Youssef Elazhary

Vaccination of cattle with a DNA plasmid encoding the bovine viral diarrhoea virus major glycoprotein E2.

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle that is ubiquitously distributed worldwide. In this study, cattle were immunized by intramuscular injections with plasmid DNA expressing the BVDV type 1 major glycoprotein E2. Animals either received injections of naked DNA (N-DNA) or DNA in cationic liposomes (L-DNA). Both DNA preparations induced virus-specific neutralizing antibodies in vaccinates, although the response was much lower in N-DNA-immunized animals. N-DNA-vaccinated animals also showed virus-specific lymphocyte proliferation responses to type 1, live BVDV in vitro, whereas L-DNA vaccination induced no such responses. After 16 weeks, DNA-vaccinated and mock-vaccinated animals were challenged with a USDA-certified BVDV type 1 strain. Four significant observations were made: (1) N-DNA-vaccinated calves showed limited protection from virus challenge, (2) L-DNA-vaccinated animals did not show any signs of protection, (3) the challenge induced strong memory responses in the production of serum neutralizing antibodies to both genotypes (type 1 and 2 of BVDV), and (4) the challenge induced a mucosal memory response in nasal secretions of both L- and N-DNA-vaccinated animals.

The 5'-untranslated region sequence of a potential new genotype of bovine viral diarrhea virus.

SummaryThe 5′ untranslated region (UTR) of several bovine viral diarrhea virus (BVDV) isolates from the severe Quebec outbreak was amplified by polymerase chain reaction (PCR) and sequenced. Sequences revealed the loss, for the BVDV type II isolates, of an internal PstI restriction site, which is present in all known BVDV type 1 5′UTR sequences. A single restriction enzyme digestion (PstI) of an aliquot of PCR product allowed us to differentiate BVDV type I and BVDV type II.

Recent H3N2 swine influenza virus with haemagglutinin and nucleoprotein genes similar to 1975 human strains

Of the four pandemic strains of human influenza A virus observed this century, the 1977 virus strain was very similar in all genes to a 1950 isolate. Since mammalian influenza A viruses change annually by genetic drift, this reappearance could only be attributed at that time to conservation of the virus in a frozen state. We report here the isolation of swine influenza A viruses with haemagglutinin and nucleoprotein genes which are virtually identical to those of the human virus that circulated in 1975. We have also found serological evidence that this virus is circulating extensively in Quebec swine herds. We propose that human-like H3N2 influenza A strains may remain invariant for long periods in swine, which may serve as a reservoir for human pandemics.

Immunological response to recombinant VP8* subunit protein of bovine rotavirus in pregnant cattle

Bovine rotavirus VP8*, N-terminal trypsin cleavage product of VP4, was produced in Escherichia coli. To examine if this antigen could induce neutralizing antibody responses, different species of animals were immunized with the recombinant VP8* protein. The VP8* antigen was found to stimulate a neutralizing immune response in rabbits. When VP8*-immunized mice were exposed to bovine rotavirus strain C486, significantly higher antibody responses were observed than if they were only exposed to C486. To stimulate a current vaccination protocol in the field with livestock, mice were exposed to live C486 virus first and then to VP8*. These mice had the elevated immune responses indicating that VP8* could boost immunity in primed mice. The immune response to VP8* was also tested in pregnant cows. The efficacy of VP8* in stimulating milk antibody was compared with a commercial inactivated vaccine. Differences in colostral antibody titres between VP8*-vaccinated and unvaccinated cows were statistically significant (P < 0.05) and equivalent to the commercial vaccine (P = 0.0569). The milk antibody titres on day 10 were comparable between VP8*- and commercial vaccine-vaccinated animals and were significantly higher (P < 0.05) than in unvaccinated controls. Furthermore, rabbit and bovine antibodies induced by VP8* were able to neutralize different P types of bovine rotaviruses to varying degrees, suggesting that serotype-specific and cross-reactive epitope(s) are present on the VP8* protein of rotavirus. Taken together, E. coli-expressed VP8* may be useful as a subunit vaccine candidate for bovine rotavirus.

Purification of IgG from serum with caprylic acid and ammonium sulphate precipitation is not superior to ammonium sulphate precipitation alone

Immunoglobulin G (IgG) from bovine serum raised against Aeromonas Salmonicida was purified by ammonium sulphate precipitation (ASP) or caprylic acid treatment followed by ammonium sulphate precipitation (CAAS). Purity of IgG samples prepared by both methods were examined by High Performance Gel Permeation Chromatography, electrophoresis and antibody activity assay. Results suggest that IgG prepared by ASP is better than that obtained by CAAS method in terms of the yield of the IgG monomers and the recovery of the antibody activity.

Persistence of a 1930 swine influenza A (H1N1) virus in Quebec.

Two antigenically distinct H1N1 influenza A viruses were isolated during an outbreak of respiratory disease in Quebec swine in 1990/91. Analysis of haemagglutinin and partial nucleoprotein sequences indicated that one was a variant of the swine H1N1 influenza virus circulating in the American Midwest whereas the other was very similar to virus isolated from swine in 1930. The existence of this latter isolate supports the concept that influenza viruses can be maintained for long periods in swine, perhaps in geographically limited pockets. Serological evidence indicates that these distinct strains continued to circulate widely in south-central Quebec until at least 1993.

Investigation of the immunological properties of the bovine viral diarrhea virus protein NS3 expressed by an adenovirus vector in mice

SummaryTwo replication-defective human adenovirus recombinants encoding the NS3 protein (p80) of bovine viral diarrhea virus (BVDV) under the control of a modified adenovirus major later promoter (BM5), rAdBM5/NS3, and human cytomegalovirus promoter (CMV5), rAdCMV5/NS3, were constructed. These two recombinant adenoviruses were tested for their expression of the NS3 protein in vitro in three different cell lines and also in vivo for the induction of BVDV-specific immune responses in mice. The recombinant adenoviruses containing two different promoters induced different levels of humoral responses to the NS3 protein. The rAdBM5/NS3 was used to vaccinate mice in order to evaluate the ability of the NS3 protein in the induction of cellular immune responses. The rAdBM5/NS3 did not cause a stimulation of cell proliferation but caused a very strong increase in production of IFN-γ in murine mononuclear cells stimulated in vivo by BVDV strains of genotype 1 and 2.

Comparison of polymerase chain reaction and monoclonal antibodies for G-typing of group A bovine rotavirus directly from fecal material

A reverse transcriptase and polymerase chain reaction (RT-PCR)-based assay for G-typing of bovine rotaviruses (BRV) was compared with a monoclonal antibody-based immunoassay (MAbs-ELISA) in the characterization of BRV field strains obtained from calves in different regions of Quebec between 1992 and 1994. The strains were analysed for two G types (G6 and G10) which are the most predominate BRV field strains. Fecal samples positive for BRV by polyacrylamide gel electrophoresis (n = 74) were typed by both methods revealing 77% correlation. RT-PCR detected 10 more G6 and 2 more G10 serotypes than MAbs-ELISA. Nine of the 12 discrepant samples could be cultivated and were confirmed as G6 (8) or G10 (1) by both methods. RT-PCR was able to efficiently detect artificial mixes of G6 and G10 and detected two mixed field infections. Four additional infections considered as mixed by MAbs-ELISA and as only G6 by RT-PCR were possibly MAbs-ELISA cross-reactions. RT-PCR provided a very sensitive method for typing BRV field isolates.

Maternal Immunization of Pregnant Cattle with Recombinant VP8* Protein of Bovine Rotavirus Elicits Neutralizing Antibodies to Multiple Serotypes

Neonatal calf diarrhoea caused by bovine rotavirus is one of the most common diseases in cattle. VP8 portion of the rotavirus VP4 protein is known to contain neutralizing epitopes and hemagglutination activity. We expressed the VP8* portion of bovine rotavirus strain C486 (G6P1 serotype) in E. coli, and examined potential of the recombinant VP8* protein for induction of neutralizing antibody responses in host animals. One hundred twenty pregnant beef cows were selected and immunized eight weeks prior to parturition with the recombinant VP8* protein. Colostral and milk samples were collected 12 hours and 10 days post-calving, respectively, and the virus neutralizing titers elicited by the recombinant subunit antigen were determined by plaque reduction assay. Colostral antibody titres of the vaccinated group were significantly higher than those of the unvaccinated control group, and these titers were equivalent to the titers elicited by a commercial vaccine. While titers of the control group rapidly decreased to 220 after 10 days of calving, neutralizing titers in the milk of the vaccinees remained 510. Rabbit and bovine antibodies induced by the recombinant VP8* protein were also able to neutralize bovine rotavirus P5 serotype (B641) at significant level and P11 serotype (B223) moderately. Our results suggest that the E. coli-produced recombinant VP8* protein can be an useful subunit vaccine candidate to prevent rotavirus infection in new-born calves.

Influence of immunomodulatory agents on bovine humoral and cellular immune responses to parenteral inoculation with bovine rotavirus vaccines.

Sodium diethyldithiocarbamate (DTC), mycobacterium cell wall extract (MCWE, Regressin), killed Corynebacterium parvum (C. parvum, Immunoregulin) and muramyldipeptide (MDP) were each combined with purified, live bovine rotavirus and inoculated into 3 month-old Holstein-Friesian calves in order to examine their ability to potentiate specific humoral and cellular immune responses. The vaccinated calves were boosted twice at 3 and 6 weeks after initial vaccine inoculation. The rotavirus was administered intramuscularly either in an aqueous suspension or in a water-in-oil (WIO) emulsion, prepared with incomplete Freunds adjuvant (IFA). DTC and C. parvum were given by the intravenous route, while MCWE and MDP were incorporated directly in the rotavirus suspension. Two groups of calves were also vaccinated either with rotavirus and IFA or with rotavirus emulsified in mineral oil and a mannide oleate compound (MOC, Montanide 888). A control group of calves was given phosphate-buffered saline (PBS) solution emulsified with IFA. The different vaccine preparations were then compared by studying the kinetics of serum rotavirus-neutralizing antibody production and of proliferative response by blood lymphocytes following in vitro stimulation with bovine rotavirus. The results showed that: (1) the bovine rotavirus should be incorporated in a WIO emulsion in order to induce a cell-mediated immune response as detected by the rotavirus-specific in vitro stimulation test with blood lymphocytes, and to produce higher neutralizing antibody titers in the serum; (2) the vaccines prepared with the mineral oil-MOC complex or IFA both induced comparable levels of humoral and cellular immune responses. The use of mineral oil and MOC as adjuvant may be preferred to IFA, because of the facility of preparing the vaccine and of the low viscosity of the resulting WIO emulsion: (3) the addition of MDP to the WIO emulsion prepared with IFA resulted in a higher cell-mediated immune response as determined by the in vitro blood lymphocyte transformation index specific for bovine rotavirus.