Surveys of horse serum are a useful method of surveillance for Brazilian spotted fever in areas where humans are exposed to Amblyomma cajennense ticks.

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.

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.Key words: Rickettsia -spotted fever -fleas -ticks -opossum -domestic animals Rickettsiae are obligate intracellular bacteria that have been classically divided into two groups: the typhus group (TG), composed of Rickettsia prowazekii and Rickettsia typhi, which are associated with lice and fleas, respectively; and the spotted fever group (SFG), which includes more than 20 valid species, mostly associated with ticks (e.g., Rickettsia rickettsii, Rickettsia parkeri), and at least one species associated with fleas (Rickettsia felis). Other species, such as Rickettsia bellii and Rickettsia canadensis, both associated with ticks, have been considered a distinct group sharing antigenic, genetic and ecological characters with both TG and SFG rickettsiae (Parola et al. 2005b). Since all SFG Rickettsia share common outer membrane antigens, conventional serological assays employing a single Rickettsia antigen (e.g., R. rickettsii) allow detecting a SFG infection, but they do not identify the Rickettsia species responsible for the infection, even in the presence of high titers (La Scola & Raoult 1997). For a more specific serological diagnosis, it is recommended techniques such as testing serum against all known Rickettsia species present in a given area, or serum cross-absorption tests (La Scola & Raoult 1997, Parola et al. 2005b).For many years, SFG rickettsiosis in the Americas was thought to be caused only by R. rickettsii, transmitted by ticks (Weiss & Moulder 1984). This rickettsia has been reported in several countries from North, Central, and South America causing an acute, highly lethal febrile disease receiving different names according to the country...

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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).Rickettsia felis is a bacterium belonging to the spotted fever group of the genus Rickettsia. It was first observed by Adams et al. in 1990 (1) within midgut cells of cat fleas (Ctenocephalides felis felis) in the United Sates and was later described as a new Rickettsia species (2, 10). The organism has been detected, by molecular assays, infecting the flea C. felis felis in different countries of the world, suggesting a cosmopolitan distribution (5,7,8,13). There are only a few reports of R. felis infecting other flea species, including Ctenocephalides canis, Pulex irritans, and Anomiopsyllus nudata (15).R. felis has been reported as the causative agent of the flea-borne spotted fever, an emerging human rickettsiosis that has been diagnosed in Mexico, the United States, Brazil, France, Germany, and Thailand (12,13,14,17). Diagnosis of human infection by R. felis has been performed by molecular assays or serological testing. The latter has been limited by the difficulties of producing R. felis antigens, since only one laboratory has been able to establish R. felis in tissue culture, using the XTC-2 cell line (10, 13). Here we report the isolation and establishment of R. felis in tissue culture, using C6/36 cells, a mosquito cell line derived from Aedes albopictus (6).In this experiment, uninfected and infected cells were incubated at 28 and 25°C, respectively, and the level of infected cells was determined visually by Gimenez staining (3). Fleas (C. felis felis) were collected from naturally infested dogs in a farm at Pedreira, São Paulo State, Brazil, where a previous study detected up to 80% of the fleas infected with R. felis (5). In the laboratory, four pools of five live fleas were formed. Each pool was disinfected for 10 min in iodine alcohol; washed twice in sterile water; triturated in 600 l of Leibovitz-15 medium with amphotericin B (Cultilab, Brazil), 2.5% bovine calf serum (HyClone), and antibiotics (penicillin [100 U/ml] and streptomycin [0.5 mg/ml]), and inoculated into four shell vials (Ϸ150 l per shell vial) containing a monolayer of C6/36 cells. After inoculation, the shell vials were centrifuged for 1 h at 700 ϫ g and 22°C (12). Thereafter, the monolayer was washed and 1 ml of the same medium was added to each shell vial, which was incubated at 25°C. On the third day, the medium was changed to antibiotic-free medium. On the fifth day, a new change of medium was made, but the aspirated medium was centrifuged and the pellet was examined by Gimenez staining. Typical Rickettsia-like organisms (Fig. 1A) were observed within cells from the four inoculated shell vials. The monolayers of these infected shell vials were harvested and inoculated into a 25-cm ...

The applicability of laboratory bioassays to diagnose ivermectin (IVM) resistance in Rhipicephalus microplus was evaluated. Adult immersion tests (AITs), larval immersion tests (LITs) and larval packet tests (LPTs) were performed to characterise the effects of ivermectin toxicity on adults and larvae of a susceptible reference strain. The AIT was determined to be a reasonable assay but requires a large number of individuals to attain interpretable results. The LIT and LPT were validated with an IVM resistant strain, revealing resistance ratios (RRs) of 6.73 and 1.49, respectively. In a field survey, nine different populations of cattle tick from the states of São Paulo and Mato Grosso do Sul, Brazil, were analysed with the LIT. Populations without previous exposure to ivermectin exhibited RRs between 0.87 and 1.01. Populations previously exposed to IVM showed RRs between 1.83 and 4.62. The LIT was more effective at discriminating between resistant and susceptible populations than the LPT. The use of the LIT is recommended for the diagnosis of ivermectin resistance in R. microplus.

Fipronil is a phenylpyrazolic insecticide that is widely used in agriculture and has been recently used to control the cattle tick, Rhipicephalus (Boophilus) microplus. Because of the serious problems associated with resistance to the available acaricides, this product has been used as an important alternative to control acaricide-resistant ticks. The objective of this work was to analyse the fipronil sensitivity of ticks that were collected from farms with a history of fipronil use by larval bioassays. A total of 11 Brazilian tick populations were studied: one population from Rio Grande do Sul, one population from Mato Grosso do Sul and nine populations from São Paulo. To validate the assays, susceptible reference strains, POA (Porto Alegre, Brazil) and Mozo (Dilave, Uruguay), and ticks from six different farms that never used fipronil were tested. The resistance of various tick populations to technical grade fipronil (95.3%) was primarily evaluated using the larvae immersion test (LIT) and the larval packet test (LPT), when a sufficient number of larvae was collected. Using the LIT, the resistance ratios (RR(50)) of the tick populations from Rio Grande do Sul and Mato Grosso do Sul were 14.9 and 2.6, respectively, and the populations derived from São Paulo had RR(50)s ranging from 2.5 to 6.9. Four populations were evaluated with the LPT, and two populations displayed lower RR(50), while other populations displayed higher RR(50) than those determined by the LIT. This article reports the first cases of fipronil resistance in Brazil and highlights the LIT as a more sensitive technique for the evaluation of fipronil resistance in R. (B) microplus ticks. We suggest the use of the LIT as an evaluation tool for monitoring fipronil resistance in the control programmes of R. (B) microplus.