Publications by authors named "Heike Köhler"

42 Publications

Detection of Paratuberculosis in Dairy Herds by Analyzing the Scent of Feces, Alveolar Gas, and Stable Air.

Molecules 2021 May 11;26(10). Epub 2021 May 11.

Institute of Molecular Pathogenesis at 'Friedrich-Loeffler-Institut' (Federal Research Institute for Animal Health), Naumburgerstr. 96a, 07743 Jena, Germany.

Paratuberculosis is an important disease of ruminants caused by ssp. (MAP). Early detection is crucial for successful infection control, but available diagnostic tests are still dissatisfying. Methods allowing a rapid, economic, and reliable identification of animals or herds affected by MAP are urgently required. This explorative study evaluated the potential of volatile organic compounds (VOCs) to discriminate between cattle with and without MAP infections. Headspaces above fecal samples and alveolar fractions of exhaled breath of 77 cows from eight farms with defined MAP status were analyzed in addition to stable air samples. VOCs were identified by GC-MS and quantified against reference substances. To discriminate MAP-positive from MAP-negative samples, VOC feature selection and random forest classification were performed. Classification models, generated for each biological specimen, were evaluated using repeated cross-validation. The robustness of the results was tested by predicting samples of two different sampling days. For MAP classification, the different biological matrices emitted diagnostically relevant VOCs of a unique but partly overlapping pattern (fecal headspace: 19, alveolar gas: 11, stable air: 4-5). Chemically, relevant compounds belonged to hydrocarbons, ketones, alcohols, furans, and aldehydes. Comparing the different biological specimens, VOC analysis in fecal headspace proved to be most reproducible, discriminatory, and highly predictive.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/molecules26102854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8150929PMC
May 2021

Engineering Synthetic Lipopeptide Antigen for Specific Detection of subsp. Infection.

Front Vet Sci 2021 23;8:637841. Epub 2021 Apr 23.

INRAE, Université de Tours, ISP, Nouzilly, France.

Unlike other MAC members, subsp. (MAP) does not produce glycopeptidolipids (GPL) on the surface of the cell wall but a lipopentapeptide called L5P (also termed Lipopeptide-I or Para-LP-01) characterized in C-type (bovine) strains. This lipopeptide antigen contains a pentapeptide core, D-Phenylalanine-N-methyl-L-Valine-L-Isoleucine-L-Phenylalanine-L-Alanine, in which the N-terminal D-Phenylalanine is amido-linked with a fatty acid (C18-C20). The molecular and genetic characterization of this antigen demonstrated that L5P is unique to MAP. Knowledge of the structure of L5P enabled synthetic production of this lipopeptide in large quantities for immunological evaluation. Various studies described the immune response directed against L5P and confirmed its capability for detection of MAP infection. However, the hydrophobic nature of lipopeptide antigens make their handling and use in organic solvents unsuitable for industrial processes. The objectives of this study were to produce, by chemical synthesis, a water-soluble variant of L5P and to evaluate these compounds for the serological diagnosis of MAP using well-defined serum banks. The native L5P antigen and its hydrosoluble analog were synthesized on solid phase. The pure compounds were evaluated on collections of extensively characterized sera from infected and non-infected cattle. ROC analysis showed that L5P and also its water-soluble derivative are suitable for the development of a serological test for Johne's disease at a population level. However, these compounds used alone in ELISA have lower sensitivity (Se 82% for L5P and Se 62% for the water-soluble variant of L5P) compared to the Se 98% of a commercial test. Advantageously, these pure synthetic MAP specific antigens can be easily produced in non-limiting quantities at low cost and in standardized batches for robust studies. The fact that L5P has not been validated in the context of ovine paratuberculosis highlights the need to better characterize the antigens expressed from the different genetic lineages of MAP to discover new diagnostic antigens. In the context of infections due to other mycobacteria such as or the more closely related species subsp. , the L5P did not cross react and therefore may be a valuable antigen to solve ambiguous results in other tests.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fvets.2021.637841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8103206PMC
April 2021

Interferon-γ Response of subsp. Infected Goats to Recombinant and Synthetic Mycobacterial Antigens.

Front Vet Sci 2021 26;8:645251. Epub 2021 Mar 26.

Animal and Plant Health Agency, Addlestone, United Kingdom.

Despite its potential for early diagnosis of subsp. (MAP) infection, the IFN-γ release assay is not used routinely, because of low specificity of the established crude antigen preparation Johnin (PPDj). Limited data are available assessing the potential of MAP-derived protein and lipopeptide antigens to replace PPDj in assays for goats, while cattle and sheep have been studied more extensively. Furthermore, MAP infection is claimed to interfere with the diagnosis of bovine tuberculosis when other crude antigen preparations (PPDb, PPDa) are applied. In this study, the diagnostic potential of MAP-derived recombinant protein antigens, synthetic MAP lipopentapeptides and of specific peptide cocktails was assessed compared to crude mycobacterial antigen preparations in experimentally infected goats. Goats were inoculated with MAP, or subsp. (MAH) as surrogate for environmental mycobacteria, non-exposed animals served as controls. Complex-specific antibody and PPDj-induced IFN-γ responses were monitored . Infection status was assessed by pathomorphological findings and bacteriological tissue culture at necropsy 1 year after inoculation. The IFN-γ response to 13 recombinant protein antigens of MAP, two synthetic MAP lipopentapeptides and three recombinant peptide cocktails of was investigated at three defined time points after infection. At necropsy, MAP or MAH infection was confirmed in all inoculated goats, no signs of infection were found in the controls. Antibody formation was first detected 3-6 weeks post infection (wpi) in MAH-inoculated and 11-14 wpi in the MAP-inoculated goats. Maximum PPDj-induced IFN-γ levels in MAH and MAP exposed animals were recorded 3-6 and 23-26 wpi, respectively. Positive responses continued with large individual variation. Antigens Map 0210c, Map 1693c, Map 2020, Map 3651cT(it), and Map 3651c stimulated increased whole blood IFN-γ levels in several MAP-inoculated goats compared to MAH inoculated and control animals. These IFN-γ levels correlated with the intensity of the PPDj-induced responses. The two synthetic lipopentapeptides and the other MAP-derived protein antigens had no discriminatory potential. Stimulation with peptide cocktails ESAT6-CFP10, Rv3020c, and Rv3615c did not elicit IFN-γ production. Further work is required to investigate if test sensitivity will increase when mixtures of the MAP-derived protein antigens are applied.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fvets.2021.645251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034290PMC
March 2021

Detection of ssp. in Cultures From Fecal and Tissue Samples Using VOC Analysis and Machine Learning Tools.

Front Vet Sci 2021 3;8:620327. Epub 2021 Feb 3.

National Reference Laboratory for Paratuberculosis, Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut, Jena, Germany.

Analysis of volatile organic compounds (VOCs) is a novel approach to accelerate bacterial culture diagnostics of subsp. (MAP). In the present study, cultures of fecal and tissue samples from MAP-infected and non-suspect dairy cattle and goats were explored to elucidate the effects of sample matrix and of animal species on VOC emissions during bacterial cultivation and to identify early markers for bacterial growth. The samples were processed following standard laboratory procedures, culture tubes were incubated for different time periods. Headspace volume of the tubes was sampled by needle trap-micro-extraction, and analyzed by gas chromatography-mass spectrometry. Analysis of MAP-specific VOC emissions considered potential characteristic VOC patterns. To address variation of the patterns, a flexible and robust machine learning workflow was set up, based on random forest classifiers, and comprising three steps: variable selection, parameter optimization, and classification. Only a few substances originated either from a certain matrix or could be assigned to one animal species. These additional emissions were not considered informative by the variable selection procedure. Classification accuracy of MAP-positive and negative cultures of bovine feces was 0.98 and of caprine feces 0.88, respectively. Six compounds indicating MAP presence were selected in all four settings (cattle vs. goat, feces vs. tissue): 2-Methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, heptanal, isoprene, and 2-heptanone. Classification accuracies for MAP growth-scores ranged from 0.82 for goat tissue to 0.89 for cattle feces. Misclassification occurred predominantly between related scores. Seventeen compounds indicating MAP growth were selected in all four settings, including the 6 compounds indicating MAP presence. The concentration levels of 2,3,5-trimethylfuran, 2-pentylfuran, 1-propanol, and 1-hexanol were indicative for MAP cultures before visible growth was apparent. Thus, very accurate classification of the VOC samples was achieved and the potential of VOC analysis to detect bacterial growth before colonies become visible was confirmed. These results indicate that diagnosis of paratuberculosis can be optimized by monitoring VOC emissions of bacterial cultures. Further validation studies are needed to increase the robustness of indicative VOC patterns for early MAP growth as a pre-requisite for the development of VOC-based diagnostic analysis systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fvets.2021.620327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887282PMC
February 2021

Acid-base variables in acute and chronic form of nontuberculous mycobacterial infection in growing goats experimentally inoculated with Mycobacterium avium subsp. hominissuis or Mycobacterium avium subsp. paratuberculosis.

PLoS One 2020 14;15(12):e0243892. Epub 2020 Dec 14.

Institute of Molecular Pathogenesis at 'Friedrich-Loeffler-Institut' (Federal Research Institute for Animal Health), Jena, Germany.

In current literature, data assessing the acid-base equilibrium in animals and humans during bacterial infection are rare. This study aimed to evaluate acid-base deteriorations in growing goats with experimentally induced NTM (nontuberculous mycobacteria) infections by application of the traditional Henderson-Hasselbalch approach and the strong ion model. NTM-challenged animals were orally inoculated with either Mycobacterium avium subsp. hominissuis (MAH; n = 18) or Mycobacterium avium subsp. paratuberculosis (MAP; n = 48). Twenty-five goats served as non-infected controls. Until 51st week post-inoculation (wpi), blood gas analysis, serum biochemical analysis, and serum electrophoresis were performed on venous blood. Fifty percent (9/18) of goats inoculated with MAH developed acute clinical signs like apathy, fever, and diarrhea. Those animals died or had to be euthanized within 11 weeks post-inoculation. This acute form of NTM-infection was characterized by significantly lower concentrations of sodium, calcium, albumin, and total protein, as well as significantly higher concentrations of gamma globulin, associated with reduced albumin/globulin ratio. Acid-base status indicated alkalosis, but normal base excess and HCO3- concentrations, besides significantly reduced levels of SID (strong ion difference), Atot Alb (total plasma concentration of weak non-volatile acids, based on albumin), Atot TP (Atot based on total protein) and markedly lower SIG (strong ion gap). The remaining fifty percent (9/18) of MAH-infected goats and all goats challenged with MAP survived and presented a more sub-clinical, chronic form of infection mainly characterized by changes in serum protein profiles. With the progression of the disease, concentrations of gamma globulin, and total protein increased while albumin remained lower compared to controls. Consequently, significantly reduced albumin/globulin ratio and lower Atot Alb as well as higher Atot TP were observed. Changes were fully compensated with no effect on blood pH. Only the strong ion variables differentiated alterations in acid-base equilibrium during acute and chronic NTM-infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0243892PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7735625PMC
February 2021

Core profile of volatile organic compounds related to growth of Mycobacterium avium subspecies paratuberculosis - A comparative extract of three independent studies.

PLoS One 2019 15;14(8):e0221031. Epub 2019 Aug 15.

Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal Health, Jena, Germany.

Analysis of volatile organic compounds (VOC) derived from bacterial metabolism during cultivation is considered an innovative approach to accelerate in vitro detection of slowly growing bacteria. This applies also to Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of paratuberculosis, a debilitating chronic enteritis of ruminants. Diagnostic application demands robust VOC profiles that are reproducible under variable culture conditions. In this study, the VOC patterns of pure bacterial cultures, derived from three independent in vitro studies performed previously, were comparatively analyzed. Different statistical analyses were linked to extract the VOC core profile of MAP and to prove its robustness, which is a prerequisite for further development towards diagnostic application. Despite methodical variability of bacterial cultivation and sample pre-extraction, a common profile of 28 VOCs indicating cultural growth of MAP was defined. The substances cover six chemical classes. Four of the substances decreased above MAP and 24 increased. Random forest classification was applied to rank the compounds relative to their importance and for classification of MAP versus control samples. Already the top-ranked compound alone achieved high discrimination (AUC 0.85), which was further increased utilizing all compounds of the VOC core profile of MAP (AUC 0.91). The discriminatory power of this tool for the characterization of natural diagnostic samples, in particular its diagnostic specificity for MAP, has to be confirmed in future studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0221031PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695172PMC
March 2020

Crowd monitoring in dairy cattle-real-time VOC profiling by direct mass spectrometry.

J Breath Res 2019 07 11;13(4):046006. Epub 2019 Jul 11.

Department of Anaesthesiology and Intensive Care Medicine, Rostock Medical Breath Research Analytics and Technologies (ROMBAT), University Medicine Rostock, Schillingallee 35, 18057 Rostock, Germany.

Volatile organic compounds (VOCs) emitted from breath, faeces or skin may reflect physiological and pathological processes in vivo. Our setup employs real-time proton-transfer-reaction time-of-flight mass spectrometry (PTR-TOF-MS) to explore VOC emissions of dairy cows in stable air under field conditions. Within one herd of 596 cows, seven groups (8-117 cows per group) were assessed. Groups differed in milk yield and health status (two contained cows with paratuberculosis, a chronic intestinal infection). Each group arrived one after another in the area of air measurement in front of the milking parlour. A customised PTR-TOF-MS system with a 6 m long and heated transfer line, was used for measuring VOCs continuously for 7 h, 1.5 m above the cows. Three consecutive time periods were investigated. Twenty-seven VOCs increased while the animals were gathering in the waiting area, and decreased when the animals entered the milking parlour. Linear correlations between the number of animals present and VOC concentrations were found for (CH)H and (CHO)H. A relatively high concentration of acetone above the cows that had recently given birth to a calf might be related to increased fat turnover due to calving and different nutrition. Changes in VOC emissions were related to the presence of animals with paratuberculosis, to different average milk yields per group and to the time of the day (morning versus noon milking time). We found that VOC monitoring of stable air may provide additional immediate information on an animal's metabolic or health status and foster novel applications in the field of breath research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1088/1752-7163/ab269fDOI Listing
July 2019

Evaluation of needle trap micro-extraction and solid-phase micro-extraction: Obtaining comprehensive information on volatile emissions from in vitro cultures.

Biomed Chromatogr 2018 Oct 5;32(10):e4285. Epub 2018 Jun 5.

Department of Anesthesiology and Intensive Care Medicine, Rostock Medical Breath Research Analytics and Technologies, University Medical Center Rostock, Rostock, Germany.

Volatile organic compounds (VOCs) emitted from in vitro cultures may reveal information on species and metabolism. Owing to low nmol L concentration ranges, pre-concentration techniques are required for gas chromatography-mass spectrometry (GC-MS) based analyses. This study was intended to compare the efficiency of established micro-extraction techniques - solid-phase micro-extraction (SPME) and needle-trap micro-extraction (NTME) - for the analysis of complex VOC patterns. For SPME, a 75 μm Carboxen®/polydimethylsiloxane fiber was used. The NTME needle was packed with divinylbenzene, Carbopack X and Carboxen 1000. The headspace was sampled bi-directionally. Seventy-two VOCs were calibrated by reference standard mixtures in the range of 0.041-62.24 nmol L by means of GC-MS. Both pre-concentration methods were applied to profile VOCs from cultures of Mycobacterium avium ssp. paratuberculosis. Limits of detection ranged from 0.004 to 3.93 nmol L (median = 0.030 nmol L ) for NTME and from 0.001 to 5.684 nmol L (median = 0.043 nmol L ) for SPME. NTME showed advantages in assessing polar compounds such as alcohols. SPME showed advantages in reproducibility but disadvantages in sensitivity for N-containing compounds. Micro-extraction techniques such as SPME and NTME are well suited for trace VOC profiling over cultures if the limitations of each technique is taken into account.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bmc.4285DOI Listing
October 2018

Continuous real-time breath analysis in ruminants: effect of eructation on exhaled VOC profiles.

J Breath Res 2018 05 14;12(3):036014. Epub 2018 May 14.

Department of Anesthesiology and Intensive Care Medicine, Rostock Medical Breath Research Analytics and Technologies (ROMBAT), University Medicine Rostock, Schillingallee 35, D-18057 Rostock, Germany.

Background: The analysis of volatile organic compounds (VOCs) in breath allows non-invasive investigations of diseases. Animal studies are conducted as a model to perform research of VOCs and their relation to diseases. In large animal models ruminants were often used as experimental targets. The effect of their physiological eructation on VOC exhalation has not been examined yet and is the objective of this study.

Methods: Continuous breath profiles of two young cattle, four adult goats and four adult sheep were measured through a mask, covering mouth and nose, in real-time (200 ms) by means of proton transfer reaction time of flight mass spectrometry. Each animal was analysed twelve times for 3 consecutive minutes.

Results: Real-time monitoring yielded a distinction of different episodes in the breath profiles of ruminants. An algorithm to separate eructation episodes and alveolar breath was established. In the first exhalation after eructation at least 19 VOC concentrations increased (up to 36-fold) and went back to initial levels in subsequent exhalations in all investigated ruminants. Decay of concentrations was substance specific. In goats, less VOCs were affected by the eructation compared to cattle and sheep. Breath profiles without exclusion of eructation episodes showed higher variations and median values than profiles where eructation episodes were excluded.

Conclusion: Real-time breath analysis of ruminants enables the discrimination and characterisation of alveolar breath and eructation episodes. This leads to a better understanding of variation in breath data and possible origins of VOCs: breath or digestion related. To avoid impairment of breath gas results and to gain further information on bacterial products from the rumen, eructation and alveolar breath data should be analysed separately.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1088/1752-7163/aabdafDOI Listing
May 2018

Comparative analysis of volatile organic compounds for the classification and identification of mycobacterial species.

PLoS One 2018 20;13(3):e0194348. Epub 2018 Mar 20.

Institute of Molecular Pathogenesis at the 'Friedrich-Loeffler-Institut' (Federal Research Institute for Animal Health), Jena, Germany.

Background: Species of Mycobacteriaceae cause serious zoonotic diseases in mammals, for example tuberculosis in humans, dogs, parrots, and elephants (caused by Mycobacterium tuberculosis) and in ruminants and humans (caused by M. bovis and M. caprae). Pulmonary diseases, lymphadenitis, skin diseases, and disseminated diseases can be caused by non-tuberculous mycobacteria (NTM). Diagnosis and differentiation among Mycobacterium species are currently done by culture isolation. The established diagnostic protocols comprise several steps that allow species identification. Detecting volatile organic compounds (VOCs) above bacterial cultures is a promising approach towards accelerating species identification via culture isolation. The aims of this project were to analyse VOCs in the headspace above 13 different species of mycobacteria, to define VOC profiles that are unique for each species, and to compile a set of substances that indicate the presence of growing mycobacteria in general.

Materials & Methods: VOCs were measured in the headspace above 17 different mycobacterial strains, all cultivated on Herrold's Egg Yolk Medium and above pure media slants that served as controls. For pre-concentration of VOCs, needle-trap micro-extraction was employed. Samples were subsequently analysed using gas chromatography-mass spectrometry. All volatiles were identified and calibrated by analysing pure reference substances.

Results: More than 130 VOCs were detected in headspace above mycobacteria-inoculated and control slants. Results confirmed significant VOC emissions above all mycobacterial species that had grown well. Concentration changes were measurable in vials with visually assessed bacterial growth and vials without apparent growth. VOCs above mycobacterial cultures could be grouped into substances that were either higher or equally concentrated, lower or equally concentrated, or both as those above control slants. Hence, we were able to identify 17 substances as potential biomarkers of the presence of growing mycobacteria in general.

Conclusions: This study revealed species-specific VOC profiles for eleven species of mycobacteria that showed visually apparent bacterial growth at the time point of analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194348PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5860768PMC
July 2018

Complete Genome Sequence of JII-1961, a Bovine subsp. Field Isolate from Germany.

Genome Announc 2017 Aug 24;5(34). Epub 2017 Aug 24.

Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Jena, Germany.

subsp. causes Johne's disease in ruminants and was also detected in nonruminant species, including human beings, and in milk products. We announce here the 4.829-Mb complete genome sequence of the cattle-type strain JII-1961 from Germany, which is very similar to cattle-type strains recovered from different continents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/genomeA.00870-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5571421PMC
August 2017

Evaluation of different diagnostic methods for the detection of Mycobacterium avium subsp. paratuberculosis in boot swabs and liquid manure samples.

BMC Vet Res 2017 Aug 18;13(1):259. Epub 2017 Aug 18.

Institute for Molecular Pathogenesis, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Jena, Thuringia, Germany.

Background: Environmental sampling based on boot swabs and/or liquid manure samples is an upcoming strategy for the identification of paratuberculosis (paraTB) positive herds, but only limited data are available regarding the diagnostic performance of molecular detection methods (qPCR) versus faecal culture (FC) for this purpose. In the present study, the test characteristics of two different qPCR protocols (A and B) and a standardized FC protocol, for the detection of Mycobacterium avium subsp. paratuberculosis in boot swabs and liquid manure samples were evaluated.

Results: In 19 paraTB unsuspicious and 58 paraTB positive herds boot swabs and liquid manure were sampled simultaneously and analyzed in three different diagnostic laboratories. Using boot swabs and liquid manure, a substantial to excellent accordance was found between both qPCRs, for boot swabs also with culture, while for liquid manure the detection rate of culture was decreased after prolonged storage at -20 °C. The quantitative results of both qPCR methods correlated well for the same sample and also for boot swabs and liquid manure from the same herd. When cut-off threshold cycle (C-)-values were applied as recommended by the manufacturers, herd level specificity (Sp) of qPCR B was below 100% for boot swabs and for both qPCRs for liquid manure. A decreased herd level sensitivity was encountered after adjustment of Sp to 100% and re-calculation of the cut-off C-values.

Conclusions: qPCR is equally suitable as bacterial culture for the detection of Mycobacterium avium subsp. paratuberculosis in boot swabs and liquid manure samples. Both matrices represent easily accessible composite environmental samples which can be tested with reliable results. The data encourage qPCR testing of composite environmental samples for paraTB herd diagnosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12917-017-1173-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563032PMC
August 2017

Strategies for the identification of disease-related patterns of volatile organic compounds: prediction of paratuberculosis in an animal model using random forests.

J Breath Res 2017 Nov 1;11(4):047105. Epub 2017 Nov 1.

Institute of Epidemiology, Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany. Department of Mathematics and Computer Science, University of Greifswald, Germany.

Modern statistical methods which were developed for pattern recognition are increasingly being used for data analysis in studies on emissions of volatile organic compounds (VOCs). With the detection of disease-related VOC profiles, novel non-invasive diagnostic tools could be developed for clinical applications. However, it is important to bear in mind that not all statistical methods are equally suitable for the investigation of VOC profiles. In particular, univariate methods are not able to discover VOC patterns as they consider each compound separately. The present study demonstrates this fact in practice. Using VOC samples from a controlled animal study on paratuberculosis, the random forest classification method was applied for pattern recognition and disease prediction. This strategy was compared with a prediction approach based on single compounds. Both methods were framed within a cross-validation procedure. A comparison of both strategies based on these VOC data reveals that random forests achieves higher sensitivities and specificities than predictions based on single compounds. Therefore, it will most likely be more fruitful to further investigate VOC patterns instead of single biomarkers for paratuberculosis. All methods used are thoroughly explained to aid the transfer to other data analyses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1088/1752-7163/aa83bbDOI Listing
November 2017

Evaluation of associations between genotypes of Mycobacterium avium subsp. paratuberculsis and presence of intestinal lesions characteristic of paratuberculosis.

Vet Microbiol 2017 Mar 22;201:188-194. Epub 2017 Jan 22.

Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Institute of Molecular Pathogenesis, 07743 Jena, Naumburger Str. 96a, Germany. Electronic address:

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis affecting ruminants worldwide. Depending on the MAP-Type (MAP-C or MAP-S, cattle or sheep type), strains differ in virulence and host preference. There is not yet any strong evidence indicating that individual field strains of the same MAP-subgroup exhibit differences in virulence. The aim of this study was to evaluate a potential association between the genotype of individual field strains belonging to the MAP-C group and the presence of macroscopic intestinal lesions characteristic of paratuberculosis in the infected animals. 88 MAP-C isolates were sampled from clinically healthy cows at slaughter. Cows were grouped as A (n=46) with, and B (n=42) without macroscopic intestinal lesions. Sampled cows from both the A and B groups came from different farms and had a similar age distribution. MAP isolates were characterized by MIRU-VNTR and IS900-RFLP analysis. Resulting genotypes were examined for an association with the presence of macroscopic intestinal lesions characteristic of paratuberculosis. MAP isolates from groups A and B exhibited similar strain diversity: 20 and 18 combined genotypes, altogether 32 genotypes. Six of these genotypes were detected in both groups. Although no association was found between individual combined genotypes and presence of macroscopic intestinal lesions, IS900-RFLP-(BstEII)-Type-C1 (the most common type worldwide) was found more often in group A (p<0.01). The data give only weak indication for the existence of differences in virulence among MAP-cattle type isolates. Differences in the development and severity of lesions may rather depend on unknown host factors or inoculation dose. Virulence properties of IS900-RFLP-(BstEII)-Type-C1 isolates should be examined in more detail.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vetmic.2017.01.026DOI Listing
March 2017

Detection of mycobacteria by volatile organic compound analysis of invitro cultures using differential ion mobility spectrometry.

J Med Microbiol 2017 Mar 23;66(3):276-285. Epub 2017 Mar 23.

Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Naumburger Str. 96a, 07743 Jena, Germany.

Purpose: Differential ion mobility spectrometry (DMS) is an analytical technique used to detect volatile organic compounds (VOCs) in gaseous samples at very low concentration ranges from ppb to ppt. The aim of this study was to investigate whether VOC analysis by DMS is capable of detecting Mycobacterium avium subsp. paratuberculosis (MAP).

Methodology: Headspaces of in vitro cultures of two different MAP strains at 1, 2, 3, 4 and 6 weeks after inoculation (each at two dilutions) were analysed with DMS in comparison to control samples without viable bacteria [(i) blank medium, (ii) medium inoculated with heat-inactivated MAP and (iii) sterile-filtered MAP culture broth]. Furthermore, VOC patterns in the headspace over cultures of six non-tuberculous mycobacterial species were compared to MAP-derived VOC patterns. Data analysis included peak detection, cluster analysis, identification of discriminating VOC features (Mann-Whitney U test) and different cross-validated discriminant analyses.

Results: VOC analysis resulted in up to 127 clusters and revealed highly significant differences between MAP strains and controls at all time points. In addition, few clusters allowed differentiation between MAP and other non-tuberculous mycobacteria and even between different MAP strains. Compounds have not been characterized. VOC analysis by DMS was able to identify MAP-positive samples after 1 week of in vitro growth.

Conclusions: This study provides strong evidence that VOC analysis of headspace over mycobacterial cultures in combination with appropriate data analysis has the potential to become a valuable method to identify positive samples much earlier than with current standard procedures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/jmm.0.000410DOI Listing
March 2017

Effects of biological and methodological factors on volatile organic compound patterns during cultural growth of Mycobacterium avium ssp. paratuberculosis.

J Breath Res 2016 09 8;10(3):037103. Epub 2016 Sep 8.

Institute of Molecular Pathogenesis at the 'Friedrich-Loeffler-Institut' (Federal Research Institute for Animal Health), Naumburger Str. 96a, 07743 Jena, Germany.

Mycobacterium avium ssp. paratuberculosis (MAP) causes chronic granulomatous enteritis in ruminants. Bacterial growth is still the diagnostic 'gold standard', but is very time consuming. MAP-specific volatile organic compounds (VOCs) above media could accelerate cultural diagnosis. The aim of this project was to assess the kinetics of a VOC profile linked to the growth of MAP in vitro. The following sources of variability were taken into account: five different culture media, three different MAP strains, inoculation with different bacterial counts, and different periods of incubation. Needle-trap microextraction was employed for pre-concentration of VOCs, and gas chromatography-mass spectrometry for subsequent analysis. All volatiles were identified and calibrated by analysing pure references at different concentration levels. More than 100 VOCs were measured in headspaces above MAP-inoculated and control slants. Results confirmed different VOC profiles above different culture media. Emissions could be assigned to either egg-containing media or synthetic ingredients. 43 VOCs were identified as potential biomarkers of MAP growth on Herrold's Egg Yolk Medium without significant differences between the tree MAP strains. Substances belonged to the classes of alcohols, aldehydes, esters, ketones, aliphatic and aromatic hydrocarbons. With increasing bacterial density the VOC concentrations above MAP expressed different patterns: the majority of substances increased (although a few decreased after reaching a peak), but nine VOCs clearly decreased. Data support the hypotheses that (i) bacteria emit different metabolites on different culture media; (ii) different MAP strains show uniform VOC patterns; and (iii) cultural diagnosis can be accelerated by taking specific VOC profiles into account.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1088/1752-7155/10/3/037103DOI Listing
September 2016

Characterization of tuberculous granulomas in different stages of progression and associated tertiary lymphoid tissue in goats experimentally infected with Mycobacterium avium subsp. hominissuis.

Comp Immunol Microbiol Infect Dis 2016 Aug 26;47:41-51. Epub 2016 May 26.

Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Naumburger Str. 96a, 07743 Jena, Germany. Electronic address:

Oral infection of goats with Mycobacterium avium subsp. hominissuis (MAH) resulted in a large variety of granulomas in organized gut-associated lymphatic tissues and intestinal lymph nodes. To characterize the cellular composition of granulomas, CD4(+), CD8(+), γδ, B lymphocytes and plasma, CD25(+), CD68(+), MHC-II(+), Ki67(+) and endothelial cells were labeled in consecutive frozen sections by immunohistochemistry and acid fast bacilli (AFB) by Kinyoun stain. Granulomas with extensive necrosis, little mineralization and variable numbers of AFB surrounded by many CD4(+) T cells, but only few epitheloid macrophages were observed in severely sick goats at 2-3mpi. They were interpreted as exuberant immune reaction. Organized granulomas with very few AFB were seen in clinically healthy goats at 13mpi. The necrotic cores were surrounded by a zone of granulomatous infiltrate with many epitheloid macrophages and few lymphocytes. This zone was initially wide and highly vascularized and became progressively smaller. It was enclosed by an increasing layer of connective tissue. All organized granulomas were surrounded by compartimentalized tertiary lymphoid tissue. The granulomas in experimental infection of goats with MAH reflect the heterogeneity of lesions seen in mycobacterial infections of humans and ruminants and are therefore valuable for comparative research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cimid.2016.05.006DOI Listing
August 2016

Management of the calving pen is a crucial factor for paratuberculosis control in large dairy herds.

J Dairy Sci 2016 May;99(5):3744-3752

Institute of Epidemiology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Südufer 10, 17493 Greifswald-Insel Riems, Germany.

Improvement of hygiene and herd management to reduce the contact of calves with adult cow feces to prevent new infections is one of the basic strategies to manage paratuberculosis-affected dairy herds. Control programs should recommend an evidence-based selection of factors that demonstrably reduce the transmission of the infectious agent and decrease the prevalence of infected cattle to improve acceptance and implementation of the recommended measures among farmers. This study aimed to assess the influence of several management measures on control success in a longitudinal study in 28 large dairy herds with a median size of 415 cows in Thuringia, Germany. The cumulative incidence of cows shedding Mycobacterium avium ssp. paratuberculosis (MAP) per year was determined by individual fecal culture of all cows during 5 consecutive years. Relevant management practices as well as herd size, milk yield, and purchase of cattle were recorded by on-farm risk assessment. Mean holding time of MAP shedders within the herd was calculated from individual data of each shedding cow. Using multiple regression models, separate calving pens for shedders and disinfection of the pen after use were identified as significant risk factors that reduced the cumulative incidence of MAP shedders per year on the herd level. The results provide evidence that, in addition to other factors, calving hygiene and management of the calving pens are crucial for paratuberculosis control, particularly in large dairy herds. Considered together with the outcome from other studies, these results might be important to weight various risk factors and to avoid overburdening and overwhelming farmers and keeping them committed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3168/jds.2015-10625DOI Listing
May 2016

Impact of food intake on in vivo VOC concentrations in exhaled breath assessed in a caprine animal model.

J Breath Res 2015 Dec 15;9(4):047113. Epub 2015 Dec 15.

Institute of Molecular Pathogenesis at 'Friedrich-Loeffler-Institut' (Federal Research Institute for Animal Health), Naumburger Str. 96a, 07743 Jena, Germany.

Physiological processes within the body may change emitted volatile organic compound (VOC) composition, and may therefore cause confounding biological background variability in breath gas analyses. To evaluate the effect of food intake on VOC concentration patterns in exhaled breath, this study assessed the variability of VOC concentrations due to food intake in a standardized caprine animal model. VOCs in (i) alveolar breath gas samples of nine clinically healthy goats and (ii) room air samples were collected and pre-concentrated before morning feeding and repeatedly after (+60 min, +150 min, +240 min) using needle trap microextraction (NTME). Analysis of VOCs was performed by gas chromatography and mass spectrometry (GC-MS). Only VOCs with significantly higher concentrations in breath gas samples compared to room air samples were taken into consideration. Six VOCs that belonged to the chemical classes of hydrocarbons and alcohols were identified presenting significantly different concentrations before and after feeding. Selected hydrocarbons showed a concentration pattern that was characterized by an initial increase 60 min after food intake, and a subsequent gradual decrease. Results emphasize consideration of physiological effects on exhaled VOC concentrations due to food intake with respect to standardized protocols of sample collection and critical evaluation of results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1088/1752-7155/9/4/047113DOI Listing
December 2015

Comprehensive insights in the Mycobacterium avium subsp. paratuberculosis genome using new WGS data of sheep strain JIII-386 from Germany.

Genome Biol Evol 2015 09 17;7(9):2585-2601. Epub 2015 Sep 17.

RNA Bioinformatics and High Throughput Analysis, Faculty of Mathematics and Computer Science, Friedrich Schiller University Jena, Leutragraben 1, 07743 Jena, Germany.

Mycobacterium avium (M. a.) subsp. paratuberculosis (MAP) - the etiologic agent of Johne's disease - affects cattle, sheep and other ruminants worldwide. To decipher phenotypic differences among sheep and cattle strains (belonging to MAP-S [Type-I/III] respectively MAP-C [Type-II]) comparative genome analysis needs data from diverse isolates originating from different geographic regions of the world. The current study presents the so far best assembled genome of a MAP-S-strain: sheep isolate JIII-386 from Germany. One newly sequenced cattle isolate (JII-1961, Germany), four published MAP strains of MAP-C and MAP-S from U.S. and Australia and M. a. subsp. hominissuis (MAH) strain 104 were used for assembly improvement and comparisons. All genomes were annotated by BacProt and results compared with NCBI annotation. Corresponding protein-coding sequences (CDSs) were detected, but also CDSs that were exclusively determined either by NCBI or BacProt. A new Shine-Dalgarno sequence motif (5'AGCTGG3') was extracted. Novel CDSs including PE-PGRS family protein genes and about 80 non-coding RNAs exhibiting high sequence conservation are presented. Previously found genetic differences between MAP-types are partially revised. Four out of ten assumed MAP-S-specific large sequence polymorphism regions (LSPs) are still present in MAP-C strains; new LSPs were identified. Independently of the regional origin of the strains, the number of individual CDSs and single nucleotide variants confirm the strong similarity of MAP-C strains and show higher diversity among MAP-S strains. This study gives ambiguous results regarding the hypothesis that MAP-S is the evolutionary intermediate between MAH and MAP-C, but it clearly shows a higher similarity of MAP to MAH than to M. intracellulare.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/gbe/evv154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4607514PMC
September 2015

Physiological variability in volatile organic compounds (VOCs) in exhaled breath and released from faeces due to nutrition and somatic growth in a standardized caprine animal model.

J Breath Res 2015 May 14;9(2):027108. Epub 2015 May 14.

Institute of Molecular Pathogenesis at 'Friedrich-Loeffler-Institut' (Federal Research Institute for Animal Health), Naumburger Str. 96a, 07743 Jena, Germany.

Physiological effects may change volatile organic compound (VOC) concentrations and may therefore act as confounding factors in the definition of VOCs as disease biomarkers. To evaluate the extent of physiological background variability, this study assessed the effects of feed composition and somatic growth on VOC patterns in a standardized large animal model. Fifteen clinically healthy goats were followed during their first year of life. VOCs present in the headspace over faeces, exhaled breath and ambient air inside the stable were repeatedly assessed in parallel with the concentrations of glucose, protein, and albumin in venous blood. VOCs were collected and analysed using solid-phase or needle-trap microextraction and gas chromatograpy together with mass spectroscopy. The concentrations of VOCs in exhaled breath and above faeces varied significantly with increasing age of the animals. The largest variations in volatiles detected in the headspace over faeces occurred with the change from milk feeding to plant-based diet. VOCs above faeces and in exhaled breath correlated significantly with blood components. Among VOCs exhaled, the strongest correlations were found between exhaled nonanal concentrations and blood concentrations of glucose and albumin. Results stress the importance of a profound knowledge of the physiological backgrounds of VOC composition before defining reliable and accurate marker sets for diagnostic purposes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1088/1752-7155/9/2/027108DOI Listing
May 2015

In Vivo Volatile Organic Compound Signatures of Mycobacterium avium subsp. paratuberculosis.

PLoS One 2015 27;10(4):e0123980. Epub 2015 Apr 27.

Department of Anaesthesia and Intensive Care, University Medicine Rostock, Rostock, Germany.

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of a chronic enteric disease of ruminants. Available diagnostic tests are complex and slow. In vitro, volatile organic compound (VOC) patterns emitted from MAP cultures mirrored bacterial growth and enabled distinction of different strains. This study was intended to determine VOCs in vivo in the controlled setting of an animal model. VOCs were pre-concentrated from breath and feces of 42 goats (16 controls and 26 MAP-inoculated animals) by means of needle trap microextraction (breath) and solid phase microextraction (feces) and analyzed by gas chromatography/ mass spectrometry. Analyses were performed 18, 29, 33, 41 and 48 weeks after inoculation. MAP-specific antibodies and MAP-specific interferon-γ-response were determined from blood. Identities of all marker-VOCs were confirmed through analysis of pure reference substances. Based on detection limits in the high pptV and linear ranges of two orders of magnitude more than 100 VOCs could be detected in breath and in headspace over feces. Twenty eight substances differed between inoculated and non-inoculated animals. Although patterns of most prominent substances such as furans, oxygenated substances and hydrocarbons changed in the course of infection, differences between inoculated and non-inoculated animals remained detectable at any time for 16 substances in feces and 3 VOCs in breath. Differences of VOC concentrations over feces reflected presence of MAP bacteria. Differences in VOC profiles from breath were linked to the host response in terms of interferon-γ-response. In a perspective in vivo analysis of VOCs may help to overcome limitations of established tests.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123980PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4411140PMC
January 2016

Characterization of a caprine model for the subclinical initial phase of Mycobacterium avium subsp. paratuberculosis infection.

BMC Vet Res 2015 Mar 24;11:74. Epub 2015 Mar 24.

Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Jena, Germany.

Background: Paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (MAP) is difficult to control due to a long phase of clinically non-apparent (latent) infection for which sensitive diagnostics are lacking. A defined animal model for this phase of the infection can help to investigate host-MAP interactions in apparently healthy animals and identify surrogate markers for disease progress and might also serve as challenge model for vaccines. To establish such a model in goats, different age at inoculation and doses of oral inoculum of MAP were compared. Clinical signs, faecal shedding as well as MAP-specific antibody, IFN-γ and IL-10 responses were used for in vivo monitoring. At necropsy, about one year after inoculation (pi), pathomorphological findings and bacterial organ burden (BOB) were scored.

Results: MAP infection manifested in 26/27 inoculated animals irrespective of age at inoculation and dose. Clinical signs developed in three goats. Faecal shedding, IFN-γ and antibody responses emerged 6, 10-14 and 14 wpi, respectively, and continued with large inter-individual variation. One year pi, lesions were detected in 26 and MAP was cultured from tissues of 23 goats. Positive animals subdivided in those with high and low overall BOB. Intestinal findings resembled paucibacillary lesions in 23 and multibacillary in 4 goats. Caseous and calcified granulomas predominated in intestinal LNN. BOB and lesion score corresponded well in intestinal mucosa and oGALT but not in intestinal LNN.

Conclusions: A defined experimental infection model for the clinically non-apparent phase of paratuberculosis was established in goats as suitable basis for future studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12917-015-0381-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4404677PMC
March 2015

Facts, myths and hypotheses on the zoonotic nature of Mycobacterium avium subspecies paratuberculosis.

Int J Med Microbiol 2014 Oct 25;304(7):858-67. Epub 2014 Jul 25.

Helmholtz Centre for Infection Research, Molecular Immunology, Inhoffenstraße 7, 38124 Braunschweig, Germany.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis (Johne's disease [JD]), a chronic granulomatous enteritis in ruminants. JD is one of the most widespread bacterial diseases of domestic animals with significant economic impact. The histopathological picture of JD resembles that of Crohn's disease (CD), a human chronic inflammatory bowel disease of still unresolved aetiology. An aetiological relevance of MAP for CD has been proposed. This and the ambiguity of other published epidemiological findings raise the question whether MAP represents a zoonotic agent. In this review, we will discuss evidence that MAP has zoonotic capacity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijmm.2014.07.006DOI Listing
October 2014

Stability of genotyping target sequences of Mycobacterium avium subsp. paratuberculosis upon cultivation on different media, in vitro- and in vivo passage, and natural infection.

Vet Microbiol 2013 Dec 13;167(3-4):573-83. Epub 2013 Sep 13.

Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Naumburger Str. 96a, 07743 Jena, Germany.

Mycobacterium (M.) avium subsp. paratuberculosis - the causative agent of paratuberculosis (Johne's disease) - affects domestic and wild ruminants worldwide. Recently, different typing techniques have been combined to provide sufficient discriminatory power for the differentiation of isolates and for epidemiological studies. In order to challenge the reliability of this approach the stability of different M. avium subsp. paratuberculosis genotypes determined after primary isolation was investigated after sub-cultivation on six different media (A), twelve in vitro passages (B), or a singular in vivo passage (C). In addition, different isolates from a single animal or herd were investigated (D). Sub-cultures of type- and reference strains, re-isolated inoculation strain after in vivo passage, and 23 field isolates were genotyped by mycobacterial interspersed repetitive unit-variable-number of tandem-repeat (MIRU-VNTR)-, short-sequence-repeat (SSR)-, and IS900-based restriction-fragment length-polymorphism (IS900-RFLP)-analyses and compared with initial genotypes. MIRU-VNTR-alleles (at loci 292, X3, 25, 47, 7, and 32) were stable after in vitro cultivations and after animal passage. Results of SSR analysis at Locus 1 with 7G nucleotides and at Loci 8 and 9 (tri-nucleotides) were also stable. At Locus 2 9G repeats changed into 10G after goat passage. After in vitro subculture (A+B) but not after animal passage (C) IS900-RFLP-typing revealed changes of BstEII-patterns for 3 of 23 strains (including ATCC 19698). Multiple isolates from individual animals or from a single cattle herd with natural infection (D) which exhibited identical IS900-RFLP- and MIRU-VNTR- genotypes, showed different G repeat numbers at SSR locus 2. This implies strand slippage events during chromosomal duplication of bacteria in the course of bacterial spreading within hosts and herds. Consequently, SSR-Locus 2 is not suitable as genome marker for epidemiological studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vetmic.2013.09.008DOI Listing
December 2013

[Herd prevalence studies of Mycobacterium avium ssp. paratuberculosis (MAP) in cattle using serological tests: opportunities, limitations and costs].

Berl Munch Tierarztl Wochenschr 2012 Sep-Oct;125(9-10):361-70

Thüringer Tierseuchenkasse, Jena.

Infection status of cattle herds concerning Mycobacterium avium spp. paratuberculosis (MAP) is based on direct MAP detection or detection of MAP specific antibodies. This study aimed to calculate laboratory costs for a representative herd prevalence survey of MAP infections in Thuringian cattle herds.Therefore, sensitivity and specificity of ELISA-tests for detection of MAP antibodies which are licensed in Germany had to be evaluated using the target population to calculate the sample size which allows to classify a herd as MAP positive or MAP negative at a 95 % probability level. A total of 460 fecal culture positive cattle from 16 MAP positive herds and 344 fecal culture negative individuals from seven MAP negative herds were tested in each of the four ELISA's. In relation to fecal culture, diagnostic sensitivity ranged between 23.3% and 32.2%, and diagnostic specificity between 96.8% and 98.6%. A minimum sample size of 610 individuals per herd and 1.69 Mio Euro laboratory costs were calculated for the best performing ELISA test. Using the fecal culture based approach a maximum sample size of 41 cattle and 0.47 Mio Euro were necessary. Costs of serological testing exceeded costs for a culture based testing for all ELISA-tests. Considering limitations of required sample size and difficult assessment of positive test results, serological testing is suitable to evaluate MAP infection status in large cattle herds, but not in small herds.
View Article and Find Full Text PDF

Download full-text PDF

Source
October 2013

Suspicion of Mycobacterium avium subsp. paratuberculosis transmission between cattle and wild-living red deer (Cervus elaphus) by multitarget genotyping.

Appl Environ Microbiol 2012 Feb 16;78(4):1132-9. Epub 2011 Dec 16.

Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Jena.

Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n = 13), six cattle herds living in the area of this park (n = 23), and five cattle herds without any contact with these red deer (n = 17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable interpretations of epidemiological studies on paratuberculosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AEM.06812-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3273033PMC
February 2012

Divergent cytokine responses of macrophages to Mycobacterium avium subsp. paratuberculosis strains of Types II and III in a standardized in vitro model.

Vet Microbiol 2011 Aug 12;152(1-2):101-11. Epub 2011 Apr 12.

Friedrich-Loeffler-Institut, Institute of Molecular Pathogenesis, Naumburger Str. 96 a, D-07743 Jena, Germany.

Based on epidemiological and clinical observations, different strains of Mycobacterium avium subsp. paratuberculosis (MAP) are suspected to significantly differ in their virulence for ruminants. In the pathogenesis of paratuberculosis, macrophages represent the principal target cell for MAP. In order to judge the ability of different MAP-genotypes to modulate macrophage responses, the cytokine responses of the monocyte cell line THP-1 were studied after challenge with three different MAP strains under standardized conditions. The bovine field isolate J1961 (major Type II) and the ovine field isolate JIII-86 (Type III) were compared with the laboratory adapted reference strain ATCC 19698 (Type II). Strains were shown by three different typing methods (IS900-RFLP-, MIRU-VNTR-, and SSR-analysis) to substantially differ in several genotypic features. Macrophage function was assessed by quantifying mRNA of the cytokines TNF-α, IL-1ß, and IL-10 by quantitative RT-PCR. Secreted TNF-α protein was measured by a cytotoxicity test, IL-1ß and IL-10 using ELISA tests. The three MAP strains of various genotypes differ in their effect on human macrophages depending on challenge dose and infection time. These differences concerned both the mRNA level and secreted protein amounts of proinflammatory cytokines TNF-α, IL-1β and anti-inflammatory cytokine IL-10. Type III strain produced less IL-10 and IL-1β mRNA and protein but more TNF-α protein at 2h than the Type II strains. In summary, our results support the hypothesis that strain characteristics might have relevance for the host response towards MAP and, consequently, for the pathogenesis of paratuberculosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vetmic.2011.04.002DOI Listing
August 2011

Chronic intestinal Mycobacteria infection: discrimination via VOC analysis in exhaled breath and headspace of feces using differential ion mobility spectrometry.

J Breath Res 2011 Jun 21;5(2):027103. Epub 2011 Apr 21.

Institute of Molecular Pathogenesis at the 'Friedrich-Loeffler-Institut' (Federal Research Institute for Animal Health), Jena, Germany.

Differential ion mobility spectrometry (DMS) is a method to detect volatile organic compounds (VOC) in the ppt range. This study assessed whether VOC analysis using DMS could discriminate subjects with an experimentally induced chronic intestinal infection caused by Mycobacteria from non-infected controls. The animal model consisted of two groups of goats orally infected with two different doses of Mycobacterium avium subspecies paratuberculosis (MAP) and one group of non-infected healthy controls (each group: n = 6). Using DMS, exhaled breath and headspace of feces were analyzed on-line on an individual basis 9 months after inoculation of MAP. Data analysis included peak detection, cluster analysis, selection of discriminating VOC features (Mann-Whitney U test), and classification using a support-vector-machine. Taking the background of ambient air conditions into account, VOC analysis of exhaled breath as well as of feces revealed significant differences between chronically infected animals and non-infected controls. In both specimens, increasing as well as decreasing VOC features could be attributed to infection. Discrimination between infected and non-infected animals was sharper analyzing exhaled breath compared to headspace of feces. In exhaled breath, at least two VOC features were found to increase in a dose-dependent manner with increasing doses of MAP inoculated. Results of this study provide strong evidence that DMS analysis of exhaled breath has the potential to become a valuable tool for non-invasive assessment of VOC specifically related to certain diseases or infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1088/1752-7155/5/2/027103DOI Listing
June 2011

Unique genotypes of Mycobacterium avium subsp. paratuberculosis strains of Type III.

Vet Microbiol 2009 Nov 21;139(3-4):398-404. Epub 2009 Jun 21.

Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Naumburger Str. 96a, 07743 Jena, Germany.

Mycobacterium avium subsp. paratuberculosis (MAP) strains with two new IS900 restriction fragment length polymorphism (RFLP) BstEII types intermediate suspected to belong to the MAP Type III group were isolated from migrating sheep in Germany. Such strains have only been sporadically identified in a few studies. For a better understanding of the genomic diversity of MAP with regard to specific host associations, geographic origin, and the discussed classification into Type I, Type II and Type III, these isolates were further characterized. Using IS900-RFLP, the isolates showed unique fingerprint patterns after BstEII-, PstI-, PvuII- and BamHI-digestion which had not been published before. Additionally, using gyrB-PCR-restriction endonuclease analysis (PCR/REA) and mycobacterial interspersed repetitive unit (MIRU)-PCR, the two strains showed differences to known patterns of the Type I as well as the Type II group. Unique genotypes were also obtained with multilocus short sequence repeat (MLSSR) sequencing and MIRU-variable-number tandem-repeat (VNTR) typing. As expected, genomic profiles identical to the Type I and different from the Type II group were detected by IS1311-PCR/REA, IS1311 sequencing as well as by Large Sequence Polymorphism analysis (LSP(A) 8, 17, 20, 4-II, and 18). In addition to distinct growth characteristics, the unique genotypes of the studied sheep strains support their affiliation to the assumed third group within the MAP subspecies and suggest the existence of different genotypes within this Type III group. The results could serve as further evidence that Type I and Type III groups are more closely related to each other than to the bovine Type II group.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vetmic.2009.06.011DOI Listing
November 2009