Natural infection, transovarial transmission, and transstadial survival of Rickettsia bellii in the Tick Ixodes loricatus (Acari: Ixodidae) from Brazil.

An Ixodes loricatus engorged female, infected with Rickettsia bellii, was collected from an opossum (Didelphis aurita) in Mogi das Cruzes, São Paulo State, Brazil. Two consecutive laboratory tick generations (F(1) and F(2)) reared from this single engorged female were evaluated for Rickettsia infection by polymerase chain reaction (PCR) targeting specific Rickettsia genes. Immature ticks fed on naïve Wistar rats (Rattus norvegicus) and adult ticks fed on opossum (D. aurita), both free of ticks and rickettsial infection. PCR performed on individual ticks from the F(1) (20 larvae, 10 nymphs, and 10 adults) and the F(2) (30 larvae, 30 nymphs, and 15 adults) yielded expected bands compatible with Rickettsia. All the PCR products that were sequenced, targeting gltA gene, resulted in sequences identical to each other and 99.7% (349/350) similar to the corresponding sequence of R. bellii in GenBank. The R. bellii infection on ticks from the second laboratory generation (F(2)) was confirmed by other PCR protocols and successful isolation of R. bellii in cell culture. We report for the first time a Rickettsia species infecting I. loricatus, and the first report of R. bellii in the tick genus Ixodes. We conclude that there was an efficient transovarial transmission and transstadial survival of this Rickettsia species in the tick I. loricatus. Our results suggest that R. bellii might be maintained in nature solely by transovarial transmission and transstadial survival in ticks (no amplifier vertebrate host is needed), since there has been no direct or indirect evidence of infection of vertebrate hosts by R. bellii.

Prevalence of Rickettsia felis in the fleas Ctenocephalides felis felis and Ctenocephalides canis from two Indian villages in Sao Paulo Municipality, Brazil.

We evaluated the presence of Rickettsia infection among fleas collected on domestic dogs in two Guarani Indian communities in the suburban area of São Paulo Municipality, Brazil. A total of 114 Ctenocephalides felis felis and 47 Ctenocephalides canis were collected from 40 dogs. A total of 41 C. felis felis (36.0%) and 9 C. canis (19.1%) fleas yielded expected bands by PCR, which were all shown by DNA sequencing to be indentical to the corresponding sequence of a fragment of the Rickettsia felis gltA gene deposited in GenBank. The overall prevalence of R. felis was 31.0% (49/161).

Larval immersion tests with ivermectin in populations of the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) from State of Sao Paulo, Brazil.

Larval immersion tests (LIT) with commercial formulation of ivermectin were carried out with larvae of two field populations of Rhipicephalus (Boophilus) microplus from commercial dairy farms of the State of Sao Paulo, Brazil and a susceptible strain (Porto Alegre) to differentiate resistant-suspected and susceptible strains. One of the populations tested (Barra Alegre) showed a LC(50) value significantly higher than the susceptible strain and a resistance ratio (CI95%) of 3.78 (3.47-4.12), leading to suspect that this population shows traces of resistance to ivermectin. Population Sao Francisco, with no records of ivermectin injections on cattle, showed no difference on the ivermectin response in relation to Porto Alegre strain characterizing it as susceptible. Although LIT is not yet recommended by FAO to diagnose resistance to acaricides, this technique was successful on the differentiation of resistance-suspected population and a susceptible strain and can be used to detect populations of R. (B.) microplus resistant to ivermectin. This is the first report of R. (B.) microplus resistant to ivermectin detected by an in vitro bioassay.

Prevalence of antibodies to spotted fever group rickettsiae in humans and domestic animals in a Brazilian spotted fever-endemic area in the state of São Paulo, Brazil: serologic evidence for infection by Rickettsia rickettsii and another spotted fever group Rickettsia.

In serum samples obtained from all the healthy humans, horses, dogs, and donkeys present on three farms in the Pedreira Municipality, an endemic area for Brazilian spotted fever, an indirect immunofluorescence assay (IFA) detected antibodies against Rickettsia rickettsii in 17 (77.3%) horses, 5 (31.3%) dogs (titers ranging from 64 to 4,048), and none of 4 donkeys or 50 humans. Five canine and eight equine sera with high antibody titers to R. rickettsii were also tested by IFA against R. bellii, R. akari, and R. africae antigens. Sera from two horses and two dogs that showed similar high antibody titers against two rickettsial antigens were evaluated after cross-absorption. Sera from seven horses and two dogs contained antibodies specific for R. rickettsii, and one dog serum had antibodies against a Rickettsia species very closely related to R. africae. The latter may have been caused by infection with the recently identified COOPERI strain.

Parasitism of domestic swine (Sus scrofa) by amblyomma ticks (Acari: Ixodidae) on a farm at Monte Negro, western Amazon, Brazil.

ABSTRACT In January 2001, while conducting a survey of the tick fauna of the State of Rondjnia, Brazil, a rural area within Monte Negro county was visited. On one farm within the county the producer maintained a herd of crossbred swine, Sus scrofa L., that was reared under unconfined conditions, with unrestrained access to the pasture and adjacent native Amazon equatorial forest. Inspection of the swine herd produced a total of 77 ticks collected from eight adult pigs (mean, 9.6 ticks per pig) that were identified as Amblyomma naponense (Packard) (26 males, eight females), A. oblongoguttatum Koch (five males, three females), A. ovale Koch (one female) and A. scalpturatum Neumann (one male). One Amblyomma larva and 32 Amblyomma nymphs also were collected from the pigs. Of these, six nymphs were reared in the laboratory until they reached the adult stage, one being an A. oblongoguttatum female and five being A. scalpturatum females.

Notes on parasitism by Amblyomma humerale (Acari: Ixodidae) in the state of Rondônia, western Amazon, Brazil.

The tick Amblyomma humerale Koch is endemic to South America. All host records refer to the adult stage parasitizing tortoises, mostly yellow-footed tortoise, Geochelone denticulata (L.), and red-footed tortoise, Geochelone carbonaria (Spix). The current study reports the presence of A. humerale in the state of Rondônia, Brazil. A total of 215 adult ticks (201 males, 14 females) was collected from six G denticulata in an Indian reserve and nine Geochelone sp. in rural Monte Negro County, giving an overall mean infestation of 14.3 +/- 12.0 (range: 2-44) ticks per tortoise. Male ticks always outnumbered females on the host and nine tortoises had only male ticks. Male ticks were mostly attached in clusters on the ventral sides of the carapace near the anterior and posterior margins, and more rarely on the outer margin of the plastron. All females were found attached to the tortoise skin, at different sites such as head, neck, shoulders or legs. Male ticks were rarely observed attached to the body skin. Seven engorged nymphs collected on small vertebrates from Monte Negro County molted to adults of A. humerale. This included one nymph each on the seven-colored lizard, Plica plica (L), green tree climber, Plica umbra (L.), and wide-foraging lizard, Kentropyx calcarata Spix,three nymphs on the common opossum, Didelphis marsupialis L., and one nymph on the silky anteater, Cyclopes didactylus L. These constitute the first host records for the immature stages of the tick A. humerale.

Taxonomic status of Ixodes didelphidis (Acari: Ixodidae).

Ixodes didelphilis Fonseca & Aragão was described in Brazil in 1952 as a new tick species that differed from Ixodes loricatus Neumann by the spiracular plate pattern. We have reared four tick colonies from different geographic areas in the laboratory that were started from single engorged females originally identified as I. didelphidis (BMG, colony ) and I. Iocicatus (CSP, PSP, and TRJ colonies). We analyzed the spiracular plate morphology of F1 adult ticks from each tick colony, compared their biological 11th, and performed a molecular analysis of the second internal transcribed rDNA spacer (ITS2) to test the validity of the species I. didelphidis. The spiracular plate analysis of laboratory F1 adult ticks yielded single females from the four colonies showed variations that invalidate morphological parameters for differentiation of loricatus and I. didelphidis. Biological data of the BMC, CSP, and TRJ colonies were similar. The biology ofthe PSP colony was not evaluated. The ITS2 sequence variations observed between the tick colonies ranged from 1.3 to 4.9%, and the similarity tree constructed by the neighbor-joining method with nucleotide distances showerl that the distances between the samples were similar to what is expected for intraspecific variations found in other ticks species. The morphological and biological results, in conjunction with the ITS2 analysis, supported the conspecificity I. loricatus and I. didelphidis.

Molecular characterization of mediterranean spotted fever rickettsia isolated from a European traveler in the state of São Paulo, Brazil.

Rickettsial spotted fever is common in southeastern Brazil. Differential diagnosis of pathogens can be performed with proper laboratory methods. A traveler arriving from Portugal developed a fatal febrile hemorrhagic syndrome diagnosed as spotted fever rickettsiosis. We isolated the agent, which was identified as Rickettsia conorii conorii by sequencing rickettsial genes.

Rickettsia infection in five areas of the state of São Paulo, Brazil.

This study investigated rickettsial infection in animals, humans, ticks, and fleas collected in five areas of the state of São Paulo. Eight flea species (Adoratopsylla antiquorum antiquorum, Ctenocephalides felis felis, Polygenis atopus, Polygenis rimatus, Polygenis roberti roberti, Polygenis tripus, Rhopalopsyllus lugubris, and Rhopalopsyllus lutzi lutzi), and five tick species (Amblyomma aureolatum, Amblyomma cajennense, Amblyomma dubitatum, Ixodes loricatus, and Rhipicephalus sanguineus) were collected from dogs, cats, and opossums. Rickettsia felis was the only rickettsia found infecting fleas, whereas Rickettsia bellii was the only agent infecting ticks, but no animal or human blood was shown to contain rickettsial DNA. Testing animal and human sera by indirect immunofluorescence assay against four rickettsia antigens (R. rickettsii, R. parkeri, R. felis, and R. bellii), some opossum, dog, horse, and human sera reacted to R. rickettsii with titers at least four-fold higher than to the other three rickettsial antigens. These sera were considered to have a predominant antibody response to R. rickettsii. Using the same criteria, opossum, dog, and horse sera showed predominant antibody response to R. parkeri or a very closely related genotype. Our serological results suggest that both R. rickettsii and R. parkeri infected animals and/or humans in the studied areas.

Isolation of Rickettsia felis in the mosquito cell line C6/36.

We report the isolation and establishment of Rickettsia felis in the C6/36 cell line. Rickettsial growth was intense, always with 90 to 100% of cells being infected after few weeks. The rickettsial isolate was confirmed by testing infected cells by PCR and sequencing fragments of three major Rickettsia genes (gltA, ompB, and the 17-kDa protein gene).

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.

Rickettsia infection in five areas of the state of São Paulo, Brazil

This study investigated rickettsial infection in animals, humans, ticks, and fleas collected in five areas of the state of São Paulo. Eight flea species (Adoratopsylla antiquorum antiquorum, Ctenocephalides felis felis, Polygenis atopus, Polygenis rimatus, Polygenis roberti roberti, Polygenis tripus, Rhopalopsyllus lugubris, and Rhopalopsyllus lutzi lutzi), and five tick species (Amblyomma aureolatum, Amblyomma cajennense, Amblyomma dubitatum, Ixodes loricatus, and Rhipicephalus sanguineus) were collected from dogs, cats, and opossums. Rickettsia felis was the only rickettsia found infecting fleas, whereas Rickettsia bellii was the only agent infecting ticks, but no animal or human blood was shown to contain rickettsial DNA. Testing animal and human sera by indirect immunofluorescence assay against four rickettsia antigens (R. rickettsii, R. parkeri, R. felis, and R. bellii), some opossum, dog, horse, and human sera reacted to R. rickettsii with titers at least four-fold higher than to the other three rickettsial antigens. These sera were considered to have a predominant antibody response to R. rickettsii. Using the same criteria, opossum, dog, and horse sera showed predominant antibody response to R. parkeri or a very closely related genotype. Our serological results suggest that both R. rickettsii and R. parkeri infected animals and/or humans in the studied areas.