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.     

Selective isolation of Yersinia pestis from plague-infected fleas

We evaluated Yersinia CIN agar for the isolation of Yersinia pestis from infected fleas. CIN media is effective for the differentiation of Y. pestis from flea commensal flora and is sufficiently inhibitory to other bacteria that typically outcompete Y. pestis after 48 h of growth using less selective media.     

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.     

An additional step in the transmission of Yersinia pestis?

Plague, caused by the bacterium Yersinia pestis, is a mammalian vector-borne disease, transmitted by fleas that serve as the vector between rodent hosts. For many pathogens, including Y. pestis, there are strong evolutionary pressures that lead to a reduction in ‘useless genes'', with only those retained that reflect function in the specific environment inhabited by the pathogen. Genetic traits critical for survival and transmission between two environments, the rodent and the flea, are conserved in epizootic/epidemic plague strains. However, there are genes that remain conserved for which no function in the flea–rodent cycle has yet been observed, indicating an additional environment may exist in the transmission cycle of plague. Here, we present evidence for highly conserved genes that suggests a role in the persistence of Y. pestis after death of its host. Furthermore, maintenance of these genes points to Y. pestis traversing a post-mortem path between, and possibly within, epizootic periods and offering insight into mechanisms that may allow Y. pestis an alternative route of transmission in the natural environment.     

Characterization of the rat pneumonic plague model: infection kinetics following aerosolization of Yersinia pestis CO92

Yersinia pestis, the causative agent of human bubonic and pneumonic plague, is spread during natural infection by the fleas of rodents. Historically associated with infected rat fleas, studies on the kinetics of infection in rats are surprisingly few, and these reports have focused mainly on bubonic plague. Although the natural route of primary infection results in bubonic plague in humans, it is commonly thought that aerosolized Y. pestis will be utilized during a biowarfare attack. Accordingly, based on our previous characterization of the mouse model of pneumonic plague, we sought to examine the progression of infection in rats exposed in a whole-body Madison chamber to aerosolized Y. pestis CO92. Following an 8.6 LD₅₀ dose of Y. pestis, injury was apparent in the rat tissues based on histopathology, and chemokines and cytokines rose above control levels (1 h post infection [p.i.]) in the sera and organ homogenates over a 72-h infection period. Bacteria disseminated from the lungs to peripheral organs, with the largest increases in the spleen, followed by the liver and blood at 72 h p.i. compared to the 1 h controls. Importantly, rats were as sensitive to pneumonic plague as mice, having a similar LD₅₀ dose by the intranasal and aerosolized routes. Further, we showed direct transmission of plague bacteria from infected to uninfected rats. Taken together, the data allowed us to characterize for the first time a rat pneumonic plague model following aerosolization of Y. pestis.     

The Deer Mouse (<Emphasis Type="Italic">Peromyscus maniculatus</Emphasis>) as an Enzootic Reservoir of Plague in California

It has long been theorized that deer mice (Peromyscus maniculatus) are a primary reservoir of Yersinia pestis in California. However, recent research from other parts of the western USA has implicated deer mice as spillover hosts during epizootic plague transmission. This retrospective study analyzed deer mouse data collected for plague surveillance by public health agencies in California from 1971 to 2016 to help elucidate the role of deer mice in plague transmission. The fleas most commonly found on deer mice were poor vectors of Y. pestis and occurred in insufficient numbers to maintain transmission of the pathogen, while fleas whose natural hosts are deer mice were rarely observed and even more rarely found infected with Y. pestis on other rodent hosts. Seroprevalence of Y. pestis antibodies in deer mice was significantly lower than that of several chipmunk and squirrel species. These analyses suggest that it is unlikely that deer mice play an important role in maintaining plague transmission in California. While they may not be primary reservoirs, results supported the premise that deer mice are occasionally exposed to and infected by Y. pestis and instead may be spillover hosts.     

Similarities and seasonal variations in bacterial communities from the blood of rodents and from their flea vectors

Vector-borne microbes are subject to the ecological constraints of two distinct microenvironments: that in the arthropod vector and that in the blood of its vertebrate host. Because the structure of bacterial communities in these two microenvironments may substantially affect the abundance of vector-borne microbes, it is important to understand the relationship between bacterial communities in both microenvironments and the determinants that shape them. We used pyrosequencing analyses to compare the structure of bacterial communities in Synosternus cleopatrae fleas and in the blood of their Gerbillus andersoni hosts. We also monitored the interindividual and seasonal variability in these bacterial communities by sampling the same individual wild rodents during the spring and again during the summer. We show that the bacterial communities in each sample type (blood, female flea or male flea) had a similar phylotype composition among host individuals, but exhibited seasonal variability that was not directly associated with host characteristics. The structure of bacterial communities in male fleas and in the blood of their rodent hosts was remarkably similar and was dominated by flea-borne Bartonella and Mycoplasma phylotypes. A lower abundance of flea-borne bacteria and the presence of Wolbachia phylotypes distinguished bacterial communities in female fleas from those in male fleas and in rodent blood. These results suggest that the overall abundance of a certain vector-borne microbe is more likely to be determined by the abundance of endosymbiotic bacteria in the vector, abundance of other vector-borne microbes co-occurring in the vector and in the host blood and by seasonal changes, than by host characteristics.     

Understanding the Persistence of Plague Foci in Madagascar

Plague, a zoonosis caused by Yersinia pestis, is still found in Africa, Asia, and the Americas. Madagascar reports almost one third of the cases worldwide. Y. pestis can be encountered in three very different types of foci: urban, rural, and sylvatic. Flea vector and wild rodent host population dynamics are tightly correlated with modulation of climatic conditions, an association that could be crucial for both the maintenance of foci and human plague epidemics. The black rat Rattus rattus, the main host of Y. pestis in Madagascar, is found to exhibit high resistance to plague in endemic areas, opposing the concept of high mortality rates among rats exposed to the infection. Also, endemic fleas could play an essential role in maintenance of the foci. This review discusses recent advances in the understanding of the role of these factors as well as human behavior in the persistence of plague in Madagascar.     

Feeding Behavior Modulates Biofilm-Mediated Transmission of Yersinia pestis by the Cat Flea,Ctenocephalides felis

Background

The cat flea, Ctenocephalides felis, is prevalent worldwide, will parasitize animal reservoirs of plague, and is associated with human habitations in known plague foci. Despite its pervasiveness, limited information is available about the cat flea’s competence as a vector for Yersinia pestis. It is generally considered to be a poor vector, based on studies examining early-phase transmission during the first week after infection, but transmission potential by the biofilm-dependent proventricular-blocking mechanism has never been systematically evaluated. In this study, we assessed the vector competence of cat fleas by both mechanisms. Because the feeding behavior of cat fleas differs markedly from important rat flea vectors, we also examined the influence of feeding behavior on transmission dynamics.

Methodology/Principal Findings

Groups of cat fleas were infected with Y. pestis and subsequently provided access to sterile blood meals twice-weekly, 5 times per week, or daily for 4 weeks and monitored for infection, the development of proventricular biofilm and blockage, mortality, and the ability to transmit. In cat fleas allowed prolonged, daily access to blood meals, mimicking their natural feeding behavior, Y. pestis did not efficiently colonize the digestive tract and could only be transmitted during the first week after infection. In contrast, cat fleas that were fed intermittently, mimicking the feeding behavior of the efficient vector Xenopsylla cheopis, could become blocked and regularly transmitted Y. pestis for 3–4 weeks by the biofilm-mediated mechanism, but early-phase transmission was not detected.

Conclusions

The normal feeding behavior of C. felis, more than an intrinsic resistance to infection or blockage by Y. pestis, limits its vector competence. Rapid turnover of midgut contents results in bacterial clearance and disruption of biofilm accumulation in the proventriculus. Anatomical features of the cat flea foregut may also restrict transmission by both early-phase and proventricular biofilm-dependent mechanisms.     

Dynamics of Yersinia pestis and Its Antibody Response in Great Gerbils (Rhombomys opimus) by Subcutaneous Infection

Background

Rhombomys opimus (great gerbil) is a reservoir of Yersinia pestis in the natural plague foci of Central Asia. Great gerbils are highly resistant to Y. pestis infection. The coevolution of great gerbils and Y. pestis is believed to play an important role in the plague epidemics in Central Asia plague foci. However, the dynamics of Y. pestis infection and the corresponding antibody response in great gerbils have not been evaluated. In this report, animal experiments were employed to investigate the bacterial load in both the liver and spleen of infected great gerbils. The dynamics of the antibody response to the F1 capsule antigen of Y. pestis was also determined.

Methodology

Captured great gerbils that tested negative for both anti-F1 antibodies and bacterial isolation were infected subcutaneously with different doses (10⁵ to 10¹¹ CFU) of a Y. pestis strain isolated from a live great gerbil during routine plague surveillance in the Junggar Basin, Xinjiang, China. The clinical manifestations, changes in body weight, anal temperature, and gross anatomy of the infected animals were observed. The blood cell count, bacterial load, and anti-F1 antibody titers were determined at different time points after infection using a blood analyzer, plate counts, and an indirect hemagglutination assay, respectively.

Conclusions/Significance

The dynamics of bacterial load and the anti-F1 antibody concentration in great gerbils are highly variable among individuals. The Y. pestis infection in great gerbils could persist as long as 15 days. They act as an appropriate reservoir for plague in the Junggar Basin, which is part of the natural plague foci in Central Asia. The dynamics of the Y. pestis susceptibility of great gerbil will improve the understanding of its variable resistance, which would facilitate the development of more effective countermeasures for controlling plague epidemics in this focus.     

Seasonal dynamics of epizootic process of the plague agent <Emphasis Type="Italic">Yersinia pestis</Emphasis> transmission to the long-tailed suslik <Emphasis Type="Italic">Citellus undulatus</Emphasis> by the flea <Emphasis Type="Italic">Citellophilus tesquorum</Emphasis> in Tuva

Transmission of Yersinia pestis to the long-tailed suslik (Citellus undulatus) by fleas (Citellophilus tesquorum) in the Tuva natural plague focus in different seasons (spring, summer, and autumn) was studied experimentally. Between feeding periods, insects were kept in an artificial nest under temperature and humidity closely corresponding to seasonal ones. The character of the agent transmission was estimated according to the fraction of fleas with the agent in the aggregated state (bacterial lumps, partial blocks of proventriculus), the fraction of blocked individuals, and the fraction of infected susliks and of those with the generalized form of infection. Seasonal dynamics of epizootic process of the Y. pestis transmission corresponded to the results obtained in the epizootic examination of the Tuva natural plague focus and reflected the dynamics of the epizootic process (increase-peak-decline). The activity of the formation of a proventriculus block in C. t. altaicus, the infection ability of the fleas, and the sensitivity of long-tailed Siberian susliks to Y. pestis were the highest in mid-summer (July-first ten days of August), during the period of epizooty activation in the focus. The maximal number of C. t. altaicus with the plague agent at the aggregated state was observed in the cold period, before wintering of insects and after their hibernation.     

Relationship between the Presence of Bartonella Species and Bacterial Loads in Cats and Cat Fleas (Ctenocephalides felis) under Natural Conditions

Cats are considered the main reservoir of three zoonotic Bartonella species: Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae. Cat fleas (Ctenocephalides felis) have been experimentally demonstrated to be a competent vector of B. henselae and have been proposed as the potential vector of the two other Bartonella species. Previous studies have reported a lack of association between the Bartonella species infection status (infected or uninfected) and/or bacteremia levels of cats and the infection status of the fleas they host. Nevertheless, to date, no study has compared the quantitative distributions of these bacteria in both cats and their fleas under natural conditions. Thus, the present study explored these relationships by identifying and quantifying the different Bartonella species in both cats and their fleas. Therefore, EDTA-blood samples and fleas collected from stray cats were screened for Bartonella bacteria. Bacterial loads were quantified by high-resolution melt real-time quantitative PCR assays. The results indicated a moderate correlation between the Bartonella bacterial loads in the cats and their fleas when both were infected with the same Bartonella species. Moreover, a positive effect of the host infection status on the Bartonella bacterial loads of the fleas was observed. Conversely, the cat bacterial loads were not affected by the infection status of their fleas. Our results suggest that the Bartonella bacterial loads of fleas are positively affected by the presence of the bacteria in their feline host, probably by multiple acquisitions/accumulation and/or multiplication events.     

Responses of the coastal bacterial community to viral infection of the algae Phaeocystis globosa

The release of organic material upon algal cell lyses has a key role in structuring bacterial communities and affects the cycling of biolimiting elements in the marine environment. Here we show that already before cell lysis the leakage or excretion of organic matter by infected yet intact algal cells shaped North Sea bacterial community composition and enhanced bacterial substrate assimilation. Infected algal cultures of Phaeocystis globosa grown in coastal North Sea water contained gamma- and alphaproteobacterial phylotypes that were distinct from those in the non-infected control cultures 5 h after infection. The gammaproteobacterial population at this time mainly consisted of Alteromonas sp. cells that were attached to the infected but still intact host cells. Nano-scale secondary-ion mass spectrometry (nanoSIMS) showed ∼20% transfer of organic matter derived from the infected ¹³C- and ¹⁵N-labelled P. globosa cells to Alteromonas sp. cells. Subsequent, viral lysis of P. globosa resulted in the formation of aggregates that were densely colonised by bacteria. Aggregate dissolution was observed after 2 days, which we attribute to bacteriophage-induced lysis of the attached bacteria. Isotope mass spectrometry analysis showed that 40% of the particulate ¹³C-organic carbon from the infected P. globosa culture was remineralized to dissolved inorganic carbon after 7 days. These findings reveal a novel role of viruses in the leakage or excretion of algal biomass upon infection, which provides an additional ecological niche for specific bacterial populations and potentially redirects carbon availability.     

Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus)

Bacterial gut symbiont communities are critical for the health of many insect species. However, little is known about how microbial communities vary among host species or how they respond to anthropogenic disturbances. Bacterial communities that differ in richness or composition may vary in their ability to provide nutrients or defenses. We used deep sequencing to investigate gut microbiota of three species in the genus Bombus (bumble bees). Bombus are among the most economically and ecologically important non-managed pollinators. Some species have experienced dramatic declines, probably due to pathogens and land-use change. We examined variation within and across bee species and between semi-natural and conventional agricultural habitats. We categorized as ‘core bacteria'' any operational taxonomic units (OTUs) with closest hits to sequences previously found exclusively or primarily in the guts of honey bees and bumble bees (genera Apis and Bombus). Microbial community composition differed among bee species. Richness, defined as number of bacterial OTUs, was highest for B. bimaculatus and B. impatiens. For B. bimaculatus, this was due to high richness of non-core bacteria. We found little effect of habitat on microbial communities. Richness of non-core bacteria was negatively associated with bacterial abundance in individual bees, possibly due to deeper sampling of non-core bacteria in bees with low populations of core bacteria. Infection by the gut parasite Crithidia was negatively associated with abundance of the core bacterium Gilliamella and positively associated with richness of non-core bacteria. Our results indicate that Bombus species have distinctive gut communities, and community-level variation is associated with pathogen infection.     

Caspase-3 Mediates the Pathogenic Effect of Yersinia pestis YopM in Liver of C57BL/6 Mice and Contributes to YopM's Function in Spleen

The virulence protein YopM of the plague bacterium Yersinia pestis has different dominant effects in liver and spleen. Previous studies focused on spleen, where YopM inhibits accumulation of inflammatory dendritic cells. In the present study we focused on liver, where PMN function may be directly undermined by YopM without changes in inflammatory cell numbers in the initial days of infection, and foci of inflammation are easily identified. Mice were infected with parent and ΔyopM-1 Y. pestis KIM5, and effects of YopM were assessed by immunohistochemistry and determinations of bacterial viable numbers in organs. The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids. A new in-vivo phenotype of YopM was revealed: death of inflammatory cells, evidenced by TUNEL staining beginning at d 1 of infection. Based on distributions of Ly6G⁺, F4/80⁺, and iNOS⁺ cells within foci, the cells that were killed could have included both PMNs and macrophages. By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells. The destruction depended on the presence of both PMNs in the mice and YopM in the bacteria. In mice that lacked the apoptosis mediator caspase-3 the infection dynamics were novel: the parent Y. pestis was limited in growth comparably to the ΔyopM-1 strain in liver, and in spleen a partial growth limitation for parent Y. pestis was seen. This result identified caspase-3 as a co-factor or effector in YopM''s action and supports the hypothesis that in liver YopM''s main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.