GENOTYPE CHARACTERIZATION OF THE Haematobia irritans (DIPTERA: MUSCIDAE) FROM BRAZIL, DOMINICAN REPUBLIC AND COLOMBIA BASED ON RANDOMLY AMPLIFIED POLYMORPHIC DNA (RAPD) ANALYSIS

Blood-sucking flies are important parasites in animal production systems, especially regarding confinement conditions. Haematobia irritans, the horn fly, is one of the most troublesome species within bovine production systems, due to the intense stress imposed to the animals. H. irritans is one of the parasites of cattle that cause significant economic losses in many parts of the world, including South America. In the present work, Brazilian, Colombian and Dominican Republic populations of this species were studied by Random Amplified Polymorphic DNA(RAPD) to assess basically genetic variability between populations. Fifteen different decamer random primers were employed in the genomic DNA amplification, yielding 196 fragments in the three H. irritans populations. Among h. irritans samples, that from Colombia produced the smallest numbers of polymorphic hands. This high genetic homogeneity may be ascribed to its geographic origin, which causes high isolation, low gene flow, unlike the other American populations, from Brazil and Dominican Republic. Molecular marker fragments, which its produced exclusive bands, detected in every sample enabled the population origin to be characterized, but they are also potentially useful for further approaches such as the putative origin of Brazilian, Colombian and Dominican Republic populations of horn fly from South America. Similarity indices produced by chemo metric analysis showed the closest relationships between flies from Brazil and Dominican Republic, while flies from Colombia showed the greatest genotypic differentiation relative to the others populations.CAPESCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single- Domain Antibody Fragments.

Submitted by EMERSON LEAL ([email protected]) on 2018-07-12T14:10:06Z No. of bitstreams: 1 Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.PDF: 2085939 bytes, checksum: ee0b2f63f746338bb86bb2d5e895816d (MD5)Approved for entry into archive by EMERSON LEAL ([email protected]) on 2018-07-12T20:22:29Z (GMT) No. of bitstreams: 1 Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.PDF: 2085939 bytes, checksum: ee0b2f63f746338bb86bb2d5e895816d (MD5)Made available in DSpace on 2018-07-12T20:22:29Z (GMT). No. of bitstreams: 1 Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.PDF: 2085939 bytes, checksum: ee0b2f63f746338bb86bb2d5e895816d (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Universidade Federal Fluminense. Rio de Janeiro, RJ, Brasil.Universidade Federal de Rondônia. Porto Velho, RO, Brasil.Empresa Brasileira de Pesquisa Agropecuária. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil. / Universidade Federal de Rondônia. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil. / Universidade Federal de Rondônia. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil. / Universidade Federal de Rondônia. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil. / Centro de Pesquisa em Medicina Tropical. Porto Velho, RO, Brasil.Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenomtherapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I byWestern blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) inmice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem

Selection of anti-BthTX-I and anti-BthTX-II VHHs.

<p>VHHs from the recombinant phage displayed library were submitted separately against immobilized BthTX-I or BthTX-II. After one round of panning, 82 and 28 clones selected for BthTX-I and BthTX-II, respectively, presented VHHs. ELISA assays were performed to verify clone reactivity. Twenty-eight (28) and six (06) clones recognized BthTX-I and BthTX-II. (A) Clonal reactivity of VHHs (1–40) selected against BthTX-I, NC–negative control; (B) Clonal reactivity of VHHs (41–83) selected against BthTX-I, Cross-reactivity of VHH 82 with BthTX-II, NC–negative control; (C) Clonal reactivity of VHHs (1–37) selected against BthTX-II, Cross-reactivity of 02, 09, 20, 21, 28 and 30 VHHs with BthTX-I, NC–negative control. All clones that showed an absorbance value (OD 450nm) higher than the stipulated cut-off point (2 mean OD from negative samples plus 2 standard deviations) were considered positive. All measurements were performed in triplicate. The negative control was performed using the llama pre-immune serum. Error bars represent standard deviation.</p

<i>In vitro</i> inhibition of phospholipase activity.

<p>The quantification of phospholipase activity inhibition by selected VHHs was assayed using synthetic fluorescent phospholipid. To verify the ability of VHHs to inhibit the phospholipase activity of BthTX-II, the toxin was pre-incubated with selected VHHs for 30 minutes at 37°C in different proportions (1:5; 1:10 and 1:40 w/w). Inhibition of phospholipase activity by (A) KF498607; (B) KF498608; (C) KF329715; (D) KC329718. BthTX-II activity on the phospholipid, in the absence of VHH, was used as a positive control, and considered as having 100% activity. The negative controls were carried out using medium reaction without BthTX-II. All measurements were performed in duplicate. Error bars represent standard deviation.</p

Interaction analysis by SPR.

<p>Interaction analysis by SPR.</p

<i>In vivo</i> neutralization of <i>B</i>. <i>jararacussu</i> venom and PLA₂-induced myotoxicity by VHHs.

<p>Plasma creatine kinase (CK) levels were measured at 340 nm to determine VHH ability to inhibit the myotoxicity caused by <i>B</i>. <i>jararacussu</i> venom or PLA₂s, or by a related toxin, MTX-I, a PLA₂ isolated from <i>B</i>. <i>brazili</i>. For this, groups of 5 animals were injected with PBS, VHH KF498607, VHH KC329718, BthTX-I, BthTX-I + VHH KF498607, BthTX-I + VHH KC329718, BthTX-II, BthTX-II + VHH KF498607, BthTX-II + VHH KC329718, <i>B</i>. <i>jararacussu</i> venom, <i>B</i>. <i>jararacussu</i> venom + VHH KF498607, <i>B</i>. <i>jararacussu</i> venom + VHH KC329718, MTX-I, MTX-I + VHH KC329718, MTX-I + VHH KC329718. Before administration to mice, venom or PLA₂s and antibody preparations were pre-incubated at 37°C for 1 h, in a proportion 1:5 or 1:10 (w/w). The negative control was performed with PBS or VHHs, and as a positive control, animals were injected with <i>B</i>. <i>jararacussu</i> venom, BthTX-I, BthTX-II or MTX-I without the addition of VHH. Bonferroni’s test was used for significance analysis. (*) P <0.05. Error bars represent standard deviation.</p

Docking results showing binding sites of VHHs on surfaces of PLA₂s.

<p>(A) KF498607 (blue ribbon) and BthTX-I (red surface); (B) KC329718 (blue ribbon) and BthTX-I (red surface); (C) KF498607 (blue ribbon) and BthTX-II (orange surface); (D) KC329718 (blue ribbon) and BthTX-II (orange surface). The interaction sites were enlarged to show hydrogen bonds formed between the amino acid residues. Both VHHs covered the enzymatic groove surface areas of the modeled BthTX-II and inserted the CDR3 into the catalytic cleft, as well as being able to interact with amino acid residues of the PLA₂ C-termini.</p

VHHs affinity to BthTX-I and BthTX-II.

<p>Representative sensorgrams of the interaction were measured in a Biacore T200 system (GE Healtcare). Purified VHHs (KF498607, KF498608 and KC329718) at concentrations of 6.25 to 0.049 μg/mL were flowed over a BthTX-I-coated CM5 chip and 25 to 0.006 μg/mL were used for immobilized BthTX-II. (A) KF498607 x BthTX-I; (B) KF498607 x BthTX-II; (C) KF498608 x BthTX-I; (D) KF498608 x BthTX-II; (E) KC329718 x BthTX-I; (F) KC329718 x BthTX-II. Assays were injected in a flow rate of 30 μl/min at 37°C. RU indicates resonance units.</p

PDB code, resolution, references, percent similarity and RMSD of templates for modeled VHHs.

<p>PDB code, resolution, references, percent similarity and RMSD of templates for modeled VHHs.</p

Western blot of purified VHHs against BthTX-I.

<p>15% SDS-PAGE was carried out to resolve BthTX-I and BthTX-II under reducing conditions. After electrophoresis, BthTX-I and BthTX-II were electrotransferred to a nitrocellulose membrane and probed with selected VHHs (KF498607, KF498608, KC329715, and KC329718). Samples were incubated with mouse anti-His antibody followed by HRP-conjugated anti-mouse IgG produced in goat. Reactive signals were detected by DAB staining in the presence of hydrogen peroxide. <i>Lama glama</i> pre-immune serum was used as negative control (-), and <i>Lama glama</i> immune serum, as positive control (+).</p