Rapidly growing populations of South American camelids (SAC), introduced to Europe as non-indigenous species, have increased contacts at the livestock and human interfaces. This study assessed the occurrence of epizootic and zoonotic viral and bacterial pathogens of prime importance on 10 farms, selected to mirror the diversity of German SAC holdings in size and animal use. Farms were visited four times at approximately 6-months intervals, with 20 animals sampled per visit, resulting in 739 blood and 723 fecal samples from 449 animals (292 alpacas, 156 llamas, one huarizo). Wherever possible, diagnostic methods applied followed national or international recommendations. Antibodies against Schmallenberg virus were detected in 54.6% of animals. Only three animals showed reactivity against Borna disease Virus 1 and one against bovine viral diarrhea virus (BVDV) 1. All animals tested negative for BVDV-2, border disease virus and bovine herpesvirus 1. Thirty-six samples from 30 SAC yielded a non-negative, presumably false-positive results for antibodies against the Mycobacterium tuberculosis complex. Ten samples from six animals were non-negative in an iELISA for brucellae antibodies, but all tested negative by Complement Fixation Test. Coxiella burnetii-specific antibodies were detected in three individuals from two different flocks, and a single fecal sample tested PCR-positive for C. burnetii. Chlamydiaceae-specific antibodies were found in 9 of 10 flocks and in 13.6% of the animals, with chlamydial fecal shedding observed in 8 of 10 flocks and in 29.6% of the animals. The animal positivity rate for Candidatus Mycoplasma haemolamae was 31.6%. Mycobacterium avium subsp. paratuberculosis was isolated from 0.34% and C. difficile from 2.7% of the animals. While Salmonella Typhimurium was found in only 1 of 719 fecal samples, thermotolerant Campylobacter (16 C. jejuni and 7 C. coli) were isolated from 4.8% of the animals. The overall rate of stx-positive samples, indicative of Shiga toxin-producing Escherichia coli (STEC) shedding, was 32.4%. Based on these detection rates, SAC do not appear to pose a greater risk of infection than other German livestock species at present. However, SAC represent a novel reservoir host that could disturb established and monitored indigenous epizootic transmission networks including those of enteric and abortifacient zoonotic agents.

Mycobacterium avium ssp. paratuberculosis (MAP) is an intestinal pathogen which is excreted fecally and can be spread in the environment through contaminated manure. Lactic acid fermentation (LAF) was evaluated as a method to inactivate MAP in cattle manure (CM). As carbohydrate (CHO) sources oats and saccharose (Sac) were used. After mixing, manure was incubated at 21°C for 8 weeks. The microbial shift was determined using cultural methods. The results showed different suitability of the selected CHOs for inactivation of MAP by LAF. Using squeezed oats as an additive, culturable MAP was reduced to below the detection limit after 35 and 42 days of fermentation. Additional Sac decreased the reduction time to 21 days. With Sac only addition, inactivation of MAP was not successful and bacterial counts did not differ from the negative control. Detection of IS900 genome fragments using real time-polymerase chain-reaction (PCR) showed that the number of gene copies in the manure did not decrease during the course of the experiments. This study showed that LAF is a valuable option for decontaminating CM with natural resources in the case of paratuberculosis.

This study investigated the genetic diversity of Mycobacterium avium subsp. paratuberculosis (Map)-the causative agent of paratuberculosis-isolated from different host species in Germany. A total of 500 isolates from 243 cattle herds and 9 other host species originating from 13 federal states were genotyped. A multi-target approach was applied, comprising IS900-RFLP with BstEII and PstI digestion; MIRU-VNTR; and SSR1, SSR8, and SSR9 analysis. In total, 93 combined genotypes were identified, 84 in cattle and 21 in non-cattle isolates. Ninety genotypes were assigned to the C-type group, and three genotypes (three from sheep and one from cattle) were assigned to the S-type/subtype III group. Cluster analysis divided genotypes into subgroups similar to those shown for WGS-SNP-based phylogenetic trees. New genotypes were revealed, including INMV262-267 and a specific sequence at locus VNTR7. Five genotypes that were predominant in cattle were also detected in sheep, goats, and deer. The majority of genotypes [61%] were identified only once. Polyclonal infections were observed in individual animals and herds, and various potential Map transmission linkages were uncovered. This high genotype richness of Map reflects the long history of paratuberculosis in Germany and intensive nationwide animal movement and international trading activity.

Inter-laboratory trials were carried out to evaluate the reproducibility and estimate test performance characteristics of the Phagomagnetic separation (PhMS)-qPCR assay, a novel phage-based assay recently developed as a rapid alternative to culture for detecting viable Mycobacterium avium subsp. paratuberculosis (MAP) in bovine milk and feces. Unique reagents and a detailed instruction manual required for the PhMS-qPCR assay were provided to five European veterinary diagnostic laboratories by Rapid-Myco Technologies Limited. Milk and feces test panels were prepared at NEIKER and distributed to participant laboratories between April–June 2023 and March–May 2024, respectively. Each test panel comprised of MAP-spiked and/or naturally infected bovine milk or feces samples (18 samples per panel on two separate occasions for each sample matrix). The six participant laboratories (including organizer) performed automated or manual PhMS and used whatever qPCR instrument they had available. All laboratories used the IDEXX RealPCR MAP DNA test for the qPCR part of the assay. Generally, substantial agreement was observed overall between PhMS-qPCR results and reference culture results for spiked milk (Kappa value 0.5982) and naturally MAP-infected feces (Kappa value 0.7780 using an amended protocol). Preliminary estimates of the detection (analytical) sensitivity (Se), detection specificity (Sp) and trueness (T) of the PhMS-qPCR assay applied to bovine milk and feces were obtained. The mean Se, Sp, and T values across six laboratories were 93.1%, 67.9%, and 88.7% when milk was tested and 84.1%, 93.7%, and 88.9% when feces was tested. Overall, the PhMS-qPCR assay performed well in multiple laboratories and test reproducibility was demonstrated (Cohen's Kappa ≥0.6–1.000). The estimates of performance characteristics of the PhMS-qPCR assay are generally acceptable for a potential diagnostic test. Hence the PhMS-qPCR assay shows considerable promise as a rapid test to detect viable MAP in veterinary specimens such as milk and feces. Further and fuller validation of the assay will continue to assess its diagnostic potential.

Zusammenfassung Gegenstand und Ziel In Deutschland liegen über die derzeitige Verbreitung der Paratuberkulose in Schaf- und Ziegenherden nur wenige Informationen vor. Diese Studie gibt einen Überblick bezüglich der Verbreitung von Mycobacterium avium ssp. paratuberculosis (MAP) in 165 Thüringer Schaf- und Ziegenherden und untersucht Beziehungen zwischen dem MAP-Status der Herde und herdenspezifischen Merkmalen sowie dem Ergebnis der serologischen Einzeltieruntersuchung und den tierspezifischen Merkmalen Alter, Ernährungszustand, Geschlecht und Tierart. Material und Methoden Zur Untersuchung der Verbreitung von MAP wurde im Jahr 2021 eine Stichprobe von 2550 Schafen und 1171 Ziegen aus 165 Herden mit einer Größe von 2 bis 2879 Tieren serologisch auf MAP untersucht. Zusätzlich wurden je nach Herdengröße in jedem Betrieb eine bis 6 Umgebungskotproben entnommen und mittels bakteriologischer Kultur und einer kommerziellen real-time-PCR untersucht. Ergebnisse In 20,6 % der 165 untersuchten Herden wurden MAP-Antikörper nachgewiesen, davon bei 41 Schafen (1,6 %) und 29 Ziegen (2,5 %). Die Symptome der Paratuberkulose – Abmagerung bei erhaltener Fresslust und veränderte Kotbeschaffenheit oder Durchfall – wurden nur in 4 Herden festgestellt. Es konnte ein positiver Zusammenhang zwischen dem Nachweis von MAP oder MAP-spezifischen Antikörpern und der Herdengröße sowie eine positive Beziehung zwischen dem Messwert im ELISA (P/PK-Verhältnis) und dem Alter des Tieres festgestellt werden. Des Weiteren konnte eine Assoziation zwischen einem ansteigenden Messwert im ELISA (P/PK-Verhältnis) und einem abnehmenden Ernährungszustand des Tieres ermittelt werden. Schlussfolgerung und klinische Relevanz Unter Berücksichtigung der Kenntnis über die Verbreitung der Paratuberkulose bei kleinen Wiederkäuern sollte diese Erkrankung bei Abmagerung und Durchfall immer in die differentialdiagnostische Abklärung einbezogen werden. Bei hoher Verbreitung innerhalb der Herde sollte eine Sanierung erwogen werden. Ein stichprobenbasiertes serologisches Herdenmonitoring auf Paratuberkulose sollte risikobasiert erfolgen, indem Tiere mit schlechtem Ernährungszustand bevorzugt in die Stichprobe einbezogen werden.

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.

BackgroundParatuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic granulomatous enteritis that affects domestic and wild ruminants and camelids. The disease has rarely been reported in alpacas in Germany. This publication describes epidemiologically independent cases of paratuberculosis in two alpacas in Germany.Case presentationTwo alpacas, a 26-year-old female zoo animal (case 1) and a 2.5-year-old breeding stallion from a private owner (case 2), presented with progressive emaciation, leading to death (case 2) or euthanasia (case 1) because of deteriorating general condition. In both cases typical granulomatous lesions in the intestinal mucosa and mesenteric lymph nodes were found. In case 2, other lymph nodes were severely enlarged and MAP was detected in the mandibular lymph node, lung, and liver by qPCR. The MAP isolates differed between the alpacas, with two distinct phylogenetic clades (Clade 1 and 8) within Subgroup A of the MAP-C type group and two distinct INMV profiles (INMV 2 and 1) found. These genotypes have been identified in cattle and goats in different regions in Germany. The genotype isolated from case 1 has been detected in goats from the zoo since 2011, indicating transmission between these species.ConclusionsMAP can cause severe clinical disease in alpacas of variable age and under different husbandry conditions. Therefore, paratuberculosis should be considered for differential diagnosis in alpacas with emaciation and poor general condition. Although not definitely shown, cross-species infection between ruminant species and camelids is exceedingly likely.

Goats are natural hosts of Mycobacterium (M.) bovis, and affected herds can be the cause of significant economic losses. Similarites in disease course and lesions of M. bovis infections in goats and M. tuberculosis in humans make goats good models for human tuberculosis. The aim of this investigation was to characterize M. bovis challenge models in goats. For this, goats were endobronchially inoculated with three doses of M. bovis or culture medium. Clinical signs, shedding, and immune responses were monitored until 146 days post inoculation (dpi). At necropsy, lesions were examined by computed tomography, histology, and bacteriological culture. Infected goats did not develop clinical signs. M. bovis was cultured from feces, but never from nasal swabs. IGRAs were positive from 28 dpi onwards, antibodies at 140 dpi, and SICCT at 146 dpi. The increase in CD25+, IFN-γ+, and IFN-γ-releasing T-cell subpopulations was time-related, but not dose-dependent. All infected goats developed paucibacillary granulomas in the lungs and regional lymph nodes. M. bovis was regularly cultured. Dose-dependent effects included the size of pulmonary lesions, caverns, intestinal lesions, and early generalization in the high-dose group. In summary, reproducible challenge models with dose-dependent differences in lesions were established, which may serve for testing vaccines for veterinary or medical use.

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.

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.

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.

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.

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.

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.

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.

South American camelids (SAC) are increasingly popular in Germany. Although frequently in close contact to livestock and humans, data on the prevalence of epizootic and zoonotic bacteria and of bacteria exhibiting antimicrobial resistance (AMR) in SAC is sparse. Therefore, 94 composite faecal samples collected in 43 private SAC holdings in the German states Saxony, Thuringia and Saxony-Anhalt were tested for the presence of Clostridium difficile, Mycobacterium avium subsp. paratuberculosis (MAP), Salmonella spp. and Chlamydia spp. Escherichia coli served as an AMR-indicator and for detecting Shiga toxin (Stx)-producers (STEC). Chlamydial presence was also probed with 136 vaginal swabs and twelve placental tissue samples. Most owners (n = 41/43) replied to a questionnaire providing information on animals and husbandry conditions. The questionnaire covered 974 animals, among them 20 llamas, mostly kept in smallholdings with less than 20 animals (75%). Frequently (n = 30/41), SAC were co-housed with other animal species. Most holdings (76%, n = 31/41) welcomed visitors regularly. Clostridium difficile was detected in 8.5% (n = 8/94) of the faecal samples, Chlamydia spp. in 7.4% (n = 7/94) of the faecal samples, 4.4% (n = 6/136) of the vaginal swabs and 8.3% (n = 1/12) of the placental samples. MAP and Salmonella spp. were not detected. Samples from 28% of the holdings were stx-positive. STEC strains isolated belonged to eae-negative, non-O157 serovars. All E. coli isolates were resistant to at least five antibiotics with 46% possible ESBL-producers. Even though shedding of bacteria of public health concern by SAC at livestock-livestock and livestock-human interfaces was found to be less common, positive results indicate the need for establishing continuous surveillance measures.

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.

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.

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.