... 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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