A concept for international societally relevant microbiology education and microbiology knowledge promulgation in society.

EXECUTIVE SUMMARY: Microbes are all pervasive in their distribution and influence on the functioning and well-being of humans, life in general and the planet. Microbially-based technologies contribute hugely to the supply of important goods and services we depend upon, such as the provision of food, medicines and clean water. They also offer mechanisms and strategies to mitigate and solve a wide range of problems and crises facing humanity at all levels, including those encapsulated in the sustainable development goals (SDGs) formulated by the United Nations. For example, microbial technologies can contribute in multiple ways to decarbonisation and hence confronting global warming, provide sanitation and clean water to the billions of people lacking them, improve soil fertility and hence food production and develop vaccines and other medicines to reduce and in some cases eliminate deadly infections. They are the foundation of biotechnology, an increasingly important and growing business sector and source of employment, and the centre of the bioeconomy, Green Deal, etc. But, because microbes are largely invisible, they are not familiar to most people, so opportunities they offer to effectively prevent and solve problems are often missed by decision-makers, with the negative consequences this entrains. To correct this lack of vital knowledge, the International Microbiology Literacy Initiative-the IMiLI-is recruiting from the global microbiology community and making freely available, teaching resources for a curriculum in societally relevant microbiology that can be used at all levels of learning. Its goal is the development of a society that is literate in relevant microbiology and, as a consequence, able to take full advantage of the potential of microbes and minimise the consequences of their negative activities. In addition to teaching about microbes, almost every lesson discusses the influence they have on sustainability and the SDGs and their ability to solve pressing problems of societal inequalities. The curriculum thus teaches about sustainability, societal needs and global citizenship. The lessons also reveal the impacts microbes and their activities have on our daily lives at the personal, family, community, national and global levels and their relevance for decisions at all levels. And, because effective, evidence-based decisions require not only relevant information but also critical and systems thinking, the resources also teach about these key generic aspects of deliberation. The IMiLI teaching resources are learner-centric, not academic microbiology-centric and deal with the microbiology of everyday issues. These span topics as diverse as owning and caring for a companion animal, the vast range of everyday foods that are produced via microbial processes, impressive geological formations created by microbes, childhood illnesses and how they are managed and how to reduce waste and pollution. They also leverage the exceptional excitement of exploration and discovery that typifies much progress in microbiology to capture the interest, inspire and motivate educators and learners alike. The IMiLI is establishing Regional Centres to translate the teaching resources into regional languages and adapt them to regional cultures, and to promote their use and assist educators employing them. Two of these are now operational. The Regional Centres constitute the interface between resource creators and educators-learners. As such, they will collect and analyse feedback from the end-users and transmit this to the resource creators so that teaching materials can be improved and refined, and new resources added in response to demand: educators and learners will thereby be directly involved in evolution of the teaching resources. The interactions between educators-learners and resource creators mediated by the Regional Centres will establish dynamic and synergistic relationships-a global societally relevant microbiology education ecosystem-in which creators also become learners, teaching resources are optimised and all players/stakeholders are empowered and their motivation increased. The IMiLI concept thus embraces the principle of teaching societally relevant microbiology embedded in the wider context of societal, biosphere and planetary needs, inequalities, the range of crises that confront us and the need for improved decisioning, which should ultimately lead to better citizenship and a humanity that is more sustainable and resilient. ABSTRACT: The biosphere of planet Earth is a microbial world: a vast reactor of countless microbially driven chemical transformations and energy transfers that push and pull many planetary geochemical processes, including the cycling of the elements of life, mitigate or amplify climate change (e.g., Nature Reviews Microbiology, 2019, 17, 569) and impact the well-being and activities of all organisms, including humans. Microbes are both our ancestors and creators of the planetary chemistry that allowed us to evolve (e.g., Life's engines: How microbes made earth habitable, 2023). To understand how the biosphere functions, how humans can influence its development and live more sustainably with the other organisms sharing it, we need to understand the microbes. In a recent editorial (Environmental Microbiology, 2019, 21, 1513), we advocated for improved microbiology literacy in society. Our concept of microbiology literacy is not based on knowledge of the academic subject of microbiology, with its multitude of component topics, plus the growing number of additional topics from other disciplines that become vitally important elements of current microbiology. Rather it is focused on microbial activities that impact us-individuals/communities/nations/the human world-and the biosphere and that are key to reaching informed decisions on a multitude of issues that regularly confront us, ranging from personal issues to crises of global importance. In other words, it is knowledge and understanding essential for adulthood and the transition to it, knowledge and understanding that must be acquired early in life in school. The 2019 Editorial marked the launch of the International Microbiology Literacy Initiative, the IMiLI. HERE, WE PRESENT: our concept of how microbiology literacy may be achieved and the rationale underpinning it; the type of teaching resources being created to realise the concept and the framing of microbial activities treated in these resources in the context of sustainability, societal needs and responsibilities and decision-making; and the key role of Regional Centres that will translate the teaching resources into local languages, adapt them according to local cultural needs, interface with regional educators and develop and serve as hubs of microbiology literacy education networks. The topics featuring in teaching resources are learner-centric and have been selected for their inherent relevance, interest and ability to excite and engage. Importantly, the resources coherently integrate and emphasise the overarching issues of sustainability, stewardship and critical thinking and the pervasive interdependencies of processes. More broadly, the concept emphasises how the multifarious applications of microbial activities can be leveraged to promote human/animal, plant, environmental and planetary health, improve social equity, alleviate humanitarian deficits and causes of conflicts among peoples and increase understanding between peoples (Microbial Biotechnology, 2023, 16(6), 1091-1111). Importantly, although the primary target of the freely available (CC BY-NC 4.0) IMiLI teaching resources is schoolchildren and their educators, they and the teaching philosophy are intended for all ages, abilities and cultural spectra of learners worldwide: in university education, lifelong learning, curiosity-driven, web-based knowledge acquisition and public outreach. The IMiLI teaching resources aim to promote development of a global microbiology education ecosystem that democratises microbiology knowledge.

Risk factors associated with the infection of sheep with Dichelobacter nodosus.

Ovine footrot is a highly contagious foot disease caused by the gram-negative bacterium Dichelobacter nodosus (D. nodosus). In a recent report, we showed a prevalence of 42.9% D. nodosus positive swabs across Germany. In this follow-up study, we used real-time PCR results for D. nodosus and footrot scores of 9297 sheep from 208 flocks and collated these data with survey data on herd and animal characteristics and herd management. The aims of the present study were to investigate herd and animal factors associated with D. nodosus infection and footrot scores in individual sheep. Multivariable analyses with generalized mixed models showed that month of recording, breed, herdbook membership, use of antibiotics, and footbaths in the past 3-10 years, signs of footrot in the past 12 months and flock environment of the sheep, modelled as a random farm effect within region, were significant risk factors. Among the 21 different breeds, Romney had the lowest risk of D. nodosus infection, while Swifter had the highest risk and German Merino and German White Heath were the next breeds at highest risk of D. nodosus infection. The variance between farms in the prevalence of D. nodosus was large and accounted for 84% of the total variance in the mixed model analysis. We conclude that specific and as yet unknown effects influencing D. nodosus infections in flocks, as well as breed and weather, are the most important effects on D. nodosus infection in sheep, pointing towards the need to establish adequate infection control at farm level.

Corynebacterium uberis sp. nov. frequently isolated from bovine mastitis.

Several strains belonging to the genus Corynebacterium, but not to any described species of the genus were isolated from bovine mastitic milk samples over the past five years in the diagnostic unit of the University of Bern. Six of these strains (18M0132T, 17M2518, 18M0913, 19M0083, 20M1046 and 20M1090) that were phenotypically similar were further characterized genotypically. Gram-positive coryneform rods were catalase positive, facultative anaerobe and CAMP-test negative. Whole genome sequencing and subsequent phylogenetic analysis revealed their genome size to be 2.53 Mb and their G + C content to be between 65.4 and 65.5 mol%. Digital DNA-DNA hybridisation (dDDH) showed the highest similarity of only less than 20% with Corynebacterium mastitidis and Corynebacterium frankenforstense, which indicated that the isolates belong to an undescribed Corynebacterium species. This was confirmed by studying the average nucleotide identity (ANI) where the accepted species boundary is around 95% and which ranged between 70.3% and 74.9% with the most closely related species C. mastitidis. We established MALDI-TOF fingerprints of the species, which allows a clear separation from related species and can be used by other laboratories for diagnostic purposes. Based on our analyses we conclude that the selected strains belong to a previously undescribed species and propose the name Corynebacterium uberis sp. nov. The proposed type strain is 18M0132T (=DSM 111922T, = CCOS 1972T).

Serological Diversity of Dichelobacter nodosus in German Sheep Flocks.

Footrot is one of the major causes of lameness in sheep and leads to decreased animal welfare and high economic losses. The causative agent is the Gram-negative anaerobic bacterium Dichelobacter nodosus. The prevalence of D. nodosus in 207 sheep flocks across Germany was 42.9%. Based on the sequence variation in the type IV fimbrial gene fimA, D. nodosus can be subdivided into ten serogroups (A-I and M). There are commercially available vaccines covering nine serogroups, but the efficacy is low compared to bivalent vaccines. The aim of this study was to investigate the diversity of serogroups in Germany at the flock and animal levels. In total, we detected at least one serogroup in 819 samples out of 969 D. nodosus-positive samples from 83 flocks using serogroup-specific singleplex PCR for the serogroups A-I. Serogroup A was most prevalent at the animal level, followed by serogroups B, H and C. At the flock level, serogroups A and B had the highest prevalence, each with 64%, but only 40% of flocks had both. The average number of serogroups per animal was 1.42 (range one to five) and, per flock, 3.10 (range one to six). The serogrouping showed within-flock specific clusters but were widely distributed, with 50 different combinations across the flocks. The factors associated with the number of serogroups per animal and single serogroups were the load of D. nodosus, footrot score, sheep breed and flock. Our results indicate that efficient vaccination programs would benefit from tailor-made flock-specific vaccines and regular monitoring of circulating serotypes in the flock to be able to adjust vaccine formulations for nationwide progressive control of footrot in Germany.

Enhanced fitness of SARS-CoV-2 variant of concern Alpha but not Beta.

Emerging variants of concern (VOCs) are driving the COVID-19 pandemic1,2. Experimental assessments of replication and transmission of major VOCs and progenitors are needed to understand the mechanisms of replication and transmission of VOCs3. Here we show that the spike protein (S) from Alpha (also known as B.1.1.7) and Beta (B.1.351) VOCs had a greater affinity towards the human angiotensin-converting enzyme 2 (ACE2) receptor than that of the progenitor variant S(D614G) in vitro. Progenitor variant virus expressing S(D614G) (wt-S614G) and the Alpha variant showed similar replication kinetics in human nasal airway epithelial cultures, whereas the Beta variant was outcompeted by both. In vivo, competition experiments showed a clear fitness advantage of Alpha over wt-S614G in ferrets and two mouse models-the substitutions in S were major drivers of the fitness advantage. In hamsters, which support high viral replication levels, Alpha and wt-S614G showed similar fitness. By contrast, Beta was outcompeted by Alpha and wt-S614G in hamsters and in mice expressing human ACE2. Our study highlights the importance of using multiple models to characterize fitness of VOCs and demonstrates that Alpha is adapted for replication in the upper respiratory tract and shows enhanced transmission in vivo in restrictive models, whereas Beta does not overcome Alpha or wt-S614G in naive animals.

Ovine footrot: A review of current knowledge.

Footrot is a contagious foot disease mainly affecting sheep. It is caused by the Gram-negative anaerobic bacterium Dichelobacter nodosus. Warm, wet environmental conditions favour development of footrot, and under perfect conditions, it takes just 2-3 weeks from infection to manifestation of clinical signs. Affected sheep show lameness of various degrees and often graze while resting on their carpi. Local clinical signs vary in severity and extent from interdigital inflammation (benign footrot) to underrunning of the complete horn shoe in advanced stages of virulent footrot. Laboratory diagnosis ideally involves collection of four-foot interdigital swab samples followed by competitive real time PCR, allowing for detection of the presence of D. nodosus and differentiation between benign and virulent strains. Laboratory-based diagnostics at the flock level based on risk-based sampling and pooling of interdigital swab samples are recommended. The list of treatment options of individual sheep includes careful removal of the loose undermined horn, local or systemic administration of antimicrobials, systemic administration of non-steroidal anti-inflammatories (NSAIDs) and disinfectant footbathing. Strategies for control at the flock level are manifold and depend on the environmental conditions and the procedures traditionally implemented by the respective country. Generally, measures consist of treatment/culling of infected sheep, vaccination and prevention of reinfection of disease-free flocks. Gaining deeper insight into the beneficial effects of NSAIDs, screening for eco-friendly footbath solutions, developing better vaccines, including the development of a robust, reproducible infection model and elucidation of protective immune responses, as well as the elaboration of effective awareness training programs for sheep farmers, are relevant research gaps.

Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV.

The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1-10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-ß pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.

Prevalence of Dichelobacter nodosus and Ovine Footrot in German Sheep Flocks.

The bacterium Dichelobacter nodosus (D. nodosus) is the causative agent of ovine footrot. The aim of this field study was to determine the prevalence of D. nodosus in German sheep flocks. The sheep owners participated voluntarily in the study. More than 9000 sheep from 207 flocks were screened for footrot scores using a Footrot Scoring System from 0 to 5 and sampling each sheep using one interdigital swab for all four feet of the sheep. The detection and discrimination between benign and virulent strains was done employing a real-time PCR. Our results showed a mean prevalence of 42.93% of D. nodosus in German sheep on an animal level. Underrunning of hoof horn on at least one foot (Scores 3-5) was detected in 567 sheep (6.13%). Sheep with four clinically healthy feet were found through visual inspection in 47.85% of all animals included in this study. In total, 1117 swabs from sheep with four clinically healthy feet tested positive for D. nodosus. In 90.35% of the positive swabs, virulent D. nodosus were detected. Benign D. nodosus were detected in 4.74% of the D. nodosus-positive swabs while 4.91% tested positive for both, benign and virulent D. nodosus. In 59 flocks D. nodosus were not detected and in 115 flocks only virulent D. nodosus were found while seven flocks tested positive for benign strains.

Antimicrobial resistant and extended-spectrum ß-lactamase (ESBL) producing Escherichia coli isolated from fecal samples of African dromedary camels.

This study was conducted to determine the distribution of antimicrobial resistance among Escherichia coli isolated from feces of healthy dromedary camels in Kenya. A total of 162 fecal samples were cultivated for E. coli. Samples were also subcultivated to detect E. coli with extended-spectrum ß-lactamases (ESBLs). Antimicrobial susceptibility testing (AST) was performed by disk diffusion using a panel of 16 antimicrobials. In addition, isolates were screened for the presence of the plasmid-mediated colistin resistance genes mcr-1 to mcr-5. Samples from 20 (12.4%) of the camels contained antimicrobial resistant (AMR) E. coli, and 85% of the AMR isolates were multidrug resistant (MDR). The highest frequency of resistance was observed to tetracycline (11.7%), followed by ampicillin and streptomycin (both 10.5%), and sulfamethoxazole/trimethoprim (9.9%). Two (1.2%) of the isolates showed intermediate resistance to cefazolin and streptomycin, respectively. All the isolates were susceptible to amoxycillin/clavulanic acid, ciprofloxacin, fosfomycin, aztreonam and kanamycin, and 86.4% of the isolates were susceptible to all 16 antimicrobials used in this study. The prevalence of fecal carriage of ESBL producing E. coli was 0.6%. PCR and amplicon sequencing showed that the ESBL producer belonged to E. coli phylogenetic group A, sequence type (ST) 48, and harbored bla CTX-M-15. None of the isolates contained mcr genes. The results indicate that dromedary camels in Kenya may be reservoirs of AMR E. coli, including ESBL producers, that could potentially be transmitted to humans by direct contact or via the food chain.

An unusual case of bovine anthrax in the canton of Jura, Switzerland in 2017.

BACKGROUND: Anthrax caused by Bacillus anthracis is a zoonotic disease mainly affecting herbivores. The last Swiss outbreak was over 20 years ago. We describe a recent anthrax outbreak involving two cows from the same herd. One cow was designated as a peracute clinical case with sudden death and typical lung lesions, while the other cow presented with protracted fever and abortion. CASE PRESENTATION: On April 29th 2017, a 3.5-year-old Montbéliard dairy cow was found dead while out at pasture with haemorrhage from the nose. The veterinarian suspected pneumonia and performed a necropsy on site. Subsequently, a lung and liver sample were sent to the laboratory. Unexpectedly, Bacillus anthracis was isolated, a pathogen not found in Switzerland for decades. Several days later, a second cow from the same farm showed signs of abortion after protracted fever. Since these symptoms are not typical for anthrax, and the bacteria could not be demonstrated in blood samples from this animal, a necropsy was performed under appropriate biosafety measures. Subsequently, Bacillus anthracis could be isolated from the placenta and the sublumbal lymph nodes but not from the blood, liver, spleen and kidney. The outbreak strain (17OD930) was shown to belong to the lineage B.Br.CNEVA, the same as Swiss strains from previous outbreaks in the region. We speculate that the disease came from a temporarily opened cave system that is connected to an old carcass burial site and was flushed by heavy rainfall preceding the outbreak. CONCLUSION: Even in countries like Switzerland, where anthrax is very rare, new cases can occur after unusual weather conditions or ground disturbance. It is important for public officials to be aware of this risk to avoid possible spread.

Shiga toxin-producing Escherichia coli (STEC) isolated from fecal samples of African dromedary camels.

Shiga toxin-producing Escherichia coli (STEC) cause gastrointestinal illnesses including non-bloody or bloody diarrhoea, haemorrhagic colitis (HC), and the haemolytic uremic syndrome (HUS). To investigate the occurrence of STEC among grazing dromedaries from Kenya, E. coli isolated from fecal matter collected from 163 dromedaries on a large ranch were screened for the presence of stx1 and stx2. STEC strains were isolated and serotyped. Isolates were subjected to PCR for the subtyping of stx genes and for the detection of eae and ehx. In addition, whole genome sequencing (WGS) was carried out to detect further virulence genes and to determine the multilocus sequence types (MLST). Antimicrobial resistance profiles were determined by disk diffusion. STEC was isolated from 20 (12.3%) of the fecal samples. Thereof, nine (45%) isolates were STEC O156:H25, three (15%) isolates typed STEC O43:H2. The remaining isolates occurred as single serotypes or were O non-typeable. Eleven (55%) of the isolates harboured stx2a, nine (45%) eae, and 14 (70%) ehx, respectively. WGS revealed the presence of iss in 16 (80%), subAB in four (20%) and astA in two (10%) of the isolates, Furthermore, espA, tccP, nleA, nleB, tccP, and tir were found exclusively among STEC O156:H25. Eleven different sequence types (ST) were detected. The most prominent was ST300/ST5343, which comprised STEC O156:H25. All STEC isolates were pan susceptible to a panel of 16 antimicrobial agents. Overall, the results indicate that dromedary camels in Kenya may be reservoirs of STEC, including serotypes possessing virulence markers associated to disease in humans, such as STEC O156:H25. STEC in camels may represent a health hazard for humans with close contact to camels or to consumers of camel derived foodstuffs, such as unpasteurised camel milk.

Otitis in a cat associated with Corynebacterium provencense.

BACKGROUND: The role of corynebacteria in canine and feline otitis has not been investigated in detail; however, members of this genus are increasingly recognized as pathogens of otitis in both human and veterinary medicine. CASE PRESENTATION: Here we report the first case of feline otitis associated with the recently described species Corynebacterium provencense. A seven-month old cat presented with a head tilt and ataxia was diagnosed with peripheral vestibular syndrome associated with an otitis media/interna. This took place 6 weeks after resection of a polyp, having initially shown a full recovery with topical ofloxacin and glucocorticoid treatment. Bacteriology of an ear swab yielded a pure culture of corynebacteria, which could not be identified at the species level using routine methods. However, the 16S rRNA gene sequence was 100% identical to the recently published novel corynebacterium species, Corynebacterium provencense. Whole genome sequencing of the cat isolate and calculation of average nucleotide identity (99.1%) confirmed this finding. The cat isolate was found to contain additional presumptive iron acquisition genes that are likely to encode virulence factors. Furthermore, the strain tested resistant to clindamycin, penicillin and ciprofloxacin. The cat was subsequently treated with chloramphenicol, which lead to clinical improvement. CONCLUSION: Corynebacteria from otitis cases are not routinely identified at the species level and not tested for antimicrobial susceptibility in veterinary laboratories, as they are not considered major pathogens. This may lead to underreporting of this genus or animals being treated with inappropriate antimicrobials since corynebacteria are often resistant to multiple drugs.

Serological testing of cattle experimentally infected with Mycoplasma mycoides subsp. mycoides Small Colony using four different tests reveals a variety of seroconversion patterns.

BACKGROUND: To study the specific antibody response to infection with Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), the agent of Contagious Bovine Pleuropneumonia (CBPP), we examined three panels of sera collected during three experimental infection trials in African cattle. The methods used included an in-house complement fixation test (CFT), a commercially available CFT, a competitive antibody ELISA (cELISA) and the immunoblotting test (IBT). In addition, lung tissue samples were examined by culture. RESULTS: A total of 89% (51/59) of all experimentally infected animals tested positive on at least one of the serological tests throughout the trial. The specific antibody titres to the MmmSC infection became positive first by CFT (6 to 9 days post infection [dpi]), followed by IBT (9 to 13 dpi) and cELISA (13 to 16 dpi). Individual animals were found to display remarkably distinct seroconversion patterns, which allowed their classification into i) early high responders, ii) late high responders, and iii) low responders. In accordance with other studies, none of the present serological tests was capable of detecting all CBPP infected animals. CONCLUSION: Comparison of the assays' performance in terms of sensitivity and specificity raises serious questions as to their reliability for identification of infected individuals in the field. In view of these limitations, a combination of CFT and cELISA can markedly improve CBPP diagnosis at single-animal level.

Assessment of a novel multiplex real-time PCR assay for the detection of the CBPP agent Mycoplasma mycoides subsp. mycoides SC through experimental infection in cattle.

BACKGROUND: Mycoplasma mycoides subsp. mycoides SC is the pathogenic agent of contagious bovine pleuropneumonia (CBPP), the most important disease of cattle in Africa causing significant economic losses. The re-emergence of CBPP in Europe in the 1980s and 1990s illustrates that it is still a threat also to countries that have successfully eradicated the disease in the past. Nowadays, probe-based real-time PCR techniques are among the most advanced tools for a reliable identification and a sensitive detection of many pathogens, but only few protocols have been published so far for CBPP diagnosis. Therefore we developed a novel TaqMan®-based real-time PCR assay comprising the amplification of two independent targets (MSC_0136 and MSC_1046) and an internal exogenous amplification control in a multiplex reaction and evaluated its diagnostic performance with clinical samples. RESULTS: The assays detected 49 MmmSC strains from diverse temporal and geographical origin, but did not amplify DNA from 82 isolates of 20 non-target species confirming a specificity of 100%. The detection limit was determined to be 10 fg DNA per reaction for the MSC_0136 assay and 100 fg per reaction for the MSC_1046 assay corresponding to 8 and 80 genome equivalents, respectively. The diagnostic performance of the assay was evaluated with clinical samples from 19 experimentally infected cattle and from 20 cattle without CBPP and compared to those of cultivation and a conventional PCR protocol. The two rt-PCR tests proved to be the most sensitive methods and identified all 19 infected animals. The different sample types used were not equally suitable for MmmSC detection. While 94.7% of lung samples from the infected cohort were positively tested in the MSC_0136 assay, only 81% of pulmonal lymph nodes, 31% of mediastinal lymph nodes and 25% of pleural fluid samples gave a positive result. CONCLUSIONS: The developed multiplex rt-PCR assay is recommended as an efficient tool for rapid confirmation of a presumptive CBPP diagnosis in a well-equipped laboratory environment.

Isolation and characterization of intestinal Escherichia coli clones from wild boars in Germany.

Our understanding of the composition of Escherichia coli populations in wild boars is very limited. In order to obtain insight into the E. coli microflora of wild boars, we studied E. coli isolates from the jejunums, ileums, and colons of 21 wild boars hunted in five geographic locations in Germany. Ten isolates per section were subjected to clonal determination using pulsed-field gel electrophoresis. One representative isolate per clone was further investigated for virulence traits, phylogenetic affiliation, and antimicrobial susceptibility. Macrorestriction analysis of 620 isolates revealed a range of clone diversity among the sections and animals, with up to 9 and 16 different clones per section and animal, respectively. Most of the clones for a given animal were shared between two adjacent intestinal sections. The overall highest clonal diversity was observed within the colon. While the astA gene was present in a large number of clones, other virulence genes and hemolytic ability were detected only sporadically. Clones of all four ECOR groups dominated the intestinal sections. Phylogenetic analysis and the occurrence of virulence genes correlated with the isolation frequencies for clones. All E. coli clones from wild boars were susceptible to all antimicrobial agents tested. In conclusion, though several parameters (including an animal-specific and highly diverse E. coli clone composition, the simultaneous occurrence of single clones in two adjacent intestinal sections of a given animal, and a higher E. coli diversity in the large intestine than in the small intestine) of E. coli populations of wild boars were similar to those of previously described E. coli populations of conventionally reared domestic pigs, our data also indicate possible differences, as seen for the E. coli diversity in the large intestine, the occurrence of certain virulence genes and phylogenetic groups, and antimicrobial susceptibilities.

Multilocus sequence typing (MLST) of Mycoplasma hyopneumoniae: a diverse pathogen with limited clonality.

A multilocus sequence typing (MLST) scheme was established and evaluated for Mycoplasma hyopneumoniae, the etiologic agent of enzootic pneumonia in swine with the aim of defining strains. Putative target genes were selected by genome sequence comparisons. Out of 12 housekeeping genes chosen and experimentally validated, the 7 genes efp, metG, pgiB, recA, adk, rpoB, and tpiA were finally used to establish the MLST scheme. Their usefulness was assessed individually and in combination using a set of well-defined field samples and strains of M. hyopneumoniae. A reduction to the three targets showing highest variation (adk, rpoB, and tpiA) was possible resulting in the same number of sequence types as using the seven targets. The established MLST approach was compared with the recently described typing method using the serine-rich repeat motif-encoding region of the p146 gene. There was coherence between the two methods, but MLST resulted in a slightly higher resolution. Farms recognized to be affected by enzootic pneumonia were always associated with a single M. hyopneumoniae clone, which in most cases differed from farm to farm. However, farms in close geographic or operational contact showed identical clones as defined by MLST typing. Population analysis showed that recombination in M. hyopneumoniae occurs and that strains are very diverse with only limited clonality observed. Elaborate classical MLST schemes using multiple targets for M. hyopneumoniae might therefore be of limited value. In contrast, MLST typing of M. hyopneumoniae using the three genes adk, rpoB, and tpiA seems to be sufficient for epidemiological investigations by direct amplification of target genes from lysate of clinical material without prior cultivation.

Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine.

In vitro studies on the pathogenesis in swine have been hampered by the lack of relevant porcine cell lines. Since many bacterial infections are swine-specific, studies on pathogenic mechanisms require appropriate cell lines of porcine origin. We have characterized the permanent porcine intestinal epithelial cell line, IPEC-J2, using a variety of methods in order to assess the usefulness of this cell line as an in vitro infection model. Electron microscopic analyses and histochemical staining revealed the cells to be enterocyte-like with microvilli, tight junctions and glycocalyx-bound mucin. The functional integrity of monolayers was determined by transepithelial electrical resistance (TEER) measurements. Both commensal bacteria and important bacterial pathogens were chosen for study based on their principally different infection mechanisms: obligate extracellular Escherichia coli, facultative intracellular Salmonella and obligate intracellular Chlamydia. We determined the colonization and proliferation of the bacteria on and within the host cells and monitored the host cell response. We verified the expression of mRNAs encoding the cytokines IL-1alpha, -6, -7, -8, -18, TNF-alpha and GM-CSF, but not TGF-beta or MCP-1. IL-8 protein expression was enhanced by Salmonella invasion. We conclude that the IPEC-J2 cell line provides a relevant in vitro model system for porcine intestinal pathogen-host cell interactions.

Long-term clonal lineages within Campylobacter jejuni O:2 strains from different geographical regions and hosts.

The genomic diversity of 11 epidemiologically unrelated strains of Campylobacter jejuni (serotype O:2) isolated from different hosts and different geographical regions in Germany over a period of 15 years was studied and results were compared with the reference strain NCTC11168. By flagellin PCR-RFLP typing six fla types were identified, while macrorestriction analysis with three different restriction enzymes revealed almost identical patterns for two human and one bovine strain, even though they were isolated between 1984 and 1996. Interestingly, the PFGE and fla profiles from strain NCTC11168, which was originally isolated in 1977 from an outbreak case in Worcester (UK), were highly similar or even identical to the profiles of these strains. Besides this, mapping of selective genetic markers to the obtained restriction fragments by Southern blot hybridization showed a conserved localization of the investigated sequences in several strains. Our data confirmed considerable degrees of genomic conservation and the occurrence of long-term O:2 serotype-associated clonal lineages in C. jejuni in different geographical regions and hosts.

[Plasticity of bacterial genomes: pathogenicity islands and the locus of enterocyte effacement (LEE)]. / Plastizität bakterieller Genome: Pathogenitätsinseln und der Locus of Enterocyte Effacement (LEE).

Many bacterial virulence attributes, like toxins, adhesins, invasins, iron uptake systems, are encoded within specific regions of the bacterial genome. These in size varying regions are termed pathogenicity islands (PAIs) since they confer pathogenic properties to the respective micro-organism. Per definition PAIs are exclusively found in pathogenic strains and are often inserted near transfer-RNA genes. Nevertheless, non-pathogenic bacteria also possess foreign DNA elements that confer advantageous features, leading to improved fitness. These additional DNA elements as well as PAIs are termed genomic islands and were acquired during bacterial evolution. Significant G+C content deviation in pathogenicity islands with respect to the rest of the genome, the presence of direct repeat sequences at the flanking regions, the presence of integrase gene determinants as other mobility features,the particular insertion site (tRNA gene) as well as the observed genetic instability suggests that pathogenicity islands were acquired by horizontal gene transfer. PAIs are the fascinating proof of the plasticity of bacterial genomes. PAIs were originally described in human pathogenic Escherichia (E.) coli strains. In the meantime PAIs have been found in various pathogenic bacteria of humans, animals and even plants. The Locus of Enterocyte Effacement (LEE) is one particular widely distributed PAI of E coli. In addition, it also confers pathogenicity to the related species Citrobacter (C.) rodentium and Escherichia (E.) alvei. The LEE is an important virulence feature of several animal pathogens. It is an obligate PAI of all animal and human enteropathogenic E. coli (EPEC), and most enterohaemorrhegic E. coli (EHEC) also harbor the LEE. The LEE encodes a type III secretion system, an adhesion (intimin) that mediates the intimate contact between the bacterium and the epithelial cell, as well as various proteins which are secreted via the type III secretion system. The LEE encoded virulence features are responsible for the formation of so called attaching and effacing (AE) lesions in the intestinal epithelium. Due to its wide distribution in animal pathogens, LEE encoded antigens are suitable vaccine antigens. Acquisition and structure of the LEE pathogenicity island is the crucial point of numerous investigations. However, the evolution of the LEE, its origin and further spread in E. coli, are far from being resolved.