Comparative analysis of the interactions of different Streptococcus suis strains with monocytes, granulocytes and the complement system in porcine blood.

Streptococcus suis (S. suis) is an important porcine pathogen causing meningitis, arthritis, and septicemia. Serotypes 2 and 14 are the most common zoonotic ones worldwide, whereas serotypes 2, 9, and 7 are very important in pigs in Europe. To cause invasive infections S. suis needs to enter the bloodstream. Consequently, the immune response in blood represents an important line of defense and bacteremia plays a key role in the pathogenesis of invasive S. suis infections. We investigated the working hypothesis that S. suis strains of the same serotype but different clonal complex (CC) might exhibit substantial differences in the interaction with components of the immune system in porcine blood. The experimental design of this study includes comparative analysis of 8 virulent strains belonging to 4 serotypes with strains of the same serotype being genetically not closely related. Significant differences between two strains of the same serotype but different clonal complex were recorded in the flow cytometric analysis of association with different leukocytes for serotype 9 and 14. Our results demonstrate that the serotype 9 strain of CC94 shows significantly increased association with monocytes and survival in porcine blood of conventional piglets as well as a tendency towards decreased composition of C3 in plasma of these piglets in comparison to the serotype 9 strain of CC16. Correlation analysis of C3 deposition on the bacterial surface and survival in respective blood samples of 8-week-old piglets demonstrated a negative correlation indicating that C3 deposition is a crucial step to limit bacterial survival and proliferation of different S. suis pathotypes in the blood of these piglets. In summary, our results indicate that the capsule composition of a S. suis strain is not alone sufficient to determine association with leukocytes, activation of complement, induction of proinflammatory cytokines, oxidative burst, and bacterial survival in porcine blood. In this study, substantial differences in these host-pathogen interactions were observed between strains of the same serotype. Therefore, a more comprehensive characterization of the field isolates, including at least MLST analysis to determine the sequence type/clonal complex, is recommended.

Invasive Bacterial Infections of the Musculoskeletal and Central Nervous System during Pig Rearing: Detection Frequencies of Different Pathogens and Specific Streptococcus suis Genotypes.

Locomotor and central nervous system disorders occur during pig rearing, but there is no systematic recording of the different causative agents in Germany. Joint and meningeal swabs, kidneys, lungs, and eight different lymph nodes per pig were cultured, and isolated pathogens were identified using polymerase chain reactions (PCRs). The cps and pathotype of Streptococcus suis (S. suis) isolates were determined using multiplex-PCR. S. suis was the most important pathogen in the infected joints (70.8%) and meningeal swabs (85.4%) and was most frequently detected in both sites in suckling and weaning piglets. To elucidate the possible portal of entry of S. suis, eight different lymph nodes from 201 pigs were examined in a prospective study. S. suis was detected in all examined lymph nodes (n = 1569), including the mesenteric lymph nodes (15.8%; n = 121/765), with cps 9 (37.2%; n = 147) and cps 2 (24.3%; n = 96) being the most dominating cps types. In piglets with a systemic S. suis infection, different lymph nodes are frequently infected with the invasive S. suis strain, which does not help clarify the portal of entry for S. suis.

Streptococcus equi subspecies zooepidemicus - a case report of sudden death in a German sow farm.

A farm in North-West Germany experienced a high morbidity and mortality in their sow herd. Sows showed fever, lethargy, oedema, mucosal discharge and dyspnoea. Necropsy revealed a severe fibrinous and purulent polyserositis. Haematological and histological examinations confirmed septicaemia. Streptococcus equi subspecies zooepidemicus was isolated in high yields from major organs. Sequence typing of this isolate (21/455) revealed a new sequence type showing a significantly higher proliferation rate in comparison to two other isolates. Other infectious agents (influenza A virus, Porcine Reproductive and Respiratory Syndrome Virus, Porcine Circovirus 2, african swine fever virus, classical swine fever virus, Actinobacillus pleuropneumoniae) were excluded by routine diagnostic examinations. A climate check revealed an insufficient air supply in the area for the gestating sows. This case describes the first disease outbreak in swine due to S. zooepidemicus in Germany.

A distinct variant of the SzM protein of Streptococcus equi subsp. zooepidemicus recruits C1q independent of IgG binding and inhibits activation of the classical complement pathway.

Streptococcus equi subsp. zooepidemicus (SEZ) is a major equine pathogen that causes pneumonia, abortion, and polyarthritis. It can also cause invasive infections in humans. SEZ expresses the M-like protein SzM, which recruits host proteins such as fibrinogen to the bacterial surface. Equine SEZ strain C2, which binds only comparably low amounts of human fibrinogen in comparison to human SEZ strain C33, was previously shown to proliferate in equine and human blood. As the expression of SzM_C2 was necessary for survival in blood, this study investigated the working hypothesis that SzM_C2 inhibits complement activation through a mechanism other than fibrinogen and non-immune immunoglobulin binding. Loss-of-function experiments showed that SEZ C2, but not C33, binds C1q via SzM in IgG-free human plasma. Furthermore, SzM C2 expression is necessary for recruiting purified human or equine C1q to the bacterial surface. Flow cytometry analysis demonstrated that SzM expression in SEZ C2 is crucial for the significant reduction of C3b labelling in human plasma. Addition of human plasma to immobilized rSzM_C2 and immobilized aggregated IgG led to binding of C1q, but only the latter activated the complement system, as shown by the detection of C4 deposition. Complement activation induced by aggregated IgG was significantly reduced if human plasma was pre-incubated with rSzM_C2. Furthermore, rSzM_C2, but not rSzM_C33, inhibited the activation of the classical complement pathway in human plasma, as determined in an erythrocyte lysis experiment. In conclusion, the immunoglobulin-independent binding of C1q to SzM_C2 is associated with complement inhibition.

Immunoglobulin M-degrading enzyme of Streptococcus suis (Ide Ssuis ) impairs porcine B cell signaling.

Streptococcus suis (S. suis) is an important porcine pathogen, causing severe disease like meningitis and septicemia primarily in piglets. Previous work showed that the IgM-degrading enzyme of S. suis (Ide Ssuis ) specifically cleaves soluble porcine IgM and is involved in complement evasion. The objective of this study was to investigate Ide Ssuis cleavage of the IgM B cell receptor and subsequent changes in B cell receptor mediated signaling. Flow cytometry analysis revealed cleavage of the IgM B cell receptor by recombinant (r) Ide Ssuis _homologue as well as Ide Ssuis derived from culture supernatants of S. suis serotype 2 on porcine PBMCs and mandibular lymph node cells. Point-mutated rIde Ssuis _homologue_C195S did not cleave the IgM B cell receptor. After receptor cleavage by rIde Ssuis _homologue, it took at least 20 h for mandibular lymph node cells to restore the IgM B cell receptor to levels comparable to cells previously treated with rIde Ssuis _homologue_C195S. B cell receptor mediated signaling after specific stimulation via the F(ab')2 portion was significantly inhibited by rIde Ssuis _homologue receptor cleavage in IgM+ B cells, but not in IgG+ B cells. Within IgM+ cells, CD21+ B2 cells and CD21- B1-like cells were equally impaired in their signaling capacity upon rIde Ssuis _homologue B cell receptor cleavage. In comparison, intracellular B cell receptor independent stimulation with tyrosine phosphatase inhibitor pervanadate increased signaling in all investigated B cell types. In conclusion, this study demonstrates Ide Ssuis cleavage efficacy on the IgM B cell receptor and its consequences for B cell signaling.

An Explorative Study of the Causal Pathogenesis of Green Liver Discoloration in Organically Reared Female Bronze Turkeys (Meleagris gallopavo) Considering the Infectious Risk Factors.

A recent study revealed that organically raised Bronze turkeys showed a high prevalence of green liver discoloration. This alteration is commonly associated with the Turkey Osteomyelitis Complex and potentially caused by opportunistic bacteria. Therefore, 360 organically fattened Bronze turkeys were examined post-mortem throughout two fattening trials with two examinations each to determine possible infectious risk factors and reduce disease prevalence. Clinical and pathoanatomical examinations were performed on every hen. Histopathological, bacteriological, parasitological, and virological examinations were performed on at least six hens without and, if applicable, six hens with green livers on each examination date. Overall, 9.0% of all hens had a green liver without a correlation with bacterial or parasitological findings but multiple health impairments. The discoloration correlated significantly with the detection of immunosuppressive turkey hemorrhagic enteritis virus at the early stage and macro- and histological joint/bone lesions at the late fattening stage, indicating the presence of two different predisposing pathogeneses. Flocks not being vaccinated against hemorrhagic enteritis but having a virus-positive sample showed the highest prevalence of green liver discoloration and developed worse in various parameters. In conclusion, an adequate vaccination schedule and the prevention of field infections may lead to a decreased risk of performance reduction and improved animal health.

d-Alanylation of Lipoteichoic Acids in Streptococcus suis Reduces Association With Leukocytes in Porcine Blood.

Streptococcus suis (S. suis) is a common swine pathogen but also poses a threat to human health in causing meningitis and severe cases of streptococcal toxic shock-like syndrome (STSLS). Therefore, it is crucial to understand how S. suis interacts with the host immune system during bacteremia. As S. suis has the ability to introduce d-alanine into its lipoteichoic acids (LTAs), we investigated the working hypothesis that cell wall modification by LTA d-alanylation influences the interaction of S. suis with porcine blood immune cells. We created an isogenic mutant of S. suis strain 10 by in-frame deletion of the d-alanine d-alanyl carrier ligase (DltA). d-alanylation of LTAs was associated with reduced phagocytosis of S. suis by porcine granulocytes, reduced deposition of complement factor C3 on the bacterial surface, increased hydrophobicity of streptococci, and increased resistance to cationic antimicrobial peptides (CAMPs). At the same time, survival of S. suis was not significantly increased by LTA d-alanylation in whole blood of conventional piglets with specific IgG. However, we found a distinct cytokine pattern as IL-1ß but not tumor necrosis factor (TNF)-α levels were significantly reduced in blood infected with the ΔdltA mutant. In contrast to TNF-α, activation and secretion of IL-1ß are inflammasome-dependent, suggesting a possible influence of LTA d-alanylation on inflammasome regulation. Especially in the absence of specific antibodies, the association of S. suis with porcine monocytes was reduced by d-alanylation of its LTAs. This dltA-dependent phenotype was also observed with a non-encapsulated dltA double mutant indicating that it is independent of capsular polysaccharides. High antibody levels caused high levels of S. suis-monocyte-association followed by inflammatory cell death and strong production of both IL-1ß and TNF-α, while the influence of LTA d-alanylation of the streptococci became less visible. In summary, the results of this study expand previous findings on d-alanylation of LTAs in S. suis and suggest that this pathogen specifically modulates association with blood leukocytes through this modification of its surface.

Porcine iucA+ but rmpA- Klebsiella pneumoniae strains proliferate in blood of young piglets but are killed by IgM and complement dependent opsonophagocytosis when these piglets get older.

Klebsiella (K.) pneumoniae causes different diseases in humans and animals including the life-threatening liver abscess syndrome and septicemia, respectively. However, host-pathogen interactions of K. pneumoniae in porcine blood have not been studied. We investigated the working hypothesis that only distinct K. pneumoniae strains have the capacity to survive in porcine blood and that this feature is associated with specific molecular markers such as sequence type, profile of siderophore genes and the regulator of the mucoid phenotype (rmp). Furthermore, we characterize the immune response in growing piglets leading to killing of an invasive K. pneumoniae strain. The veterinary isolates showed great diversity in sequence types and profile of siderophore genes. Porcine isolates were mainly positive for the aerobactin gene iucA but did not carry rmpA and this genotype was associated with proliferation in blood of 4-week-old piglets. Supernatants of an iucA+ but not an iucA- strain boosted growth in porcine serum. Between four and eight weeks of age, piglets showed a prominent increase of IgM binding to K. pneumoniae. Immunglobulin M and complement were crucial for killing of a serum-resistant iucA+ porcine K. pneumoniae strain at eight weeks of age. Flow cytometry analysis confirmed induction of phagocytosis and oxidative burst mediated by serum samples of 8-week-old piglets. Based on our in vitro findings we propose that many porcine iucA+ rmpA- K. pneumoniae strains have the ability to cause bacteremia in young piglets in association with aerobactin-mediated iron acquisition and that this phenotype is lost as specific IgM increases after weaning.

Immunogenicity and protective efficacy of a Streptococcus suis vaccine composed of six conserved immunogens.

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.

Identification of Zoophilic Dermatophytes Using MALDI-TOF Mass Spectrometry.

Dermatophytoses represent a major health burden in animals and man. Zoophilic dermatophytes usually show a high specificity to their original animal host but a zoonotic transmission is increasingly recorded. In humans, these infections elicit highly inflammatory skin lesions requiring prolonged therapy even in the immunocompetent patient. The correct identification of the causative agent is often crucial to initiate a targeted and effective therapy. To that end, matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) represents a promising tool. The objective of this study was to evaluate the reliability of species identification of zoophilic dermatophytes using MALDI-TOF MS. The investigation of isolates from veterinary clinical samples suspicious of dermatophytoses suggests a good MALDI-TOF MS based identification of the most common zoophilic dermatophyte Microsporum canis. Trichophyton (T.) spp. usually achieved scores only around the cutoff value for secure species identification because of a small number of reference spectra. Moreover, these results need to be interpreted with caution due to the close taxonomic relationship of dermatophytes being reflected in very similar spectra. In our study, the analysis of 50 clinical samples of hedgehogs revealed no correct identification using the provided databases, nor for zoophilic neither for geophilic causative agents. After DNA sequencing, adaptation of sample processing and an individual extension of the in-house database, acceptable identification scores were achieved (T. erinacei and Arthroderma spp., respectively). A score-oriented distance dendrogram revealed clustering of geophilic isolates of four different species of the genus Arthroderma and underlined the close relationship of the important zoophilic agents T. erinacei, T. verrucosum and T. benhamiae by forming a subclade within a larger cluster including different dermatophytes. Taken together, MALDI-TOF MS proofed suitable for the identification of zoophilic dermatophytes provided fresh cultures are used and the reference library was previously extended with spectra of laboratory-relevant species. Performing independent molecular methods, such as sequencing, is strongly recommended to substantiate the findings from morphologic and MALDI-TOF MS analyses, especially for uncommon causative agents.

A Comparative Transcriptome Analysis of Human and Porcine Choroid Plexus Cells in Response to Streptococcus suis Serotype 2 Infection Points to a Role of Hypoxia.

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.

Complete Genome Sequences of Streptococcus suis Pig-Pathogenic Strains 10, 13-00283-02, and 16085/3b.

Streptococcus suis is an important pathogen of pigs that, as a zoonotic agent, can also cause severe disease in humans, including meningitis, endocarditis, and septicemia. We report complete and annotated genomes of S. suis strains 10, 13-00283-02, and 16085/3b, which represent the highly prevalent serotypes cps2, cps7, and cps9, respectively.

Identification of a large repetitive RTX immunogen in a highly virulent Rodentibacter heylii strain.

Rodentibacter (R.) heylii is frequently detected in laboratory rodents. Repeats in toxin (RTX) toxins are considered important virulence factors of this major murine pathogen. We evaluated the virulence of a R.heylii strain negative for all known RTX toxin genes and Muribacter (M.) muris, a commensal in mice, in experimental infections of C57BL/6 and BALB/c mice. Experimental intranasal infection with 108 CFU of the pnxI-, pnxII- and pnxIII- R. heylii strain resulted in 75% and 100% mortality in C57BL/6 and BALB/c mice, respectively. In early losses, multiple internal organs were infected and purulent bronchopneumonia was the main pathology. Intranasal application of M. muris did not result in mortality or severe weight loss. Immunoproteomics led to the identification of a surface-associated and specific immunogen, which was designated as R. heylii immunogen A (RhiA) and which was exclusively recognised by sera obtained from mice infected with this R. heylii pathotype. RhiA is a 262.6 kDa large protein containing long imperfect tandem repeats and C-terminal RTX consensus sequences. Immunohistochemical analysis confirmed that this R.heylii pathotype expresses RhiA in the lower respiratory tract. In summary, this study describes a specific immunogen in a virulent R. heylii, strain which is an excellent antigen for pathotype-specific serological screenings and which might carry out RTX-related functions.

Proline-rich antimicrobial peptide Api137 is bactericidal in porcine blood infected ex vivo with a porcine or human Klebsiella pneumoniae strain.

OBJECTIVES: Klebsiella pneumoniae is an emerging invasive pathogen in humans and pigs. Resistance against multiple antibiotics in this species is a major health concern and the development of new antibiotics is urgently needed. The objective of this study was to investigate the effects of proline-rich antimicrobial peptides (PrAMPs) on the survival of K. pneumoniae strains in porcine blood. METHODS: We established a bactericidal assay with K. pneumoniae in fresh blood drawn from 4-week-old piglets. PrAMPs, namely the apidaecins Api137 and Api802 as well as the oncocin Onc112, were added to ex vivo-infected whole blood samples in order to study their bactericidal effects and, in the case of Api137, also immune responses. RESULTS: A porcine invasive and a human iucA+rmpA+ K. pneumoniae strain showed prominent proliferation in porcine blood. Application of Api137 resulted in a dose-dependent prominent bactericidal effect killing the invasive porcine K. pneumoniae strain. Addition of 8 µg/mL Api137 also resulted in complete killing of the human iucA+rmpA+ strain. Cytotoxicity, haemolysis and induction of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNFα) in K. pneumoniae-infected porcine blood treated with Api137 was comparable with values obtained after application of 10 µg/mL cefquinome. CONCLUSION: We describe a new non-rodent model for invasive K. pneumoniae bacteraemia and present promising data for the PrAMP Api137 for the control of infection with hypervirulent K. pneumoniae strains.

Low-Energy Electron Irradiation Efficiently Inactivates the Gram-Negative Pathogen Rodentibacter pneumotropicus-A New Method for the Generation of Bacterial Vaccines with Increased Efficacy.

Bacterial pathogens cause severe infections worldwide in livestock and in humans, and antibiotic resistance further increases the importance of prophylactic vaccines. Inactivated bacterial vaccines (bacterins) are usually produced via incubation of the pathogen with chemicals such as formaldehyde, which is time consuming and may cause loss of immunogenicity due to the modification of structural components. We evaluated low-energy electron irradiation (LEEI) as an alternative method to generate a bacterin. Rodentibacter pneumotropicus, an invasive Gram-negative murine pathogen, was inactivated with LEEI and formaldehyde. LEEI resulted in high antigen conservation, and LPS activity was significantly better maintained when compared with formaldehyde treatment. Immunization of mice with LEEI-inactivated R. pneumotropicus elicited a strong immune response with no detectable bacterial burden upon sublethal challenge. The results of this study suggest the inactivation of bacteria with LEEI as an alternative, fast and efficient method to generate bacterial vaccines with increased efficacy.

Differentiation of Rodentibacter pneumotropicus, Rodentibacter heylii and Muribacter muris by MALDI-TOF MS.

The pathogens Rodentibacter (R.) pneumotropicus and R. heylii as well as the commensal Muribacter (M.) muris are frequently isolated in mice. In this study, a MALDI-TOF MS database was extended with spectra of well characterized strains of these species. Compared to a multiplex PCR, all examined out-of-sample isolates were correctly identified.

Survival of Streptococcus suis in Porcine Blood Is Limited by the Antibody- and Complement-Dependent Oxidative Burst Response of Granulocytes.

Bacteremia is a hallmark of invasive Streptococcus suis infections of pigs, often leading to septicemia, meningitis, or arthritis. An important defense mechanism of neutrophils is the generation of reactive oxygen species (ROS). In this study, we report high levels of ROS production by blood granulocytes after intravenous infection of a pig with high levels of S. suis-specific antibodies and comparatively low levels of bacteremia. This prompted us to investigate the working hypothesis that the immunoglobulin-mediated oxidative burst contributes to the killing of S. suis in porcine blood. Several S. suis strains representing serotypes 2, 7, and 9 proved to be highly susceptible to the oxidative burst intermediate hydrogen peroxide, already at concentrations of 0.001%. The induction of ROS in granulocytes in ex vivo-infected reconstituted blood showed an association with pathogen-specific antibody levels. Importantly, inhibition of ROS production by the NADPH oxidase inhibitor apocynin led to significantly increased bacterial survival in the presence of high specific antibody levels. The oxidative burst rate of granulocytes partially depended on complement activation, as shown by specific inhibition. Furthermore, treatment of IgG-depleted serum with a specific IgM protease or heat to inactivate complement resulted in >3-fold decreased oxidative burst activity and increased bacterial survival in reconstituted porcine blood in accordance with an IgM-complement-oxidative burst axis. In conclusion, this study highlights an important control mechanism of S. suis bacteremia in the natural host: the induction of ROS in blood granulocytes via specific immunoglobulins such as IgM.

Prominent Binding of Human and Equine Fibrinogen to Streptococcus equi subsp. zooepidemicus Is Mediated by Specific SzM Types and Is a Distinct Phenotype of Zoonotic Isolates.

Streptococcus equi subsp. zooepidemicus is an important pathogen in horses that causes severe diseases such as pneumonia and abortion. Furthermore, it is a zoonotic agent, and contact with horses is a known risk factor. In this study, we investigated the working hypothesis that the zoonotic potential varies among S. equi subsp. zooepidemicus strains in association with differences in M-like protein-mediated binding of host plasma proteins. We demonstrate via in-frame deletion mutagenesis of two different S. equi subsp. zooepidemicus strains that the M-like protein SzM is crucial for the binding of fibrinogen to the bacterial surface and for survival in equine and human blood. S. equi subsp. zooepidemicus isolates of equine and human origins were compared with regard to SzM sequences and binding of equine and human fibrinogens. The N-terminal 216 amino acids of the mature SzM were found to exhibit a high degree of diversity, but the majority of human isolates grouped in three distinct SzM clusters. Plasma protein absorption assays and flow cytometry analysis revealed that pronounced binding of human fibrinogen is a common phenotype of human S. equi subsp. zooepidemicus isolates but much less so in equine S. equi subsp. zooepidemicus isolates. Furthermore, binding of human fibrinogen is associated with specific SzM types. These results suggest that SzM-mediated binding of human fibrinogen is an important virulence mechanism of zoonotic S. equi subsp. zooepidemicus isolates.

Sensitive and immunogen-specific serological detection of Rodentibacter pneumotropicus infections in mice.

BACKGROUND: Rodentibacter (R.) pneumotropicus colonizes the respiratory and urogenital tracts of laboratory mice with a reported moderate serological prevalence from 4 to 13%. Thus, regular tests to identify this pathogen in mice are recommended for animal facilities. However, a recent study indicated that current serological assays are partly insensitive, as C57BL/6 and BALB/c mice infected with R. pneumotropicus were incorrectly screened as seronegative. RESULTS: Here, we report a systematic analysis of protein and lipopolysaccharides antigens by immunoblot and ELISA that allowed establishing a sensitive test system able to differentiate between R. pneumotropicus and the closely related species R. heylii. Furthermore, the main immunogen, designated as 'characteristic antigen for Rodentibacter of laboratory origin 1' (CARLO-1), was identified by two-dimensional gel electrophoresis followed by immunoblot and tandem mass spectrometry in a preparation of outer membrane proteins. An indirect ELISA relying on the recombinantly expressed protein provided high sensitivity, specificity, and selectivity. The corresponding carlo1 gene was highly conserved (> 97%) among 21 isolates of R. pneumotropicus and R. heylii. CONCLUSION: The newly identified protein CARLO-1 is well suited for the sensitive and specific serological detection of Rodentibacter infections in mice. Indirect differentiation of R. pneumotropicus and R. heylii infections may be possible using an ELISA based on a whole-cell antigen preparation. All four established ELISA systems using a whole-cell preparation, lipopolysaccharides, outer-membrane proteins and protein CARLO-1 as antigen, respectively, outperformed a commercial ELISA in terms of sensitivity.