Experimental Chlamydia gallinacea infection in chickens does not protect against a subsequent experimental Chlamydia psittaci infection.

Chlamydia psittaci was considered the predominant chlamydial species in poultry until Chlamydia gallinacea was discovered in 2009. C. psittaci is a zoonotic obligate intracellular bacterium reported in more than 465 bird species including poultry. In poultry, infections can result in asymptomatic disease, but also in more severe systemic illness. The zoonotic potential of C. gallinacea has yet to be proven. Infections in poultry appear to be asymptomatic and in recent prevalence studies C. gallinacea was the main chlamydial species found in chickens. The high prevalence of C. gallinacea resulted in the question if an infection with C. gallinacea might protect against an infection with C. psittaci. To investigate possible cross protection, chickens were inoculated with C. gallinacea NL_G47 and subsequently inoculated with either a different strain of C. gallinacea (NL_F725) or C. psittaci. Chickens that had not been pre-inoculated with C. gallinacea NL_G47 were used as a C. gallinacea or C. psittaci infection control. In the groups that were inoculated with C. psittaci, no difference in pharyngeal or cloacal shedding, or in tissue dissemination was observed between the control group and the pre-inoculated group. In the groups inoculated with C. gallinacea NL_F725, shedding in cloacal swabs and tissues dissemination was lower in the group pre-inoculated with C. gallinacea NL_G47. These results indicate previous exposure to C. gallinacea does not protect against an infection with C. psittaci, but might protect against a new infection of C. gallinacea.

Pathogenicity of Chlamydia gallinacea in chickens after oral inoculation.

Chlamydia gallinacea is a recently discovered and widespread obligate intracellular bacterium in chickens. In chickens, infections appear to be asymptomatic, but can result in reduced weight gain in broilers. Molecular typing revealed C. gallinacea is genetically diverse which might lead to differences in pathogenic potential between strains. However, studies about the pathogenesis of different C. gallinacea strains are still limited. In this study, the pathogenesis of C. gallinacea strain NL_G47 was investigated in three consecutive animal experiments. The first experiment served as a pilot in which a maximum culturable dose was administered orally to 13 chickens. Excretion of chlamydial DNA in cloacal swabs was measured during 11 days post infection, but no clinical signs were observed. The second and third experiment were a repetition of the first experiment, but now chickens were sacrificed at consecutive time points to investigate tissue dissemination of C. gallinacea. Again excretion of chlamydial DNA in cloacal swabs was detected and no clinical signs were observed in line with the results of the first experiment. PCR and immunohistochemistry of tissue samples revealed C. gallinacea infected the epithelium of the jejunum, ileum and caecum. Furthermore, C. gallinacea could be detected in macrophages in the lamina propria and in follicular dendritic cells (FDCs) of the B cell follicles in the caecal tonsil. Results of serology showed a systemic antibody response from day seven or eight and onward in all three experiments. The experiments with strain NL_G47 confirmed observations from field studies that C. gallinacea infection does not result in acute clinical disease and mainly resides in the epithelium of the gut. Whether the presence of C. gallinacea results in chronic persistent infections with long term and less obvious health effects in line with observations on other infections caused by Chlamydiae, needs further investigation.

In vivo transcriptomes of Streptococcus suis reveal genes required for niche-specific adaptation and pathogenesis.

Streptococcus suis is a Gram-positive bacterium and a zoonotic pathogen residing in the nasopharynx or the gastrointestinal tract of pigs with a potential of causing life-threatening invasive disease. It is endemic in the porcine production industry worldwide, and it is also an emerging human pathogen. After invasion, the pathogen adapts to cause bacteremia and disseminates to different organs including the brain. To gain insights in this process, we infected piglets with a highly virulent strain of S. suis, and bacterial transcriptomes were obtained from blood and different organs (brain, joints, and heart) when animals had severe clinical symptoms of infection. Microarrays were used to determine the genome-wide transcriptional profile at different infection sites and during growth in standard growth medium in vitro. We observed differential expression of around 30% of the Open Reading Frames (ORFs) and infection-site specific patterns of gene expression. Genes with major changes in expression were involved in transcriptional regulation, metabolism, nutrient acquisition, stress defenses, and virulence, amongst others, and results were confirmed for a subset of selected genes using RT-qPCR. Mutants were generated in two selected genes, and the encoded proteins, i.e., NADH oxidase and MetQ, were shown to be important virulence factors in coinfection experiments and in vitro assays. The knowledge derived from this study regarding S. suis gene expression in vivo and identification of virulence factors is important for the development of novel diagnostic and therapeutic strategies to control S. suis disease.

Pneumococcal colonization and invasive disease studied in a porcine model.

BACKGROUND: Streptococcus pneumoniae, a Gram-positive bacterium carried in the human nasopharynx, is an important human pathogen causing mild diseases such as otitis media and sinusitis as well as severe diseases including pneumonia, meningitis and sepsis. There is a strong resemblance between the anatomy, immunology and physiology of the pig and human species. Furthermore, there are striking similarities between S. suis pathogenesis in piglets and S. pneumoniae pathogenesis in humans. Therefore, we investigated the use of piglets as a model for pneumococcal colonization and invasive disease. RESULTS: Intravenous inoculation of piglets with an invasive pneumococcal isolate led to bacteraemia during 5 days, showing clear bacterial replication in the first two days. Bacteraemia was frequently associated with fever and septic arthritis. Moreover, intranasal inoculation of piglets with a nasopharyngeal isolate led to colonization for at least six consecutive days. CONCLUSIONS: This demonstrates that central aspects of human pneumococcal infections can be modelled in piglets enabling the use of this model for studies on colonization and transmission but also on development of vaccines and host-directed therapies. Moreover this is the first example of an animal model inducing high levels of pneumococcal septic arthritis.

Invasive pneumococcal disease leads to activation and hyperreactivity of platelets.

Using a novel porcine model of intravenous Streptococcus pneumoniae infection, we showed that invasive pneumococcal infections induce marked platelet activation and hyperreactivity. This may contribute to the vascular complications seen in pneumococcal infection.

A naturally occurring nucleotide polymorphism in the orf2/folc promoter is associated with Streptococcus suis virulence.

BACKGROUND: Streptococcus suis is a major problem in the swine industry causing meningitis, arthritis and pericarditis in piglets. Pathogenesis of S. suis is poorly understood. We previously showed that introduction of a 3 kb genomic fragment from virulent serotype 2 strain 10 into a weakly virulent serotype 2 strain S735, generated a hypervirulent isolate. The 3 kb genomic fragment contained two complete open reading frames (ORF) in an operon-structure of which one ORF showed similarity to folylpolyglutamate synthetase, whereas the function of the second ORF could not be predicted based on database searches for protein similarity. RESULTS: In this study we demonstrate that introduction of orf2 from strain 10 into strain S735 is sufficient to dramatically increase the virulence of S735 in pigs. This increase in virulence could not be associated with changes in pro-inflammatory responses of porcine blood mononucleated cells in response to S. suis in vitro. Sequence analysis of the orf2-folC-operon of S. suis isolates 10 and S735 revealed an SNP in the -35 region of the putative promoter sequence of the operon, as well as several SNPs resulting in amino acid substitutions in the ORF2 protein. Transcript levels of orf2 and folC were significantly higher in the virulent strain 10 than in the weakly virulent strain S735 and in vitro mutagenesis of the orf2 promoter confirmed that this was due to a SNP in the predicted -35 region upstream of the orf2 promoter. In this study, we demonstrated that the stronger promoter was present in all virulent and highly virulent S. suis isolates included in our study. This highlights a correlation between high orf2 expression and virulence. Conversely, the weaker promoter was present in isolates known to be weakly pathogenic or non-pathogenic. CONCLUSION: In summary, we demonstrate the importance of orf2 in the virulence of S. suis.

Lipoprotein signal peptidase of Streptococcus suis serotype 2.

This paper reports the complete coding sequence for a proliprotein signal peptidase (SP-ase) of Streptococcus suis, Lsp. This is believed to be the first SP-ase described for S. suis. SP-ase II is involved in the removal of the signal peptide from glyceride-modified prolipoproteins. By using in vitro transcription/translation systems, it was shown that the lsp gene was transcribed in vitro. Functionality of Lsp in Escherichia coli was demonstrated by using an in vitro globomycin resistance assay, to show that expression of Lsp in E. coli increased the globomycin resistance. An isogenic mutant of S. suis serotype 2 unable to produce Lsp was constructed and shown to process lipoproteins incorrectly, including an S. suis homologue of the pneumococcal PsaA lipoprotein. Five piglets were inoculated with a mixture of both strains in an experimental infection, to determine the virulence of the mutant strain relative to that of the wild-type strain in a competitive challenge experiment. The data showed that both strains were equally virulent, indicating that the knockout mutant of lsp is not attenuated in vivo.

Response regulator important in pathogenesis of Streptococcus suis serotype 2.

In this study, we describe the first response regulator of Streptococcus suis serotype 2, designated RevS. RevS was cloned and the sequence was determined. No histidine kinase was found in the vicinity ofrevS, therefore RevS was considered to be an orphan response regulator. An isogenic knock-out mutant of RevS was shown to be attenuated in colonising the S. suis specific organs in a competitive assay in four piglets, indicating thatrevS plays a role in the pathogenesis of S. suis infections. We analysed the protein expression profiles of various fractions of the wild-type strain 10 and the mutant strain 10DeltaRevS. The expression of known virulence factors ofS. suis by wild-type and the mutant strain, was not different. However, one protein in the protoplast fraction, with unknown identity was shown to be repressed by revS in the exponential growth phase of the mutant.

Distribution of environmentally regulated genes of Streptococcus suis serotype 2 among S. suis serotypes and other organisms.

The occurrence of 36 environmentally regulated genes of Streptococcus suis strain 10 among all 35 S. suis serotypes was determined by using hybridization with the amplified genes as probes. In addition, the distribution of these genes among the virulence phenotypes of serotypes 1 and 2 was assessed. Hybridization was also performed with various other streptococcal species and nonstreptococcal bacterial species which may be present in pigs. Interestingly, probe ivs-25/iri-1, similar to agrA and sapR, hybridized only with S. suis serotype 1 and 2 strains with virulent phenotypes and is therefore suitable as a diagnostic parameter. Only one probe was specific for S. suis. This probe's sequence was identical to the epf gene, a putative virulence factor of S. suis. Probe ivs-31 was similar to a virulence factor of S. suis, namely, a gene encoding a fibronectin- and fibrinogen-binding protein. This probe hybridized only with oral streptococci. Nearly half of the probes (45%) hybridized with the oral streptococci (S. oralis, S. milleri, S. sanguis, S. gordonii, and S. mitis) and with Streptococcus pneumoniae. This indicates a close relationship between S. suis, the oral streptococci, and S. pneumoniae with respect to the selected environmentally regulated genes. One probe only hybridized with gram-negative species and therefore seems to be obtained by S. suis from a gram-negative organism by horizontal transfer.

Contribution of fibronectin-binding protein to pathogenesis of Streptococcus suis serotype 2.

In the present study we investigated the role of the fibronectin (FN)- and fibrinogen (FGN)-binding protein (FBPS) in the pathogenesis of Streptococcus suis serotype 2 in piglets. The complete gene encoding FBPS from S. suis serotype 2 was cloned in Escherichia coli and sequenced. The occurrence of the gene in various serotypes was analyzed by hybridization studies. The FBPS protein was expressed in E. coli and purified, and binding to human FN and FGN was demonstrated. The induction of antibodies in piglets was studied upon infection. An isogenic mutant unable to produce FBPS was constructed, and the levels of virulence of the wild-type and mutant strains were compared in a competitive infection model in young piglets. Organ cultures showed that FBPS was not required for colonization of the tonsils but that FBPS played a role in the colonization of the specific organs involved in an S. suis infection. Therefore, the FBPS mutant was considered as an attenuated mutant.