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Anthrax is an infectious disease of relevance for military forces. Although spores of Bacillus anthracis obiquitously occur in soil, reports on soil-borne transmission to humans are scarce. In this narrative review, the potential of soil-borne transmission of anthrax to humans is discussed based on pathogen-specific characteristics and reports on anthrax in the course of several centuries of warfare. In theory, anthrax foci can pose a potential risk of infection to animals and humans if sufficient amounts of virulent spores are present in the soil even after an extended period of time. In praxis, however, transmissions are usually due to contacts with animal products and reported events of soil-based transmissions are scarce. In the history of warfare, even in the trenches of World War I, reported anthrax cases due to soil-contaminated wounds are virtually absent. Both the perspectives and the experience of the Western hemisphere and of former Soviet Republics are presented. Based on the accessible data as provided in the review, the transmission risk of anthrax by infections of wounds due to spore-contaminated soil is considered as very low under the most circumstance. Active historic anthrax foci may, however, still pose a risk to the health of deployed soldiers.

Aim: The aim of the study was to further clarify the origin of Escherichia coli O104:H4 outbreak in Germany in 2011 (German Ec) as the likelihood of a deliberate act has not been excluded in previous analyses., Methods: We use an original and the most detailed scoring method so far, with 33 parameters pertaining to the source of infection/reservoir or possible perpetrator, pathogen or biological agent, transmission mechanism/factors or means/media of delivery, and population at risk or target., Results: Total scores for a deliberate or accidental epidemic indicate that the outbreak was more probably caused unintentionally, presumably due to technical accidents or hygienic shortcomings in the food chain., Conclusions: The validity of the present assessment is limited by the lack of data on the reservoir of the pathogen, the source of infection, and the mode of food contamination. Conclusive evidences on these parameters are essential for the final clarification of the outbreak origin., (Copyright© by the National Institute of Public Health, Prague 2015.)

Background: In 2011, Germany was hit by one of its largest outbreaks of acute gastroenteritis and haemolytic uraemic syndrome caused by a new emerging enterohaemorrhagic Escherichia coli O104:H4 strain. The German Haemolytic Uraemic Syndrome/Enterohaemorrhagic E. coli (GHUSEC) outbreak had unusual microbiological, infectiological and epidemiological features and its origin is still only partially solved. The aim of this article is to contribute to the clarification of the origin of the epidemic., Methods: To retrospectively assess whether the GHUSEC outbreak was natural, accidental or a deliberate one, we analysed it according to three published scoring and differentiation models. Data for application of these models were obtained by literature review in the database Medline for the period 2011-13., Results: The analysis of the unusual GHUSEC outbreak shows that the present official assumption of its natural origin is questionable and pointed out to a probability that the pathogen could have also been introduced accidentally or intentionally in the food chain., Conclusion: The possibility of an accidental or deliberate epidemic should not be discarded. Further epidemiological, microbiological and forensic analyses are needed to clarify the GHUSEC outbreak., (© The Author 2014. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

A tularaemia outbreak was investigated involving 188 suspected cases in the Kocaeli region of Turkey between December 2004 and April 2005. A case-control study comprising 135 laboratory-confirmed cases and 55 controls was undertaken to identify risk factors for the development of the outbreak and to evaluate laboratory diagnostic methods. Tularaemia was confirmed by a microagglutination test (MAT) titre of >or=1 : 160 in 90 of the patients. In MAT-negative sera, 23/44 (52 %) were positive by ELISA with Francisella tularensis LPS and 1/9 (11 %) by Western blotting with this antigen. A species-specific PCR was positive in 16/25 (64 %) throat swabs and 8/13 (62 %) lymph node aspirates. Multivariate analysis showed that drinking natural spring water was the leading risk factor for the development of tularaemia (P=0.0001, odds ratio 0.165, 95 % CI 0.790-0.346). The outbreak ceased after abandonment of the suspected natural water springs.

Tularemia outbreaks have occurred in various regions of Turkey in recent years. In this study, clinical (145 patients) and laboratory (97 patients) features of patients with oropharyngeal tularemia were evaluated during the tularemia outbreak in the district of Gölcük in Kocaeli, Turkey. We analyzed the risk factors for therapeutic failure and prolonged recovery time, and compared the efficacy of three antibiotic groups, namely aminoglycoside, tetracycline and quinolone. The most common physical sign and laboratory findings in patients were lymphadenopathy (LAP) and increased erythrocyte sedimentation rate, respectively. Treatment failure was observed in 55 of the 145 (38%) patients during one-year follow-up and the most successful results were obtained in the quinolone group. It was determined that antimicrobial therapy initiated 14 days after onset of symptoms was a statistically significiant risk factor, reducing the success rate (p=0.0001, OR=13.10, 95% CI=5.69-30.15) and prolonging the recovery period (p=0.001, OR=3.23, 95% CI=1.63-6.40) in oropharyngeal tularemia cases. These results suggest that antimicrobial treatment should be started early, and quinolones such as moxifloxacin and ciprofloxacin seem to be new alternatives in the treatment of oropharyngeal tularemia.

Ebola virus (EBOV) has caused outbreaks of severe viral hemorrhagic fever in regions of Central Africa where medical facilities are ill equipped and diagnostic capabilities are limited. To obtain a reliable test that can be implemented easily under these conditions, monoclonal antibodies to the EBOV matrix protein (VP40), which previously had been found to work in a conventional enzyme-linked immunosorbent assay, were used to develop an immunofiltration assay for the detection of EBOV antigen in chemically inactivated clinical specimens. The assay was evaluated by use of defined virus stocks and specimens from experimentally infected animals. Its field application was tested during an outbreak of Ebola hemorrhagic fever in 2003. Although the original goal was to develop an assay that would detect all EBOV species, only the Zaire and Sudan species were detected in practice. The assay represents a first-generation rapid field test for the detection of EBOV antigen that can be performed in 30 min without electrical power or expensive or sensitive equipment.

Francisella tularensis was identified as the cause of a die-off which occurred among a colony of semi-free-living common marmosets (Callithrix jacchus). During the outbreak 5 out of 62 animals died of tularaemia in a research facility located in the district of Goettingen, Germany. All animals had been born at the facility suggesting an endemic infection. A total of five culture isolates were recovered and characterized as F. tularensis holarctica, biovar I. These cultures represent the first isolates obtained in the Federal Republic of Germany for more than 45 years. The outbreak area shows several geographical and ecological characteristics known to favour long-term presence of F. tularensis. Persistence of the pathogen in the remote region along the former German-German border, continuous re-introduction from eastern European countries after destruction of the 'Iron curtain' or introduction through migrating birds are testable hypotheses which could explain the emergence of tularaemia in this particular region.

The human anthrax vaccines currently licensed contain the protective antigen (PA) of Bacillus anthracis as main antigen together with traces of some other bacillus components, e.g. lethal factor (LF). The present study aimed at monitoring the course of specific antibody titres against PA and LF by enzyme linked immunosorbent assays (ELISA), as well as the levels of toxin-neutralising antibodies, in 11 volunteers vaccinated with the human anthrax vaccine UK. After an initial seroconversion in all vaccinees, a significant reduction of both antibody titres against PA and LF, and of neutralising antibodies, was detected just prior to a vaccine boost 6 months after completion of the basic immunisation. Following the booster injection, titres increased again to levels comparable to those after the fourth immunisation. ELISA titres against PA correlated significantly with neutralising antibodies (r=0.816, p<0.001). Therefore, the less work- and time-consuming ELISA should be favoured to monitor the efficacy of an anthrax vaccination.

Tularemia is a highly contagious infectious zoonosis, transmissible by inoculation, ingestion, or inhalation of the infectious agent Francisella tularensis. The disease is perpetuated by infected rodents, blood-sucking arthropods, and by contaminated water. Therefore, nonhuman primates housed outdoors may be at risk for exposure. An epizootic of F. tularensis occurred in an indoor/outdoor-housed group of cynomolgus monkeys (Macaca fascicularis) at the German Primate Center. Tularemia was diagnosed in 18 out of 35 animals within a period of 2 years. Six animals died with unspecific clinical symptoms; 12 animals developed seroconversion and were still alive. Pathologic findings were similar in all monkeys that died and resembled the clinical picture of the human disease, including an ulceroglandular syndrome with local lymphadenopathy, gingivostomatitis, and systemic spread, with manifestations such as subacute necrotizing hepatitis, granulomatous splenitis, and pneumonia. Tularemia was diagnosed by culture, real-time polymerase chain reaction, and ELISA techniques. This is the largest outbreak in nonhuman primates and the first report of tularemia in cynomolgus monkeys. An overview of the recent literature about tularemia in nonhuman primates is given.

We have sequenced fragments of five metabolic housekeeping genes and two genes encoding outer membrane proteins from 81 isolates of Francisella tularensis, representing all four subspecies. Phylogenetic clustering of gene sequences from F. tularensis subsp. tularensis and F. tularensis subsp. holarctica aligned well with subspecies affiliations. In contrast, F. tularensis subsp. novicida and F. tularensis subsp. mediasiatica were indicated to be phylogenetically incoherent taxa. Incongruent gene trees and mosaic structures of housekeeping genes provided evidence for genetic recombination in F. tularensis.

A novel enzyme-linked immunosorbent assay (ELISA) and a confirmatory Western blot (WB) to detect human antibodies against Francisella tularensis were evaluated. The ELISA was based on partially purified lipopolysaccharide (LPS), the WB on whole antigen of F. tularensis. Positive WB showed a typical LPS ladder. Sensitivity and specificity of the ELISA, as assessed in 104 positive sera and 1149 'normal' sera from healthy young adults, were 99.0% and 97.1% respectively. Sensitivity of the WB was close to 100%, whereas specificity was 99.6%. Antibodies against the LPS of F. tularensis were detected in four of the 'normal' sera in both ELISA and WB. The assays were further evaluated using sera of individuals from Norway, Sweden and Kosovo suspected to be infected in tularemia outbreaks. Results revealed that the combination of ELISA and WB is suitable for laboratory confirmation of tularemia as well as for large-scale epidemiological studies.

Melioidosis is a severe gram-negative infection caused by the facultative intracellular bacterium Burkholderia pseudomallei, which is responsible for a broad spectrum of symptoms in both humans and animals. No licensed vaccine currently exists. This study evaluated the protective effect of a monoclonal antibody (Mab Ps6F6) specific to B. pseudomallei exopolysaccharide in an outbred murine model of sub-acute melioidosis. When administered before the infectious challenge, Ps6F6 significantly increased resistance to infection and restrained bacterial burden in the spleen over a 30-days period. Patterns of IFN-gamma production were similar in the treated and non treated groups of mice. However, Ps6F6 lowered IFN-gamma levels over the duration of the assay period, except on day 1, suggesting a transient and rapid production of IFN-gamma under Ps6F6 control. Minor but persisting increases occurred in IL-12 levels while TNF-alpha was detected only in the controls at the later stages of infection. No IL-10 secretion was detected in both groups of mice. These data suggest that passive prophylaxis with Mab Ps6F6 provide a moderate and transient induction of inflammatory responses in infected mice but failed to trigger a sterilizing protective immunity.

The serodiagnostic efficiencies of five different approaches to detecting antibodies (immunoglobulins G, A, and M) developed in clinically proven infections with Francisella tularensis have been assessed. Fifty serum samples from patients suffering from tularemia during an outbreak in Sweden were compared with samples from 50 healthy blood donors (controls) by using an enzyme-linked immunosorbent assay (ELISA), microagglutination (MA), Western blotting (WB), an indirect immunofluorescence assay (IIFA), and flow cytometry (FC). ELISA, WB, and FC were based on the use of preparations of lipopolysaccharides (LPS) of the live vaccine strain of Francisella tularensis subsp. holarctica (ATCC 29684) as a capture antigen. Whole methanol-fixed bacteria were used for IIFA and MA. Optimized protocols yielded a diagnostic sensitivity and specificity of 100% for WB, MA, and FC, 98% for ELISA, and 93% for IIFA. A total of 6,632 serum samples from individuals between the ages of 18 and 79 years, representatively recruited from all regions of Germany, were screened to estimate and confirm the positive predictive value (PVpos) of the ELISA. Serum samples from 15 (0.226%) individuals tested positive for F. tularensis-specific antibodies by ELISA and confirmatory WB. The resulting prevalence-dependent PVpos of 10.2% and specificity of 98.1% were consistent with our findings for tularemia patients and controls. We conclude that the combined usage of a screening ELISA and a confirmatory WB based on LPS as a common antigen, as well as the MA, is a suitable serodiagnostic tool, while the quality of the IIFA is hampered by subjective variations of the results. FC is a promising new approach that might be improved further in terms of multiplex analyses or high-throughput applications.

The risk of terrorist attacks with weapons of mass destruction like biological agents is increasing. Biological agents can be disseminated as aerosols or by contaminating food and beverages. The multitude of agents and the different pathways of transmission cause very different clinical presentations. Natural infections with potential biological agents in Germany are rare and in most cases imported from endemic areas abroad. It is crucial to include these diseases in the spectrum of differential diagnosis. Local and state health departments have to be notified as early as possible in dubious cases. Public health management can be efficient only, if there is high reporting discipline and all epidemic measures are well coordinated., (© Springer-Verlag 2003.)

Recently, several cases of melioidosis imported to Europe have been reported. The diagnosis of the acute or chronic infection remains challenging. This report describes an optimised protocol for fast and reliable DNA preparation for use in two different polymerase chain reaction (PCR) assays, namely: (1) a seminested PCR assay targeting a genus specific sequence of the ribosomal protein subunit 21 (rpsU) gene and (2) a nested PCR assay targeting the gene encoding the filament forming flagellin (fliC). Various strains of Burkholderia spp, strains of closely related genera, and spleen tissue samples of experimentally infected mice were investigated. The combination of PCR and sequencing of the amplicons resulted in high sensitivity and specificity. These procedures may allow rapid, sensitive, and reliable detection of B pseudomallei DNA in routinely formalin fixed and paraffin wax embedded samples, thus providing a safe diagnostic tool and avoiding the cultivation of a risk group 3 agent. In addition, this method could be useful for retrospective histopathological investigations.

A pandemic appearance of influenza A virus must be expected at any time. The limitations of health preserving and life-saving resources, which will inevitably be reached in the event of a pandemic, will be accompanied by ethical and possibly social conflicts, which can be lessened or resolved only through precautionary planning, clearly specified competencies and transparent decisions within a social consensus. In case of a shortage of vaccines and virostatic agents, decisions will have to be made with regard to the segment of the population that absolutely must be vaccinated. It is currently estimated that a (monovalent) vaccine developed for a new pandemic strain would only suffice for the single vaccination of approximately half of the German population after a year; only 10-14 million vaccine dosages would be available to provide basic immunization and single boosters to personnel required to maintain basic medical care and essential infrastructure after half a year. In the event of local influenza outbreaks, antiviral chemotherapeutic agents could be used to close the gap until a vaccine can become effective. Even if suitable influenza vaccines and virostatic agents are not sufficiently available at the start of a pandemic, it is still possible to at least prevent an outbreak of two of the most feared secondary infections that accompany influenza: pneumococcal pneumonia or meningitis and illnesses resulting from Haemophilus influenzae. Agreement still needs to be reached with manufacturers for guaranteeing the necessary vaccine production or ensuring that they have a sufficient stock to meet the minimum demand for antiviral agents and agents for symptomatic treatment.

The events of 11 September and the subsequent anthrax outbreaks in the USA have opened the world's eyes to the threat posed by terrorist groups, criminal organizations and lone operators who will stop at nothing to achieve their goals. The open or covert use of pathogens and toxins as biological warfare agents can no longer be ruled out. Against this background, the appearance of an unusual disease must be studied in order to clarify whether it is a natural or artificially caused occurrence. This issue was recently raised in discussions with local representatives and relief organizations during a tularemia epidemic in Kosovo from October 1999 to May 2000. This paper will present a procedure which attempts to use certain criteria to identify or rule out the use of biological warfare agents in the event of an unusual outbreak of disease. Data and findings gathered by routine epidemiologic and microbiological studies often provide only an indirect answer to this problem. For this reason, various criteria were formulated and points allocated to represent their importance, allowing us to deduce in a semiquantitative manner the degree of possibility of an artificial genesis of outbreaks. The significance and characterization of each criterion are discussed. An analysis of the tularemia epidemic in Kosovo based on the procedure described here indicates that a deliberate release of the causative agent of tularemia, Francisella tularensis, as a biological warfare agent is doubtful. In this paper, an approach is described to discriminate between the intentional use of biological warfare agents and natural outbreaks of infectious diseases. The developed model is flexible and considers the political, military and social analysis of the crisis-afflicted region, the specific features of the pathogen, and the epidemiologic and clinical characteristics of the epidemic.

The following conceptual framework formed the basis for a common decision made by the health ministers of Germany's 16 federal states to set up an influenza pandemic preparedness plan. The worst case scenario was used, on the basis of the data from the pandemic of 'Spanish flu', in 1918-20. The priority groups for vaccination were assessed, as well as the potentially available antiviral treatments. National policies could be highly improved by a common European view.

Burkholderia pseudomallei is the etiological agent of melioidosis, a potentially fatal disease occurring in man and animals. The aim of this study was to investigate the pathophysiological course of experimental melioidosis, and to identify the target organs, in an animal model. For this purpose SWISS mice were infected intraperitoneally with the virulent strain B. pseudomallei 6068. The bacterial load of various organs was quantified daily by bacteriological analysis and by an enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody specific to B. pseudomallei exopolysaccharide (EPS). Electron microscopic investigation of the spleen was performed to locate the bacteria at the cellular level. In this model of acute melioidosis, B. pseudomallei had a marked organ tropism for liver and spleen, and showed evidence of in vivo growth with a bacterial burden of 1.6x10(9) colony forming units (CFU) per gram of spleen 5 days after infection with 200 CFU. The highest bacterial loads were detected in the spleen at all time points, in a range from 2x10(6) to 2x10(9) CFU g(-1). They were still 50-80 times greater than the load of the liver at the time of peak burden. Other investigated organs such as lungs, kidneys, and bone marrow were 10(2)-10(4)-fold less infected than the spleen, with loads ranging from 3x10(2) to 3x10(6) CFU g(-1). The heart and the brain were sites of a delayed infection, with counts in a range from 10(3) to 10(7) times lower than bacterial counts in the spleen. The EPS-specific ELISA proved to be highly sensitive, particularly at the level of those tissues in which colony counting on agar revealed low contamination. In the blood, EPS was detected at concentrations corresponding to bacterial loads ranging from 8x10(3) to 6x10(4) CFU ml(-1). Electron microscopic examination of the spleen revealed figures of phagocytosis, and the presence of large numbers of intact bacteria, which occurred either as single cells or densely packed into vacuoles. Sparse figures suggesting bacterial replication were also observed. In addition, some bacteria could be seen in vacuoles that seemed to have lost their membrane. These observations provide a basis for further investigations on the pathogenesis of the disease.

The species Yersinia (Y.) enterocolitica consists of biochemically and serologically heterogeneous strains. A vernacular nomenclature divides these strains in 'European' and 'American' bioserotypes. We investigated six strains of each group by DNA-DNA hybridization, determination of G + C mol% content and sequence alignment studies. Based on different DNA-DNA hybridization values and the 16S rRNA gene sequences a division into two Yersinia enterocolitica subspecies is justified. We propose the names Yersinia enterocolitica subsp. enterocolitica for strains belonging to the 16S rRNA gene type represented by the Type strain ATCC 9610 and Yersinia enterocolitica subsp. palearctica for strains belonging to the 16S rRNA gene type of strain Y11 (DSMZ13030).

The early detection of Francisella tularensis, the causative agent of tularemia, is important for adequate treatment by antibiotics and the outcome of the disease. Here we describe a new capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies specific for lipopolysaccharide (LPS) of Francisella tularensis subsp. holarctica and Francisella tularensis subsp. tularensis. No cross-reactivity with Francisella tularensis subsp. novicida, Francisella philomiragia, and a panel of other possibly related bacteria, including Brucella spp., Yersinia spp., Escherichia coli, and Burkholderia spp., was observed. The detection limit of the assay was 10(3) to 10(4) bacteria/ml. This sensitivity was achieved by solubilization of the LPS prior to the cELISA. In addition, a novel immunochromatographic membrane-based handheld assay (HHA) and a PCR, targeting sequences of the 17-kDa protein (TUL4) gene of F. tularensis, were used in this study. Compared to the cELISA, the sensitivity of the HHA was about 100 times lower and that of the PCR was about 10 times higher. All three techniques were successfully applied to detect F. tularensis in tissue samples of European brown hares (Lepus europaeus). Whereas all infected samples were recognized by the cELISA, those with relatively low bacterial load were partially or not detected by PCR and HHA, probably due to inhibitors or lack of sensitivity. In conclusion, the HHA can be used as a very fast and simple approach to perform field diagnosis to obtain a first hint of an infection with F. tularensis, especially in emergent situations. In any suspect case, the diagnosis should be confirmed by more sensitive techniques, such as the cELISA and PCR.

Four identification systems were used to type Yersinia enterocolitica strain Y11 and Yersinia enterocolitica sensu strictoT. Two systems based on biochemical reaction patterns identified both strains as Yersinia enterocolitica. Two molecular assays targeting the 16S rRNA gene failed to identify either strain Y11 or the type strain. Therefore, both strains were typed by the classical taxonomical approach requiring a determination of the overall base composition and the base sequence similarity using hybridization. Again both strains were classified as Yersinia enterocolitica isolates. Consequently, the sequences of the 16S rRNA gene of both strains were determined and compared. The strains differed in a region where nucleotide changes between species of the genus Yersinia had been described earlier. These differences may explain the failure of the molecular assays to identify the strains. They also demonstrate an independent evolution of the 16S rRNA genes in the species Yersinia enterocolitica sensu stricto suggesting an amendment to the nomenclature to be used in the future.

Monoclonal antibodies (MAbs) to Burkholderia cepacia were produced from mice immunized with inactivated whole-cell antigen. For screening of resulting MAbs an enzyme-linked immunosorbent assay (ELISA) was used. A stable hybridoma cell line (BC-2) producing specific antibodies to a 64 kDa somatic antigen from B. cepacia was established. In ELISA and immunoblotting analysis the MAb BC-2 recognized all tested strains of B. cepacia whereas no cross-reaction with 32 Pseudomonas aeruginosa strains was found. From a wide range of other bacteria only strains of the species Burkholderia mallei, Burkholderia pseudomallei, and Burkholderia gladioli showed cross-reactions. The MAb BC-2 will be used to develop a diagnostic assay for the identification of B. cepacia and B. gladioli, important agents of nosocomial infections in immunocompromised patients suffering especially from cystic fibrosis (CF).

360 volunteers were recruited for the investigation from a homologous collective. 174 were immunized parenterally with "Influmun" from SSW Dresden, GDR. 176 volunteers were immunized twice orally with an interval of 60 days with an influenza vaccine inactivated by x-ray using enteric-coated capsules. In an interval of six months ARI-symptoms were investigated. Between 13th and 17th week 1988 an increased ARI-morbidity in the Greifswald-region was observed in which influenza A viruses were involved. In comparison with 312 non-immunized persons of the same age, sex and living area the immunized volunteers of the two groups showed 83.4% less sickness days. 30 persons of the non-immunized group got ill for totally 217 days, whereas only nine persons of the two immunized groups were put on the sicklist for totally 36 days. No significant differences concerning the occurrence and duration of acute respiratory infections (ARI) between the two differently immunized groups could be observed.

In a multicentric trial 350 persons (19-24 years) were immunized with influenza vaccines containing the following virus antigens: A/Singapore/6/86, (H1N1); A/Mississippi/1/85, (H3N2); B/Ann Arbor/1/86. 174 received an i.m. injection of 0.5 ml "Influmun" vaccine from SSW Dresden/GDR. 176 persons were immunized twice within 60 days with enteric-wated capsules each containing approximately 60 micrograms hemagglutinin of all three virus strains. The volunteers were clinically observed in an interval of 6 months. The majority of orally immunized persons with low pre-immunization titers responded with fourfold or higher IgA antibody titer rises in nasal secretions, but no significant IgG antibody titer increase in sera could be observed. Secretory antibody titers remained elevated for 2 to 4 months. Parenterally immunized persons showed antibody titer rises in sera but not in nasal secretions. In both groups the highest antibody titer increases were observed after application of the A/Singapore/6/86 virus antigen. Volunteers with high pre-immunization titer did not show an antibody increase neither after parenteral nor oral immunization.

A further case of subsepsis allergica Wissler in a 35-year-old woman is reported. The problems of the differential diagnosis of this rare disease are discussed. In the present case the disease could successfully be controlled using imuran in combination with corticosteroids.