Yersinia pestis Δail Mutants Are Not Susceptible to Human Complement Bactericidal Activity in the Flea.

Ail confers serum resistance in humans and is a critical virulence factor of Y. pestis, the causative agent of plague. Here, the contribution of Ail for Y. pestis survival in the flea vector was examined. Rat or human but not mouse sera were bactericidal against a Y. pestis Δail mutant at 28°C in vitro. Complement components deposited rapidly on the Y. pestis surface as measured by immunofluorescent microscopy. Ail reduced the amount of active C3b on the Y. pestis surface. Human sera retained bactericidal activity against a Y. pestis Δail mutant in the presence of mouse sera. However, in the flea vector, the serum protective properties of Ail were not required. Flea colonization studies using murine sera and Y. pestis KIM6+ wild type, a Δail mutant, and the Δail/ail+ control showed no differences in bacterial prevalence or numbers during the early stage of flea colonization. Similarly, flea studies with human blood showed Ail was not required for serum resistance. Finally, a variant of Ail (AilF100V E108_S109insS) from a human serum-sensitive Y. pestis subsp. microtus bv. Caucasica 1146 conferred resistance to human complement when expressed in the Y. pestis KIM6+ Δail mutant. This indicated that Ail activity was somehow blocked, most likely by lipooligosaccharide, in this serum sensitive strain. IMPORTANCE This work contributes to our understanding of how highly virulent Y. pestis evolved from its innocuous enteric predecessor. Among identified virulence factors is the attachment invasion locus protein, Ail, that is required to protect Y. pestis from serum complement in all mammals tested except mice. Murine sera is not bactericidal. In this study, we asked, is bactericidal sera from humans active in Y. pestis colonized fleas? We found it was not. The importance of this observation is that it identifies a protective niche for the growth of serum sensitive and nonsensitive Y. pestis strains.

Drivers of flea (Siphonaptera) community structure in sympatric wild carnivores in northwestern Mexico.

Host identity, habitat type, season, and interspecific interactions were investigated as determinants of the community structure of fleas on wild carnivores in northwestern Mexico. A total of 540 fleas belonging to seven species was collected from 64 wild carnivores belonging to eight species. We found that the abundances of some flea species are explained by season and host identity. Pulex irritans and Echidnophaga gallinacea abundances were significantly higher in spring than in fall season. Flea communities on carnivore hosts revealed three clusters with a high degree of similarity within each group that was explained by the flea dominance of E. gallinacea, P. simulans, and P. irritans across host identity. Flea abundances did not differ statistically among habitat types. Finally, we found a negative correlation between the abundances of three flea species within wild carnivore hosts. Individual hosts with high loads of P. simulans males usually had significantly lower loads of P. irritans males or tend to have lower loads of E. gallinacea fleas and vice-versa. Additionally, the logistic regression model showed that the presence of P. simulans males is more likely to occur in wild carnivore hosts in which P. irritans males are absent and vice-versa. These results suggest that there is an apparent competitive exclusion among fleas on wild carnivores. The study of flea community structure on wild carnivores is important to identify the potential flea vectors for infectious diseases and provide information needed to design programs for human health and wildlife conservation.

Small-Scale Die-Offs in Woodrats Support Long-Term Maintenance of Plague in the U.S. Southwest.

Our longitudinal study of plague dynamics was conducted in north-central New Mexico to identify which species in the community were infected with plague, to determine the spatial and temporal patterns of the dynamics of plague epizootics, and to describe the dynamics of Yersinia pestis infection within individual hosts. A total of 3156 fleas collected from 535 small mammals of 8 species were tested for Y. pestis DNA. Nine fleas collected from six southern plains woodrats (Neotoma micropus) and from one rock squirrel (Otospermophilus variegatus) were positive for the pla gene of Y. pestis. None of 127 fleas collected from 17 woodrat nests was positive. Hemagglutinating antibodies to the Y. pestis-specific F1 antigen were detected in 11 rodents of 6 species. All parts of the investigated area were subjected to local disappearance of woodrats. Despite the active die-offs, some woodrats always were present within the relatively limited endemic territory and apparently were never exposed to plague. Our observations suggest that small-scale die-offs in woodrats can support maintenance of plague in the active U.S. Southwestern focus.

Infection Prevalence, Bacterial Loads, and Transmission Efficiency in Oropsylla montana (Siphonaptera: Ceratophyllidae) One Day After Exposure to Varying Concentrations of Yersinia pestis in Blood.

Unblocked fleas can transmit Yersinia pestis, the bacterium that causes plague, shortly (≤4 d) after taking an infectious bloodmeal. Investigators have measured so-called early-phase transmission (EPT) efficiency in various fleas following infection with highly bacteremic blood (≥108 cfu/ml). To date, no one has determined the lower limit of bacteremia required for fleas to acquire and transmit infection by EPT, though knowing this threshold is central to determining the length of time a host may be infectious to feeding fleas. Here, we evaluate the ability of Oropsylla montana (Baker) to acquire and transmit Y. pestis after feeding on blood containing 103 to 109 cfu/ml. We evaluated the resulting infection prevalence, bacterial loads, and transmission efficiency within the early-phase time period at 1 d postinfection. Fleas acquired infection from bacteremic blood across a wide range of concentrations, but transmission was observed only when fleas ingested highly bacteremic blood.

Molecular Survey of Bartonella Species and Yersinia pestis in Rodent Fleas (Siphonaptera) From Chihuahua, Mexico.

Rodent fleas from northwestern Chihuahua, Mexico, were analyzed for the presence of Bartonella and Yersinia pestis. In total, 760 fleas belonging to 10 species were tested with multiplex polymerase chain reaction analysis targeting the gltA (338-bp) and pla genes (478-bp) of Bartonella and Y. pestis, respectively. Although none was positive for Y. pestis, 307 fleas were infected with Bartonella spp., resulting in an overall prevalence of 40.4%. A logistic regression analysis indicated that the presence of Bartonella is more likely to occur in some flea species. From a subset of Bartonella-positive fleas, phylogenetic analyses of gltA gene sequences revealed 13 genetic variants clustering in five phylogroups (I­V), two of which were matched with known pathogenic Bartonella species (Bartonella vinsonii subsp. arupensis and Bartonella washoensis) and two that were not related with any previously described species or subspecies of Bartonella. Variants in phylogroup V, which were mainly obtained from Meringis spp. fleas, were identical to those reported recently in their specific rodent hosts (Dipodomys spp.) in the same region, suggesting that kangaroo rats and their fleas harbor other Bartonella species not reported previously. Considering the Bartonella prevalence and the flea genotypes associated with known pathogenic Bartonella species, we suggest that analysis of rodent and flea communities in the region should continue for their potential implications for human health. Given that nearby locations in the United States have reported Y. pestis in wild animals and their fleas, we suggest conducting larger-scale studies to increase our knowledge of this bacterium.

Seasonal fluctuations of small mammal and flea communities in a Ugandan plague focus: evidence to implicate Arvicanthis niloticus and Crocidura spp. as key hosts in Yersinia pestis transmission.

BACKGROUND: The distribution of human plague risk is strongly associated with rainfall in the tropical plague foci of East Africa, but little is known about how the plague bacterium is maintained during periods between outbreaks or whether environmental drivers trigger these outbreaks. We collected small mammals and fleas over a two year period in the West Nile region of Uganda to examine how the ecological community varies seasonally in a region with areas of both high and low risk of human plague cases. METHODS: Seasonal changes in the small mammal and flea communities were examined along an elevation gradient to determine whether small mammal and flea populations exhibit differences in their response to seasonal fluctuations in precipitation, temperature, and crop harvests in areas within (above 1300 m) and outside (below 1300 m) of a model-defined plague focus. RESULTS: The abundance of two potential enzootic host species (Arvicanthis niloticus and Crocidura spp.) increased during the plague season within the plague focus, but did not show the same increase at lower elevations outside this focus. In contrast, the abundance of the domestic rat population (Rattus rattus) did not show significant seasonal fluctuations regardless of locality. Arvicanthis niloticus abundance was negatively associated with monthly precipitation at a six month lag and positively associated with current monthly temperatures, and Crocidura spp. abundance was positively associated with precipitation at a three month lag and negatively associated with current monthly temperatures. The abundance of A. niloticus and Crocidura spp. were both positively correlated with the harvest of millet and maize. CONCLUSIONS: The association between the abundance of several small mammal species and rainfall is consistent with previous models of the timing of human plague cases in relation to precipitation in the West Nile region. The seasonal increase in the abundance of key potential host species within the plague focus, but not outside of this area, suggests that changes in small mammal abundance may create favorable conditions for epizootic transmission of Y. pestis which ultimately may increase risk of human cases in this region.

LPS modification promotes maintenance of Yersinia pestis in fleas.

Yersinia pestis, the causative agent of plague, can be transmitted by fleas by two different mechanisms: by early-phase transmission (EPT), which occurs shortly after flea infection, or by blocked fleas following long-term infection. Efficient flea-borne transmission is predicated upon the ability of Y. pestis to be maintained within the flea. Signature-tagged mutagenesis (STM) was used to identify genes required for Y. pestis maintenance in a genuine plague vector, Xenopsylla cheopis. The STM screen identified seven mutants that displayed markedly reduced fitness in fleas after 4 days, the time during which EPT occurs. Two of the mutants contained insertions in genes encoding glucose 1-phosphate uridylyltransferase (galU) and UDP-4-amino-4-deoxy-l-arabinose-oxoglutarate aminotransferase (arnB), which are involved in the modification of lipid A with 4-amino-4-deoxy-l-arabinose (Ara4N) and resistance to cationic antimicrobial peptides (CAMPs). These Y. pestis mutants were more susceptible to the CAMPs cecropin A and polymyxin B, and produced lipid A lacking Ara4N modifications. Surprisingly, an in-frame deletion of arnB retained modest levels of CAMP resistance and Ara4N modification, indicating the presence of compensatory factors. It was determined that WecE, an aminotransferase involved in biosynthesis of enterobacterial common antigen, plays a novel role in Y. pestis Ara4N modification by partially offsetting the loss of arnB. These results indicated that mechanisms of Ara4N modification of lipid A are more complex than previously thought, and these modifications, as well as several factors yet to be elucidated, play an important role in early survival and transmission of Y. pestis in the flea vector.

Evaluation of the effect of host immune status on short-term Yersinia pestis infection in fleas with implications for the enzootic host model for maintenance of Y. pestis during interepizootic periods.

Plague, a primarily flea-borne disease caused by Yersinia pestis, is characterized by rapidly spreading epizootics separated by periods of quiescence. Little is known about how and where Y. pestis persists between epizootics. It is commonly proposed, however, that Y pestis is maintained during interepizootic periods in enzootic cycles involving flea vectors and relatively resistant host populations. According to this model, while susceptible individuals serve as infectious sources for feeding fleas and subsequently die of infection, resistant hosts survive infection, develop antibodies to the plague bacterium, and continue to provide bloodmeals to infected fleas. For Y. pestis to persist under this scenario, fleas must remain infected after feeding on hosts carrying antibodies to Y. pestis. Studies of other vector-borne pathogens suggest that host immunity may negatively impact pathogen survival in the vector. Here, we report infection rates and bacterial loads for fleas (both Xenopsylla cheopis (Rothschild) and Oropsylla montana (Baker)) that consumed an infectious bloodmeal and subsequently fed on an immunized or age-matched naive mouse. We demonstrate that neither the proportion of infected fleas nor the bacterial loads in infected fleas were significantly lower within 3 d of feeding on immunized versus naive mice. Our findings thus provide support for one assumption underlying the enzootic host model of interepizootic maintenance of Y. pestis.

Yersinia murine toxin is not required for early-phase transmission of Yersinia pestis by Oropsylla montana (Siphonaptera: Ceratophyllidae) or Xenopsylla cheopis (Siphonaptera: Pulicidae).

Plague, caused by Yersinia pestis, is characterized by quiescent periods punctuated by rapidly spreading epizootics. The classical 'blocked flea' paradigm, by which a blockage forms in the flea's proventriculus on average 1-2 weeks post-infection (p.i.), forces starving fleas to take multiple blood meals, thus increasing opportunities for transmission. Recently, the importance of early-phase transmission (EPT), which occurs prior to blockage formation, has been emphasized during epizootics. Whilst the physiological and molecular mechanisms of blocked flea transmission are well characterized, the pathogen-vector interactions have not been elucidated for EPT. Within the blocked flea model, Yersinia murine toxin (Ymt) has been shown to be important for facilitating colonization of the midgut within the flea. One proposed mechanism of EPT is the regurgitation of infectious material from the flea midgut during feeding. Such a mechanism would require bacteria to colonize and survive for at least brief periods in the midgut, a process that is mediated by Ymt. Two key bridging vectors of Y. pestis to humans, Oropsylla montana (Siphonaptera: Ceratophyllidae) or Xenopsylla cheopis (Siphonaptera: Pulicidae), were used in our study to test this hypothesis. Fleas were infected with a mutant strain of Y. pestis containing a non-functional ymt that was shown previously to be incapable of colonizing the midgut and were then allowed to feed on SKH-1 mice 3 days p.i. Our results show that Ymt was not required for EPT by either flea species.

Effects of low-temperature flea maintenance on the transmission of Yersinia pestis by Oropsylla montana.

Yersinia pestis, the causative agent of plague, is primarily a rodent-associated, flea-borne zoonosis maintained in sylvatic foci throughout western North America. Transmission to humans is mediated most commonly by the flea vector Oropsylla montana and occurs predominantly in the southwestern United States. With few exceptions, previous studies showed O. montana to be an inefficient vector at transmitting Y. pestis at ambient temperatures, particularly when such fleas were fed on susceptible hosts more than a few days after ingesting an infectious blood meal. We examined whether holding fleas at subambient temperatures affected the transmissibility of Y. pestis by this vector. An infectious blood meal containing a virulent Y. pestis strain (CO96-3188) was given to colony-reared O. montana fleas. Potentially infected fleas were maintained at different temperatures (6°C, 10°C, 15°C, or 23°C). Transmission efficiencies were tested by allowing up to 15 infectious fleas to feed on each of 7 naïve CD-1 mice on days 1-4, 7, 10, 14, 17, and 21 postinfection (p.i.). Mice were monitored for signs of infection for 21 days after exposure to infectious fleas. Fleas held at 6°C, 10°C, and 15°C were able to effectively transmit at every time point p.i. The percentage of transmission to naïve mice by fleas maintained at low temperatures (46.0% at 6°C, 71.4% at 10°C, 66.7% at 15°C) was higher than for fleas maintained at 23°C (25.4%) and indicates that O. montana fleas efficiently transmit Y. pestis at low temperatures. Moreover, pooled percent per flea transmission efficiencies for flea cohorts maintained at temperatures of 10°C and 15°C (8.67% and 7.87%, respectively) showed a statistically significant difference in the pooled percent per flea transmission efficiency from fleas maintained at 23°C (1.94%). This is the first comprehensive study to demonstrate efficient transmission of Y. pestis by O. montana fleas maintained at temperatures as low as 6°C. Our findings further contribute to the understanding of plague ecology in temperate climates by providing support for the hypothesis that Y. pestis is able to overwinter within the flea gut and potentially cause infection during the following transmission season. The findings also might hold implications for explaining the focality of plague in tropical regions.

Combining real-time polymerase chain reaction using SYBR Green I detection and sequencing to identify vertebrate bloodmeals in fleas.

Programs that aim to control vector-borne zoonotic diseases require information on zoonotic hosts and on the feeding behavior of bridging vectors that are capable of transmitting pathogens from those hosts to humans. Here we describe an assay developed to identify bloodmeals in field-collected cat fleas (Ctenocephalides felis Bouché) to assess this species' potential role as a Yersinia pestis bridging vector in a plague-endemic region of Uganda. Our assay uses a single primer set and SYBR Green I-based real-time polymerase chain reaction to amplify a segment of the 12S mitochondrial ribosomal RNA gene for identification by sequencing. The assay capitalizes on the sensitivity of real-time polymerase chain reaction and the specificity of sequencing and can be used to differentiate vertebrate bloodmeals to the genus or species level without a priori knowledge of the host community. Because real-time assays that detect vertebrate DNA are highly sensitive to human DNA contamination, we analyzed detection in artificially fed and unfed fleas to establish a Ct cutoff that optimized specificity without completely sacrificing sensitivity. Using the established cutoff, our assay detected human, rat, and goat DNA in artificially fed C. felis up to 72 h postfeeding.

Evaluation of the infectiousness to mice of soil contaminated with Yersinia pestis-infected blood.

Plague, an often-fatal zoonotic disease caused by Yersinia pestis, is characterized by epizootic and quiescent periods. How Y. pestis is maintained during inter-epizootic periods is poorly understood, but soil has been implicated as a potential reservoir. Although previous studies have suggested that Y. pestis is able to survive in soil for weeks or months, it is unclear whether or not it is infectious to susceptible hosts. Here we investigate the potential for Y. pestis to infect mice through close contact with contaminated soil under laboratory conditions. In an attempt to approximate the natural conditions under which animals would be exposed to Y. pestis-contaminated soil, mouse cages filled with soil from a plague-endemic region were held at temperature and humidity ranges observed in ground squirrel burrows. These laboratory "burrows" were contaminated with highly bacteremic blood (>10(8) cfu/mL) to simulate the introduction of infectious material from a dying animal during an epizootic. Outbred Swiss-Webster mice with scarified skin patches were held on contaminated soil for 10 days and monitored for signs of illness. Following exposure to contaminated soil, one animal of 104 became infected with Y. pestis. None of the remaining animals seroconverted following a 21-day holding period. Under our experimental conditions, which maximized the likelihood of contact between susceptible mice and contaminated soil, transmission efficiency from soil to mice was 0.96% (95% CI 0.17, 5.25%). This suggests that although transmission of Y. pestis from contaminated soils is possible, it is not likely a major transmission route under natural conditions.

Changing socioeconomic indicators of human plague, New Mexico, USA.

Socioeconomic indicators associated with temporal changes in the distribution of human plague cases in New Mexico were investigated for 1976-2007. In the 1980s, cases were more likely in census block groups with poor housing conditions, but by the 2000s, cases were associated with affluent areas concentrated in the Santa Fe-Albuquerque region.

Flea diversity as an element for persistence of plague bacteria in an East African plague focus.

Plague is a flea-borne rodent-associated zoonotic disease that is caused by Yersinia pestis and characterized by long quiescent periods punctuated by rapidly spreading epidemics and epizootics. How plague bacteria persist during inter-epizootic periods is poorly understood, yet is important for predicting when and where epizootics are likely to occur and for designing interventions aimed at local elimination of the pathogen. Existing hypotheses of how Y. pestis is maintained within plague foci typically center on host abundance or diversity, but little attention has been paid to the importance of flea diversity in enzootic maintenance. Our study compares host and flea abundance and diversity along an elevation gradient that spans from low elevation sites outside of a plague focus in the West Nile region of Uganda (~725-1160 m) to higher elevation sites within the focus (~1380-1630 m). Based on a year of sampling, we showed that host abundance and diversity, as well as total flea abundance on hosts was similar between sites inside compared with outside the plague focus. By contrast, flea diversity was significantly higher inside the focus than outside. Our study highlights the importance of considering flea diversity in models of Y. pestis persistence.

Evaluation and modification of off-host flea collection techniques used in northwest Uganda: laboratory and field studies.

Quantifying the abundance of host-seeking fleas is critical for assessing risk of human exposure to flea-borne disease agents, including Yersinia pestis, the etiological agent of plague. Yet, reliable measures of the efficacy of existing host-seeking flea collection methods are lacking. In this study, we compare the efficacy of passive and active methods for the collection of host-seeking fleas in both the laboratory and human habitations in a plague-endemic region of northwest Uganda. In the laboratory, lighted "Kilonzo" flea traps modified with either blinking lights, the creation of shadows or the generation of carbon dioxide were less efficient at collecting Xenopsylla cheopis Rothchild and Ctenocephalides felis Bouché fleas than an active collection method using white cotton socks or cotton flannel. Passive collection using Kilonzo light traps in the laboratory collected significantly more X. cheopis than C. felis and active collection, using white socks and flannel, collected significantly more C. felis than X. cheopis. In field studies conducted in Uganda, Kilonzo traps using a flashlight were similar in their collection efficacy to Kilonzo traps using kerosene lamps. However, in contrast to laboratory studies, Kilonzo flea traps using flashlights collected a greater number of fleas than swabbing. Within human habitations in Uganda, Kilonzo traps were especially useful for collecting C. felis, the dominant species found in human habitations in this area.

Effects of temperature on the transmission of Yersinia Pestis by the flea, Xenopsylla Cheopis, in the late phase period.

BACKGROUND: Traditionally, efficient flea-borne transmission of Yersinia pestis, the causative agent of plague, was thought to be dependent on a process referred to as blockage in which biofilm-mediated growth of the bacteria physically blocks the flea gut, leading to the regurgitation of contaminated blood into the host. This process was previously shown to be temperature-regulated, with blockage failing at temperatures approaching 30°C; however, the abilities of fleas to transmit infections at different temperatures had not been adequately assessed. We infected colony-reared fleas of Xenopsylla cheopis with a wild type strain of Y. pestis and maintained them at 10, 23, 27, or 30°C. Naïve mice were exposed to groups of infected fleas beginning on day 7 post-infection (p.i.), and every 3-4 days thereafter until day 14 p.i. for fleas held at 10°C, or 28 days p.i. for fleas held at 23-30°C. Transmission was confirmed using Y. pestis-specific antigen or antibody detection assays on mouse tissues. RESULTS: Although no statistically significant differences in per flea transmission efficiencies were detected between 23 and 30°C, efficiencies were highest for fleas maintained at 23°C and they began to decline at 27 and 30°C by day 21 p.i. These declines coincided with declining median bacterial loads in fleas at 27 and 30°C. Survival and feeding rates of fleas also varied by temperature to suggest fleas at 27 and 30°C would be less likely to sustain transmission than fleas maintained at 23°C. Fleas held at 10°C transmitted Y. pestis infections, although flea survival was significantly reduced compared to that of uninfected fleas at this temperature. Median bacterial loads were significantly higher at 10°C than at the other temperatures. CONCLUSIONS: Our results suggest that temperature does not significantly effect the per flea efficiency of Y. pestis transmission by X. cheopis, but that temperature is likely to influence the dynamics of Y. pestis flea-borne transmission, perhaps by affecting persistence of the bacteria in the flea gut or by influencing flea survival. Whether Y. pestis biofilm production is important for transmission at different temperatures remains unresolved, although our results support the hypothesis that blockage is not necessary for efficient transmission.

Effects of temperature on early-phase transmission of Yersina pestis by the flea, Xenopsylla cheopis.

Sharp declines in human and animal cases of plague, caused by the bacterium Yersinia pestis (Yersin), have been observed when outbreaks coincide with hot weather. Failure of biofilm production, or blockage, to occur in the flea, as temperatures reach 30 degrees C has been suggested as an explanation for these declines. Recent work demonstrating efficient flea transmission during the first few days after fleas have taken an infectious blood meal, in the absence of blockage (e.g., early-phase transmission), however, has called this hypothesis into question. To explore the potential effects of temperature on early-phase transmission, we infected colony-reared Xenopsylla cheopis (Rothchild) fleas with a wild-type strain of plague bacteria using an artificial feeding system, and held groups of fleas at 10, 23, 27, and 30 degrees C. Naive Swiss Webster mice were exposed to fleas from each of these temperatures on days 1-4 postinfection, and monitored for signs of infection for 21 d. Temperature did not significantly influence the rates of transmission observed for fleas held at 23, 27, and 30 degrees C. Estimated per flea transmission efficiencies for these higher temperatures ranged from 2.32 to 4.96% (95% confidence interval [CI]: 0.96-8.74). In contrast, no transmission was observed in mice challenged by fleas held at 10 degrees C (per flea transmission efficiency estimates, 0-1.68%). These results suggest that declines in human and animal cases during hot weather are not related to changes in the abilities of X. cheopis fleas to transmit Y. pestis infections during the early-phase period. By contrast, transmission may be delayed or inhibited at low temperatures, indicating that epizootic spread of Y. pestis by X. cheopis via early-phase transmission is unlikely during colder periods of the year.

Evaluation of rodent bait containing imidacloprid for the control of fleas on commensal rodents in a plague-endemic region of northwest Uganda.

In recent decades, the majority of human plague cases (caused by Yersinia pestis) have been reported from Africa. In an effort to reduce the risk of the disease in this area, we evaluated the efficacy of a host-targeted rodent bait containing the insecticide imidacloprid for controlling fleas on house-dwelling commensal rodents in a plague-endemic region of northwestern Uganda. Results demonstrated that the use of a palatable, rodent-targeted, wax-based bait cube was effective at reducing the prevalence of fleas on commensal rodents and flea burdens on these animals at day 7 postbait exposure, but lacked significant residual activity, allowing flea populations to rebound in the absence of additional bait applications. Our results indicate the use of a palatable host-targeted bait block containing imidacloprid was an effective technique for quickly reducing flea numbers on rodents in northwest Uganda and, thus, could be useful for lowering the potential risk of human flea bite exposures during plague outbreaks if applied continuously during the period of risk.

Biofilm formation is not required for early-phase transmission of Yersinia pestis.

Early-phase transmission (EPT) is a recently described model of plague transmission that explains the rapid spread of disease from flea to mammal host during an epizootic. Unlike the traditional blockage-dependent model of plague transmission, EPT can occur when a flea takes its first blood meal after initially becoming infected by feeding on a bacteraemic host. Blockage of the flea gut results from biofilm formation in the proventriculus, mediated by the gene products found in the haemin storage (hms) locus of the Yersinia pestis chromosome. Although biofilms are required for blockage-dependent transmission, the role of biofilms in EPT has yet to be determined. An artificial feeding system was used to feed Xenopsylla cheopis and Oropsylla montana rat blood spiked with the parental Y. pestis strain KIM5(pCD1)+, two different biofilm-deficient mutants (Delta hmsT, Delta hmsR), or a biofilm-overproducer mutant (Delta hmsP). Infected fleas were then allowed to feed on naïve Swiss Webster mice for 1-4 days after infection, and the mice were monitored for signs of infection. We also determined the bacterial loads of each flea that fed upon naïve mice. Biofilm-defective mutants transmitted from X. cheopis and O. montana as efficiently as the parent strain, whereas the EPT efficiency of fleas fed the biofilm-overproducing strain was significantly less than that of fleas fed either the parent or a biofilm-deficient strain. Fleas infected with a biofilm-deficient strain harboured lower bacterial loads 4 days post-infection than fleas infected with the parent strain. Thus, defects in biofilm formation did not prevent flea-borne transmission of Y. pestis in our EPT model, although biofilm overproduction inhibited efficient EPT. Our results also indicate, however, that biofilms may play a role in infection persistence in the flea.

Vector control improves survival of three species of prairie dogs (Cynomys) in areas considered enzootic for plague.

Plague causes periodic epizootics that decimate populations of prairie dogs (PDs) (Cynomys), but the means by which the causative bacterium (Yersinia pestis) persists between epizootics are poorly understood. Plague epizootics in PDs might arise as the result of introductions of Y. pestis from sources outside PD colonies. However, it remains possible that plague persists in PDs during interepizootic periods and is transmitted at low rates among highly susceptible individuals within and between their colonies. If this is true, application of vector control to reduce flea numbers might reduce mortality among PDs. To test whether vector control enhances PD survival in the absence of obvious plague epizootics, we reduced the numbers of fleas (vectors for Y. pestis) 96-98% (1 month posttreatment) on 15 areas involving three species of PDs (Cynomys leucurus, Cynomys parvidens in Utah, and Cynomys ludovicianus in Montana) during 2000-2004 using deltamethrin dust delivered into burrows as a pulicide. Even during years without epizootic plague, PD survival rates at dusted sites were 31-45% higher for adults and 2-34% higher for juveniles compared to survival rates at nondusted sites. Y. pestis was cultured from 49 of the 851 flea pools tested (6882 total fleas) and antibodies against Y. pestis were identified in serum samples from 40 of 2631 PDs. Although other explanations are possible, including transmission of other potentially fatal pathogens by fleas, ticks, or other ectoparasites, our results suggest that plague might be maintained indefinitely in PD populations in the absence of free epizootics and widespread mortality among these animals. If PDs and their fleas support enzootic cycles of plague transmission, there would be important implications for the conservation of these animals and other species.