In Vivo Expression of Chicken Gut Anaerobes Identifies Carbohydrate- or Amino Acid-Utilising, Motile or Type VI Secretion System-Expressing Bacteria.

Complex gut microbiota increases chickens' resistance to enteric pathogens. However, the principles of this phenomenon are not understood in detail. One of the possibilities for how to decipher the role of gut microbiota in chickens' resistance to enteric pathogens is to systematically characterise the gene expression of individual gut microbiota members colonising the chicken caecum. To reach this aim, newly hatched chicks were inoculated with bacterial species whose whole genomic sequence was known. Total protein purified from the chicken caecum was analysed by mass spectrometry, and the obtained spectra were searched against strain-specific protein databases generated from known genomic sequences. Campylobacter jejuni, Phascolarctobacterium sp. and Sutterella massiliensis did not utilise carbohydrates when colonising the chicken caecum. On the other hand, Bacteroides, Mediterranea, Marseilla, Megamonas, Megasphaera, Bifidobacterium, Blautia, Escherichia coli and Succinatimonas fermented carbohydrates. C. jejuni was the only motile bacterium, and Bacteroides mediterraneensis expressed the type VI secretion system. Classification of in vivo expression is key for understanding the role of individual species in complex microbial populations colonising the intestinal tract. Knowledge of the expression of motility, the type VI secretion system, and preference for carbohydrate or amino acid fermentation is important for the selection of bacteria for defined competitive exclusion products.

Probiotic bacteria of wild boar origin intended for piglets - An in vitro study.

Using probiotics represents a potential solution to post-weaning diarrheal diseases in piglets on commercial farms. The gastrointestinal tract of wild boars serves as a promising reservoir of novel lactic acid bacteria with suitable probiotic characteristics. In this study, we isolated eight bacterial strains from the intestinal content of wild boars identified as representatives of the species Bifidobacterium apri, Lactobacillus amylovorus, and Ligilactobacillus salivarius. These isolates underwent in vitro analysis and characterisation to assess their biological safety and probiotic properties. Analysis of their full genome sequences revealed the absence of horizontally transferrable genes for antibiotic resistance. However, seven out of eight isolates harboured genes encoding various types of bacteriocins in their genomes, and bacteriocin production was further confirmed by mass spectrometry analysis. Most of the tested strains demonstrated the ability to inhibit the growth of selected pathogenic bacteria, produce exopolysaccharides, and stimulate the expression of interleukin-10 in porcine macrophages. These characteristics deem the isolates characterised in this study as potential candidates for use as probiotics for piglets during the post-weaning period.

River Sediments Downstream of Villages in a Karstic Watershed Exhibited Increased Numbers and Higher Diversity of Nontuberculous Mycobacteria.

The impact of residential villages on the nontuberculous mycobacteria (NTM) in streams flowing through them has not been studied in detail. Water and sediments of streams are highly susceptible to anthropogenic inputs such as surface water flows. This study investigated the impact of seven residential villages in a karst watershed on the prevalence and species spectrum of NTM in water and sediments. Higher NTM species diversity (i.e., 19 out of 28 detected) was recorded downstream of the villages and wastewater treatment plants (WWTPs) compared to sampling sites upstream (i.e., 5). Significantly, higher Zn and lower silicon concentrations were detected in sediments inside the village and downstream of the WWTP's effluents. Higher phosphorus concentration in sediment was downstream of WWTPs compared to other sampling sites. The effluent from the WWTPs had a substantial impact on water quality parameters with significant increases in total phosphorus, anions (Cl-and N-NH3-), and cations (Na+ and K+). The results provide insights into NTM numbers and species diversity distribution in a karst watershed and the impact of urban areas. Although in this report the focus is on the NTM, it is likely that other water and sediment microbes will be influenced as well.

Succession, Replacement, and Modification of Chicken Litter Microbiota.

Chickens are in constant interaction with their environment, e.g., bedding and litter, and their microbiota. However, how litter microbiota develops over time and whether bedding and litter microbiota may affect the cecal microbiota is not clear. We addressed these questions using sequencing of V3/V4 variable region of 16S rRNA genes of cecal, bedding, and litter samples from broiler breeder chicken flocks for 4 months of production. Cecal, bedding, and litter samples were populated by microbiota of distinct composition. The microbiota in the bedding material did not expand in the litter. Similarly, major species from litter microbiota did not expand in the cecum. Only cecal microbiota was found in the litter forming approximately 20% of total litter microbiota. A time-dependent development of litter microbiota was observed. Escherichia coli, Staphylococcus saprophyticus, and Weissella jogaejeotgali were characteristic of fresh litter during the first month of production. Corynebacterium casei, Lactobacillus gasseri, and Lactobacillus salivarius dominated in a 2-month-old litter, Brevibacterium, Brachybacterium, and Sphingobacterium were characteristic for 3-month-old litter, and Salinococcus, Dietzia, Yaniella, and Staphylococcus lentus were common in a 4-month-old litter. Although the development was likely determined by physicochemical conditions in the litter, it might be interesting to test some of these species for active modification of litter to improve the chicken environment and welfare. IMPORTANCE Despite intimate contact, the composition of bedding, litter, and cecal microbiota differs considerably. Species characteristic for litter microbiota at different time points of chicken production were identified thus opening the possibility for active manipulation of litter microbiota.

The Quality of DNA Isolated from Processed Food and Feed via Different Extraction Procedures.

The extraction of DNA is a critical step for species identification by PCR analysis in processed food and feed products. In this study, eight DNA extraction procedures were compared-DNeasy Blood and Tissue Kit, DNeasy mericon Food Kit, chemagic DNA Tissue 10 Kit, Food DNA Isolation Kit, UltraPrep Genomic DNA Food Mini Prep Kit, High Pure PCR Template Preparation Kit, phenol-chloroform extraction, and NucleoSpin Food-Using self-prepared samples from both raw and heat-processed and/or mechanically treated muscles and different types of meat products and pet food (pork, beef, and chicken). The yield, purity, and suitability of DNA for PCR amplification was evaluated. Additionally, comparisons between the effectiveness of various extraction methods were made with regard to price, and labor- and time-intensiveness. It was found that the DNeasy mericon Food Kit was the optimal choice for the extraction of DNA from raw muscle, heat-treated muscle, and homemade meat products from multiple and single species.

Serological Prevalence of Enteropathogenic Yersinia spp. in Pigs and Wild Boars from Different Production Systems in the Moravian Region, Czech Republic.

Human yersiniosis caused by pathogenic Yersinia spp. is one of the most common reported zoonoses in the European Union and pigs are considered as the major reservoir of these bacteria. Serological testing represents a suitable method to obtain information about the prevalence of enteropathogenic Yersinia spp. in food animals. The prevalence of antibodies against enteropathogenic Yersinia spp. was studied in 319 slaughtered pigs and 135 wild boars from different production systems in the Moravian region (Czech Republic) using a commercially available ELISA test (an apparent prevalence). The seroprevalence was significantly associated with the type of breeding system, with the lowest seroprevalence being observed in household-raised pigs (13/29, 44.8%). No significant difference between the prevalence of anti-Yersinia antibodies in conventional (146/180, 81.1%) and organic pigs (92/110, 83.6%) was found. Antibodies were found in 65.9% (89/135) of wild boars without a significant difference between adult (23/41, 56.1%) and young (66/94, 70.2%) animals. Seropositivity was significantly higher in domestic (251/319, 78.7% in total) compared to feral pigs. A Bayesian approach taking into account the sensitivity and specificity of the ELISA test was used to estimate the true prevalence of anti-Yersinia antibodies in pigs and wild boars. According to our results, domestic pigs and wild boars proved to be an important reservoir of enteropathogenic Yersinia in the Czech Republic. Attention should be paid to good hygienic practice during slaughtering and handling of meat to prevent meat contamination and subsequently human infection.

Comparison of methods for the isolation of mycobacteria from water treatment plant sludge.

Nontuberculous mycobacteria (NTM) are ubiquitous organisms in all natural ecosystems, including water environments. Several of these species are potential pathogens which affect human health. NTM most commonly cause pulmonary, skin or soft tissue infections. Primary sludge obtained from the water treatment plants of four drinking water reservoirs were subjected to analysis for mycobacteria. Five decontamination methods (5% oxalic acid, modified Petroff, HCl-NaOH, N-acetyl-L-cysteine-sodium hydroxide and 0.05% cetylpyridinium chloride), three growth media (Herrold's egg yolk medium with and without the antibiotic cocktail PANTA and Löwenstein-Jensen medium with sodium pyruvate) and three incubation temperatures (25, 30 and 37 °C) for isolation of mycobacteria were compared in the analysis of 18 sludge samples. To evaluate examined methods, the overall positive, negative, and contamination rate, and these rates in respect to localities are taken into account. Statistical analysis demonstrated that the best combination for the recovery of mycobacteria with the minimum number of contaminating microorganisms is 5% oxalic acid decontamination cultured on Herrold's egg yolk medium with the antibiotic cocktail PANTA at 25 °C. The least suitable is N-acetyl-L-cysteine-sodium hydroxide decontamination cultured on Löwenstein-Jensen medium with sodium pyruvate at 25 °C. From 18 sludge samples we isolated 27 mycobacterial species or groups; Mycobacterium algericum, M. arabiense, M. heraklionense, M. minnesotense, M. moriokaense, M. salmoniphilum and M. vulneris were isolated from the natural water environment for the first time. Because the natural water environment is the main source of potentially pathogenic mycobacteria for humans, it is important to direct particular focus to newly described mycobacterial species.

The water environment as a source of potentially pathogenic mycobacteria.

Nontuberculous mycobacteria (NTM) are ubiquitous organisms of a wide variety of environmental reservoirs, including natural and municipal water, soil, aerosols, protozoans, animals and humans. Several of these species are potential pathogens which affect human health. The aim of this study was to determine the occurrence of NTM in the water environment. Samples were taken from 13 water-related facilities including fish ponds, storage ponds, drinking water reservoirs and an experimental recirculation system. Altogether, 396 samples of water, sediment and aquatic plants were collected and analysed. All samples were examined using conventional culture methods. Suspected microbial isolates were subjected to polymerase chain reaction analysis and identified using partial sequence analysis of the 16S rDNA gene. The culture revealed 94/396 samples (23.7%) that contained mycobacteria. Among known NTM we identified potentially pathogenic mycobacteria isolated from the fresh water environment for the first time: Mycobacterium asiaticum, M. chimaera, M. interjectum, M. kumamotonense, M. lentiflavum, M. montefiorense, M. nebraskense, M. paraffinicum and M. simiae. Epidemiologic studies suggest that the natural water environment is the principal source of human exposure. Our results indicate that besides the well-known potentially pathogenic mycobacteria it is important to observe occurrence, proliferation and persistence of newly discovered mycobacterial species.

Ovine and Caprine Strains of Corynebacterium pseudotuberculosis on Czech Farms-A Comparative Study.

Caseous lymphadenitis (CLA) is a worldwide disease of small ruminants caused by Corynebacterium pseudotuberculosis, a facultative intracellular pathogen that is able to survive and multiply in certain white blood cells of the host. In this study, 33 strains of C. pseudotuberculosis were isolated from sheep and goats suffering from CLA on nine farms in the Czech Republic. All these strains were tested for their antibiotic susceptibility, ability to form a biofilm and resistance to the effects of commonly used disinfectant agents. To better understand the virulence of C. pseudotuberculosis, the genomes of strains were sequenced and comparative genomic analysis was performed with another 123 genomes of the same species, including ovis and equi biovars, downloaded from the NCBI. The genetic determinants for the virulence factors responsible for adherence and virulence factors specialized for iron uptake and exotoxin phospholipase D were revealed in every analyzed genome. Carbohydrate-Active Enzymes were compared, revealing the presence of genetic determinants encoding exo-α-sialidase (GH33) and the CP40 protein in most of the analyzed genomes. Thirty-three Czech strains of C. pseudotuberculosis were identified as the biovar ovis on the basis of comparative genome analysis. All the compared genomes of the biovar ovis strains were highly similar regardless of their country of origin or host, reflecting their clonal behavior.

Comparative Genome Analysis and Characterization of the Probiotic Properties of Lactic Acid Bacteria Isolated from the Gastrointestinal Tract of Wild Boars in the Czech Republic.

Probiotics are crucial components for maintaining a healthy gut microbiota in pigs, especially during the weaning period. Lactic acid bacteria (LAB) derived from the gastrointestinal tract of wild boars can serve as an abundant source of beneficial probiotic strains with suitable properties for use in pig husbandry. In this study, we analyzed and characterized 15 strains of Limosilactobacillus mucosae obtained from the gut contents of wild boars to assess their safety and suitability as probiotic candidates. The strains were compared using pan-genomic analysis with 49 L. mucosae strains obtained from the NCBI database. All isolated strains demonstrated their safety by showing an absence of transferrable antimicrobial resistance genes and hemolysin activity. Based on the presence of beneficial genes, five candidates with probiotic properties were selected and subjected to phenotypic profiling. These five selected isolates exhibited the ability to survive conditions mimicking passage through the host's digestive tract, such as low pH and the presence of bile salts. Furthermore, five selected strains demonstrated the presence of corresponding carbohydrate-active enzymes and the ability to utilize various carbohydrate substrates. These strains can enhance the digestibility of oligosaccharide or polysaccharide substrates found in food or feed, specifically resistant starch, α-galactosides, cellobiose, gentiobiose, and arabinoxylans. Based on the results obtained, the L. mucosae isolates tested in this study appear to be promising candidates for use as probiotics in pigs.

Contact with adult hens affects the composition of skin and respiratory tract microbiota in newly hatched chicks.

Chickens in commercial production are hatched in hatcheries without any contact with their parents and colonization of their skin and respiratory tract is therefore dependent on environmental sources only. However, since chickens evolved to be hatched in nests, in this study we evaluated the importance of contact between hens and chicks for the development of chicken skin and tracheal microbiota. Sequencing of PCR amplified V3/V4 variable regions of the 16S rRNA gene showed that contact with adult hens decreased the abundance of E. coli, Proteus mirabilis and Clostridium perfringens both in skin and the trachea, and Acinetobacter johnsonii and Cutibacterium acnes in skin microbiota only. These species were replaced by Lactobacillus gallinarum, Lactobacillus aviarius, Limosilactobacillus reuteri, and Streptococcus pasterianus in the skin and tracheal microbiota of contact chicks. Lactobacilli can be therefore investigated for their probiotic effect in respiratory tract in the future. Skin and respiratory microbiota of contact chickens was also enriched for Phascolarctobacterium, Succinatimonas, Flavonifractor, Blautia, and [Ruminococcus] torque though, since these are strict anaerobes from the intestinal tract, it is likely that only DNA from nonviable cells was detected for these taxa.

Immunoglobulin secretion influences the composition of chicken caecal microbiota.

The chicken caecum is colonised by hundreds of different bacterial species. Which of these are targeted by immunoglobulins and how immunoglobulin expression shapes chicken caecal microbiota has been addressed in this study. Using cell sorting followed by sequencing of V3/V4 variable region of 16S rRNA, bacterial species with increased or decreased immunoglobulin coating were determined. Next, we determined also caecal microbiota composition in immunoglobulin knockout chickens. We found that immunoglobulin coating was common and major taxa were coated with immunoglobulins. Similarly, more taxa required immunoglobulin production for caecum colonisation compared to those which became abundant in immunoglobulin-deficient chickens. Taxa with low immunoglobulin coating such as Lactobacillus, Blautia, [Eubacterium] hallii, Megamonas, Fusobacterium and Desulfovibrio all encode S-layer proteins which may reduce interactions with immunoglobulins. Although there were taxa which overgrew in Ig-deficient chickens (e.g. Akkermansia) indicating immunoglobulin production acted to exclude them from the chicken caecum, in most of the cases, immunoglobulin production more likely contributed to fixing the desired microbiota in the chicken caecum.

Modulation of Gut Microbiome and Autism Symptoms of ASD Children Supplemented with Biological Response Modifier: A Randomized, Double-Blinded, Placebo-Controlled Pilot Study.

The etiology and mechanisms of autism and autism spectrum disorder (ASD) are not yet fully understood. There is currently no treatment for ASD for providing significant improvement in core symptoms. Recent studies suggest, however, that ASD is associated with gut dysbiosis, indicating that modulation of gut microbiota in children with ASD may thus reduce the manifestation of ASD symptoms. The aim of this pilot study (prospective randomized, double-blinded, placebo-controlled) was to evaluate efficacy of the biological response modifier Juvenil in modulating the microbiome of children with ASD and, in particular, whether Juvenil is able to alleviate the symptoms of ASD. In total, 20 children with ASD and 12 neurotypical children were included in our study. Supplementation of ASD children lasted for three months. To confirm Juvenil's impact on the gut microbiome, stool samples were collected from all children and the microbiome's composition was analyzed. This pilot study demonstrated that the gut microbiome of ASD children differed significantly from that of healthy controls and was converted by Juvenil supplementation toward a more neurotypical microbiome that positively modulated children's autism symptoms.

Detecting horizontal gene transfer among microbiota: an innovative pipeline for identifying co-shared genes within the mobilome through advanced comparative analysis.

Horizontal gene transfer (HGT) is a key driver in the evolution of bacterial genomes. The acquisition of genes mediated by HGT may enable bacteria to adapt to ever-changing environmental conditions. Long-term application of antibiotics in intensive agriculture is associated with the dissemination of antibiotic resistance genes among bacteria with the consequences causing public health concern. Commensal farm-animal-associated gut microbiota are considered the reservoir of the resistance genes. Therefore, in this study, we identified known and not-yet characterized mobilized genes originating from chicken and porcine fecal samples using our innovative pipeline followed by network analysis to provide appropriate visualization to support proper interpretation.

Chicken innate immune response to oral infection with Salmonella enterica serovar Enteritidis.

The characterization of the immune response of chickens to Salmonella infection is usually limited to the quantification of expression of genes coding for cytokines, chemokines or antimicrobial peptides. However, processes occurring in the cecum of infected chickens are likely to be much more diverse. In this study we have therefore characterized the transcriptome and proteome in the chicken cecum after infection with Salmonella Enteritidis. Using a combination of 454 pyrosequencing, protein mass spectrometry and quantitative real-time PCR, we identified 48 down- and 56 up-regulated chicken genes after Salmonella Enteritidis infection. The most inducible gene was that coding for MMP7, exhibiting a 5952 fold induction 9 days post-infection. An induction of greater than 100 fold was observed for IgG, IRG1, SAA, ExFABP, IL-22, TRAP6, MRP126, IFNγ, iNOS, ES1, IL-1ß, LYG2, IFIT5, IL-17, AVD, AH221 and SERPIN B. Since prostaglandin D2 synthase was upregulated and degrading hydroxyprostaglandin dehydrogenase was downregulated after the infection, prostaglandin must accumulate in the cecum of chickens infected with Salmonella Enteritidis. Finally, above mentioned signaling was dependent on the presence of a SPI1-encoded type III secretion system in Salmonella Enteritidis. The inflammation lasted for 2 weeks after which time the expression of the "inflammatory" genes returned back to basal levels and, instead, the expression of IgA and IgG increased. This points to an important role for immunoglobulins in the restoration of homeostasis in the cecum after infection.

Chicken faecal microbiota and disturbances induced by single or repeated therapy with tetracycline and streptomycin.

BACKGROUND: In this study, we characterised the microbiota present in the faeces of 15- and 46-week-old egg laying hens before and after tetracycline or streptomycin therapy. In the first experiment, the layers were subjected to 7 days of therapy. In the second experiment, the hens were subjected to two days of therapy, which was repeated for an additional two days after 12 days of antibiotic withdrawal. This enabled us to characterise dynamics of the changes after antibiotic administration and withdrawal, and to identify genera repeatedly resistant to tetracycline and streptomycin. RESULTS: Real-time PCRs specific for Enterobacteriales, Lactobacillales, Clostridiales and Bifidobacteriales showed that changes in the microbiota in response to antibiotic therapy and antibiotic withdrawal were quite rapid and could be observed within 24 hours after the change in therapy status. Pyrosequencing of PCR amplified V3/V4 variable regions of 16S rRNA genes showed that representatives of the orders Clostridiales, Lactobacillales, Bacteroidales, Bifidobacteriales, Enterobacteriales, Erysipelotrichales, Coriobacteriales, Desulfovibrionales, Burkholderiales, Campylobacterales and Actinomycetales were detected in the faeces of hens prior to the antibiotic therapy. Tetracycline and streptomycin therapies decreased the prevalence of Bifidobacteriales, Bacteroidales, Clostridiales, Desulfovibrionales, Burkholderiales and Campylobacterales in faecal samples in both experiments. On the other hand, Enterobacteriales and Lactobacillales always increased in prevalence in response to both therapies. Within the latter two orders, Escherichia and Enterococcus were the genera prevalence of which increased after all the antibiotic treatments. CONCLUSIONS: The changes in microbiota composition induced by the antibiotic therapy were rapid and quite dramatic and only representatives of the genera Enterococcus and Escherichia increased in response to the therapy with both antibiotics in both experiments.

Nontuberculous mycobacteria in freshwater fish and fish products intended for human consumption.

Nontuberculous mycobacteria (NTM) are potentially pathogenic agents commonly found in natural ecosystems, while food is considered to be another source of NTM for humans. We investigated a total of 92 tissue samples of freshwater fish and fish products: fish directly obtained from ponds (n=25), retail fresh (n=23) and frozen fish (n=23) and smoked fish products (n=21). Culture examination for the presence of mycobacteria was positive in 11 (11.9%) from all the examined samples. The 15 obtained isolates were identified as Mycobacterium fortuitum (n=5), M. immunogenum (n=2), M. phocaicum/ mucogenicum (n=1), M. neoaurum (n=2), M. peregrinum (n=2), M. porcinum (n=1) and M. senegalense/houstonense/conceptionense (n=2). NTM DNA was found in one (4.0%) sample of fresh fish from ponds and in 60.9% and 91.3% of retail fresh and frozen fish, respectively. None of the smoked fish products contained NTM DNA. The results of our study suggest that freshwater fish and fish products, especially retail frozen fish, might be a reservoir of NTM for humans, and proper handling and treatment before consumption of such products is recommended.

Microbial Succession in the Cheese Ripening Process-Competition of the Starter Cultures and the Microbiota of the Cheese Plant Environment.

A large variety of cheeses can be produced using different manufacturing processes and various starter or adjunct cultures. In this study, we have described the succession of the microbial population during the commercial production and subsequent ripening of smear-ripened cheese using 16S rRNA gene sequencing. The composition of the microbiota during the first 6 days of production was constant and consisted mainly of LAB (lactic acid bacteria) originating from the starter culture. From day 7, the proportion of LAB decreased as other bacteria from the production environment appeared. From the 14th day of production, the relative proportion of LAB decreased further, and at the end of ripening, bacteria from the environment wholly dominated. These adventitious microbiota included Psychrobacter, Pseudoalteromonas haloplanktis/hodoensis, Vibrio toranzoniae, and Vibrio litoralis (Proteobacteria phylum), as well as Vagococcus and Marinilactibacillus (Firmicutes phylum), Psychrilyobacter (Fusobacteria phylum), and Malaciobacter marinus (Campylobacterota phylum), all of which appeared to be characteristic taxa associated with the cheese rind. Subsequent analysis showed that the production and ripening of smear-ripened cheese could be divided into three stages, and that the microbiota compositions of samples from the first week of production, the second week of production, and supermarket shelf life all differed.

Effective Control of Johne's Disease in Large Czech Dairy Herds.

Introduction: Johne's disease, caused by infection with Mycobacterium avium subsp. paratuberculosis (MAP), causes economic losses in dairy herds due to reduced milk production and premature culling. A test-and-cull strategy coupled with changes in calf rearing management preventing new infections has been introduced into infected herds to control MAP prevalence. This study appraised the effectiveness of these practice changes. Material and Methods: In 19 large dairy herds (of a median 470 milk-producing cows), implementing MAP control measures for 3-7 years, a serum ELISA was used to detect infected cows in their dry-off period. The number of ELISA-positive animals per year (EPAY) was calculated and statistical analysis was used to test whether the EPAY total decreased during the control period and to analyse the EPAY in relationship to the duration of the control programme. Results: Statistical support was found for a decrease of EPAY over time (P < 0.01, odds ratio 0.756) and in 14 herds a significant fall in the percentages of EPAY during the test period (P ≤ 0.05) was noted. Conclusion: Our results demonstrated the effectiveness of the control measures in place to reduce MAP infection in herds with initial EPAY ≥3.36%. The missing decreasing trend in the remaining five herds with low average initial EPAY suggested the need for additional measures to reduce the number of infected animals in these herds.

A viability assay combining palladium compound treatment with quantitative PCR to detect viable Mycobacterium avium subsp. paratuberculosis cells.

Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogenic bacterium causing the paratuberculosis, chronic and infectious disease common particularly in wild and domestic ruminants. Currently, culture techniques to detect viable MAP are still used most commonly, although these require a long incubation period. Consequently, a faster molecular method for assessing MAP cell viability based on cell membrane integrity was introduced consisting of sample treatment with the intercalation dye propidium monoazide (PMA) followed by quantitative PCR (qPCR). However, the PMA-qPCR assay is complicated by demanding procedures involving work in a darkroom and on ice. In this study, we therefore optimized a viability assay combining sample treatment with palladium (Pd) compounds as an alternative viability marker to PMA, which does not require such laborious procedures, with subsequent qPCR. The optimized Pd-qPCR conditions consisting of 90 min exposure to 30 µM bis(benzonitrile)dichloropalladium(II) or 30 µM palladium(II)acetate at 5 °C and using ultrapure water as a resuspension medium resulted in differences in quantification cycle (Cq) values between treated live and dead MAP cells of 8.5 and 7.9, respectively, corresponding to approximately 2.5 log units. In addition, Pd-qPCR proved to be superior to PMA-qPCR in distinguishing between live and dead MAP cells. The Pd-qPCR viability assay thus has the potential to replace time-consuming culture methods and demanding PMA-qPCR in the detection and quantification of viable MAP cells with possible application in food, feed, clinical and environmental samples.