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.Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Estadual Norte Fluminense, Lab Biol Recognit, Rio de Janeiro, BrazilUniv Estadual Norte Fluminense, Lab Anim Morphol & Pathol, Rio de Janeiro, BrazilInst Butantan, Lab Immunochem, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilFAPERJ: E-26/111.6111CNPq: 410555Web of Scienc

Development of Equine IgG Antivenoms against Major Snake Groups in Mozambique.

BACKGROUND:Snake envenoming is a significant public health problem in underdeveloped and developing countries. In sub-Saharan Africa, it is estimated that 90,000-400,000 envenomations occur each year, resulting in 3,500-32,000 deaths. Envenomings are caused by snakes from the Viperidae (Bitis spp. and Echis spp.) and Elapidae (Naja spp. and Dendroaspis spp.) families. The African continent has been suffering from a severe antivenom crisis and current antivenom production is only sufficient to treat 25% of snakebite cases. Our aim is to develop high-quality antivenoms against the main snake species found in Mozambique. METHODS:Adult horses primed with the indicated venoms were divided into 5 groups (B. arietans; B. nasicornis + B. rhinoceros; N. melanoleuca; N. mossambica; N. annulifera + D. polylepis + D. angusticeps) and reimmunized two times for antivenom production. Blood was collected, and plasma was separated and subjected to antibody purification using caprylic acid. Plasmas and antivenoms were subject to titration, affinity determination, cross-recognition assays and in vivo venom lethality neutralization. A commercial anti-Crotalic antivenom was used for comparison. RESULTS:The purified antivenoms exhibited high titers against B. arietans, B. nasicornis and B. rhinoceros (5.18 x 106, 3.60 x 106 and 3.50 x 106 U-E/mL, respectively) and N. melanoleuca, N. mossambica and N. annulifera (7.41 x 106, 3.07 x 106 and 2.60 x 106 U-E/mL, respectively), but lower titers against the D. angusticeps and D. polylepis (1.87 x 106 and 1.67 x 106 U-E/mL). All the groups, except anti-N. melanoleuca, showed significant differences from the anti-Crotalic antivenom (7.55 x 106 U-E/mL). The affinity index of all the groups was high, ranging from 31% to 45%. Cross-recognition assays showed the recognition of proteins with similar molecular weight in the venoms and may indicate the possibility of paraspecific neutralization. The three monospecific antivenoms were able to provide in vivo protection. CONCLUSION:Our results indicate that the anti-Bitis and anti-Naja antivenoms developed would be useful for treating snakebite envenomations in Mozambique, although their effectiveness should to be increased. We propose instead the development of monospecific antivenoms, which would serve as the basis for two polyvalent antivenoms, the anti-Bitis and anti-Elapidae. Polyvalent antivenoms represent an increase in treatment quality, as they have a wider range of application and are easier to distribute and administer to snake envenoming victims

The complementarity-determining region sequences in IgY antivenom hypervariable regions

The data presented in this article are related to the research article entitled ''Development of IgY antibodies against anti-snake toxins endowed with highly lethal neutralizing activity'' (da Rocha et al., 2017) [1]. Complementarity-determining region (CDR) sequences are variable antibody (Ab) sequences that respond with specificity, duration and strength to identify and bind to antigen (Ag) epitopes. B lymphocytes isolated from hens immunized with Bitis arietans (Ba) and anti-Crotalus durissus terrificus (Cdt) venoms and expressing high specificity, affinity and toxicity neutralizing antibody titers were used as DNA sources. The VLF1, CDR1, CDR2, VLR1 and CDR3 sequences were validated by BLASTp, and values corresponding to IgY VL and VH anti-Ba or anti-Cdt venoms were identified, registered [Gallus gallus IgY Fv Light chain (GU815099)/Gallus gallus IgY Fv Heavy chain (GU815098)] and used for molecular modeling of IgY scFv anti-Ba. The resulting CDR1, CDR2 and CDR3 sequences were combined to construct the three - dimensional structure of the Ab paratope. Keywords: PCR, Sequencing, Modeling of biomolecule

African adders: partial characterization of snake venoms from three Bitis species of medical importance and their neutralization by experimental equine antivenoms.

BACKGROUND:An alarming number of fatal accidents involving snakes are annually reported in Africa and most of the victims suffer from permanent local tissue damage and chronic disabilities. Envenomation by snakes belonging to the genus Bitis, Viperidae family, are common in Sub-Saharan Africa. The accidents are severe and the victims often have a poor prognosis due to the lack of effective specific therapies. In this study we have biochemically characterized venoms from three different species of Bitis, i.e., Bitis arietans, Bitis gabonica rhinoceros and Bitis nasicornis, involved in the majority of the human accidents in Africa, and analyzed the in vitro neutralizing ability of two experimental antivenoms. METHODOLOGY/PRINCIPAL FINDINGS:The data indicate that all venoms presented phospholipase, hyaluronidase and fibrinogenolytic activities and cleaved efficiently the FRET substrate Abz-RPPGFSPFRQ-EDDnp and angiotensin I, generating angiotensin 1-7. Gelatinolytic activity was only observed in the venoms of B. arietans and B. nasicornis. The treatment of the venoms with protease inhibitors indicated that Bitis venoms possess metallo and serinoproteases enzymes, which may be involved in the different biological activities here evaluated. Experimental antivenoms produced against B. arietans venom or Bitis g. rhinoceros plus B. nasicornis venoms cross-reacted with the venoms from the three species and blocked, in different degrees, all the enzymatic activities in which they were tested. CONCLUSION:These results suggest that the venoms of the three Bitis species, involved in accidents with humans in the Sub-Saharan Africa, contain a mixture of various enzymes that may act in the generation and development of some of the clinical manifestations of the envenomations. We also demonstrated that horse antivenoms produced against B. arietans or B. g. rhinoceros plus B. nasicornis venoms can blocked some of the toxic activities of these venoms

Electrophoretic profile and cross-recognition by antivenoms as assessed by western blotting (WB).

<p>(a) SDS-Page, upper gel 5%, lower gel 12.5%, stained with silver. (b) WB using anti-<i>B arietans</i> antivenom. (c) WB using anti-<i>B nasicornis + B</i>. <i>rhinoceros</i> antivenom. (d) WB using anti-<i>N</i>. <i>melanoleuca</i> antivenom. (e) WB using anti-<i>N</i>. <i>mossambica</i> antivenom. (f) WB using anti-<i>D</i>. <i>angusticeps + D</i>. <i>polylepis + N</i>. <i>annulifera</i> antivenom. In all experiments, 10 μg of crude venom was resolved under non-reducing conditions. The antibody dilution was fixed at 1:20,000.</p

Affinity of experimental and standard (anti-<i>Crotalic</i>) antivenoms after IgG purification.

<p>(a) Affinity curves and (b) affinity index comparison. The plates were sensitized with 1 μg of antigen/well and the plasma dilutions fixed at 1:20,000. The KSCN dilution ranged from 0 M to 5 M in 1 M intervals. The affinity was calculated as the percentage of antibodies bound at KSCN equal to 5 M; the dotted line represents 100%. The medians were compared using a one-way ANOVA followed by the Dunnett’s Multiple Comparison Test (standard antivenom as comparison), ***P < 0.05. The data represent three independent experiments.</p