Serosurvey of Leptospira interrogans, Brucella abortus and Chlamydophila abortus infection in free-ranging giant anteaters (Myrmecophaga tridactyla) from Brazil / Inquérito sorológico de Leptospira interrogans, Brucella abortus e Chlamydophila abortus em tamanduás-bandeira (Myrmecophaga tridactyla) de vida livre no Brasil

A serological survey for antibodies against Leptospira interrogans, Brucella abortus, and Chlamydophila abortus was conducted in 21 clinically healthy, free-ranging giant ant- eaters (Myrmecophaga tridactyla) from Parque Nacional das Emas (Goiás State, Brazil; n=6), Parque Nacional da Serra da Canastra (Minas Gerais State, Brazil; n=9), and RPPN SESC Pantanal (Mato Grosso State, Brazil; n=6) between July 2001 and September 2006. Sera were screened for antibodies against 22 serovars of Leptospira interrogans with a microscopic agglutination test. Twelve tested positive for L. interrogansserovars sentot (n=5 in PN Emas, n=2 in PN Serra da Canastra), butembo (n=2 in PN Serra da Canastra), autumnalis, bataviae, and shermani/icterohaemorrhagiae(n=1 each in SESC Pantanal)One adult female tested positive for B. abortus with the buffered plate antigen test. All sera were negative for C. abortususing the complement fixation text. This is the first report of pathogens that may interfere with the reproduction and population dynamics of free-ranging giant anteaters.

Inquéritos sorológicos para detecção de anticorpos contra Leptospira interrogans, Brucella abortus, e Chlamydophila abortus foram realizados em 21 tamanduás-bandeira (Myrmecophaga tridactyla) de vida livre do Parque Nacional das Emas (Goiás, Brasil, n=6), o Parque Nacional da Serra da Canastra (Minas Gerais, Brasil, n=9) e RPPN SESC Pantanal (Mato Grosso, Brasil, n=6) entre julho de 2001 e setembro de 2006. Os sor os foram testados para anticorpos contra 22 sorotipos de Leptospira interrogans com um teste de aglutinação microscópica. Doze animais foram considerados positivos para L. interrogans sorovares sentot (n=5 em PN Emas, n=2 em PN Serra da Canastra), butembo (n=2 em PN Serra da Canastra), autumnalis, bataviae e shermani/icterohaemorrhagiae(n=1 para cada sorovar em SESC Pantanal). Uma fêmea adulta testou positivo para B. abortuscom o teste do antígeno tamponado. Todos os soros se mostraram negativos para C. abortusatravés do teste de fixação do complemento. Este é o primeiro relato de patógenos que podem interferir na dinâmica reprodutiva de populações de tamanduás em estado selvagem.

Distinct intensity of host-pathogen interactions in Chlamydia psittaci- and Chlamydia abortus-infected chicken embryos.

Factors and mechanisms determining the differences in virulence and host specificity between the zoonotic agents Chlamydia psittaci and Chlamydia abortus are still largely unknown. In the present study, two strains were compared for their invasiveness, virulence, and capability of eliciting an immune response in chicken embryos. On breeding day 10, embryonated chicken eggs were inoculated with 5 × 10(4) inclusion-forming units. As shown by immunohistochemistry and quantitative real-time PCR, C. psittaci displayed a significantly better capability of disseminating in the chorioallantoic membrane (CAM) and internal organs than C. abortus. The higher infectious potential of C. psittaci in birds was underlined by significantly higher mRNA expression rates of essential chlamydial genes, such as incA, groEL (in CAM, liver, and spleen), cpaf, and ftsW (in CAM). Although the immune responses to both pathogens were similar, C. psittaci elicited higher macrophage numbers and a stronger expression of a subset of immune-related proteins. The data imply that invasiveness of Chlamydia spp. and propagation in the host are not solely dependent on the level of host immune response but, even to a greater extent, on the expression of bacterial factors related to virulence. The fact that C. psittaci has coped far better than C. abortus with the avian embryo's response by upregulating essential genes may be a key to understanding the mechanisms underlying host adaptation and etiopathology.

Co-administration of the polysaccharide of Lycium barbarum with DNA vaccine of Chlamydophila abortus augments protection.

Lycium barbarum polysaccharides (LBP) can stimulate moderate immune responses therefore could potentially be used as a substitute for oil adjuvants in veterinary vaccines. In the present study, it was shown that the isolated active component of LBP3a, combined with a DNA vaccine encoding the major outer membrane protein (MOMP) of Chlamydophila abortus, induced protection in mice against challenge. Sixty BALB/c mice were randomly assigned to 5 groups. Sub-fractions of polysaccharide LBP3a, at 12.5, 25 and 50 mg/kg concentrations, respectively, were mixed with a pCI-neo::MOMP (pMOMP) vaccine. Mice administrated with pCI-neo + LBP3a were served as a control. All mice were inoculated at day 0, 14, and 28, and challenged on day 44. The effects of LBp3a on serum antibody levels, in vitro lymphocyte proliferation, the activity of interleaukin-2 (IL-2), interferon-γ (IFN-γ), tumor necrosis factor α(TNF-α)and chlamydia clearance were determined. A combination of DNA vaccine and LBP3a induced significantly higher antibody levels in mice, higher T cell proliferation and higher levels of IFN-γ and IL-2. Mice immunized with DNA and LBP3a also showed significantly higher levels of chlamydia clearance in mice spleens and a greater Th1 immune response. The immunoenhancement induced by 25 mg/kg LBP3a is more effective than that induced by a 12.5 and 50 mg/kg. This implies that LBP3a at 25 mg/kg has a high potential to be used as an effective adjuvant with a DNA vaccine against swine Chlamydophila abortus.

Efficacy of a new inactivated Chlamydophila felis vaccine in experimentally-infected cats.

A new inactivated and adjuvanted Chlamydophila felis vaccine was developed and its efficacy in cats was compared with that of commercially available inactivated and live vaccines. Two commercial vaccines conferred insufficient immunity on inoculated cats, as evaluated by antibody production and a challenge experiment, whereas cats administered the newly generated vaccine produced high-titre antibodies and acquired sufficient immunity. The cats immunised with the new vaccine revealed no or only mild clinical signs, and no chlamydiae were recovered from their tissue samples after exposure to a virulent C felis. However, they shed chlamydiae in their nasal and conjunctival secretions after challenge, as did those immunised with the commercial vaccines and the non-vaccinated controls. The newly developed vaccine caused no adverse reaction in the inoculated cats. These findings suggest that the new vaccine prepared here may be promising for practical use in controlling C felis infection in cats.

Involvement of CD252 (CD134L) and IL-2 in the expression of cytotoxic proteins in bacterial- or viral-activated human T cells.

Regulation of cytotoxic effector molecule expression in human CTLs after viral or bacterial activation is poorly understood. By using human autologous dendritic cells (DCs) to prime T lymphocytes, we found perforin only highly up-regulated in virus- (HSV-1, vaccinia virus) but not in intracellular bacteria- (Listeria innocua, Listeria monocytogenes, Mycobacterium tuberculosis, Chlamydophila pneumoniae) activated CTLs. In contrast, larger quantities of IFN-gamma and TNF-alpha were produced in Listeria-stimulated cultures. Granzyme B and granulysin were similarly up-regulated by all tested viruses and intracellular bacteria. DCs infected with HSV-1 showed enhanced surface expression of the costimulatory molecule CD252 (CD134L) compared with Listeria-infected DC and induced enhanced secretion of IL-2. Adding blocking CD134 or neutralizing IL-2 Abs during T cell activation reduced the HSV-dependent up-regulation of perforin. These data indicate a distinct CTL effector function in response to intracellular pathogens triggered via differing endogenous IL-2 production upon costimulation through CD252.

Chlamydophila felis infection. ABCD guidelines on prevention and management.

OVERVIEW: Chlamydophila felis is a Gram-negative bacterium and its primary target is the conjunctiva. The bacterium does not survive outside the host. INFECTION: Transmission requires close contact between cats; ocular secretions are probably the most important body fluid for infection. Most cases occur in cats under 1 year of age. Chlamydophila felis is the infectious organism most frequently associated with conjunctivitis. DISEASE SIGNS: Unilateral ocular disease generally progresses to become bilateral. There can be intense conjunctivitis with extreme hyperaemia of the nictitating membrane, blepharospasm and ocular discomfort. Transient fever, inappetence and weight loss may occur shortly after infection, although most cats remain well and continue to eat. DIAGNOSIS: PCR techniques are now preferred for diagnosing C felis infection. Ocular swabs are generally used. In unvaccinated cats, antibody detection can be used to indicate infection. DISEASE MANAGEMENT: Tetracyclines are generally regarded as the antibiotics of choice. Doxycycline has the advantage of requiring only single daily administration and is given at a dose of 10 mg/kg orally. Vaccination should be considered if there is a history of confirmed chlamydial disease in a shelter. Single housing and routine hygiene measures should suffice to avoid cross-infection. Cats maintained together for longer terms should be vaccinated regularly. In breeding catteries where C felis infection is endemic, the first step should be to treat all cats with doxycycline for at least 4 weeks. Once clinical signs have been controlled, the cats should be vaccinated. VACCINATION RECOMMENDATIONS: Vaccination should be considered for cats at risk of exposure to infection. Vaccination generally begins at 8-10 weeks of age, with a second injection 3-4 weeks later. Annual boosters are recommended for cats at continued risk of exposure.

Ovine trophoblast is a primary source of TNFalpha during Chlamydophila abortus infection.

Chlamydophila abortus is a Gram-negative obligate intracellular bacterium that causes infectious abortion in sheep (ovine enzootic abortion, OEA) and humans. Infected placentas recovered from sheep that experience OEA have thickened membranes, contain dense inflammatory cellular infiltrates and show evidence of intravascular thrombosis. Despite widespread inflammation, chlamydial multiplication is restricted to the chorionic trophoblast cells. To investigate the potential role of trophoblast in the initiation and propagation of placental inflammation during OEA, the AH-1 ovine trophoblast cell line was experimentally infected with C. abortus and analysed for the release of pro-inflammatory mediators. C. abortus was found to induce the release of both tumour necrosis factor-alpha (TNFalpha) and CXCL8 (interleukin-8) from AH-1 cells in a dose- and time-dependent manner. Ultra-violet (UV)-killed organisms did not elicit this profile, indicating that intracellular multiplication of C. abortus was required for release of these pro-inflammatory mediators. Exposure of AH-1 cells to recombinant ovine TNFalpha alone resulted in the release of CXCL8, suggestive of a self-propagating inflammatory cytokine and chemokine cascade. These data indicate a primary role for trophoblast in the initiation and propagation of placental inflammation during chlamydial abortion.

Evaluation of the health of free-ranging greater rheas (Rhea americana) in Argentina.

The health of 22 free-ranging adult rheas (Rhea americana) examined and sampled during a translocation/reintroduction project and six juvenile rheas kept in semicaptivity was investigated, and details of their haematology and plasma biochemistry are presented. Serological testing for antibodies to infectious agents was negative for infectious laryngotracheitis, avian adenovirus, avian influenza, avian reovirus, infectious bursal disease, infectious bronchitis virus, paramyxovirus types 1, 2, and 3, fowlpox and Salmonella Pullorum. Antibodies to Chlamydophila species were found in 25 of 27 of the birds, and 22 of 25 had antibodies to Aspergillus species. Ova of gastrointestinal nematodes of the genus Capillaria were identified, and the anoplocephalid cestode Monoecocestus cf rheiphilus was identified in R americana for the first time.

Antibody induction after combined application of an adjuvanted recombinant FeLV vaccine and a multivalent modified live virus vaccine with a chlamydial component.

The compatibility, safety and interaction on antibody induction of a combined vaccine application were assessed. Specific pathogen-free cats were vaccinated with either a modified live virus vaccine containing feline calici- (FCV), herpes- (FHV-1), parvovirus (FPV) and Chlamydophila felis (C. felis), an adjuvanted recombinant feline leukaemia virus (FeLV) vaccine or both vaccines in one syringe. After combined application, FeLV ELISA antibody titres were unaltered, However antibody production based on indirect immunofluorescence assay was remarkably enhanced for FCV and was at selected time points also enhanced for FHV-1 and C. felis but diminished for FPV. The use of these vaccines in combination was safe and will simplify vaccination schedules in veterinary practice.

[Antibodies against Chlamydophila in patients with acute myocardial infarction and coronary risk and their association with mortality]. / Anticuerpos contra Chlamydophila en pacientes con infarto agudo del miocardio y riesgo coronario, y su relación con la muerte.

OBJECTIVE: The primary aim of this study was to determine whether antibodies against Chlamydophila pneumoniae in patients with acute myocardial infarction (AMI) and coronary risk factors are associated with death. MATERIAL AND METHODS: A cross-sectional study was conducted among 100 patients hospitalized in the Coronary Unit of Centro Medico La Raza Hospital of the Mexican Institute of Social Security, between 1999 and 2000. Subjects were males and females older than 18 years, diagnosed with AMI and coronary risk. Antibodies against Chlamydophila pneumoniae, Chlamydophila psitacii and Chlamydia trachomatis were measured using an indirect microinmunofluorescence assay. In addition, blood samples from 33 patients from the original group were taken when the patients were discharged from the hospital,and 3 months after their myocardial infarction. Data analysis consisted of geometric means and standard deviations as well as odds ratios with 95% confidence intervals. RESULTS: Seventy percent of patients presented antibodies against Chlamydophila pneumoniae. Antibodies against Chlamydophila psittaci and Chlamydia trachomatis were not identified. No statistically significant association was found between antibodies and death in these patients with coronary risk factors and AMI. In the subgroup of 33 individuals 25 had antibodies against Chlamydophila pneumoniae and in 83% of them antibodies decreased three months after the AMI event. CONCLUSIONS: Even though patients with coronary risk factors and AMI had an increased seropositivity for Chlamydophila pneumoniae it was not significantly associated with death.

Anticuerpos contra Chlamydophilaen pacientes con infarto agudo del miocardio y riesgo coronario, y su relación con la muerte / Antibodies against Chlamydophila in patients with acute myocardial infarction and coronary risk and their association with mortality

OBJETIVO: Determinar si los anticuerpos contra Chlamydophila pneumoniae en pacientes con infarto agudo del miocardio y factores de riesgo coronario se asocian con la muerte. MATERIAL Y MÉTODOS: Se hizo un estudio observacional, prospectivo, transversal y comparativo. Se incluyeron en el estudio 100 sujetos que, entre 1999 y 2000, estuvieron hospitalizados en la Unidad Coronaria del Hospital de Especialidades del Centro Médico La Raza, del Instituto Mexicano del Seguro Social. Se trataba de una muestra constituida por pacientes de ambos sexos, mayores de 18 años, con infarto agudo del miocardio y riesgo coronario. Mediante microinmunofluorescencia indirecta se identificaron anticuerpos contra Chlamydophila pneumoniae, Chlamydophila psitacii y Chlamydia trachomatis. De entre los 100 sujetos, se eligieron al azar 33, a quienes se les determinaron anticuerpos contra Chlamydophila, no sólo durante su estancia en el hospital, sino también al salir de éste y a los tres meses de haber sufrido el infarto agudo del miocardio. Se calcularon las medias y las desviaciones geométricas estándares para los títulos de anticuerpos contra Chlamydophila, y se determinó la razón de momios y el intervalo de confianza al 95 por ciento entre los factores de riesgo coronario y la muerte. RESULTADOS: Setenta por ciento de los pacientes de la muestra inicial presentaron anticuerpos contra Chlamydophila pneumoniae; no se identificaron anticuerpos contra Chlamydophila psitacii y Chlamydia trachomatis. No se observó una fuerza de asociación estadísticamente significativa con la muerte en pacientes con infarto agudo del miocardio y factores de riesgo coronario. De los 33 individuos de la submuestra, 25 presentaron anticuerpos contra Chlamydophila pneumoniae, y en 83 por ciento de estos últimos casos, se registró un descenso de dichos anticuerpos a los tres meses de haberse presentado el infarto agudo del miocardio. CONCLUSIONES: A pesar de que en pacientes con infarto agudo del miocardio y riesgo coronario se presentó un incremento en la frecuencia de seropositividad a Chlamydophila pneumoniae, no se observó una fuerza de asociación estadísticamente significativa de ello con la muerte.

Molecular cloning of the Chlamydophila abortus groEL gene and evaluation of its protective efficacy in a murine model by genetic vaccination.

The immunogenicity and protective effect of a DNA vaccine encoding the heat-shock protein (Hsp) GroEL of Chlamydophila abortus AB7, an obligate intracellular bacterium that causes abortion in sheep, was evaluated in pregnant and non-pregnant mouse models. The C. abortus groEL gene was cloned by screening a genomic library constructed in lambdaFIX II arms with a nucleic acid probe corresponding to the central portion of the groEL gene from C. abortus. Sequence analysis of a positive clone revealed an open reading frame of 1632 bp encoding a 544 amino acid polypeptide with a predicted molecular mass of 58 256 Da and highly similar to GroEL of Chlamydia trachomatis (93 %) and Chlamydophila pneumoniae (94 %). As observed in other sequenced chlamydial genomes, the groEL gene belongs to an operon comprising another gene encoding the Hsp GroES. OF1 outbred mice were immunized intramuscularly with plasmid DNA carrying the groEL gene three times at 3 week intervals and challenged 2 weeks after the last DNA injection. In pregnant mice, no reduction in abortion was observed and the DNA vaccination failed to reduce the bacterial infection in the placenta and spleen of mice. Nevertheless, partial protection of fetuses was obtained. Immunization of non-pregnant mice with the groEL gene resulted in a specific humoral response with the predominant IgG2a isotype, suggesting a Th1-type immune response. The anti-GroEL antibodies showed no neutralizing effect in vitro on C. abortus infectivity. Although the DNA vaccine induced a delayed-type hypersensitivity response, it failed to elicit an efficient cellular immune response since the mice were not protected against bacterial challenge.

Induction of inflammatory host immune responses by organisms belonging to the genera Chlamydia/Chlamydophila.

Chlamydia/Chlamydophila are a family of intracellular gram-negative bacteria that infect their hosts primarily via mucosal epithelia. Chronic disease associated with bacterial persistence, inflammation and tissue damage are common sequelae of infection with these organisms. Human epithelial cell lines respond to infection by releasing pro-inflammatory cytokines and chemokines such as interleukin (IL)-6 and IL-8, and upregulating the expression of mRNA encoding Ikappa-Balpha, the endogenous inhibitor of NF-kappaB. However, Ikappa-Balpha is not upregulated in response to bacterial lipopolysaccharide (LPS). The failure of epithelial cells to respond to LPS is associated with the absence of surface expression of CD14. Identification of the components of Chlamydia/Chlamydophila that can induce pro-inflammatory mediators coupled with the mechanisms by which epithelial cells detect infection and respond accordingly will advance the development of preventative strategies.

An epizootic of chronic regurgitation associated with chlamydophilosis in recently imported emerald tree boas (Corallus caninus).

One hundred and five wild-caught emerald tree boas (Corallus caninus) were added to a collection of 15 others. in Central Florida, during a 4-mo period. Eighty-one boas (67%) developed repetitive regurgitation during the 23-mo period after the initial introduction, and 61 (75%) of these died. Regurgitation occurred 3-4 days after feeding. Prevalence of regurgitation in this population of snakes was 25%/mo (range 0-42%), and incidence was 3.52/mo (range 0-13/mo). The cumulative mortality for those boas developing repetitive regurgitation (61 of 120) during the 23-mo epizootic was 51%. Hematologic findings included anemia and leukocytosis, with lymphocytosis, monocytosis, and azurophilia. Histologic evaluation of the gastrointestinal tract showed multifocal to diffuse lymphoplasmacytic inflammation with granuloma formation and positive immunohistochemical staining for chlamydial antigen. Electron microscopic evaluation of granulomas showed organisms consistent with Chlamydophila sp.

Detection of Chlamydophila (Chlamydia) abortus and Toxoplasma gondii in smears from cases of ovine and caprine abortion by the streptavidin-biotin method.

Fetal membranes from 59 cases of ovine and six of caprine abortion from a total of 52 flocks or herds were collected. Immunohistochemical examination of cotyledons fixed in formalin and embedded in paraffin wax detected Chlamydophila abortus in 44 (68%) cases. Immunocytochemical examination of smears made from the surface of fetal membranes detected the organism in 37 (57%) cases. Light microscopical examination of such smears stained by Stamp's method detected C. abortus in 26 (40%) cases. The streptavidin-biotin method described proved to have 100% specificity and 84% sensitivity in the detection of C. abortus in cotyledon smears. Sensitivity could probably be increased still further by the simultaneous examination of smears made from the cut surface of several cotyledons. In five cases Toxoplasma gondii was detected in the cotyledons by immunohistochemical examination. In three of these cases the presence of T. gondii was revealed also by immunocytological examination. In four cases, simultaneous C. abortus and T. gondii infection of the cotyledons was observed. The two pathogens and the lesions caused by them occurred in separate locations.

Polymorphic outer-membrane proteins of Chlamydophila abortus are glycosylated.

Antigenic profiles of mono-, bi- and poly-specific monoclonal antibodies against 90 kDa polymorphic outer-membrane proteins (POMPs) and a 105 kDa POMP-related protein of Chlamydophila abortus ATCC VR 656(T), after one- and two-dimensional electrophoretic analysis, helped identify each one of the triplets POMP 90, 91A and 91B, and a POMP-related protein at 85 kDa. The lectin concanavalin A bound to the four POMPs and the POMP-related protein in a specific manner and the binding was sensitive to treatment with the amidase N-endoglycosidase F, suggesting the presence of small asparagine-linked oligosaccharide chains. The exposure of the five proteins on the chlamydial surface and the orientation of the attached oligosaccharide chains was examined by protease and endoglycosidase treatments of intact bacteria. The results were consistent with the concept that some of the oligosaccharides in the POMPs face outwards, possibly protecting the polypeptides from proteolytic enzymes, whereas the oligosaccharides in the 105 kDa POMP-related protein are oriented inwards, thereby rendering the polypeptide chain accessible to proteases. A possible role for the N-linked oligosaccharides in the POMPs might be the promotion of the proper folding and processing of these proteins.

Limited role of polymorphonuclear neutrophils in a pregnant mouse model of secondary infection by Chlamydophila abortus (Chlamydia psittaci serotype 1).

The aim of this work was to study the role of polymorphonuclear neutrophils (PMNs) in the clearance of infection, and in the development of specific immunity against Chlamydophila abortus (Chlamydia psittaci serotype 1) secondary infection. A pregnant mouse model depleted of neutrophils by the RB6-8C5 monoclonal antibody was used. No clinical signs were observed in depleted or non-depleted mice after secondary infection and no significant differences were observed in the litter size between the infected and control groups. In PMN-depleted mice C. abortus was not detected in the materno-fetal unit but merely produced low, persistent levels of infection in spleen and liver. In the non-depleted mice the level of infection was significantly lower, being resolved during the first few days post-reinfection. In both infected mice groups the immune response in the liver was quickly established and was seen to be composed mainly of CD4(+)T lymphocytes and macrophages. A Th1 response characterized by the presence of IFN-gamma and TNF-alpha in serum was observed during early infection, with significantly higher levels in the non-depleted animals. Our results suggest that PMNs have little influence on the control of C. abortus secondary infection, although they are a first line of defense and may influence the early production of TNF-alpha and IFN-gamma.