Viable Campylobacter jejuni on Eggshells and Its Potential to Cross-contaminate Egg White and Yolk When Using a Manual Separation Technique, Determined by Culture and Propidium Monoazide (PMA) qPCR.

Manual separation of egg yolk from egg white using the eggshell is common practice in private households. For this, the egg is cracked and both components are separated by passing the egg yolk back and forth between the two halves of the eggshell, allowing the egg white to drip down while the egg yolk remains in the shell. During this process, the egg content naturally gets in contact with the outside of the eggshell, which might lead to a cross-contamination with its microorganisms, thus was correspondingly assessed in this study. Campylobacter jejuni is one of the most important zoonotic pathogens that can be found on eggshells. Therefore, this bacterium was used to artificially contaminate the eggshells (n = 22) with concentrations of 3.1 ± 0.6 log10 cfu/g. After separating the egg yolk from the egg white, cross-contamination was determined using culture and qPCR. Altogether, cross-contaminations with C. jejuni were found in 15 egg white (68%) and in three egg yolk (14%) samples. Afterward, 90 eggs from 30 egg packs from different producers in and around Munich (Germany) were obtained for field study purposes. To address the problem of culturing due to a possible viable but nonculturable (VBNC) status of C. jejuni, a method to differentiate viable and dead C. jejuni on eggshell using 10 µM propidium monoazide (PMA) and qPCR was developed. As a result, seven egg packs (23%) were positive for C. jejuni. Of these, only one (3%) was contaminated with viable cells, but still in a concentration of 3.3 log10 cells/g shell. According to these results and considering that eggshells might also be naturally contaminated with other pathogens, the authors recommend avoiding the manual separation technique of egg white and yolk by the eggshell. Especially if raw egg white or yolk is used for preparation of not sufficiently heated foods, where contaminating pathogens are not inactivated during processing, this technique might be a safety hazard for the consumer.

Changes in the Microbiota from Fresh to Spoiled Meat, Determined by Culture and 16S rRNA Analysis.

Growth of meat microbiota usually results in spoilage of meat that can be perceived by consumers due to sensory changes. However, a high bacterial load does not necessarily result in sensory deviation of meat; nevertheless, this meat is considered unfit for human consumption. Therefore, the aims of this study were to investigate changes in the microbiota from fresh to spoiled meat and whether the proportions of certain bacteria can probably be used to indicate the hygiene status of meat. For this purpose, 12 fresh pork samples were divided into two groups, and simultaneously aerobically stored at 4°C and 22°C. At each time-temperature point (fresh meat, days 6, 13, and 20 at 4°C, and days 1, 2, 3, and 6 at 22°C), 12 meat subsamples were investigated. Sequences obtained from next-generation sequencing (NGS) were further analyzed down to species level. Plate counting of six bacterial groups and NGS results showed that Pseudomonas spp. and lactic acid bacteria (LAB) were found in a high proportion in all stored meat samples and can therefore be considered as important "spoilage indicator bacteria". On the contrary, sequences belonging to Staphylococcus epidermidis were found in a relatively high proportion in almost all fresh meat samples but were less common in stored meat. In this context, they can be considered as "hygiene indicator bacteria" of meat. Based on these findings, the proportion of the "hygiene indicator bacteria" in relation to the "spoilage indicator bacteria" was calculated to determine a "hygiene index" of meat. This index has a moderate to strong correlation to bacterial loads obtained from culture (p < 0.05), specifically to Pseudomonas spp., LAB and total viable counts (TVCs). Knowledge of the proportions of hygiene and spoilage indicator bacteria obtained by NGS could help to determine the hygiene status even of (heat-) processed composite meat products for the first time, thus enhancing food quality assurance and consumer protection.

Impact of the Revision of European Food Hygiene Legislation and the Introduction of Convenience-based Food on Food Safety in the German Military.

The implementation of the European Food Regulation in the German military started in 2003 and was fully implemented in 2006. In addition, in 2003 the German military introduced the concept of using convenience-based foods targeted to improve the safety of food served to the troops. The aim of this study was to evaluate the impact of these changes on food safety and the occurrence of food-borne disease outbreaks in the German military. For this purpose, data from a total of 517 food-borne outbreaks that occurred between 1995 and 2019 in the responsible areas of the German military both within the country and abroad were subjected to a retrospective analysis. As a result, a significant decrease (p = 2.47 × 10-5) in the number of the food-borne outbreak was observed in the second observation period (2003-2019) compared to the first period (1995-2002). Food groups often found contaminated with pathogens were desserts and prepared dishes (first period), fresh produce, soups, and sauces (second period). Bacillus cereus, Enterobacteriaceae, Salmonella spp., and Staphylococcus aureus were dominant pathogens isolated from suspected foods during disease outbreaks in both periods, however, the absolute number of isolates reduced significantly in the second period. Therefore it can be concluded that the implementation of European food hygiene regulations together with the introduction of convenience-based foods had a significant positive impact on food safety in the German military.

Cold-tolerant microorganisms causing spoilage of vacuum-packed beef under time-temperature abuse determined by culture and qPCR.

Vacuum packaging and storage conditions at chilled temperatures are commonly used in order to prolong the shelf life of meat. Under these conditions and time-temperature abuse, cold-tolerant (facultatively) anaerobic spoilage microorganisms can continue growing. This study investigated growth of six relevant spoilage microorganisms in vacuum-packed beef (n = 12, 72 subsamples, stored at 10 °C for 28 days) using culture and qPCR methods. Correspondingly, six qPCRs were newly developed/modified (for total bacteria, lactic acid bacteria (LAB), Enterobacterales, total fungi, Kazachstania psychrophila, and cold-tolerant Clostridium spp.). Besides microbial quantification, four spoilage appearances of meat (gas production, spoilage odor, % drip loss, and meat color) were observed. Results obtained from culture and qPCR show that total bacteria, LAB, and Enterobacterales reached their stationary phase at day 7 when spoilage parameters such as gas production were statistically increased and a deviation of odor was detected. Fastidious cold-tolerant Clostridium spp. and K. psychrophila could be detected from day 7. Based on microbiological and sensory analysis results, the maximum shelf life of vacuum-packed beef stored at 10 °C is 7 days. The developed qPCR has the potential to be used as an alternative method to culturing for determination of microbial growth.

Symbiotic Husbandry of Chickens and Pigs Does Not Increase Pathogen Transmission Risk.

A symbiotic or mixed animal husbandry (e.g., pigs and chickens) is considered to have a positive effect for animal welfare and sustainable agriculture. On the other hand, a risk of infection and transmission of microorganisms, especially of zoonotic pathogens, between animal species may potentially occur and thus might increase the risk of foodborne illnesses for consumers. To prove these assumptions, two groups of animals and their environmental (soil) samples were investigated in this study. Animals were kept in a free-range system. In the first group, pigs and chickens were reared together (pasture 1), while the other group contained only pigs (pasture 2). During a one-year study, fecal swab samples of 240 pigs and 120 chickens, as well as 120 ground samples, were investigated for the presence of Campylobacter spp., Salmonella spp. and E. coli. Altogether, 438 E. coli and 201 Campylobacter spp. strains were isolated and identified by MALDI-TOF MS. Salmonella spp. was not isolated from any of the sample types. The prevalences of Campylobacter coli and C. jejuni in pigs were 26.7% and 3.3% in pasture 1 and 30.0% and 6.7% in pasture 2, while the prevalences of C. coli and C. jejuni in chickens from pasture 1 were 9.2% and 78.3%, respectively. No correlation between the rearing type (mixed vs. pigs alone) and the prevalence of Campylobacter spp. was observed. All swab samples were positive for E. coli, while the average prevalences in soil samples were 78.3% and 51.7% in pasture 1 and 2, respectively. Results of similarity analysis of the MALDI-TOF MS spectra (for C. coli, C. jejuni and E. coli) and FT-IR spectra (for E. coli) of the same bacterial species showed no recognizable correlations, no matter if strains were isolated from chickens, pig or soil samples or isolated at different sampling periods. The results of the study indicate that the symbiotic husbandry of pigs and chickens neither results in an increased risk of a transmission of Campylobacter spp. or E. coli, nor in a risk of bacterial alteration, as shown by MALDI-TOF MS and FT-IR spectra. In conclusion, the benefits of keeping pigs and chickens together are not diminished by the possible transmission of pathogens.

Investigation of Meat from Ostriches Raised and Slaughtered in Bavaria, Germany: Microbiological Quality and Antimicrobial Resistance.

Ostrich meat is characterized by high nutritional value; however, it remains an exotic product in most countries worldwide. In Europe, only few data are available regarding its microbial contamination, prevalence of antimicrobial-resistant bacteria, and safety. Therefore, this study aimed to investigate the microbiological quality and safety of ostrich meat samples (n = 55), each from one animal, produced in Bavaria, Germany. The provided microbiological status of ostrich meat included mesophilic aerobic bacteria, Enterobacteria, and mesophilic yeast and molds. In terms of food safety, all meat samples were negative for Salmonella spp. and Trichinella spp. Additionally, meat samples and a further 30 stool samples from 30 individuals were investigated for Shiga toxin-producing Escherichia coli genes, with two meat samples that were qPCR-positive. Antimicrobial-resistant Enterobacteriaceae, Enterococcus faecalis, and Enterococcus faecium strains were from meat and stool samples also analyzed; 13 potentially resistant Enterobacteriaceae (meat samples) and 4 Enterococcus faecium (stool samples) were isolated, and their susceptibility against 29 and 14 antimicrobials, respectively, was characterized. The results of this study provide an overview of microbial loads and food safety aspects that may be used as baseline data for the ostrich meat industry to improve their hygienic quality. However, the implementation of monitoring programs is recommended, and microbiological standards for ostrich meat production should be established.

A simple method for the isolation of cold-tolerant Clostridium spp. from meat samples.

Pure isolates of Clostridium spp. are required when further investigations are needed, such as the characterisation of the colonies, sporulation and germination, biochemical analysis, growth rate and growth conditions, toxin production, spoilage potential and genomic profile. However, the isolation of cold-tolerant Clostridium spp. from suspicious meat samples is challenging due to their slow growth rates and overgrowth with less fastidious meat microbiota. Therefore, this study aimed to establish a practical method for the isolation of psychrophilic and psychrotolerant Clostridium spp. Primarily, meat drip samples (n = 3), enriched in Peptone Yeast Glucose Starch (PYGS) broth, were heated with different temperatures and durations, before they were subcultured on Columbia blood agar (CBA). The treatment procedure of heating samples at 80 °C for 5 min was evaluated as effective and was further validated using pure cultures of lactic acid bacteria (n = 5), bacteria belonging to the family of Enterobacteriaceae (n = 5), and cold-tolerant Clostridium spp. (n = 34). Almost all other meat microbiota was inactivated by heating at 80 °C for 5 min, while 28 strains of cold-tolerant Clostridium spp. survived. Finally, the treatment procedure was applied with drip of meat samples (n = 41) previously tested positive for 49 cold-tolerant Clostridium spp. using specific multiplex qPCR. All meat drip samples were enriched in PYGS broth by incubating them at 4 °C for 3 weeks, to ensure growth of Clostridium spp. before the enrichments were proceeded to heat treatment and to culturing on CBA. A total of 35 (71 %) from 49 Clostridium spp. strains were isolated using this culturing procedure. The accuracy of the recovery rates obtained from two replicates was 68 %. The method of detection and isolation applied in this study is easy and resulted in a high isolation rate of cold-tolerant Clostridium spp.

Unknown cold-tolerant Clostridium spp.: Characteristics and potential to cause meat spoilage.

Clostridium spp. are ubiquitous bacteria and often found in foods and animals. Some species are pathogenic, others food spoiling or commensals. In this study, 65 cold-tolerant Clostridium spp. strains isolated from variable samples (beef, lamb, venison, feces/skin of wild boars) were investigated. Fifty strains were lecithinase positive; six additionally produced ß-hemolysis. By applying specific qPCR, 16S rRNA gene analysis, RFLP method, and MALDI-TOF MS, they were classified into two major groups: 29 strains were identified as C. tagluense-like, while the other 36 remained unidentified. Subsequently, twenty-two vacuum-packed beef samples were spiked with a single strain from both groups and stored at 4 °C for 8 weeks. The odor of challenged samples was variable (from unchanged, sour/musty, to sulfurous), while color, meat consistency and drip loss were similar to the control group. The ability to produce gas of all tested strains was lower than of C. estertheticum. Even though both groups of cold-tolerant clostridia exhibited similar 16S rRNA genes and biochemical activities, RFLP methods and MALDI-TOF MS are sufficient to differentiate them. In terms of food safety, strains producing lecithinase and hemolysin should be further investigated for their potential to produce substances affecting human and animal health.

Antimicrobial Effects of Plant Extracts against Clostridium perfringens with Respect to Food-Relevant Influencing Factors.

ABSTRACT: The application of plant extracts (PEs) could be a promising option to satisfy consumers' demand for natural additives to inhibit growth of variable pathogenic bacteria. Thus, the aim of this study was to develop a standardized microdilution method to examine the antimicrobial effects of 10 hydrophilic PEs against two strains of Clostridium perfringens facing various food-relevant influencing factors. Because of the high opacity of PEs, resazurin was used as an indicator for bacterial growth instead of pellet formation. The highest value of the MIC of the replications of each PE was defined as effective plant extract concentration (EPC), whereas the next concentration beneath the lowest MIC was defined as the ineffective plant extract concentration (IEPC). The EPCs of seven PEs, allspice, cardamom, cinnamon, clove, coriander, ginger, and mace, were between 0.625 and 10 g/kg, whereas extracts of caraway, nutmeg, and thyme showed no antimicrobial activity up to the maximum concentration tested (10 g/kg) against C. perfringens in vitro. Two intrinsic factors, sodium chloride (NaCl) and sodium nitrite (NaNO2), displayed either synergistic or additive effects or no interaction with most PEs. By combination with PEs at their IEPC (0.08 to 1.25 g/kg), MIC of NaCl and NaNO2 decreased from between 25 and 50 g/kg to between 6 and 25 g/kg and from more than 200 mg/kg to between 0.2 and 100 mg/kg, respectively. In contrast, lipid (sunflower oil) at a low concentration inhibited the antimicrobial effects of all tested PEs. For extrinsic factors, only allspice, ginger, and coriander could maintain their antimicrobial effects after being heated to 78°C for 30 min. The synergistic effect between PEs and pH values (5.0 and 5.5) was also found for all PEs. The established screening method with resazurin and defining EPC and IEPC values allows the verification of antimicrobial effects of PEs under various food-relevant influencing factors in a fast and reproducible way.

High incidence of cold-tolerant Clostridium frigoriphilum and C. algidicarnis in vacuum-packed beef on retail sale in Germany.

Sixty vacuum-packed beef samples retailed in Germany were investigated for the occurrence of cold-tolerant Clostridium spp. After a storage period at 4 °C for eight weeks, meat juice from all samples was processed for culturing, DNA extraction and SYBR green qPCR for Clostridium species. After that, a previously developed multiplex qPCR, sequence analysis of the 16S rRNA gene, and MALDI-TOF MS were applied in order to identify Clostridium spp. found in samples. Subsequently, 23 samples were found positive for C. frigoriphilum (n = 19), C. estertheticum (n = 2), C. tagluense (n = 1) and C. lacusfryxellense/C. frigoris (n = 1). By using a new multiplex qPCR and a new RFLP method developed in this study, a further 15 meat juice samples were revealed to be contaminated with C. algidicarnis. With some samples being co-contaminated with two different species, 53% (n = 32) of all investigated vacuum-packed beef samples were found to be positive for cold-tolerant clostridia. This is the first report of detection and identification of C. algidicarnis in meat samples in Germany and Central Europe.

Development of two specific multiplex qPCRs to determine amounts of Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta and Staphylococcus in meat and heat-treated meat products.

Culturing methods are conventionally applied to investigate the contamination of food with several microorganisms after heat processing. However, with these methods, it is not possible to evaluate whether heat-treated meat products, such as cooked sausages, contained parts of spoiled meat. Therefore, two specific multiplex qPCRs were developed in this study in order to determine the microbiological quality of the raw materials used for these products. The PCR targets focused on four bacterial groups often found on meat (family Enterobacteriaceae, genus Pseudomonas, genus Staphylococcus and species Brochothrix thermosphacta). Specificity as well as sensitivity of the developed multiplex qPCRs, validated by using 68 microbial species, were 100%. The applicability of both multiplex qPCRs compared to culturing methods was performed using 96 meat samples (fresh and naturally spoiled) and 12 inhouse-made "Lyoner" sausages containing variable ratios of spoiled meat (0%, 5%, 12% and 25%; n = 3 for each group). Both methods showed similar results by evaluating the ∆log10 cfu/g, the relative accuracy and the t-test analysis (p > 0.05). Comparing qPCR results of the different sausage groups, a significant difference between sausages containing fresh meat and sausages containing spoiled meat (12% and 25%) was found only for Pseudomonas and B. thermosphacta in both raw and cooked sausages. The statistical difference between 5% vs. 12% and 25% spoiled meat in cooked sausages, was also found only for these two bacterial groups. The developed multiplex qPCRs were further applied to 30 commercially available "Bologna-type" sausages. The results showed a total of 14 sausages considered to be suspicious for Food Fraud. While the role of Staphylococcus spp. in meat spoilage remains unclear, Pseudomonas, Enterobacteriaceae and B. thermosphacta could together be used as an indicator for "spoiled meat" used in sausages. The developed qPCR systems in this study allow the detection of four relevant bacterial groups in the heated Bologna-type sausages and provide information about the hygienic quality of raw materials used. This method could thus be helpful for screening food suspected of Food Fraud.

Shedding of Mycobacterium caprae by wild red deer (Cervus elaphus) in the Bavarian alpine regions, Germany.

The number of natural infections with Mycobacterium caprae in wildlife and in cattle in the Bavarian and Austrian alpine regions has increased over the last decade. Red deer (Cervus elaphus) have been recognized as maintenance reservoir; however, the transmission routes of M. caprae among and from naturally infected red deer are unknown. The unexpected high prevalence in some hot spot regions might suggest an effective indirect transmission of infection. Therefore, this study was undertaken to diagnose the occurrence of M. caprae in faeces and secretions of red deer in their natural habitat. A total of 2,806 red deer hunted in this region during 2014-2016 were included in this study. After pathological examination, organs (lymph nodes, lung, heart), excretions and secretions (faeces, urine, saliva and tonsil swabs) were further investigated by qPCR specific for Mycobacterium tuberculosis complex (MTC), M. bovis and M. caprae. Samples tested positive by qPCR were processed for culturing of mycobacteria. In total, 55 (2.0%) animals were confirmed positive for M. caprae by pathological examination, PCR and culturing of the affected organ material. With the exception of one sample, all of the secretion and excretion samples were negative for mycobacteria of the Mycobacterium tuberculosis complex (MTC). From one red deer, M. caprae could be isolated from the heart sac as well as from the faeces. Whole-genome sequencing confirmed that both strains were clonally related. This is the first confirmation that M. caprae can be shed with the faeces of a naturally infected red deer. However, further studies focusing on a higher number of infected animals, sample standardization and coordinated multiple sampling are necessary to improve the understanding of transmission routes under natural conditions.

Differentiation of live and dead Mycobacterium tuberculosis complex in meat samples using PMA qPCR.

The causative agents of zoonotic bovine tuberculosis (bTB), Mycobacterium bovis and M. caprae, are members of the M. tuberculosis complex (MTC). Wildlife such as red deer infected with bTB are often without pathological findings, thus meat thereof may be classified as safe for human consumption. The culturing of MTC is time consuming and inappropriate to be applied with fresh meat and food. Therefore, a rapid method "PMA qPCR" to differentiate living and dead cells of MTC was developed in this study. By treating with 50 µM PMA™ dye, dead M. bovis BCG (≤104 cells/ml meat suspension) could be completely discriminated and was not detected by specific MTC PCR. The limit of detection of MTC without treatment with PMA™ dye was 10 cells/ml. All 50 venison samples obtained for field study purposes were negative for MTC. However, 40% were slightly PCR positive for non-TBC mycobacteria. By culturing using selective enrichment, one single colony of M. avium was isolated. This is the first report on the isolation of M. avium from venison. Considering the difficulties of diagnosing mycobacteria in various matrices, the developed PMA qPCR is applicable for the differentiation of dead and living cells of MTC in meat samples.

Antimicrobial resistance of Enterobacter cloacae complex isolates from the surface of muskmelons.

The increasing antimicrobial resistance (AMR) among pathogenic and opportunistic pathogenic microorganisms is one of the main global public health problems. The consumption of food contaminated with such bacteria (ARB), especially of raw products, might result in the direct acquisition of ARB and in a spread of resistant bacteria along the food chain. The aim of the study was to characterize the antimicrobial susceptibility of potentially extended spectrum ß-lactamase (ESBL) producing or AmpC resistant Enterobacteriaceae isolated from the surface of 147 muskmelons from wholesale and retail. A phenotypic analysis was carried out by using minimum inhibitory concentration (MIC) test strips for ESBL detection and MIC susceptibility plates against 14 antimicrobials. Furthermore, ESBL genes, sul-genes and plasmid-mediated AmpC resistance were analyzed by real-time PCR. Additionally, a further insight in the AmpC resistance of isolates of the Enterobacter cloacae complex (ECC) was obtained by analyzing the sequence of the ampC regulatory region (n = 15). A total of 73 potentially resistant Enterobacteriaceae were isolated from 56 muskmelons. Of these, 15 isolates of the ECC were suspicious for ESBL/AmpC resistance, and eleven thereof were positive for the AmpC family EBC. Phenotypic analysis showed diminished susceptibility against "critically" and "highly important" antimicrobials, according to the WHO classification. Furthermore, divergence in the ampC regulatory region was detected between the 15 isolates. These findings highlight the important role that raw produce might play in the transmission of antimicrobial resistances along the food chain.

Development of a multiplex qPCR for the species identification of Clostridium estertheticum, C. frigoriphilum, C. bowmanii and C. tagluense-like from blown pack spoilage (BPS) meats and from wild boars.

Psychrophilic and psychrotolerant clostridia (n = 110) were isolated from vacuum-packed meat (beef and lamb), fresh venison and from skin and fecal samples of wild boars. They were identified to species level using MALDI-TOF MS, sequence and phylogeny analysis of the 16S rRNA and species specific multiplex qPCR. The results of all three methods were concordant. The majority of isolates were identified as C. tagluense-like Group I (n = 34) and Group II (n = 42). Thirty-five isolates could be identified to species level as follows: C. estertheticum (n = 15), C. frigoriphilum (n = 13), C. frigidicarnis (n = 1) and C. bowmanii (n = 5). This is the first report of detection and identification of C. frigoriphilum and C. tagluense-like Group II as causative agents of blown pack spoilage of beef. The species specific multiplex qPCR developed in this study could be applied to identify and to quantify the Clostridium species described above in suspicious meat juice samples.

Accelerated biogas production from lignocellulosic biomass after pre-treatment with Neocallimastix frontalis.

Two Neocallimastix frontalis strains, isolated from rumen fluid of a cow and of a chamois, were assessed for their ability to degrade lignocellulosic biomass. Two independent batch experiments were performed. Each experiment was split into two phases: hydrolysis phase and batch fermentation phase. The hydrolysis process during the N. frontalis incubation led to an initial increase of biogas production, an accelerated degradation of dry matter and an increased concentration of volatile fatty acids. As monitored by quantitative PCR, the applied N. frontalis strains were present and transcriptionally active during the hydrolysis phase but were fading during the batch fermentation phase. Thus, a separate hydrolytic pretreatment phase with anaerobic fungi, such as N. frontalis, represents a feasible strategy to improve biogas production from lignocellulosic substrates.

Hepatitis E Virus in Wild Boar in Northwest Poland: Sensitivity of Methods of Detection.

In northwest Poland, 163 blood and 53 fecal samples of wild boars were collected in winter 2012/13 and 2013/14. All blood samples were tested for the presence of hepatitis E virus (HEV) ribonucleic acid (RNA) by two reverse transcription-polymerase chain reaction (RT-PCR) based methods and by anti-HEV IgG enzyme-linked immunosorbent assay (ELISA). About 17.2% of blood samples were seropositive. One-step nested RT-PCR turned out to be too insensitive (11.6% were positive). Therefore a two-step nested RT-PCR was applied where 25.8% of the blood samples were tested positive for HEV RNA. About 50.0% of blood samples positive in ELISA were also positive in two-step nested RT-PCR. The prevalence of HEV RNA in feces was 9.4%. Based on the results of blood (ELISA, PCR) and fecal (PCR) tests, the overall prevalence of HEV in wild boars in northwest Poland was 36.8%. There was no correlation between the ELISA results and the presence of HEV RNA in plasma or in feces. According to the sequencing results of 348 bp PCR products of HEV, there were four different subtypes identified. Reports on the prevalence of HEV in wild boar populations are varying due to different sensitivities of the detection methods. However, this study reveals based on a highly sensitive method that HEV is widely spread in wild boar populations in the northwestern region of Poland and posing a potential risk to the consumer of game meat.

Development of three specific PCR-based tools to determine quantity, cellulolytic transcriptional activity and phylogeny of anaerobic fungi.

Anaerobic fungi (AF) decompose plant material with their rhizoid and multiple cellulolytic enzymes. They disintegrate the complex structure of lignocellulosic substrates, making them more accessible and suitable for further microbial degradation. There is also much interest in their use as biocatalysts for biotechnological applications. Here, three novel polymerase chain reaction (PCR)-based methods for detecting AF and their transcriptional activity in in vitro cultures and environmental samples were developed. Two real-time quantitative PCR (qPCR)-based methods targeting AF were developed: AF-SSU, was designed to quantify the 18S rRNA genes of AF. AF-Endo, measuring transcripts of an endoglucanase gene from the glycoside hydrolase family 5 (GH5), was developed to quantify their transcriptional cellulolytic activity. The third PCR based approach was designed for phylogenetical analysis. It targets the 28S rRNA gene (LSU) of AF revealing their phylogenetic affiliation. The in silico-designed primer/probe combinations were successfully tested for the specific amplification of AF from animal and biogas plant derived samples. In combination, these three methods represent useful tools for the analysis of AF transcriptional cellulolytic activity, their abundance and their phylogenetic placement.

Specific amplification of bacterial DNA by optimized so-called universal bacterial primers in samples rich of plant DNA.

Universal primers targeting the bacterial 16S-rRNA-gene allow quantification of the total bacterial load in variable sample types by qPCR. However, many universal primer pairs also amplify DNA of plants or even of archaea and other eukaryotic cells. By using these primers, the total bacterial load might be misevaluated, whenever samples contain high amounts of non-target DNA. Thus, this study aimed to provide primer pairs which are suitable for quantification and identification of bacterial DNA in samples such as feed, spices and sample material from digesters. For 42 primers, mismatches to the sequence of chloroplasts and mitochondria of plants were evaluated. Six primer pairs were further analyzed with regard to the question whether they anneal to DNA of archaea, animal tissue and fungi. Subsequently they were tested with sample matrix such as plants, feed, feces, soil and environmental samples. To this purpose, the target DNA in the samples was quantified by qPCR. The PCR products of plant and feed samples were further processed for the Single Strand Conformation Polymorphism method followed by sequence analysis. The sequencing results revealed that primer pair 335F/769R amplified only bacterial DNA in samples such as plants and animal feed, in which the DNA of plants prevailed.

Reconstruction of the original mycoflora in pelleted feed by PCR-SSCP and qPCR.

Ground feeds for pigs were investigated for fungal contamination before and after pelleting (subsamples in total n = 24) by cultural and molecular biological methods. A fungal-specific primer pair ITS1/ITS5.8R was used to amplify fungal DNA; PCR products were processed for the PCR-SSCP method. In the resulting acrylamide gel, more than 85% of DNA bands of ground feeds were preserved after pelleting. Twenty-two DNA bands were sequenced; all represented fungal DNA. The level of fungal DNA in ground feed samples was equivalent to 4.77-5.69 log10  CFU g(-1) , calculated by qPCR using a standard curve of Aspergillus flavus. In pelleted feed, the level of fungal DNA was in average ± 0.07 log10 different from ground feed. Quantified by cultural methods, the fresh ground feeds contained up to 4.51 log10  CFU g(-1) culturable fungi, while there was < 2.83 log10  CFU g(-1) detected in pelleted feeds. This result shows that, while the process of pelleting reduced the amount of living fungi dramatically, it did not affect the total fungal DNA in feed. Thus, the described methodology was able to reconstruct the fungal microbiota in feeds and reflected a considerable fungal contamination of raw materials such as grains.