Evaluation of a Respiration Rate Sensor for Recording Tidal Volume in Calves under Field Conditions.

In the assessment of pulmonary function in health and disease, both respiration rate (RR) and tidal volume (Vt) are fundamental parameters of spontaneous breathing. The aim of this study was to evaluate whether an RR sensor, which was previously developed for cattle, is suitable for additional measurements of Vt in calves. This new method would offer the opportunity to measure Vt continuously in freely moving animals. To measure Vt noninvasively, the application of a Lilly-type pneumotachograph implanted in the impulse oscillometry system (IOS) was used as the gold standard method. For this purpose, we applied both measuring devices in different orders successively, for 2 days on 10 healthy calves. However, the Vt equivalent (RR sensor) could not be converted into a true volume in mL or L. For a reliable recording of the Vt equivalent, a technical revision of the RR sensor excluding artifacts is required. In conclusion, converting the pressure signal of the RR sensor into a flow equivalent, and subsequently into a volume equivalent, by a comprehensive analysis, provides the basis for further improvement of the measuring system.

Video Endoscopy-Guided Intrabronchial Spray Inoculation of Mycobacterium bovis in Goats and Comparative Assessment of Lung Lesions With Various Imaging Methods.

Bovine tuberculosis (bTB) not only poses a zoonotic threat to humans but also has a significant economic impact on livestock production in many areas of the world. Effective vaccines for humans, livestock, and wildlife are highly desirable to control tuberculosis. Suitable large animal models are indispensable for meaningful assessment of vaccine candidates. Here, we describe the refinement of an animal model for bTB in goats. Intrabronchial inoculation procedure via video-guided endoscopy in anesthetized animals, collection of lungs after intratracheal fixation in situ, and imaging of lungs by computed tomography (CT) were established in three goats using barium sulfate as surrogate inoculum. For subsequent infection experiments, four goats were infected with 4.7 × 102 colony-forming units of M. bovis by intrabronchial inoculation using video-guided endoscopy with spray catheters. Defined amounts of inoculum were deposited at five sites per lung. Four age-matched goats were mock-inoculated. None of the goats developed clinical signs until they were euthanized 5 months post infection, but simultaneous skin testing confirmed bTB infection in all goats inoculated with M. bovis. In tissues collected at necropsy, M. bovis was consistently re-isolated from granulomas in lymph nodes, draining the lungs of all the goats infected with M. bovis. Further dissemination was observed in one goat only. Pulmonary lesions were quantified by CT and digital 2D radiography (DR). CT revealed mineralized lesions in all the infected goats ranging from <5 mm to >10 mm in diameter. Small lesions <5 mm predominated. The DR failed to detect small lesions and to determine the exact location of lesions because of overlapping of pulmonary lobes. Relative volume of pulmonary lesions was low in three but high in one goat that also had extensive cavitation. CT lesions could be correlated to gross pathologic findings and histologic granuloma types in representative pulmonary lobes. In conclusion, video-guided intrabronchial inoculation with spray catheters, mimicking the natural way of infection, resulted in pulmonary infection of goats with M. bovis. CT, but not DR, presented as a highly sensitive method to quantify the extent of pulmonary lesions. This goat model of TB may serve as a model for testing TB vaccine efficacy.

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

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.

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

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.

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.

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.

Regeneration of Pulmonary Tissue in a Calf Model of Fibrinonecrotic Bronchopneumonia Induced by Experimental Infection with Chlamydia Psittaci.

Pneumonia is a cause of high morbidity and mortality in humans. Animal models are indispensable to investigate the complex cellular interactions during lung injury and repair in vivo. The time sequence of lesion development and regeneration is described after endobronchial inoculation of calves with Chlamydia psittaci. Calves were necropsied 2-37 days after inoculation (dpi). Lesions and presence of Chlamydia psittaci were investigated using histology and immunohistochemistry. Calves developed bronchopneumonia at the sites of inoculation. Initially, Chlamydia psittaci replicated in type 1 alveolar epithelial cells followed by an influx of neutrophils, vascular leakage, fibrinous exudation, thrombosis and lobular pulmonary necrosis. Lesions were most extensive at 4 dpi. Beginning at 7 dpi, the number of chlamydial inclusions declined and proliferation of cuboidal alveolar epithelial cells and sprouting of capillaries were seen at the periphery of necrotic tissue. At 14 dpi, most of the necrosis had been replaced with alveoli lined with cuboidal epithelial cells resembling type 2 alveolar epithelial cells and mild fibrosis, and hyperplasia of organized lymphoid tissue were observed. At 37 dpi, regeneration of pulmonary tissue was nearly complete and only small foci of remodeling remained. The well-defined time course of development and regeneration of necrotizing pneumonia allows correlation of morphological findings with clinical data or treatment regimen.

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

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.

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

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.

Acid-base assessment of post-parturient German Holstein dairy cows from jugular venous blood and urine: A comparison of the strong ion approach and traditional blood gas analysis.

Evaluating acid-base status is important for monitoring dairy herd health. In a field study, we aimed to compare the acid-base status measured by net acid-base excretion (NABE) in urine with results of venous blood analysis in clinically healthy, but possibly metabolically burdened cows in their transition period. For this, we sampled blood from the jugular vein and urine from 145 German Holstein cows within 1 to 76 days post-partum. In blood, the metabolic parameters non-esterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB), as well as numerous parameters of the acid-base status were measured. The traditional approach, based on bicarbonate concentration, base excess (BE) and anion gap (AG), was compared to the strong ion approach variables, e.g. acid total (Atot), measured strong ion difference (SIDm), strong ion gap (SIG), and unmeasured anions (XA), respectively. Results of both approaches were set against the outcome of urine analysis, i.e. the NABE, base-acid ratio and pH of urine, in a cluster analysis, which provided 7 moderately stable clusters. Evaluating and interpreting these 7 clusters offered novel insights into the pathophysiology of the acid-base equilibrium in fresh post-partum dairy cows. Especially in case of subclinical acid-base disorders, the parameters of the strong ion difference theory, particularly SIDm, Atot and SIG or XA, provided more in-depth information about acid-base status than the traditional parameters BE, bicarbonate or AG in blood. The acid-base status of fresh cows with protein aberrations in blood could be differentiated in a much better way using the strong ion approach than by traditional blood gas analysis or by the measurement of urinary excretion. Therefore, the strong ion approach seems to be a suitable supplement for monitoring acid-base balance in dairy cattle.

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

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.

Optimising experimental research in respiratory diseases: an ERS statement.

Experimental models are critical for the understanding of lung health and disease and are indispensable for drug development. However, the pathogenetic and clinical relevance of the models is often unclear. Further, the use of animals in biomedical research is controversial from an ethical perspective.The objective of this task force was to issue a statement with research recommendations about lung disease models by facilitating in-depth discussions between respiratory scientists, and to provide an overview of the literature on the available models. Focus was put on their specific benefits and limitations. This will result in more efficient use of resources and greater reduction in the numbers of animals employed, thereby enhancing the ethical standards and translational capacity of experimental research.The task force statement addresses general issues of experimental research (ethics, species, sex, age, ex vivo and in vitro models, gene editing). The statement also includes research recommendations on modelling asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, lung infections, acute lung injury and pulmonary hypertension.The task force stressed the importance of using multiple models to strengthen validity of results, the need to increase the availability of human tissues and the importance of standard operating procedures and data quality.

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

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.

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

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.

Circulating and broncho-alveolar interleukin-6 in relation to body temperature in an experimental model of bovine Chlamydia psittaci infection.

In rodent models of experimentally induced fever, the important role of interleukin-6 (IL-6) as a circulating endogenous pyrogen is well established. Studies employing larger animal species and real infections are scarce. Therefore, we assessed bioactive IL-6 in peripheral blood and in broncho-alveolar lavage fluid (BALF) of calves after intra-bronchial inoculation with vital Chlamydia psittaci (Cp), with inactivated Cp, or with BGM cells. Only calves inoculated with vital Cp developed fever (peak at 2-3 days after challenge) and significantly increased IL-6 activity. Controls inoculated with either inactivated Cp or BGM cells also expressed increased bioactive IL-6, but no fever developed. Activity of IL-6 in BALF was significantly higher compared to blood serum. This experimental model of Cp infection revealed no apparent relation between IL-6 in blood and body temperature, but did reveal a relation between IL-6 and other markers of inflammation in BALF. We conclude that a local inflammatory response in the lungs of infected calves caused fever, which developed by mechanisms including other mediators besides IL-6.

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

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.

A European Respiratory Society technical standard: exhaled biomarkers in lung disease.

Breath tests cover the fraction of nitric oxide in expired gas (FeNO), volatile organic compounds (VOCs), variables in exhaled breath condensate (EBC) and other measurements. For EBC and for FeNO, official recommendations for standardised procedures are more than 10 years old and there is none for exhaled VOCs and particles. The aim of this document is to provide technical standards and recommendations for sample collection and analytic approaches and to highlight future research priorities in the field. For EBC and FeNO, new developments and advances in technology have been evaluated in the current document. This report is not intended to provide clinical guidance on disease diagnosis and management.Clinicians and researchers with expertise in exhaled biomarkers were invited to participate. Published studies regarding methodology of breath tests were selected, discussed and evaluated in a consensus-based manner by the Task Force members.Recommendations for standardisation of sampling, analysing and reporting of data and suggestions for research to cover gaps in the evidence have been created and summarised.Application of breath biomarker measurement in a standardised manner will provide comparable results, thereby facilitating the potential use of these biomarkers in clinical practice.

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

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.

Hydrogen peroxide in exhaled air: a source of error, a paradox and its resolution.

The concentration of hydrogen peroxide (H2O2) in exhaled air has been reported to be elevated in asthma and chronic obstructive pulmonary disease (COPD), but results are inconsistent and difficult to reproduce. As H2O2 occurs in ambient air, we examined its association with exhaled H2O2 in human subjects. Exhaled breath condensate (EBC) of 12 COPD patients and nine healthy control subjects was collected either with an inhalation filter (efficiency 81%) or without. Ambient air condensate (AAC) was collected in parallel and samples were analysed for H2O2. Additionally, ambient H2O2 was recorded by an atmospheric measuring device (online fluorometric measurement). H2O2 concentration in AAC was significantly higher (p<0.001) than in EBC. AAC variations were concordant with the data from the atmospheric measuring instrument. In both subjects' groups, the inhalation filter reduced H2O2 values (p<0.01). Despite generally low levels in exhaled air, analysis by a mathematical model revealed a contribution from endogenous H2O2 production. The low H2O2 levels in exhaled air are explained by the reconditioning of H2O2-containing inhaled air in the airways. Inhaled H2O2 may be one factor in the heterogeneity and limited reproducibility of study results. A valid determination of endogenous H2O2 production requires inhalation filters.

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

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.

Acute phase proteins as promising biomarkers: Perspectives and limitations for human and veterinary medicine.

Acute phase proteins (APPs) are highly conserved plasma proteins that are increasingly secreted by the liver in response to a variety of injuries, independently of their location and cause. APPs favor the systemic regulation of defense, coagulation, proteolysis, and tissue repair. Various APPs have been applied as general diagnostic parameters for a long time. Through proteomic techniques, more and more APPs have been discovered to be differentially altered. Since they are not consistently explainable by a stereotypic hepatic expression of sets of APPs, most of these results have unfortunately been neglected or attributed to the nonspecificity of the acute phase reaction. Moreover, it appears that various extrahepatic tissues are also able to express APPs. These extrahepatic APPs show focally specific roles in tissue homeostasis and repair and are released primarily into interstitial and distal fluids. Since these focal proteins might leak into the circulatory system, mixtures of hepatic and extrahepatic APP species can be expected in blood. Hence, a selective alteration of parts of APPs might be expected. There are several hints on multiple molecular forms and fragments of tissue-derived APPs. These differences offer the chance for multiple selective determinations. Thus, specific proteoforms might indeed serve as tissue-specific disease indicators.