Development of a Novel Method for Identification of Alaria alata Mesocercariae by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Alaria (A.) alata mesocercariae (AM) have increasingly appeared as incidental findings during the mandatory inspection of wild boars for Trichinella in many European countries. An Alaria spp.-specific PCR is available for the identification of AM; however, it is time- and cost-intensive. Therefore, we propose a rapid and cost-efficient MALDI-TOF assay for the identification of AM in wild boar meat that can be applied in routine diagnostics. In this study, a fast and methodologically simple protocol for the protein extraction of AM from different host species in different countries was established, and an AM-specific reference spectra database was created as part of the ongoing development of an existing Trichinella spp. database. A formic acid protein extraction was performed after pooling 10 AM from the same host individual. In total, 61 main spectra profiles (MSPs) from different host individuals were stored in an AM-specific MSP library. The cluster analysis of these 61 MSPs indicated a possible variation within the A. alata species with a tentative association with the geographical origin of the host, but not the host species. This MALDI-TOF assay allows for a fast verification of the AM isolates, which is the next step in the development of a universal database for the identification of several parasites isolated from meat.

Species identification of Trichinella originated from various host and different geographical location by MALDI-TOF.

The foodborne zoonotic nematode Trichinella spp. can cause human trichinellosis when raw or undercooked contaminated meat is ingested. To date, twelve Trichinella species/genotypes have been described. According to EU regulation any Trichinella larvae detected during mandatory routine examinations need to be identified at a species level by a competent laboratory. Currently, Trichinella species identification is performed using molecular biology tools such as multiplex PCR, PCR-sequencing or PCR-RFLP. These techniques require high level of skills for good interpretation of the results. Due to its rapidness and ease of use a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protocol was previously developed for the identification of Trichinella species. Using this method, spectra from different Trichinella species and strains were acquired allowing to generate new Main Spectra (MSP). Finally a new MSP database from Trichinella spp. Samples of different countries (France, Germany and Poland), including field samples, was generated. Comparing the different main spectra, Trichinella worms were identified at the species level and differences in the genetic diversities within the different species are discussed. In conclusion, using the previously described method on field samples is a reliable, rapid, easy-to-use and cheap tool for Trichinella species identification. The new Trichinella database could be incremented with new samples. It constitutes a tool, which could be used as an alternative method to replace the actual molecular methods for Trichinella species identification.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples.

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses. There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Rapid Identification of the Foodborne Pathogen Trichinella spp. by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Human trichinellosis occurs through consumption of raw or inadequately processed meat or meat products containing larvae of the parasitic nematodes of the genus Trichinella. Currently, nine species and three genotypes are recognized, of which T. spiralis, T. britovi and T. pseudospiralis have the highest public health relevance. To date, the differentiation of the larvae to the species and genotype level is based primarily on molecular methods, which can be relatively time consuming and labor intensive. Due to its rapidness and ease of use a matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS) reference spectra database using Trichinella strains of all known species and genotypes was created. A formicacid/acetonitrile protein extraction was carried out after pooling 10 larvae of each Trichinella species and genotype. Each sample was spotted 9 times using α-cyano 4-hydoxy cinnamic acid matrix and a MicroFlex LT mass spectrometer was used to acquire 3 spectra (m/z 2000 to 20000 Da) from each spot resulting in 27 spectra/species or genotype. Following the spectra quality assessment, Biotyper software was used to create a main spectra library (MSP) representing nine species and three genotypes of Trichinella. The evaluation of the spectra generated by MALDI-TOF MS revealed a classification which was comparable to the results obtained by molecular methods. Also, each Trichinella species utilized in this study was distinct and distinguishable with a high confidence level. Further, different conservation methods such as freezing and conservation in alcohol and the host species origin of the isolated larvae did not have a significant influence on the generated spectra. Therefore, the described MALDI-TOF MS can successfully be implemented for both genus and species level identification and represents a major step forward in the use of this technique in foodborne parasitology.

Estimation of the sensitivity and specificity of a Leptospira spp. in-house ELISA through Bayesian modelling.

The microscopic agglutination test (MAT) is still considered the gold standard for the diagnosis of leptospirosis, although studies have shown that the test is an imperfect gold standard for clinical samples and unsuitable for epidemiological studies. Here, test characteristics of an in-house ELISA were identified for both subclinical and clinical populations by Bayesian latent class models. A conditional dependence model for two diagnostic tests and two populations was adapted to analyse a clinical and a subclinical scenario, respectively. These Bayesian models were used to estimate the sensitivity and specificity of the in-house ELISA and the MAT as well as the prevalences. The Bayesian estimates of the in-house ELISA were: clinical sensitivity=83.0%, clinical specificity=98.5%, subclinical sensitivity=85.7% and subclinical specificity=99.1%. In contrast, the estimates of the MAT were: clinical sensitivity=65.6%, clinical specificity=97.7%, subclinical sensitivity 54.9% and subclinical specificity=97.3%. The results show the suitability of the in-house ELISA for both clinical investigations and epidemiological studies in mildly endemic areas.

Differential phenotyping of Brucella species using a newly developed semi-automated metabolic system.

BACKGROUND: A commercial biotyping system (Taxa Profile™, Merlin Diagnostika) testing the metabolization of various substrates by bacteria was used to determine if a set of phenotypic features will allow the identification of members of the genus Brucella and their differentiation into species and biovars. RESULTS: A total of 191 different amines, amides, amino acids, other organic acids and heterocyclic and aromatic substrates (Taxa Profile™ A), 191 different mono-, di-, tri- and polysaccharides and sugar derivates (Taxa Profile™ C) and 95 amino peptidase- and protease-reactions, 76 glycosidase-, phosphatase- and other esterase-reactions, and 17 classic reactions (Taxa Profile™ E) were tested with the 23 reference strains representing the currently known species and biovars of Brucella and a collection of 60 field isolates. Based on specific and stable reactions a 96-well "Brucella identification and typing" plate (Micronaut™) was designed and re-tested in 113 Brucella isolates and a couple of closely related bacteria.Brucella species and biovars revealed characteristic metabolic profiles and each strain showed an individual pattern. Due to their typical metabolic profiles a differentiation of Brucella isolates to the species level could be achieved. The separation of B. canis from B. suis bv 3, however, failed. At the biovar level, B. abortus bv 4, 5, 7 and B. suis bv 1-5 could be discriminated with a specificity of 100%. B. melitensis isolates clustered in a very homogenous group and could not be resolved according to their assigned biovars. CONCLUSIONS: The comprehensive testing of metabolic activity allows cluster analysis within the genus Brucella. The biotyping system developed for the identification of Brucella and differentiation of its species and biovars may replace or at least complement time-consuming tube testing especially in case of atypical strains. An easy to handle identification software facilitates the applicability of the Micronaut™ system for microbiology laboratories.

Brucella inopinata sp. nov., isolated from a breast implant infection.

A Gram-negative, non-motile, non-spore-forming coccoid bacterium (strain BO1(T)) was isolated recently from a breast implant infection of a 71-year-old female patient with clinical signs of brucellosis. Affiliation of strain BO1(T) to the genus Brucella was confirmed by means of polyamine pattern, polar lipid profile, fatty acid profile, quinone system, DNA-DNA hybridization studies and by insertion sequence 711 (IS711)-specific PCR. Strain BO1(T) harboured four to five copies of the Brucella-specific insertion element IS 711, displaying a unique banding pattern, and exhibited a unique 16S rRNA gene sequence and also grouped separately in multilocus sequence typing analysis. Strain BO1(T) reacted with Brucella M-monospecific antiserum. Incomplete lysis was detected with bacteriophages Tb (Tbilisi), F1 and F25. Biochemical profiling revealed a high degree of enzymic activity and metabolic capabilities. In multilocus VNTR (variable-number tandem-repeat) analysis, strain BO1(T) showed a very distinctive profile and clustered with the other 'exotic' Brucella strains, including strains isolated from marine mammals, and Brucella microti, Brucella suis biovar 5 and Brucella neotomae. Comparative omp2a and omp2b gene sequence analysis revealed the most divergent omp2 sequences identified to date for a Brucella strain. The recA gene sequence of strain BO1(T) differed in seven nucleotides from the Brucella recA consensus sequence. Using the Brucella species-specific multiplex PCR assay, strain BO1(T) displayed a unique banding pattern not observed in other Brucella species. From the phenotypic and molecular analysis it became evident that strain BO1( T) was clearly different from all other Brucella species, and therefore represents a novel species within the genus Brucella. Because of its unexpected isolation, the name Brucella inopinata with the type strain BO1(T) (=BCCN 09-01(T)=CPAM 6436(T)) is proposed.

Brucella microti sp. nov., isolated from the common vole Microtus arvalis.

Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915(T) and CCM 4916), isolated from clinical specimens of the common vole Microtus arvalis during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA (rrs) and recA gene sequence similarities, both isolates were allocated to the genus Brucella. Affiliation to Brucella was confirmed by DNA-DNA hybridization studies. Both strains reacted equally with Brucella M-monospecific antiserum and were lysed by the bacteriophages Tb, Wb, F1 and F25. Biochemical profiling revealed a high degree of enzyme activity and metabolic capabilities not observed in other Brucella species. The omp2a and omp2b genes of isolates CCM 4915(T) and CCM 4916 were indistinguishable. Whereas omp2a was identical to omp2a of brucellae from certain pinniped marine mammals, omp2b clustered with omp2b of terrestrial brucellae. Analysis of the bp26 gene downstream region identified strains CCM 4915(T) and CCM 4916 as Brucella of terrestrial origin. Both strains harboured five to six copies of the insertion element IS711, displaying a unique banding pattern as determined by Southern blotting. In comparative multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) with 296 different genotypes, the two isolates grouped together, but formed a separate cluster within the genus Brucella. Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS711-based AMOS PCR, a 1900 bp fragment was generated with the Brucella ovis-specific primers, revealing that the insertion element had integrated between a putative membrane protein and cboL, encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915(T) and CCM 4916 could be clearly distinguished from all known Brucella species and their biovars by means of both their phenotypic and molecular properties, and therefore represent a novel species within the genus Brucella, for which the name Brucella microti sp. nov. with the type strain CCM 4915(T) (=BCCN 07-01(T)=CAPM 6434(T)) is proposed.

Protocell-like microspheres from thermal polyaspartic acid.

One of the most prominent amino acids to appear in monomer-generating origin-of-life experiments is aspartic acid. Hugo Schiff found in 1897 that aspartic acid polymerizes when heated to form polyaspartylimide which hydrolyzes in basic aqueous solution to form thermal polyaspartic acid which is a branched polypeptide. We recently reported at the ISSOL 2005 Conference that commercially made thermal polyaspartic acid forms microspheres when heated in boiling water and allowed to cool. In a new experiment we heated aspartic acid at 180 degrees C for up to 100 h to form thermal polyaspartylimide which when heated in boiling water without addition of base hydrolyzed to form thermal polyaspartic acid which upon cooling formed microspheres. Thermal polyaspartic acid microspheres appear protocell-like in the sense of being prebiotically plausible lattices or containers that could eventually have been filled with just the right additions of primordial proteins, nucleic acids, lipids, and metabolites so as to constitute protocells capable of undergoing further chemical and biological evolution. Thermal polyaspartic acid microspheres are extremely simple models of protocells that are more amenable to precise quantitative experimental investigation than the proteinoid microspheres of Sidney W. Fox. We present here scanning electron microscope images of such thermal polyaspartic acid microspheres. Figure 1 shows thermal polyaspartic acid microspheres from L: -aspartic acid heated at 180 degrees C for 50 h, at a magnification of 3,500x. Figure 2 shows thermal polyaspartic acid microspheres from the same sample at a magnification of 7,000x. The thermal polyaspartic acid microspheres have a diameter of approximately 1 mum These images were viewed with a Hitachi S2460N scanning electron microscope at 20 kV acceleration voltage. Figure 1 Thermal polyaspartic acid microspheres from L: -aspartic acid heated at 180 degrees C for 50 h, at a magnification of 3,500x. Figure 2 Thermal polyaspartic acid microspheres from L: -aspartic acid heated at 180 degrees C for 50 h, at a magnification of 7,000x.

[Validation of the Fluorescence Polarisation Assay (FPA) for the serological diagnosis of brucellosis]. / Validierung des Fluoreszenz-Polarisations- Assay (FPA) für die Serodiagnostik der Brucellose.

In order to evaluate suitability of Fluorescence Polarisation Assay (FPA) for serological Brucella diagnostic, 1739 samples of sera from cattle, pigs, sheep and goats (65 Brucella-positive, 960-negative and 714 false-positive sera) were investigated at a dilution of 1:10. The cut-off was adjusted by means of ROC analysis. Furthermore, the serum samples were examined for Brucella antibodies using SAT, CFT and ELISA and the results were evaluated regarding sensitivity and specificity. FPA, SAT, CFT and ELISA attained a sensitivity of 92.3, 98.5, 84.6 and 86.2%. In comparison, specificity varied with 87.8, 72.6, 92.5 and 85.8%, respectively. Accordingly, FPA is a suitable test for serodiagnosis of brucellosis.

[Canine brucellosis--a case report]. / Brucellose des Hundes--eine Fallbeschreibung.

Brucella (B.) canis was isolated from ejaculate of a 4-year old Korthals-Griffon male dog after occurrence of epididymitis and orchitis. Despite several trials of therapy with different antibiotics relapes occurred, with B. canis being isolated from ejaculate, blood and urine samples, respectively. Bacteriological examinations were added by serological testing over a period of about 1.5 years. During the study SAT serum titre steadily dropped from 1:200 to 1:50. By CFT, B. canis antibodies were detectable at the beginning with a titre of 1:320 and to the end of the study with titres between 1:80 and 1:160.