in a laboratory experiment previously reported ( 9 ) , the first generation ( f1 ) larval progeny of engorged a. aureolatum female ticks collected in atibaia , so paulo state , brazil , were allowed to feed on 4 r. rickettsii  infected guinea pigs ( infected group ) and 2 uninfected guinea pigs ( control group ) . these guinea pigs were infected by intraperitoneal inoculation of a homogenate of r. rickettsii  infected guinea pig organs , as detailed previously ( 9 ) . the taiau strain of r. rickettsii used in this experiment was the first guinea pig passage from a naturally infected a. aureolatum tick , which was cryopreserved before this strain was adapted for in vitro growth ( 5 ) . engorged tick larvae obtained from the infected and control groups of guinea pigs were held in an incubator at 23c and 90% relative humidity for molting to nymphs . as reported ( 9 ) , 100% of the resultant nymphs from the infected group were shown to be infected by r. rickettsii , whereas no nymph from the control group was infected by rickettsiae . the f1 unfed nymphs of infected and control groups were reared separately in the laboratory for 4 consecutive generations until they were f4 unfed adults . throughout the experiment , infestations with infected ticks and control groups were done in parallel ; infested animals were held in the same room under the same environmental conditions as the controls . male guinea pigs and female rabbits were infested with larvae and nymphs , and dogs and rabbits were infested with adult ticks . however , in each infestation with a given tick stage , different individuals of the same host species were used at the same time for infected and control groups . every guinea pig or rabbit used in this study was tick naive ; these animals were provided by a private laboratory that raised the animals under proper sanitary conditions . the dogs used for adult infestations had been infested by a. aureolatum ticks in a previous study ( 10 ) , when they were also infected with r. rickettsii ; the dogs were therefore immune to rmsf . infestation of each animal was performed inside a feeding chamber glued to its shaved dorsum , as previously described ( 11 ) . all infested animals had their temperature rectally measured daily from the day of infestation ( day 0 ) to 21 days postinfestation . when present , skin lesions ( e.g. , scrotal reactions ) during this period were also noted . naturally detached engorged larvae , nymphs , or female ticks were recovered daily from the feeding chambers of the infested animals of both groups and immediately taken to a single incubator adjusted to 23c and 95% relative humidity for molting ( for engorged larvae and nymphs ) or for egg laying and incubation ( for engorged females ) . engorged females had their individual weight measured the day they detached from the host . in addition , the total egg mass deposited by each female was weighed on the day of the end of oviposition and a conversion efficiency index ( cei  =  mg egg mass / mg engorged female  100 ) , which measures the efficiency with which a female tick converts body weight into eggs ( 12 ) , was determined for each female that oviposited . percentage of egg hatching for each egg mass was visually estimated ( 13 ) . during the experiment , random samples of unfed f1 nymphs and adults ; f2 eggs and nymphs ; and f4 eggs , larvae , nymphs , and adults from both infected and control groups were submitted for dna extraction as described ( 9 ) and tested by pcr targeting a 401-bp fragment of the rickettsial glta gene , as described ( 9 ) . eggs were tested in pools ( 1 egg pool containing 510 eggs / female ) , whereas larvae , nymphs , and adults were tested individually . at the end of the study , pcr products from 3 f4 nymphs underwent dna sequencing ( 5 ) , and the resultant sequence was compared with genbank data by blast analysis ( www.ncbi.nlm.nih.gov/blast ) . all tick - infested guinea pigs and rabbits were tested for seroconversion to r. rickettsii antigens . for this purpose , blood samples were collected at 0 days postinfestation and 21 days postinfestation ; these samples were tested for anti rickettsii reactive antibodies by immunofluorescence assay ( ifa ) , as previously described ( 14 ) . some infested guinea pigs that died before the 21 days postinfestation were not tested by ifa because a second blood sample was not obtained ; however , a fragment of their lungs was submitted to dna extraction by using the dneasy tissue kit ( qiagen , chatsworth , ca , usa ) and tested by the same pcr protocol referenced above . during the experiment , tick biologic parameters were compared between infected and control groups . for this purpose , larval and nymphal molting success and female oviposition success ( i.e. , death of engorged ticks ) were compared by the  test . in addition , weight of engorged females and their corresponding egg masses , percentage of egg hatching , and cei values were compared by student t test . the study was approved by the bioethical committee in animal research of the faculty of veterinary medicine of the university of so paulo ( protocol no . the infected tick group remained infected by rickettsiae through 4 consecutive generations , until the end of the experiment ( f4 unfed adults ) . in all infestations performed with ticks from this group , fever developed in all guinea pigs and rabbits 59 days postinfestation . guinea pigs also had scrotal reactions characterized by swelling , followed by necrosis in most animals . among the 16 guinea pigs infested with infected larvae or nymphs , 8 died during the febrile period . of the 8 dead guinea pigs , 6 had their lungs tested by pcr , which detected rickettsial dna . the remaining guinea pigs plus the rabbits showed seroconversion for r. rickettsii at 21 days postinfestation ( table 1 ) . in contrast , no guinea pig or rabbit infested with ticks from the control group experienced fever or seroconverted ; i.e. , they were nonreactive for r. rickettsii at both 0 days postinfestation and 21 days postinfestation . fever did not develop in the dogs infested with generations f1 and f3 adult ticks , and serum was not tested on 0 days postinfestation and 21 days postinfestation because it was already known that the dogs were seroreactive to r. rickettsii because they had been previously infected in another study ( 10 ) . * dpi , days postinfestation ; ifa , immunofluorescence assay ; pos , positive ; nd , not done ;  , data not collected . pcr performed on lung - extracted dna from guinea pigs that died during the febrile period . a positive result means seroconversion , namely nonreactive ( titer < 64 ) at 0 dpi , and reactive ( titer > 1,024 ) at 21 dpi . all pcrs performed on unfed ticks from the infected group resulted in amplicons compatible with r. rickettsii . these amplicons resulted from 45 f1 nymphs , 10 f1 adults ( 5 males , 5 females ) , 30 f2 nymphs , 100 f4 larvae , 30 f4 nymphs , and 6 f4 adults ( 3 males , 3 females ) , which were all tested individually ( table 1 ) . in addition , 12 f2 egg pools and 5 f4 egg pools , derived from all f1 and f3 infected engorged females , also yielded pcr amplicons compatible with r. rickettsii . the 100 pcr - positive f4 larvae cited above encompassed 5 groups of 20 larvae , each derived from a different f3 engorged female . pcr products from 3 f4 nymphs underwent dna sequencing , and the resultant sequence was 100% identical to r. rickettsii strain taiau , available in genbank ( accession no . no visible amplicon was generated by all ticks tested by pcr , which included 20 f1 nymphs , 6 f1 adults ( 3 males , 3 females ) , 10 f2 nymphs , 30 f4 larvae , 20 f4 nymphs , and 6 f4 adults ( 3 males , 3 females ) , which were all tested individually , and 13 f2 egg pools and 6 f4 egg pools , derived from all f1 and f3 control engorged females . the 30 pcr - negative f4 larvae cited above encompassed 3 groups of 10 larvae , with each group derived from a different f3 engorged female . although the mortality rate for engorged f4 larvae and f3 nymphs was higher for the infected group than the control group , overall molting success of engorged larvae and nymphs was similar between infected and control ticks because there was no significant difference in the molting success of these ticks when the 4 generations were grouped . however , there was an overall lower egg - laying success of engorged females from the infected group when compared with the control group ( table 2 ) ; i.e. , although 89.7% of the control engorged females successfully oviposited in the incubator , only 66.7% of the infected females oviposited in the same incubator . regarding the reproductive performance of these females , a few significant differences were observed between infected and control females , generally in favor of control ticks ( table a1 ) . * molting or oviposition success values for infected and control ticks of the same tick stage were significantly different ( p<0.05 ) . in the study reported here , r. rickettsii was preserved by transstadial maintenance and transovarial transmission in a. aureolatum ticks for 4 consecutive generations , because all tested eggs , larvae , nymphs , and adults from the infected group were shown by pcr to contain rickettsial dna , from the first to the fourth generation . in addition , infestations of guinea pigs and rabbits with larvae and nymphs from the 4 generations and adults from the third generation confirmed that ticks of these 3 developmental stages from the 4 generations were infected by r. rickettsii because all infested guinea pigs and rabbits became infected by r. rickettsii , which was confirmed by seroconversion through ifa or pcr in addition to compatible clinical data . these results also confirm that larvae , nymphs , and adults of a. aureolatum ticks are competent vectors of r. rickettsii . together , our results strongly support clinical and epidemiologic data that have implicated the a. aureolatum tick as the main vector of r. rickettsii in the metropolitan area of so paulo ( 4,5,9,15 ) . all infected egg pools tested by pcr yielded rickettsial dna , indicating a transovarial transmission rate ( the proportion of infected females giving rise to at least 1 infected egg or larva ) of 100% among r. rickettsii  infected females . because all the individual larvae from the infected group tested by pcr also yielded rickettsial dna , a filial infection rate ( proportion of infected eggs or larvae obtained from an infected female ) of 100% is also likely to have occurred . in contrast , no egg pool or individual larva from the control group yielded rickettsial dna by pcr . these results are in agreement with the larval infestations , which resulted in confirmed rickettsiosis in all guinea pigs or rabbits infested by larvae from the infected group and with no rickettsiosis in animals infested with control group larvae . although transovarial transmission of spotted fever rickettsiae in ticks seems to occur worldwide ( 16 ) , including r. rickettsii in new world ticks ( 1723 ) , few studies have quantified this perpetuation route . previous studies in the united states demonstrated a 100% transovarial transmission rate and a 100% filial infection rate of r. rickettsii in d. variabilis naturally infected female ticks and in d. andersoni female ticks , either naturally infected or experimentally infected during the larval stage ( 20,24 ) . in the study reported here , although f2 female ticks fed on susceptible rabbits , f1 and f3 females fed on dogs previously infected by r. rickettsii . as expected , these dogs showed no clinical signs during the study , a condition certainly related to their immune status because they were shown to have high antibody titers against r. rickettsii ( data not shown ) . regardless of feeding on immune ( dogs ) or susceptible ( rabbits ) hosts , 100% transovarial transmission rates and filial infection rates were found for all generations evaluated . similarly , burgdorfer and brinton ( 24 ) observed 100% transovarial transmission rates and filial infection rates for d. andersoni female ticks that fed on either immune or susceptible hosts , resulting in no alteration of biological characteristics of the bacterium . thus , the host immune status to r. rickettsii does not seem to alter the transovarial transmission of r. rickettsii in ticks . overall , no expressive differences in mortality rates were observed between engorged immature ticks from the infected and control groups . these results contrast with those of a previous study ( 21 ) that reported much higher mortality rates for infected d. andersoni engorged larvae and nymphs ( 34.9%97.7% ) than those observed for uninfected sibling ticks ( 1.4%2.1% ) and also with those of experimental studies on r. conorii in r. sanguineus ticks , in which significant mortality rates of immature ticks were observed when compared with uninfected ticks ( 25,26 ) . on the other hand , our results demonstrated that the mortality of r. rickettsii  infected engorged females was 3 higher than the mortality of uninfected females ; i.e. , oviposition success was only 66.7% in the former and 89.7% in the latter . in addition , the reproductive performance of uninfected females was significantly higher than that of infected females , as demonstrated by higher egg mass weight for control ticks . because both the infected and control ticks were siblings derived from the same field - engorged females used to start a laboratory colony , reared under the same laboratory conditions during the whole study , we conclude that the lower survival and reproductive performance of infected females was a result of a deleterious effect caused by the r. rickettsii infection . these results are in agreement with those of a previous study ( 24 ) , which reported higher mortality of r. rickettsii infected d. andersoni and d. variabilis engorged female ticks and lower egg masses oviposited by these females than by uninfected females . this higher mortality and lower egg mass was attributed to massive rickettsial development in the female body , including the ovaries ( 24 ) . tick mortality is much more influential on the tick population when it occurs in engorged females ; i.e. , although each dead egg , larva , or nymph is only less 1 subsequent larva , nymph , or adult , respectively , in the tick population , a dead engorged female represents thousands of eggs fewer in the following generation . different field studies in brazil and in the united states have reported that < 1% of the dermacentor spp . and a. aureolatum ticks , respectively , are found naturally infected by r. rickettsii within rmsf - endemic areas ( 1,5,27 ) . on the other hand , the present study demonstrated that besides being highly susceptible to r. rickettsii infection , a. aureolatum ticks are highly efficient in maintaining the infection through 100% transstadial transmission , transovarial transmission , and filial infection rates . the main reason for these contrasting findings is the deleterious effect the r. rickettsii infection causes in ticks , as previously demonstrated for dermacentor spp . ticks ( 21,24,27 ) . therefore , despite of the high susceptibility of a. aureolatum ticks to r. rickettsii infection , the higher mortality and reduced reproductive performance of infected engorged females may contribute to low infection rates among a. aureolatum tick field populations in rmsf - endemic areas of the so paulo metropolitan area , such as the 0.9% infection rate previously reported ( 5 ) . on the basis of our results , it seems unlikely that a. aureolatum could sustain r. rickettsii infection over multiple successive generations solely by vertical transmission because the number of infected ticks would gradually decrease after each generation . thus , horizontal transmission through the participation of amplifier vertebrate hosts in the formation of new lineages of infected ticks seems to be crucial for maintenance of r. rickettsii in the rmsf - endemic areas where the a. aureolatum tick is implicated as the principal vector , just as has been reported for rmsf - endemic areas in the united states , where dermacentor spp . ticks are the vector ( 21,27 ) . although natural amplifier hosts are known for dermacentor spp . ticks in rmsf - endemic areas in the united states ( 27 ) , and for a. cajennense ticks in areas endemic for brazilian spotted fever in brazil ( 1 ) , they are not known for a. aureolatum ticks . further studies should test the natural hosts of a. aureolatum immature tick stages , which are some passerine birds and small rodents ( 6 ) , for their competence to act as amplifier hosts of r. rickettsii to a. aureolatum ticks . regarding dogs , the main host for the adult stage of a. aureolatum ticks within the so paulo metropolitan area , even though it has been shown that dogs are capable of having rickettsial infections sufficient to infect other tick species ( i.e. , they are a competent amplifier host ) ( 23 ) , only adult - stage a. aureolatum ticks feed on dogs . because transovarial transmission rates are likely to be low or absent when the primary infection of ticks occurs during adult feeding ( 18,23,24 ) , the epidemiologic influence of dogs as amplifier hosts of r. rickettsii for a. aureolatum ticks is questionable . therefore , future studies should target potential hosts of the immature stages of a. aureolatum ticks that could act as amplifier hosts .
we experimentally infected amblyomma aureolatum ticks with the bacterium rickettsia rickettsii , the etiologic agent of rocky mountain spotted fever ( rmsf ) . 
 these ticks are a vector for rmsf in brazil . 
 r. rickettsii was efficiently conserved by both transstadial maintenance and vertical ( transovarial ) transmission to 100% of the ticks through 4 laboratory generations . 
 however , lower reproductive performance and survival of infected females was attributed to r. rickettsii infection . 
 therefore , because of the high susceptibility of a. aureolatum ticks to r. rickettsii infection , the deleterious effect that the bacterium causes in these ticks may contribute to the low infection rates ( < 1% ) usually reported among field populations of a. aureolatum ticks in rmsf - endemic areas of brazil . because the number of infected ticks would gradually decrease after each generation 
 , it seems unlikely that a. aureolatum ticks could sustain r. rickettsii infection over multiple successive generations solely by vertical transmission .