Nicola Casson

Lausannevirus, a giant amoebal virus encoding histone doublets

Large viruses infecting algae or amoebae belong to the NucleoCytoplasmic Large DNA Viruses (NCLDV) and present genotypic and phenotypic characteristics that have raised major interest among microbiologists. Here, we describe a new large virus discovered in Acanthamoeba castellanii co-culture of an environmental sample. The virus, referred to as Lausannevirus, has a very limited host range, infecting Acanthamoeba spp. but being unable to infect other amoebae and mammalian cell lines tested. Within A. castellanii, this icosahedral virus of about 200 nm exhibits a development cycle similar to Mimivirus, with an eclipse phase 2 h post infection and a logarithmic growth leading to amoebal lysis in less than 24 h. The 346 kb Lausannevirus genome presents similarities with the recently described Marseillevirus, sharing 89% of genes, and thus belongs to the same family as confirmed by core gene phylogeny. Interestingly, Lausannevirus and Marseillevirus genomes both encode three proteins with predicted histone folds, including two histone doublets, that present similarities to eukaryotic and archaeal histones. The discovery of Lausannevirus and the analysis of its genome provide some insight in the evolution of these large amoebae-infecting viruses.

Parachlamydia spp. and related Chlamydia-like organisms and bovine abortion.

Chlamydophila abortus and Waddlia chondrophila cause abortion in ruminants. We investigated the role of Parachlamydia acanthamoebae in bovine abortion. Results of immunohistochemical analyses were positive in 30 (70%) of 43 placentas from which Chlamydia-like DNA was amplified, which supports the role of Parachlamydia spp. in bovine abortion.

Protochlamydia naegleriophila as Etiologic Agent of Pneumonia

Using ameba coculture, we grew a Naegleria endosymbiont. Phenotypic, genetic, and phylogenetic analyses supported its affiliation as Protochlamydia naegleriophila sp. nov. We then developed a specific diagnostic PCR for Protochlamydia spp. When applied to bronchoalveolar lavages, results of this PCR were positive for 1 patient with pneumonia. Further studies are needed to assess the role of Protochlamydia spp. in pneumonia.

New Diagnostic Real-Time PCR for Specific Detection of Parachlamydia acanthamoebae DNA in Clinical Samples

ABSTRACT Given the low sensitivity of amoebal coculture, we developed a specific real-time PCR for the detection of Parachlamydia. The analytical sensitivity was high, and the inter- and intrarun variabilities were low. When the PCR was applied to nasopharyngeal aspirates, it was positive for six patients with bronchiolitis. Future studies should assess the role of Parachlamydia in bronchiolitis.

Evidence for Parachlamydia in bovine abortion

Bovine abortion of unknown infectious aetiology still remains a major economic problem. In this study, we focused on a new possible abortigenic agent called Parachlamydia acanthamoebae. Retrospective samples (n=235) taken from late-term abortions in cattle were investigated by real-time diagnostic PCR for Chlamydiaceae and Parachlamydia spp., respectively. Histological sections of cases positive by real-time PCR for any Chlamydia-related agent were further examined by immunohistochemistry using specific antibodies. Chlamydophila abortus was detected only in three cases (1.3%) by real-time PCR and ArrayTube Microarray playing a less important role in bovine abortion compared to the situation in small ruminants in Switzerland. By real-time PCR as many as 43 of 235 (18.3%) cases turned out to be positive for Parachlamydia. The presence of Parachlamydia within placental lesions was confirmed in 35 cases (81.4%) by immunohistochemistry. The main histopathological feature in parachlamydial abortion was purulent to necrotizing placentitis (25/43). Parachlamydia should be considered as a new abortigenic agent in Swiss cattle. Since Parachlamydia may be involved in lower respiratory tract infections in humans, bovine abortion material should be handled with care given the possible zoonotic risk.

Murine model of pneumonia caused by Parachlamydia acanthamoebae

The role of Parachlamydia acanthamoebae as an agent of pneumonia is suggested by sero-epidemiological studies, molecular surveys and by the permissivity of macrophages, lung fibroblasts and pneumocytes to this obligate intracellular bacteria. We thus developed a murine model of pneumonia due to Parachlamydia. Mice were inoculated intratracheally with Parachlamydia acanthamoebae. Pneumonia-associated mortality was of 50% 5 days post-inoculation. Lungs histopathology was characterized by purulent and interstitial pneumonia. The presence of Parachlamydia in the lesions was demonstrated by PCR, immunohistochemistry and electron microscopy. Moreover, living Parachlamydia could be recovered from the lungs of infected mice using amoebal co-culture. All control mice inoculated with heat-inactivated bacteria were free of symptoms and survived. Thus, we demonstrated that Parachlamydia induce a severe pneumonia in mice. This animal model, which confirms the third and fourth Koch postulates, may be suitable to test in vivo efficient therapeutic regimens against Parachlamydia.

Serological Cross-Reactivity between Different Chlamydia-Like Organisms

ABSTRACT The serological cross-reactivity between different recently described Chlamydia-related organisms was determined. Mouse sera exhibited a strong reactivity against autologous antigen and closely related heterologous antigen but no cross-reactivity with distantly related species. These results are important to better interpret serological studies and assess the pathogenic role of these obligate intracellular bacteria.

Parachlamydia acanthamoebae infection and abortion in small ruminants.

To the Editor: Abortion in ruminants is of worldwide economic importance. Moreover, several abortigenic agents have a zoonotic potential, i.e., Brucella abortus, Coxiella burnetii, and Chlamydophila abortus. C. abortus, which causes ovine enzootic abortion, may also infect pregnant women who have had contact with C. abortus–infected sheep and goats, and such infection can lead to miscarriage (1). Parachlamydia acanthamoebae (2) is a Chlamydia-related organism considered as an emerging agent of pneumonia in humans. Recently, we reported its role in the setting of bovine abortion (3). Here, we investigated the prevalence of C. abortus and P. acanthamoebae infections in abortions in small ruminants. Formalin-fixed placenta, fetal lung and liver, or both, were available from abortion products from 144 goats and 86 sheep (n = 211). These specimens had previously been investigated for several abortigenic agents (4). Placentas and fetal organs were analyzed by histopathologic examination and by specific real-time PCR and immunohistochemical protocols that detect members of the Chlamydiaceae family and P. acanthamoebae. DNA from paraffin blocks was extracted as described (5) by using the DNeasy Tissue kit (QIAGEN, Hilden, Germany). The real-time PCR for Chlamydiaceae was conducted on an ABI 7500 (Applied Biosystems, Foster City, CA, USA) by using a modified version of Everett’s PCR (6). Primers Ch23S-F (5′-CTGAAACCAGTAGCTTATAAGCGGT-3′), Ch23S-R (5′-ACCTCGCCGTTTAACTTAACTCC-3′), and probe Ch23S-p (5′-FAM-CTCATCA TGCAAAAGGCACGCCG-TAMRA-3′) were used to amplify and detect a 111-bp product specific for members of the family Chlamydiaceae. Chlamydial species identification of real-time PCR positive cases was performed with the ArrayTube Microarray (Clondiag, Jena, Germany) as described (7). The Parachlamydia-specific real-time PCR was performed with the ABI Prism 7000 sequence detection system (Applied Biosystems), as reported (8). This PCR is genus-specific, as demonstrated by the absence of PCR positivity with DNA extracted from other Parachlamydiaceae (Protochlamydia spp./Neochlamydia hartmannellae). To confirm positive results, another specific PCR, which targeted the tlc gene, was performed (9). Paraffin sections from specimens positive in real-time PCR were further examined by immunohistochemical tests. A Chlamydiaceae-specific mouse monoclonal antibody directed against the chlamydial lipopolysaccharide (Progen, Heidelberg, Germany) and a specific mouse polyclonal antibody against Parachlamydia spp. was used as described (3,5,10). These antibodies were applied at dilutions of 1:200 and 1:1,000, respectively. Detection was performed with a detection kit (ChemMate; Dako, Glostrup, Denmark). Antigen retrieval was performed by enzyme digestion for 10 minutes (Pronase; Dako) for the Chlamydiaceae antibody and repeated microwave treatment in citrate buffer (ChemMate; Dako) for the Parachlamydia antibody, respectively. Double immunohistochemical labeling was performed on the sheep abortion specimen identified as simultaneously infected with Chlamydiaceae and Parachlamydia spp. Immunohistochemical analysis for both pathogens was performed subsequently by using diaminobenzidine as substrate for the Chlamydiaceae antibody (brown labeling) and by using 3-amino-9-ethylcarbazole as substrate for the Parachlamydia antibody (red labeling). Specificity of PCR and immunohistochemical tests for Chlamydiaceae and Parachlamydia spp., respectively, was assessed by using negative control placentas taken from 2 healthy ruminants (both specimens were negative in all tests). Results of real-time PCR showed that 55 (26.1%) of 211 specimens were positive for Chlamydiaceae. All 55 cases could be identified as C. abortus by ArrayTube Microarray (Clondiag). Of these, 42 (76.4%) could be confirmed by immunohistochemical analysis with the anti-Chlamydiaceae antibody. Of the 211 specimens, only 2 (0.9%) were positive for Parachlamydia spp. by real-time PCR, and both cases could be confirmed by immunohistochemical testing with the parachlamydial antibody. These 2 specimens were negative for other common abortigenic agents such as Toxoplasma gondii, C. burnetii, and border disease virus (data not shown). One case was recorded among the 144 goat samples investigated. This placenta displayed necrotizing placentitis and was positive for Parachlamydia spp. by 16S rRNA-specific real-time PCR (cycle threshold [Ct] 40.5) and immunohistochemical testing, but negative for Chlamydiaceae. Results of this PCR was confirmed by another PCR, targeting the tlc gene (Ct 36.7), which excluded false-positive results because of amplicon contamination. The second case was identified among the 86 sheep investigated. Placenta and fetal lung and liver exhibited necrotizing placentitis and vasculitis (Figure, panel A), interstitial pneumonia (Figure, panel B), and mixed cellular periportal hepatitis. Fetal liver was negative by parachlamydial 16S rRNA real-time PCR and immunohistochemical analysis, but the fetal lung was positive by parachlamydial 16S rRNA real-time PCR (Ct 40.7) and immunohistochemical tests (Figure, panel C), but negative with the tlc PCR. Fetal lung and liver were positive by real-time PCR for Chlamydiaceae (mean Ct for both organs 36.7), but negative by immunohistochemical tests. The placenta was positive for Chlamydiaceae by immunohistochemical tests and real-time PCR (mean Ct 23.3), and C. abortus was identified by ArrayTube Microarray. Brown (Chlamydiaceae) and red (Parachlamydia spp.) granular reaction was demonstrated within the necrotic lesions of the placenta by double immunohistochemical labeling (Figure, panel D). Figure A) Sheep placenta positive by real-time PCR and immunohistochemistry for Parachlamydia spp. and Chlamydiaceae. Chlamydophila abortus was identified by ArrayTube Microarray. Necrotizing placentitis and vasculitis are shown (hematoxylin and eosin stain; ... We report Parachlamydia infection in small ruminant abortion. C. abortus and Parachlamydia spp. were simultaneously present in an aborted sheep placenta. Parachlamydia spp. could be further detected in the lung of the aborted sheep fetus by real-time PCR and immunohistochemistry. Parachlamydia was also detected in a goat placenta. Thus, Parachlamydia spp. should be considered as a new abortigenic agent in sheep and goats. Persons in contact with small ruminants should be informed about the zoonotic potential of these abortigenic agents.

Role of MyD88 and Toll-Like Receptors 2 and 4 in the Sensing of Parachlamydia acanthamoebae

ABSTRACT Parachlamydia acanthamoebae is a Chlamydia-related organism whose pathogenic role in pneumonia is supported by serological and molecular clinical studies and an experimental mouse model of lung infection. Toll-like receptors (TLRs) play a seminal role in sensing microbial products and initiating innate immune responses. The aim of this study was to investigate the roles of MyD88, TLR2, and TLR4 in the interaction of Parachlamydia with macrophages. Here, we showed that Parachlamydia entered bone-marrow derived macrophages (BMDMs) in a TLR-independent manner but did not multiply intracellularly. Interestingly, compared to live bacteria, heat-inactivated Parachlamydia induced the production of substantial amounts of tumor necrosis factor alpha (TNF), interleukin-6 (IL-6), and IL-12p40 by BMDMs and of TNF and IL-6 by peritoneal macrophages as well as RAW 264.7 and J774 macrophage cell lines. Cytokine production by BMDMs, which was partially inhibited upon trypsin treatment of Parachlamydia, was dependent on MyD88, TLR4, and, to a lesser extent, TLR2. Finally, MyD88−/−, TLR4−/−, and TLR2−/− mice were as resistant as wild-type mice to lung infection following the intratracheal instillation of Parachlamydia. Thus, in contrast to Chlamydia pneumoniae, Parachlamydia acanthamoebae weakly stimulates macrophages, potentially compensating for its low replication capacity in macrophages by escaping the innate immune surveillance.

Tissue microarray and immunohistochemistry as tools for evaluation of antibodies against Chlamydia-like bacteria.

Tissue microarray technology was used to establish immunohistochemistry protocols and to determine the specificity of new antisera against various Chlamydia-like bacteria for future use on formalin-fixed and paraffin-embedded tissues. The antisera exhibited strong reactivity against autologous antigen and closely related heterologous antigen, but no cross-reactivity with distantly related species.