Paratuberculosis, a chronic disease affecting ruminant livestock, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has direct and indirect economic costs, impacts animal welfare and arouses public health concerns. In a survey of 48 countries we found paratuberculosis to be very common in livestock. In about half the countries more than 20% of herds and flocks were infected with MAP. Most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the major objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. An holistic approach across all ruminant livestock industries and long-term commitment is required for control of paratuberculosis.

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.

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

Analysis of volatile organic compounds (VOCs) is a novel approach to accelerate bacterial culture diagnostics ofMycobacterium aviumsubsp.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.

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