J Vet Sci.  2013 Sep;14(3):263-270. 10.4142/jvs.2013.14.3.263.

Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

• Rossi L
• Di Giancamillo A
• Reggi S
• Domeneghini C
• Baldi A
• Sala V
• Dell'Orto
• Coddens A
• Cox E
• Fogher C

• KMID: 1705556
• DOI: http://doi.org/10.4142/jvs.2013.14.3.263

Abstract

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.

Keyword

Agrobacterium tumefaciens; edible vaccines; Escherichia coli; pig; verocytotoxins

MeSH Terms

Administration, Oral
Agrobacterium tumefaciens
Animals
Antigens, Bacterial/genetics/metabolism
Bacterial Vaccines/administration & dosage/adverse effects/*pharmacology
Edema Disease of Swine/*immunology/microbiology
Escherichia coli Infections/immunology/microbiology/*veterinary
Escherichia coli Proteins/*genetics/metabolism
Female
Fimbriae Proteins/genetics/metabolism
Genetic Engineering
Intestines/immunology/microbiology/pathology
Mice
Mice, Inbred BALB C
Models, Animal
Plants, Genetically Modified/*genetics/metabolism
Seeds/genetics/metabolism
Shiga Toxin 2/genetics/metabolism
Shiga-Toxigenic Escherichia coli/genetics/immunology/*pathogenicity
Swine
Tobacco/*genetics/metabolism
Virulence Factors/genetics/metabolism
Antigens, Bacterial
Bacterial Vaccines
Escherichia coli Proteins
Fimbriae Proteins
Shiga Toxin 2
Virulence Factors

Figure

• 

  Fig. 1 Chimeric constructs used for Agrobacterium tumefaciens EHA105 transformations. Transgenes carrying the kanamycin resistance gene were inserted under control of the GLOB promoter and NOS terminator. (A) pBIpGLOB-VT2eB was 13800 bp. (B) pBIpGLOB-F18 was 14049 bp.

• 

  Fig. 2 Western blot of VT2-B. Lane 1: positive control represented by 200 ng of VT2e-B expressed through the pET-system in BL21 Escherichia. coli strain, lane 2: wild type (WT), corresponding to non-transformed tobacco seed proteins, lanes 3 and 4: samples.

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  Fig. 3 Northern blot analyses for F18 mRNA detection. Lane WT: total mRNA extracted from wild-type tobacco seeds, lanes 1~4: samples positive for the presence of mRNA corresponding to F18 fimbriae, lane 5: negative sample, lane 6: positive sample.

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  Fig. 4 Agglutination on slides with F18+ polyclonal serum. (A) Total protein extracted from wild-type seeds. (B) Total protein extracted from F18+ seeds.

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  Fig. 5 Duodenum. IgA-producing cells can be seen in the intestinal tunica propria of a CG and TG mouse (A and B, respectively arrows). Scale bars = 50 µm.

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