Abortion and subsequent excretion of chlamydiae from the reproductive tract of sheep during estrus.

Chlamydia psittaci serovar 1 infection in pregnant sheep typically causes abortion or the birth of weak lambs. Eight sheep that experienced chlamydia-induced abortion during their first pregnancy were successfully rebred yearly for the past 2 years. Chlamydia-specific lipopolysaccharide was detectable for approximately 3 weeks in vaginal swabs taken from the experimentally infected sheep following abortion. There was no evidence of chlamydiae in vaginal, placental, or neonatal samples obtained immediately after each subsequent successful pregnancy. Sera collected from the experimentally infected sheep had persistent, high antibody levels to C. psittaci, suggesting continued exposure of the immune system to the organism. Examination of vaginal specimens obtained during various stages of the estrus cycle revealed detectable levels of chlamydiae only when the animal was near ovulation. Chlamydiae were not detected in swabs from sheep that did not experience abortion. Enhanced chlamydial excretion during the periovulation period of sheep may provide sufficient stimulation of the immune system to account for the persistent antibody response. Furthermore, the association between estrus and chlamydial shedding has important implications for transmission of infection to other ewes during breeding.     

Lymphocyte subpopulations in the blood of sheep persistently infected with border disease virus

The surface phenotypes of peripheral blood lymphocytes in groups of lambs and adult sheep persistently infected with Border disease virus (P-I BD) were compared with those of healthy controls. The proportion and number of lymphocytes bearing surface immunoglobulin (sIg+) and expressing class II MHC antigen (B cells) were significantly increased. A significant increase in CD1+ lymphocytes was also evident. Conversely, the proportion of T lymphocytes in P-I BD lambs was reduced. A marked reduction in the proportion of circulating lymphocytes expressing class I MHC antigen was also observed. These findings were not affected by differences in the strain of the virus responsible for the persistent infection.     

Expression of lymphotoxin-alpha by keratinocytes: a further mediator for the lichenoid reaction.

OBJECTIVE: Lichen planus (LP) represents a disease in which autoimmune mechanisms mediated by Th1 T cells are involved. Lymphotoxin-alpha (LT-alpha) represents a Th1 cytokine with proinflammatory activities in LP, as has recently been demonstrated for interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). METHODS: Expression of LT-alpha mRNA was investigated by RT-PCR and nonradioactive in situ hybridization. Double staining methods were applied to characterize the phenotype of cells expressing LT-alpha. Cell stimulation experiments were performed on the transformed squamous cell line HaCaT. RESULTS: In contrast to normal skin, LT-alpha-specific RT-PCR products were found in all cases of LP. Cells in the inflammatory infiltrate expressing LT-alpha were identified as mainly T cells and mast cells, as shown by in situ hybridization. Furthermore, predominant LT-alpha mRNA expression could be observed in lesional keratinocytes adjacent to the band-like inflammatory infiltrate. In cell stimulation experiments, it could be shown that IFN-gamma induces LT-alpha and TNF-alpha mRNA in the human squamous cell line HaCaT, concomitant with upregulation of MHC class II and intercellular adhesion molecule-1, which could also be observed on lesional keratinocytes in LP. CONCLUSIONS: In LP, LT-alpha mRNA is predominantly expressed by lesional keratinocytes and to a lesser extent by inflammatory cells. Induction of LT-alpha in keratinocytes is closely related to the expression of TNF-alpha and MHC class II. The loci of TNF-alpha and LT-alpha map to MHC class III on chromosome 6, which is closely linked to the MHC class II gene locus. Our results suggest that stimulation of keratinocytes with IFN-gamma results in the upregulation of proinflammatory cytokines such as LT-alpha and TNF-alpha as well as MHC class II, which map to the same gene region of immunoregulatory genes on chromosome 6 and may be involved in the induction and maintenance of the disease.     

Abortive potency of Chlamydophila abortus in pregnant mice is not directly correlated with placental and fetal colonization levels

Abortion, placental and fetal colonization, and levels of gamma interferon were analyzed for four Chlamydophila abortus strains presenting antigenic variations in a mouse model. Expression of virulence of these strains varied and indicated that abortion was not directly related to the number of bacteria in the placenta, and thus, other factors may have an important role in activating the abortion process.     

Veterinary and medical aspects of abortion in Danish sheep

The Danish sheep population totals around 144,000 animals, but little is known of the causes and prevalance of diseases. This study focuses on the causes of abortion in Danish sheep. During one breeding season, aborted foetuses and stillbirths with signs of intrauterine death or malformation were submitted for laboratory examination from a population of 3,758 breeding ewes. Samples from 24 incidents of abortion and 21 ewes delivering malformed lambs or lambs with ante partum decomposition were submitted. A specific aetiology was established in 66.7% and 14.3% of the cases, respectively. Bacterial pathogens were the most prevalent cause of abortion. Several of the abortifacients were zoonotic microorganisms, for example Listeria monocytogenes, Campylobacter fetus subsp. fetus, Yersinia pseudotuberculosis and Toxoplasma gondii. The identified microorganisms probably represent the most common causes of abortion in Danish sheep but occurrence in Denmark of other pathogens such as Coxiella burnetii and Chlamydophila abortus cannot be excluded. Due to the high prevalence of zoonotic microorganisms, precautions must be taken in handling abortions or assisting lambing, especially for pregnant women.     

Comparison of fetal and maternal inflammatory responses in the ovine placenta after experimental infection with Chlamydophila abortus

Placentae from 13 pregnant ewes infected intravenously with Chlamydophila abortus, together with placentae from nine uninfected control ewes, were examined at 14, 21 or 28 days post-inoculation (p.i.). Chlamydial inclusions were present in the trophoblast at 14 days p.i. and were widespread by 21 days p.i. Chorioallantoic lesions (oedema, arteritis and thrombosis) were severe at 28 days p.i., the changes being particularly marked in the membrane surrounding placentomes. Lymphocytes constituted only a small proportion of the cellular infiltrate in the chorioallantois; neutrophil infiltration of the chorionic surface was evident where the trophoblast layer had sloughed, whereas macrophages represented the predominant cell type in the deeper stroma. In contrast, on the maternal side of the placenta, chlamydial inclusions were sparse at all timepoints, and even at 28 days p.i., lesions were restricted to focal endometritis at the placentomal limbus and occasional foci of septal necrosis. T lymphocytes were numerous within endometrial and septal lesions, the infiltrate consistently containing more CD8(+) than CD4(+) cells. The fetal response to chlamydial invasion of the placenta was innate in character, whereas the maternal response appeared to represent an acquired, chlamydia-specific immune response.     

Differential peripheral expansion and in vivo antigen reactivity of alpha/beta and gamma/delta T cells emigrating from the early fetal lamb thymus

The concentrations of different lymphocyte subsets in the blood of lambs which had been thymectomized (Tx) in utero between days 67-75 of fetal gestation were measured at birth and at various intervals during the first year of life. Compared to thymus-intact (Ti) controls, Tx lambs were severely depleted of both alpha/beta and gamma/delta T cells at birth (less than 10% of control levels). The majority of the residual alpha/beta T cells present in Tx lambs at birth were CD4+CD8-. As the Tx lambs aged, the concentration of alpha/beta T cells in blood increased steadily to reach levels around 50% of control values. In contrast, the circulating gamma/delta T cells did not expand in Tx animals and remained barely detectable throughout the observation period, although these cells accounted for 30%-60% of the T cells in the blood of Ti lambs. The expansion of alpha/beta but not gamma/delta T cells was also reflected in changes in the cellular composition of solid lymphoid organs in Tx lambs. B cell numbers were similar in both groups at birth but Tx lambs were persistently B lymphopenic from 3 weeks of age onwards. The alpha/beta T cells that had expanded in Tx lambs responded to stimulation with bacterial antigens in a way that was qualitatively similar to the response in Ti lambs. By contrast, the few gamma/delta T cells in Tx lambs responded abnormally. Our results show that although sheep alpha/beta and gamma/delta T cells are equally thymus dependent during ontogeny, the early fetal thymic emigrants which establish the two T cell lineages in the periphery have strikingly different antigen reactivities and capacities for self-renewal and expansion.     

Serological diagnosis of ovine enzootic abortion by enzyme-linked immunosorbent assay with a recombinant protein fragment of the polymorphic outer membrane protein POMP90 of Chlamydophila abortus

Ovine enzootic abortion (OEA) resulting from infection of sheep and goats with Chlamydophila abortus is of major economic importance worldwide. Over the last 50 years the serological diagnosis of infection has been based mainly on the complement fixation test (CFT), which lacks both sensitivity and specificity because of cross-reactive antibodies to other gram-negative bacteria, including another common chlamydial pathogen of sheep, Chlamydophila pecorum. In the present study, a series of overlapping recombinant antigens representing the polymorphic outer membrane protein POMP90 of C. abortus was assessed by enzyme-linked immunosorbent assay (ELISA) with a panel of 143 serum samples from sheep experimentally infected with C. abortus, from sheep clinically free of OEA, and from specific-pathogen-free lambs experimentally infected with different subtypes of C. pecorum. The results were compared to those obtained by CFT and another recently described test, an indirect ELISA (iELISA) with the recombinant OMP91B (rOMP91B) fragment (rOMP91B iELISA) (D. Longbottom, E. Psarrou, M. Livingstone, and E. Vretou, FEMS Microbiol. Lett. 195:157-161, 2001). The rOMP90-3 and rOMP90-4 ELISAs were identified as being more sensitive and specific than CFT. Assays with both fragments were evaluated further with a panel of 294 field serum samples from flocks with documented histories of abortion, from flocks with no clinical histories of abortion but which had a high proportion of samples seropositive by CFT, and from animals with no histories of abortion but from which various C. pecorum subtypes had been isolated. ELISAs with both POMP90 fragments outperformed CFT with serum samples from C. pecorum-infected animals, producing no false-positive results. However, the ELISA with the rOMP90-4 fragment appeared to be more sensitive than the one with rOMP90-3, as it identified more of the OEA-positive samples. The ELISA with the rOMP90-4 fragment was also able to identify apparently healthy animals that were infected with an enteric strain of C. abortus in flocks that were probably infected with both enteric C. abortus and C. pecorum strains. The identification of animals infected with enteric C. abortus is extremely important in controlling the spread of OEA. Overall, the new rOMP90-4 ELISA was found to be a more sensitive and specific test than CFT for differentiating animals infected with C. abortus from those infected with C. pecorum.     

Characterization of the immune response in the placenta of cattle experimentally infected with Neospora caninum in early gestation

A serial examination of three groups of cattle infected intravenously (iv) (Group 1, n=8) or subcutaneously (sc) (Group 2, n=8) with live Neospora caninum tachyzoites or with VERO cells (Group 3, n=8) at 70 days' gestation was carried out and the nature of the inflammatory responses in the placenta and the presence of parasite antigen were analysed. Immune cells expressing CD3, CD4, CD8, gamma delta (gammadelta) T-cell receptors (TCR), CD79alpha cytoplasmic (cy) (B cells) and NKp46 [natural killer (NK) cells] antigens were identified immunohistochemically and cells expressing mRNA for interferon-gamma (IFN-gamma) were labelled by in-situ hybridization. Intravenous inoculation caused mortality in all fetuses from 28 days post-inoculation (dpi) onwards. Subcutaneous inoculation caused mortality in 50% of the animals by 28dpi. Pathological changes in the placenta consisted of necrosis of fetal placental villi, necrosis and inflammation in adjacent areas of the maternal septum and inflammation at the base of the maternal caruncle. The inflammatory infiltrate consisted mainly of CD3(+) lymphocytes, dominated by CD4(+) and gammadelta TCR(+) cells, with CD8(+) cells present to a lesser extent. The results from the control group indicated fewer NK cells than those occurring in the placenta of human beings or mice. Infiltration of CD4(+) cells and NKp46(+) cells was observed in the caruncular base and septa 14 days after infection, whereas infiltration of gammadelta TCR(+) cells was observed from 28 dpi onwards. To our knowledge this is the first report on the presence and distribution of NK cells in the bovine placenta. Maternal inflammatory cells expressing mRNA for IFN-gamma were identified in animals inoculated with parasites iv or sc at 14 and 28 dpi, respectively. In the sc-inoculated dams with live fetuses at 28, 42 and 56dpi, there was no evidence of parasite antigen, infiltration of immune cells or production of IFN-gamma, suggesting that the parasite had not reached the placenta. The exact cause of fetal death was not established. Tissue destruction by the parasite may have occurred; in addition, there may have been a T helper 1 (Th-1) immune response to the neospora infection at the materno-fetal interface, resulting in infiltrations of CD4T cells, gammadelta T cells and NK cells and the subsequent production of IFN-gamma. It is possible that a pro-inflammatory Th-1 response early in gestation protects the dam by eliminating the parasite; however, it may lead to destruction of the placental tissues themselves and thus be incompatible with fetal survival.     

Phenotypic characterisation of intestinal dendritic cells in sheep

The present study was undertaken to identify dendritic cells (DCs) in the ileum and rectum of lambs and adult sheep. The distribution of these cells in four different intestinal compartments, i.e. lamina propria, lymphoid follicles, domes and interfollicular areas was assessed, and the presence of these cells in lambs and adult sheep was compared. Specimens were examined by using a number of potential DC markers (CD11c, CD205, MHC class II (MHCII), CD1b and CD209) in immunohistochemical and multicolour immunofluorescent procedures. The ovine ileal and rectal mucosa contain many CD11c+/CD205+ cells with a dendritic morphology, and the majority of these cells co-expressed MHCII. These double-positive cells were also labelled with the CD209 antibody in the lamina propria and interfollicular regions. Only very few cells expressed CD1b. In conclusion, a major DC population in ileum and rectum of sheep co-expressed the CD11c, CD205 and MHCII molecules. The CD209 antibody appeared to be a novel marker for a subpopulation of ovine intestinal DCs.     

Ontogeny of ovine lymphocytes. I. An immunohistological study on the development of T lymphocytes in the sheep embryo and fetal thymus

The time of appearance of lymphocytes expressing T-cell markers and the subsequent development of the fetal thymus were studied in ovine embryos using a panel of monoclonal antibodies. Leucocyte common antigen (LCA) and major histocompatibility complex class I (MHCI) antigens were seen on a small number of cells within the ovine embryo at Day 19 of gestation. SBU-T6 (CD1)-positive cells were found at Day 22 of gestation, while major histocompatibility complex class II (MHC II) antigens were first observed at Day 25 of gestation. Large basophilic cells, weakly staining for SBU-T1 (CD5), were present in the mesenchyme of the neck and in the dorsal mediastinum and mesentery of embryonic sheep of 33 days gestational age (g.a.); however, no SBU-T1-positive cells were detected in the thymus at this time. No SBU-T4 (CD4)- or SBU-T8 (CD8)-positive cells were detected in any organs of embryos of this age. SBU-T4- and SBU-T8-positive cells were first seen in fetal thymi, and elsewhere within the fetus, at 35-38 days g.a. SBU-T19-positive cells were first seen within the fetal thymus at 50-58 days g.a.     

In vivo inhibition by a monoclonal antibody to CD4+ T cells of humoral and cellular immunity in sheep.

The ability of intravenously injected anti-CD4 and anti-CD8 monoclonal antibody (mAb) to deplete specific lymphocyte subsets in vivo and their effects on antibody responses to ovalbumin (OVA) and Brucella abortus, and skin reactivity to T-cell mitogens was examined in merino lambs. Repeated administration of anti-CD4 or anti-CD8 mAb caused a specific and sustained depletion of target cells from peripheral blood. Anti-CD4 mAb significantly inhibited the in vivo antibody response to OVA but had no effect on the antibody response to LPS of B. abortus. In contrast, antibody responses to both OVA and B. abortus lipopolysaccharides (LPS) remained unaffected in lambs depleted of their CD8+ T lymphocytes. These results confirm the T-cell dependence and independence of antibody responses to OVA and LPS, respectively. Skin reactions elicited by intradermal injections of phytohaemagglutinin (PHA) and concanavalin A (Con A) were also significantly suppressed in lambs depleted of their CD4+ T cells, but treatment with anti-CD8 mAb had no effect on skin responsiveness. Together, these results suggest that mAb can be extremely effective at selectively depleting lymphocyte subsets in vivo and can be used for studying various aspects of immunoregulation and immunity in sheep.     

T-cell receptor-gamma delta association with lymphocyte populations in sheep intestinal mucosa.

T cells expressing T-cell receptor (TcR)-gamma delta and CD8 represent a significant population in mouse and chicken intra-epithelial lymphocytes (IEL) but represent a minor population in human IEL. We examined the TcR-gamma delta usage and co-expression of CD5, CD4, CD8 and major histocompatibility complex (MHC) class II on isolated sheep IEL and lamina propria lymphocytes (LPL), and compared them with the TcR-gamma delta + cells in peripheral blood, intestinal lymph and jejunal Peyer's patches (PP). There were a number of notable differences. TcR-gamma delta + cells comprised 18% of IEL and 10% of LPL. Among the population of TcR-gamma delta + IEL, 24% were CD8+ and 54% were CD5+, which contrasts with the TcR-gamma delta + cells in blood and intestinal lymph that were universally CD5+ CD4- CD8-. A notable feature of the IEL was the presence of distinct CD8+ and TcR-gamma delta + populations that lacked CD5. Also a high percentage of IEL and LPL were CD2+ and MHC class II+. Analysis of the expression of MHC class II on T-cell subsets, as an indicator of activation, showed that 60-95% of the various IEL and LPL subsets were MHC class II+ compared with only 5-40% in jejunal PP, lymph nodes, spleen and blood. Therefore, it is possible that the circulating TcR-gamma delta + and CD8+ cells that localize in the gut epithelium might become activated and stop the expression of CD5 under the influence of the local microenvironment. These cells appear not to emigrate while still expressing the TcR-gamma delta + (CD8+) CD5- MHC class II+ phenotype. Our data, together with those from other studies, show that there is much heterogeneity in the use of TcR-gamma delta and accessory T-cell molecules by IEL.     

Hereford cattle protected against Boophilus microplus with antigens purified by immunoaffinity chromatography from larval and adult ticks.

The subpopulations of lymphocytes in the pregnant and non-pregnant mammary glands of the sheep were delineated by a panel of monoclonal antibodies. The most striking feature observed was that in the mammary gland of both pregnant and non-pregnant sheep the great majority of the lymphocytes in the ductal and alveolar epithelium were agranulated CD8+ CD5- cells. A small subpopulation of granulated lymphocytes in the epithelium expressed the CD45R antigen but not the major histocompatibility complex (MHC) class II molecules. Other subpopulations, especially B lymphocytes, were present in much lower concentrations and were located mainly in the periductal and intralobular connective tissues. Patches of lymphocytes clustering around venules were observed and the majority of them were shown to be CD5+ CD4+, while some were CD5+ CD8+ but none were CD45R+ (B cell). It is suggested that selective traffic of T cells occurs at these sites.     

Phenotypic analysis of the murine CD4-related glycoprotein,CD223 (LAG-3)

CD223 (LAG-3) is an activation-induced cell surface molecule, structurally similar to the T cell coreceptor CD4, that binds MHC class II molecules with high affinity. Little is known about the expression and function of murine CD223. Here, we show that mRNA expression is restricted to the thymic medulla, splenic red pulp and sparse cells in the adult brain cortex. In contrast, surprisingly high expression was seen in defined tracts at the base of the cerebellum and in the choroid plexus of day 7 postnatal brain. mCD223:Ig, but not CD4:Ig, fusion proteins stained cells expressing MHC class II molecules. Analysis of mCD223 cell surface expression was performed with a new monoclonal antibody (mAb) that recognizes an epitope in the D2 domain. Although it blocked mCD223 function in vitro, it did not block binding of mCD223 to MHC class II molecules. While very few TCRalpha beta T cells in the spleen and thymus of naive mice express surface mCD223 (<3 %), approximately 18 % TCR gamma delta T cells and approximately 10 % NK cells are positive. This small population of TCRalpha beta T cells are cycling memory T cells (BrdU(+), CD44(hi), CD62L(lo)). In contrast, all T cells express mCD223 2-3 days post activation. This study and the anti-CD223 mAb should greatly assist in the elucidation of CD223 function.     

Cyclosporin A reduces expression of adhesion molecules in the kidney of rats with chronic serum sickness

Treatment with cyclosporin A (CsA) improves proteinuria and reduces renal cellular infiltration in chronic serum sickness (CSS). We examined if these effects were associated with a reduced renal expression of CD54 and its ligands, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and MHC class II molecules. We studied two groups of rats in which CSS was induced by daily injections of ovalbumin (OVA): a group treated with CsA (OVA.CsA group, n = 11) and a group that received no treatment (OVA.CSS group, n = 11). An additional group of five rats (control group) received only phosphate buffer. Immunostaining techniques were used to follow CSS and to study the expression of CD54, CD18, CD11b/c, IFN-gamma, TNF-alpha and MHC class molecules. Proteinuria (mg/24 h) was reduced from 248.2 +/- 73.1 (OVA.CCS group) to 14.5 +/- 13.1 with CsA treatment (P < 0.0001). The renal expression of CD54 and its ligands (CD18 and CD11b/c) was reduced by 50% to 75%. Correspondingly, there was a 60% to 85% reduction in the number of infiltrating leucocytes. The number of cells expressing TNF-alpha, IFN-gamma and MHC II molecules was also reduced. CsA reduces expression of CD54 and its ligands. This effect is associated with a reduction of cellular infiltration, IFN-gamma, TNF-alpha-producing cells and with MHC II expression in the kidney. These findings suggest that expression of adhesion molecules plays a critical role in CSS and underline the importance of cellular immunity in this experimental model.