern portion of the state of Rio Grande do Sul, Brazil, close to the border with Uruguay (Table 1; Figure 1). Dogs were sampled primarily for another project on the epidemiology of neosporosis (NA Cunha-Filho, unpublished data), and thereafter, the stored samples were made available for this study. Blood serum samples were obtained from 87 dogs from September 2002 to September 2003 and from 302 dogs from May to December 2006. None of the sampled dogs presented clinical signs of acute infectious diseases when the blood was collected.

Each canine serum was tested by IFA using crude antigens derived from five Rickettsia isolates from Brazil (R. bellii strain Mogi, R. amblyommii strain Ac37, R. rhipicephali strain HJ5, R. rickettsii strain Taiaçu, and R. parkeri strain At24), as previously described.11 Canine sera were diluted in 2-fold increments with phosphate-buffered saline (PBS), starting from the 1:64 dilution. For each sample, the endpoint IgG titer reacting with each of the five Rickettsia antigens was determined. Serum showing for a Rickettsia species titer at least 4-fold higher than that observed for any other Rickettsia species was considered homologous to the first Rickettsia species or to a very closely related species.11,22 In each slide, a serum previously shown to be non-reactive (negative control) and a known reactive serum (positive control) were tested at the 1:64 dilution. This positive control serum was previously shown to react with R. rickettsii, R. parkeri, R. bellii, R. amblyommii, and R. rhipicephali at endpoint titers of 8,192, 2,048, 1,024, 1,024, and 1,024, respectively.11

Serum samples were also tested by IFA using E. canis–infected DH82 cells as antigen, performed with the Jaboticabal strain of E. canis from Brazil as previously described.21,23 Serum was considered to contain antibodies reactive to E. canis if it displayed a reaction at the 1:40 dilution.24 In each slide, a serum previously shown to be non-reactive (negative control) and a known reactive canine serum (positive control; endpoint titer 1,280) were tested at the 1:40 dilution.21 Samples that reacted at the screening dilution (1:40) were titrated using serial 2-fold dilutions to determine endpoint titers.

For each sampled dog, a questionnaire was given to the dog owner with the purpose of gaining information about independent variables that could be associated with seroreactivity to Rickettsia spp. or E. canis (dependent variables). For statistical analysis, seroreactivity to Rickettsia spp. or E. canis was analyzed qualitatively at a dichotomous level (0, non-reactive for Rickettsia spp. or E. canis; 1, reactive at the 1:64 dilution for any of the five Rickettsia species or reactive at the 1:40 dilution for E. canis). First, the independent variables were subjected to univariate analysis, and those with statistical association (P < 0.20, χ2 test or Fisher exact test) were tested in the multivariate model by the stepwise forward method. The variables were included in the multivariate model if they displayed statistical significance of P < 0.05. The odds ratio (OR) was calculated for the independent variable that showed statistical significance in the multivariate analysis. All analyses were performed using SPSS for Windows.25

RESULTS

Serologic results for dogs sampled in 2002–2003 were not compared with results for dogs samples in 2006 because samples were collected in different localities. However, because the procedures for collecting, storing, and testing canine sera obtained in 2002–2003 and 2006 were the same, the results were pooled for presentation. A total of 389 dogs from 33 localities were tested by IFA for five Rickettsia species. Overall, 42.4% (165/389) of the dogs were seroreactive (titer ≥ 64) to at least one Rickettsia species. Only 8 of the 33 localities had all dogs seronegative for Rickettsia spp. The remaining localities had 12.5–100% of the dogs reactive to at least one Rickettsia species. A total of 154 (39.6%), 137 (35.2%), 131 (33.7%), 132 (33.9%), and 18 (4.6%) dogs were reactive to R. parkeri, R. rickettsii, R. amblyommii, R. rhipicephali, and R. bellii, respectively (Table 2). Only 11 sera reacted with another Rickettsia species without reacting with R. parkeri.

A total of 100 (25.7%) canine sera from 23 localities showed titers to R. parkeri at least 4-fold higher than those to any of the other four antigens. The antibody titers in these 100 dogs were considered to have been stimulated by R. parkeri or a very closely related species (Table 2). Based on this criteria, only one serum was considered to have been stimulated by R. amblyommii (IFA titers: 128 for R. amblyommii, seronegative for the other antigens), and another serum was considered to have been stimulated by R. rickettsii (IFA titers: 256 for R. rickettsii, 64 for R. rhipicephali, seronegative for the remaining antigens). For the remaining 63 seroreactive dogs, it was not possible to discriminate the infection agent, because they displayed similar titers (< 4-fold difference) for two or more Rickettsia species or had a single titer of 64 for a single Rickettsia species. IFA titers for R. parkeri varied from 64 to 65,536 (Table 3). Among the 154 R. parkeri–seroreactive dogs, 117 animals (76.0%) had titers ≥ 1,024, and 84 animals (54.5%) had titers ≥ 4,096 to R. parkeri. Titers for the other Rickettsia species ranged as follows: R. rickettsii, 64 to 8,192; R. amblyommii, 64 to 8,192; R. rhipicephali, 64 to 4,096; R. bellii, 64 to 1,024.

By univariate analysis, the dependent variable canine seroreactivity was statistically coupled (P < 0.20) with the independent variables (1) canine age, (2) living place, (3) direct contact with pastures or forests, and (4) dog purpose (Table 4). However, when these four independent variables were submitted for multivariate analysis, only the direct contact with pastures or forests was statistically associated with canine seroreactivity (model significance, P = 0.002). Dogs that had direct contact with pastures or forests were 2.138 times more likely to be seroreactive to Rickettsia spp. than dogs that had no direct contact with pastures or forests (Table 5).

Only 19 (4.8%) of the 389 dogs were seroreactive to E. canis, with the following IFA titers: 40 (six dogs), 80 (nine dogs), 160 (two dogs), 320 (one dog), and 5,120 (one dog). These E. canis–reactive dogs were from localities 3 (one dog), 4 (one dog), 7 (three dogs), 8 (two dogs), 9 (two dogs), 10 (two dogs), 11 (three dogs), 12 (four dogs), and 27 (one dog). Among these localities, only localities 11 and 12 were urban areas (Table 1). There was no statistically significant association between any of the independent variables with the canine seroreactivity (titer ≥ 40) for E. canis (Table 4). Only 6 of the 19 E. canis–reactive dogs were also reactive for Rickettsia, with titers ranging from 256 to 16,384 for R. parkeri.

DISCUSSION

This study reports, for the first time, serologic evidence of infection by Rickettsia spp. on dogs in the state of Rio Grande do Sul. Notably, 42.4% of the dogs were seroreactive to Rickettsia spp., with at least 100 dogs showing serologic evidence (possibly homologous reaction) of infection caused by R. parkeri or a very closely related species. The infection by R. rickettsii (the agent of BSF in southeastern Brazil) can be ignored in these 100 dogs, because their antibody titers to R. rickettsii was at least 4-fold lower that that for R. parkeri. The geographic distribution of these 100 dogs encompassed 23 different localities in eight municipalities (Morro Redondo, Pelotas, Pedro Osório, Piratini, Dom Pedrito, Canguçu, Rio Grande, Capão do Leão), suggesting a widespread distribution of R. parkeri in the southern portion of the state of Rio Grande do Sul. However, it is noteworthy that, in all seven localities sampled in Don Pedrito Municipality, 75–100% of the dogs from each locality were seroreactive, with at least 1–10 dogs showing serologic evidence of R. parkeri infection in each locality (Table 2).

In a previous study, dogs experimentally infected with R. rickettsii (highly pathogenic for dogs) or R. montanensis (non-pathogenic for dogs) developed high antibody titers to the former (mean titers ~4,096) but low antibody titers (mean titers ~64) to the latter.7 Because no clinical abnormality was observed in R. montanensis–infected dogs, in contrast to the severe disease developed by R. rickettsii,12 high antibody titers could be indicative of rickettsial pathogenicity. Similarly, we have observed in our laboratory that, when exposed to R. rickettsii infection, guinea pigs that developed fever presented much higher antibody titers to R. rickettsii than guinea pigs that remained non-febrile (MBL, unpublished data). The pathogenicity of R. parkeri for dogs is unknown; however, our serologic analyses suggested that R. parkeri is pathogenic for dogs, because very high titers for this agent were found (84 dogs [54.5%] had antibody titers ranging from 4,096 to 65,536). Among the 84 dogs with antibody titers ranging from 4,096 to 65,536 for R. parkeri, exactly 42 animals were sampled during 2002–2003, and 42 animals were sampled in 2006. This pattern indicates establishment of the agent in the region, because high antibody titers is indicative of recent rickettsial infection.12

Rickettsia parkeri is a recognized human pathogen with several confirmed cases in the United States. It has been suggested with convincing evidence that R. parkeri is the agent responsible for previously reported cases of SFG rickettsiosis transmitted by the tick Amblyomma triste in Canelones, southern Uruguay,26,27 which is 400–450 km away from our study area. Interestingly, previously reported cases of rickettsiosis (reported as BSF) in southern Brazil were mild febrile illness, with some of them presenting lymphadenopathy,7 a clinical feature commonly caused by R. parkeri but rarely caused by R. rickettsii.26,28 In addition, two genotypes of R. parkeri were recently isolated from the ticks Amblyomma dubitatum15 and A. triste16 in the states of São Paulo, Brazil; these tick species are also established in the state of Rio Grande do Sul9 and should be considered potential vectors of R. parkeri in that state.

Two dogs from the same locality in Pelotas Municipality showed serologic (possibly homologous reaction) evidence of infection caused by R. rickettsii and R. amblyommii, respectively (Table 2). Because these were single cases, it does not suggest establishment of these agents in the state of Rio Grande do Sul. R. rickettsii is a known canine pathogen,12 whereas canine infection caused by R. amblyommii has been restricted to a study showing serologic evidence in northern Brazil.11 On the other hand, there has been no serologic evidence of R. bellii infection in dogs; most of the Amblyomma tick species that feed on dogs in rural areas of Brazil are infected with R. bellii.11,29 These facts suggest that the R. bellii–reacting antibodies detected in 18 (4.6%) dogs in this study were all cross-reacting antibodies, and in this case, mostly elicited by R. parkeri.

Previous canine serosurveys in two BSF-endemic areas in southeastern Brazil found 31.2% and 64% seroreactive dogs to R. rickettsii, indicating dogs as a suitable sentinel for human infection by R. rickettsii in these areas.29,30 Because R. parkeri is a human pathogen, the results of this study suggest that at least in the southern part of the state of Rio Grande do Sul (where 39.6% of the dogs were R. parkeri reactive), dogs should be a efficient sentinel for human infection by R. parkeri.

Our multivariate analysis showed that dogs that had direct contact with pasture or forest areas were > 2 more times likely to be seroreactive to Rickettsia than dogs with no such direct contact. Additionally, univariate analyses indicated a significant association (P < 0.05) of Rickettsia seroreactivity with dogs living in rural areas and with dogs used for herding or hunting (Table 4). These results indicate that the tick vector of R. parkeri in the study region prefers to live in rural areas, especially in areas with vegetation (forest, pastures). In contrast, our statistical analysis did not show a major role of R. sanguineus as a vector of R. parkeri to dogs, because this tick species prefers to live in urban areas, inside households, and is not adapted to areas with vegetation in Brazil.31

The low-frequency value for E. canis–infected dogs (4.8%) is corroborated by a previous study that found 0.7–1.7% of seroreactive dogs in the states of Rio Grande do Sul and Santa Catarina, the southernmost states of Brazil.32 These results contrast with the southeastern, northeastern, and central regions of the country, where 15.5–54.5% of seroreactive dogs have been reported.32 The reasons for such contrasting differences are unknown, because R. sanguineus (the vector of E. canis) is abundant through all urban areas of Brazil, including the state of Rio Grande do Sul,9,31 where clinical CME has seldom been reported (NA Cunha-Filho, unpublished data). Interestingly, in Uruguay, where R. sanguineus is also abundant, CME has never been reported.33

We found six dogs that were reactive for both E. canis and Rickettsia spp. Two of these dogs had R. parkeri antibody titers of 8,192 and 16,384, respectively, and a titer of 40 for E. canis (data not shown). Because it has been considered that little cross-reactivity of Rickettsia with Ehrlichia species seems to occur,28 we can not discard the possibility that some of the reactivity for E. canis seen in this study was a result of cross-reactivity with Rickettsia.

The current distribution of R. parkeri includes southern United States,26 Uruguay,27,34 and southeastern Brazil,16 where the agent has been isolated from ticks or/and humans. Serologic evidence for canine infection by R. parkeri exists in northern,11 southeastern,19,22 and southern Brazil (this study). These findings present the possibility that the distribution of R. parkeri in the American continent is much broader than currently known. In addition, it is possible that unrecognized clinical cases of SFG rickettsiosis caused by R. parkeri are occurring in numerous regions of this continent, because of the characteristic mild febrile illness that this agent causes and the unavailability of specific reagents for diagnosis of rickettsiosis in almost all clinical laboratories in Latin America.

Table 1

Localities in the state of Rio Grande do Sul, Brazil, where dogs were sampled for rickettsial infection

Locality number in Figure 1Main characteristicMunicipality
1RuralArroio do Padre
2,6RuralMorro Redondo
3–5,7,8,15,24,29RuralPelotas
11,12UrbanPelotas
13RuralPedro Osorio
14,30RuralPiratini
16–22RuralDom Pedrito
23RuralCanguçu
26RuralRio Grande
27,28RuralChuí
9,10,25,32,33RuralCapão do Leão
31UrbanCapão do Leão
Table 2

Results of indirect IFA for five Rickettsia species in dogs from 33 localities in the southern portion of the state of Rio Grande do Sul, Brazil

No. seroreactive dogs to each of the Rickettsia species (% seroreactivity for the locality)
Locality*No. dogs testedRickettsia parkeriRickettsia rickettsiiRickettsia amblyommiiRickettsia rhipicephaliRickettsia belliiNo. dogs with determined homologous reaction (PAIHR in parentheses)†
* See Table 1 and Figure 1.
† A homologous reaction was determined when an endpoint titer to a Rickettsia species was at least 4-fold higher than those observed for the other Rickettsia species. In this case, the Rickettsia species involved in the highest endpoint titer was considered the possible antigen involved in a homologous reaction (PAIHR).
180 (0)0 (0)0 (0)0 (0)0 (0)0
210 (0)0 (0)0 (0)0 (0)0 (0)0
320 (0)0 (0)0 (0)0 (0)0 (0)0
441 (25.0)1 (25.0)1 (25.0)2 (50.0)1 (25.0)1 (R. parkeri)
5103 (33.3)2 (20.0)2 (20.0)2 (20.0)0 (0)2 (R. parkeri)
681 (12.5)0 (0)0 (0)0 (0)0 (0)1 (R. parkeri)
7509 (18.0)6 (12.0)8 (16.0)6 (12.0)2 (4.0)6 (R. parkeri)
8289 (32.1)10 (35.7)7 (25.0)8 (28.6)0 (0)6 (R. parkeri)
92217 (77.3)17 (77.3)17 (77.3)15 (68.2)4 (18.2)15 (R. parkeri)
102414 (58.3)11 (45.8)11 (45.8)11 (45.8)0 (0)8 (R. parkeri)
116219 (30.6)16 (25.8)16 (25.8)17 (27.4)3 (4.8)9 (7 R. parkeri, 1 R. amblyommii, 1 R. rickettsii)
125615 (26.8)13 (23.2)11 (19.6)14 (25.0)2 (3.6)9 (R. parkeri)
1384 (50.0)3 (37.5)2 (25.0)1 (12.5)0 (0)2 (R. parkeri)
1430 (0)0 (0)0 (0)0 (0)0 (0)0
1532 (66.7)1 (33.3)1 (33.3)1 (33.3)0 (0)1 (R. parkeri)
1611 (100)1 (100)1 (100)1 (100)0 (0)1 (R. parkeri)
1799 (100)9 (100)9 (100)9 (100)0 (0)7 (R. parkeri)
1822 (100)2 (100)2 (100)2 (100)0 (0)1 (R. parkeri)
1966 (100)5 (83.3)6 (100)5 (83.3)1 (16.7)4 (R. parkeri)
2021 (50.0)1 (50.0)1 (50.0)1 (50.0)1 (50.0)1 (R. parkeri)
211413 (92.9)13 (92.9)12 (85.7)13 (92.9)0 (0)10 (R. parkeri)
2286 (75.0)6 (75.0)5 (62.5)5 (62.5)0 (0)5 (R. parkeri)
2375 (71.4)5 (71.4)5 (71.4)5 (71.4)0 (0)5 (R. parkeri)
2421 (50.0)1 (50.0)1 (50.0)1 (50.0)0 (0)0
2520 (0)0 (0)0 (0)0 (0)0 (0)0
2633 (100)3 (100)3 (100)3 (100)0 (0)3 (R. parkeri)
2750 (0)0 (0)0 (0)0 (0)0 (0)0
2820 (0)0 (0)0 (0)0 (0)0 (0)0
2910 (0)0 (0)0 (0)0 (0)0 (0)0
30259 (36.0)9 (36.0)8 (32.0)9 (36.0)3 (12.0)2 (R. parkeri)
3161 (16.7)0 (0)0 (0)0 (0)0 (0)1 (R. parkeri)
3242 (50.0)1 (25.0)1 (25.0)0 (0)1 (25.0)2 (R. parkeri)
3311 (100)1 (100)1 (100)1 (100)0 (0)0
Total389154 (39.6)137 (35.2)131 (33.7)132 (33.9)18 (4.6)102 (100 R. parkeri, 1 R. amblyommii, 1 R. rickettsii)
Table 3

Titers of indirect IFA for R. parkeri in dogs from the southern portion of the state of Rio Grande do Sul, Brazil

No. dogs showing the following endpoint titers to R. parkeri
Locality*641282565121,0242,0484,0968,19216,38432,76865,536Total
* See Table 1 and Figure 1.
411
5123
611
711113119
8111339
931344217
10233122114
1122356119
1221322515
132114
15112
1611
1725119
18112
1911316
2011
21453113
2211316
232215
2411
261113
30123219
3111
32112
3311
Total69913161736281541154
Table 4

Dichotomous categories of each independent variable used in the univariate analysis with seronegative (code 0) and seroreactive (code 1) dogs and their corresponding P values for statistical association by the χ2 test or Fisher exact test

Independent variablesCode 0Code 1P values for seroreactivity to E. canis*P values for seroreactivity to Rickettsia spp.*
* Dog was considered seroreactive if displayed a titer ≥ 40 for E. canis or ≥ 64 for Rickettsia spp.
† Variable selected for the multivariate analysis (see Table 5).
Canine age≤ 2 years old> 2 years old1.0000.192†
Canine sexMaleFemale1.0000.415
Total number of dogs in the farm or household≤ 3> 30.9250.458
Living placeRuralUrban0.8230.007†
Direct contact with pastures or forestsNoYes0.7890.004†
Dog purposeCompany or guardHerding or hunting1.0000.026†
Table 5

Results of multivariate analysis

95% Confidence interval
Independent variableCoefficientSEP value*Odds ratioLowerUpper
Dependent variable is seroreactivity (titer ≥ 64) for Rickettsia spp. Independent variables used for analyses were selected based on univariate analyses (P < 0.20), as shown in Table 4.
*Model significance: P = 0.002.
Constant−0.9240.2270.001
Direct contact with pastures or forests0.7600.2560.0032.1381.2953.530
Figure 1.
Figure 1.

The southern portion of the state of Rio Grande do Sul, Brazil, indicating 33 localities where dogs were sampled for rickettsial infection.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 1; 10.4269/ajtmh.2008.79.102

*

Address correspondence to Marcelo B. Labruna, Faculty of Veterinary Medicine, University of São Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitária, São Paulo, SP 05508-270, Brazil. E-mail: labruna@usp.br

Authors’ addresses: Taís B. Saito, Faculty of Veterinary Medicine, University of São Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitária, São Paulo, SP 05508-270, Brazil, Tel: 55-11-3091-1403, E-mail: tbsaito@gmail.com. Nilton A. Cunha-Filho, Institute of Biology, Federal University of Pelotas, Pelotas, RS, Brazil, Tel: 55-53-3275-7381, E-mail: niltonacfilho@hotmail.com. Richard C. Pacheco, Faculty of Veterinary Medicine, University of São Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitária, São Paulo, SP 05508-270, Brazil, Tel: 55-11-3091-7701, E-mail: pachecorichard@yahoo.com.br. Fernando Ferreira, Faculty of Veterinary Medicine, University of São Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitária, São Paulo, SP 05508-270, Brazil, Tel: 55-11-3091-9937, E-mail: fernando@vps.fmvz.usp.br. Felipe G. Pappen, Faculty of Veterinary Medicine, Federal University of Pelotas, Pelotas, RS, Brazil, Tel: 55-53-3275-7381, E-mail: felipepappen@hotmail.com. Nara A. R. Farias, Institute of Biology, Federal University of Pelotas, Pelotas, RS, Brazil, Tel: 55-53-3275-7381, E-mail: nafarias@ufpel.tche.br. Carlos E. Larsson, Faculty of Veterinary Medicine, University of São Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitária, São Paulo, SP 05508-270, Brazil, Tel: 55-11-3091-1276, E-mail: larsderm@usp.br. Marcelo B. Labruna, Faculty of Veterinary Medicine, University of São Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitária, São Paulo, SP 05508-270, Brazil, Tel: 55-11-3091-1394, Fax: 55-11-3091-7928, E-mail: labruna@usp.br.

Financial support: This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP research grant to MBL), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq academic career scholarship to MBL and FF), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES graduating scholarships to TBS and NAC).

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