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The domestic cat is not considered a primary host for any specific tick species; however, it can be affected by some Ixodidae species, such as Rhipicephalus sanguineus sensu lato and Amblyomma spp. The study reports parasitism by Amblyomma auricularium and the detection of anti-Rickettsia spp. antibodies in domestic cats from a rural property in the Afrânio municipality, Pernambuco, Brazil. Amblyomma auricularium (24 nymphs, six females, and four males) and Amblyomma sp. (42 larvae) parasitized three cats, and 73 free-living ticks were captured in armadillo burrows: A. auricularium (36 nymphs, six females, five males) and Amblyomma sp. (26 larvae). Blood samples from cats were collected and the obtained plasma were subjected to indirect immunofluorescence assay (IFA) to detect antibodies against Rickettsia antigens. Thus, anti-Rickettsia spp. antibodies were determined (titers ranging from 128 to 512) and showed a predominant antibody response to Rickettsia amblyommatis or a very closely related genotype. This study reports the first infestation of nymphs and adults of A. auricularium on cats in a new area of occurrence in the semi-arid region of Northeastern Brazil and reports for the first time the presence of anti-Ricketsia antibodies in cats in the region, with R. amblyommatis as the probable infectious agent.

Caatinga and Atlantic Forest are Brazilian biomes threatened by anthropogenic disturbances, which are a serious threat to biodiversity and favor the emergence of zoonotic pathogens. Capybara populations are increasingly present in urban and rural areas due to the loss of their natural habitats. There are no studies on the rickettsial infection in these rodents in Northeastern Brazil, although there are case records of Brazilian Spotted Fever (BSF) in this region. In this context, we evaluated rickettsial infection in capybaras and their ticks in four areas of the Atlantic Forest and Caatinga biomes in Northeastern Brazil. Blood sera from 21 capybaras were tested by indirect immunofluorescence assay (IFA) using Rickettsia rickettsii, R. parkeri, and R. amblyommatis antigens. Blood samples and ticks (Amblyomma sp., Amblyomma dubitatum and Amblyomma sculptum) were tested by PCR, targeting a fragment of the rickettsial gltA gene and R. bellii primers. Overall, 15 out of 21 capybaras (71.4%) reacted positively (titer ≥64) to at least one of the three Rickettsia antigens tested, with final titers ranging from 64 to 1024. No rickettsial DNA was found in capybara blood samples. Rickettsial DNA was detected in 22 of 31 adult tick pools using gltA gene, being all nine pools of A. sculptum and 16 of the 19 of A. dubitatum, but all samples were negative to R. bellii specific primers. All attempts to sequence the amplicon were unsuccessful, indicating a possible low rickettsial load in the tick samples. The present study reports for the first time the serological evidence of Rickettsia spp. infection in free-ranging capybaras and suggest the rickettsial infection in A. sculptum and A. dubitatum in the Caatinga and Atlantic Forest biomes in Northeastern Brazil., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Caatinga is a biome exclusive to the semiarid zone of Brazil, where studies on ticks and tick-borne diseases are scarce. Herein, we investigated the occurrence of Rickettsia, Ehrlichia, and Coxiella in wild mammals, domestic dogs and their ectoparasites using molecular and serological techniques. During 2014-2016, blood samples and ectoparasites were collected from 70 small mammals (51 rodents, 18 marsupials, 1 wild canid) and 147 domestic dogs in three areas of the Caatinga. Through serological analyses of domestic dogs of the three areas, 8 to 11 % were seropositive for Rickettsia rickettsii, 9 to 37 % for Rickettsia amblyommatis, 61 to 75 % for Ehrlichia canis, and 0-5% for Coxiella burnetii. All wild mammals were seronegative for Rickettsia spp. and C. burnetii, except for one rodent (Wiedomys pyrrhorhinos) and one marsupial (Didelphis albiventris) that were seroreactive to C. burnetii, one wild canid (Cerdocyon thous) for R. amblyommatis, and two Rattus rattus for Rickettsia spp. Through PCR targeting DNA of Rickettsia, Ehrlichia or Coxiella, all blood samples were negative, except for the presence of Ehrlichia canis DNA in 8.8 % of the domestic dogs, and a recently reported novel agent, Ehrlichia sp. strain Natal, in one marsupial (Gracilinanus agilis). A total of 222 ticks, 84 fleas, and six lice were collected. Ticks were mostly Rhipicephalus sanguineus sensu lato, some Ixodes loricatus, Ornithodoros rietcorreai, Haemaphysalis sp., and Amblyomma spp.; fleas were Ctenocephalides felis felis, Pulex sp. and Polygenis (Polygenis) bohlsi jordani; and lice were Polyplax sp. and Gyropus sp. Through molecular detection of microorganisms, 9% of C. felis felis contained Rickettsia felis, 20 % of A. auricularium contained R. amblyommatis and 13 % of A. parvum contained 'Candidatus Rickettsia andeanae', whereas Ehrlichia canis DNA was detected in at least 6% of the R. sanguineus s.l. from one area. We report a variety of ectoparasites infesting small mammals and domestic dogs in the Caatinga biome, where these ectoparasites probably act as vectors of rickettsiae, ehrlichial agents (E. canis and Ehrlichia sp. strain Natal) and C. burnetii. Our results highlight to the potential risks of human infection by these tick-borne agents in the Caatinga biome., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

The genetic structure of populations of the tick Amblyomma ovale from five distinct areas of the Brazilian Atlantic rainforest was evaluated via DNA sequencing and associated with the presence of domestic dogs acting as hosts at the edge of forest fragments. Ticks were collected from domestic dogs and from the environment between 2015 and 2017. Four collection areas were located in the surroundings and within the Serra do Mar State Park, São Paulo State (23°37'21"S, 45°24'43"W), where dogs were bimonthly monitored along 2 years using camera traps and GSM trackers. To determine the spatial limits of genetic structure, ticks collected upon dogs living near the Serra do Baturié, Ceará State (4°15'40"S, 38°55'54"W) were included as well. A total of 39 haplotypes of 16S rRNA and Cox 1 mitochondrial genes sequences were observed, with 27 of them coming from areas within the Serra do Mar State Park. No haplotype was shared between the Serra do Mar and the Serra do Baturié indicating isolation of tick populations at the scale of 2000 km. Although three different haplotype lineages of A. ovale occurred within the Serra do Mar State Park, no genetic structure was found across the study sites within this park, suggesting high tick gene flow across a range of 45 km. Monitoring data from domestic dogs and wild carnivores showed that these species share the same habitats at the forest edge, with dogs playing a likely limited role in tick dispersal. Our findings have important implications for understanding the genetic structure of wide spread A. ovale along Brazilian rainforest remnants, which can further be associated to tick-borne infectious agents, such as Rickettsia parkeri, and used for predicting future patterns of tick diversity in the Brazilian Atlantic rainforest.

Amblyomma rotundatum is an ixodid tick strictly associated with cold-blooded animals, among them the toad Rhinella jimi. This work aimed to evaluate the seasonal dynamics of larvae, nymphs, and adults of A. rotundatum parasitizing R. jimi in an area within the semi-arid Caatinga Biome of northeastern Brazil. Monthly sampling from February 2014 to January 2016 resulted in a total of 592 R. jimi toads captured and inspected for infestation by ticks, which were counted and identified. After the procedure, the toads were released with their ticks at the same capture site. During the study period, a total of 658 A. rotundatum were counted, comprising 497 larvae, 110 nymphs, and 51 females. The two-year mean abundances of larvae, nymphs, and adults were 0.84, 0.19, and 0.09, respectively. The two-year mean infestation intensities of larvae, nymphs, and adults were 3.65, 1.45, and 1.34, respectively. Comparing the monthly values of prevalence, mean abundance, and mean infestation intensity of A. rotundatum life stages, differences were observed between the first and second year of study; however, with no clear seasonal pattern., (Copyright © 2018. Published by Elsevier GmbH.)

In this study, Amblyomma ovale Koch ticks were collected from domestic dogs in two localities of the Atlantic rainforest biome of Brazil: 1) the Paty Valley of the Chapada Diamantina National Park, Bahia state (northeastern Brazil), and 2) Adrianópolis, Paraná state (southern Brazil). Ticks were screened for the presence of Rickettsia-like structures by the hemolymph test with Giménez staining, and then processed for isolation of rickettsiae in Vero cell culture by the shell-vial technique. Rickettsiae were isolated from one A. ovale tick of each of the two localities. The two isolates were successfully established in the laboratory with several passages, each one reaching >90% infection of the cells. The two isolates were identified as the spotted fever group (SFG) agent Rickettsia sp. strain Atlantic rainforest, as their gltA (350 bp), ompB (781 bp), and ompA (567 bp) gene fragments were 100% equal to GenBank corresponding sequences of the original strain Atlantic rainforest, reported to be infecting a human in southeastern Brazil, and also 100% equal to the available ompA sequence of strain Bahia, reported to be infecting a human in Paty Valley, the same area of the present study in Bahia state. Ten dogs from Paty Valley were serologically tested against rickettsial antigens by the indirect immunofluorescence antibody test. At least 60% of them were seroreactive to SFG rickettsiae. The role of A. ovale as vector of Rickettsia sp. strain Atlantic rainforest in the Paty Valley area, as well as in other parts of Latin America, is discussed., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Until recently, Amblyomma cajennense (Fabricius, 1787) was considered to represent a single tick species in the New World. Recent studies have split this taxon into six species. While the A. cajennense species complex or A. cajennense (sensu lato) (s.l.) is currently represented by two species in Brazil, A. cajennense (sensu stricto) (s.s.) and Amblyomma sculptum Berlese, 1888, their geographical distribution is poorly known., Methods: The distribution of the A. cajennense (s.l.) in Brazil was determined by morphological examination of all lots of A. cajennense (s.l.) in two large tick collections of Brazil, and by collecting new material during three field expeditions in the possible transition areas between the distribution ranges of A. cajennense (s.s.) and A. sculptum. Phylogenetic analysis inferred from the ITS2 rRNA gene was used to validate morphological results. Morphological description of the nymphal stage of A. cajennense (s.s.) is provided based on laboratory-reared specimens., Results: From the tick collections, a total 12,512 adult ticks were examined and identified as 312 A. cajennense (s.s.), 6,252 A. sculptum and 5,948 A. cajennense (s.l.). A total of 1,746 ticks from 77 localities were collected during field expeditions, and were identified as 249 A. cajennense (s.s.), 443 A. sculptum, and 1,054 A. cajennense (s.l.) [these A. cajennense (s.l.) ticks were considered to be males of either A. cajennense (s.s.) or A. sculptum]. At least 23 localities contained the presence of both A. cajennense (s.s.) and A. sculptum in sympatry. DNA sequences of the ITS2 gene of 50 ticks from 30 localities confirmed the results of the morphological analyses. The nymph of A. cajennense (s.s.) is morphologically very similar to A. sculptum., Conclusion: Our results confirmed that A. cajennense (s.l.) is currently represented in Brazil by only two species, A. cajennense (s.s.) and A. sculptum. While these species have distinct distribution areas in the country, they are found in sympatry in some transition areas. The current distribution of A. cajennense (s.l.) has important implications to public health, since in Brazil A. sculptum is the most important vector of the bacterium Rickettsia rickettsii, the etiological agent of Brazilian spotted fever.

This study evaluated rickettsial infection in Amblyomma rotundatum ticks collected from toads (Rhinella jimi) in the Brazilian Caatinga biome, an unique semiarid region of South America. Tick infestations were observed in 57.8% toads (26/45); mean infestation: 1.6 ticks/toad. DNA extraction from 42 ticks (6 larvae, 22 nymphs and 11 female adults) was tested by polymerase chain reaction (PCR) targeting Rickettsia organisms, which were detected in 100% of the ticks. Amplicons' DNA sequences were identical to each other and 99% identical to Rickettsia bellii from GenBank. DNA samples extracted from the blood of the 45 toads were negative by rickettsia-PCR protocols., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

This study investigated rickettsial infection in Amblyomma auricularium ticks from the state of Pernambuco, northeastern Brazil. An engorged female of A. auricularium collected from a skunk (Conepatus semistriatus) was sent alive to the laboratory, where the female was found through molecular analysis to be infected by Rickettsia amblyommii. This engorged female oviposited, and its offspring was reared through three consecutive generations, always using tick-naïve rabbits to feed the ticks. PCR performed on five egg pools, 10 larvae, 10 nymphs, and 10 adults of each of the three generations always yielded rickettsial DNA, indicating maintenance of rickettsial infection in the ticks by transstadial and transovarial passages. DNA sequences of random PCR products from eggs, larvae, nymphs, and adults were identified as R. amblyommii. All infested rabbits seroconverted to R. amblyommii antigens at the 21(st) day after infestation, indicating that larvae, nymphs, and adults transmitted R. amblyommii through parasitism. However, no infested rabbit presented fever or any clinical alteration during the experimental period. Rickettsiae were successfully isolated from the two A. auricularium females, and the isolates were established in Vero cell culture. Molecular characterization of the isolates confirmed R. amblyommii by sequencing partial gltA, ompA, and ompB genes. From another sample of 15 A. auricularium adult ticks collected from two armadillos (Euphractus sexcinctus), eight (53.3%) were infected by R. amblyommii. This study reports R. amblyommii infecting the tick A. auricularium for the first time. This is also the first report of rickettsia infecting ticks in the northeastern region of Brazil.

This work evaluated the infection of opossums (Didelphis aurita) by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and their role as amplifier hosts for horizontal transmission to Amblyomma cajennense and/or Amblyomma dubitatum ticks. Infection in D. aurita was induced by intraperitoneal inoculation with R. felis (n = 4 opossums), R. bellii (n = 4), and R. parkeri (n = 2). Another group of six opossums were inoculated intraperitoneally with Leibovitz-15 sterile culture medium, representing the uninfected groups (n = 2 opossums simultaneously to each infected group). Opossum blood samples collected during the study were used for DNA extraction, followed by real-time polymerase chain reaction targeting the rickettsial gene gltA, hematology, and detection of Rickettsia spp.-reactive antibodies by indirect immunofluorescence assay. Opossums were infested with uninfected A. cajennense and/or A. dubitatum for 30 days postinoculation (DPI). Flat ticks molted from ticks fed on opossums were allowed to feed on uninfected rabbits, which were tested for seroconversion by immunofluorescence assay. Samples of flat ticks were also tested by real-time polymerase chain reaction. Inoculated opossums showed no clinical abnormalities. Antibodies to Rickettsia spp. were first detected at the second to fourth DPI, with detectable titers until the 150th DPI. Rickettsemia was detected only in one opossum inoculated with R. parkeri, at the eighth DPI. Only one A. cajennense tick (2.0%) previously fed on a R. parkeri-inoculated opossum became infected. None of the rabbits infested with opossum-derived ticks seroconverted. The study demonstrated that R. felis, R. bellii, and R. parkeri were capable to produce antibody response in opossums, however, with undetectable rickettsemia for R. felis and R. bellii, and very low rickettsemia for R. parkeri. Further studies must be done with different strains of these rickettsiae, most importantly the strains that have never gone through in vitro passages.

The presence of rickettsial infection was surveyed in 3,545 Amblyomma cajennense ticks and 2,666 Amblyomma dubitatum ticks. Using the hemolymph test, polymerase chain reaction and isolation of Rickettsia in cell cultures, all of the Amblyomma cajennense were negative, whereas 634 (23.8%) of the Amblyomma dubitatum ticks were shown to be infected with Rickettsia bellii.

The present study evaluated the infection of capybaras (Hydrochoerus hydrochaeris) by Rickettsia rickettsii and their role as amplifier hosts for horizontal transmission of R. rickettsii to Amblyomma cajennense ticks. Two groups of two capybaras each were evaluated: on day 0, group 1 (G1) was infested by R. rickettsii-infected ticks, and group 2 (G2) was inoculated intraperitoneally with R. rickettsii. Two additional groups were control groups, not exposed to R. rickettsii, being CG1 group the control of G1, and CG2 group the control of G2. Capybara rectal temperature was measured daily. Blood samples were collected every 3 days during 30 days, and used to (i) inoculate guinea pigs intraperitoneally; (ii) DNA extraction followed by real-time PCR targeting the rickettsial gene gltA; (iii) hematology; (iv) detection of R. rickettsii-reactive antibodies by indirect immunofluorescence assay (IFA). Blood was also collected from G1 capybaras every approximately 10-30 days till the 146th day, to be tested by serology. Capybaras were infested by uninfected A. cajennense nymphs from the 3rd to the 18th day. Engorged nymphs were collected, allowed to molt to adults in an incubator. Thereafter, the subsequent flat ticks were tested by PCR. All G1 and G2 capybaras became infected by R. rickettsii, as demonstrated by guinea pig inoculation and seroconversion, but they showed no fever. Rickettsemia was continually detected from the 6th (G2 capybaras) or 9th (G1 capybaras) to the 18th day post inoculation or infestation with R. rickettsii-infected ticks. A total of 20-25% and 30-35% of the flat ticks previously fed on G1 and G2 capybaras, respectively, became infected by R. rickettsii. The study demonstrated that R. rickettsii was capable to infect capybaras without causing clinical illness, inducing rickettsemia capable to cause infection in guinea pigs and ticks. Our results indicate that capybaras act as amplifier host of R. rickettsii for A. cajennense ticks in Brazil.

The present study evaluated the infection of opossums (Didelphis aurita) by Rickettsia rickettsii and their role as amplifier hosts for horizontal transmission of R. rickettsii to Amblyomma cajennense ticks. Three groups of opossums were evaluated: on day 0, group 1 (G1) was inoculated intraperitoneally with R. rickettsii; group 2 (G2) was infested by R. rickettsii-infected ticks; and group 3 (G3) was the uninfected control group. Opossum rectal temperature was measured daily. Blood samples were collected every 2 to 4 days during 30 days, and used to (1) inoculate guinea pigs intraperitoneally; (2) extract DNA followed by real-time polymerase chain reaction (PCR) targeting the rickettsial gene gltA; (3) study hematology; (4) detect R. rickettsii-reactive antibodies by indirect immunofluorescence assay (IFA). Blood was also collected every 10 days from days 30 to 180, to be tested by serology. Opossums were infested by uninfected A. cajennense larvae and nymphs from days 3 to 15. Engorged ticks were collected and allowed to molt in an incubator. Thereafter, the subsequent flat ticks were allowed to feed on uninfected rabbits, which were tested for seroconversion by IFA. Samples of flat ticks were also tested by real-time PCR. All G1 and G2 opossums became infected by R. rickettsii, as demonstrated by realtime PCR or/and guinea pig inoculation, but they showed no clinical abnormality. Rickettsemia was first detected at days 2 to 8, lasting intermittently till days 1 to 30. Approximately 18% and 5% of the flat ticks previously fed on G1 and G2 opossums, respectively, became infected by R. rickettsii, but only the rabbits infested with G1-derived ticks seroconverted. The study demonstrated that R. rickettsii was capable of infecting opossums without causing illness and developing rickettsemia capable of causing infection in guinea pigs and ticks, although the infection rate in ticks was low.

The current study compared the susceptibility of larval stages of Amblyomma cajennense (F.), Amblyomma aureolatum (Pallas), and Rhipicephalus sanguineus (Latreille) to infection by a Brazilian strain of Rickettsia rickettsii. Guinea pigs experimentally infected by R. rickettsii were simultaneously infested by larvae of the three tick species. Recovered engorged larvae were allowed to molt to nymphs and held in an incubator at 23 degrees C and 85-90% RH. Subsequent flat nymphs were tested for rickettsial infection by polymerase chain reaction (PCR). Concomitant infestations with sibling ticks on noninfected guinea pigs (control) were done. While 10-60% of the A. cajennense nymphs were shown to be infected by R. rickettsii, both A. aureolatum and R. sanguineus were highly susceptible to R. rickettsii, since 80-100% of their nymphs were shown to be infected in the corresponding trials. Most of the engorged larvae (approximately 70-95%), regardless of being infected or not, successfully molted to nymphs. Mortality rates for engorged larvae tended to be statistically similar (P > 0.05) for ticks recovered from R. rickettsii-infected and noninfected guinea pigs, within each tick species. The only exceptions were the significantly higher mortalities (P < 0.05) for engorged A. cajennense larvae recovered from two infected guinea pigs. Therefore, A. cajennense was less susceptible to R. rickettsii infection than A. aureolatum and R. sanguineus, while feeding on rickettsemic guinea pigs. These two later species were similarly highly susceptible.

Biological data of three generations of Amblyomma tigrinum in the laboratory are reported and the suitability of different host species for immature ticks are compared. Grouping the three generations, infestations by both the larval and nymphal stages were performed on chickens (Gallus gallus), wistar rats (Rattus norvegicus), rabbits (Orytolagus cuniculus), wild mice (Calomys callosus), dogs (Canis familiaris) and opossums (Didelphis albiventris). Only dogs were used for infestations by adult ticks. Tick developmental periods were observed in an incubator at 27 degrees C and RH 90%. The proportion of engorged larvae recovered from chickens (21.7% of the exposed larvae) was significantly larger (p<0.001) than those from the five mammal species used in the infestations (maximum of 3.1%). A significant larger (p<0.01) proportion of engorged larvae successfully molted after being fed on chickens than on mammal hosts. The proportion of engorged nymphs recovered from chickens (28.8% of the exposed nymphs) was significantly larger (p0.001) than those from mammal hosts (range: 0-2.1%). Larvae showed similar feeding periods on exposure to different host species, except for those larvae fed on C. callosus, which showed significantly longer (p<0.001) feeding periods. Engorged larvae detachment peaked on the 5th feeding day, followed by the 6th day, on all hosts except for C. callosus. Larval premolt periods were similar for engorged ticks exposed to different host species, except for larvae fed on dogs, which showed significantly longer (p<0.001) premolt periods. Host detachment of engorged nymphs peaked on the 6th feeding day on chickens. Although nymphal detachment on rats peaked on the 8th day, only 15 nymphs were recovered from this host species. In a sample of 144 F3 nymphs fed on chickens no significant difference (p>0.10) was found between the feeding or premolt periods of 82 males and 62 females, but female nymphs were significantly heavier (p<0.005) than male nymphs. Sixteen engorged females (61.5% of the exposed ticks) were recovered after being fed on dogs. and all these females laid viable eggs. Chickens, the only avian host, were the most suitable host when compared with the five mammal species. Dogs were demonstrated to be a suitable host for adults of A. tigrinum, which is consistent with, several reports of adult A. tigrinum ticks parasitizing dogs in different areas of South America. Our results reinforce that in these same areas avian species are the major hosts for immature stages of this tick species.