Serosurvey of Rickettsia spp. in dogs and humans from an endemic area for Brazilian spotted fever in the State of São Paulo, Brazil

Sorologia para Rickettsia spp. em cães e humanos de uma área endêmica para febre maculosa brasileira no Estado de São Paulo, Brasil

Abstracts

The present study provides a rickettsial serosurvey in 25 dogs and 35 humans in an endemic area for Brazilian spotted fever in the State of São Paulo, where the tick Amblyomma aureolatum is the main vector. Testing canine and human sera by indirect immunofluorescence against four Rickettsia antigens (R. rickettsii, R. parkeri, R. felis and R. bellii) showed that 16 (64%) of canine sera and 1 (2.8%) of human sera reacted to at least one of these rickettsial antigens with titers <FONT FACE=Symbol>³</FONT> 64. Seven canine sera and the single reactive human serum showed titers to R. rickettsii at least four times those of any of the other three antigens. The antibody titers in these 7 animals and 1 human were attributed to stimulation by R. rickettsii infection. No positive canine or human serum was attributed to stimulation by R. parkeri, R. felis, or R. bellii. Our serological results showed that dogs are important sentinels for the presence of R. rickettsii in areas where the tick A. aureolatum is the main vector of Brazilian spotted fever.

Rickettsia; Spotted Fever; Tick-Borne Diseases; Dogs

Este estudo avaliou a ocorrência de anticorpos anti-Rickettsia em 25 cães e 35 humanos, em uma área endêmica para a febre maculosa brasileira no Estado de São Paulo, onde o principal vetor é o carrapato Amblyomma aureolatum. Soros dos cães e humanos foram testados pela técnica de imunofluorescência indireta contra quatro antígenos de riquétsias (R. rickettsii, R. parkeri, R. felis, R. bellii), mostrando que soros de 16 (64%) cães e 1 (2,8%) humano reagiram com títulos <FONT FACE=Symbol>³</FONT> 64 para pelo menos um dos antígenos de riquétsias. Sete soros caninos e o único soro humano reativo demonstraram títulos para R. rickettsii no mínimo quatro vezes maior do que aqueles para os outros antígenos de riquétsias. Os títulos de anticorpos nesses cães e um humano foram considerados homólogos a R. rickettsii, enquanto que nenhum soro de cão ou humano foi considerado reativamente homólogo para R. parkeri, R. felis ou R. bellii. Os resultados sorológicos mostraram que cães são importantes sentinelas para a presença da bactéria R. rickettsii em áreas onde o carrapato A. aureolatum é o principal vetor da febre maculosa brasileira.

Rickettsia; Febre Maculosa; Doenças Transmitidas por Carrapatos; Cães

ARTIGO ARTICLE

Serosurvey of Rickettsia spp. in dogs and humans from an endemic area for Brazilian spotted fever in the State of São Paulo, Brazil

Sorologia para Rickettsia spp. em cães e humanos de uma área endêmica para febre maculosa brasileira no Estado de São Paulo, Brasil

Adriano Pinter^{I, II}; Maurício C. Horta^I; Richard C. Pacheco^I; Jonas Moraes-Filho^I; Marcelo B. Labruna^I

^IFaculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, Brasil

^IISuperintendência de Controle de Endemias, São Paulo, Brasil

^{Correspondence}

ABSTRACT

The present study provides a rickettsial serosurvey in 25 dogs and 35 humans in an endemic area for Brazilian spotted fever in the State of São Paulo, where the tick Amblyomma aureolatum is the main vector. Testing canine and human sera by indirect immunofluorescence against four Rickettsia antigens (R. rickettsii, R. parkeri, R. felis and R. bellii) showed that 16 (64%) of canine sera and 1 (2.8%) of human sera reacted to at least one of these rickettsial antigens with titers ³ 64. Seven canine sera and the single reactive human serum showed titers to R. rickettsii at least four times those of any of the other three antigens. The antibody titers in these 7 animals and 1 human were attributed to stimulation by R. rickettsii infection. No positive canine or human serum was attributed to stimulation by R. parkeri, R. felis, or R. bellii. Our serological results showed that dogs are important sentinels for the presence of R. rickettsii in areas where the tick A. aureolatum is the main vector of Brazilian spotted fever.

Rickettsia; Spotted Fever; Tick-Borne Diseases; Dogs

RESUMO

Este estudo avaliou a ocorrência de anticorpos anti-Rickettsia em 25 cães e 35 humanos, em uma área endêmica para a febre maculosa brasileira no Estado de São Paulo, onde o principal vetor é o carrapato Amblyomma aureolatum. Soros dos cães e humanos foram testados pela técnica de imunofluorescência indireta contra quatro antígenos de riquétsias (R. rickettsii, R. parkeri, R. felis, R. bellii), mostrando que soros de 16 (64%) cães e 1 (2,8%) humano reagiram com títulos ³ 64 para pelo menos um dos antígenos de riquétsias. Sete soros caninos e o único soro humano reativo demonstraram títulos para R. rickettsii no mínimo quatro vezes maior do que aqueles para os outros antígenos de riquétsias. Os títulos de anticorpos nesses cães e um humano foram considerados homólogos a R. rickettsii, enquanto que nenhum soro de cão ou humano foi considerado reativamente homólogo para R. parkeri, R. felis ou R. bellii. Os resultados sorológicos mostraram que cães são importantes sentinelas para a presença da bactéria R. rickettsii em áreas onde o carrapato A. aureolatum é o principal vetor da febre maculosa brasileira.

Rickettsia; Febre Maculosa; Doenças Transmitidas por Carrapatos; Cães

Introduction

Brazilian spotted fever (BSF) is an acute, febrile, tick-borne disease caused by the bacterium Rickettsia rickettsii. In the State of São Paulo, Brazil, two tick species have been implicated in the transmission of BSF to humans: Amblyomma cajennense in the central part of the State and A. aureolatum in the eastern part, where the Atlantic Rainforest is preponderant ¹. R. rickettsii is classified in the spotted fever group of Rickettsiae, which includes more than 20 Rickettsia species ². These include the pathogens R. parkeri and R. felis, reported infecting ticks and fleas, respectively, in the State of São Paulo ^3,4,5. Besides the spotted fever group of Rickettsiae, R. bellii has been reported frequently infecting ticks in the State of São Paulo ^3,6,7. Although strictly associated with ticks, R. bellii is not a spotted fever group Rickettsia. In addition, the pathogenicity of R. bellii in humans and dogs has never been demonstrated ⁸.

The few available records of natural hosts for sub-adults of A. aureolatum have included mostly a few bird and rodent species ^9,10,11,12. Meanwhile, it has been reported that A. aureolatum adult ticks feed mainly on dogs in rural areas close to remnants of rainforest ^11,13,14. Additionally, only adult ticks have been collected parasitizing humans ^11,14. Since A. aureolatum needs moist habitats like inner rainforests ¹, dogs may play an important role in carrying A. aureolatum adult ticks from inside the forest to the human environment. This situation potentially poses an increased risk of dog owners acquiring BSF in areas where A. aureolatum is the main vector.

In the pre-antibiotic era, case-fatality rates of BSF among humans were nearly 80% ¹⁵. The case-fatality rate still reaches 40% in the State of São Paulo ¹⁶. In contrast, canine infection by R. rickettsii appears to be much less fatal. Nevertheless, R. rickettsii-infected dogs develop an anamnestic IgG response, detectable by indirect immunofluorescence ¹⁷.

In order to determine which areas are endemic for BSF, it is necessary to detect either a R. rickettsii-infected tick population or R. rickettsii-seropositive sentinel hosts. The infection rate by R. rickettsii within an A. aureolatum population has been reported at less than 1% ⁷, which makes a survey for R. rickettsii in a tick population an exhaustive and sometimes fruitless task. Thus, the aim of this study was to use serology to investigate whether dogs are important sentinels for BSF transmitted by A. aureolatum. The study was performed in a BSF-endemic area of the State of São Paulo where Pinter & Labruna ⁷ found dogs parasitized by R. ricketsii-infected ticks.

Methods

The study was performed in the rural area of the Taiaçupeba District in the county (municipality) of Mogi das Cruzes, State of São Paulo (23°38'54.9"S, 46°11'0.3"W), a well-known endemic area for Brazilian spotted fever. In Taiaçupeba, several fatal human cases of BSF have been reported in recent years ¹⁴. The canine serosurvey in the present study was conducted during an A. aureolatum seasonality study ¹⁴ and a Rickettsia-infection survey on ticks collected on dogs ⁷ in the same area.

Dogs were sampled in 8 small family farms (< 10ha) located in an area originally consisting of Atlantic rainforest, at 800m altitude on the Serra do Mar mountain chain, next to the Jundiai River Dam. The agriculture was mostly vegetable farming, with no livestock. Most of the farm families owned one or more domestic dogs, mostly raised unrestrained with free access to forest areas. All the dogs were sampled, except for a minority that were raised completely restrained on the farms. Blood samples were drawn from the dogs on two visits to the farms, in August 2001 and April 2002. A total of 19 and 19 dogs were sampled during the first and second visits, respectively. Since only 13 individual dogs were sampled on both visits, 6 dogs were sampled on the first visit only and another 6 dogs were sampled on the second visit only. Thus, a total of 25 dogs were sampled in the study. The age of each dog was noted. Each dog sample was individually identified with a capital letter (referring to the farm where the dog lived) followed by an Arabic numeral (referring to the individual dog present on that farm) (Table 1). A total of 35 humans living on the farms, in direct contact with the dogs, had blood samples drawn. Eighteen humans were sampled on both visits, while 7 and 10 were sampled only on the first and second visits, respectively.

The blood samples were taken to the laboratory at room temperature and were centrifuged (1,500g for 10 minutes), and serum aliquots were stored at -20ºC until tested. Serum samples were processed by indirect immunofluorescence as described elsewhere ^8,18, using crude antigens derived from four Rickettsia isolates from Brazil: R. rickettsii strain Taiaçu, R. bellii strain Mogi das Cruzes, R. felis strain Pedreira, and R. parkeri strain At24. While the first three Rickettsia species constitute the Rickettsiae known to occur in the study site ^4,7, the latter species is known to occur in other parts of the State of São Paulo ^3,5. Human and canine sera were diluted in twofold increments with PBS starting from a 1:64 dilution. Serum was considered to contain antibodies against the Rickettsiae if it displayed a reaction at 1:64. Endpoint titers against each Rickettsia strain were determined by testing serial twofold serum dilutions. Reactive sera were tested in two or three replications before determining the endpoint titer. On each slide, a serum previously shown to be non-reactive (negative control) and a known reactive serum (positive control) were tested. A serum showing a titer for a Rickettsia species at least fourfold that observed for any other Ricketttsia species was considered homologous for the first Rickettsia species or a very closely related genotype ^8,19.

During a two-year study (December 2000 to November 2002) of rickettsial infection in A. aureolatum ticks collected on some of the dogs sampled in the present study ⁷, R. rickettsii and R. bellii were found infecting 0.9 and 1.5% of the A. aureolatum ticks, respectively. Since our blood samples were drawn on dates (August 2001 and April 2002) within this two-year period, we compared the data of infected ticks reported by Pinter & Labruna ⁷ with the serological results of the present study.

Before starting, the present study was submitted to and approved by the Research Ethics Committee of the Universidade de São Paulo [University of São Paulo].

Results

Indirect immunofluorescence detected antibodies reactive to R. rickettsii (titer ³ 64) in 16 (64%) out of 25 dogs. Of those, 14 (56%) also reacted to R. parkeri, 6 (24%) to R. felis, and 3 (12%) to R. bellii. No serum reacted to any of these three Rickettsiae without reacting to R. rickettsii. The serum endpoint titers ranged from 64 to 8,192 for R. rickettsii, 64 to 2,048 for R. parkeri, 64 to 1,024 for R. felis, and 256 to 1,024 for R. bellii (Table 1). Seven canine sera (A3, B1, C1, C3, E1, E2, and F3) showed titers to R. rickettsii at least four times higher than those of any of the other three antigens. The antibody titers in these 7 animals were attributed to stimulation by R. rickettsii infection. For another 9 animals, it was not possible to determine whether R. rickettsii had been the infecting agent, since they also showed high titers for R. parkeri (C2, D1, F1, F2, G2, H1, and H2) or R. felis (D2), or showed the same titer for the four antigens (G1) (Table1).

The overall proportion of dogs that were reactive to R. rickettsii was 64% (16/25). However, this frequency increased to 77.7% (14/18) for dogs older than 12 months and 100% (9/9) for those older than 36 months.

Canine blood samples were collected twice (August 2001 and April 2002), i.e., with an 8-month interval. Among the 13 dogs sampled on both occasions, 8 sera were reactive to R. rickettsii. Of these, 5 sera (A3, C2, D1, D2, G1) showed stable titers, whereas 2 dogs (C1, C3) showed ³ fourfold rise and 1 serum (F2) showed a twofold decrease in R. rickettsii antibody titers (Table 1).

According to data reported by Pinter & Labruna ⁷ in January 2001, one R. rickettsii-infected tick and one R. bellii-infected tick were taken from dog E2. Seven months later (August 2001), this dog had a titer of 8,192 for R. rickettsii and was not reactive to R. bellii (Table 1). Dog E2 was not sampled on the second occasion, in April 2002. In June 2001, a R. bellii-infected tick was taken from dog C4 ⁷. Two months later (August 2001), this dog was not reactive to any of the four rickettsial antigens, a condition detected again in April 2002, when blood was drawn the second time (Table 1). The remaining R. rickettsii or R. bellii-infected ticks reported by Pinter & Labruna ⁷ were collected from other dogs not sampled in the present study, or were collected from our sampled dogs at dates after our second samples.

Only 1 (2.8%) of 35 humans showed antibodies to R. rickettsii (titer: 256). No human serum reacted to R. parkeri, R. felis, or R. bellii. This single reactive serum was collected from the only person with a previous history of BSF, which was confirmed by checking the files of the São Paulo State Health Secretariat.

Discussion

Indirect immunofluorescence is currently the test of choice for serological diagnosis of rickettsial infection in humans and animals ^17,20,21. However, cross-reactive antibodies between Rickettsia species are often observed, thus hindering the serological identification of the Rickettsia species involved in an infection. Testing a clinical serum against the possible Rickettsia species known to occur in a given area is ideal, because homologous antibody titers are often higher than heterologous antibody titers. In some cases, the differences in titers may be great enough to differentiate the rickettsial species potentially stimulating the immune response ^20,21. Since our study tested serum samples against the Rickettsia antigens known to occur in the study area (R. rickettsii, R. bellii, and R. felis) plus one species yet to be reported (R. parkeri), we can technically assume that R. rickettsii was the Rickettsia species responsible for infection in seven dogs. In addition, we present no serological evidence of canine infection due to R. felis, R. bellii, or R. parkeri in the study area.

No dog appeared to have been infected by R. felis, even though natural R. felis-infected fleas were found parasitizing dogs in the study area ⁴. Beyond the widespread occurrence of R. felis-infected fleas in the world, there has been no evidence for the vectorial competence of fleas for R. felis. Similarly, no dog appeared to have been infected by R. bellii in the present study, although 1.5% of the A. aureolatum ticks collected on dogs in the study area were shown to be infected by R. bellii ⁷. For instance, interestingly, a R. bellii-infected tick was removed from dog C4 in June 2001 ⁷, but this dog was serologically negative for Rickettsia in August 2001 and April 2002 (Table 1). These results corroborate a recent study in Northern Brazil in which no dog was shown to have R. bellii antigen-stimulating antibodies, despite the high frequency of R. bellii-infected ticks parasitizing the dogs ⁸. Thus, these results indicate that either R. bellii is not infective for dogs or that R. bellii-infected ticks are not able to transmit the agent to dogs.

The proportion of seropositive dogs increased with age (100% of dogs > 36 months of age were R. rickettsii-seropositive). Since puppies (< 6 months of age) are not likely to be physically capable of circulating extensively inside the forest, they are probably much less exposed to A. aureolatum ticks. Thus, the older the dog the higher the odds that the animal has been parasitized at least once by a R. rickettsii-infected tick.

Interestingly, dog C3 appeared to have been infected by R. rickettsii at least twice: the first contact with R. rickettsii is believed to have occurred before 18 months of age, when the first blood sample was drawn and the dog had an IFA titer of 1:512 for R. rickettsii. When the same animal was 26 months old it had a 1:4,096 titer for R. rickettsii (Table 1). This supposed re-infection is corroborated by laboratory data on experimental infection of dogs with R. rickettsii ¹⁷, showing that once inoculated with the agent, dogs elicit indirect immunofluorescence peak titers (between 2,048 and 4,096) around 3 weeks after inoculation, after which they gradually decreased in the following weeks reaching 128 to 512 at around 7 months post-inoculation. A similar supposition of re-infection can be inferred for dog C1, which showed titers of 64 and 256 for the first and second samples, respectively (Table 1).

Sangioni et al. ²² proposed surveys of horse sera as a useful method for BSF surveillance in areas where humans are exposed to A. cajennense ticks. This procedure was based on the fact that horses are primary hosts for A. cajennense, and thus that the seroprevalence of R. rickettsii-reactive horses in BSF-endemic areas has varied from 57.1% to 90% ^19,23. In contrast, the following seroprevalence values of R. rickettsii-reactive dogs have been reported in areas where A. cajennense is the vector: 8% ²⁴, 13.7% ²⁵, 31.2% ¹⁹, and 36.4% ²³. These lower values for dogs are due to the fact that unlike horses, dogs are not primary hosts for A. cajennense (they are merely accidental hosts). Meanwhile, the present study showed an overall seroprevalence of 64% of R. rickettsii-reactive dogs in a BSF-endemic area, where the A. aureolatum tick is the vector. This higher value is due to the fact that dogs are primary hosts for the adult stage of A. aureolatum. We thus recommend surveys of dog sera as a useful method for Brazilian spotted fever surveillance in areas where humans are potentially exposed to A. aureolatum ticks. This procedure would be much more useful and productive than a direct tick assay targeting R. rickettsii. For a survey of dog sera, the sample to be tested should include mainly dogs older than 36 months, born and raised in the target region.

Contributors

A. Pinter participated in the field collection of biological material, laboratory tests, and elaboration of the manuscript. M. C. Horta, R. C. Pacheco, and J. Moraes-Filho contributed to the field collection of biological material, laboratory tests, analysis and interpretation of data, and critical revision and approval of the manuscript. M. B. Labruna oriented the research work, contributed to the elaboration of the manuscript, and final revision of the text.

Acknowledgments

We wish to thank nurse Laura Freitas for drawing the human blood samples and Renato Caraviere for technical support during the fieldwork. The research was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo [FAPESP; São Paulo State Research Foundation] and the Conselho Nacional de Desenvolvimento Científico e Tecnológico [CNPq; Brazilian National Research Council].

Submitted on 19/Mar/2007

Final version resubmitted on 10/Jun/2007

Approved on 29/Jun/2007

References

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Publication Dates

• Publication in this collection
  01 Feb 2008
• Date of issue
  Feb 2008

History

• Accepted
  29 June 2007
• Received
  19 Mar 2007
• Reviewed
  10 June 2007