Improved Discrimination of Bacillus anthracis from Closely Related Species in the Bacillus cereus Sensu Lato Group Based on Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Discrimination of highly pathogenic bacteria, such as Bacillus anthracis, from closely related species based on molecular biological methods is challenging. We applied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to a collection of B. anthracis strains and close relatives in order to significantly improve the statistical confidence of identification results for this group of bacteria. Protein mass spectra of 189 verified and diverse Bacillus strains of the Bacillus cereus sensu lato group were generated using MALDI-TOF MS and subsequently analyzed with supervised and unsupervised statistical methods, such as shrinkage discriminant analysis (SDA) and principal-component analysis (PCA). We aimed at identifying specific biomarkers in the protein spectra of B. anthracis not present in closely related Bacillus species. We could identify 7, 10, 18, and 14 B. anthracis-specific biomarker candidates that were absent in B. cereus, B. mycoides, B. thuringiensis, and B. weihenstephanensis strains, respectively. Main spectra (MSP) of a defined collection of Bacillus strains were compiled using the Bruker Biotyper software and added to an in-house reference library. Reevaluation of this library with 15 hitherto untested strains of B. anthracis and B. cereus yielded improved score values. The B. anthracis strains were identified with score values between 2.33 and 2.55 using the in-house database, while the same strains were identified with scores between 1.94 and 2.37 using the commercial database, and no false-positive identifications occurred using the in-house database.

High seroprevalence for indigenous spotted fever group rickettsiae in forestry workers from the federal state of Brandenburg, Eastern Germany.

In the last decade six Rickettsia species, including Rickettsia slovaca have been characterized in Germany. All of these species could be linked to distinct clinical syndromes in humans. However, due to lack of seroepidemiological data an estimation of the prevalence and the public health impact of rickettsial infections in Germany is difficult. The aim of the present study was to determine the seroprevalence of spotted fever group (SFG) rickettsiae in a population with an elevated exposure risk to ticks. For that purpose, 559 sera of forestry workers in the federal state of Brandenburg, Eastern Germany, were screened for SFG-rickettsiae reactive IgG antibodies. Positive sera were subsequently titrated by microimmunofluorescence assay against R. helvetica, R. raoultii, R. felis, "R. monacensis" and R. slovaca. The total average IgG seroprevalence rate against SFG rickettsiae of 27.5% was found to be represented by 9.7% R. helvetica, 5% R. raoultii, 2.7% R. felis, 0.5% "R. monacensis" and 0.5% R. slovaca. The remaining 9.1% positive test results were of non-differentiable origin. IgG seroprevalences ranged from 11% to 55% in the different forestry districts. Older and male participants had a significantly higher probability for seropositivity and higher anti-rickettsia antibody titer level. In addition, the number of recent as well as the recalled lifetime tick bites was significantly associated with seropositivity and higher titers against SFG rickettsiae. In conclusion, we found an unexpected high total seroprevalence against SFG rickettsiae in forestry workers and serological evidence confirming the occurrence of R. raoultii, R. felis, "R. monacensis" and R. helvetica in the federal State of Brandenburg.

Hantavirus cardiopulmonary syndrome due to Puumala virus in Germany.

In Germany Puumala virus (PUUV), known to cause mild forms of hemorrhagic fever with renal syndrome (HFRS), is the predominating endemic hantavirus. We herein describe an unusually severe case of a PUUV infection that occurred in summer 2015 in South Eastern Germany in a region known to be endemic for PUUV since over ten years. A 54-year-old female gardener was admitted to hospital with fever, cough and dyspnea. Within 48hours the patient developed a rapid progressive adult respiratory distress syndrome (ARDS) with circulatory failure and required ECMO (extracorporeal membrane oxygenation) treatment. Serological and molecular biological examinations of serum samples confirmed an infection with PUUV. Partial sequences of the S- and M-segment clustered to a strain previously described in South Eastern Germany. Our reported case highlights, that in rare incidents PUUV can cause hantavirus cardiopulmonary syndrome, a syndrome that is usually found after infections with New World hantaviruses, and neurological symptoms.

Identification of factors influencing the Puumala virus seroprevalence within its reservoir in aMontane Forest Environment.

Puumala virus (PUUV) is a major cause of mild to moderate haemorrhagic fever with renal syndrome and is transmitted by the bank vole (Myodes glareolus). There has been a high cumulative incidence of recorded human cases in South-eastern Germany since 2004 when the region was first recognized as being endemic for PUUV. As the area is well known for outdoor recreation and the Bavarian Forest National Park (BFNP) is located in the region, the increasing numbers of recorded cases are of concern. To understand the population and environmental effects on the seroprevalence of PUUV in bank voles we trapped small mammals at 23 sites along an elevation gradient from 317 to 1420m above sea level. Generalized linear mixed effects models(GLMEM) were used to explore associations between the seroprevalence of PUUV in bank voles and climate and biotic factors. We found that the seroprevalence of PUUV was low (6%-7%) in 2008 and 2009, and reached 29% in 2010. PUUV seroprevalence was positively associated with the local species diversity and deadwood layer, and negatively associated with mean annual temperature, mean annual solar radiation, and herb layer. Based on these findings, an illustrative risk map for PUUV seroprevalence prediction in bank voles was created for an area of the national park. The map will help when planning infrastructure in the national park (e.g., huts, shelters, and trails).

In Search for Factors that Drive Hantavirus Epidemics.

In Europe, hantaviruses (Bunyaviridae) are small mammal-associated zoonotic and emerging pathogens that can cause hemorrhagic fever with renal syndrome (HFRS). Puumala virus, the main etiological agent carried by the bank vole Myodes glareolus is responsible for a mild form of HFRS while Dobrava virus induces less frequent but more severe cases of HFRS. Since 2000 in Europe, more than 3000 cases of HFRS have been recorded, in average, each year, which is nearly double compared to the previous decade. In addition to this upside long-term trend, significant oscillations occur. Epidemic years appear, usually every 2-4 years, with an increased incidence, generally in localized hot spots. Moreover, the virus has been identified in new areas in the recent years. A great number of surveys have been carried out in order to assess the prevalence of the infection in the reservoir host and to identify links with different biotic and abiotic factors. The factors that drive the infections are related to the density and diversity of bank vole populations, prevalence of infection in the reservoir host, viral excretion in the environment, survival of the virus outside its host, and human behavior, which affect the main transmission virus route through inhalation of infected rodent excreta. At the scale of a rodent population, the prevalence of the infection increases with the age of the individuals but also other parameters, such as sex and genetic variability, interfere. The contamination of the environment may be correlated to the number of newly infected rodents, which heavily excrete the virus. The interactions between these different parameters add to the complexity of the situation and explain the absence of reliable tools to predict epidemics. In this review, the factors that drive the epidemics of hantaviruses in Middle Europe are discussed through a panorama of the epidemiological situation in Belgium, France, and Germany.