Involvement of Parachlamydia in bovine abortions in Scotland.

Bovine abortion represents a major animal welfare issue and a cause of substantial economic loss yet the rate of successful diagnosis remains low. Chlamydia-related organisms including Parachlamydia have recently emerged as putative cattle abortifacients. Placental tissue samples and fetal lung from bovine abortion submissions across Scotland in Spring 2011 were investigated by histopathology for the presence of suspect Chlamydia-related organisms. Evidence of Chlamydia-related organisms was observed in 21/113 (18.6%) placenta samples. Thirteen of the suspect cases and 18 histopathology negative cases were analysed by molecular and immunohistochemical methods. All samples were PCR positive for Parachlamydia but sequencing revealed high homology between identified environmental 16S sequences in all but three cases. Parachlamydial antigen was detected in 10/31 placental samples (32.2%) with pathology consistent with chlamydial infection. This work supports the need for further surveillance investigations and experimental studies to determine the role of Parachlamydia in bovine abortion.

Waddlia, Parachlamydia and Chlamydiaceae in bovine abortion.

The etiology remains unknown in many cases of bovine abortion in Switzerland. Bacteria of the Chlamydiales order are known abortive agents, therefore cases of bovine abortion from three representative regions of Switzerland were investigated in this study. Particularly Chlamydiaceae as well as the Chlamydia-like organisms Waddlia and Parachlamydia were of interest, especially because of their possible zoonotic potential. Placenta samples (n=343) were tested for these bacteria by different PCR-methods, immunohistochemistry and serology for Chlamydia abortus. Additionally an attempt for the isolation of Waddlia and Parachlamydia was made by co-cultivation in amoebae. In 67.3% of the 343 cases a necrotizing and/or purulent placentitis was found histologically. By real-time PCR, 0.9% (3/343) of the cases were positive for Waddlia, 13.4% (46/343) positive for Parachlamydia and 14.6% (50/343) positive or questionable positive for Chlamydiaceae. Of these samples, confirmation by immunohistochemistry was possible in 2/3 cases for Waddlia, 25/46 for Parachlamydia and 4/50 for Chlamydiaceae. Of the 50 cases positive or questionable positive for Chlamydiaceae, species-identification by ArrayTube Microarray or 16S rRNA PCR resulted in 41 cases positive for C. abortus whereas the presence of Chlamydia suis was confirmed in four and Chlamydia pecorum in one case. This study brought evidence for the importance of different members of Chlamydiales in different regions of Switzerland although Waddlia is not occurring in a high prevalence. On the other hand mixed infections with different Chlamydiales as well as with other abortigenic agents could be found.

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.

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.

Biofilm formation by staphylococci on fresh, fresh-frozen and processed human and bovine bone grafts.

Biofilm formation is a multi-step process influenced by surface properties. We investigated early and mature biofilm of Staphylococcus aureus on 4 different biological calcium phosphate (CaP) bone grafts used for filling bone defects. We investigated standardised cylinders of fresh and fresh-frozen human bone grafts were harvested from femoral heads; processed humanand bovine bone grafts were obtained preformed. Biofilm formation was done in tryptic soy broth (TSB) using S. aureus (ATCC 29213) with static conditions. Biofilm density after 3 h (early biofilm) and 24 h (mature biofilm) was investigated by sonication and microcalorimetry. After 3 h, bacterial density was highest on fresh-frozenandfresh bone grafts. After 24 h, biofilm density was lowest on freshbone grafts (p < 0.001) compared to the other 3 materials, which did not differ quantitatively (p > 0.05). The lowest increase in bacterial density was detected on fresh bone grafts (p < 0.001). Despite normal shaped colonies, we found additional small colonies on the surface of the fresh and fresh-frozen samples by sonication. This was also apparent in microcalorimetric heat-flow curves. The four investigated CaP bone grafts showed minor structural differences in architecture but marked differences concerning serum coverage and the content of bone marrow, fibrous tissue and bone cells. These variations resulted in a decreased biofilm density on freshand fresh-frozenbone grafts after 24 h, despite an increased early biofilm formation and might also be responsible for the variations in colony morphology (small colonies). Detection of small colony variants by microcalorimetry might be a new approach to improve the understanding of biofilm formation.

Une IgA à coefficient de sédimentation 11 S dans les sécrétions externes de l'espèce bovine

The sedimentation coefficient of a secretory IgA found in bovine colostrum and saliva is compared with that of IgG and IgM from the same colostrum. The IgA fraction gives a value of 10.8 S, whereas the major part of the IgG has a value of 7.1 S and the IgM 19.2 S. The sedimentation coefficient of the free secretory piece has also been determined: its value is 4.95 S.

Survey of Infectious Etiologies of Bovine Abortion during Mid- to Late Gestation in Dairy Herds.

Bovine abortion of unknown infectious etiology still remains a major economic problem. Thus, we investigated whether Brucella spp., Listeria monocytogenes, Salmonella spp., Campylobacter spp. and Coxiella burnetii are associated with abortion and/or stillbirth in Tunisian dairy cattle. Using a pan-Chlamydiales PCR, we also investigated the role of Chlamydiaceae, Waddlia chondrophila, Parachlamydia acanthamoebae and other members of the Chlamydiales order in this setting. Veterinary samples taken from mid to late-term abortions from twenty dairy herds were tested. From a total of 150 abortion cases collected, infectious agents were detected by PCR in 73 (48.66%) cases, 13 (8.66%) of which represented co-infections with two infectious agents. Detected pathogens include Brucella spp (31.3%), Chlamydiaceae (4.66%), Waddlia chondrophila (8%), Parachlamydia acanthamoebae (5.33%), Listeria monocytogenes (4.66%) and Salmonella spp. (3.33%). In contrast, Campylobacter spp. and Coxiella burnetii DNA were not detected among the investigated veterinary samples. This demonstrates that different bacterial agents may cause bovine abortion in Tunisia. This is the first report suggesting the role of Parachlamydia acanthamoebae in bovine abortion in Africa. Further studies with a larger number of samples are necessary to confirm whether this emerging pathogen is directly linked to abortion in cattle.

Expression of inducible nitric oxide synthase in bovine corneal endothelial cells and keratocytes in vitro after lipopolysaccharide and cytokines stimulation.

PURPOSE: To determine whether bovine corneal endothelial (BCE) cells and keratocytes express the inducible form of nitric oxide synthase (NOS) after exposure to cytokines and lipopolysaccharide (LPS), and to study the regulation of NOS by growth factors. METHODS: Cultures of bovine corneal endothelial cells and keratocytes were exposed to increasing concentrations of LPS, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). At selected intervals after exposure, nitrite levels in the supernatants were evaluated by the Griess reaction. Total RNA was extracted from the cell cultures, and messenger RNA levels for inducible NOS (NOS-2) were measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Exposure of BCE cells and keratocytes to LPS and IFN-gamma resulted in an increase of nitrite levels that was potentiate by the addition of TNF-alpha. Analysis by RT-PCR demonstrated that nitrite release was correlated to the expression of NOS-2 messenger RNA in BCE cells and keratocytes. Stereoselective inhibitors of NOS and cycloheximide inhibited LPS-IFN-gamma-induced nitrite release in both cells, whereas transforming growth factor-beta (TGF-beta) slightly potentiated it. Fibroblast growth factor-2 (FGF-2) inhibited LPS-IFN-gamma-induced nitrite release and NOS-2 messenger RNA accumulation in keratocytes but not in BCE cells. CONCLUSIONS: The results demonstrate that in vitro activation of keratocytes and BCE cells by LPS and cytokines induces NOS-2 expression and release of large amounts of NO. The high amounts of NO could be involved in inflammatory corneal diseases in vivo.

Role of Waddlia chondrophila placental infection in miscarriage.

Waddlia chondrophila is an intracellular bacterium suspected to cause human and bovine abortion. We confirmed an association between antibodies against W. chondrophila and human miscarriage and identified this organism in placenta or genital tract of women who had had miscarriages. These results suggest a possible role of W. chondrophila infection in miscarriage.

Imprinting of tumor-suppressor genes in human placenta.

Transcriptional deregulation in cancer has been shown to be associated with epigenetic alterations, in particular to tumor-suppressor- gene (TSG) promoters. In contrast, DNA methylation of TSGs is not considered to be present in normal differentiated cells. Nevertheless, we previously showed that the promoter of the tumor-suppressor gene APC is methylated, for one allele only, in normal gastric cells. Recently, RASSF1A has been shown to be imprinted in normal human placenta. To clarify putative TSG methylation in the placenta, 23 normal placental tissues from the first trimester, both decidua and villi, and four normal non-gestational endometrium were screened for DNA methylation by methylation-sensitive single-strand conformation analysis (MS-SSCA) and sequencing after bisulfite modification, on a panel of 12 genes known to be implicated in carcinogenesis. In all placental villi, four TSG promoters-APC, SFRP2, RASSF1A and WIF1-were hypermethylated, whereas all decidua and normal endometrium did not show any methylation. Allele-specific methylation analysis revealed that this methylation was monoallelic. Furthermore, comparison with maternal DNA indicated that APC and WIF1 were methylated on the maternal allele, whereas SFRP2 was methylated on the paternal allele. Sequence analysis of WIF1 mRNA revealed that only the unmethylated paternal allele was transcribed. The imprinting status of these TSGs is conserved during pregnancy. These results indicate that TSG imprinting is pre-existent in normal human placenta and should not be confused with carcinogenesis or pathology-induced methylation.

Histological behavior of glutaraldehyde cross-linked bovine collagen injected into the human bladder for the treatment of vesicoureteral reflux

Endoscopic subureteral collagen injection has become an accepted means for the treatment of vesicoureteral reflux in children. The aim of this study was to evaluate the histological behavior of glutaraldehyde cross-linked bovine collagen implants. The specimens were harvested from 29 patients who underwent reimplant surgery 2 to 30 months (mean 9.5) after unsuccessful subureteral injection therapy. In addition to routine hematoxylin and eosin staining, a new staining method (solophenyl red 3BL) able to demonstrate selectively neoformation of types I and III human collagen, was applied. Invasion of host fibroblasts into the bovine implant and the formation of endogenous types I and III collagen were demonstrated in all 29 cases. Adverse histological reactions were rare and, if present, they were predominantly of an inflammatory nature.

Involvement of connexin 43 in meiotic maturation of bovine oocytes.

In ovarian follicles, cumulus cells provide the oocyte with small molecules that permit growth and control maturation. These nutrients reach the germinal cell through gap junction channels, which are present between the cumulus cells and the oocyte, and between the cumulus cells. In this study the involvement of intercellular communication mediated by gap junction channels on oocyte maturation of in vitro cultured bovine cumulus-oocyte complexes (COCs) was investigated. The stages of oocyte maturation were determined by Hoechst 33342 staining, which showed that 90% of COCs placed in the maturation medium for 24 h progress to the metaphase II stage. Bovine COC gap junction communication was disrupted initially using n-alkanols, which inhibit any passage through gap junctions. In the presence of 1-heptanol (3 mmol l(-1)) or octanol (3.0 mmol l(-1) and 0.3 mmol l(-1)), only 29% of the COCs reached metaphase II. Removal of the uncoupling agent was associated with restoration of oocyte maturation, indicating that treatment with n-alkanols was neither cytotoxic nor irreversible. Concentrations of connexin 43 (Cx43), the major gap junction protein expressed in the COCs, were decreased specifically using a recombinant adenovirus expressing the antisense Cx43 cDNA (Ad-asCx43). The efficacy of adenoviral infection was > 95% in cumulus cells evaluated after infection with recombinant adenoviruses expressing the green fluorescence protein. RT-PCR performed on total RNA isolated from Ad-asCx43-infected COCs showed that the rat Cx43 cDNA was transcribed. Western blot analysis revealed a three-fold decrease in Cx43 expression in COCs expressing the antisense RNA for Cx43. Injection of cumulus cells with Lucifer yellow demonstrated further that the resulting lower amount of Cx43 in infected COCs is associated with a two-fold decrease in the extent of coupling between cumulus cells. In addition, oocyte maturation was decreased by 50% in the infected COC cultures. These results indicate that Cx43-mediated communication between cumulus cells plays a crucial role in maturation of bovine oocytes.

Development of a well-defined medium for the in vitro maturation of immature bovine cumulus-oocyte complexes.

PURPOSE: Our purpose was to develop a well-defined medium for the in vitro maturation (IVM) of immature bovine cumulus-oocyte complexes (COC). METHODS: The COC were cultured in the presence of three protein supplementations: fetal bovine serum (FBS), bovine serum albumin, and Synthetic Serum Substitute. The embryos obtained after in vitro fertilization of IVM oocytes were cocultured with Vero cells and their development to the morula and blastocyst stages was studied. RESULTS: When FBS was absent from the IVM medium, a significantly lower fertilization rate was observed, followed by a decrease in the percentage of embryos reaching the blastocyst stage. When FBS was replaced by a defined protein supplementation, the best results were obtained with Synthetic Serum Substitute. CONCLUSIONS: Adequate protein supplementation of the IVM medium optimizes the fertilization rate and the development of bovine IVM oocytes. The implication of these results in the human field is discussed.

Increased endometrial placenta growth factor (PLGF) gene expression in women with successful implantation.

Objective: To analyze the vascularization of the endometrium via hysteroscopy and to assess its correlation with angiogenic factor gene expression and embryo implantation rate.Design: Cross-sectional study.Setting: Public university hospital.Patient(s): Patients undergoing hysteroscopy for supposed infertility.Intervention(s): Endometrial quality assessment according to Sakumoto-Masamoto, performed in the early secretory phase of the cycle. Collection of an endometrial tissue biopsy.Main Outcome Measure(s): RNA extraction, reverse transcription, and determination of gene expression of angiogenesis- and implantation-relevant factors using quantitative polymerase chain reaction. Retrieval of pregnancy information from the medical records.Result(s): Good quantity/quality RNA with infertility history was obtained from 63 participating women. Those with a "good" endometrium and subsequent pregnancy showed increased gene expression for placenta growth factor when compared with patients with a "bad" endometrium and who did not succeed with pregnancy to date. Nonpregnant women with a "good" endometrium presented an intermediate result. No significant differences were observed for several other genes tested, but trends in the same direction were observed.Conclusion(s): This study demonstrates for the first time that endometrial PLGF expression corresponds to the hysteroscopic appearance of the endometrium, and therefore has potential as a clinically relevant prognosticator for infertility treatment success. (Fertil Steril (R) 2011;96:663-8. (C)2011 by American Society for Reproductive Medicine.)