Background: Mice are a natural host for Rodentibacter (R.) pneumotropicus. Despite specific monitoring, it is still one of the most important infectious agents in laboratory animals. The objective of this study was to determine the virulence of a prevalent pathotype of R. pneumotropicus and characterize the host response in a new animal model. Results: Intranasal infection of C57BL/6 and BALB/c mice with a R. pneumotropicus strain (JF4Ni) bearing the genes of the three known repeats in toxin (RTX) toxins resulted in an unprecedented high mortality and morbidity above 50 and 80%, respectively. Morbidity was associated with severe weight loss as well as conjunctivitis and dyspnea. A main pathology was a catarrhal purulent to necrotic bronchopneumonia. Specific immune globuline (Ig) A was detected in tracheonasal lavages of most surviving mice which were still colonized by R. pneumotropicus. Furthermore, all surviving animals showed a distinct production of IgG antibodies. To differentiate T-helper cell (Th) 1 and Th2 immune responses we used subclasses of IgGs as indicators. Mean ratios of IgG2b to IgG1 were below 0. 8 in sera drawn from both mice strains prior infection and from BALB/c mice post infection. In contrast, C57BL/6 mice had a mean IgG2b/IgG1 ratio of 1.6 post infection indicating a Th1 immune response in C57BL/6 versus a Th2 response in BALB/c mice associated with a tenfold higher bacterial load in the lung. In accordance with a Th1 response high antigen-specific IgG2c titers were detected in the majority of surviving C57BL/6 mice. Conclusions: R. pneumotropicus JF4Ni is a highly virulent strain causing severe pneumonia and septicemia after intranasal infection of C57BL/6 and BALB/c mice. Persisting infections in the two mice strains are associated with Th1 and Th2 immune responses, respectively, and differences in the bacterial burden of the lung. The described model is ideally suited for future vaccination studies using the natural host. Keywords: Bronchopneumonia, RTX toxins, Th1/Th2 responses, Colonization, Animal model * Correspondence: firstname.lastname@example.org Institute for Bacteriology and Mycology, Faculty of Veterinary Medicine, University Leipzig, An den Tierkliniken 29, 04103 Leipzig, Germany Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fornefett et al. BMC Microbiology (2018) 18:45 Page 2 of 11 Background septicaemia but also to persisting infections is import- Pasteurella (P.) pneumotropica was thought to be a species ant for future studies on virulence and protection. occurring mainly in two different biotypes: Jawetz and Heyl [1, 2]. However, P. pneumotropica was very recently reclas- Results sified and these two biotypes belong now to two different Distribution of pnxIA, pnxIIA and pnxIIIA in R. species, namely Rodentibacter (R.) pneumotropicus and R. pneumotropicus and R. heylii heylii, respectively . The differentiation of the two Different genes encoding RTX-toxins have been identi- biotypes is based on the phenotype of the colony colour fied in P. pneumotropica, namely pnxIA, pnxIIA and (grey and yellow, respectively), but polymerase chain reac- pnxIIIA. By PCRs targeting these pnx genes, we investi- tion (PCR)-based differentiation is also possible [4–6]. In a gated recently collected 27 R. pneumotropicus and 26 R. recent study  differences in distribution of virulence fac- heylii strains. This profiling revealed that the distribution tors between the two biotypes are described. Three differ- of pnxIA, pnxIIA and pnxIIIA differs substantially be- ent repeats-in-toxin (RTX) toxins, designated PnxI, PnxII tween the two species (Table 1). In 46% of the R. heylii and PnxIII, have been identified in P. pneumotropica. strains only pnxI was detected. The gene pnxII was not Whilst PnxI and PnxII are secreted and act as haemolysins found at all in this species. Twelve percent of the R. heylii or cytotoxins , PnxIII is associated with the bacterial strains carried both pnxIII and pnxI, whereas 43% were membrane. Specifically, PnxIII interacts with the extracellu- PCR negative for all RTX genes. In contrast, all three RTX lar matrix [9, 10] but can also induce host cell cytotoxicity genes were found in 74% of the R. pneumotropicus [9, 10]. Therefore, these RTX toxins are considered as im- strains. Only 7% of the R. pneumotropicus strains did portant virulence factors . not carry any of the pnx genes. For further investiga- P. pneumotropica is among the most important pathogens tion, a recently isolated pnxIA+, pnxIIA+ and pnxIIIA in laboratory animal populations with a reported prevalence +R. pneumotropicus strain (JF4Ni) was chosen, because of 4 to 13% in Europe and North America, respectively . of its prevalent genotype identified by the screening of It is described as an opportunistic pathogen with low viru- strains collected in Germany. lence in immunocompetent mice  but clinical signs were recorded in immunodeficient and –suppressed mice or in Morbidity, mortality and histopathology of co-infections with Mycoplasma pulmonis or Pneumo- experimentally infected animals and sentinels cystis carinii . Infected animals are generally unsuitable Intranasal infection with 10 CFU R. pneumotropicus for scientific research due to suppurative to necrotizing le- JF4Ni resulted in 100% (16/16) morbidity in BALB/c and sions in various organs  and modulation of the immune 87.5% (14/16) morbidity in C57BL/6 mice within one response . The Federation of Laboratory Animal Science day. Fifty-six percent (9/16) of BALB/c died or had to be Association (FELASA) lists P. pneumotropica as an import- euthanized within 2–4 days after infection and 50% (8/ ant pathogen in mice, rats and hamsters and recommends 16) of C57BL/6 within 3–6 days after infection (Fig. 1a). theexamination every3months . Various monitoring Diseased mice showed unspecific signs such as ruffled methods are described including PCR [4, 5, 17]and indirect coat, bended back, heavy weight loss (Fig. 1b), dehydra- enzyme-linked immunosorbent assays (ELISAs) [18–20]. tion as well as specific signs such as dyspnoea, conjunc- It is common practice to use soiled bedding sentinels tivitis and mild incoordination in two cases. Significant for health monitoring of laboratory animals. Nevertheless, differences in mortality and morbidity were only re- studies revealed a limited survival of P. pneumotropica in corded between infection animals and controls, but not the environment [21, 22] and the failure of detecting P. between the two mouse strains. All early deceased mice pneumotropica infections by bedding sentinels . showed multifocal moderate to severe catarrhal-purulent In this study, we evaluated the pathologies and immune bronchopneumonia, in some cases with necrosis (Table 2 responses induced by experimental infection of BALB/c and Fig. 1c). These pathologies were not recorded in and C57BL/6 mice with a R. pneumotropicus pathotype mice which survived the experimental R. pneumotropi- emerging in German laboratory animal facilities. The cus infection. A mild to moderate interstitial pneumonia new intranasal model leading to severe pneumonia, was found in all mice including controls and sentinels. Table 1 Distribution of RTX genes pnxIA, pnxIIA and pnxIIIA in R. pneumotropicus and R. heylii only pnxIA only pnxIIA only pnxIIIA pnxIA + IIA pnxIA + IIIA pnxIIA + IIIA pnxIA + IIA + IIIa none R. pneumotropicus (n = 27) 7% 0% 0% 4% 4% 4% 74% 7% R. heylii (n = 26) 46% 0% 0% 0% 12% 0% 0% 42% in total (n = 53) 26% 0% 0% 2% 8% 2% 38% 24% Fornefett et al. BMC Microbiology (2018) 18:45 Page 3 of 11 Table 2 Degree and extent of catarrhal-purulent bronchopneumonia in R. pneumotropicus infected mice (for definition of scores see Additional file 5: Table S5) BALB/c C57BL/6 01–34–70 1–34–7 Controls 10/10 0/10 0/10 9/9 0/9 0/9 Losses 0/9 1/9 8/9 1/8 0/8 7/8 Survivors 7/7 0/7 0/7 8/8 0/8 0/8 Contact sentinels 4/4 0/4 0/4 4/4 0/4 0/4 Furthermore, this pathogen disseminated into non-respiratory internal organs in every experimentally infected mouse (Fig. 2). Bacterial loads in the brains, lungs, L n. tracheobronchiales, livers, spleens, kidneys and genito-urinary tracts were significantly higher in BALB/c than in C57BL/6 mice as assessed by semi-quantitative scoring (mean bacteriological scores for mice succumbing to infection: BALB/c: 11.6 with SD 3.0 and C57BL/6: 6.1 with SD 1.9; p =0.002; Additional file 1: Table S1; Fig. 2). In contact sentinels R. pneumotropicus was mainly de- tected in the lungs and TNLs (Fig. 2), indicating that dis- semination occurred mainly in experimentally infected animals but not in contact sentinels. Additionally, R. pneu- motropicus was not detected in bedding sentinels at all. BALB/c and C57BL/6 mice succumbing to infection within 2 to 6 days post infection (dpi) had high specific bacterial loads in TNL in most cases (mean of 2.6 × 10 colony forming units (CFU)/ml with SD 3.1 × 10 and 5 5 3.4 × 10 CFU/ml with SD 3.7 × 10 , respectively). At the end of the observation period, the bacterial load of the lung was significantly higher in surviving BALB/c mice than in C57BL/6 mice (mean of 4.4 × 10 CFU per g tissue with 4 3 SD 3.2 × 10 and mean of 3.9 × 10 CFU per g tissue with SD 5.0 × 10 , respectively, Fig. 3a). In contact sentinels, mean R. pneumotropicus loads in TNL of 2.8 × 10 CFU Fig. 1 Mortality (a) and body weight (b) of the indicated mice infected with R. pneumotropicus JF4Ni (n = 16 per mouse strain) or per ml TNL in BALB/c and 2.6 × 10 CFU per ml TNL in 4 4 treated with PBS as control (n = 10 for BALB/c and n = 9 for C57BL/ C57BL/6 were recorded (SD 3.9 × 10 and 1.9 × 10 ,re- 6, one control died during anaesthesia). Data of contact sentinels are spectively). Noteworthy, the mean bacteriological score not included. Bronchopneumonia was a main pathology. A based on semi-quantitative assessment of bacterial loads multifocal severe catarrhal-purulent bronchopneumonia of a BALB/c in the brains, lungs, lymphonodi tracheobronchiales, mouse 2 days after intranasal infection is shown (c). Alveoli and bronchioles of this mouse were infiltrated with high numbers of livers, spleens, kidneys and genito-urinary tracts was sig- neutrophilic granulocytes (200 x magnification). The log rank test nificantly higher in surviving BALB/c than in surviving was used to analyse differences between the two mice strains and C57BL/6 mice (mean bacteriological scores of 5.7 (SD 2.7) the groups (a) and 2.4 (SD 3.4) in BALB/c and C57BL/6 mice, respect- ively; p =0.036; Additional file 2: Table S2; Fig. 2). Contact and bedding sentinels showed no clinical signs and pathologies related to R. pneumotropicus infection Antigen-specific IgA in TNL of experimentally infected (data not shown). animals and sentinels As R. pneumotropicus was isolated in TNL of experi- Detection of R. pneumotropicus in tracheonasal lavages mentally infected mice surviving to the end of the obser- (TNL) and internal organs vation period, we asked if this colonization occurred in R. pneumotropicus was detected in TNL and various in- the presence of specific IgA. R. pneumotropicus-specific ternal organs of experimentally infected animals. IgA was not detected in TNL of controls and mice that Fornefett et al. BMC Microbiology (2018) 18:45 Page 4 of 11 Fig. 2 Semi-quantitative determination of R. pneumotropicus in the indicated tissues of BALB/c (a) and C57BL/6 (b) mice either infected experimentally (losses and survivors) or used as contact sentinels. Losses occured 2–6 dpi, survivors were sampled 28 dpi and contact sentinels 56 dpi. A low grade is equal to less than 20 CFU per plate; a middle grade refers to 20–70 CFU and a high grade to more than 70 CFU per plate died within 6 days following experimental infection. On animals (n = 19) consistently gave negative results. Fur- the other hand, TNLs collected 4 weeks post infection thermore, R. pneumotropicus-specific IgG was not re- from experimentally infected mice of both strains and corded in sera from mice succumbing to infection within BALB/c contact sentinels revealed mean titers above 80 the first 6 dpi or in sera from bedding sentinels. ELISA units and mainly positive IgA-titers against R. pneumotropicus (Fig. 3b). In contrast, C57BL/6 contact IgG subclass differentiation indicate differences in the sentinels had rather low specific IgA titers (mean titer of 8 immune response of BALB/c and C57BL/6 mice to R. ELISA units with a SD of 5.9). In summary P. pneumotropi- pneumotropicus infection cus was found to colonize efficiently respiratory mucosa As the specific bacterial load of the lung of surviving despite the presence of specific IgA in mice surviving ex- BALB/c mice was significantly higher than the load of perimental infection. C57BL/6 mice (Fig. 3a), we investigated putative differ- ences in the immune response of the two mice strains. Serum IgG-levels in experimentally infected animals and We used levels of IgG subclasses as indicators for the sentinels kind of immune response as IgG2a and IgG2b are asso- Experimental infection of BALB/c and C57BL/6 mice elic- ciated with a Th1 response while IgG1 is associated with ited specific IgG-titers in all surviving animals (sampled a Th2 response. Upon infection, not only the ratio of 28 dpi) as well as in contact sentinels (sampled 56 dpi) as antigen-specific IgG subtypes might change in serum in shown by ELISA using whole cell extract or a concen- association with a Th1 or Th2 response, but also the trated culture supernatant as antigen (Fig. 4a and b). Sera overall ratios of IgG subtypes . Differentiation of IgG collected prior to infection (n =59) and from control subclasses was conducted in 7 BALB/c and 8 C57BL/6 Fornefett et al. BMC Microbiology (2018) 18:45 Page 5 of 11 Fig. 3 Quantitative determination of R. pneumotropicus in TNL and lungs of the indicated mice surviving experimental infection (a) and pathogen-specific IgA-levels in TNL (b). Medians are marked by the horizontal line. The non-parametric Mann-Whitney test was used for statistical analysis (** for P ≤ 0.01) mice showing a distinct antibody response to R. pneu- IgG2ctiters(above50ELISA Units),whereas theseanti- motropicus antigen. In BALB/c mice, we measured the bodies were not recorded in sera drawn from these mice ratios of IgG2atoIgG1andIgG2btoIgG1. Duetothe prior infection or from control mice (Fig. 5c). lack ofIgG2ainC57BL/6miceonlythe ratioof IgG1 to IgG2 in was determined in this strain. Prior to Discussion infection, the mean IgG2/IgG1 ratios were 0.5 for Experimental infections with P. pneumotropica have IgG2a and 0.4 for IgG2b in BALB/c and 0.8 for IgG2b been conducted in vaccination studies to investigate the in C57BL/6 mice (Fig. 5a and b). Post infection, the protective efficacies of different recombinant proteins mean IgG2a/IgG1 ratio in BALB/c mice increased such as the RTX toxin PnxIII  and different outer slightly to 0.7, while the IgG2b/IgG1 ratio remained membrane proteins . In these studies, protective effi- constant. These results indicate a balanced immune re- cacies were assessed by determining specific bacterial sponse with a tendency to Th2 in BALB/c mice. In loads at different mucosal sites such as the nasal con- C57BL/6 the IgG2btoIgG1ratio increasedto1.6 in- chae, lung and conjunctivae. However, clinical read out dicating a Th1 prone immune response (Fig. 5a). Ac- parameters were not included. Here, an infection model cordingly, the majority of surviving, experimentally with high rates of morbidity and mortality in immuno- infected C57BL/6 mice had high R. pneumotropicus-specific competent BALB/c and C57BL/6 wildtype mice was Fornefett et al. BMC Microbiology (2018) 18:45 Page 6 of 11 Fig. 4 IgG-levels against the indicated R. pneumotropicus antigens in intranasally infected BALB/c (a) and C57BL/6 (b) mice and the respective contact and bedding sentinels in sera drawn at the indicated time points. A serum pool from mice naturally infected with R. pneumotropicus was used as reference serum. The culture supernatant had a concentration factor of 100. Early losses refer to mice killed for animal welfare reasons after developing severe signs of sepsis. Statistical analysis with the Mann-Whitney test was performed to analyse differences between the different groups. The Wilcoxon test was used to compare different time point values within the same groups. The star in panel (b)(whole cellextract)indicates significance (* for P ≤ 0.05) established for the first time, enabling the usages of be relevant for these pathogens as well. The fact that mice respective read out parameters in future vaccination are the natural host of R. pneumotropicus is an important studies. The high morbidity was associated with se- advantage, especially as members of the Pasteurellaceae vere lung pathologies and dissemination of R. pneu- show substantial host adaptation. motropicus to extra-respiratory sites such as liver and The infection dose of 1 × 10 CFU used in this study is brain. Based on the presented data we consider these high. However, previous studies have used similar or models as ideal for investigating protection against severe even higher [15, 27, 28] doses without induction of mor- pneumonia and associated sepsis. Importantly, numerous bidity and mortality. For example, Chapes et al. could immunogens of R. pneumotropicus are homologous to not induce any clinical signs or pathologies in immuno- proteins expressed by Haemophilus influenza and P. competent C57BL/6 wildtype mice with a 400 times multocida , two important pneumonia and sepsis higher dose of the R. pneumotropicus type strain ATCC agents in humans and livestock, respectively. Thus, vac- 35149 . Furthermore, immunocompetent Crlj:CD1 cination trials using the described murine models might mice did not develop lung lesions or clinical signs of disease Fornefett et al. BMC Microbiology (2018) 18:45 Page 7 of 11 might be related to differences in virulence between R. pneumotropicus strains. The R. pneumotropicus JF4Ni strain used in this study is regarded as highly virulent based on the results of the experimental infections and the substantial health issues of the animal facility it was detected in. The fact that this strain bears all three known RTX toxin genes of this pathogen might be re- lated to its virulent phenotype as the toxins are considered important virulence factors [8–10]. Specifically, PnxIII is cytotoxic to macrophages , which are crucial for clearance of R. pneumotropicus in the lungs as shown by transfer of Toll-like receptor 4-positive macro- phages to knockout mice . Noteworthy, more than 70% of the R. pneumotropicus strains investigated in this study shared this RTX toxin genotype, namely pnxIA+ pnxIIA+ pnxIIIA+. As these strains were recently isolated, putative emergence of this important pathotype should be further investigated. All surviving animals of both mouse strains and their contact sentinels produced high levels of R. pneumotropi- cus-specific IgG antibodies, as shown by ELISA using two different antigens. Sera drawn prior to infection and from control animals consistently gave negative results which indicates high specificities of the established ELISAs. Noteworthy, a commercially available ELISA for the de- tection of P. pneumotropica-specific IgG failed to detect specific antibodies in the sera of infected animals surviving until the end of the experiment (results not shown). In agreement to previous studies, all bedding sentinels were serologically negative, rendering this approach in- appropriate for R. pneumotropicus health monitoring [21, 23]. On the other hand, serological screenings of con- tact sentinels for R. pneumotropicus-specific serum IgG using the described ELISA seems to be rather sensitive as an indirect indicator of infection and specific based on the results shown in Fig. 4. Furthermore, contact sentinels might be useful to study clinically inapparent colonization, as none of these mice became morbid at any time post Fig. 5 Ratios of the indicated IgG-subclasses in surviving C57BL/6 (a) infection despite colonisation of the lungs in numerous and BALB/c (b) mice and antigen-specific IgG2c titers in respective C57BL/6 (c). Seven infected BALB/c and 8 C57BL/6 mice with a animals (Fig. 2). distinct antibody answer to R. pneumotropicus antigen (Fig. 4) were This study shows for the first time that R. pneumotropi- chosen for the determination of IgG subclasses in (a) and (b), cus infection results in a mean ratio of IgG2b to IgG1 respectively. In BALB/c mice the ratios between IgG2a to IgG1 and above 1 in C57BL/6 but not in BALB/c mice indicating a IgG2b to IgG1 were determined. As C57BL/6 mice lack IgG2a, only Th1-prone immune response. In contrast, IgG2/IgG1 in IgG1 and IgG2b were measured. Medians are marked by the horizontal line. Differentiation of a Th1 and Th2 immune response is indicated by BALB/c mice remained below 1 post infection suggesting the dashed line. IgG2c-titers in C57BL/6 mice against whole cell extract a more balanced response. It is known already that BALB/ of R. pneumotropicus (c). Titers were determined as indicated either in c and C57BL/6 mice exhibit distinct genetically deter- experimentally infected survivors or respective controls (fbl = final mined differences in their immune systems under physio- bleeding). A serum pool from C57BL/6 mice naturally infected with R. logical conditions, which includes higher amounts of pneumotropicus was used as reference serum. The non-parametric Mann- Whitney test was used for statistical analysis. The P-values are indicated interleukin (IL)-12 in C57BL/6  and associated higher productions of IgG2c and 2b  inducing a Th1 immune after experimental infection with the R. pneumotropicus response. However, based on the rapid progress of disease type strain in contrast to immunodeficient NOD/ShiJic- starting very early after infection, it is reasonable to scid/Jcl mice . This difference to previous studies hypothesize that other mechanisms but Th1 and Th2 Fornefett et al. BMC Microbiology (2018) 18:45 Page 8 of 11 immune responses were crucial for survival in the im- were collected from the submandibular vein and stored munologically naïve mice of the described experimental in- at − 20 °C. fection. This is in agreement with the finding that the more Th1-prone C57BL/6 and the more Th2-prone BALB/c Bacterial strains and culture media mice show comparable morbidities and pathologies. R. pneumotropicus and R. heylii were cultivated as ap- We found high titres of R. pneumotropicus-specific propriate overnight at 37 °C on Columbia Blood Agar IgA (above 150 Elisa) in the TNL of two surviving (COB) or in Brain Heart Infusion (BHI). Experimental BALB/c mice 28 dpi in association with high numbers of infection was conducted with R. pneumotropicus strain this pathogen (above 10 CFU/ml, results not shown). JF4Ni grown in BHI until a concentration of 10 colony These results suggest maintenance of mucosal colonization forming units (CFU) per millilitre was reached. This of R. pneumotropicus despite high titers of specific IgA. As strain was recently isolated from a German research facil- the specific bacterial load in the lung of the investigated ity with severe health problems associated with dyspnoea Dbl−/− survivors was significantly lower in C57BL/6 than in and increased mortality in P2X /P2X knockout but 2 3 BALB/c mice, it is reasonable to hypothesize that the puta- also wt mice . Furthermore, R. pneumotropicus refer- tive Th1-prone immune response in C57BL/6 mice is more ence strain ATCC 35149 and 27 Biotype Jawetz-as well as efficient in restricting persisting lung infection. 26 Biotype Heyl strains were included in this study (Add- itional file 3: Table S3). Conclusions Many R. pneumotropicus strains recently isolated from Infection laboratory mice in Germany carry the genes of the three For experimental infection/mock treatment mice were known RTX toxins of this pathogen. Intranasal applica- anaesthetised by isoflurane inhalation. Mice were experi- tion of the pnxIA+ pnxIIA+ pnxIIIA+ R. pneumotropicus mentally infected by intranasal application of 1 × 10 JF4Ni strain results in invasive and fatal infections in wt CFU R. pneumotropicus JF4Ni in 25 μlphosphate BALB/c and C57BL/6 mice associated with severe pneu- buffered saline (PBS, 12.5 μl per nostril). Controls monia and dissemination to extra-respiratory sites. In were inoculated with PBS only. Contact sentinels remained contrast to BALB/c mice, surviving C57BL/6 mice show untreated, bedding sentinels were turned over to used bed- a Th1-prone immune response and a bacterial load of ding 7 days after inoculation. Experimentally infected and the lung below 10 CFU per g tissue after 28 dpi. The sentinel animals were sacrificed 4 and 8 weeks after described model is ideal for future studies on virulence experimental infection, respectively. For this, mice were and protection elicited by vaccination. anaesthesized through intraperitoneal application of 100 mg ketamin per kg body weight and 5 mg xylazin Methods per kg body weight. They were bled by heart punc- Animals ture and finally killed by cervical dislocation. Eight-week-old female wild type BALB/c mice obtained from Charles River laboratory (Sulzfeld Germany) and Clinical examination and treatment C57BL/6 mice from Janvier Labs (Le Genest-Saint-Isle Every 12 h thorough adspection and weighing of mice was France), both specific pathogen free, were caged ran- performed. Based on predefined criteria (Additional file 4: domly in 6 groups per strain including 2 uninfected and Table S4) clinical signs were scored. Mice with a cumula- 4 infected groups. Each group contained 4 infection/ tive clinical score of 3 or more were treated with flunixin placebo treated animals and one contact sentinel. Add- meglumine for animal welfare reasons (5 mg per kg body itionally, 10 week old female P. pneumotropica-free CD1 weight every 12 h subcutaneously). The following end outbred mice (raised by the Fraunhofer Institute for Cell points led to euthanasia of respective animals: bleeding Therapy and Immunology, Leipzig, Germany) were in- from orifices, paralysis, acute respiratory distress, cyanosis cluded in this study as bedding sentinels (2 cages with 4 and 20% weight loss. Mice with a cumulative score above mice each). Each bedding-group was assigned to one or equal 9 or a score above or equal 6 for 24 h were also mouse strain. The number of animals included in this killed for animal welfare reasons. study was based on the objectives to reveal putative differences between BALB/c and C57BL/6 mice in suscep- tibility, to evaluate sentinel monitoring and to obtain con- Pathological examinations valescence sera for a further immunoproteomics study. For pathological studies brain, nasal conchae, lung, liver, All animals were housed as described in a previous study spleen and kidney were collected, macroscopically exam- in detail , which included ventilated cages with HEPA ined and fixed in 4% CaCO buffered formalin. Tissues filters and air conditioning as well as ad libitum feeding were embedded in paraffin, sectioned and stained with and drinking. One week before infection blood samples haematoxylin and eosin for light microscopy. Scoring of Fornefett et al. BMC Microbiology (2018) 18:45 Page 9 of 11 histopathological findings was conducted as specified in Absorbance was measured at 450 nm (reference at Additional file 5: Table S5. 630 nm). The samples and the controls were measured in a du- Cultural examinations plicate series of four (seven for reference sera) twofold For semi quantitative cultural examinations, lung, brain, dilutions (starting with 1:200 for IgG and 1:50 for IgA). lymphonodi tracheobronchiales, liver, spleen, kidney and Sera collected from mice of a laboratory animal facility the genito-urinary tract of all BALB/c and C57BL/6 mice infected with the R. pneumotropicus strain JF4Ni were were collected and a fresh cut side was pressed on a pooled and served as reference in the IgG and IgGc COB plate, which was incubated for 24 h at 37 °C after ELISA, TNL of an experimentally infected mouse from streaking. For comparative analysis low, middle and high this experiment with a mean antibody titre as reference grades of detection of the typical colonies were scored for the IgA ELISA. These reference samples were de- with 1, 2 and 3 for each investigated tissue, respectively. fined to include 100 ELISA units. Pooled sera and TNL The sum of the scores for all investigated tissues constitutes samples from R. pneumotropicus-free mice were used as the total bacteriological score of each animal. Furthermore, negative controls. Calculation of ELISA units for IgG quantitative cultural examinations were performed with the and IgA was conducted as previously described . TNLs (300 μl PBS). For this, TNLs were serially diluted, spread on COB in duplicates and incubated for 24 h Determination of concentrations of IgG subtypes and at 37 °C. Colonies were differentiated by MALDI- respective ratios in sera of experimentally infected mice TOF-MS (Bruker microflex LT, Bremen). Determination of IgG subtypes and calculation of respect- ive ratios was conducted as described previously . Antigen preparation and ELISA for detection of antigen- specific serum-IgG, serum-IgG2c and mucosal IgA Purification of DNA ELISAs were established for detection of serum IgG and The DNeasy Blood & Tissue Kit (Qiagen) was used ac- mucosal IgA directed against R. pneumotropicus antigen. cording to the manufacturer’s instruction to purify DNA Either a whole cell extract or a concentrated culture super- from R. pneumotropicus and R. heylii cultures. natant of R. pneumotropicus JF4Ni was used as antigen. For preparation of the antigens, 100 ml R. pneumotro- PCR screening for RTX genes pnxIA, pnxIIA and pnxIIIA picus culture were centrifuged at 2000 g for 15 min at To detect the RTX toxin genes pnxIA, pnxIIA and 4 °C. The supernatant was concentrated 100 fold with a pnxIIIA the primer pairs pnxIAF/pnxIAR, pnxIIAF/ 30 kDa centrifugal filter unit (Amicon ultra), mixed with pnxIIAR and pnxIIIAF/pnxIIIARs (Additional file 6: 0.5 ml 10 x protease inhibitor (Protease Inhibitor Cock- Table S6) were used. The pnxIA and pnxIIA genes were tail with EDTA by Sigma Aldrich) per g cell pellet and amplified in full length from the DNA purified from dif- dialyzed against 0.9% NaCl. ferent isolates (Additional file 3: Table S3), including Whole cell extract was prepared from pelleted bacteria strains genotyped in previous studies through 16S–23S as described for Streptobacillus moniliformis previously rRNA internal transcribed spacer analysis [6, 35]. For . Three hundred nanogramms of P. pneumotropica detection of pnxIIIA amplification of an internal 1 kbp antigen (either whole cell extract or a concentrated cul- fragment was conducted. The pnx sequences were amp- ture supernatant) or casein (background measurement) lified by PCR with 30 cycles and Taq-polymerase (Invi- were used per well to coat Corning Costar® assay plates trogen Thermo Scientific Fisher) as recommended by in carbonate buffer (pH 8.1). Blocking with casein and the manufacturer using the following conditions: de- washing of ELISA plates was conducted as described naturation at 95 °C for 30 s, annealing at 58 °C (pnxIA . Twofold serial dilutions of sera and TNLs in PBS and pnxIIIA) or 61 °C (pnxIIA) for 30 s and elongation with 2 mM EDTA, 0.1% Tween20 and 0.1% bovine casein at 72 °C for 3:30 min (pnxIA), 6:30 min (pnxIIA) or were applied to the ELISA plates including also reference 2min(pnxIIIA). sera/reference TNLs as well as negative controls. For detec- tion of P. pneumotropica-specific serum-IgG, serum-IgG2c or TNL-IgA, plates were incubated for 1 h at RT with a Statistical analysis 1:10,000 dilution of a HRP-conjugated goat anti-mouse IgG The Mann-Whitney test was performed to analyse differ- antibody (Jackson Immuno Research Laboratories), or a ences between the different groups of mice. The Wilcoxon 1:10,000 dilution of a HRP-conjugated goat anti-mouse test was used for comparison of different time point values IgG2c antibody (Biorad; C57BL/6 mice only) or a 1:5000 di- within the same group. The data in the Kaplan-Meyer sur- lution of HRP-conjugated goat anti-mouse IgA antibody vival and morbidity diagrams were analysed with the log (BIOMOL GmbH), respectively. ELISA plates were devel- rank test. Probabilities lower than 0.05 were considered oped with 3,3′,5,5′-tetramethylbenzidine as described . significant, lower than 0.001 highly significant. Fornefett et al. BMC Microbiology (2018) 18:45 Page 10 of 11 Additional files Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany. Institute for Veterinary Pathology, Faculty of Veterinary Medicine, University Leipzig, Leipzig, Germany. GVG Diagnostics GmbH, Leipzig, Germany. Additional file 1: Table S1. Scoring of semiquantitive bacteriological Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy findings in R. pneumotropicus infected mice of the indicated strains and Centre for Biotechnology and Biomedicine, University Leipzig, Leipzig, succumbing to infection within the first week. (PDF 12 kb) Germany. Central Unit for Animal Research and Animal Welfare Affairs, Additional file 2: Table S2. Scoring of semiquantitive bacteriological Heinrich-Heine-University, University Hospital, Düsseldorf, Germany. Institute findings in R. pneumotropicus infected mice of the indicated strains of Immunology/Molecular Pathogenesis, Faculty of Veterinary Medicine and surviving until the end of the observation period (4 weeks). (PDF 33 kb) Centre for Biotechnology and Biomedicine, University Leipzig, Leipzig, Germany. Additional file 3: Table S3. Scoring of clinical signs in mice infected with R. pneumotropicus. (PDF 10 kb) Received: 23 August 2017 Accepted: 15 May 2018 Additional file 4: Table S4 R. pneumotropicus and R. heylii strains used in this study. (PDF 10 kb) Additional file 5: Table S5 Scoring of catarrhal - purulent inflammations References in mice infected with R. pneumotropicus. (PDF 4 kb) 1. Boot R, Bisgaard M. Reclassification of 30 Pasteurellaceae strains isolated Additional file 6: Table S6: Oligonucleotide primers used in this study. from rodents. Lab Anim. 1994;29:314–9. (PDF 16 kb) 2. Hedrich HJ. The laboratory mouse. 2nd ed; 2012. 3. 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P. pneumotropica: Pasteurella pneumotropica; PBS: Phosphate buffered saline; 5. Hayashimoto N, Ueno M, Takakura A, Itoh T. A specific polymerase chain PCR: Polymerase chain reaction; R. pneumotropicus/heylii: Rodentibacter reaction based on the gyrB gene sequence and subsequent restriction pneumotropicus/heylii; RTX: Repeats in toxin; SD: Standard deviation; Th: T-helper fragment length polymorphism analysis of Pasteurella pneumotropica cell; TNL: Tracheonasal lavage isolates from laboratory mice. J Am Assoc Lab Anim Sci. 2007;46:54–8. 6. Benga L, Peter W, Benten M, Engelhardt E, Bleich A, Gougoula C, et al. Funding Development of a multiplex PCR assay based on the 16S–23S rRNA internal This work was financially supported by the European Funds of Regional transcribed spacer for the detection and identification of rodent Development (EFRE). The funding body had no role in the design of the Pasteurellaceae. J Microbiol Methods. 2013;95:256–61. study and collection, analysis, and interpretation of data as well as in writing 7. Sasaki H, Ishikawa H, Terayama H, Asano R, Kawamoto E, Ishibashi H, et al. the manuscript. Identification of a virulence determinant that is conserved in the Jawetz and Heyl biotypes of Pasteurella pneumotropica. Pathog Dis. 2016;74:1–4. Availability of data and materials 8. Sasaki H, Kawamoto E, Tanaka Y, Sawada T, Kunita S, Yagami KI. The datasets analysed during this current study are available from the Identification and characterization of hemolysin-like proteins similar to RTX corresponding author upon a reasonable request. toxin in Pasteurella pneumotropica. J Bacteriol. 2009;191:3698–705. 9. Sasaki H, Ishikawa H, Kojima K, Itoh M, Matsumoto T, Itoh T, et al. Intranasal Authors’ contributions immunization with a non-adjuvanted adhesive protein descended from CB and TG conceived the study. 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Published: May 30, 2018