Safety and Immunogenicity of Recombinant Bacille Calmette-Guérin Strain VPM1002 and Its Derivatives in a Goat Model.

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.

Vaccine-Induced Subcutaneous Granulomas in Goats Reflect Differences in Host-Mycobacterium Interactions between BCG- and Recombinant BCG-Derivative Vaccines.

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.

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

Paratuberculosis is an important disease of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP). Early detection is crucial for successful infection control, but available diagnostic tests are still dissatisfying. Methods allowing a rapid, economic, and reliable identification of animals or herds affected by MAP are urgently required. This explorative study evaluated the potential of volatile organic compounds (VOCs) to discriminate between cattle with and without MAP infections. Headspaces above fecal samples and alveolar fractions of exhaled breath of 77 cows from eight farms with defined MAP status were analyzed in addition to stable air samples. VOCs were identified by GC-MS and quantified against reference substances. To discriminate MAP-positive from MAP-negative samples, VOC feature selection and random forest classification were performed. Classification models, generated for each biological specimen, were evaluated using repeated cross-validation. The robustness of the results was tested by predicting samples of two different sampling days. For MAP classification, the different biological matrices emitted diagnostically relevant VOCs of a unique but partly overlapping pattern (fecal headspace: 19, alveolar gas: 11, stable air: 4-5). Chemically, relevant compounds belonged to hydrocarbons, ketones, alcohols, furans, and aldehydes. Comparing the different biological specimens, VOC analysis in fecal headspace proved to be most reproducible, discriminatory, and highly predictive.

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

Volatile organic compounds (VOCs) emitted from in vitro cultures may reveal information on species and metabolism. Owing to low nmol L-1 concentration ranges, pre-concentration techniques are required for gas chromatography-mass spectrometry (GC-MS) based analyses. This study was intended to compare the efficiency of established micro-extraction techniques - solid-phase micro-extraction (SPME) and needle-trap micro-extraction (NTME) - for the analysis of complex VOC patterns. For SPME, a 75 µm Carboxen®/polydimethylsiloxane fiber was used. The NTME needle was packed with divinylbenzene, Carbopack X and Carboxen 1000. The headspace was sampled bi-directionally. Seventy-two VOCs were calibrated by reference standard mixtures in the range of 0.041-62.24 nmol L-1 by means of GC-MS. Both pre-concentration methods were applied to profile VOCs from cultures of Mycobacterium avium ssp. paratuberculosis. Limits of detection ranged from 0.004 to 3.93 nmol L-1 (median = 0.030 nmol L-1 ) for NTME and from 0.001 to 5.684 nmol L-1 (median = 0.043 nmol L-1 ) for SPME. NTME showed advantages in assessing polar compounds such as alcohols. SPME showed advantages in reproducibility but disadvantages in sensitivity for N-containing compounds. Micro-extraction techniques such as SPME and NTME are well suited for trace VOC profiling over cultures if the limitations of each technique is taken into account.

Evaluation of different diagnostic methods for the detection of Mycobacterium avium subsp. paratuberculosis in boot swabs and liquid manure samples.

BACKGROUND: Environmental sampling based on boot swabs and/or liquid manure samples is an upcoming strategy for the identification of paratuberculosis (paraTB) positive herds, but only limited data are available regarding the diagnostic performance of molecular detection methods (qPCR) versus faecal culture (FC) for this purpose. In the present study, the test characteristics of two different qPCR protocols (A and B) and a standardized FC protocol, for the detection of Mycobacterium avium subsp. paratuberculosis in boot swabs and liquid manure samples were evaluated. RESULTS: In 19 paraTB unsuspicious and 58 paraTB positive herds boot swabs and liquid manure were sampled simultaneously and analyzed in three different diagnostic laboratories. Using boot swabs and liquid manure, a substantial to excellent accordance was found between both qPCRs, for boot swabs also with culture, while for liquid manure the detection rate of culture was decreased after prolonged storage at -20 °C. The quantitative results of both qPCR methods correlated well for the same sample and also for boot swabs and liquid manure from the same herd. When cut-off threshold cycle (CT-)-values were applied as recommended by the manufacturers, herd level specificity (Sp) of qPCR B was below 100% for boot swabs and for both qPCRs for liquid manure. A decreased herd level sensitivity was encountered after adjustment of Sp to 100% and re-calculation of the cut-off CT-values. CONCLUSIONS: qPCR is equally suitable as bacterial culture for the detection of Mycobacterium avium subsp. paratuberculosis in boot swabs and liquid manure samples. Both matrices represent easily accessible composite environmental samples which can be tested with reliable results. The data encourage qPCR testing of composite environmental samples for paraTB herd diagnosis.

Management of the calving pen is a crucial factor for paratuberculosis control in large dairy herds.

Improvement of hygiene and herd management to reduce the contact of calves with adult cow feces to prevent new infections is one of the basic strategies to manage paratuberculosis-affected dairy herds. Control programs should recommend an evidence-based selection of factors that demonstrably reduce the transmission of the infectious agent and decrease the prevalence of infected cattle to improve acceptance and implementation of the recommended measures among farmers. This study aimed to assess the influence of several management measures on control success in a longitudinal study in 28 large dairy herds with a median size of 415 cows in Thuringia, Germany. The cumulative incidence of cows shedding Mycobacterium avium ssp. paratuberculosis (MAP) per year was determined by individual fecal culture of all cows during 5 consecutive years. Relevant management practices as well as herd size, milk yield, and purchase of cattle were recorded by on-farm risk assessment. Mean holding time of MAP shedders within the herd was calculated from individual data of each shedding cow. Using multiple regression models, separate calving pens for shedders and disinfection of the pen after use were identified as significant risk factors that reduced the cumulative incidence of MAP shedders per year on the herd level. The results provide evidence that, in addition to other factors, calving hygiene and management of the calving pens are crucial for paratuberculosis control, particularly in large dairy herds. Considered together with the outcome from other studies, these results might be important to weight various risk factors and to avoid overburdening and overwhelming farmers and keeping them committed.

Characterization of a caprine model for the subclinical initial phase of Mycobacterium avium subsp. paratuberculosis infection.

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.

Facts, myths and hypotheses on the zoonotic nature of Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis (Johne's disease [JD]), a chronic granulomatous enteritis in ruminants. JD is one of the most widespread bacterial diseases of domestic animals with significant economic impact. The histopathological picture of JD resembles that of Crohn's disease (CD), a human chronic inflammatory bowel disease of still unresolved aetiology. An aetiological relevance of MAP for CD has been proposed. This and the ambiguity of other published epidemiological findings raise the question whether MAP represents a zoonotic agent. In this review, we will discuss evidence that MAP has zoonotic capacity.

[Prevalence of paratuberculosis in Thuringian sheep and goat flocks]. / Verbreitung der Paratuberkulose in Thüringer Schaf- und Ziegenherden.

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.

Mycobacterium avium subsp. Paratuberculosis in Different Environmental Samples from a Dairy Goat Barn-Implications for Sampling Strategies for Paratuberculosis Diagnostic and Prevention.

Environmental samples are often used to classify the paratuberculosis status of cattle herds. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), predominantly through oral ingestion during infancy. In this explorative study, the presence of MAP was determined in the barn environment of a paratuberculosis-infected vaccinated dairy goat herd. A total of 256 bedding, dust, feed, and water samples were collected at eight time points and examined using culture and qPCR. Detection rates of both methods were compared, and factors determining MAP confirmation were identified. MAP was cultured from 28 bedding and one dust sample, while MAP DNA was detected in all materials (117/256). Samples from high animal traffic areas and those collected during the indoor season were more likely to yield positive culture and qPCR results. Cultivation of MAP from kidding pens indicated this area as a possible infection site. Dust proved to be the most suitable material for detecting MAP DNA, as bedding was for MAP culture. Environmental sampling was demonstrated to be an effective way to detect MAP in a dairy goat herd. qPCR results could confirm herd infection, while culture results provided insight into crucial areas for MAP transmission. These findings should be considered when designing farm-specific paratuberculosis control plans.

Molecular Diversity of Mycobacterium avium subsp. paratuberculosis in Four Dairy Goat Herds from Thuringia (Germany).

This study investigated the intra- and inter-herd diversity of Mycobacterium avium subsp. paratuberculosis (MAP) isolates from four goat herds in Thuringia (Germany) that were affected by paratuberculosis for several years. The main focus was on the characterization and distribution of genotypes among animals and the environment of goat herd 1. This study included 196 isolates from the feces of 121 infected goats, various tissues from 13 clinically diseased goats, 29 environmental samples from herd 1, and additionally, 22 isolates of different origin from herds 2 to 4. The isolates, sampled between 2018 and 2022, were genotyped using short-sequence-repeat (SSR) analysis, mycobacterial-interspersed repetitive units-variable-number tandem repeat (MIRU-VNTR) analysis, and a single nucleotide polymorphism (SNP)-based assay for phylogenetic grouping. All the isolates belonged to the MAP-C group. In herd 1, one predominant genotype was determined, while two other genotypes were identified very rarely and only in fecal and environmental samples. One of three further genotypes was found in each of herds 2 to 4. The assignment of genotypes to different phylogenetic clades suggested six different infection strains. The results indicated no epidemiological links between the examined herds. Based on the current MAP genotyping data from Germany, possible sources of infection are MAP-contaminated barns previously used by infected cattle and the purchase of sub-clinically infected goats.

Suspicion of Mycobacterium avium subsp. paratuberculosis transmission between cattle and wild-living red deer (Cervus elaphus) by multitarget genotyping.

Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n = 13), six cattle herds living in the area of this park (n = 23), and five cattle herds without any contact with these red deer (n = 17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable interpretations of epidemiological studies on paratuberculosis.

Variation in the Performance of Different Batches of Two Mycobacterium avium Subspecies paratuberculosis Antibody ELISAs Used for Pooled Milk Samples.

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

Mycobacteriosis in Various Pet and Wild Birds from Germany: Pathological Findings, Coinfections, and Characterization of Causative Mycobacteria.

A total of 50 birds diagnosed with mycobacteriosis were examined for pathomorphological lesions, coinfections, and causative agents. Mycobacterial species were identified and isolates differentiated using multilocus sequence typing (MLST) and mycobacterial interspersed repetitive-unit variable-number of tandem-repeat (MIRU-VNTR) analysis. Possible associations between mycobacterial species, pathomorphological findings, coinfections, bird orders, and husbandry conditions were evaluated statistically. Mycobacteria were isolated from 34 birds (13 of 22 Psittaciformes, 12 of 18 Passeriformes, five of six Columbiformes, and four other orders) belonging to 26 species in total. Mycobacterium genavense (Mg) was cultured from 15 birds, Mycobacterium avium subsp. avium (Maa) from 20 birds, and Mycobacterium avium subsp. hominissuis (Mah) from three birds; hence, four birds had mixed infections. About equal numbers of psittacines and passerines were infected with Ma and Mg. The genetic diversity differed; Mg isolates belonged to one MLST type, Maa to six, and Mah to three combined genotypes. Several coinfections were detected; viruses and/or endoparasites affected 44%, fungi 38%, and bacteria 29% of the birds. Pathological findings and mycobacteriosis-affected organs were independent of coinfections. Overall, gross pathological findings were more often seen in mycobacteriosis caused by Ma (95%) compared with Mg (66%). Organ distribution of mycobacteriosis was independent of the mycobacterial species. Pathomorphological changes were seen in the small intestine of 71% and the lung of 65% of the birds, suggesting oral or pulmonal ingestion of mycobacteria. There were no associations between mycobacterial species and bird orders or bird husbandry conditions. Not only Mg, but also Maa and Mah, were clearly identified as primary cause of mycobacteriosis in pet birds. IMPORTANCE In this study, the causative agents and confounding factors of mycobacteriosis in a set of pet and some wild birds from Germany were examined. Not only Mycobacterium genavense, but also M. avium subsp. avium and M. avium subsp. hominissuis, contributed to mycobacteriosis in these birds. Various coinfections did not affect the manifestation of mycobacteriosis. Due to different gross necropsy findings, however, a different pathogenicity of the two species was assumed. New strains of M. avium subsp. hominissuis originating from birds were identified and characterized, which is important for epidemiological studies and for understanding the zoonotic role of this pathogen, as the subsp. hominissuis represents an increasing public health concern. The study provides some evidence of correlation between M. avium subsp. avium genotypes and virulence which will have to be confirmed by broader studies.

Analysis of Colostrum and Udder Skin Swabs from a Dairy Goat Herd in Germany regarding the Occurrence of Mycobacterium avium Subsp. paratuberculosis.

Oral intake of Mycobacterium avium subspecies paratuberculosis (MAP) in first days of life is considered to be the main route of infection for paratuberculosis. This can be related to a direct contact to contaminated feces or feeding of MAP containing colostrum. Colostrum is believed to become contaminated either by lactogenic shedding or introduction of MAP from environmental sources. In this pilot study, the presence of MAP in individual and bulk colostrum samples from a paratuberculosis-infected, vaccinated dairy goat herd in Germany and the effect of udder skin disinfection on the MAP load of colostrum were examined. In order to distinguish between lactogenic shedding and fecal contamination, 49 udder skin swabs were cultivated on solid medium whereas 29 swabs were additionally analyzed by qPCR. qPCR was applied on 110 individual colostrum samples collected from 55 goats, one before and one after disinfection with a mycobactericidal disinfectant, and 14 bulk colostrum samples. MAP DNA was detected in 10.3% (3/29) of the swab samples, but no viable MAP was cultivated from any sample. These results indicate a low-level MAP contamination of the udder skin and colostrum of milking goats suggesting a low risk of MAP transmission via these routes.

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

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

Interferon-γ Response of Mycobacterium avium subsp. paratuberculosis Infected Goats to Recombinant and Synthetic Mycobacterial Antigens.

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.

Engineering Synthetic Lipopeptide Antigen for Specific Detection of Mycobacterium avium subsp. paratuberculosis Infection.

Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis (MAP) does not produce glycopeptidolipids (GPL) on the surface of the cell wall but a lipopentapeptide called L5P (also termed Lipopeptide-I or Para-LP-01) characterized in C-type (bovine) strains. This lipopeptide antigen contains a pentapeptide core, D-Phenylalanine-N-methyl-L-Valine-L-Isoleucine-L-Phenylalanine-L-Alanine, in which the N-terminal D-Phenylalanine is amido-linked with a fatty acid (C18-C20). The molecular and genetic characterization of this antigen demonstrated that L5P is unique to MAP. Knowledge of the structure of L5P enabled synthetic production of this lipopeptide in large quantities for immunological evaluation. Various studies described the immune response directed against L5P and confirmed its capability for detection of MAP infection. However, the hydrophobic nature of lipopeptide antigens make their handling and use in organic solvents unsuitable for industrial processes. The objectives of this study were to produce, by chemical synthesis, a water-soluble variant of L5P and to evaluate these compounds for the serological diagnosis of MAP using well-defined serum banks. The native L5P antigen and its hydrosoluble analog were synthesized on solid phase. The pure compounds were evaluated on collections of extensively characterized sera from infected and non-infected cattle. ROC analysis showed that L5P and also its water-soluble derivative are suitable for the development of a serological test for Johne's disease at a population level. However, these compounds used alone in ELISA have lower sensitivity (Se 82% for L5P and Se 62% for the water-soluble variant of L5P) compared to the Se 98% of a commercial test. Advantageously, these pure synthetic MAP specific antigens can be easily produced in non-limiting quantities at low cost and in standardized batches for robust studies. The fact that L5P has not been validated in the context of ovine paratuberculosis highlights the need to better characterize the antigens expressed from the different genetic lineages of MAP to discover new diagnostic antigens. In the context of infections due to other mycobacteria such as M. bovis or the more closely related species M. avium subsp. hominissuis, the L5P did not cross react and therefore may be a valuable antigen to solve ambiguous results in other tests.

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

Analysis of volatile organic compounds (VOCs) is a novel approach to accelerate bacterial culture diagnostics of Mycobacterium avium subsp. paratuberculosis (MAP). In the present study, cultures of fecal and tissue samples from MAP-infected and non-suspect dairy cattle and goats were explored to elucidate the effects of sample matrix and of animal species on VOC emissions during bacterial cultivation and to identify early markers for bacterial growth. The samples were processed following standard laboratory procedures, culture tubes were incubated for different time periods. Headspace volume of the tubes was sampled by needle trap-micro-extraction, and analyzed by gas chromatography-mass spectrometry. Analysis of MAP-specific VOC emissions considered potential characteristic VOC patterns. To address variation of the patterns, a flexible and robust machine learning workflow was set up, based on random forest classifiers, and comprising three steps: variable selection, parameter optimization, and classification. Only a few substances originated either from a certain matrix or could be assigned to one animal species. These additional emissions were not considered informative by the variable selection procedure. Classification accuracy of MAP-positive and negative cultures of bovine feces was 0.98 and of caprine feces 0.88, respectively. Six compounds indicating MAP presence were selected in all four settings (cattle vs. goat, feces vs. tissue): 2-Methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, heptanal, isoprene, and 2-heptanone. Classification accuracies for MAP growth-scores ranged from 0.82 for goat tissue to 0.89 for cattle feces. Misclassification occurred predominantly between related scores. Seventeen compounds indicating MAP growth were selected in all four settings, including the 6 compounds indicating MAP presence. The concentration levels of 2,3,5-trimethylfuran, 2-pentylfuran, 1-propanol, and 1-hexanol were indicative for MAP cultures before visible growth was apparent. Thus, very accurate classification of the VOC samples was achieved and the potential of VOC analysis to detect bacterial growth before colonies become visible was confirmed. These results indicate that diagnosis of paratuberculosis can be optimized by monitoring VOC emissions of bacterial cultures. Further validation studies are needed to increase the robustness of indicative VOC patterns for early MAP growth as a pre-requisite for the development of VOC-based diagnostic analysis systems.

Acid-base variables in acute and chronic form of nontuberculous mycobacterial infection in growing goats experimentally inoculated with Mycobacterium avium subsp. hominissuis or Mycobacterium avium subsp. paratuberculosis.

In current literature, data assessing the acid-base equilibrium in animals and humans during bacterial infection are rare. This study aimed to evaluate acid-base deteriorations in growing goats with experimentally induced NTM (nontuberculous mycobacteria) infections by application of the traditional Henderson-Hasselbalch approach and the strong ion model. NTM-challenged animals were orally inoculated with either Mycobacterium avium subsp. hominissuis (MAH; n = 18) or Mycobacterium avium subsp. paratuberculosis (MAP; n = 48). Twenty-five goats served as non-infected controls. Until 51st week post-inoculation (wpi), blood gas analysis, serum biochemical analysis, and serum electrophoresis were performed on venous blood. Fifty percent (9/18) of goats inoculated with MAH developed acute clinical signs like apathy, fever, and diarrhea. Those animals died or had to be euthanized within 11 weeks post-inoculation. This acute form of NTM-infection was characterized by significantly lower concentrations of sodium, calcium, albumin, and total protein, as well as significantly higher concentrations of gamma globulin, associated with reduced albumin/globulin ratio. Acid-base status indicated alkalosis, but normal base excess and HCO3- concentrations, besides significantly reduced levels of SID (strong ion difference), Atot Alb (total plasma concentration of weak non-volatile acids, based on albumin), Atot TP (Atot based on total protein) and markedly lower SIG (strong ion gap). The remaining fifty percent (9/18) of MAH-infected goats and all goats challenged with MAP survived and presented a more sub-clinical, chronic form of infection mainly characterized by changes in serum protein profiles. With the progression of the disease, concentrations of gamma globulin, and total protein increased while albumin remained lower compared to controls. Consequently, significantly reduced albumin/globulin ratio and lower Atot Alb as well as higher Atot TP were observed. Changes were fully compensated with no effect on blood pH. Only the strong ion variables differentiated alterations in acid-base equilibrium during acute and chronic NTM-infection.