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.

Analysis of clonal type-specific antibody reactions in Toxoplasma gondii seropositive humans from Germany by peptide-microarray.

BACKGROUND: Different clonal types of Toxoplasma gondii are thought to be associated with distinct clinical manifestations of infections. Serotyping is a novel technique which may allow to determine the clonal type of T. gondii humans are infected with and to extend typing studies to larger populations which include infected but non-diseased individuals. METHODOLOGY: A peptide-microarray test for T. gondii serotyping was established with 54 previously published synthetic peptides, which mimic clonal type-specific epitopes. The test was applied to human sera (n = 174) collected from individuals with an acute T. gondii infection (n = 21), a latent T. gondii infection (n = 53) and from T. gondii-seropositive forest workers (n = 100). FINDINGS: The majority (n = 124; 71%) of all T. gondii seropositive human sera showed reactions against synthetic peptides with sequences specific for clonal type II (type II peptides). Type I and type III peptides were recognized by 42% (n = 73) or 16% (n = 28) of the human sera, respectively, while type II-III, type I-III or type I-II peptides were recognized by 49% (n = 85), 36% (n = 62) or 14% (n = 25) of the sera, respectively. Highest reaction intensities were observed with synthetic peptides mimicking type II-specific epitopes. A proportion of the sera (n = 22; 13%) showed no reaction with type-specific peptides. Individuals with acute toxoplasmosis reacted with a statistically significantly higher number of peptides as compared to individuals with latent T. gondii infection or seropositive forest workers. CONCLUSIONS: Type II-specific reactions were overrepresented and higher in intensity in the study population, which was in accord with genotyping studies on T. gondii oocysts previously conducted in the same area. There were also individuals with type I- or type III-specific reactions. Well-characterized reference sera and further specific peptide markers are needed to establish and to perform future serotyping approaches with higher resolution.

Peptide microarray analysis of in silico-predicted epitopes for serological diagnosis of Toxoplasma gondii infection in humans.

Toxoplasma gondii infections occur worldwide in humans and animals. In immunocompromised or prenatally infected humans, T. gondii can cause severe clinical symptoms. The identification of specific epitopes on T. gondii antigens is essential for the improvement and standardization of the serological diagnosis of toxoplasmosis. We selected 20 peptides mimicking linear epitopes on GRA1, GRA2, GRA4, and MIC3 antigenic T. gondii proteins in silico using the software ABCpred. A further 18 peptides representing previously published epitopes derived from GRA1, SAG1, NTPase1, and NTPase2 antigens were added to the panel. A peptide microarray assay was established to prove the diagnostic performance of the selected peptides with human serum samples. Seropositive human serum samples (n = 184) were collected from patients presenting with acute toxoplasmosis (n = 21), latent T. gondii infection (n = 53), and inactive ocular toxoplasmosis (n = 10) and from seropositive forest workers (n = 100). To adjust the cutoff values for each peptide, sera from seronegative forest workers (n = 75) and patients (n = 65) were used. Univariate logistic regression suggested the significant diagnostic potential of eight novel and two previously published peptides. A test based on these peptides had an overall diagnostic sensitivity of 69% (100% in ocular toxoplasmosis patients, 86% in acutely infected patients, 81% in latently infected patients, and 57% in seropositive forest workers). The analysis of seronegative sera performed with these peptides revealed a diagnostic specificity of 84%. The results of our study suggest that the use of a bioinformatic approach for epitope prediction in combination with peptide microarray testing is a powerful method for the selection of T. gondii epitopes as candidate antigens for serological diagnosis.

Seroprevalence study in forestry workers from eastern Germany using novel genotype 3- and rat hepatitis E virus-specific immunoglobulin G ELISAs.

Hepatitis E virus (HEV) is the causative agent of an acute self-limiting hepatitis in humans. In industrialized countries, autochthonous cases are linked to zoonotic transmission from domestic pigs, wild boar and red deer. The main route of human infection presumably is consumption of contaminated meat. Farmers, slaughterers and veterinarians are expected to be risk groups as they work close to potentially infected animals. In this study, we tested four Escherichia coli-expressed segments of the capsid protein (CP) of a German wild boar-derived HEV genotype 3 strain for their diagnostic value in an indirect immunoglobulin G (IgG) ELISA. In an initial validation experiment, a carboxy-terminal CP segment spanning amino acid (aa) residues 326-608 outperformed the other segments harbouring aa residues 112-608, 326-660 and 112-335. Based on this segment, an indirect ELISA for detection of anti-HEV IgG antibodies in human sera was established and validated using a commercial line immunoassay as reference assay. A total of 563 sera from forestry workers of all forestry offices of Brandenburg, eastern Germany and 301 sera of blood donors from eastern Germany were surveyed using these assays. The commercial test revealed seroprevalence rates of 11% for blood donors and 18% for forestry workers. These rates are in line with data obtained by the in-house test (12 and 21%). Hence, the in-house test performed strikingly similar to the commercial test (sensitivity 0.9318, specificity 0.9542). An initial screening of forestry worker and blood donor sera with a corresponding CP segment of the recently discovered Norway rat-associated HEV revealed several strong positive sera exclusively in the forestry worker panel. Future investigations have to prove the performance of this novel IgG ELISA in large-scale seroepidemiological studies. In addition, the observed elevated seroprevalence in a forestry worker group has to be confirmed by studies on groups of forestry workers from other regions. The epidemiological role of ratHEV in human disease should be assessed in a large-scale study of risk and non-risk groups.

Seroprevalence study in forestry workers of a non-endemic region in eastern Germany reveals infections by Tula and Dobrava-Belgrade hantaviruses.

Highly endemic and outbreak regions for human hantavirus infections are located in the southern, southeastern, and western parts of Germany. The dominant hantavirus is the bank vole transmitted Puumala virus (PUUV). In the eastern part of Germany, previous investigations revealed Tula virus (TULV) and Dobrava-Belgrade virus (DOBV) infections in the respective rodent reservoirs. Here, we describe a seroprevalence study in forestry workers from Brandenburg, eastern Germany, using IgG ELISA and immunoblot tests based on recombinant TULV, DOBV, and PUUV antigens. Out of the 563 sera tested, 499 from male and 64 from female workers, we found 41 out of the 499 (8.2%) sera from men (mean age 47 years) and 10 out of 64 (15.6%) from the women (mean age 48 years) anti-hantavirus-positive. The majority of the 51 seropositive samples reacted exclusively in the TULV (n=22) and DOBV tests (n=17). Focus reduction neutralization assay investigations on selected sera confirmed the presence of TULV- and DOBV-specific antibodies in the forestry workers. These investigations demonstrated a potential health threat for forestry workers and also the average population in non-endemic geographical regions where TULV and DOBV are circulating in the corresponding reservoir hosts. The infections in this region might be frequently overlooked due to their unspecific and mild symptoms.