Ticks and tick-borne rickettsiae from dogs in El Salvador, with report of the human pathogen Rickettsia parkeri.

Twelve tick species have been reported in El Salvador; however, information regarding ticks infesting domestic dogs is lacking, and pathogenic tick-borne Rickettsia species have never been reported in El Salvador. This work evaluated ticks infesting 230 dogs from ten municipalities in El Salvador from July 2019 to August 2020. A total of 1,264 ticks were collected and identified into five species: Rhipicephalus sanguineus sensu lato (s.l.), Rhipicephalus microplus, Amblyomma mixtum, Amblyomma ovale, and Amblyoma cf. parvum. The tick R. sanguineus s.l. was the most frequent species in all localities (81.3% of sampled dogs), followed by Amblyomma mixtum (13.0%), Amblyomma ovale (10.9%) and Amblyomma cf. parvum (10.4%). The overall mean intensity of tick infestation was 5.5 ticks/dog. The highest specific mean intensity value was for R. sanguineus s.l. (4.8 ticks/dog), varying from 1.6 to 2.7 ticks/dog for the three Amblyomma species. From a random sample of 288 tick specimens tested molecularly for the presence of rickettsial agents, three spotted fever group Rickettsia were detected: Rickettsia amblyommatis in 90% (36/40) A. mixtum, 46% (11/24) A. cf. parvum, 4% (7/186) R. sanguineus s.l., and 17% Amblyomma spp.; Rickettsia parkeri strain Atlantic rainforest in 4% (1/25) A. ovale; and an unnamed rickettsia agent, designated as 'Rickettsia sp. ES-A.cf.parvum', in 4% (1/24) A. cf. parvum. Our finding of R. parkeri strain Atlantic rainforest in A. ovale is highly relevant because this agent has been associated to spotted fever illness in other Latin American countries, where A. ovale is implicated as its main vector. These findings suggest that spotted fever cases caused by R. parkeri strain Atlantic rainforest could be occurring in El Salvador.

'Candidatus Rickettsia andeanae' and Probable Exclusion of Rickettsia parkeri in Ticks from Dogs in a Natural Area of the Pampa Biome in Brazil.

Spotted fever illness caused by the tick-borne pathogen Rickettsia parkeri has emerged in the Pampa biome in southern Brazil, where the tick Amblyomma tigrinum is implicated as the main vector. Because domestic dogs are commonly parasitized by A. tigrinum, this canid is also a suitable sentinel for R. parkeri-associated spotted fever. Herein, we investigate rickettsial infection in ticks, domestic dogs and small mammals in a natural reserve of the Pampa biome in southern Brazil. The ticks A. tigrinum, Amblyomma aureolatum and Rhipicephalus sanguineus were collected from dogs. Molecular analyses of ticks did not detect R. parkeri; however, at least 34% (21/61) of the A. tigrinum ticks were infected by the non-pathogenic agent 'Candidatus Rickettsia andeanae'. Serological analyses revealed that only 14% and 3% of 36 dogs and 34 small mammals, respectively, were exposed to rickettsial antigens. These results indicate that the study area is not endemic for R. parkeri rickettsiosis. We tabulated 10 studies that reported rickettsial infection in A. tigrinum populations from South America. There was a strong negative correlation between the infection rates by R. parkeri and 'Candidatus R. andeanae' in A. tigrinum populations. We propose that high infection rates by 'Candidatus R. andeanae' might promote the exclusion of R. parkeri from A. tigrinum populations. The mechanisms for such exclusion are yet to be elucidated.

Rickettsial infection in ticks from a natural area of Atlantic Forest biome in southern Brazil.

From June 2013 to January 2014, blood sera samples and ticks were collected from domestic dogs and wild small mammals, and ticks from the vegetation in a preservation area of the Atlantic Forest biome (Turvo State Park), and the rural area surrounding the Park in Derrubadas municipality, state of Rio Grande do Sul, southern Brazil. Dogs were infested by Amblyomma ovale and Amblyomma aureolatum adult ticks, whereas small mammals were infested by immature stages of A. ovale, Amblyomma yucumense, Amblyomma brasiliense, Ixodes loricatus, and adults of I. loricatus. Ticks collected on vegetation were A. brasiliense, A. ovale, A. yucumense, Amblyomma incisum, and Haemaphysalis juxtakochi. Three Rickettsia species were molecularly detected in ticks: Rickettsia bellii in I. loricatus (also isolated through cell culture inoculation), Rickettsia amblyommatis in A. brasiliense, and Rickettsia rhipicephali in A. yucumense. The latter two are tick-rickettsia associations reported for the first time. Seroreactivity to Rickettsia antigens were detected in 33.5% (55/164) small mammals and 8.3% (3/36) canine sera. The present study reveals a richness of ticks and associated-rickettsiae in the largest Atlantic Forest Reserve of the state of Rio Grande do Sul, which is characterized by a rich fauna of wild mammals, typical of more preserved areas of this biome. Noteworthy, none of the detected Rickettsia species have been associated to human or animal diseases. This result contrasts to other areas of this biome in Brazil, which are endemic for tick-borne spotted fever caused by Rickettsia rickettsii or Rickettsia parkeri.

Seroprevalence and detection of Rickettsia spp. in wild birds of Arauca, Orinoquia region, Colombia.

Wild birds have an important role as hosts of ticks infected by rickettsiae. However, the role of birds as reservoirs of tick-borne rickettsiae is unknown and poorly understood. This is particularly relevant in several tropical and subtropical areas, where migration influences the global spread of ectoparasites and pathogens of public health importance. This research aimed to detect and evaluate the exposure to spotted fever group rickettsiae in wild birds that could represent reservoirs in the Department of Arauca in the Colombian Orinoquia region. Sampling was conducted in three municipalities of the Department of Arauca (Colombia). Blood samples were collected from 255 birds and processed to obtain serum (n = 155) and DNA (n = 255) samples. The serum samples were processed for indirect immunofluorescence assays (IFA) for the detection of antibodies to Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommatis, Rickettsia rhipicephali, and Rickettsia bellii. Additionally, we investigated rickettsiae DNA in blood samples by amplification of the citrate synthase gene (gltA). The IFA results revealed seropositivity in 97 samples from 54 species of resident and migratory birds. No sample was positive for rickettsial DNA. The presence of antibodies in 62.5% of the sera indicates previous exposure of these birds to rickettsiae. The null detection of rickettsiae in the blood of seropositive birds is possibly due to a short period of bacteremia. Experimental studies are required to improve our understanding of the role of wild birds as sources of rickettsial infections in ticks.

Domestic dogs as amplifying hosts of Rickettsia rickettsii for Amblyomma aureolatum ticks.

Brazilian spotted fever (BSF) is an acute infectious disease caused by the bacterium Rickettsia rickettsii, which is transmitted by different tick species. Due to deleterious effects caused on ticks, the horizontal transmission of R. rickettsii through amplifying hosts is crucial for its maintenance in tick populations among BSF-endemic areas. The tick Amblyomma aureolatum is the main vector of R. rickettsii in the São Paulo metropolitan area; nevertheless, it is not known which vertebrate could act as an amplifying host for this tick species. Herein, we evaluated the potential of domestic dogs - primary hosts for A. aureolatum adults in BSF-endemic areas - to act as amplifying hosts. For this purpose, A. aureolatum non-infected adults were allowed to feed on two groups of dogs: the control group (G1), composed of one dog not exposed to R. rickettsii; and, the infected group (G2), composed of three dogs infected with R. rickettsii via tick parasitism. All G2-dogs became ill, seroconverted to R. rickettsii, and rickettsial DNA was detected in 87% of the engorged females that fed on them. Transovarial transmission rate was estimated to be 25% and infected larvae successfully transmitted R. rickettsii to guinea-pigs, confirming transovarial transmission and vector competence. No rickettsial DNA was detected in individual samples of eggs or larvae, which precluded the estimation of filial infection rate, but implies that it was low. Our results suggest that domestic dogs act as amplifying hosts of R. rickettsii for A. aureolatum ticks in BSF-endemic areas in Brazil.

Exploring the ecological and evolutionary relationships between Rickettsia and hard ticks in the Neotropical region.

This study addresses a meta-analysis of the distribution of Rickettsia spp. in the Neotropical region, as well as their associations with ticks and vertebrates. A total of 219 published reports on Rickettsia in ticks in the target region were compiled, providing 599 records of 31 species of Rickettsia recorded in 50 species of Ixodidae. The aim is to capture the phylogenetic relationships between rickettsiae and the ticks carrying them in the target region, with a focus on the co-speciation ticks-rickettsiae. We compared the phylogeny of ticks, the records of rickettsiae, the environmental gradients colonized by ticks and the effect of the phylogenetic composition of vertebrates feeding ticks on the detection of Rickettsia in ticks. Results show that differences in rickettsial composition in ticks do not depend on the vertebrate's blood-source. This is the first time this result is demonstrated. This study pinpoints that some Neotropical rickettsial organisms are associated with well-defined phylogenetical clusters of ticks. Secondarily, and probably only in a few cases, rickettsiae share species of phylogenetically distant ticks distributed along a gradient of environmental traits in which the ticks overlap (i.e., the different strains of Rickettsia parkeri sensu lato). We outline the importance of some ticks that share hosts and habitat: these ticks may act as "bridges" for the circulation of rickettsial species. There are also many species of Rickettsia that have been detected so far in only one tick species, pointing to a tight relationship or to the lack of data preventing conclusions about the detection of these bacteria in other ticks. Two species, namely Rickettsia amblyommatis and Rickettsia bellii have been recorded in the majority of ticks in the region (mainly Amblyomma spp.) and seem to be not associated with definite tick species because they may be an essential symbiont of the ticks. We conclude that an adequate analysis of rickettsiae-ticks-habitat is necessary to address the human health issues derived from the infections by rickettsiae.

Maintenance of the infection by Rickettsia amblyommatis in Amblyomma cajennense sensu stricto ticks and evaluation of vector competence.

Rickettsia amblyommatis has been reported infecting various tick species throughout the western hemisphere, including Amblyomma cajennense sensu stricto (s.s.). The present study aimed to evaluate whether R. amblyommatis can be maintained by transovarial transmission and transstadial passage in A. cajennense s.s. ticks, and whether these ticks are competent vectors of this rickettsial agent. We selected engorged females that were naturally infected or uninfected by R. amblyommatis, and reared their offspring (infected and control groups, respectively). Immature ticks were allowed to feed on guinea pigs, whereas adults fed on rabbits. All stages and individuals of each generation of the infected group yielded rickettsial DNA, which was not detected in any tick from the control group. No host from the control group seroconverted to R. amblyommatis. Among 16 guinea pigs and eight rabbits infested with R. amblyommatis-infected ticks, only one guinea pig seroconverted to R. amblyommatis. Some unfed adult ticks of the infected group were dissected, and DNA was extracted from their salivary glands and from their carcasses. DNA of R. amblyommatis was detected in all carcasses, but not in the salivary glands. Results of this study indicate very low vector competence of A. cajennense s.s. for R. amblyommatis. Previous studies reported domestic animals with high titers to R. amblyommatis in areas where these animals are naturally infested chiefly by A. cajennense s.s. ticks. One may consider that the vector competence of A. cajennense s.s. for R. amblyommatis could vary among tick populations and/or rickettsial strains.

Capybaras (Hydrochoerus hydrochaeris) as amplifying hosts of Rickettsia rickettsii to Amblyomma sculptum ticks: Evaluation during primary and subsequent exposures to R. rickettsii infection.

Rickettsia rickettsii is the agent of Brazilian spotted fever (BSF), the most lethal tick-borne disease in the western hemisphere. Amblyomma sculptum is the main vector of R. rickettsii in southeastern Brazil. Capybaras act as primary hosts for this tick species, and as amplifying hosts for R. rickettsii, creating new infected lineages of A. sculptum in BSF-endemic areas. In a recent study, we demonstrated that capybaras successively exposed to R. rickettsii-infected A. sculptum ticks developed a mean rickettsemic period of 9.2 days during primary infection, but no rickettsemia during subsequent expositions, when capybaras were immune to R. rickettsii. During the primary and subsequent infections, capybaras were also infested with uninfected A. sculptum ticks. These infestations compose the present study, which aimed to evaluate: (i) if either rickettsemic or non-rickettsemic capybaras could serve as infection sources of R. rickettsii for A. sculptum larvae and nymphs; (ii) the vector competence of the subsequent nymphs and adults (molted from the engorged ticks collected from capybaras); and (iii) if there were R. rickettsii-acquisition by uninfected ticks co-feeding with infected ticks on immune capybaras (without rickettsemia). Through experimental infection of capybaras with R. rickettsii via tick feeding, simulating a natural condition, we demonstrated that primarily infected capybaras developed rickettsemia that resulted in successful acquisition feeding of larvae and nymphs of A. sculptum, since part of these ticks maintained R. rickettsii transstadially, and the resultant molted ticks (either nymphs or adults) successfully transmitted the bacterium by feeding on susceptible rabbits. Contrastingly, all rabbit infestations with ticks derived from acquisition feeding on R. rickettsii-immune capybaras (including when acquisition ticks fed in direct contact with donor ticks) evidenced absence of R. rickettsii transmission due to lack of clinical signs and antibody response in those rabbits. Our results indicate that capybaras could serve as R. rickettsii-amplifying hosts for A. sculptum ticks only during the capybara's primary infection, but not during subsequent infections on immune capybaras. Finally, the probable co-feeding nonsystemic transmission of R. rickettsii seems to be irrelevant in the context of BSF epidemiology, in areas where capybaras are incriminated as main amplifying hosts of R. rickettsii for A. sculptum ticks.

Author Correction: Clinical and serological evaluation of capybaras (Hydrochoerus hydrochaeris) successively exposed to an Amblyomma sculptum-derived strain of Rickettsia rickettsii.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Clinical and serological evaluation of capybaras (Hydrochoerus hydrochaeris) successively exposed to an Amblyomma sculptum-derived strain of Rickettsia rickettsii.

Brazilian spotted fever (BSF), caused by Rickettsia rickettsii, is the most lethal tick-borne disease in the western hemisphere. In Brazil, Amblyomma sculptum ticks are the main vector. Capybaras (Hydrochoerus hydrochaeris), the largest living rodents of the world (adults weighing up to 100 Kg), have been recognized as amplifying hosts of R. rickettsii for A. sculptum in BSF-endemic areas; i.e., once primarily infected, capybaras develop bacteremia for a few days, when feeding ticks acquire rickettsial infection. We conducted experimental infections of five capybaras with an A. sculptum-derived strain of R. rickettsii and performed clinical and bacteremia evaluation during primary and subsequent infections. Bacteremia was detected in all capybaras during primary infection, but not in subsequent infections. All animals seroconverted to R. rickettsii (titres range: 64-32,768), and remained seropositive throughout the study. Primary infection resulted in clinical spotted fever illness in four capybaras, of which two had a fatal outcome. Subsequent infections in seropositive capybaras resulted in no clinical signs. Capybaras developed a sustained immune response that prevented a second bacteremia. This condition may imply a high reproduction rate of capybaras in BSF-endemic areas, in order to continuously generate capybaras susceptible to bacteremia during primary infection.

Epidemiology of capybara-associated Brazilian spotted fever.

BACKGROUND: Brazilian spotted fever (BSF), caused by the bacterium Rickettsia rickettsii, has been associated with the transmission by the tick Amblyomma sculptum, and one of its main hosts, the capybara (Hydrochoerus hydrochaeris). METHODS: During 2015-2019, we captured capybaras and ticks in seven highly anthropic areas of São Paulo state (three endemic and four nonendemic for BSF) and in two natural areas of the Pantanal biome, all with established populations of capybaras. RESULTS: The BSF-endemic areas were characterized by much higher tick burdens on both capybaras and in the environment, when compared to the BSF-nonendemic areas. Only two tick species (A. sculptum and Amblyomma dubitatum) were found in the anthropic areas; however, with a great predominance of A. sculptum (≈90% of all ticks) in the endemic areas, in contrast to a slight predominance of A. dubitatum (≈60%) in the nonendemic areas. Tick species richness was higher in the natural areas, where six species were found, albeit with a predominance of A. sculptum (≈95% of all ticks) and environmental tick burdens much lower than in the anthropic areas. The BSF-endemic areas were characterized by overgrowth populations of A. sculptum that were sustained chiefly by capybaras, and decreased populations of A. dubitatum. In contrast, the BSF-nonendemic areas with landscape similar to the endemic areas differed by having lower tick burdens and a slight predominance of A. dubitatum over A.sculptum, both sustained chiefly by capybaras. While multiple medium- to large-sized mammals have been incriminated as important hosts for A. sculptum in the natural areas, the capybara was the only important host for this tick in the anthropic areas. CONCLUSIONS: The uneven distribution of R. rickettsii infection among A. sculptum populations in highly anthropic areas of São Paulo state could be related to the tick population size and its proportion to sympatric A. dubitatum populations.

Comparative Susceptibility of Different Populations of Amblyomma sculptum to Rickettsia rickettsii.

The bacterium Rickettsia rickettsii is the etiological agent of Brazilian spotted fever (BSF), which is transmitted in Brazil mainly by the tick Amblyomma sculptum. Herein, larvae and nymphs of six populations of A. sculptum were exposed to R. rickettsii by feeding on needle-inoculated guinea pigs, and thereafter reared on uninfected guinea pigs or rabbits. Two tick populations were exposed to autochthone R. rickettsii strains, whereas four tick populations were exposed to non-autochthone strains. The six geographically different populations of A. sculptum showed different susceptibilities to R. rickettsii, higher among the two tick populations that were exposed to their autochthone R. rickettsii strain. In addition, higher rates of transovarial transmission of R. rickettsii and vector competence success also included the two tick populations that were exposed to autochthone R. rickettsii strains. These results indicate that the susceptibility of A. sculptum to R. rickettsii varies among different tick populations, with a clear bias for higher susceptibility to an autochthone R. rickettsii strain that has already coevolved with a tick population for some time. Our results demonstrated that the R. rickettsii infection induces higher mortality of engorged larvae and nymphs, and tend to reduce the reproductive fitness of engorged females. All together, these results might explain the low R. rickettsii-infection rates of A. sculptum under natural conditions (usually <1%), and indicate that an A. sculptum population should not be able to sustain a R. rickettsii infection for successive tick generations without the creation of new cohorts of infected ticks via horizontal transmission on vertebrate rickettsemic hosts (amplifying hosts). Finally, despite of the ubiquitous distribution of A. sculptum in southeastern and central-western Brazil, most of the populations of this tick species are devoid of R. rickettsii infection. This scenario might be related to two major factors: (i) insufficient numbers of susceptible amplifying hosts; and (ii) lower susceptibilities of many tick populations. While the first factor has been demonstrated by mathematical models in previous studies, the second is highlighted by the results observed in the present study.

Isolation of Rickettsia rickettsii from the tick Amblyomma sculptum from a Brazilian spotted fever-endemic area in the Pampulha Lake region, southeastern Brazil.

Brazilian spotted fever (BSF) is a tick-borne disease caused by the bacterium Rickettsia rickettsii, the deadliest spotted fever of the world, transmitted in southeastern Brazil mainly by the tick Amblyomma sculptum, a member of the Amblyomma cajennense species complex. In the present study, over 5000 adults of A. sculptum ticks were collected by dry ice traps in the Municipal Ecological Park, alongside the Pampulha Lake region, a BSF-endemic area of Belo Horizonte city, state of Minas Gerais, southeastern Brazil. Ticks were taken alive to the laboratory, where a sample of 2100 specimens was processed for isolation of R. rickettsii. For this purpose, ticks were macerated and intraperitoneally inoculated into guinea pigs. Only one out of 21 inoculated guinea pigs presented high fever within 21days post inoculation with tick homogenates. This febrile animal was euthanized and its internal organs were macerated and inoculated into additional guinea pigs (guinea pig passage). A spleen sample from a febrile guinea pig was used to inoculate Vero cells, resulting in a successful isolation and in vitro establishment of rickettsiae. Rickettsia-infected Vero cells were used for molecular characterization of the rickettsial isolate through PCR and DNA sequencing of fragments of three rickettsial genes (gltA, ompA, and ompB), which were all 100% identical to corresponding sequences of R. rickettsii from GenBank. The present R. rickettsii isolate was designated as strain Pampulha. A minimal infection rate of 0.05% R. rickettsii-infected ticks was estimated for A. sculptum population of the Pampulha Lake region. Our results, coupled with epidemiological evidences, suggest that R. rickettsii strain Pampulha, isolated from A. sculptum ticks in the present study, is the strain responsible for human clinical cases of BSF in the Pampulha Lake region of Belo Horizonte city.

Ecology of a tick-borne spotted fever in southern Brazil.

Rio Grande do Sul is the southernmost state of Brazil, bordering Uruguay. Clinical cases of spotted fever group (SFG) rickettsiosis were recently reported in Rio Grande do Sul. None of these cases was lethal, and all were confirmed by seroconversion to R. rickettsii antigens. Because serological cross-reactions are well known to occur between different SFG agents, the SFG agent responsible for the clinical cases remains unknown in Rio Grande do Sul, where no rickettsial agent is known to infect ticks. During 2013-2014, ticks and blood sera samples were collected from domestic dogs and wild small mammals, and from the vegetation in a SFG-endemic area of Rio Grande do Sul. Dogs were infested by Amblyomma ovale adult ticks, whereas small mammals were infested by immature stages of A. ovale, Ixodes loricatus, and adults of I. loricatus. Ticks collected on vegetation were adults of A. ovale, and immature stages of A. ovale, Amblyomma dubitatum, and Amblyomma longirostre. Three Rickettsia species were detected: Rickettsia bellii in I. loricatus, Rickettsia amblyommii in A. longirostre, and a Rickettsia parkeri-like agent (Rickettsia sp. strain Atlantic rainforest) in A. ovale. Seroreactivity to SFG antigens were detected in 19.7 % (27/137) canine and 37.5 % (15/40) small mammal sera, with highest titers to R. parkeri. Results indicate that the R. parkeri-like agent, strain Atlantic rainforest, is circulating between A. ovale ticks, dogs and small mammals in the study area, suggesting that this SFG pathogen could be one of the etiological agents of SFG clinical cases in Rio Grande do Sul.

Amblyomma yucumense n. sp. (Acari: Ixodidae), a Parasite of Wild Mammals in Southern Brazil.

During 2013-2014, adult ticks were collected on the vegetation and subadult ticks were collected from small mammals [Didelphis aurita Wied-Neuwied, Sooretamys angouya (Fischer), Euryoryzomys russatus (Wagner), Akodon montensis Thomas, Oxymycterus judex Thomas] in an Atlantic rainforest reserve in southern Brazil. Analyses of the external morphology of the adult ticks revealed that they represent a new species, Amblyomma yucumense n. sp. Partial 16S rRNA sequences generated from males, females, and nymphs were identical to each other and closest (95% identity) to corresponding sequences of Amblyomma dubitatum Neumann. A. yucumense is morphologically and genetically closest related to A. dubitatum. Dorsally, male of these species can be separated by major longitudinal pale orange stripes associated with a pseudoscutum indicated by a pale stripe in A. yucumense, in contrast to pale creamy longitudinal stripes and absence of pseudoscutum in A. dubitatum. Ventrally, male coxal I spurs are separated by a space narrower than external spur width in A. yucumense, and wider than external spur width in A. dubitatum. Females of the two species can be separated by coxal I spurs, longer in A. yucumense than in A. dubitatum. In addition, the adult capitulum and ventral idiosoma of A. yucumense are generally dark brown colored, while A. dubitatum is yellowish or light brown colored. The nymph of A. yucumense differs from A. dubitatum by the scutal cervical groove length, slightly shorter in the former species. Currently, A. yucumense is restricted to southern Brazil.