several species of spotted fever group ( sfg ) rickettsiae have been isolated from ticks in the united states ; however , the only species considered to cause human disease in maryland is rickettsia rickettsii , the causative agent of rocky mountain spotted fever ( rmsf ) . the potential pathogenicity of rickettsial organisms is most often predicted by the ability of the species to cause disease in guinea pigs . the reliability of this method has been debated , and researchers have suggested that " every rickettsial species may have pathogenic potential , provided that its reservoir arthropod is capable of biting humans " ( 1,2 ) . the prevalence of sfg rickettsia infection in dermacentor variabilis , the primary vector of r. rickettsii in the eastern united states , has been estimated in several studies . prevalences from 0.2% in ohio ( 3 ) to 8.6% in ( table 1 ) maryland ( 4 ) have been reported . many studies have implied that these infections were r. rickettsii , but few have confirmed these identities ( 5 ) . numerous sfg - rickettsial species have been isolated or partially characterized from molecular evidence in the eastern united states ; these species include r. rickettsii , r. rhipicephali , r. montanensis ( = r . these species have been identified , either together or separately , in areas where rmsf is endemic ( table 2 ) . as the distributions of different sfg - species in disease - endemic areas become better understood , determining the relationship between the rickettsiae involved in human disease and those isolated from vector ticks and mammal and tick reservoirs may be necessary . ci , confidence interval ; sfg , spotted fever group .  fisher exact p values .  ticks with unknown month of collection were excluded from the statistical analyses for this characteristic . ci , confidence interval . differentiating the tick - borne sfg rickettsia before the 1990s depended largely on culture and epitope recognition techniques , such as immunoflourescence and agglutination tests and mouse serotyping with monoclonal antibodies . genotypic studies of rickettsiae conducted during the 1990s led to two rickettsial genes that can be used to identify rickettsial infections : citrate synthase ( glta ) and rompa ( 9 ) . citrate synthase encodes the first enzyme of the tricarboxylic acid cycle and is highly conserved among all rickettsia species , serving as a polymerase chain reaction ( pcr ) target to identify any rickettsial infection . rompa encodes a surface - expressed protein of sfg - rickettsiae that is important for adhesion to host cells ( 10 ) . only sfg rickettsia contain the rompa gene ( 11 ) , making it an ideal pcr target to identify sfg rickettsia infections . approximately 35 cases of rmsf are reported annually in maryland . from 1994 through 1998 , maryland ranked 8th nationally , reporting 112 cases . these cases , confirmed by the maryland department of health and mental hygiene , meet the centers for disease control and prevention ( cdc ) case definition , yet not much information exists to characterize the infection rate of sfg rickettsiae in d. variabilis in the state . this cross - sectional study examined the prevalence and composition of sfg rickettsia in d. variabilis in maryland . in 2002 , genomic dna was extracted from 392 adult d. variabilis collected by flagging in anne arundel , baltimore , calvert , charles , prince george 's , and st . quality of the modified hexadecyltrimethylammonium bromide ( ctab ) dna extractions was verified by amplifying a tick 16s mtdna fragment ( 12 ) . modifying the existing extraction procedure involved an additional phenol : chloroform : isoamyl alcohol ( 25:24:1 ) extraction step to further stabilize the extracted dna . tick extractions were screened by pcr for evidence of infection with rickettsia by using primers specific to the rickettsia citrate synthase gene ( 9 ) . the rickettsia infection rate was 6.1% ( 24/392 , 95% confidence interval [ ci ] 4.0%9.0% ) . all rickettsia - positive tick extractions were subsequently screened by pcr for sfg rickettsia by using primers for the rompa gene of sfg - rickettsia ( 9 ) . the prevalence of sfg rickettsia infection was 3.8% ( 15/392 , 95% ci 2.2%6.2% ) . single strand conformational polymorphism ( sscp ) banding patterns were identical for all tick - derived rompa pcr amplicons . similarly , sscp banding patterns of the tick - derived citrate synthase amplicons for the sfg - rickettsia  positive samples were monomorphic . these results suggest that these tick infections represent a single sfg rickettsia taxon ( 13 ) . citrate synthase and rompa pcr products from three ticks were sequenced with the citrate synthase and shortened rompa pcr primers , respectively . sequences of each respective gene fragment derived from these ticks were identical and confirm the sscp findings ( genbank accession no . the derived sequences were also compared to rickettsiae sequences in the public domain and were identical to those derived from r. montanensis from d. andersoni ( genbank accession no . prevalence estimates were reported as percentages with exact 95% ci based on the binomial distribution . fisher exact test was used to compare infection prevalence across the strata of selected characteristics . the association between each characteristic and the prevalence of infection was quantified as odds ratios ( or ) , calculated with logistic regression or exact methods for categorical data when the data were highly unbalanced . all statistical analyses were performed with stata ( version 7.0 ; stata corporation , college station , tx ) or statxact ( version 5.0.3 ; cytel software corporation , cambridge , ma ) . the variation in prevalence of rickettsia - positive ticks across all counties was marginally significant ( p = 0.052 ) , with a higher prevalence in st . mary 's county compared to all other counties ( or 5.1 , 95% ci 0.527.2 , p value = 0.08 ) . in contrast to the equivocal results for the geographic distribution of rickettsia - positive ticks , temporal heterogeneity was evident , as the prevalence of rickettsia - positive ticks varied significantly with month of collection ( p = 0.007 ) . risk for infection was significantly elevated for any rickettsia organism in ticks collected in july or august ( or 4.1 , 95% ci 1.511.5 ) compared to those collected in april . further analyses combining the data from the spring and early summer months showed that the risk for infection with any rickettsia organism in july or august was even higher ( or 4.7 , 95% ci 2.011.3 ) . the risk for infection with r. montanensis with the late summer months , compared to the spring and early summer months , was somewhat less but still approached statistical significance ( or 3.0 , 95% ci 0.810.2 , p value = 0.06 ) . this observation may be an artifact of diminishing tick abundance later in the summer months . the prevalence of sfg rickettsia in d. variabilis estimated from this study ( 3.8% ) was lower than that in previous reports from maryland . however , in regions where rmsf is observed annually , prevalence estimates range widely , from 2% in connecticut to 10% in alabama , with intermediate prevalences in new york , kentucky , tennessee , and arkansas ( 5 ) . in addition , r. montanensis had not been previously recognized in maryland . most earlier studies of sfg rickettsia infection prevalence did not identify the rickettsia to the species level , although the sfg - positive samples were sometimes assumed to represent r. rickettsii . one study in maryland in which 26 rickettsia isolates were obtained from d. variabilis determined the species composition of the rickettsiae . two isolates were r. rickettsii , 1 isolate was r. bellii ( non - sfg ) , and 23 ( 88% ) were identified as wb-8 - 2 , a then - unnamed sfg - rickettsia ( 5 ) . weller et al . performed a phylogenetic analysis and found wb-8 - 2 ( " r . amblyommii " ) to be closely related to r. montanensis ( 14 ) , although they can be differentiated by serotyping . r. montanensis has been isolated from ticks in other eastern states . during the 1980s , feng et al . reported that r. montanensis represented 41 ( 91% ) of 45 of the sfg isolates from d. variabilis collected in cape cod , massachusetts ( 7 ) . anderson et al . reported isolation of r. montanensis from d. variabilis in connecticut ( 6 ) , and in 1990 , pretzman et al . reported that most sfg rickettsia isolated from dermacentor ticks throughout ohio was r. montanensis ( 3 ) . further , these researchers noted that r. rickettsii were not isolated from ticks collected in several ohio counties where rmsf was considered endemic . these studies illustrate that the rickettsial composition and dynamics within the rmsf - endemic areas are complex and need to be addressed with greater scrutiny . the role of sfg rickettsia in human health is largely unknown , and many are considered to be nonpathogenic either because the bacteria have not been isolated from humans or they do not demonstrate pathogenicity in animal models . for example , r. montanensis is avirulent in guinea pigs but virulent in voles ( 15 ) . r. montanensis and other " nonpathogenic " sfg rickettsia  infected ticks may also benefit human health by decreasing r. rickettsii in tick populations as a result of the " interference " phenomenon ( 15 ) . the findings of this study and others raise important questions . in 2000 , a total of 495 cases of rmsf were reported to cdc and 4 deaths were attributed to spotted fever caused by rickettsia rickettsii . the extent to which r. rickettsii is the agent responsible for reported cases of rmsf should be reevaluated , considering the number of studies completed in rmsf - endemic regions , including this one , that have found non ( u50/ccu319554 ) and niehs training awards ( t32es07141 ) to j.m.a . and k.i.s .