Microbial community structure of plant-based meat alternatives.

A reduction in animal-based diets has driven market demand for alternative meat products, currently raising a new generation of plant-based meat alternatives (PBMAs). It remains unclear whether these substitutes are a short-lived trend or become established in the long term. Over the last few years, the trend of increasing sales and diversifying product range has continued, but publication activities in this field are currently limited mainly to market research and food technology topics. As their popularity increases, questions emerge about the safety and nutritional risks of these novel products. Even though all the examined products must be heated before consumption, consumers lack experience with this type of product and thus further research into product safety, is desirable. To consider these issues, we examined 32 PBMAs from Austrian supermarkets. Based on 16S rRNA gene amplicon sequencing, the majority of the products were dominated by lactic acid bacteria (either Leuconostoc or Latilactobacillus), and generally had low alpha diversity. Pseudomonadota (like Pseudomonas and Shewanella) dominated the other part of the products. In addition to LABs, a high diversity of different Bacillus, but also some Enterobacteriaceae and potentially pathogenic species were isolated with the culturing approach. We assume that especially the dominance of heterofermentative LABs has high relevance for the product stability and quality with the potential to increase shelf life of the products. The number of isolated Enterobacteriaceae and potential pathogens were low, but they still demonstrated that these products are suitable for their presence.

Capture and transport of white rhinoceroses (Ceratotherium simum) cause shifts in their fecal microbiota composition towards dysbiosis.

Translocations of Rhinocerotidae are commonly performed for conservation purposes but expose the animals to a variety of stressors (e.g. prolonged fasting, confinement, novel environment, etc.). Stress may change the composition of gut microbiota, which can impact animal health and welfare. White rhinoceroses in particular can develop anorexia, diarrhea and enterocolitis after translocation. The aim of this study was to investigate the associations of age, sex and translocation on the rhinoceros' fecal bacterial microbiota composition. fecal samples were collected from rhinoceroses at capture (n = 16) and after a >30-hour road transport (n = 7). DNA was isolated from these samples and submitted for 16S rRNA V3-V4 phylotyping. Alpha diversity indices of the rhinoceros' fecal microbiota composition of different age, sex and before and after transport were compared using non-parametric statistical tests and beta diversity indices using Permutational Multivariate Analysis Of Variance (PERMANOVA). Resulting P-values were alpha-corrected (Padj.). Alpha and beta diversity did not differ between rhinoceroses of different age and sex. However, there was a significant difference in beta diversity between fecal samples collected from adult animals at capture and after transport. The most abundant bacterial phyla in samples collected at capture were Firmicutes and Bacteroidetes (85.76%), represented by Lachnospiraceae, Ruminococcaceae and Prevotellaceae families. The phyla Proteobacteria (Padj. = 0.009) and Actinobacteria (Padj. = 0.012), amongst others, increased in relative abundance from capture to after transport encompassing potentially pathogenic bacterial families such as Enterobacteriaceae (Padj. = 0.018) and Pseudomonadaceae (Padj. = 0.022). Important commensals such as Spirochaetes (Padj. = 0.009), Fibrobacteres (Padj. = 0.018) and Lachnospiraceae (Padj. = 0.021) decreased in relative abundance. These results indicate that the stressors associated with capture and transport cause an imbalanced fecal microbiota composition in white rhinoceroses that may lead to potentially infectious intestinal disorders. This imbalance may result from recrudescence of normally innocuous pathogens, increased shedding of pathogens or increased vulnerability to new pathogens.

Existence and Quality of Data on Control Programs for EU Non-regulated Cattle Diseases: Consequences for Estimation and Comparison of the Probability of Freedom From Infection.

Some European countries have successfully implemented country-specific control programs (CPs) for infectious cattle diseases that are not regulated or are regulated only to a limited extent at the European Union (EU) level. Examples of such diseases include bovine viral diarrhea (BVD), infectious bovine rhinotracheitis (IBR), and Johne's disease (JD). The CPs vary between countries in the design and quality of collected data as well as methods used to detect infection and estimate prevalence or probability of freedom from infection. Differences in disease status between countries and non-standardized approaches to assess freedom from infection pose a risk for countries with CPs for non-regulated diseases as infected animals may influence the progress of the disease control or eradication program. The implementation of output-based standards allows estimation and comparison of the probability of freedom for non-regulated cattle diseases in European countries. The aim of the current study was to assess the existence and quality of data that could be used for estimating freedom from infection in European countries. The online data collection tool was sent to 32 countries participating in the SOUND control COST Action and was completed by 24 countries. Data on cattle demographics and data from CPs of IBR and BVD exist in more than 50% of the response countries. However, data describing risk factors and CP of JD was reported as existing in <25% of the countries. The overall quality of data in the sections on demographics and CPs of IBR and BVD were evaluated as "good", but risk factors and JD data were mostly evaluated as "fair." Data quality was considered less good mainly due to two quality criteria: accessibility and accuracy. The results of this study show that the quantity and quality of data about cattle populations and CPs are relatively similar in many surveyed countries. The outcome of this work provides an overview of the current situation in the European countries regarding data on EU non-regulated cattle diseases and will further assist in the development and implementation of output-based standards.

Overview of Mitigation Programs for Non-EU-Regulated Cattle Diseases in Austria.

Background: The non-regulation of animal diseases due to missing regulation at the European Union (EU) level enables member states to implement mitigation programs based on their own country-specific conditions such as priority settings of the governments, availability of financial resources, and epidemiological situation. This can result in a heterogeneous distribution of mitigation activities and prevalence levels within and/or between countries, which can cause difficulties for intracommunity trade. This article aims to describe the past, current, and future mitigation activities and associated prevalence levels for four non-regulated animal diseases, i.e., enzootic bovine leukosis (EBL), infectious bovine rhinotracheitis/infectious pustular vulvovaginitis (IBR/IPV), bovine viral diarrhea (BVD), and bluetongue disease (BT) for Austria. Over a period of 40 years (1978-2020), regulations concerning EBL, IBR/IPV, BVD, and BT were retraced to analyze the changes of legislation, focusing on sampling, testing, and mitigation activities in Austria, and were linked to the collected diagnostic testing results. The study results clearly demonstrate the adoption of the legislation by the Austrian governments in dependency of the epidemiological situations. Furthermore, our study shows that, related to the forthcoming Animal Health Law on April 21, 2021, Austria has a good initial situation to achieve disease-free status and/or free from infection status based on the current available epidemiological situation and previously implemented mitigation activities. The study results presented here are intended to contribute to a better comparison of the eradication status across the European countries for non-EU-regulated cattle diseases by providing information about the mitigation activities and data of testing results over a period of 40 years.

Cryptosporidium spp. Infections in Combination with Other Enteric Pathogens in the Global Calf Population.

The most common worldwide diarrhoea-causing agents in neonatal calves are Cryptosporidium spp. (Crypto), bovine rotavirus (BRV), bovine coronavirus (BCoV), and enterotoxigenic Escherichia coli F5 (K99) (ETEC). Crypto is a zoonotic pathogen of diarrhoea in humans, particularly for children and immunocompromised adults. Four weighted-stratified random-effect meta-analyses including meta-regression analyses were performed to calculate the worldwide mean prevalence of Crypto and associated concurrent infections with BRV, BCoV and ETEC, as well as their potential influencing factors. The meta-analysis incorporated 28 studies (56 substudies) in 17 countries that determined the presence or absence of concurrent infections with Crypto in the global calf population. Approximately half of all considered studies presented here were conducted in Europe independently of the type of infections with Crypto. Within Europe, the highest estimated mean Crypto-BRV prevalence was identified in Ireland (16.7%), the highest estimated mean Crypto-BCoV prevalence was detected in the United Kingdom (4.3%), and the highest estimated mean Crypto-ETEC prevalence across the literature was determined in Turkey (4.7%). The chance of detecting BRV, BCoV, and ETEC in calves with diarrhoea was 0.8 (confidence interval (CI): 0.6-1.0), 0.7 (CI: 0.5-1.0) and 0.6 (CI: 0.4-0.9) lower in the presence of Crypto compared to calves without Crypto. This may indicate an inhibitory effect between BRV, BCoV, ETEC, and Crypto in calves. The variance in the published prevalence across the literature can mainly be explained by the "diagnostic" factor (R2 min-max: 0.0-40.3%), followed by the "health status of the sampled animals" (R2 min-max: 1.4-27.3%) and "geographical region" (R2 min-max: 5.9-23.6%).

Animal-related factors predicting fatal cases of blackleg and gas gangrene in cattle.

BACKGROUND: Blackleg and gas gangrene are acute clostridial infections primarily affecting cattle. The objectives of this study were to identify (i) animal-related factors influencing the occurrence and (ii) prognostic pathological findings supporting the differentiation of fatal blackleg and gas gangrene cases in the cattle population from 1998 to 2018 in Styria, Austria. METHODS: Two binomial logistic models were applied to analyse the research questions. Additionally, cross-validations were performed to estimate the accuracy of the predictive models. RESULTS: Model results show that animal-related factors (i.e., age, geographical discovery location of dead cattle, vaccination status) significantly influence the occurrence of blackleg when compared to gas gangrene. Pathological findings are similar for both diseases. CONCLUSIONS: Model results reveal that using animal-related factors has a better accuracy to predict the fatal cases caused by both pathogens. Thus, the authors recommend not relying on pathological findings as predictive factors in the differentiation between blackleg and gas gangrene in cattle.

Bacteria of eleven different species isolated from biofilms in a meat processing environment have diverse biofilm forming abilities.

Biofilms are formed by microorganisms protected by a self-produced matrix, most often attached to a surface. In the food processing environments biofilms endanger the product safety by the transmission of spoilage and pathogenic bacteria. In this study, we characterised the biofilm formation of the following eleven strains isolated from biofilms in a meat-processing environment: Acinetobacter harbinensis BF1, Arthrobacter sp. BF1, Brochothrix thermosphacta BF1, Carnobacterium maltaromaticum BF1, Kocuria salsicia BF1, Lactococcus piscium BF1, Microbacterium sp. BF1, Pseudomonas fragi BF1, Psychrobacter sp. BF1, Rhodococcus erythropolis BF1, Stenotrophomonas sp. BF1. We applied whole- genome sequencing and subsequent genome analysis to elucidate genetic features associated with the biofilm lifestyle. We furthermore determined the motility and studied biofilm formation on stainless steel using a static mono-species biofilm model mimicking the meat processing environment. The biomass and the EPS components carbohydrates, proteins and extracellular DNA (eDNA) of the biofilms were investigated after seven days at 10 °C. Whole-genome analysis of the isolates revealed that all strains except the Kocuria salsicia BF1 isolate, harboured biofilm associated genes, including genes for matrix production and motility. Genes involved in cellulose metabolism (present in 82% of the eleven strains) and twitching motility (present in 45%) were most frequently found. The capacity for twitching was confirmed using plate assays for all strains except Lactococcus piscium BF1, which showed the lowest motility behaviour. Differences in biofilm forming abilities could be demonstrated. The bacterial load ranged from 5.4 log CFU/cm2 (Psychrobacter sp. isolate) to 8.7 log CFU/cm2 (Microbacterium sp. isolate). The amount of the matrix components varied between isolates. In the biofilm of six strains we detected all three matrix components at different levels (carbohydrates, proteins and eDNA), in two only carbohydrates and eDNA, and in three only carbohydrates. Carbohydrates were detected in biofilms of all strains ranging from 0.5 to 4.3 µg glucose equivalents/cm2. Overall, the Microbacterium sp. strain showed the highest biofilm forming ability with high bacterial load (8.7 log CFU/cm2) and high amounts of carbohydrates (2.2 µg glucose equivalents/cm2), proteins (present in all experiments) and eDNA (549 ng/cm2). In contrast, Brochothrix thermosphacta was a weak biofilm former, showing low bacterial load and low levels of carbohydrates in the matrix (6.2 log CFU/cm2 and 0.5 µg glucose equivalents/cm2). This study contributes to our understanding of the biofilm forming ability of bacteria highly abundant in the meat processing environment, which is crucial to develop strategies to prevent and reduce biofilm formation in the food producing environment.

Prevalence of Worldwide Neonatal Calf Diarrhoea Caused by Bovine Rotavirus in Combination with Bovine Coronavirus, Escherichia coli K99 and Cryptosporidium spp.: A Meta-Analysis.

Multiple enteropathogens such as bovine rotavirus (BRV), bovine coronavirus (BCoV), Escherichia coli K99 (ETEC) and Cryptosporidium spp. (Crypto) are the most common causes of calf diarrhoea during the first 30 days of animal age. Three weighted-stratified random-effects meta-analyses were performed to calculate the worldwide prevalence of mixed infections of the causative agents (i.e., BRV-BCoV, BRV-ETEC, BRV-Crypto) and their potential influencing factors. The meta-analysis covered 41 studies (94 sub-studies) in 21 countries that determined the presence or absence of mixed infections in global calf populations. The highest worldwide estimated pooled prevalence was identified for BRV-Crypto (6.69%), followed by BRV-BCoV (2.84%), and BRV-ETEC (1.64%). The chance of detecting BCoV in calves with diarrhoea was 1.83 higher in the presence of BRV compared to calves without BRV, whereby an inhibition effect (odds ratio: 0.77) was determined between BRV and Crypto infections. The diagnostic methods were identified as a significant influencing factor in the detection of all considered mixed infections, while the other analysed factors differed in relation to their effect on prevalence. In contrast to BRV-BCoV, the prevalence of BRV-ETEC and BRV-Crypto mixed infections followed the course of individual ETEC and Crypto prevalence related to the age class of the sampled animals.

Corrigendum: Overview of Mitigation Programs for Cattle Diseases in Austria.

[This corrects the article DOI: 10.3389/fvets.2021.689244.].

Detection of Various Streptococcus spp. and Their Antimicrobial Resistance Patterns in Clinical Specimens from Austrian Swine Stocks.

Knowledge of pathogenic potential, frequency and antimicrobial resistance patterns of porcine Streptococcus (S.) spp. other than S. suis is scarce. Between 2016 and 2020, altogether 553 S. spp. isolates were recovered from clinical specimens taken from Austrian swine stocks and submitted for routine microbiological examination. Antimicrobial susceptibility testing towards eight antimicrobial substances was performed using disk diffusion test. All isolates from skin lesions belonged to the species S. dysgalactiae subspecies equisimilis (SDSE). S. hyovaginalis was mainly isolated from the upper respiratory tract (15/19) and S. thoraltensis from the genitourinary tract (11/15). The majority of S. suis isolates were resistant to tetracycline (66%), clindamycin (62%) and erythromycin (58%). S. suis isolates from the joints had the highest resistance rates. S. suis and SDSE isolates resistant to tetracycline were more likely to be resistant to erythromycin and clindamycin (p < 0.01). Results show that different species of Streptococcus tend to occur in specific body sites. Nevertheless, a statement whether these species are colonizers or potential pathogens cannot be given so far. High resistance rates of S. suis towards tetracyclines and erythromycin and high recovery rates of S. suis from lung tissue should be considered when treating pigs with respiratory diseases.

PacBio and Illumina MiSeq Amplicon Sequencing Confirm Full Recovery of the Bacterial Community After Subacute Ruminal Acidosis Challenge in the RUSITEC System.

The impact of subacute rumen acidosis (SARA) on the rumen bacterial community has been frequently studied in in vivo trials. Here we investigated whether these alterations can be mirrored by using the rumen simulation technique (RUSITEC) as an in vitro model for this disease. We hypothezised that the bacterial community fully recovers after a subacute ruminal acidosis challenge. We combined a PacBio nearly full-length 16S rRNA gene analysis with 16S rRNA gene Illumina MiSeq sequencing of the V4 hypervariable region. With this hybrid approach, we aimed to get an increased taxonomic resolution of the most abundant bacterial groups and an overview of the total bacterial diversity. The experiment consisted of a control period I and a SARA challenge and ended after a control period II, of which each period lasted 5 d. Subacute acidosis was induced by applying two buffer solutions, which were reduced in their buffering capacity (SARA buffers) during the SARA challenge. Two control groups were constantly infused with the standard buffer solution. Furthermore, the two SARA buffers were combined with three different feeding variations, which differed in their concentrate-to-hay ratio. The induction of SARA led to a decrease in pH below 5.8, which then turned into a steady-state SARA. Decreasing pH values led to a reduction in bacterial diversity and richness. Moreover, the diversity of solid-associated bacteria was lower for high concentrate groups throughout all experimental periods. Generally, Firmicutes and Bacteroidetes were the predominant phyla in the solid and the liquid phase. During the SARA period, we observed a decrease in fibrolytic bacteria although lactate-producing and -utilizing families increased in certain treatment groups. The genera Lactobacillus and Prevotella dominated during the SARA period. With induction of the second control period, most bacterial groups regained their initial abundance. In conclusion, this in vitro model displayed typical bacterial alterations related to SARA and is capable of recovery from bouts of SARA. Therefore, this model can be used to mimic SARA under laboratory conditions and may contribute to a reduction in animal experiments.

The sources and transmission routes of microbial populations throughout a meat processing facility.

Microbial food spoilage is responsible for a considerable amount of waste and can cause food-borne diseases in humans, particularly in immunocompromised individuals and children. Therefore, preventing microbial food spoilage is a major concern for health authorities, regulators, consumers, and the food industry. However, the contamination of food products is difficult to control because there are several potential sources during production, processing, storage, distribution, and consumption, where microorganisms come in contact with the product. Here, we use high-throughput full-length 16S rRNA gene sequencing to provide insights into bacterial community structure throughout a pork-processing plant. Specifically, we investigated what proportion of bacteria on meat are presumptively not animal-associated and are therefore transferred during cutting via personnel, equipment, machines, or the slaughter environment. We then created a facility-specific transmission map of bacterial flow, which predicted previously unknown sources of bacterial contamination. This allowed us to pinpoint specific taxa to particular environmental sources and provide the facility with essential information for targeted disinfection. For example, Moraxella spp., a prominent meat spoilage organism, which was one of the most abundant amplicon sequence variants (ASVs) detected on the meat, was most likely transferred from the gloves of employees, a railing at the classification step, and the polishing tunnel whips. Our results suggest that high-throughput full-length 16S rRNA gene sequencing has great potential in food monitoring applications.

Prevalence of Virulence Genes and Antimicrobial Resistances in E. coli Associated with Neonatal Diarrhea, Postweaning Diarrhea, and Edema Disease in Pigs from Austria.

Increasing numbers of multi-resistant Escherichia (E.) coli from clinical specimens emphasize the importance of monitoring of their resistance profiles for proper treatment. Furthermore, knowledge on the presence of virulence associated genes in E. coli isolates from European swine stocks is scarce. Consequently, a total of 694 E. coli isolated between 2016 and 2018 from diarrheic piglets of Austrian swine herds were investigated. The isolates were tested for their susceptibility to twelve antibiotics using agar disk diffusion test and for the presence of 22 virulence associated genes via PCR. Overall, 71.9, 67.7, and 49.5% of all isolates were resistant to ampicillin, tetracycline, and trimethoprim-sulfamethoxazole, while resistance levels to gentamicin and fosfomycin were 7.7 and 2.0%, respectively. Resistance frequency to ciprofloxacin was higher than in previous studies. Isolates were more likely to be resistant to ampicillin if they were also resistant to ciprofloxacin. No isolate was resistant to meropenem or amikacin. Virulence genes were detected more frequently in isolates expressing hemolytic activity on blood agar plates. The detection rate of faeG was increased in fimH negative isolates. We assume, that hemolytic activity and absence of fimH could be considered as potential indicators for the virulence of E. coli in piglets.

Comparison of Defined Course Doses (DCDvet) for Blanket and Selective Antimicrobial Dry Cow Therapy on Conventional and Organic Farms.

Antimicrobial use in livestock production is a controversial subject. While antimicrobials should be used as little as possible, it is still necessary, from both an animal health and welfare point of view, to treat infected animals. The study presented here aimed to analyse antimicrobial use on Austrian dairy farms by calculating the number of Defined Course Doses (DCDvet) administered per cow and year for dry cow therapy. Antimicrobial use was analysed by production system and whether farmers stated that they used blanket dry cow therapy (i.e., all cows in the herd were treated) or selective dry cow therapy (i.e., only cows with a positive bacteriological culture or current/recent history of udder disease were treated). A statistically significant difference (p < 0.001) was determined between antimicrobial use for blanket (median DCDvet/cow/year: 0.88) and selective dry cow therapy (median DCDvet/cow/year: 0.41). The difference between antimicrobial use on conventional and organic farms for dry cow therapy as a whole, however, was not statistically significant (p = 0.22) (median DCDvet/cow/year: 0.68 for conventional; 0.53 for organic farms). This analysis demonstrates that selective dry cow therapy leads to a lower overall use of antimicrobials and can assist in a more prudent use of antimicrobials on dairy farms.

A meta-analysis of bovine viral diarrhoea virus (BVDV) prevalences in the global cattle population.

A random effect meta-analysis was performed to estimate the worldwide pooled bovine viral diarrhoea virus (BVDV) prevalences of persistently infected (PI), viraemic (VI) and antibody-positive (AB) animals and herds. The meta-analysis covered 325 studies in 73 countries that determined the presence or absence of BVDV infections in cattle from 1961 to 2016. In total, 6.5 million animals and 310,548 herds were tested for BVDV infections in the global cattle population. The worldwide pooled PI prevalences at animal level ranged from low (≤0.8% Europe, North America, Australia), medium (>0.8% to 1.6% East Asia) to high (>1.6% West Asia). The PI and AB prevalences in Europe decreased over time, while BVDV prevalence increased in North America. The highest mean pooled PI prevalences at animal level were identified in countries that had failed to implement any BVDV control and/or eradication programmes (including vaccination). Our analysis emphasizes the need for more standardised epidemiological studies to support decision-makers implementing animal health policies for non-globally-regulated animal diseases.

An RNA Hybridization Assay for Screening Influenza A Virus Polymerase Inhibitors Using the Entire Ribonucleoprotein Complex.

Novel antiviral drugs, which are less prone to resistance development, are desirable alternatives to the currently approved drugs for the treatment of potentially serious influenza virus infections. The viral polymerase is highly conserved and serves as an attractive target for antiviral drugs since potent inhibitors would directly stop viral replication at an early stage. Recent structural studies on the functional domains of the heterotrimeric influenza polymerase, which comprises subunits PA, PB1, and PB2, opened the way to a structure-based approach for optimizing inhibitors of viral replication. These strategies, however, are limited by the use of isolated protein fragments instead of employing the entire ribonucleoprotein complex (RNP), which represents the functional form of the influenza polymerase in infected cells. In this study, we have established a screening assay for efficient and reliable analysis of potential influenza polymerase inhibitors of various molecular targets such as monoselective polymerase inhibitors targeting the endonuclease site, the cap-binding domain, and the polymerase active site, respectively. By utilizing whole viral RNPs and a radioactivity-free endpoint detection with the capability for efficient compound screening while offering high-content information on potential inhibitors to drive medicinal chemistry program in a reliable manner, this biochemical assay provides significant advantages over the currently available conventional assays. We propose that this assay can eventually be adapted for coinstantaneous analysis and subsequent optimization of two or more different chemical scaffold classes targeting multiple active sites within the polymerase complex, thus enabling the evaluation of drug combinations and characterization of molecules with dual functionality.