Phenotypic and genotypic assessment of iron acquisition in diverse bovine-associated non-aureus staphylococcal strains.

Although the role of iron in bacterial infections has been well described for Staphylococcus (S.) aureus, iron acquisition in (bovine-associated) non-aureus staphylococci and mammaliicocci (NASM) remains insufficiently mapped. This study aimed at elucidating differences between four diverse bovine NASM field strains from two species, namely S. chromogenes and S. equorum, in regards to iron uptake (with ferritin and lactoferrin as an iron source) and siderophore production (staphyloferrin A and staphyloferrin B) by investigating the relationship between the genetic basis of iron acquisition through whole genome sequencing (WGS) with their observed phenotypic behavior. The four field strains were isolated in a previous study from composite cow milk (CCM) and bulk tank milk (BTM) in a Flemish dairy herd. Additionally, two well-studied S. chromogenes isolates originating from a persistent intramammary infection and from a teat apex were included for comparative purpose in all assays. Significant differences between species and strains were identified. In our phenotypical iron acquisition assay, while lactoferrin had no effect on growth recovery for all strains in iron deficient media, we found that ferritin served as an effective source for growth recovery in iron-deficient media for S. chromogenes CCM and BTM strains. This finding was further corroborated by analyzing potential ferritin iron acquisition genes using whole-genome sequencing data, which showed that all S. chromogenes strains contained hits for all three proposed ferritin reductive pathway genes. Furthermore, a qualitative assay indicated siderophore production by all strains, except for S. equorum. This lack of siderophore production in S. equorum was supported by a quantitative assay, which revealed significantly lower or negligible siderophore amounts compared to S. aureus and S. chromogenes. The WGS analysis showed that all tested strains, except for S. equorum, possessed complete staphyloferrin A (SA)-synthesis and export operons, which likely explains the phenotypic absence of siderophore production in S. equorum strains. While analyzing the staphyloferrin A and staphyloferrin B operon landscapes for all strains, we noticed some differences in the proteins responsible for iron acquisition between different species. However, within strains of the same species, the siderophore-related proteins remained conserved. Our findings contribute valuable insights into the genetic elements associated with bovine NASM pathogenesis.

The role of Helicobacter suis, Fusobacterium gastrosuis, and the pars oesophageal microbiota in gastric ulceration in slaughter pigs receiving meal or pelleted feed.

This study investigated the role of causative infectious agents in ulceration of the non-glandular part of the porcine stomach (pars oesophagea). In total, 150 stomachs from slaughter pigs were included, 75 from pigs that received a meal feed, 75 from pigs that received an equivalent pelleted feed with a smaller particle size. The pars oesophagea was macroscopically examined after slaughter. (q)PCR assays for H. suis, F. gastrosuis and H. pylori-like organisms were performed, as well as 16S rRNA sequencing for pars oesophagea microbiome analyses. All 150 pig stomachs showed lesions. F. gastrosuis was detected in 115 cases (77%) and H. suis in 117 cases (78%), with 92 cases (61%) of co-infection; H. pylori-like organisms were detected in one case. Higher infectious loads of H. suis increased the odds of severe gastric lesions (OR = 1.14, p = 0.038), while the presence of H. suis infection in the pyloric gland zone increased the probability of pars oesophageal erosions [16.4% (95% CI 0.6-32.2%)]. The causal effect of H. suis was mediated by decreased pars oesophageal microbiome diversity [-1.9% (95% CI - 5.0-1.2%)], increased abundances of Veillonella and Campylobacter spp., and decreased abundances of Lactobacillus, Escherichia-Shigella, and Enterobacteriaceae spp. Higher infectious loads of F. gastrosuis in the pars oesophagea decreased the odds of severe gastric lesions (OR = 0.8, p = 0.0014). Feed pelleting had no significant impact on the prevalence of severe gastric lesions (OR = 1.72, p = 0.28). H. suis infections are a risk factor for ulceration of the porcine pars oesophagea, probably mediated through alterations in pars oesophageal microbiome diversity and composition.

Presence of potentially novel Helicobacter pylori-like organisms in gastric samples from cats and dogs.

While seven gastric non-Helicobacter pylori Helicobacter (NHPH) species are known to commonly colonize the stomach of cats and dogs, the potential of H. pylori and H. pylori-like organisms to infect animals remains controversial and was investigated in this study using gastric samples of 20 cats and 27 dogs. A Helicobacter genus-specific 16 S rRNA PCR assay, H. pylori-specific ureAB and glmM PCR assays and a nested PCR detecting 23 S rRNA in a Helicobacter genus-specific manner in a first round of PCR and a H. pylori-specific manner in a second round, were performed in combination with sequencing. Histopathological and anti-Helicobacter immunohistochemical evaluations were also performed. Based on 16 S rRNA sequence analysis, 39/47 animals (83%) appeared infected with canine/feline gastric NHPHs in the corpus and/or antrum. H. pylori-specific ureAB amplicons were obtained in samples of 22 stomachs (47%). One canine antrum sample positive in the ureAB assay was also positive in the H. pylori-specific glmM assay. While 36/47 (77%) animals had a positive sample in the first round of the nested 23 S rRNA PCR assay, all samples were negative in the second round. Sequence analysis of obtained amplicons and immunohistochemistry point towards the presence of unidentified H. pylori-like organisms in cats and dogs. Histopathological examination suggests a low pathogenic significance of the gastric Helicobacter spp. present in these animals. In conclusion, cats and dogs may be (co-)infected with gastric Helicobacter organisms other than the known gastric NHPHs. Culture and isolation should be performed to confirm this hypothesis.

Whole genome sequencing to study antimicrobial resistance and RTX virulence genes in equine Actinobacillus isolates.

Actinobacillus equuli is mostly associated with disease in horses and is most widely known as the causative agent of sleepy foal disease. Even though existing phenotypic tools such as biochemical tests, 16S rRNA gene sequencing, and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) can be used to identify members of the Actinobacillus genus, these methods struggle to differentiate between certain species and do not allow strain, virulence, and antimicrobial susceptibility typing. Hence, we performed in-depth analysis of 24 equine Actinobacillus isolates using phenotypic identification and susceptibility testing on the one hand, and long-read nanopore whole genome sequencing on the other hand. This allowed to address strain divergence down to the whole genome single nucleotide polymorphism (SNP) level. While lowest resolution was observed for 16S rRNA gene classification, a new multi-locus sequence typing (MLST) scheme allowed proper classification up to the species level. Nevertheless, a SNP-level analysis was required to distinguish A. equuli subspecies equuli and haemolyticus. Our data provided first WGS data on Actinobacillus genomospecies 1, Actinobacillus genomospecies 2, and A. arthritidis, which allowed the identification of a new Actinobacillus genomospecies 1 field isolate. Also, in-depth characterization of RTX virulence genes provided information on the distribution, completeness, and potential complementary nature of the RTX gene operons within the Actinobacillus genus. Even though overall low prevalence of acquired resistance was observed, two plasmids were identified conferring resistance to penicillin-ampicillin-amoxicillin and chloramphenicol in one A. equuli strain. In conclusion our data delivered new insights in the use of long-read WGS in high resolution identification, virulence gene typing, and antimicrobial resistance (AMR) of equine Actinobacillus species.

Review on the methodology to assess respiratory tract lesions in pigs and their production impact.

Porcine respiratory disease is one of the most important health problems in pig production worldwide. Cranioventral pulmonary consolidation (CVPC) and pleurisy are the two most common lesions in the respiratory tract of slaughtered pigs. The present review paper discusses pathogens involved in the lesions, lesion prevalence, scoring systems, advantages and disadvantages of slaughterhouse examination, and the impact of CVPC and pleurisy on performance, carcass, and meat quality. Cranioventral pulmonary consolidation and pleurisy in slaughter pigs are characteristic for infections with Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae, respectively, although other pathogens may cause similar lesions and/or be involved in their development. The overall prevalence of CVPC and pleurisy in slaughter pigs are still high, being the prevalence of CVPC generally higher than that of chronic pleurisy. The advantages and disadvantages of slaughterhouse examination are discussed in relation to practical aspects, the assessment of lesions, the number and representativeness of the examined animals and the interpretation and value of the results for the stakeholders. The main scoring methods for CVPC and pleurisy are shortly reviewed. In general, scoring methods can be applied rapidly and easily, although significant variation due to abattoir and observer remains. Artificial intelligence-based technologies that automatically score lesions and facilitate processing of data may aid solving these problems. Cranioventral pulmonary consolidation and pleurisy have a major negative impact on pig performance, and the effects increase the extension of the lesions and/or presence of multiple lesions. The performance losses caused by these lesions, however, vary significantly between studies and farms, possibly due to differences in study population and used methodology. Both lesions also have a negative impact on different carcass and meat quality parameters, leading to increased risk for poor processing and storage of the carcasses. Monitoring lung lesions of slaughter pigs should be optimized and implemented routinely; however, it is recommended to complement this information with farm data and laboratory results for specific pathogens.

Long-term follow-up of Mycoplasma hyopneumoniae-specific immunity in vaccinated pigs.

Mycoplasma hyopneumoniae is the primary agent of enzootic pneumonia in pigs. To minimize the economic losses caused by this disease, M. hyopneumoniae vaccination is commonly practiced. However, the persistence of M. hyopneumoniae vaccine-induced immunity, especially the cell-mediated immunity, till the moment of slaughter has not been investigated yet. Therefore, on two commercial farms, 25 pigs (n = 50) received a commercial bacterin intramuscularly at 16 days of age. Each month, the presence of M. hyopneumoniae-specific serum antibodies was analyzed and the proliferation of and TNF-α, IFN-γ and IL-17A production by different T cell subsets in blood was assessed using recall assays. Natural infection with M. hyopneumoniae was assumed in both farms. However, the studied pigs remained M. hyopneumoniae negative for almost the entire trial. Seroconversion was not observed after vaccination and all pigs became seronegative at two months of age. The kinetics of the T cell subset frequencies was similar on both farms. Mycoplasma hyopneumoniae-specific cytokine-producing CD4+CD8+ T cells were found in blood of pigs from both farms at one month of age but decreased significantly with increasing age. On the other hand, T cell proliferation after in vitro M. hyopneumoniae stimulation was observed until the end of the fattening period. Furthermore, differences in humoral and cell-mediated immune responses after M. hyopneumoniae vaccination were not seen between pigs with and without maternally derived antibodies. This study documents the long-term M. hyopneumoniae vaccine-induced immune responses in fattening pigs under field conditions. Further research is warranted to investigate the influence of a natural infection on these responses.

Metabolites of non-aureus staphylococci affect the ability of Staphylococcus aureus to adhere to and internalize into bovine mammary epithelial cells.

This study investigated whether cell-free supernatants (SN) from four bovine non-aureus staphylococcal (NAS) isolates prevent Staphylococcus aureus adhesion to and internalization into bovine mammary epithelial cells (MAC-T cells) and if so, to determine whether such effects were potentially associated with the S. aureus accessory gene regulator (agr) system. Overall, we demonstrated that all SN obtained from the NAS isolates promoted adhesion of a S. aureus agr+ strain to, yet reduced the internalization into MAC-T cells, while similar effects were not observed for its agr- mutant strain. Our findings provide novel anti-virulence strategies for treating and controlling bovine S. aureus mastitis.

Priming of the murine mammary gland with Staphylococcus chromogenes IM reduces bacterial growth of Streptococcus uberis: a proof-of-concept study.

Streptococcus uberis is a major causative agent of bovine mastitis, an inflammation of the mammary gland with substantial economic consequences. To reduce antibiotic use in animal agriculture, alternative strategies to treat or prevent mastitis are being investigated. Bovine-associated non-aureus staphylococci are proposed in that respect due to their capacity to inhibit the in vitro growth of S. uberis. We demonstrate that priming the murine mammary gland with Staphylococcus chromogenes IM reduces S. uberis growth in comparison with non-primed glands. The innate immune system is activated by increasing IL-8 and LCN2, which may explain this decreased growth.

Drivers of salamander extirpation mediated by Batrachochytrium salamandrivorans.

The recent arrival of Batrachochytrium salamandrivorans in Europe was followed by rapid expansion of its geographical distribution and host range, confirming the unprecedented threat that this chytrid fungus poses to western Palaearctic amphibians. Mitigating this hazard requires a thorough understanding of the pathogen's disease ecology that is driving the extinction process. Here, we monitored infection, disease and host population dynamics in a Belgian fire salamander (Salamandra salamandra) population for two years immediately after the first signs of infection. We show that arrival of this chytrid is associated with rapid population collapse without any sign of recovery, largely due to lack of increased resistance in the surviving salamanders and a demographic shift that prevents compensation for mortality. The pathogen adopts a dual transmission strategy, with environmentally resistant non-motile spores in addition to the motile spores identified in its sister species B. dendrobatidis. The fungus retains its virulence not only in water and soil, but also in anurans and less susceptible urodelan species that function as infection reservoirs. The combined characteristics of the disease ecology suggest that further expansion of this fungus will behave as a 'perfect storm' that is able to rapidly extirpate highly susceptible salamander populations across Europe.

Helicobacter bizzozeronii infection in a girl with severe gastric disorders in México: case report.

BACKGROUND: Gastric non-Helicobacter pylori helicobacters (NHPH) naturally colonize the stomach of animals. In humans, infection with these bacteria is associated with chronic active gastritis, peptic ulceration and MALT-lymphoma. H. bizzozeronii belongs to these NHPH and its prevalence in children is unknown. CASE PRESENTATION: This case report describes for the first time a NHPH infection in a 20-month-old girl with severe gastric disorders in Mexico. The patient suffered from melena, epigastric pain, and bloating. Gastroscopy showed presence of a Hiatus Hill grade I, a hemorrhagic gastropathy in the fundus and gastric body, and a Forrest class III ulcer in the fundus. Histopathologic examination revealed a chronic active gastritis with presence of long, spiral-shaped bacilli in the glandular lumen. Biopsies from antrum, body and incisure were negative for presence of H. pylori by culture and PCR, while all biopsies were positive for presence of H. bizzozeronii by PCR. Most likely, infection occurred through intense contact with the family dog. The patient received a triple therapy consisting of a proton pump inhibitor, clarithromycin, and amoxicillin for 14 days, completed with sucralfate for 6 weeks, resulting in the disappearance of her complaints. CONCLUSION: The eradication could not be confirmed, although it was suggested by clear improvement of symptoms. This case report further emphasizes the zoonotic importance of NHPH. It can be advised to routinely check for presence of both H. pylori and NHPH in human patients with gastric complains.

Gastric Helicobacter species associated with dogs, cats and pigs: significance for public and animal health.

This article focuses on the pathogenic significance of Helicobacter species naturally colonizing the stomach of dogs, cats and pigs. These gastric "non-Helicobacter (H.) pylori Helicobacter species" (NHPH) are less well-known than the human adapted H. pylori. Helicobacter suis has been associated with gastritis and decreased daily weight gain in pigs. Several studies also attribute a role to this pathogen in the development of hyperkeratosis and ulceration of the non-glandular stratified squamous epithelium of the pars oesophagea of the porcine stomach. The stomach of dogs and cats can be colonized by several Helicobacter species but their pathogenic significance for these animals is probably low. Helicobacter suis as well as several canine and feline gastric Helicobacter species may also infect humans, resulting in gastritis, peptic and duodenal ulcers, and low-grade mucosa-associated lymphoid tissue lymphoma. These agents may be transmitted to humans most likely through direct or indirect contact with dogs, cats and pigs. Additional possible transmission routes include consumption of water and, for H. suis, also consumption of contaminated pork. It has been described that standard H. pylori eradication therapy is usually also effective to eradicate the NHPH in human patients, although acquired antimicrobial resistance may occasionally occur and porcine H. suis strains are intrinsically less susceptible to aminopenicillins than non-human primate H. suis strains and other gastric Helicobacter species. Virulence factors of H. suis and the canine and feline gastric Helicobacter species include urease activity, motility, chemotaxis, adhesins and gamma-glutamyl transpeptidase. These NHPH, however, lack orthologs of cytotoxin-associated gene pathogenicity island and vacuolating cytotoxin A, which are major virulence factors in H. pylori. It can be concluded that besides H. pylori, gastric Helicobacter species associated with dogs, cats and pigs are also clinically relevant in humans. Although recent research has provided better insights regarding pathogenic mechanisms and treatment strategies, a lot remains to be investigated, including true prevalence rates, exact modes of transmission and molecular pathways underlying disease development and progression.

Molecular detection of Helicobacter spp. and Fusobacterium gastrosuis in pigs and wild boars and its association with gastric histopathological alterations.

Besides Helicobacter pylori, a Gram-negative bacterium that may cause gastric disorders in humans, non-Helicobacter pylori helicobacters (NHPH) may also colonize the stomach of humans and animals. In pigs, H. suis can induce gastritis and may play a role in gastric ulcer disease, possibly in association with Fusobacterium gastrosuis. In the present study, gastric samples from 71 slaughtered pigs and 14 hunted free range wild boars were tested for the presence of DNA of F. gastrosuis and gastric Helicobacter species associated with pigs, dogs cats and humans, using species-specific PCR assays, followed by sequencing of the amplicon. These gastric samples were also histopathologically evaluated. Almost all the pigs presented gastritis (95.8%). Helicobacter spp. were detected in 78.9% and F. gastrosuis in 35.2% of the animals. H. suis was the most frequently identified Helicobacter species (57.7% of the animals), followed by a H. pylori-like species (50.7%) and less often H. salomonis and H. felis (each in 2.8% of the animals). H. suis was most often detected in the glandular (distal) part of the stomach (pars oesophagea 9.9%, oxyntic mucosa 35.2%, antral mucosa 40.8%), while the H. pylori-like species was mainly found in the non-glandular (proximal) part of the stomach (pars oesophagea 39.4%, oxyntic mucosa 14.1%, antral mucosa 4.2%). The great majority of wild boars were also affected with gastritis (71.4%) and Helicobacter spp. and F. gastrosuis were detected in 64.3% and 42.9% of the animals, respectively. H. bizzozeronii and H. salomonis were the most frequently detected Helicobacter species, while a H. pylori-like species and H. suis were only occasionally identified. These findings suggest that these microorganisms can colonize the stomach of both porcine species and may be associated with gastric pathology. This should, however, be confirmed through bacterial isolation. This is the first description of the presence of F. gastrosuis DNA in the stomach of wild boars and a H. pylori-like species in the pars oesophagea of the porcine stomach.

Influence of Mycoplasma hyopneumoniae natural infection on the respiratory microbiome diversity of finishing pigs.

Mycoplasma (M.) hyopneumoniae interacts with the respiratory microbiota and facilitates colonization of other pathogens. The present study investigated the pulmonary and nasal microbiota of M. hyopneumoniae-infected and M. hyopneumoniae-free pigs. Sixty-six pigs from three commercial herds were selected at the end of the finishing phase: 44 originated from two M. hyopneumoniae-positive herds and 22 from a M. hyopneumoniae-negative farm. At the slaughterhouse, samples of nasal turbinate (NT) and bronchus-alveolar lavage fluid (BALF) were collected. DNA was extracted with a commercial kit and the infection status was confirmed by qPCR. All samples from the same herd were pooled, and next-generation sequencing based on the hypervariable region V3-V4 of the 16 s bacterial rDNA was performed. Data analysis included the taxonomic analysis, Alpha diversity indexes, and Principal coordinates analysis (Pcoa) using Jaccard, Bray-Curtis, Weighted Unifrac, and Unweighted Unifrac distances. All pigs from the infected herds tested PCR positive for M. hyopneumoniae, whereas all pigs from the negative farm were negative. There was a greater diversity of microorganisms in BALF when compared to NT samples in all the farms. BALF samples from infected animals showed higher abundance of M. hyopneumoniae than NT samples and a predominance of Pasteurella multocida among the main species identified, which was also abundant in the M. hyopneumoniae-free herd. PCoa diagrams indicated that for most of the samples, dissimilarity on bacterial composition was observed, regardless of infection status and sample type. Therefore, the lung microbiota was modulated by M. hyopneumoniae infection, which could play a role in the pathogenesis of M. hyopneumoniae-disease.

Helicobacter species binding to the human gastric mucosa.

Helicobacter pylori infects half of the world population, being associated with several gastric disorders, such as chronic gastritis and gastric carcinoma. The Helicobacter genus also includes other gastric helicobacters, such as H. heilmannii¸ H. ailurogastricus, H. suis, H. felis, H. bizzozeronii, and H. salomonis. These gastric helicobacters colonize both the human and animal stomach. The prevalence of gastric non-Helicobacter pylori Helicobacter (NHPH) species in humans has been described as low, and the in vitro binding to the human gastric mucosa was never assessed. Herein, human gastric tissue sections were used for the evaluation of the tissue glycophenotype and for the binding of gastric NHPH strains belonging to different species. Histopathological evaluation showed that 37.5% of the patients enrolled in our cohort presented chronic gastritis, while the presence of neutrophil or eosinophilic activity (chronic active gastritis) was observed in 62.5% of the patients. The secretor phenotype was observed in 68.8% of the individuals, based on the expression of Lewis B antigen and binding of the UleX lectin. The in vitro binding assay showed that all the NHPH strains evaluated were able to bind, albeit in low frequency, to the human gastric mucosa. The H. heilmannii, H. bizzozeronii, and H. salomonis strains displayed the highest binding ability both to the gastric superficial epithelium and to the deep glands. Interestingly, we observed binding of NHPH to the gastric mucosa of individuals with severe chronic inflammation and intestinal metaplasia, suggesting that NHPH binding may not be restricted to the healthy gastric mucosa or slight chronic gastritis. Furthermore, the in vitro binding of NHPH strains was observed both in secretor and non-secretor individuals in a similar frequency. In conclusion, this study is the first report of the in vitro binding ability of gastric NHPH species to the human gastric mucosa. The results suggest that other glycans, besides the Lewis antigens, could be involved in the bacterial adhesion mechanism; however, the molecular intervenients remain unknown.

Brachyspira Species Avidity to Colonic Mucins from Pigs with and without Brachyspira hyodysenteriae Infection Is Species Specific and Varies between Strains.

Brachyspira hyodysenteriae is commonly associated with swine dysentery (SD), a disease that has an economic impact on the swine industry. B. hyodysenteriae infection results in changes to the colonic mucus niche with massive mucus induction, which substantially increases the number of B. hyodysenteriae binding sites in the mucus. We previously determined that a B. hyodysenteriae strain binds to colon mucins in a manner that differs between pigs and mucin types. Here, we investigated if adhesion to mucins is a trait observed across a broad set of B. hyodysenteriae strains and isolates and furthermore at a genus level (B. innocens, B. pilosicoli, B. murdochii, B. hampsonii, and B. intermedia strains). Our results show that binding to mucins appears to be specific to B. hyodysenteriae, and within this species, the binding ability to mucins varies between strains/isolates, increases for mucins from pigs with SD, and is associated with sialic acid epitopes on mucins. Infection with B. hyodysenteriae strain 8dII results in mucin glycosylation changes in the colon, including a shift in sialic acid-containing structures. Thus, we demonstrate through hierarchical cluster analysis and orthogonal projections to latent structures discriminant analysis (OPLS-DA) models of the relative abundances of sialic acid-containing glycans that sialic acid-containing structures in the mucin O-glycome are good predictors of B. hyodysenteriae strain 8dII infection in pigs. The results emphasize the role of sialic acids in governing B. hyodysenteriae interactions with its host, which may open perspectives for therapeutic strategies.

Evaluation of Nanopore Sequencing as a Diagnostic Tool for the Rapid Identification of Mycoplasma bovis from Individual and Pooled Respiratory Tract Samples.

Rapid identification of Mycoplasma bovis infections in cattle is a key factor to guide antimicrobial therapy and biosecurity measures. Recently, Nanopore sequencing became an affordable diagnostic tool for both clinically relevant viruses and bacteria, but the diagnostic accuracy for M. bovis identification is undocumented. Therefore, in this study Nanopore sequencing was compared to rapid identification of M. bovis with matrix-assisted laser desorption ionization-time of flight mass spectrometry (RIMM) and a triplex real-time PCR assay in a Bayesian latent class model (BLCM) for M. bovis in bronchoalveolar lavage fluid (BALf) samples obtained from calves. In practice, pooling of samples is often used to save money, but the influence on diagnostic accuracy has not been described for M. bovis. Therefore, a convenience sample of 17 pooled samples containing 5 individual BALf samples per farm was analyzed as well. The results for the pooled samples were compared with those for the individual samples to determine sensitivity and specificity. The BLCM showed good sensitivity (77.3% [95% credible interval, 57.8 to 92.8%]) and high specificity (97.4% [91.5 to 99.7%]) for Nanopore sequencing, compared to RIMM (sensitivity, 93.0% [76.8 to 99.5%]; specificity, 91.3% [82.5 to 97.0%]) and real-time PCR (sensitivity, 94.6% [89.7 to 97.7%]; specificity, 86.0% [76.1 to 93.6%]). Sensitivity and specificity of pooled analysis for M. bovis were 85.7% (95% confidence interval, 59.8 to 111.6%) and 90.0% (71.4 to 108.6%%), respectively, for Nanopore sequencing and 100% (100% to 100%) and 88.9% (68.4 to 109.4%) for RIMM. In conclusion, Nanopore sequencing is a rapid, reliable tool for the identification of M. bovis. To reduce costs and increase the chance of M. bovis identification, pooling of 5 samples for Nanopore sequencing and RIMM is possible.

Perspectives for improvement of Mycoplasma hyopneumoniae vaccines in pigs.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the primary agents involved in the porcine respiratory disease complex, economically one of the most important diseases in pigs worldwide. The pathogen adheres to the ciliated epithelium of the trachea, bronchi, and bronchioles, causes damage to the mucosal clearance system, modulates the immune system and renders the animal more susceptible to other respiratory infections. The pathogenesis is very complex and not yet fully understood. Cell-mediated and likely also mucosal humoral responses are considered important for protection, although infected animals are not able to rapidly clear the pathogen from the respiratory tract. Vaccination is frequently practiced worldwide to control M. hyopneumoniae infections and the associated performance losses, animal welfare issues, and treatment costs. Commercial vaccines are mostly bacterins that are administered intramuscularly. However, the commercial vaccines provide only partial protection, they do not prevent infection and have a limited effect on transmission. Therefore, there is a need for novel vaccines that confer a better protection. The present paper gives a short overview of the pathogenesis and immune responses following M. hyopneumoniae infection, outlines the major limitations of the commercial vaccines and reviews the different experimental M. hyopneumoniae vaccines that have been developed and tested in mice and pigs. Most experimental subunit, DNA and vector vaccines are based on the P97 adhesin or other factors that are important for pathogen survival and pathogenesis. Other studies focused on bacterins combined with novel adjuvants. Very few efforts have been directed towards the development of attenuated vaccines, although such vaccines may have great potential. As cell-mediated and likely also humoral mucosal responses are important for protection, new vaccines should aim to target these arms of the immune response. The selection of proper antigens, administration route and type of adjuvant and carrier molecule is essential for success. Also practical aspects, such as cost of the vaccine, ease of production, transport and administration, and possible combination with vaccines against other porcine pathogens, are important. Possible avenues for further research to develop better vaccines and to achieve a more sustainable control of M. hyopneumoniae infections are discussed.

Transfer of Mycoplasma hyopneumoniae-specific cell mediated immunity to neonatal piglets.

Mycoplasma hyopneumoniae is the primary agent of enzootic pneumonia in pigs. Although cell mediated immunity (CMI) may play a role in protection against M. hyopneumoniae, its transfer from sows to their offspring is poorly characterized. Therefore, maternally-derived CMI was studied in piglets from vaccinated and non-vaccinated sows. The potential influence of cross-fostering before colostrum ingestion on the transfer of CMI from dam to piglets was also investigated. Six M. hyopneumoniae vaccinated sows from an endemically infected herd and 47 of their piglets, of which 24 piglets were cross-fostered, were included, as well as three non-vaccinated control sows from an M. hyopneumoniae-free herd and 24 of their piglets. Vaccinated sows received a commercial bacterin intramuscularly at 6 and 3 weeks prior to farrowing. The TNF-α, IFN-γ and IL-17A production by different T-cell subsets in blood of sows, colostrum and blood of piglets was assessed using a recall assay. In blood of sows cytokine producing T-cells were increased upon M. hyopneumoniae vaccination. Similarly, M. hyopneumoniae-specific T-cells were detected in blood of 2-day-old piglets born from these vaccinated sows. In contrast, no M. hyopneumoniae-specific cytokine producing T-cells were found in blood of piglets from control sows. No difference was found in M. hyopneumoniae-specific CMI between cross-fostered and non-cross-fostered piglets. In conclusion, different M. hyopneumoniae-specific T-cell subsets are transferred from the sow to the offspring. Further studies are required to investigate the role of these transferred cells on immune responses in piglets and their potential protective effect against M. hyopneumoniae infections.

Metabolites of bovine-associated non-aureus staphylococci influence expression of Staphylococcus aureus agr-related genes in vitro.

Communications via quorum sensing (QS) between non-aureus staphylococci (NAS) and Staphylococcus (S.) aureus in the bovine mammary gland remains largely unexplored. We determined whether 34 S. chromogenes, 11 S. epidermidis, and 14 S. simulans isolates originating from bovine milk samples and teat apices were able to regulate the QS of S. aureus, and if so, how in vitro growth inhibition of S. aureus by NAS, or NAS metabolites, or NAS cells themselves play a role in this process. In co-culture with S. aureus we observed that these 3 NAS species in general downregulated the expression of rnaIII, the effector molecule of the QS system, but this effect was more pronounced in S. chromogenes and S. simulans isolates than in S. epidermidis isolates. In vitro growth inhibition of S. aureus by NAS resulted in a small underestimation of the downregulating effect of NAS on rnaIII expression of S. aureus. Additionally, the culture supernatant of these NAS isolates and supernatant treated with proteinase K expressed greater regulatory activity over S. aureus virulence genes rnaIII, hla, and spa than washed NAS cells suspended in sterile water. These microbial interactions may influence S. aureus virulence and pathogenesis within the host. Isolation and identification of NAS metabolites affecting the QS system of S. aureus might help to develop alternative strategies for treatment and control of S. aureus mastitis.

Bovine-associated non-aureus staphylococci suppress Staphylococcus aureus biofilm dispersal in vitro yet not through agr regulation.

Biofilm formation is a significant virulence factor in Staphylococcus (S.) aureus strains causing subclinical mastitis in dairy cows. A role of environmental signals and communication systems in biofilm development, such as the agr system in S. aureus, is suggested. In the context of multispecies biofilm communities, the presence of non-aureus staphylococci (NAS) might influence S. aureus colonization of the bovine mammary gland, yet, such interspecies interactions have been poorly studied. We determined whether 34 S. chromogenes, 11 S. epidermidis, and 14 S. simulans isolates originating from bovine milk samples and teat apices (TA) were able to affect biofilm formation and dispersion of S. aureus, and if so, how isolate traits such as the capacity to regulate the S. aureus agr quorum sensing system are determinants in this process. The capacity of an agr-positive S. aureus strain to form biofilm was increased more in the presence of S. chromogenes than in the presence of S. simulans and S. epidermidis isolates and in the presence of NAS isolates that do not harbor biofilm related genes. On the other hand, biofilm dispersion of this particular S. aureus strain was suppressed by NAS as a group, an effect that was more pronounced by isolates from TA. Furthermore, the observed effects on biofilm formation and dispersion of the agr-positive S. aureus strain as well as of an agr-negative S. aureus strain did not depend on the capacity of NAS to repress the agr system.