Document Type

Dissertation - University Access Only

Award Date

2012

Degree Name

Doctor of Philosophy (PhD)

Department / School

Veterinary and Biomedical Sciences

First Advisor

David H. Francis

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains are the most common cause of diarrhea in the developing world and a major cause of severe diarrheal disease in neonatal and weanling animals. The majority of such strains express the fimbrial adhesion K88 (F4), plus heat labile enterotoxin (LT) and heat stable enterotoxin type b (STb ). Previous research supports the proposition that inoculation with heat labile toxin (LT) increases ETEC attachment to and colonization of IPEC-12 cells and piglet intestines respectively, probably by altering host epithelium such that it is more susceptible to colonization. Other studies support that neutralizing immunity stimulated by LT toxin also protects against heat stable toxin (ST)-expressing ETEC and unrelated enteric pathogens. In this study, we test in an experimental pig model whether LT enhances the colonization of bacteria not expressing LT. Gnotobiotic piglets were subjected to laparotomy and inoculated in the jejunum with LT (pretreatment). Six hours post-surgery, piglets were challenge with ETEC strain MUN300 expressing K88 fimbriae adhesion but not LT or ST. We demonstrated that LT pretreatment enhanced the colonization of strain MUN300. We further investigated whether anti-LT antibody protected against LT-enhanced colonization by ETEC strains producing LT and or STb. Three isogenic strains used in this study were WAM23 l 7 (K88+, LT+, STb+), MUN 297 (K88+, LT+, STb), MUN 299 (K88+, LT-, STb-). Active immunization of pigs failed to elicit high anti-LT antibody titers in serum. Therefore, the immunization strategy was revised from active immunization to passive transfer of LT antibodies. We injected our principle group of piglets with LT-specific hyperimmune serum followed by intestinal inoculation with LT, then oral bacterial challenge. We demonstrated that pretreatment with LT increases the binding of strain MUN299 which expresses STb, but not LT. We further showed that treatment with immunization with hyperimmune anti-LT specific serum significantly reduced the bacteria colonization of strain MUN299 , thus establishing a mechanism for the observation that vaccination with LT can reduce colonization by non-LT producing bacteria. It has been demonstrated that oral administration of purified K88 (F4) fimbriae induces a mucosa} immune response in piglets that express receptors for K88 fimbriae. vi To assess whether expression of K88 fimbriae and genetic modified enterotoxin antigens in oral live vaccine strains protect pigs from diarrhea disease caused by wild type ETEC infection, three isogenic strains were constructed using a non-toxigenic K88+ E. coli strain into which were transformed the plasmid vector pBR322 with or without genes for the non-toxigenic LT mutant (LT R192G) or LT Rl92G fused with the gene for STb. Vaccine test animals were thirty-nine naturally farrowed piglets and suckled to their dams for 5 days then weaned to milk replacer. Piglets were orally vaccinated with the living vaccine strains or a placebo at 7 days of age and again at 14 days of age. At 21 days of age, piglets were challenged with wild type virulent ETEC strain 3030-2 (K88/LT/STb). Following challenge, all receptor positive pigs in the placebo control group developed diarrhea and became dehydrated. These pigs also exhibited a significant weight loss due to the diarrhea and subsequent dehydration. None of the piglets inoculated with any of three isogenic strains exhibited any clinical signs of disease following ETEC challenge. There was no mortality among any of the pigs inoculated with any of the three isogenic vaccine strains when piglets were challenged with a wild-type ETEC strain. There was a significantly greater K88-specific antibody response (IgG and IgA) in serum as well as mucosa} lgA in cecal content in all vaccinates subsequent to vaccination. Mucosa} LTspecific lgA titers measured in cecal content are significantly higher in pigs vaccinated with K88+vaccine strain expressing a non-toxic form of LTR192G or LTR192G-STb fusion protein. This study indicates that oral isogenic vaccine strains expressing adhesive fimbriae K88 (F4) and a non-toxic form of LTR192G or LTR192G-STb protein can elicit an appropriate immune response and provide significant protection of pigs of weaning age from challenge by ETEC. Swine influenza virus (SIV) is both a pathogen of economic significance to the swine industry and a potential zoonotic organism that may be transmitted to humans. A vaccine that can effectively prevent or eliminate spread of influenza virus within and between swine herds is urgently needed, as currently vaccines only prevent clinical disease. We describe here for the first time the characterization of an intranasally delivered vaccine consisting of an inactivated SIV HIN I vaccine combined with an enterotoxigenic E. coli (ETEC) subunit vaccine consisting of K88 fimbriae and wild-type heat labile enterotoxin (LT). The LT serves both as an antigen and an adjuvant. Intranasal immunization with the inactivated HIN 1 vaccine elicited a robust virus-specific IgA response and provided protection from detectable infection by a homologous virus challenge as measured by RT-PCR of lung tissue samples and virus culture analyses of nasal swabs. This level of protection was observed in each of the twelve vaccinated piglets intranasally challenged with 1 x 106 TC ID of homologous swine HlN l virus. In contrast, the seventeen unvaccinated controls, either inoculated with LT or PBS then subjected to virus challenge, developed infection of the challenge virus as detected in MDC K cultures of nasal swabs. Further, five out of eight unvaccinated controls, when euthanized at 3 days post-challenge, were positive for detectable virus in their lung tissues by RT-PCR assay. Thus, these data demonstrate that intranasal immunization with an inactivated SIV H 1 N 1 vaccine administered in conjunction with an ETEC subunit vaccine that includes LT can prevent virus infection and virus shedding in pigs exposed to a homologous virus strain. Further, because the vaccine was shown highly protective against ETEC, it was confirmed that the addition of influenza virus antigens to the vaccine did not diminish protection from ETEC infection.

Library of Congress Subject Headings

Escherichia coli infections in swine
Swine influenza
Swine -- Diseases
Vaccines

Publisher

South Dakota State University

Download

Share

COinS
 

Rights Statement

In Copyright