Publications by authors named "Dimitrios Frangoulidis"

34 Publications

Small Mammals as Reservoir for Zoonotic Agents in Afghanistan.

Mil Med 2021 Jan 19. Epub 2021 Jan 19.

Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Greifswald 17493, Germany.

Introduction: Rodents and other small mammals can serve as reservoirs for a large number of zoonotic pathogens. A higher risk of infection with rodent-borne pathogens exists for humans with direct contact to rodents and/or their excretions, e.g., soldiers in operation areas. To date, little is known about endemic human pathogenic disease agents that are naturally associated with small mammals in Afghanistan. The aim of this study was to screen abundant rodents and insectivores collected from 2009 to 2012 in four field camps of the German Federal Armed Forces (Bundeswehr) in Northern Afghanistan for the presence of different pathogens.

Materials And Methods: Isolated nucleic acids from ear pinna were screened by real-time PCR for spotted fever group (SFG) rickettsiae and from liver samples for Francisella spp., Coxiella burnetii, Brucella spp., Yersinia pestis, and poxvirus. Chest cavity lavage (CCL) samples were tested for antibodies against SFG and typhus group (TG) rickettsiae, as well as against flaviviruses using an indirect immunofluorescence assay.

Results: Rickettsial DNA was detected in 7/750 (1%) ear pinna samples with one being identified as Rickettsia conorii. Antibodies against SFG rickettsiae were detected in 15.3% (n = 67/439) of the small mammals; positive samples were only from house mice (Mus musculus). Antibodies against TG rickettsiae were found in 8.2% (n = 36/439) of the samples, with 35 from house mice and one from gray dwarf hamster (Cricetulus migratorius). Flavivirus-reactive antibodies were detected in 2.3% (n = 10/439) of the investigated CCL samples; again positive samples were exclusively identified in house mice. All 199 investigated liver-derived DNA preparations were negative in the Francisella spp., C. burnetii, Brucella spp., Y. pestis, and poxvirus-specific PCRs.

Conclusions: Further investigations will have to prove the potential value of rodents in army camps as sentinel animals.
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http://dx.doi.org/10.1093/milmed/usab008DOI Listing
January 2021

Ticks (Acari: Ixodidae) on birds migrating to the island of Ponza, Italy, and the tick-borne pathogens they carry.

Ticks Tick Borne Dis 2021 01 9;12(1):101590. Epub 2020 Oct 9.

Bundeswehr Institute of Microbiology, Munich, Germany.

Seasonal migration of birds between breeding and wintering areas can facilitate the spread of tick species and tick-borne diseases. In this study, 151 birds representing 10 different bird species were captured on Ponza Island, an important migratory stopover off the western coast of Italy and screened for tick infestation. Ticks were collected and identified morphologically. Morphological identification was supported through sequencing a fragment of the 16S mitochondrial gene. In total, 16 captured birds carried ticks from four tick species: Hyalomma rufipes (n = 14), Amblyomma variegatum (n = 1), Amblyomma sp. (n = 1), and Ixodes ventalloi (n = 2). All specimens were either larvae (n = 2) or nymphs (n = 16). All ticks were investigated for tick-borne pathogens using published molecular methods. Rickettsia aeschlimannii was detected in six of the 14 collected H. rufipes ticks. Additionally, the singular A. variegatum nymph tested positive for R. africae. In all 14 H. rufipes specimens (2 larvae and 12 nymphs), Francisella-like endosymbionts were detected. Four H. rufipes ticks tested positive for Borrelia burgdorferi sensu lato in a screening PCR but did not produce sufficient amplicon amounts for species identification. All ticks tested negative for tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, Coxiella burnetii, Coxiella-like organisms, Babesia spp., and Theileria spp. This study confirms the role of migratory birds in the spread and establishment of both exotic tick species and tick-borne pathogens outside their endemic range.
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http://dx.doi.org/10.1016/j.ttbdis.2020.101590DOI Listing
January 2021

Comparison of Excretion between Sheep and Goats Naturally Infected with One Cattle-Associated Genotype.

Pathogens 2020 Aug 13;9(8). Epub 2020 Aug 13.

Clinic for Swine and Small Ruminants, Forensic Medicine and Ambulatory Service, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany.

The main reservoir of are ruminants. They shed the pathogen through birth products, vaginal mucus, faeces and milk. A direct comparison of excretions between naturally infected sheep and goats was performed on the same farm to investigate species-specific differences. The animals were vaccinated with an inactivated phase I vaccine at the beginning of the study period for public health reasons. Vaginal and rectal swabs along with milk specimens were taken monthly during the lambing period and once again at the next lambing season. To estimate the environmental contamination of the animals' housings, nasal swabs from every animal were taken simultaneously. Moreover, dust samples from the windowsills and straw beddings were collected. All samples were examined by qPCR targeting the IS gene and the MLVA/VNTR typing method was performed. Whole genome sequencing was applied to determine the number of IS copies followed by a calculation of genome equivalents of each sample. The cattle-associated genotype C7 was detected containing 29 IS copies. Overall, goats seem to shed more through vaginal mucus and in particular shed more and for longer via the rectal route than sheep. This is supported by the larger quantities of DNA detected in caprine nasal swabs and environmental samples compared to the ovine ones. Transmission of from cattle to small ruminants must also be considered.
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http://dx.doi.org/10.3390/pathogens9080652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459479PMC
August 2020

Imported Hyalomma ticks in Germany in 2018.

Parasit Vectors 2019 Mar 26;12(1):134. Epub 2019 Mar 26.

Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, 80937, Munich, Germany.

Background: Hyalomma marginatum and Hyalomma rufipes are two-host tick species, which are mainly distributed in southern Europe, Africa and middle-eastern Asia. They are well-known vectors of Crimean Congo hemorrhagic fever (CCHF) virus and other viruses as well as Rickettsia aeschlimannii. In recent years, these tick species have been found sporadically in Germany, but they do not belong to the autochthonous tick fauna in Germany.

Methods: Ticks with unusual morphology were collected and sent from private persons or public health offices to involve institutions for morphological identification and further testing. All ticks identified as Hyalomma spp. were tested using molecular detection methods for CCHF virus, Rickettsia spp., Coxiella burnetii and Coxiella-like organisms, Babesia spp. and Theileria spp.

Results: Thirty-five ticks with an unusual appearance or behaviour were reported to us during summer-autumn 2018. For 17 of them, the description or photos implied that they belong to the hard tick genus Hyalomma. The remaining 18 ticks were sent to us and were identified as adult Hyalomma marginatum (10 specimens) or adult Hyalomma rufipes (8 specimens). All ticks tested negative for CCHF virus, Coxiella burnetii, Coxiella-like organisms, Babesia spp. and Theileria spp. The screening for rickettsiae gave positive results in 9 specimens . The Rickettsia species in all cases was identified as R. aeschlimannii.

Conclusions: These results show that exotic tick species imported into Germany were able to develop from the nymphal to the adult stage under appropriate weather conditions. Fifty percent of the ticks carried R. aeschlimannii, a human pathogen, while CCHF virus or other pathogens were not detected. Imported Hyalomma ticks may be the source of exotic diseases acquired in Germany.
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http://dx.doi.org/10.1186/s13071-019-3380-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6434826PMC
March 2019

Current perspectives on the transmission of Q fever: Highlighting the need for a systematic molecular approach for a neglected disease in Africa.

Acta Trop 2019 May 1;193:99-105. Epub 2019 Mar 1.

School of Applied Sciences, Edinburgh Napier University, Sighthill Court, Edinburgh, EH11 4BN, UK. Electronic address:

Q fever is a bacterial worldwide zoonosis (except New Zealand) caused by the Gram-negative obligate intracellular bacterium Coxiella burnetii (C. burnetii). The bacterium has a large host range including arthropods, wildlife and companion animals and is frequently identified in human and livestock populations. In humans, the disease can occur as either a clinically acute or chronic aetiology, affecting mainly the lungs and liver in the acute disease, and heart valves when chronic. In livestock, Q fever is mainly asymptomatic; however, the infection can cause abortion, and the organism is shed in large quantities, where it can infect other livestock and humans. The presence of Q fever in Africa has been known for over 60 years, however while our knowledge of the transmission routes and risk of disease have been well established in many parts of the world, there is a significant paucity of knowledge across the African continent, where it remains a neglected zoonosis. Our limited knowledge of the disease across the African sub-continent have relied largely upon observational (sero) prevalence studies with limited focus on the molecular epidemiology of the disease. This review highlights the need for systematic studies to understand the routes of C. burnetii infection, and understand the disease burden and risk factors for clinical Q fever in both humans and livestock. With such knowledge gaps filled, the African continent could stand a better chance of eradicating Q fever through formulation and implementation of effective public health interventions.
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http://dx.doi.org/10.1016/j.actatropica.2019.02.032DOI Listing
May 2019

Molecular survey of neglected bacterial pathogens reveals an abundant diversity of species and genotypes in ticks collected from animal hosts across Romania.

Parasit Vectors 2018 03 20;11(1):144. Epub 2018 Mar 20.

Institute for Diagnosis and Animal Health, Bucharest, Romania.

Background: Ticks are transmitting a wide range of bacterial pathogens that cause substantial morbidity and mortality in domestic animals. The full pathogen burden transmitted by tick vectors is incompletely studied in many geographical areas, and extensive studies are required to fully understand the diversity and distribution of pathogens transmitted by ticks.

Results: We sampled 824 ticks of 11 species collected in 19 counties in Romania. Ticks were collected mainly from dogs, but also from other domestic and wild animals, and were subjected to molecular screening for pathogens. Rickettsia spp. was the most commonly detected pathogen, occurring in 10.6% (87/824) of ticks. Several species were detected: Rickettsia helvetica, R. raoultii, R. massiliae, R. monacensis, R. slovaca and R. aeschlimannii. A single occurrence of the zoonotic bacterium Bartonella vinsonii berkhoffii was detected in a tick collected from a dog. Anaplasma phagocytophilum occurred in four samples, and sequences similar to Anaplasma marginale/ovis were abundant in ticks from ruminants. In addition, molecular screening showed that ticks from dogs were carrying an Ehrlichia species identical to the HF strain as well as the enigmatic zoonotic pathogen "Candidatus Neoehrlichia mikurensis". An organism similar to E. chaffeensis or E. muris was detected in an Ixodes ricinus collected from a fox.

Conclusions: We describe an abundant diversity of bacterial tick-borne pathogens in ticks collected from animal hosts in Romania, both on the level of species and genotypes/strains within these species. Several findings were novel for Romania, including Bartonella vinsonii subsp. berkhoffii that causes bacteremia and endocarditis in dogs. "Candidatus Neoehrlichia mikurensis" was detected in a tick collected from a dog. Previously, a single case of infection in a dog was diagnosed in Germany. The results warrant further studies on the consequences of tick-borne pathogens in domestic animals in Romania.
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http://dx.doi.org/10.1186/s13071-018-2756-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5859542PMC
March 2018

New records and host associations of the tick Ixodes apronophorus and the first detection of Ehrlichia sp. HF in Romania.

Parasitol Res 2018 Apr 16;117(4):1285-1289. Epub 2018 Feb 16.

Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, D-80937, Munich, Germany.

Ixodes (Ixodes) apronophorus is a neglected tick species and its geographical distribution, host associations, and role as a disease vector are not well known. We collected I. apronophorus from several locations in Romania. Morphological identification of ticks was confirmed by analysis of 16S rDNA and 12S rDNA gene sequences. We report new host associations of I. apronophorus, which was collected from dogs, foxes, and a hare-all new hosts for this tick species in Romania. Furthermore, we report for the first time occurrence of Ehrlichia sp. HF in I. apronophorus. Ehrlichia sp. HF was identified by sequencing a part of the 16S rDNA gene and was found in 16% (3/19) of the tested ticks. Ehrlichia sp. HF has not been previously reported in Eastern Europe and seems to have a much larger geographic distribution than previously known. Currently, it is unknown whether I. apronophorus is a competent vector for Ehrlichia sp. HF, or if the findings in this study represent infection in the hosts, namely dogs and fox.
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http://dx.doi.org/10.1007/s00436-018-5800-3DOI Listing
April 2018

The Intervening Sequence of Coxiella burnetii: Characterization and Evolution.

Front Cell Infect Microbiol 2016 19;6:83. Epub 2016 Aug 19.

Program in Cellular, Molecular and Microbial Biology, Division of Biological Sciences, University of Montana Missoula, MT, USA.

The intervening sequence (IVS) of Coxiella burnetii, the agent of Q fever, is a 428-nt selfish genetic element located in helix 45 of the precursor 23S rRNA. The IVS element, in turn, contains an ORF that encodes a hypothetical ribosomal S23 protein (S23p). Although S23p can be synthesized in vitro in the presence of an engineered E. coli promoter and ribosome binding site, results suggest that the protein is not synthesized in vivo. In spite of a high degree of IVS conservation among different strains of C. burnetii, the region immediately upstream of the S23p start codon is prone to change, and the S23p-encoding ORF is evidently undergoing reductive evolution. We determined that IVS excision from 23S rRNA was mediated by RNase III, and IVS RNA was rapidly degraded, thereafter. Levels of the resulting 23S rRNA fragments that flank the IVS, F1 (~1.2 kb) and F2 (~1.7 kb), were quantified over C. burnetii's logarithmic growth phase (1-5 d). Results showed that 23S F1 quantities were consistently higher than those of F2 and 16S rRNA. The disparity between levels of the two 23S rRNA fragments following excision of IVS is an interesting phenomenon of unknown significance. Based upon phylogenetic analyses, IVS was acquired through horizontal transfer after C. burnetii's divergence from an ancestral bacterium and has been subsequently maintained by vertical transfer. The widespread occurrence, maintenance and conservation of the IVS in C. burnetii imply that it plays an adaptive role or has a neutral effect on fitness.
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http://dx.doi.org/10.3389/fcimb.2016.00083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990558PMC
September 2017

Coxiella-like endosymbiont in argasid ticks (Ornithodoros muesebecki) from a Socotra Cormorant colony in Umm Al Quwain, United Arab Emirates.

Ticks Tick Borne Dis 2016 Feb 19;7(1):166-171. Epub 2015 Oct 19.

Department of Biology, United Arab Emirates University, PO Box 15551, Al Ain, United Arab Emirates. Electronic address:

Coxiella burnetii is a pathogen causing Q fever in domestic animals and humans. Seabirds have been implicated as possible reservoirs of this bacterium in the Arabian Gulf and in the Western Indian Ocean. Recently, Coxiella species closely related to C. burnetii was detected from ticks collected from oil rigs used as roosting areas by Socotra Cormorants (Phalacrocorax nigrogularis) in the western Arabian Gulf. We collected ticks from the largest breeding colony of Socotra Cormorants in the United Arab Emirates on the eastern extreme of the species' breeding range to determine the prevalence of C. burnetii and evaluate its role as a wild reservoir. All ticks were identified as Ornithodoros muesebecki and genomic DNA was extracted from larval and nymph/adult tick pools. Multiplex PCR tests were performed targeting three C. burnetii specific genes. C. burnetii was not detected although a Coxiella-like endosymbiont was identified that was closely related to Coxiella symbionts from Ornithodoros capensis ticks. Because domestic and wild ungulates are the primary source of C. burnetii, we suggest that the presence of free-ranging, native and non-native ungulates in some off-shore islands in the Arabian Gulf could disseminate C. burnetii to seabirds. More comprehensive studies on seabird colonies are needed to better understand the diversity and prevalence of Coxiella symbionts and to establish if C. burnetii is endemic on some of these islands.
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http://dx.doi.org/10.1016/j.ttbdis.2015.10.012DOI Listing
February 2016

[Q fever].

Dtsch Med Wochenschr 2015 Aug 11;140(16):1206-8. Epub 2015 Aug 11.

Landesgesundheitsamt Baden-Württemberg, Stuttgart.

The article summarizes some important recently identified findings about the Coxiella burnetii disease, Q fever. Beside new diagnostic parameters for follow-up issues, the importance of a timely identification of chronic Q fever and the peculiarities of the post Q fever fatigue syndrome are depicted.
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http://dx.doi.org/10.1055/s-0041-103640DOI Listing
August 2015

Cell-free propagation of Coxiella burnetii does not affect its relative virulence.

PLoS One 2015 20;10(3):e0121661. Epub 2015 Mar 20.

Department of Infection Biology, Central Veterinary Institute part of Wageningen UR, Lelystad, The Netherlands.

Q fever is caused by the obligate intracellular bacterium Coxiella burnetii. In vitro growth of the bacterium is usually limited to viable eukaryotic host cells imposing experimental constraints for molecular studies, such as the identification and characterisation of major virulence factors. Studies of pathogenicity may benefit from the recent development of an extracellular growth medium for C. burnetii. However, it is crucial to investigate the consistency of the virulence phenotype of strains propagated by the two fundamentally different culturing systems. In the present study, we assessed the viability of C. burnetii and the lipopolysaccaride (LPS) encoding region of the bacteria in both culture systems as indirect but key parameters to the infection potential of C. burnetii. Propidium monoazide (PMA) treatment-based real-time PCR was used for enumeration of viable C. burnetii which were validated by fluorescent infectious focus forming unit counting assays. Furthermore, RNA isolated from C. burnetiipropagated in both the culture systems was examined for LPS-related gene expression. All thus far known LPS-related genes were found to be expressed in early passages in both culturing systems indicating the presence of predominantly the phase I form of C. burnetii. Finally, we used immune-competent mice to provide direct evidence, that the relative virulence of different C. burnetii strains is essentially the same for both axenic and cell-based methods of propagation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121661PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4368683PMC
February 2016

Genome sequence of Coxiella burnetii strain Namibia.

Stand Genomic Sci 2014 29;9:22. Epub 2014 Dec 29.

Bundeswehr Institute of Microbiology (BwIM), Munich, Germany.

We present the whole genome sequence and annotation of the Coxiella burnetii strain Namibia. This strain was isolated from an aborting goat in 1991 in Windhoek, Namibia. The plasmid type QpRS was confirmed in our work. Further genomic typing placed the strain into a unique genomic group. The genome sequence is 2,101,438 bp long and contains 1,979 protein-coding and 51 RNA genes, including one rRNA operon. To overcome the poor yield from cell culture systems, an additional DNA enrichment with whole genome amplification (WGA) methods was applied. We describe a bioinformatics pipeline for improved genome assembly including several filters with a special focus on WGA characteristics.
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http://dx.doi.org/10.1186/1944-3277-9-22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286197PMC
January 2015

Genome Sequence of Coxiella burnetii Strain AuQ01 (Arandale) from an Australian Patient with Acute Q Fever.

Genome Announc 2014 Oct 2;2(5). Epub 2014 Oct 2.

Bundeswehr Institute of Microbiology, Munich, Germany

Coxiella burnetii strain AuQ01 was isolated from the serum of an Australian acute Q fever patient and represents the first whole genome from this historical Q fever country. This new genome shows distinct differences from existing genomic data and will enhance the understanding of this query pathogen.
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http://dx.doi.org/10.1128/genomeA.00964-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4183872PMC
October 2014

Molecular analysis of Coxiella burnetii in Germany reveals evolution of unique clonal clusters.

Int J Med Microbiol 2014 Oct 27;304(7):868-76. Epub 2014 Jun 27.

Bundeswehr Institute of Microbiology, Munich, Germany.

The causative agent of Q fever, Coxiella burnetii, is a query agent occurring naturally all over the world. We studied 104 German Coxiella burnetii strains/DNA samples obtained between 1969 and 2011 using a 14 microsatellite marker Multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA) technique. We were able to divide our collection into 32 different genotypes clustered into four major groups (A-D). Two of these (A and C) formed predominant clonal complexes that covered 97% of all studied samples. Group C consisted exclusively of cattle-associated isolates/DNA specimens, while group A comprised all other affected species including all sheep-derived strains/DNA samples. Within this second cluster, two major genotypes (A1, A2) were identified. Genotype A2 occurred in strains isolated from ewes in northern and central Germany, whereas genotype A1 was found in most areas of Germany. MLVA analysis of C. burnetii strains from neighbouring countries revealed a close relationship to German strains. We thus hypothesize that there is a western and central European cluster of C. burnetii. We identified predominant genotypes related to relevant host species and geographic regions which is in line with findings of the Dutch Q fever outbreak (2007-2010). Furthermore three of our analyzed German strains are closely related to the Dutch outbreak clone. These findings support the theory of predominant genotypes in the context of regional outbreaks. Our results show that a combination of 8 MLVA markers provides the highest discriminatory power for attributing C. burnetii isolates to genotypes. For future epidemiological studies we propose the use of three MLVA markers for easy and rapid classification of C. burnetii into 4 main clusters.
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http://dx.doi.org/10.1016/j.ijmm.2014.06.011DOI Listing
October 2014

Eight new genomes and synthetic controls increase the accessibility of rapid melt-MAMA SNP typing of Coxiella burnetii.

PLoS One 2014 21;9(1):e85417. Epub 2014 Jan 21.

CBRN Defence and Security, Swedish Defence Research Agency, Umeå, Sweden.

The case rate of Q fever in Europe has increased dramatically in recent years, mainly because of an epidemic in the Netherlands in 2009. Consequently, there is a need for more extensive genetic characterization of the disease agent Coxiella burnetii in order to better understand the epidemiology and spread of this disease. Genome reference data are essential for this purpose, but only thirteen genome sequences are currently available. Current methods for typing C. burnetii are criticized for having problems in comparing results across laboratories, require the use of genomic control DNA, and/or rely on markers in highly variable regions. We developed in this work a method for single nucleotide polymorphism (SNP) typing of C. burnetii isolates and tissue samples based on new assays targeting ten phylogenetically stable synonymous canonical SNPs (canSNPs). These canSNPs represent previously known phylogenetic branches and were here identified from sequence comparisons of twenty-one C. burnetii genomes, eight of which were sequenced in this work. Importantly, synthetic control templates were developed, to make the method useful to laboratories lacking genomic control DNA. An analysis of twenty-one C. burnetii genomes confirmed that the species exhibits high sequence identity. Most of its SNPs (7,493/7,559 shared by >1 genome) follow a clonal inheritance pattern and are therefore stable phylogenetic typing markers. The assays were validated using twenty-six genetically diverse C. burnetii isolates and three tissue samples from small ruminants infected during the epidemic in the Netherlands. Each sample was assigned to a clade. Synthetic controls (vector and PCR amplified) gave identical results compared to the corresponding genomic controls and are viable alternatives to genomic DNA. The results from the described method indicate that it could be useful for cheap and rapid disease source tracking at non-specialized laboratories, which requires accurate genotyping, assay accessibility and inter-laboratory comparisons.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085417PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3897454PMC
October 2014

The impact of Q fever-phase-specific milk serology for the diagnosis of puerperal and chronic milk shedding of C. burnetii in dairy cows.

Berl Munch Tierarztl Wochenschr 2013 Sep-Oct;126(9-10):427-35

Bavarian Animal Health Service, Poing, Germany.

C. burnetii infection might be associated with puerperal shedding; additionally, the chronic shedding of this pathogen in milk has been observed in individual animals. A longitudinal survey was performed in an endemically infected dairy cow herd with 100 cows in order to compare phase-specific milk-serology with pathogen shedding. From March 2010 through December 2011, 870 individual milk samples from 212 cows were analysed using both quantitative (q) PCR and phase-specific antibody-ELISA. The mean milk-shedding/cow was calculated for 137 cows with > or = 3 milk samples per cow. In addition, 110 puerperal swabs were collected after August 2010. The cows yielding three successive qPCR-positive milk samples or > 3 qPCR-positive milk samples, irrespective of the sequence of positive/negative results, were classified as chronic shedders (CS). Milk shedding was observed during the entire study, but a major period of puerperal shedding occurred from February through October 2011; 35/52 swabs tested positive, whereas only 3/58 swabs collected outside this period were positive. The PhI/PhII(+)-pattern in primiparous cows (< 36 months old) was consistent with puerperal shedding in the herd, but not at the individual level. This pattern was observed in older cows, irrespective of the period of puerperal shedding. Four primiparous CS-cows showed low-level mean shedding < 100 C.b./ml milk, and the PhI-titre increased from negative or weakly positive to more than 500 at the end of the first lactation. Puerperal shedding during the second parturition was observed in three of these cows. Six multiparous CS-cows with mean shedding exceeding 100 C.b./ml milk were characterised with stable PhI-titres of > or = 500. The three available puerperal swabs tested negative. Only one multiparous CS-cow showed low-level shedding and a PhI-titre below 500 for the entire study. In conclusion, the PhI-/PhII(+)-pattern in primiparous cows indicated puerperal shedding at the herd level, and a PhI-titre > or = 500 is a suitable screening method for the detection of chronic shedding in milk.
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April 2015

HoPaCI-DB: host-Pseudomonas and Coxiella interaction database.

Nucleic Acids Res 2014 Jan 16;42(Database issue):D671-6. Epub 2013 Oct 16.

CNRS/Aix-Marseille University, Laboratoire d'Ingénierie des Systèmes Macromoléculaires (UMR7255), Institut de Microbiologie de la Méditerranée (IMM), 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France, Institute for Bioinformatics and Systems Biology (MIPS), Helmholtz Zentrum München - German Research Center for Environmental Health (GmbH), Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany, Department of Genome-Oriented Bioinformatics, Center of Life and Food Science Weihenstephan, Technische Universität München, Freising, Germany and Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, 80937 Munich, Germany.

Bacterial infectious diseases are the result of multifactorial processes affected by the interplay between virulence factors and host targets. The host-Pseudomonas and Coxiella interaction database (HoPaCI-DB) is a publicly available manually curated integrative database (http://mips.helmholtz-muenchen.de/HoPaCI/) of host-pathogen interaction data from Pseudomonas aeruginosa and Coxiella burnetii. The resource provides structured information on 3585 experimentally validated interactions between molecules, bioprocesses and cellular structures extracted from the scientific literature. Systematic annotation and interactive graphical representation of disease networks make HoPaCI-DB a versatile knowledge base for biologists and network biology approaches.
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http://dx.doi.org/10.1093/nar/gkt925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965080PMC
January 2014

Microevolution of the chromosomal region of acute disease antigen A (adaA) in the query (Q) fever agent Coxiella burnetii.

PLoS One 2013 3;8(1):e53440. Epub 2013 Jan 3.

Bundeswehr Institute of Microbiology, Munich, Germany.

The acute disease antigen A (adaA) gene is believed to be associated with Coxiella burnetii strains causing acute Q fever. The detailed analysis of the adaA genomic region of 23 human- and 86 animal-derived C. burnetii isolates presented in this study reveals a much more polymorphic appearance and distribution of the adaA gene, resulting in a classification of C. burnetii strains of better differentiation than previously anticipated. Three different genomic variants of the adaA gene were identified which could be detected in isolates from acute and chronic patients, rendering the association of adaA positive strains with acute Q fever disease disputable. In addition, all adaA positive strains in humans and animals showed the occurrence of the QpH1 plasmid. All adaA positive isolates of acute human patients except one showed a distinct SNP variation at position 431, also predominant in sheep strains, which correlates well with the observation that sheep are a major source of human infection. Furthermore, the phylogenetic analysis of the adaA gene revealed three deletion events and supported the hypothesis that strain Dugway 5J108-111 might be the ancestor of all known C. burnetii strains. Based on our findings, we could confirm the QpDV group and we were able to define a new genotypic cluster. The adaA gene polymorphisms shown here improve molecular typing of Q fever, and give new insights into microevolutionary adaption processes in C. burnetii.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053440PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536764PMC
August 2013

Q fever in pregnant goats: pathogenesis and excretion of Coxiella burnetii.

PLoS One 2012 9;7(11):e48949. Epub 2012 Nov 9.

Department of Bacteriology and TSEs, Central Veterinary Institute, Wageningen University and Research Centre, Lelystad, The Netherlands.

Coxiella burnetii is an intracellular bacterial pathogen that causes Q fever. Infected pregnant goats are a major source of human infection. However, the tissue dissemination and excretion pathway of the pathogen in goats are still poorly understood. To better understand Q fever pathogenesis, we inoculated groups of pregnant goats via the intranasal route with a recent Dutch outbreak C. burnetii isolate. Tissue dissemination and excretion of the pathogen were followed for up to 95 days after parturition. Goats were successfully infected via the intranasal route. PCR and immunohistochemistry showed strong tropism of C. burnetii towards the placenta at two to four weeks after inoculation. Bacterial replication seemed to occur predominantly in the trophoblasts of the placenta and not in other organs of goats and kids. The amount of C. burnetii DNA in the organs of goats and kids increased towards parturition. After parturition it decreased to undetectable levels: after 81 days post-parturition in goats and after 28 days post-parturition in kids. Infected goats gave birth to live or dead kids. High numbers of C. burnetii were excreted during abortion, but also during parturition of liveborn kids. C. burnetii was not detected in faeces or vaginal mucus before parturition. Our results are the first to demonstrate that pregnant goats can be infected via the intranasal route. C. burnetii has a strong tropism for the trophoblasts of the placenta and is not excreted before parturition; pathogen excretion occurs during birth of dead as well as healthy animals. Besides abortions, normal deliveries in C. burnetii-infected goats should be considered as a major zoonotic risk for Q fever in humans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048949PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494687PMC
May 2013

Molecular typing of Coxiella burnetii (Q fever).

Adv Exp Med Biol 2012 ;984:381-96

Bundeswehr Institute of Microbiology, Neuherbergstr. 11, 80937, Munich, Germany.

Although we live in the age of genomics and the availability of complete genome sequences of Coxiella burnetii has increased our understanding of the genomic diversity of the agent, it is still somewhat a "query" microorganism. The epidemiology of Q fever is complex due to the worldwide distribution, reservoir and vector diversity, and a lack of studies defining the dynamic interaction between these factors. In addition Coxiella is an agent that could be used as a bioterror weapon. Therefore, typing methods that can discriminate strains and be used to trace back infections to their source are of paramount importance. In this chapter we provide an overview of historical and current typing methods and describe their advantages and limitations. Recently developed techniques such as MLVA and SNP typing have shown promise and improved the discrimination capacity and utility of genotyping methods for molecular epidemiologic studies of this challenging pathogen.
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http://dx.doi.org/10.1007/978-94-007-4315-1_19DOI Listing
January 2013

'Real-time' PCR-based detection of Coxiella burnetii using conventional techniques.

FEMS Immunol Med Microbiol 2012 Feb 8;64(1):134-6. Epub 2011 Dec 8.

Bundeswehr Institute of Microbiology, Munich, Germany.

The diagnosis of Q fever (Coxiella burnetii infection) relies primarily on the serological detection of specific antibodies. Recently, PCR-based methods have been introduced in diagnostic laboratories. Unfortunately, the fastest and most reliable 'real-time' detection method, which employs the 'online' detection of target nucleotide sequences while the amplification process is still in progress, requires expensive devices and consumables. In this study, we present a simple method that combines the simplicity of conventional PCR with new technical and methodical enhancements, resulting in a fast, specific and easy method for the molecular detection of C. burnetii. A collection of C. burnetii reference strains was tested with the modified conventional gel-based PCR approach applying a particluar PCR buffer (QIAGEN(®) Fast Cycling PCR kit) and using a closed ready-to-use gel-cassette-system (FlashGel(®)) for the visualization of specific PCR products. The modified conventional PCR method reached nearly the speed of the LightCycler(®) HybProbe real-time PCR assay (120 vs. 90 min) and showed equal sensitivity and specificity. The general cost per PCR run was 25% less than that for the LightCycler method. These improvements make this method suitable for small laboratories with limited resources and for deployable PCR diagnostics in field laboratories.
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http://dx.doi.org/10.1111/j.1574-695X.2011.00900.xDOI Listing
February 2012

Immunologic response of unvaccinated workers exposed to anthrax, Belgium.

Emerg Infect Dis 2009 Oct;15(10):1637-40

Veterinary and Agro-chemical Research Centre, Department of Bacterial Diseases, Brussels, Belgium.

To determine immunologic reactivity to Bacillus anthrax antigens, we conducted serologic testing of workers in a factory that performed scouring of wool and goat hair. Of 66 workers, approximately 10% had circulating antibodies or T lymphocytes that reacted with anthrax protective antigen. Individual immunity varied from undetectable to high.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2866386PMC
http://dx.doi.org/10.3201/eid1510.081717DOI Listing
October 2009

Validation and comparison of an extrapolysaccharide (EPS)-based in-house ELISA and the PanBio melioidosis rapid cassette test-kits for serodiagnosis of melioidosis in a non-endemic area.

Trans R Soc Trop Med Hyg 2008 Dec;102 Suppl 1:S45-6

Bundeswehr Institute of Microbiology, Neuherbergstr. 11, 80937 Munich, Germany.

Detection of anti-Burkholderia pseudomallei antibodies in sera from melioidosis patients still represents a keystone in the confirmation of the clinical diagnosis, especially in non-endemic areas. An in-house assay was compared to lateral flow assays for the rapid detection of melioidosis-specific IgG or IgM. Employing 50 positive sera from patients and 200 negative sera from blood donors, sensitivity of the ELISA, the IgG and IgM assay were 84.0%, 90.0% and 84.0%, respectively. Specificity ranged from 98.0% (ELISA) to 99.5% (IgM assay). The application of the described diagnostic assays is a suitable method for the serodiagnosis of melioidosis in a non-endemic area.
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http://dx.doi.org/10.1016/S0035-9203(08)70013-0DOI Listing
December 2008

Real-time PCR system targeting a chromosomal marker specific for Bacillus anthracis.

Mol Cell Probes 2008 Oct-Dec;22(5-6):313-5. Epub 2008 Jun 17.

Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, D-80937 Munich, Germany.

Specific identification of Bacillus anthracis and differentiation from closely related Bacillus cereus and Bacillus thuringiensis strains is still a major diagnostic problem. Commercially available diagnostic kits targeting plasmid-markers cannot differentiate between B. anthracis, non-anthracis Bacillus species harbouring anthrax-specific virulence plasmids, and plasmidless B. anthracis strains. A TaqMan PCR assay was designed targeting sequences of gene locus BA_5345 of the B. anthracis strain Ames. Specificity was determined by using a panel of 328 Bacillus strains; sensitivity was determined by probit analysis. All B. anthracis isolates (n=92) were specifically detected by using the genomic TaqMan PCR assay whereas 236 strains belonging to 19 Bacillus species other than B. anthracis were PCR negative. The detection limit was determined to be 12.7 copies per reaction (95% confidence interval 10.2-17.5 copies). Here we present an extensively evaluated and - to our current knowledge - specific TaqMan PCR assay for the detection of B. anthracis based on a chromosomal marker.
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http://dx.doi.org/10.1016/j.mcp.2008.06.001DOI Listing
March 2009

High throughput detection of Coxiella burnetii by real-time PCR with internal control system and automated DNA preparation.

BMC Microbiol 2008 May 19;8:77. Epub 2008 May 19.

Clinical Virology, Bernhard-Nocht Institute for Tropical Medicine, Bernhard Nocht Str, 74, 20359 Hamburg, Germany.

Background: Coxiella burnetii is the causative agent of Q-fever, a widespread zoonosis. Due to its high environmental stability and infectivity it is regarded as a category B biological weapon agent. In domestic animals infection remains either asymptomatic or presents as infertility or abortion. Clinical presentation in humans can range from mild flu-like illness to acute pneumonia and hepatitis. Endocarditis represents the most common form of chronic Q-fever. In humans serology is the gold standard for diagnosis but is inadequate for early case detection. In order to serve as a diagnostic tool in an eventual biological weapon attack or in local epidemics we developed a real-time 5'nuclease based PCR assay with an internal control system. To facilitate high-throughput an automated extraction procedure was evaluated.

Results: To determine the minimum number of copies that are detectable at 95% chance probit analysis was used. Limit of detection in blood was 2,881 copies/ml [95%CI, 2,188-4,745 copies/ml] with a manual extraction procedure and 4,235 copies/ml [95%CI, 3,143-7,428 copies/ml] with a fully automated extraction procedure, respectively. To demonstrate clinical application a total of 72 specimens of animal origin were compared with respect to manual and automated extraction. A strong correlation between both methods was observed rendering both methods suitable. Testing of 247 follow up specimens of animal origin from a local Q-fever epidemic rendered real-time PCR more sensitive than conventional PCR.

Conclusion: A sensitive and thoroughly evaluated real-time PCR was established. Its high-throughput mode may show a useful approach to rapidly screen samples in local outbreaks for other organisms relevant for humans or animals. Compared to a conventional PCR assay sensitivity of real-time PCR was higher after testing samples from a local Q-fever outbreak.
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http://dx.doi.org/10.1186/1471-2180-8-77DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2397412PMC
May 2008

Monitoring of ELISA-reactive antibodies against anthrax protective antigen (PA), lethal factor (LF), and toxin-neutralising antibodies in serum of individuals vaccinated against anthrax with the PA-based UK anthrax vaccine.

Vaccine 2007 May 23;25(18):3679-83. Epub 2007 Jan 23.

Institut fuer Mikrobiologie der Bundeswehr, D-80937 Munich, Germany.

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.
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http://dx.doi.org/10.1016/j.vaccine.2007.01.056DOI Listing
May 2007

Comparison of four commercially available assays for the detection of IgM phase II antibodies to Coxiella burnetii in the diagnosis of acute Q fever.

Ann N Y Acad Sci 2006 Oct;1078:561-2

Bundeswehr Institute of Microbiology, Neuherbergstr. 11, 80937 Munich, Germany.

Four commercially available serological assays for the detection of IgM phase II antibodies in patients with acute Q fever infection were compared using a panel of 23 serum samples from patients with acute Q fever and 88 control sera from blood donors.
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http://dx.doi.org/10.1196/annals.1374.110DOI Listing
October 2006

Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing.

BMC Microbiol 2006 Apr 26;6:38. Epub 2006 Apr 26.

INRA, Pathologie Infectieuse et Immunologie, 37380 Nouzilly, France.

Background: Coxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA).

Results: By applying IRS-PCR, 14 C. burnetii isolates could be divided into six groups containing up to five different isolates. Clustering as deduced from MLVA typing with 17 markers provided an increased resolution with an excellent agreement to IRS-PCR, and with the plasmid type of each strain. MLVA was then applied to 28 additional C. burnetii isolates of different origin and 36 different genotypes were identified among the 42 isolates investigated. The clustering obtained is in agreement with published Multiple Locus Sequence Typing (MLST) data. Two panels of markers are proposed, panel 1 which can be confidently typed on agarose gel at a lower cost and in any laboratory setting (10 minisatellite markers with a repeat unit larger than 9 bp), and panel 2 which comprises 7 microsatellites and provides a higher discriminatory power.

Conclusion: Our analyses demonstrate that MLVA is a powerful and promising molecular typing tool with a high resolution and of low costs. The consistency of the results with independent methods suggests that MLVA can be applied for epidemiological studies. The resulting data can be queried on a dedicated MLVA genotyping Web service.
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http://dx.doi.org/10.1186/1471-2180-6-38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1488860PMC
April 2006

Real-time PCR assay for the detection of tanapox virus and yaba-like disease virus.

J Virol Methods 2005 Dec 28;130(1-2):149-53. Epub 2005 Jul 28.

Bundeswehr Institute of Microbiology, Neuherbergstr. 11, D-80937 Munich, Germany.

The yatapoxvirus genus contains three members: tanapox virus (TPV), yaba-like disease virus (YLDV) and yaba monkey tumor virus (YMTV), two of which (TPV and YLDV) may infect humans. However, only a very small number of patients have been diagnosed with TPV outside Africa. Given the increased international travel and the similarity of clinical signs during the early stages of a TPV/YLDV infection as compared to diseases caused by agents of potential biological warfare, such as smallpox, monkeypox, tularemia and anthrax, the rapid and reliable recognition of a TPV/YLDV infection is crucial. A real-time PCR assay using TaqManchemistry was developed in order to identify unambiguously TPV/YLDV. Primers and probe targeting a 101bp region of the PstI L fragment of TPV, initial optimisations steps were carried out with YLDV DNA as template. Using probit regression analysis, the lower limit of detection was calculated to be ca. 8 copies per assay. A total of five TPV strains, one YDLV strain and scab-derived DNA from a patient with a TPV infection yielded specific amplification, whereas the DNA of YMTV was not amplified. Various viral and bacterial pathogens (n=29) associated with rash-causing illnesses were not detected using this assay.
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http://dx.doi.org/10.1016/j.jviromet.2005.06.015DOI Listing
December 2005