An outbreak of oropharyngeal tularaemia linked to natural spring water.

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.

Epizootic of tularemia in an outdoor housed group of cynomolgus monkeys (Macaca fascicularis).

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.

Re-emergence of Francisella tularensis in Germany: fatal tularaemia in a colony of semi-free-living marmosets (Callithrix jacchus).

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.

A novel screening ELISA and a confirmatory Western blot useful for diagnosis and epidemiological studies of tularemia.

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.

Management in der Behandlung von Patienten nach Einsatz biologischer Agenzien.

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.

Conceptional considerations for a German influenza pandemic preparedness plan.

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.

Strategies for PCR based detection of Burkholderia pseudomallei DNA in paraffin wax embedded tissues.

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 procedure for differentiating between the intentional release of biological warfare agents and natural outbreaks of disease: its use in analyzing the tularemia outbreak in Kosovo in 1999 and 2000.

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.

Influenza pandemic: preparedness planning in Germany.

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.

Study on the pathophysiology of experimental Burkholderia pseudomallei infection in mice.

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.

Yersinia enterocolitica 16S rRNA gene types belong to the same genospecies but form three homology groups.

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).

Detection of Francisella tularensis in biological specimens using a capture enzyme-linked immunosorbent assay, an immunochromatographic handheld assay, and a PCR.

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.

Variations in the 16S rRNA gene sequence of Yersinia enterocolitica isolates influence the specificity of molecular identification systems.

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.

Development and characterization of a murine monoclonal antibody reactive with a 64 kDa somatic antigen of Burkholderia cepacia.

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).

[Comparative study of parenteral and oral immunization to influenza in a large clinical trial. 1. Results of clinico-epidemiologic studies]. / Vergleichende Untersuchung einer parenteralen und oralen Immunisierung gegen Influenza im klinischen Grossversuch. 1. Mitteilung: Ergebnisse der klinisch-epidemiologischen Untersuchungen.

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.

[Comparative study of parenteral and oral immunization against influenza in a large clinical trial. 2. Results of immunologic studies]. / Vergleichende Untersuchung einer parenteralen und oralen Immunisierung gegen Influenza im klinischen Grossversuch. 2. Mitteilung: Ergebnisse der immunologischen Untersuchungen.

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 Wissler's allergic subsepticemia in an adult]. / Ein weiterer Fall von Subsepsis allergica Wissler im Erwachsenenalter

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.