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Mycobacteriumavium(M.a.) subsp. paratuberculosis(MAP)--the etiologic agent of Johne's disease--affects cattle, sheep, and other ruminants worldwide. To decipher phenotypic differences among sheep and cattle strains (belonging to MAP-S [Type-I/III], respectively, MAP-C [Type-II]), comparative genome analysis needs data from diverse isolates originating from different geographic regions of the world. This study presents the so far best assembled genome of a MAP-S-strain: Sheep isolate JIII-386 from Germany. One newly sequenced cattle isolate (JII-1961, Germany), four published MAP strains of MAP-C and MAP-S from the United States and Australia, and M. a. subsp. hominissuis (MAH) strain 104were used for assembly improvement and comparisons. All genomes were annotated by BacProt and results compared with NCBI (National Center for Biotechnology Information) annotation. Corresponding protein-coding sequences (CDSs) were detected, but also CDSs that were exclusively determined by either NCBI or BacProt. A new Shine-Dalgarno sequence motif (5'-AGCTGG-3') was extracted. Novel CDSs including PE-PGRS family protein genes and about 80 noncoding RNAs exhibiting high sequence conservation are presented. Previously found genetic differences between MAP-types are partially revised. Four of ten assumed MAP-S-specific large sequence polymorphism regions (LSPSs) are still present in MAP-C strains; new LSPSs were identified. Independently of the regional origin of the strains, the number of individual CDSs and single nucleotide variants confirms the strong similarity of MAP-C strains and shows higher diversity among MAP-S strains. This study gives ambiguous results regarding the hypothesis that MAP-S is the evolutionary intermediate between MAH and MAP-C, but it clearly shows a higher similarity of MAP to MAH than to Mycobacterium intracellulare. [ABSTRACT FROM AUTHOR]

In weaners, combined administration of fumonisin, deoxynivalenol and T2 together with ochratoxin A in quantities expected to be present in feeds of central European origin resulted, as a rule, in changes identical to those observed after single administration of ochratoxin A. Such reactions were partly compensated. Synergistic amplification of immunosuppressive changes due to the simultaneous intake of these mycotoxins in low concentrations is not to be expected. [ABSTRACT FROM AUTHOR]

Even in subtoxic amounts, the mycotoxin, ochratoxin A, produced immunomodulation in weaner pigs in a dose-dependent mode. In addition to increased counts of total leukocytes and neutrophils in the blood, reduced lymphocyte levels were observed. There was a striking increase in the counts of eosinophils and of apoptotic phagocytes. Functionally, there was a predominance of the production of reactive oxygen radicals in whole blood, reduced phagocytosis performance and reduced expression of a swine-specific surface marker (SWC1) on lymphocytes. In a few single experiments, clinical manifestations could be demonstrated. Lung clearance and the degree of severity of experimental pneumonia as well as cutaneous hypersensitization may be influenced by ochratoxin A. [ABSTRACT FROM AUTHOR]

Objective: In Germany, only few data on the current distribution of paratuberculosis in sheep and goat flocks is available. The present study provides an overview of the distribution of Mycobacterium avium ssp. paratuberculosis (MAP) in 165 Thuringian sheep and goat flocks. Also, the study investigated the association between the MAP status of the flock and herd specific factors as well as the association between the individual measured value of ELISA and animal specific factors like age, body condition, sex, and animal species., Material and Methods: To investigate the prevalence of MAP, serum samples from 2550 sheep and 1171 goats from 165 flocks (flock size 2 to 2879 animals) were serologically examined for MAP antibodies in 2021. Additionally, 1 to 6 environmental faecal samples were collected from every flock depending on the flock size. They were examined for the presence of MAP by using both bacteriological cultivation and a commercially available real-time-PCR., Results: MAP antibodies were detected in 41 sheep (1.6%) and 29 goats (2.5%), which accounts to a detection of MAP antibodies in 20.6% of the 165 flocks (on herd level). The symptoms of paratuberculosis, weight loss with preserved appetite and altered fecal consistency, were observed in only four of the flocks. A positive association was identified between the detection of MAP or MAP-specific antibodies in a flock and flock size, as well as positive association between the measured value in the Elisa (s/p ratio) and the age of the animal. Furthermore, an association between an increasing s/p ratio of the ELISA and a decreasing body condition was found., Conclusion and Clinical Relevance: Given what is known about the distribution of paratuberculosis in small ruminants, this disease should always be considered as a possible cause of weight loss and diarrhea. In case of high within-herd prevalence herd-specific control measures should be considered. In serological herd monitoring, animals with poor body condition should preferably be included in the sample, as the probability of being able to identify MAP positive animals is higher here., 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.)

Despite its potential for early diagnosis of Mycobacterium avium subsp. paratuberculosis (MAP) infection, the IFN-γ release assay is not used routinely, because of low specificity of the established crude antigen preparation Johnin (PPDj). Limited data are available assessing the potential of MAP-derived protein and lipopeptide antigens to replace PPDj in assays for goats, while cattle and sheep have been studied more extensively. Furthermore, MAP infection is claimed to interfere with the diagnosis of bovine tuberculosis when other crude antigen preparations (PPDb, PPDa) are applied. In this study, the diagnostic potential of MAP-derived recombinant protein antigens, synthetic MAP lipopentapeptides and of Mycobacterium bovis- specific peptide cocktails was assessed compared to crude mycobacterial antigen preparations in experimentally infected goats. Goats were inoculated with MAP, or Mycobacterium avium subsp. hominissuis (MAH) as surrogate for environmental mycobacteria, non-exposed animals served as controls. Mycobacterium avium Complex-specific antibody and PPDj-induced IFN-γ responses were monitored in vivo . Infection status was assessed by pathomorphological findings and bacteriological tissue culture at necropsy 1 year after inoculation. The IFN-γ response to 13 recombinant protein antigens of MAP, two synthetic MAP lipopentapeptides and three recombinant peptide cocktails of Mycobacterium bovis was investigated at three defined time points after infection. At necropsy, MAP or MAH infection was confirmed in all inoculated goats, no signs of infection were found in the controls. Antibody formation was first detected 3-6 weeks post infection (wpi) in MAH-inoculated and 11-14 wpi in the MAP-inoculated goats. Maximum PPDj-induced IFN-γ levels in MAH and MAP exposed animals were recorded 3-6 and 23-26 wpi, respectively. Positive responses continued with large individual variation. Antigens Map 0210c, Map 1693c, Map 2020, Map 3651cT(it), and Map 3651c stimulated increased whole blood IFN-γ levels in several MAP-inoculated goats compared to MAH inoculated and control animals. These IFN-γ levels correlated with the intensity of the PPDj-induced responses. The two synthetic lipopentapeptides and the other MAP-derived protein antigens had no discriminatory potential. Stimulation with Mycobacterium bovis peptide cocktails ESAT6-CFP10, Rv3020c, and Rv3615c did not elicit IFN-γ production. Further work is required to investigate if test sensitivity will increase when mixtures of the MAP-derived protein antigens are applied., Competing Interests: HV is co-inventor on patents protecting the application of Rv3615c for the diagnosis of tuberculosis. FB and SB are co-inventors on patents WO 2009/053844 A1 (2008) and WO 2018/115183 (2017). 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 © 2021 Köhler, Liebler-Tenorio, Hughes, Stevenson, Bakker, Willemsen, Bay, Ganneau, Biet and Vordermeier.)

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.

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

Five commercially available ELISA tests for the detection of antibodies against Mycobacterium avium subsp. paratuberculosis in bovine serum were evaluated at the individual animal level using sera from 286 paratuberculosis-free and 110 paratuberculosis-infected dairy cattle. Sensitivity (Se) and specificity (Sp) of the tests were estimated after determination of the cut-off dtheta by TG-ROC analysis or using the cut-off values recommended by the manufacturers, respectively. When the dtheta cut-off values were applied, the five ELISA tests showed sub-optimal Se and Sp. Adopting the cut-offs recommended by the manufacturers, the Sp of four of the five ELISA increased, two tests reaching Sp > or = 99.0%. Test sensitivity clearly depended on the disease state of the animals examined. Se was significantly higher in clinically diseased than in latently infected dairy cattle. Calculation of the positive and negative predictive values indicated that, depending on the test, a considerable proportion of false positive and false negative results have to be expected. Therefore, the suitability of antibody detection for the diagnosis of paratuberculosis in individual animals is questioned.

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease and is endemic to the national cattle herds of many countries. Because of the very low level of genetic heterogeneity of this organism, it is difficult to select a workable procedure for strain differentiation at a resolution sufficient to investigate epidemiological links between herds or different ruminant species and the suggested zoonotic potential of M. avium subsp. paratuberculosis for Crohn's disease. Analysis of restriction fragment length polymorphisms (RFLPs) based on the insertion element IS900 (IS900 RFLP) with four restriction enzymes and 10 markers of specific mycobacterial interspersed repetitive units (MIRUs) and variable-number tandem repeats (VNTRs) was applied to 71 bovine M. avium subsp. paratuberculosis isolates originating from 14 herds from different regions in Germany. Among these isolates, all of which belonged to the M. avium subsp. paratuberculosis type II group, 17 genotypes were detected by IS900 RFLP and consisted of a combination of seven BstEII, eight PstI, nine PvuII, and four BamHI restriction patterns. Novel RFLP types were found. The diversity of the M. avium subsp. paratuberculosis isolates inside the herds was different depending on the frequency of animal purchase. The results of typing by IS900 RFLP and MIRU-VNTR analyses were not associated. Fifteen MIRU-VNTR patterns were identified with a discriminatory index of 0.905. The most common BstEII-based IS900 RFLP type, type C1 (72%), was subdivided into 14 types by MIRU-VNTR analysis. A combination of fingerprinting and PCR-based techniques resulted in 24 M. avium subsp. paratuberculosis genotypes and achieved a discriminatory index of 0.997. By using only BstEII and PstI digestion together with typing by MIRU-VNTR analysis, a discriminatory index of 0.993 was achieved. This is high enough to support epidemiological studies on a national as well as a global scale.

Paratuberculosis or Johne's Disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a notifiable disease in Germany which produces enormous economical losses in dairy farms. At present,there is no confirmed data about the actual number of infected livestock herds in Germany. A countrywide monitoring program to evaluate the prevalence in dairy herds would only be economically feasible on the basis of bulk milk testing. In this study, we evaluated two ELISA test kits (SVANOVIR Ptb-ELISA, IDEXX-M.pt. Milk test kit) for the detection of antibodies against MAP in bulk milk. First, the Paratuberculosis-status of the herd derived from the history of the farm was used as a gold standard. Paratuberculosis-negative farms were tested negative with each test, but paratuberculosis-positive or Paratuberculosis-serologically-positive farms were detected only in one case (Svanovir) or three cases (IDEXX), respectively. Even if inconclusive results are counted as positive, 82.9 % (Svanovir) or 80 % (IDEXX) of the paratuberculosis-positive or serologically paratuberculosis positive farms were not detected. Nevertheless, a re-validation of both ELISAs by means of ROC and TG-ROC analyses was attempted by searching for ideal cut-offs, optimised for bulk milk. If a high specificity was selected, no acceptable sensitivity could be reached. The best results were obtained using a sensitivity of 32.3 % at a specificity of 100 % (Svanovir). With a small change of the cut-off value, the sensitivity increased to still 57 %, but this reduced the specificity to 67 %. Similar results were obtained with the IDEXX-ELISA. We then evaluated the Svanovir-ELISA for the detection of bulk milk samples on the basis of the current paratuberculosis prevalence within 69 dairy herds from Rhineland-Palatinate using individual milk samples.When the bulk milk samples were tested in two different laboratories using the same ELISA, considerable differences in the results became evident. Nearly all samples were tested with a higher relative test result in one laboratory, which often led to differences in the classification of the prevalence levels. The estimated within-herd seroprevalences ranged between 0 % and 37 %. There was little agreement between the historical paratuberculosis herd status and the within-herd prevalence in milk serum, as reflected in a kappa-index of 0.146. To determine the sensitivity and specificity of the bulk milk ELISA by ROC and TG-ROC analysis, 116 bulk milk samples were used that had been obtained from the 69 dairy herds participating in the study. The optimal ratio of sensitivity (81 %) and specificity (77 %) relative to a "gold standard" was obtained when the cut-off was set at the 10 % level. These values for sensitivity and specificity were better than those obtained in an evaluation of the same ELISA in which the historical Paratuberculosis herd-status was used as a "gold standard." The results of this study question the suitability of the available ELISAs for bulk milk testing. Taking into account that the Svanovir-ELISA for individual milk samples has a sensitivity of 60 96% relative to the blood serum variant of the test, and that the latter has also a limited sensitivity due to the pathogenesis of paratuberculosis, the available test systems examined in this Study do not seem to be suitable for herd diagnosis by using bulk milk samples.

The Friedrich-Loeffler-Institut, founded in 1910 by Friedrich Loeffler, the discoverer of the first animal virus, foot-and-mouth disease virus, is the oldest virological research facility in the world. Beyond viruses, its area of competence has significantly expanded since its foundation and now also covers bacterial, parasitic and prion diseases of livestock, poultry and aquatic animals. Presently located at four sites within Germany (Insel Riems, Jena,Tübingen,Wusterhausen) the tasks of the institute as delineated in the Animal Disease Act encompass research on infectious animal diseases including zoonoses, import/export examinations, epidemiological studies in case of outbreaks of notifiable animal diseases, acting as reference laboratory for notifiable animal diseases and nationwide quality management of diagnosis of notifiable animal diseases. It is obliged to publish and maintain up-to-date diagnostic regimes for notifiable animal diseases, and it publishes a yearly report on animal health in Germany. With the increasing importance of infectious diseases of animals, in particular those potentially harmful to man (zoonoses), the Friedrich-Loeffler-Institut will be moving into new facilities including laboratories and animal facilities up to the highest biosafety level at its main site Insel Riems on the occasion of its 100th anniversary.