Phenotypic and genotypic approach to characterize a Trueperella pecoris strain isolated from necrotic vestibulitis of a camel (Camelus dromedarius).

The present study was designed to characterize phenotypically and genotypically a Trueperella (T.) pecoris strain isolated from necrotic vestibulitis of a 10-year-old camel (Camelus dromedarius). The species identity of T. pecoris 203/7 investigated in the present study could be confirmed by phenotypic properties and by phylogenetic analyses based on partial sequencing of the 16S ribosomal RNA (rRNA) gene, the 16S-23S rDNA intergenic spacer region, the glyceraldehyde 3-phosphate dehydrogenase encoding gene gap, elongation factor Tu encoding gene tuf and the target gene rpoB encoding the ß-subunit of bacterial RNA polymerase. T. pecoris strain 203/7 was grouped within the genus Trueperella in the family Arcanobacteriaceae. The 16S rRNA gene analysis showed a sequence identity of 99·9% to reference strain T. pecoris DSM 111392T . The present isolate was clearly identified as T. pecoris, the most recently described species of the genus Trueperella. Strain T. pecoris 203/7 was isolated in moderate numbers from necrotic vestibulitis of the camel and could be of some importance for the infectious process. However, the investigated strain represents the first isolation of T. pecoris from a camel.

Suitability of lactic acid bacteria and deriving antibacterial preparations to enhance shelf-life and consumer safety of emulsion type sausages.

Ready-to-eat (RTE) sliced emulsion type sausages are sensitive to recontamination with Listeria (L.) monocytogenes during processing and packaging steps. Since Listeria spp. are able to grow on those products under cold storage conditions, taking steps to reduce the recontamination risk and implementing antibacterial hurdles contribute to consumer safety and increase the product quality. With this study data about the suitability of culture broth, cell-free supernatant (CFS) or concentrated bacteriocin preparations (CFSconc) of bacteriocin-producing lactic acid bacteria (LAB) obtained from fermented sausages from Germany as protective culture or antibacterial additive were provided. In different challenge tests, the potential of selected LAB or their preparations were investigated for their potential to reduce growth of L. monocytogenes and/or Brochothrix (B.) thermosphacta on sliced RTE emulsion type sausages under modified atmosphere or vacuum during refrigerated storage for a 21-day period. Applied LAB culture broth and CFS could not reduce the growth of L. monocytogenes or B. thermosphacta. On the other hand, samples treated with CFSconc obtained from Pediococcus spp. strains showed a significant inhibition (p < 0.05) of more than 1.5 log10 of the applied L. monocytogenes strains during the storage period. The growth of B. thermosphacta could not be influenced. Thereby, the need for concentrating preparations was shown to be important to obtain a suitable antibacterial preparation that would contribute to consumer safety and food quality when applied as a protective additive.

Evaluation of different target genes for the detection of Salmonella sp. by loop-mediated isothermal amplification.

The loop-mediated isothermal amplification (LAMP) technique was used to investigate six salmonella-specific sequences for their suitability to serve as targets for the pathogen identification. Sequences selected for designing LAMP primers were genes invA, bcfD, phoP, siiA, gene62181533 and a region within the ttrRSBCA locus. Primers including single nucleotide polymorphisms were configured as degenerate primers. Specificity of the designed primer sets was determined by means of 46 salmonella and 32 other food- and waterborne bacterial reference species and strains. Primers targeting the ttrRSBCA locus showed 100 % inclusivity of target and exclusivity of other test species and strains. Other primer sets revealed deficiencies, especially regarding Salmonella enterica subsp. II-IV and Salmonella bongori. Additionally, primers targeting the siiA gene failed to detect S. enterica subsp. enterica serotypes Newport and Stanley, whereas bcfD primers did not amplify DNA of S. enterica subsp. enterica serotype Schleissheim. TtrRSBCA primers, providing short detection times and constant melting temperatures of amplification products, achieved best overall performance.

Loop-mediated isothermal amplification: Development, validation and application of simple and rapid assays for quantitative detection of species of Arcobacteraceae family- and species-specific Aliarcobacter faecis and Aliarcobacter lanthieri.

AIM: The family Arcobacteraceae formerly genus Arcobacter has recently been reclassified into six genera. Among nine species of the genus Aliarcobacter, Aliarcobacter faecis and Aliarcobacter lanthieri have been identified as emerging pathogens potentially cause health risks to humans and animals. This study was designed to develop/optimize, validate and apply Arcobacteraceae family- and two species-specific (A. faecis and A. lanthieri) loop-mediated isothermal amplification (LAMP) assays to rapidly detect and quantify total number of cells in various environmental niches. METHODS AND RESULTS: Three sets of LAMP primers were designed from conserved and variable regions of 16S rRNA (family-specific) and gyrB (species-specific) genes. Optimized Arcobacteraceae family-specific LAMP assay correctly amplified and detected 24 species, whereas species-specific LAMP assays detected A. faecis and A. lanthieri reference strains as well as 91 pure and mixed culture isolates recovered from aquatic and faecal sources. The specificity of LAMP amplification of A. faecis and A. lanthieri was further confirmed by restriction fragment length polymorphism analysis. Assay sensitivities were tested using variable DNA concentrations extracted from simulated target species cells in an autoclaved agricultural water sample by achieving a minimum detection limit of 10 cells mL-1 (10 fg). Direct DNA-based quantitative detection, from agricultural surface water, identified A. faecis (17%) and A. lanthieri (1%) at a low frequency compared to family-level (93%) with the concentration ranging from 2·1 × 101 to 2·2 × 105 cells 100 mL-1 . CONCLUSIONS: Overall, these three DNA-based rapid and cost-effective novel LAMP assays are sensitive and can be completed in less than 40 min. They have potential for on-site quantitative detection of species of family Arcobacteraceae, A. faecis and A. lanthieri in food, environmental and clinical matrices. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly developed LAMP assays are specific, sensitive, accurate with higher reproducibility that have potential to facilitate in a less equipped lab setting and can help in early quantitative detection and rate of prevalence in environmental niches. The assays can be adopted in the diagnostic labs and epidemiological studies.

Development and validation of a loop-mediated isothermal amplification assay-a rapid and sensitive detection tool for Mycobacterium avium subsp. paratuberculosis in small ruminants.

AIMS: The aim of this study was to design an assay for the identification of Mycobacterium avium subsp. paratuberculosis (MAP) to be used in faeces and milk samples of small ruminants with a loop-mediated isothermal amplification (LAMP) system, as a time-saving and user-friendly method in contrast to real-time PCR. METHODS AND RESULTS: For the detection of MAP in milk and faeces of small ruminants, we developed a set of primers, specific for the target gene ISMap02. The analytical sensitivity of LAMP, when targeting ISMap02, showed a DNA detection limit of 10 fg µl-1 . After performing spiking experiments with two MAP reference strains, DSM 44133 and ATCC 19698T , the limit of detection, using the LAMP protocol described herein were 3·8 MAP CFU per ml milk and 12·5 MAP CFU per gram faeces. All LAMP results during the establishment of the assay were compared to those of the real-time PCR results. An internal amplification control was incorporated into the assay to exclude false-negative results produced and had no significant negative impact on the analytical sensitivity. Validation of the assay was confirmed by testing field samples of faeces and revising the results with real-time PCR. CONCLUSION: Our study conducted the first MAP detection system with a LAMP targeting ISMap02. Due to the positive results we encourage the use of LAMP in combination with ISMap02, when detecting MAP in faeces samples, as an alternative to targeting other genes as f57 or IS900. Further research on MAP detection in different matrices like raw milk, tissue or sperm with this system is recommended. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new achievements in MAP diagnostic. Especially small ruminants do not show signs of diarrhoea until the terminal stage of the illness. The greatest task in fighting MAP is to rule out animals, which shed MAP with faeces and milk before showing symptoms of Johne's disease. Worldwide there is a need to eradicate animals, which are low MAP shedders to stop the illness spreading in animal holdings. MAP detection with LAMP is time saving, easy to use, does not need expensive equipment, as, for example, PCR kits and can be used without access to laboratories. The target gene ISMap02 was shown to be a specific insertion element for MAP and is a reliable aim in future MAP detection studies.

Characterisation of mecA gene negative Staphylococcus aureus isolated from bovine mastitis milk from Northern Germany.

Staphylococcus aureus (S. aureus) is an important causative agent of contagious intermammary infections in dairy cattle. S. aureus is also considered as an important foodborne pathogen and cause of food poisoning cases and outbreaks worldwide. In order to understand the molecular ecology of S. aureus, the present study compared phenotypic and genotypic characteristics of 70 S. aureus isolates from bovine mastitis milk samples collected during the period from August 2001 to March 2014 in different regions of Northern Germany. The S. aureus isolates were characterised phenotypically, as well as genotypically for their genetic diversity using multi-locus sequence typing (MLST), spa typing and the presence of virulence genes encoding 16 staphylococcal enterotoxins (sea-selu), toxic shock syndrome toxin (tst), thermonuclease (nuc), clumping factor (clfA and clfB), coagulase (coa) and the methicillin resistance gene mecA. A total of 16 sequence types were grouped into eight clonal complexes (CCs), and 17 spa types were identified. These included six novel sequence types and one novel spa type. The majority of bovine mastitis milk-associated sequence types belonged to the clonal complex CC5, CC97, CC133, and CC151 and showed closely related genotypes or lineages with sequence types of human origin. The genotype CC133 (ST133-t1403) was predominant, constituting 27.1% of the isolates. In addition, the S. aureus isolates displayed nine different enterotoxigenic profiles. All S. aureus were methicillin-susceptible (MSSA). The current study provides new information on phenotypic and genotypic traits of S. aureus isolates from bovine mastitis. The comparison of characteristics of isolates from the present study originating from mastitis milk showed similarities with human isolates. This might help to better understand the distribution of S. aureus in the one health context.

First report on the isolation of Trueperella abortisuis from companion animals.

The present study gives a detailed phenotypic and genotypic characterization of three Trueperella abortisuis strains isolated from a ten year old male Hovawart dog with an abscess of anal sac, from urine of an eight year old European shorthair cat with urolithiasis and nephrolithiasis and from a 14year old Maine Coon cat with a perianal abscess, respectively. All three strains could be identified phenotypically, by MALDI-TOF MS analysis and genotypically by sequencing the 16S rDNA and the molecular target genes gap and tuf. The present study gives a first description of T. abortisuis of this origin.

Identification of Arcanobacterium hippocoleae by MALDI-TOF MS analysis and by various genotypical properties.

In the present study an Arcanobacterium hippocoleae strain isolated from a uterus swab of an apparently healthy mare could be identified by phenotypic properties, by MALDI-TOF MS analysis and genotypically by investigating the molecular targets 16S rDNA, 16S-23S rDNA intergenic spacer region and the genes encoding the ß subunit of bacterial RNA polymerase (rpoB), elongation factor tu (tuf) and glyceraldehyde 3-phosphate dehydrogenase (gap). The presented data are one of the few reports about the species A. hippocoleae and might help to elucidate the role this species plays in infections of horses.

Development and validation of a loop mediated isothermal amplification (LAMP) assay for the detection of Staphylococcus aureus in bovine mastitis milk samples.

Staphylococcus (S.) aureus is one of the most important animal pathogens causing bovine mastitis. Also, it is a major human pathogen that may produce a variety of toxins which cause staphylococcal food poisoning. In the present study a LAMP assay based on gene nuc to identify S. aureus was developed and validated. The specificity of the LAMP assay was confirmed by using 70 S. aureus isolates and 21 non-S. aureus strains. The optimal temperature-time combination to amplify gene nuc successfully was 65 °C and 30 min. The analytical sensitivity of the developed LAMP assay was 0.26 pg of S. aureus DNA per reaction. The limit of detection evaluated with milk spiked with S. aureus was 9 × 102 CFU mL-1. The final results of this assay were available within less than 2 h. The present study showed that the LAMP assay based on gene nuc appeared to be rapid and simple, and could also be used to identify S. aureus isolates from mastitis milk of dairy cows.

Application of a loop-mediated isothermal amplification (LAMP) assay for molecular identification of Trueperella pyogenes isolated from various origins.

In the present study 28 Trueperella pyogenes strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene cpn60 encoding chaperonin. No cross reaction could be observed with control strains representing four species of genus Trueperella and seven species of closely related genus Arcanobacterium. The present cpn60 LAMP assay might allow a reliable and low cost identification of T. pyogenes also in laboratories with less specified equipment.

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of Arcanobacterium pluranimalium.

In the present study 28 Arcanobacterium pluranimalium strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene pla encoding pluranimaliumlysin. No comparable reaction could be observed with control strains representing five species of genus Arcanobacterium and five species of genus Trueperella. The presented pla LAMP assay might allow a reliable and low cost identification of this bacterial pathogen also in laboratories with less specified equipment.

Investigation of persistence of infectious Toxoplasma gondii in raw sausages using in-house developed and validated real time-PCR.

The question, if and to what extent raw-sausage-products represent a possible source of infection for the globally distributed and potentially health-threatening toxoplasmosis gave reason for this study. For this, the survival capability of Toxoplasma gondii in relation to the raw-sausage-manufacturing-process including different ripening-processes was investigated. To enable a fast and reliable parasite-detection, a real time-PCR-system based on a specific 529-bp-fragment of T. gondii and an internal amplification control (IAC) was developed and established. The applicability was tested in various experiments where T. gondii-tachyzoites were mixed into different types of raw-sausages and then investigated by using the real time-PCR-system. The latter was also used to investigate the possible infection-risk of raw-sausages. For this, two pigs were intravenously infected with T. gondii-tachyzoites and after having reached the typical slaughtering age, their meat was manufactured to different raw-sausage-products ("Mett"- and "Teewurst" as well as "Salami"). In order to prove the potential infectivity of these products under conditions close to reality, sausages in different ripening stages were fed to laboratory mice. The animals' organs (brain, heart and spleen) were examined employing the real time-PCR. T. gondii-DNA was detected in four out of 288 (1.4%) mice indicating that marketable raw-sausage-products generally bear a risk for consumers. However, the probability of an infection seems to be quite marginal.

Identification of Arcanobacterium pluranimalium by matrix-assisted laser desorption ionization-time of flight mass spectrometry and, as novel target, by sequencing pluranimaliumlysin encoding gene pla.

In the present study 13 Arcanobacterium pluranimalium strains isolated from various animal origin could successfully be identified phenotypically by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and the pluranimaliumlysin encoding gene pla. The detection of mass spectra by MALDI-TOF MS and the novel genotypic approach using gene pla might help to identify A. pluranimalium in future and might elucidate the role this species plays in infections of animals.

Further characteristics of Arcanobacterium canis, a novel species of genus Arcanobacterium.

Comparable to previously conducted phenotypical and genotypical investigations characterizing Arcanobacterium canis, a newly described species with the type strain A. canis DSM 25104 isolated from an otitis externa of a dog, four additional A. canis strains isolated from infections of three dogs and one cat could reliably be identified by phenotypic properties, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and by sequencing the genomic targets 16S rDNA, 16S-23S rDNA intergenic spacer region, 23S rDNA, and the genes rpoB and gap. All four A. canis investigated in the present study were isolated from the infected animals together with several other bacterial species indicating that the pathogenic importance of A. canis remains unclear. However, the detection of peptidic spectra by MALDI-TOF MS and the presented phenotypic and genotypic approaches might help to identify A. canis in future and might elucidate the role this species plays in infections of dogs and cats.

Determination of pig sex in meat and meat products using multiplex real time-PCR.

For specific production lines, European retail companies demand exclusively female pork meat. To control the quality of their suppliers the identification and a quantitative detection of the animal sex origin of the meat is therefore of importance for meat processors. To enable a fast and reliable detection of male pig meat, a real time-PCR-system was designed in the present study. This was based on the genes AMEL-X and AMEL-Y. The real time-PCR assay allowed the detection of male pig meat at a concentration of 1% yielding a detection probability of 100% while the detection probability investigating meat samples containing 0.1% male pig meat was 44.4%. The analytic sensitivity of this system was assessed to be <5 pg DNA per PCR reaction. The assessment of the accuracy of the real time-PCR assay to correctly identify sex individuals was investigated with 62 pigs including males (n=29) and females (n=33) belonging to different breeds/lines. With the newly designed test all analysed animals were correctly sexed. No amplification was obtained with cow, goat, sheep, turkey and chicken genomic DNA. The presented assay can be used for sex diagnosis, for the detection of male pig meat and for meat quality control.

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis at a regional scale in Germany.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of Johne's disease in dairy cattle. Genotyping of MAP is useful to gain a better understanding of the origin of infection, to evaluate regional control programs, to improve diagnostics, and to develop vaccines. In this study 91 MAP isolates mainly from symptomatic dairy cattle in Rhineland-Palatinate (RP, Germany), its neighbor federal states, and Luxembourg were genotyped using Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeat (MIRU-VNTR) and Multilocus Short Sequence Repeats (MLSSR). MIRU-VNTR and MLSSR produced 11 and 6 different genotypes among the 91 isolates, respectively. The combined analysis of both methods produced 25 genotypes with an index of discrimination (D) of 0.93 (95% CI: 0.91-0.95). The results revealed the genetic diversity of MAP and the dominance of two MAP genotypes commonly found in Europe, showed the usefulness of MAP genotyping in studies at a regional scale, and provided useful information for control initiatives in RP.

Diagnosis and Molecular Characterization of Mycobacterium avium subsp. paratuberculosis from Dairy Cows in Colombia.

The objective of this study was the serological, bacteriological and molecular diagnosis, as well as the molecular characterization of Mycobacterium avium subsp. paratuberculosis (Map) in adult cows of five Colombian dairy herds. Serum samples were tested by an indirect absorbed enzyme-linked immunosorbent assay (ELISA-C). All fecal samples were tested by pooled culture. After that, fecal samples of Map positive pools were tested individually by culture and polymerase chain reaction (PCR). In one herd, slurry and tissue samples from one animal were also taken and tested by PCR and culture. Map isolates were analyzed by the Multilocus Short Sequence Repeat (MLSSR) and the Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) methods. ELISA produced positive results in 1.8% (6/329) of the animals and 40% (2/5) of the herds. Four fecal, two tissue, and two slurry samples from a herd were Map positive by culture and PCR. MLSSR and MIRU-VNTR revealed two different strain profiles among eight Map isolates recovered. This study reports the first molecular characterization of Map in one dairy herd in Colombia, the limitations for individual diagnosis of subclinical Map infections in cattle, and the usefulness of pooled fecal samples and environmental sampling for Map diagnosis.

Molecular identification and further characterization of Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins.

The present study was designed to identify phenotypically and genotypically 61 Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins. The A. pyogenes isolates showed the typical cultural and biochemical properties of this species and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the superoxide dismutase A encoding gene sodA of reference strains representing 8 species of genus Arcanobacterium and subsequent design of A. pyogenes sodA gene-specific oligonucleotide primer. The A. pyogenes sodA gene-specific oligonucleotide primer allowed, together with previously described A. pyogenes 16S-23S rDNA intergenic spacer region-specific oligonucleotide primer, a reliable molecular identification of all 61 A. pyogenes of various origins. The additionally performed PCR-mediated amplification of 5 known and putative virulence factor encoding genes revealed that 100, 20, 87, 75, and 98% of the A. pyogenes carried the genes plo, cbpA, nanH, nanP, and fimA, which allowed an individual strain characterization. This might help to elucidate the role the putative virulence factors play in bovine mastitis and in various other infections caused by this bacterial pathogen.

Characterization of Arcanobacterium abortisuis by phenotypic properties and by sequencing the 16S-23S rDNA intergenic spacer region.

The present study was designed to characterize phenotypically and genotypically nine Arcanobacterium abortisuis strains collected from specimen of pigs in a period of nine years. All nine A. abortisuis strains and A. abortisuis reference strain DSM 19515 displayed a synergistic hemolytic reaction with Staphylococcus aureus ß-hemolysin, Rhodococcus equi, and Arcanobacterium haemolyticum indicator strains and showed the typical biochemical properties of this species. The species identity could be confirmed by identification and sequencing of the 16S-23S rDNA intergenic spacer region (ISR), which appeared to be a useful target for genotypic characterization of this bacterial species. The A. abortisuis strains of the present study were isolated from specimen of pigs together with various other bacterial species indicating that the pathogenic importance of this newly described species remains to be elucidated.

Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties.

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens.