Reevaluation of the Role of Blocked Oropsylla hirsuta Prairie Dog Fleas Siphonaptera: Ceratophyllidae in Yersinia pestis Enterobacterales: ...
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Journal of Medical Entomology, 59(3), 2022, 1053–1059
https://doi.org/10.1093/jme/tjac021
Advance Access Publication Date: 5 April 2022
Research
Vector/Pathogen/Host Interaction, Transmission
Reevaluation of the Role of Blocked Oropsylla hirsuta
Prairie Dog Fleas (Siphonaptera: Ceratophyllidae) in
Yersinia pestis (Enterobacterales: Enterobacteriaceae)
Transmission
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Adélaïde Miarinjara,1,4 David A. Eads,2, David M. Bland,1 Marc R. Matchett,3
Dean E. Biggins,2 and B. Joseph Hinnebusch1,5,
1
Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, NIH, Hamilton, MT,
USA, 2U.S. Geological Survey, Fort Collins Science Center, Fort Collins, CO, USA, 3U.S. Fish and Wildlife Service, Lewistown, MT,
USA, 4Present address: Department of Environmental Sciences, Emory University, Atlanta, GA, USA, and 5Corresponding author,
e-mail: jhinnebusch@niaid.nih.gov
Subject Editor: Janet Foley
Received 14 December 2021; Editorial decision 26 January 2022
Abstract
Prairie dogs in the western United States experience periodic epizootics of plague, caused by the flea-borne
bacterial pathogen Yersinia pestis. An early study indicated that Oropsylla hirsuta (Baker), often the most
abundant prairie dog flea vector of plague, seldom transmits Y. pestis by the classic blocked flea mechanism.
More recently, an alternative early-phase mode of transmission has been proposed as the driving force be-
hind prairie dog epizootics. In this study, using the same flea infection protocol used previously to evaluate
early-phase transmission, we assessed the vector competence of O. hirsuta for both modes of transmission.
Proventricular blockage was evident during the first two weeks after infection and transmission during this
time was at least as efficient as early-phase transmission 2 d after infection. Thus, both modes of transmission
likely contribute to plague epizootics in prairie dogs.
Key words: plague, Yersinia pestis, prairie dog, Oropsylla hirsuta, vector-borne transmission
Plague, caused by the bacterium Yersinia pestis and transmitted pri- North America. It and other prairie dog species are subject to spo-
marily by fleas, was first introduced into the west coast of North radic plague epizootics that can decimate colonies, with downstream
America around the year 1900 after human cases were found on ships effects on the prairie ecosystem (Eads and Biggins 2015). For ex-
arriving from the Asian focus of the third pandemic. The disease man- ample, epizootic and persistent enzootic plague have hampered ef-
aged to enter urban rat populations, coinciding with human plague forts to restore populations of the endangered black-footed ferret
outbreaks. In total, 450 human plague cases and 288 deaths were re- (Mustela nigripes) (Audubon & Bachman; Carnivora: Mustelidae),
ported between 1900 and 1920, all emanating from port cities of the which depends on prairie dogs as its primary food source and for
United States (Link 1955). As early as 1903, the disease had spread burrow habitat. The conditions that give rise to an epizootic are not
to ground squirrel populations around the San Francisco Bay area well understood, and the extent to which plague can be enzootic
(Eskey and Haas 1940). Much of the western United States proved in prairie dogs during interepizootic periods is not entirely clear
to be fertile ecological ground for Y. pestis, and by 1950 plague had (Cully and Williams 2001, Holmes et al. 2006, Hanson et al. 2007,
been documented in several species of woodrats, mice, ground squir- Matchett et al. 2010, Salkeld et al. 2010, St Romain et al. 2013,
rels, prairie dogs, other wild rodents, and their fleas, as far east as Biggins and Eads 2019, Colman et al. 2021). However, it is clear that
Kansas (Eskey and Haas 1940, Adjemian et al. 2007). fleas play a prominent role in transmitting plague to prairie dogs, be-
The black-tailed prairie dog (Cynomys ludovicianus) (Ord; cause insecticide treatments suppress or eliminate epizootics and can
Rodentia: Sciuridae) is a keystone species of the Great Plains of increase prairie dog and ferret survival during inter-epizootic periods
Published by Oxford University Press on behalf of Entomological Society of America 2022. 1053
This work is written by (a) US Government employee(s) and is in the public domain in the US.
1054 Journal of Medical Entomology, 2022, Vol. 59, No. 3
(Biggins et al. 2010, Matchett et al. 2010, Biggins et al. 2021). Prairie the sample were not plague-infected. Prior to infection, fleas were fed
dogs are hosts for two main flea vector species, Oropsylla hirsuta, sterile rat blood every other day for 7–10 d, using an artificial mem-
which predominates during summer, and Oropsylla tuberculata brane feeder and standard protocols (Bland et al. 2017). For the two
cynomuris (Jellison; Siphonaptera: Ceratophyllidae), predominant experiments used to assess early-phase transmission only, fleas were
in winter (Salkeld and Stapp 2008, Wilder et al. 2008b, Tripp et al. either fed sterile rat blood upon arrival and again 2 d later before
2009). While both are competent vectors of Y. pestis, a 1940 study infection on day 4; or infected on the day of arrival. For infections,
indicated that their ability to transmit via the classic proventricular Y. pestis KIM6+ containing the plasmid pAcGFP1, which encodes a
blockage mechanism is poor relative to other rodent fleas such as green fluorescent protein (Clontech/Takara Bio; San Jose, CA) was
Xenopsylla cheopis (Rothschild; Siphonaptera: Pulicidae) (Eskey grown from −80°C stock in 5 ml brain heart infusion (BHI) broth con-
and Haas 1940). Mathematical modeling based on this study con- taining 10 μg/ml hemin and 100 μg/ml carbenicillin. After overnight
cluded that transmission by blocked fleas would be insufficient to incubation at 28°C without aeration, 1 ml of this culture was added to
drive an epizootic (Webb et al. 2006), and subsequent evaluations of 100 ml of BHI/carbenicillin, which was incubated overnight at 37°C
early-phase transmission, which does not depend on proventricular without aeration. Bacteria were recovered from the 100 ml culture by
blockage, suggested that this alternate mode of transmission might centrifugation, resuspended in 1 ml phosphate-buffered saline (PBS),
be much more important (Wilder et al. 2008a,b; Buhnerkempe et al. and added to 5 ml of defibrinated rat blood (Bio IVT; Westbury, NY),
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2011, Richgels et al. 2016). resulting in ~108–109 colony-forming units (CFU)/ml, a concentra-
In this study we evaluated important aspects of the vector com- tion Y. pestis can achieve in the blood of mice with terminal plague
petence of O. hirsuta. Infection rates and the ability to transmit by (Wheeler and Douglas 1945, Bosio et al. 2012). The blood source and
both the early-phase and proventricular blockage modes of trans- bacteremia level match the conditions of the previous study of early-
mission were assayed in cohorts of fleas after they had taken an in- phase transmission by O. hirsuta (Wilder et al. 2008a). The infected
fectious bloodmeal, which has not been done previously. The results blood was added to an artificial feeding device fitted with a stretched
indicate a higher potential for blockage-mediated transmission than Parafilm M membrane covered by a single layer of coarse gauze that
was reported previously and prompt a reevaluation of epizootic the fleas could cling to while feeding. Fleas were allowed to feed for
transmission pathways of plague in prairie dog populations. 1 h and then collected, immobilized by cold and CO2 exposure, and
examined using a dissecting microscope. Only those fleas that took an
infectious bloodmeal, evidenced by the presence of fresh red blood in
Materials and Methods the midgut, were included in the experiments. A sample of 10 fleas was
Fleas immediately placed at −80°C for later determination of the initial in-
fectious dose, and the remainder kept and maintained in flea capsules
Fleas were from black-tailed prairie dog colonies at Buffalo Gap
at 21°C, 75% relative humidity. Dilutions of the infectious bloodmeal
National Grasslands, Pennington County, South Dakota. Prairie dog
were spread on blood agar plates to determine the bacteremia level of
epizootics have historically occurred in some portions of the grass-
the blood the fleas had fed upon. When the number of fleas in a cohort
land (Eads et al. 2018), but all fleas were collected from colonies with
was sufficient, a portion of them was fed on sterile rat blood on the
no history or evidence of plague epizootics or detection. Fleas were
same day to serve as uninfected controls.
collected by swabbing prairie dog burrows or by combing fleas from
live-trapped prairie dogs as described (Eads 2017, Eads et al. 2018,
Eads et al. 2021). During a given collection day, fleas (≤200) were Infection and Transmission Monitoring
added to a 50 ml conical tube (perforated cap). By late-morning to After infection, fleas were allowed to feed every other day for 1 h
early-afternoon, fleas were transferred to a new tube containing saw- on sterile rat blood. The remaining blood was then collected from
dust or shredded paper to facilitate flea movement, while reducing the feeding reservoir and spread in 200 µl portions onto blood agar/
entanglement and stress (Hinnebusch et al. 2017b). The new tubes carbenicillin plates. Colony-forming units were counted after 48 h
were capped with a cell strainer insert (Falcon, 70 µm nylon mesh; incubation at 28°C to determine the total number of Y. pestis that
Corning, NY), and then enclosed in a Saf-T-Pak STP-100 Category had been transmitted by the fleas during feeding. All fleas were exam-
A Biosafety shipping container (Inmark Life Sciences, Austell, GA) ined microscopically after the feeding period to determine how many
lined with paper towels moistened with 22–23 ml saturated NaCl had fed normally (fresh red blood in the midgut only), were partially
to maintain approximately 75% relative humidity during shipment. blocked (blood in the esophagus and midgut), or were completely
The container unit was placed in an insulated cooler with 1 or 2 blocked (blood in the esophagus only, anterior to the proventriculus).
plastic ice packs, secondarily sealed in a corrugated box, and shipped Mortality was also recorded. The average number of CFU trans-
overnight to Rocky Mountain Laboratories. Collections were mor- mitted per flea was calculated by dividing the total number of CFU
phologically homogeneous and were identified as O. hirsuta using recovered from the bloodmeal reservoir by the total number of fleas
standard classification keys (Lewis 2002); and in a previous study, that fed from it. At the end of the experiment, surviving fleas were
~99% of fleas collected from the Buffalo Gap prairie dog colonies in placed at −80°C for later determination of their infection rate and
summer-fall were identified as O. hirsuta (Eads et al. 2018). bacterial load. Fleas stored at −80°C were thawed, surface-sterilized,
and individually triturated and plated as described to determine the
Infections infection rate and the bacterial load per flea (Bland et al. 2017).
Upon arrival, fleas were transferred to standard flea capsules con-
taining a layer of sawdust and placed at 10° or 21°C and 75% relative
Results
humidity (Bland et al. 2017). Fecal spots in the shipping tubes were
swabbed and resuspended in a small volume of 10 mM Tris-1 mM Fleas were collected from black-tailed prairie dogs in South Dakota
EDTA pH 8 buffer. The fecal suspension and triturates of pooled fleas during the summer to early fall (July–October) in 2017–2019. The
that had died during shipping were tested by PCR using primers for results of three independent infection experiments with cohorts
the Y. pestis virulence plasmid gene yopH to determine that fleas in of wild-caught O. hirsuta fleas are shown in Fig. 1. In the firstJournal of Medical Entomology, 2022, Vol. 59, No. 3 1055
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Fig. 1. Results of three independent experiments (A–C, corresponding to experiments 1–3 described in the text) that monitored Oropsylla hirsuta fleas following
a single infectious bloodmeal (on day 0). Left panels show mortality of infected fleas (red lines) and, when included, uninfected control fleas (blue lines). Center
panels show bacterial loads in individual infected fleas immediately following their infectious bloodmeal (day 0) and at the end of the experiments. The overall
infection rates (%) are indicated. Right panels show the average number of Yersinia pestis colony-forming units (CFU) transmitted per feeding flea (blue lines;
left y axis) at different times after infection. The number of fleas that fed during each transmission trial is indicated. The incidence of blocked and partially blocked
(PB) fleas that fed during the trials (black and white bars, respectively; right y-axis) is also shown. ND; not determined due to accidental loss of blood sample.
experiment (Fig. 1A), 56 fleas (46 females and 10 males) that had Y. pestis/ml were monitored for 18 d, when only 9 fleas remained
taken a bloodmeal containing 5.1 × 108 Y. pestis/ml were monitored alive. Six of these (67%) were still infected with large numbers of
for 24 d. Ninety-five percent of the 28 fleas alive at the end of the ex- Y. pestis. Even though the median infectious dose (Y. pestis CFU/flea
periment were still infected, verifying that Y. pestis is well able to mul- on day 0) was ten-fold lower than in the first experiment, early-phase
tiply in and stably colonize this flea. Partial and complete blockage transmission by 35 fleas that fed on day 2 after infection was greater.
was evident during the first two weeks after infection. In the 11 post- A lack of positive correlation between bacterial load and early-phase
infection feeds during the 24-day experiment, the 56 fleas fed a cu- transmission efficiency has been observed previously, likely because
mulative total of 403 times, of which 8 feeds (2%) were by blocked the relative degree of localization to the foregut is more important
fleas and 19 feeds (5%) by partially blocked fleas. The 8 blocked flea than the total number of bacteria (Eisen et al. 2015, Hinnebusch
bites were from a minimum of 5 different fleas, so the overall rate et al. 2017a). Partially and completely blocked fleas were again ob-
of complete blockage was ~10% (5–8 blocked fleas out of 56 total served during the first two weeks after infection in this experiment.
fleas infected). Examples of blocked O. hirsuta, showing the classic Of 136 cumulative flea bites over 8 post-infection feeds, 1 (0.7%)
picture of blood prevented from entering the midgut during feeding was by a blocked flea (1/43 fleas blocked = 2% blockage rate) and 12
due to occlusion of the proventricular valve by a Y. pestis biofilm ag- by partially blocked fleas (9%). After the early-phase transmission
gregate, are shown in Fig. 2. Both early-phase transmission (on day on day 2, transmission was again detected on day 9 after infection.
2, the first feeding after the infectious bloodmeal) and later transmis- In a third experiment (Fig. 1C), transmission by 32 fleas (18 fe-
sion (on days 6–16 after infection) were detected, with transmission males and 14 males) that had taken a bloodmeal containing 2.0 × 109
maxima occurring after the early phase (Fig. 1A). Y. pestis/ml was monitored for only 14 d after infection due to small
Two other experiments lasted for only about two weeks each due numbers of fleas and high mortality. Early-phase transmission was
to high mortality of the fleas. In one of these (Fig. 1B), 43 fleas (29 fe- not assessed, but transmission occurred on days 6 and 8 after in-
males and 14 males) that had taken a bloodmeal containing 1.1 – 108 fection. Of 82 total flea bites in the 7 feeds during days 2–14 after1056 Journal of Medical Entomology, 2022, Vol. 59, No. 3
infection, 1 (1.2%) was by a blocked flea (1/32 fleas blocked = 3% In a previous study in which O. hirsuta were also infected using
blockage rate) and 16 (19.5%) by fleas that appeared to be partially highly bacteremic rat blood, early-phase transmission efficiency
blocked. (the probability that an individual flea will transmit during its
Lastly, two other infection experiments assessed early-phase first bloodmeal 1–4 d after infection) was estimated to be 0–4.5%
transmission only, because of rapid high mortality and poor feeding (Wilder et al. 2008a). Proventricular biofilm-dependent transmission
rate of these groups of fleas. The results, along with the early-phase by blocked or partially blocked fleas was not examined in that study.
transmission data from the other experiments, are given in Table 1. Our early-phase transmission results were compatible with the pre-
vious estimate. Transmission was detected in three of four trials in
which 35–66 fleas fed simultaneously from a sterile rat blood reser-
Discussion
voir 2 d after their infectious bloodmeal.
Two aspects of the vector competence of O. hirsuta were evaluated The most significant finding of this study was that O. hirsuta de-
in this study. The first was the ability of Y. pestis to infect, multiply veloped proventricular blockage and was able to transmit Y. pestis
in, and stably colonize this flea species after a single infectious rat by that mechanism much more rapidly and efficiently than was re-
bloodmeal. The O. hirsuta digestive tract proved to be a permissive ported in the only previous study that examined this. Several dif-
environment for Y. pestis, as a high percentage of fleas remained ferences between the two studies could account for the disparate
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colonized by large numbers of bacteria for at least 2–3 wk after in- results. In the earlier study (Eskey and Haas 1940), a total of 70
fection. A second aspect concerned the ability of infected fleas to O. hirsuta were infected by feeding on guinea pigs with different
transmit Y. pestis during this period. Cohorts of fleas were allowed levels of bacteremia. More than one infectious bloodmeal was often
to feed once on highly bacteremic rat blood, and then early-phase required, as less than 1/3rd of the fleas were infected after a single
transmission at the first maintenance feed 2 d after infection as feed, indicating that the guinea pig bacteremia was only about 107
well as later, proventricular blockage-dependent transmission were Y. pestis/ml (Engelthaler et al. 2000, Lorange et al. 2005). After
monitored. O. hirsuta proved to be a competent vector by both their positive infection status was confirmed, fleas were fed individ-
mechanisms. ually on naïve guinea pigs daily. No early-phase transmission was
Fig. 2. Typical examples of complete proventricular blockage in Oropsylla hirsuta fleas. (A, B) Two fleas photographed immediately after attempting to feed on
day 8 after infection. Low and higher magnification images are shown. White arrows delineate the fresh red blood in the esophagus that was blocked from en-
tering the midgut. (C, D) Fluorescence microscopy images of the digestive tract of two other O. hirsuta fleas dissected 8 d or 16 d after infection with Yersinia
pestis expressing green fluorescent protein. Both fleas had dense bacterial aggregates in the proventriculus (PV) and esophagus (E). White arrows indicate por-
tions of bloodmeal that were unable to enter the midgut (MG), consistent with complete proventricular blockage.
Table 1. Summary of results of four experiments evaluating early-phase transmission (2 days post-infection) of Yersinia pestis by
Oropsylla hirsuta prairie dog fleas
Expt.a No. fleas fed No. blocked fleas No. partially blocked fleas Total CFU transmitted CFU transmitted per flea (avg.)
1 (Fig. 1A) 47 1 1 5 0.1
2 (Fig. 1B) 35 0 6 610 17
4 66 1 6 612 9
5 49 0 1 0 0
Cumulative: 197 2 14 1,227 6
a
Expt. 3 (Fig. 1C) not listed because of accidental loss of the day 2 blood sample. The average number of colony-forming units (CFU) transmitted per flea was
calculated by dividing the total CFU transmitted by the number of fleas that fed.Journal of Medical Entomology, 2022, Vol. 59, No. 3 1057
observed, which is not unexpected because multiple flea bites by fleas induce PIER-associated enhancement of early-phase transmission;
that had previously fed on blood containing ≥108 Y. pestis/ml is re- and 2) the number of CFU transmitted by early-phase O. hirsuta
quired for efficient transmission by this mode (Boegler et al. 2016). fleas is below the LD50 of Y. pestis to prairie dogs. For example, we
Subsequently, only three instances of transmission were recorded, found that, on average, 100 d after infection. Chronic infection and flea bite. Plague epizootics can decimate colonies even though prairie
proventricular blockage rates were not assessed. In contrast, we rou- dogs can be more resistant to plague and require transmission of a
tinely detected proventricular blockage and transmission during the much larger infectious dose, ranging from 50,000 CFU in
first two weeks after O. hirsuta were infected by feeding on rat blood different populations (Cully and Williams 2001, Rocke et al. 2012,
containing 108 to 109 Y. pestis/ml. Busch et al. 2013, Russell et al. 2019). Thus, unless the flea index was
Different characteristics of mammalian blood types and how high, early-phase transmission might frequently lead to seroconver-
they are digested in the flea gut can influence initial infection and sion and protective immunity, removing individuals from the suscep-
early-phase transmission. Notably, an infectious rat or guinea pig tible population rather than driving an epizootic. On the other hand,
bloodmeal leads to post-infection esophageal reflux (PIER), in which our results of up to 10% blockage rate within two weeks after infec-
incompletely digested blood and hemoglobin crystals mixed with tion indicate that transmission by blocked O. hirsuta may be much
Y. pestis is refluxed into the flea foregut (proventriculus and esoph- more significant than heretofore appreciated. Other considerations
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agus) (Bland et al. 2018). PIER rather than true biofilm-dependent supporting this are that 1) lower bacteremia levels in the host are
blockage is likely responsible for the foregut obstruction diagnosed needed for blockage development than for early-phase transmission
as partial or complete blockage that we observed in early-phase (Engelthaler et al. 2000, Lorange et al. 2005, Boegler et al. 2016);
transmission trials 2 d after infection. In the rat flea X. cheopis and and 2) blocked fleas transmit greater CFU numbers than early-phase
the ground squirrel flea Oropsylla montana (Baker: Siphonaptera: fleas (Lorange et al. 2005, Hinnebusch et al. 2017b, Bland et al.
Ceratophyllidae), PIER correlates with enhanced early-phase trans- 2018). For example, we found that on average O. hirsuta beyond
mission following an infectious rat bloodmeal but does not influ- the early phase transmitted up to ~100 CFU (Fig. 1). It is important
ence chronic infection or blockage rates, although it may shorten to note that the number of CFU transmitted by individual fleas can
the time required for biofilm-dependent blockage to develop (Bland be highly variable and does not exhibit a normal (Gaussian) distri-
et al. 2018). Early-phase transmission efficiency of O. hirsuta has bution (Lorange et al. 2005). The few blocked and partially blocked
only been assessed using rat blood [this study and (Wilder et al. fleas in the groups that fed in the later transmission trials probably
2008a)]; it is not known if prairie dog blood would induce early- account for virtually all the CFU transmitted. Thus, averages based
phase transmission-enhancing PIER. on group feedings show general relative efficiencies of the two trans-
O. hirsuta was somewhat problematic to maintain in the labo- mission modes; it would be necessary to determine transmission ef-
ratory, as others have found previously (Wilder et al. 2008a). They ficiencies at the individual flea level for statistics-based comparisons
required maintenance feeds at least every other day and, even so, of absolute efficiencies. Given the significant effect of blood source
the background mortality was high even for uninfected control fleas. on early-phase transmission (Bland et al. 2018), it would also be of
Whether this was due to stress experienced during collection and interest to compare relative transmission efficiencies of O. hirsuta
shipping or to maladaption to our laboratory conditions (e.g., the infected using prairie dog blood.
artificial feeding device and a different blood source than they are Although O. hirsuta is clearly a competent vector, its vector
used to), or both, is not known. Uninfected X. cheopis and O. mon- efficiency appears to be less than that of other rodent fleas such as
tana from established laboratory colonies exhibit much lower back- X. cheopis and O. montana. Early-phase transmission efficiency
ground mortality under the same experimental conditions. We have of the latter two flea species is about twice that of O. hirsuta (Eisen
found previously that high background mortality of uninfected con- et al. 2006, Eisen et al. 2007, Wilder et al. 2008a). In addition,
trol fleas is associated with even greater mortality of the infected fleas although O. hirsuta becomes chronically infected at comparable
and a significant underestimation of the proventricular blockage rate rates and bacterial loads as X. cheopis and O. montana infected
(Engelthaler et al. 2000, Hinnebusch et al. 2017b). In this regard, the similarly, both the blockage rate and numbers of CFU transmitted
results of the first experiment (Fig. 1A) are probably the most reli- by O. hirsuta were less than what has been documented for the
able in terms of proventricular blockage-dependent transmission, as other two flea species (blockage rates ~40%, for X. cheopis and
this group of fleas was the healthiest. ~20% for O. montana; and an average of 250 to >10,000 CFU
Based on the previous study of early-phase transmission efficiency transmitted per flea by groups of X. cheopis and O. montana 1–4
of O. hirsuta (Wilder et al. 2008a) and the reported low incidence of weeks after infection) (Lorange et al. 2005, Hinnebusch et al.
later-phase, proventricular blockage dependent transmission (Eskey 2017b). Nevertheless, because blocked fleas attempt to feed re-
and Haas 1940), recent models of the role of flea-borne transmission peatedly during the few days before they starve to death, the cu-
in prairie dog epizootics have assumed that only early-phase trans- mulative dose delivered by a single blocked O. hirsuta is probably
mission would be important (Webb et al. 2006, Buhnerkempe et al. high enough to result in disease even among moderately resistant
2011, Richgels et al. 2016). Our results demonstrate that O. hirsuta prairie dog populations. The early-phase transmission efficiency
can become blocked and transmit Y. pestis by this mechanism as of the other prominent prairie dog flea, O. tuberculata cynomuris,
efficiently as early-phase transmission, if not more so, and should was found to be greater than that of O. hirsuta (Wilder et al.
be incorporated in revised models. Several other considerations are 2008b), and its ability to block also deserves to be reexamined.
related to the relative importance of the two transmission modes Pulex simulans (Baker; Siphonaptera: Pulicidae), a generalist
in prairie dog plague dynamics. The previous early-phase study of flea that parasitizes different mammals can also be abundant on
O. hirsuta calculated efficiency based on transmission to highly prairie dogs (Tripp et al. 2009, Eads et al. 2015), but its vector
susceptible laboratory mice (LD50 < 10 Y. pestis CFU), and mice competence is unknown. Finally, although there is good evidence
that developed terminal plague as well as mice that seroconverted that prairie dog epizootics require a normal flea burden (Tripp
without developing disease were included. This may be an over- et al. 2017, Biggins et al. 2021), non-flea-borne transmission path-
estimate of the natural situation if 1) prairie dog blood does not ways may also be important. For example, oral transmission via1058 Journal of Medical Entomology, 2022, Vol. 59, No. 3
direct contact with heavily contaminated respiratory secretions Eads, D. A., D. E. Biggins, M. F. Antolin, D. H. Long, K. P. Huyvaert, and
among these highly social animals and cannibalism of plague- K. L. Gage. 2015. Prevalence of the generalist flea Pulex simulans on black-
infected burrow mates also likely contribute to driving epizootics tailed prairie dogs (Cynomys ludovicianus) in New Mexico, USA: the im-
portance of considering imperfect detection. J. Wildl. Dis. 51: 498–502.
(Richgels et al. 2016, Melman et al. 2018, Russell et al. 2021).
Eads, D. A., D. E. Biggins, J. Bowser, J. C. McAllister, R. L. Griebel, E. Childers,
T. M. Livieri, C. Painter, L. S. Krank, and K. Bly. 2018. Resistance to
deltamethrin in prairie dog (Cynomys ludovicianus) fleas in the field and
Acknowledgments
in the laboratory. J. Wildl. Dis. 54: 745–754.
This work was supported by the Intramural Research Program of the NIAID, Eads, D. A., M. R. Matchett, J. E. Poje, and D. E. Biggins. 2021. Comparison
NIH. Field work was supported by the National Park Service, U.S. Fish and of flea sampling methods and Yersinia pestis detection on prairie dog col-
Wildlife Service, U.S. Geological Survey, U.S. Forest Service, Prairie Wildlife onies. Vector Borne Zoonotic Dis. 21: 753–761.
Research, World Wildlife Fund, and Colorado State University and was con- Eisen, R. J., S. W. Bearden, A. P. Wilder, J. A. Montenieri, M. F. Antolin,
ducted under U.S. Geological Survey IACUC protocol 2015-07 (Fort Collins and K. L. Gage. 2006. Early-phase transmission of Yersinia pestis by un-
Science Center, Colorado). Field work was also supported by Grant/Cooperative blocked fleas as a mechanism explaining rapidly spreading plague epizo-
Agreement Number G14AC00403 from the U.S. Geological Survey. We thank otics. Proc. Natl. Acad. Sci. U. S. A. 103: 15380–15385.
Travis Livieri, Jeff Shannon, Chris Bosio, and Clay Jarrett for manuscript re- Eisen, R. J., A. P. Wilder, S. W. Bearden, J. A. Montenieri, and K. L. Gage.
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view. The findings and conclusions in this article are those of the authors and 2007. Early-phase transmission of Yersinia pestis by unblocked Xenopsylla
do not necessarily represent the views of the U.S. Fish and Wildlife Service. Any cheopis (Siphonaptera: Pulicidae) is as efficient as transmission by blocked
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