Early-Phase Transmission of Yersinia pestis by Unblocked Xenopsylla cheopis (Siphonaptera: Pulicidae) Is as Efficient as Transmission by Blocked Fleas

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1 VECTOR/PATHOGEN/HOST INTERACTION, TRANSMISSION Early-Phase Transmission of Yersinia pestis by Unblocked Xenopsylla cheopis (Siphonaptera: Pulicidae) Is as Efficient as Transmission by Blocked Fleas REBECCA J. EISEN, 1 ARYN P. WILDER, SCOTT W. BEARDEN, JOHN A. MONTENIERI, AND KENNETH L. GAGE Bacterial Diseases Branch, Division of Vector Borne Infectious Diseases, National Center for Zoonotic, Enteric and Vector-Borne Diseases, Centers for Disease Control and Prevention, P.O. Box 2087, Fort Collins, CO J. Med. Entomol. 44(4): 678Ð682 (2007) ABSTRACT For almost a century, the oriental rat ßea, Xenopsylla cheopis (Rothschild) (Siphonaptera: Pulicidae), was thought to be the most efþcient vector of the plague bacterium Yersinia pestis (Yersin). Approximately 2 wk after consuming an infectious bloodmeal, a blockage often forms in the ßeaÕs proventriculus, which forces the ßea to increase its biting frequency and consequently increases the likelihood of transmission. However, if ßeas remain blocked and continue to feed, they usually die within 5 d of blocking, resulting in a short infectious window. Despite observations of X. cheopis transmitting Y. pestis shortly after pathogen acquisition, early-phase transmission (e.g., transmission 1Ð4 d postinfection [ p.i.]) by unblocked ßeas was viewed as anomalous and thought to occur only by mass action. We used an artiþcial feeding system to infect colony-reared X. cheopis with a fully virulent strain of Y. pestis, and we evaluated transmission efþciency 1Ð4 d p.i. We demonstrate 1) that a single infected and unblocked X. cheopis can infect a susceptible host as early as 1 d p.i., 2) the number of ßeas per host required for unblocked ßeas to drive a plague epizootic by early-phase transmission is within the ßea infestation range observed in nature, and 3) early-phase transmission by unblocked ßeas in the current study was at least as efþcient as transmission by blocked ßeas in a previously published study using the same colony of ßeas and same bacterial strain. Furthermore, transmission efþciency seemed to remain constant until block formation, resulting in an infectious period considerably longer than previously thought. KEY WORDS transmission, plague, Yersinia pestis, Xenopsylla cheopis, ßea 1 Corresponding author, dyn2@cdc.gov. Throughout its vast geographical range, the oriental rat ßea, Xenopsylla cheopis (Rothschild) (Siphonaptera: Pulicidae), has been implicated as the primary vector of Yersinia pestis (Yersin) during plague epidemics and epizootics (Eskey and Haas 1940, Gratz 1999, Gage and Kosoy 2005). Although transmission of Y. pestis by X. cheopis is considered to be inefþcient (Lorange et al. 2005), comparative studies have revealed that it is more efþcient than most other ßea species evaluated 5 d postinfection (p.i.) (Eskey and Haas 1940; Burroughs 1947; Pollitzer 1954; Kartman et al. 1958a, 1958b; Engelthaler et al. 2000). After an infectious bloodmeal, plague bacilli multiply and can form a blockage in the proventriculus of X. cheopis. The blockage prevents fresh blood from reaching the midgut. As a result, the blocked ßea increases its feeding attempts, and occasionally newly synthesized bio- Þlm and multiplying bacteria are regurgitated into the bite site, resulting in infection in the susceptible host (Bacot and Martin 1914, Hinnebusch 2005). This mechanism of transmission requires a lengthy extrinsic incubation period before the bioþlm completely occludes the proventriculus (typically 12Ð18 d p.i.). Although the frequency of biting increases after block formation, blocked ßeas transmit with fairly low efþciency, and if they remain blocked and continue to attempt to feed, usually die within 5 d of blocking, resulting in a short infectious window (Eskey and Haas 1940, Burroughs 1947, Engelthaler et al. 2000). Although transmission by blocked ßeas was the dominant paradigm in Y. pestis transmission dynamics, recent mathematical models have indicated that blocked ßea transmission failed to explain rapid rates of spread that typify plague epidemics and epizootics (Eskey and Haas 1940, Pollitzer 1954, Barnes 1982, Gage and Kosoy 2005, Lorange et al. 2005, Eisen et al. 2006, Webb et al. 2006). However, insecticidal treatment during plague epidemics where X. cheopis is the most abundant ßea has been effective at halting pathogen dissemination within affected populations (Pollitzer 1954, Gratz 1999). This suggests that an alternative ßea-borne mode of transmission (e.g., transmission by unblocked ßeas) is important for amplifying Y. pestis within susceptible host populations /07/0678Ð0682$04.00/ Entomological Society of America

2 July 2007 EISEN ET AL.: EARLY-PHASE TRANSMISSION OF Y. pestis BY X. cheopis 679 A recent study with Oropsylla montana (Baker) (Siphonaptera: Ceratophyllidae), a ßea that commonly infests ground squirrels in North America, demonstrated that early-phase transmission (i.e., transmission 1Ð4 d p.i.) by unblocked ßeas is highly efþcient and could drive rapidly spreading plague epizootics (Eisen et al. 2006). Although other ßeas, including X. cheopis, have been reported to transmit Y. pestis shortly after pathogen acquisition, early-phase transmission was viewed as anomalous and thought to occur only by mass action (i.e., with exceptionally high ßea loads) (Verjbitski 1908, McCoy 1910, Wheeler and Douglas 1945, Burroughs 1947, Holdenried 1952, Quan et al. 1953, Engelthaler et al. 2000). Here, we demonstrate that 1) X. cheopis can transmit plague bacteria as early as 1 d p.i., and 2) transmission by unblocked ßeas 1Ð4 d p.i. is at least as efþcient as transmission by blocked ßeas. We also explore how these parameters affect estimates of the numbers of ßeas per host required for enzootic maintenance and epizootic spread of Y. pestis. Materials and Methods Methods for infecting X. cheopis ßeas with Y. pestis (CO ), conþrming transmission from ßeas to naõ ve Swiss Webster mice, quantiþcation of bacterial loads in ßeas and evaluation of vector competency 1Ð4 d p.i. were described in detail previously (Eisen et al. 2006). Brießy, on day 0, four batches of colony-reared female X. cheopis (n 50Ð60 ßeas per feeder) were allowed to feed for 1honartiÞcial feeders containing deþbrinated Sprague-Dawley strain rat blood (Bioreclamation, Jericho, NY) containing a fully virulent North American strain of Y. pestis, biovar orientalis, designated CO ( Ð colonyforming units/ml). Aliquots of bacteria used in the current study came from the same source population as those used previously (Eisen et al. 2006). Fed ßeas containing red blood in the proventriculus or midgut were differentiated from unfed ßeas by using light microscopy (Eisen et al. 2006). Any ßea that did not take a potentially infectious bloodmeal was discarded; the remaining ßeas were held at 23 C and 85% RH for 1Ð4 d p.i. On each of the 4-d p.i., pools of 10 potentially infectious ßeas were placed in feeding capsules for 1 h on anesthetized 6-wk-old Swiss Webster strain mice. After 1 h, ßeas were removed from the feeding capsule by using a mechanical aspirator. Flea feeding success was determined using microscopy (Eisen et al. 2006); fed ßeas were stored in individual microcentrifuge tubes at 80 C until infection status and bacterial loads were determined by serial dilutions of ßeas triturated in heart infusion broth supplemented with 10% glycerol and plated in duplicate on blood agar plates containing 6% sheep blood (Eisen et al. 2006). After exposure to ßeas, recipient mice were housed individually in Þlter-top cages and monitored daily. They were euthanized when signs of Y. pestis exposure were evident (e.g., slow response to stimuli, shivering, rufßed fur). Mice surviving to 21 d p.i. were euthanized, and blood was collected for testing for serologic evidence of exposure. Animal procedures were approved by the Division of Vector-Borne Infectious Diseases (Centers for Disease Control and Prevention) Institutional Animal Care and Use Committee. For each time point (1Ð4 d p.i.), transmission efþciency per individual ßea was estimated using maximum likelihood estimates based on the number of infected ßeas that fed on an individual mouse and whether or not transmission was observed for that recipient mouse by using the Microsoft Excel Add-In PooledInfRate, version 3.0 (Biggerstaff 2006). Mean maximum bacterial loads for infected ßeas fed per animal at each time point were compared using analysis of variance (ANOVA) based on log 10 -transformed values. KruskalÐWallis and Wilcoxon rank sums tests were used to compare median numbers of colonyforming units per ßea among and between treatments, respectively. Flea feeding success among time points was compared using ANOVA with TukeyÐKramer post hoc pairwise comparisons. Logistic regression was used to evaluate the relationships between number of infected ßeas fed per host or log 10 maximum bacterial load per ßea pool and transmission success (e.g., transmission occurred or not). All statistical comparisons were run using JMP statistical software (SAS Institute, Cary, NC). The number of ßeas per host required for enzootic or epizootic transmission of Y. pestis was determined following two recently published studies of vectorial capacity (Lorange et al. 2005, Eisen et al. 2006). The model assumes that 1) host density is sufþciently high for every infected ßea to Þnd a susceptible host upon which to feed, and 2) the ßea attempts to feed on the new host at least twice. The resulting model predicts the number of ßeas required per host to maintain an infection in a population as follows: m R 0 r/abp n where R 0 represents vectorial capacity or force of infection (e.g., the number of secondary infections arising from a focal infection); R 0 1 during enzootic conditions and R 0 2 during plague epizootics, m is the ßea density per host (e.g., host or nest infestation), a is the daily biting rate of infected ßeas, b is the probability of a ßea acquiring an infection after feeding on a septicemic host and transmitting the infection during a subsequent feeding on a susceptible host, p n is the probability of the ßea surviving the extrinsic incubation period (deþned as the duration of time from which a ßea is infected until it can transmit), and r deþnes the reciprocal life expectancy of the host after it reaches threshold septicemia. Results Transmission of Y. pestis by unblocked X. cheopis was observed for each of the four time points (1, 2, 3, and 4 d p.i.) (Table 1). Because transmission efþciency was similar from 1 to 4 d p.i. (Table 1), we estimated the per ßea transmission efþciency for the entire early-phase observation period by using maxi-

3 680 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 4 Table 1. Bacterial loads and transmission efficiency of X. cheopis for Y. pestis Time point (days after infectious feed) and mouse no. No. infected ßeas fed on naõ ve mouse (total no. fed of total no. exposed to mouse) Median (range) bacterial load per ßea fed on naive mouse (cfu/ßea) Transmission from ßea to mouse Estimated transmission efþciency (95% CI)/ time point 1 a 4.71 (0.34Ð21.06) 1 1 (1 of 10) Yes 2 5 (5 of 10) ( Ð ) No 3 8 (8 of 10) ( Ð ) No 4 4 (4 of 10) ( Ð ) No (0.38Ð28.54) 1 4 (5 of 10) ( Ð ) No 2 1 (4 of 10) Yes 3 2 (3 of 10) ( Ð ) No 4 2 (2 of 10) ( Ð ) No 5 2 (2 of 10) ( Ð ) No (2.65Ð25.94) 1 7 (8 of 10) ( Ð ) Yes 2 7 (8 of 10) ( Ð ) Yes 3 9 (9 of 10) ( Ð ) No 4 8 (8 of 10) ( Ð ) No 5 7 (7 of 10) (50.00Ð ) Yes (0.16Ð12.47) 1 6 (8 of 10) ( Ð ) Yes 2 6 (6 of 10) ( Ð ) No 3 5 (6 of 10) ( Ð ) No 4 9 (9 of 10) ( Ð ) No 5 10 (10 of 10) ( Ð ) No Fleas were infected in artiþcial feeders containing deþbrinated rat blood infected with Y. pestis at a concentration of Ð cfu/ml. a Median bacterial loads were higher on day 1 compared with day 3 or day 4 ( , df 3, P 0.03). mum likelihood, which yielded an estimate for transmission efþciency of 6.4% (95% CI: 2.8Ð12.8). For each time point except 1 d p.i., Þve mice were exposed to 10 ßeas. Among pools of ßeas feeding on mice, ßea feeding success ranged from 10 to 100%. Feeding success (percentage of ßeas fed per time point) was similar between days 1 (mean 45.0%, SD 28.9%) and 2 (mean 32.0%, SD 13.0%) and between days 3 (mean 80.0%, SD 7.1%) and 4 (mean 78.0% and SD 17.9%), but it was signiþcantly higher 3 and 4 d p.i. relative to 1 and 2 d p.i. (F 9.05; df 3, 18; P ) (Table 1). Only a single infected ßea per host was necessary for early-phase transmission by X. cheopis (Table 1). The number of infected ßeas fed per host was not signiþcantly associated with transmission success. Among time points, log 10 maximum bacterial loads were similar, and no association was detected between maximum bacterial load and transmission success. Median bacterial loads differed signiþcantly among time points ( , df 3, P 0.009). SpeciÞcally, median bacterial loads were higher on day 1 compared with day 3 or day 4 ( , df 3, P 0.03); all other pairwise comparisons by day p.i. were similar. Based on early-phase transmission rates for X. cheopis, daily biting rates, duration of septicemia in the host, and the probability of a ßea surviving the extrinsic incubation period (Table 2), we estimated the number of ßeas per host needed to maintain Y. pestis in a susceptible host population. With a daily biting rate of 0.38, enzootic maintenance requires 20.8 ßeas per host (95% CI: 10.4Ð50.0). However, in our study ßeas were exposed to mice for only 1 h to determine this daily biting rate. Also, our 1-d p.i. assessment is based on colony-reared ßeas that typically are fed only twice weekly, perhaps decreasing the daily feeding rate compared with natural populations. If ßeas are in regular contact with a host throughout the day, a daily biting rate of 1.0 may be more realistic. Burroughs (1947) noted that wild-caught X. cheopis would feed daily for a short time, and then they refuse to feed for one or more days. Under these daily feeding conditions, 7.8 ßeas per host (95% CI: 3.93Ð17.9) are required for enzootic maintenance. In contrast, transmission by blocked X. cheopis (Engelthaler et al. 2000) would require 26.3 ßeas per host (95% CI: 8.29Ð146.19) (Table 2) if a daily biting rate of 1.0 is assumed (Lorange et al. 2005). Discussion Previously, it was assumed that individual blocked X. cheopis transmit Y. pestis after an extrinsic incubation period of Þve or more days or that unblocked ßeas transmit shortly after pathogen acquisition but only by mass action requiring exceptionally high ßea loads (Eskey and Haas 1940, Burroughs 1947, Pollitzer 1954, Gage and Kosoy 2005, Hinnebusch 2005). In our study, we demonstrated that a single infected and unblocked X. cheopis can infect a susceptible host as early as 1 d p.i. Furthermore, unblocked ßeas seem to be able to transmit plague bacteria at least as efþciently as blocked ßeas from the same colony infected with the

4 July 2007 EISEN ET AL.: EARLY-PHASE TRANSMISSION OF Y. pestis BY X. cheopis 681 Table 2. Terms used to determine flea loads required for enzootic maintenance and epizootic spread derived experimentally or from published literature for X. cheopis Variable DeÞnition Early-phase transmission Blocked ßea transmission a Biting rate/day of infected ßea 0.38Ð1.0 a 1.0 b b Probability of a ßea becoming infected and (0.0278Ð0.1284) c (0.0038Ð0.067) c infectious after feeding on a host with septicemia at the threshold or greater 1/r Life expectancy (in days) of host after reaching 2 2 threshold septicemia b p n Probability of ßea surviving the extrinsic incubation period d a A value of 0.38 is the average feeding success for all replicates of the 24-h time point; based on previous studies (Burroughs 1947, Quan et al. 1953), it is possible that the daily biting rate is a high as 1.0 (Lorange et al. 2005). b Data from Lorange et al. (2005). c Maximum likelihood estimate (95% CI) of per ßea transmission efþciency for early-phase and blocked ßea transmission. Early-phase estimates from 1 to 4 d p.i. were based on the present study. Estimates of transmission efþciency by blocked ßeas consider only visibly blocked ßeas that transmit infection based on Engelthaler et al. 2000). ConÞdence limits were generated by maximum likelihood. d Assumes a mean extrinsic incubation period of 16.5 d (Engelthaler et al. 2000) and from Þg. 5 in Engelthaler et al. (2000) assume that 27 of 260 infected X. cheopis died by week 2 postinfection. same strain of Y. pestis after a very short extrinsic incubation period (Engelthaler et al. 2000). By reducing the extrinsic incubation period and eliminating block-induced mortality, a higher proportion of unblocked ßeas, relative to blocked ßeas, should survive up to the infectious period and continue to transmit over many days to a few weeks (Burroughs 1947, Engelthaler et al. 2000). As a result of this longer duration of infectiousness, the number of times an unblocked infectious ßea feeds may be at least as high as for an infectious blocked ßea, which is thought to attempt to feed frequently but to survive only 5 d after blockage (Eskey and Haas 1940, Wheeler and Douglas 1945, Burroughs 1947, Pollitzer 1954). Therefore, relative to transmission by blocked ßeas, transmission of Y. pestis by unblocked ßeas that are infectious as early as 1 d p.i. should require comparable or fewer numbers of ßeas per host to maintain or amplify Y. pestis within a susceptible host population (Macdonald 1961, Lorange et al. 2005). Indeed, our model estimates predict that only 7.9 ßeas per host are required for enzootic maintenance when transmission occurs as early as 1 d p.i. compared with 26.3 ßeas per host if transmission is by blocked ßeas with a similar biting rate of 1.0 per day (Table 2). As summarized previously (Eskey and Haas 1940, Hirst 1953, Pollitzer 1954, Traub 1972, Schwan 1986, Lorange et al. 2005), X. cheopis loads per host typically range from 1 to 12, but many more infectious ßeas may remain in rodent nests or burrows and have daily access to hosts. Thus, our estimates of 7.9 or 15.8 ßeas per host required for enzootic maintenance or epizootic spread, respectively, by early-phase transmission are within the range observed in nature. Compared with most other ßea species, X. cheopis is less likely to clear infections, develops proventricular blockages at higher frequencies, and requires less time for block formation to occur after initial exposure to Y. pestis (Bacot and Martin 1914, Eskey and Haas 1940, Burroughs 1947, Pollitzer 1954, Kartman and Prince 1956, Engelthaler et al. 2000). Our observation of earlyphase transmission coupled with what may be a unique ability of this ßea species to frequently block and efþciently transmit when blocked (Bacot and Martin 1914; Eskey and Haas 1940; Wheeler and Douglas 1945; Burroughs 1947; Pollitzer 1954; Kartman and Prince 1956; Kartman et al. 1956, 1958a, 1958b; Engelthaler et al. 2000; Lorange et al. 2005) suggests that the duration of infectiousness of X. cheopis after a single infectious bloodmeal may exceed 1 mo. Although often overlooked because of a focus on transmission by blocked ßeas, previous data suggest that transmission efþciency by unblocked X. cheopis 6Ð32 d p.i. (Burroughs 1947, Engelthaler et al. 2000) is within a range similar to values 1Ð4 d p.i. reported in the current study. Thus, transmission efþciency of unblocked X. cheopis seems to remain constant from pathogen acquisition up to block formation. This long duration of infectiousness may compensate for moderate vector efþciency. Early-phase transmission of Y. pestis by O. montana, a common North American ground squirrel ßea, has been demonstrated to be more efþcient than transmission by X. cheopis during the early phase (Eisen et al. 2006), but in the absence of secondary infectious feeds transmission efþciency of O. montana wanes 6 d p.i. (Eisen et al. 2007). Unlike X. cheopis, O. montana rarely becomes blocked (Eskey and Haas 1940, Burroughs 1947, Engelthaler et al. 2000), and 80% of ßeas clear infections by 35 d p.i. (Engelthaler et al. 2000), yielding a short infectious period after a single infectious bloodmeal. Compared with a previous study using the same colony of ßeas and same isolate of Y. pestis, our study revealed that vector efþciency was similar between unblocked X. cheopis 1Ð4 d p.i. and blocked ßeas transmitting Y. pestis 15Ð18 d p.i. (Engelthaler et al. 2000). However, it is important to note that others (Eskey and Haas 1940, Burroughs 1947, Kartman and Prince 1956, Kartman et al. 1958a, Lorange et al. 2005) have reported higher vector efþciency by blocked X. cheopis than Engelthaler et al. (2000). It is possible that differences among these studies in sources of ßeas, strains of bacteria, or feeding method could account for the observed variation in transmission rates. The ability of unblocked X. cheopis to transmit Y. pestis as early as 1 d p.i. and this ßeaÕs tendency to

5 682 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 4 abandon its host shortly after feeding (Quan et al. 1953, Pollitzer 1954) suggest that contact between infectious ßeas and susceptible hosts could be substantial. When ßea and host densities are sufþciently high, our model of vectorial capacity indicates that early-phase transmission by unblocked ßeas could drive rapidly spreading epizootics. It is likely that transmission efþciency remains high throughout the life of an infected ßea and that this long duration of infectiousness could serve to maintain the infection when host density declines. To accurately assess how transmission dynamics affect rates of spread of Y. pestis in host populations, future studies are needed to evaluate temporal changes in transmission efþciency, daily biting rates, and survivorship throughout the period from initial exposure to death of the ßea. Acknowledgments We thank L. Eisen for comments on the manuscript and R. Pappert for technical assistance. 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