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Object and Purpose: Acid-base disorders in dairy herds can be diagnosed by determining urinary net base excretion (NBE). Modifications of this method are the differential NBE (dNBE) with determination of the urinary concentration-independent base-acid ratio (BAR) and the simplified NBE test with reduced urine volume (sNBE). The aim of this study was to compare these methods among themselves and as a pooled test, in their assessment of cow group acid base status as well as to derive recommendations for practical use., Animals, Material, and Methods: The concentrations of NBE, dNBE, and sNBE were measured in urine samples derived from 855 German-Holstein cows in 127 cow groups at different stages of lactation. BAR was then calculated. dNBE and BAR were determined both individually per cow and as a pool of a group. Mixed linear models were used to examine the relationship between the mean of the individual animal values and the pool sample result of a group for these two parameters. In addition, all groups were evaluated with respect to acidotic or alkalotic load based on their single animal results of the respective methods, the mean values formed from them, and the measured pool result. By using the single animal BAR as reference, the sensitivity and specificity of the different methods were calculated., Results: The calculated mean values of the individual measurements of dNBE and BAR differed from the measured value in the pool sample, especially in low and high measurement ranges. In the group assessment, NBE showed the best combined sensitivity and specificity for the detection of acid base disorders. The dNBE pool assay showed satisfactory specificity with respect to acidosis and alkalosis, while the sNBE on an individual animal basis and the BAR determination in the pool showed satisfactory sensitivity with respect to acidosis., Conclusions and Clinical Relevance: It was shown that NBE determined in individual animal samples is well suited for the assessment of acid base status of cow groups and can therefore be recommended for practical use. The determination of dNBE as well as BAR as a pool test is not sufficient for the detection of alkalotic load in cow groups but may help to confirm an existing acidotic load., Competing Interests: Die Autoren erklären, dass keine geschützten, finanziellen, beruflichen oder anderweitigen Interessen an einem Produkt oder einer Firma bestehen, welche die in dieser Veröffentlichung genannten Inhalte oder Meinungen beeinflussen können., (Thieme. All rights reserved.)

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.

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 × 10 2 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., Competing Interests: LG was employed by Vakzine Projekt Management GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wedlich, Figl, Liebler-Tenorio, Köhler, von Pückler, Rissmann, Petow, Barth, Reinhold, Ulrich, Grode, Kaufmann and Menge.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vitense, Kasbohm, Klassen, Gierschner, Trefz, Weber, Miekisch, Schubert, Möbius, Reinhold, Liebscher and Köhler.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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., Competing Interests: The authors have declared that no competing interests exist.

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 (C 4 H 6 )H + and (C 3 H 6 O)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.

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., (Copyright © 2018 John Wiley & Sons, Ltd.)

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., Competing Interests: Conflict of interest: P. Bonniaud reports personal fees (board/advice) from Roche, Boehringer, Novartis, TEVA and AstraZeneca, and travel reimbursement (ERS meetings) from Chiesi, outside the submitted work. Conflict of interest: A. Fabre reports grants from Pfizer, reagents from Roche, and personal fees from MSD (Merck Sharp & Dohme Corp.), outside the submitted work. Conflict of interest: T. Maes reports grants from Belgian Science Policy (Federal Government Belgium-Interuniversity attraction Poles), Ghent University (Concerted Research Action and Spearhead Immunology) and Novartis, and personal fees (GSK Clinical Science award: prize for best abstract presentation at Belgian Pulmonology Society Meeting 2015) and non-financial support (travel/accommodation/meeting expenses) from GlaxoSmithKline, outside the submitted work; and is shareholder of Oryzon Genomics. Conflict of interest: W. Shi reports grants from National Institute of Health and Department of Defense, outside the submitted work. Conflict of interest: M. Stampfli reports grants from RespiVert and MedImmune, and personal fees from AstraZeneca and Boehringer Ingelheim, outside the submitted work. Conflict of interest: S. Uhlig reports grants from Germany Research Foundation (DFG), during the conduct of the study. Conflict of interest: E. White reports grants and other support from NIH, personal fees for consulting from Akcea Therapeutics and Kadmon Pharmaceuticals, and research grants and personal fees for consulting from Boehringer-Ingelheim, outside the submitted work. Conflict of interest: M. Witzenrath reports grants and personal fees from Bayer Health Care, Boehringer Ingelheim, Biotest and Vaxxilon, and personal fees from Actelion, Berlin Chemie, AstraZeneca, GlaxoSmithKline and Novartis, outside the submitted work. Conflict of interest: B. Crestani has receieved an honorarium for speaking from Aventis, honoraria for speaking, grants for research and congress travel support grants from Boehringer Ingelheim and Roche, grants for research from CARDIF and LVL, an honorarium for speaking and congress travel support from AstraZeneca, and a grant for research and congress travel support from MedImmune, outside the submitted work. Conflict of interest: O. Eickelberg reports grants from the Helmholtz Association, the German Center of Lung Research, Roche, and Bayer and consultancy/lecture fees from BMS, Novartis, Bayer, Intermune, McKinsey, and MorphoSys. Conflict of interest: A. Guenther reports research funding, and compensation for lectures and consulting from Roche, compensation for lectures and consulting from Boehringer Ingelheim and Teva, and research funding from Sanofi, outside the submitted work. Conflict of interest: G. Jenkins reports grants from GlaxoSmithKline (institutional funding for the PROFILE study) and the Medical Research Council (co-funder of the PROFILE study with GSK through MICA award), during the conduct of the study; grants from Biogen (SRA for work on integrins in IPF), personal fees (consulting and data monitoring committees on lung fibrosis) from Boehringer Ingelheim, grants (PhD studentship) from Galecto, personal fees (advisory board on IPF) from GlaxoSmithKline and Intermune, grants and personal fees (consulting on IPF, PhD studentship) from MedImmune, personal fees (consulting on IPF) from PharmAkea, personal fees (advisory board on IPF and lecture fees) from Roche, consulting on IPF (with no payment received to date) for Pliant Therapeutics, participating in scientific advisory boards (with no payment received to date) for NuMedii, and personal fees from Pulmatrix, outside the submitted work; and is a trustee for the charities Action for Pulmonary Fibrosis and the British Thoracic Society. Conflict of interest: G. Joos reports grants, personal fees and non-financial support from AstraZeneca, grants and personal fees from Boehringer Ingelheim, Chiesi, GlaxoSmithKline and Novartis, and personal fees from Mundipharma, Sandoz and Teva, outside the submitted work. Conflict of interest: U.A. Maus reports grants from Federal Ministry of Education and Research, German Research Foundation and Lower Saxony Society for the Control of Tuberculosis and Bronchial Diseases, outside the submitted work. Conflict of interest: L. Richeldi reports grants and personal fees (for advisory board membership) from InterMune, personal fees (for advisory board membership) from Medimmune, Roche and FibroGen, personal fees (for consulting activity) from Biogen, Sanofi-Aventis, ImmuneWorks, Celgene and Nitto, personal fees (for speaking) from Shionogi, and personal fees (for membership of steering committee) from Boehringer Ingelheim, outside the submitted work. Conflict of interest: M. Kolb reports grants and personal fees from Roche, Boehringer Ingelheim, GSK, Gilead, Prometic and Alkermes, grants from Actelion, Respivert and Synairgen, and personal fees from AstraZeneca and Genoa, outside the submitted work., (Copyright ©ERS 2018.)

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.

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.

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.

Breath tests cover the fraction of nitric oxide in expired gas ( F eNO ), volatile organic compounds (VOCs), variables in exhaled breath condensate (EBC) and other measurements. For EBC and for F eNO , 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 F eNO , 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., Competing Interests: Conflict of interest: T. Tonia acts as a methodologist for the European Respiratory Society. Further disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2017.)

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.

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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

The concentration of hydrogen peroxide (H 2 O 2 ) 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 H 2 O 2 occurs in ambient air, we examined its association with exhaled H 2 O 2 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 H 2 O 2 . Additionally, ambient H 2 O 2 was recorded by an atmospheric measuring device (online fluorometric measurement). H 2 O 2 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 H 2 O 2 values (p<0.01). Despite generally low levels in exhaled air, analysis by a mathematical model revealed a contribution from endogenous H 2 O 2 production. The low H 2 O 2 levels in exhaled air are explained by the reconditioning of H 2 O 2 -containing inhaled air in the airways. Inhaled H 2 O 2 may be one factor in the heterogeneity and limited reproducibility of study results. A valid determination of endogenous H 2 O 2 production requires inhalation filters., Competing Interests: can be found alongside this article at openres.ersjournals.com

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.

Infection of cattle with chlamydiae is ubiquitous and, even in the absence of clinical sequeleae, has a quantifiable negative impact on livestock productivity. Despite recent progress, our knowledge about immune response mechanisms capable of counteracting the infection and preventing its detrimental effects is still limited. A well-established model of bovine acute respiratory Chlamydia (C.) psittaci infection was used here to characterize the kinetics of the local and systemic immune reactions in calves. In the course of two weeks following inoculation, leukocyte surface marker expression was monitored by flow cytometry in blood and bronchoalveolar lavage fluid (BALF). Immune-related protein and receptor transcription were determined by quantitative real-time reverse transcription PCR in blood, BALF and lung tissue. An early increase of IL2RA, IL10 and HSPA1A mRNA expressions was followed by a rise of lymphocytes, monocytes, and granulocytes exhibiting activated phenotypes in blood. Monocytes showed elevated expression rates of CD11b, CD14 and MHC class II. The rates of CD62L expression on CD8hi T cells in blood and on CD4+ T cells in BALF were also augmented and peaked between 2 and 4 dpi. Notably, CD25 antigen expression was significantly elevated, not only on CD8dim/CD62L+ and CD8-/CD62L+ cells in blood, but also on granulocytes in blood and BALF between 2-3 dpi. From 4 dpi onwards, changes declined and the calves recovered from the infection until 10 dpi. The findings highlight the effectiveness of rapid local and systemic immune reaction and indicate activated T cells, monocytes and granulocytes being essential for rapid eradication of the C. psittaci infection.

Background: Cumulating reports suggest that acute phase proteins (APPs) do not only play a role as systemic inflammatory mediators, but are also expressed in different tissues as local reaction to inflammatory stimuli. The present study aimed to evaluate presence and changes in luminal lung concentrations of the APPs haptoglobin (Hp), lipopolysaccharide binding protein (LBP), C-reactive protein (CRP), and lactoferrin (Lf) in calves with an acute respiratory disease experimentally induced by Chlamydia (C.) psittaci., Results: Intra-bronchial inoculation of the pathogen resulted in a consistent respiratory illness. In venous blood of the infected calves (n = 13), concentrations of plasma proteins and serum LBP were assessed (i) before exposure and (ii) 8 times within 14 days after inoculation (dpi). Increasing clinical illness correlated significantly with increasing LBP-and decreasing albumin concentrations in blood, both verifying a systemic acute phase response. Broncho-alveolar lavage fluid (BALF) was obtained from all 13 calves experimentally infected with C. psittaci at 4, 9 and 14 dpi, and from 6 uninfected healthy calves. Concentrations of bovine serum albumin (BSA), Hp, LBP, CRP and Lf in BALF were determined by ELISA. In infected animals, absolute concentrations of LBP and Hp in BALF correlated significantly with the respiratory score. The quotient [LBP]/[BSA] in BALF peaked significantly in acutely infected animals (4 dpi), showed a time-dependent decrease during the recovery phase (9-14 dpi), and was significantly higher compared to healthy controls. Concentrations of Hp and Lf in BALF as well as [Hp]/[BSA]--and [Lf]/[BSA]-quotients decreased during the study in infected animals, but were never higher than in healthy controls. CRP concentrations and [CRP]/[BSA]-quotient did not express significant differences between infected and healthy animals or during the course of infection., Conclusion: In conclusion, absolute concentrations of LBP in blood and BALF as well as the quotient [LBP]/[BSA] in BALF perfectly paralleled the clinical course of respiratory illness after infection. Beside LBP, the suitability of Hp and Lf as local biomarkers of respiratory infections in cattle and their role in the local response to pathogens is worth further investigation, while CRP does not seem to play a role in local defense mechanisms of the bovine lung.

In this overview, we aim to outline the development of German morphology and comparative anatomy, concentrating on the "Jena school" since it played the crucial role in the growth of these disciplines. We highlight the outstanding role of Johann Wolfgang von Goethe in this process and exemplify the ways he exercised influence on theoretical and applied biosciences. Goethe crucially influenced all methodological currents of comparative anatomy, and laid foundations for both theoretical and applied research programmes. The latter included the whole range of biosciences and medical sciences including veterinary medicine. Goethe's idea to concentrate at a single location both fundamental biological research and practical medical and veterinary studies was crucial for the success of the Jena school.

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.

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.

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.

Chlamydia psittaci is a zoonotic bacterium with a wide host range that can cause respiratory disease in humans and cattle. In the present study, effects of treatment with macrolides and quinolones applied alone or in combination with rifampicin were tested in a previously established bovine model of respiratory C. psittaci infection. Fifty animals were inoculated intrabronchially at the age of 6-8 weeks. Seven served as untreated controls, the others were assigned to seven treatment groups: (i) rifampicin, (ii) enrofloxacin, (iii) enrofloxacin + rifampicin, (iv) azithromycin, (v) azithromycin + rifampicin, (vi) erythromycin, and (vii) erythromycin + rifampicin. Treatment started 30 hours after inoculation and continued until 14 days after inoculation (dpi), when all animals were necropsied. The infection was successful in all animals and sufficient antibiotic levels were detected in blood plasma and tissue of the treated animals. Reisolation of the pathogen was achieved more often from untreated animals than from other groups. Nevertheless, pathogen detection by PCR was possible to the same extent in all animals and there were no significant differences between treated and untreated animals in terms of local (i.e., cell count and differentiation of BALF-cells) and systemic inflammation (i.e. white blood cells and concentration of acute phase protein LBP), clinical signs, and pathological findings at necropsy. Regardless of the reduced reisolation rate in treated animals, the treatment of experimentally induced respiratory C. psittaci infection with enrofloxacin, azithromycin or erythromycin alone or in combination with rifampicin was without obvious benefit for the host, since no significant differences in clinical and pathological findings or inflammatory parameters were detected and all animals recovered clinically within two weeks.

The aim of this study was to evaluate the pathogenicity of Parachlamydia (P.) acanthamoebae as a potential agent of lower respiratory tract disease in a bovine model of induced lung infection. Intrabronchial inoculation with P. acanthamoebae was performed in healthy calves aged 2-3 months using two challenge doses: 10(8) and 10(10) bacteria per animal. Controls received 10(8) heat-inactivated bacteria. Challenge with 10(8) viable Parachlamydia resulted in a mild degree of general indisposition, whereas 10(10) bacteria induced a more severe respiratory illness becoming apparent 1-2 days post inoculation (dpi), affecting 9/9 (100%) animals and lasting for 6 days. The extent of macroscopic pulmonary lesions was as high as 6.6 (6.0)% [median (range)] of lung tissue at 2-4 dpi and correlated with parachlamydial genomic copy numbers detected by PCR, and with bacterial load estimated by immunohistochemistry in lung tissue. Clinical outcome, acute phase reactants, pathological findings and bacterial load exhibited an initial dose-dependent effect on severity. Animals fully recovered from clinical signs of respiratory disease within 5 days. The bovine lung was shown to be moderately susceptible to P. acanthamoebae, exhibiting a transient pneumonic inflammation after intrabronchial challenge. Further studies are warranted to determine the precise pathophysiologic pathways of host-pathogen interaction., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Antimicrobial treatment of chlamydial infections is known to be of limited efficacy. In this study, effects of doxycycline (D), usually the drug of choice, were compared with the combined therapy of doxycycline and rifampicin (R) in a bovine model of respiratory Chlamydia psittaci infection. After intrabronchial inoculation of the pathogen, 30 animals were assigned to five groups (n = 6 per group): untreated controls, monotherapy with D (5 mg kg(-1)day(-1) or 10 mg kg(-1)day(-1)), and combination therapy of D and R (600 mg day(-1)). Treatment continued until day 14 post inoculation (d.p.i.). Clinical signs, inflammatory markers, and pathological findings confirmed successful infection in all animals. Reisolation of the pathogen was possible in 4/6 untreated animals and in 4/12 animals treated with D alone until 4 d.p.i., but in none of the calves of the two D + R groups. Pathogen detection was possible in all animals without significant differences among groups. Severity of disease and time course of its resolution, assessed by clinical and pathological findings as well as inflammatory parameters, were not significantly different between untreated controls and calves receiving D alone or in combination with R. Regardless of the treatment regimen, all groups recovered clinically and cleared the infection within 2 weeks., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

The distinctive and unique features of the avian and mammalian zoonotic pathogen Chlamydia (C.) psittaci include the fulminant course of clinical disease, the remarkably wide host range and the high proportion of latent infections that are not leading to overt disease. Current knowledge on associated diseases is rather poor, even in comparison to other chlamydial agents. In the present paper, we explain and summarize the major findings of a national research network that focused on the elucidation of host-pathogen interactions in vitro and in animal models of C. psittaci infection, with the objective of improving our understanding of genomics, pathology, pathophysiology, molecular pathogenesis and immunology, and conceiving new approaches to therapy. We discuss new findings on comparative genome analysis, the complexity of pathophysiological interactions and systemic consequences, local immune response, the role of the complement system and antigen presentation pathways in the general context of state-of-the-art knowledge on chlamydial infections in humans and animals and single out relevant research topics to fill remaining knowledge gaps on this important yet somewhat neglected pathogen., (Copyright © 2014 The Authors. Published by Elsevier GmbH.. All rights reserved.)

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals. The aim was to establish in vivo sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions. Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.

Background: Chlamydia psittaci (Cp) is a respiratory pathogen capable of inducing acute pulmonary zoonotic disease (psittacosis) or persistent infection. To elucidate the pathogenesis of this infection, a translational large animal model was recently introduced by our group. This study aims at quantifying and differentiating pulmonary dysfunction and acid-base imbalances induced by Cp., Methods: Forty-two calves were grouped in (i) animals inoculated with Cp (n = 21) and (ii) controls sham-inoculated with uninfected cell culture (n = 21). For pulmonary function testing, impulse oscillometry, capnography, and FRC (functional residual capacity) measurement were applied to spontaneously breathing animals. Variables of acid-base status were assessed in venous blood using both (i) traditional Henderson-Hasselbalch and (ii) strong ion approach., Results: Both obstructive and restrictive pulmonary disorders were induced in calves experimentally inoculated with Cp. Although disorders in respiratory mechanics lasted for 8-11 days, the pattern of spontaneous breathing was mainly altered in the period of acute illness (until 4 days post inoculation, dpi). Expiration was more impaired than inspiration, resulting in elevated FRC. Ventilation was characterised by a reduction in tidal volume (-25%) combined with an increased percentage of dead space volume and a significant reduction of alveolar volume by 10%. Minute ventilation increased significantly (+50%) due to a compensatory doubling of respiratory rate. Hyperventilatory hypocapnia at 2-3 dpi resulted in slightly increased blood pH at 2 dpi. However, the acid-base equilibrium was additionally influenced by metabolic components, i.e. the systemic inflammatory response, all of which were detected with help of the strong ion theory. Decreased concentrations of albumin (2-10 dpi), a negative acute-phase marker, resulted in a decrease in the sum of non-volatile weak acids (Atot), revealing an alkalotic effect. This was counterbalanced by acidic effects of decreased strong ion difference (SID), mediated by the interplay between hypochloraemia (alkalotic effect) and hyponatraemia (acidic effect)., Conclusions: This bovine model was found to be suitable for studying pathophysiology of respiratory Cp infection and may help elucidating functional host-pathogen interactions in the mammalian lung.

Control of paratuberculosis in livestock is hampered by the low sensitivity of established direct and indirect diagnostic methods. Like other bacteria, Mycobacterium avium subsp. paratuberculosis (MAP) emits volatile organic compounds (VOCs). Differences of VOC patterns in breath and feces of infected and not infected animals were described in first pilot experiments but detailed information on potential marker substances is missing. This study was intended to look for characteristic volatile substances in the headspace of cultures of different MAP strains and to find out how the emission of VOCs was affected by density of bacterial growth. One laboratory adapted and four field strains, three of MAP C-type and one MAP S-type were cultivated on Herrold's egg yolk medium in dilutions of 10(-0), 10(-2), 10(-4) and 10(-6). Volatile substances were pre-concentrated from the headspace over the MAP cultures by means of Solid Phase Micro Extraction (SPME), thermally desorbed from the SPME fibers and separated and identified by means of GC-MS. Out of the large number of compounds found in the headspace over MAP cultures, 34 volatile marker substances could be identified as potential biomarkers for growth and metabolic activity. All five MAP strains could clearly be distinguished from blank culture media by means of emission patterns based on these 34 substances. In addition, patterns of volatiles emitted by the reference strain were significantly different from the field strains. Headspace concentrations of 2-ethylfuran, 2-methylfuran, 3-methylfuran, 2-pentylfuran, ethyl acetate, 1-methyl-1-H-pyrrole and dimethyldisulfide varied with density of bacterial growth. Analysis of VOCs emitted from mycobacterial cultures can be used to identify bacterial growth and, in addition, to differentiate between different bacterial strains. VOC emission patterns may be used to approximate bacterial growth density. In a perspective volatile marker substances could be used to diagnose MAP infections in animals and to identify different bacterial strains and origins.

The respiratory pathogen Chlamydia psittaci naturally occurs in bovine herds and was recently shown to impair calf health in a dose-dependent manner. The aim of this study was to determine whether the functional consequences and immunological reactions of infection were dose related by quantifying the consequences of acute respiratory chlamydial infection on respiratory signs, disturbances of pulmonary gas exchange, response of the innate immune system, and acute-phase reaction. Fourteen calves were challenged intrabronchially with different C. psittaci doses (from 10(6) to 10(9)inclusion-forming units (ifu) per animal). Ten controls received either UV-inactivated chlamydiae or cell culture medium. Compared to the controls, all animals challenged with live C. psittaci developed hypoxaemia linked to reduced haemoglobin oxygen saturation, increased alveolar-arterial oxygen partial pressure difference (A-aO2) and pulmonary shunt, with symptoms following a dose-dependent pattern. Increases in lipopolysaccharide-binding protein (LBP) and leukocytes were also dose-dependent and accompanied by a regenerative left shift in neutrophil granulocytes. With the exception of LBP, which reflected the load of chlamydial cell components in the host, pathophysiological reactions were only detected in calves challenged with viable chlamydiae. These results indicate that the pathophysiological consequences of respiratory C. psittaci infections are strongly dependent on the challenge dose of chlamydiae. For further studies, challenge doses between 10(6) and 10(8)ifu/calf are recommended., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Chlamydia (C.) psittaci is the causative agent of psittacosis, a zoonotic disease in birds and man. In addition, C. psittaci has been repeatedly found in domestic animals and is, at least in calves, also able to induce respiratory disease. Knowledge about transmission routes in cattle herds is still deficient, and nothing is known about differences in host response after either experimental or natural exposure to C. psittaci. Therefore, our recently developed respiratory infection model was exploited to evaluate (i) the presence of the pathogen in blood, excretions and air, (ii) the possibility of transmission and (iii) clinical symptoms, acute phase and immune response until 5 weeks after exposure. In this prospective study, intrabronchial inoculation of 10(8) inclusion-forming units of C. psittaci (n = 21 calves) led to reproducible acute respiratory illness (of approximately one week), accompanied by a systemic inflammatory reaction with an innate immune response dominated by neutrophils. Excretion and/or exhalation of the pathogen was sufficient to transmit the infection to naïve sentinel calves (n = 3) co-housed with the infected animals. Sentinel calves developed mild to subclinical infections only. Notably, excretion of the pathogen, predominantly via feces, occurred more frequently in animals naturally exposed to C. psittaci (i.e. sentinels) as compared to experimentally-inoculated calves. The humoral immune response was generally weak, and did not emerge regularly following experimental infection; however, it was largely absent after naturally acquired infection.

Prenatal maternal stress (PMS) programs dysregulation of the hypothalamus-pituitary-adrenal axis (HPAA) in postnatal life, though time periods vulnerable to PMS, are still unclear. We evaluated in pregnant sheep the effect of PMS during early gestation [30-100 days of gestation (dGA); term is 150 dGA] or late gestation (100-120 dGA) on development of fetal HPAA function. We compared the effects of endogenous cortisol with synthetic glucocorticoid (GC) exposure, as used clinically to enhance fetal lung maturation. Pregnant sheep were exposed to repeated isolation stress twice per week for 3 h in a separate box with no visual, tactile, or auditory contact with their flock-mates either during early (n = 7) or late (n = 7) gestation. Additional groups received two courses of betamethasone (BM; n = 7; 2 × 110 μg kg(- 1) body weight, 24 h apart) during late gestation (106/107 and 112/113 dGA, n = 7) or acted as controls (n = 7). Fetal cortisol responses to hypotensive challenge, a physiological fetal stressor, were measured at 112 and 129 dGA, i.e. before and during maturation of the HPAA. Hypotension was induced by fetal infusion of sodium nitroprusside, a potent vasodilator. At 112 dGA, neither PMS nor BM altered fetal cortisol responses. PMS, during early or late gestation, and BM treatment increased fetal cortisol responses at 129 dGA with the greatest increase achieved in stressed early pregnant sheep. Thus, development of the HPAA is vulnerable to inappropriate levels of GCs during long periods of fetal life, whereas early gestation is most vulnerable to PMS.

This study aimed to establish and evaluate a bovine respiratory model of experimentally induced acute C. psittaci infection. Calves are natural hosts and pathogenesis may resemble the situation in humans. Intrabronchial inoculation of C. psittaci strain DC15 was performed in calves aged 2-3 months via bronchoscope at four different challenge doses from 10(6) to 10(9) inclusion-forming units (ifu) per animal. Control groups received either UV-inactivated C. psittaci or cell culture medium. While 10(6) ifu/calf resulted in a mild respiratory infection only, the doses of 10(7) and 10(8) induced fever, tachypnea, dry cough, and tachycardia that became apparent 2-3 days post inoculation (dpi) and lasted for about one week. In calves exposed to 10(9) ifu C. psittaci, the respiratory disease was accompanied by severe systemic illness (apathy, tremor, markedly reduced appetite). At the time point of most pronounced clinical signs (3 dpi) the extent of lung lesions was below 10% of pulmonary tissue in calves inoculated with 10(6) and 10(7) ifu, about 15% in calves inoculated with 10(8) and more than 30% in calves inoculated with 10(9) ifu C. psittaci. Beside clinical signs and pathologic lesions, the bacterial load of lung tissue and markers of pulmonary inflammation (i.e., cell counts, concentration of proteins and eicosanoids in broncho-alveolar lavage fluid) were positively associated with ifu of viable C. psittaci. While any effect of endotoxin has been ruled out, all effects could be attributed to infection by the replicating bacteria. In conclusion, the calf represents a suitable model of respiratory chlamydial infection. Dose titration revealed that both clinically latent and clinically manifest infection can be reproduced experimentally by either 10(6) or 10(8) ifu/calf of C. psittaci DC15 while doses above 10(8) ifu C. psittaci cannot be recommended for further studies for ethical reasons. This defined model of different clinical expressions of chlamydial infection allows studying host-pathogen interactions.

Epidemiological data indicate that infection of cattle with chlamydiae such as Chlamydophila (C.) pecorum, C. abortus, C. psittaci and Chlamydia suis, is ubiquitous with mixed infections occurring frequently. The apparent lack of association between infection and clinical disease has resulted in debate as to the pathogenic significance of these organisms, and their tendency to sub-clinical and/or persistent infection presents a challenge to the study of their potential effects. However, recent evidence indicates that chlamydial infections have a substantial and quantifiable impact on livestock productivity with chronic, recurrent infections associated with pulmonary disease in calves and with infertility and sub-clinical mastitis in dairy cows. Data also suggest these infections manifest clinically when they coincide with a number of epidemiological risk factors. Future research should: (1) use relevant animal models to clarify the pathogenesis of bovine chlamydioses; (2) quantify the impact of chlamydial infection at a herd level and identify strategies for its control, including sub-unit vaccine development; and (3) evaluate the zoonotic risk of bovine chlamydial infections which will require the development of species-specific serodiagnostics., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

The effect of short-term antimicrobial treatment on natural excretion of Chlamydia suis in rectal swabs and C. suis and Chlamydophila psittaci in nasal swabs was investigated in 47 clinically normal piglets by quantitative real-time PCR. Pigs were treated IM with 4 mg/kg enrofloxacin for 5 days (n = 22) or 2.5mg/kg enrofloxacin for 3 days followed by 100mg/mL tiamulin (n = 25). Antimicrobial treatment reduced the number of pigs positive for chlamydiae and the quantity of chlamydial DNA in positive swabs for a few days, but chlamydial excretion recurred in both groups. Short-term antimicrobial treatment at dosages recommended for treatment of other bacterial infections in pig herds was not effective in eliminating naturally occurring subclinical chlamydial infection in pigs., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Pulmonary dysfunction was evaluated in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV, isolate VR-2332) and compared to clinical and pathological findings. Infected pigs developed fever, reduced appetite, respiratory distress and dullness at 9 days post-inoculation (dpi). Non-invasive pulmonary function tests using impulse oscillometry and rebreathing of test gases (He, CO) revealed peripheral airway obstruction, reduced lung compliance and reduced lung CO-transfer factor. PRRSV-induced pulmonary dysfunction was most marked at 9-18 dpi and was accompanied by a significantly increased respiratory rate and decreased tidal volume. Expiration was affected more than inspiration. On histopathological examination, multifocal areas of interstitial pneumonia (more severe and extensive at 10 dpi than 21 dpi) were identified as a possible structural basis for reduced lung compliance and gas exchange disturbances., (Copyright © 2009 Elsevier Ltd. All rights reserved.)

This study characterises the acid-base abnormalities in pigs experimentally infected with Chlamydia suis (Henderson-Hasselbalch equation and Constable's simplified strong ion equation). Eight pigs were challenged with the respiratory pathogen C. suis and four pigs served as non-infected controls. Pigs were monitored from 7 days before challenge to 8 days post-inoculation. Clinical examination was performed twice daily and venous blood samples were collected every two days. Blood-gas analysis, haemoxymetry, serum biochemical analysis and electrophoresis were performed in order to characterise the acid-base derangement. Aerosol challenge with C. suis resulted in severe acid-base disturbance characterised by acute respiratory acidosis and strong ion (metabolic) acidosis secondary to anaerobic metabolism and hyper L-lactataemia. Maximal changes were seen at day 3 post-inoculation when severe clinical signs of respiratory dysfunction were evident. The results of the study provide new information regarding the pathophysiology of respiratory infection caused by C. suis and the applicability and diagnostic utility of different approaches for assessing acid-base status in pigs., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Exhaled breath condensate (EBC) describes any sample collected by cooling exhaled breath. Because the method of condensate collection is simple, non-invasive, repeatable and does not necessarily require patient cooperation, EBC is not only an interesting, but also challenging, biological sample. Despite a period of EBC research lasting for more than 15 years, there are still many open questions with respect to EBC collection and analysis, and many biomarkers are still awaiting careful validation. In veterinary research, EBC collection has been described in conscious animals including calves, pigs, horses, cats and dogs. Numerous studies performed in these domestic animals not only contributed substantially to the current knowledge about the potentials of EBC-based diagnoses but also demonstrated pitfalls in EBC collection, analysis and interpretation. This review summarizes information about the collection of EBC and the interpretation of EBC results, particularly with respect to proteins, leukotrienes, hydrogen peroxide, urea, ammonia and pH. Published data emphasize the need to standardize approaches to produce reproducible EBC data. Quantifying the concentration of the EBC component of interest exhaled in a defined volume of exhaled breath (instead of comparing concentrations of this component analysed in liquid EBC) is an important step of standardization that might help to overcome methodological limitations deriving from the EBC collection process. Although information is based on domestic animal studies, it contributes to the general understanding in EBC research-independent of any particular mammalian species-and opens new perspectives for further studies.

Background: Zoonoses were already a subject of intense interest even before the SARS and avian influenza epidemics arose. For many years, chlamydiae have been hypothesized to be important zoonotic pathogens, because of their wide distribution and their infectious cycle. This article provides an overview of the current state of knowledge on this subject., Methods: The authors present a selective review of the literature as well as their own findings., Results: The scientific knowledge of the distribution and infectious cycle of chlamydiae is still inadequate. The laboratory diagnosis of chlamydial zoonoses remains unsatisfactory in both human and veterinary medicine, as there are no commercially available sensitive and species-specific tests. Acute chlamydial infections are usually treated with macrolides, tetracyclines, or quinolones. Persistent varieties are not covered by standard therapy., Conclusions: There is a considerable need for research on chlamydial infections, especially with regard to the diagnosis and treatment of persistent varieties.

This review focuses on the availability, advantages and non-advantages of asthma models in non-laboratory animals (cats, dogs, sheep, swine, cattle, horses, and monkey). Physiology and pathophysiology of the respiratory system as well as methodological aspects differ significantly between species and must be taken into account before evaluating the usefulness of a single species to serve as model for either asthma or chronic airway obstruction. Allergic asthma models have been described in cats, dogs, pigs, sheep, and monkeys. Among these species, the feline one is of particular interest because cats spontaneously develop idiopathic asthma. Currently available allergic feline models are well characterized with respect to lung function, bronchial responsiveness, airway inflammation and lung morphology (remodeling). Other species lacking for collateral airways (i.e. porcine and bovine lungs) are most sensitive to functional consequences of airway obstruction and are therefore suitable to study any obstructive lung disease. Animals of body weights comparable to humans (pigs, sheep, calves) offer the possibility to evaluate pulmonary functions using the same principles and techniques that are applicable to either children or adults during spontaneous breathing (generating lung function data in a directly comparable range). Despite the known disadvantages of being expensive and time consuming and despite limited availability of immunological or molecular tools, large animal models offer the great potential to perform long-term functional studies allowing a simultaneous within-subject approach of functional, inflammatory and morphological changes. This may add valuable information to the present knowledge about the complexity of asthma or other chronic airway diseases.

This veterinary study is aimed at further standardization of H(2)O(2) and pH measurements in exhaled breath condensate (EBC). Data obtained in the study provide valuable information for many mammalian species including humans, and may help to avoid general pitfalls in interpretation of EBC data. EBC was sampled via the 'ECoScreen' in healthy calves (body weight 63-98 kg). Serum samples and condensates of ambient (indoor) air were collected in parallel. In the study on H(2)O(2), concentrations of H(2)O(2) in EBC, blood and ambient air were determined with the biosensor system 'ECoCheck'. In EBC, the concentration of H(2)O(2) was found to be dependent on food intake and increased significantly in the course of the day. Physiologically, lowest H(2)O(2) concentrations at 06:00 varied within the range 138-624 nmol l(-1) EBC or 0.10-0.94 nmol per 100 l exhaled breath and individual concentrations were significantly different indicating a remarkable intersubject variability. Highly reproducible results were seen within each subject (three different days within 4 weeks). No correlation existed between H(2)O(2) concentrations in EBC and blood, and EBC-H(2)O(2) was not influenced by variables of spontaneous breathing. Further results confirmed that standardization of H(2)O(2) measurements in EBC requires (1) the re-calculation of the concentration exhaled per 100 l exhaled breath (because the analyzed concentration in the liquid condensate underlies multiple methodological sources of variability given by the collection process), and (2) subtracting the concentration of inspired indoor H(2)O(2). In the study on pH use of the ISFET electrode (Sentron, the Netherlands) and a blood gas analyzer ABL 550 (Radiometer, Denmark) led to comparable results for EBC-pH (r=0.89, R(2)=79.3%, p

This study was aimed at evaluating the pathophysiology of pulmonary dysfunctions and inflammatory consequences of an acute respiratory chlamydial infection induced experimentally in conventionally raised pigs (aged 39-44 days). Eight animals were exposed to Chlamydia suis (C. suis) and four non-infected animals served as controls. The total observation period was from seven days before challenge to seven days post exposure. While non-infected control pigs did not exhibit any clinical symptoms, animals exposed to C. suis developed fever and were severely respiratory distressed within the first week after exposure. After C. suis infection, pulmonary dysfunctions were characterised by a significant decrease in the diffusion capacity of the lung (i.e. transfer factor of the lung for carbon monoxide; TL CO), a significant increase in the functional residual capacity (FRC), and significant changes in the pattern of ventilation (respiratory rate increased while the tidal volume decreased). In exhaled breath condensate (EBC), leukotriene B(4) (LTB(4)) and interleukin 6 (IL-6) showed a tendency to increase after infection. In the broncho-alveolar lavage fluid (BALF) of C. suis infected pigs, the activity of matrix metalloprotease 9 (MMP-9) was found to be increased compared to controls. BALF cytology was characterised by increased numbers of granulocytes and activated lymphocytes. Pulmonary inflammation in infected pigs was confirmed by post mortem histology. A prominent dissemination of chlamydial bodies in the lung was accompanied by an influx of macrophages, granulocytes and activated T-cells. Data obtained in this study provide new insight into the pathogenesis of acute respiratory chlamydial infections in pigs.

To assess long-term effects of naturally occurring infection with Chlamydophila spp. on animal health, 25 calves were grouped according to their chlamydial carrier status and checked for health parameters from 2 to 7 months of age. Monthly PCR testing revealed persistent or frequently recurring infections with Chlamydophila pecorum and Chlamydophila abortus in Group 2 (Chl+, n=13), but not in Group 1 (Chl-, n=12). Despite the absence of any clinical illness, calves in Group 2 showed significantly higher body temperatures (subfebrile), lower bodyweights, reduced serum iron concentrations, lower total haemoglobin and haematocrit values. Counting and flow cytometric differentiation of peripheral white blood cells revealed a general decrease in leukocytes in Group 2. At necropsy, follicular bronchiolitis was found in 10/13 calves in Group 2 but in none of Group 1, and the weight of pharyngeal tonsils was significantly higher in Group 2. In conclusion, naturally occurring infections with Chlamydophila species in calves were found to be associated with chronic effects on animal health at a subclinical level.

This study was aimed at evaluating functional and inflammatory consequences of persistent chlamydial infections on the respiratory system in clinically inconspicuous calves aged 2-7 months. Thirteen calves persistently infected with Chlamydophila (C.) abortus and/or C. pecorum (Chl+) were compared to 12 calves without chlamydial infections (Chl-). In order to evaluate lung function, 36 non-invasive impulse oscillometry tests were performed per animal within 6 months. The group of chronically infected animals was distinguished by significantly higher peripheral airway resistance (indicating peripheral airway obstruction), significantly higher respiratory rates, and significantly higher minute volumes of ventilation. At the age of seven months, all calves were necropsied, broncho-alveolar lavage fluid (BALF) was obtained ex vivo, and lungs were examined histologically. Significantly higher concentrations of total protein and 8-iso-prostane (8-IP), as well as higher activities of matrix metalloprotease 2 were measured in BALF samples of Chl+ calves. Histologically, markedly activated bronchus-associated lymphoid tissue (BALT) causing partial obstruction of bronchiolar lumina was found in the apical pulmonary lobes of Chl+ calves. Chlamydial DNA was detected in the lung tissue of 7 out of 13 Chl+ calves by real-time PCR. In conclusion, respiratory chlamydial infection appeared to be associated with chronic inflammation of the lungs and airways. Despite the lack of clinical symptoms, pulmonary dysfunctions persisted in calves until the age of seven months. Data obtained in this study provide new insight illustrating the impact of nearly ubiquitous subclinical infections on the respiratory system.