Pesquisa de Rickettsia spp em carrapatos Amblyomma cajennense e Amblyomma dubitatum no Estado de São Paulo

Foi pesquisada a presença de riquétsias em 3.545 carrapatos Amblyomma cajennense e 2.666 Amblyomma dubitatum. Através do teste de hemolinfa, reação em cadeia pela polimerase e isolamento de rickettsia em cultivo celular, todos os Amblyomma cajennense foram negativos, sendo que 634 (23,8%) Amblyomma dubitatum mostraram-se infectados com Rickettsia bellii.

The expression of genes coding for distinct types of glycine-rich proteins varies according to the biology of three metastriate ticks, Rhipicephalus (Boophilus) microplus, Rhipicephalus sanguineus and Amblyomma cajennense

Background: Ticks secrete a cement cone composed of many salivary proteins, some of which are rich in the amino acid glycine in order to attach to their hosts' skin. Glycine-rich proteins (GRPs) are a large family of heterogeneous proteins that have different functions and features; noteworthy are their adhesive and tensile characteristics. These properties may be essential for successful attachment of the metastriate ticks to the host and the prolonged feeding necessary for engorgement. In this work, we analyzed Expressed Sequence Tags (ESTs) similar to GRPs from cDNA libraries constructed from salivary glands of adult female ticks representing three hard, metastriate species in order to verify if their expression correlated with biological differences such as the numbers of hosts ticks feed on during their parasitic life cycle, whether one (monoxenous parasite) or two or more (heteroxenous parasite), and the anatomy of their mouthparts, whether short (Brevirostrata) or long (Longirostrata). These ticks were the monoxenous Brevirostrata tick, Rhipicephalus (Boophilus) microplus, a heteroxenous Brevirostrata tick, Rhipicephalus sanguineus, and a heteroxenous Longirostrata tick, Amblyomma cajennense. To further investigate this relationship, we conducted phylogenetic analyses using sequences of GRPs from these ticks as well as from other species of Brevirostrata and Longirostrata ticks. Results: cDNA libraries from salivary glands of the monoxenous tick, R. microplus, contained more contigs of glycine-rich proteins than the two representatives of heteroxenous ticks, R. sanguineus and A. cajennense (33 versus, respectively, 16 and 11). Transcripts of ESTs encoding GRPs were significantly more numerous in the salivary glands of the two Brevirostrata species when compared to the number of transcripts in the Longirostrata tick. The salivary gland libraries from Brevirostrata ticks contained numerous contigs significantly similar to silks of true spiders (17 and 8 in, respectively, R. microplus and R. sanguineus), whereas the Longirostrata tick contained only 4 contigs. The phylogenetic analyses of GRPs from various species of ticks showed that distinct clades encoding proteins with different biochemical properties are represented among species according to their biology. Conclusions: We found that different species of ticks rely on different types and amounts of GRPs in order to attach and feed on their hosts. Metastriate ticks with short mouthparts express more transcripts of GRPs than a tick with long mouthparts and the tick that feeds on a single host during its life cycle contain a greater variety of these proteins than ticks that feed on several hosts.

Experimental Infection of the Opossum Didelphis aurita by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and Evaluation of the Transmission of the Infection to Ticks Amblyomma cajennense and Amblyomma dubitatum

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.

Experimental Infection of Opossums Didelphis aurita by Rickettsia rickettsii and Evaluation of the Transmission of the Infection to Ticks Amblyomma cajennense

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 direct 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 real-time 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.

Effect of Amblyomma cajennense Ticks on the Immune Response of BALB/c Mice and Horses

This work evaluated the effect of the Amblyomma cajennense tick on the immune response of BALB/c mice and on horse lymph node cell proliferation. We observed that mice do not develop resistance to nymphs of this tick species and that lymphocyte proliferation of this host is inhibited by tick saliva, nymphal extract, or infestations. Horse lymph node cell proliferation is inhibited by tick saliva as well. Mice lymphocytes under the effect of tick saliva, nymphal extract, or infestations display a predominantly. p Th-2 cytokine production pattern. Observed results partially explain this tick`s disease vectoring capacity and broad host range.

Antigen-presenting cells in draining lymph nodes of goats repeatedly infested by the Cayenne tick Amblyomma cajennense nymphs

Resistance to tick feeding has been previously shown to be an acquired, immunologically mediated phenomenon in goats, associated with cutaneous basophilia to nymphs of Amblyomma cajennense, the Cayenne tick, after repeated infestations. On the other hand, it is well known that antigen-presenting cells (APCs) play an important role in the host immune reaction to tick infestations. The most able APCs for Th cells are the well defined dendritic cells, mononuclear phagocytes and B-lymphocytes. Immunohistochemical analysis of draining lymph nodes of goats repeatedly infested with nymphs of the ixodid tick A. cajennense to search for APCs was done. Pre-scapular lymph nodes draining the tick attachment sites were collected 15 days after both the first and third infestations. Tick infestations resulted in increased number of CD21(+) B lymphocytes in lymph nodes after the tertiary infestation. However, the number of CD11b(+) and CD11c(+) cells were not altered after the successive infestations. Lower numbers of CD11c(+) cells had infiltrated lymph nodes responsible for draining the tick infested skin. These findings suggest that acquired immunity of goats against nymphs of A. cajennense is possibly established by B lymphocytes during the first infestation and that APCs may play a key role in this mechanism.

Experimental infection of capybaras Hydrochoerus hydrochaeris by Rickettsia rickettsii and evaluation of the transmission of the infection to ticks Amblyomma cajennense

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 G I capybaras every approximate to 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. (c) 2008 Elsevier B.V. All rights reserved.

Expressed sequence tags (ESTs) from the salivary glands of the tick Amblyomma cajennense (Acari : Ixodidae)

The neotropical tick Amblyomma cajennense is a significant pest to domestic animals, the most frequently human-biting tick in South America and the main vector of Brazilian spotted fever (caused by Rickettsia rickettsii), a deadly human disease. The purpose of this study is to characterize the adult A. cajennense salivary gland transcriptome by expressed sequence tags (ESTs). We report the analysis of 1754 clones obtained from a cDNA library, which reveal mainly transcripts related to proteins involved in the hemostatic processes, especially proteases and their inhibitors. Remarkably, five types of possible serine protease inhibitors were found, including a molecule with a distinguished structure that contains repeats of the active motif of hirudin inhibitors. Besides, other components that may be active over the host immune system or acting as defensins against infecting microorganisms were also described, including a molecule similar to insect venom allergens. The conjunction of components from this transcriptome suggests a diverse strategy of A. cajennense tick during feeding, but emphasized in the coagulation system. (c) 2008 Published by Elsevier Ltd.

Cross-mating experiments with geographically different populations of Amblyomma cajennense (Acari: Ixodidae)

The present study evaluated the reproductive compatibility of the crosses between adult ticks of the following three geographically different populations of Amblyomma cajennense: State of So Paulo (SP), southeastern Brazil; State of Rondnia (RO), northern Brazil; and Colombia (CO). In addition, crosses between A. cajennense ticks from Argentina (AR) and SP ticks were also performed. The Argentinean population (AR) was compatible with SP because their crosses resulted in high % egg hatching (mean values ranging from 71.5 to 93.5%), similarly to all homologous (intrapopulational) crosses. In contrast, the tick populations SP, RO, and CO were shown to be incompatible with each other, since their heterologous (interpopulational) crosses always resulted in very low % egg hatching (range: 0-5%). The F(1) larval offspring derived from some of these females that yielded 5% egg hatching were reared until the F(1) adult stage. In all cases, only adult females molted from engorged nymphs. These F(1) females were likely to be a product of thelytokous parthenogenesis of the SP, RO, and CO females that were used in the heterologous crosses. Reproductive incompatibility is not expected to occur between different populations of a single species. Thus, our results suggest that the taxon A. cajennense might be represented by a complex of different species, whereas SP and AR ticks might represent a single species. Further populational genetic studies, coupled with extensive morphological analyses, are needed to clarify and determine a possible complex of valid species that might have been classified under the taxon A. cajennense.

Influence of Photoperiod and Temperature on the Larval Behavioral Diapause of Amblyomma cajennense (Acari: Ixodidae)

Larval behavioral diapause was shown to be the major factor controlling the 1-yr generation pattern of Amblyomma cajennense (F.) (Acari: Ixodidae) in Brazil. During fieldwork, this behavior was shown to coincide with long daylength (>12 h) and high mean ground temperature (approximate to 25 degrees C), which prevail during spring-summer in Brazil. The current study evaluated biological parameters of engorged females, their eggs, and the resultant larvae inside plastic pots planted with the grass Brachiaria decumbens Stapf. held in incubators set with different combinations of temperature and photoperiod. Both the long daylength (photoperiod 14:10 [L:D]h) and high temperature (25 degrees C) during larval hatching induced larval behavioral diapause, characterized by the confinement of hatched larvae on the ground below the vegetation for many weeks. When long daylength was present during hatching, but temperature was low (15 degrees C), larvae did not enter diapause. Similarly, when short daylength (10:14 or 12:12) was present during larval hatching, larvae did not enter diapause regardless whether temperature was high (25 degrees C). Termination of diapause was induced by shifting photoperiod from 14:10 to 12:12 or the temperature from 25 to 15 degrees C. When applied to field conditions, the present results indicate that both high ground mean temperature (approximate to 25 degrees C) and long daylength (>12 h) during spring-summer (October-March) are responsible for the induction and maintenance of A. cajennense larval behavioral diapause in the field. Furthermore, both the low ground mean temperature (-20 degrees C) and the short daylength (<12h) during autumn (April-May) are responsible for termination of larval behavioral diapause in the field.

Study of chelicerae in the tick Amblyomma cajennense (Fabricius 1787) by scanning electron microscopy

Chelicerae de Amblyomma cajennense foram examinados em microscopia de varredura. Esta técnica mostrou detalhes não evidenciáveis ao poder de resolução do microscópio ótico.

Pesquisa de Rickettsia spp em carrapatos Amblyomma cajennense e Amblyomma dubitatum no Estado de São Paulo

Foi pesquisada a presença de riquétsias em 3.545 carrapatos Amblyomma cajennense e 2.666 Amblyomma dubitatum. Através do teste de hemolinfa, reação em cadeia pela polimerase e isolamento de rickettsia em cultivo celular, todos os Amblyomma cajennense foram negativos, sendo que 634 (23,8%) Amblyomma dubitatum mostraram-se infectados com Rickettsia bellii.

Poderá o carrapato transmitir a lepra?: isolamento e cultura dum bacilo acido-álcool resistente de sedimento de "Amblyomma cajennense" capturado em leproso: 2ª nota

In this 2nd note upon the possibility of transmission of human leprosy by ticks, the A. relates his stepps to obtain the collaboration of his colleagues working in leprosaria in various States of Brazil, Argentina and Paraguay in such studies. Firstly the A. describes the positive results of examination of sediment of ticks, the cattle tick Boophilus microplus (Canestrini, 1888), received from Paraná (Leprosário São Roque) , which were put on active lepers, two of them sucking during 9 days and one during 7 days. Two out of three were killed for examination and were very strongly positive for acid-fast bacilli. A series of tubes of Loewenstein medium was smeared with the sediment of such ticks. Secondly the A. relates his personnal experiment, carried out in Rio de Janeiro, trying to infect normal ticks in lepers. The experiment with Boophilus microplus was negative and was twicely positive the experiment with Amblyomma cajennense Fabricius, 1794. The experiment is being in progress and will be continued in other places of Brazil. Finally, after being given the general characteristics of Boophilus microplus, the A. describes the non-chromogenic culture of a acid-fast bacillus isolated by him from sediment of ticks (Amblyomma cajennense) captured in lepers from Colônia Santa Isabel (Minas gerais), which parasitism was spontaneous. The first isolation was obtained in Loewenstein medium after 62 days incubation at 37°C. The culture is pure and the bacillus is permanent acid-fast. The plate1, in full color, represents this culture in its four generations. The colonies are pearl-white in color, dry, elevated and rough, developing slowly and beginning as white pinhead points scattered upon the surface of the medium. The culture is not yet rich enough to be inoculated into laboratory animals, which will be done when possible.

Poderá o carrapato transmitir a lepra?: mais quatro amostras de culturas de bacilos acido-alcool resistentes obtidas de carrapatos (2 de "Amblyomma cajennense"e 2 de "Boophilus microplus") infectados em leprosos do Paraná: 3ª nota

The AA. carried out experiments in the leprosarium São Roque, State of Paraná, South Brazil, to verify if the cattle tick Boophilus microplus could be experimentally infected in lepers, which was true. The AA. Tried also to be ascertained if Boophilus microplus and Amblyomma cajennense could change of hosts during their feedings which was true, both ticks continue feeding, the last species for many days, after being transferred from one to another leper. The junior A. describes in full their experiments and also a dermatites caused by tick bites. The senior A. brought to Rio de Janeiro most of the infected ticks for examination, which revealed a very high positivity. He smeared the sediments of lots of both species of ticks in Loewenstein medium and after a variable periode of incubation at 37° C. he obtained four new samples of cultures of acid-fast organisms, two from Amblyomma cajennense and two from Boophilus microplus. These cultures are being studied and will be inoculated into laboratory animals. The senior A. inoculated new batches of white rats with sediments of many ticks infected in lepers. Various hypotheses of both previous notes upon the subject now are verified facts. The A. is accumulating facts to draw the conclusions in the future. He also suggested the leprosy workers in the interior of the country to cooperate with him in such important studies, specially in the habitat of lepers in the rural zones of various States.

Infecção experimental de carrapatos (Amblyomma cajennense) em ratos com lepra Stefansky

The A. succeeded, after various experiments, to infect ticks (Amblyoma cajennense) with STEFANSKY bacillus, upon feeding them in rats suffering from murine leprosy, in advanced stages. The A. thinks that will be ease to transfer the sickness from rat to rat by means of inoculation of triturate of infected ticks, as did MARCHOUX with Laelaps echdninus.