and A.S.V.-S.; Methodology, N.A., J.V.S., C.E.C., A.-M.L.R., A.E.C., G.F.F. for optimal signal-to-noise ratio and low coefficient of variation% values. We used a guinea pig model as it is a part of our overall research efforts to understand the immunological and clinical response to SFGR CITED2 species after tick transmission. Guinea pigs are a useful INT-767 model to study SFR and show clinical signs of SFR, such as fever and eschars. We anticipate that this assay will be easily adapted to other hosts, including humans and other SFGR species. (SFGR) species. There are more than 20 known SFGR species worldwide. the agent of Rocky Mountain spotted fever (RMSF), is the most virulent SFGR species in North America and was long considered the only pathogenic SFGR species in the US. This belief changed in 2004 when the index case of a rickettsiosis caused by was described in a patient from VA, USA . is an emerging pathogenic SFGR species and agent of SFR; it is primarily transmitted via (Gulf Coast tick) in the southern USA . A closely related tick species in the group, in SFR cases from Arizona . In 2010 2010, 364D became the third known tick-borne pathogenic SFGR species in the US; it is transmitted to humans through along the West Coast . Additionally, there are presumably non-pathogenic species in the SFGR, most notably (lone star tick) [7,8]. Seroepidemiologic studies consistently show exposure to SFGR species among a significant percentage of the US population. The largest serological survey of rickettsial contamination in children living in the southeastern and south-central INT-767 US found that 12% of children tested were seropositive; that is, they had rickettsial antibody titers of at least 64 . Another seroepidemiologic study in North Carolina identified antibodies to one or more SFGR species, including system, correctly monitoring exposure to tick-transmitted rickettsiae in guinea pigs is critical for understanding the immunological and clinical response to SFGR. Clinical symptoms of SFR in humans are flu-like, including fever and headache, and often include a characteristic rash or, in the case of and 364D, an eschar at the site of the tick bite [6,19]. Symptoms may mimic other illnesses, especially in the absence INT-767 of dermal lesions or a history of tick bite. Specific detection of SFGR in clinical specimens (e.g., biopsies) by PCR or immunohistochemistry is not always possible and can be challenging. While confirmation of SFR can be made by detecting rickettsial DNA or antigen, or by culture isolation of rickettsiae, the presence of a four-fold increase in antibodies in paired serum samples is also sufficient to diagnose SFR . However, one should note that serodiagnosis of SFR is not as useful in clinical decision-making because detectable antibodies appear late relative to disease development and the need for rapid treatment. Currently, the immunofluorescence antibody assay (IFA) is considered the gold standard for serodiagnosis of SFR. However, the IFA may be subjective, requiring a well-trained professional to interpret the results appropriately, and remains a semi-quantitative, low-throughput diagnostic assay. To overcome the shortcomings of the IFA, we developed an enzyme-linked immunosorbent assay (ELISA) for SFR diagnosis as the ELISA is usually objective, quantitative, has higher throughput, and is more sensitive compared to the IFA. As serologic assays are more useful for epidemiologic studies, an improved assay remains critical to assess population exposure and make informed public health decisions. 2. Results and Discussion Here, we addressed the need for an improved method of identifying SFGR exposure using the guinea pig model for SFR. Through methodically optimizing each parameter in sequence, we developed an ELISA for detecting exposure with a higher level of sensitivity and confidence of positivity than is possible with an IFA. First, through an initial titration, we decided the secondary antibody concentration that would produce an acceptable signal-to-noise ratio (S/N). Other parameters were subsequently optimized, after which a final optimization of the secondary antibody became feasible. We performed the initial secondary antibody titration by preparing a serial dilution of 1 1:500 to 1 1:32,000 on a plate with antigen only and selecting the dilution where an inflection point around the curve of optical density (OD) versus dilution occurred. This value was 1:8000; we used this dilution to optimize other parameters and then decided the.