Virulence difference between the prototypic Schu S4 strain (A1a) and Francisella tularensis A1a, A1b, A2 and type B strains in a murine model of infection.

BACKGROUND: The use of prototypic strains is common among laboratories studying infectious agents as it promotes consistency for data comparability among and between laboratories. Schu S4 is the prototypic virulent strain of Francisella tularensis and has been used extensively as such over the past six decades. Studies have demonstrated virulence differences among the two clinically relevant subspecies of F. tularensis, tularensis (type A) and holarctica (type B) and more recently between type A subpopulations (A1a, A1b and A2). Schu S4 belongs to the most virulent subspecies of F. tularensis, subspecies tularensis. METHODS: In this study, we investigated the relative virulence of Schu S4 in comparison to A1a, A1b, A2 and type B strains using a temperature-based murine model of infection. Mice were inoculated intradermally and a hypothermic drop point was used as a surrogate for death. Survival curves and the length of temperature phases were compared for all infections. Bacterial burdens were also compared between the most virulent type A subpopulation, A1b, and Schu S4 at drop point. RESULTS: Survival curve comparisons demonstrate that the Schu S4 strain used in this study resembles the virulence of type B strains, and is significantly less virulent than all other type A (A1a, A1b and A2) strains tested. Additionally, when bacterial burdens were compared between mice infected with Schu S4 or MA00-2987 (A1b) significantly higher burdens were present in the blood and spleen of mice infected with MA00-2987. CONCLUSIONS: The knowledge gained from using Schu S4 as a prototypic virulent strain has unquestionably advanced the field of tularemia research. The findings of this study, however, indicate that careful consideration of F. tularensis strain selection must occur when the overall virulence of the strain used could impact the outcome and interpretation of results.

Lack of antimicrobial resistance in Yersinia pestis isolates from 17 countries in the Americas, Africa, and Asia.

Yersinia pestis is the causative agent of plague, a fulminant disease that is often fatal without antimicrobial treatment. Plasmid (IncA/C)-mediated multidrug resistance in Y. pestis was reported in 1995 in Madagascar and has generated considerable public health concern, most recently because of the identification of IncA/C multidrug-resistant plasmids in other zoonotic pathogens. Here, we demonstrate no resistance in 392 Y. pestis isolates from 17 countries to eight antimicrobials used for treatment or prophylaxis of plague.

Epidemiology, Ecology and Prevention of Plague in the West Nile Region of Uganda: The Value of Long-Term Field Studies.

Plague, a fleaborne rodent-associated zoonosis, is a neglected disease with most recent cases reported from east and central Africa and Madagascar. Because of its low incidence and sporadic occurrence, most of our knowledge of plague ecology, prevention, and control derives from investigations conducted in response to human cases. Long-term studies (which are uncommon) are required to generate data to support plague surveillance, prevention, and control recommendations. Here we describe a 15-year, multidisciplinary commitment to plague in the West Nile region of Uganda that led to significant advances in our understanding of where and when persons are at risk for plague infection and how to reduce morbidity and mortality. These findings provide data-driven support for several existing recommendations on plague surveillance and prevention and may be generalizable to other plague foci.

An Evaluation of the Flea Index as a Predictor of Plague Epizootics in the West Nile Region of Uganda.

Plague is a low incidence flea-borne zoonosis that is often fatal if treatment is delayed or inadequate. Outbreaks occur sporadically and human cases are often preceded by epizootics among rodents. Early recognition of epizootics coupled with appropriate prevention measures should reduce plague morbidity and mortality. For nearly a century, the flea index (a measure of fleas per host) has been used as a measure of risk for epizootic spread and human plague case occurrence, yet the practicality and effectiveness of its use in surveillance programs has not been evaluated rigorously. We sought to determine whether long-term monitoring of the Xenopsylla flea index on hut-dwelling rats in sentinel villages in the plague-endemic West Nile region of Uganda accurately predicted plague occurrence in the surrounding parish. Based on observations spanning ~6 yr, we showed that on average, the Xenopsylla flea index increased prior to the start of the annual plague season and tended to be higher in years when plague activity was reported in humans or rodents compared with years when it was not. However, this labor-intensive effort had limited spatial coverage and was a poor predictor of plague activity within sentinel parishes.

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.

Two Distinct Yersinia pestis Populations Causing Plague among Humans in the West Nile Region of Uganda.

BACKGROUND: Plague is a life-threatening disease caused by the bacterium, Yersinia pestis. Since the 1990s, Africa has accounted for the majority of reported human cases. In Uganda, plague cases occur in the West Nile region, near the border with Democratic Republic of Congo. Despite the ongoing risk of contracting plague in this region, little is known about Y. pestis genotypes causing human disease. METHODOLOGY/PRINCIPAL FINDINGS: During January 2004-December 2012, 1,092 suspect human plague cases were recorded in the West Nile region of Uganda. Sixty-one cases were culture-confirmed. Recovered Y. pestis isolates were analyzed using three typing methods, single nucleotide polymorphisms (SNPs), pulsed field gel electrophoresis (PFGE), and multiple variable number of tandem repeat analysis (MLVA) and subpopulations analyzed in the context of associated geographic, temporal, and clinical data for source patients. All three methods separated the 61 isolates into two distinct 1.ANT lineages, which persisted throughout the 9 year period and were associated with differences in elevation and geographic distribution. CONCLUSIONS/SIGNIFICANCE: We demonstrate that human cases of plague in the West Nile region of Uganda are caused by two distinct 1.ANT genetic subpopulations. Notably, all three typing methods used, SNPs, PFGE, and MLVA, identified the two genetic subpopulations, despite recognizing different mutation types in the Y. pestis genome. The geographic and elevation differences between the two subpopulations is suggestive of their maintenance in highly localized enzootic cycles, potentially with differing vector-host community composition. This improved understanding of Y. pestis subpopulations in the West Nile region will be useful for identifying ecologic and environmental factors associated with elevated plague risk.

Use of temperature for standardizing the progression of Francisella tularensis in mice.

The study of infectious agents, their pathogenesis, the host response and the evaluation of newly developed countermeasures often requires the use of a living system. Murine models are frequently used to undertake such investigations with the caveat that non-biased measurements to assess the progression of infection are underutilized. Instead, murine models predominantly rely on symptomology exhibited by the animal to evaluate the state of the animal's health and to determine when euthanasia should be performed. In this study, we used subcutaneous temperature as a non-subjective measurement to follow and compare infection in mice inoculated with Francisella tularensis, a Gram-negative pathogen that produces an acute and fatal illness in mice. A reproducible temperature pattern defined by three temperature phases (normal, febrile and hypothermic) was identified in all mice infected with F. tularensis, regardless of the infecting strain. More importantly and for the first time a non-subjective, ethical, and easily determined surrogate endpoint for death based on a temperature, termed drop point, was identified and validated with statistical models. In comparative survival curve analyses for F. tularensis strains with differing virulence, the drop point temperature yielded the same results as those obtained using observed time to death. Incorporation of temperature measurements to evaluate F. tularensis was standardized based on statistical models to provide a new level of robustness for comparative analyses in mice. These findings should be generally applicable to other pathogens that produce acute febrile disease in animal models and offers an important tool for understanding and following the infection process.

Virulence differences among Francisella tularensis subsp. tularensis clades in mice.

Francisella tularensis subspecies tularensis (type A) and holarctica (type B) are of clinical importance in causing tularemia. Molecular typing methods have further separated type A strains into three genetically distinct clades, A1a, A1b and A2. Epidemiological analyses of human infections in the United States suggest that A1b infections are associated with a significantly higher mortality rate as compared to infections caused by A1a, A2 and type B. To determine if genetic differences as defined by molecular typing directly correlate with differences in virulence, A1a, A1b, A2 and type B strains were compared in C57BL/6 mice. Here we demonstrate significant differences between survival curves for infections caused by A1b versus A1a, A2 and type B, with A1b infected mice dying earlier than mice infected with A1a, A2 or type B; these results were conserved among multiple strains. Differences were also detected among type A clades as well as between type A clades and type B with respect to bacterial burdens, and gross anatomy in infected mice. Our results indicate that clades defined within F. tularensis subsp. tularensis by molecular typing methods correlate with virulence differences, with A1b strains more virulent than A1a, A2 and type B strains. These findings indicate type A strains are not equivalent with respect to virulence and have important implications for public health as well as basic research programs.