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A more effective vaccine against tuberculosis than Bacille Calmette-Guérin (BCG) is urgently needed. BCG derived recombinant VPM1002 has been found to be more efficacious and safer than the parental strain in mice models. Newer candidates, such as VPM1002 Δpdx1 (PDX) and VPM1002 ΔnuoG (NUOG), were generated to further improve the safety profile or efficacy of the vaccine. Herein, we assessed the safety and immunogenicity of VPM1002 and its derivatives, PDX and NUOG, in juvenile goats. Vaccination did not affect the goats’ health in regards to clinical/hematological features. However, all three tested vaccine candidates and BCG induced granulomas at the site of injection, with some of the nodules developing ulcerations approximately one month post-vaccination. Viable vaccine strains were cultured from the injection site wounds in a few NUOG- and PDX- vaccinated animals. At necropsy (127 days post-vaccination), BCG, VPM1002, and NUOG, but not PDX, still persisted at the injection granulomas. All strains, apart from NUOG, induced granuloma formation only in the lymph nodes draining the injection site. In one animal, the administered BCG strain was recovered from the mediastinal lymph nodes. Interferon gamma (IFN-γ) release assay showed that VPM1002 and NUOG induced a strong antigen-specific response comparable to that elicited by BCG, while the response to PDX was delayed. Flow cytometry analysis of IFN-γ production by CD4+, CD8+, and γδ T cells showed that CD4+ T cells of VPM1002- and NUOG-vaccinated goats produced more IFN-γ compared to BCG-vaccinated and mock-treated animals. In summary, the subcutaneous application of VPM1002 and NUOG induced anti-tuberculous immunity, while exhibiting a comparable safety profile to BCG in goats.

Tuberculous granulomas are highly dynamic structures reflecting the complex host–mycobacterium interactions. The objective of this study was to compare granuloma development at the site of vaccination with BCG and its recombinant derivatives in goats. To characterize the host response, epithelioid cells, multinucleated giant cells (MNGC), T cell subsets, B cells, plasma cells, dendritic cells and mycobacterial antigen were labelled by immunohistochemistry, and lipids and acid-fast bacteria (AFB) were labelled by specific staining. Granulomas with central caseous necrosis developed at the injection site of most goats though lesion size and extent of necrosis differed between vaccine strains. CD4+ T and B cells were more scarce and CD8+ cells were more numerous in granulomas induced by recombinant derivatives compared to their parental BCG strain. Further, the numbers of MNGCs and cells with lipid bodies were markedly lower in groups administered with recombinant BCG strains. Microscopic detection of AFB and mycobacterial antigen was rather frequent in the area of central necrosis, however, the isolation of bacteria in culture was rarely successful. In summary, BCG and its recombinant derivatives induced reproducibly subcutaneous caseous granulomas in goats that can be easily monitored and surgically removed for further studies. The granulomas reflected the genetic modifications of the recombinant BCG-derivatives and are therefore suitable models to compare reactions to different mycobacteria or TB vaccines.

Regionally, the monitoring of paratuberculosis at the herd level is performed by the detection of specific antibodies in pooled milk samples by ELISA. The negative/positive cut-off S/P values applied for pooled milk samples are low and particularly vulnerable to variation in the test performance. In this study, a batch variation in the test performance of two ELISA tests was assessed to identify consequences for sample classification. A total of 72 pooled milk samples (50 from MAP-infected herds, 22 from one MAP-non-infected herd) were analyzed using three different batches, each of two different MAP antibody ELISA tests (A and B). Receiver operating characteristic (ROC) analysis was performed, with the results of each batch, S/P values of the samples and optical density (OD) readings of the negative and positive control samples included in the kits being compared between the batches of one test. ROC analysis revealed a considerable variation in the test performance of the batches of the two individual tests, caused by differences in the S/P values of the samples and resulting in different sensitivities at a specificity of 100%. Major sources of variation originate from the manufacturing processes of test batches. These sources have to be better controlled, and the test performance has to be revisited regularly.

Paratuberculosis is an important disease of ruminants caused by Mycobacterium avium ssp. paratuberculosis (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.

Analysis of volatile organic compounds (VOCs) is a novel approach to accelerate bacterial culture diagnostics of Mycobacterium avium subsp. paratuberculosis (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.

Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis (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 M. bovis or the more closely related species M. avium subsp. hominissuis, the L5P did not cross react and therefore may be a valuable antigen to solve ambiguous results in other tests.

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.

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 eitherMycobacterium aviumsubsp.hominissuis(MAH; n = 18) orMycobacterium aviumsubsp.paratuberculosis(MAP; n = 48). Twenty-five goats served as non-infected controls. Until 51stweek 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(Atotbased 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 Albas well as higher Atot TPwere 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.

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 (C4H6)H+ and (C3H6O)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.

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.

Germany was declared officially free from bovine tuberculosis (bTB) effective from 1 July 1996. After the occurrence of several Mycobacterium (M.) bovis outbreaks in north-western Germany in recent years with high intraherd prevalence at the time of detection, the reliability of abattoir surveillance as the principal component of the national bTB control programme was debated by veterinary public health officials. Rising numbers of wildlife-associated outbreaks caused by M. caprae in southern Germany eventually prompted a nationwide cross-sectional study on bTB. A total of 51 999 cattle, that is 0.41% of the national herd kept on 1.73% of German cattle farms, were tested. Despite 4 positive and 152 inconclusive single intradermal comparative cervical test results, none of the animals was confirmed as bTB-positive by a subsequent interferon-release assay or by post-mortem PCR testing. The estimated prevalence of bTB in Germany was thus calculated as 0.0% (CI 0.0000-0.0064%) affirming that Germany still qualifies as an officially tuberculosis-free (OTF) country. Occasional randomized nationwide testing can be an appropriate tool to reassure the OTF status and may also help to maintain an appropriate training level for the diagnostic procedures and for supporting sustained disease awareness among stakeholders.

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.

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.

Volatile organic compounds (VOCs) emitted from in vitro cultures may reveal information on species and metabolism. Owing to low nmol L-1 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-1 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-1 (median = 0.030 nmol L-1 ) for NTME and from 0.001 to 5.684 nmol L-1 (median = 0.043 nmol L-1 ) 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.

Mycobacterium avium subsp. paratuberculosis (MAP) causes lesions in naturally and experimentally infected ruminants which greatly differ in severity, cellular composition and number of mycobacteria. Morphologically distinct lesions are already found during the clinically inapparent phase of infection. The complex local host response and number of MAP were characterized at the initial sites of lesions, organized gut-associated lymphoid tissue, in experimentally infected goats. Tissues were collected at 3, 6, 9 and 12 month post-inoculation (mpi) from goat kids that had orally received 10 times 10 mg of bacterial wet mass of MAP (JII-1961). The cellular composition of lesions in Peyer's patches in the jejunum and next to the ileocecal valve was evaluated in 21 MAP-inoculated goats, where lesions were compared with unaltered tissue of six control goats. CD68 + , CD4 + , CD8 + , γδ T lymphocytes, B lymphocytes and plasma cells, MHC class II + and CD25 + cells were demonstrated by immunohistochemistry in serial cryostat sections. At 3 mpi, extensive granulomatous infiltrates predominated, consisting of numerous epitheloid cells admixed with many CD4 and γδ T lymphocytes. Only single MAP were detected. This indicates a strong cellular immune reaction able to control MAP infection. γδ T lymphocytes were markedly increased in this type of lesion which may reflect their important role early in the pathogenesis of paratuberculosis. At 9 and 12 mpi, divergent lesions were observed which may reflect different outcomes of host–pathogen interactions. In five goats, minimal granulomatous lesions were surrounded by extensive lymphoplasmacytic infiltrates and no MAP were detected by immunohistochemistry. This was interpreted as effective host response that was able to eliminate MAP locally. In three goats, decreased numbers of lymphocytes, but extensive granulomatous infiltrates with numerous epitheloid cells containing increased numbers of mycobacteria were seen. This shift of the immune response resulted in uncontrolled mycobacterial multiplication. Focal and multifocal circumscribed granulomatous infiltrates of mainly epitheloid cells may represent sites of new infection, since they were observed in goats at all times after inoculation. Their presence in goats with minimal granulomatous lesions surrounded by extensive lymphoplasmacytic infiltrates may indicate that despite the local clearance, the infection may be perpetuated. The complex cellular immune reactions postulated for the pathogenesis of paratuberculosis were demonstrated at the local sites of infection. These early host–pathogen interactions are most likely essential for the eventual outcome of the MAP infection.

Mycobacterium avium subsp. paratuberculosis 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.

Mycobacterium avium subsp. paratuberculosis 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.

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 (CT-)-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 CT-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.

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.

There is a paucity of published data reporting acid–base equilibrium in goats, and no information is available on how the acid–base complexity changes when suckling goat kids become ruminants. The aims of this study were to evaluate young healthy goats for age-related changes in serum proteins, metabolites, and electrolytes; differences in results when the Henderson–Hasselbalch equation or strong ion approaches were used were also assessed. To assess biological variability and reproducibility, two consecutive long-term studies, each lasting from the 6th to 56th week of life (wl), were performed in 15 (Study 1) and 10 (Study 2) animals. Blood gas analysis, serum biochemical analysis, and electrophoresis were performed on venous blood, and acid–base information was obtained using the traditional Henderson–Hasselbalch approach, Stewart’s strong ion model, and Constable’s simplified strong ion model. In all goats within the first 4–5 months, serum concentrations of glucose, L-lactate, and inorganic phosphate decreased significantly, while serum concentrations of total protein, albumin, and gamma globulin increased. Consequently, nonvolatile weak acids (Atot Alb and Atot TP) increased. At the end of this ‘adaptation period’, i.e. when milk was replaced by purely plant-based food, significantly lower bicarbonate and base excess values were accompanied by blood pH that shifted towards acidosis. Electrolytes (Na+, K+, Ca2+, and Cl−), anion gap, strong ion difference, and strong ion gap did not show age-dependent trends. In conclusion, somatic growth and development of gastro-intestinal fermentation in growing goats act as complex sources of physiological variability on acid–base equilibrium that was not reflected by the Henderson–Hasselbalch equation only.

ZusammenfassungDie Tuberkulose des Rindes, hervorgerufen durch Mycobacterium (M.) bovis/M. caprae, ist weltweit eine der wichtigsten Rinderkrankheiten mit zoonotischem Potenzial und war bis in die zweite Hälfte des20. Jahrhunderts hinein auch in Deutschland weit verbreitet. Durch effektive Bekämpfungsmaßnahmen in Deutschland, Ost wie West, wurde die Seuche in den Rinderbeständen nahezu getilgt, sodass Deutschland seit Ende 1961 (West) bzw. Ende 1978 (Ost) als praktisch tuberkulosefrei gelten konnte. Durch Zuerkennung des Status „officially free of tuberculosis“ (OTF) im Jahr 1996 wurde Deutschland nach der Vereinigung die Freiheit von Rindertuberkulose durch die Europä ische Kommission offiziell bestätigt. Die Voraussetzung hierfür war, dass jährlich bei weniger als 0,1% der Rinderhaltungsbetriebe in Deutschland Tuberkulose festgestellt wurde. Dieser Status konnte bisher ohne Unterbrechung aufrechterhalten werden, sodass die Rindertuberkulose für viele Jahrzehnte aus dem Blickfeld der Tierärzte, der Landwirte und der Öffentlichkeit verschwand. Seit dem Jahr 2008 steigt die Zahl der angezeigten Ausbrüche, die über viele Jahre bei einer Gesamtzahl von etwa 200000 Rinderhaltungsbetrieben (mit abnehmender Tendenz) bei durchschnittlich unter 10 Ausbrüchen pro Jahr lag, deutlich an. Aufgrund verstärkter Überwachungsintensität waren im Jahr 2013 sogar 46 Ausbrüche zu verzeichnen, wobei diese besonders häufig die Bundesländer Bayern und Niedersachsen betrafen. Diese Entwicklung hatte zur Folge, dass die Tuberkulose-Verordnung in den Jahren 2009, 2012 und 2013 überarbeitet wurde, um die Grundlage zu legen für eine nun notwendig erachtete Veränderung der Tuberkulose-Überwachung und, regional, sogar der Bekämpfung der Tuberkulose. Neben den traditionell angewandten Untersuchungs- und Nachweismethoden, dem Tuberkulin-Hauttest (ante mortem) und dem bakteriologischen Nachweis (post mortem), wurden der Gamma-Interferon-Freisetzungstest (ante mortem) und der molekulare Erregernachweis (post mortem) in das zugelassene Methodenspektrum aufgenommen. Durch Hinzunahme dieser Methoden ist die Tuberku lose-Diagnostik schneller, in vielen Fällen jedoch nicht einfacher geworden.

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.

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.

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.

Bartonella species are Gram-negative, fastidious bacteria. Bartonella henselae is found in cats and transmitted to humans via cat scratches or bites causing cat-scratch disease, characterized by clinical symptoms with varying severity. The prevalence of bartonellosis among humans in Germany appears to be high, and severe clinical cases have been described. However, epidemiological data of B. henselae in cats are rare. In this study we determined the detection rates of Bartonella ssp. in cats by culture and real-time PCR. Furthermore, B. henselae isolates were genetically characterized by highly discriminatory amplified fragment length polymorphism (AFLP) and multilocus sequence typing (MLST). Bartonella spp. were isolated by culture from 11 (2.2%) of 507 blood samples. Out of 169 blood samples additionally analyzed by PCR, 28 (16.6%) were found positive for Bartonella spp., illustrating the advantage of PCR in Bartonella spp. detection. PCR-REA identified B. henselae in 27 cats and Bartonella clarridgeiae in one cat. B. henselae isolates from different geographical regions in Germany were genetically characterized by AFLP and MLST. Both methods confirmed genetic diversity of B. henselae on the strain level. MLST identified 11 new sequence types, all of them assigned to three clonal complexes as determined by eBURST. AFLP typing revealed genetic relation among the B. henselae isolates from the same geographical region. Combining AFLP typing and MLST/eBURST analyses revealed that B. henselae of the same AFLP subcluster belonged to the same clonal complex. Altogether these results indicate that B. henselae may evolve clonally.

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 (LSPA 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. (c) 2009 Elsevier B.V. All rights reserved

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease and is endemic to the national cattle herds of many countries. Because of the very low level of genetic heterogeneity of this organism, it is difficult to select a workable procedure for strain differentiation at a resolution sufficient to investigate epidemiological links between herds or different ruminant species and the suggested zoonotic potential of M. avium subsp. paratuberculosis for Crohn's disease. Analysis of restriction fragment length polymorphisms (RFLPs) based on the insertion element IS 900 (IS 900 RFLP) with four restriction enzymes and 10 markers of specific mycobacterial interspersed repetitive units (MIRUs) and variable-number tandem repeats (VNTRs) was applied to 71 bovine M. avium subsp. paratuberculosis isolates originating from 14 herds from different regions in Germany. Among these isolates, all of which belonged to the M. avium subsp. paratuberculosis type II group, 17 genotypes were detected by IS 900 RFLP and consisted of a combination of seven BstEII, eight PstI, nine PvuII, and four BamHI restriction patterns. Novel RFLP types were found. The diversity of the M. avium subsp. paratuberculosis isolates inside the herds was different depending on the frequency of animal purchase. The results of typing by IS 900 RFLP and MIRU-VNTR analyses were not associated. Fifteen MIRU-VNTR patterns were identified with a discriminatory index of 0.905. The most common BstEII-based IS 900 RFLP type, type C1 (72%), was subdivided into 14 types by MIRU-VNTR analysis. A combination of fingerprinting and PCR-based techniques resulted in 24 M. avium subsp. paratuberculosis genotypes and achieved a discriminatory index of 0.997. By using only BstEII and PstI digestion together with typing by MIRU-VNTR analysis, a discriminatory index of 0.993 was achieved. This is high enough to support epidemiological studies on a national as well as a global scale.

For molecular biological detection of Mycobacterium avium subsp. paratuberculosis (MAP), PCR methods with primers targeting different regions specific for MAP are used worldwide. However, some uncertainties exist concerning the specificity of certain target regions and the sensitivity. To identify the methods which are best suited for diagnostics, 8 single-round and 5 nested PCR systems including 12 different primer pairs based on IS900 (9 x), ISMav2 (1 x), 1757 (1 x), and locus 255 (1 x) sequences were compared regarding their analytical sensitivity and specificity under similar PCR conditions. Reference strains and field isolates of 17 Mycobacterium species and subspecies, 16 different non-mycobacterial bovine pathogens and commensals were included in this study. Single-round PCR resulted in a detection limit of 100 fg to 1 pg, and nested PCR in 10 fg or below. Depending on the specific primer sequences targeting IS900, false positive results occurred with one of the five single-round and two of the four nested PCR systems. This also applied to the single-round PCR based on ISMav2 and the nested PCR based on f57. A high number of non-specific products were primarily detected for the single-round PCR assay based on ISMav2, but also for a single-round PCR targeting the IS900 and the locus 255. In conclusion, stringent selection of IS900-specific primers ensures that IS900 remains a favourite target sequence for amplification of MAP specific loci. The studied PCR systems based on f57, and locus 255 can also be recommended. Revision of ISMav2 primers is necessary. Single-round PCR systems are very reliable. Nested PCR assays were occasionally disturbed by contaminations, thus bearing a risk for routine diagnostics. (c) 2007 Elsevier B.V. All rights reserved

Campylobacter (C.) jejuni and C. coli can cause gastrointestinal disorders in humans characterized by acute inflammation. Inflammatory signals are initiated during interaction between these pathogens and human intestinal cells, but nothing is known about the stimulation of avian intestinal cells by Campylobacter. Interleukin-8 (IL-8) as a proinflammatory chemokine plays an important role in mobilizing cellular defence mechanism. IL-8 mRNA expression in both human intestinal cells (INT 407) and primary intestinal chick cells (PIC) was determined by quantitative real-time RT-PCR. The secretion of IL-8 protein by INT407 was measured using ELISA. Although C. jejuni and C. coli are considered to be harmless commensals in the gut of birds, the avian Campylobacter isolates investigated were able to induce the proinflammatory IL-8 in PIC as well as in INT407. In an in vitro system, C. jejuni as well as C. coli were able to induce IL-8 mRNA in PIC. Relation between the virulence properties like toxin production, the ability to invade and to survive in Caco-2 cells and the level of IL-8 mRNA produced by INT 407 and PIC after infection with Campylobacter strains was also investigated.

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.

Summary Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic pathogen that causes infections in man and animals. In this study, 18 goat kids were inoculated orally with a high dose of MAH. One group of goats (n = 9) developed severe clinical disease for up to 2–3 months post inoculation (mpi). At necropsy examination, there were ulcerative and granulomatous lesions in gut-associated lymphoid tissue and granulomas with extensive necrosis in the lymph nodes (LNs) of the cranial mesenteric lymphocentre (CMLNs). Culture revealed growth of MAH in all lesions with systemic spread. A second group of goats were healthy at the end of the trial (13 mpi); however, all had extensive granulomas in the CMLNs, but no extra-intestinal spread of bacteria. Moderate faecal shedding occurred in all goats up to 2 mpi. Microscopical characterization of the granulomas revealed solid non-necrotic, necrotic, calcified and fibrocalcified granulomas with resemblance to those seen in human and bovine tuberculosis. The two different courses of disease, with highly heterogenic lesions, systemic spread in goats with severe clinical disease and the development of granulomas of all stages in the surviving goats, makes the experimental infection of goats with MAH a valuable model for tuberculosis research. This model might allow new insights into host–pathogen interaction and anti-mycobacterial compound testing.