Genotyping of the R. heylii challenge strain SF27GVG
For this study, we selected a recent R. heylii isolate (SF27GVG), which was PCR negative for all three known pnx genes encoding RTX-toxins [8]. We conducted an additional independent PCR for each pnx gene to verify the genotype. As shown in Fig. 1, the gene encoding the “characteristic antigen for Rodentibacter of laboratory origin 1“ (CARLO-1 [12]) was detected but none of the three known pnx genes using DNA of strain R. heylii SF27GVG in contrast to the R. pneumotropicus strain JF4Ni previously characterized in experimental infections [8].
R. heylii strain SF27GVG carrying no known pnx gene is highly virulent
As R. heylii is an opportunistic pathogen in rodents known to cause severe systemic infections in immunodeficient mice but much less so in wildtype mice, we expected only mild to moderate clinical signs after experimental infection of wildtype mice. However, intranasal application of 108 CFU resulted in a mortality of 100% (8/8) in BALB/c and 75% (6/8) in C67BL/6 mice due to severe weight loss above 20% or reaching other termination criteria (Fig. 2A). These mice showed unspecific signs such as ruffled coat and bended back as well as specific signs such as conjunctivitis and dyspnoea. Mortality was only observed on the first two days following experimental infection. Lungs of early euthanized mice infected with 108 CFU of R. heylii showed mild, moderate or severe catarrhal-purulent bronchopneumonia (Fig. 2B), which was not observed in control mice (Tab. 1). Within inflamed tissue larger aggregations of bacteria were found (Fig. 2C). All mice surviving the experiment until the end of the observation period including those infected with M. muris did not show any histopathological findings except for an interstitial pneumonia also recorded in control animals.
Moderate weight loss was observed in 3 of 4 C57BL/6 (5-11% weight loss) and 4 of 4 BALB/c mice (5-9% weight loss) infected with a lower dose of 106 CFU (Fig. 3). Mice infected with the lowest dose of 104 CFU of R. heylii SF27GVG did not show a weight loss exceeding 1% or any other clinical signs.
Infection with M. muris 694/11 did not result in any mortality. As shown in Fig. 3, after infection with 108 CFU per mouse, mice showed a moderate decrease in body weight with a loss of 11-13% in C57BL/6 mice and 3-8% in BALB/c mice. These mice showed mildly ruffled coat and depression.
In conclusion, R. heylii SF27GVG but not M. muris 694/11 is capable of inducing weight loss above 20% associated with purulent bronchopneumonia in C57BL/6 and BALB/c wildtype mice after intranasal application of 108 CFU. The known RTX toxins of the genus Rodentibacter are not necessary for this virulence potential.
R. heylii strain SF27GVG disseminates in various internal organs of wildtype mice
R. heylii was detected in various internal organs of early euthanized mice as specified in Fig. 4. In 3 of 6 C57BL/6 mice and 6 of 8 BALB/c mice R. heylii was detected in at least two internal organs including liver, spleen, kidney, genito-urinary tract and brain. Except in one C57BL/6 mouse, these mice showed dissemination of R. heylii to the genito-urinary tract. In the two surviving C57BL/6 mice infected with 108 CFU R. heylii was isolated from the lymphonodus (Ln.) tracheobronchialis and the genito-urinary tract. Infection with 106 CFU was associated with cultural detection of R. heylii in lungs, lymphonodi tracheobronchiales, and the genito-urinary tract but no other internal organs except for the liver of one animal (Fig. 4, Fig. 5). In contrast, a lower dose of 104 CFU of R. heylii did not resulted in detection of this pathogen in the genito-urinary tract but in the tracheo-nasal lavage with a specific bacterial load comparable to loads in animals infected with higher doses.
M. muris was identified in tracheo-nasal lavages (TNL) of all mice experimentally infected with M. muris (Fig. 6B). Furthermore, in 11/32 mice M. muris was also isolated from the genito-urinary tracts and in spleens of 4/32 mice (Fig. 6A). In contrast, M. muris was not detected in mock-infected mice. Based on semi-quantitative assessments the specific bacterial load of M. muris in the genito-urinary tract was very high in 7/32 mice.
In summary, mortality in R. heylii infected mice was associated with dissemination of the pathogen to internal organs. The genito-urinary tract is a main target organ of R. heylii and M. muris.
PCR-based detection of R. heylii and M. muris in swabs
PCRs for R. heylii and M. muris DNA were conducted with swabs taken post infection from the ano-genital region and the pharynx as described before [13]. Ano-genital swabs exhibited positive PCR results at 7, 14, 21 and 28 days after infection in 5/8, 7/8, 6/8 and 4/8 mice, respectively, infected with 104 CFU and 106 CFU of R. heylii. Surviving mice infected with 108 CFU of R. heylii exhibited positive PCR results at 21 and 28 days after infection. Furthermore, R. heylii DNA was detected in all pharynx swabs taken from infected animals 28 days after experimental infection. Four of eight tested mice infected with 106 CFU or 108 CFU of M. muris exhibited positive PCR results in ano-genital swabs 28 days after infection. All placebo-treated mice remained PCR negative for R. heylii and M. muris throughout the experiment. In conclusion, swabs from the pharynx and ano-genital region are suitable samples for detection of R. heylii and M. muris infection using PCR.
Seroconversion of R. heylii and M. muris infected mice
As M. muris is considered to be apathogenic and mice infected with lower doses of R. heylii and M. muris did not show any clinical signs, we asked if these mice developed an adaptive serum IgG immune responsive against these bacteria. Therefore, an in-house whole cell extract ELISA was developed for both species (Fig. 7). Both ELISA were specific and sensitive (R. heylii-ELISA: sensitivity = 94.4%, specificity = 81.5%; M. muris-ELISA: sensitivity = 93.8%, specificity = 100%). Mice infected with R. heylii or M. muris showed increased serum IgG levels against the respective antigen 28 days after infection in comparison to phosphate buffered saline (PBS) treated mice. Specific IgG were not detected in early losses after R. heylii infection (Fig. 6A-B). One mouse showed elevated specific IgG levels against M. muris before experimental infection. Additionally, sera from mice of recent experimental infections were also investigated in the two ELISAs. None of the mice infected experimentally with R. heylii or R. pneumotropicus showed values above the cut-off in the in-house ELISA detecting IgG antibodies against M. muris. In contrast, 11 of 20 tested sera from R. pneumotropicus infected mice [8] showed positive results in the IgG anti-R. heylii ELISA. In summary, most of the intranasally with R. heylii or M. muris infected C57BL/6 and BALB/c wildtype mice showed specific IgG antibodies against the homologous antigen.