Development and validation of a competitive enzyme-linked immunosorbent assay for detection of type A influenza antibodies in avian sera.

Serologic screening of avian sera for group-specific antibodies to type A influenza is currently accomplished by using the avian influenza (AI) agar gel immunodiffusion (AGID) test. A competitive enzyme-linked immunosorbent assay (CELISA) was developed using a baculovirus vector, Autographa californica nuclear polyhedrosis virus, expressing the nucleoprotein (NP) gene of A/Ann Arbor/6/60 influenza virus. The recombinant NP was obtained by inoculation of Spodoptera frugiperda (Sf9) insect cells or Trichoplusia ni insect larvae with the recombinant baculovirus. A hybridoma cell line producing monoclonal antibody against influenza virus A nucleoprotein was used to generate mouse ascitic fluid for the CELISA. The nucleoprotein and the monoclonal antibody were used without further purification in a CELISA for detection of avian-origin serum antibodies to type A influenza. The AI AGID and CELISA tests were compared for sensitivity and specificity using 1651 experimental and reference antisera. Samples discrepant in AGID and CELISA test results were further evaluated by the AI indirect fluorescent antibody (IFA), hemagglutination-inhibition (HI), and neuraminidase-inhibition (NI) tests. The results demonstrated a high degree of correlation between the AGID and CELISA test results, with the IFA, HI, and NI tests offering additional support of CELISA test specificity. The CELISA is a rapid, economical, sensitive, and specific serodiagnostic method for screening large numbers of avian sera for antibodies to avian influenza virus.

Susceptibility of pigeons to avian influenza.

Susceptibility to infection with avian influenza virus (AIV) was studied in pigeons inoculated via oculonasal (Experiment 1) or intravenous (Experiment 2) route. Chickens were included as susceptible hosts in both experiments. Two subtypes each of the highly pathogenic AIV (HPAIV; HP CK/PA H5N2 and HP CK/Australia H7N7) and non-pathogenic AIV (NPAIV; NP CK/PA H5N2 and NP emu/TX H7N1) at a dose of 10(5) embryo infective dose per bird were used as inoculum. The pigeons inoculated with HP CK/PA H5N2 or HP CK/Australia H7N7 remained apparently healthy throughout the 21-day observation period, did not shed viruses on 3, 7, 14, and 21 days postinoculation (DPI), and had no demonstrable levels of antibodies on 21 DPI. On the other hand, 9 of 12 chickens inoculated with the HPAIV died of highly pathogenic avian influenza; the viruses were recovered from their respiratory and intestinal tissues, and the surviving chickens had antibodies to AIV. Regarding responses of pigeons to inoculation with NP CK/PA H5N2 or NP emu/TX H7N1, the pigeons remained clinically healthy throughout the 21-day observation period and did not have detectable levels of antibodies on 21 DPI; only one pigeon yielded the NP emu/TX H7N1 on 3 DPI. The virus was isolated from a tracheal swab and was believed to be the residual inoculum virus. Based on the responses of pigeons to NPAIV and HPAIV, it was concluded that the pigeons were resistant or minimally susceptible to infection with HPAIV or NPAIV.

Construction and insect larval expression of recombinant vesicular stomatitis nucleocapsid protein and its use in competitive ELISA.

The gene encoding the nucleocapsid (N) protein of Indiana 1 serotype vesicular stomatitis virus (VSV-IN1) was transferred into the genome of Autographa californica nuclear polyhedrosis virus (baculovirus) as a full-length non-fusion construct under the control of the polyhedrin gene promoter. Recombinant N protein was obtained from Trichoplusia ni insect larvae inoculated 72-96 h previously with the recombinant baculovirus. Polyclonal antibody (PAB) against VSV-IN1 was produced in mice using VSV-IN1 whole virus antigen concentrated from virus-infected cell culture fluids. The N protein and the PAB were used without further purification in a competitive enzyme-linked immunosorbent assay (C-ELISA) for detection of bovine, porcine, and equine origin serum antibodies against VSV-IN1. A limited number of field origin, experimental, and reference VSV antisera were evaluated using the C-ELISA and with a standard serum neutralization (SN) procedure. Sensitivity of the C-ELISA was comparable to the serotypically homologous SN procedure. Subject to further validation, similar C-ELISA tests for the other VSV serotypes, used in conjunction with the test described here, may offer the best combination of rapidity, sensitivity, simplicity, economy, and laboratory biosafety of any of the methods yet developed for VSV serodiagnosis.

Antigenic differences between European and American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) are encoded by the carboxyterminal portion of viral open reading frame 3.

Antigenic differences between European and American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were revealed by serologic analysis of a recombinant protein derived from PRRSV open reading frame 3 (ORF 3). The hydrophilic carboxyterminal 199 amino acids encoded by the ORF 3 of a European (Lelystad) isolate of PRRSV were expressed as a recombinant fusion protein (BP03-P) in a baculovirus gene expression system. Sera from gnotobiotic swine exposed to prototypic reference European and American isolates of PRRSV and sera from conventionally reared European and American swine convalescing from naturally acquired PRRSV infections were used to characterize the BP03-P protein. Sera from gnotobiotic and conventionally reared swine exposed to European isolates of PRRSV were significantly more reactive (P < 0.01) with BP03-P than were the corresponding American PRRSV antisera using the indirect immunoperoxidase monolayer assay (IPMA). Prototypic European, but not American, PRRSV antisera also recognized BP03-P using western immunoblotting and radioimmunoprecipitation assay (RIPA) procedures. However, gnotobiotically derived antiserum to an atypical American-origin PRRSV was reactive with BP03-P by both IPMA and western immunoblot. Despite a predicted potential for N-linked glycosylation, studies with tunicamycin and peptide-N-glycosidase F (PNGase F) indicated that BP03-P was not N-glycosylated in either insect cell cultures or Trichoplusia ni larvae infected with the recombinant baculovirus. Sera from rabbits inoculated with BP03-P failed to neutralize both the European (Lelystad) and American (ATCC VR-2332) reference isolates of PRRSV and did not react by IPMA with PRRSV-infected cell cultures. Taken together, the data suggest that the carboxyterminal portion of PRRSV ORF 3 encodes a non-neutralizing viral peptide that is partially responsible for the serologic differences noted between European and most American isolates of PRRSV.

Management of postoperative pain by continuous epidural infusion of analgesics.

The anatomy and physiology of the epidural space and the mechanism of action, sites of action, and pharmacokinetics of analgesics administered by continuous epidural infusion are reviewed, and the efficacy, adverse effects, and postoperative indications for use of analgesics administered by this route are discussed. Narcotics selectively block pain conduction by occupying specific opiate receptors in the spinal cord. Local anesthetics provide analgesia by axonal membrane blockade; they also can produce nonselective sympathetic and somatic (sensory and motor) blockade in addition to analgesia. A narcotic-local anesthetic mixture should provide an additive analgesic effect, without an increase in the incidence of adverse effects. Comparative efficacy studies have shown that continuous epidural infusions of narcotics, local anesthetics, and narcotic-local anesthetic combinations, when used appropriately, may produce better analgesia than conventional bolus methods of pain relief. Continuous epidural infusions also offer a safety advantage over intermittent epidural injections because peak and trough levels of the analgesic agent are avoided. Adverse effects of epidurally administered narcotics include respiratory depression, pruritus, urinary retention, nausea and vomiting, and sedation. Adverse effects of epidurally administered local anesthetics include urinary retention, hypotension, numbness, motor weakness, tachyphylaxis, and, rarely, systemic toxicity. The cost of epidurally administered drugs is substantially higher than that for i.m. or i.v. narcotic analgesia, but this cost may be offset by other benefits such as a shorter hospital stay. Current studies suggest superior analgesia for the majority of surgical procedures with continuous epidural analgesia infusions compared with more traditional methods of providing analgesia.

Development of a comprehensive operating room pharmacy.

The justification, implementation, and services of a comprehensive on-site operating room (OR) pharmacy are reviewed. Justification was accomplished through audits of controlled substance accountability, drug preparation, labeling and storage, and cost analyses of drug waste and potential savings. Implementation was achieved through the joint efforts of the Department of Anesthesia, Pharmacy, and Nursing. Services of the OR pharmacy are dynamic and include standardized drug preparation, case-by-case drug distribution and patient billing, controlled substance accountability, provision of drug information, and clinical research support. This pharmacy has proven cost effective and has become integral to the daily function of the OR.