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.

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.

Evaluation of a Yersinia pestis mutant impaired in a thermoregulated type VI-like secretion system in flea, macrophage and murine models.

Type VI secretion systems (T6SSs) have been identified recently in several Gram-negative organisms and have been shown to be associated with virulence in some bacterial pathogens. A T6SS of Yersinia pestis CO92 (locus YPO0499-YPO0516) was deleted followed by investigation of the phenotype of this mutation. We observed that this T6SS locus of Y. pestis was preferentially expressed at 26 degrees C in comparison to 37 degrees C suggesting a possible role in the flea cycle. However, we found that the deletion of T6SS locus YPO0499-YPO0516 in Y. pestis CO92 had no effect on the ability of this strain to infect the oriental rat flea, Xenopsylla cheopis. Nevertheless, this mutant displayed increased intracellular numbers in macrophage-like J774.A1 cells after 20 h post-infection for bacterial cells pre-grown at 26 degrees C indicating that expression of this T6SS locus limited intracellular replication in macrophages. In addition, deletion of the YPO0499-YPO0516 locus reduced the uptake by macrophages of the Y. pestis mutant pre-grown at 37 degrees C, suggesting that this T6SS locus has phagocytosis-promoting activity. Further study of the virulence of the T6SS mutant in murine bubonic and inhalation plague models revealed no attenuation in comparison with the parental CO92 strain.

Flea abundance on black-tailed prairie dogs (Cynomys ludovicianus) increases during plague epizootics.

Black-tailed prairie dogs (Cynomys ludovicianus) on the Great Plains of the United States are highly susceptible to plague, caused by the bacterium Yersinia pestis, with mortality on towns during plague epizootics often approaching 100%. The ability of flea-borne transmission to sustain disease spread has been questioned because of inefficiency of flea vectors. However, even with low individual efficiency, overall transmission can be increased if flea abundance (the number of fleas on hosts) increases. Changes in flea abundance on hosts during plague outbreaks were recorded during a large-scale study of plague outbreaks in prairie dogs in north central Colorado during 3 years (2004-2007). Fleas were collected from live-trapped black-tailed prairie dogs before and during plague epizootics and tested by PCR for the presence of Y. pestis. The predominant fleas were two prairie dog specialists (Oropsylla hirsuta and Oropsylla tuberculata cynomuris), and a generalist flea species (Pulex simulans) was also recorded from numerous mammals in the area. The three species differ in seasonal abundance, with greatest abundance in spring (February and March) and fall (September and October). Flea abundance and infestation intensity increased during epizootics and were highest on prairie dogs with Y. pestis-infected fleas. Seasonal occurrence of epizootics among black-tailed prairie dogs was found to coincide with seasonal peaks in flea abundance. Concentration of infected fleas on surviving animals may account for rapid spread of plague during epizootics. In particular, the role of the generalist flea P. simulans was previously underappreciated.

Identification of flea blood meals using multiplexed real-time polymerase chain reaction targeting mitochondrial gene fragments.

Human plague is found in the West Nile region of Uganda and Democratic Republic of the Congo where flea vectors are often found inhabiting homes. We have developed a multiplexed, real-time polymerase chain reaction assay targeting mitochondrial genes that is capable of detecting blood meal sources in fleas collected off-host in East Africa. Laboratory tests showed that the assay is specific for the intended targets and has a detection limit below one picogram of DNA. Testing of wild-caught fleas from the Democratic Republic of Congo suggests that humans are at significant risk from flea-borne disease and implicates domestic animals including cats, chickens, and the black rat as potential sources of human exposure to fleas and flea-borne diseases. Future application of the assay will help us better define the ecology of plague in East Africa to implement effective control measures to combat the spread of disease.

Colorado animal-based plague surveillance systems: relationships between targeted animal species and prediction efficacy of areas at risk for humans.

Human plague risks (Yersinia pestis infection) are greatest when epizootics cause high mortality among this bacterium's natural rodent hosts. Therefore, health departments in plague-endemic areas commonly establish animal-based surveillance programs to monitor Y. pestis infection among plague hosts and vectors. The primary objectives of our study were to determine whether passive animal-based plague surveillance samples collected in Colorado from 1991 to 2005 were sampled from high human plague risk areas and whether these samples provided information useful for predicting human plague case locations. By comparing locations of plague-positive animal samples with a previously constructed GIS-based plague risk model, we determined that the majority of plague-positive Gunnison's prairie dogs (100%) and non-prairie dog sciurids (85.82%), and moderately high percentages of sigmodontine rodents (71.4%), domestic cats (69.3%), coyotes (62.9%), and domestic dogs (62.5%) were recovered within 1 km of the nearest area posing high peridomestic risk to humans. In contrast, the majority of white-tailed prairie dog (66.7%), leporid (cottontailed and jack rabbits) (71.4%), and black-tailed prairie dog (93.0%) samples originated more than 1 km from the nearest human risk habitat. Plague-positive animals or their fleas were rarely (one of 19 cases) collected within 2 km of a case exposure site during the 24 months preceding the dates of illness onset for these cases. Low spatial accuracy for identifying epizootic activity prior to human plague cases suggested that other mammalian species or their fleas are likely more important sources of human infection in high plague risk areas. To address this issue, epidemiological observations and multi-locus variable number tandem repeat analyses (MLVA) were used to preliminarily identify chipmunks as an under-sampled, but potentially important, species for human plague risk in Colorado.