In pig livestock, pathogenic E. coli can contain different combinations of virulence factors such as fimbriae, adhesins, and toxins characterizing many pathotypes and virotypes (this term is used to describe strains characterized by different combinations of toxins and fimbr iae). Verocytotoxic and enterotoxigenic Escherichia coli strains (VTEC and ETEC), major pathotypes involved in piglets’ diseases such as Post Weaning Disease (PWD), enterotoxaemia, and Oedema Disease (OD), are responsible for important economic losses due to mortality, reduced pig performance, and the cost of therapeutical treatments, which includes antimicrobials. Considering the global emergence of antibiotic resistance, alternatives to antibiotics, capable of promoting health status and preventing diseases, are urgently needed. In this scenario, vaccines can impact antibiotic-resistant infections through both a reduction of pathogenic microorganisms specifically target and a prevention of the dysbiosis process. In recent years, oral vaccines have been investigated as an interesting strategy to control E. coli infection at the intestinal level due to the induction of mucosal immune response, where the microorganisms gain access to the body. Plant-based oral vaccines, currently developed also for several human and animal diseases and also in the recent pandemic situation against the SARS-CoV-2, offer a cost-effective, needleless, convenient, safe alternative to vaccine delivery. The general aim of this study was to develop tobacco plants for the seed-specific expression of antigens of major E. coli pathotypes of pigs, as a model of edible vaccines against VTEC and ETEC strains. In particular, the genes codifing for FedF (F18 adhesive fimbriae), FaeG (F4 adhesive fimbriae), and the B subunit of the verocytotoxin-e (Vte2-B), optimized for the expression in tobacco by codon adaptation index, were included in the plant genome of different lines of Nicotiana tobacco cv Xhanti by agroinfection. Tobacco leaf discs were transformed via Agrobacterium tumefaciens (EHA105) with chimeric constructs containing structural parts of the selected genes under control of the beta-conglycinin promoter, isolated from leaves of soybean (Glycine max cv. Richland). Regenerated transformed plants, not showing morphological modifications or retarded germination compared to the wild type, were evaluated by blotting techniques in order to verify the integration of the exogenous genes and the level of expression. Southern blot analyses showed the engineered plants carried more copies of the exogenous genes and that all exogenous genes have stably integrated into the genome of the tobacco plants. The foreign FedF, FaeG, and Vte2-B genes were incorporated in the plant genome and expressed in the seeds in the mid-late maturation. The western blotting carried out on the leaf, culm, and roots were negative. The seed-specific expression guarantees the natural encapsulation of antigens that can be included in the feed for the vaccine delivery to pigs with a reduced risk of gastric degradation. Moreover, the simultaneous inclusion in the animal diet of the seeds from the obtained three transformed lines can be considered as interesting multivalent oral vaccine candidates against the major virulence factor of the main E. coli pathotype in the weaning period. Moreover, the use of tobacco seed for the delivering of antigenic proteins offers advantages related to the reduction of the possibility of accidental entry of pharmaceuticals into the human food chain. In fact, tobacco seeds are not utilized in human nutrition and tobacco seed cake, even if it is included in the catalogue of Feed Materials, is not largely used. Our findings encouraged scientific studies with the goal of evaluating whether the feed administration of the combination of the three lines of tobacco seeds, transformed for the expression of the main virulence factors of E. coli pathotypes is protective against the wild-type E. coli infection in weaned piglets.

Multivalent seed-based oral vaccines against major swine E. coli pathotypes / S. Reggi, M. Dell’Anno, L. Rossi. ((Intervento presentato al 4. convegno ISPMF Conference tenutosi a Online nel 2021.

Multivalent seed-based oral vaccines against major swine E. coli pathotypes

S. Reggi;M. Dell’Anno;L. Rossi
2021

Abstract

In pig livestock, pathogenic E. coli can contain different combinations of virulence factors such as fimbriae, adhesins, and toxins characterizing many pathotypes and virotypes (this term is used to describe strains characterized by different combinations of toxins and fimbr iae). Verocytotoxic and enterotoxigenic Escherichia coli strains (VTEC and ETEC), major pathotypes involved in piglets’ diseases such as Post Weaning Disease (PWD), enterotoxaemia, and Oedema Disease (OD), are responsible for important economic losses due to mortality, reduced pig performance, and the cost of therapeutical treatments, which includes antimicrobials. Considering the global emergence of antibiotic resistance, alternatives to antibiotics, capable of promoting health status and preventing diseases, are urgently needed. In this scenario, vaccines can impact antibiotic-resistant infections through both a reduction of pathogenic microorganisms specifically target and a prevention of the dysbiosis process. In recent years, oral vaccines have been investigated as an interesting strategy to control E. coli infection at the intestinal level due to the induction of mucosal immune response, where the microorganisms gain access to the body. Plant-based oral vaccines, currently developed also for several human and animal diseases and also in the recent pandemic situation against the SARS-CoV-2, offer a cost-effective, needleless, convenient, safe alternative to vaccine delivery. The general aim of this study was to develop tobacco plants for the seed-specific expression of antigens of major E. coli pathotypes of pigs, as a model of edible vaccines against VTEC and ETEC strains. In particular, the genes codifing for FedF (F18 adhesive fimbriae), FaeG (F4 adhesive fimbriae), and the B subunit of the verocytotoxin-e (Vte2-B), optimized for the expression in tobacco by codon adaptation index, were included in the plant genome of different lines of Nicotiana tobacco cv Xhanti by agroinfection. Tobacco leaf discs were transformed via Agrobacterium tumefaciens (EHA105) with chimeric constructs containing structural parts of the selected genes under control of the beta-conglycinin promoter, isolated from leaves of soybean (Glycine max cv. Richland). Regenerated transformed plants, not showing morphological modifications or retarded germination compared to the wild type, were evaluated by blotting techniques in order to verify the integration of the exogenous genes and the level of expression. Southern blot analyses showed the engineered plants carried more copies of the exogenous genes and that all exogenous genes have stably integrated into the genome of the tobacco plants. The foreign FedF, FaeG, and Vte2-B genes were incorporated in the plant genome and expressed in the seeds in the mid-late maturation. The western blotting carried out on the leaf, culm, and roots were negative. The seed-specific expression guarantees the natural encapsulation of antigens that can be included in the feed for the vaccine delivery to pigs with a reduced risk of gastric degradation. Moreover, the simultaneous inclusion in the animal diet of the seeds from the obtained three transformed lines can be considered as interesting multivalent oral vaccine candidates against the major virulence factor of the main E. coli pathotype in the weaning period. Moreover, the use of tobacco seed for the delivering of antigenic proteins offers advantages related to the reduction of the possibility of accidental entry of pharmaceuticals into the human food chain. In fact, tobacco seeds are not utilized in human nutrition and tobacco seed cake, even if it is included in the catalogue of Feed Materials, is not largely used. Our findings encouraged scientific studies with the goal of evaluating whether the feed administration of the combination of the three lines of tobacco seeds, transformed for the expression of the main virulence factors of E. coli pathotypes is protective against the wild-type E. coli infection in weaned piglets.
16-set-2021
Settore AGR/18 - Nutrizione e Alimentazione Animale
Multivalent seed-based oral vaccines against major swine E. coli pathotypes / S. Reggi, M. Dell’Anno, L. Rossi. ((Intervento presentato al 4. convegno ISPMF Conference tenutosi a Online nel 2021.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/910285
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