Detection of influenza A virus nucleoprotein antibodies in oral fluid specimens from pigs infected under experimental conditions using a blocking ELISA.

In commercial swine populations, influenza is an important component of the porcine respiratory disease complex (PRDC) and a pathogen with major economic impact. Previously, a commercial blocking ELISA (FlockChek(™) Avian Influenza Virus MultiS-Screen(®) Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME, USA) designed to detect influenza A nucleoprotein (NP) antibodies in avian serum was shown to accurately detect NP antibodies in swine serum. The purpose of this study was to determine whether this assay could detect NP antibodies in swine oral fluid samples. Initially, the procedure for performing the NP-blocking ELISA on oral fluid was modified from the serum testing protocol by changing sample dilution, sample volume, incubation time and incubation temperature. The detection of NP antibody was then evaluated using pen-based oral fluid samples (n = 182) from pigs inoculated with either influenza A virus subtype H1N1 or H3N2 under experimental conditions and followed for 42 days post inoculation (DPI). NP antibodies in oral fluid were detected from DPI 7 to 42 in all inoculated groups, that is, the mean sample-to-negative (S/N) ratio of influenza-inoculated pigs was significantly different (P < 0.0001) from uninoculated controls (unvaccinated or vaccinated-uninoculated groups) through this period. Oral fluid versus serum S/N ratios from the same pen showed a correlation of 0.796 (Pearson's correlation coefficient, P < 0.0001). The results showed that oral fluid samples from influenza virus-infected pigs contained detectable levels of NP antibodies for ≥42 DPI. Future research will be required to determine whether this approach could be used to monitor the circulation of influenza virus in commercial pig populations.

Detection of PrPsc in blood from sheep infected with the scrapie and bovine spongiform encephalopathy agents.

The role of blood in the iatrogenic transmission of transmissible spongiform encephalopathy (TSE) or prion disease has become an increasing concern since the reports of variant Creutzfeldt-Jakob disease (vCJD) transmission through blood transfusion from humans with subclinical infection. The development of highly sensitive rapid assays to screen for prion infection in blood is of high priority in order to facilitate the prevention of transmission via blood and blood products. In the present study we show that PrP(sc), a surrogate marker for TSE infection, can be detected in cells isolated from the blood from naturally and experimentally infected sheep by using a rapid ligand-based immunoassay. In sheep with clinical disease, PrP(sc) was detected in the blood of 55% of scrapie agent-infected animals (n = 80) and 71% of animals with bovine spongiform encephalopathy (n = 7). PrP(sc) was also detected several months before the onset of clinical signs in a subset of scrapie agent-infected sheep, followed from 3 months of age to clinical disease. This study confirms that PrP(sc) is associated with the cellular component of blood and can be detected in preclinical sheep by an immunoassay in the absence of in vitro or in vivo amplification.

Cloning and cDNA sequence analysis of Lys(49) and Asp(49) basic phospholipase A(2) myotoxin isoforms from Bothrops asper.

Snake venom myotoxic phospholipases A(2) contribute to much of the tissue damage observed during envenomation by Bothrops asper, the major cause of snake bites in Central America. Several myotoxic PLA(2)s have been identified, but their mechanism of myotoxicity is still unclear. To aid in the molecular characterization of these venom toxins, the complete open reading frames encoding two Lys(49) and one Asp(49) basic PLA(2) myotoxins from the Central American snake B. asper (terciopelo) were obtained by cDNA cloning from venom gland poly-adenylated RNA. The amino acid sequence deduced from the myotoxins II and III open reading frames corresponded in each case to one of the reported amino acid sequence isoforms. The sequence of a new myotoxin IV-like sequence (MT-IVa) contains conservative Val-->Leu(18) and Ala-->Val(23) substitutions when compared with the reported N-terminus of the native myotoxin IV, suggesting minor isoform variations among specimens of a single species. Sequence alignment studies indicated significant (>75% sequence identity) identities with other crotalid venom Lys(49) PLA(2)s, particularly bothropstoxin I/Ia isoforms of B. jararacussu and myotoxin II of B. asper.

Immunochemical properties of the N-terminal helix of myotoxin II, a lysine-49 phospholipase A(2) from Bothrops asper snake venom.

Myotoxic class II phospholipases A(2) from snake venoms can be divided into Asp49 and Lys49 types. The latter, including Bothrops asper myotoxin II, exert membrane damage despite lacking catalytic activity. A heparin-binding, hydrophobic/cationic region, near the C-terminus of myotoxin II (115-129) has been shown to be relevant in its membrane-damaging actions. However, some observations suggest also a potential participation of its N-terminal region. An immunochemical approach was utilized to examine the properties and possible role in toxicity of the N-terminal helix of myotoxin II. Rabbit antibodies raised to a synthetic peptide comprising residues 1-15 recognized the native protein. These antibodies were utilized to compare the antigenic characteristics of the N-terminal helix of several myotoxic phospholipases A(2), showing generally stronger binding to Lys49 myotoxins, in comparison to Asp49 counterparts. However, three Lys49 myotoxins (Cerrophidion godmani myotoxin II, Atropoides nummifer myotoxin II, and Trimeresurus flavoviridis basic protein I) were not recognized by the antibodies, revealing a significant antigenic variability of the N-terminal region within this group of toxins. In neutralization experiments, pre-incubation of myotoxin II with affinity-purified antibodies to the N-terminal helix did not inhibit its myotoxic activity in mice, nor its cytotoxic effect upon cultured muscle cells. These findings argue against a critical role of the N-terminal region of this protein in toxicity. Thus, the precise role of the N-terminal helix of myotoxin II and related Lys49 phospholipases A(2), regarding their toxic mechanisms, remains controversial, and requires further experimental study to be clarified.

Two phospholipase A2 inhibitors from the plasma of Cerrophidion (Bothrops) godmani which selectively inhibit two different group-II phospholipase A2 myotoxins from its own venom: isolation, molecular cloning and biological properties.

Myotoxic phospholipases A(2) (PLA(2)s; group II) account for most of the muscle-tissue damage that results from envenomation by viperid snakes. In the venom of the Godman's viper (Cerrophidion godmani, formerly Bothrops godmani), an enzymically active PLA(2) (myotoxin I) and an inactive, Lys-49 variant (myotoxin II) induce extensive muscle damage and oedema. In this study, two distinct myotoxin inhibitor proteins of C. godmani, CgMIP-I and CgMIP-II, were purified directly from blood plasma by selective binding to affinity columns containing either myotoxin I or myotoxin II, respectively. Both proteins are glycosylated, acidic (pI=4) and composed of 20-25-kDa subunits that form oligomers of 110 kDa (CgMIP-I) or 180 kDa (CgMIP-II). In inhibition studies, CgMIP-I specifically neutralized the PLA(2) and the myotoxic, oedema-forming and cytolytic activities of myotoxins I, whereas CgMIP-II selectively inhibited the toxic properties of myotoxin II. N-terminal amino acid sequence analysis and sequencing of cDNAs encoding the two inhibitors revealed that CgMIP-I is similar to gamma-type inhibitors, which share a pattern of cysteine residues present in the Ly-6 superfamily of proteins, whereas CgMIP-II shares sequence identity with alpha-type inhibitors that contain carbohydrate-recognition-like domains, also found in C-type lectins and mammalian PLA(2) receptors. N-terminal sequencing of myotoxin I revealed a different primary structure from myotoxin II [De Sousa, Morhy, Arni, Ward, Díaz and Gutiérrez (1998) Biochim. Biophys. Acta 1384, 204-208], which provides insight into the nature of such pharmacological specificity.

Effects of aqueous extract of Casearia sylvestris (Flacourtiaceae) on actions of snake and bee venoms and on activity of phospholipases A2.

The crude aqueous extract from the leaves of Casearia sylvestris, a plant found in Brazilian open pastures, was assayed for its ability to inhibit phospholipase A2 (PLA2) activity and some biological activities of bee and several snake venoms, and of a number of isolated PLA2s. The extract induced partial inhibition of the PLA2 activity of venoms containing class I, II and III PLA2s. When tested against the purified toxins, it showed the highest efficacy against class II PLA2s from viperid venoms, being relatively ineffective against the class I PLA2 pseudexin. In addition, C. sylvestris extract significantly inhibited the myotoxic activity of four Bothrops crude venoms and nine purified myotoxic PLA2s, including Lys-49 and Asp-49 variants. The extract was able to inhibit the anticoagulant activity of several isolated PLA2s, with the exception of pseudexin. Moreover, it partially reduced the edema-inducing activity of B. moojeni and B. jararacussu venoms, as well as of myotoxins MjTX-II and BthTX-I. The extract also prolonged the survival time of mice injected with lethal doses of several snake venoms and neutralized the lethal effect induced by several purified PLA2 myotoxins. It is concluded that C. sylvestris constitutes a rich source of PLA2 inhibitors.

A randomized blinded clinical trial of two antivenoms, prepared by caprylic acid or ammonium sulphate fractionation of IgG, in Bothrops and Porthidium snake bites in Colombia: correlation between safety and biochemical characteristics of antivenoms.

A randomized blinded clinical trial was performed in 53 patients bitten by Bothrops sp. and Porthidium sp. in Antioquia and Chocó, Colombia, in order to compare the efficacy and safety of two antivenoms made of whole IgG obtained by either ammonium sulphate (monovalent anti-B. atrox) or caprylic acid (polyvalent) fractionation. Additionally, antivenoms were compared by electrophoretic and chromatographic analyses and anticomplementary activity in vitro. With a protocol of 2, 4 and 6 antivenom vials for the treatment of mild, moderate and severe envenomings, respectively, both antivenoms were equally efficient to neutralize the most relevant signs of envenoming and to clear serum venom levels in patients from the first hour and later on. Three patients with severe envenoming and initially treated with less than six vials on admission had persistent or recurrent venom antigenemia within 12-48 h. Monovalent antivenom fractionated by ammonium sulphate precipitation had higher amounts of protein aggregates and nonimmunoglobulin proteins than polyvalent antivenom fractionated by caprylic acid precipitation. Both antivenoms presented anticomplementary activity in vitro, being higher in the monovalent product. In agreement, monovalent antivenom induced a significantly higher incidence of early antivenom reactions (52%) than polyvalent antivenom (25%).

Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper.

A protein that neutralizes the biological activities of basic phospholipase A2 (PLA2) myotoxin isoforms from the venom of the snake Bothrops asper was isolated from its blood by affinity chromatography with Sepharose-immobilized myotoxins. Biochemical characterization of this B. asper myotoxin inhibitor protein (BaMIP) indicated a subunit molecular mass of 23-25 kDa, an isoelectric point of 4, and glycosylation. Gel-filtration studies revealed a molecular mass of 120 kDa, suggesting that BaMIP possesses an oligomeric structure composed of five 23-25 kDa subunits. Functional studies indicated that BaMIP inhibits the PLA2 activity of B. asper basic myotoxins I and III, as well as the myotoxicity and edema-forming activity in vivo and cytolytic activity in vitro towards cultured endothelial cells, of all four myotoxin isoforms (I-IV) tested. Sequence analysis of the first 63 amino acid residues from the N-terminus of BaMIP indicated more than 65% sequence similarity to the PLA2 inhibitors isolated from the blood of the crotalid snakes Trimeresurus flavoviridis and Agkistrodon blomhoffii siniticus. These inhibitors also share sequences similar to the carbohydrate-recognition domains of human and rabbit cellular PLA2 receptors, suggesting a common domain evolution among snake plasma PLA2 inhibitors and mammalian PLA2 receptors. Despite this similarity, this is the first description of a natural anti-myotoxic factor from snake blood.