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... J Osteopath Med 2022; 122(6): 289295 Medical Education Original Article Tyler Hamby*, PhD, Don P. Wilson, MD, Priya Bui, DO, Jonathan Lowery, PhD and Riyaz Basha, PhD Medical student research opportunities: a survey of osteopathic medical schools in the United States https://doi.org/10.1515/jom-2021-0242 Received September 30, 2021; accepted January 27, 2022; published online March 2, 2022 Abstract Context: It is important for colleges of osteopathic medicine (COMs) to provide opportunities for osteopathic medical students (OMSs) to conduct research under the guidance of professional researchers. However, COMs historically lag behind allopathic medical schools in research offerings for medical students. The literature would benefit from a synopsis of research opportunities for OMSs at COMs. Objectives: This study aims to assess the availability of research opportunities currently offered to OMSs and to identify structured research programs (SRPs) to provide data that may help COMs expand such opportunities. Methods: Two online surveys were developed. The General Survey asked about general research opportunities, research requirements, and SRPs, which we define as optional, intramural, and mentored research programs. The follow-up SRP Survey sought to understand the history, funding, and organizational structure of SRPs. Between February and June 2021, the General and SRP *Corresponding author: Tyler Hamby, PhD, Department of Research Operations, Cook Childrens Health Care System, 801 7th Avenue, Fort Worth, TX 76104, USA; and Texas College of Osteopathic Medicine at The University of North Texas Health Science Center, Fort Worth, TX, USA, E-mail: tyler.hamby@cookchildrens.org. https://orcid.org/ 0000-0002-0523-8769 Don P. Wilson, MD, Department of Pediatric Endocrinology and Diabetes, Cook Childrens Health Care System, Fort Worth, TX, USA Priya Bui, DO, Texas College of Osteopathic Medicine at The University of North Texas Health Science Center, Fort Worth, TX, USA Jonathan Lowery, PhD, College of Osteopathic Medicine at Marian University, Indianapolis, IN, USA Riyaz Basha, PhD, Department of Research Operations, Cook Childrens Health Care System, Fort Worth, TX, USA; and Texas College of Osteopathic Medicine at The University of North Texas Health Science Center, Fort Worth, TX, USA Open Access. 2022 Tyler Hamby et al., published by De Gruyter. International License. Surveys were sent to all COMs in the United States. Response data were analyzed descriptively. Results: Responses were received from 32 (84.2%) of 38 COMs. Nearly all COMs offered research symposia, offered third- or fourth-year research elective rotations, and provided some form of funding for OMSs to participate in research. Fourteen (43.8%) COMs had mandatory research requirements. Twenty COMs (62.5%) offered 31 SRPs, and surveys were completed for 25 (80.6%) SRPs. SRPs were founded a median (range) of 7 (143) years prior and accommodated 20 (450) OMSs annually. Among the responding SRPs, 12.0% had external funding, 96.0% required applications, 50.0% interviewed applicants prior to acceptance into the program, 72.0% required OMSs to identify their own mentors, 68.0% offered stipends to OMSs, 28.0% offered course credits, 96.0% had clinical research opportunities, and 68.0% offered research-oriented didactics. In 84.0% of SRPs, OMSs worked predominantly in the summer after OMS-I; for these SRPs, students had 410 weeks of dedicated time for participation in research. Conclusions: Findings from our surveys provide a synopsis of the research opportunities currently provided by COMs in the United States. Our data demonstrated wide variability of research opportunities among COMs. Keywords: colleges of osteopathic medicine; medical education; medical student research; survey study. Several studies have indicated that conducting research during medical school improves students medical writing, ability to critically evaluate the literature, and knowledge of and skills for research processes [1]. The declining proportion of physician-scientists provides another reason to provide medical students with research opportunities [2], and a meta-analysis of three studies showed that research exposure in medical school increased medical students interest in conducting research in their future careers [3]. More specic to colleges of osteopathic medicine (COMs), the adoption of a single accreditation system for all medical This work is licensed under the Creative Commons Attribution 4.0 290 Hamby et al.: Medical student research opportunities at COMs students in the United States, which was implemented in 2020, places osteopathic medical students (OMSs) from COMs in direct competition with medical students from US allopathic medical schools (USMD) for residencies [4]. Because residency programs often consider publications and research experience to be important credentials when selecting residencies, it is more important than ever that COMs provide research opportunities to their OMSs [5]. Indeed, a study of 2016 and 2018 National Resident Matching Program data showed that medical students who matched in their preferred specialties had signicantly more research accomplishments (abstracts, presentations, and publications) on average than did medical students who did not match in their preferred specialties. Unfortunately, the study also showed that COM graduates had signicantly fewer research accomplishments on average than USMD graduates [6]. Opportunities for COMs to encourage student research include funding research expenses, organizing research symposia, adding mandatory research requirements to curricula, or offering third- or fourth-year research rotations. They may also offer dual-degree programs that require research, although these attract only a small proportion of the OMS population due to the heavy time and financial investments involved. Alternatively, COMs may offer structured research programs (SRPs), which we define as optional, extracurricular programs that allow OMSs to conduct research under mentors. Developing SRPs may be challenging for faculty and staff at COMs, owing to COMs relative lack of research infrastructure [4, 7]. Descriptive examples of SRPs may serve as models for individuals who wish to develop an SRP. However, only a few articles have been published that describe COM SRPs [810]. In the present study, we surveyed all US COMs about the research opportunities being provided to OMSs. The purpose of this study is to assess the present state of research opportunities being offered to OMSs. Methods Survey development An online survey (Supplemental Material) was developed by a member of the study team (T.H.) in consultation with five faculty and staff members who served as a focus group. These faculty and staff included two PhDs and two MDs from a COM and a nonprofit, nonacademic, nonteaching childrens hospital. They included an oncologist and chair of a pediatrics department at the COM, an endocrinologist and director of medical education at the hospital, a biomedical sciences associate professor and vice chair for research at the COM, a research director at the hospital, and a research coordinator at the COM. The focus group discussed relevant survey topics and reviewed potential survey items iteratively, which enhanced content validity and face validity. The study consisted of two separate surveys (Supplementary Material): the General Survey and the SRP Survey. Each survey began by defining SRPs as being optional intramural programs that allow OMSs to conduct research under mentors, and it was explained that elective research rotations and ad hoc research should not be included. The General Survey contained items concerning the presence of and contacts for an SRP, and the presence of other mandatory and optional research opportunities for OMSs. The SRP Survey asked respondents to confirm whether the program is an SRP, and it contains items about the SRPs history, funding, and organization. Each COM completed the General Survey once at most, but the SRP Survey was conducted for each SRP listed in responses to the General Survey. The 9-item General Survey and 24-item SRP Survey each included checklist, categorical multiple-choice, and free-response item formats. Typically, the General Survey took less than 10 min to complete, and the SRP Survey took 1015 min to complete. Participants All 43 COMs listed in the 20202021 American Association of Colleges of Osteopathic Medicine (AACOM) Student Guide to Osteopathic Medical Colleges were surveyed [11]. For the General Survey, contact information was garnered from the websites for COMs; potential respondents were generally contacted in the following sequence: (1) research directors; (2) deans of research; and (3) COM deans. For the SRP Survey, potential respondents included those provided by respondents to the General Survey, but other potential respondents associated with the SRP were identied on websites for COMs as needed. Survey completion was voluntary, and no nancial compensation or other incentives were provided for respondents. Data collection The online survey was developed in and administered via REDCap, which housed the responses in a secure database. Links to the REDCap surveys were provided in emails to potential respondents [12]. A single potential respondent was emailed up to 3 times over 3 weeks for each survey. If no response was received after 4 weeks, this process was repeated with another potential respondent twice more for each General Survey and once more for each SRP Survey. General Surveys were emailed between February and May 2021, and SRP Surveys were emailed between April and June 2021. After the General Survey was completed, the COMs SRP Survey(s) was emailed simultaneously with the next set of initial emails to potential respondents beginning in April. Each survey could be taken only once and was closed after completion. Data collection was complete in June 2021. Attempts were made to clarify missing or ambiguous responses via email. Data analysis For COMs with multiple affiliated campuses (e.g., A.T. Still University COM, Edward Via COM), each campus was sent the General Survey and then the responses were compared. If the responses were similar across campuses, they were combined into one COM; otherwise, each campus was reported separately. When estimates were given for the 291 Hamby et al.: Medical student research opportunities at COMs year that the SRP was developed (e.g., before 2003, prior to 2015), the most recent year was utilized (e.g., 2003, 2015). When ranges were provided for the average number of students in the SRP per year (e.g., 3040 students), the average value was utilized (e.g., 35). Responses to the quantitative items were described with frequencies and percentages for categorical variables; medians and ranges were utilized for numerical variables because they were skewed and non-normally distributed. Relationships between numerical variables were examined with Pearsons correlation. Data were analyzed in SAS Enterprise (version 6.1; SAS Institute Inc, Cary, NC). Graphs were developed utilizing RStudio (RStudio, Inc, Boston, MA). This study was approved by the Cook Childrens Health Care System Institutional Review Board (IRB) as exempt and completing the surveys implied consent. Results Table : Results of general survey (n=). Item Research day or symposium Funding: travel for presentations Funding: printing posters Funding: research projects Funding: publication costs Elective research rotation Mandatory research requirements Structured research program(s) There was a total of 43 COMs surveyed: 27 single-campus COMs; three COMs with two campuses; two COMs with three campuses; and one COM with four campuses. Two multi-campus COMs were identified as providing similar research opportunities across campuses, and responses were combined for both COMs. Of the remaining 38 COMs, 32 (84.2%) responses were received. Table 1 summarizes the results of the General Survey. All COMs reported having a designated research day or symposium in which OMSs may present research. Most COMs provided OMSs with funding for travel for presentations (29 [90.6%]), costs for printing posters (29 [90.6%]), research projects (24 [75.0%]), and publication costs (22 [68.8%]). Almost all (31 [96.9%]) COMs, provided funding for one or more of the above. Thirty-one (96.9%) COMs offered elective research rotations in OMS-III and/or OMS-IV. Only 14 (43.8%) COMS had mandatory research requirements for OMSs. Twentythree (71.9%) COMs reported having a total of 41 SRPs. However, 10 (24.4%) SRPs were excluded from further analysis: one was a dual-degree program; one was an elective research rotation; and eight were later verified not to be SRPs by respondents. After these exclusions, 20 (62.5%) COMs reported having 31 SRPs: 13 (65.0%) COMs had one SRP; 4 (20.0%) COMS had two SRPs; 2 (10.0%) COMs had three SRPs; and 1 (5.0%) COM had four SRPs. SRP survey Responses were received for 25 (80.6%) of the 31 SRPs reported. Table 2 summarizes the results of the SRP Survey. (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) Table : Results of structured research program survey (n=). Item General Survey n (%) External funding Require student applications Require student interviews Students select mentors Course credit Stipends or nancial support for some or all students Research option: clinical research Research option: public health, health services, and/or epidemiology Research option: basic or translational science Research option: ethics, humanities, and/or social science Didactic lectures on research Students work predominantly in summer after OMS I n (%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) These programs were founded a median (range) of 7 (143) years prior and all were ongoing. These SRPs have existed for a median (range) of 20.7% (4.493.5%) of their respective COMs existence [11]. There was a strong, signicant correlation between the years since the COMs and the SRPs were founded (Figure 1). Only 3 (12.0%) SRPs from two COMs were supported by external funding. For OMSs to participate, they were required to submit applications for 24 (96.0%) SRPs and be interviewed for 12 (50.0%) SRPs. Students selected their own mentors for 18 (72.0%) SRPs and were assigned their mentors in 7 (28.0%) SRPs. A median (range) of 20 (450) OMSs participated in the SRPs each year. Taken as proportions of 2019 OMS-I enrollment [13], these programs accommodated a median (range) of 7.6% (1.321.1%) of students enrolled per class. There was a negligible correlation between the numbers of participants in SRPs and enrollment in the corresponding 292 Hamby et al.: Medical student research opportunities at COMs Figure 1: Scatterplot of association between years since the colleges of osteopathic medicine and structured research programs were founded. COM, college of osteopathic medicine; SRP, structured research program; R, Pearsons correlation; p, p-value. Figure 2: Scatterplot of association between enrollments at colleges of osteopathic medicine and in structured research programs each year. COM, college of osteopathic medicine; SRP, structured research program; R, Pearsons correlation; p, p-value. COMs (Figure 2). Course credit was provided for only 7 (28.0%) SRPs. Eight (32.0%) SRPs did not provide stipends or nancial support to students; the others provided funding for some (4 [16.0%]) or all (13 [52.0%]) students. The research opportunities offered included clinical research (24 [96.0%]); public health, health services, and/or epidemiology (22 [88.0%]); basic or translational science (21 [84.0%]); and ethics, humanities, and/or social sciences (15 [60.0%]). Seventeen (68.0%) programs provided didactic lectures on research. For 21 (84.0%) SRPs, OMSs did most of their work in the summer after OMS-I, although these programs typically had requirements beyond the summer. Seventeen (68.0%) SRPs required that OMSs dedicate a median (range) of 7 (410) weeks to research in the summer: 4 weeks (1 [4.0%]), 6 weeks (7 [28.0%]), 7 weeks (1 [4.0%]), 8 weeks (6 [24.0%]), and 10 weeks (2 [8.0%]). One (4.0%) SRP permitted up to 240 h of work to be completed by the end of OMS-II, and 2 (8.0%) SRPs required that OMSs dedicate a specied number of hours (80 and 200) to research between the summer and a specied date in the following winter. Two (8.0%) SRPs had requirements for each year of medical school but specied neither the numbers of weeks dedicated in summer nor hours required. For 4 (16.0%) SRPs, OMSs predominantly worked outside of the summer between OMS-I and OMS-II. One (4.0%) SRP required that OMSs dedicate 3 months to research any time from the summer after OMS-I to graduation, 1 (4.0%) SRP required that OMSs dedicate 35 hours per week to research throughout OMS-II, and 2 (8.0%) SRPs required that OMSs take a fth year of training for research. Discussion The present study provides, to our knowledge, the first overview of research opportunities being offered to OMSs at COMs across the United States; and with response rates >80% for both the General and SRP Surveys, the results are likely representative. The data on structural characteristics of the SRPs may prove to be useful to faculty and staff at COMs who seek to develop an SRP. Administrators may select the SRP characteristics that fit best with their COMs administrative capacity, faculty, and student curricula. In general, the results show that US COMs provided a variety of research opportunities for OMSs. All COMs sponsored annual research symposia to allow students to present their research findings, and nearly all COMs offered elective research rotations during OMSs clerkship years. Although the numbers differed by category, nearly all COMs funded OMSs for at least one research activity. The proportion of COMs with mandatory research requirements was comparable to that reported in 2017 for USMDs (43.8 vs. 44.2%) [14]. However, survey items were not included to determine what COMs required students to do, and the free-text descriptions of these requirements Hamby et al.: Medical student research opportunities at COMs showed great variability between COMs. There are benets of requiring all OMSs to conduct research. For example, some OMSs, who may not have opted for voluntary research experiences, may discover that they enjoy research. Additionally, all OMSs can benet from learning those research skills (e.g., literature review, hypothesis generation) that have applications to clinical practice, and research experiences improve OMSs resumes for residencies [15]. However, a survey study of OMS-I students at four COMs suggests that not all OMSs (177/328 [54.0%]) are interested in research [16], and medical students have been shown to report greater satisfaction with voluntary, compared to mandatory, research programs [17]. Further, requiring disinterested OMSs to conduct research under faculty mentorship is a drain on nancial resources and faculty members time [15]. Among the numerous options for voluntary research programs, dual-degree PhD programs provide the most rigorous training for prospective clinician-scientists. Recent data show that only 18.6% of COMs [18], compared with 71.0% of USMDs [19], offer dual-degree PhD programs. In light of the obstacles that COMs face in providing research opportunities to OMSs [4, 7], voluntary SRPs may be more appropriate than either curricular research requirements or PhD programs. Encouragingly, most COMs offered at least one SRP. The finding that 33.3% of SRPs were developed in the prior 2 years suggests a recent push by COMs to provide OMSs with research opportunities. Still, most SRPs had been active for 5 years, which speaks to these programs sustainability. Critically, 90% of COMs with SRPs supported these programs without extramural funding. Most SRPs provided some or all OMSs with stipends, and some programs offered course credit. Although a survey of OMS-I students at four COMs showed both monetary compensation (213/328 [64.9%]) and extra credit in courses (195/328 [59.5%]) to be strong incentives for OMSs to participate in research, the same study also showed that a slim majority of respondents were either currently doing research or planning to do research while in medical school [16]. In the present study, seven SRPs provided neither incentive, including the three SRPs with the highest proportions of participating students relative to total enrollment. Still, in unreported analyses, neither the number of SRP participants nor the SRP student participation as a proportion of annual enrollment statistically signicantly differed based on whether either incentive was offered. The effects of nancial and course credit incentives on participation in SRPs should be empirically examined. If students are willing to participate in SRPs without incentives, those resources may be 293 more efciently utilized to provide administrative and research support for SRPs. Nearly all SRPs required students to apply to participate, but only half interviewed applicants. Most SRPs had students select mentors. Interestingly, of the seven SRPs that assigned mentors to students, only one SRPthe Pediatric Research Program, which some of the authors oversee (T.H., D.P.W., P.B., and R.B.)interviewed applicants. In our experience, interviews provide an opportunity to probe further into students interests and to determine compatibility with potential mentors and projects. Nearly all SRPs offered clinical research options, which 3 prior survey studies have shown to be the most popular type of research for OMSs (65.582.0%) [16, 20, 21]. A majority of SRPs provided research lectures or didactics. Although most SRPs were longitudinal to some extent, the typical SRP had students work primarily in a 4- to 10-week period in the summer after OMS-I. However, some programs lasted 1 year or all 4 years. Although numerous descriptions of SRPs at USMDs and some at COMs have been published, no study has systematically surveyed them for comparison. However, in one study, investigators surveyed all summer SRPs in Canadian medical schools and affiliated institutions; although the response rate was 50.5% and SRPs with only undergraduate students were included, the results may be compared [22]. Canadian summer SRPs had a much larger average number of students per year than COM SRPs (40.04 vs. 20.76). Compared to COMs SRPs, Canadian summer SRPs were more likely to have been active for 5 years (78.3 vs. 58.3%), were less likely to have clinical opportunities (87.0 vs. 96.0%), and were similar in the likelihood of assigning students to mentors (81.8 vs. 72.0%) and providing didactic lessons (61.4 vs. 68.0%). Probably owing to the inclusion of undergraduate students with longer summer breaks, Canadian summer SRPs tended to have a greater duration in weeks than summer COM SRPs. Canadian summer SRPs were far more likely to have external fundingfrom private investigator grants (45.7%), private donations (43.5%), or government support (13.0%)than were COM SRPs (12.0%). Limitations and directions for further research Although the present results provide valuable information about research opportunities for OMSs at US COMs, limitations must be acknowledged. The primary limitation of the present study was that respondents may not have had all information required to complete the survey. A lack of 294 Hamby et al.: Medical student research opportunities at COMs readily available information may have dissuaded some potential respondents from participating. Lack of information and the social desirability bias could potentially have produced inaccurate responses. To address these concerns, survey responses were compared with information from respective COMs websites when available, although survey responses were unaltered when inconsistencies were noted. Overall, inconsistencies between responses and COMs websites were rare. Importantly, respondents were asked not to submit the survey until all information was verified and to forward the survey to more appropriate respondents if applicable; therefore, we believe inaccurate responses to be minimal. The present study offers a broad overview of research programs for OMS at COMs, and the surveys were purposefully brief to achieve a high response rate. Although a high response rate was achieved, the resulting data are limited in depth. Perhaps a lengthier survey conducted under the auspices of the AACOM would produce more detailed data while still retaining a high response rate. The General Survey provided no quantitative data about research symposia, research funding, elective research rotations, and mandatory research requirements beyond their presence or absence. A direction for further research would be to conduct a more detailed survey study focusing on these questions. The SRP Survey provided more detailed quantitative data on SRPs, but it lacked the qualitative data necessary to understand the practical management of SRPs. It is important that more researchers publish their COMs experiences with SRPs. Additionally, it would be useful to conduct a multicenter study that contrasts several SRPs both quantitatively and qualitatively. Such studies provide usable templates for faculty and staff at COMs to develop research opportunities for OMSs. Lastly, it would be interesting to conduct a survey of research opportunities for medical students at all USMDs and COMs to allow for comparisons. Conclusions The present survey study provides a generalizable overview of the research opportunities that US COMs currently offer OMSs. Results demonstrated the variability of research opportunities among COMs. All COMs attempted to provide their OMSs with research opportunities. Most COMs offered SRPs, and there is evidence of a recent push to expand these opportunities. The majority of SRPs took place in the summer after OMS-I. Acknowledgments: This study was conducted as part of the University of North Texas Health Science Center and Cook Childrens Pediatric Research Program. The authors thank the College of Osteopathic Medicine personnel who responded to the surveys and graciously provided the information. Research funding: None reported. Dr. Basha is supported by grants from the National Institute on Minority Health and Health Disparities (#1S21MD012472-01; 2U54 MD006882-06), the National Cancer Institute (#P20CA233355-01), and the Cancer Prevention and Research Institute of Texas (RP170301; RP210046). Author contributions: All authors provided substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; all authors drafted the article or revised it critically for important intellectual content; all authors gave nal approval of the version of the article to be published; and all authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Competing interests: None reported. 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... education sciences Article Addressing Motivations and Barriers to Research Involvement during Medical School among Osteopathic Medical Students in the United States Krista L. Jackson 1,2 , Oladipupo Ogunbekun 1,2 , Benjamin Nick 1,2 , Nicole Griffin 1,2 , Tyler Hamby 3,4 , Jake Herber 1,2 , Julia M. Hum 1,2 , Sarah Zahl 5,6 , Michael Baumann 7 and Jonathan W. Lowery 1,2,8, * 1 2 3 4 5 6 7 8 * Citation: Jackson, K.L.; Ogunbekun, O.; Nick, B.; Griffin, N.; Hamby, T.; Herber, J.; Hum, J.M.; Zahl, S.; Baumann, M.; Lowery, J.W. Addressing Motivations and Barriers to Research Involvement during Medical School among Osteopathic Medical Students in the United States. Educ. Sci. 2022, 12, 407. https:// doi.org/10.3390/educsci12060407 Academic Editors: Maria Alessandra Sotgiu and Bernard John Moxham Received: 25 April 2022 Accepted: 10 June 2022 Published: 15 June 2022 Publishers Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Division of Biomedical Science, Marian University College of Osteopathic Medicine, Indianapolis, IN 46222, USA; kjackson466@marian.edu (K.L.J.); oogunbekun797@marian.edu (O.O.); bnick087@marian.edu (B.N.); ngriffin860@marian.edu (N.G.); jherber626@marian.edu (J.H.); jmhum@marian.edu (J.M.H.) Bone & Muscle Research Group, Marian University, Indianapolis, IN 46222, USA Texas College of Osteopathic Medicine, University of North Texas Health Science Center, Fort Worth, TX 76107, USA; tyler.hamby@cookchildrens.org Department of Research Operations, Cook Childrens Health Care System, Fort Worth, TX 76104, USA Division of Clinical Affairs, Marian University College of Osteopathic Medicine, Indianapolis, IN 46222, USA; szahl@marian.edu Office of the Dean, Marian University College of Osteopathic Medicine, Indianapolis, IN 46222, USA Department of Communication, College of Arts & Sciences, Marian University, Indianapolis, IN 46222, USA; mbaumann@marian.edu Office of the Provost, Marian University, Indianapolis, IN 46222, USA Correspondence: jlowery@marian.edu Abstract: Involvement in research is regarded as a high-impact educational practice, which, for medical professionals, is associated with sharpened critical thinking and life-long learning skills, greater appreciation for evidence-based medicine, and better clinical competence scores. However, there are limited data regarding the research experience and/or interest among osteopathic medical students in the United States despite a rapidly increasing enrollment and expansion of the number of osteopathic medical schools. Thus, we administered an electronic survey examining prior research experience, interests, and perceptions about research participation during medical school to four successive classes of incoming first-year osteopathic medical students. We also performed focus groups with rising third-year osteopathic medical students around the topic of perceived barriers to and potential enablers of promoting research participation. This yielded a survey addendum where first-year osteopathic medical students provided feedback on the likelihood of specific incentives/enablers to encourage participation in research during medical school. Overall, osteopathic medical students are interested in research, view research experience as valuable, and perceive research experience as beneficial to future career development. Students perceive that the primary barrier to involvement in research is a potential negative impact upon performance in coursework. Feedback on the likelihood of specific enablers/incentives was also garnered. Our findings from a single institution may have important implications in defining the prior experiences and perceptions held by first-year osteopathic medical students. Specifically, our study indicates that research experiences intentionally designed with (1) a strong likelihood of gaining a publication, (2) financial compensation, and (3) the opportunity for short-term involvement, a flexible time commitment, and/or a dedicated time period are most likely to encourage research participation by osteopathic medical students. Copyright: 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article Keywords: undergraduate medical education; osteopathic medical school; research; students; survey; focus groups; barrier; enabler; incentive distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Educ. Sci. 2022, 12, 407. https://doi.org/10.3390/educsci12060407 https://www.mdpi.com/journal/education Educ. Sci. 2022, 12, 407 2 of 12 1. Introduction Involvement in research is regarded as a high-impact educational practice. For medical professionals, it is associated with sharpened critical thinking and life-long learning skills [14], greater appreciation for evidence-based medicine [5], and better clinical competence scores [6]. Additionally, medical students engaging in original research may obtain advantages in their professional pathways through stronger academic portfolios for United States (US) residency programs [7]especially for more competitive programsand stronger performance in required research projects during residency. Consistent with this, the accrediting body for colleges of osteopathic medicine (COMs) in the United States requires COMs to provide instruction in the basic scientific principles of research and support research involvement among students [8]. However, according to 2020 National Residency Match Program data, while 80% of allopathic medical students self-reported a research experience leading to a demonstrable product (e.g., publication, abstract, etc.), only 59% of osteopathic medical students do so [9]. This is notable because research by us and others indicates that the majority of osteopathic medical students express interest in clinical research [1013]. Thus, it is important to identify osteopathic medical students motivations for participating in research as well as the real and/or perceived barriers preventing participation in research during osteopathic medical school. In the present study, we administered an electronic survey examining prior research experience, interests, and perceptions about research participation during medical school to four successive classes of incoming first-year osteopathic medical students. We also performed focus groups with rising third-year osteopathic medical students around the topic of perceived barriers to and potential enablers for promoting research participation. This yielded a survey addendum where first-year osteopathic medical students provided feedback on the likelihood of specific incentives/enablers to encourage participation in research during medical school. 2. Methods 2.1. Main Survey For our main survey, we used a descriptive survey study design to investigate the previous research experiences and current perceptions of research among first-year osteopathic medical students at Marian University College of Osteopathic Medicine (MU-COM) in Indianapolis, Indiana. The main survey instrument (Supplemental File S1), which was used in our prior publication [10], consisted of fifteen items: fourteen multiple-choice questions, with select questions allowing for participants to write in responses, and one question asking participants to input their age. Osteopathic medical students in the classes of 2022, 2023, 2024, and 2025 who had entered medical school three weeks earlier, were sent an email inviting them to voluntarily participate in an anonymous survey on research interests. The invitation provided basic information about the study as well as a statement indicating that submission of the survey by a student constituted informed consent. A hyperlink to the survey was provided at the end of the e-mail invitation. The survey was administered by the online survey service Qualtrics, which allowed for anonymous data collection and concealment of the participants identities. The survey remained open for two weeks, after which the data were recorded. One reminder e-mail was sent on the last day of the survey. Incomplete responses were excluded from the analysis. 2.2. Focus Groups Five semi-structured focus groups (one-hour duration each) were conducted with two cohorts of rising third-year students using open-ended discussion prompts in order to provide a convenience sampling of student perceptions. Third-year students were sent an email inviting them to voluntarily participate in focus groups along with basic information about the study and a hyperlink to a sign-up page. Participation was incentivized by providing lunch for participants and a raffle for one US$25 gift card per session. The Educ. Sci. 2022, 12, 407 3 of 12 focus groups were facilitated by pairs of second-year osteopathic medical students (KJJ and OO for the Class of 2021, BN and NG for the Class of 2023) who had been trained on best practices and strategies for focus group facilitation by SZ. Focus groups were audio-recorded and transcribed by a third-party vendor (Rev). Participant identity was protected by redaction and any statements that included student-specific information were de-identified prior to analysis. Transcripts were coded and analyzed for themes by the research team. 2.3. Survey Addendum A one-question electronic survey addendum (Supplemental File S2) was generated for the class of 2025 and seamlessly administered at the end of the main survey. This contained a list of fifteen specific incentives/enablers aimed at encouraging participation in research during medical school. Respondents ranked items using a Likert scale from 15 as follows: (1) definitely will not; (2) probably will not; (3) might or might not; (4) probably will; (5) definitely will. 2.4. Statistical Analyses Data were summarized in aggregate form by class and overall average. Where indicated in the text or figure legend, some items were analyzed by unpaired t test or linear regression. 2.5. Regulatory Compliance This study was approved by the Institutional Review Board of Marian University (protocols S17.018 and S18.060). 3. Results 3.1. Participant Demographics In this study, a total of 272 students participated from MU-COM (Table 1). Of the 272 students that responded, 56 (20.6%) were from the Class of 2022, 84 (30.9%) were from the Class of 2023, 58 (21.3%) were from the Class of 2024, and 74 (27.2%) were from the Class of 2025. The overall response rate was 45% (272 out of 604) while the response rate for each Class ranged from 37% to 56% (Table 1). The age of all students ranged from 21 to 34 years (Table 1). As shown in Table 2, for the majority of students (overall average: 71% per class; range: 60.883.9%), a baccalaureate degree was the highest degree earned with basic science the most prevalent field of study (overall average: 79% per class; range: 74.182.1%); seventy-eight students (27.8%) had a graduate or professional degree. One student reported a graduate certificate in lieu of a baccalaureate degree (Table 2). No students reported holding a PhD. As indicated in Table 3, the majority of students across all four incoming classes reported participation in research before entering osteopathic medical school (overall average: 79.9% per class; range: 73.286.9%). Most of those with research experience (overall average: 62.7% per class; range: 57.465.5%) had published or presented their research findings (Table 3). Table 1. Demographics of survey respondents. For respondents, percentage refers to the percentage of total respondents. For response rate, percentage refers to overall or each class year as indicated. Total Respondents Response Rate Age Range, years Overall Class of 2022 No. (%) Class of 2023 No. (%) Class of 2024 No. (%) Class of 2025 No. (%) 272 56 (20.6) 84 (30.9) 58 (21.3) 74 (27.2) 45% 37% 56% 39% 48% 2134 2233 2134 2129 2233 Educ. Sci. 2022, 12, 407 4 of 12 Table 2. Demographics of survey respondents. Percentages refer to within each column grouping with the exception of respondents where referring to the percentage of the average grouping. Field of Bachelors Degree Applied science Basic science Social science Liberal Arts Other Highest Degree Earned Graduate Certificate Bachelors Masters PhD Professional Average Class of 2022 No. (%) Class of 2023 No. (%) Class of 2024 No. (%) Class of 2025 No. (%) 3.4% 79% 4.8% 2.9% 9.6% 0 (0) 46 (82.1) 4 (7.1) 1 (1.8) 5 (8.9) 3 (3.6) 66 (78.6) 1 (1.2) 4 (4.8) 9 (10.7) 1 (1.7) 43 (74.1) 4 (6.9) 3 (5.2) 7 (12.1) 6 (8.1) 60 (81.1) 3 (4.1) 0 (0) 5 (6.8) 0.3% 71% 27.8% 0% 1% 0 (0) 47 (83.9) 9 (16.1) 0 (0) 0 (0) 1 (1.2) 56 (66.7) 26 (31) 0 (0) 1 (1.2) 0 (0) 42 (72.4) 16 (27.6) 0 (0) 0 (0) 0 (0) 45 (60.8) 27 (36.5) 0 (0) 2 (2.7) Table 3. Previous research experience among survey respondents. Percentages refer to within each column grouping. Previous Research Experience Yes Published or presented Not published or presented No Average Class of 2022 No. (%) Class of 2023 No. (%) Class of 2024 No. (%) Class of 2025 No. (%) 79.9% 41 (73.2) 73 (86.9) 47 (81) 58 (78.4) 62.7% 26 (63.4) 47 (64.4) 27 (57.4) 38 (65.5) 31.1% 13 (31.7) 23 (31.5) 15 (31.9) 17 (29.3) 20.1% 15 (26.8) 11 (13.1) 11 (19) 16 (21.6) 3.2. Research Interest and Perception of Opportunities When asked if they were interested in participating in research during medical school, a majority of students (overall average: 70.6% per class; range: 53.582.8%) either expressed interest in or were currently doing research (Table 4). Just over a quarter of entering medical students (n = 70, 26.2%) also indicated that they might be interested in participating in research (Table 4). Nine students (overall: 3.2% per class; range: 05.4%) indicated no interest in research (Table 4). We subsequently surveyed perceptions regarding research opportunities available during medical school (Table 4). Over half (overall: 53.2%; range: 5058.3%) identified that there were some opportunities available at MU-COM. 26.3% (range: 22.428.6%) upgraded the extent of available opportunities to many. 20.1% (range: 14.325.9%) stated that they dont know if research opportunities exist at MU-COM. One student (overall: 0.34%) perceived that there were no research opportunities available at MU-COM during medical school. We expected that the majority of osteopathic medical students would be interested in clinical research; this was supported by the finding that an average of 88.2% of students per class (range: 85.793.2%) affirmed interest in clinical research (Table 5). As indicated in Table 5, this was followed by interest in basic science (overall average: 61.2% per class; range: 55.465.5%), anatomical research (overall average: 43.9% per class; range: 35.754.1%), social science (overall average: 38.7% per class; range: 30.444.8%), osteopathic manipulative medicine (overall average: 29.6% per class; range 25.733.9%), translational research (overall average: 17.8% per class; range: 8.927.4%), applied science (overall average: 9.7% per class; range: 7.113.1%), and other (overall average: 1.7% per class; range: Educ. Sci. 2022, 12, 407 5 of 12 03.6%). Regarding other, students listed genetic engineering, nutrition, orthopedics, neuro, and case studies as their research interests. Table 4. Interest in participating in research during osteopathic medical school and perception of research opportunities among survey respondents. For the type of research, participants were allowed to choose one or more factors. Percentages refer to within each column grouping. Interested in Participating in Research Yes Maybe Currently participating No/No Response Do opportunities exist at MU-COM for students to participate in research during medical school? Yes, many Yes, some No Dont know Average Class of 2022 No. (%) Class of 2023 No. (%) Class of 2024 No. (%) Class of 2025 No. (%) 66.7% 26.2% 3.9% 3.2% 25 (44.6) 23 (41.1) 5 (8.9) 3 (5.4) 58 (69) 21 (25) 1 (1.2) 4 (4.8) 48 (82.8) 10 (17.2) 0 (0) 0 (0) 52 (70.3) 16 (21.6) 4 (5.4) 2 (2.7) 26.3% 53.2% 0.3% 20.1% 16 (28.6) 28 (50) 0 (0) 12 (21.4) 23 (27.4) 49 (58.3) 0 (0) 12 (14.3) 13 (22.4) 30 (51.7) 0 (0) 15 (25.9) 20 (27) 39 (52.7) 1 (1.4) 14 (18.9) Table 5. Types of research interests and perception of research opportunities in areas of interest among survey respondents. For the type of research, participants were allowed to choose one or more factors. Percentages refer to within each column grouping. Type of research interest Clinical Research Basic Science Anatomical Research Social Science Osteopathic Manipulative Medicine Translational Research Applied Science Other Not interested in Research Do research opportunities exist at MU-COM in your area of interest? Yes, many Yes, some No Dont know Average Class of 2022 No. (%) Class of 2023 No. (%) Class of 2024 No. (%) Class of 2025 No. (%) 88.2% 61.2% 43.9% 38.7% 48 (85.7) 34 (60.7) 20 (35.7) 17 (30.4) 72 (85.7) 53 (63.1) 36 (42.9) 34 (40.5) 51 (87.9) 38 (65.5) 25 (43.1) 26 (44.8) 69(93.2) 41 (55.4) 40 (54.1) 29 (39.2) 29.6% 19 (33.9) 22 (26.2) 19 (32.8) 19 (25.7) 17.8% 9.7% 1.7% 2.6% 5 (8.9) 4 (7.1) 0 (0) 3 (5.4) 23 (27.4) 11 (13.1) 3 (3.6) 2 (2.4) 10 (17.2) 6 (10.3) 1 (1.7) 0 (0) 13 (17.6) 6 (8.1) 1 (1.4) 2 (2.7) 9.2% 35.4% 3.9% 51.5% 3 (5.4) 23 (41.1) 3 (5.4) 27 (48.2) 12 (14.6) 34 (41.5) 1 (1.2) 35 (42.7) 5 (8.6) 21 (36.2) 2 (3.5) 30 (51.7) 6 (8.1) 17 (23) 4 (5.4) 47 (63.5) A query regarding the availability of research opportunities at MU-COM in respondents areas of interest during medical school yielded a significant shift in responses (Table 5). Approximately half of all respondents (overall: 51.5%; range: 42.763.5%) indicated that they dont know if such opportunities exist. 35.4% (range: 2341.1%) believed there to be some research opportunities available at MU-COM in their area of interest. Only 9.2% (range: 5.414.6%) felt there were many such opportunities available. Meanwhile, 3.9% (range: 1.25.4%) perceived there to be no research opportunities in their area of interest available at MU-COM during medical school. Educ. Sci. 2022, 12, 407 6 of 12 3.3. Perceived Importance and Benefits of Research Participation Osteopathic medical students were also queried as to the importance and benefits of participating in research while in osteopathic medical school (Table 6). An overwhelming majority of students (overall average: 97.3% per class; range: 96.498.8%) indicated some level of importance and 50.2% indicated that research participation was very or extremely important (range average: 41.162.1%). Only seven students (2.7% overall) indicated that research participation during medical school was not important. Regarding the benefits of participating in research during medical school (Table 6), a majority of students (overall average: 95.3% per class; range: 9496.6%) believed that research participation would enhance their competitiveness in residency applications. This was followed by an opportunity to interact with current faculty (overall average: 78.9% per class; range: 73.882.8%), to deepen understanding of curricular concepts (overall average: 68% per class; range: 62.271.4%) and to develop skills for conducting research as a physician (overall average: 67.1% per class; range: 60.774.1%). Some students (overall average: 3.5% per class; range: 1.84.8%) reported other benefits, including the following: understanding foundational research concepts & the benefits of research on career/medical field; advancing medical research; helping to answer specific questions; becoming an expert in one area; critical thinking skills; relate to others in the medical field; and further understanding of research & biological processes. Only one respondent indicated that research participation produced no benefit. Table 6. Survey respondents perceptions of the importance and benefits of participating in research during osteopathic medical school. For the perceived benefits, participants were allowed to choose one or more factors. Percentages refer to within each column grouping. Average Class of 2022 No. (%) Class of 2023 No. (%) Class of 2024 No. (%) Class of 2025 No. (%) Importance of participating in research experience Extremely important 18.5% 7 (12.5) 18 (21.4) 13 (22.4) 13 (17.6) Very important 31.7% 16 (28.6) 22 (26.2) 23 (39.7) 24 (32.4) Moderately Important 37.4% 23 (41.1) 35 (41.7) 16 (27.6) 29 (39.2) Slightly important 9.7% 8 (14.3) 8 (9.5) 4 (6.9) 6 (8.1) Not important 2.7% 2 (3.6) 1 (1.2) 2 (3.5) 2 (2.7) Enhancing competitiveness for residency slots 95.3% 53 (94.6) 79 (94) 56 (96.6) 71 (95.9) Engaging with faculty members 78.9% 43 (76.8) 62 (73.8) 48 (82.8) 61 (82.4) Deepening understanding of curricular concepts 68% 40 (71.4) 57 (67.9) 41 (70.7) 46 (62.2) Developing skills for doing research as a physician 67.1% 34 (60.7) 53 (63.1) 43 (74.1) 52 (70.3) Other 3.5% 1 (1.8) 4 (4.8) 2 (3.5) 3 (4.1) No benefit 0.3% 0 (0) 1 (1.2) 0 (0) 0 (0) Benefits of participating in research Educ. Sci. 2022, 12, 407 7 of 12 3.4. Potential Barriers Preventing and Enablers Encouraging Research Participation Osteopathic medical students were also asked to choose one or more factors that might prevent them from participating in research during medical school (Table 7). We expected that their prevailing concern would be a possible negative impact upon performance in coursework; indeed, an overwhelming majority of students (overall average: 86.7% per class; range: 79.791.4%) expressed this concern. Relatively fewer students indicated that a preference for other extracurricular activities (overall average: 26.5% per class; range: 1936.5%) might be a reason to not participate in research. Lack of opportunity for a specific kind of research was a less prevalent concern (overall average: 13% per class; range: 10.814.3%). Some students (overall average: 10.9% per class; range: 8.114.9%) reported additional concerns. A majority of these concerns revolved around the constraints of the time commitment involved in research. Other key concerns included a prior lack of experience, uncertainty on how to get involved, and a general lack of interest in research itself. Intriguingly, two students listed apprehension about the possibility of not gaining a publication from their research efforts as a concern. Table 7. Survey respondents perceptions of the reasons to not participate in and possible enablers to encourage participating in research during osteopathic medical school. Participants were allowed to choose one or more factors. Percentages refer to within each column grouping. Average Class of 2022 No. (%) Class of 2023 No. (%) Class of 2024 No. (%) Class of 2025 No. (%) Concern about academic performance 86.7% 49 (87.5) 74 (88.1) 53 (91.4) 59 (79.7) Prefer other extracurricular activities 26.5% 15 (26.8) 20 (23.8) 11 (19) 27 (36.5) Lack of opportunity for a specific kind of research 13% 8 (14.3) 11 (13.1) 8 (13.8) 8 (10.8) Other 10.9% 6 (10.7) 8 (9.5) 5 (8.1) 11 (14.9) Not applicable 6.2% 5 (8.9) 6 (7.1) 2 (3.5) 4 (5.4) Monetary compensation 81.9% 40 (71.4) 74 (88.1) 48 (82.8) 63 (85.1) Extra credit 61% 39 (69.6) 47 (56) 35 (60.3) 43 (58.1) Specific type of research 39.7% 20 (35.7) 31 (36.9) 28 (48.3) 28 (37.8) Other 9.4% 3 (5.4) 5 (5.6) 5 (8.6) 13 (17.6) Nothing 5.1% 5 (8.9) 3 (3.6) 2 (3.5) 1 (1.4) Reasons to not participate Possible enablers We also surveyed osteopathic medical students about the potential incentives that might encourage them to participate in research during medical school (Table 7). An overwhelming majority indicated a positive perception toward monetary compensation (overall average: 81.9% per class; range: 71.488.1%) and extra credit in coursework (overall average: 61% per class; range: 5669.6%). Some students (overall average: 39.7% per class; range: 35.748.3%) reported that an opportunity for a specific type of research might encourage their participation. Relatively few students (overall average: 5.1%; range: 1.48.9%) reported none of the listed incentives would encourage them to participate in research during medical school. Educ. Sci. 2022, 12, 407 8 of 12 3.5. Focus Groups on Perceived Barriers and Potential Enablers to Research Participation To enhance our understanding of osteopathic medical students perceptions of research, we carried out a series of voluntary focus groups with rising third-year students. This time point in training was an advantageous opportunity for examining how perceptions may be influenced by the conclusion of preclinical coursework prior to initiating clinical clerkships and how those perceptions may have shifted throughout their preclinical experience. Three focus groups and two focus groups (average attendance of seven per session) were held with the Class of 2021 and Class of 2023, respectively. Focus groups with the Class of 2022 were not possible due to restrictions of the COVID-19 pandemic. The focus groups were facilitated by rising second-year students (KLJ, OG, BN, NG) and formatted to be open-ended discussions around the topic of perceived barriers to and potential enablers for promoting research participation by osteopathic medical students. Analysis of the transcripts from these sessions identified several broad themes, some of which are outlined in Table 8. Table 8. Representative themes identified from focus groups with rising third-year osteopathic medical students. Themes Desire for earlier and more intentional connections with faculty to match research interests Interest in educational/instructional overview of research process before getting started Guidance/instruction about integrating research with other activities to help with time management and prioritization of research Mentors style and availability are one of the most important factors to research success A desire for allowing students to shape their own research experience with mentors guidance A strong desire in gaining publication(s) Some students are intrinsically motivated to participate in research while others are not 3.6. Likelihood of Specific Incentives/Enablers to Encourage Research Participation Using the thematic information gleaned from focus groups, we designed a survey addendum for the Class of 2025 to gather feedback on the likelihood of specific incentives/enablers to encourage students to participate in research during medical school. This quantitative approach helped address the potential impact of reflexivity, sample size, convenience sampling, and other confounding variables on the semi-structured focus groups. Respondents were allowed to rate each item on a Likert scale from definitely will not through definitely will. This revealed striking differences between students who affirmed interest or were currently participating versus those who indicated they might be interested in participating in research during medical school (Table 9). For instance, each potential incentive/enabler was scored higher among those students who affirmed interest or were currently participating in research as compared to those who indicated potential interest. Among the former group, the highest-ranked incentive/enabler (mean: 4.64; mode: 5) was strong likelihood of gaining a publication, whereas, among the latter group, this incentive/enabler ranked third (mean: 3.81; mode: 4). For students affirming interest in or currently participating in research, four other items also had a mode of five (Table 9). In contrast, the highest-ranked incentive/enabler (mean: 4; mode: 4) for those who indicated potential interest in participating in research was opportunity for flexible time commitment during research involvement and no item had a mode of five. Educ. Sci. 2022, 12, 407 9 of 12 Table 9. Class of 2025 feedback on the likelihood of certain incentives/enablers to encourage participation in research during medical school. Respondents ranked items using a Likert scale from 15 as follows: (1) definitely will not; (2) probably will not; (3) might or might not; (4) probably will; (5) definitely will. SD, standard deviation. Affirmed Interest or Currently Participating Potential Interest Possible Enablers Mean SD Mode Mean SD Mode Strong likelihood of gaining a publication 4.6 0.6 5 3.8 0.8 4 Opportunity for flexible time commitment during research involvement 4.4 0.7 5 4 0.6 4 Opportunity for short-term involvement in research projects 4.1 0.9 5 3.9 0.7 4 Availability of a dedicated period of time to be involved in research 4.1 0.9 5 3.6 1.0 3 Opportunity for a dual degree program (such as DO/MS, DO/PhD, etc.) 3.2 1.5 5 2.2 1.3 1 Financial compensation 4.3 0.7 4 3.5 0.6 3 A formalized Distinction in Research designation 4.1 0.8 4 3.3 0.9 4 Opportunity for a specific kind of research (certain topic area, clinical, translational, etc.) 4.0 0.8 4 3.4 0.9 3 Availability of a point person for connecting students with available research opportunities 3.9 0.8 4 3 1.0 3 Likelihood of travel opportunities to conferences, meetings, etc. 3.6 1.1 4 3.1 1.0 3 Transcript credit 3.5 1.0 4 3 0.8 3 Improved communication of available research opportunities 3.8 0.9 3 2.9 0.8 3 Improved research facilities and instrumentation 3.5 1.1 3 2.4 0.7 3 Instruction in how to do research 3.5 1.1 3 3.4 0.9 3 Opportunity for funding of student-initiated research projects 3.3 1.0 3 2.4 0.6 2 We were intrigued by the findings with regard to the opportunity for a dual-degree program. As shown in Table 9, this item ranked the lowest among all the items for both students affirming interest or currently participating in research (mean: 3.18) and those indicating potential interest (2.19) but was statistically different between these groups (Figure 1). That said, for the former group, this item had a mode of five (Table 9) and a relatively large standard deviation while it had a mode of one for the latter group (Table 9). Given that some respondents already held graduate degrees, we hypothesized this might influence the perception of a dual-degree program. On average, however, this was not supported by the data as there was no statistically significant difference in mean score between those students with or without graduate degrees (Figure 2). We also performed linear regression to examine if age influenced respondents scoring of the dual-degree program item, however, for both students affirming interest in or currently performing research and those who expressed potential interest, this was not statistically significant 2022, 12, x FOR PEER REVIEW Educ. Sci. 2022, 12, 407 Instruction in how to do research 3.5 1.1 3 3.4 0.9 Opportunity funding of 10 of 12 0.9 Instruction in how for to do research 3.53.3 1.1 33 3.4 1.0 2.4 0.6 student-initiated research projects Opportunity for funding of 3.3 1.0 3 2.4 0.6 student-initiated research projects (affirmed interest or currently participating: R2 = 0.012, F(1.54) = 0.66, p = 0.422; potential interest: R2 = 0.018, F(1.14) = 0.25, p = 0.624). Figure 1. Ranking for dual-degree program among students affirming interest or curren pating in research and those indicating potential interest. Individual responses are in 1. Ranking dual-degree program students affirming interest or currently openFigure circles. Linesfor represent mean +/among standard deviation. * indicates p < participat0.05 by unpair Figure 1. among students affirming interest or curren ingRanking in researchfor and dual-degree those indicating program potential interest. Individual responses are indicated by open circles. Lines represent mean +/ standard deviation. * indicates p < 0.05 by unpaired t test. pating in research and those indicating potential interest. Individual responses are in open circles. Lines represent mean +/ standard deviation. * indicates p < 0.05 by unpaire Figure 2. Ranking for dual-degree program among students with or without a prior graduate degree. Results are separated by students affirming interest or currently participating in research and those Figure 2. Ranking for dual-degree program among students with or without a prior g indicating potential interest. Individual responses are indicated by open circles. Lines represent mean gree.+/Results are separated by students affirming interest or currently participating in re standard deviation. those indicating potential interest. Individual responses are indicated by open circles. L 4. Discussion sent2. mean +/ standard deviation. Figure Ranking for dual-degree program among students with or without a prior gr Our findings have important implications in defining the prior experiences and per- gree. Results areheld separated by students interestThat or currently participating ceptions by first-year osteopathicaffirming medical students. said, we recognize that in re 4. Discussion those indicating potential interest. Individual responses are indicated by open circles. L the generalizability of this study is potentially limited by several factors, including (1) its 45% overall response rate per class, (2) our ability to perform longitudinal studies at sent mean +/ findings standardhave deviation. Our important implications in defining the prior experience ceptions held by first-year osteopathic medical students. That said, we recogni 4. Discussion generalizability of this study is potentially limited by several factors, including overall responsehave rate important per class, (2) our ability in to perform studies a Our findings implications defininglongitudinal the prior experience Educ. Sci. 2022, 12, 407 11 of 12 only one COM, (3) our reliance on self-reported information, and (4) the potential for regional and/or institution-specific factors (such as admissions practices). While these factorswhich are beyond the scope of our current studyand other important considerations such as reflexivity may influence the findings, our results are remarkably consistent with prior results using the same survey instrument at four additional COMs in other regions of the US. [10,11] Thus, we cautiously generalize our findings and interpret them in the context of undergraduate osteopathic medical education overall. This study advances our previous work in which we examined research experience and attitudes toward research among first-year osteopathic medical students across five locations of four COMs. Here, examining four successive cohorts of first-year students, our findings are strikingly similar to our earlier work with the vast majority of students reporting prior research experience (79.9% overall average) and affirming interest in participating in research during medical school (70.6% overall average). The majority of students (88.2% overall average) reported interest in clinical research and/or basic science research (61.2% overall average), which is consistent with our prior report and several additional reports [1013] Additionally, approximately one-third of students (29.6% overall average) reported interest in osteopathic manipulative medicine research, which is also similar to our prior report. [10] An overwhelming majority of students hold the perception that involvement in research during medical school is important (97.3% overall average), with more than half reporting it is very or extremely important. The primary benefits that students perceive to be garnered from involvement in research are as follows: enhanced competitiveness in residency applications (95.3%), opportunity to interact with faculty (78.9%), deepening of curricular concepts (68%), and developing skills for conducting research as a physician (67.1%). Students perceive that the primary barrier to involvement in research is a potential negative impact upon performance in coursework (86.7%) with no other potential barrier being affirmed by a majority of students. To add greater understanding to the perceptions held by students, we carried out a series of focus groups with third-year osteopathic medical students, which identified several broad themes that were utilized for designing a survey addendum to gather feedback on the likelihood of specific incentives/enablers to encourage students to participate in research during medical school. Strikingly, responses to specific incentives/enablers differed greatly between students who affirmed interest or were currently participating in research as compared to those indicating potential interest. Moreover, across all items, the score was consistently higher among those affirming interest or currently participating. To us, this suggests that intrinsic motivation has a strong influence on the perception of additional motivators. In other words, students who are interested in research participation may be further encouraged to do so but students who are uncertain are less likely to be encouraged to participate. For those affirming interest or already participating, the strongest incentive/enabler was a strong likelihood of gaining a publication. That said, both groups of students responded favorably to research experiences with an opportunity for short-term involvement, a flexible time commitment, and/or a dedicated time period. Taken together, these findings are consistent with the primary expressed concern of a potential negative impact upon coursework by (a) limiting the time involved in research while (b) increasing the likelihood of a demonstrable outcome. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/educsci12060407/s1, Supplemental File S1: Main Survey; Supplemental File S2: Survey Addendum. Author Contributions: Conceptualization: T.H., J.M.H., S.Z., M.B. and J.W.L.; Data curation, K.L.J., B.N., J.M.H. and J.W.L.; Formal analysis, K.L.J., O.O., T.H., J.M.H., S.Z., M.B. and J.W.L.; Funding acquisition, S.Z. and J.W.L.; Investigation, K.L.J., O.O., B.N., N.G., T.H., J.H., J.M.H., S.Z., M.B. and J.W.L.; Methodology, T.H., J.M.H., S.Z., M.B. and J.W.L.; Project administration, J.M.H., S.Z. and J.W.L.; Resources, S.Z. and J.W.L.; Supervision, J.M.H., S.Z. and J.W.L.; Visualization, K.L.J., B.N. and J.W.L.; Writingoriginal draft, K.L.J., B.N. and J.W.L.; Writingreview & editing, K.L.J., O.O., B.N., Educ. Sci. 2022, 12, 407 12 of 12 N.G., T.H., J.H., J.M.H., S.Z., M.B. and J.W.L. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the American Association of Colleges of Osteopathic Medicine (issued to S.Z. and J.W.L.) and intramural funds from the Marian University College of Osteopathic Medicine. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of Marian University (protocols S17.018 and S18.060, approved 5/11/2017 and 1/2/2019, respectively). Informed Consent Statement: Informed consent was obtained from all subjects involved in this study. Data Availability Statement: The data associated with this study will be made available upon reasonable request. Acknowledgments: The authors wish to acknowledge helpful feedback from members of the MU Bone & Muscle Research Group. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Alguire, P.C.; Anderson, W.A.; Albrecht, R.R.; Poland, G.A. Resident research in internal medicine training programs. Ann. Intern. Med. 1996, 124, 321328. [CrossRef] [PubMed] Rivera, J.A.; Levine, R.B.; Wright, S.M. Completing a scholarly project during residency training. Perspectives of residents who have been successful. J. Gen. Intern. Med. 2005, 20, 366369. [CrossRef] [PubMed] Hamann, K.L.; Fancher, T.L.; Saint, S.; Henderson, M.C. Clinical research during internal medicine residency: A practical guide. Am. J. Med. 2006, 119, 277283. [CrossRef] [PubMed] Potti, A.; Mariani, P.; Saeed, M.; Smego, R.A., Jr. Residents as researchers: Expectations, requirements, and productivity. Am. J. Med. 2003, 115, 510514. [CrossRef] [PubMed] Smith, M. Research in residency: Do research curricula impact post-residency practice? Fam. Med. 2005, 37, 322327. [PubMed] Kohlwes, R.J.; Shunk, R.L.; Avins, A.; Garber, J.; Bent, S.; Shlipak, M.G. The PRIME curriculum. Clinical research training during residency. J. Gen. Intern. Med. 2006, 21, 506509. [CrossRef] [PubMed] Matthews, C.N.; Estrada, D.C.; George-Weinstein, M.; Claeson, K.M.; Roberts, M.B. Evaluating the Influence of Research on Match Success for Osteopathic and Allopathic Applicants to Residency Programs. J. Am. Osteopath. Assoc. 2019, 119, 588596. [CrossRef] [PubMed] Commission on Osteopathic College Accreditation, American Osteopathic Association. Accreditation of Colleges of Osteopathic Medicine: COM Continuing Accreditation Standards. Available online: https://osteopathic.org/wp-content/uploads/2018/02/ com-continuing-accreditation-standards.pdf (accessed on 12 March 2019). National Resident Matching Program. Charting Outcomes in the Match: Senior Students of U.S. Osteopathic Medical Schools. Available online: https://mk0nrmpcikgb8jxyd19h.kinstacdn.com/wp-content/uploads/2018/06/Charting-Outcomes-in-theMatch-2018-Osteo.pdf (accessed on 7 January 2019). Nguyen, V.; Kaneshiro, K.; Nallamala, H.; Kirby, C.; Cho, T.; Messer, K.; Zahl, S.; Hum, J.; Modrzakowski, M.; Atchley, D.; et al. Assessment of the Research Interests and Perceptions of First-Year Medical Students at 4 Colleges of Osteopathic Medicine. J. Am. Osteopath. Assoc. 2020, 120, 236244. [CrossRef] [PubMed] Carter, J.O.I.; McClellan, N.O.I.; McFaul, D.O.I.; Massey, B.L.O.I.; Guenther, E.; Kisby, G. Assessment of Research Interests of First-Year Osteopathic Medical Students. J. Am. Osteopath. Assoc. 2016, 116, 472478. [CrossRef] [PubMed] Pheley, A.M.; Lois, H.; Strobl, J. Interests in research electives among osteopathic medical students. J. Am. Osteopath. Assoc. 2006, 106, 667670. [PubMed] Licciardone, J.C.; Fulda, K.G.; Smith-Barbaro, P. Rating interest in clinical research among osteopathic medical students. J. Am. Osteopath. Assoc. 2002, 102, 410412. [PubMed]  ...

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... pathogens Article A Murine Model of Waning Scrub Typhus Cross-Protection between Heterologous Strains of Orientia tsutsugamushi Nicole L. Mendell 1 , Guang Xu 2 , Thomas R. Shelite 3 , Donald H. Bouyer 4 1 2 3 4 * Citation: Mendell, N.L.; Xu, G.; Shelite, T.R.; Bouyer, D.H.; Walker, D.H. A Murine Model of Waning Scrub Typhus Cross-Protection between Heterologous Strains of Orientia tsutsugamushi. Pathogens 2022, 11, 512. https://doi.org/ 10.3390/pathogens11050512 Academic Editor: Keun Hwa Lee Received: 2 March 2022 Accepted: 21 April 2022 Published: 26 April 2022 Publishers Note: MDPI stays neutral and David H. Walker 4, * Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USA; nlmendel@utmb.edu College of Osteopathic Medicine, Marian University, Indianapolis, IN 46222, USA; guangxu@marian.edu Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77555, USA; trshelit@utmb.edu Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, Center for Tropical Diseases, Sealy Institute for Vaccine Development, Institute of Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555, USA; dobouyer@utmb.edu Correspondence: dwalker@utmb.edu; Tel.: +409-772-3990; Fax: +409-772-1850 Abstract: Orientia tsutsugamushi, the etiologic agent of the life-threatening febrile disease scrub typhus, is an obligately intracellular small coccobacillary bacterium belonging to the family Rickettsiaceae and is transmitted by the parasitic larval stage of trombiculid mites. Progress towards a vaccine for protection against scrub typhus has been impeded by characteristics of the pathogen and the infection. There are numerous strains of O. tsutsugamushi in the Asia-Pacific region with geographical overlap. In human cases immunity has been described as poor against heterologous strains of the pathogen, as well as short-lived against the homologous strain, with a mean antibody reversion rate of less than one year. Animal models of cross-protection as well as of deterioration of this crossprotection are needed to enhance understanding of transient immunity to scrub typhus. To build upon current understanding of this ineffective protection we sought to utilize our recently developed models, sublethal intradermal infection followed by challenge via ordinarily lethal hematogenous dissemination. Mice that were initially infected sublethally with O. tsutsugamushi Gilliam strain and were challenged with an ordinarily lethal dose of heterologous Karp strain were protected from death by a robust immune response at one month after the primary infection as evidenced by an abundance of mononuclear cellular infiltrates in target organs such as lung, liver, and kidney; maintenance of body weight; and low bacterial loads in the organs. Waning protection from lethal Karp strain challenge indicated by weight loss mirroring that observed in nave mice was observed as early as 9 months after primary Gilliam strain infection, and higher bacterial loads, severe disease, and eventual death in some mice was observed after challenge with Karp strain at 14 months post-initial heterologous infection. Keywords: Orientia; scrub typhus; intradermal; hematogenous model; sublethal; immunity; cross-protection with regard to jurisdictional claims in published maps and institutional affiliations. 1. Introduction Copyright: 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Naturally acquired immunity to scrub typhus in humans was reported to be suboptimal as early as 1917, and reinfection was suggested by 1946 [1,2]. Strain heterogeneity of the causative agent, Orientia tsutsugamushi, was addressed as early as 1939, when human studies utilizing the administration of live Pescadores strain afforded short-term protection against the virulent Niigata strain [3]. A better understanding that heterologous protection was short-lived emerged in 1950 when human volunteers who had previously had scrub typhus, which was unlikely to have been caused by Gilliam strain due to the geographic strain distribution, were inoculated intradermally with O. tsutsugamushi Gilliam strain. Eight volunteers who had scrub typhus within the previous two months did not develop Pathogens 2022, 11, 512. https://doi.org/10.3390/pathogens11050512 https://www.mdpi.com/journal/pathogens Pathogens 2022, 11, 512 2 of 12 disease, whereas 11 out of 16 volunteers in whom 1125 months had elapsed since their prior scrub typhus diagnosis developed disease of similar severity to nave controls [4]. Two volunteers who were challenged with the homologous strain after three years, one natural-route and one needle-challenge, did not develop scrub typhus illness [4,5]. Immunity of human volunteers generated by infection with O. tsutsugamushi Gilliam strain and subsequent treatment after one week with chloramphenicol was shown to be effective to prevent symptomatic infection for up to 14 months against homologous challenge, but 2 of the 13 volunteers had detectable rickettsemia [6]. However, heterologous strain challenge caused mild illness with rickettsemia one month after primary infection, and when challenged after one year all volunteers developed scrub typhus disease [5]. Vaccines for scrub typhus which have been tested in humans and animal models do not stimulate heterologous strain protection, and provide only limited short-lived protection from death, but not protection from illness, upon homologous strain challenge [5,79]. Limited homologous strain protection from illness was observed during operation Tyburn, the attempt in the United Kingdom to propagate a scrub typhus vaccine for extensive field-trials during the Second World War. Accidental laboratory-acquired infection and subsequent illness were reported in laboratory personnel who had received full courses of the formalin inactivated vaccine, which was prepared from cotton rat lung infected with O. tsutsugamushi Karp strain [1012]. This project emphasized the importance of antigenic conformation in protection and implied poor homologous protection by denatured antigens. However, the authors of that report asserted that the absence of lethality in the four vaccinated cases was an improvement from the three fatalities among five unimmunized laboratory infections observed during vaccine propagation. Field trials of the same scrub typhus Karp strain vaccine in present-day Myanmar did not decrease the incidence of scrub typhus [13]. These infections were likely caused by heterologous strains [14,15]. A formalin inactivated rat lungspleen vaccine utilizing O. tsutsugamushi Volner strain (isolated in the Philippines) proved ineffective against heterologous strains during a human field trial in Japan as evidenced by the isolation of 17 strains of O. tsutsugamushi from scrub typhus cases (9 Volner vaccine/11 murine typhus vaccine recipients) during the course of the study [16]. Vaccines that have been tested in animal models for scrub typhus utilizing live, fixed, or replication-deficient O. tsutsugamushi have resulted in variable, time-dependent protection against homologous challenge and inadequate, waning protection against heterologous strains. Complete homologous protection of Balb/c mice immunized with gamma-irradiated O. tsutsugamushi was accomplished, whereas incomplete heterologous protection lasted only six months. Trivalent vaccination containing multiple strains of irradiated O. tsutsugamushi (KarpGilliamKato) elicited longer-term protection that lasted 6 months against Kato and Buie strain challenges and 12 months against Karp and Gilliam strain challenges [1719]. Gerbils whose initial O. tsutsugamushi infection was treated with para-aminobenzoic acid were protected from both homologous and heterologous challenge 6-9 months later [20]. In a guinea pig model, cross-protection was observed after recovery from primary challenge with a heterologous strain of O. tsutsugamushi; however, duration of this immunity was not addressed [21]. Currently, the mechanism of short-lived heterologous immunity is poorly understood. The objective of this study was to establish and characterize a murine model of solid immunity to ordinarily lethal heterologous challenge and subsequent time-dependent waning protection for future use in mechanistic studies. 2. Results To establish a model of heterologous cross-protection between different strains of Orientia tsutsugamushi, mice were initially infected with a sublethal dose of O. tsutsugamushi Gilliam strain. Later, mice were challenged with an ordinarily lethal dose (5 lethal doses 50% (LD50 )) of O. tsutsugamushi Karp strain one, three, nine, 14, and 19 months following the initial Gilliam strain infection and were observed for signs of illness, and samples Pathogens 2022, 11, x FOR PEER REVIEW Pathogens 2022, 11, 512 3 of 13 3 of 12 doses 50% (LD50)) of O. tsutsugamushi Karp strain one, three, nine, 14, and 19 months following the initial Gilliam strain infection and were observed for signs of illness, and samples collected to assess bacterial loads and physiologic responses to infection to evaluate bacterial loads and physiologic responses to infection to evaluate the thecollected durationtoofassess this protective immunity. duration of this protective immunity. 2.1. Loss of Protection and Development of Illness 2.1. Loss of Protection and Development of Illness Mice were protected from illness with no overt signs of illness or decrease in activity Mice were protected from illness with no overt signs of illness or decrease in activity when challenged with an ordinarily lethal dose of O. tsutsugamushi Karp strain one month when challenged with an ordinarily lethal dose of O. tsutsugamushi Karp strain one month after infection with O. tsutsugamushi Gilliam strain. Body weight was maintained throughafter infection with O. tsutsugamushi Gilliam strain. Body weight was maintained throughout the course of infection (Figure 1A). Three months after primary Gilliam strain infecout the course of infection (Figure 1A). Three months after primary Gilliam strain infection, tion, mice challenged with an ordinarily lethal dose of O. tsutsugamushi Karp strain exhibmice challenged with an ordinarily lethal dose of O. tsutsugamushi Karp strain exhibited ited labored breathing and slightly ruffled fur from six to nine days post-infection (dpi) labored breathing and slightly ruffled fur from six to nine days post-infection (dpi) as well as well as progressive weight loss from five to 10 dpi (Figure 1B). as progressive weight loss from five to 10 dpi (Figure 1B). Figure 1. Weight change following challenge with a heterologous strain of Orientia tsutsugamushi. Figure 1. Weight change following challenge with a heterologous strain of Orientia tsutsugamushi. Percent body weight change animals inoculated intravenously with 5 lethal doses 50% (LD Percent body weight change of of animals inoculated intravenously with 5 lethal doses 50% (LD 50)50 of) of O. tsutsugamushi Karp strain at 1 (A), 3 (B), 9 (C), or 14/19 (D) month(s) after initial O. tsutsugamushi O. tsutsugamushi Karp strain at 1 (A), 3 (B), 9 (C), or 14/19 (D) month(s) after initial O. tsutsugamushi Gilliam (open blue circles) sham inoculation (red squares and month(s)/black asterisks Gilliam (open blue circles) oror sham inoculation (red squares 1, 1, 3, 3, 9, 9, and 1414 month(s)/black asterisks 1919 months) asas compared toto sham/sham inoculated (closed black circles) or or O. O. tsutsugamushi Gilliam months) compared sham/sham inoculated (closed black circles) tsutsugamushi Gilliam strain/sham inoculated controls (green triangles). strain/sham inoculated controls (green triangles). When mice were heterologously challenged with ordinarily lethal dose Karp When mice were heterologously challenged with anan ordinarily lethal dose of of Karp strain nine months after primary Gilliam strain infection, they developed significant body strain nine months after primary Gilliam strain infection, they developed significant body weight loss concomitant with overt signs illness including decreased activity, labored weight loss concomitant with overt signs ofof illness including decreased activity, labored breathing, ruffled fur, hunched posture, and erythema beginning dpi and, conjuncbreathing, ruffled fur, hunched posture, and erythema beginning at at sixsix dpi and, conjunctivitis beginning seven dpi (Figure 1C). The mean percent weight change observed tivitis beginning at at seven dpi (Figure 1C). The mean percent weight change observed at at six dpi was 12.58% (SD = 3.99) and the mean nadir of 19.35% (SD = 4.64) on 13 dpi. six dpi was 12.58% (SD = 3.99) and the mean nadir of 19.35% (SD = 4.64) on 13 dpi. Similar signs of illness were observed during this period in nave lethally infected mice Similar signs of illness were observed during this period in nave lethally infected mice with a greater degreeofofdecreased decreasedactivity. activity.The The mean percent weight change nave with a greater degree mean percent weight change forfor nave lethally infected miceononsix sixdpi dpiwas was10.42% 10.42% (SD = 4.77) with the mean nadirofof17.60% 17.60% lethally infected mice (SD = 4.77) with the mean nadir (SD = 4.42) observed eight dpi. While all nave mice succumbed to illness by nine dpi, heterologously challenged mice sustained erythema through 12 dpi, and ruffled fur and hunched posture through 14 dpi, and returned to normal activity by 16 dpi coincident with Pathogens 2022, 11, x FOR PEER REVIEW Pathogens 2022, 11, 512 4 of 13 (SD = 4.42) observed eight dpi. While all nave mice succumbed to illness by nine dpi, heterologously challenged mice sustained erythema through 12 dpi, and ruffled fur4 and of 12 hunched posture through 14 dpi, and returned to normal activity by 16 dpi coincident with onset of weight gain. All heterologously challenged mice survived infection with Karp strain at one, three and nine months post primary infection with Gilliam strain, onset of weight gain. All heterologously challenged mice survived infection with Karp whereas all Gilliam strain nave mice succumbed to infection between eight to nine days strain at one, three and nine months post primary infection with Gilliam strain, whereas post Karp strain infection (Figure 2AC). all Gilliam strain nave mice succumbed to infection between eight to nine days post Karp strain infection (Figure 2AC). Figure 2. 2. Time-dependent Time-dependent susceptibility susceptibility to to challenge challenge with withaaheterologous heterologousstrain strainof ofO. O.tsutsugamushi. tsutsugamushi. Figure Percent survival following sham inoculation (black circle) or inoculation with 5 LD O. 5050 O.tsutsugamushi tsutsugamuPercent survival following sham inoculation (black circle) or inoculation with 5 LD Karp strain 1 (A), 3 (B), 9 (C), 14 (D), or 19 (black asterisk, D) month(s) after primary sham (red (red shi Karp strain 1 (A), 3 (B), 9 (C), 14 (D), or 19 (black asterisk, D) month(s) after primary sham square) or primary infection with O. tsutsugamushi Gilliam strain (blue circle). square) Heterologous challenge months post-Gilliam infection resulted in ruffled fur (10/10) Heterologous challenge1414 months post-Gilliam infection resulted in ruffled fur and conjunctivitis (4/10) in Gilliam strain immunized mice beginning at four dpi, earlier (10/10) and conjunctivitis (4/10) in Gilliam strain immunized mice beginning at four dpi, than onset overt in nave mice, mice, whichwhich was observed at five On five dpi earlier than of onset of illness overt illness in nave was observed at dpi. five dpi. On five signs of illness included erythema andand hunched posture in all mice inoculated with O. dpi signs of illness included erythema hunched posture in all mice inoculated with tsutsugamushi Karp strain, but also labored breathing in heterologous challenged mice as O. tsutsugamushi Karp strain, but also labored breathing in heterologous challenged mice well. AtAt this timepoint, and as well. this timepoint,weight weightloss lossonset onsetwas wasobserved observedby byday day three three post-infection post-infection and the rate of decline matched for both groups infected with O. tsutsugamushi Karp strain the rate of decline matched for both groups infected with O. tsutsugamushi Karp strain and and heterologously challenged that survived challenge had not recovered 21 heterologously challenged micemice that survived challenge had not recovered by dayby21day postpost-infection (Figure 1D). Two (20%) of the heterologously challenged mice succumbed infection (Figure 1D). Two (20%) of the heterologously challenged mice succumbed after after inoculation with O. tsutsugamushi Karp strain 14 months after Gilliam strain infection inoculation with O. tsutsugamushi Karp strain 14 months after Gilliam strain infection (Fig(Figure 2B). ure 2B). All of the mice succumbed (n = 9) to heterologous Karp strain challenge 19 months All of the mice succumbed (n = 9) to heterologous Karp strain challenge 19 months after the initial Gilliam strain infection. Decreased activity accompanied by ruffled fur was after the initial Gilliam strain infection. Decreased activity accompanied by ruffled fur was observed by day four following ordinarily lethal Karp strain challenge, and these observaobserved by day four following ordinarily lethal Karp strain challenge, and these obsertions expanded to include labored breathing, hunched back, erythema, and conjunctivitis vations expanded to include labored breathing, hunched back, erythema, and conjunctiby day five. The rate of weight loss for this group of mice typified lethal challenge of nave vitis by day five. The rate of weight loss for this group of mice typified lethal challenge of mice (Figure 1D). All heterologously challenged mice succumbed to illness between days six and 18 following challenge (Figure 2B). Pathogens 2022, 11, x FOR PEER REVIEW Pathogens 2022, 11, 512 5 of 13 nave mice (Figure 1D). All heterologously challenged mice succumbed to illness between 5 of 12 days six and 18 following challenge (Figure 2B). 2.2. 2.2. Bacterial BacterialDissemination Disseminationin inPreviously Previously Challenged Challenged Mice Mice Tissue-specific bacterial loads indicate disseminated infection to spleen, kidney, Tissue-specific bacterial loads indicate disseminated infection to spleen, kidney, liver, liver, and after lunginfection after infection an ordinarily lethal dose strain of Karp strain3).(Figure 3). and lung with anwith ordinarily lethal dose of Karp (Figure However, However, in the previously Gilliam strain-infected group, the mean bacterial loads in the in the previously Gilliam strain-infected group, the mean bacterial loads in the protected protected were markedly lower compared to the infected naveAt mice. thethree-, one-, mice weremice markedly lower compared to the infected nave mice. the At one-, three-, and nine-month timepoints when animals were completely protected from death, and nine-month timepoints when animals were completely protected from death, tissue tissue bacterial loadthe was the lowest liver (Figure and highest in the lung 3D). (Figure bacterial load was lowest in liverin(Figure 3B) and3B) highest in the lung (Figure An 3D). An average of 1308-fold lowerspleen mean spleen bacterial load was observed mice preaverage of 1308-fold lower mean bacterial load was observed in miceinpreviously viously with Gilliam strainthree-, one-, three-, or months nine- months prior (1,3,9 months-proinfectedinfected with Gilliam strain one-, or nineprior (1,3,9 months-protected) tected) than in infected nave age-matched mice whereas the mean splenic bacterial loads than in infected nave age-matched mice whereas the mean splenic bacterial loads for for mice that succumbed to infection 14 months post-Gilliam strain infection (14-months mice that succumbed to infection 14 months post-Gilliam strain infection (14-months succumbed) 4.83-fold (Figure 3A). TheThe same trends of reduction in bacsuccumbed)was wasonly onlyreduced reduced 4.83-fold (Figure 3A). same trends of reduction in terial loads for for prior Gilliam strain infected mice were bacterial loads prior Gilliam strain infected mice wereobserved observedininthe thekidney kidney(1020-fold (1020-fold 1-,3-,914-months succumbed, Figure 3B), liver (13551-fold 11-,3-,9-months monthsprotected/5.51-fold protected/5.51-fold 14-months succumbed, Figure 3B), liver (13551-fold ,3-,9months protected/20.4-fold 14-months succumbed, Figure 3C), andand lung (313-fold 11-,3-,9months protected/20.4-fold 14-months succumbed, Figure 3C), lung (313-fold ,3-,9months protected/14-fold 14-months succumbed, Figure 3D). Mice that survived het1-,3-,9- months protected/14-fold 14-months succumbed, Figure 3D). Mice that survived erologous O. tsutsugamushi Karp strain challenge after heterologous O. tsutsugamushi Karp strain challenge after1414months monthscleared clearedthe the infection infection more thanmice micethat that survived heterologous challenge months as evimore slowly than survived heterologous challenge afterafter nine nine months as evidenced denced by significantly higher bacterial load (nine-month M56 = 9.72 kDa copies/mg tisby significantly higher bacterial load (nine-month M = 9.72 kDa 56 copies/mg tissue and 14-month M = 181.50, = 0.012) days21after in the in lung, major sue and 14-month M = p181.50, p =21 0.012) daysKarp afterstrain Karp challenge strain challenge the alung, a target target organ organ (Figure 4). major (Figure 4). Figure 3. Orientia tsutsugamushi Karp bacterial burden following challenge. O. tsutsugamushi Karp specific bacterial loads expressed as genome copies (56 kDa gene) per milligram (mg) of tissue of Figure 3. Orientia tsutsugamushi Karp bacterial burden following challenge. O. tsutsugamushi Karp spleen (A), kidney (B), liver (C), and lung (D) after O. tsutsugamushi Karp strain infection in nave specific bacterial loads expressed as genome copies (56 kDa gene) per milligram (mg) of tissue of mice (red = 6, day(B), 79)liver or mice 1 (n = 4),(D) 3 (n = 6),O.9 (n = 3), or 14 months (n = 2,infection day 8, 14)inafter O. spleen (A),nkidney (C), at and lung after tsutsugamushi Karp strain nave tsutsugamushi Gilliam strain infection (blue). *, p < 0.05; **, p < 0.01. mice (red n = 6, day 79) or mice at 1 (n = 4), 3 (n = 6), 9 (n = 3), or 14 months (n = 2, day 8, 14) after O. tsutsugamushi Gilliam strain infection (blue). *, p < 0.05; **, p < 0.01. Pathogens2022, 2022,11, 11,512 x FOR PEER REVIEW Pathogens Pathogens 2022, 11, x FOR PEER REVIEW Figure 4. 6 of 13 6 of 13 6 of 12 Orientia tsutsugamushi Karp bacterial burden following ordinarily lethal challenge. Figure 4. Orientia tsutsugamushi Karp bacterial burden following ordinarily lethal challenge. O. tsuO. 4. tsutsugamushi Karpbacterial specific bacterial loads,following as genome (56milligram kDa Figure Orientia tsutsugamushi Karp loads, bacterial burden challenge. O. tsu-gene) per miltsugamushi Karp specific expressed asexpressed genomeordinarily copies (56lethal kDa copies gene) per tsugamushi Karp specific bacterial loads, expressed as genome copies (56 kDa gene) per milligram ligram (mg)ofofspleen tissue,(A), of kidney spleen(B), (A),liver kidney (B), (D), liverand (C), lung andafter brain 21 days after O. (mg) of tissue, (C), lung brain (E)(D), 21 days O.(E) tsutsuga(mg) of tissue, of spleen (A), kidney (B),atliver (C), (D), and brain (E) 21 days after O. tsutsugamushi Karp strain challenge of challenge mice 9 (nof = mice 3) lung or 14 = 8) O. tsutsugamushi tsutsugamushi Karp strain at months 9 (n = 3)(nor 14after months (n = 8) afterGilliam O. tsutsugamushi mushi Karp strain challenge mice at 9 (n = 3) or 14 months (n = 8) after O. tsutsugamushi Gilliam strain infection (blue). *, pof < 0.05. Gilliam strain infection (blue). *, p < 0.05. strain infection (blue). *, p < 0.05. 2.3. Response to Heterologous Challenge 2.3.Physiologic Physiologic Response to Heterologous Challenge 2.3. Physiologic Response to Heterologous Challenge Hematologic analysis of mice between eight and nine days following heterologous Hematologic analysis ofbetween mice between eight nine days heterologous following heterologous Hematologic analysis of mice eight and nine and daysincrease following challenge with an ordinarily lethal dose revealed a significant in white blood cells challenge with an ordinarily lethal dose revealed a significant increase in white challenge with an dose revealed significant in whiteboth bloodelevated cells blood cells as compared to ordinarily nave Karplethal strain infected micea (Figure 5A)increase that comprised as compared to nave Karp strain infected mice (Figure 5A) that comprised both elevated as levels compared to nave Karp strain5B) infected mice (Figure 5A) that of lymphocytes (Figure and neutrophils (Figure 5C).comprised both elevated levels of lymphocytes and neutrophils (Figure 5C). levels of lymphocytes (Figure(Figure 5B) and5B) neutrophils (Figure 5C). Figure 5. 5. Hematologic responses to O.to tsutsugamushi infection or heterologous strain challenge. ToFigure Hematologic responses O. tsutsugamushi infection or heterologous strain challenge. Total Figure 5. Hematologic responses to O. tsutsugamushi infection heterologous strain challenge. To-per tal white blood cell (A), lymphocyte (B), and neutrophil (C)or counts expressed as thousand cells white blood cell (A), lymphocyte (B), and neutrophil (C) counts expressed as thousand cells per tal microliter white blood cell (A), lymphocyte and neutrophil (C) counts expressed as (D) thousand cells (K/L) in whole blood (B), or total spleen weight in milligrams (mg) of mice 89per days microliter (K/L) in whole blood or total spleen weight in milligrams (mg) (D) of mice 89 days microliter (K/L) with in whole blood or total spleen in milligrams (mg) (D) of mice 89 days post-infection O. tsutsugamushi Karp (redweight squares) or heterologously challenged (open blue post-infection with O. tsutsugamushi Karp (red squares) heterologously challenged (open blue (open blue post-infection with O. tsutsugamushi Karp (red or squares) or heterologously challenged circles) with 5 LD50 O. tsutsugamushi Karp strain as compared to sham (closed black circles) or O. tsutsugamushi Gilliam followed by time-indicated sham inoculated controls (green triangles). Grey shaded region indicates normal range. *, p < 0.05; **, p < 0.01, *** or p < 0.001. Pathogens 2022, 11, x FOR PEER REVIEW Pathogens 2022, 11, 512 7 of 13 circles) with 5 LD50 O. tsutsugamushi Karp strain as compared to sham (closed black circles) or O. of 12 tsutsugamushi Gilliam followed by time-indicated sham inoculated controls (green triangles). 7Grey shaded region indicates normal range. *, p < 0.05; **, p < 0.01, *** or p < 0.001. Significant Significant splenomegaly splenomegaly was was observed observedin in all all mice mice that that survived survived heterologous heterologouschalchallenge as compared to nave mice that succumbed to lethal O. tsutsugamushi lenge as compared to nave mice that succumbed to lethal O. tsutsugamushi Karp Karp infection infection (Figure 5D).Histopathologic Histopathologicobservations observations in lethally challenged micesuccumbed that suc(Figure 5D). in lethally challenged navenave mice that cumbed to O. tsutsugamushi Karp strain lymphohistiocytic included lymphohistiocytic vascular inflammato O. tsutsugamushi Karp strain included vascular inflammation and intion and pneumonitis interstitial pneumonitis and 7B). Although miceinfected previously terstitial (Figures 6B (Figures and 7B). 6B Although mice previously withinfected Gilliam with strain were protected death, histopathologic changes, lesions with constrainGilliam were protected from death, from histopathologic changes, and lesionsand consistent sistent with scrub typhus such as perivascular inflammation, multifocal lesions, and polyscrub typhus such as perivascular inflammation, multifocal lesions, and polymononuclear mononuclear cellular observed in liver, lungs,and kidney, and6CE brainand (Figures cellular infiltrates wereinfiltrates observedwere in lungs, kidney, brainliver, (Figures 7CE 6CE, 7CEWe and 8C,D). We observed coagulative in the liver of micechallenged heteroloand 8C,D). observed coagulative necrosis in the necrosis liver of mice heterologously gously challenged initial Gilliam strain7D) infection (Figureand 7D)steatosis and nethree months afterthree initialmonths Gilliamafter strain infection (Figure and necrosis crosis the liver of mice to which succumbed to an ordinarily lethal Karp in the and liversteatosis of mice in which succumbed an ordinarily lethal Karp strain challenge 14 months after Gilliam strainafter infection (Figure Meningoencephalitis with perivascular strain challenge 14 months Gilliam strain7F). infection (Figure 7F). Meningoencephalitis inflammation wasinflammation observed in the brains of heterologously mice one and three with perivascular was observed in the brainschallenged of heterologously challenged months strain infection (Figure 8C,D). mice onefollowing and threeprimary months Gilliam following primary Gilliam strain infection (Figure 8C,D). Figure 6. 6. Histopathologic Histopathologic changes changes of of lung lung one one month month following following sublethal sublethal O. O. tsutsugamushi tsutsugamushi Gilliam Gilliam Figure strain 50 inoculation strain infection infection (A) (A) or or day 89 after 5 LD50 inoculation with with O. O. tsutsugamushi tsutsugamushi Karp Karp strain strain in in nave nave mice (E), or or 14 14 (F) (F) months months after after sublethal sublethal infection infection with with Gilliam Gilliam strain. strain. Bar mice (B) (B) or or 11 (C), (C), 33 (D), (D), 99 (E), Bar represents 100). represents 200 200 m m (original (original magnification magnification 100 ). Pathogens 2022, 11, x FOR PEER REVIEW Pathogens 2022, 11, 512 8 of 13 8 of 12 Figure7.7.Histopathologic Histopathologicchanges changesof ofliver liverone onemonth monthfollowing followingsublethal sublethalO. O.tsutsugamushi tsutsugamushiGilliam Gilliam Figure straininfection infection(A) (A)or orday day 89 89 after after 55 LD LD5050inoculation inoculationwith withO. O.tsutsugamushi tsutsugamushiKarp Karpstrain strainin innave nave strain mice mice(B) (B) or or 11 (C), (C), 33 (D), (D), 99 (E), (E), or or 14 (F) months after sublethal sublethal infection infection with with Gilliam Gilliam strain. strain. Bar Bar represents 200 m (original magnification 100). represents 200 m (original magnification 100). Pathogens 2022, 2022, 11, 11, 512 x FOR PEER REVIEW Pathogens 99 of 13 of 12 Figure 8. 8. Histopathologic changes of brain one month following sublethal O. tsutsugamushi Gilliam Gilliam Figure strain infection infection (A) or or day day 89 89 after after 55 LD LD50 50 inoculation with O. tsutsugamushi tsutsugamushi Karp strain in nave nave mice (B) or 1 (C), 3 (D), 9 (E), or 14 (F) months after sublethal infection with Gilliam strain. Bar mice (B) or 1 (C), 3 (D), 9 (E), or 14 (F) months after sublethal infection with Gilliam strain. Bar represents 200 m (original magnification 100). represents 200 m (original magnification 100). 3. Discussion We We developed developed aa murine murine model model demonstrating demonstrating waning waning protection protection against against challenge challenge with with aa heterologous heterologous strain strain of of Orientia Orientia tsutsugamushi tsutsugamushi utilizing utilizing C57Bl/6 C57Bl/6 strain mice and aa primary primary O. O. tsutsugamushi tsutsugamushi Gilliam Gilliam strain straininfection infectionfollowed followedby byordinarily ordinarilylethal lethalO. O.tsutsugtsutsuamushi Karp strain challenge. In this model we observed no overt signs of illness gamushi Karp strain challenge. In this model we observed no overt signs of illness after after challenge with an ordinarily lethal dose of O. O. tsutsugamushi tsutsugamushi Karp strain one month month after after Gilliam Gilliam strain strain infection; infection; however, however, signs signs of of illness illness were were observed observed at three months following following Gilliam Gilliam strain strain infection infection and and progressed progressed in in correlation correlation with with the the period period since since the the initial initial Gilliam strain infection. In addition to the loss of heterologous protection observed in Gilliam strain infection. In addition to the loss of heterologous protection observed in this this study, study,the the use use of of age-matched age-matched mice mice at at each each challenge challenge timepoint timepoint allowed allowed us us to to observe observe that that the dissemination of lethal infection withwith O. tsutsugamushi Karp the kinetics kineticsand andhematogenous hematogenous dissemination of lethal infection O. tsutsugamushi was not affected by the increased mouse age. Karp was not affected by the increased mouse age. Previous Previous O. O. tsutsugamushi tsutsugamushi Gilliam Gilliam strain strain infection infection does does not not result result in in sterile sterile protection protection from Karp strain challenge in the animals that remained healthy as well as those from Karp strain challenge in the animals that remained healthy as well as those thatthat bebecame fataloutcome outcomewas wasassociated associatedwith withgreater greaterbacterial bacterial loads. loads. Mice Mice that that survived came ill.ill. AA fatal survived an ordinarily lethal lethalchallenge challengewith withO.O. tsutsugamushi Karp strain by having a previous an ordinarily tsutsugamushi Karp strain by having a previous hetheterologous infection had a robust immune response, characterized by reported pathoerologous infection had a robust immune response, characterized by reported pathologic logic manifestations of scrub typhus disease including splenomegaly and hepatomegaly. manifestations of scrub typhus disease including splenomegaly and hepatomegaly. AltAlthough bacterial loads were relatively low in protected mice, an abundance of cellular hough bacterial loads were relatively low in protected mice, an abundance of cellular ininfiltrates was observed in the tissues of protected mice which inversely correlated with filtrates was observed in the tissues of protected mice which inversely correlated with the Pathogens 2022, 11, 512 10 of 12 the amount of time passed since the primary Gilliam strain infection. Immune-mediated damage and vascular inflammation have been described in a lethal murine scrub typhus model in the absence of high bacterial loads in tissues such as the brain [22]. Further investigation is warranted in this heterologous challenge model to understand the significance and mechanism of the histopathologic observations. This model of waning protection between heterologous strains of O. tsutsugamushi will be used by our laboratory to study the immune components and cell subsets important for cross-protection. This model may also be employed to evaluate the difference between immunity to heterologous and homologous strains of O. tsutsugamushi Karp. In our laboratory, mice originally infected with a sublethal dose of O. tsutsugamushi Karp strain exhibited no overt signs of disease or significant weight loss upon re-challenge with a lethal dose (1.25 106 organisms) of the homologous strain of O. tsutsugamushi 165, 180, 240, 292, or 430 days post-primary infection. In contrast, mice that survived Karp strain were heterologously challenged after 240 days with O. tsutsugamushi Gilliam strain which resulted in illness and weight loss. The protection observed from prior homologous or heterologous strain infection in these animal models indicate that native antigen is important to mount an effective immune response. For future vaccine development, consideration of current circulating human scrub typhus field isolates is paramount. This animal model provides the framework to develop future strategies for testing these isolates. We hypothesize that mouse strains that are less resistant to challenge with O. tsutsugamushi than C57Bl/6 may provide an opportunity to shorten the timeline of this model. Additionally, we will use the knowledge obtained from this model as a baseline to evaluate vaccine effectiveness and durability against heterologous strain challenge. 4. Materials and Methods 4.1. Stock Propagation Orientia tsutsugamushi Gilliam and Karp strains were used in this study due to the availability of well-characterized sublethal and lethal murine models. The bacterial strains were obtained from the Rickettsial and Ehrlichial Species Collection at the University of Texas Medical Branch. Orientia tsutsugamushi was cultivated in L929 cells or harvested from mouse liver in vivo and stored at 80 C in sucrosephosphateglutamate (SPG) buffer (218 mM sucrose, 3.8 mM KH2PO4, 7.1 mM K2HPO4, 4.9 mM monosodium L-glutamic acid, pH 7.0) until used as previously described [23,24]. The L-929 cell line was obtained from American Type Culture Collection (ATCC, catalog number CCL-1). 4.2. Bacterial Viability and Load Determination A quantitative viability assay was utilized to enumerate viable Orientia for inoculation as previously described [25]. Bacterial loads and dissemination to selected organs were assessed by qPCR. Strain-specific primers were designed utilizing the variable domain IV of the 56 kDa gene (accession numbers DQ485289, AY956315, M33004) using PrimerSelect from Lasergene software suite version 12 (DNASTAR, Inc., Madison, WI, USA) (O. tsutsugamushi Gilliam OtG56.729 [50 -TCGTGATGTGGGGGTTGATAC-30 ], OtG56.873 [50 TTCTGAGGATCTGGGACCATATAG-30 ], O. tsutsugamushi Karp 56 kDa OtK56.877 [50 GATCCTAATGGGCCTATGGTTATA-30 ], and [OtK56.982 50 -AACCTGCAGGCGGATTTG30 ]). DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) from bead-homogenized tissue samples according to the manufacturers instructions. Tissue samples were normalized using tissue wet weight, and bacterial loads were expressed as the number of O. tsutsugamushi Karp strain or Gilliam strain 56 kDa copies per milligram (mg) of tissue. 4.3. Mouse Infection This animal study was approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch-Galveston (protocol number 1302003 and approved 1 February 2013). Female C57BL/6 (B6) mice, six to eight weeks of age, Pathogens 2022, 11, 512 11 of 12 were purchased from Envigo Laboratories (Indianapolis, IN, USA) and were housed in an animal biosafety level 3 facility (ABSL3) under specific pathogen-free conditions. The mice were allowed to acclimate for at least seven days prior to experimental use and then were inoculated intradermally in the lateral ear with 2.5 105 O. tsutsugamushi Gilliam strain organisms as determined by viability assay and monitored twice daily for signs of illness for 2 weeks, and then weekly until secondary challenge. Mice were inoculated by intravenous tail vein at indicated timepoints post-Gilliam strain infection with 1.3 106 viable O. tsutsugamushi Karp strain (approximately 5 LD50 ). Mice were monitored twice daily for signs of illness for up to three weeks. When mice were moribund, they were sacrificed humanely and necropsied along with matched mice from the other experimental groups, and their tissues were weighed, tested for bacterial loads, and prepared for histology. The remaining animals were observed for veterinary-accepted signs of illness (ruffled fur, hunched posture, erythema, lethargy, conjunctivitis, and weight loss). Moribund mice euthanized according to animal welfare criteria were counted as deceased for statistical analyses. All animal experiments were conducted twice. 4.4. Hematologic Analyses Blood samples were collected at experimental endpoints in K2 EDTA-coated BD microtainer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) for hematologic analyses followed by centrifugation at 1300 RCF for 10 min. Blood cell counts were performed on whole blood using a calibrated 950FS HemaVet apparatus (Drew Scientific, Waterbury, CT, USA) using the FS-Pak reagent kit, and white blood cell count (WBC) and differential leukocyte (%) count were measured. 4.5. Histology Tissue samples were fixed in 10% neutral buffered formalin (NBF) and embedded in paraffin. Tissue sections (5 m thickness) were stained with hematoxylin and eosin and examined with an Olympus BX51 microscope (Olympus Scientific, Waltham, MA, USA). 4.6. Statistical Analysis Values are reported as mean standard deviation (SD). The data were analyzed using a one-way ANOVA with Tukeys multiple comparison as post hoc analysis or a two-way ANOVA with Bonferronis post-tests (GraphPad Prism, San Diego, CA, USA) and are reported at statistical significance levels of *, p < 0.05; **, p < 0.01; or ***, p < 0.001. Author Contributions: Conceptualization, N.L.M. and D.H.W.; Data curation, N.L.M.; Formal analysis, N.L.M. and D.H.W.; Funding acquisition, D.H.W.; Investigation, N.L.M., G.X. and T.R.S.; Methodology, N.L.M., G.X., T.R.S., D.H.B. and D.H.W.; Project administration, D.H.W.; Resources, D.H.B. and D.H.W.; Supervision, D.H.B. and D.H.W.; Validation, N.L.M., G.X. and T.R.S.; Visualization, G.X., T.R.S., D.H.B. and D.H.W.; Writingoriginal draft, N.L.M.; Writingreview and editing, N.L.M., G.X., T.R.S. and D.H.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by The Carmage and Martha Walls Distinguished University Chair in Tropical Diseases. Institutional Review Board Statement: The animal study protocol was approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch-Galveston (protocol code 1302003 and approved 1 February 2013). Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in the article. Acknowledgments: The authors wish to express gratitude to Lucas Blanton and Patricia CrocquetValdes for critical comments and to Tuha Ha for histological assistance. Pathogens 2022, 11, 512 12 of 12 Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. Kawamura, R. Studies on Tsutsushi Disease (Japanese flood fever). Med. Bull. Coll. Med. Univ. Cincinnati. 1926, 4, 1229. Romeo, B.J. Convalescence from scrub typhus. Bull. United States Army Med. Dep. 1946, 6, 167173. Kawamura, R.; Kasahara, S.; Toyama, T.; Nishinarita, F.; Tsubaki, S. On the prevention of tsutsugamushi. 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