An Outbreak Investigation of Salmonella Typhimurium Illnesses in the United States Linked to Packaged Leafy Greens Produced at a Controlled Environment Agriculture Indoor Hydroponic Operation - 2021.

In 2021, the U.S. Food and Drug Administration (FDA), the Centers for Disease Control and Prevention (CDC), and state partners investigated a multistate outbreak of Salmonella Typhimurium illnesses linked to packaged leafy greens from a controlled environment agriculture (CEA) operation in Illinois. Thirty-one illnesses and four hospitalizations were reported in four states, with a significant epidemiologic signal for packaged leafy greens from Farm A. A traceback investigation for leafy greens included seven points of service (POS) with food exposure data from eight ill people. Each POS was supplied leafy greens by Farm A. FDA investigators observed operations at Farm A and noted that 1) the firm did not consider their indoor hydroponic pond water as agricultural water, 2) condensate dripping from the chiller water supply line inside the building, and 3) unprotected outdoor storage of packaged soilless growth media and pallets used for finished product. FDA collected 25 product, water, and environmental samples from Farm A. The outbreak strain was recovered from a water sample collected from a stormwater drainage basin located on the property adjacent to Farm A. In addition, an isolate of Salmonella Liverpool was recovered from two indoor growing ponds within the same growing house, but no illnesses were linked to the isolate. Farm A voluntarily recalled all implicated products and provided their root cause analysis (RCA) and return-to-market plan to FDA. While the source and route of the contamination were not determined by the RCA, epidemiologic and traceback evidence confirmed the packaged salads consumed by ill persons were produced by Farm A. This was the first investigation of a multistate foodborne illness outbreak associated with leafy greens grown in a CEA operation. This outbreak demonstrated the need for growers using hydroponic methods to review their practices for potential sources and routes of contamination and to reduce food safety risks when identified.

Mosquito Control Activities during Local Transmission of Zika Virus, Miami-Dade County, Florida, USA, 2016.

In 2016, four clusters of local mosquitoborne Zika virus transmission were identified in Miami-Dade County, Florida, USA, generating "red zones" (areas into which pregnant women were advised against traveling). The Miami-Dade County Mosquito Control Division initiated intensive control activities, including property inspections, community education, and handheld sprayer applications of larvicides and adulticides. For the first time, the Mosquito Control Division used a combination of areawide ultralow-volume adulticide and low-volume larvicide spraying to effectively control Aedes aegypti mosquitoes, the primary Zika virus vector within the county. The number of mosquitoes rapidly decreased, and Zika virus transmission was interrupted within the red zones immediately after the combination of adulticide and larvicide spraying.

Transmission of the relapsing fever spirochete, Borrelia miyamotoi, by single transovarially-infected larval Ixodes scapularis ticks.

The relapsing fever spirochete, Borrelia miyamotoi, is increasingly recognized as a cause of human illness (hard tick-borne relapsing fever) in the United States. We previously demonstrated that single nymphs of the blacklegged tick, Ixodes scapularis, can transmit B. miyamotoi to experimental hosts. However, two recent epidemiological studies from the Northeastern United States indicate that human cases of hard tick-borne relapsing fever peak during late summer, after the spring peak for nymphal tick activity but coincident with the peak seasonal activity period of larval ticks in the Northeast. These epidemiological findings, together with evidence that B. miyamotoi can be passed from infected I. scapularis females to their offspring, suggest that bites by transovarially-infected larval ticks can be an important source of human infection. To demonstrate experimentally that transovarially-infected larval I. scapularis ticks can transmit B. miyamotoi, outbred Mus musculus CD1 mice were exposed to 1 or 2 potentially infected larvae. Individual fed larvae and mouse blood taken 10 d after larvae attached were tested for presence of B. miyamotoi DNA, and mice also were examined for seroreactivity to B. miyamotoi 8 wk after tick feeding. We documented B. miyamotoi DNA in blood from 13 (57%) of 23 mice exposed to a single transovarially-infected larva and in 5 (83%) of 6 mice exposed to two infected larvae feeding simultaneously. All 18 positive mice also demonstrated seroreactivity to B. miyamotoi. Of the 11 remaining mice without detectable B. miyamotoi DNA in their blood 10 d after infected larvae attached, 7 (64%) had evidence of spirochete exposure by serology 8 wk later. Because public health messaging for risk of exposure to Lyme disease spirochetes focuses on nymphal and female I. scapularis ticks, our finding that transovarially-infected larvae effectively transmit B. miyamotoi should lead to refined tick-bite prevention messages.

Prevalence and distribution of seven human pathogens in host-seeking Ixodes scapularis (Acari: Ixodidae) nymphs in Minnesota, USA.

In the north-central United States, the blacklegged tick (Ixodes scapularis) is currently known to vector seven human pathogens. These include five bacteria (Borrelia burgdorferi sensu stricto, Borrelia mayonii, Borrelia miyamotoi, Anaplasma phagocytophilum, Ehrlichia muris eauclairensis), one protozoan (Babesia microti) and one virus (Powassan). We sought to assess the prevalence and distribution of these pathogens in host-seeking nymphs collected throughout Minnesota, a state on the northwestern edge of the tick's expanding range, where reported cases of I. scapularis-borne diseases have increased in incidence and geographic range over the past decade. Among the 1240 host-seeking I. scapularis nymphs that we screened from 64 sites, we detected all seven pathogens at varying frequencies. Borrelia burgdorferi s.s. was the most prevalent and geographically widespread, found in 25.24% of all nymphs tested. Anaplasma phagocytophilum and Babesia microti were also geographically widespread, but they were less prevalent than Bo. burgdorferi s.s. (detected in 6.29% and 4.68% of ticks, respectively). Spatial clusters of sites with high prevalence for these three pathogens were identified in the north-central region of the state. Prevalence was less than 1.29% for each of the remaining pathogens. Two or more pathogens were detected in 90 nymphs (7.26%); coinfections with Bo. burgdorferi s.s. and either A. phagocytophilum (51 nymphs, 4.11%) or Ba. microti (43 nymphs, 3.47%) were the most common combinations. The distribution and density of infected ticks mirrors the distribution of notifiable tick-borne diseases in Minnesota and provides information on the distribution and prevalence of recently described human pathogens.

An Evaluation of Removal Trapping to Control Rodents Inside Homes in a Plague-Endemic Region of Rural Northwestern Uganda.

Rodents pose a significant threat to human health, particularly in rural subsistence farming communities in Africa, where rodents threaten food security and serve as reservoirs of human pathogens, including the agents of plague, leptospirosis, murine typhus, rat-bite fever, Lassa fever, salmonellosis, and campylobacteriosis. Our study focused on the plague-endemic West Nile region of Uganda, where a majority of residents live in Uganda government-defined poverty, rely on subsistence farming for a living, and frequently experience incursions of rodents into their homes. In this study, we show that rodent removal was achieved in a median of 6 days of intensive lethal trapping with multiple trap types (range: 0-16 days). However, rodent abundance in 68.9% of homesteads returned to pretreatment levels within a median of 8 weeks (range 1-24 weeks), and at least a single rodent was captured in all homesteads by a median of 2 weeks (range 1-16 weeks) after removal efforts were terminated. Results were similar between homesteads that practiced rodent control whether or not their neighbors implemented similar strategies. Overall, intensive lethal trapping inside homes appears to be effective at reducing rodent abundance, but control was short lived after trapping ceased.

An Acarological Risk Model Predicting the Density and Distribution of Host-Seeking Ixodes scapularis Nymphs in Minnesota.

Ixodes scapularis is the vector of at least seven human pathogens in Minnesota, two of which are known to cause Lyme disease (Borrelia burgdorferi sensu stricto and Borrelia mayonii). In Minnesota, the statewide incidence of Lyme disease and other I. scapularis-borne diseases and the geographic extent over which cases have been reported have both increased substantially over the last two decades. These changes correspond with an expanding distribution of I. scapularis over a similar time frame. Because the risk of exposure to I. scapularis-borne pathogens is likely related to the number of ticks encountered, we developed an acarological risk model predicting the density of host-seeking I. scapularis nymphs (DON) in Minnesota. The model was informed by sampling 81 sites located in 42 counties in Minnesota. Two main foci were predicted by the model to support elevated densities of host-seeking I. scapularis nymphs, which included the seven-county Minneapolis-St. Paul metropolitan area and counties in northern Minnesota, including Lake of the Woods and Koochiching counties. There was substantial heterogeneity observed in predicted DON across the state at the county scale; however, counties classified as high risk for I. scapularis-borne diseases and counties with known established populations of I. scapularis had the highest proportion of the county predicted as suitable for host-seeking nymphs (≥ 0.13 nymphs/100 m2). The model provides insight into areas of potential I. scapularis population expansion and identifies focal areas of predicted suitable habitat within counties where the incidence of I. scapularis-borne diseases has been historically low.

Evaluating acarological risk for exposure to Ixodes scapularis and Ixodes scapularis-borne pathogens in recreational and residential settings in Washington County, Minnesota.

The distribution of I. scapularis, the tick vector of the bacteria that cause Lyme disease, has been expanding over the last two decades in the north-central United States in parallel with increasing incidence of human cases of Lyme disease in that region. However, assessments of residential risk for exposure to ticks are lacking from this region. Here, we measured the density of host-seeking I. scapularis nymphs in two suburban and two rural public recreational sites located in Washington County, Minnesota as well as in nearby residential properties. We sought to compare tick densities across land use types and to identify environmental factors that might impact nymphal density. We also assessed the prevalence of infection in the collected ticks with Lyme disease spirochetes (Borrelia burgdorferi sensu stricto, B. mayonii), and other I. scapularis-borne pathogens including B. miyamotoi, Babesia microti and Anaplasma phagocytophilum. Similar to studies from the eastern United States, on residential properties, I. scapularis nymphal densities were highest in the ecotonal areas between the forest edge and the lawn. Residences with the highest densities of nymphs were more likely to have a higher percentage of forest cover, log piles, and signs of deer on their property. In recreational areas, we found the highest nymphal densities both in the wooded areas next to trails as well as on mowed trails. Among the 303 host-seeking I. scapularis nymphs tested for pathogens, B. burgdorferi sensu stricto, A. phagocytophilum and B. miyamotoi were detected in 42 (13.8%), 14 (4.6%), and 2 (0.6%) nymphs, respectively.

Rat Fall Surveillance Coupled with Vector Control and Community Education as a Plague Prevention Strategy in the West Nile Region, Uganda.

Plague, primarily a disease of rodents, is most frequently transmitted by fleas and causes potentially fatal infections in humans. In Uganda, plague is endemic to the West Nile region. Primary prevention for plague includes control of rodent hosts or flea vectors, but targeting these efforts is difficult given the sporadic nature of plague epizootics in the region and limited resource availability. Here, we present a community-based strategy to detect and report rodent deaths (rat fall), an early sign of epizootics. Laboratory testing of rodent carcasses is used to trigger primary and secondary prevention measures: indoor residual spraying (IRS) and community-based plague education, respectively. During the first 3 years of the program, individuals from 142 villages reported 580 small mammal deaths; 24 of these tested presumptive positive for Yersinia pestis by fluorescence microscopy. In response, for each of the 17 affected communities, village-wide IRS was conducted to control rodent-associated fleas within homes, and community sensitization was conducted to raise awareness of plague signs and prevention strategies. No additional presumptive Y. pestis-positive carcasses were detected in these villages within the 2-month expected duration of residual activity for the insecticide used in IRS. Despite comparatively high historic case counts, no human plague cases were reported from villages participating in the surveillance program; five cases were reported from elsewhere in the districts. We evaluate community participation and timeliness of response, report the frequency of human plague cases in participating and surrounding villages, and evaluate whether a program such as this could provide a sustainable model for plague prevention in endemic areas.

Comparison of Zoonotic Bacterial Agents in Fleas Collected from Small Mammals or Host-Seeking Fleas from a Ugandan Region Where Plague Is Endemic.

Fleas (n = 407) were collected from small mammals trapped inside huts and surroundings of homesteads in five villages within the Arua and Zombo districts of Uganda. The most common flea species were Dinopsyllus lypusus (26%) and Xenopsylla cheopis (50%). Off-host fleas (n = 225) were collected inside huts by using Kilonzo flea traps. The majority of the off-host fleas were Ctenocephalides felis (80%). All fleas were examined for the presence of Bartonella spp., Rickettsia spp., and Yersinia spp. Bartonella DNA was detected in 91 fleas, with an overall prevalence of 14%. Bartonella prevalence was significantly higher in rodent or shrew fleas than in off-host fleas (22% versus 1%). The majority of Bartonella-positive fleas were of the species D. lypusus (61%), X. cheopis (20%), and Ctenophthalmus calceatus (14%). Sequencing analysis identified 12 Bartonella genetic variants, 9 of which belonged to the zoonotic pathogen B. elizabethae species complex. Rickettsia DNA was detected in 143 fleas, giving an overall prevalence of 23%, with a significantly higher prevalence in off-host fleas than in rodent or shrew fleas (56% versus 4%). The majority (88%) of Rickettsia-positive fleas were C. felis and were collected from Kilonzo traps, while a small portion (10%) were X. cheopis collected from rodents. Sequencing analysis identified six Rickettsia genogroups that belonged either to zoonotic R. felis or to the closely related "Candidatus Ricksettia asemboensis" and "Candidatus Ricksettia sengalensis." Yersinia DNA was not detected in the fleas tested. These observations suggested that fleas in northwestern Uganda commonly carry the zoonotic agents B. elizabethae and R. felis and potentially play an important role in transmitting these infections to humans. IMPORTANCE Fleas play critical roles in transmitting some infections among animals and from animals to humans. Detection of pathogens in fleas is important to determine human risks for flea-borne diseases and can help guide diagnosis and treatment. Our findings of high prevalence rates of B. elizabethae and R. felis in fleas in the Arua and Zombo districts of Uganda implicate these agents as potential causative agents of undiagnosed febrile illnesses in this area.

Transmission of the Lyme Disease Spirochete Borrelia mayonii in Relation to Duration of Attachment by Nymphal Ixodes scapularis (Acari: Ixodidae).

The recently recognized Lyme disease spirochete, Borrelia mayonii, has been detected in host-seeking Ixodes scapularis Say ticks and is associated with human disease in the Upper Midwest. Although experimentally shown to be vector competent, studies have been lacking to determine the duration of time from attachment of a single B. mayonii-infected I. scapularis nymph to transmission of spirochetes to a host. If B. mayonii spirochetes were found to be transmitted within the first 24 h after tick attachment, in contrast to Borrelia burgdorferi spirochetes (>24 h), then current recommendations for tick checks and prompt tick removal as a way to prevent transmission of Lyme disease spirochetes would need to be amended. We therefore conducted a study to determine the probability of transmission of B. mayonii spirochetes from single infected nymphal I. scapularis ticks to susceptible experimental mouse hosts at three time points postattachment (24, 48, and 72 h) and for a complete feed (>72-96 h). No evidence of infection with or exposure to B. mayonii occurred in mice that were fed upon by a single infected nymph for 24 or 48 h. The probability of transmission by a single infected nymphal tick was 31% after 72 h of attachment and 57% for a complete feed. In addition, due to unintended simultaneous feeding upon some mice by two B. mayonii-infected nymphs, we recorded a single occasion in which feeding for 48 h by two infected nymphs resulted in transmission and viable infection in the mouse. We conclude that the duration of attachment of a single infected nymphal I. scapularis tick required for transmission of B. mayonii appears to be similar to that for B. burgdorferi: transmission is minimal for the first 24 h of attachment, rare up to 48 h, but then increases distinctly by 72 h postattachment.

Pathogenesis of oral FIV infection.

Feline immunodeficiency virus (FIV) is the feline analogue of human immunodeficiency virus (HIV) and features many hallmarks of HIV infection and pathogenesis, including the development of concurrent oral lesions. While HIV is typically transmitted via parenteral transmucosal contact, recent studies prove that oral transmission can occur, and that saliva from infected individuals contains significant amounts of HIV RNA and DNA. While it is accepted that FIV is primarily transmitted by biting, few studies have evaluated FIV oral infection kinetics and transmission mechanisms over the last 20 years. Modern quantitative analyses applied to natural FIV oral infection could significantly further our understanding of lentiviral oral disease and transmission. We therefore characterized FIV salivary viral kinetics and antibody secretions to more fully document oral viral pathogenesis. Our results demonstrate that: (i) saliva of FIV-infected cats contains infectious virus particles, FIV viral RNA at levels equivalent to circulation, and lower but significant amounts of FIV proviral DNA; (ii) the ratio of FIV RNA to DNA is significantly higher in saliva than in circulation; (iii) FIV viral load in oral lymphoid tissues (tonsil, lymph nodes) is significantly higher than mucosal tissues (buccal mucosa, salivary gland, tongue); (iv) salivary IgG antibodies increase significantly over time in FIV-infected cats, while salivary IgA levels remain static; and, (v) saliva from naïve Specific Pathogen Free cats inhibits FIV growth in vitro. Collectively, these results suggest that oral lymphoid tissues serve as a site for enhanced FIV replication, resulting in accumulation of FIV particles and FIV-infected cells in saliva. Failure to induce a virus-specific oral mucosal antibody response, and/or viral capability to overcome inhibitory components in saliva may perpetuate chronic oral cavity infection. Based upon these findings, we propose a model of oral FIV pathogenesis and suggest alternative diagnostic modalities and translational approaches to study oral HIV infection.

Transmission of Borrelia miyamotoi sensu lato relapsing fever group spirochetes in relation to duration of attachment by Ixodes scapularis nymphs.

Borrelia miyamotoi sensu lato relapsing fever group spirochetes are emerging as causative agents of human illness (Borrelia miyamotoi disease) in the United States. Host-seeking Ixodes scapularis ticks are naturally infected with these spirochetes in the eastern United States and experimentally capable of transmitting B. miyamotoi. However, the duration of time required from tick attachment to spirochete transmission has yet to be determined. We therefore conducted a study to assess spirochete transmission by single transovarially infected I. scapularis nymphs to outbred white mice at three time points post-attachment (24, 48, and 72h) and for a complete feed (>72-96h). Based on detection of B. miyamotoi DNA from the blood of mice fed on by an infected nymph, the probability of spirochete transmission increased from 10% by 24h of attachment (evidence of infection in 3/30 mice) to 31% by 48h (11/35 mice), 63% by 72h (22/35 mice), and 73% for a complete feed (22/30 mice). We conclude that (i) single I. scapularis nymphs effectively transmit B. miyamotoi relapsing fever group spirochetes while feeding, (ii) transmission can occur within the first 24h of nymphal attachment, and (iii) the probability of transmission increases with the duration of nymphal attachment.

Isolation of the Lyme Disease Spirochete Borrelia mayonii From Naturally Infected Rodents in Minnesota.

Borrelia mayonii is a newly described member of the Borrelia burgdorferi sensu lato complex that is vectored by the black-legged tick (Ixodes scapularis Say) and a cause of Lyme disease in Minnesota and Wisconsin. Vertebrate reservoir hosts involved in the enzootic maintenance of B. mayonii have not yet been identified. Here, we describe the first isolation of B. mayonii from naturally infected white-footed mice (Peromyscus leucopus Rafinesque) and an American red squirrel (Tamiasciurus hudsonicus Erxleben) from Minnesota, thus implicating these species as potential reservoir hosts for this newly described spirochete.

Prevalence and Diversity of Tick-Borne Pathogens in Nymphal Ixodes scapularis (Acari: Ixodidae) in Eastern National Parks.

Tick-borne pathogens transmitted by Ixodes scapularis Say (Acari: Ixodidae), also known as the deer tick or blacklegged tick, are increasing in incidence and geographic distribution in the United States. We examined the risk of tick-borne disease exposure in 9 national parks across six Northeastern and Mid-Atlantic States and the District of Columbia in 2014 and 2015. To assess the recreational risk to park visitors, we sampled for ticks along frequently used trails and calculated the density of I. scapularis nymphs (DON) and the density of infected nymphs (DIN). We determined the nymphal infection prevalence of I. scapularis with a suite of tick-borne pathogens including Borrelia burgdorferi, Borrelia miyamotoi, Anaplasma phagocytophilum, and Babesia microti. Ixodes scapularis nymphs were found in all national park units; DON ranged from 0.40 to 13.73 nymphs per 100 m2. Borrelia burgdorferi, the causative agent of Lyme disease, was found at all sites where I. scapularis was documented; DIN with B. burgdorferi ranged from 0.06 to 5.71 nymphs per 100 m2. Borrelia miyamotoi and A. phagocytophilum were documented at 60% and 70% of the parks, respectively, while Ba. microti occurred at just 20% of the parks. Ixodes scapularis is well established across much of the Northeastern and Mid-Atlantic States, and our results are generally consistent with previous studies conducted near the areas we sampled. Newly established I. scapularis populations were documented in two locations: Washington, D.C. (Rock Creek Park) and Greene County, Virginia (Shenandoah National Park). This research demonstrates the potential risk of tick-borne pathogen exposure in national parks and can be used to educate park visitors about the importance of preventative actions to minimize tick exposure.

Notes from The Field: Ebola Virus Disease Cluster - Northern Sierra Leone, January 2016.

On January 14, 2016, the Sierra Leone Ministry of Health and Sanitation was notified that a buccal swab collected on January 12 from a deceased female aged 22 years (patient A) in Tonkolili District had tested positive for Ebola virus by reverse transcription-polymerase chain reaction (RT-PCR). The most recent case of Ebola virus disease (Ebola) in Sierra Leone had been reported 4 months earlier on September 13, 2015 (1), and the World Health Organization had declared the end of Ebola virus transmission in Sierra Leone on November 7, 2015 (2). The Government of Sierra Leone launched a response to prevent further transmission of Ebola virus by identifying contacts of the decedent and monitoring them for Ebola signs and symptoms, ensuring timely treatment for anyone with Ebola, and conducting an epidemiologic investigation to identify the source of infection.

Reported Distribution of Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus in the United States, 1995-2016 (Diptera: Culicidae).

Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) albopictus (Skuse) transmit arboviruses that are increasing threats to human health in the Americas, particularly dengue, chikungunya, and Zika viruses. Epidemics of the associated arboviral diseases have been limited to South and Central America, Mexico, and the Caribbean in the Western Hemisphere, with only minor localized outbreaks in the United States. Nevertheless, accurate and up-to-date information for the geographical ranges of Ae. aegypti and Ae. albopictus in the United States is urgently needed to guide surveillance and enhance control capacity for these mosquitoes. We compiled county records for presence of Ae. aegypti and Ae. albopictus in the United States from 1995-2016, presented here in map format. Records were derived from the Centers for Disease Control and Prevention ArboNET database, VectorMap, the published literature, and a survey of mosquito control agencies, university researchers, and state and local health departments. Between January 1995 and March 2016, 183 counties from 26 states and the District of Columbia reported occurrence of Ae. aegypti, and 1,241 counties from 40 states and the District of Columbia reported occurrence of Ae. albopictus. During the same time period, Ae. aegypti was collected in 3 or more years from 94 counties from 14 states and the District of Columbia, and Ae. albopictus was collected during 3 or more years from 514 counties in 34 states and the District of Columbia. Our findings underscore the need for systematic surveillance of Ae. aegypti and Ae. albopictus in the United States and delineate areas with risk for the transmission of these introduced arboviruses.

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.

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.

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.

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.