Streptococcus suis (S. suis) expresses a specific immunoglobulin M (IgM) protease, designated IdeSsuis. Vaccination with recombinant IdeSsuis elicits protection against S. suis serotype 2. This study was initiated by the finding that the isogenic deletion mutant ΔideSsuis shows a hypervirulent phenotype in 4-week-old piglets, presumably in association with maternally derived antibodies against IdeSsuis. This prompted us to conduct a field study to test the working hypothesis that maternal αIdeSsuis IgG antibodies are biologically relevant. Investigations in one herd with autogenous S. suis vaccination pre-farrowing showed a significant correlation of IgG levels against IdeSsuis in serum of 2-week-old-piglets and levels in colostrum. These antibody levels declined over the subsequent weeks of life. Levels of IgM binding to S. suis in sera of 2-week-old piglets did not correlate with respective levels in colostrum but with the amount of colostrum taken up. Bactericidal assays with wild type (wt) and ideSsuis mutants demonstrated that expression of functional or nonfunctional full-length IdeSsuis results in increased killing of S. suis serotype 2 in the blood of 2-week-old piglets. This phenotype disappeared as piglets aged. Linear mixed-effects models confirmed that serum IgG against IdeSsuis exerts a significant, time-independent effect on survival of S. suis wt and ∇ideSsuis_C195S, expressing a nonfunctional variant of IdeSsuis, but not ΔideSsuis. Furthermore, IgM binding to S. suis wt influenced survival of wt and ∇ideSsuis_C195S in a time-dependent manner. This is in line with the concept that αIdeSsuis IgG are protective through induction of fragment crystallizable (Fc)-mediated opsonophagocytosis rather than neutralization of the IgM protease, at least in suckling piglets.

Bordetella bronchiseptica is a gram-negative bacterium contributing to respiratory diseases in many different animal species. In the swine population, it occurs frequently and plays a role in the Porcine Respiratory Disease Complex as well as in the pathogenesis of atrophic rhinitis. The dermonecrotic toxin (DNT) is involved in the destruction of the nasal conchae, a hallmark of atrophic rhinitis, and several studies have shown the effects of DNT on osteoblastic cells. Surprisingly, only little is known about the interactions of DNT and respiratory epithelial cells. Thus, we investigated the influence of DNT on porcine respiratory epithelial cells during mono- and co-infections in vitro. For this, we infected porcine precision-cut lung slices and air-liquid interface cultures with a DNT-positive B. bronchiseptica wild-type strain and its isogenic DNT-deficient mutant strain. For co-infection experiments, a Streptococcus suis serotype 2 wild-type strain was used. We evaluated cytotoxic effects and colonization of both pathogens, as well as the pro-inflammatory cytokine response of the host cells. Remarkably, DNT neither contributed to the cytotoxic effects of B. bronchiseptica nor did it affect bacterial colonization. Regarding the cytokine response, pro-inflammatory cytokines were expressed mainly upon infection with B. bronchiseptica but hardly after infection with S. suis, whereas co-infection with both pathogens had an amplifying effect on cytokine expression after prolonged infection, independently of DNT. Concluding, we found no evidence that DNT contributes to the early stages of infection with B. bronchiseptica and S. suis in in vitro models of the porcine respiratory tract.

Respiratory infections of livestock represent a major health issue for the animals and cause high economic losses for the farmers. Still, little is known about the intricate interactions between host cells and the many different pathogens that cause respiratory diseases, leaving a substantial knowledge gap to be filled in order to develop effective therapies. Immortalized cell lines and two-dimensional cultures of primary respiratory epithelial cells do not reflect the complex architecture and functionality of the respiratory tract tissues. Thus, it is essential to develop and apply appropriate primary cell culture systems to study respiratory diseases. In human research, the use of complex cell culture systems, such as air-liquid interface (ALI) cultures, organoids and lung-on-chip, has proceeded significantly during the last years, whereas in veterinary research, these models are only rarely used. Nevertheless, there are several three-dimensional, primary cell culture systems available to study respiratory infections of livestock. Here, we give an overview on models that are currently used in this field: nasal mucosa explants, tracheal organ cultures, ALI cultures, and precision-cut lung slices. All these models align with the 3R principle, as they can replace animal experiments to some extent and the tissue material for these culture systems can be obtained from abattoirs or veterinary research facilities. We aim to encourage other researchers to use these versatile cell culture systems to drive investigations of respiratory tract infections of livestock forward. Finally, these models are not limited to infection research, but can also be applied in other research fields and can be transferred to other animal species than livestock.

BackgroundThe interconnectedness of human, animal, and environmental health drives emerging threats, such as antimicrobial-resistant pathogens. The widespread use of the same antimicrobials in both human and livestock may play a role in interspecies bacterial transmission by disrupting natural microbial communities and creating an environment favouring resistant bacteria. Pigs and poultry receive high levels of antimicrobials and are reservoirs of multidrug-resistant bacteria, including Streptococcus suis, a zoonotic pig pathogen. S. suis detection in non-porcine hosts, particularly poultry, raises a critical question: is this due to transient spillover or does it represent sustained host jumps and adaptation?ResultsAnalysing over 3000 S. suis genomes from a diverse range of hosts-including pigs, wild boar, humans, cats, dogs, cattle, fish, otter, and birds-we identify a multidrug-resistant lineage, distinct from the lineage responsible for most zoonoses, that has undergone multiple host jump events into birds. Unlike transmission to humans, which is exclusively derived through contacts with pigs, we find evidence of S. suis adaptation to birds. This includes phylogenetic persistence, independent acquisition of bird-specific mobile genomic islands, enhanced survival in chicken versus pig blood, and subsequent transmission from poultry to wild birds.ConclusionsWhile chickens may not be a source of zoonotic S. suis infections, shared antibiotic usage in pigs and poultry may have promoted host jumps of multidrug-resistant S. suis, leading to onward transmission to wild bird populations. Our results suggest that antibiotic use in livestock production may promote transmission of antimicrobial-resistant bacteria to other hosts, thereby expanding the ecological range of bacterial pathogens.

Marine mammals are sentinel species representing the "health" of our oceans on which we are dependent. There are many threats to marine mammals including infectious diseases that increase with climate change and pollution of the marine environment. Streptococcus phocae has frequently been isolated from diseased or dead marine mammals. However, its pathogenicity and contribution to disease in marine mammals is still unknown. As bacteria including (potential) pathogens has to deal with different host environments during colonization or infection, we investigated the survival of S. phocae in fresh porcine and phocid blood, in seawater and in the presence of macrophages and (epithelial) cells from harbor seals and pigs. Furthermore, we tested adherence on and invasion of different (marine) mammalian cells by S. phocae. Our results showed that S. phocae can survive in seawater for at least 11 and 28 days at 16°C and 4°C, respectively. It is able to grow in blood of harbor and grey seals, but not in porcine blood. Furthermore, S. phocae is adherent and invasive to cells from seals and pigs, while the portion of invasive cells was higher in seal derived cells. Macrophages of harbor seals were more efficient in killing S. phocae than porcine macrophages. Our results indicate that S. phocae has strategies enabling it to adapt to the marine environment and seal hosts.

Introduction: Streptococcus pneumoniae is the most common cause of bacterial meningitis and meningoencephalitis in humans. The bacterium produces numerous virulence determinants, among them hydrogen peroxide (H2O2) and pneumolysin (Ply), which contribute to bacterial cytotoxicity. Microglia, the resident phagocytes in the brain, are distinct from other macrophages, and we thus compared their susceptibility to pneumococcal toxicity and their ability to phagocytose pneumococci with those of bone marrow-derived macrophages (BMDM). Methods: Microglia and BMDM were co-incubated with S. pneumoniae D39 to analyze survival of phagocytes by fluorescence microscopy, bacterial growth by quantitative plating, and phagocytosis by an antibiotic protection assay. Ply was detected by hemolysis assay and Western blot analysis. Results: We found that microglia were killed during pneumococcal infection with a wild-type and an isogenic ply-deficient mutant, whereas viability of BMDM was not affected by pneumococci. Treatment with recombinant Ply showed a dose-dependent cytotoxic effect on microglia and BMDM. However, high concentrations of recombinant Ply were required and under the chosen experimental conditions, Ply was not detectable in the supernatant during infection of microglia. Inactivation of H2O2 by exogenously added catalase abolished its cytotoxic effect. Consequently, infection of microglia with pneumococci deficient for the pyruvate oxidase SpxB, primarily producing H2O2, resulted in reduced killing of microglia. Conclusion: Taken together, in the absence of Ply, H2O2 caused cell death in primary phagocytes in concentrations produced by pneumococci.

Streptococcus suis, both a common colonizer of the porcine upper respiratory tract and an invasive pig pathogen, successfully adapts to different host environments encountered during infection. Whereas the initial infection mainly occurs via the respiratory tract, in a second step, the pathogen can breach the epithelial barrier and disseminate within the whole body. Thereby, the pathogen reaches other organs such as the heart, the joints, or the brain. In this review, we focus on the role of S. suis metabolism for adaptation to these different in vivo host niches to encounter changes in nutrient availability, host defense mechanisms and competing microbiota. Furthermore, we highlight the close link between S. suis metabolism and virulence. Mutants deficient in metabolic regulators often show an attenuation in infection experiments possibly due to downregulation of virulence factors, reduced resistance to nutritive or oxidative stress and to phagocytic activity. Finally, metabolic pathways as potential targets for new therapeutic strategies are discussed. As antimicrobial resistance in S. suis isolates has increased over the last years, the development of new antibiotics is of utmost importance to successfully fight infections in the future.

Streptococcus suis is a porcine and zoonotic pathogen in the upper respiratory tract, expressing different capsular serotypes and virulence-associated factors. Given its genomic and phenotypic diversity, the virulence potential of S. suis cannot be attributed to a single factor. Since strong inflammatory response is a hallmark of S. suis infection, the objective of this study was to investigate the differences in transcriptional host responses to two serotype 2 and one serotype 9 strains. Both serotypes are frequently found in clinical isolates. We infected porcine precision-cut lung slices (PCLSs) with two serotype 2 strains of high (strain S10) and low (strain T15) virulence, and a serotype 9 strain 8067 of moderate virulence. We observed higher expression of inflammation-related genes during early infection with strains T15 and 8067, in contrast to infection with strain 10, whose expression peaked late. In addition, bacterial gene expression from infected PCLSs revealed differences, mainly of metabolism-related and certain virulence-associated bacterial genes amongst these strains. We conclude that the strain- and time-dependent induction of genes involved in innate immune response might reflect clinical outcomes of infection in vivo, implying rapid control of infection with less virulent strains compared to the highly virulent strain S10.

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis , a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized.

The Gram-negative, obligate intracellular bacterium Coxiella burnetii is the causative organism of the zoonosis Q fever and is known for its resistance toward various intra- and extracellular stressors. Infected ruminants such as cattle, sheep, and goats can shed the pathogen in their milk. Pasteurization of raw milk was introduced for the inactivation of C. burnetii and other milk-borne pathogens. Legal regulations for the pasteurization of milk are mostly based on recommendations of the Codex Alimentarius. As described there, C. burnetii is considered as the most heat-resistant non-spore-forming bacterial pathogen in milk and has to be reduced by at least 5 log10-steps during the pasteurization process. However, the corresponding inactivation data for C. burnetii originate from experiments performed more than 60 years ago. Recent scientific findings and the technological progress of modern pasteurization equipment indicate that C. burnetii is potentially more effectively inactivated during pasteurization than demanded in the Codex Alimentarius. In the present study, ultra-high heat-treated milk was inoculated with different C. burnetii field isolates and subsequently heat-treated in a pilot-plant pasteurizer. Kinetic inactivation data in terms of D- and z-values were determined and used for the calculation of heat-dependent log reduction. With regard to the mandatory 5 log10-step reduction of the pathogen, the efficacy of the established heat treatment regime was confirmed, and, in addition, a reduction of the pasteurization temperature seems feasible.

Highly pathogenic avian influenza viruses of the H5N8 subtype have been circulating in Europe and Asia since 2016, causing huge economic losses to the poultry industry. A new wave of H5Nx infections has begun in 2020. The viruses mainly infect wild birds and waterfowl; from there they spread to poultry and cause diseases. Previous studies have shown that the H5N8 viruses have seldom spread to mammals; however, reports in early 2021 indicate that humans may be infected, and some incident reports indicate that H5Nx clade 2.3.4.4B virus may be transmitted to wild mammals, such as red foxes and seals. In order to get more information on how the H5N8 virus affects seals and other marine animals, here, we used primary cultures to analyze the cell tropism of the H5N8 virus, which was isolated from an infected grey seal (H5N8/Seal-2016). Primary tracheal epithelial cells were readily infected by H5N8/Seal -2016 virus; in contrast, the commonly used primary seal kidney cells required the presence of exogenous trypsin to initiate virus infection. When applied to an ex vivo precision-cut lung slice model, compared with recombinant human H3N2 virus or H9N2 LPAI virus, the H5N8/Seal-2016 virus replicated to a high titre and caused a strong detrimental effect; with these characteristics, the virus was superior to a human H3N2 virus and to an H9N2 LPAI virus. By using well-differentiated air-liquid interface (ALI) cultures, we have observed that ALI cultures of canines, ferrets, and harbour seals are more sensitive to H5N8/Seal-2016 virus than are human or porcine ALI cultures, which cannot be fully explained by sialic acid distribution. Our results indicate that the airway epithelium of carnivores may be the main target of H5N8 viruses. Consideration should be given to an increased monitoring of the distribution of highly pathogenic avian influenza viruses in wild animals.

Streptococcus suis , a common member of the porcine respiratory microbiota, can cause life-threatening diseases in pigs as well as humans. A previous study identified the gene trpX as conditionally essential for in vivo survival by intrathecal infection of pigs with a transposon library of S. suis strain 10. Here, we characterized trpX, encoding a putative tryptophan/tyrosine transport system substrate-binding protein, in more detail. We compared growth capacities of the isogenic trpX -deficient mutant derivative strain 10∆ trpX with its parent. Growth experiments in chemically defined media (CDM) revealed that growth of 10∆ trpX depended on tryptophan concentration, suggesting TrpX involvement in tryptophan uptake. We demonstrated that trpX is part of an operon structure and co-transcribed with two additional genes encoding a putative permease and ATPase, respectively. Bioinformatics analysis identified a putative tryptophan T-box riboswitch in the 5′ untranslated region of this operon. Finally, qRT-PCR and a reporter activation assay revealed trpX mRNA induction under tryptophan-limited conditions. In conclusion, our study showed that TrpX is part of a putative tryptophan ABC transporter system regulated by a T-box riboswitch probably functioning as a substrate-binding protein. Due to the tryptophan auxotrophy of S. suis , TrpX plays a crucial role for metabolic adaptation and growth during infection.

Glaesserella (G.) parasuis is one of the most important porcine pathogens causing Glaesser’s disease. Neutrophil granulocytes are the major counteracting cell type of the innate immune system, which contribute to the host defense by phagocytosis or the formation of neutrophil extracellular traps (NETs). Recently, NET-formation has been shown to facilitate the survival of bacteria from the Pasteurellaceae family. However, the interaction of NETs and G. parasuis is unclear so far. In this study, we investigated the interplay of three G. parasuis serotypes with porcine neutrophils. The production of reactive oxygen species by neutrophils after G. parasuis infection varied slightly among the serotypes but was generally low and not significantly influenced by the serotypes. Interestingly, we detected that independent of the serotype of G. parasuis, NET formation in neutrophils was induced to a small but significant extent. This phenomenon occurred despite the ability of G. parasuis to release nucleases, which can degrade NETs. Furthermore, the growth of Glaesserella was enhanced by external DNases and degraded NETs. This indicates that Glaesserella takes up degraded NET components, supplying them with nicotinamide adenine dinucleotide (NAD), as this benefit was diminished by inhibiting the 5′-nucleotidase, which metabolizes NAD. Our results indicate a serotype-independent interaction of Glaesserella with neutrophils by inducing NET-formation and benefiting from DNA degradation.

Marine mammals are sentinels for the marine ecosystem and threatened by numerous factors including infectious diseases. One of the most frequently isolated bacteria are beta-hemolytic streptococci. However, knowledge on ecology and epidemiology of streptococcal species in marine mammals is very limited. This review summarizes published reports on streptococcal species, which have been detected in marine mammals. Furthermore, we discuss streptococcal transmission between and adaptation to their marine mammalian hosts. We conclude that streptococci colonize and/or infect marine mammals very frequently, but in many cases, streptococci isolated from marine mammals have not been further identified. How these bacteria disseminate and adapt to their specific niches can only be speculated due to the lack of respective research. Considering the relevance of pathogenic streptococci for marine mammals as part of the marine ecosystem, it seems that they have been neglected and should receive scientific interest in the future.

Streptococcus suis is a common pathogen colonising the respiratory tract of pigs. It can cause meningitis, sepsis and pneumonia leading to economic losses in the pig industry worldwide. Cyclooxygenase-2 (COX-2) and its metabolites play an important regulatory role in different biological processes like inflammation modulation and immune activation. In this report we analysed the induction of COX-2 and the production of its metabolite prostaglandin E2 (PGE2) in a porcine precision-cut lung slice (PCLS) model. Using Western blot analysis, we found a time-dependent induction of COX-2 in the infected tissue resulting in increased PGE2 levels. Immunohistological analysis revealed a strong COX-2 expression in the proximity of the bronchioles between the ciliated epithelial cells and the adjacent alveolar tissue. The morphology, location and vimentin staining suggested that these cells are subepithelial bronchial fibroblasts. Furthermore, we showed that COX-2 expression as well as PGE2 production was detected following infection with two prevalent S. suis serotypes and that the pore-forming toxin suilysin played an important role in this process. Therefore, this study provides new insights in the response of porcine lung cells to S. suis infections and serves as a basis for further studies to define the role of COX-2 and its metabolites in the inflammatory response in porcine lung tissue during infections with S. suis.

A vaccine protecting against different Streptococcus suis serotypes is highly needed in porcine practice to improve animal welfare and reduce the use of antibiotics. We hypothesized that immunogens prominently recognized by convalescence sera but significantly less so by sera of susceptible piglets are putative protective antigens. Accordingly, we investigated immunogenicity and protective efficacy of a multicomponent vaccine including six main conserved immunogens, namely SSU0934, SSU1869, SSU0757, SSU1950, SSU1664 and SSU0187. Flow cytometry confirmed surface expression of all six immunogens in S. suis serotypes 2, 9 and 14. Although prime-booster vaccination after weaning resulted in significantly higher specific IgG levels against all six immunogens compared to the placebo-treated group, no significant differences between bacterial survival in blood from either vaccinated or control animals were recorded for serotype 2, 9 and 14 strains. Furthermore, vaccinated piglets were not protected against morbidity elicited through intranasal challenge with S. suis serotype 14. As ~50% of animals in both groups did not develop disease, we investigated putative other correlates of protection. Induction of reactive oxygen species (ROS) in blood granulocytes was not associated with vaccination but correlated with protection as all piglets with >5% ROS survived the challenge. Based on these findings we discuss that the main immunogens of S. suis might actually not be a priori good candidates for protective antigens. On the contrary, expression of immunogens that evoke antibodies that do not mediate killing of this pathogen might constitute an evolutionary advantage conserved in many different S. suis strains.

Salmonella pathogenicity island (SPI) 2 type three secretion system (T3SS)-mediated effector molecules facilitate bacterial survival in phagocytes but their role in the intestinal epithelium in vivo remains ill-defined. Using our neonatal murine infection model in combination with SPI2 reporter technology and RNA-Seq of sorted primary enterocytes, we demonstrate expression of SPI2 effector molecules by intraepithelial Salmonella Typhimurium (S. Typhimurium). Contrary to expectation, immunostaining revealed that infection with SPI2 T3SS-mutants resulted in significantly enlarged intraepithelial Salmonella-containing vacuoles (SCV) with altered cellular positioning, suggesting impaired apical to basolateral transmigration. Also, infection with isogenic tagged S. Typhimurium strains revealed a reduced spread of intraepithelial SPI2 T3SS mutant S. Typhimurium to systemic body sites. These results suggest that SPI2 T3SS effector molecules contribute to enterocyte apical to basolateral transmigration of the SCV during the early stage of the infection.

Serotyping is the most common method to characterize field isolates of Actinobacillus (A.) pleuropneumoniae , the etiological agent of porcine pleuropneumonia. Based on serology, many farms seem to be infected and antibodies against a wide variety of serovars are detectable, but, so far it is unknown to what degree respective serovars contribute to outbreaks of clinical manifest disease. In this study, 213 German A. pleuropneumoniae field isolates retrieved for diagnostic purposes from outbreaks of porcine pleuropneumonia between 2010 and 2019 were genetically serotyped and analyzed regarding their apx -toxin gene profile using molecular methods. Serotyping revealed a prominent role of serovar 2 in clinical cases (64% of all isolates) and an increase in the detection of this serovar since 2010 in German isolates. Serovar 9/11 followed as the second most frequent serovar with about 15% of the isolates. Furthermore, very recently described serovars 16 (n = 2) and 18 (n = 8) were detected. Most isolates (93.4%) showed apx -profiles typical for the respective serovar. However, this does not hold true for isolates of serovar 18, as 75% (n = 6) of all isolates of this serovar deviated uniformly from the “typical” apx -gene profile of the reference strain 7311555. Notably, isolates from systemic lesions such as joints or meninges did not harbor the complete apxICABD operon which is considered typical for highly virulent strains. Furthermore, the extremely low occurrence (n = 1) of NAD independent (biovar II) isolates in German A. pleuropneumoniae was evident in our collection of clinical isolates.

Streptococcus suis ( S. suis ) is an important opportunistic pathogen, which can cause septicemia and meningitis in pigs and humans. Previous in vivo observations in S. suis -infected pigs revealed lesions at the choroid plexus (CP). In vitro experiments with primary porcine CP epithelial cells (PCPEC) and human CP epithelial papilloma (HIBCPP) cells demonstrated that S. suis can invade and traverse the CP epithelium, and that the CP contributes to the inflammatory response via cytokine expression. Here, next generation sequencing (RNA-seq) was used to compare global transcriptome profiles of PCPEC and HIBCPP cells challenged with S. suis serotype (ST) 2 infected in vitro , and of pigs infected in vivo . Identified differentially expressed genes (DEGs) were, amongst others, involved in inflammatory responses and hypoxia. The RNA-seq data were validated via quantitative PCR of selected DEGs. Employing Gene Set Enrichment Analysis (GSEA), 18, 28, and 21 enriched hallmark gene sets (GSs) were identified for infected HIBCPP cells, PCPEC, and in the CP of pigs suffering from S. suis ST2 meningitis, respectively, of which eight GSs overlapped between the three different sample sets. The majority of these GSs are involved in cellular signaling and pathways, immune response, and development, including inflammatory response and hypoxia. In contrast, suppressed GSs observed during in vitro and in vivo S. suis ST2 infections included those, which were involved in cellular proliferation and metabolic processes. This study suggests that similar cellular processes occur in infected human and porcine CP epithelial cells, especially in terms of inflammatory response.