Biomedical subjects

John Pasick

Publications and source records attributed to John Pasick.

8 recordsLinked to original sources

PubMed2006

A microsphere immunoassay for detection of antibodies to avian influenza virus.

A microsphere immunoassay (MIA) was developed for the detection of serum antibodies to avian influenza virus. A recombinant influenza A nucleoprotein expressed in baculovirus was conjugated to microspheres and incubated with antibodies. High median fluorescent intensities (MFIs) were obtained with a monoclonal antibody and positive chicken sera. Chickens were inoculated with 10 strains of avian influenza virus representing different subtypes, including high and low pathogenic H5 and H7 subtypes. Three hundred and fifty-four samples from experimentally infected chickens and controls were tested with a competitive ELISA (cELISA) and the MIA. MFIs were converted to positive/negative (PN) ratios. The results of both tests, as percentage inhibition and PN ratio, showed a high correlation (R2 = 0.77). From the comparison data, a ratio of > or =4.5 was selected as the cut-off value for positivity in the MIA. Using this cut-off value, the sensitivity and specificity of the MIA relative to the cELISA when all discordant experimental samples were retested was 99.3 and 93.1%, respectively. The relative specificity increased to 94.7% when additional negative sera (n = 68) were tested. The MIA may be useful for surveillance testing and as a screening test for flocks infected with low pathogenic avian influenza virus and could be expanded for simultaneous detection of antibodies against other avian infectious disease agents.

Animals↗

PubMed2006

A multiplex DNA suspension microarray for simultaneous detection and differentiation of classical swine fever virus and other pestiviruses.

An oligonucleotide suspension microarray (Luminex microsphere system) was developed for detection and differentiation of animal pestiviruses: classical swine fever virus (CSFV), bovine viral diarrhea virus types 1 and 2 (BVDV1 and BVDV2), and border disease virus (BDV). Species-specific and pestivirus-common oligonucleotide probes were designed to the 5' UTR region and conjugated to individual color-coded Luminex carboxy beads (probe beads). Target pestivirus sequences were amplified by asymmetric PCR using a biotinylated reverse primer and a forward and reverse primer ratio of 1:5. The biotinylated products were hybridized to eight probe beads in a multiplex assay and analyzed using streptavidin conjugated to a fluorescent reporter molecule. The assay was able to detect and differentiate all 40 strains of CSFV, BVDV1, BVDV2 and BDV tested. The analytical sensitivity was determined to be 0.2-10 TCID50/ml. The major advantages of the DNA-microsphere suspension microarray, as a low density array, are its ease of handling and ability to simultaneously detect and type multiple infectious agents.

5' Untranslated Regions↗

PubMed2005

Intersegmental recombination between the haemagglutinin and matrix genes was responsible for the emergence of a highly pathogenic H7N3 avian influenza virus in British Columbia.

In February 2004 a highly pathogenic avian influenza (HPAI) outbreak erupted in British Columbia. Investigations indicated that the responsible HPAI H7N3 virus emerged suddenly from a low pathogenic precursor. Analysis of the haemagglutinin (HA) genes of the low and high pathogenic viruses isolated from the index farm revealed the only difference to be a 21 nt insert at the HA cleavage site of the highly pathogenic avian influenza virus. It was deduced that this insert most probably arose as a result of non-homologous recombination between the HA and matrix genes of the same virus. Over the course of the outbreak, a total of 37 isolates with, and 3 isolates without inserts were characterized. The events described here appear very similar to those which occurred in Chile in 2002 where the virulence shift of another H7N3 virus was attributed to non-homologous recombination between the HA and nucleoprotein genes.

Animals↗

PubMed2005

Antibody responses of pigs to defined Erns fragments after infection with classical swine fever virus.

Antibody responses of pigs to defined Erns fragments, after classical swine fever virus (CSFV) infection, were studied by using an enzyme-linked immunosorbent assay (ELISA). Selection of various E(rns) fragments was based on an immunodominant Erns region encompassing three overlapping antigenic regions, amino acids 65 to 145 (Erns(aa)65-145) (AR1), 84 to 160 (Erns(aa)84-160) (AR2), and 109 to 220 (Erns(aa)109-220) (AR3), identified earlier by our group (M. Lin, E. Trottier, J. Pasick, and M. Sabara, J. Biochem., in press). Defined Erns fragments, including AR1, AR2, AR3, Erns(aa)65-160 (AR12), Erns(aa)84-220 (AR23), Erns(aa)65-220 (AR123), Erns(aa)109-145 (the consensus region defined by the three overlapping regions), and Erns(aa)109-160 (a fragment 15 amino acids larger than the consensus region), were expressed in Escherichia coli, purified by nickel chelate affinity chromatography, and used to measure antibody responses in 20 sera serially collected from pigs experimentally infected with CSFV. Based on the optimum cutoffs determined by receiver operating characteristic analysis after testing 238 negative field sera from Canadian sources, all the Erns fragments were capable of distinguishing positive from negative antibody responses with sensitivities ranging between 75 and 90% and specificities ranging between 83.2 and 100%. Detection of antibody responses to refolded Erns(aa)109-145 and Erns(aa)109-160 by ELISA (this study) but not by Western blots (Lin et al., in press) indicated that the epitopes within the consensus region are conformational. When cutoff values were raised to give a specificity of 100%, four Erns fragments (AR2, AR23, Erns(aa)109-145, and Erns(aa)109-160) offered much higher sensitivities (75 to 90%) than those obtained with other fragments (20 to 65%). Erns(aa)109-145 and Erns(aa)109-160 were capable of detecting antibody responses in infected pigs as early as 7 days postinfection. Demonstration of antibody responses to either one of the four fragments can thus be an alternative to use of the full-length protein in ELISA for serological diagnosis of CSFV infection. An advantage of such a test would be its utilization for serological survey in a classical swine fever-free country (e.g., Canada) in biocontainment level 2 laboratories.

Animals↗

PubMed2004

Application of DIVA vaccines and their companion diagnostic tests to foreign animal disease eradication.

The risk of foreign animal disease introduction continues to exist despite Canada's strict regulations concerning the importation of animals and animal products. Given the rapidity with which these diseases can spread, especially in areas with dense livestock populations, eradication efforts which rely solely on quarantine and stamping-out measures can present a formidable undertaking. This, combined with growing economic and ethical considerations, has led to renewed interest in the use of vaccination as a tool in controlling foreign animal disease outbreaks. Vaccination has effects at the individual and population levels. Efficacious vaccines reduce or prevent clinical signs without necessarily preventing virus replication. They may also increase the dose of virus needed to establish an infection and/or reduce the level and duration of virus shedding following infection. Vaccine effectiveness within a population is a function of its ability to reduce virus transmission. Transmission is best described by the reproductive ratio, R, which is defined as the average number of new infections caused by one infectious individual. By helping to reduce the R-value below 1, vaccination can be an effective adjunct in abbreviating an outbreak. Nevertheless, vaccination can also complicate serological surveillance activities that follow eradication, if the antibody response induced by vaccination is indistinguishable from that which follows infection. This disadvantage can be overcome by the use of DIVA vaccines and their companion diagnostic tests. The term DIVA (differentiating infected from vaccinated individuals) was coined in 1999 by J. T. van Oirschot of the Central Veterinary Institute, in The Netherlands. It is now generally used as an acronym for 'differentiating infected from vaccinated animals'. The term was originally applied to the use of marker vaccines, which are based on deletion mutants of wild-type microbes, in conjunction with a differentiating diagnostic test. The DIVA strategy has been extended to include subunit and killed whole-virus vaccines. This system makes possible the mass vaccination of a susceptible animal population without compromising the serological identification of convalescent individuals. The DIVA approach has been applied successfully to pseudorabies and avian influenza eradication, and has been proposed for use in foot-and-mouth disease and classical swine fever eradication campaigns. This paper will survey current vaccine technology, the host immune response, and companion diagnostic tests that are available for pseudorabies, foot-and-mouth disease, classical swine fever and avian influenza.

Animals↗

PubMed2004

Identification of antigenic regions of the Erns protein for pig antibodies elicited during classical swine fever virus infection.

The structural glycoprotein E(rns) of classical swine fever virus (CSFV) is one of the major antibody targets upon infection of pigs with the virus. Molecular dissection of the structure of E(rns) would define the minimal immunodominant regions that induce antibody responses after infection and may thus help design an effective diagnostic reagent or vaccine. In this study, deletion analysis was made within amino acids (aa) 297 to 776 of the CSFV Alfort/187 polyprotein containing the large C-terminal portion of the E(rns) protein (aa 27 to 227), the entire E1 protein (aa 1 to 195), and the N-terminal portion of the E2 protein (aa 1 to 87). Various protein fragments with target deletions from N- or/and C-terminal ends were constructed with pET30, expressed in Escherichia coli and probed on Western blots with antisera from pigs infected with CSFV. This has resulted in the identification within E(rns) of three overlapping antigenic regions: AR1(E(rns)aa 65-145), AR2 (E(rns)aa 84-160) and AR3 (E(rns)aa 109-220). N- or C-terminal deletions as small as 3 residues introduced into these regions disrupt their reactivity with antibodies, indicating that they are the minimum requirements for recognition by pig antibodies. The three minimal antigenic regions correlated well with the hydropathy profiles and the 3D structural model of E(rns). Each individual region and a protein fragment containing AR1, AR2 and AR3 reacted equally well with pig anti-CSFV sera. Since variable and conserved sequences are present within the three overlapping antigenic regions of E(rns) of different pestiviruses, specific serological detection of CSFV infection or broad detection of pestivirus infections may be achieved with the use of a single E(rns) region or a combination of two or three E(rns) regions.

Amino Acid Sequence↗

PubMed2004

Comparison of reverse transcriptase-polymerase chain reaction, virus isolation, and immunoperoxidase assays for detecting pigs infected with low, moderate, and high virulent strains of classical swine fever virus.

Pigs were experimentally inoculated with Glentorf, Lelystad/97, and Alfort/187: representative low, moderate, and high virulent strains of classical swine fever virus (CSFV). Animals were tested for viremia using virus isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) assays run under routine diagnostic conditions. The virus was detected in the peripheral blood by virus isolation and RT-PCR assays of all Glentorf- and Lelystad/97-infected pigs beginning at 3 days postinoculation (dpi) and in all Alfort/187-infected pigs beginning at 2 dpi. Viremia, as determined by virus isolation, remained detectable in Lelystad/97- and Alfort/187-infected pigs until the last animal within each cohort was euthanized on days 12 and 7 postinoculation, respectively. In contrast, the virus could be isolated from the blood of all Glentorf-infected pigs between 3 and 7 dpi but not from 10 to 21 dpi when the experiment was concluded. Viremia, as determined by RT-PCR, became apparent in Alfort/187-infected pigs at 2 dpi and in Glentorf- and Lelystad/97-infected pigs at 3 dpi. All pigs, regardless of the CSFV strain used, remained RT-PCR positive until they were euthanized. Tonsils were harvested from all the pigs and frozen sections tested for the presence of the CSFV antigen using polyclonal pestivirus and monoclonal CSFV horseradish peroxidase (HRPO) conjugates. Immunostaining reactions were positive for all the Alfort/187- and Lelystad/97-infected pigs. By contrast, tonsils from the Glentorf-infected pigs gave negative to equivocal results. These data suggest that an RT-PCR assay performed on blood may be the best test when dealing with pigs infected with low virulent strains of CSFV.

Animals↗

PubMed2004

Susceptibility of pigs and chickens to SARS coronavirus.

An outbreak of severe acute respiratory syndrome (SARS) in humans, associated with a new coronavirus, was reported in Southeast Asia, Europe, and North America in early 2003. To address speculations that the virus originated in domesticated animals, or that domestic species were susceptible to the virus, we inoculated 6-week-old pigs and chickens intravenously, intranasally, ocularly, and orally with 106 PFU of SARS-associated coronavirus (SARS-CoV). Clinical signs did not develop in any animal, nor were gross pathologic changes evident on postmortem examinations. Attempts at virus isolation were unsuccessful; however, viral RNA was detected by reverse transcriptase-polymerase chain reaction in blood of both species during the first week after inoculation, and in chicken organs at 2 weeks after inoculation. Virus-neutralizing antibodies developed in the pigs. Our results indicate that these animals do not play a role as amplifying hosts for SARS-CoV.

Animals↗