Literature Review
Infectious, gastrointestinal diseases are the leading cause of morbidity and mortality in pigs. Most disease is experienced by neonatal pigs or post weaning, nursery age pigs. Edema disease is a condition of weaned pigs that was first reported in Ireland in 1938, but the disease etiology was unknown (Uemura, Sueyoshi et al., 2004). It is now known that toxigenic strains of Escherichia coli bacteria are responsible disease symptoms that characterize porcine edema disease. Virulent E. coli strains are transmitted via the fecal oral route of infection. Once these organisms have been ingested, then they can attach to the small intestine. Colonization is facilitated by attachment fimbriae or pili expressed on the surface of the bacterial cell. Then a variety of toxins cause different disease symptoms.

Escherichia coli bacteria are normal, commensal inhabitants of the mammalian gastrointestinal tract. However, many strains of E. coli express virulence factors that cause gastrointestinal disease, as in the case of edema disease (Moxley, 2000). Some virulence factors only cause disease in certain species. For example, Strains positive for K88 fimbriae cause disease in pigs but not in calves. Serotype O157:H7 causes disease in humans but not calves (Matise, Cornick et al., 2003). There are a variety of different toxin virulence factors: Stabile toxin (STa), Labile Toxin (LT), Shiga-like Toxin I (SLT I), Shiga-like Toxin II (SLT II), Shiga-like and Shiga-like Toxin IIv (SLTIIv) (Bosworth, Samuel et al., 1996). Also, there are a number of different colonizing virulence factors: intimin, K88, K99, F41, 987P, F4 and F18 fimbriae (Bosworth, Samuel et al., 1996 and Imbrechts, Deprez et al., 1997). Different combinations of attachment and toxin virulence factors create different disease syndromes in different species and ages of animals. Escherichia coli strains require a combination of toxin and attachment virulence factors to become pathogenic. The general E. coli pathogenesis sequence of events requires bacterial attachment to the intestinal wall in conjunction with the production of toxin (Bosworth, Samuel et al. 1996). Many pathogenic strains carry more than one of each type of virulence factors (Matise, Cornick et al., 2003).

Neonatal animals are the most susceptible population to disease caused by pathogenic E. coli strains. For example, neonatal calves born to naïve dams are very susceptible to K99 diarrheal disease. These calves can exhibit symptoms within hours of birth and die of dehydration within 24 hours. However, calves living past 7 days are no longer susceptible to infection. Children and the elderly encompass the human population most susceptible to O157:H7 induced disease (Matise, Cornick et al., 2003). Combinations of certain E. coli virulence factors each reside in a unique disease causing niche. Like other disease strains, edema disease producing E. coli isolates also carry a signature set of virulence factors (Smeds, Pertovaara et al., 2003). These bacteria produce a variant type of Shiga-toxin 2 (Stx2) called Stx2e, also known as vero-toxin. Edema disease causing strains are referred to as vero-toxigenic E. coli or VTEC. The attachment fimbriae can be one of two types; F4 or F18 (Imbrechts, Cornick et al., 1997). Isolates expressing the F4 pilus have a tendency to induce disease in pigs about 1 week after weaning while the F18 strains cause disease in pigs that are about 2 weeks past weaning (Verdonck, Cox et al., 2002). The F18 pilus is further characterized by the differentiation of F18ac vs. F18ab fimbriae. Escherichia coli strains expressing F18ab fimbriae and Stx2e toxin are capable of inducing edema disease in post weaning pigs (Moxley, 2003). These strains also carry genes that express LT and STa of which are responsible for inducing diarrheal symptoms associated with the edema disease syndrome (Verdonck, Cox et al., 2002).

Vero-toxin damages smooth muscle cells and vascular endothelial cells. Nervous tissue blood vessels and intestinal blood vessels are particularly sensitive to damage (Bosworth, Samuel et al., 1996). The clinical symptoms as a result of blood vessel necrosis in the brain are central nervous system disorders (ataxia and lateral recumbency) (Verdonck, Cox et al., 2002). Necrotic blood vessels become "leaky" causing cerebral swelling, hence the term "edema disease" (Bosworth, Samuel et al., 1996). These symptoms can become apparent in as few as 7 hours after ingestion of the pathogenic bacteria. In severe cases, death can occur within 24 hours (Uemura, Sueyoshi et al., 2003). Diarrhea is also a clinical sign of disease. Tissue pathology can exhibit gross lesions in the brain from clinical specimens. Post mortem tissue samples from sub-clinical specimens may exhibit only microangiopathy of brain capillaries upon histological evaluations. Histological observations of intestinal tissue may show colonized bacteria or mesenteric blood vessel necrosis (Bosworth, Samuel et al., 1996).

Severity of disease symptoms is multi-factorial. The amount of toxin present in the feces enables some prediction of disease outcome. Infected pigs having high amounts of vero-toxin present in the feces are likely to succumb to severe neurological damage and death. However, the outcome of disease in pigs with low amounts of toxin present is not as predictable, as some of these pigs will also die (Matise, Cornick et al., 2003). This suggests that there are variables affecting intestinal permeability which could affect toxemia burden. It has been shown that porcine red blood cells have a receptor for this particular toxin molecule. Red blood cells could be the vehicle by which toxin is delivered to sensitive epithelial cells. Pigs could be more, or less, genetically susceptible to disease in accordance with RBC receptor expression (Matise, Cornick et al., 2003). Also, some pigs do not express the receptors necessary for attachment. Diet also plays a role in disease susceptibility. Pigs that don’t get enough dietary roughage are much more susceptible to disease. Some producers have eliminated herd disease by changing the daily ration formulation (Bosworth, Samuel et al., 1996).

Edema disease-causing strains of E. coli have found a unique way of evading the immune system. The evasion tactic is founded on timing rather than some other biochemical mechanism. There is no cross placental transfer of antibodies in pigs. Neonates are born without any circulating protective antibodies. Instead, protective antibodies are passively transferred to the newborn piglet via the colostrum. Upon ingestion, antibodies are absorbed across the intestinal wall into the neonatal blood stream (Makino, Watarai et al., 2001). The absorptive capacity of the neonate gut is most pronounced during the first 24 hours of life and diminishes significantly thereafter. Also, as time passes, the concentration of colostral antibodies decreases as the colostrum turns to milk. This is a very important process in animals as evidenced by the difficulty in keeping colostrum deprived pigs alive. After weaning, pigs no longer have access to the immunological benefits of milk (Makino, Watarai et al., 2001). Nor have the pigs’ immune systems been challenged by excess amounts of toxin antigens because the colostrum and milk has kept pathogenic organisms like VTEC strains of E. coli in check. Therefore, the weaned pigs have not developed any active immunity. The time period between colostrum/milk consumption and development of active immune responses provides a window of opportunity for edema disease to strike.

Disease susceptibility is also a function of challenge load. If pigs are raised in an environment where edema disease strains of E. coli are not endemic, then disease will not be prevalent. Nursing pigs that are never exposed to a challenge load will never actively develop any immunity to these organisms. Therefore, when naïve weaned pigs are commingled with carrier pigs, then the naïve pigs can become infected. Challenge load also plays a role in the quality of passive protection provided by the sow. If there is no challenge load present, then it is unlikely that the sow will develop a Stx2e specific antitoxin titer. Therefore, pigs will not be passively immunized with an antitoxic specific antibody titer.

The process of weaning can provide an immunological niche that can be filled with edema disease strains of E. coli. Some animals like nursing range calves regularly forage on whatever they can find in the pasture for an extended period of time (6 months) before weaning. Pigs, on the other hand, are abruptly weaned at about 19 days of age. While nursing pigs are provided a source of creep feed to nibble on ad libitum, the primary food source for nursing pigs is maternal milk. When the pigs are weaned, then they must survive on the food that is offered to them. This abrupt dietary change affects the normal flora of the gut allowing the opportunistic colonization of pathogens like E. coli. The weaning process also induces a lot of stress (Makino, Watarai et al., 2001). Stress is known to have an effect on immuno-competence leaving animals susceptible to disease. Edema disease strains of E. coli take advantage of this immunological window of opportunity.

Vaccine Description
Indications: This product induces mucosal immunity against Stx2e toxin, and F4 and F18 fimbrial antigens in post weaning pigs providing protection against porcine edema disease induced by vero-toxigenic strains of E. coli. Vaccine immunogens consist of inactivated Stx2e (toxoid) antigen, and crudely purified F4 and F18 fimbrial antigens. Adjuvant consists of the B unit of Escherichia coli Labile Toxin.
Directions and Dosage: Administer 1, 2 ml (one ml per nare) intranasal dose to nursing piglets one week prior to weaning.
Precautions: Pigs may experience anaphylaxis within 30 minutes following administration. Treat symptoms with inject able epinephrine.

There are multiple commercial vaccines available for the prevention of colibacillosis disease in pigs. Traditional colibacillosis in newborn pigs is caused by infection with strains of enterotoxigenic Escherichia coli (ETEC) strains. The colibacillosis infectious process is similar to that of edema disease. Bacteria are transmitted via the fecal, oral route of infection. Once ingested, ETEC strain fimbriae like K99, K88 or 987P facilitate attachment of the bacteria to the intestinal wall. Then Stabile (STa) and Labile (LT) toxins produced by the E. coli strains induce diarrhea resulting in severe dehydration and death (Prager, Bauerfeind et al., 2004). Commercial vaccines that protect against this disease target an immune response to fimbrial antigens. Pregnant sows are vaccinated with fimbrial antigen well in advance of farrowing. Antibodies developed in the sow are then passively transferred to the newborn pig through the colostrum. This is an appropriate and effective immunization strategy for ETEC diseases because they strike within the first hours of life. Colostral delivery of fimbrial specific antibodies to the site of infection (intestine) neutralizes the binding capacity of these bacteria, thereby preventing disease. This vaccine design is partially applicable to the described edema disease vaccine design. Like ETEC disease, VTEC strains of E. coli must attach to the intestinal wall to cause symptoms. Therefore, immunity generated against edema disease fimbrial antigens will prevent colonization and subsequent disease. Passive protection against edema disease has also been demonstrated through the administration of anti-fimbrial antibodies produced in chicken eggs (Imbrechts, Deprez et al., 1997). That is why this vaccine is formulated to contain fimbrial antigen.

Colonization of the intestinal wall in and of itself is not necessarily damaging. The intestinal lining is normally colonized by billions of bacteria. Fimbrial expression on VTEC strains is a virulence factor only because it provides a colonization mechanism. The damaging effects of infection are due to the actions of Stx2e. Therefore, it is necessary that the vaccine contain Stx2e antigen to elicit an anti-toxin specific response.

The timing of edema disease onset had to be taken into consideration for proper vaccine design. Currently available ETEC vaccines protect against a disease that occurs in new- born pigs. Edema disease occurs in pigs that have been weaned, and are 4-5 weeks old. Therefore, pigs are no longer getting any passive, maternal protection. Loss of maternal protection comes multi-factorially. First, even nursing pigs are not getting any passive antibody protection just prior to weaning because the gut is no longer open to maternal antibody absorption, as previously discussed, and weaned pigs get no milk at all. Second, milk may provide some non-antibody mediated antitoxic activity of which weaned pigs will no longer have. Third, passively transferred antibodies diminish over time in the piglet. Sows immunized with Stx2e could passively transfer Stx2e specific antibodies to the newborn. But, the piglet antibody titer will be reduced by the time edema disease susceptibility is approached. Passive immunization with fimbrial antigens is inappropriate in the case of edema disease. This is so because edema disease strains do not colonize until after weaning. Even if sows were adequately immunized with F4 and F18 antigens, the resulting antibodies could not be delivered to the site of colonization in a non-nursing pig. Therefore, active immunization is required at the right time. Vaccine administration 1 week prior to weaning is necessary to account for the following variables. Newborn pigs are not immuno-competent and would not respond very well to vaccination. Therefore, pigs should not be vaccinated until they can age as much as possible. Contrarily, vaccination on the day of weaning would not allow adequate time for a maximum immune response because disease can strike as early as one week post weaning, and a good antibody response requires at least 10 days. For these reasons, the vaccine is to be administered 1 week prior to weaning.

The intranasal administration of the vaccine is a key element to the elicitation of a protective immune response. Studies have been done to investigate the efficacy of parenteral (injectable) routes of administration in pigs. However, Stx2e and fimbrial antigens presented to the immune system this way were proven un-efficacious against a virulent challenge (Bianchi, Scholten et al. 1996 and Verdonck, Cox et al., 2002). Foreign antigen in muscle tissue is processed through the regional, draining lymph nodes. This type of antigen processing drives the production IgM but primarily an IgG antibody response. It seems reasonable that a circulating IgG antibody titer specific to Stx2e would neutralize the toxin and protect pigs from edema disease symptoms. However, researchers have shown that pigs vaccinated with Stx2e toxoid responded with an IgG response but were not protected from challenge (Bianchi, Scholten et al. 1996 and Verdonck, Cox et al., 2002). Perhaps this data supports why pigs are not effectively immunized passively. There is evidence to suggest that mucosal immunity is protective as convalescent pigs are immune to edema disease recurrence (Verdonck, Cox et al., 2002). Pigs that become immune to disease recurrence would have developed mucosal immunity rather than parenteral immunity. This evidence suggests that mucosal presentation of the vaccine should be protective (Verdonck, Cox et al., 2002).

Pathogenic antigens processed by the mucosal immune system drain to mucosal associated lymph nodes. These lymph nodes drive an IgA antibody response rather than an IgG response. Anti-fimbrial IgA secreted into the intestinal lumen can then work against VTEC strain colonization. The mucosal immune system encompasses all epithelial surfaces of the body that secrete mucous (upper respiratory system, GI tract and vaginal surfaces). Immune responses are not localized. For example, antigen processed by a draining lymph node in the leg will disseminate immunity throughout the body’s tissues. Likewise, antigen processed by mucosally associated lymph nodes in the lower GI tract will ultimately cause IgA secretion throughout the mucosal immune system. Therefore, intranasal vaccine administration is theoretically appropriate to induce a GI tract immune response. Furthermore, intranasal administered antigen will be swallowed and presented to the site of pending infection any how. For these reasons the intranasal route of vaccine administration was selected.

Most inactivated vaccines require a two dose regime to elicit protective immunity. However, this vaccine was designed as a one dose product for multiple reasons. The previously described time window only allows for single dosing. Vaccine administration prior to 2 weeks of age (1 week before weaning) would be fruitless because pigs would not respond very well due to immuno-incompetence. Disease strikes as soon as 1 week after weaning and as late as 2 weeks after weaning. Good antibody responses require 10-14 days. So, a second dose would be redundant if given only 7 days after weaning and second dosing 2 or 3 weeks after initial dosing would be past the window of disease onset. Pigs that make it past the 2 week susceptibility window usually never experience disease.

The economics of pig production were also taken into consideration upon vaccine design. Pig producers today have integrated tracking systems into their operations today helping them to identify very subtle costs. For example, the cost/benefit ratio of clipping needle teeth on baby pigs has been evaluated. Piglet needle teeth are very sharp, causing damage to the lactating sow which could affect piglet weight gain. Also, litter mates have a habit of tail biting and fighting. Needle teeth can cause cuts leading to infection. Then pigs require additional treatment. To avoid these problems, needle teeth are removed sometime shortly after birth. It takes approximately 4 seconds to remove the needle teeth from one pig. Every day, large pig operations have thousands of pigs requiring tooth removal. This costs producers labor (money), thus exemplifying the desire to limit time spent on piglet processing. Nursing pigs require processing at approximately one week prior to weaning for other reasons. Therefore, the vaccination protocol was designed to incorporate administration of product while pigs are being processed for other reasons thereby minimizing labor costs.

Vaccine antigens are prepared from live E. coli cultures in broth medium. Following peak growth, the bacterial cells are heated to 60 C. Heavy agitation will release fimbrial antigens into the culture supernatant. Also, as the culture grows, Stx2e is released into the culture supernatant. After releasing fimbrial antigens, then the cells are centrifuged out leaving behind soluble fimbrial and toxin antigen. This processing can yield a significant vaccine safety problem. Escherichia coli is a gram negative bacterium. The cell wall of gram negative bacteria is partly composed of lipo-polysacharride (LPS). When exposed to the immune system, LPS induces a potentially lethal immune response. LPS causes release of the cytokine TNF-a. TNF-a triggers inflammatory responses like vasodilation and increased vascular permeablization. If tissue is infected with gram negative bacteria, then the LPS/TNF-a activated inflammatory response is beneficial toward containing the infection and recruiting other effector cells and molecules to the site of infection. However, if LPS is disseminated throughout the body, then TNF-a is released systemically. This causes not local, but a systemic inflammatory cascade resulting in shock, organ failure and death. LPS that is contained in living gram negative cells does not cause this to happen. However, when infectious gram negative organisms die, then LPS is released and can induce the inflammatory response. Raw culture fluids containing soluble fimbrial and toxin antigens will also contain soluble LPS meaning that LPS will end up in final vaccine product. Vaccinated pigs could experience a fatal, systemic, TNF-a induced inflammatory response. Epinephrine is the antidote that interrupts the inflammatory response cascade preventing LPS induced death. Since E. coli is a gram negative bacterium, it was deemed necessary to include a precautionary statement.

An adjuvant is a compound that enhances the immune response to an antigen. There are two main categories of vaccine adjuvants; depot effect adjuvants and effector adjuvants. Depot effect adjuvants are compounds that increase the immune response through the slow, sustained presentation of antigen to the immune system over time. Different depot effect adjuvants include; oil emulsions, aluminum hydroxide and alum. Effector adjuvants stimulate an enhanced immune response via some biochemical pathway. This vaccine is formulated to contain the B unit of E. coli Labile Toxin (LT) as an effector adjuvant. The mucosal immune system does not normally respond very well to soluble antigens such as E. coli fimbrial and toxin antigens (Apostolaki and Williams, 2004). However, researchers have shown that E. coli LT can boost immune mucosal immune responses to such antigens (Apostolaki and Williams, 2004). More importantly, mammals can develop immune tolerance to orally administered, soluble antigens thereby preventing any immune response. LT may also work to prevent antigen immune tolerance (Bagley, Abdelwahab et al., 2003). As previously described, LT is a toxin that is produced by toxigenic strains of E. coli, including edema disease strains. This toxin molecule has been shown to have adjuvant potential similar to that of Cholera toxin (Bagley, Abdelwahab et al., 2003). This phenomenon has been investigated numerous times. While the LT toxin molecule has adjuvant potential, it is also toxic. It was included as a human flu vaccine adjuvant, but the toxin caused facial paralysis in some of the vaccine recipients. Therefore, the product was discontinued. Since that time, it has been discovered that removal of the A subunit of the molecule renders it non-toxic while the B subunit keeps its adjuvant potential. One group of scientists evaluated the LT B subunit as a mucosal adjuvant in mice. As compared to mice immunized with ovalbumin alone, ovalbumin/LT B vaccinated mice exhibited higher IgG antibody responses and higher rates of ovalbumin specific T cell proliferation (Apostolaki and Williams, 2004). This data supports intranasal administration to pigs.

Vaccine Trial
Summary
A total of 14 bred gilts will be procured for the study. Gilts will have no pre-existing, blood serum antibody titers to F4, F18 or Stx2e antigens. Animals will be randomly numbered and assigned to test groups. The 14 animals will be assigned to one of 4 different test groups; non-vaccinated positive control group, non-vaccinated negative control group, high dose group, and low dose group. After farrowing, piglets will be allowed to nurse on the sow ad libitum. Weaning will be scheduled on day 21 after birth. Vaccine will be administered to respective test group animals on day 14 after birth. Pre-vaccination and post-vaccination blood samples will be drawn from the piglets. Blood samples will also be taken from the sow at these times. All piglets will be challenged intra-gastrically with live, vero-toxigenic E. coli (Stx2e positive and F18 positive) on day 35 after birth (except negative control group). Piglets will be observed daily for clinical signs of disease following challenge. Any pig that dies during the 7 day observation period will be evaluated for cause of death. All surviving piglets will be euthanized on day 42 after birth and evaluated for edema disease pathology. For a valid study, non-vaccinated controls must experience a significant rate of disease as compared to the negative control group as evidenced by an increased rate of clinical disease, pathology or disease induced mortality.

Vaccine
The vaccine will be a combination of VTEC culture supernatants. One culture will be grown to contain F18 fimbriae. Another will contain F4 fimbriae. A third culture will contain Stx2e and the last culture will contain LT B. ELISA plates coated with antigen specific monoclonal antibodies to F18, F4, Stx2e and LT, respectively, will be used to quantify these antigens. Vaccine will be formulated to contain 10 ELISA units of antigen per dose in the high dose vaccine and 1 ELISA unit of antigen per dose in the low dose vaccine.

It is important to titrate the antigen input per dose for two reasons. First, it is economically unproductive to formulate vaccine with too much antigen. Second, lower dose vaccines will also have less LPS, making a safer product.

Test Groups
Gilts (first time mothers) are used as the subject population source for multiple reasons. Piglets from gilts are most likely to be the most susceptible group. Gilts don’t always produce good colostrum, therefore, piglets may not receive maximum, good health inducing colostral attributes. Gilts are not as likely as sows to have developed natural VTEC exposure immunity. Therefore, piglets will not carry non-detectable VTEC specific antibodies of which could compromise the study. If the vaccine will protect litters from gilts, then it will most certainly protect piglets from the more immuno-competent sow population.

Non-vaccinated positive control group: this group consists of 3 litters of piglets. None of these piglets will receive any vaccination. All of these piglets will be challenged with live, virulent VTEC bacteria.
Non-vaccinated negative control group: This group consists of 3 litters of piglets. None of these piglets will receive any vaccination. None of these piglets will be challenged.

High dose vaccine group: This group consists of 5 litters of piglets. These piglets will receive one, 2 ml intranasal dose (1ml per nare) of high dose vaccine on day 14 after birth. All of these piglets will then be challenged with live, VTEC bacteria.

Low dose vaccine group: This group consists of 3 litters of piglets. These piglets will receive one, 2 ml intranasal dose (1ml per nare) of low dose vaccine on day 14 after birth. All of these piglets will then be challenged with live, VTEC bacteria.

Vaccination
Piglets designated to receive vaccine will receive one, 2 ml dose (1ml per nare) of vaccine on day 14 after birth.

Blood Samples
Blood samples will be taken from an inventory of bred gilts prior to purchase. Only negative dams will be selected for the study.

Gilts will also be bled on the day of piglet vaccination (14 days post farrowing) and on the day of piglet challenge.

Piglets will be bled on the day of, but just prior to vaccination (day 14 after birth).

Non-vaccinated piglets will be bled on day 14 after birth.

All piglets will be bled on day 35 after birth just prior to challenge.

All piglets will be bled on day 42 after birth (day of euthanizaiton).

Blood serum will be harvested and stored for future antibody testing. Testing of blood serum will enable detection of naïve bred gilts and sero-conversion to vaccine and/or challenge fimbrial and toxin antigens in piglets.

Challenge
All piglets designated to be challenged will receive 1x1011 cfu/ml of live VTEC bacteria.
Live bacteria will be administered intra-gastrically.

Observations
Following challenge, piglets will be observed for the following clinical signs of disease; diarrhea and ataxia.

Piglets that die will be necropsied to determine cause of death.

Post mortem observations will include: gross pathology (cerebral lesions) and histology (microscopic cerebral and mesenteric lesions and intestinal, bacterial colonization).

Euthanization
All piglets will be euthanized and evaluated post mortem for signs of disease on day 42 (7 days post challenge) after birth.

ELISA Testing
ELISA tests will be used to test both vaccine antigen and blood serum antibodies. Dimeric IgA is secreted into the intestinal lumen. However, monomeric IgA does get into the circulating blood stream.
To test vaccine antigen, 96 well ELISA plates are coated with mono-clonal antibodies specific to either F4, F18, Stx2e or LT antigens, respectively. Then antigen to be tested is 2-fold, serially diluted across the plate. Following incubation for 1 hour at 37 degrees C, the plates are washed and a standard dilution of polyclonal rabbit (serum from rabbits vaccinated with the respective antigen) serum that has been tagged with horse radish peroxidase conjugate is added. Following incubation for 1 hour at 37 degrees C, substrate is added and relative antigen content is calculated base on color change (optical density).
The following procedure is used to test for IgG and IgA antibodies specific for F4, F18 and Stx2e antigens in pig serum. Ninety-six well ELISA plates are coated with mono-clonal antibodies specific to either F4, F18, or Stx2e antigens, respectively. Following incubation and washing, a standard dilution of antigen is added to each well of the plate. Then following incubation and washing, pig serum to be tested is added to the plate and 2-fold serially diluted across the wells. Following incubation and washing, a conjugated goat-anti pig specific to either IgG or IgA is added to the plate. Following incubation and washing, substrate is added and relative specific antibody content is calculated based on color change (optical density).

When testing pigs for antigen specific antibodies, it is important to test for both IgG and IgA. The vaccine is intended to elicit a mucosal immune response. Therefore, if only IgG is present, then mucosal immunity may not have been achieved. If post challenge, non-vaccinated control pigs produce IgA but post vaccinated, pre-challenge test pigs do not, then it might be concluded that the vaccine does not induce mucosal immunity.

Results
The following criteria will be used as indicators of mucosally induced vaccine efficacy: positive control pigs must experience a significant rate of disease as evidenced by mortality, clinical and post mortem signs of disease as compared to the negative control group. Vaccinated pigs must experience a statistically significant reduced rate of disease symptoms as compared to the positive control group. Also, induction of an IgA immune response must be measured.

Problems
The vaccine is intended to yield a mucosal immune response. This study does not include any variables that would evaluate whether or not the vaccine antigen and adjuvant must make it past the stomach and into the gut to be protective. As previously described, immunity generated in the nasal mucosal immune system should provide GI tract immunity as well. However, maybe the nasal mucous membranes cannot process enough antigen to elicit a strong enough response. If so, then antigen must make to the small intestine for processing. That may cause a problem because antigen and adjuvant may be diminished by the harsh stomach environment. This could contribute to an vaccine failure. To correct this, the antigen input level per dose could be increased. Or, the antigen/adjuvant complex could be delivered in time released capsule format.

Having the vaccine as a one-dose product could cause vaccine failure. Most vaccines, particularly killed vaccines, are two dose products. The immune system responds best to active infection where invading organisms actively reproduce and provide sustained presentation to the immune system. Two-dose vaccination regimes compensate for this by first priming the immune system and then boosting it. However, as previously described, a two dose regime does not fit this protection protocol very well. It is attempted here to compensate for the one-dose deficiency by maximizing antigen input in conjunction with the LT adjuvant.

References
Aitken R and Hirst TR. Recombinant Enterotoxins as Vaccines Against Escherichia coli-mediated Diarrhoea. Vaccine 1993;11(2):227-233.

Apostolaki M and Williams NA. Nasal Delivery of Antigen With the B Subunit of Escherichia coli Heat-Labile Enterotoxin Augments Antigen-Specific T-Cell Clonal Expansion and Differentiation. Infection and Immunity 2004;72(7):4072-4080.

Bagley KC, Abdelwahab SF, Tuskan RG and Lewis GK. An Enzymatically Active A Domain is Required for Cholera-Like Enterotoxins To Induce a Long-Lived Blockade on the Induction of Oral Tolerance: New Method for Screening Mucosal Adjuvants. Infection and Immunity 2003;71(12):6850-6856.

Bertschinger HU, Nief V and Tschape H. Active Oral Immunization of Suckling Piglets to Prevent Colonization After Weaning by Enterotoxigenic Escherichia coli with Fimbriae F18. Infection and Immunity 2000;71:255-267.

Bianchi ATJ, Scholten J W, van Zijderveld AM, van Zijderveld FG, Bokhout BA. Parental vaccination of mice and piglets with F4+ Escherichia coli Suppress the enteric anti-F4 response upon oral infection. Vaccine 1996;14:199-206.

Bosworth BT, Samuel JE, Moon HW, O’Brien AD, Gordon VM and Whipp SC. Vaccination with Genetically Modified Shiga-Like Toxin IIe Prevents Edema Disease in Swine. Infection and Immunity 1996;64(1):55-60.

Imbrechts H, Deprez P, Van Driessche E and Pohl P. Chicken Egg Yolk Antibodies against F18ab Fimbriae on Escherichia coli Inhibit Shedding of F18 Positive E. coli by Experimentally Infected Pigs. Veterinary Microbiology 1997;54:329-341.

Makino S I, Watarai M, Tabuchi H, Shirahata T, Furoka H, Kobayashi Y and Takeda Y. Genetically Modified Shiga Toxin 2e (Stx2e) Producing Escherichia coli is a Vaccine Candidate for Porcine Edema Disease. Microbial Pathogenesis 2001;31:1-8.

Matise I, Cornick NA, Samuel JE and Moon HW. Binding of Shiga Toxin 2e to Porcine Erythrocytes In Vivo and In Vitro. Infection and Immunity 2003;71(9):5194-5201.

Moxley RA. Edema Disease. Vet Clin North Am Food Anim Pract. 2000;16(1):175-185.

Prager R, Bauerfeind R, Tietze E, Behrend J, Fruth A and Tschape H. Prevalence and Deletion Types of the Pathogenicity Island ETT2 Among Escherichia coli Strains From Oedema Disease and Colibacillosis in Pigs. Veterinary Microbiology 2004;99:287-294.

Smeds A, Pertovaara M, Timonen T, Pohjanvirta T, Pelkonen S and Palva A. Mapping the Binding Domain of the F18 Fimbrial Adhesin. Infection and Immunity 2003;71(4):2163-2172.

Uemura R, Sueyoshi M, Taura Y and Nagatomo H. Effect of Antimicrobial Agents on the Production and Release of Shiga Toxin by Enteroxaemic Escherichia coli Isolates from Pigs. J. Vet. Med. Sci. 2004;66(8):899-903.

Uemura R, Sueyoshi M, Nagayoshi M, and Nagatomo H. Antimicrobial Susceptibilities of Shiga Toxin-Producing Escherichia coli Isolates From Pigs With Edema Disease in Japan. Microbiol. Immunol. 2003;47(1):57-61.

Verdonck F, Cox E, van Gog K, Vander Stede Y, Duchateau L, Deprez P, Goddeeris BM. Different kinetic of antibody responses following infection of newly weaned pigs with an F4 enterotoxigenic Escherichia coli strain of an F18 vertoxigenic Escherichia coli strain. Vaccine 2002;20:2995-3004.

Van den Broeck W, Cox E, Goddeeris BM. Receptor-Dependent Immune Responses in Pigs after Oral Immunization with F4 Fimbriae. Infection and Immunity 1999;67(2):520-526.

Informed Consent

Enclosed you will find experimental vaccine for administration to nursing piglets for the purpose of evaluating adverse reactions. Vaccine efficacy was tested and approved satisfactorily by the USDA. This trial is to evaluate only the safety of the vaccine.

The product contains inactivated, subunit components derived from live Escherichia coli organisms. The vaccine has passed the following quality control tests as prescribed by The Federal Code of Regulations, Volume 9 (9CFR): sterility testing, safety testing in laboratory animals and in-vitro potency testing (by ELISA antigen quantitation).

Anaphylactic shock is the only known possible side effect that could accompany administration of this product. Symptoms of anaphylaxis include malaise, heavy breathing, vomiting, increased heart rate, appearance of choking, laying on side and paddling or death. These kinds of reactions occur within the first 30 minutes following vaccination. Treat symptoms immediately with injectable epinephrine as per label recommendations. Do not administer the vaccine if there epinephrine is not available.
It is not known what other side effects this product could cause. Observe animals for adverse reactions according to the following schedule: every 15 minutes for the first 3 hours, twice daily for 1 week, and once weekly for one month thereafter. Record any signs of adverse reactions.

The product is to be administered by a licensed veterinarian. Observations are to be recorded by the same veterinarian. Data should be summarized and reported by the same, attending veterinarian. The number of doses administered should be documented.

Any loss of piglet productivity should be accounted for and validated by the attending veterinarian. Monetary compensation will be provided according to current market price of market weight animals.

Administer product according to the following recommendations:

Shake well before using. Administer one, 2ml (one ml per nare) intranasal dose to nursing piglets one week (7 days) prior to the scheduled weaning date.

Use entire contents upon opening, or return the used bottle. Do not store opened bottles. Send back any remaining inventory.

Call 1-800-ddekker in case of emergency.

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