New developments in the diagnostic procedures for zoonotic brucellosis in humans.

At present, laboratory diagnosis of human brucellosis is based on isolation of the bacteria from clinical samples followed by standard microbiological tube testing, detection of anti-Brucella antibodies using various serological tests, and the use of molecular methods for the detection of Brucella DNA. None of these diagnostic tools can be used on its own to reliably detect the causative agent. Cultures give a low yield and subsequent phenotypic characterisation is time consuming, meaning that the initiation of adequate antibiotic therapy is frequently delayed. Serological tests seem to be more effective but are not internationally standardised. Moreover, antibodies can remain detectable despite successful therapy, cross-reacting antibodies may occur, and variable cut-offs for different levels of endemicity are lacking. Molecular assays may reduce diagnostic delays in clinical laboratories, but diagnostic criteria for active infection have not yet been defined. This article reviews the latest microbiological methods for the diagnosis of human brucellosis and outlines developments for the future.

Antimicrobial resistance of Yersinia enterocolitica and presence of plasmid pYV virulence genes in human and animal isolates.

Interactions between bacterial virulence and antimicrobial resistance are of increasing interest in clinical microbiology. On this account, antimicrobial resistance of Yersinia enterocolitica O:3 strains isolated from humans (n = 55), food-chain animals (n = 58) and companion animals (n = 13) was determined in relation to the absence or presence of the pYV plasmid-encoded virulence genes yadA and virF. There were no statistically significant associations between the rate of antimicrobial resistance and the presence or absence of the plasmid, in either human-derived or animal-derived strains. Therefore, it can be concluded that response to conventionally used antimicrobials in Y. enterocolitica O:3 strains is not dependent on pYV-encoded virulence determinants.

Acute Q fever infection in Thuringia, Germany, after burial of roe deer fawn cadavers (Capreolus capreolus): a case report.

We report on a case of a 48-year-old man who presented with acute Q fever infection after burying two fawn cadavers (Capreolus capreolus). Recent outbreaks of Q fever in Europe have been traced back to intensive goat breeding units, sheep flocks in the proximity of highly populated urban areas or to farmed deer. To our knowledge, this is the first case report describing Q fever infection in a human linked to roe deer as a source of infection.

First Draft Genome Sequence of a Human Coxiella burnetii Isolate, Originating from the Largest Q Fever Outbreak Ever Reported, the Netherlands, 2007 to 2010.

In 2009, Coxiella burnetii caused a large regional outbreak of Q fever in South Limburg, the Netherlands. Here, we announce the genome draft sequence of a human C. burnetii isolate, strain NL-Limburg, originating from this outbreak, including a brief summary of the genome's general features.

Specific detection of plasmid bearing Yersinia isolates by PCR.

A total of 210 isolates belonging to 9 different species of the genus Yersinia (Y.) was investigated with three different PCR assays targeting two plasmoidal genes, the Yersinia adhesin gene (yadA) and the V-antigen gene. The yadA PCR assay described in 1995 by Blais and Phillipe, targeting a Y. enterocolitica specific gene region and a newly designed assay targeting the gene region functionally responsible for autoagglutination, were compared. Both assays identified the same Y. enterocolitica strains. To exclude the possibility that false negative results were obtained due to mutations that had occurred in parallel in both gene regions, a third PCR assay by Neubauer et al. (2000) targeting a conserved region of the V-antigen gene was used as control. Again, DNA of the same Y. enterocolitica strains was amplified. In contrast to the yadA PCR assay described by Blais and Phillipe, the newly established yadA and the V-antigen PCR assays amplified DNA from Y. pseudotuberculosis strains. Therefore, by using the PCR technique as a molecular tool spontaneous mutations could be excluded as the cause of anomalous reactions in PCR assays targeting genes of the Yersinia virulence plasmid. Based on these results, it can be assumed that all presumptive pathogenic Yersinia isolates can be identified on the basis of PCR analysis. These molecular assays may also produce fewer false positive reactions in comparison to phenotypic tests such as the autoagglutination test which depend heavily on the handler's experience. It has to be stressed that the PCR assays used in this study have not been evaluated for routine use. Therefore, standardization of the PCR methodology including sample preparation, primer target sequences and PCR reagents is needed for the reliable and safe diagnosis of pathogenic Yersinia spp. in future.

Glanders in animals: a review on epidemiology, clinical presentation, diagnosis and countermeasures.

Glanders or farcy, caused by Burkholderia mallei, is an infectious and zoonotic disease of solipeds. Horses, donkeys and mules are the only known natural reservoir of B. mallei. Although glanders has been eradicated from most countries, it has regained the status of a re-emerging disease because of the numerous recent outbreaks. Pre-symptomatic or carrier animals are the potential source of infection for the healthy equine population and play a crucial role in the spreading of the infectious agent. Glanders is characterized by ulcerating nodular lesions of the skin and mucous membrane. Generalized symptoms include fever, malaise, depression, cough, anorexia and weight loss. Burkholderia mallei can invade its host through mucous membranes, gastrointestinal tract and the integument. Its virulence mechanisms and pathogenesis are not yet completely understood. A major problem when using serological tests for diagnosing glanders is the occurrence of false-positive and false-negative results leading to difficulties in international trade with equids and to the spread of glanders to disease-free regions. Moreover, poor tests critically result in poor control of disease. These tests are not only incapable of discriminating between B. mallei and B. pseudomallei antibodies, they are also unable to differentiate between malleinized and naturally infected animals. Combined use of both serological and molecular detection methods increases the detection rate of glanders. Countermeasures against glanders include early detection of disease in susceptible animals, stringent quarantine measures, testing and safe destruction of infected carcasses, adequate compensation to the animal owners, disinfection of infected premises and awareness about glanders and the zoonotic implications through veterinary extension services. An account of the clinical picture and successful experimental therapy of spontaneous equine glanders is also given.

Development and clinical evaluation of a PCR assay targeting the metalloprotease gene (mprA) of B. pseudomallei.

A PCR assay targeting the metalloprotease gene (mprA) of Burkholderia pseudomallei was developed for the specific detection of this organism in pure cultures and clinical samples. All other closely related organisms including B. mallei the causative agent of glanders, and B. thailandensis tested negative. Burkholderia pseudomallei DNA was successfully amplified from paraffin-embedded lung tissue of a camel with a generalized B. pseudomallei infection. The developed PCR assay can be used as a simple tool for the specific and sensitive detection of B. pseudomallei.

Detection of Burkholderia cepacia DNA from artificially infected EDTA-blood and lung tissue comparing different DNA isolation methods.

Bacterial DNA (Burkholderia cepacia) was prepared from artificially infected equine ethylenediaminetetraacetic acid (EDTA)-blood and lung tissue by using four standard methods (lysis buffer containing proteinase K, phenol/chloroform/isoamylalcohol-extraction, microwave-treatment, heat treatment) and six commercially available kits (Puregene, High Pure PCR Template Preparation Kit, InstaGene, QiaAmp Tissue Kit, DNAzol and Elu-Quik). After a subsequent polymerase chain reaction (PCR), their efficacy and sensitivity were compared. Concerning the detection limits, the simple lysis with a proteinase K-containing buffer led to the best results for EDTA-blood as well as for artificially infected lung tissue.

Serodiagnosis of Burkholderia mallei infections in horses: state-of-the-art and perspectives.

Burkholderia mallei causes glanders or farcy in solipeds, a disease that must be reported to the OIE (Office International des Epizooties, Paris, France). The number of reported outbreaks has increased steadily during the last decade. Serodiagnosis is hampered by the considerable number of false-positives and -negatives of the internationally prescribed tests. The major problem leading to low sensitivity and specificity of complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e. crude preparations of whole cells. Future perspectives for the development and evaluation of serological test kits using well-characterized single antigens are discussed in the light of recent molecular research on B. mallei and the closely related saprozoonotic agent B. pseudomallei.

Melioidosis in animals: a review on epizootiology, diagnosis and clinical presentation.

Melioidosis, an infectious disease caused by Burkholderia pseudomallei is an emerging disease with high impact on animals and man. In different animal species, the clinical course varies and delayed diagnosis poses risks for the dissemination of the agent in non-endemic areas. Not only migration and transport of animals around the world but also tourism increases the risk that melioidosis can leave its endemic boundaries and establish itself elsewhere. Detection of the agent is a major challenge, as the agent has to be handled in laboratories of biosafety level 3 and test kits are not yet commercially available. Veterinarians and doctors should be aware of melioidosis not only as an agent of public interest but also in terms of a bioterrorist attack. The aim of this review is to describe the agent, its aetiology, the manifestation in a variety of animal species as well as to describe diagnostic procedures, typing techniques and countermeasures.

[Diagnosis o Yersinia enterocolitica infections: a review on classical identification techniques and new molecular biological methods]. / Die Diagnostik der Yersinia enterocolitica-Infektionen: Eine Ubersicht über klassische Nachweistechniken und neue molekularbiologische Methoden.

Only three of the eleven species of the genus Yersinia are associated with disease. Y. pestis is the causative agent of plague, Y. pseudotuberculosis and several pathogenic bio/serovars of the species Y. enterocolitica cause yersiniosis. New Y. enterocolitica subspecies with diagnostic relevance have been proposed allowing the differentiation of European and American isolates. The ISO-standard (ISO 102739) summarizes the knowledge gained from enrichment and isolation of Y. enterocolitica from food and feed samples. The final biochemical identification must be carried out by classical tube testing, as commercially available test-systems are not sensitive and specific. For the assessment of the presumptive pathogenicity of a Y. enterocolitica isolate empiric virulence markers can be replaced by PCR assays targeting plasmoidal or chromosomal genes. Their evaluation in terms of routine diagnostic procedures is still missing. The definite identification of Y. enterocolitica isolates can also be achieved by sequencing the 16S rRNA gene. Immunoblot based on plasmoidal encoded Yersinia proteins enables the serological determination of animal and human infections. The development of simple, sensitive and specific rapid identification systems applicable for the direct and indirect diagnosis for veterinary use is a challenge for the future.

[Yersinia enterocolitica infections: 1. Impact on animal health]. / Yersinia enterocolitica-Infektionen: 1. Bedeutung bei Tieren.

In Europe, yersiniosis is mainly caused by the bacterial species Yersinia enterocolitica. The clinical picture of yersiniosis in farm animals including pig, chinchilla, cattle, small ruminants and the pet animals dog and cat is described in detail. However, data on seroprevalence and prevalence in these animals are not available. Therefore, further research is needed in the fields of epidemiology and epizootiology.

[Yersinia enterolitica infections: 2. Impact on human health]. / Yersinia-enterocolitica-Infektionen: 2. Bedeutung bei Menschen.

The clinical picture of yersiniosis in humans and its prevalence in the human population is described in detail. Mass production of animals, development of meat factories based on sophisticated chains of cold storage units and international trade of meat products and animals are believed to be the reasons for the increasing prevalence of yersiniosis in humans. In Germany, anti-Yersinia antibodies are found in up to 40% of the average population. The financial losses for the national economy cannot be judged. Of special interest for industrial medicine are sequelae-like reactive arthritis in exposed occupational groups such as veterinarians or butchers. However, the lack of national and international data makes the assessment of the potential of yersiniosis as a zoonosis difficult. Therefore, intensive and interdisciplinary research is needed to close the gaps described. Already proven and proposed countermeasures at the different stages of mass production of animals and reglementations for international trade of meat products and animals are introduced. The need for development not only of cheap and rapid diagnostic tools but also for countermeasures and treatment strategies is discussed.

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.