Difference in virulence of Mycobacterium avium isolates sharing indistinguishable DNA fingerprint determined in murine model of lung infection.

BACKGROUND: Opportunistic Mycobacterium avium typically causes disease in immunocompromised patients and in some groups of apparently healthy individuals. The high virulence of some bacterial lineages increases the disease risk. High-resolution molecular genotyping studies of M. avium clinical isolates demonstrated that some genotype patterns were more prevalent than others, suggesting that close genetic relatedness of these successful isolates sharing a similar genotype could determine similar biological properties associated with high virulence. METHODS AND FINDINGS: In this study, we aimed to compare the virulence and pathogenic properties of two epidemiologically unrelated M. avium isolates sharing an indistinguishable DNA fingerprint in a well-characterized model of pulmonary infection in mice, resistant or susceptible to mycobacteria. The mice, C57BL/6 wild- type or IFN-gamma gene disrupted (GKO), respectively, were intratracheally infected with two isolates, H27 (human blood isolate) and P104 (pig lymph node isolate), and the lungs were examined for bacterial loads, histopathology and cytokine gene expression. The obtained data demonstrated significant differences in the virulence properties of these strains. Although the H27 strain grew significantly faster than P104 in the early stage of infection, this bacterium induced protective immunity that started to reduce bacterial numbers in the wild-type mice, whereas the P104 strain established a chronic infection. In the GKO mice, both strains were capable of causing a chronic infection, associated with higher bacterial burdens and severe lung pathology, in a similar manner. CONCLUSIONS/SIGNIFICANCE: The results demonstrated that the studied isolates differed in the pathogenic properties although were indistinguishable by actually widely used genotyping techniques demonstrating that the genotype similarity does not predict similarity in virulence of M. avium isolates.

Avaliação dos anticorpos séricos totais antiglicolipídeo fenólico de Mycobacterium leprae em indivíduos portadores de hanseníase e seus contatos domiciliares no estado de Goiás, Brasil / Evaluation of total serum antibodies against phenolic glycolipid-1 (PGL-1) of Mycobacterium leprae in individuals with Hansen disease and their house contacts in the State of Goias, Brazil

A hanseníase é uma doença infecciosa, de evolução crônica, cujo agente causal é o Mycobacterium leprae, um bacilo intracelular obrigatório que infecta mais frequentemente macrófagos e células nervosas periféricas de Schwann. O diagnóstico da hanseníase é complexo, visto que a doença evolui para diferentes formas clínicas e histopatológicas. Novos testes diagnósticos têm sido propostos com o objetivo de identificar possíveis fontes de contágio e controlar a transmissão da doença. Dentre eles, destaca-se a dosagem de anticorpos IgM antiglicolipídeo fenólico (PGL-1), específico de Mycobacterium leprae, para vigilância de doentes e seus contatos. Neste trabalho, foram avaliados os níveis séricos de anticorpos totais antiPGL-1 em 102 indivíduos portadores de hanseníase (formas multibacilar e paucibaciliar) e 65 contatos domiciliares destes indivíduos, por meio de ensaio imunoenzimático. Tanto os pacientes quanto seus contatos apresentaram níveis séricos detectáveis de anticorpos antiPGL-1, o que indica potencial risco de transmissão entre eles.

Monocytes/macrophages infected with Toxoplasma gondii do not increase co-stimulatory molecules while maintaining their migratory ability.

Toxoplasma gondii is an obligate intracellular parasite that is able to disseminate into deep tissues and cross biological barriers, reaching immunoprivileged sites such as the brain and retina. The parasite is able to infect macrophages and dendritic cells and use them for dispersal throughout the body, but the activation state of those cells is unknown. We investigated the ability of human and murine cells from monocytic/macrophage lineages that had not previously been exposed to inflammatory cytokines to up-regulate co-stimulatory and adhesion molecules upon infection. Toxoplasma gondii-infected human monocytes (freshly isolated and THP1 lineage) were unable to up-regulate CD86, CD83, CD40 or CD1a. CD80 expression increased in infected cells but expression of l-selectin and beta2 integrin was unaltered. We evaluated the ability of infected macrophages from wild type C57/BL/6 or CD14(-/-) mice to migrate in 8 mum transwells. Infected cells from CD14(-/-) mice were more likely to de-adhere than infected cells from wild type mice but they did not show any increase in migratory ability. The non-stimulatory profile of these infected cells may contribute to parasite spread throughout the lymphatic circulation in the initial phases of infection.

Development of process to produce polyvalent IgY antibodies anti-African snake venom.

Polyvalent anti-Bitis and anti-Naja antivenom IgY antibodies were prepared using B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca, and N. mossambica venoms to immunize chickens. Blood and eggs were collected before and during the 10-month immunization period; the sera and yolk extracts were then prepared and assayed for the presence of antivenom antibodies by ELISA and Western blot methods. ELISA Antivenom antibody titers, referred to as U-ELISA/ml of serum or egg yolk extracts, absent in pre-immunization sera or yolk, increased sharply during the 4 weeks after immunization, reaching a plateau thereafter. Yolk extracts with high antivenom titers, as detected by ELISA were used to isolate and purify IgY. Purified IgY preparations recognized venom protein bands from 10 to 20 kDa to 60 and 70 kDa, as shown by Western blot. Recovery of antivenom antibodies from the whole yolk was over 80%. Final preparations exhibited high antivenom activity (>100,000 U-ELISA/ml) as well as efficacy in neutralizing venom lethality (1,440 microg of IgY neutralize 62.2 LD(50) of venom), and were free of toxic products, pyrogen or bacterial and fungal contaminations.

Neutrophils regulate the expression of cytokines, chemokines and nitric oxide synthase/nitric oxide in mice injected with Bothrops atrox venom.

Bothrops atrox crude venom injected intraperitoneal (i.p.) into BALB/c mice induced local afflux of inflammatory cells, one neutrophil-rich peak after 6h and another macrophage-rich peak after 48 h. A similar pattern of local cell afflux plus edema, Delta lesions of some skeletal muscle cells, and hemorrhage were observed in mice intramuscular (i.m.) injected with the venom. Measurement of serum cytokines in neutrophil-depleted (by anti-mouse rat monoclonal antibody (mAb) RB6-8C5) and non-depleted BALB/c mice was performed by ELISA. With the exception of IL-1beta (78 pg/ml), higher levels of IL-6 (1348 pg/ml), MIP-1beta (437 pg/ml) and MIP-2 (904 pg/ml) were observed in neutrophil-depleted mice, in comparison to the values found in non-neutrophil depleted mice: IL-1beta (437 pg/ml), IL-6 (750 pg/ml), MIP-1beta (165 pg/ml) and MIP-2 (90 pg/ml). TNF-alpha was not detected. NO was detected (18 microM) 24h after venom injection in neutrophil-depleted mice. RT-PCR using representative primers detected expression of mRNA in cells from BALB/c mice injected with B. atrox venom: (a) for IL-1beta, IL-6, inducible nitric oxide synthase (iNOS), CXCR2, MIP-2 and RANTES in cells from mice that were neutrophil-depleted or not; (b) for CCR1, CCR5 and MIP-1beta in cells from neutrophil-depleted mice; (c) for MIP-1alpha in cells from non-neutrophil-depleted mice; (d) TNF-alpha and TGF-beta were not detected in either of the mice. These results indicate that neutrophils play a role in regulating the production of some cytokines and chemokines as well as locally expressed or liberated iNOS/NO in tissues injected with B. atrox crude venom.

Microbiota fungica na pelagem de camundongos de linhagens isogênicas e congênicas mantidos em biotério de criaçäo / Fungal microbiota of isogenic and congenic strains on nice in animal holding rooms

Com o desenvolvimento da tecnologia e do manejo de animais de Laboratório, mantidos em biotérios que seguem diferentes normas de trabalho, de acordo com os objetivos a que se propöem, é mister que se avalie a microbiota dos ambientes de criaçäo, bem como dos animais que deles fazem parte. Estudou-se a freqüência de fungos na pelagem de 95 camundongos (47 machos e 48 fêmeas de Linhagem isogênicas (A/Sn (10), Balb/c (16), CBA (15), C57BL/10J (10) e congênitas (B10. A (14), B10.d2/o (15) e B 10.D2/n, com idades que variavam entre 37 55 dias. A coleta de material foi realizada pela fricçäo de um pedaço de tapete esterilizado, em toda regiäo corpórea dos camundongos, seguida de compressäo do material em placas de Petri, contendo agar Sabouraud dextrose acrescido de coranfenicol (100 microng/ ml) - ASD e Mycobiotic agar' (MA), para o isolamento dos fungos. Verificou-s, maior número de isolamentos no primeiro (224) do que na segunda (61). As freqüências de isolamento foram, respectivamente: Penicillium sp (93,68%), Trichoderma sp (92,63%), Cladosporium sp (38,95%), torulapsis sp (7,37%, Rhizapus sp (4, 21%), Aureobasidium sp (3, 16%), Absidia sp (1,05%), Asperigillus sp (1,05%), Mycellia sterilia (1,05%), Trichotecium sp (1,05%), Candida sp (1,05%) e Rhadotorula sp (1,05%). Maior número de gênero fúngicos foi isolado de Linhagens isogênicas e de camundongos do sexo feminino. Estes resultados foram estatisticamente significativos para p = 0,05 na prova dos sinais. Entre os fungos isolados nos ambientes de criaçäo incluiram-se os seguintes gêneros Absidia, Aureobasidium. Cladosporium, Cepnalosporium, Ebicoccum, Geotrichum, Mycelia sterilia, Panicillium e Trichoderma. ªNäo foram encontrados fungos considerados patogênicos para as Linhagens estudadas, tanto nos animais como nas diferentes salas de criaçäo