Experimental infection and vector competence of Amblyomma patinoi, a member of the Amblyomma cajennense species complex, for the human pathogen Rickettsia rickettsii.

Amblyomma patinoi ticks infected with Rickettsia rickettsii are present in Colombia, but its vector competence is unknown. Hence, we evaluated the vector competence of A. patinoi with R. rickettsii under laboratory conditions. Experimental guinea pigs and rabbits (males and females) were separated in the infected group (IG) and the control group (CG). In the IG, the filial 1 (F1) larvae (R. rickettsii-free) from Colombian A. patinoi engorged female specimens were exposed to R. rickettsii (ITU strain) by feeding on infected guinea pigs. Next, F1 nymphs and adults, and F2 larvae were allowed to feed on uninfected guinea pigs or rabbits and tested by qPCR targeting the gltA rickettsial gene. All animals used to feed the IG F1 ticks became febrile and had R. rickettsii infection (89% fatality rate) detected through serological or molecular techniques. After the F1 larvae ticks became R. rickettsii infected, subsequent IG tick stages were able to maintain the rickettsial infection by transstadial maintenance to all infested animals, indicating A. patinoi vector competence. Subsequently, almost 31% of the F1 female egg masses and only 42% of their F2 larvae were infected. Less than 50% of the infected females transmitted R. rickettsii transovarially, and only a part of the offspring were infected. This study demonstrated that A. patinoi might not be able to sustain R. rickettsii infection by transovarial transmission for successive tick generations without horizontal transmission via rickettsemic hosts. This condition might result in low R. rickettsii-infection rates of A. patinoi under natural conditions.

The Distinct Transcriptional Response of the Midgut of Amblyomma sculptum and Amblyomma aureolatum Ticks to Rickettsia rickettsii Correlates to Their Differences in Susceptibility to Infection.

Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain Spotted Fever (RMSF). In Brazil, two species of ticks in the genus Amblyomma, A. sculptum and A. aureolatum, are incriminated as vectors of this bacterium. Importantly, these two species present remarkable differences in susceptibility to R. rickettsii infection, where A. aureolatum is more susceptible than A. sculptum. In the current study, A. aureolatum and A. sculptum ticks were fed on suitable hosts previously inoculated with R. rickettsii, mimicking a natural infection. As control, ticks were fed on non-infected animals. Both midgut and salivary glands of all positively infected ticks were colonized by R. rickettsii. We did not observe ticks with infection restricted to midgut, suggesting that important factors for controlling rickettsial colonization were produced in this organ. In order to identify such factors, the total RNA extracted from the midgut (MG) was submitted to next generation RNA sequencing (RNA-seq). The majority of the coding sequences (CDSs) of A. sculptum differentially expressed by infection were upregulated, whereas most of modulated CDSs of A. aureolatum were downregulated. The functional categories that comprise upregulated CDSs of A. sculptum, for instance, metabolism, signal transduction, protein modification, extracellular matrix, and immunity also include CDSs of A. aureolatum that were downregulated by infection. This is the first study that reports the effects of an experimental infection with the highly virulent R. rickettsii on the gene expression of two natural tick vectors. The distinct transcriptional profiles of MG of A. sculptum and A. aureolatum upon infection stimulus strongly suggest that molecular factors in this organ are responsible for delineating the susceptibility to R. rickettsii. Functional studies to determine the role played by proteins encoded by differentially expressed CDSs in the acquisition of R. rickettsii are warranted and may be considered as targets for the development of strategies to control the tick-borne pathogens as well as to control the tick vectors.

Association of the occurrence of Brazilian spotted fever and Atlantic rain forest fragmentation in the São Paulo metropolitan region, Brazil

Brazilian Spotted Fever (BSF) is a zoonotic disease caused by the bacterium Rickettsia rickettsii. In the São Paulo Metropolitan Region (SPMR) it is transmitted by Amblyomma aureolatum ticks. In this region, annual lethality of the disease can reach 80% and spatial occurrence depends on environmental factors and more particularly on the presence and interaction of domestic and wild carnivores as well as the presence and characteristics of the remnant Atlantic Rain Forest patches. This study analyzed the association between forest fragmentation and its influence on the risk of occurrence of the disease in the human population. Domestic dogs tested for R. rickettsii antibodies in nine different areas under the influence of different patterns of Rain Forest fragmented landscapes and human occupancy. Landscape metrics were obtained by analyzing satellite images and high-resolution orthophotos. Principal component analysis (PCA) was used to determine among the different landscape variables the one that could best explain the data variance, and the results were tested against canine seroprevalence in order to address disease occurrence risk levels. From 270 canine samples, the seroprevalence ranged from 0 to 37%...

High Seroprevalence for Rickettsia rickettsii in Equines Suggests Risk of Human Infection in Silent Areas for the Brazilian Spotted Fever

Equines play a role in the epidemiology of Brazilian spotted fever (BSF) since they are a primary host for the tick Amblyomma sculptum.We studied the seroprevalence for three species of Rickettsia in equines in four endemic (with human cases) and in four non-endemic areas (no human cases) in the Piracicaba River Basin, São Paulo, Brazil. A serological survey of 504 equines was performed: around 63 animals were sampled in each area and tested through indirect immunofluorescence assay for R. rickettsii, R. parkeri, and R. bellii in2012–2013. Blood samples were seropositive for 183 equines (36.3%) in which 73 (39.9%) were from non-endemic areas. In the studied sites equines were highly exposed to Rickettsia infection ranging from 6.1% to 54.7%, with Geometric Mean Titers greater in endemic area (p = 0.012). Results suggest that Rickettsia may be more widespread than the surveillance of BSF has detected. These results highlight the need to include data on the seroprevalence of sentinel animals to improve human diagnoses and surveillance in areas with no reported human cases...

Frequência de anticorpos da classe IgG anti-Rickettsia rickettsiiem equinos na Universidade Federal Rural do Rio de Janeiro, Campus Seropédica

O objetivo do presente estudo foi verificar,através da reação de imunofluorescência indireta (RIFI), a frequência de anticorpos anti--Rickettsia rickettsii em equinos na Universidade Federal Rural do Rio de Janeiro (UFRRJ) campus Seropédica, estado do Rio de Janeiro. Foram analisadas amostras de soro de 42 equinos do Setor de Equinocultura da UFRRJ. Todas as amostras foram testadas utilizando lâminas fixadas com antígenos para R. rickettsii, Rickettsia rhipicephali e Rickettsia parkeri. Foi observada uma prevalência geral para Rickettsia spp. de 83,33% (35/42). Para o agente R. rickettsii observou-se uma soroprevalência de 66,67% (28/42), sendo ainda categorizados em títulos de 1:64 (19/28) e 1:128 (9/28). Nove dos 28 equinos positivos para R. rickettsii (21,43%) não foram reativos para os demais agentes, apresentando títulos de 1:64 (8/9) e 1:128 (1/9). As únicas espécies de carrapatos encontradas parasitando os equinos no campus da UFRRJ durante o período de coleta foram Amblyomma cajennense e Dermacentor nitens. O campus da UFRRJ apresenta um ambiente que propicia um nicho epidemiológico ideal para a circulação de bactérias do gênero Rickettsia. A alta prevalência encontrada no presente estudo indica uma provável circulação de Rickettsia spp., havendo assim, risco para infecção humana na área estudada...

The aim of this study was to verify, through the indirect immunofluorescence assay (IFA), the frequency of anti-Rickettsia rickettsii antibodies in horses at Universidade Federal Rural do Rio de Janeiro (UFRRJ) Seropédica campus, state of Rio de Janeiro. We analyzed serum samples from 42 horses from Department of Breeding Equine of UFRRJ. All samples were tested using fixed slides with antigens for R. rickettsii, Rickettsia rhipicephali and Rickettsia parkeri. We observed an overall prevalence of Rickettsia spp. 83.33% (35/42). For the agent R. rickettsii revealed a prevalence of 66.67% (28/42), still being categorized in titers of 1:64 (19/28) and 1:128 (9/28). Nine of the 28 positives horses for R. rickettsii (21.43%) were no reactive to other agents, with titers 1:64 (8/9) and 1:128 (1/9). The only tick species found parasitizing horses on the campus of UFRRJ during the collection period were Amblyomma cajennense and Dermacentor nitens. The UFRRJ presents an environment that provides a ideal epidemiological niche for the permanence of Rickettsia bacteria. The high prevalence found in this study indicates that attention to epidemiological agent of Brazilian Spotted Fever in the study area is of utmost importance...

Isolation and characterization of microsatellite loci from the tick Amblyomma aureolatum (Acari: Ixodidae)

Amblyomma aureolatum (Pallas) is the main vector of the bacterium Rickettsia rickettsii, the etiological agent of Brazilian spotted fever. This disease is the most lethal human spotted fever rickettsiosis in the world. Microsatellite loci were isolated from a dinucleotide-enriched library produced from A. aureolatum sampled in Southeastern Brazil. Eight polymorphic microsatellites were further characterized among 38 individuals sampled from São Paulo metropolitan region. The number of observed alleles ranged from 2 to 9, observed heterozygosity was 0.184-0.647, and expected heterozygosity was 0.251-0.747. Cross-species amplifications suggested that these loci will be useful for other Amblyomma species...

Natural Blood Feeding and Temperature Shift Modulate the Global Transcriptional Profile of Rickettsia rickettsii Infecting Its Tick Vector

Rickettsia rickettsii is an obligate intracellular tick-borne bacterium that causes Rocky Mountain Spotted Fever(RMSF), the most lethal spotted fever rickettsiosis. When an infected starving tick begins blood feeding from a vertebrate host, R. rickettsii is exposed to a temperature elevation and to components in the blood meal. These two environmental stimuli have been previously associated with the reactivation of rickettsial virulence in ticks, but the factors responsible for this phenotype conversion have not been completely elucidated. Using customized oligonucleotide microarrays and high-throughput microfluidic qRT-PCR, we analyzed the effects of a 10°C temperature elevation and of a blood meal on the transcriptional profile of R. rickettsii infecting the tick Amblyommaaureolatum. This is the first study of the transcriptome of a bacterium in the genus Rickettsia infecting a natural tickvector. Although both stimuli significantly increased bacterial load, blood feeding had a greater effect, modulating five-fold more genes than the temperature up shift. Certain components of the Type IV Secretion System (T4SS) were up-regulated by blood feeding. This suggests that this important bacterial transport system may be utilized to secrete effectors during the tick vector’s blood meal. Blood feeding also up-regulated the expression of antioxidant enzymes,which might correspond to an attempt by R. rickettsii to protect itself against the deleterious effects of free radicals produced by fed ticks. The modulated genes identified in this study, including those encoding hypothetical proteins, require further functional analysis and may have potential as future targets for vaccine development...

Ecology, biology and distribution of spotted-fever tick vectors in Brazil

Spotted-fever-caused Rickettsia rickettsii infection is in Brazil the major tick-borne zoonotic disease. Recently, a second and milder human rickettsios is caused by an agent genetically related to R. parkeri was discovered in the country (Atlantic rainforest strain). Both diseases clearly have an ecological background linked to a few tick species and their environment. Capybaras (Hydrochoerus hydrochaeris) and Amblyomma cajennense ticks in urban and rural areas close to water sources are the main and long-known epidemiological feature behind R. rickettsii-caused spotted-fever. Unfortunately, this ecological background seems to be increasing in the country and disease spreading may be foreseen. Metropolitan area of São Paulo, the most populous of the country, is embedded in Atlantic rain forest that harbors another important R. rickettsii vector, the tick Amblyomma aureolatum. Thus, at the city-forest interface, dogs carry infected ticks to human dwellings and human infection occurs. A role for R. rickettsii vectoring to humans of a third tick species, Rhipicephalus sanguineus in Brazil, has not been proven; however, there is circumstantial evidence for that. A R. parkeri-like strain was found in A. ovale ticks from Atlantic rain forest and was shown to be responsible for a milder febrile human disease...

Epidemiology of Brazilian spotted fever in the Atlantic Forest, state of São Paulo, Brazil

The tick-borne bacterium Rickettsia rickettsii is the a etiological agent of Brazilian spotted fever (BSF). The present study evaluated tick infestations on wild and domestic animals, and the rickettsial infection in these animals and their ticks in 7 forest areas adjacent to human communities in the São Paulo Metropolitan Area (SPMA). The results were compared to ecological traits of each sampled area. Two main tick species, Amblyomma aureolatum and Rhipicephalus sanguineus, were collected from dogs. The major ticks found on small mammals and birds were Ixodes loricatus and Amblyomma longirostre, respectively. Both anti-R. rickettsii antibodies and R. rickettsii-infected ticks were detected on dogs from only 2 areas in the southern part of the SPMA, which were considered to be endemic for BSF; the remaining 5 areas were considered to be non endemic. Ecologically, the BSF-endemic areas clearly differed from the non-endemic areas by the presence of significantly more degraded forest patches in the former. The present results corroborate historical observations that have indicated that all human cases of BSF in the SPMA were contracted in the southern part of this metropolitan area. However, not all forest patches in the southern part of the SPMA were shown to be associated with BSF endemism...

Experimental infection of Rhipicephalus sanguineus ticks with the bacterium Rickettsia rickettsii, using experimentally infected dogs

We evaluated if Rickettsia rickettsii-experimentally infected dogs could serve as amplifier hosts for hipicephalus sanguineus ticks. In addition, we checked if Rh. sanguineus ticks that acquired Ri. rickettsii from dogs could transmit the bacterium to susceptible hosts (vector competence), and if these ticks could maintain the bacterium by transstadial and transovarial transmissions. Uninfected larvae, nymphs, and adults of Rh. sanguineus were allowed to feed upon three groups of dogs: groups 1 (G1) and 2 (G2) composed of Ri. rickettsii-infected dogs, infected intraperitoneally and via tick bites, respectively, and group 3 composed of uninfected dogs. After larval and nymphal feeding on rickettsemic dogs, 7.1-15.2% and 35.8-37.9% of the molted nymphs and adults, respectively, were shown by polymerase chain reaction (PCR) to be infected by Ri. rickettsii, confirming that both G1 and G2 dogs were efficient sources of rickettsial infection (amplifier host), resulting in transstadial transmission of the agent...

Amblyomma imitator Ticks as Vectorsof Rickettsia rickettsii, Mexico

Real-time PCR of Amblyomma imitator tick egg massesobtained in Nuevo Leon State, Mexico, identifi ed a Rickettsiaspecies. Sequence analyses of 17-kD common antigenand outer membrane protein A and B gene fragmentsshowed to it to be R. rickettsii, which suggested a potentialnew vector for this bacterium...

Rickettsial infection in animals and Brazilian spotted fever endemicity.

We compared the rickettsial infection status of Amblyomma cajennense ticks, humans, dogs, and horses in both Brazilian spotted fever (BSF)-endemic and -nonendemic areas in the state of Sao Paulo, Brazil. Most of the horses and few dogs from BSF-endemic areas had serologic titers against Rickettsia rickettsii antigens. In contrast, no dogs or horses from BSF-nonendemic areas had serologic titers against R. rickettsii antigens, although they were continually exposed to A. cajennense ticks. All human serum samples and ticks from both areas were negative by serologic assay and polymerase chain reaction, respectively. Our results indicate that surveys of horse serum are a useful method of BSF surveillance in areas where humans are exposed to A. cajennense ticks. In addition, we successfully performed experimental infection of A. cajennense ticks with R. parkeri.

Isolamento de Rickettsia em cultura de células vero / Isolation of Rickettsia in vero cell culture

Embora o diagnóstico da febre maculosa baseie-se em sinais e sintomas característicos, o mesmo requer confirmaçäo laboratorial, pois existem alguns diagnósticos diferenciais possíveis como meningococcemia, leptospirose, infecçäo por enterovírus e febre tifóide. A confirmaçäo laboratorial pode ser feita através da pesquisa de anticorpos específicos, possível somente alguns dias após o aparecimento da doença, através do isolamento do agente em amostras de sangue e/ou biópsia de pele, e ainda, de amostras de carrapatos coletados do paciente ou de animais reservatório. O isolamento a partir de sangue ou biópsia de pele resulta em diagnóstico precoce da doença, pois na fase de rickettsemia ainda näo há anticorpos detectáveis no sangue. Assim, com o objetivo de facilitar o diagnóstico precoce da febre maculosa, estabelecemos um método de isolamento de rickettsia em cultura de células vero. Para a padronizaçäo foi inoculada amostra padräo de Rickettsia rickettsii, cepa Sheyla Smith, cedida pelo CDC. A identificaçäo foi feita através da reaçäo de imunofluorescência indireta. A presença de microrganismos verdes fluorescentes visualizados no interior do citoplasma das células caracterizou o crescimento do agente. Posteriormente, a metodologia foi confirmada pelo isolamento do agente da febre maculosa em amostras de biópsia de pele de paciente proveniente de área endêmica no Estado de Säo Paulo, bem como, de amostras de carrapato do gênero Amblyomma, considerado o reservatório e transmissor da doença no Brasil