Presence of Non-Tuberculous Mycobacteria Including Mycobacterium avium subsp. paratuberculosis Associated with Environmental Amoebae.

One of the obstacles to eradicating paratuberculosis or Johne's Disease (JD) seems to be the persistence of Mycobacterium avium subsp. paratuberculosis (Map) in the environment due to its ability to survive alone or vectorized. It has been shown that Map is widely distributed in soils and water. Previously, we isolated amoebae associated with Map strains in the environment of bovines from an infected herd. This work aims to verify our working hypothesis, which suggests that amoebae may play a role in the transmission of JD. In this study, we sampled water in the vicinity of herds infected with Map or Mycobacterium bovis (M. bovis) and searched for amoebae and mycobacteria. Live amoebae were recovered from all samples. Among these amoebae, four isolates associated with the presence of mycobacteria were identified and characterized. Map and other mycobacterial species were detected by qPCR and, in some cases, by culture. This study suggests that amoebae and Map may be found in the same environment and might represent a risk of exposure of animals to pathogenic mycobacteria. These data open up new perspectives on the control measures to be put in place to prevent contamination by Map.

Characterization of Rosculus vilicus sp. nov., a rhizarian amoeba interacting with Mycobacterium avium subsp. paratuberculosis.

Free-living amoebae are described as potential reservoirs for pathogenic bacteria in the environment. It has been hypothesized that this might be the case for Mycobacterium avium subsp. paratuberculosis, the bacterium responsible for paratuberculosis. In a previous work, we isolated an amoeba from a water sample in the environment of infected cattle and showed that this amoeba was associated with Mycobacterium avium subsp. paratuberculosis. While a partial 18S rRNA gene has allowed us to suggest that this amoeba was Rosculus-like, at that time we were not able to sub-cultivate it. In the present study, we succeeded in cultivating this strain at 20-25°C. This amoeba is among the smallest (5-7 µm) described. The sequencing of the whole genome allowed us to extract the full 18S rRNA gene and propose this strain as a new species of the Rosculus genus, i.e., R. vilicus. Of note, the mitochondrial genome is particularly large (184,954 bp). Finally, we showed that this amoeba was able to phagocyte Mycobacterium avium subsp. paratuberculosis and that the bacterium was still observed within amoebae after at least 3 days. In conclusion, we characterized a new environmental amoeba species at the cellular and genome level that was able to interact with Mycobacterium avium subsp. paratuberculosis. As a result, R. vilicus is a potential candidate as environmental reservoir for Mycobacterium avium subsp. paratuberculosis but further experiments are needed to test this hypothesis.

Towards delimitation of the Echinococcus multilocularis parasite's southernmost range in France.

Alveolar echinococcosis is a severe, potentially fatal, parasitic disease caused by ingestion of microscopic eggs of Echinococcus multilocularis. The lifecycle of the parasite is essentially sylvatic, and based on a prey-predator relationship between red foxes and small rodents. A westward expansion from the eastern historical focus has been reported in France, though the parasite has also been detected in the southern Alps. While the focus in the Auvergne region (central France) was described in the 1980s, the southern delimitation of the actual endemic area, especially in the south, was unknown in the absence of dedicated surveys. Red fox samples were collected from 2013 to 2020 in the framework of other transversal epidemiological studies in five sampling areas from southwestern and southeastern France. One hundred and seven intestines were analysed by SSCT, and 221 faecal samples from intestines were analysed by copro-qPCR. None of the 328 foxes exhibited E. multilocularis worms or DNA. Although the presence of E. multilocularis cannot be totally excluded in the departments from the study areas, the sample size tested argues for an absence of the parasite in these studied areas, which is in accordance with the currently known endemic situation in France. These new data are helpful in determining the southernmost limit of E. multilocularis distribution in France. The warm, dry Mediterranean climate in the southeastern areas is less favourable to the transmission of E. multilocularis and especially to the survival of eggs in the environment than the climate in the French Alps or Liguria (Italy) climate where the parasite is present. The intermediate area between the southwestern study areas and the historical focus of Auvergne, which is separated by around 150 km, will be investigated in the coming years. Moreover, an ongoing national surveillance programme on E. multilocularis in foxes is targeting French departements along the edge of the known endemic area both in the southeast and southwest. The data produced will supplement the results of this study, thus greatly helping to define the current distribution of E. multilocularis in France and to target prevention measures to reduce human exposure.

Mycobacterium microti Infection in Red Foxes in France.

Mycobacterium microti, member of the Mycobacterium tuberculosis, complex is known to interfere in the screening and diagnosis of bovine tuberculosis. This pathogen is increasingly detected in the frame of surveillance programs for tuberculosis in livestock and wildlife. Recently, red foxes (Vulpes vulpes) were found infected by Mycobacterium bovis in four French endemic areas. M. microti infection was concomitantly found during this investigation. Rates of infection by M. microti and M. bovis are not different except in one of the four areas (lower prevalence for M. microti in Charente). As for M. bovis infection, none of the infected foxes presented gross TB-like lesions. Infection of red foxes by M. microti seems to occur by ingestion of contaminated food, as mesenteric lymph nodes are mostly infected albeit no fecal excretion could be detected. Red foxes appear to be susceptible to Mycobacterium microti infection but seem to play a role of dead-end host for the transmission of this bacillus.

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.

Mycobacterium bovis Infection in Red Foxes in Four Animal Tuberculosis Endemic Areas in France.

In France, animal tuberculosis (TB) due to Mycobacterium bovis (M. bovis) affects a multi-host community that include cattle and wildlife species such as wild boars (Sus scrofa), badgers (Meles meles), or wild deer (Cervus elaphus, Capreolus capreolus). The involvement of foxes in the epidemiology of TB is fairly described in countries facing multispecies concerns. After the discovery of grouped cases of TB in foxes in a French TB endemic region, a study was implemented in the core of four TB endemic areas in Dordogne, Charente, Landes (departments of Nouvelle-Aquitaine region), and Côte-d'Or (Burgundy-Franche-Comté region). No infected fox was found in Côte-d'Or (n = 146), where in parallel TB in cattle and other wild species became sparse in the last years. In contrast, in Dordogne, Charente, and Landes, 13 (n = 184), 9 (n = 98) and 7 (n = 140) foxes were found infected by M. bovis, respectively, corresponding to 7.1% (CI95% 3.8-11.8%), 9.2% (4.3-16.7%) and 5.0% (CI95% 2.0-10.0%) prevalence rates, respectively. These infection rates are comparable with those observed in badgers and wild boar in these same three areas (ranging from 9 to 13.2% and 4.3 to 17.9%, respectively), where the number of cattle outbreaks has increased in the last 10-15 years. In each area, the genotypes of foxes' M. bovis isolates were the same as those in local cattle and other wildlife species. None of the infected foxes presented TB-like gross lesions. M. bovis was found in the mesenteric lymph nodes of 28 foxes (68%). For the 12 foxes where retropharyngeal and respiratory lymph nodes were analyzed separately, M. bovis was present in the respiratory lymph nodes of eight individuals. With regard to excretion, appropriate samples were available for 12 infected foxes from Dordogne. M. bovis DNA was detected in the feces of five of these animals, four of which were infected in the mesenteric lymph nodes. Combined with the knowledge on the biology and ecology of foxes, the results of this study suggest that in areas where infection in cattle is still active in France, foxes might play a role of spillover host in the epidemiology of M. bovis.

Tuberculosis in the wild boar: Frequentist and Bayesian estimations of diagnostic test parameters when Mycobacterium bovis is present in wild boars but at low prevalence.

The Eurasian wild boar (Sus scrofa) is increasingly considered as a relevant actor in the epidemiology of animal tuberculosis (TB). Therefore, monitoring TB in this species is key when establishing comprehensive control schemes for this disease still present in Europe. No data are available on direct and indirect TB diagnostic methods in wild boars in epidemiological contexts where TB is endemic in cattle and detected in wild boars at low prevalence. We aimed to estimate and compare sensitivity and specificity values for bacterial culture, PCR and three commercial ELISAs, i.e. the TB ELISA-VK (using the bPPD antigen), INgezim TB Porcine and IDEXX M. bovis Ab Test (both using the MPB83 and MPB70 antigens), under field conditions in France. We used frequentist methods, with bacteriology as the gold standard, and a Bayesian formulation of the latent class analysis (LCA), without using a gold standard. Submandibular lymph nodes and sera from 495 wild boars hunter-harvested in three endemic areas (Aquitaine region, Côte d'Or region, and Corsica region) were collected between 2014 and 2016. Only eight individuals were positive for M. bovis by bacteriology (1.61%; CI95% 0.70-3.51%). The LCA method provided high specificities (99.2%; CI95% 98.2-99.8% for INgezim TB Porcine and 99.7%; CI95% 98.8-100% for IDEXX M. bovis Ab Test) and sensitivities (78.5%; CI95% 65.1-88.8% for INgezim TB Porcine and 83.9%; CI95% 58.9-97.2% for IDEXX M. bovis Ab Test) for both ELISAs using the MPB83 and MPB70 antigens. Bacterial culture showed limited sensitivity (42.8%; CI95% 19.0-70.6%), estimated as the probability of a positive result in an animal exposed to M. bovis. PCR and ELISA using the bPPD antigens demonstrated high specificities, and sensitivities intermediates between culture and the ELISAs using the MPB83 and MPB70 antigens. These results suggest that ELISA tests using the MPB83 and MPB70 antigens are useful to detect and monitor TB exposure of wild boar populations in field conditions in France.

Second line molecular diagnosis for bovine tuberculosis to improve diagnostic schemes.

Surveillance of bovine tuberculosis (bTB) is partly based on the sanitary inspection of carcasses at the abattoir to detect bTB-like lesions which, in compliance with EU recommendations, are analysed by bacteriology and histopathology to disclose Mycobacterium bovis (or M. caprae) infection. Moreover, since 2012, a PCR method with similar sensitivity and specificity values of histopathology and bacteriology respectively is additionally employed in France, partially compensating for the weaknesses of classical diagnostic methods. We analysed a collection of bTB-like lesions from cattle presenting positive histological results albeit with negative PCR results. We present here the results of these samples, recovered from 292 animals culled between 2013 and 2016, analysed with a second line molecular diagnosis approach that consists in a combination of PCRs targeting the M. tuberculosis-M. avium complexes as well as the Mycobacterium genus and sequencing of hsp65 gene. These molecular analyses disclosed to identify the presence of non-tuberculous bacteria which could be responsible for most of these non-specific TB lesions: non tuberculous mycobacteria (24%) or Actinomycetales (56%) such as Rhodococcus equi (53%); 24% of the samples were negative. M. bovis -or any other MTBC members- was neither detected by molecular methods nor isolated in any of them at the end of the 3 months of culture. In conclusion, these results highlight the lack of specificity of histopathology and the usefulness of a first line PCR with a second line molecular diagnostic test to circumvent it. This diagnostic strategy makes it possible to reduce the number of suspect bTB cases raised at the abattoir or shortening their lock-up periods. By simplifying diagnostic schemes, the use of this tool could improve bTB surveillance and make eradication programs more efficient in the future.

Mycobacterium bovis Infection of Red Fox, France.

Mycobacterium bovis infection in wild red foxes was found in southern France, where livestock and other wildlife species are infected. Foxes frequently interact with cattle but have been underestimated as a reservoir of M. bovis. Our results suggest a possible role of the red fox in the epidemiology of bovine tuberculosis.

Successful Application of the Gamma-Interferon Assay in a Bovine Tuberculosis Eradication Program: The French Bullfighting Herd Experience.

In the French Camargue region, where bovine tuberculosis had been enzootic for several years in bullfighting cattle herds, the gamma-interferon (IFN) assay was used since 2003 in parallel with the intradermal test in order to increase overall disease detection sensitivity in infected herds. This study presents the results of a field-evaluation of the assay during a 10-year period (2004-2014) of disease control and surveillance program and explores the particular pattern of IFN assay results in bullfight herds in comparison to cattle from other regions of France. The low sensitivity [59.2% (50.6; 67.3)] of IFN assay using the tuberculin stimulation could be related to the poor gamma-IFN production from bullfight cattle blood cells which is significantly lower than in animals of conventional breeds. The characteristics of the assay were progressively adapted to the epidemiological situation and the desired strategic applications. Data analysis with a receiver operating characteristic curve based on a simple S/P value algorithm allowed for the determination of a new cutoff adapted for a global screening, giving a high specificity of 99.9% results and a high accuracy of the assay. Having regularly risen to above 5% since 2005, with a peak around 10% in 2010, the annual incidence dropped to under 1% in 2014. The positive predictive value relative to the bacteriological confirmation evolved during the years, from 33% in 2009 to 12% during the last screening period, a normal trend in a context of decreasing prevalence. The estimated rate of false-positive reactions during screening campaigns was 0.67%, confirming the high specificity of the test, measured in bTB negative herds, in this epidemiological context. The proportion of false-positive reactions decreased with the age and was higher in males than in females. Although these results indicate that the IFN assay is accurate in the field, it also emphasizes great differences between interferon quantities produced by bullfight cattle blood samples compared to those of classical bovine breeds, which underlines the necessity to adapt the algorithms and combinations of the assay according to local epidemiological contexts.

Multilaboratory Evaluation of a Novel Lateral Flow Immunochromatographic Assay for Confirming Isolation of Mycobacterium bovis from Veterinary Diagnostic Specimens.

A novel lateral flow immunochromatographic device (LFD) was evaluated in several veterinary diagnostic laboratories. It was confirmed to be specific for Mycobacterium bovis and M.caprae cells. The performance of the novel LFD was assessed relative to the confirmatory tests routinely applied after culture (spoligotyping or quantitative PCR [qPCR]) in each laboratory; liquid (MGIT or BacT/Alert) and/or solid (Stonebrink, Coletsos, or Lowenstein-Jensen) cultures were tested. In comparison to spoligotyping of acid-fast-positive MGIT cultures, percent agreement between positive LFD and spoligotyping results was excellent in two United Kingdom laboratories (97.7 to 100%) but lower in the Spanish context (76%), where spoligotyping was applied to MGIT cultures previously confirmed to be positive for M. tuberculosis complex (MTBC) by qPCR. Certain spoligotypes of M. bovis and M. caprae were not detected by the LFD in Spanish MGIT cultures. Compared to qPCR confirmation, the agreement between positive LFD and qPCR results was 42.3% and 50% for BacT/Alert and MGIT liquid cultures, respectively, and for solid cultures, it ranged from 11.1 to 89.2%, depending on the solid medium employed (Coletsos, 11.1%; Lowenstein-Jensen, 55.6%; Stonebrinks, 89.2%). Correlation between the novel LFD and BD MGIT TBc Identification test results was excellent when 190 MGIT cultures were tested (r = 0.9791; P < 0.0001), with the added benefit that M. bovis was differentiated from another MTBC species in one MGIT culture by the novel LFD. This multilaboratory evaluation demonstrated the novel LFD's potential utility as a rapid test to confirm isolation of M. bovis and M. caprae from veterinary specimens following culture.

Interferon gamma response to Mycobacterium avium subsp. paratuberculosis specific lipopentapeptide antigen L5P in cattle.

After Mycobacterium avium subsp. paratuberculosis (Map) infection the cell-mediated immune (CMI) response indicative of early Th1 activation may be detected using interferon-gamma release assay (IGRA). Currently, the purified protein derivatives (PPDs), i.e., the total extract of mycobacteria antigens are used to recall CMI responses against Map. This study aimed to assess the ability of the chemically synthesized Map specific cell wall lipopentapeptide L5P to induce CMI response in cows infected by Map compared to PPD. L5P and PPD elicited an IFN-γ response in 12 and 35 animals from two Map infected herds respectively, but IFN-γ was not detected in the 13 cows recruited from a non-infected herd. Levels of IFN-γ detected were higher with PPD than with L5P. There was no correlation between the IFN-γ response and the humoral response to Map or faecal culture.

Estimation of sensitivity and specificity of bacteriology, histopathology and PCR for the confirmatory diagnosis of bovine tuberculosis using latent class analysis.

Bacteriology and histopathology are the most commonly used tests used for official confirmatory diagnosis of bovine tuberculosis (bTB) in cattle in most countries. PCR is also being used increasingly because it allows a fast diagnosis. This test could be applied as a supplement to or replacement for current bTB confirmatory diagnostic tests but its characteristics have first to be evaluated. The aim of this study was to estimate and compare sensitivities and specificities of bacteriology, histopathology and PCR under French field conditions, in the absence of a gold standard using latent class analysis. The studied population consisted of 5,211 animals from which samples were subjected to bacteriology and PCR (LSI VetMAX™ Mycobacterium tuberculosis Complex PCR Kit, Life Technologies) as their herd of origin was either suspected or confirmed infected with bTB or because bTB-like lesions were detected during slaughterhouse inspection. Samples from 697 of these animals (all with bTB-like lesions) were subjected to histopathology. Bayesian models were developed, allowing for dependence between bacteriology and PCR, while assuming independence from histopathology. The sensitivity of PCR was higher than that of bacteriology (on average 87.7% [82.5-92.3%] versus 78.1% [72.9-82.8%]) while specificity of both tests was very good (on average 97.0% for PCR [94.3-99.0%] and 99.1% for bacteriology [97.1-100.0%]). Histopathology was at least as sensitive as PCR (on average 93.6% [89.9-96.9%]) but less specific than the two other tests (on average 83.3% [78.7-87.6%]). These results suggest that PCR has the potential to replace bacteriology to confirm bTB in samples submitted from suspect cattle.

Bovine tuberculosis in Europe from the perspective of an officially tuberculosis free country: trade, surveillance and diagnostics.

Switzerland has been officially free of bovine tuberculosis (OTF) since 1960. Since 1980 the control of bovine tuberculosis (bTB) has been reduced to passive abattoir surveillance. Isolated cases of bTB, partly due to reactivation of human Mycobacterium bovis infections with subsequent transmission to cattle, have been noticed in the last years. In Europe, the overall prevalence of bTB is slightly increasing. Both OTF and non-OTF countries report increases in the proportion of bTB positive cattle herds. Current bTB eradication and control programs in Europe are facing a range of challenges. Whole herd depopulation is becoming a less attractive option for economic reasons and due to animal welfare concerns. Live animal trade is increasing both at national and international levels. Regarding these tendencies and taking into account the chronicity of bTB infection, pre-movement testing is becoming increasingly important as a central tool for eradication and for protection against re-introduction of bTB. Pre-movement testing, however specifically focuses on the infection status in individuals, requiring a high level of diagnostic accuracy to correctly diagnose infected animals. Current screening tests for bTB, however, have been designed to meet demands as herd tests. This illustrates that the modification of existing and/or the development of new diagnostics for bTB might be needed. The tuberculin skin test (TST), the primary screening test for bTB may in certain situations have low sensitivity. The interferon gamma (IFN-γ) assay is accepted to be more sensitive compared to TST. Reduced specificity, however, especially in areas of low bTB prevalence raises concerns. New antigen combinations including Rv3615c, OmpATb and others have been shown to complement ESAT-6 and CFP-10 in the whole blood IFN-γ assay and resulted in improved sensitivity (compared to ESAT-6 and CFP-10) and specificity (compared to tuberculins). Lesion detection after slaughter represents a cost-effective procedure for passive surveillance of bTB, especially in areas of low prevalence or in regions free of bTB; however, its sensitivity is very low. This illustrates that trade is linked with a certain risk to re-introduce bTB in OTF regions or countries and that there may be delays in detecting a re-introduction of bTB. In conclusion, regarding the fact that some parameters linked with bTB programs are changing, the development of improved diagnostic tests with a high reliability for use as individual animal tests will be important for future eradication of bTB, in line with international commitment to high standard animal health programs.

Determination of decisional cut-off values for the optimal diagnosis of bovine tuberculosis with a modified IFNγ assay (Bovigam®) in a low prevalence area in France.

The Bovigam(®) gamma interferon (IFNγ) assay was used to complement official skin-test screening in a low bovine tuberculosis (bTB) prevalence region in France. The aim of our work was to determine decisional cut-off values for protein purified derivatives (PPD) and ESAT6-CFP10 antigens (R) in order to optimize the efficacy of the modified Bovigam(®) test, in this low-prevalence area, for optimal classification of infected or non-infected herds following positive skin tests. The sensitivity of the IFNγ assay relative to post-mortem bTB-positive animals (Se(r)) was studied in 60 cattle from 20 bTB-infected herds. Its absolute specificity (Sp) was studied in 492 cattle from 25 bTB-free herds from a bTB-free zone. Its operational specificity (relative to the positive skin test) (Sp(r)) was also studied in 547 skin-test positive cattle from 172 bTB-free herds from an infected zone. Using normalized interpretations for individual (PPD or R) results, the cut-off values at 0.02 for PPD and 0.01 for R were obtained with a view to employ them in low prevalence areas with no previously observed non-specific reactions to SITT. Concerning its use after positive skin tests, cut-off values were set at 0.05 for PPD and at 0.03 for R. The choice of an interpretation method considering positive results with PPD and/or R (PPDUR), justified in a high risk context, provided a test Se(r) of 93% [84-98] and Sp(r) of 71.8% [67.9-75.6]. Analysis of positive results with PPD and R (PPDUR), ideal for low-risk contexts, provided a test Sp(r) of 94.3% [92.0-96.1] and Se(r) of 77% [64-87]. Thus, adapting the criteria to the region's infection status and to the conditions for its application is essential for the appropriate use of the IFNγ assay.