A.J. Buendía

Polymorphonuclear neutrophils are necessary for the recruitment of CD8+ T cells in the liver in a pregnant mouse model of Chlamydophila abortus (Chlamydia psittaci serotype 1) infection

ABSTRACT The role of polymorphonuclear neutrophils (PMNs) in the development of the specific immune response against Chlamydophila abortus (Chlamydia psittaci serotype 1) infection was studied in a pregnant mouse model involving treatment with RB6-8C5 monoclonal antibody. PMN depletion significantly affected the immune response in the liver, in which the T-lymphocyte and F4/80+cell populations decreased, particularly the CD8+ T-cell population. A Th1-like response, characterized by high levels of gamma interferon without detectable levels of interleukin 4 (IL-4) in serum, was observed in both depleted and nondepleted mice, although an increased production of IL-10 was detected in the depleted group. Our results suggest that PMNs play a very important role in the recruitment of other leukocyte populations to the inflammatory foci but have little influence in the polarization of the immune specific response toward a Th1-like response.

Experimental Coxiella burnetii infection in pregnant goats: a histopathological and immunohistochemical study.

Pregnant goats were inoculated subcutaneosly with Coxiella burnetii and the course of infection was studied. Abortion in the last third of pregnancy occurred in all infected animals. Tissues from the placenta and other organs were studied before and after abortion by immunohistochemistry and PCR analysis. After infection, mild lesions were observed in several maternal organs, mainly the mammary gland but also the lung and the liver. The trophoblast cells of the choriallantoic membrane were the first target cells of the placenta; there was, however, a substantial delay between initial infection and placental colonization. In the last weeks of pregnancy, just before abortion, massive bacterial multiplication was detected in the placenta. In this stage of infection a necrotic and suppurative placentitis separated the fetal trophoblast cells from maternal syncytial epithelium. Vasculitis was observed in the fetal mesenchyme. A strong maternal T-cell response was detected in the inter-placentomal areas but not in the placentomes, where only neutrophils and smaller numbers of macrophages were associated with the lesions. Neither lesions nor C. burnetii DNA were found in maternal organs in animals maintained until day 120 post-abortion.

Field evaluation of a new commercially available ELISA based on a recombinant antigen for diagnosing Chlamydophila abortus (Chlamydia psittaci serotype 1) infection.

A new commercially available ELISA (ELISAr-Chlamydia) for detecting antibodies against Chlamydophila abortus has been evaluated using sheep field serum samples. The ELISA is based on a recombinant antigen which expresses part of a protein from the 80-90kDa family that is specific to C. abortus. Sera (105) from six flocks with confirmed ovine chlamydial abortion (OEA) outbreaks were used in this study, as well as sera (258) from 18 flocks which had suffered no OEA in the last lambing. The ELISAr-Chlamydia was compared with the complement fixation test (CFT) and with an ELISA using purified C. abortus elementary bodies (ELISA-EB), employing as reference technique a comparative microimmunofluorescence test that differentiates C. abortus infection from Chlamydophila pecorum infection. The results showed that the sensitivity of ELISAr-Chlamydia was 90.9% with a specificity of 85.9%, the sensitivity of CFT was 71.0% with a specificity of 83.6%, while the sensitivity of ELISA-EB was 95.2% and the specificity was 54.2%. Furthermore, ELISAr-Chlamydia was the test with fewer false positives resulting from positive reactivity to C. pecorum, although 15% of the sera positive for C. pecorum but negative for C. abortus antibodies reacted positively. This study demonstrated with field material that ELISAr-Chlamydia provides the most balanced results between sensitivity and specificity, especially in flocks with no clinical OEA but reactivity to C. abortus.

Endogenous Interleukin-12 Is Not Required for Resolution of Chlamydophila abortus (Chlamydia psittaci Serotype 1) Infection in Mice

ABSTRACT A Th1 immune response involving gamma interferon (IFN-γ) production is required to eliminate Chlamydophila abortusinfections. In this study, the role of interleukin-12 (IL-12) in protecting against C. abortus infection was investigated using IL-12−/− and wild-type (WT) C57BL/6 mice to determine the role of this Th1-promoting cytokine. IL-12−/− mice were able to eliminate the C. abortus infection in a primary infection. However, there was a delay in the clearance of bacteria when IL-12−/− mice were infected with a sublethal dose of C. abortus, the delay being associated with a lower production of IFN-γ. The low level of IFN-γ was essential for survival of IL-12−/−infected mice. Both WT and IL-12−/− mice developed a Th1 immune response against C. abortus infection, since they both produced IFN-γ and immunoglobulin G2a antibody isotype. In addition, when mice were given a secondary infectious challenge withC. abortus, a protective host response which resolved the secondary infection was developed by both WT and IL-12−/−mice. The lack of IL-12 resulted in few infiltrating CD4+ T cells in the liver relative to the number in WT mice, although the number of CD8+ T cells was slightly higher. The more intense Th1 response presented by WT mice may have a pathogenic effect, as the animals showed higher morbidity after the infection. In conclusion, these results suggest that although IL-12 expedites the clearance of C. abortus infection, this cytokine is not essential for the establishment of a protective host response against the infection.

Histopathological Lesions in 15 Cats with Leishmaniosis

Recent research into the prevalence of Leishmania infantum infection in the Mediterranean basin points to the involvement of cats as a reservoir host, but only sporadic cases of feline leishmaniosis have been reported. Feline leishmaniosis presents primarily as cutaneous disease and diagnosis is based on the demonstration of the parasite by skin biopsy. The present report describes the microscopical changes in tissue biopsies from 15 cats with leishmaniosis. The biopsies were derived from the skin, ocular tissue and mucocutaneous junctions. The most common histopathological feature was diffuse granulomatous inflammation with macrophages containing numerous amastigotes. Other patterns included granulomatous perifolliculitis and lichenoid interface dermatitis, where there were fewer parasitized macrophages. The presence of amastigotes was confirmed by immunohistochemistry in each case. The results of the study confirm the value of histopathological and immunohistochemical techniques for the diagnosis of feline leishmaniosis.

Relationship between the immune response and protection conferred by new designed inactivated vaccines against ovine enzootic abortion in a mouse model.

Chlamydophila abortus is a gram-negative obligate intracellular bacterium and the etiological agent of ovine enzootic abortion (OEA), an economically important disease in many countries. Inactivated vaccines have been reported to induce immunity in ewes and they have been used for many years. However, some outbreaks have been reported in correctly vaccinated flocks, so it is clear that new vaccines are necessary to address adequate protection and to avoid the shedding of the microorganism. This idea lead us to design inactivated vaccines, in a previously established mouse model, evaluating different inactivation procedures and new adjuvants. To assess the protection conferred, the results were analyzed on the basis of clinical signs and the isolation of C. abortus from spleen. These findings were correlated with the immune response induced by the vaccines, as determined by the production of C. abortus-specific IFN-gamma and IL-4 from splenocyte cultures and the detection of IgG isotypes in serum. BEI was found to be the best C. abortus-inactivation procedure. The inactivated vaccines adjuvated with QS-21 (QS) or Montanide 773 (M7) induced the best protection both against homologous and heterologous challenge, with an adequate (Th1-like) immune response. Finally, these selected vaccines were evaluated in a pregnant mouse model, in which they were seen to confer good protection and to avoid the C. abortus persistence in uterus after delivery. With these results, this mouse model could be considered as an adequate tool for selecting and optimizing effective vaccines against OEA.

High prevalence of antibodies against Chlamydiaceae and Chlamydophila abortus in wild ungulates using two “in house” blocking-ELISA tests

Few data are available on the prevalence and relevance of chlamydiae in wild mammals, and even fewer studies have been conducted to determine the prevalence of Chlamydophila abortus in wildlife hosts, most probably due to the absence of suitable species-specific serological assays for testing sera from wild animals. In light of this, we have developed two in-house blocking-ELISA tests for detection of antibodies against Chlamydiaceae and C. abortus in wild ungulates, and analyzed the relationship between geographical and biological factors and the prevalence of antibodies against Chlamydiaceae and C. abortus in 434 wild ungulates from Spain, including sera from European wild boar, Red deer, Fallow deer, Roe deer, Mouflon, Barbary sheep, Southern chamois, and Iberian ibex. Serology revealed that 41.7+/-4% of the sera were positive for the b-ELISA-LPS (Chlamydiaceae-specific) and 18.9+/-3% for the b-ELISA-rPOMP (C. abortus-specific). Antibodies against Chlamydiaceae lipopolysaccharide (LPS) were detected in sera from all eight ungulate species, the prevalence ranging from 23 to 60%. Iberian ibex was the only wild ungulate not showing seropositivity to the C. abortus specific polymorphic outer membrane protein (POMP). The prevalence of anti-POMP antibodies in the other seven wild ungulate species ranged from 7 to 40%. While significant seroprevalence differences were detected among species and among sampling regions, no effect of age and sex was observed. The high prevalence levels found should be considered with regards to livestock and human health, and warrant further research.

B-Cell-Deficient Mice Show an Exacerbated Inflammatory Response in a Model of Chlamydophila abortus Infection

ABSTRACT The resolution of Chlamydophila abortus (Chlamydia psittaci serotype 1) infection is dependent on gamma interferon and CD8+ T cells, and classically, B cells have been considered to play a minimal role in host defense. The role of B cells in the immune response was studied by using a model of infection in mice with genetically modified immunoglobulin M transmembrane domains (μMT). In the absence of B cells, infection with C. abortus leads to an acute severe fatal disease that involves a disseminated intravascular coagulation syndrome. μMT mice displayed an increased level of proinflammatory cytokines in serum, and an increased number of neutrophils was observed in the lesions. The possible deleterious role of neutrophils in the pathogenesis of disease in μMT mice was determined by depletion of the neutrophils with the monoclonal antibody RB6-8C5. This led to an enhancement of the bacterial burden and early mortality in both μMT and wild-type mice, while necrotic lesions remained. Analysis of the presence of immunoregulatory cytokines showed significantly lower levels of transforming growth factor β in the sera of μMT mice. However, mice lacking mature B cells were able to establish a specific immune response that protected them from a secondary challenge. Taken together, these data suggest an immunomodulatory role for B cells in the early events of C. abortus primary infection that can protect mice against an exaggerated inflammatory response.

Immunohistochemical Characterization and Evaluation of Prognostic Factors in Canine Oral Melanomas with Osteocartilaginous Differentiation

Melanomas are the most common malignant oral neoplasm in dogs. Osteocartilaginous differentiation in oral melanomas is a rare feature described both in veterinary and human medicine. Here, 10 cases of this type of neoplasm were used to study their immunohistochemical, biological, and clinical characteristics. Reactivity for S100 and melan A antigen was evaluated, and 4 prognosis factors (mitotic index, invasiveness of epithelium, nuclear atypia, and proliferation index) were analyzed and correlated with the clinical course of the neoplasms after diagnosis. Immunohistochemical analysis of the studied neoplasms, including the osteocartilaginous areas, showed positive immunoreaction for S100 and melan A, except in one dog, which was negative for melan A. Analysis of the results showed that oral melamonas with osteocartilaginous differentiation have a clinical course similar to that of other melanomas in the oral cavity. Analysis of the mitotic index and the expression of proliferation marker Ki-67 could be useful tools for predicting the biological behavior of these neoplasms.

Natural killer (NK) cells play a critical role in the early innate immune response to Chlamydophila abortus infection in mice

Chlamydophila abortus, the aetiological agent of ovine enzootic abortion, induces a strong inflammatory reaction that leads to the T helper cell (Th1) specific immune response necessary for the clearance of infection. Because the role of natural killer (NK) cells during the first stages of this response has received little attention, this study focused on determining the function of these cells in a mouse model of infection. The location of NK cells in the liver and spleen of infected mice was examined immunohistochemically with an anti-Ly49G monoclonal antibody. The number of NK cells increased during the infection both in spleen and liver. In subsequent experiments, an anti-asialo GM1 polyclonal antibody was injected to deplete the NK cells. NK-depleted mice showed a substantial increase in their susceptibility to C. abortus infection, with high mortality rates and an increased burden of bacteria in the liver. Histopathological studies showed that inflammatory foci, composed mainly of neutrophils, were greater in size and number in depleted mice, while numerous chlamydial inclusions were associated with the foci. Serum concentrations of IFN-gamma, a key cytokine in the control of C. abortus infection, were substantially reduced in the NK-depleted mice. To establish the relationship between NK cells and other components of the innate immune response, neutrophils were depleted with the RB6-8C5 antibody. These cells were shown to be crucial in the recruitment of NK cells to the inflammatory foci.