Mixed infections with Chlamydia and porcine epidemic diarrhea virus - a new in vitro model of chlamydial persistence.

BACKGROUND: Chlamydiae induce persistent infections, which have been associated with a wide range of chronic diseases in humans and animals. Mixed infections with Chlamydia and porcine epidemic diarrhea virus (PEDV) may result in generation of persistent chlamydial infections. To test this hypothesis, an in vitro model of dual infection with cell culture-adapted PEDV and Chlamydia abortus or Chlamydia pecorum in Vero cells was established. RESULTS: Infected cultures were investigated by immunofluorescence (IF), transmission electron microscopy (TEM) and re-infection experiments. By IF, Chlamydia-infected cells showed normal inclusions after 39 hpi. Dual infections with Chlamydia abortus revealed a heterogenous mix of inclusion types including small inclusions consisting of aberrant bodies (ABs), medium-sized inclusions consisting of ABs and reticulate bodies and normal inclusions. Only aberrant inclusions were observable in dual infection experiments with Chlamydia pecorum and PEDV. TEM examinations of mixed infections with Chlamydia abortus and Chlamydia pecorum revealed aberrant chlamydial inclusions containing reticulate-like, pleomorphic ABs, which were up to 2 microm in diameter. No re-differentiation into elementary bodies (EBs) was detected. In re-infection experiments, co-infected cells produced fewer EBs than monoinfected cells. CONCLUSIONS: In the present study we confirm that PEDV co-infection alters the developmental cycle of member species of the family Chlamydiaceae, in a similar manner to other well-described persistence induction methods. Interestingly, this effect appears to be partially species-specific as Chlamydia pecorum appears more sensitive to PEDV co-infection than Chlamydia abortus, as evidenced by TEM and IF observations of a homogenous population of aberrant inclusions in PEDV - Chlamydia pecorum co-infections.

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.

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.

Identification of farnesoid X receptor beta as a novel mammalian nuclear receptor sensing lanosterol.

Nuclear receptors are ligand-modulated transcription factors. On the basis of the completed human genome sequence, this family was thought to contain 48 functional members. However, by mining human and mouse genomic sequences, we identified FXRbeta as a novel family member. It is a functional receptor in mice, rats, rabbits, and dogs but constitutes a pseudogene in humans and primates. Murine FXRbeta is widely coexpressed with FXR in embryonic and adult tissues. It heterodimerizes with RXRalpha and stimulates transcription through specific DNA response elements upon addition of 9-cis-retinoic acid. Finally, we identified lanosterol as a candidate endogenous ligand that induces coactivator recruitment and transcriptional activation by mFXRbeta. Lanosterol is an intermediate of cholesterol biosynthesis, which suggests a direct role in the control of cholesterol biosynthesis in nonprimates. The identification of FXRbeta as a novel functional receptor in nonprimate animals sheds new light on the species differences in cholesterol metabolism and has strong implications for the interpretation of genetic and pharmacological studies of FXR-directed physiologies and drug discovery programs.

Ovine Enzootic Abortion (OEA): a comparison of antibody responses in vaccinated and naturally-infected swiss sheep over a two year period.

BACKGROUND: Prevention and control of ovine enzootic abortion (OEA) can be achieved by application of a live vaccine. In this study, five sheep flocks with different vaccination and infection status were serologically tested using a competitive enzyme-linked immunosorbent assay (cELISA) specific for Chlamydophila (Cp.) abortus over a two-year time period. RESULTS: Sheep in Flock A with recent OEA history had high antibody values after vaccination similar to Flock C with natural Cp. abortus infections. In contrast, OEA serology negative sheep (Flock E) showed individual animal-specific immunoreactions after vaccination. Antibody levels of vaccinated ewes in Flock B ranged from negative to positive two and three years after vaccination, respectively. Positive antibody values in the negative control Flock D (without OEA or vaccination) are probably due to asymptomatic intestinal infections with Cp. abortus. Excretion of the attenuated strain of Cp. abortus used in the live vaccine through the eye was not observed in vaccinated animals of Flock E. CONCLUSION: The findings of our study indicate that, using serology, no distinction can be made between vaccinated and naturally infected sheep. As a result, confirmation of a negative OEA status in vaccinated animals by serology cannot be determined.

Tissue MicroArray (TMA) analysis of normal and persistent Chlamydophila pneumoniae infection.

BACKGROUND: Chlamydophila pneumoniae infection has been implicated as a potential risk factor for atherosclerosis, however the mechanism leading to persistent infection and its role in the disease process remains to be elucidated. METHODS: We validated the use of tissue microarray (TMA) technology, in combination with immunohistochemistry (IHC), to test antibodies (GroEL, GroES, GspD, Ndk and Pyk) raised against differentially expressed proteins under an interferon-gamma (IFN-gamma) induced model of chlamydial persistence. RESULTS: In the cell pellet array, we were able to identify differences in protein expression patterns between untreated and IFN-gamma treated samples. Typical, large chlamydial inclusions could be observed in the untreated samples with all antibodies, whereas the number of inclusions were decreased and were smaller and atypical in shape in the IFN-gamma treated samples. The staining results obtained with the TMA method were generally similar to the changes observed between normal and IFN-gamma persistence using proteomic analysis. Subsequently, it was shown in a second TMA including archival atheromatous heart tissues from 12 patients undergoing heart transplantation, that GroEL, GroES, GspD and Pyk were expressed in atheromatous heart tissue specimens as well, and were detectable morphologically within lesions by IHC. CONCLUSION: TMA technology proved useful in documenting functional proteomics data with the morphologic distribution of GroEL, GroES, GspD, Ndk and Pyk within formalin-fixed, paraffin-embedded cell pellets and tissues from patients with severe coronary atherosclerosis. The antibodies GroEL and GroES, which were upregulated under persistence in proteomic analysis, displayed positive reaction in atheromatous heart tissue from 10 out of 12 patients. These may be useful markers for the detection of persistent infection in vitro and in vivo.

Chlamydiaceae family, Parachlamydia spp., and Waddlia spp. in porcine abortion.

At present, despite extensive laboratory investigations, most cases of porcine abortion remain without an etiological diagnosis. Due to a lack of recent data on the abortigenic effect of order Chlamydiales, 286 fetuses and their placentae of 113 abortion cases (1-5 fetuses per abortion case) were investigated by polymerase chain reaction (PCR) methods for family Chlamydiaceae and selected Chlamydia-like organisms such as Parachlamydia acanthamoebae and Waddlia chondrophila. In 0.35% of the cases (1/286 fetuses), the Chlamydiaceae real-time PCR was positive. In the Chlamydiaceae-positive fetus, Chlamydia abortus was detected by a commercial microarray and 16S ribosomal RNA PCR followed by sequencing. The positive fetus had a Porcine circovirus-2 coinfection. By the Parachlamydia real-time PCR, 3.5% (10/286 fetuses of 9 abortion cases) were questionable positive (threshold cycle values: 35.0-45.0). In 2 of these 10 cases, a confirmation by Chlamydiales-specific real-time PCR was possible. All samples tested negative by the Waddlia real-time PCR. It seems unlikely that Chlamydiaceae, Parachlamydia, and Waddlia play an important role as abortigenic agents in Swiss sows.

Evidence for Chlamydia in wild mammals of the Serengeti.

Only limited information is available on the presence of Chlamydiaceae in wildlife, a deficit that is particularly acute concerning mammalian wildlife in Africa. In a retrospective analysis of organ material from an earlier study on wild mammals from the Seregenti National Park, 521 samples from 54 animals of 14 mammalian species were investigated. The presence of Chlamydiaceae was analyzed using molecular methods and immunohistochemistry. Chlamydial DNA was detected by real-time polymerase chain reaction in formalin-fixed and paraffin-embedded tissues from large ruminants (African buffaloes, Syncerus caffer, n=4) and a large predator (spotted hyena, Crocuta crocuta, n=1). Microarray results revealed Chlamydia abortus in all cases, confirmed by sequencing of selected samples, and a mixed infection with Chlamydia abortus and Chlamydia pneumoniae in an African buffalo. This is the first report of Chlamydiaceae in African wildlife of the Serengeti area.

Chlamydia in canine or feline coronary arteriosclerotic lesions.

BACKGROUND: There are numerous reports linking Chlamydia infection to human coronary atherosclerosis. However, there is a lack of data regarding this correlation in dogs and cats, and there are no reports investigating coronary arteriosclerosis and Chlamydia in these species. The aim of the present study was to examine whether there is a correlation between canine and feline spontaneous atherosclerosis or arteriosclerosis and the presence of Chlamydia. Archived histopathological samples of dogs (n = 16) and cats (n = 13) with findings of atherosclerosis or arteriosclerosis in heart tissue were examined for the presence of Chlamydiaceae using real-time PCR, ArrayTube Microarray and immunohistochemistry. Additionally, arteriosclerotic lesions of all cases were histologically classified and graded. RESULTS: Both canine atherosclerotic cases, and all 14 canine arteriosclerotic cases were negative for Chlamydia. Only one of the 13 arteriosclerotic feline cases was positive for Chlamydia by real-time PCR, revealing C. abortus by ArrayTube Microarray. To our knowledge, this is the first description of C. abortus in a cat. Overall, the type and grade of canine and feline arteriosclerotic lesions revealed similarities, and were predominantly moderate and hyperplastic. CONCLUSIONS: These findings suggest that there is no obvious correlation between canine and feline coronary arteriosclerosis and the presence of Chlamydia. In order to draw final conclusions about the correlation between Chlamydia and canine atherosclerosis, examination of more samples is required.

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.

Automated yeast two-hybrid screening for nuclear receptor-interacting proteins.

High throughput analysis of protein-protein interactions is an important sector of hypothesis-generating research. Using an improved and automated version of the yeast two-hybrid system, we completed a large interaction screening project with a focus on nuclear receptors and their cofactors. A total of 425 independent yeast two-hybrid cDNA library screens resulted in 6425 potential interacting protein fragments involved in 1613 different interaction pairs. We show that simple statistical parameters can be used to narrow down the data set to a high confidence set of 377 interaction pairs where validated interactions are enriched to 61% of all pairs. Within the high confidence set, there are 64 novel proteins potentially binding to nuclear receptors or their cofactors. We discuss several examples of high interest, and we expect that communication of this huge data set will help to complement our knowledge of the protein interaction repertoire of this family of transcription factors and instigate the characterization of the various novel candidate interactors.

RGS21 is a novel regulator of G protein signalling selectively expressed in subpopulations of taste bud cells.

Abstract G-protein-mediated signalling processes are involved in sweet and bitter taste transduction. In particular, the G protein alpha-subunit gustducin has been implicated in these processes. One of the limiting factors for the time-course of cellular responses induced by tastants is therefore the intrinsic GTPase activity of alpha-gustducin, which determines the lifetime of the active G protein complex. In several signalling systems specific 'regulator of G protein signalling' (RGS) proteins accelerate the GTPase activity of G protein alpha-subunits. Using differential screening approaches, we have identified a novel RGS protein termed RGS21, which represents the smallest known member of this protein family. Reverse transcription polymerase chain reaction and in situ hybridization experiments demonstrated that RGS21 is expressed selectively in taste tissue where it is found in a subpopulation of sensory cells. Furthermore, it is coexpressed in individual taste cells with bitter and sweet transduction components including alpha-gustducin, phospholipase Cbeta2, T1R2/T1R3 sweet taste receptors and T2R bitter taste receptors. In vitro binding assays demonstrate that RGS21 binds alpha-gustducin in a conformation-dependent manner and has the potential to interact with the same Galpha subtypes as T1R receptors. These results suggest that RGS21 could play a regulatory role in bitter as well as sweet taste transduction processes.