Host traits,identity, and ecological conditions predict consistent flea abundance and prevalence on free-living California ground squirrels

Understanding why some individuals are more prone to carry parasites and spread diseases than others is a key question in biology. Although epidemiologists and disease ecologists increasingly recognize that individuals of the same species can vary tremendously in their relative contributions to the emergence of diseases, very few empirical studies systematically assess consistent individual differences in parasite loads within populations over time. Two species of fleas (Oropsylla montana and Hoplopsyllus anomalous) and their hosts, California ground squirrels (Otospermophilus beecheyi), form a major complex for amplifying epizootic plague in the western United States. Understanding its biology is primarily of major ecological importance and is also relevant to public health. Here, we capitalize on a long-term data set to explain flea incidence on California ground squirrels at Briones Regional Park in Contra Costa County, USA. In a 7 year study, we detected 42,358 fleas from 2,759 live trapping events involving 803 unique squirrels from two free-living populations that differed in the amount of human disturbance in those areas. In general, fleas were most abundant and prevalent on adult males, on heavy squirrels, and at the pristine site, but flea distributions varied among years, with seasonal conditions (e.g., temperature, rainfall, humidity), temporally within summers, and between flea species. Although on-host abundances of the two flea species were positively correlated, each flea species occupied a distinctive ecological niche. The common flea (O. montana) occurred primarily on adults in cool, moist conditions in early summer whereas the rare flea (H. anomalous) was mainly on juveniles in hot, dry conditions in late summer. Beyond this, we uncovered significantly repeatable and persistent effects of host individual identity on flea loads, finding consistent individual differences among hosts in all parasite measures. Taken together, we reveal multiple determinants of parasites on free-living mammals, including the underappreciated potential for host heterogeneity – within populations – to structure the emergence of zoonotic diseases such as bubonic plague.     

Local factors associated with on‐host flea distributions on prairie dog colonies

Outbreaks of plague, a flea‐vectored bacterial disease, occur periodically in prairie dog populations in the western United States. In order to understand the conditions that are conducive to plague outbreaks and potentially predict spatial and temporal variations in risk, it is important to understand the factors associated with flea abundance and distribution that may lead to plague outbreaks. We collected and identified 20,041 fleas from 6,542 individual prairie dogs of four different species over a 4‐year period along a latitudinal gradient from Texas to Montana. We assessed local climate and other factors associated with flea prevalence and abundance, as well as the incidence of plague outbreaks. Oropsylla hirsuta, a prairie dog specialist flea, and Pulex simulans, a generalist flea species, were the most common fleas found on our pairs. High elevation pairs in Wyoming and Utah had distinct flea communities compared with the rest of the study pairs. The incidence of prairie dogs with Yersinia pestis detections in fleas was low (n = 64 prairie dogs with positive fleas out of 5,024 samples from 4,218 individual prairie dogs). The results of our regression models indicate that many factors are associated with the presence of fleas. In general, flea abundance (number of fleas on hosts) is higher during plague outbreaks, lower when prairie dogs are more abundant, and reaches peak levels when climate and weather variables are at intermediate levels. Changing climate conditions will likely affect aspects of both flea and host communities, including population densities and species composition, which may lead to changes in plague dynamics. Our results support the hypothesis that local conditions, including host, vector, and environmental factors, influence the likelihood of plague outbreaks, and that predicting changes to plague dynamics under climate change scenarios will have to consider both host and vector responses to local factors.     

Field efficacy of rodent bait containing the systemic insecticide imidacloprid against the fleas of California ground squirrels

The efficacy of rodent bait containing the insecticide imidacloprid was evaluated for controlling fleas on the California ground squirrel, Spermophilus beecheyi. The bait was designed to deliver an oral dose of insecticide resulting in flea mortality when obtaining a blood meal. During the five‐week trial, performed at Vandenberg Air Force Base, Santa Barbara County, CA, a spot‐baiting technique was used to apply bait to ground squirrel burrows. Bait was applied six times throughout the trial. Results indicated that the use of a host‐targeted bait was effective in significantly reducing the flea burden on S. beecheyi. Efficacy at reducing flea abundance was near 100% at both day 15 and day 29 of the trial. Use of the bait also reduced the prevalence of flea‐infested S. beecheyi. Our results indicate that the use of rodent bait containing insecticide could provide an effective, economical method of controlling the fleas of S. beecheyi, the primary vectors of human plague in California.     

Dermal Neutrophil,Macrophage and Dendritic Cell Responses to Yersinia pestis Transmitted by Fleas

Yersinia pestis, the causative agent of plague, is typically transmitted by the bite of an infected flea. Many aspects of mammalian innate immune response early after Y. pestis infection remain poorly understood. A previous study by our lab showed that neutrophils are the most prominent cell type recruited to the injection site after intradermal needle inoculation of Y. pestis, suggesting that neutrophil interactions with Y. pestis may be important in bubonic plague pathogenesis. In the present study, we developed new tools allowing for intravital microscopy of Y. pestis in the dermis of an infected mouse after transmission by its natural route of infection, the bite of an infected flea. We found that uninfected flea bites typically induced minimal neutrophil recruitment. The magnitude of neutrophil response to flea-transmitted Y. pestis varied considerably and appeared to correspond to the number of bacteria deposited at the bite site. Macrophages migrated towards flea bite sites and interacted with small numbers of flea-transmitted bacteria. Consistent with a previous study, we observed minimal interaction between Y. pestis and dendritic cells; however, dendritic cells did consistently migrate towards flea bite sites containing Y. pestis. Interestingly, we often recovered viable Y. pestis from the draining lymph node (dLN) 1 h after flea feeding, indicating that the migration of bacteria from the dermis to the dLN may be more rapid than previously reported. Overall, the innate cellular host responses to flea-transmitted Y. pestis differed from and were more variable than responses to needle-inoculated bacteria. This work highlights the importance of studying the interactions between fleas, Y. pestis and the mammalian host to gain a better understanding of the early events in plague pathogenesis.     

Changes in Flea (Siphonaptera) Vector Activity in the Siberian Natural Plague Foci

Comparative analysis of vector activity of fleas in the Siberian natural plague foci was carried out during two long-term periods of experimental studies: 1967–1980 and 1983–2007. The data on block formation frequency in adult fleas infected with Yersinia pestis were analyzed for 127 experiments with 15 flea species and subspecies. The vector activity of fleas in all the Siberian plague foci (Altai, Tuva, and Transbaikalia) has increased over a rather short time period of 30–40 years. The frequencies of flea blocking were significantly different (P < 0.001) between the analyzed periods in all the three plague foci.

     

Flea-Associated Bacterial Communities across an Environmental Transect in a Plague-Endemic Region of Uganda

The vast majority of human plague cases currently occur in sub-Saharan Africa. The primary route of transmission of Yersinia pestis, the causative agent of plague, is via flea bites. Non-pathogenic flea-associated bacteria may interact with Y. pestis within fleas and it is important to understand what factors govern flea-associated bacterial assemblages. Six species of fleas were collected from nine rodent species from ten Ugandan villages between October 2010 and March 2011. A total of 660,345 16S rRNA gene DNA sequences were used to characterize bacterial communities of 332 individual fleas. The DNA sequences were binned into 421 Operational Taxonomic Units (OTUs) based on 97% sequence similarity. We used beta diversity metrics to assess the effects of flea species, flea sex, rodent host species, site (i.e. village), collection date, elevation, mean annual precipitation, average monthly precipitation, and average monthly temperature on bacterial community structure. Flea species had the greatest effect on bacterial community structure with each flea species harboring unique bacterial lineages. The site (i.e. village), rodent host, flea sex, elevation, precipitation, and temperature also significantly affected bacterial community composition. Some bacterial lineages were widespread among flea species (e.g. Bartonella spp. and Wolbachia spp.), but each flea species also harbored unique bacterial lineages. Some of these lineages are not closely related to known bacterial diversity and likely represent newly discovered lineages of insect symbionts. Our finding that flea species has the greatest effect on bacterial community composition may help future investigations between Yersinia pestis and non-pathogenic flea-associated bacteria. Characterizing bacterial communities of fleas during a plague epizootic event in the future would be helpful.     

Transmission Efficiency of Two Flea Species (<Emphasis Type="Italic">Oropsylla tuberculata cynomuris</Emphasis> and <Emphasis Type="Italic">Oropsylla hirsuta</Emphasis>) Involved in Plague Epizootics among Prairie Dogs

Plague, caused by Yersinia pestis, is an exotic disease in North America circulating predominantly in wild populations of rodents and their fleas. Black-tailed prairie dogs (Cynomys ludovicianus) are highly susceptible to infection, often experiencing mortality of nearly all individuals in a town as a result of plague. The fleas of black-tailed prairie dogs are Oropsylla tuberculata cynomuris and Oropsylla hirsuta. We tested the efficiency of O. tuberculata cynomuris to transmit Y. pestis daily from 24 to 96 h postinfection and compared it to previously collected data for O. hirsuta. We found that O. tuberculata cynomuris has over threefold greater transmission efficiency (0.18 infected fleas transmit Y. pestis at 24 h postinfection) than O. hirsuta (0.05 fleas transmit). Using a simple model of flea-borne transmission, we combine these laboratory measurements with field data on monthly flea loads to compare the seasonal vectorial capacity of these two flea species. Coinciding with seasonal patterns of flea abundance, we find a peak in potential for flea-borne transmission in March, during high O. tuberculata cynomuris abundance, and in September–October when O. hirsuta is common. Our findings may be useful in determining the timing of insecticidal dusting to slow plague transmission in black-tailed prairie dogs.     

The absence of concordant population genetic structure in the black‐tailed prairie dog and the flea,Oropsylla hirsuta,with implications for the spread of Yersinia pestis

The black‐tailed prairie dog (Cynomys ludovicianus) is a keystone species on the mid‐ and short‐grass prairies of North America. The species has suffered extensive colony extirpations and isolation as a result of human activity including the introduction of an exotic pathogen, Yersinia pestis, the causative agent of sylvatic plague. The prairie dog flea, Oropsylla hirsuta, is the most common flea on our study colonies in north‐central Montana and it has been shown to carry Y. pestis. We used microsatellite markers to estimate the level of population genetic concordance between black‐tailed prairie dogs and O. hirsuta in order to determine the extent to which prairie dogs are responsible for dispersing this potential plague vector among prairie dog colonies. We sampled fleas and prairie dogs from six prairie dog colonies in two regions separated by about 46 km. These colonies were extirpated by a plague epizootic that began months after our sampling was completed in 2005. Prairie dogs showed significant isolation‐by‐distance and a tendency toward genetic structure on the regional scale that the fleas did not. Fleas exhibited higher estimated rates of gene flow among prairie dog colonies than the prairie dogs sampled from the same colonies. While the findings suggested black‐tailed prairie dogs may have contributed to flea dispersal, we attributed the lack of concordance between the population genetic structures of host and ectoparasite to additional flea dispersal that was mediated by mammals other than prairie dogs that were present in the prairie system.     

Nutrient depletion may trigger the Yersinia pestis OmpR‐EnvZ regulatory system to promote flea‐borne plague transmission

The flea’s lumen gut is a poorly documented environment where the agent of flea‐borne plague, Yersinia pestis, must replicate to produce a transmissible infection. Here, we report that both the acidic pH and osmolarity of the lumen’s contents display simple harmonic oscillations with different periods. Since an acidic pH and osmolarity are two of three known stimuli of the OmpR‐EnvZ two‐component system in bacteria, we investigated the role and function of this Y. pestis system in fleas. By monitoring the in vivo expression pattern of three OmpR‐EnvZ‐regulated genes, we concluded that the flea gut environment triggers OmpR‐EnvZ. This activation was not, however, correlated with changes in pH and osmolarity but matched the pattern of nutrient depletion (the third known stimulus for OmpR‐EnvZ). Lastly, we found that the OmpR‐EnvZ and the OmpF porin are needed to produce the biofilm that ultimately obstructs the flea’s gut and thus hastens the flea‐borne transmission of plague. Taken as a whole, our data suggest that the flea gut is a complex, fluctuating environment in which Y. pestis senses nutrient depletion via OmpR‐EnvZ. Once activated, the latter triggers a molecular program (including at least OmpF) that produces the biofilm required for efficient plague transmission.     

Potential Effects of Environmental Conditions on Prairie Dog Flea Development and Implications for Sylvatic Plague Epizootics

Fleas are common ectoparasites of vertebrates worldwide and vectors of many pathogens causing disease, such as sylvatic plague in prairie dog colonies. Development of fleas is regulated by environmental conditions, especially temperature and relative humidity. Development rates are typically slower at low temperatures and faster at high temperatures, which are bounded by lower and upper thresholds where development is reduced. Prairie dogs and their associated fleas (mostly Oropsylla spp) live in burrows that moderate outside environmental conditions, remaining cooler in summer and warmer in winter. We found burrow microclimates were characterized by stable daily temperatures and high relative humidity, with temperatures increasing from spring through summer. We previously showed temperature increases corresponded with increasing off-host flea abundance. To evaluate how changes in temperature could affect future prairie dog flea development and abundance, we used development rates of O. montana (a species related to prairie dog fleas), determined how prairie dog burrow microclimates are affected by ambient weather, and combined these results to develop a predictive model. Our model predicts burrow temperatures and flea development rates will increase during the twenty-first century, potentially leading to higher flea abundance and an increased probability of plague epizootics if Y. pestis is present.

     

Efficacy of a fipronil bait in reducing the number of fleas (Oropsylla spp.) infesting wild black‐tailed prairie dogs

Bubonic plague (Yersinia pestis) is a deadly zoonosis with black‐tailed prairie dogs (Cynomys ludovicianus) as a reservoir host in the United States. Systemic insecticides are a promising means of controlling the vectors, Oropsylla spp. fleas, infesting these prairie dogs, subsequently disrupting the Y. pestis cycle. The objective of this study was to conduct a field trial evaluating the efficacy of a grain rodent bait containing fipronil (0.005%) against fleas infesting prairie dogs. The study was performed in Larimer County, CO, where bait was applied to a treatment area containing a dense prairie dog population, three times over a three‐week period. Prairie dogs were captured and combed for fleas during four study periods (pre‐, mid‐, 1^st post‐, and 2^nd post‐treatment). Results indicated the use of bait containing fipronil significantly reduced flea burden. The bait containing fipronil was determined to reduce the mean number of fleas per prairie dog >95% for a minimum of 52 days post‐initial treatment application and 31 days post‐final treatment application. These results suggest the potential for this form of treatment to reduce flea population density on prairie dogs, and subsequently plague transmission, among mammalian hosts across the United States and beyond.     

Plague studies in California: a review of long‐term disease activity,flea‐host relationships and plague ecology in the coniferous forests of the Southern Cascades and northern Sierra Nevada mountains

We review 28 years of long‐term surveillance (1970–1997) for plague activity among wild rodents from ten locations within three coniferous forest habitat types in the northern Sierra Nevada and the Southern Cascade mountains of northeastern California. We identify rodent hosts and their fleas and document long‐term plague activity in each habitat type. The highest seroprevalence for Yersinia pestis occurred in the chipmunks, Tamias senex and T. quadrimaculatus, and the pine squirrel, Tamiasciurus douglasii. The most commonly infected fleas were Ceratophyllus ciliatus and Eumolpianus eumolpi from chipmunks and Oropsylla montana and O. idahoensis from ground squirrels. Serological surveillance demonstrated that populations of T. senex, T. quadrimaculatus and T. douglasii are moderately resistant to plague, survive infection, and are, therefore, good sentinels for plague activity. Recaptured T. senex and T. quadrimaculatus showed persistence of plague antibodies and evidence of re‐infection over a two year period. These rodent species, their fleas, and the ecological factors common to the coniferous forest habitats likely promote the maintenance of plague foci in northeastern California.     

Yersinia pestis infection and laboratory conditions alter flea-associated bacterial communities

We collected Oropsylla montana from rock squirrels, Spermophilus varigatus, and infected a subset of collected fleas with Yersinia pestis, the etiological agent of plague. We used bar-tagged DNA pyrosequencing to characterize bacterial communities of wild, uninfected controls and infected fleas. Bacterial communities within Y. pestis-infected fleas were substantially more similar to one another than communities within wild or control fleas, suggesting that infection alters the bacterial community in a directed manner such that specific bacterial lineages are severely reduced in abundance or entirely eliminated from the community. Laboratory conditions also significantly altered flea-associated bacterial communities relative to wild communities, but much less so than Y. pestis infection. The abundance of Firmicutes decreased considerably in infected fleas, and Bacteroidetes were almost completely eliminated from both the control and infected fleas. Bartonella and Wolbachia were unaffected or responded positively to Y. pestis infection.     

The Effect of Initial Infection of <Emphasis Type="Italic">Citellophilus tesquorum altaicus</Emphasis> Ioff, 1936 (Siphonaptera: Ceratophyllidae) with <Emphasis Type="Italic">Yersinia pestis</Emphasis> on Its Alimentary Activity,Mortality, and Biofilm Formation

Alimentary activity and mortality was assessed in fleas Citellophilus tesquorum altaicus non-infected with Yersinia pestis and those with initial infection levels 50 and 100% during feeding on a non-specific host (white mice). The presence of the plague pathogen in fleas significantly stimulated their feeding activity, especially in females. No effect of infection on flea mortality was observed. At the same time, male fleas died more frequently than females.     

Rodent-flea-plague relationships at the higher elevations of San Diego County, California.

Rodent-flea-plague relationships were examined at sites located at higher elevations of San Diego County during 1991-2002. The most frequently sampled rodents were the California ground squirrel, Spermophilus beecheyi, and white-footed mice, Peromyscus spp. Higher seasonal mean dependent variables (prevalences of squirrels and indices of O. montana), higher mean amounts of seasonal rainfall, and lower seasonal mean ambient temperatures were found at sites during 1991--1993 compared with 2000--2002. Levels of plague antibody indicate that squirrels seroconverted at most sites during spring/early summer when exposed to infected fleas when cleaning out burrows following hibernation, when interacting more with other squirrels occurred establishing home ranges, and when dispersing juveniles occupied abandoned burrows containing old nests. The majority of squirrels (35/41) were reported seropositive at Heise County Park (elev. 1,281 m) during 1996, 2001, and 2002. This may reflect fewer fleas biting squirrels more often when squirrels spent more time below-ground in order to avoid higher ambient temperatures. In contrast, most squirrels (12/13) were reported seropositive at two higher sites (elev. 1,584 and 1,641 m) on Palomar Mountain during 1993--1996 when there were higher mean dependent variables and amounts of seasonal rainfall compared with 1999--2002. This increased rainfall may have benefited host and flea populations and thus plague transmission. Seasonal indices were usually reduced after dusting squirrel burrows with 2% diazinon dust. Findings also indicated that S. beecheyi is a primary reservoir for plague at the higher elevations of these and other sites in southern California, and that the deer mouse, Peromyscus maniculatus, and other Peromyscus spp. were not major host reservoirs at the sites sampled.     

Evaluation of the Murine Immune Response to Xenopsylla cheopis Flea Saliva and Its Effect on Transmission of Yersinia pestis

Background/Aims

Arthropod-borne pathogens are transmitted into a unique intradermal microenvironment that includes the saliva of their vectors. Immunomodulatory factors in the saliva can enhance infectivity; however, in some cases the immune response that develops to saliva from prior uninfected bites can inhibit infectivity. Most rodent reservoirs of Yersinia pestis experience fleabites regularly, but the effect this has on the dynamics of flea-borne transmission of plague has never been investigated. We examined the innate and acquired immune response of mice to bites of Xenopsylla cheopis and its effects on Y. pestis transmission and disease progression in both naïve mice and mice chronically exposed to flea bites.

Methods/Principal Findings

The immune response of C57BL/6 mice to uninfected flea bites was characterized by flow cytometry, histology, and antibody detection methods. In naïve mice, flea bites induced mild inflammation with limited recruitment of neutrophils and macrophages to the bite site. Infectivity and host response in naïve mice exposed to flea bites followed immediately by intradermal injection of Y. pestis did not differ from that of mice infected with Y. pestis without prior flea feeding. With prolonged exposure, an IgG1 antibody response primarily directed to the predominant component of flea saliva, a family of 36–45 kDa phosphatase-like proteins, occurred in both laboratory mice and wild rats naturally exposed to X. cheopis, but a hypersensitivity response never developed. The incidence and progression of terminal plague following challenge by infective blocked fleas were equivalent in naïve mice and mice sensitized to flea saliva by repeated exposure to flea bites over a 10-week period.

Conclusions

Unlike what is observed with many other blood-feeding arthropods, the murine immune response to X. cheopis saliva is mild and continued exposure to flea bites leads more to tolerance than to hypersensitivity. The immune response to flea saliva had no detectable effect on Y. pestis transmission or plague pathogenesis in mice.     

The phenomenon of <Emphasis Type="Italic">Yersinia pestis</Emphasis> biofilm formation in the organism of fleas

The paper considers, for the first time, the formation of the extracellular matrix envelope (EME), or the biofilm, by Yersinia pestis as the basis determining the nature of interaction of the plague agent with the flea organism. The significance of the insect proventriculus in the process of biofilm formation is shown. The ultrastructure of the conglomerates of the plague microbe in the flea proventriculus and midgut was studied and the uniform mechanism of their formation was established. The role of Yersinia pestis biofilm in preservation of the plague microbe in the intestine of ectoparasites and in the soil of rodent burrows was discussed. PCR analysis confirmed the presence of the agent in plague infected corpses and flea feces stored at +8−10°C for 7 years and 9 months.