MSU LIBRARIES “ RETURNING MATERIALS: PIace in book drop to remove this checkout from your record. FINES wiII be charged if book is returned after the date stamped below. THE EFFECT OF THE ORAL ADMINISTRATION OF 987P MONOCLONAL ANTIBODY ON GNOTOBIOTIC PIGS CHALLENGED WITH 987P-POSITIVE ESQHEBIQELA COLI BY Sheila D. Grimes A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1988 ABSTRACT THE EFFECT OF THE ORAL ADMINISTRATION OF 987P MONOCLONAL ANTIBODY ON GNOTOBIOTIC PIGS CHALLENGED WITH 987P'POSITIVE W13 QQLI. BY Sheila D. Grimes This study determined the effect of 987P monoclonal antibody (MCA) on the in yitrg adhesion of 987P-positive Egghgrighig col; (E; ggli) to pig intestinal brush borders and the effect of direct passive oral immunization with 987P HCA on clinical signs, lesions, in 2129 adhesion and enterotoxin production in gnotobiotic pigs challenged with 987P-positive E; 9911. Using a brush border adhesion test, ileal samples from 29 pigs were used to determine if in yitrg adhesion of 987P-positive E; 9911 to brush borders could be prevented by 987P MCA. Brush borders and bacteria were mixed with several concentrations of 987P MCA, titer of 10,000, for a total of 8 treatments per sample. Adhesion was present in all 29 samples tested in the absence of MCA. A statistically significant inhibition of adhesion Of 987P-positive L go}; to brush border fragments by 987P MCA was present with all treatments. Sheila D. Grimes Neonatal gnotobiotic pigs from 8 litters were given 0.5 to 12 ml of 987P MCA, challenged with 987P-positive E; 9911 0.5 to 2 hours later and observed clinically for signs of enteric colibacillosis. Clinical signs, bacterial counts, and gross, histologic and scanning electron microscopic lesions were similar in control and treated animals. All, except 2 animals, had diarrhea, and gross lesions were minimal. Adhesion and colonization based on ‘microbiologic, histologic and scanning electron microscopic examinations were limited in both control and treated animals, making it difficult to assess the efficacy of the MCA. Enterotoxin production was determined in 2 of the 8 litters of gnotobiotic pigs. Pig ileal mucosa cyclic guanosine monophosphate (cGMP) content was determined using a cGMP radioimmunoassay. No statistically significant difference in cGMP values was present between treated and control animals. Using a suckling' mouse assay, all intestinal content filtrates tested were negative for heat-stable enterotoxin. At the time of euthanasia, substantial amounts of enterotoxin were not present in the pigs. .13 gigrg, the 987P MCA was effective in the inhibition of adhesion of 987P-positive E; ggli to brush borders. However, the 987? MCA was ineffective in the Sheila D. Grimes prevention of diarrhea in gnotobiotic pigs challenged with 987P-positive E; coli. DEDICATION To the three Maes in my life, Julia Mae Grimes, Willie Mae Moss and Eula Mae Grimes and my father, George Washington Grimes ACKNOWLEDGEMENTS I wish to express my sincere thanks to Dr. Glenn L. Waxler, my major advisor, for his guidance, friendship, and unwavering support and encouragement during the course of this study. I would also like to thank the members of my guidance committee, Dr. Robert Bull, Dr. Roger Maes, Dr. Thomas Mullaney and Dr. Allan Trapp, for their advice, assistance and constructive suggestions during this study. Special thanks are extended to Dr. Robert Bull for the use of his laboratory and to Ms. Peggy Coffman, Mr. John Allen and Ms. Irene Brett for their technical assistance. I would also like to thank Dr. David Benfield for the hybridoma used in this study. I am indebted to Dr. E. Terence Adams, Dr. Calvert Louden, Mr. Joe Manning, Dr. Margit Rezabek, Mr. Dale Rezabek, Dr. Robert Sills and Ms. Lisa Thomas for their help during my gnotobiotic pig surgeries. I would like to thank Dr. Margit Rezabek, Dr. Joyce Bowling, Dr. Robert Sills, Dr. Robert Holland, my Tuskegee comrades, and my family and friends, whose moral support, encouragement and understanding were invaluable in the completion of this degree. vi I greatly appreciate the financial support which I received from the Equal Opportunity Program Programmatic Fund during my graduate studies at Michigan State University. vii TABLE OF CONTENTS Page LIST OF TABLES..........................................Xi LIST OF FIGURES....... .......... .. ...... . ......... .....xii LIST OF ABBREVIATIONS.. .............. ..... ..... . ...... .xiv INTRODUCTION.............................................1 LITERATURE REVIEW........................................5 Clinical Signs......................................6 Lesions.............................................7 Pathogenesis............................ .......... ..8 Pili...........................................8 The K88 Pilus............................12 The K99 Pilus............................16 The 987P Pilus...........................18 The F41 Pilus............................20 Type 1 Pili..............................21 Receptors.....................................23 Enterotoxins..................................34 The Heat-Labile Enterotoxin..............35 The Heat-Stable Enterotoxin..............39 Innate Resistance to E; 9911 Enteric Infections....45 Immunoprophylaxis..................................51 E; 9911 Vaccines..............................51 Pilus Vaccines...........................51 Enterotoxin Vaccines.....................53 Passive Immunization..........................55 Serum, Colostrum and Milk................55 Monoclonal Antibody......................64 CHAPTER 1: THE EFFECT OF 987P MONOCLONAL ANTIBODY ON THE ADHESION OF 987P-POSITIVE EfigflEBLQflLA QOLI TO PIG INTESTINAL BRUSH BORDERS INTRODUCTIONOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.000000070 viii Page “TERI” ANDETHODSOOOOOOOOOOOOOOOOO00.0.0000000076 Specimens.....................................76 Brush Border Preparations.....................76 Protein Assay.................................78 E; 9911 Strains...............................80 Slide Agglutination...........................81 Monoclonal Antibody Production................82 Brush Border Adhesion Test....................85 Statistical Analysis..........................88 RESULTSOOOOOOOO..OOOOOOOOOOOOOCOOO0.0.0.0.0.000000089 Brush Border Preparations.....................89 Protein Concentrations of the Brush Borders...89 Monoclonal Antibody Production................91 Adhesion......................................91 DISCUSSIONCOOOOOOOOOOOOO0.0.0.0.0.0...00.000.00.00102 SUWYOOOOOOOOOOOOOOOOOOO..OOOOOOOOOOOOO00.00.00.106 CHAPTER 2: THE EFFECT OF THE ORAL ADMINISTRATION OF 987P MONOCLONAL ANTIBODY ON THE CLINICAL SIGNS, LESIONS AND ADHESION OF 987P-POSITIVE EfigfiEBIQfllA QQLI IN GNOTOBIOTIC PIGS INTRODUWIONCOCOCOOOOO0.0.0.0000... ...... 0.0.0....109 MATERIALS AND METHODS.............................112 E; c011 Strains..............................112 Quantitation of E; 9911 in Inocula...........113 Monoclonal Antibody..........................113 Experimental Animals.........................114 Technique for Obtaining Gnotobiotic Pigs.....1l4 Bacteriologic Monitoring of Isolators........118 Treatment of Pigs............................119 Determination of Clinical Signs..............1l9 Necropsy Procedure...........................121 Preparation of Histologic Specimens..........121 Preparation of Scanning Electron Microscopic Sections..................................122 Microbiologic Quantitation of E; co11........123 MCA Activity in Gastric and Cecal Contents...124 Statistical Analysis.........................125 RESULTS...........................................126 Bacteriologic Monitoring.....................126 Clinical Signs...............................126 Gross Lesions................................130 Mistopathologic Lesions......................131 ix Page Scanning Electron Microscopic Lesions........138 Microbiologic Quantitation...................138 MCA Activity in Gastric and Cecal Contents...145 DISCIJSSIONOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0.0000000146 SUWY...O...OOOOOOOOOOOOOOOOOOOOOOO0.0.00.000000170 CHAPTER 3: THE EFFECT OF THE ORAL ADMINISTRATION OF 987P MONOCLONAL ANTIBODY ON ENTEROTOXIN PRODUCTION OF 987P-POSITIVE W QQLI IN GNOTOBIOTIC PIGS INTRODUflION.....OCCCCOOCOOCCOOOO0.0...0.0...0.0.0.0...173 MATERIALS ANDMETHODSOOOOOOCOOOOOOOOOOOOOOOOOOOO00175 Experimental AnimaISOOOOOOOOOOOO0.0.0.0000000175 cGMP Radioimmunoassay........................175 suCklinguouseAssaYOOICOOOO0.0.0.00000000000177 Statistical Analysis.........................178 RESULTSOOOOOOOOOO..OOOOOOOOOOOOOOOOOO00.00.00.0000178 cGMP Radioimmunoassay........................178 suCklingMouse AssaYOOOOOOOOIOOOOOOOO...0.00.180 DISCUSSION...0.00.00.00.000......O...0.0.000000000180 SUWYOOOOOOOOOOOOOOOOOO00......0.0.0.00000000000185 BIBLIOGRAPHY.OOOOOOOOOOOOOOOOOOOOOOIOO0.0.00.0000000000186 VITAOOOOOO.....OOOOOOOOOOOOOOOO0.0.0.0....00.0.00000000229 Table LIST OF TABLES Page The effect of 987P monoclonal antibody on the adhesion of 987P-positive E; 9911_to brush borders from porcine ileal samples........ ...... 93 Treatment protocol for the 8 litters of gnotObiotic pigSOOOOOOOOOOOOOOOOOOOO... ......... 120 Onset of clinical signs, morbidity, mortality and intestinal colonization in gnotobiotic pigs given 987P monoclonal antibody and exposed to 987P-positive E; c011............127 Adhesion index for 987P-positive E; coli to gnotobiotic pig small intestinal sections.. ..... 133 Numbers of viable E; 9911 in the luminal contents of 10 cm segments from pigs in litters 1-4.0.00000000000000000...... ........... 141 Numbers of viable E; 9911 associated with the intestinal wall in 10 cm segments of ileum from pigs in litters 1-4..................142 Numbers of viable E; 9911 in the luminal contents of 10 cm segments from pigs in litters 5-80.000000000000000000000000000000 ..... 143 Numbers of viable E; 9911 associated with the intestinal wall in 10 cm segments of ileum from pigs in litters 5-8............ ...... 144 Cyclic GMP radioimmunoassay results from ileal samples of gnotobiotic pigs challenged with 987P-positive E; 9911......................179 Suckling mouse assay results from intestinal filtrates of gnotobiotic pigs challenged with 987P-positive E; 9911...... ..... 181 xi Figure 1-1 LIST OF FIGURES Page Brush Border Adhesion Test.......................87 Phase contrast photomicrograph of a brush border fragment with no adhesion of 987P- positive E; 9911. Notice the moderately sized bacterial aggregates present beside the fragment(480X)...............................90 Distribution of the degrees of adhesion of 987P-positive and K88-positive E; 9911 to brush border fragments in ileal samples from 29 pigs..........................................92 Phase contrast photomicrograph of a brush border fragment with good adhesion. Notice the bacteria adhering to the brush border surface (arrow) (480K)...........................95 Phase contrast photomicrograph of a brush border fragment with partial adhesion. Notice the small aggregates of bacteria present in the background (480M)......... ..... ... ........... 95 The effect of preincubation of 987P monoclonal antibody with 987P-positive E; 9911, prior to the addition of brush borders, on the brush border adhesion test.......................97 The effect of preincubation of 987P monoclonal antibody with brush borders, prior to the addition of 987P-positive E; 9911, on the brush border adhesion test...................99 Phase contrast photomicrograph of a brush border fragment with good adhesion. Notice the adhesion of bacteria to the brush border surface and to adjacent bacteria (480X)........101 Experimental Design.............................115 xii Figure Page Photomicrograph of a section of ileum with bacteria adherent to the small intestinal villi extending from the crypts to the tips of the villi (arrows). Notice the absence of adherent bacteria to the adjacent small intestinal villi (Giemsa stain, 120X)...........135 Photomicrograph of a section of ileum with numerous rod-shaped bacteria (arrows) adherent to the lateral surfaces of the intestinal villi (Giemsa stain, 480K)......................135 Photomicrograph of a section of ileum with few bacteria adherent to the lateral surfaces of the villi (arrows) (Giemsa stain, 480X)......137 Photomicrograph of a section of ileum with bacteria entrapped in mucus (arrow) but not contiguous to the brush border surface (Giemsa stain, 480X)............................137 Scanning electron micrograph of a section of ileum with bacteria adherent to the lateral surface of the intestinal villi (arrow) (320X)..140 Scanning electron micrograph of a section of ileum with bacteria adherent to the brush border surface of the intestinal epithelium (5,300).........................................14o xiii LIST OF ABBREVIATIONS ADP... ................. . ........ ...adenosine 5'diphosphate cAMP.... ............ ..cyclic adenosine 3',5'-monophosphate cGMP........................cyclic guanosine monophosphate ELISA....................enzyme linked immunosorbent assay ETEC......................enterotoxigenic Escgegich1a c011 GalNac. ................... ...........N-acetylgalactosamine GluNac.... ............. . ........... ....N-acetylglucosamine GDP... .............. ..............guanosine 5'-diphosphate Gs- ........... guanyl nucleotide binding regulatory protein GTP..............................guanosine 5'-triphosphate Ig.......................... ...... ..........immunoglobulin KDa.......... ............ . ........ ..............kilodalton LT.................................heat-labile enterotoxin LTh..........................human heat-labile enterotoxin LTD........................porcine heat-labile enterotoxin MCA.. ........ ..........................monoclonal antibody MRHA...........mannose-resistant hemagglutinating activity MSHA...........mannose-sensitive hemagglutinating activity nm..............................................nanometers pI.......................................isoelectric point RIA.......................................radioimmunoassay STOOOOOOOOOOOOOOOOOOOO......OOOOOOOheat-stable enterotOXin xiv INTRODUCTION Worldwide, enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea in neonatal pigs, calves and lambs, as well as in children. Two virulence determinants are necessary for Escherichia 9911 (E; go_li_) to cause diarrhea: 1) enterotoxin production and 2) pilus production. Four antigenically distinct pili, K88, K99, 987P and F41, are present on separate strains of E; 9911 capable of infecting pigs (¢rskov 9_t._ _a_]_._;, 1961; Moon 99 91;, 1977; Nagy 9; 91;, 1976; Morris t a1., 1983). Pili allow E; 9911 to attach to specific receptors on the mucosal surface of the pig's small intestine. Once attached E;, 9911_ produce enterotoxins which cause hypersecretion, diarrhea, dehydration and death (Moon 93; 91;, 1979). Since adhesion of ETEC to the intestine is an essential prerequisite for the development of enteric colibacillosis, antibody-mediated prevention of bacterial adhesion is a potential defense mechanism against E; 9911, In neonatal animals, such antibody may be acquired from colostrum and milk. Vaccination of sows prior to farrowing helps to induce protective levels of antibodies 1 2 in the sow's colostrum and milk, thus providing passive immunity to the newborn pig. Currently, several vaccines have been developed for the protection of pigs against neonatal enteric colibacillosis. Research has demonstrated that protection correlates with anti-pilus antibodies in the colostrum and milk (Acres 99 91;, 1979; Nagy 99' (91;, 1978; Rutter 99: 91;, 1976). Several researchers believe that colostral antibodies prevent the colonization of ETEC by blocking adhesion (Nagy 99 1., 1978; Rutter, 1975; Rutter 99 91;, 1973, 1976). Recently, an alternative method for direct passive immunization of newborn animals against ETEC, the oral administration of E; 9911 pilus-specific monoclonal antibody (MCA) , has been developed. However, knowledge concerning the effectiveness of pilus-specific MCAs is limited. One of the first reports involved the oral administration of E; 9911 K99 MCA to calves (Sherman 99 91;, 1983). In that study, there was a statistically significant reduction in the mortality rate and the severity and duration of diarrhea in the treated versus the control calves. HOwever, there was no difference in the incidence of diarrhea. Explanations postulated for the diarrhea were: 1) the amount of MCA used may not have completely blocked colonization and 2) the number of bacteria in the challenge inoculum might have been large 3 enough to produce sufficient enterotoxin, without adhesion to the mucosa, to cause diarrhea. Studies with pigs using K88- (Foged 99 91;, 1986; Sadowski, 1984), K99- (Mainil 99 91;, 1987: Sadowski 99 91;, 1983) and 987P MCAs have also been performed. Few animals were used in the studies which were primarily clinical in nature. The effectiveness of the antibody varied from protection against challenge, based on an increased survival rate and a delayed onset of diarrhea, to no prophylactic effect. In the present study, 987P MCAs were given to gnotobiotic pigs in an attempt to protect the pigs from diarrhea due to 987P-positive E; 9911; The objectives of this study were: 1. To determine if the 19; 21999] adhesion of 987P- positive E; 9911 to pig intestinal brush borders could be prevented by the administration of E; 9911 987P MCA. 2. To determine if the clinical signs and lesions produced by 987P-positive E; 9911 in gnotobiotic pigs could be prevented by the oral administration of E; 9911 987P MCA. 3. To determine if the 19 2199 adhesion of 987P-positive E; 9911 to the intestinal mucosa of gnotobiotic pigs could be prevented by the oral administration of E; 9911 987P MCA. 4 4. To determine if enterotoxin production in gnotobiotic pigs challenged with 987P-positive E; 9911 could be affected by oral MCA administration. IJTERATURE REVIEW .Efign9119919 9911, a gram negative, motile or nonmotile, nonspore-forming rod, belonging to the family W, is a normal inhabitant of the lower intestinal tract of all warm-blooded animals (Bruner and Gillespie, 1973). However, when pathogenic strains of E; 9911 are present in the small intestine in large numbers, diarrhea occurs. Since 1899, E; 9911 has been thought to be a cause of diarrhea in neonatal pigs (Jensen, 1948). Several different types of L 9911 have been reported to cause diarrhea in domestic animals and man. M19919 9911 associated with diarrhea are divided into 5 categories: 1) enterotoxigenic, 2) enteropathogenic, 3) enteroinvasive, 4) enterohemorrhagic and 5) enteroadherent. Classifications are based on the bacterium's virulence properties, interactions with the intestinal mucosa, clinical syndromes, epidemiology and O:H serotypes (Levine, 1987). A vast amount of literature has been published regarding ETEC infections. There are several current general reviews about ETEC in the literature (Gaastra and 6 de Graaf, 1982; Gross and Rowe, 1985: Klemm, 1985) The emphasis of this literature review will be placed on ETEC infections in swine and details relevant to the present research. Clinical Signs Piglets from 12 hours of age to several weeks of age, with a peak incidence occurring at 3 days of age, are susceptible to enteric colibacillosis. Usually several or all the pigs in a litter are affected. Pigs farrowed by first litter gilts have a greater incidence of enteric colibacillosis than do pigs farrowed by sows (Blood 3 91;, 1983: Wilson, 1986). Infected piglets have yellowish or brownish feces which vary from a pasty to a watery consistency. The anal or perineal region may be inflamed from contact with the alkaline feces. The animals have rough hair coats and normal to subnormal temperatures. They are also anorectic, depressed and weak. As the condition progressively worsens, the animals become dehydrated and emaciated. Pigs commonly die within 24 hours after the onset of clinical signs (Blood 99 1., 1983; Wilson, 1986). 7 Lesions Gross lesions are minimal. The intestines of the affected pigs may be normal, hyperemic, distended with fluid or gas, or flaccid. Clotted milk may be present in dilated stomachs (Sojka, 1965). Venous infarcts may be present on the greater curvature of the stomach. Catarrhal enteritis may also be present (Blood 99 1., 1983; Jubb and Kennedy, 1985: Wilson, 1986). Histologically, few lesions are present in the intestines. Smith and. Jones (1963) reported. that no inflammatory changes were present in the intestinal tracts of pigs. Christie and Waxler (1972) reported that a mild neutrophilic infiltration of the intestinal villi, hydropic degeneration in villous epithelial cells and edema were present in gnotobiotic pigs infected with E; 9911. Bacilli may be seen attached along the margins of the small intestinal villi (Wilson, 1986). Congested blood vessels may be present in the lamina propria, and villous atrophy may be present in localized areas of the small intestine (Wilson, 1986). With both transmission and scanning electron microscopy, bacteria are seen attached on their sides and poles to small intestinal epithelial cells (Hohman and Wilson, 1975). Villous atrophy may also be seen, with the loss of degenerative epithelial cells occurring primarily at the villous extrusion zones. Moon (1982) suggested 8 that the loss of villous epithelial cells indicated an accelerated epithelial replacement rate. Pathogenesis Following oral ingestion of E; 9911, 2 important factors are required for the organism to cause diarrhea: 1) pilus production and 2) enterotoxin production. Pili Pili, a Latin term for hairs or hair-like structures, are filamentous organelles projecting from the bacterial surface. Brinton first used the term in association with these structures in 1959. Prior to that time, Duguid 99 91; (1955) had used the term fimbriae, meaning fringe, threads or fibers in Latin. Both terms are presently being used in the literature. Two important functions are associated with pili: 1) they increase the active surface area which may facilitate membrane associated activities such as respiration and nutrient uptake and 2) they act as attachment organelles (Ottow, 1975) . The latter function plays an important role in the pathogenesis of neonatal enteric colibacillosis. Structurally, the pili present on ETEC are composed of numerous repeating protein subunits (Isaacson, 1977; Mooi and de Graaf, 1979). A single pilus consists of from several hundred to a thousand identical subunits 9 (Korhonen 99 91;, 1985). The subunits are held together by noncovalent forces: no intersubunit cysteine bridges have been discovered (Klemm, 1985). The lack of sulfur- containing amino acids suggests that no disulfide bonds are present in the pili. Pili acting as adhesive organelles are peritrichously arranged on the bacterial surface (Ottow, 1975). The pili on ETEC were originally thought to be capsular (K) antigens and were designated K88 and K99. However, K antigens are polysaccharide in nature, and pili are composed almost entirely of protein. Hence their designation as K antigens is not correct. Following entry of bacteria into the small intestine, there are several non-immunological defense mechanisms, such as gut peristalsis, villous pumping, and flowing ingesta and mucus, which tend to rapidly wash bacteria out of the small intestine (Dixon, 1960: Moon, 1980). Pili, however, enable ETEC to overcome these natural defense mechanisms by allowing bacteria to attach and rapidly proliferate in the small intestine to numbers comparable to those normally found in the sluggishly motile, avillous large intestine (Bertschinger 99 91;, 1972: Jones and Rutter, 1972). Evidence supporting the fact that pili on ETEC facilitate adhesion has been demonstrated 19 2199 and 19 11999. Several early reports demonstrated the close microscopic association of ETEC with small intestinal 10 villi (Arbuckle, 1970, 1971: Drees and Waxler, 1970: Moon 99 91;, 1971: Staley 99 91;, 1969). Bertschinger 99 91; (1972) , showed that enteropathogenic E; 9911 (EEC) are more likely to be associated with the intestinal epithelium than nonenteropathogenic E; 9911 (NEEC) . The EEC were present adjacent to the brush border along the entire villus, but the NEEC were randomly distributed in the central lumen. In conventionally reared pigs, Jones and Rutter (1972) demonstrated that the K88—positive E; 9911 was able to adhere to the mucosa of the small intestine and cause diarrhea, compared to the K88-negative mutant which was unattached, was distributed throughout the lumen and did not cause diarrhea. These results indicated that the K88 pilus is responsible for adhesion of K88-positive bacteria to the small intestinal mucosa and that adhesion is required for the virulence of K88-positive bacteria in pigs. Supportive evidence for pili facilitating adhesion was also provided by Nagy 99 91; (1976, 1977) when strain 987P, a K88-negative piliated strain of ETEC, was also shown to adhere to the small intestine. The 111 11999 adhesion of piliated ETEC has been demonstrated with the brush border technique (Sellwood 99 91;, 1975), intestinal epithelial cell technique (Isaacson 99 91;, 1978: Nagy 99 91;, 1977; Wilson and Hohman, 1974), 11 the intestinal villus technique (Girardeau, 1980) and hemagglutination studies (Evans 99 91;, 1979). The pili most commonly present on strains of E; 9911 capable of causing diarrhea in neonatal pigs are K88, K99 and 987P. The F41 pilus maybe present on piglet strains of ETEC (Morris 99 91;, 1983). A pilus distinct from the former pili, with characteristics of :mannose-resistant hemagglutinating activity (MRHA) to porcine erythrocytes, has also been described (Aning and Thomlinson, 1983) . Recently, another adhesive factor, designated F42, with MRHA to human, sheep, guinea pig and chicken erythrocytes has been isolated from pigs (Yano 99 91;, 1986). It has also been suggested that the common or type 1 pili may also be important in the pathogenesis of enteric colibacillosis (Jayappa 99 91;, 1983, 1985). Strains of ETEC may produce more than 1 type of pilus antigen. Morris 99 91; (1980a) described a bipiliated strain producing both the K99 and F41 pilus antigens. Also, ETEC strains have been shown to produce both K88 and 987P pili (Schneider and To, 1982: Suarez 99 91;, 1987). Pili are also present on calf, lamb and human strains of E; 9911. The pili present on calf strains of ETEC are K99 (prskov 99 91;, 1975), F41 (Morris 99 91;, 1980a: To, 1984b), F(Y) (Morris 99 91;, 1985: Pohl 99 91;, 1982: Shimizu 99 91;, 1987) and 31A (Contrepois 99 91;, 1986: Shimizu 99 91;, 1987) . Lamb ETEC strains possess the K99 12 pilus (¢rskov 99 91;, 1975). Human ETEC strains possess colonization factor adhesin (CFA)/I (Evans 99 91;, 1975), CPA/II (Evans and Evans, 1978) and putative colonization factor (PCF) 8775 (Thomas 99 91;, 1982). In an attempt to standardize pilus terminology, ¢rskov and prskov (1983) have suggested a new nomenclature system. In their system, the previously mentioned pili are named the following: F1 (type 1), F2 (CFA/I), F3 (CPA/II), F4 (K88), F5 (K99), and F6 (987P). The new terminology has not been universally accepted. Only those pili present on pig strains of ETEC will be discussed. The K88 Pilus. Of the 3 most commonly found pili, the K88 pilus was the first to be discovered and consequently has had the most written about it. ¢rskov 99 ‘91; first described the K88 pilus in 1961. The extrachromosomally transferred K88 pilus is encoded for by a 50 megadalton plasmid (¢rskov 99 91;, 1966: Shipley 99 91;, 1978). Chemically, the K88 pilus has been identified as a protein, containing all the common amino acids except cysteine, with no carbohydrate present (Anderson 99 91;, 1980: Mooi and de Graaf, 1979; Stirm 99 91;, 1967b). Structurally, Stirm 99 91; (1967a) described it as a flexible, filamentous antigen with a diameter of 7-11 nanometers (nm) and a length of 100 to 150 nm. wadstrém 99 91; (1979) estimated the K88 pilus's diameter to be 2.1 :mm. The K88 pilus is a homopolymer of approximately 100 l3 hydrophobically linked subunits whose monomeric molecular ‘weight ranges from approximately 23,000 to 27,000, depending upon the serologic variant (Anderson 99 91;, 1980: Klemm, 1981: Mooi and de Graaf, 1979). Serologically, there are 3 variants of the K88 antigen, K88ab, K88ac and K88ad (Guinee and Jansen, 1979a): prskov 99 91;, 1964). The isoelectric point (pI) of the 3 K88 variants pooled together is 4.2 (Mooi and de Graaf, 1979). The common antigenic determinant, a, combines with the variable antigenic factor designated b, c, or d. The variants are antigenically different due to amino acid sequence differences in the pilus proteins. Amino acid sequences corresponding to the antigenic variables a, b, c, and d have been predicted (Gaastra and de Graaf, 1982: Gaastra 99 91;, 1979, 1981, 1983: Klemm, 1981: Klemm and Mikkelsen, 1982). Regardless of the antigenic variant, the K88 subunit consists of 264 amino acid residues. The existence of the K88ab and K88ac variants has been known since 1964, but the K88ad variant was discovered recently. The K88ab variant was common in the 19608, but its presence is rare today. Now, most K88- positive E; 9911 isolated from infected pigs possess either the K88ac of K88ad variant (Gaastra and de Graaf, 1982: Guinee and Jansen, 1979b). A recent survey suggests that the K88ac variant is the predominant variant in the 14 major pork-producing region of the United States based on a study examining 415 K88-positive strains of E; 9911 present in 9 states from 1976 to 1985 (Westerman 99 91;, 1988). In fact, all 415 strains of E; 9911 were found to possess the K88ac pilus. No other serologic variants were found. It is unclear whether the amino acid substitutions present in the K88 pilus variants indicate an attempt of the bacteria to evade the host's immune system or whether they indicate bacterial adaptation to altered receptor sites on pig small intestinal brush borders. It has been suggested that the K88ac variant was derived from the K88ab variant and that the K88ab variant has undergone selective pressure by large increases in the amount of antibody directed against it on pig farms (Guinee and Jansen, 1979a). The very low number of base substitutions not resulting in amino acid substitutions, only 5 out of 47, suggests that the K88ab and K88ad variants have not evolved from each other in a normal way but that they must have been subject to intensive selective pressure, such as might result. from an intensive vaccination program (Gaastra 99 91;, 1983). The other possibility is that the number of resistant pigs (pigs without receptors for the K88 pilus) may have caused a selection toward E; 9911 with an alteration in their pili to match receptor sites 15 already present on the pigs' small intestinal epithelium (Gaastra and de Graaf, 1982). Molecular analysis of pilus determinants has shown that they encode multiple gene systems with the interaction of several genes being required for the assembly of the pili (Dougan 99 91;, 1983; Kehoe 99 91;, 1981: Mooi 99 91;, 1979). Six structural genes are involved in the production of the K88 pilus. Mooi 99 91; (1979) isolated the genetic determinant for K88ab by molecular cloning. The resultant recombinant plasmid appeared to code for 5 polypeptides, with molecular weights of 17 kilodaltons (KDa), 26 Kda, 27 KDa, 27.5 KDa and 81 KDa, which were precursors in the biosynthesis of K88ab (Mooi 99 91;, 1981, 1982). The 17, 27 and 27.5 KDa polypeptides are located in the periplasmic space, the 81 KDa polypeptide in the outer membrane and the 26 KDa polypeptide (the K88ab pilus subunit) is located on the cell surface, probably attached to the outer membrane (van Doorn 99 91;, 1982). The 81 KDa probably serves to anchor the pilus subunits to the outer membrane (Mooi 99 91;, 1983). The sixth polypeptide, A, a pilus-like protein, appears to be necessary for the export of the K88ab subunits, and it has also been suggested that polypeptide A and the K88ab pilus are part of the same structure, with the K88ab pilus subunit probably representing the major component. Polypeptide A might serve as a link 16 between the K88ab pilus and the anchorage protein. .Alternatively, the 2 polypeptides might form separate structures on the cell surface, the synthesis being interdependent. Thus, the colonization of some surfaces by K88-positive E;, 9911 might require at least 2 extracellular proteins (Mooi 99 91;, 1984). E9999919n19 9911 hearing the K88 pilus show MRHA for guinea pig (Jones and Rutter, 1974; Stirm 99 91;, 1967) and chicken erythrocytes. The K88 pilus is species specific. Strains of E; 9911 that are K88-positive are able to colonize pig intestine 19 9199 and cause diarrhea, but K88-positive ETEC have not been reported as enterotoxigenic in other species (Moon 99 91;,1977; Smith and Halls, 1967). Deneke 99 91; (1984) showed that ETEC with K88ac or K88ab pili bind to isolated human small intestinal epithelial cells. Also, Tzipori 99 91; (1984) have determined that K88 ab- and K88ac-positive E; 9911 attach 19 11999 to epithelial cells from both foals and adult horses. E99n9x19n19 9911 bearing K88 pili are found in pigs up to several weeks of age. The K99 Pilus. prskov 99 91 first described the Kco antigen present on calf and lamb enterotoxigenic strains of E; 9911 in 1975. In 1977, the K99 pilus was described on pig strains of ETEC (Moon 99 91;, 1977). The K99 pilus is encoded for' by a ‘transmissible plasmid (Smith and Linggood, 1972) with a molecular weight of 57 megadaltons 17 (So 99 91;, 1976). Isaacson (1977) concluded that the K99 antigen was a pilus based on its subunit structure, external surface location and rod-like shape. It has been described as a thin, flexible filament with an open structure and stretched subunits (Jones and Isaacson, 1983). The pilus's length was reported to be 130 to 160 nm, and its diameter was reported to be approximately 8 nm (Altman 99 91;, 1982: Isaacson, 1977). Isaacson 99 91; (1981) have since reported the pilus's diameter to be 4.8 nm. The K99 pilus is protein in nature and lacks the sulfur-containing amino acids, cysteine and. methionine (Altman 99 91;, 1982). In the past, there has been a great deal of controversy concerning the molecular weight and characterization of the K99 pilus with disparate reports based on different isolation procedures originating from several laboratories (Altman ‘99 J91;, 1982: Chantner, 1982: de Graaf 99 91;, 1980; Isaacson, 1977, 1978; Morris 99 91;, 1977, 1978a,b). Morris 99 91;, (1980a) have reported that there are 2 pilus subunits present in serogroups 09 and 0101 with different molecular weights and ionic charges. The cationic and lower molecular weight component was the K99 pilus, and the high molecular weight, anionic component was the F41 pilus which has been shown to be a pilus with adhesive abilities (Morris 99 91;, 1982). The molecular weight of the K99 18 pilus subunit was reported by de Graaf (1980) to be 18,500 and by Chanter (1982) to be 19,000. Horse and sheep erythrocytes show MRHA with K99- positive E; 9911 (Burrows 99 91;, 1976, Gaastra and de Graaf, 1982: Isaacson, 1978: Morris 99 91;, 1980a). With a pI of 9.5, K99 is the only pilus antigen with a basic pI (de Graaf 99 91;, 1980). The nucleotide (Roosendaal 99 91;, 1984) and amino acid sequences (de Graaf 99 91;, 1980 and de Graaf and Roorda, 1982) of the K99 pilus have been determined. Structural genes encoding 7 polypeptides involved in K99 biosynthesis have also been described (de Graaf 99 91;, 1984). The K99 pilus is not species specific. Calf, lamb and pig strains of E; 9911 bearing K99 pili are pathogenic (Moon 99 (91;, 1977: prskov 99 91;, 1975: Smith and Linggood, 1972). The K99-positive E; 9911 are found in pigs less than 2-weeks-old (Moon 99 91;, 1980). The 987P Pilus. The 987P pilus was first determined to facilitate intestinal adhesion and colonization in 1976 (Nagy 99 91;, 1976). Composed primarily of protein, the 987P pilus has also been shown to possess an amino sugar component (Fusco 99_ 91;, 1978: Isaacson and. Richter, 1981). The 987P pili are morphologically indistinguishable from type 1 pili. The 2 types of pili have a tight, rigid structure and are composed of fewer subunits per turn than K88 and K99 pili, creating an 19 axial hole as determined by electron microscopy (Jones and Isaacson, 1983) . Strong hydrogen bonds appear to be present between the 987P subunits (Schifferli, 1987). The pili have diameters of 7 nm and obtain lengths up to 1 um (Fusco 99 91;, 1978: Isaacson and Richter, 1981). The molecular weight of the 987P pilus's subunit was first reported as 18.9 KDa by Fusco 99 91;, (1978) and has since then been reported to be 20 KDa by Isaacson and Richter (1981). The 987P pilus is not plasmid determined (Isaacson 99 91;, 1977: Nagy 99 91;, 1977) but is believed to be genetically encoded for by a chromosome (Gaastra and de Graaf, 1982). The pilus has a pI of 3.7 (Isaacson and Richter, 1981). Mannose-resistant. hemagglutination of horse, guinea pig, sheep, rabbit, pig and cow erythrocytes does not occur with 987P-positive E; 9911 (Isaacson 99 .91;, 1977: Isaacson and Richter, 1981), but weak MRHA of chicken erythrocytes has been demonstrated by Awad- Masalmeh 99 91; (1982). In a study by Moon 99 91; (1980) to determine the prevalence of pili in ETEC isolated from neonatal pigs with diarrhea, 76% of the E; 9911 strains were able to produce 987P pili. In a later study by Brinton 99 91; (1983), 987P pili were also reported to be the most common pili, excluding type 1 pili, in the United States. The more common occurrence of the 987P pilus compared to the 20 other type of pili is probably due to the chromosomal locus of the pilus's gene (Brinton 99_91;, 1983). The 987P pilus is considered to be species specific as a virulence factor in pigs, even though it has been found on calf strains of E;_‘9911, In calves, 987P piliated strains of E; 9911 appear to be harmless (Moon, 1978b). The 987P pilus is found on strains of ETEC present in pigs less than 2 weeks of age. The F41 Pilus. The F41 pilus was first described by Morris 99 91; in 1980 and referred to as F41 in 1982. The pilus was first found in association with K99-producing ETEC of serogroups 09 and 0101 (Morris 99 91;, 1980a, 1983). The F41 pilus appears to be the same pilus that was described by Mbon 99 91; (1980) and Awad-Masalmeh 99 91; (1982). The F41 pilus is thin and flexible (Moon 99 91;, 1980) and has a diameter of 3.2 nm, a'pI of 4.6 and a molecular weight of 29.5 KDa (de Graaf and Roorda, 1982). It has the largest fimbrial subunit that has been found to date. E9999919919, 9911_ possessing the F41 pilus hemagglutinate guinea pig and sheep erythrocytes strongly and horse erythrocytes to a lesser degree (Gaastra and de Graaf, 1982: Morris 99 91;, 1980a). However, de Graaf and Roorda (1982) indicated that the F41 pilus caused strong MRHA of human and guinea pig erythrocytes and weak MRHA of sheep and horse erythrocytes. To (1984b) reported 21 that the F41 pilus agglutinated chicken, goat and pig erythrocytes. The amino acid sequence of the F41 pilus has been determined (de Graaf and Roorda, 1982). The F41 antigen is encoded for by a chromosome, and the genes required for F41 production have been cloned (Moseley 99 91;, 1986) . Morris 99 91; (1983) were the first to discover the F41 pilus on piglet strains of ETEC. Strains of E; 9911 possessing F41 alone have been shown to be virulent in pigs (Awad-Masalmeh, 1982: Morris 99 91;, 1982, 1983; To, 1984b) and calves (To, 1984b). E99999191119 9911 hearing the F41 pilus also colonize the intestinal epithelium of lambs (Morris 99 91;, 1980b). Type 1 P111. The type 1 pili are generally not believed to be involved in the pathogenesis of enteric colibacillosis. However, some authors have suggested that type 1 pili may indeed play a role in the colonization of the small intestine by ETEC. The previously-described pili present on ETEC are distinctly different from the common or type 1 pili of E; 9911. A major distinction is that ETEC pili have MRHA, compared to the mannose-sensitive hemagglutinating activity (MSHA) of type 1 pili (Duguid 99 91;, 1955: Evans 99 91;, 1979). Type 1 pili of enterobacteria, including E; 9911, show the same pattern of hemagglutination specificity for erythrocytes of different animal species. 22 They show strong MSHA for guinea pig, chicken, horse and monkey erythrocytes, moderate MSHA for human erythrocytes, weak MSHA for sheep erythrocytes and no MSHA for cow erythrocytes (Duguid 99 91;, 1955, 1979: Duguid and Old, 1980). Type 1 pili measure 7-8 nm in width and 0.2 to 2 um in length (Brinton, 1959, 1967, 1978). The pili are chromosomally encoded (Brinton, 1959, 1965, 1967). It has been determined that the type 1 pilus family consists of at least 12 different types of pili (Brinton 99_ 91;, 1983). However, all serotyped type 1 pili isolated from neonatal swine with diarrhea have been serotype 2, type 1 pili. Some experiments suggest that K88-positive E; 9911 may require serotype 2, type 1 pili for virulence. Also, a porcine isolate of E; 9911 phase cloned to express only serotype 2, type 1 pili caused experimental colibacillosis in a significant portion of pigs, indicating that serotype 2, type 1 pili may act as independent pilus virulence factors for swine neonatal enteric colibacillosis (Jayappa 99 91;, 1983). Jayappa 99 91; (1983, 1985) have reported that ETEC with type 1 pili adhere to and colonize the small intestine of pigs and that this adhesion could be prevented by type 1 antiserum. Immunofluorescence demonstrated the presence of E; 9911 expressing type 1 pili on the brush borders of colostrum deprived newborn pigs, thus suggesting the involvement of the pili in colonization. Anti-type 1 serum was given to 23 newborns prior to challenge, and the number of gut associated E; 9911 compared to controls was decreased. In another study, a strain of E; 9911 possessing type 1 pili, but no other known swine pili, with MSHA to guinea pig erythrocytes caused diarrhea in hysterectomy ‘derived colostrum-deprived pigs (Nakazawa 99 91, 1986). Type 1 pili have also been shown to adhere 19 91999 to porcine intestinal epithelial cells (Isaacson 99 91;, 1978). A type 1 pilus vaccine has been shown to be protective in humans challenged with the same serotype expressed in the vaccine (Levine 99 91;, 1982) . Reports have indicated that a vaccine containing type 1, serotype 2 pili, as well as K88, K99 and 987P pili, is much more effective in the prevention of swine enteric colibacillosis than similar vaccines which lack the type 1 pili (Brinton 99 91;, 1983: Jayappa 99 91;, 1983). However, in a study by To 99 91; (1984) it was reported that not only were type 1 pili not protective antigens for ETEC infections, but also that the challenge strain didn't produce type 1 pili in the small intestine of the pigs during colonization. Receptors Pili facilitate attachment of bacteria to specific receptors present on the small intestinal mucosa (Paris 99 91;, 1980: Evans and Evans, 1978: Smit 99 91;, 1984). Ultrastructural observations indicate that strong mucosal attachment of ETEC, sufficient to overcome host 24 peristaltic clearing mechanisms, can be achieved by the formation of a large number of bonds between pili and host mucosal receptors (Knutton 99 91;, 1984). Less specific hydrophobic and ion electrostatic interactions between the bacterial glycocalyx and intestinal epithelium are also involved in the attachment of bacteria to the small intestine (Wadstrém 99_91;, 1979). Present knowledge about the actual nature of the receptors to which K88, K99, 987P (and F41 attach is limited. Research is currently ongoing to determine the chemical composition of the receptors. Reports indicate that chemically the receptors are composed of glycolipids or glycoproteins (Gibbons 99 91;, 1975: Kearns and Gibbons, 1979). Depending upon the assay involved, various sugars, glycoproteins or glycolipids may inhibit the binding of piliated E; 9911 to brush borders, epithelial cells or erythrocytes. Gibbons 99 91; (1975) determined that glycoproteins with a terminal fi-D-galactosyl structure were able to inhibit the hemagglutination of guinea pig erythrocytes by K88 pili, suggesting that the glycoprotein might bind with the K88 pilus and inhibit hemagglutination due to its resemblance to the K88 receptor. The IB-galactosyl residues have also been shown to play an important role in the binding of K88 pili to brush borders (Sellwood, 1980b) . Binding of K88 pili to brush borders occurs from 25 4'C to 37'C, while binding to the guinea pig erythrocytes only occurs at 4'C, indicating' that the fit for the natural receptors is better than that of the guinea pig erythrocyte receptors (Sellwood, 1980b: Gibbons 99 919, 1975). Glycoproteins with N-acetylglucosamine (GluNac) and N-acetylgalactosamine (GalNac) as terminal sugars inhibit binding of the K88 antigen to brush borders and, to a lesser degree, sugars alone, such as GluNac, GalNac and N- acetylmannosamine, can also inhibit binding (Anderson 99 91;, 1980). The sugar' D-galactosamine has also ibeen reported to inhibit binding of the K88 pilus to brush borders (Sellwood, 1984a,b). Glycolipids have also been shown to have K88 receptor activity (Sellwood and Kearns, 1979: Kearns and Gibbons, 1979) . The brush border receptor for the K88 pilus reacts chemically in a way similar to that of the cholera toxin receptor which is a G141 ganglioside. However, according to Sellwood and Kearns (1979), it doesn't really appear to be a ganglioside. Hemagglutination tests have been performed on erythrocytes from several species in an attempt to identify specific receptor sites. Parry and Porter (1978) demonstrated that K88ab adhered strongly to chicken erythrocytes but not to the extent of that seen on the brush borders cf intestinal epithelial cells. Guinea pig 26 erythrocytes were agglutinated by both K88ab and K88ac pilus preparations and live organisms. They concluded that both the a and the b or c determinants of the K88 pilus for K88ab and K88ac are involved in adhesion to intestinal epithelial cells and guinea pig erythrocytes. However, in the chicken erythrocytes, adhesion of the pilus appears to involve only the K88b determinant's receptor on the erythrocyte (Parry and Porter, 1978) . The hemagglutination reaction is easier to study, but the adhesion is not as specific as that seen with the natural receptors on epithelial cells. There may not be a separate brush border receptor for each of the serologic variants of K88-positive E; 9911. Bijlsma 99 91; (1982) showed that blocking the receptor site with 1 serologic variant of the K88 antigen also inhibited the adhesion of other K88 variants. For example, when brush borders from pigs susceptible to all 3 serologic variants of E; 9911 were exposed to the K88ab antigen and the receptor sites were blocked, K88ad- and K88ac-positive strains of E; 9911 were no longer able to adhere. However, adhesion of K88ad to brush borders from 1 pig susceptible to all 3 serologic variants of K88 was not inhibited by K88ab or K88ac antigen. This suggests that the receptor site for K88ad in brush border from pigs susceptible to all 3 serologic variants may have 2 configurations which can only be detected by receptor 27 blocking. Bijlsma 99 91; (1982) consider that only 1 receptor, depending upon its modifications, will allow attachment of 1, 2 or all 3 of the K88 serologic variants. Wilson and Hohmann (1974) believe that the K88a antigenic component doesn't affect the ability of bacteria to attach to intestinal mucosal cells. They found that the antigen necessary for blocking adhesion was the c or b component. Bijlsma 99 91; (1982) say that it is unlikely that the antigenic components b, c, or d are exactly the same as the adhesion component of the K88 pilus because K88ac antigen is able to block K88ab and K88ad receptors of phenotype A brush border, yet K88c antibody doesn't react with K88b and K88d. Conversely, Parry and Porter (1978) were able to block the adhesion of K88ab- and K88ac-positive strains of E; 9911 to brush borders by using antisera specific for the K88a determinant. Antibodies against K88b and K88c antigens only inhibited homologous strains of K88-positive E; 9911. Intestinal epithelial cells are believed to have different receptors for different pili. Using strains of E; 9911 with K99 and 987P pili, binding of piliated E; 9911 to brush borders was inhibited by homologous pili, but not heterologous pili, which indicated that a difference in receptors existed (Isaacson 99 91;, 1978). The K99 antigen has been shown to be specific for sialic acid (Faris 99 1., 1980: Lindahl and Wadstrém, 28 1984: Smit 99 91;, 1984). Substances with sialic acid as a major component were able to inhibit the binding of calf K99-positive ETEC to sheep erythrocytes using a MRHA test. The K99 surface hemagglutinin of ETEC recognize terminal GalNac and sialic acid residues of glycophorin and other complex glycoconjugates. The K99 pilus has also been shown to bind to glycophorin A on human erythrocytes (Lindahl 99 91;, 1984) and to bind to NeuGc (2»3) lactosylceramide, a glycolipid, on equine erythrocytes (Smit 99 91;, 1984). Lindahl and Wadstrém (1984) have suggested that the K99 erythrocyte receptor may be a glycophorin with a N-acetylneuramic acid 2-6 GalNac structure. Earlier, Morris 99 91; (1977) demonstrated with hemagglutination experiments that the K99 pilus was sensitive to the GalNac structure. Human, porcine and equine erythrocytes which are hemagglutinated by K99 pili all have similar glycophorin oligosaccharide structures (Lindahl and Wadstrém, 1984). Hemagglutination inhibition of K99-positive ETEC has also been inhibited with a glycoconjugate resembling the GMz-ganglioside (Paris 99 91;, 1980). The N-acetylneuramic acid portion appeared to play an important role in the inhibition of binding. The F41 pilus may be specific for GalNac and have some affinity for GluNac (Lindahl and Wadstrém, 1984). It appears the receptors for pili are capable of releasing 19 91999 from brush borders or intestinal cell 29 membranes. Kearns and Gibbons (1979) reported that plasma membranes prepared from positive brush borders lost 98% of their receptor activity. They were able to demonstrate that a supernatant fraction, obtained during the preparation of K88-positive brush borders, was able to enhance the adhesion of K88-positive E; 99191 to both positive and negative brush borders. Supernatant fractions obtained from negative brush borders did not enhance adhesion. {Hfljizing pig mucosal organ cultures, other researchers have also demonstrated the release of receptors for K88-positive E; 9911 into culture media (Staley and Wilson, 1983: Wilson 99 91;, 1984). Dean and Isaacson (1982) have identified a soluble 987P pilus receptor-containing fraction which was released from adult rabbit brush borders stored at 4‘C. They have shown that the receptor is a low molecular weight, less than 14 KDa, acidic glycoprotein based on SDS gel electrophoresis (Dean and Isaacson, 1985b) . The 987P receptor of adult rabbits was present along the entire small intestinal villous surface and in goblet cells, but in infant rabbits it was present only in goblet cells. The receptors in adult rabbits were spread equally throughout the jejunum and ileum. Material antigenically similar to the rabbit 987P receptor has been demonstrated in goblet cells in neonatal pig ileum. The identification of the 987P receptor in what appeared to be goblet cells 30 was consistent with previous thought that the 987P receptor was a component of the glycocalyx. Support for the receptor being a component of glycocalyx is based on the acidic nature of the glycoprotein 987P receptor (Dean and Isaacson, 1985a), its release from brush borders upon storage (Dean and Isaacson, 1982) and the coadherence, or multilayered adherence, of 987P-positive bacteria to epithelial brush borders (Dean and Isaacson, 1982). It has been suggested that the 987P receptor is produced and secreted by goblet cells and that the 987P receptor is a lectin that must bind to brush borders to function as a receptor for the 987P pilus (Dean and Isaacson, 1985a). The 987P receptor is multivalent (Dean and Isaacson, 1985b). Charge, salting effects and perhaps a free amino group may be important factors in the 987P pilus-receptor interaction since the 987P pilus can be inhibited from binding to its receptor by high concentration of NaCl and by compounds containing a free amino group, such as NH4C1, amino sugar, lysine or ethanolamine, but not by neutral sugars or by glycine. Periodate oxidation of the 987P pilus also inhibits the pilus-receptor interaction which indicates that the pilus's carbohydrate may play a role in the interaction (Dean and Isaacson, 1985b). Evidence also exists that receptors for K99 pili may also be released from brush borders 19 21999 upon storage at 4’C (Grimes 99 91;,1986). 31 The presence of receptors for K88 pili in pigs is genetically determined. Pigs lacking the receptor are resistant to diarrhea caused by K88-positive E; 9911, whereas those with the receptor are susceptible (Sellwood 99 91;, 1975). Moon 99 91; (1979) have stated that they have not encountered pigs congenitally resistant to colonization by E; 9911 with K99 pili or 987P pili, indicating that all pigs have receptors for the K99 and 987P pili. In a study by Grimes 99 91; (1986) it was reported that most, if not all, pigs had intestinal receptors for K99 pili. Receptors for the K88 pilus are located throughout the small intestine in pigs (Hohman and Wilson, 1975: Kohler 99 91;, 1975: Moon 99 91;, 1979: Smith and Linggood, 1971a). However, the association of K88-positive E; 9911 with villi occurs most commonly in the ileum and least commonly in the duodenum (Arbuckle, 1976). Receptors for the K88 pilus are also present on mouse and calf epithelial cells (Runnels 99 91;, 1980). 19 91999, receptors for 987P (Dean and Isaacson, 1982: Isaacson 99 91;, 1978; Nagy 99 91;, 1977) and K99 (Isaacson 99 91;, 1978) pili are present on pig jejunal and ileal brush borders. 19 9199, in natural infections with 987P- positive E; 9911, colonization appears to be confined to the ileum and large intestine (Isaacson 99 91;, 1978: Moon 99 91;, 1979) , but ligated gut experiments have shown 32 attachment to the jejunum also (Moon 99 91;, 1979: Nagy 99 91;, 1976). 19 1119, K99-positive E; 9911 adhere to the ileum of pigs (Isaacson 99 91;, 1978: Moon 99 91;, 1977, 1979). ‘ Mucus may play an important role in bacterial attachment to the small intestine (Forstner, 1978: Hoskins, 1978) . Mucus contains mucin, which is composed of glycoproteins and glycolipids (Mantle 99 91;, 1981: Marshall and Allen, 1978; Schacter and Williams, 1982: Slomiany and Slomiany, 1984) . As mentioned previously, some sugar structures of glycoproteins may serve as receptors for bacterial pili. Bacteria must pass through the mucus covering intestinal epithelial cells prior to their attachment to the cell, so glycoproteins present in mucus may act as false receptors for bacterial pili. This may prevent colonization by interfering with adhesion of the bacteria to the underlying epithelial cells. In one study, when mucus was removed, there was a significant increase in the number of E; 9911 adherent to ileal and colonic segments. Ileal mucin material, containing galactose and galactosamine, decreased the adhesion of E; 9911 to the ileal segments (Golderman 99 91;, 1985). Mouricout and Julien (1986, 1987) have suggested that in calves, diarrhea due to ETEC involves a bacterium-mucin recognition phenomenon in which bacterial pili and specific glycoprotein mucus receptors are important. 33 sialic acid and galactose seemed to be partially responsible for the attachment of K99 pili, whereas F41 pili recognized desialylated receptors. It has been suggested by Freter (1981) that to resist the flux of intestinal secretions and to colonize tissue, it may be sufficient for bacteria to bind to mucus alone. Even in cases where bovine ETEC bound to enterocytes, the E; 9911 also interacted with the mucus (Freter, 1981: Laux 99 91;, 1984: Mouricout 99 91;, 1986). Thus, mucus may act as a site for replication and colonization before bacteria actually adhere to the small intestinal epithelial cells. Laux 99 91; (1986) have identified 2 glycoprotein receptors, with molecular weights of 57 and 64 KDa, for the K88ab pilus in mouse small intestine. The receptors are present in both mucus and brush border membranes. A 91 KDa glycoprotein receptor present only in the brush border membranes was also identified. Adhesion of K88- positive E; 9911 to the mouse small intestine was inhibited primarily by D-galactosamine. The presence of receptors for the K88ab pilus in mucus as well as on the brush border membrane may represent secreted mucus glycoproteins, and it has also been reported by Weiser (1984) that brush border membrane components are often released into the gut lumen. (Laux 99 91;, 1986). Receptors specific for the K88 pilus have been shown to 34 exist in mouse, rat, rabbit and pig mucus (Laux 99 91;, 1984: Slomiany and Slomiany, 1984). Enterotoxins Once attachment and colonization have occurred, ETEC are able produce enterotoxins. Enterotoxin production is the second important virulence determinant (Smith and Halls, 1967). Enterotoxins, when given by the intraluminal route, cause the net movement of fluid and electrolytes from plasma to the intestinal lumen (Dorner 99 91;, 1976). Two types of enterotoxins, based on their thermolability, antigenicity and molecular weight, are produced by ETEC: 1) a heat-stable toxin (ST) and 2) a heat-labile toxin (LT). The heat-labile toxin is inactivated by heat at 60'C for 30 minutes, has a high molecular weight, and is antigenic (Gyles, 1974a: Gyles and Barnum, 1969). The heat-stable toxin is resistant to temperatures of 100'C for 30 minutes, has a low molecular weight, and is nonantigenic (Smith and Halls, 1967: Smith and Gyles, 1970). Both types of enterotoxin production are controlled by transmissible plasmids (Skerman 99 91;, 1972: Smith and Halls, 1968). Strains of ETEC may produce only 1 or both types of enterotoxin. Strains of ETEC that possess the K88 pilus may be associated with the production of LT, ST or both (Guinee and Jansen, 1979b: Gyles and Barnum, 1969: Smith and Gyles, 1970: Séderlind and Méllby, 1979). The 987P 35 pilus is associated with the production of ST (Guinee and Jansen, 1979b: Moon 99 91;, 1980). The K99 pilus is also associated with ST production (Guinee and Jansen, 1979b: Moon 99 91;, 1976). The Heat-Labile Enterotoxin. Although many different strains of ETEC produce LT, it is a highly homologous substance in all the strains. The LT toxin is divided into 2 types based on the origin of the isolate, human (LTh) or porcine (LTp) (Geary 99 91;, 1982: Honda 99 91;: 1981c: Takeda 99 91;, 1983: Tsuji 99 91;, 1982: Yamamoto and Yokota, 1983). The 2 types of toxins have demonstrated minor antigenic and structural differences (Honda 99 91;, 1981c: Tsuji 99 91;, 1982), but the genes for the toxins are virtually identical (Dallas, 1983). The LT was considered by Finkelstein 99 91; (1976) to be a heterogenous molecule whose molecular weight ranged from 35 KDa to 100 KDa, but Dorner 99 91; (1976) and Evans 99 91; (1976) reported a molecular weight of approximately 100 KDa. Finkelstein 99 91; (1976) suggested that the heterogeneity was due to proteolytic splitting of the molecule during purification. The toxin has now been shown to have a molecular weight of 86.5 KDa. Purification of the toxin may also have been complicated because LT is a cell-associated toxin, located in the periplasmic space (Wensink 99 91;, 1978) . 36 The LT toxin is similar, antigenically, structurally and functionally, to the cholera toxin of 219919 9991999 (Clements and Finkelstein, 1978a,b: Gill 99 1981: Gyles, 1974a,b: Gyles and Barnum, 1969: LindhoLm 99 91;, 1983). structurally LT, like cholera toxin, consists of 2 subunit fragments, 1 A subunit, with a molecular weight of 28 KDa, and 5 B subunits, with individual molecular weights of 11.5 KDa (Clements and Finkelstein, 1979). The B subunits are aggregated in a ring by tight noncovalent bonds. The A subunit is linked to and partially inserted in the B ring through weaker non-covalent interactions (Clements and Finkelstein, 1979: Dafni and Robbins, 1976: Gill 99 91;, 1981: Kunkel and Robertson, 1979: Walk a .91;, 1980). The LT B subunit. has a DNA. nucleotide sequence of 103 amino acid residues (Dallas and Falkow, 1980), and the. A. subunit consists of 236 amino acid residues (Spicer 99 91;, 1981, Spicer and Noble, 1982). The B subunit pentamer binds the LT toxin to a GMl ganglioside (Donta and Viner, 1975: Moss 99 91;, 1980: Svennerholm and Holmgren, 1978) in the mucosal cell membrane and creates a functional pore through which the A subunit enters the cell's cytosol (Holmgren, 1981) . Also, in rabbit intestinal mucosal cells a glycoprotein, structurally related to the ganglioside, has been shown to act as an additional receptor for LT, but not cholera toxin (Holmgren 99 91;, 1982: Holmgren, 1981) . The action 37 of the toxin has a lag time of greater than 30 minutes, but its effect is prolonged and unaffected by washing. The LT toxin is not tissue specific, it has a wide range of cells which it is capable of binding to and affecting (Guerrant 99 91;, 1974: Kantor 99 91;, 1974: Kwan 99_91;, 1974: Mashiter 99 91;, 1973: Zenser and Metzger, 1974). The LT toxin, like cholera toxin, activates membrane bound adenyl cyclase which subsequently leads to an increase in intracellular cyclic adenosine 3',5'- monophosphate (cAMP) in epithelial cells of the small intestine (Evans 99 (91;, 1972: Hewlett, 1974). The activation of adenyl cyclase occurs after the movement of the A subunit of LT across the membrane and into the cytoplasm of the intestinal epithelial cell. A catalytically active fragment of the A subunit then splits intracellular nicotinamide adenine dinucleotide (NAD). If guanosine 5'-triphosphate (GTP) is present, the adenine 5'-diphosphate (ADP) ribose portion of NAD then covalently links onto the guanyl nucleotide binding regulatory protein (Gs) of the adenylate cyclase complex at the inner side of the basolateral membrane of the mucosal cell. Adenylate cyclase is then locked in an active form by the inhibition of an inherent feedback regulatory mechanism which normally involves the hydrolysis of GTP to guanosine 5'-diphosphate (GDP) and inorganic phosphorus, and this results in the net 38 accumulation of intracellular cAMP (Gill 99 91;, 1976,: Moss 99 91;, 1979: Moss and Richardson, 1978). Choleragen, and possibly LT catalyzed ADP ribosylation of G8 appears to increase adenylate cyclase activity by 2 mechanisms. The toxin inhibits the GTPase associated with Gs, thus prolonging the life of the active GTP-G8 complex (Cassel and Selinger, 1977). Second, the ADP-ribosylation of G8 decreases it affinity for GDP, resulting in acceleration of GDP release and formation of the active GTP complex (Burns 99 91;, 1982). Subsequently, there is a net secretion of an isotonic, alkaline fluid rich in electrolytes as a result of increased sodium, bicarbonate, and water secretion by crypt cells and decreased absorption of chloride coupled to sodium by the villus cells (Field, 1981: Moon, 1974:Moss and Vaughan, 1980). The increased secretion by the crypt cells leads to a secretory diarrhea (Moon 99 91;, 1978a). Field (1981) has suggested that calcium might be an important intracellular regulator of intestinal electrolyte transport, acting as the ultimate messenger for E; 9911 cAMP-induced secretion. It has been postulated that increased levels of cAMP cause a release of intracellular calcium which triggers activation of the calcium/calmodulin complex. This complex ultimately results in increased permeability of chloride from the intestinal crypt cells. 39 Several tests have been devised to test for the presence of LT - passive immune hemolysis (Evans and Evans, 1977) , radioimmunoassay (RIA) (Greenberg a 91;, 1977), enzyme linked immunosorbent assay (ELISA) (Svennerholm and Holmgren, 1978: Yolken 99 91;, 1977), Biken test (Honda 99 91;, 1981a,b), latex particle agglutination (Finkelstein and Yang, 1983) and DNA hybridization (Moseley 99 91;, 1980). Biological assays using animals (Moon and Whipp, 1971: Hamilton 99 91;, 1978: Burgess 99 91;, 1979) and -tissue culture (Donta 99 91;, 1974: Guerrant 99 91;, 1974) have also been used to detect LT. The Heat-Stable Enterotoxin. The second type of enterotoxin is ST. There is evidence that suggests that STs from different strains of ETEC represent a heterogenous group of E; 9911 (Guerrant 99 91;, 1975) . At least 2 distinct types of ST are known: 1) ST]; or STa which is methanol soluble, active in the infant mouse model and active in ligated jejunal segments of piglets 1 to 3 days old and 2) STII or 8Tb which is methanol insoluble, inactive in the infant mouse model and active in ligated intestinal segments of 7-9-week-old weaned pigs (Burgess 99 91;, 1978: Giannella, 1976: Kapitany 99 91;, 1979a,b: Newsome 99 91;, 1978). The structure of 8Tb has been shown by nucleotide sequencing to be distinctly different from that of STa (Lee 99 91;, 1983: Picken 99 40 91;, 1983), and the two are also immunologically unrelated. The 8T3:I toxin has 2 subclasses (Moseley 99 91;, 1980, 1983: So and McCarthy, 1980), 1 human (STh) and 1 porcine (STD) , which show a high degree of homology (Aimoto 99 91;, 1982: Chan and Giannella, 1981: So and McCarthy, 1980: Takao 99 91;, 1983). The STp, also known as STIaI is produced by porcine, bovine and human strains and STh, also known as STIb' is produced only by human strains. The STas of human, porcine and bovine origin have been purified, synthesized and sequenced (Aimoto 99 91;, 1982: Chan and Giannella, 1981: Lallier 99 91;, 1982: Lazure 99 91;, 1983, Ronnberg 99 91;, 1983: Saeed 99 91;,1983). The toxins consist of 18 or 19 amino acids (Lallier 99 91;, 1982) which share common core sequences and immunological domains. Twelve amino acids appear to be conserved with respect to their positions in the toxins (Lazure 99 91;, 1983: Staples 99 91;, 1980) . The ST is considered to be nonantigenic, although it has been shown to act as a hapten. It may be made antigenic by coupling it to a carrier protein (Alderete and Robertson, 1978: Frantz and Robertson, 1981) . According to Alderete and Robertson (1978) , the toxin has a molecular weight of 4.4 KDa. Consisting of a small family of polypeptides, STa polypeptides share 13 common amino acids in the region of the active site (Aimoto 99 41 91;, 1982: Chan and Giannella, 1981: Thompson 99 91;, 1985). The STa toxin, which has a high content of half- cysteines (Alderete and Robertson, 1978), has 3 disulfide bonds, some of which are important for its biologic activity (Chan and Giannella, 1981: Dreyfus 99 91;, 1983, 1984: Greenberg 99 91;, 1983b). The action of ST is almost instantaneous, relatively short lasting and readily reversible by rinsing (Evans 99 91;, 1973; Field 99 91;, 1978: Guerrant 93; 9.1.1: 1980: Hughes 99 91;_1978). In contrast to LT, the action of ST is primarily restricted to intestinal cells (Guerrant 99 91;, 1980: Rao 99 91;, 1980), but receptors for STa have also been found on rat basophil leukemia cells (Thomas and Knoop, 1983) . The Sta toxin has a much more pronounced effect on the small intestine and cecum than the colon (Guerrant 99 91;, 1980: Rao 99 91;.: 1980). The variation in the effect on the intestine is probably due to the tissue distribution of the ST receptors, whose molecular nature is unknown. However, the STa toxin is known to bind to brush border membrane receptors (Frantz 99 91;, 1984: Giannella 99 91;, 1983). 'It has been reported that the number of receptors per microgram of membrane protein is greater in infants than in older children and that the number of receptors decreases rapidly with age (Cohen 99 91;, 1988). 42 Utilizing various assay systems, Sta does not appear to activate adenyl cyclase (Hamilton 99 9L, 1978: Sack and Sack 1975: Smith and Gyles, 1970). The mechanism of action of STa involves the activation of guanylate cyclase in small intestinal epithelial cells (Field 99 91;, 1978: Hughes 99 91;, 1978: Newsome 99 91;, 1978b). Guanylate cyclase activity in the rat small intestine occurs mainly in the intestinal microvillus, with considerably greater activity at the differentiated villus tip than in the crypts (de Jonge, 1975). The STa toxin ultimately leads to a decrease in sodium chloride absorption and an increase in chloride secretion (Guandalini 99 91;, 1982: Rao 99 91;, 1980). There are several postulated pathways for the activation of guanylate cyclase. The STa toxin may interact directly with the transmembrane guanylate cyclase receptor located in intestinal microvilli (de Jonge, 1975, 1984). Another suggested pathway involves the activation of a calcium/calmodulin-dependent phospholipase A2 via STa to produce arachidonate. Arachidonate then proceeds by' the cyclooxygenase or lipoxygenase pathways to produce a free radical that activates guanylate cyclase (Guerrant 99 91;, 1980: Greenberg 99 91;, 1980, 1982a: Knoop and Abbey, 1981: Knoop and Thomas, 1983) . A report by Dreyfus 99 91; (1984) disputes this theory however. 43 Subsequent to the activation of guanylate cyclase, a protein kinase and calcium have been implicated in the events that lead to secretion. There are 2 specific cyclic guanosine monophosphate (cGMP) -dependent protein kinases. One, G-kinase, type II described by de Jonge (1984) is found only in intestinal microvilli. The G- kinase is an 86 KDa protein which can be split into 71 and 15 KDa fragments. The larger fragment binds cGMP and catalyzes phosphorylation of specific sites on proteins. The smaller fragment contains 1 of 2 cGMP-dependent phosphorylation sites present in the intestinal microvilli. This self-phosphorylation site may provide an efficient step in the events that lead rapidly to active chloride secretion after the addition of STa (de Jonge, 1981) . The second cGMP-dependent phosphorylation site is on a 25 KDa protein that is also present on intestinal microvilli (de Jonge, 1984). This protein may be phosphorylated by either cAMP- or cGMP-dependent protein kinases. It is not known whether autophosphorylation of the 15 KDa fragment of the G-kinase or phosphorylation of the 25 KDa G-kinase substrate directly alters a nearby anion channel or whether cGMP, the G-kinase or the phosphorylated G-kinase substrate then subsequently changes calcium balance in the intestinal brush border to cause secretion. Some agents that affect calcium and calmodulin also affect secretory responses to ST or cGMP 44 analogues (Guerrant 99 91;, 1980: Greenberg 99 91;, 1980, l982a,b). The 8Tb toxin's mechanism of action is believed to be cyclic nucleotide independent (Kennedy' 99_ 91;, 1984). Like STa, STb has a rapid but reversible onset of action. 19 2199, 8Tb causes a significant increase in the amount of bicarbonate, sodium and chloride in the intestinal contents (Weikel 99 91;, 1986a) . The 8Tb producing E; 9911 strains are not believed to be a major cause of diarrhea in people (Weikel 99 91;, 1986b). Bioassays used for the detection of STa include the suckling mouse assay (Dean 99 91;, 1972) and the ligated pig loop assay (Smith and Gyles, 1970). Several other tests for the detection of STa have also been described, ELISA (de M01 99 91;, 1983, 1985, Klipstein 99 91;, 1984: Lockwood and Robertson, 1984: Thompson 99 91;, 1984), RIA (Dreyfus 99 91;, 1983: Frantz and Robertson, 1981: Giannella 99 91;, 1981) and DNA hybridization (Georges 99 91;, 1983: Moseley 99 91;, 1980: Seriwatana 99 91;, 1983). Also used for the detection of STa is an 19 39,919 assay which determines the activation of intestinal membrane guanylate cyclase (Waldman 99 91;, 1984). 45 Innate Resistance to E; 9911 Enteric Infections Neonatal pigs are highly susceptible to diarrhea caused by ETEC. Several innate mechanisms of resistance exist within the pig's intestine to inhibit the successful colonization of the small intestine by ETEC (Newby and Stokes, 1984). One of the first barriers that bacteria, such as E; 9911, must face is gastric acid. The low pH of the stomach acts as a bactericidal trap which limits the number of viable bacteria and viruses which can enter the intestine. At a pH of 3.0 the bactericidal effect is complete (Gray and Shiner, 1967) . The susceptibility of bacteria and viruses to low pH does vary, though. However, the pH of the neonatal pig's stomach is relatively high, it has been reported to be greater than 5.0 (Smith and Jones, 1963). Thus, in the neonatal pig more viable bacteria are able to enter the small intestine. Gut peristalsis is another important factor which limits the number of bacteria within the small intestine. Organisms entering the small intestine are rapidly removed by peristalsis (Dixon, 1960). Peristaltic contractions and villous pumping act as major defense mechanisms in the small intestine and thus it is essential, as in the case of ETEC, that bacteria adhere to the small intestinal mucosa to overcome this clearance mechanism (Moon, 1980). Mucus is also capable of acting as a protective agent 46 against bacteria in the small intestine. Flowing mucus and ingesta act to cleanse the epithelial surface (Moon, 1980). Mucus also entraps bacteria (Shrank and Verwey, 1976) which eases their removal. As mentioned previously, mucus contains substances that resemble epithelial receptor sites for bacteria, and these substances may also facilitate bacterial trapping (Strombeck and Harrold, 1974). The normal microflora of the small intestine acts to resist colonization by pathogenic bacteria. Suggested mechanisms via which normal bacteria prevent colonization by pathogenic bacteria include competition for space, nutrients or receptor sites. Also, the normal microflora may make the environment toxic to the pathogenic bacteria by altering the pH or producing toxic substances such as volatile fatty acids and colicins (Mushin 99 91;, 1970). Substances present in the intestine, such as lactoferrin and lysozyme, also have antibacterial effects. Lactoferrin, which is present in intestinal secretions (Masson 99_91;, 1966), successfully competes for iron with E; 9911 and thus has a bacteriostatic effect on E; 9911. Lysozyme, produced by the Paneth cells of the small intestine (Sandow and Whitehead, 1979), also has antibacterial effects. Interferon plays an important role in antiviral defense in the small intestine. 47 Immunoglobulins present in intestinal secretions play a role in the protection of mucosal surfaces from bacteria. The major antibody, in porcine intestinal secretions is immunoglobulin A (IgA) . Secretory IgA interferes with the binding of microorganisms and their products to epithelium. Thus, IgA could prevent diarrhea due to ETEC by inhibiting the adhesion of E; 9911 and the binding of its enterotoxins to intestinal epithelial cells. The Igs, IgG and IgM, may also play roles in the protection of the small intestine against bacteria (Newby and Stokes, 1984). As pigs age, they become resistant to ETEC (Moon and Whipp, 1970: Nielsen 99 91;, 1968: Smith and Halls,,1967). Diarrhea caused by K88-positive ETEC may occur during the neonatal and postweaning period, but diarrhea due to K99- positive ETEC is not known to occur after weaning in pigs (Moon, 1978b: Sojka, 1965). The mechanism of age resistance is unknown. Some pigs may be born resistant to diarrhea caused by K88-positive ETEC (Gibbons 99 91;, 1977: Sellwood 99 91;, 1975) . British researchers have determined that the presence or absence of receptors for the K88 pilus of E; 9911 is inherited. Two alleles are present at a single locus and are inherited in a simple Mendelian manner. One allele, the allele coding for the receptor, is dominant over the other allele. Three genotypes occur: homozygous 48 dominant (SS, susceptible, adherent), heterozygous (Ss, susceptible, adherent) and homozygous recessive (ss, resistant, nonadherent). Two phenotypes occur, susceptible and resistant. The susceptible or positive phenotype is the expression of the dominant allele (Gibbons 99 91;, 1977: Sellwood 99 91;, 1975). Phenotypic expression is independent of the age of the pig and is fully established at birth (Gibbons 99 91;, 1977). Sellwood ( 1980a) has also described another phenotype as weak adhesive which is probably coded for by a third allele. With the weak adhesive phenotype, very few K88- positive bacteria adhere to the intestinal brush border 19 11999, compared to the number of bacteria attached to brush borders of the original susceptible phenotype. The phenotype can be determined by using a simple 19 91999 technique described by Sellwood 99 91; (1975) which demonstrates the adhesion of K88-positive E; 9911 to brush borders from pig intestinal cells. Adhesion of bacteria to brush borders occurs in pigs which have the receptors (positive pigs) and no adhesion occurs in pigs lacking the receptors (negative pigs) (Sellwood 99 91;, 1975). Rutter 99 91; (1973) have shown experimentally that negative pigs are resistant to infection by K88-positive E; 9911. The K88-positive bacteria colonized the gut of positive pigs more readily than the gut of negative pigs, and the positive piglets were more likely to be 49 susceptible to diarrhea caused by E; 9911. In negative piglets, the organisms were unable to attach and rapidly disappeared from the intestines (Rutter 99 91;, 1973) . Sellwood (1979) reported that in a natural outbreak of scours, negative piglets were resistant to diarrhea caused by E; 9911. However, susceptibility to infection is more complicated in a clinical situation. Positive piglets receiving colostrum or milk from the dam that contains K88 antibodies are passively protected (Rutter and Jones, 1973). It is assumed that positive piglets are susceptible to infection, but passive protection due to K88 antibodies in the colostrum prevents ETEC attaching and multiplying to high numbers in the small intestine. The negative phenotype primarily determines whether an animal will be resistant to infection with K88-positive strains of E; 9911, but if the positive animals receive antibodies, they may also be resistant to infection (Rutter 91; all, 1973) . More recently, Bijlsma 99, 91; (1982) in the Netherlands, found that there are 5 phenotypes in swine based on the 3 serologic variants of the K88 antigen. The phenotypes differ depending upon whether or not a pig is susceptible to adhesion in the brush border test to 3 (phenotype A), 2 (phenotype B and C) or 1 (phenotype D) of the serologic variants of the K88 pilus or resistant to all 3 variants (phenotype E). Sellwood's adhesion- 50 negative phenotype corresponds to the D and E phenotypes, because the K88ad serologic variant of the E; 9911 was not recognized when he performed his study. The existence of 5 phenotypes complicates the rather simple genetic model proposed by British researchers, and additional work is needed to determine the inheritance pattern of the 5 phenotypes. WOrk by the Rapacz and Hasler-Rapacz (1986) suggests that phenotypes A and B correspond to 2 haplotypes with genes at 2 or 3 closely linked loci or that the phenotypes are expressions of alleles at 1 locus, with each allele specifying a receptor able to bind 2 or 3 different serological variants of the K88 pilus. Moon 99 91; (1979) stated that they have not encountered pigs congenitally resistant to colonization by E; 9911 carrying the K99 or 987P pili. However, using isolated small intestinal epithelial cells, from pigs 1 day, 3 weeks and 6 weeks old, Runnels 99 91; (1980) demonstrated resistance with age to K99-positive E; 9911. From 8.8 to 14.5 more K99-positive ETEC were shown to adhere to 1-day-old piglet epithelial cells than to 6- week-old pig epithelial cells. The resistance to adhesion wasn't demonstrable with 3-week-old pigs. 51 Immunoprophylaxis E; 9911 vaccines Both pili and enterotoxins are being used as immunogens for ETEC vaccine development. Pilus Vaccines. Currently, several pilus vaccines have been developed for the protection of pigs against neonatal enteric colibacillosis. Pili act as important antigens for vaccine development because they are present on the bacterial surface, thus making them readily accessible to antibody and also because pilus production is an essential step in the early pathogenesis of enteric colibacillosis. Vaccines are administered parenterally or orally to sows prior to farrowing (Moon, 1981). The vaccine induces protective levels of antibody in the sow's colostrum and milk which provide passive immunity to the newborn pig. Research has demonstrated that protection correlates with antipilus antibodies in the colostrum and milk (Acres 99 91;, 1978: Isaacson 99 91;, 1980: Morgan 99 91;, 1978: Nagy 99 91;, 1978: Rutter and Jones, 1973: Rutter 99 91;, 1976) . Several researchers believe that colostral antibodies prevent the colonization of ETEC by blocking adhesion to specific epithelial receptors (Nagy 99 91;, 1978: Rutter, 1975: Rutter and Jones, 1973: Rutter 99 91;, 1976). Without successful colonization, the ETEC are not able to produce a sufficient amount of enterotoxin to cause diarrhea. Piglets suckling 52 vaccinated dams are more resistant to infection by ETEC than control pigs when challenged by ETEC with homologous pili (Moon, 1981). According to several researchers, the mortality, morbidity and the duration of diarrhea are decreased in piglets suckling vaccinated dams (Morgan 99 91;, 1978: Nagy 99 91;, 1978: Rutter 99 91;, 1976). Also, the number of E; 9911 attached to the villous epithelium of the small intestine is decreased (Nagy 99 91;, 1978). Several vaccines which use either purified or semipurified pili (Acres 99 91;, 1979: Morgan 99 91;, 1978: Myers, 1978: Myers and Guinee, 1976: Myers 99 91;, 1973: Nagy 99 91;, 1985: Rutter and Jones, 1973: Sojka 99 91;, 1978) or whole bacterial cell preparations have been developed. Since pigs may be infected by strains of ETEC with different pilus antigens, multivalent vaccines are necessary to provide protection from enteric colibacillosis. Initially, whole cell bacterins were used to protect pigs against enteric colibacillosis (Kohler, 1974: Kohler 99 91;, 1975: Rutter and Anderson, 1972: Wilson, 1974). Pitman-Moore has developed a trivalent bacterin called Porcimune which contains K88ac, K99 and 987P piliated strains of E; 9911 (Anonymous, 1981) . Pitman-Moore has also developed an E; 9911 bacterin containing 4 piliated strains of 9; 9911, K88, K99, 9379 and F41 (To, 1984a). Recently, a multiple pilus phase cloned whole cell 53 bacterin containing K88ac, K99, 987P and type 1 pili has also been shown to be protective against ETEC under natural and laboratory conditions (Brinton 99 91;, 1983: Jayappa 99 91;, 1983, 1984). Subunit vaccines, consisting of pili only, have also been developed. A trivalent subunit vaccine, containing K88, K99 and 987P pili, which utilizes recombinant DNA gene splicing techniques has been made by Salsbury. The plasmids responsible for pilus production are introduced into a laboratory strain of E; 9911. The laboratory strain of E; 9911 then produces many more pili than are normally produced by a wild strain of E; 9911. The pili are sheared from the bacterial surface by mechanical means and incorporated into the vaccine. The endotoxins associated with bacterins, which may cause adverse effects such as allergies, shock and abortions, are excluded from subunit vaccines (Anonymous, 1983) . A subunit bacterin containing K88, K99, 987P and F41 pili has also been produced (Schlink, 1985). Enterotoxin Vaccines. Toxoid vaccines have been developed against LT and ST. For an E; 9911 toxoid vaccine to provide complete protection against any strain of ETEC, regardless of its somatic serotype or pilus antigens, it should contain both LT and ST (Klipstein 99 91;, 1982). 54 Piglets suckling sows parenterally’ immunized. with porcine LT have been protected from ETEC diarrhea (Dobrescu and Huygelen, 1976: Dorner 99 91;, 1980). A vaccine containing procholeragenoid, an attenuated form of cholera toxin, has also been shown to be protective against enteric colibacillosis (Frantz and. Mellencamp, 1984: Furer 99 91;, 1982, 1983a,b). Vaccines for STa have also been developed. The STa enterotoxin is nonantigenic, but is capable of acting as a hapten and has therefore been conjugated to carrier proteins such as bovine serum albumin or Ig (Frantz and Robertson, 1981: Giannella 99 91;, 1981). In a study by Moon 99 91; (1983) where pregnant swine were immunized with STa coupled to bovine Ig, suckling pigs were not protected against challenge with ETEC producing ST alone. The vaccine stimulated the production of antibodies with high binding activity but low neutralizing activity and thus low protective activity for suckling pigs. An experimental synthetic STa vaccine has been produced. However, protection from disease was only partial and not as effective as vaccination with whole cell bacterins (Frantz 99 91;, 1987). Immunization of animals with LT, its 8 subunit or ST provides protection against ETEC that produce homologous but not heterologous enterotoxins (Klipstein 99 91;, 1983a,b). Therefore, experimentally, vaccines containing 55 ST chemically coupled to LT have also been developed (Klipstein 99 91;, 1982, 1983a,b). A completely synthetic toxoid vaccine containing ST and antigenic determinants of the LT toxin's B subunit has recently been developed (Houghten 99 91;, 1985). Passive Immunization Immunologicallyy the newborn pig is relatively defenseless and thus highly susceptible to infection. Passive immunization, via the absorption of antibodies from colostrum and milk, of the neonatal pig prior to its exposure to an infectious agent protects the pig from infection. The correlation of the 3 ages of peak incidence of porcine enteric colibacillosis, neonatal, 3 weeks and immediately postweaning, with periods of antibody deficiency indicate that such a deficiency predisposes pigs to ETEC (Moon, 1974). Since adhesion of ETEC to the intestine is an essential prerequisite for the development of enteric colibacillosis, antibody-mediated prevention of bacterial adhesion is a potential defense mechanism against E; 9911. Such antibody’ may be acquired from colostrum and milk. Serum, Colostrum and Milk. There have been several reports in the literature demonstrating the effectiveness of hyperimmune serum, colostrum and milk against experimental challenge with ETEC. The predominant Ig in porcine serum and colostrum is IgG, however IgA is the 56 predominant Ig in milk and intestinal secretions (Porter, 1969a,b: Porter 99 91;, 1970a,b: Curtis and Bourne, 1971: Bourne, 1976). As early as 1966, Kohler and Bohl showed that orally administered serum, obtained from hyperimmunized gnotobiotic pigs, had a protective effect against E; 9911 in infected pigs. Four- to 6-day-old gnotobiotic pigs were fed 7 ml of hyperimmune serum in milk one and a half hours prior to challenge. The serum in milk was then administered every one and a half hours for 30 to 36 hours. No clinical signs of diarrhea were observed during the period the serum was administered. However, the immune serum only had a temporary protective effect: 12 to 24 hours after the serum was last given, diarrhea ensued. Heated antiserum was administered in a similar manner with similar effects. Three and a half milliliters of immune serum in milk were also administered to a few pigs during a 24 hour period with a comparable temporary protective effect. Results indicated that the hyperimmune serum's protective action was apparently not dependent upon the complement-antibody bactericidal system, but was thought at that time to be related to the inactivation of toxin in the intestinal lumen. In another study by Kohler (1967), the effects of parenterally, as well as orally, administered antisera, produced in conventional swine, were evaluated in 57 gnotobiotic pigs infected with E; 9911. Not only were the animals observed clinically, but gross, histologic and microbiologic observations were also made. The protocol for oral administration of antiserum was the same as that previously described (Kohler and .Bohl, 1966) . For the parenteral administration of antiserum, 14 ml of antiserum were injected intraperitoneally 16-20 hours prior to oral infection. Pigs were killed at predetermined time intervals between 6 and 48 hours postinfection. Orally administered antisera protected pigs from fluid loss and diarrhea. With the parenterally administered serum, diarrhea was not observed, but there was fluid loss into the intestinal lumen. The mechanism of protection did not appear to involve a marked reduction in the number of live E; 9911 in the small intestine. In 1968, Rejnek 99 91; fed colostrum and serum from immunized sows to gnotobiotic pigs infected. with the homologous E; 9911 strain. Two hours after challenge, 3- day-old pigs were given 12.5-50 ml immune colostrum or serum. Control pigs were given serum from nonimmunized sows. Pigs fed colostrum or serum from immunized sows survived whereas the controls died. Miniats 99 91; (1970) attempted to protect pigs against enteric colibacillosis by orally administering E; 9911_antisera from immunized specific pathogen free pigs. Six-day-old gnotobiotic pigs were given 4 ml of antiserum 58 or normal serum 30 minutes prior to challenge with homologous E;, 9911, Thereafter, the serum was administered at 8 hour intervals for 3 consecutive days. Antiserum directed against 1 strain of KB8ab-positive E; 9911 used was demonstrated to have a protective effect. The 3 pigs receiving specific antiserum against the homologous strain of E; 9911 with which they were infected remained healthy while the serum was administered, but 2 of the 3 pigs died 2 days following the withdrawal of the serum. Antiserum against the K88ab-positive strain of E; 9911 did not significantly decrease the number of E; 9911 present in the intestine between treated and control animals, suggesting that the protective effect of the antibody in the intestine was due to its action on the enterotoxin. Svendsen and Wilson (1971) observed that feeding colostral whey or serum from sows intramammarily or intramuscularly vaccinated with a live formalin-attenuated K88ac-positive E; 9911 vaccine to gnotobiotic pigs infected with E; 9911 was protective, as demonstrated by an increased survival time. Pigs given the colostral whey from vaccinated sows also had a delayed onset of diarrhea. However, those receiving colostrum from nonvaccinated sows did not. Ten-day-old gnotobiotic pigs were fed colostrum or serum (10 or 40 ml) 6 hours prior to challenge and at 8 hour intervals following the first feeding for 3 days. 59 Diarrhea appeared in all infected pigs except those treated with colostrum from sows vaccinated by the intramuscular route, in which it was not observed until 24 hours after the cessation of colostrum feeding. Results indicated that in order for antibody to be effective, it must be continually present in the intestinal tract of pigs. In an associated study, whey prepared from milk collected 7 days after farrowing increased the survival time but did not delay the onset of diarrhea in infected gnotobiotic pigs (Wilson and Svendsen, 1971) . Control pigs were given milk whey from nonvaccinated sows or condensed milk. The protocol was similar to the previous experiment. All the pigs developed profuse diarrhea 8 to 16 hours after infection, and the diarrhea continued throughout the experiment with no difference in the severity of diarrhea or the rapidity of onset of dehydration between pigs in the different treatment (10 or 40 ml) groups. The pigs fed 40 ml of milk whey from vaccinated sows showed an increased survival time. In another study by Wilson (1972) , colostrum from immunized sows or IgG isolated from that colostrum was orally administered to gnotobiotic pigs. Based on a prolonged survival time, significant protection of the pigs from enteric colibacillosis was reported. Smith and Linggood (1971b) parenterally administered 125 to 200 ml of E; 9911 antiserum, depending upon the 60 size of the pig, to conventionally reared weaned pigs. The administration of antiserum usually prevented the pigs from developing diarrhea or edema disease after infection with K88-positive E; 9911. Protection was only demonstrated when animals were infected with the homologous strain of E; 9911, and protection appeared to correlate with a failure of the infecting organism to proliferate in the intestine. Protection was believed to be primarily bactericidal, but an antienterotoxic effect was also suggested. In a study by Smith (1972), colostrum-deprived pigs were given 20 ml of antiserum in milk orally, and then 2 hours later the pigs were infected orally with K88-positive ETEC and a larger number of nonpathogenic E; 9911 and lactobacilli. Controls were given normal serum. Results of the study indicated that antiserum prepared against live, heat-killed or methanol- killed bacteria of the 0141:K85ab, 88ab strain inhibited the multiplication of homologous ETEC in the small intestine of pigs given the antiserum. This effect, considered to be antibacterial, was felt to be responsible for delaying or preventing the onset of diarrhea. An antienterotoxic effect didn't appear to be present since antiserum against the enterotoxin did not delay the onset of diarrhea caused by E; 9:91 0141:K85ab, 88ab. Antisera against the O or other K antigens of the strain 0141 also had no effect. However, antisera against the pathogenic 61 E; 9911 strain 08:K87, 88ab had a diarrhea-controlling effect, apparently attributable to the K88ab antigen common to both strains of E; 9911. Smith postulated that the protective effect was associated with the inhibition of adhesion by the K88 antigen of E; 9911. Antiserum was also administered subcutaneously, it had a controlling effect on diarrhea but it was less effective than antiserum administered orally. Parenteral therapy with serum has indicated that antibody can pass into the intestinal tract of pigs and possibly contribute to local immunity (Murray, 1973). In a study by Morilla 99 91; (1984) serum from adult pigs was given orally to neonatal pigs 8 hours after birth. The treated animals had a better appearance, a higher body weight, more stamina and less diarrhea than the controls. Recently, Bar-Guard-99, an oral calf serum from Anchor, has been used commercially for the prevention of enteric colibacillosis in calves. Bar-Guard-99 contains whole cell antibodies to K99-positive E; 9911 and thus provides polyclonal protection against enteric colibacillosis. A company trial indicated that the survival rate of animals receiving Bar-Guard-99 was twice that of animals receiving a K99 MCA product (Anonymous, 1987). 19 21999, the Fab fragments of antibody specific for 987P pili have been shown to block the adhesion of 987P- 62 positive ETEC to pig small intestinal cells (Isaacson 99 91;, 1978). Also using intestinal epithelial cells, Wilson and Hohman (1974) inhibited the adhesion of E; 9911 bearing the K88ab or K88ac antigen with homologous antisera against the K88 b or c antigens. Antisera against K88-positive E; 9911 also inhibited the adhesion of E; 9911 to disks of porcine intestinal mucosa obtained from gnotobiotic pigs (Jones and Rutter, 1972). Parry and Porter (1978) were able to block the adhesion of K88ab- and K88ac-positive strains of E; 9911 to brush borders by using antisera specific for the K88a determinant. Antibodies against K88b and K88c antigens only inhibited homologous strains of K88-positive E; 9911. Colostrum or milk from sows vaccinated orally with live cultures of E; 9911 also protected gnotobiotic piglets against diarrhea when challenged with the homologous ETEC (Kohler, 1974). The pigs were fed colostrum or milk, infected 26 hours after birth and then were continuously fed milk or colostrum till 48 hours of age. Results of preliminary experiments indicated that better protection was provided pigs fed mammary secretions from the first feeding than was provided by pigs initially fed the mammary secretions only 3 hours before infection. The colostrum and milk inhibited the multiplication of a large number of both the homologous 63 K88-positive and homologous K88-negative strains on the intestinal mucosa. Scoot 99 91; (1972) gave gamma globulins orally to piglets. When the globulins were given for only 1 day, the piglets' serum IgGs rose to near normal levels, but the piglets died of colibacillosis. However, when they were given gamma globulins orally for a minimum of 10 days, colibacillosis did not occur. Miler 99 91; (1975) determined the protective effect of porcine colostrum, serum, IgG, IgM and IgA against an enterotoxic strain of E; 9911 in newborn germfree pigs. They reported that IgA isolated from immune porcine sera and colostrum was effective at a lower concentration against ETEC in ligated pig loops than IgG or IgM isolated from the same sources. It has been reported that the protective effect of human milk against gastrointestinal illness in infants (Chandra, 1979: Cunningham, 1979: Gerrard, 1974: Jason 99 91;, 1984: Kovar 99 91;, 1984: Larsen and Homer, 1978) may be due to specific antibodies (Glass 99 91;, 1983: Majumdar and Ghose, 1982: Stoliar 99 91;, 1976) as well non- immunoglobulin components (Goldman and Smith, 1973: Holmgren 99 91;, 1983: Welsh and May, 1979) . 19 21919 studies have also demonstrated the anti- adhesive activity of colostrum and milk. In a study by Rutter 99 91; (1976) colostral antibody inhibited the adhesion of K88-positive E; 9911 to slices of intestinal 64 tissue. Antibody from sow's milk has been shown to inhibit the attachment of K88-positive E; 9911 to pig intestinal epithelial cells (Evans 99 91;, 1980). Using pigs from a herd which had acquired natural immunity to K88-positive E; 9911, Sellwood (1984b) reported that colostrum from genetically susceptible sows in the herd inhibited the binding of radiolabelled K88 antigens to brush borders better than colostrum from genetically resistant dams. The colostrum from susceptible dams was also more efficient in 19 21999 opsonic phagocytosis and killing of K88-positive E; 9911 than colostrum from resistant sows. Also, antibodies from sheep colostrum inhibited the attachment of K99-positive E; 9911 to sheep intestinal epithelial cells (Morris 99 91;, 1980b). Monoclonal Antibody. Recently, an alternative method for the direct passive immunization of newborn animals against ETEC, the oral administration of E; 9911 pilus- specific, hybridoma-derived MCA, has been developed. Since Kohler and Milstein (1975) first produced hybridomas, there have been amazing advances in biomedical research, immunodiagnostics and immunotherapy. In the area of infectious diseases, MCAs have been used to determine antigenic structure, virulence mechanisms, and host responses to a wide variety of bacteria, viruses and parasites. Numerous advances in immunodiagnosis, prophylaxis and therapy have resulted. A recent review by 65 Sherman and Markham (1986) describes the current and future applications of MCAs against bacteria in veterinary medicine. Disease prevention by passive immunization is one possible clinical use of MCAs in veterinary medicine. Monoclonal antibody therapy might be of particular benefit when unexpected disease outbreaks occur in susceptible herds or flocks where a vaccination program has not been undertaken, effective vaccines are not available or the cost-benefit ratio of prophylaxis was not considered (Sherman and Markham, 1986) . Recently, there have been several reports in the literature concerning the passive immunization of neonatal animals against enteric colibacillosis. One of the first reports involved the oral administration of E; 9911 K99 MCA to calves (Sherman 99 91;, 1983) . Since calves and lambs are susceptible to K99-positive ETEC only during the first few days of life (Runnels 99 91;, 1980: Smith and Halls, 1967) it was assumed that, if antibody against the K99 pilus was present in the intestine during this time, colonization and thus diarrhea could be prevented until the animal became naturally resistant to infection by ETEC. Three separate challenge trials were used to determine the efficacy of the MCA. In the first trial, newborn calves were fed colostrum, treated with 1 ml of a 1:12,000 titer 66 K99 MCA at 10 hours of age and then challenged with K99- positive E; 9911 at 12 hours of age. The protocol in the other 2 trials was similar except that the calves were colostrum deprived. In the study, there was a statistically significant reduction in the mortality rate and the severity and duration of diarrhea in the treated calves versus the control calves. However, there was no difference in the incidence of diarrhea. Several explanations were postulated for the diarrhea which occurred in animals which received the K99 MCA. It was speculated that the amount of MCA used may not have completely blocked colonization. Also, it was suggested that the number of bacteria in the challenge inoculum might have been large enough to produce sufficient enterotoxin, without adhesion to the mucosa, to cause diarrhea. The previous study led to the development of a commercially available K99 MCA for calves, Genecol 99 (Molecular Genetics, Inc. Minnetonka, MN). Using Caesarian-derived, colostrum-deprived piglets, the oral administration of K99 MCAs has also been shown to be protective against challenge with K99-positive ETEC. Pigs were fed 100 mg of MCA and then challenged with 1 x1010 K99-positive E... 9911. Eleven of 14 pigs (79%) which received the MCAs survived the challenge in comparison to only 2 of 10 survivors (20%) in the unprotected group (Sadowski 99 91;, 1983). In a similar study, newborn 67 calves were protected against challenge with K99-positive E; 9911 by the oral administration of 20 mg of K99 MCA. Six of 7 (86%) treated calves survived compared to 2 of 8 (25%) survivors in the control group (Sadowski 99 91;, 1983).. In another study,’ 8 hysterectomy derived, colostrum-deprived pigs were used: 4 pigs received K99 MCA and 4 received K88 MCA. The pigs received the MCA 3 times a day mixed with milk replacer from 2 through 7 days of age and were observed for 17 days after challenge. The pigs were challenged with a K99-positive strain of E; 9911 just before their second MCA feeding at 2 days of age. All the challenged pigs developed diarrhea and 1 pig in each group died. The diarrhea started later and ended earlier in the pigs fed K99 MCA compared to those fed K88 MCA. The mean concentration of available K99 antigen in the feces of pigs fed K99 MCA was lower than in those receiving K88 MCA in the initial days of the study (Mainil ‘99 91;, 1987). The K99 MCA used in all the previously- described studies was obtained from the same hybridoma. Neonatal, colostrum deprived pigs have been protected from fatal colibacillosis due to K88-positive ETEC through the use of ”a” and ”c" specific MCAs to the K88 pilus (Sadowski, 1984). In an experimental trial, a K88 MCA against all 3 serologic variants was orally administered to newborn pigs. A therapeutic effect was noted, but there was no prophylactic effect. Pigs given a 68 therapeutic treatment with the MCA (29 pigs), with or without a preceding prophylactic treatment, had a zero mortality rate. However, in 1 litter, where the antibody was only administered prophylactically, all of the animals died. In.the control group, which was not treated, 9 of 12 animals died (Foged 99 91; , 1986). A 987P MCA has also been used experimentally for the passive immunization of colostrum-deprived neonatal pigs. Seven of 11 treated animals survived challenge (64%) with 987P-positive E; 9911 compared'to 1 of 11 (9%) of the control animals. The 987P MCA was also used in a field outbreak of enteric colibacillosis caused by 987P- positive E; 9911. Ten litters were equally divided between placebo and control animals. The placebo treated animals had a higher mortality rate, 24 of 44 pigs (54%), than the treated animals, 8 of 44 (18%). The 987P pilus levels in the feces of the MCA-treated animals was lower than in the control animals (Sadowski 99 91;, 1986). Molecular Genetics has evaluated the efficacy of a trivalent MCA mixture containing antibodies against K88, K99 and 987P pdli against porcine enteric colibacillosis (Sherman and Markham, 1986). CHAPTER 1 THE EFFECT OF 987P MDNOCLONAL.ANTIBODY ON THE ADHESION OF 987P-POSITIVE E§QflEBlQflIA.QQL1 TO PIG INTESTINAL BRUSH BORDERS CHAPTER 1 THE EFFECT OF 987P MONOCLONAL.ANTIBODY ON THE ADHESION OF 987PbPOSITIVE E§QEEBLQEIA.QQLI TO PIG INTESTINAL BRUSH BORDERS INTRODUCTION Adhesion to the small intestinal mucosa is an essential prerequisite for the development of diarrhea due to ETEC in animals. Current knowledge indicates that pili present on the bacterial surface allow E; 9911 to attach to specific receptors present on the small intestinal mucosa. Receptors for the various pili present on strains of ETEC differ. 19 2129, antibody, both naturally acquired and passively acquired through colostrum and milk from pilus vaccinated dams, has been shown to be ‘protective against experimental and clinical ETEC infections. It is theorized that pilus specific antibodies prevent piliated bacteria from attaching to the small intestinal mucosa, thereby preventing subsequent steps in the pathogenesis of enteric colibacillosis. Evidence supporting the antiadhesive effect of pilus- specific antibodies has been demonstrated 19 21999 utilizing various assay systems. 70 71 Several 19 21999 techniques have been described which demonstrate the adhesion of piliated E; 9911 to the small intestine, as well as to oral epithelial cells and erythrocytes. Examples of the various methods are the brush border technique (Sellwood 99 91;, 1975) , the intestinal epithelial cell technique (Isaacson 99 91;, 1978: Nagy 99 91;, 1977: Wilson and Hohman, 1974), the intestinal villous technique (Girardeau, 1980), the immobilized intestinal mucosa technique (Laux 99 91;, 1986), the buccal epithelial cell technique (Gibbons and van Houte, 1971), an indirect enzyme linked immunosorbent assay (Schifferli 99 91;, 1987) and hemagglutination assays (Evans 99 91;, 1979). Several researchers have demonstrated that the 19 21999 adhesion of piliated E; 9911 to the small intestinal mucosa can be inhibited by antipilus antibodies in colostrum and milk. In a study by Rutter 99 91; (1976), colostral antibody inhibited the adhesion of K88-positive E; 9911 to discs of intestinal tissue. Antibody from sow's milk has been shown to inhibit the attachment of K88-positive E; 9911 to pig intestinal epithelial cells (Evans 99 91;, 1980). Using pigs from a herd which had acquired natural immunity to K88-positive E; 9911, Sellwood ( 1980a) reported that colostrum from genetically susceptible sows in the herd inhibited the binding of radiolabelled K88 antigens to brush borders better that 72 colostrum from genetically resistant dams. At concentrations of > 1mg/ml, colostral fractions from susceptible sows containing IgM or IgA caused almost 100% inhibition of adhesion, and fractions containing IgG were less effective in inhibiting adhesion. In a later study by Sellwood (1984b), IgG did not appear to act as an antiadhesin. The colostrum from susceptible dams was also more efficient in 19 21999 opsonic phagocytosis and killing of K88-positive (E; 9911 than colostrum from resistant sows (Sellwood, 1980a). Also, antibodies from sheep colostrum inhibited the attachment of K99-positive E; 9911 to sheep intestinal epithelial cells (Morris 99 91;, 19801)) . Antisera have been used to prevent 19 21999 adhesion. Jones and Rutter (1972) used antiserum against K88-positive E; 9911 to inhibit the adhesion of E; 9911 to disks of porcine intestinal mucosa obtained from gnotobiotic pigs. The Fab fragments of antibody specific for 987P pili have been shown to block the adhesion of 987P-positive ETEC to pig small intestinal cells when the bacteria were preincubated with the Fab fragments (Isaacson 99 91;, 1978). Using intestinal epithelial cells, Wilson and Hohman (1974) inhibited the adhesion of E; 9911 hearing the K88ab or K88ac antigen with homologous antiserum against the K88 b or c antigens. Rabbit antiserum was preincubated 73 for 30 minutes at 37 'C with the K88-positive bacteria prior to the addition of the enterocytes. No inhibition of adhesion was seen with heterologous K88 antiserum or with K99 antiserum. Parry and Porter (1978) were able to block the adhesion of K88ab- and K88ac-positive strains of E; 9911 to brush borders by using antisera specific for the K88a determinant. Antibodies against K88b and K88c antigens only inhibited homologous strains of K88-positive E; 9911. Using rabbit K88 antiserum, Sellwood .99 ‘91; (1975) also demonstrated that adhesion of K88-positive E; 9911 to brush borders could be inhibited by the addition of rabbit antisera against the K88 pilus. Several dilutions of the antisera were preincubated with the bacteria at room temperature for 1 hour prior to the addition of the brush borders. Antisera directed against type 1 pili of E; 9911 have been shown to inhibit attachment of E; 9911 to mammalian cells 19 21999 (Isaacson 99 91;, 1978: Abraham 99 91;, 1983: Guerina 99 91;, 1983: Salit and Gotschlich, 1977: Silverblatt and Cohen, 1979: Silverblatt 99 91;, 1982). Antiserum to E; 9911, mannose-sensitive pili inhibited the attachment of piliated bacteria to buccal epithelial cells (Weinstein and Silverblatt, 1983). It was assumed that the antibody inhibited adhesion by obstructing the mannose-specific binding domains of the pili. However, it was also suggested that the anti-pilus 74 antibody may not have directly inhibited adhesion but may have reduced the number of potentially adhering bacteria by agglutination. Adherence was also prevented. by monovalent Fab' fragments of IgG, and monovalent antibody fragments can bind to pili but are unable to cause agglutination. Also in this study, the inhibition of bacterial adherence was not augmented by complement- dependent bacteriolysis, opsonophagocytosis or intravascular clearance. Using antiserum prepared against type 1 pili Isaacson 99 91; (1978) inhibited the attachment of E; 9911 to porcine intestinal epithelial cells. In a study by Abraham 99 91;_(1983), MCAs to type 1 pili were shown to inhibit the attachment of type 1 pili 19 21999 to oral epithelial cells, guinea pig erythrocytes and yeast cells. One of the MCAs was quaternary structure-specific, only recognizing polymers of z 6 subunits. The other MCAs were subunit-specific and were directed toward antigenic determinants “hidden" in the quaternary structural conformation of the intact pili. Disassociation of the pili into subunits exposed the inaccessible determinants but eliminated the quaternary structural determinants. However, reassembly of the subunits 19 21999 restored the quaternary structural determinants but concealed the subunit determinants. 75 Only the quaternary structure-specific MCAs inhibited the adhesive properties of the pili to the cells 19 21999. In a study by Schifferli 99 91; (1987), an indirect ELISA adhesion assay was used to demonstrate the antiadhesive ability of 987P MCAs directed against various antigenic determinants of the pili. Three MCAs recognized exposed antigenic determinants present on intact pili. Two of the 3 antibodies demonstrated quaternary structural specificities, but one of the MCAs recognized assembled pili and individual pilus subunits. A fourth MCA was subunit-specific because it did not react with fully assembled pili. Piliated 987P-positive E; 9911 were preincubated for 1 hour with 987P MCAs at their ”lowest subagglutinating titer". The preincubated bacteria were then allowed to adhere to ileal and jejunal epithelial cells, isolated from 1- to 2-week-old pigs, immobilized in microtiter’ plate wells. Bacterial adhesion was then determined by the ELISA. Only antibodies that bound to intact pili were able to completely block 987P-mediated adhesion to piglet epithelial cells, suggesting that this characteristic may be a prerequisite to successfully prevent adhesion. In the case of 987P pili and type 1 pili, it appears that MCAs directed against pili must recognize quaternary structural antigenic determinants in order for the MCAs to successfully prevent adhesion. 76 Interestingly, the type 1 pili and 987P pili have similar helical structural conformations. The objective of this study was to determine if the 19 21999 adhesion of 987P-positive E; 9911 to pig intestinal brush borders could be prevented by the administration of E; 9911 987P MCA. MATERIALS AND METHODS Specimens Samples of ileum, each 6 to 8 inches in length, were obtained from 17 freshly killed slaughter-weight pigs at Michigan State University Meats Laboratory, East Lansing, MI. Additional ileal samples, 12 inches in length, were obtained from twelve 3-12-day-old pigs which were euthanatized with sodium pentobarbitala. Ileal samples ‘were carefully flushed 'with cold 0.15 M INaCl solution to remove mucus and ingesta. They were then placed in plastic bags and packed in crushed ice until further procedures were carried out, usually within an hour of the time of slaughter. Brush Border Preparations Intestinal specimens were processed using a modification of the procedure described by Sellwood 99 91; (1975) . In the laboratory, the intestinal specimen was a Butler Co., Columbus, OH. 77 ligated on one end, and the lumen was filled with an ethylenediaminetetracetate (EDTA) buffer solution (pH 6.8) until slight distention occurred. The other end of the intestine was then clamped with a hemostat. The buffer was at room temperature. To prevent drying of the external surface, sections of ileum were immersed in a similar solution containing 0.3 M sucrose instead of EDTA, (pH 6.8) for 15-20 minutes at room temperature. Next, the EDTA buffer in the intestinal lumen was discarded and replaced with the sucrose buffer until the lumen was half- filled. The epithelial cells were detached by gently massaging each intestinal section between the thumb and fingers. The intestinal contents were collected in 50 ml plastic centrifuge tubes. The intestine was again half- filled with sucrose buffer and massaged. The latter procedure was repeated until 25-40 ml of the epithelial cell suspension were obtained. The following steps were performed at 4°C. The epithelial cell suspension was centrifuged at 1200 x g for 15 minutesb. The supernatant was removed from each tube, and the pellet was resuspended to a volume of 45-50 ml with cold 0.005 M EDTA (pH 7.4, adjusted with 0.5 M Na2C03) . Next, the suspension was homogenized with a Teflon serrated-tipped tissue grinder (clearance - 0.15- b International Refrigerated Centrifuge, Model Pr-6, International Equipment Co. , Needham, MA. 78 0.23 mm)° by moving the pestle up and down 6 times while it rotated at approximately 900 rpm. The suspension was centrifuged at 300 x g for 5 minutes, and the supernatant was discarded. The pellet was resuspended in the EDTA buffer, then homogenization and centrifugation were repeated 3 to 5 times until the supernatant appeared clear. Next, the pellet was washed 3 times with Krebs- Henseleit (KH) buffer (pH 7.4). It was then resuspended to 8 times its volume with KH buffer and filtered through glass wool. The KH solution containing the brush borders was allowed to stand for 15 minutes to let any mucus present rise to the surface. The mucus was then discarded. For prolonged storage, part of the brush border suspension was mixed with an equal volume of glycerol and stored at -70'C (Bijlsma 99 91;, 1982) . Protein Assay A modified Lowry protein assay (Lowry 99 91;, 1951) was performed to determine the amount of protein present in the brush border preparations. Bovine serum albumin (BSA)d at a concentration of 1 mg/ml was used as a standard. Using 13 x 100 mm glass tubes, 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 ug protein were added to individual tubes. Distilled, deionized water was added to c Thomas Co., Philadelphia, PA. 9 Sigma Chemical Co., St. Louis, MO. 79 each of the tubes to give a total volume of 3.0 ml. Next, 20 pl of 2.5% sodium deoxycholate was added to each of the tubes, and the solution was well mixed. The tubes were then allowed to stand for 15 minutes. One milliliter of 24% trichloroacetic acid (TCA) was then mixed in each tube. The tubes were then centrifuged for 30 minutes at 3300 x 99. The Lowry protein assay was performed on the supernatant. Using 12 x 75 mm glass tubes, 100 pl of 1N NaOH and 100 p1 of distilled, deionized water were added to each standard sample. Distilled, deionized water was added to 25 pl and 50 pl samples of brush border suspension to produce a final volume of 100 pl. Next, 100 pl of 1N NaOH was mixed into each of the brush border samples. One milliliter of a freshly made solution, containing 98 parts 2% Na2C03, 1 part 2% sodium tartrate and 1 part 1% CuSO4, was added to each of the tubes. The tubes were then allowed to stand for 10-15 minutes. Next, 100 pl of phenol reagentf, diluted 1:1 with water, were added to each of the tubes. The phenol reagent mixture, which is light sensitive, was covered with foil while being aliquoted. The tubes were then allowed to incubate and the optical density of each tube was read in a e Beckman Model J-6B Refrigerated Centrifuge, Beckman Instruments Inc., Palo Alto, CA. f Harleco, Gibbstown, NJ. 80 spectrophotometerg set at 700 nm exactly 30 minutes after the addition of phenol reagent to that tube. E; 9911 Strains The 987P-positive E; 9911 used in the brush border adhesion test was strain 81-1469. The culture, obtained from Dr. David Francis, Department of Veterinary Science, South Dakota State University, Brookings, SD, was maintained in IMinca. broth (Guinee ‘99, 91;, 1977) with IsoVitalexh (MIB). Cultures of E; 9911 were aerobically incubated at 37‘C in a shaking water bath1 for 12-16 hours prior to testing for adhesion. The K88ac-positive strain of E; 9911, strain I 248 (0157:K?), was obtained from Dr. R.E. Isaacson, School of Public Health, University of Michigan, Ann Arbor, MI and was grown in a similar manner. Following incubation, both cultures were centrifuged at 1500 x g for 20 minutes. The supernatant was discarded, and the bacteria were washed in KH and centrifuged 3 times. The pellets were then resuspended to an optical 9 Beckman Spectrophotometer Acta III, Beckman Instruments Inc., Palo Alto, CA. h Baltimore Biological Laboratories, Cockeysville, MD. 1 Precision Scientific Co., Chicago, IL. 81 density between .075 and .085 at 560 nmj. The suspensions contained approximately 108 colony-forming units per ml. Slide Agglutination .A standard slide agglutination test was carried out to verify piliation using 987P and K88 antisera. Prior to this study, the antiserum was produced in Dr. G.L. Waxler's laboratory, Department of’ Pathology, Michigan State University, East Lansing“, MI. The antiserum was produced by hyperimmunizing rabbits with purified pili obtained from Dr. R.E. Isaacson. A 1:10 dilution of 987P or K88 antiserum was used for the tests. One drop of each of the bacterial suspensions was placed on a glass slide, a drop of antiserum was added, and the 2 drops were mixed with a wooden applicator stick. The glass slide was held near a light source with a magnifying mirror and slowly rocked and tilted in a circular motion for approximately 1 minute. Degrees of agglutination were recorded as strong, intermediate, weak or negative. Only those E; 9211 cultures showing strong or intermediate agglutination were used in the brush border adhesion test. A control slide using KH instead of antiserum was used to check each of the bacterial suspensions for autoagglutination. 3’ Shimadzu UV Visible Recording Spectrophotometer, Model UV-260, Shimadzu Corporation, Kyoto, Japan. 82 Monoclonal Antibody Product ion A 987P hybridoma, 4A3, was used in the production of 987P MCAs of the isotype 1961. Prior to this experiment, the hybridoma was produced at South Dakota State University in Dr. David A. Benfield's laboratory according to the method of Greenberg 93; 31; (1983a). Briefly, myeloma cells from the NS-l cell line were fused with spleen cells from Balb/c mice, immunized with the 987P pilus antigen. The NS-l myeloma cell line is a non- secreting clone of P3X63 Ag 8 myeloma and produces Kappa light chains (Kohler and Milstein, 1975). The hybridomas were grown as stationary suspension cultures in Dulbecco's Modified Eagle Medium containing L- glutamine and high glucosek (850 ml/l) supplemented with 10% heat-inactivated fetal bovine serumd, 0.45 mM/ml sodium pyruvated (10 ml/l), 100 units/ml penicillin Gk (5 ml/l) , 100 ug/ml streptomycink (5 ml/l) , bicarbonate (pH 7.5) (20 ml/l) and HEPESd (10 ml/l). Aseptic technique was used at all times when medium, and cultures were being handled. The medium was prepared in a horizontal laminar flow hood and filtered prior to its use with a 20 u disposable filterl. Polystyrene tissue culture flasksm k GIBCO, Grand Island, NY 1 Nalge Co., Rochester, NY. 83 containing the cell suspension were incubated in an automatic C02 incubatorn at 37'C in a humidified atmosphere with 5% C02 and 95% air. Cultures were examined using an inverted phase contrast microscope°. Under the laminar flow hood, the cell suspensions were split in half and then an equal volume of fresh medium was transferred aseptically to the culture flasks every other day. Cultures which were to be harvested were allowed to grow for ‘7 days without a change of medium and were then centrifuged for 10 minutes at 1000 x gP and the supernatant collected. The supernatant was then stored at 20'C. The specificity and titer of the antibody in the supernatant was checked at least once every 2 weeks using a bacterial slide agglutination test with 987?- positive E; coli. When a sufficient volume of supernatant was collected (approximately 1 liter) antibody in the supernatant was concentrated by an ammonium sulfate precipitation. A saturated solution of ammonium sulfate was prepared by adding approximately 1 Kg (NH4)ZSO4 crystals per liter of m Corning Glass Works, Corning, NY. n Queue Systems Dual Chamber Incubator, Model 2220, Parkersburg, West Virginia ° Model 1810, American Optical Corporation, Buffalo, NY. P Model HN-S Centrifuge, International Equipment Co., Needham Heights, MA. 84 distilled water. The solution was stirred at room temperature for 8 hours. When most of the crystals were dissolved, the solution was stored overnight at 4'C. The pH was then adjusted to 7.0 with 30% (weight/volume) NaOH. The saturated ammonimm sulfate solution was used to precipitate the MCAs from the tissue-culture medium. An equal volume of saturated ammonium sulphate solution was added to the tissue culture supernatant. The mixture was mixed immediately by swirling, and precipitation was allowed to occur for 1 hour. The entire procedure was carried out on ice at 4'C. The precipitate was collected by centrifugation at 48,000 x g for 30 minutesq. After centrifugation, the supernatant was discarded and the precipitate was dissolved in 0.14 M sodium phosphate (pH 8.0). Using cellulose dialysis tubing with a molecular weight cut off of 12,000 to 14,000r, the solution was dialyzed with 3 changes of 0.14 M sodium phosphate buffer (pl-I 8.0) . The antibody titer was then checked using a slide agglutination test. The MCA was further concentrated and purified using an affinity column chromatography procedure described by By gt a1; (1978). Protein A-Sepharose CL-4Bk was swollen in 10 mM phosphate buffered saline (PBS) (pH 8.0) q Beckman L2-65B Ultracentrifuge (SW27 rotor), Beckman Instruments Inc., Palo Alto, CA. r Spectra/Par 2 Dialysis Membranes, Spectrum Medical Industries Inc., Los Angeles, CA. 85 containing 0.1% sodium azide and packed in a column. The column, whose bed volume was approximately 6 ml (2 mg Protein A/ml gel) .was stored and used in a cold room (4'C). The buffer solutions used were 0.14 M sodium phosphate (pH 8.0) or 0.1 M sodium citrate/citric acid (pH 3.0 and pH 6.0) containing azide at 0.05%. Before using the column, it was washed with the pH 3.0 buffer to free bound material and then equilibrated at pH 8.0. The supernatant was applied to the protein A-Sepharose column. The IgG MCA was eluted with the 0.1 M citric acid buffer (pH 6.0). The eluate was monitored at an absorbance of 280 nmst. The flow rate was 0.5 ml/min and 2.0 ml fractions were collected. Appropriate fractions were pooled and neutralized with Tris buffer, pH 8.8. The antibody titer was determined using a slide agglutination test. Brush Border Adhesion Test The procedure described by Sellwood gt a1; (1975) was adapted. With Sellwood's procedure, bacteria and brush borders are incubated together to determine if the bacteria are able to adhere to the brush border fragments. The following test was used to determine if 987P MCA 3 Gilson Absorbance LC Detector, Model III, Gilson Medical Electronics Inc., Middleton, WI. t Gilson Microfractionator, Gilson Medical Electronics Inc., Middleton, WI. 86 would be able to inhibit the adhesion of 987P-positive E; 9911 to pig intestinal brush borders. The MCA was preincubated either with 987P-positive E; 9911 or with brush border fragments prior to the addition of the third component. The incubation of’ MCA ‘with brush border fragments before the addition of bacteria mimics a prophylactic situation in the live animal where the MCA would be present in the gut prior to infection. In the brush border adhesion test, pig intestinal brush borders and 987P-positive E; 9911 were mixed with several different concentrations of 987P MCA. A negative control, using K88-positive E; coli, was also used. With each ileal sample, 2 procedures were used. In the first, 0.1 ml of bacteria (987P- or K88-positive E; coli) was preincubated for 15 minutes with 0.1 ml of MCA and then mixed with the 0.1 ml of brush borders for 15 additional minutes. In the second procedure, MCA was added to the brush border preparation prior to the addition of the bacteria (Figure 1-1). The stock. MCA solution 'was diluted 1:10, 1:100, 1:1000 or 1:10,000. The titer of the MCA was redetermined prior to each test with a slide agglutination assay. The mixtures were incubated at 37°C with continuous gentle mixing. Each E; 9211 culture was also incubated with 0.1 ml of brush borders alone to determine if adhesion was present in the absence of 87 Pique 1-1. Brush mrder Acmesim Mt 987P-Specific unoclmal Antibody-t (0111111163 - 1:10, 1:100, 1:1000, 1:10.000) nggn BrushBorders K88+E;sa].i Borders 15mm kushBorders when BmshBorders [(88+E;gan 15m mimtimbymasecrntrastMicroswpy *-titerofstodcsolutimms 10,000 88 antibody. Additionally, each E; 95211 culture was incubated with 0.1 ml of KM to determine if autoagglutination was occurring. Also, known 987P- and K88-positive brush border preparations were run as positive controls on each test day. Following incubation, a drop of each of the suspensions was placed on a glass slide and coverslipped. The slide was then viewed by phase contrast microscopyu with a 40X objective. Brush border fragments were indicated as having no adhesion if no bacteria were adherent, partial adhesion if 2 or more bacteria were present and good adhesion if the entire brush border surface contained adherent bacteria. Statistical Analysis Data were analyzed using Friedman's test to determine if differences among the eight 987P MCA treatment groups existed for the 987P-positive and K88- positive E; 9911. The sign test was used to determine differences between paired treatment groups (Steel and Torie, 1980). To perform the sign test and Friedman's test the following values were assigned to the degrees of adhesion: 1 - no adhesion on all fragments, 2 - no adhesion on some fragments and partial adhesion on some fragments, 3 -no adhesion on some fragments and. good adhesion on some fragments, 4 — no adhesion on some u Zeiss Photomicroscope III, Carl Zeiss, Oberkocken, West Germany. 89 fragments, partial adhesion on some fragments and good adhesion on some fragments, 5 - partial adhesion on all fragments, 6 - partial adhesion on some fragments and good adhesion on some fragments, 7 - good adhesion on all fragments. Differences between groups were considered significant at the P g 0.05 level. RESULTS Brush Border Preparations The brush border fragments prepared were easily discernible when 'viewed by phase contrast microscopy. Varying in size, the fragments were semilunar to round in shape with a distinct microvillus border. Characteristically, a halo of bright light was present around each of the brush border fragments. Cell remnants other than the microvillus border also comprised the fragment (Figure 1-2). Protein Concentrations of the Brush Borders Five brush border samples were assayed for protein concentrations. The protein concentrations were 1.8 mg/ml, 2.2 mg/ml, 2.3 mg/ml, 2.7 mg/ml and 3.1 mg/ml. The average protein concentration was 2.42 mg/ml. 90 Figure 1-2. Phase contrast photomicrograph of a brush border fragment with no adhesion of 987P-positive E; 95111. Notice the moderately sized bacterial aggregates present beside the fragment (480K). 91 Monoclonal Antibody Production Using a slide agglutination test, the titer of the antibody in the tissue culture supernatant ranged from 1:100 to 1:1000 depending upon the day of collection. Approximately 350 ml of supernatant were collected per week. A total volume of 29.5 1 of tissue culture supernatant was collected. Following ammonium sulfate precipitation, l l of supernatant yielded approximately 100 ml of fluid. After affinity chromatography, the 100 ml of supernatant acquired from the ammonium sulfate precipitation step yielded a volume of 8-10 ml with a titer of 10,000. A total volume of 284 m1 of 987P MCA were produced with a titer of 10,000. Adhesion Prior to incubation with the MCA, ileal samples from each of the 29 pigs all had some degree of adhesion of 987P-positive E; 95211 to the brush border fragments (Figure 1-3 and Table 1-1). The adhesion ranged from individual brush border fragments with no adhesion to brush border fragments with the entire microvillus surface covered by bacteria (Figures 1-2,4 and 5). Brush border fragments were present in the same preparation with no bacteria adhering to some brush border fragments and varying numbers of bacteria adhering to other fragments. Adhesion for ileal samples incubated with the K88-positive 92 15 —"— £3" ...... 10 _— [34 'H O m H ' 2 5 '7‘ II u 5 255525: 2 . I I ig I W ' 5% mg§ 9 8 7 P+ L 2911 K8 8+ L 29;; Bacteria No adhesion - I No adhesion on some fragments: partial adhesion on some fragments - No adhesion on some fragments: good adhesion on some fragments - H No adhesion on some fragments: partial adhesion on I some fragments: good adhesion on some fragment - . Partial adhesion - 3 some fragments - g Good adhesion - I Figure 1-3. Distribution of the degrees of adhesion of 987P-positive and K88-positive E; 9911 to brush border fragments in ileal samples from 29 pigs. . 305me m .50 0.00000? 30520058 0 28.0me H. 380 0.00803 .HHunobHuHfiHm a 28.0me o 5.0 8.08008 38.83058 0 :86me m and 0.003% .HHeabHuEmHu 028.on e 8.0 2.80008 .HHflHSHuHEHm 0 286me m sea 05.8qu .HHunobHuHfiHm 0 286me m 0:0 5.0.0....” 580 8000003 58.83088 0 28.3.0. 0 as. H and 05800:. 3050305? 0 .... HEAT, Eu: coflsHHu o8.oHuH +38% ..0 .HHHdd+.6:8H§HH08oH.H+§fiH§c-u.HH&d+ 328055 8HuH+§§fiaumIJHmud+H§8H§HH0oHuH+§§c ..m .§§+ .6: 83:fl0o8.oHuH+flfl48:¢.§§+.§8§HH0¢8H“H+HH&48-H 0000802850 +A§8H§HH08HuH+Hju~.§§§+A§8H§HH0°H"H+HH~&48uH “Egg md.0.0.0 5.0.0 2.0.0 5.0.0.0 «.0.0 2.0 n n «agwuawm 33.090 93 s m o o o o o o NH 88 H a H o m m o o p 080 #0380 o o o o H H o o H H0330 m m m o m H o o s 080 .Hg .032 m o H o n o o o H 008 .082 m m HH nH 0 m H o H Hg .05: n mH «H 0H m 0H mm mm o 082 m H. 0 m a n a H .figg 803.085 58060 no 8.68 muons 00.5.80 ....003 050909 g 35 wagon £085 3 a d 0>Huflmoaum$m «0 c3850 05 8 among Hgg Ema no 000.30 05. .HIH 0.30m. 94 Figure 1-4. Phase contrast photomicrograph of a brush border fragment with good adhesion. Notice the bacteria adhering to the brush border surface (arrow) (480K). Figure 1-5. Phase contrast photomicrograph of a brush border fragment with partial adhesion. Notice the small aggregates of bacteria present in the background (480K). 95 Figure 1-4 Figure 1-5 96 Figure 1-6. The effect of preincubation of 987P monoclonal antibody with 987P-positive E; coli, prior to the addition of brush borders, on the brush border adhesion test. No adhesion - I No adhesion on some fragments; partial adhesion on some fragments - No adhesion on some fragments; good adhesion on some fragments - H No adhesion on some fragments; partial adhesion on . some fragments; good adhesion on some fragment - . Partial adhesion - E Partial adhesion 9n some fragments; good adhesion on some fragments -§g Number of Pigs 30 25 20 15 10 97 __ __ __ i_. __ I I l‘ I n E 1:10 1:100 1:1000 1:10,000 Dilutions of 987P Monoclonal Antibody Figure 1-6 98 Figure 1—7. The effect of preincubation of 987P monoclonal antibody with brush borders, prior to the addition of 987P-positive L coli, on the brush border adhesion test. No adhesion - I No adhesion (n1 some fragments; partial adhesion on some fragments - No adhesion on some fragments; good adhesion on some fragments - H No adhesion on some fragments; partial adhesion on I some fragments; good adhesion on some fragments - I Partial adhesion on some fragments; good adhesion on some fragments -§§ Good adhesion - I Number of Pigs 30 25 20 15 10 99 _— _a— _p-. 1:10. 1:100 1:1000 1:10.000 Dilutions of 987P Monoclonal Antibody Figure 1-7 100 L 9211 ranged from 7 samples with no adhesion to 22 samples with varying degrees of partial and good adhesion (Figure 1-3). Results of the brush border adhesion test for ileal samples incubated with 987P MCA and 987P-positive EL 9911 are listed in Figures 1-6 and 1-7, and Table 1-1. The 8 different 987P MCA treatment groups showed a statistically significant (P 5 0.05) inhibition of adhesion of 987P- positive 3; 2911 to the brush border fragments. There was no statistically significant difference between the extent of adhesion for K88-positive L 9911 incubated with and without the 987P MCA in the brush border adhesion test. Brush border preparations containing MCA at the 3 lowest dilutions, 1:10, 1:100 and 1:1000, had large to moderately sized bacterial aggregates present (Figure 1- 2). Brush border fragments could not be identified beneath the aggregates. Small aggregates were present at the highest dilution. No bacterial aggregates were present with the K88-positive EL 9911; Coadherence occurred with the 987P-positive E; coli. The bacteria adhered not only to the brush border membrane, but also concurrently adhered to adjacent bacteria, which in turn may have been adherent to the fragment and/or other bacteria (Figure 1-8). 101 Figure 1-8. Phase contrast photomicrograph of a brush border fragment with good adhesion. Notice the adhesion of bacteria to the brush border surface and to adjacent bacteria (480X). 102 DISCUSSION Adhesion of 987P-positive L 9911 to brush borders was present in all 29 ileal samples tested in the absence of MCA, indicating that all the pigs tested had receptors for the 987P pilus. The results of this study support the statement of Moon g; a1; (1979) who indicated that they had not encountered pigs congenitally resistant to 987P- positive L coli. Adhesion was present in some samples along the entire brush border surface, in both the neonatal pigs and adults, suggesting that age-related resistance to adhesion of 987P-positive E. coli is not the result of a decreased number of receptors, as is the case with K99-positive E; coli (Runnels gt Q1;, 1980). Also, neonatal and adult pigs had varying degrees of adhesion of 987P-positive and K88-positive EL 9911. These results are similar to the results of Sellwood (1980a) who described a weak adhesive phenotype where few K88-positive L 529;; adhered to brush borders in 21:19; Inhibition of adhesion of homologous but not heterologous pili was demonstrated by the 987P MCA. With each of the MCA treatments, adhesion to the brush border fragments by 987P-positive L 9311 was inhibited. No inhibition of adhesion was present when the 987 MCA was either initially mixed with the K88-positive E; coli or 103 brush borders and then mixed with the third component, suggesting that the MCA was specific for 987P pili. Research indicates that pilus antibodies inhibit adhesion by preventing the attachment of piliated E; coli to specific receptors. The 987P MCA used in the brush border adhesion test, whether' preincubated. with the bacteria or brush borders, did appear to prevent 987P- positive E; 9911 from adhering to brush border fragments. Preincubation of the brush borders with the MCA was not as effective as preincubation of the 987P—positive L 2911 with the MCA. Compared to the other treatment groups, inhibition of adhesion was particularly effective when the 1:10 and 1:100 dilutions of 987P MCA were preincubated with bacteria prior to the addition of brush borders. A 1:1000 dilution of MCA preincubated with 987P-positive EL 9911 was as effective as a 1:10 or 1:100 dilution of MCA preincubated with the brush borders. The inhibition of adhesion may have been associated with a decreased availability of free bacteria. The bacteria appeared to be prevented from adhering, but large aggregates of bacteria were present in the 1:10, 1:100 and 1:1000 dilutions of MCA. Since the large aggregates were not seen with K88-positive L 95111, the aggregates were probably due to the agglutination of 987P-positive bacteria by the 987P MCA. The number of bacteria available to bind to brush border fragments may be 104 severely limited due to the agglutination of bacteria. Kallenius gt; 0.1... (1985) have stated that antiserum or purified Igs, no matter how specific, should not be used as inhibitors of in 21529 bacterial adhesion since they aggregate bacteria and thus decrease the number of free bacteria. Therefore, observed adhesion values will be decreased in a non-specific manner. It has been suggested that the preferred manner of studying the inhibition of bacterial adhesion by antibodies is to use the antibody in a monovalent form. Intact Igs such as 196 would be cleaved to yield Fab fragments, which are incapable of agglutination. Thus, the inhibition of adhesion by the use of Fab fragments would not be the result of bacterial or fimbrial agglutination but due to specific blocking of the interaction between the epithelial cell receptor and the bacterial pilus (Fachon-Kalweit gt 11;, 1985). However at the 1:10:000 dilution, the aggregates either were not present or were small, and adhesion was still inhibited to some extent, indicating that inhibition of adhesion can not be attributed solely to bacterial agglutination. Some authors have used subagglutinating titers of antibody in bacterial inhibition adhesion assays to overcome the problem associated with agglutination. Sellwood gt 0.1... (1975) used a 1:80 dilution of K88 antiserum which inhibited adhesion to brush borders but did not agglutinate at that dilution. 105 Methods of quantifying the adherent bacteria were based on visual observations alone which may have limited the accuracy of bacterial quantitation. A more specific method of quantifying bacterial adhesion involves the use of radiolabelled bacteria (Hanson gt alt, 1985: Powell gt g1“ 1976) . However, such a procedure generally uses intact epithelial cells rather than brush borders. Inhibition of in yitzg attachment of 987P-positive fig ggli by 987P MCAs suggests that the MCAs should be capable of preventing the adhesion of the 987P pilus to receptors present in the pig intestine in 2129. However, responses in an in yitrg model should not be considered directly indicative of the in 1119 responses. In xittg observations should be evaluated in relation to in 211g observations. Bacteria grown in a laboratory don't always express the same surface structures as bacteria grown in animals, and this may also be true of pilim Also, isolated epithelial cells or brush borders don't necessarily retain relevant surface structures when studied in yittg (Kallenius gt 31;, 1985). 106 SUMMARY A study of 29 pigs was performed to determine if the in yittg adhesion of 987P-positive BL ggli to pig intestinal brush borders could be prevented by 987P MCA. Ileal samples, collected from 17 slaughter-weight pigs and 12 neonatal pigs, were examined using a brush border test. In the brush border adhesion test, pig intestinal brush borders and 987P-positive E; ggli were mixed with several different concentrations of 987P MCA. Two procedures were used with each ileal sample, either MCA and bacteria or MCA and brush borders were preincubated together prior to the addition of the third component for a total of eight different treatments per sample. Adhesion of 987P-positive fit ggli was present in all 29 ileal samples tested in the absence of MCA, indicating that all the pigs tested had receptors for the 987P pilus. Varying degrees of adhesion of 987P-positive E; ggli were present for the samples. A statistically significant inhibition of adhesion of 987P-positive fig ggli to brush border fragments by 987P MCA was present whether the 987P MCA was preincubated with the bacteria or brush borders. Preincubation of the brush borders with the MCA was not as effective as preincubation of the 987P-positive E; ggli with the MCA. Inhibition of adhesion was most effective at the 1:10 and 1:100 dilution of the antibody. Inhibition may have occurred because of prevention of adhesion to 107 brush border receptors or because of a decreased availability of free bacteria due to agglutination. In yitrg, the 987P MCA appeared to be effective in the inhibition of adhesion of 987P-positive L 51911 to brush borders. CHAPTERZ THE EFFECT OF THE ORAL ADMINISTRATION OF 987P HONOCLONAL ANTIBODY ON THE CLINICAL SIGNS, LESIONS AND ADHESION OP 987P-POSITIVE W QQLI IN GNOTOBIOTIC PIGS CHAPTERZ THE EFFECT OF THE ORAL AINIINISTRATION OF 987P HONOCIDNAL ANTIBODY ON THE CLINICAL SIGNS, LESIONS AND ADHESION OF 987P-POSITIVE W QQLI IN GNOTOBIOTIC PIGS INTRODUCTION Immunologically, the newborn pig is relatively defenseless and thus highly susceptible to infection. Correlation of the 3 ages of peak incidence of porcine enteric colibacillosis - neonatal, 3 weeks and immediately postweaning - with periods of antibody deficiency indicate that such a deficiency predisposes pigs to ETEC (Moon, 1974). Since adhesion of ETEC to the intestine is an essential prerequisite for the development of enteric colibacillosis, antibody-mediated prevention of bacterial adhesion is a potential defense mechanism against E; ggli. Passive immunization, via the absorption of antibodies from colostrum and milk, of the neonatal pig prior to its exposure to an infectious agent protects the pig from infection. Vaccination of sows prior to farrowing helps to induce protective levels of antibodies in the sow's colostrum and milk, thus providing passive immunity to the 109 110 newborn pig. Currently, several vaccines have been developed for the protection of pigs against neonatal enteric colibacillosis. Research has demonstrated that protection correlates with antipilus antibodies in the colostrum and milk (Acres gt 1L, 1979: Nagy gt g1_,_, 1978: Rutter gt Q1“ 1976) . Several researchers believe that colostral antibodies prevent the colonization of ETEC by blocking adhesion (Nagy gt git, 1978: Rutter, 1975; Rutter gt LL... 1973, 1976). Several early reports in the literature have demonstrated a protective effect of orally or parenterally administered hyperimmune serum, colostrum and milk against experimental challenge with ETEC in gnotobiotic and conventional pigs (Kohler, 1967, 1974: Kohler and Bohl, 1966: Miler gt g1“ 1975: Miniats gt gL, 1970: Rejnek gt alt, 1968: Smith, 1972: Smith and Linggood, 1971b: Svendsen and Wilson, 1971: Wilson and Svendsen, 1971). Recently, an alternative method for the direct passive immunization of newborn animals against ETEC, the oral administration of L ggn pilus-specific MCA, has been developed. Knowledge concerning the effectiveness of pilus-specific MCAs against ETEC in neonatal animals is limited. One of the first reports involved the oral administration of L gglj. K99 MCA to calves (Sherman gt git, 1983). In that study, there was a statistically 111 significant reduction in the mortality rate and the severity and duration of diarrhea in the treated calves versus the control calves. However, there was no difference in the incidence of diarrhea. Several explanations were postulated for the diarrhea which occurred in animals which received the K99 MCAs. It was speculated that the amount of HCA used may not have completely blocked colonization. Also, it was suggested that the number of bacteria in the challenge inoculum might have been large enough to produce sufficient enterotoxin without adhesion to the mucosa to cause diarrhea. The previous study led to the development of a commercially available K99 MCA for calves, Genecol 99 (Molecular Genetics, Inc. Minnetonka, MN). Studies with pigs using K88- (Foged gt g1“ 1986; Sadowski, 1984), K99- (Mainil gt g1_,_, 1987; Sadowski gt 31;, 1983) and 987P (Sadowski gt alt, 1986) MCAs have also been performed. Few animals were used in the studies which were primarily clinical in nature. The effectiveness of the antibody varied from protection against challenge, based on an increased survival rate and a delayed onset of diarrhea, to no prophylactic effect. Interestingly, both treated and control animals developed diarrhea in the majority of the studies. 112 In this study, MCAs specific for the 987P pilus were administered to gnotobiotic pigs in an attempt to protect the pigs from diarrhea due to 987P-positive fig ggli. The objectives of this study were: 1) To determine if the clinical signs and lesions produced by 987P- positive EL ggli in gnotobiotic pigs could be altered by the oral administration of L 9211 987P MCAs and 2) To determine if the in yiyg adhesion of 987-positive EL 9211 to the intestinal mucosa of gnotobiotic pigs could be prevented by the oral administration of at ggli 987P MCA. MATERIALS AND METHODS fit ggli Strains The 987P-positive strain of E_,_ 9211 used to infect the gnotobiotic pigs was strain 81-1469. The culture, obtained from Dr. David Francis, Department of Veterinary Science, South Dakota State University, Brookings, SD, was incubateda aerobically at 37'C for 8 hours in trypticase soy brothb prior to challenge. Following incubation, the culture was centrifuged at 1500 g for 20 minutes. The supernatant was discarded, and the bacteria were washed in phosphate buffered saline and centrifuged 1 more time. The pellet was resuspended to an optical density between a Thelco Incubator, Model 4, Precision Scientific, Chicago, IL. b Baltimore Biological Laboratories, Cockeysville, MD. 113 .075 and .085 at 560 nmc. The suspension contained approximately 108 colony-forming units per ml. Bacteria were checked for piliation using a standard slide agglutination test with 987? antiserum (refer to Chapter 1). The challenge inoculum was aseptically placed in 5 to 10 ml sterile glass ampules. The ampules were heat sealed with a propane torch and aseptically transferred into the isolators just prior to challenge of the pigs. Quantitation of L 95111 in Inocula One milliliter of the bacterial culture was pipetted into 9 ml of peptone water and ten 10-fold dilutions were made -in peptone water. Using a standard pour plate technique, 1 ml of each dilution was plated on MacConkey agar in duplicate and then incubated for 22 hours at 37’C. Colony-forming—units were counted using a digital colony counter, and the average colony count for each dilution was determined. Duplicate dilutions were done for each bacterial culture. Monoclonal Antibody The production of 987P MCA was the same as that previously described in Chapter 1. The MCA and PBS placebo were placed in 5 to 10 ml sterile glass ampules and heat sealed. The ampules were then aseptically c Shimadzu UV Visible Recording Spectrophotometer, Model UV-260, Shimadzu Coporation, Kyoto, Japan. 114 transferred along with the challenge inocula into the appropriate isolators. BXperimental Animals Eight litters of gnotobiotic pigs were used (Figure 2-1) . Pregnant sows were obtained from Michigan State University's Swine Center. Technique for Obtaining Gnotdbiotic Pigs The gnotobiotic pigs were obtained using a technique described by Waxler gt g1; (1966). All flexible, transparent, vinyl film isolators used in obtaining and rearing the gnotobiotic pigs were sterilized prior to use. Cages were sterilized in an autoclave at 121'C for 30 minutes prior to passage into detergent-cleaned isolators. The isolators and the cages were subsequently sprayed with an aerosol of 2% peracetic acidd. The air entering the sterile isolators passed through sterile air filter units. Feeding and other equipment were sterilized in an autoclave at 121'C for 30 minutes in a stainless steel, filter-equipped cylinder’ before being passed into the isolators via a transfer sleeve sterilized with peracetic acid. Cans of milk were also sprayed with peracetic acid prior to passage into the isolator. The pregnant sow was anesthetized on the 112th day of gestation. Using an 18 gauge, 6 inch needle, an d FMC Corporation, New York, NY. 115 Figure 2-1. Experimental Design gnotobiotic pigs (8 litters) 3-day-old piglets control treated PBS placebo 987P monoclonal antibody 30 to 120 minutes challenged orally with 987P+ E; ggli observed clinically 1 to 3 days necropsy ileum for 4 segments 4 segments 0 t h e r bacterial of small of small t i s s u e counts intestine intestine sections for histo- for scan- for histo- pathology ning elec- pathology (HtE and tron mi- Giemsa) croscopy 116 epidural was performed by injecting 15 ml of lidocainee at the lumbosacral articulation (Getty, 1963) . Two to 3 ml of a tranquilizerf were then intramuscularly injected into the sow. Next, the sow was secured on the surgical table in right lateral recumbency. The left flank was then clipped, shaved, and surgically prepared with Betadine Surgical Scrubg, Betadine Solutionf and 70 % ethyl alcohol. Chloroform was then applied to the left flank and dried with sterile towels. Next, the flank's surgical site and the vinyl film covering the 12-inch port in the floor of the surgical isolator were sprayed with a surgical adhesiveh. The lZ-inch port was then positioned over the surgical site and the port was secured in place with 3 cords. The surgeon, operating through shoulder-length rubber gloves, then cut through the vinyl floor of the 12-inch port and the sow's skin ‘with a cautery' unitiu The incision was approximately 9 inches in length and extended from the most dorsal aspect of the port to near the most ventral aspect. As the incision was made, the cut edges e Lid-O-Cain 2%, Butler Co., Columbus, OH. f PromAce (Acepromazine maleate), Fort Dodge Laboratories Inc., Fort Dodge, IA. 9 Purdue Frederick Co., Norwalk, CT. h Vi-Drape, Parke-Davis, Detroit, MI. 1National Electric Instrument Co., Inc., Long Island, NY. 117 of the vinyl film and the abdominal skin were clamped together with Allis tissue forceps, and the forcep handles were affixed to rubber bands extending through holes in the border of the 12-inch port. The remaining layers of the abdominal wall were then incised with scissors. Each pig was removed from the uterus by a separate incision in the uterine wall. Umbilical clampsj were placed on the umbilical cord of each pig prior to cutting the cord. The pig was then asepticalLy transferred into an attached transport isolator where it was dried and placed in a cage. When all the pigs had been removed, the sow was euthanatized by an intravenous injection of sodium pentobarbitalk. After delivery of the pigs, the transport isolator was disconnected from the sleeve attaching it to the surgical isolator. Pigs were then randomly placed in the rearing isolators and housed in individual cages within the isolators with 4 cages per isolator. Control animals were housed in separate isolators. The room temperature was 30-32'C. Equipment for feeding and for microbiologic determination was present in each isolator. The diet j Double Grip Disposable Cord-Clamp, Hollister, Inc., Chicago, IL. k Butler Company, Columbus, OH. 118 consisted of evaporated milk1 and each pig was fed ad lib 3 times a day. Bacteriologic Mbnitoring of Isolators Swabs were taken from each isolator just prior to the time the pigs were exposed to 987P-positive L 991.1 and subsequent to the termination of the experiment. Swab specimens were obtained from rectal contents, waste material from the cages, contents of feeding trays and the plastic isolator. Material from the swabs was suspended into 0.5 ml of Brain Heart Infusion (BHI) brothb, and 0.1 ml of this suspension was inoculated into enriched blood agar (BHI with, 5% defibrinated sheep blood, 5% heat- inactivated horse serum and 1% yeast extract powder) and MacConkey agar“. The cultures were then aerobically incubated at 27'C, 37'C and 56°C. The CDC anaerobic blood agar was also inoculated and anaerobically incubated at 27’C, 37°C and 56'C in a Bio-Bag environmental chamber type A“. The BHI suspension was also inoculated into thioglycollate mediumm supplemented with Hemin and vitamin K and incubated aerobically in tightly screwed capped tubes at 27'C, 37'C and 56‘C. The primary plating medium 1 Carnation Company, Los Angeles, CA. m Difco Laboratories, Detroit, MI. n Marion Laboratories, Kansas City, MO. 119 and thioglycollate medium were checked daily for 2 weeks. Isolated organisms were examined and identified. Treatment of Pigs In the initial trial, 3 litters of pigs were used to determine the optimum amount of MCA to be administered to the pigs. The study was a paired study, for each control animal there was a treated animal. Control and treated animals were randomly assigned. Three—day-old pigs were fasted 18 hours prior to the administration of the 987P MCA. In the first litter of pigs, the MCA was administered in the backs of the piglets' mouths with a syringe. In the second litter of pigs the MCA was given via a stomach tube, and in the last 6 litters of pigs, the MCA was administered via a bottle. The MCA was mixed with 5-10 ml of milk in the bottle, and the pigs were then fed milk immediately after the administration of the MCA. Control pigs were given a phosphate buffered saline placebo. One half hour to 2 hours later the pigs were challenged with 1 m1 of the bacterial culture (Table 2-1). Determination of Clinical Signs Pigs were observed 3 times daily, at each feeding, for clinical signs of anorexia, vomiting, dehydration, depression and diarrhea. .8382 62 800.0 0:5. 0 .gfiqHa you mandamus no .8952 0 fine» 8333588 830 0 R 3:838 308.3 00 0003 0 58 .68 .6838 H8388 .56 n . $68 3880 8 a 0302 08 $30 3880 3 8 80nd :83 H0880 me n 030: :83 80080 8 a 008.3 you 089.0 8328 ~58 80: 83 0008.8 8 8580 0 080 m 0.82 md 083 m3 8 0.3 name A 2 m 080 m 8.2 m6 0380 m3 8 0K 080 m S a. 080 a .88 m6 0380 «3 Ha ed 28 n m 0 080 m 8.2 m.o 0380 m3 3 0.2 260 a 3 m 080 m 008: m6 0308. «3 as 6.2 080 0 «a w m E d: 04. 1 080 n 0.82 ma 0800 SS 5 s. o... 080 m S n 003 >00 A 008: m6 8800 m3 E o.n mane n m m 5 as o; ammo n 052 m 585 mod 5 8 m6 080 n S H 00803 833805 88E 00 00 0883 008580.“. 82 no ~88 008—2 839808 050. 693 flag «a 6: 03 .oz .803 .030 0338080 «o 0802 m 05 you H8880 0850009 .Tm 03E. 121 Necropsy Procedure Twenty-four to 72 hours (Table 2-1) after exposure to 987P-positive E... 9311 the pigs were removed from the isolator and euthanatized by an overdose of sodium pentobarbital administered intravenously. A complete gross examination was performed. The small intestine was removed, and 4 equally spaced segments were fixed in 10% buffered formalin for histologic examination. Four additional segments of small intestine were taken for scanning electron microscopic evaluation from half the control and treated pigs in the litter. A 10 cm segment of ileum was taken from each pig for microbiologic culture. In addition to the small intestinal sections, sections of lung, liver, kidney, spleen, lymph node, stomach, cecum, spiral colon, pancreas and adrenal were also formalin-fixed. Preparation of Histologic Sections The formalin-fixed sections were routinely processed, embedded in paraffin, cut by' a microtome into 6 (an sections and stained with hematoxylin and 'eosin, and Giemsa stains. Giemsa stained sections were evaluated to determine the relative numbers of bacteria adherent to the mucosa based on a modified association index (Bertschinger gt glt, 1972). Two criteria were used. The first criterion was based on the tendency for bacteria to be contiguous to epithelial cells and was graded as 1 for 122 none to 5 for maximum. The second criterion was based on the number of bacteria adherent to the villi and was graded as 1 for none to 5 for maximum. The adhesion index for the histologic section was then determined by multiplying the values for the 2 criteria. Preparation of Scanning Electron Microscopic Sections One centimeter sections of the small intestine were opened along their mesenteric border and washed with cold 0.1 M PBS until the intestinal contents were removed. The intestinal sections were then fixed individually in cold 2% glutaraldehyde for 8 hours. The fixative was replaced, and fixation was continued for 48 hours at 4’C. The small intestinal segments were then washed 3 times in cold 0.1 M PBS and once with distilled water, trimmed to 3 mm, and postfixed in 1% osmium tetroxide° in 0.1 M cacodylate bufferP (pH 7.2) at 4'C overnight (Hadad gt 3L, 1982a). The tissues were then washed 6 times in distilled water during a period of 10 to 15 minutes and fixed by a modified thiocarbohydrazide (TCH) method (Malick and Wilson, 1975) . A 1% saturated solution of Tel-1° in distilled water was freshly prepared and filtered prior to its use. The tissues were incubated in the TCH solution for 20 to 30 minutes at room temperature with gentle ° Polysciences, Inc., Warrington, PA. P Electron Microscopy Sciences, Fort Washington, PA. 123 agitation. The tissues were then rinsed with 6 changes of distilled water during a period of 10 to 15 minutes. Next, the tissues were placed in a 1% osmium tetroxide solution prepared with distilled water for 2 to 3 hours at room temperature with gentle agitation. The tissues were rinsed 6 times with distilled water. A fresh TCH solution was prepared, and the tissues were incubated in the solution for 20 to 30 minutes. The tissues were rinsed 6 times with distilled water. The osmium tetroxide step was repeated and the tissues were rinsed 6 more times. A standard dehydration in alcohol was performed. The tissues were then critical point driedq and mounted on aluminum stubs. Samples were examined using a scanning electron microscoper. ‘Microbiologic Quantitation of fig ggli To further determine the extent of colonization, a 10 cm segment of ileum was removed from each pig. The luminal contents of each segment were washed out with 10 ml of a 0.3% solution of peptone waterl. The volume of each luminal washing and each intestinal wall sampling were adjusted to 30 ml with peptone water. The samples were then individually homogenized at high speed for 1 minute using a Sorvall Omni-Mixerq. One milliliter of the q Ivan Sorvall, Inc., Newton, CT. r JEOL JSM-3SC Scanning Electron Microscope, Japanese Electron Optics Laboratory, Tokyo, Japan. 124 resultant mixture was then pipetted into 9 ml of peptone water, and eight 10-fold serial dilutions were made in peptone water. Duplicate dilutions were done for each sample. Using a standard pour plate technique, 1 ml of each dilution was plated on MacConkey agar in duplicate and then incubated for 22 hours at 37'C. Colony-forming- units were counted using a digital colony counters, and the average colony count for each dilution was determined. The formula used ‘to calculate the number' of lactose- fermenting bacteria was the following: Con - 1/10’a x 30 x b. The original concentration of the numbers of lactose- fermenting bacteria per 10 cm of ileum corresponds to Con, the dilution from which the inoculum was taken corresponds to 10‘3, the total volume of peptone water into which the 10 cm segment was homogenized corresponds to 30, and the average number of colonies counted for that dilution corresponds to b (Nagy gt glt, 1976). MCA Activity in Gastric and Cecal Contents Six 3-day-old pigs, which had been removed from the sow shortly after suckling colostrum, were used. Three pigs were bottle fed 10 ml of 987P MCA mixed with 2 oz of milk. The 3 control pigs were fed 10 ml of PBS mixed with milk. The piglets were euthanatized 2 hours later. 3 Lab-Line Digimatic Colony Counter, Lab-Line, Inc., Melrose Park, IL. 125 Gastric contents were collected and centrifuged. The supernatants were diluted 1:10, 1:100, 1:1000 and 1:10,000 and used in a slide agglutination test (refer to Chapter 1) with 987P-positive and K88-positive fit ggli. Three 3-day-old pigs were also removed from another sow shortly after suckling colostrum. Two of the pigs were treated with MCA as described previously and the third pig received PBS. The piglets were euthanatized 6 hours later. The stomach and cecal contents were collected and centrifuged. The slide agglutination test was performed as described previously. Also, a brush border adhesion test (refer to Chapter 1) was performed with preincubation of the 4 dilutions of gastric and cecal supernatants with 987P-positive and K88-positive E1 9911 prior to the addition of brush borders. Statistical Analysis Data were analyzed using a Chi-square test to determine if differences in morbidity, mortality and colonization existed between the control and treated animals in each litter. A paired t test was used to determine differences between bacteriologic counts for the control and treated pigs in each litter. Bacteriologic count data were transformed using a square root transformation. 126 Differences between groups were considered significant at the P 5 0.05 level (Steel and Torie, 1980). RESULTS Bacteriologic Monitoring Prior to challenge, no bacteria were detected in any of the samples collected from the isolators housing the 8 litters. At the time of euthanasia, samples from all of the isolators except 1 yielded pure cultures of 987P- positive L 9211. No bacteria were isolated from one isolator in litter 7. Clinical Signs Before challenge with 987P-positive (3;, 9911, the gnotobiotic pigs were active, alert and healthy. Usually within 2 days the pigs were nursing well from bottles. Their yellowish to tan feces were firm to pasty in texture. Within 5 to 16 hours of challenge (Table 2-2), the pigs began to show signs typical of enteric colibacillosis. Clinical signs between the treated and control animals were very similar, particularly in litters 4-8. Clinical signs in the 8 litters of pigs consisted of diarrhea, dehydration, anorexia, depression and weakness. All of the infected pigs, both control and treated, had diarrhea. The pale to golden yellow feces ranged from watery to pasty in consistency. Usually during the day, 127 .Buflgaggmufigivflzmomgfianofiuogo .Egusxfiabouon .3080 300008 0883088 3 08800000 838880 0 o o o 0 8H 8H S 3 m o o H o 03 8 «H S H. H H o 0 8H 03 HH HH m o o o o 8H 8H «H «H m o o o 6 8H 8H HH HH H. H o as :3 «H .25» 1H0 t m :3 t R ..N.HaTHc 3 2a E n m HHs E be 8 8H SH .2518 m m 0... o o o o 8H 8H m o m o m o om 8H 8H 3 H H .H. 0 .H. 0 .H. o .H. 90 . “masons 88H8 035 8 E 203 H888 80:2 00 .8052 3238: EH38! no 0008 0.0003 13.3 an 03.3ng 3 00398 use 833:0 H8308.- mhom 536 mean 00.88008 5 833830 H.053: H8 3338 .5888 .8? H8230 no 0008 .nnm £an 128 while the pigs were being fed, diarrhea was present. However, little evidence of diarrhea was present the next morning. The diarrhea would ensue after the morning feeding. Generally, the piglets' appetites were good, but occasionally the piglets became anorectic, particularly those piglets which had severe diarrhea or were near death. At the time of euthanasia, the diarrhea had usually abated and the pigs were recovering from the infection In litter 1, initially all the pigs had similar clinical signs, but by the end of the 3 day observation period 1 of the control pigs had died and another control pig was extremely depressed, thin, anorectic and dehydrated in comparison to the other pigs. At the time of euthanasia the 8 other pigs appeared to be recovering from the infection. Sixteen hours after infection, the animals began to show signs of mild diarrhea characterized by a yellow, pasty stool which stained the piglets' hindquarters. However, the piglets remained alert and active and had good appetites. Similar clinical signs were observed the next day. One control animal was depressed though. The next morning, 44 hours after challenge, the animals' stools were firmer. One control pig was still depressed and anorectic. Four hours later, mild diarrhea was again present in all the pigs, and the control animal was dead. 129 Animals in litter 2 were observed clinically for 24 hours. Only mild diarrhea was observed in the piglets. In litter 3, within 5 hours, all of the pigs except for the pigs treated with 7 ml of MCA and 1 of the pigs treated with 4 ml of MCA had damp hindquarters. Also, 3 of the control pigs were depressed. After 10 hours all except 1 of the pigs treated with 7 ml of MCA had diarrhea. By the next morning 3 of the control pigs had died. The other 2 control pigs were weak, anorectic, depressed and dehydrated so they were euthanatized and necropsied. On the following day, 2 of 3 pigs in the group treated with 4 ml of MCA not only had diarrhea, but were depressed, anorectic, weak and 5-10% dehydrated so they were euthanatized and necropsied. On the third and final day after challenge, 1 of the pigs treated with 7 ml of MCA had died. The 3 remaining MCA treated pigs were thin and stained with feces, but the 2 pigs treated with 7 ml of MCA were also alert, active and hungry. In litters 4-8, the clinical signs were similar to those observed previously but not as severe. Signs for the treated and control animals were very similar. In litter 4, the control pigs appeared to have slightly worse diarrhea and were anorectic near the end of the observation period. Initially, the diarrhea in the control animals from litter 6 was worse than the diarrhea in the treated animals. Later, the clinical signs were 130 the same in the treated and control groups. Two control 'pigs in litter 7 remained. healthy throughout the observation period. One of the treated pigs in litter 7, the runt of the litter, died the day after it was infected. Otherwise clinical signs in the 2 groups in litter 7 were very similar. Gross LesiOns Minimal gross lesions were observed. In the 8 litters examined, the lesions differed very little between the control and treated animals. Gastrointestinal lesions consisted of variable amounts of fluid in the small intestine, cecum and colon, occasional distention of the bowel, and soft to liquid feces in the rectum. Stomachs were often distended with milk. Dehydration, urate crystals in the kidney, inflamed vulvas, anteroventral pneumonia and occasional gastric fundic hemorrhage were also present in some of the piglets. Few gross lesions were present in litter 1. Liquid contents were present in the colon of all the pigs. The control pig which died from the infection and 1 other control pig were gaunt and dehydrated. The small intestines and spiral colons of both control and treated animals were distended with fluid in litter 2. Anteroventral bronchopneumonia was also present in the piglets. 131 Grossly, in control and treated pigs in litter 3, the small intestines, primarily the distal half of the jejunum and the ileum, were distended with fluid and were hyperemic. Gastric fundic hemorrhage was present along the greater curvature of the stomachs of approximately half the control and treated animals. Gastric lesions and small intestinal hyperemia were present in animals which died from the infection or, in animals which were euthanatized because of severe illness. Petechial hemorrhages were present on the surface of the kidney of 1 treated pig. Aspiration pneumonia was present in several of the pigs. The piglets' stomachs also tended to be distended with milk, and liquid contents were present in their colons. At the end of the observation period, 1 of the pigs treated with 7 ml of MCA had grossly normal intestines, and the stool was beginning to form. Gross lesions in litters 4-8 were similar and consisted of variable amounts of fluid in the colon, soft pasty to liquid stools, milk distended stomachs, aspiration pneumonia and inflamed perineums. Histopathologic Lesions Intestinal lesions, when present, were generally confined to the distal half of the small intestines, primarily the ileum and lower jejunum. Mild to moderate neutrophilic infiltration was present in the lamina propria of some small intestinal sections. Post mortem 132 autolysis in some of the pigs which died from the infection made the interpretation of intestinal lesions difficult. The degree of adhesion of ' 987P-positive L 9911 to the intestinal mucosa varied but was generally low (Table 2-3). A few piglets, primarily animals from litter 3, had numerous bacteria adherent to the microvillous surface of intestinal villi extending from the crypts to the tips of the villi (Figures 2-2 and 2-3). In some pigs only a small number of adherent, randomly scattered bacteria were present in the small intestine (Figure 2-4). It was not uncommon to find one section of the small intestine with adherent bacteria and an area adjacent to the section with no adherent bacteria (Figure 2-2) . In most of the piglets, particularly litters 4—8, rm) adherent bacteria whatsoever were present throughout the small intestine. Often in areas where no adherent bacteria were present, large numbers of bacteria were seen adjacent to the small intestinal villi entrapped in mucus but not contiguous to the brush border surface (Figure 2-5). Various numbers of bacteria were present in the lumen of most intestinal sections. In litters 2-8, the majority of the piglets had mild to moderate, multifocal, neutrophilic bronchopneumonia consisting of neutrophils in bronchioles and surrounding alveoli. In litter 3, piglets with gross lesions of .mmfimmumugnzn gsflggflfigfigfihgégggfi Hg .mguHHfigggmgmgngHHfigHofigHfi009055.82 umom .nougl .H. .HOHuHUou O .ssmHH u v ...—Hanan H330 a n .gnoh 8&5 u N ...g s H I H I H I H mIH oHN NHIH m.m «IH m.N .H. H I H I H I H NIH H .H oNIH N.HH TH HUN 0 v H I H I H I H NIH mH . H nIH ms . H eIH N . N .H. H I H I H I H NIH 0H . H mNIH m . H. mHIH mN . m U m H I H I H I H I H I H NIH 0H .H. 3 H I H I H I H NIH N.H NHIN mNé mIH mN.m o N n H I H I H I H I H I H I H .H. I H I H I H I H I H I H I H O H m z m z m z m z m s m z z m a: h m m H. N 05303 H0852 .8qu 8:38.95” .8380 3835 HHS—m men 0303395 3 a an gHuHmonImhwm you as: 8.30504 . «IN 3an. 134 Figure 2-2. Photomicrograph of a section of ileum with bacteria adherent to the small intestinal villi extending from the crypts to the tips of the villi (arrows). Notice the absence of adherent bacteria to the adjacent small intestinal villi (Giemsa stain, 120X). Figure 2-3. Photomicrograph of a section of ileum with numerous rod-shaped bacteria (arrows) adherent to the lateral surfaces of the intestinal villi (Giemsa stain, 480K). .1. ..I .mw H V” ... w "mm. .0 w % us mwa Figure 2-2 Figure 2-3 136 Figure 2-4. Photomicrograph of a section of ileum with few bacteria adherent to the lateral surfaces of the villi (arrows) (Giemsa stain, 480K). Figure 2-5. Photomicrograph of a section of ileum with bacteria entrapped in mucus (arrow) but not contiguous to the brush border surface (Giemsa stain, 480K). Figure 2-5 138 gastric fundic hemorrhage had hyperemic blood vessels present from the tunica muscularis to the mucosa of their stomachs. A few vessels appeared to contain fibrinous thrombi. Lesions were difficult to detect in many of the stomachs because of post—mortem changes. MOderate infiltrations of neutrophils were present from the serosal to the mucosal surface of the stomachs. Also, sections of small intestine contained numerous hyperemic blood vessels in the mucosa with occasional thrombi. Generally, histopathologic lesions were not found in any of the other organs examined. Scanning Electron Microscopic Lesions In most of the intestinal samples examined via scanning electron. microscopy no ‘visible bacteria were attached to the small intestinal villi. In the segments of distal jejunum and ileum of 2 animals in litter 3, 1 control and 1 treated, bacteria were seen attached by their sides and. poles to intestinal epithelial cells, primarily on the sides of the villi (Figures 2-6 and 2-7). Microbiologic Quantitation Bacterial counts for intestinal contents and the intestinal wall are listed in Tables 2-4 - 2-7. Generally, the numbers of viable EL 9911 in the lumen and intestinal wall were low, less than 103, except for litter 3. Numbers ranged from 0 to >1012 E; coli. 139 Figure 2-6. Scanning electron micrograph of section of ileum with bacteria adherent to the lateral surface of the intestinal villi (arrow) (320X). Figure 2-7. Scanning electron micrograph of section of ileum with bacteria adherent to the brush border surface of the intestinal epithelium (5,300X). 140 Figure 2—6 Figure 2-7 141 $3335 ggfimm no isfifiuflgmflu .booflufi HabHoQaafimm no E e fifigfifi Emu .fimfifimgHflumHflHfiuflHa «Hague «Hobgnon . God w a mains 33.8 as smug :amfimn guufl “.5235? sHHSHumflBm a 1.3 x H.~ moHv 83 x fin an x 54 «3 x a.» 83 x N.m HHoHA as x m4. m3 x ....H m3 x Em boHv 83 x «6 HHS x H.N an x m4 o3 x H.H eoH x m.N moHv 83 x >4. 23 x H.H NoHv moH x 9H as x 1m. noHv 1.3 x o.m 82 x m4 m3 x >6 «3 x m6. NoHv m3 x >6 m3 x m.H 93 x NH moH x «am 83 x 3. an x NH «3v moHv m3 x m6 «2 x «6 a o .H. o a o a no 1. on N H g; .YH fiBuHHchdegmufiammmfioHuogaoHagHmflfiado§>uo§z .TNaHfia 142 $68.35 36888 .53 no 3:33 @358» Emu .3333 Ragga uoHaefiHauflngn HuguezofifioIoa OOHV mo." X 0.N OHOH N Din MOH N ¢.N wOH X ¢.H 80H X m.N HHOH N oom wOH X m.b 00H X v.0 ®OHV mo." X N.m 80H X m.m HHOH X won GOH X hom OHOH X m.m mOH X mtn FOH X mg. QQOH uh mtn NHOHA MOH X H.N FOH X n.m OHOH X 5.2—” mo." X 0.5. mOH X w.¢ 8HOH N bom HHOH X mtm MOH X N.w mOHV 00H X N.” FOH x 0.m bOHV QOHA QOHOH X 5." ”OH X Mtv MOH X m...” WOHV 60H X 0.0 00H X NEH l [I H. U .H. U .H. U .H. MU m N H Egg..— .TH 33»: fl 83 g gfiuomaflammfigfiHHEHEBmuenmflfiHsuflfioommuHHadefiguogz .mINmHnus 143 figozuuzm moHv m2 x hm m3 x .1. m.fiv moHv no? es x hm m3 x o6 m3 x as oz s3 x 3. mo? ...Sv m3 x ma 2: x we ouz m3 x s; moHv ooHv m3 x is 92 x ma hS x «4 as x H.~ moHv 93 x H4 m3 x o.~ «3 x «A «S x 9m an x HA «2 x m6 m3 x «.n 53 x in moHv as x «6 s3 x in 33 x ta ...S x 3.. moHv m3 x «4 m3 x H.H «3v 9 u a o a o a u ... a: “EH .3 gfifimfidgusaamomfifiuomflflfiogfiHmnfi Haddoguog SIN oHnaa 144 figoznozn . 2.36 w 8 29:5. Houuc8 can H3385 5253 858%? ..EmoHuHEHm zHHBHumflfim a 90H x ma soHv $3 on ea «3 x o.N «2 an ad oz moH x o.N moHv «oHv onv “.3 x o.H «oHv oz oz moH x m.N moHv an x in $2 uz «3v eoH x 9H NoHv m3 x «6 m2 x m.» «3 x m6 m3 x n4. noH x TH «.3 x in mg x m4 oz moH x m6 m2 x 9H moH x «H «S x o6 an x m... ooH x m.H NoHv oz ooH x H.m coHv a o a o a o .H. o m as o m an: E “ .mummumfiflfimoflg fiufluoflfiaumfifififigggmufifiggadflguogz .TNoHnnH 145 Usually, intestinal wall numbers were lower than luminal content numbers. MCA Activity in Gastric and Cecal Contents At dilutions of 1:10, 1:100 and 1:1000 the gastric contents of the 3 treated pigs in the first study caused agglutination of 987P-positive E; coli. No agglutination of K88-positive L 95211 occurred with the treated pigs' gastric contents. No agglutination with the control pigs' gastric contents occurred with 987P- or K88-positive L ggli. Agglutination occurred with the cecal content supernatants of both treated pigs at dilutions of 1:10 and 1:100. Cecal contents of one treated pig also agglutinated the K88-positive strain of E; 2911 at dilutions of 1:10 and 1:100. No supernatant was obtained from the stomach contents of the treated pigs, only ropy mucus was present, so an agglutination titer was not obtained. Cecal and stomach contents of the control pig were negative for agglutination with 987P- and K88- positive E; coli. With the brush border adhesion test, at a dilution of 1: 10 the cecal content supernatant of one treated pig was able to prevent the adhesion of 987P- positive E; coli, but not K88-positive E. ggli, to brush borders. large aggregates were seen in the brush border preparations with 987P-positive L $11 at a 1:10 and 1:100 dilution of the cecal content supernatant. Adhesion 146 of 987P- or K88-positive E; coli was not inhibited at any dilution of the cecal content supernatants of the other 2 pigs. DISCUSSION The first 3 litters of pigs were used to determine a protective dose of 987P MCA to be administered to the gnotobiotic pigs. Results with the initial 3 litters of pigs were somewhat promising, particularly in litter 3, indicated by the prolonged survival time in the treated animals and decreased mortality rate in the animals treated with 7 ml MCA. In litter 1, the mortality rate and number of animals colonized was lower in the treated than in the control animals. Since a 7 ml dose of MCA appeared to be palliative in the third litter, 10 m1 of HCA was decided upon as an adequate dose for the subsequent litters. Amounts of MCA administered in previous studies have ranged from single doses of 1 ml of a 1:12,000 titer K99 MCA for calves (Sherman e1; gig, 1983), 20 mg of K99 MCA for calves (Sadowski g; g1&, 1983) and 100 mg of K99 MCA for pigs (Sadowski gt 31;, 1983) to multiple doses of 10 mg of K99 MCA (Manil gt glL, 1987) or 25 mg of K88 MCA for pigs (Foged g; glL, 1986). The challenge inoculum varied in the first 3 litters before a reliable method of standardization of bacterial concentration was found. With an exposure to 101° 987P- positive §_._ coli, the greatest protective effect of the 147 MCA. as well as the :most significant clinical, gross, microscopic, scanning electron microscopic and microbiologic results were obtained. However, with this number of bacteria, signs of endotoxic shock were present in the pigs. In order to remove complicating factors which might have been associated with the endotoxemia such as disseminated intravascular coagulation, the number of E. coli was decreased to 108 bacteria. With these lower numbers, however, signs of colonization were generally not present. ' In retrospect, it appears that a more appropriate number of the bacteria might have been in the 109 range since 2 control animals were colonized in the first litter which received 109 E; 9911. Saunders gt 31. (1963) have reported that enteric colibacillosis will occur in colostrum-deprived, specific pathogen free pigs whether the infecting inoculum is 103 or 108 viable L c211. They did not indicate whether colonization would occur with these numbers. Initially, antibody and bacteria were orally administered to gnotobiotic pigs via a syringe placed in the backs of their mouths. However, it was difficult to determine if the pigs were receiving full amounts of the MCA and bacteria. Therefore, the MCA and bacteria were administered to the next litter of pigs via a stomach tube. However, some of the pigs developed aspiration pneumonia though, possibly from the incorrect placement of 148 the stomach tube. Thereafter, the last 6 litters of pigs were bottle fed the MCA and bacteria. However, because of the angle and fervor with which the pigs nursed some still developed aspiration pneumonia. Also, the first litter of pigs was challenged 2 hours after the administration of HCA compared to the last 7 litters. Since the effectiveness of protection was difficult to determine subsequent to a 2 hour time lapse, the time between challenge and treatment was shortened to 30 minutes. Challenge times after treatment in other experiments using serum, milk, colostrum or’ MCA. have varied from 30 minutes to 26 hours, but generally ranged from one half hour to 2 hours. Unlike this study though, several of the studies continued to administer the antibody after challenge (Kohler, 1974: Kohler and Bohl, 1966; Miniats gt git, 1970; Scoot gt glt, 1972; Svendsen and Wilson, 1971; Wilson and Svendsen, 1971). Like previous studies (Mainil gt alt, 1983; Sherman gt 3L, 1983; Svendsen and Wilson, 1971; Wilson, 1972: Wilson and Svendsen, 1971), no differences in the incidence of diarrhea were seen between the control and treated animals in the litters. All animals, both control and treated, had clinical signs of enteric colibacillosis. However, at the time of euthanasia, except for the first 3 litters, evidence indicative of colonization was not present. The single dose of HCA caused a temporary 149 protective effect in the onset of diarrhea in litter 3 and also limited the severity of the diarrhea in the initial stages in some of the treated pigs. The onset of clinical signs in litter 3 varied from 5 hours for the control and 2 of the animals treated with 4 ml MCA to 10 hrs for all except 1 of the other treated animals. In the other litters, the onset of clinical signs was very similar. However, initially in litter 6 the diarrhea was less severe in the treated animals than the control animals. Pigs were only observed for 2 to 3 days after challenge so the period available to determine survival time was limited. However, at the time the pigs were euthanatized they were generally recovering so a more prolonged observation period would probably have made no difference in the survival time between treated and control animals. Studies administering milk and colostrum to gnotobiotic pigs infected with E_,_ 9211 have shown a delayed onset of diarrhea and an increased survival time (Svendsen and Wilson, 1971: Wilson, 1972; Wilson and Svendsen, 1971). In one study, K99 MCA caused a statistically significant reduction in the severity and duration of diarrhea in the treated versus control calves (Sherman gt alt, 1983). Also using K99 MCA, Hainil gt 31; (1987) indicated that diarrhea started later and ended 150 earlier in the treated versus control hysterectomy- derived, colostrum-deprived pigs. Gnotobiotic pigs were used in this experiment. Jones and Rutter (1972) have shown that, even though the K88 pilus is essential for ETEC to cause diarrhea in conventional pigs, it is not required for diarrhea to occur in monoassociated gnotobiotic pigs. Therefore, a treatment directed against the pilus alone may not be effective in the prevention of diarrhea and its associated clinical signs. The effectiveness of colostrum and milk in increasing the survival time in some studies utilizing gnotobiotic pigs exposed to ETEC may be related to additional protective factors present in milk and colostrum. Under the conditions of this study, the mortality rate ranged from 90% of the pigs in litter 3 to 0% in 5 of the 8 litters. In litters 2,4,5,6, and 8, there was no difference in the mortality rate between treated and control animals. No statistically significant difference in the mortality rate was present between the treated and control pigs in any of the litters. Mortality rates in previous passive immunization studies have differed, but generally the treated animals had a lower mortality rate than the control animals (Foged gt 1L“ 1986: Rejnek gt g1“ 1968; Sadowski gt 11.4,, 1983, 1986; Sherman gt 3L, 1983). In litter 7, the only animal to die from the 151 infection was a treated animal, but it was the smallest pig in the litter. Grossly, the variable amounts of fluid present in the small and large intestines in association with liquid to semiliquid feces in the rectum correlate well with lesions associated with a hypersecretory diarrhea. The pneumonia which occurred in a large number of the piglets was probably due to the aspiration of milk while the pigs were nursing. Gastric stasis precedes the development of diarrhea in piglets (White gt 31;, 1969) and this probably accounts for the milk present in the pigs' stomachs. The gastric fundic hemorrhage and hyperemic intestines present in litter 3 were probably related to an endotoxemia associated with an extremely high E; ggli challenge inoculum. Hicroscopically, the majority of the piglets were not colonized at the time of euthanasia. Piglets in litters l and 3 were the only piglets with substantial evidence of adhesion histopathologically. When adhesion was present, it occurred in the lower half of the small intestine which is in accordance with what has been reported previously (Isaacson gt 3L, 1978; Nagy gt LL“ 1976). Bertschinger gt a]; (1972) have reported that they were unable to detect EL ggli using a fluorescent antibody test when the bacteria numbered less than 106. Also, there 152 were few adherent bacteria observed with scanning electron microscopy. The microscopic lesions correlated with the bacteriologic results since the animals were usually not colonized except for litters 1 and 3. No significant differences were noted between bacterial counts for the intestinal wall and contents between control and treated animals except for the wall in litter 7 and the intestinal contents in litter 3. Differences in the first 3 litters may have been significant if, at the time of death of the control piglet, a paired treated animal was euthanatized and bacterial counts were taken. Interestingly, all of the animals had signs of diarrhea prior to euthanasia, but at the time of euthanasia, evidence of adhesion and colonization ‘was generally not present. Therefore, it is difficult to interpret the effect of the MCA on adhesion. Theoretically, the animals could have been colonized in the early stages of the infection and have almost recovered from the infection at the time of euthanasia, usually 2 days after challenge. The MCA may have inhibited colonization in the early stages of the disease compared to the controls, but that seems unlikely since the onset and degree of clinical signs was similar in the control and treated pigs in most litters. 153 Theoretically, the 987P MCA would have been expected to decrease the adhesion of the 987P-positive E1 ggli and thus inhibit colonization. Previous reports in which serum, colostrum or' milk. have been administered have suggested a protective effect against enteric colibacillosis due to an antienterotoxic or antibacterial effect of the antibody. Hyperimmune serum's protective action was believed to be independent of the complement- antibody bactericidal system and was initially thought to be related to the inactivation of toxin in the intestinal lumen (Kohler and Bohl, 1966) . In studies by Kohler (1967) and Miniats gt git (1970), antisera orally administered to gnotobiotic pigs infected with L 9211 protected the pigs from diarrhea, but the mechanism of protection did not appear to involve a marked reduction in the numbers of live fit ggli in the small intestine. They suggested that the protective effect of the antibody was due to its action on the enterotoxin. An antienterotoxic effect was not evident based on clinical signs in the present study, and furthermore would not be expected since the MCA was specifically directed against the 987? pilus. Also, gnotobiotic pigs were used in the previous studies, and since pili appear to provide no added advantage in the multiplication of E; ggli in gnotobiotic pigs (Jones and Rutter, 1972), a reduction in the numbers of bacteria in treated versus control animals would probably not have 154 been expected. A similar situation may have occurred with the gnotobiotic pigs used in this study. However, in one (in yiyg experiment. where gnotobiotic pigs were given colostrum and milk, the protected pigs had 2 to 4 logs/gm lower numbers of E; ggli than the unprotected pigs (Wilson and Svendsen, 1971). Colostrum or’ milk. may' provide additional factors, such as lactoferrin and the lactoperoxidase-thiocyanate-Hzoz system (Contrepois gt 51‘, 1986), that are lacking in serum or MCA preparations which allow the colostrum and milk to be effective in the prevention of enteric colibacillosis in gnotobiotic pigs. Lactoferrin has been reported to have a powerful bacteriostatic effect on fit ggli (Bullen gt alt, 1972). Using conventionally-reared, weaned pigs, Smith and Linggood (1971) demonstrated that the protective effect of parenterally administered L ggli antiserum correlated with a failure of the infecting organism, the homologous strain of E; ggli, to proliferate in the intestine. The administration of 125 to 200 ml of antiserum usually prevented the pigs from developing diarrhea or edema disease after exposure to K88-positive fit ggli. Protection was believed to be primarily bactericidal, but an antienterotoxic effect was also suggested. Kohler (1967) also administered L ggu antiserum parenterally but to gnotobiotic pigs with no reduction in the numbers of bacteria. 155 In a study by Smith (1972) , colostrum-deprived pigs were given antiserum in milk orally and then challenged with K88-positive ETEC and a larger number of nonpathogenic Eh,ggli and lactobacilli. Multiplication of homologous ETEC was inhibited in the small intestine of pigs given the antiserum. This effect, considered to be antibacterial, was believed to be responsible for delaying or preventing the onset of diarrhea. An antienterotoxic effect didn't appear to be present since antiserum against the enterotoxin did not delay the onset of diarrhea caused by L 9211 0141:K85ab, 88ab. Smith believed that the protective effect was associated with the inhibition of adhesion by the K88 antigen of at ggli. In the initial days of a study by Hainil gt LL, (1987) the mean concentration of available K99 antigen in the feces of hysterectomy derived, colostrum deprived pigs fed K99 MCA was lower when measured with an ELISA test than in those receiving K88 MCA. Also, in a study by Sadowski gt 31; (1986), the 987P pilus levels in the feces of 987P HCA treated colostrum-deprived neonatal pigs were lower than in the control animals. Therefore, except for the study by Wilson and Svendsen (1971), studies reporting a reduction in the number of L 9211 in the small intestine of animals receiving MCA have not used gnotobiotic pigs. 156 With the exception of litter 3, the 987P MCA administered did not appear to be protective against enteric colibacillosis due to 987P-positive L 9911 in gnotobiotic pigs. Several reasons may explain the ineffectiveness of the antibody. First, in pigs, unlike with calves, one single dose of the antibody may not be an effective treatment. Piglets are susceptible to E1 ggli infections for several weeks compared to calves and lambs which are primarily susceptible just during the first couple of days of life (Runnels gt git, 1980: Smith and Halls, 1967). Until calves and lambs become naturally resistant to ETEC infections, antibody need only be present for a short period of time in the intestine to prevent colonization and thus diarrhea. However with pigs, pilus-specific antibody should probably be present for an extended period of time in the intestine. Also, since antibodies in suckling pigs are constantly being diluted and lost from the small intestine, they should be continuously replaced to maintain a protective level (Kohler, 1967, 1978). Porter gt 1L (1970b) showed that even after a single large feed of milk, IgA could no longer be detected in the jejunum after 3 hours in the intestines of young piglets. Several studies have shown that in pigs, both gnotobiotic and specific-pathogen-free, hyperimmune 157 serum, milk and colostrum have a temporary protective effect against ETEC (Kohler, 1967, 1974; Kohler and Bohl, 1966; Miniats gt git, 1970; Scoot gt 1L, 1972; Svendsen and Wilson, 1971). In their studies, pigs were fed the antibody prior to challenge and then for 1 to 3 days after challenge at intervals ranging from 1 1/2 to 8 hours. Generally no clinical signs of diarrhea were observed during the period the antibody was administered, but following the withdrawal of the serum, diarrhea would ensue within 12 to 24 hours. Only a slight protective effect was seen in one study where hysterectomy derived, colostrum-deprived pigs were fed K99 MCA for several days after their initial HCA feeding. All the challenged pigs developed diarrhea, and 1 pig in each group died. However, the diarrhea did start later and end earlier in the pigs fed K99 MCA compared to those fed K88 MCA (Mainil gt AL, 1987) . The previous results indicate that, in order for antibody to be effective, it must be continually present in the intestinal tract of pigs. In the preceding experiments the antibody was not only administered for a prophylactic effect but also for a treatment effect. In 1 experimental trial, a K88 MCA against all 3 serologic variants was orally administered to neonatal, colostrum deprived pigs. A therapeutic effect was noted, but there was no prophylactic effect. Pigs given a therapeutic 158 treatment with the HCA ( 29 pigs), with or without a preceding prophylactic treatment, had a zero mortality rate. However, in l litter where the antibody was only administered prophylactically, all the animals died (Foged gt 1L“ 1986). In the present study, the single dose of HCA was administered in a prophylactic manner. Possibly, directly after the administration of the MCA there was inhibition of the multiplication and colonization of the ETEC, but as the antibody passed through the small intestine the bacteria were then able to colonize the intestine and cause diarrhea. If so, the antibody had only a very slight effect since clinical signs in the treated and control animals occurred at approximately the same time. Sherman et al (1983) speculated that diarrhea occurred in calves receiving the K99 MCA because the MCA may not have completely blocked colonization. The doses of HCA administered in the present study may have been insufficient and thus colonization with subsequent clinical signs of enteric colibacillosis was able to occur. Isaacson it at; have said that, even if only 10% of a K99-piliated strain of L ggu was capable of adhering, the bacteria would be able to proliferate to a high enough concentration such that > 109 bacteria could adhere to 10 cm of ileum 16 hours after inoculation of the pig with 109 bacteria. Consequently, the MCA would have 159 to be present in a high enough concentration to prevent more than 90% of the piliated bacteria from binding. The type of antibody used in this study, IgG, may explain the ineffectiveness of treatment. It's generally believed that IgG is of minor importance compared to IgA and IgM in the protection of the intestine (Bienstock, 1975; Miler gt 31‘, 1975; Sellwood, 1984b). Porter gt alt (1970a) have suggested that secretory IgA is the first line of defense against 3;, ggli infections in pigs. Secretory IgA is highly resistant to proteases and probably persists in the intestine longer than 196 and IgM. Miler gt 31; (1975) determined the protective effect of porcine colostrum, serum, IgG, IgM and IgA against an enterotoxic strain of L 9211 in newborn germfree pigs. They reported that IgA isolated. from immune porcine sera and colostrum was effective at a lower concentration against ETEC in ligated pig loops than IgG or IgM isolated from the same sources. With the brush border adhesion inhibition assay, IgG isolated form colostrum and milk was incapable of blocking the binding of the K88 pilus to the intestinal brush border surface, unlike IgA and IgM (Sellwood, 1984b). The use of an IgA type of 987P MCA may have ‘been :more effective in the treatment of enteric colibacillosis in the pigs. Some MCAs are very susceptible to changes in pH and salt concentrations (Goding, 1986). The antibody may not 160 have been effective because of its early destruction in the gastrointestinal tract due to gastric pH or proteases. An acidic gastric pH is capable of destroying or altering antibody. However, the gastric pH of newborn pigs is relatively high (Moon, 1974; Smith and. Jones, 1963). Also, proteolytic enzymes within the stomach may not be active in newborn piglets. The pH of the gastric contents does not drop low enough soon after birth for the enzymes to be strongly hydrolytic. Additionally, milk rapidly escapes into the intestine within 5 minutes of being suckled, possibly not allowing adequate time for the globulins to be destroyed in a significant amount. In the present study 987P MCA present in the gastric contents of 3 treated pigs was still active in a slide agglutination assay, as well as MCA present in the cecal contents of another pig. Germfree animals in comparison to conventional animals do, however, have an increased number of enzymes in their intestinal contents (Kawai and Morotomi, 1978). Two explanations have been postulated for the increase. Slower peristalsis of the germfree intestine may lead to slower elimination of intestinal contents, or a slower turnover of intestinal epithelial cells may result in more mature cells with more active synthesis of certain enzymes (Savage gt alt, 1981; Wostmann and Bruckner-Kardoss, 1981) . The higher concentration of proteolytic enzymes 161 would thus lead to a more rapid destruction of intestinal Igs (Fubara and Freter, 1972) . The rapid proteolytic destruction would thus lead to the ineffectiveness of the MCA, particularly if the MCA was not replenished on a regular basis. Using a brush border adhesion test in the present study, 1 pigs' cecal content supernatant at a dilution of 1:10 was able to prevent the adhesion of 987P-positive E; ggli. Large aggregates of bacteria were seen at the 1:10 and 1:100 dilution, even though adhesion was not inhibited at 1:100. The presence of the aggregates tends to indicate that the antibody was present and capable of agglutination. In the other treated pig adhesion to the brush border’ was not inhibited at a 1:10 dilution. Therefore, in at least some instances antibody appears to be active as it passes from the small intestine and capable of preventing adhesion in an in yitzg situation. The titer of the antibody used in the present study was determined with a slide agglutination test which may not have been a good indicator of the protective ability of the antibody. Nagy gt Q; (1978) and Rutter gt g1; (1976) have observed that antibodies demonstrated by agglutination tests are not major contributory factors in protection. Nagy gt 51; (1985) noted no correlation between colostral or seroagglutinins to fimbrial antigens of B; ggli and mortality rates in litters challenged with 162 homologous pili, but good correlation was found between colostral precipitins to K88 antigens and mortality rates in litters. The effect of vaccination on antipilus antibody titers differed from almost nil (K99) to slight (K88 ab and ac) or marked (987P). However, no correlation was noted between these titers and mortality rates in litters challenged with homologously piliated E; ggli. Another factor which may have caused the MCA's ineffectiveness is the binding strength of the antibody. Some MCA show weak epitope binding (DePinho gt g1;, 1986: Young, 1985). If the MCA had a low affinity, gut peristalsis could have disassociated the pilus-MCA complex. Some environmental conditions may alter the MCA binding site and thus prevent the MCA from binding (Goding, 1986). Also, the species of origin of the MCA may have limited the antibody's effectiveness. According to Raybould (1985), in some instances passive immunity may be induced more effectively by MCA originating from the same species as the animal under treatment. The pilus subunit is composed of numerous epitopes. Monoclonal antibodies against some pilus epitopes are capable of preventing adhesion whereas others are not (Schifferli gt g1;, 1987). In 2129, the 987P MCA may not have been able to prevent adhesion because of the epitope it was directed against. A MCA directed against another pilus epitope or several different MCAs directed against 163 multiple pilus epitopes might be more effective in the prevention of adhesion. Alternate mechanisms other than the direct inhibition of adhesion via which the antibody could have prevented the bacteria from colonizing the gut may involve agglutination, opsonization, bactericidal or bacteriostatic effects, steric hindrance, altered bacterial surface charge or plasmid curing (Brinton, 1978; Linggood and Porter, 1978; To gt g1;, 1984). However, if the principal mechanism was via antibody opsonization or agglutination, a protective effect would be expected from antibodies to any pilus present on infecting bacteria, even if that pilus was not necessary for virulence (Brinton gt 31;, 1983). Jayappa gt Q1; (1983) concluded that both mechanisms probably occur in the protection of pigs. The type of experimental animals, gnotobiotic pigs, used in this study may have also affected the results. In monoassociated gnotobiotic pigs, colonization can occur with most bacteria, and the virulence factors required for colonization in a conventional animal may not be necessary. In fact, even when some animals are immunized against certain bacteria, the bacteria are still able to colonize the intestine (Shedlofsky and Freter, 1974). Several factors contribute to the ease with which gnotobiotic pigs are colonized in the intestine: 1) lack 164 of local and systemic immune mechanisms .(rev. Kim, 1981), 2) the lack of bacterial antagonism (Aly and Shinefield, 1982; Dubos, 1963; Freter, 1956: van der Waaij gt g1;, 1971), 3) slowed peristalsis. Jones and Rutter (1972) have shown that, even though the K88 pilus is essential for ETEC to cause diarrhea in conventional pigs, it is not required for diarrhea to Occur in monoassociated gnotobiotic pigs. It was assumed that the slower intestinal motility and more rapid growth of the monoassociate in the absence of competition from other microflora obviate the need for ETEC to adhere to the intestinal wall. Therefore, as appeared to be the case in the present study, 5; gg11 could cause diarrhea without significant adhesion in gnotobiotic pigs. In ligated intestinal loops, where the washout effects of intestinal motility are absent, ETEC caused fluid accumulation in the absence of extensive adhesion to the small intestinal epithelium. Also, ETEC neither multiplied faster nor attained higher total numbers than nonenterotoxigenic E; 9911 (Bertschinger gt g1;, 1972) . Thus, extensive adhesion is not essential for E; gg11 to cause fluid loss by the small intestine and therefore, diarrhea could have occurred in the gnotobiotic pigs without extensive adhesion. In fact, that appeared to be the case in the present study; Clinically, the animals had. signs of diarrhea but evidence of colonization was not present in 165 most pigs. Since colonization was limited in most litters, the value of the antibody was difficult to determine. Several reasons may exist to explain the lack of adhesion. One reason for the lack of colonization may be as simple as an inadequate challenge inoculum, since with an inoculum of 1010 E; 9911, adhesion and colonization were present. Another reason for the lack of adhesion may be associated with genetic resistance to 987P-positive L 9911. However, Moon et a1. (1979) have reported that they have not encountered pigs genetically resistant to 987P- positive L gg11 and also, in the previous chapter, all the brush border samples examined had receptors for 987P- positive L gg11. The possibility does exist, though, that some pigs may be genetically resistant to 987P— positive L gg11 since neither the control nor treated pigs were successfully colonized. Another reason for the lack of adhesion may be due to a lack of a capsule by the strain of E. 9911 used. Some reports indicate that the capsule enhances the colonizing ability of L 9311 strains that produce them (Hadad and Gyles, 1982; Nagy gt g1;, 1976; Smith and Huggins, 1978). Piliated acapsular mutants of many strains have been shown to colonize the small intestine to a smaller extent than the encapsulated parenteral strains. Isaacson gt Q1; (1977) reported that both a polysaccharide capsule and the 987P pilus are required for colonization by strain 987 to 166 occur in 2119, even though the acapsular piliated form of strain 987 adhered to isolated epithelial cells 13 y1ttg. Moon gt g1; (1979) have suggested that the capsule could be required for the expression of pili in vivo or that both structures could be required for adhesion by strain 987 in M. Moon and Runnels (1984) indicated that an encapsulated ETEC strain was more virulent in suckling neonatal pigs than was its acapsular mutant. In the present study, the bacteria were able to adhere in the first 3 litters. Unless the bacteria lost the ability to produce the capsule, this seems to be an unlikely explanation. Hewever, it is interesting to note that in the first litter, a challenge inoculum of 108 bacteria was able to cause colonization of the small intestine. However, in the last 4 litters challenged with 108 L gg11, only 1 animal was colonized. Evidence of colonization was present bacteriologically in that animal but not histologically. In gnotobiotic animals, glycoproteins that often act as receptors for bacterial adhesion are of a different composition because they are not degraded by indigenous microflora (Jones, 1977) . Thus, the receptors for the 987P pili in the present study may be incapable of binding piliated E; 9911. However, this possibility does not seem likely since in some pigs adhesion was present. 167 Even though bacterial adhesion was not seen in most of the intestinal sections, there was a close association of the bacteria with the small intestinal villi. Bacteria were often seen entrapped in mucus adjacent to the villi. The mucus may have provided binding sites for the E; 9911, and along with the decreased gut peristalsis in the gnotobiotic pig, multiplication of the bacteria may have been great enough to produce a sufficient amount of enterotoxin to cause diarrhea. Freter (1981) has suggested that to resist the flux of intestinal secretions and to colonize tissue, it may be sufficient for bacteria to bind to mucus alone. In a recent study by Broes gt Q1; (1988) it was shown that a diarrhea-causing ETEC, strain 08:KX105, colonized the neonatal pigs' small intestine to a moderate extent but did not strongly adhere to the intestinal epithelium. The n; 9911 strain was classified as a nonclassical ETEC which produced LT and 8Tb enterotoxin and usually produced 987P pili. W gg11 small intestinal colony counts of 108 or 107/10 cm were present in the ileum and jejunum respectively of all piglets with diarrhea. However, with histology, immunofluorescence microscopy and scanning electron microscopy little evidence of bacterial adhesion to the small intestinal epithelium was seen. Only a few adherent bacteria were seen on the tips of the villi or randomly scattered in the lumen. An 168 association index of 1.1 was found. In the present study in the last 5 litters, little evidence of adhesion was present on the histopathologic and scanning electron microscopic sections of intestine. It is interesting to note, that in the initial part of this study, an indirect fluorescent antibody test was performed on the first 3 litters with little evidence of positive fluorescence even though the animals were colonized microbiologically. Use of the test was subsequently discontinued since there appeared to be a technical problem with the test. Yet, year-old positive samples from a previous study were positive using the same technique. Broes gt g1; (1988) suggested that the 08:KX105 strain lacked the ability to strongly adhere to the intestinal mucosa, possibly only binding to mucus. Also, unlike the classical 987P- positive E; gg11 strains which produce STa enterotoxin and are usually isolated from pigs less than 1 week old, the ETEC used in their study produced LT and STb enterotoxin and were isolated from weaned as well as suckling pigs. They speculated that the 987P pili produced by such strains was modified leading to a decreased ability to adhere to intestinal villi 1n yiyg. They thus demonstrated that certain ETEC may cause acute diarrhea in piglets in the absence of extensive colonization of the small intestine. The serotype of the E; gg11 strain used in the present study is unknown but many parallels can be 169 drawn between the E; gg1i strain used in my study and that previously described. The results of the present study suggest that the direct passive oral immunization of gnotobiotic pigs with 987P MCA (from hybridoma 4A3) is not beneficial in the prevention of diarrhea caused by 987P-positive E; 9911 in gnotobiotic pigs. No significant differences in morbidity, mortality, gross lesions, histologic lesions and most of the bacteriologic counts were present between the treated and control animals in the 8 litters. Since pigs infected with 108 L gng, had clinical signs of diarrhea even though histologic, scanning electron microscopic and microbiologic results generally indicated that colonization was not present, it was difficult to determine the effectiveness of the antibody. Though the MCA was ineffective in gnotobiotic pigs, the efficacy of the antibody may differ in conventional animals. Therefore, in the future it may be worthwhile to determine the MCA's effect in conventional pigs. Also, in m and in 1119, it would be interesting to determine the antiadhesive ability of the MCA in comparison to colostrum, milk, a monospecific polyclonal serum and a mixture of several different 987P MCAs. Additionally, the 987P MCA could be used in the development of various immunodiagnostic tests, such as a fluorescent antibody 170 test or an ELISA, for the clinical detection of 987P- positive E; gg11. SUMMARY This study was used to determine if the clinical signs, lesions and in 1119 adhesion of 987P-positive L 9911 to the intestinal mucosa of gnotobiotic pigs could be altered by the oral administration of 987P MCA. Two to 5-day-old gnotobiotic pigs from 8 litters were each given 0.5 to 12 ml of 987P MCA, challenged 0.5 to 2 hours later and then observed clinically for signs of enteric colibacillosis. Gross, histopathologic, scanning electron microscopic and microbiologic examinations were performed. Clinical signs, bacteriologic counts, and gross, histologic and scanning electron microscopic lesions were similar in the control and treated animals. Clinically, all except two animals had diarrhea. Gross lesions were minimal and variable, consisting primarily of fluid in the small and large intestines and pasty to liquid rectal contents. Adhesion and colonization based on microbiologic, histologic and scanning electron microscopic examinations were limited. Only animals in litters 1 and 3 had substantial adhesion and colonization. Based on the results of this study the 987P MCA was ineffective in the prevention of diarrhea in gnotobiotic 171 pigs challenged with 987P-positive E; 9911. An accurate assessment of the MCA's effect on adhesion was difficult, though, because of the limited colonization of both the treated and control animals. It was also difficult to assess the efficacy of the 987P MCA because gnotobiotic pigs were used in the study and reports have indicated that pili are not a necessary virulence attribute for ETEC in gnotobiotic pigs. CHAPTER 3 THE EFFECT OF THE ORAL WHISTRATION OP 987P HONOCLONAL ANTIBODY ON ENTEROTOXIN PRODUCTION OF 987P-POSITIVE W ELI IN GNOTOBIOTIC PIGS CHAPTER 3 THE EFFECT OF THE ORAL.ADHINISTRATION OF 987P HDNOCLONAL.ANTIBODY ON ENTEROTOXIN PRODUCTION OF 987P-POSITIVE ESSBEBIQEIAHQQLI IN GNOTOBIOTIC PIGS INTRODUCTION The second virulence determinant necessary for ETEC to cause diarrhea in neonatal pigs is enterotoxin production (Smith and Halls, 1967). Two types of enterotoxins, based on their thermolability, antigenicity and molecular weight, are produced by ETEC: 1) ST and 2) LT. In the present study, 987P-positive L gg11 were used. Strains of ETEC which produce the 987P pilus are always associated with ST enterotoxin production (Guinee and Jansen, 1979b: Moon gt g1;, 1980). Two distinct types of ST are known: 1) STa which is methanol soluble, and active in the infant mouse model and in ligated jejunal segments of piglets 1-3 days old and 2) 8Tb which is methanol insoluble, inactive in the infant mouse model and active in ligated intestinal segments of 7-9-week-old weaned pigs (Burgess gt g1;, 1978; Gianella, 1976; Kapitany gt g1;, 1979a,b: Newsome gt g1;, 1978a,b). 173 174 The action of STa is almost instantaneous, relatively short lasting and readily reversible by rinsing (Evans gt 31;, 1973; Field gt 31;, 1978; Hughes gt 31;, 1978; Guerrant gt 31;, 1980). The STa toxin binds to intestinal membrane brush border receptors (Frantz gt 31;, 1984: Giannella gt 31;, 1983). The mechanism of action of STa involves the activation of guanylate cyclase in the small intestinal epithelial cell with a subsequent increase in cGMP levels (Field gt 31;, 1978; Hughes gt 31;, 1978; Newsome gt 31;, 1978a,b). The toxin causes a hypersecretory diarrhea by decreasing NaCl absorption and increasing chloride secretion (Guandalini gt 31;, 1982: Rao gt 31;, 1930). The STb toxin's mechanism of action is believed to be cyclic nucleotide independent (Kennedy (gt (31;, 1984). Like STa, STb has a rapid, but reversible, onset of action. In vivo, 8Tb causes a significant increase in the amount of bicarbonate, sodium and chloride in the intestinal contents (Weikel gt 31;, 1986). Assays for the detection of ST are limited because of its nonantigenic nature, and the more commonly used tests involve animals (Dean gt 31;, 11972). Recently, a few immunologic tests for the detection of ST have been described (de Mol gt 31;, 1983, 1985; Dreyfus gt 31;, 1983; Thompson gt 31;, 1984). 175 Sherman gt 31; (1983) observed that calves to which K99 MCA was administered and subsequently challenged with K99-positive L gg11 all had diarrhea. They speculated that the number of E; gg11 in the challenge inoculum might have been large enough to produce sufficient enterotoxin without adhesion to the mucosa to cause diarrhea. In the present study, the L 95111 inoculum was washed prior to being given to the pigs to remove the enterotoxin. The objective of this study was to determine the effects of 987P MCA on enterotoxin production in gnotobiotic pigs challenged with 987P-positive E; go 1. MATERIALS AND METHODS Experimental Animals Piglets from the last two litters, 7 and 8, were used in the this study. The experimental protocol was the same as that previously described Fin Chapter 2 with the following additions. cGMP Radioimmunoassay Piglets were anesthetized with sodium pentobarbitala. An incision was made on the ventral abdominal surface of the pigs. The small intestine was removed, and a 3-cm section of ileum near the cecum was opened and excised. The ileal section was then immediately placed on ice and a Butler Company, Columbus, OH. 176 the mucosa stripped from the intestine with a razor blade. The mucosa was then homogenized within 60 seconds of tissue removal in 5 ml of 6% TCAb with a Teflon serrated- tipped tissue grinderc rotating at approximately 900 rpm at 4'C. The protein concentration of 1 m1 of the TCA suspension was determined by the Lowry method (refer to Chapter 1) . The remaining part of the suspension was centrifuged at 10,000 x g. The TCA was removed from a 2 ml volume of the supernatant solution with 3 2-ml washings of diethyl ethemb. The 3 ether washings were pooled. The TCA-free extract, the ether layer, was dried with a nitrogen evaporatord and then reconstituted to 2 ml with 50 mM acetate buffer. The samples were then frozen at -70'C until the test was performed. The cGMP was measured using a commercial radioimmunoassay kite. Three dilutions of the sample were used. The sample dilutions were run in. duplicate and. counted in. a scintillation counterf for 5 minutes per sample (Kapitany gt 31;, 1978). b Mallinckrodt Inc., Paris, KY. c Thomas Co., Philadelphia, PA. d Model 111, Organomation Assoc. , Inc. , Shrewsbury, e Amersham Corporation, Arlington Heights, IL. f Beckman Model LS 3133P, Beckman Instruments, Inc., Palo Alto, CA. 177 Suckling Mouse Assay A section of ileum, 30 cm in length, was removed from each pig. The intestinal contents were rinsed from the lumen with 5 ml of PBS and placed in a tube on ice. The contents were centrifuged at 5,000 x g' (4'C) in a refrigerated centrifugeg for 15 minutes. The supernatant was then filtered through a 0.20 um filter. The supernatant was then frozen at -70’C for 1'to 2 days. Heat stable enterotoxin activity was then determined using a suckling mouse test. As a positive control, the 987P-positive strain of E; 9311, strain 81-1469, was grown in 10 ml trypticase soy brothh overnight at 37'C in a shaking water bathi. The culture was then filtered through a 0.20 um filter. Intestinal content supernatants were thawed. One drop of methylene blue dye was added to the broth and intestinal content filtrates to determine if the injection had been properly performed. Three-day-old mice were separated from their mothers. The mice were then given 0.1 ml of broth or intestinal content filtrate. Three mice were used for each sample. Using a syringe and a 30 gauge Ihypodermic needle, the fluid. was injected through the body wall directly into the milk-filled 9 International Refrigerated Centrifuge, Model PR-6, International Equipment Co., Needham, MA. h Baltimore Biological Laboratories, Cockeysville, MD. 1 Precision Scientific Co., Chicago, IL. 178 stomachs of the mice. The mice were kept at room temperature for 4 hours and then euthanatized with ether. The abdomen of each mouse was then opened, and the entire intestine was removed. Mice with methylene blue in the peritoneal cavity were not used. The intestine and the remainder of the body were then weighed separately. Gut weight to body weight ratios below 0.070 were considered negative for enterotoxin, ratios between 0.070 and 0.090 were considered equivocal and ratios above 0.090 were considered strongly positive for enterotoxigenic activity (Dean gt 31;, 1972; Gianella, 1976). Statistical Analysis Data were analyzed using a paired t test to determine if differences existed in cGMP values between control and treated animals. A Chi—square test was done to determine differences in STa between control and treated animals using the suckling mouse assay. Differences between groups were considered significant at the P 5_0.05 level (Steel and Torie, 1980). RESULTS cGMP Radioimmunoassay Results of the cGMP RIA are listed in Table 3-1. No statistically significant difference was present between the treated and control cGMP values for the animals in litters 7 and 8. Values ranged from 2.0 to 3.7 picomole .copguezpfifiouoa 9 7 fl 1.. Em Ea fin «a in 9 En Cm Em 2m m6. 0 m o.m fin fin Em m.~ Em .H. u «A o.m ~.~ o.~ o a. g game 298 $352 amped .28 «a gflemodnmsmm 5.? 0098.393 moan 0.33.835 mo umdwfim Hood.“ g magma momma—REG? E6 Dag ..Tn 033. 180 (pmole)/mg protein with an average value of 2.7 for the control animals and 3.0 for the treated animals. Suckling Mouse Assay Intestinal filtrates from both the control and treated animals in the 2 litters were all negative for STa enterotoxin. Results for the 987P-positive L g911 broth culture were in the equivocal range (Table 3-2). DISCUSSION No statistical difference was present in cGMP levels between the treated and control animals in the 2 litters. The cGMP level was only slightly elevated compared to reported control values, 2.3 3; 0.1 pmole/mg protein (Newsome gt 31;, 1978a,b). Values for the 2 control animals in litter 7 with no diarrhea were very similar to the values in animals with diarrhea. Average cGMP values in the treated pigs were slightly higher than the control animal cGMP values. Since diarrhea was lessening at the time of euthanasia, ST production was probably low. The 2-day observation period after challenge was probably too long for an accurate assessment of an elevations in cGMP. However, euthanasia of the animals prior to this time would have greatly limited the useful data obtained from the last two litters. In retrospect, sacrifice within. 2 hours of the onset of clinical signs would probably have been most beneficial in .mofismuommsumufimfimaoflmmn .gflagaflgflwggmuadfimuguemagnum Bod a d m mood Rod dmod dead wood a 1 Bod mmod God mead mead o m mmod Hm“ «a mood mood wood wood Sod «mod 9 mmo.o meo.o mmo.o emo.o emo.o o s nutmeg £33 5 9.8852 H393 23.3 «a «finance-meme £8 c.8556 8.3 08388.8 no 353m 3:33.: 5.8 3888 >88 «32. sauna dun «and. 182 the demonstration of cGMP levels, especially since Holmgren (1985) has suggested that diarrhea itself may reverse the ST action by "diluting" the toxin from its mucosal receptor. In one report, biopsies were taken from mice 40 minutes after dosing with toxin to determine cGMP levels (Newsome, 1978a). A report by Kapitany gt 31; (1978) indicated that cGMP was not elevated until an ED (minimum dose of protein required to give a positive response in suckling mice) dose level of 1000, where fluid secretion was most pronounced, was given. Some authors have indicated that there is a change in the ratio of cAMP to cGMP so examining the ratio of cAMP to cGMP may have been beneficial. Using freeze-clamped, full-thickness intestinal biopsies, Newsome gt 31; (1978a,b) said that STa raised the cGMP concentration and lowered the cAMP\cGMP ratio in mice. Intestinal samples were removed and placed on ice as soon as possible, but post mortem changes may have still occurred and altered the cGMP levels. Also, because of time constraints, samples for the cGMP RIA were frozen for several days prior to performance of the test which may have adversely affected the results. Since the action of STa is reversible by rinsing, it was postulated that STa bound in the piglet small intestine could be "rinsed" free and subsequently detected 183 in the suckling mouse assay. Results of the assay indicated that enterotoxin was not present in an amount large enough to cause intestinal secretion in suckling mice. Removal of the luminal contents with 5 m1 of PBS may have diluted the enterotoxin to a level to low to cause fluid secretion. Also, diarrhea was abating at the time of euthanasia so the amount of bound ST!l may have been low, particularly since .STa's action is almost instantaneous and relatively short lasting (Field gt 31;, 1978: Hughes gt 31;, 1978: Guerrant gt 31;, 1980). Freezing the intestinal filtrate samples should not have affected the results since Dean gt 31; (1972) froze toxin at -70'C for 18-20 days with no adverse effect on the enterotoxins' activity. The gut weight/body weight ratio for the 987P- positive E; gg11 broth culture was in the equivocal range for enterotoxin production (0.070 to 0.090, Dean gt 31;, 1972; Gianella, 1976). Moon gt 31; have stated that a gut to carcass weight ratio value ‘2 0.085(pmole)/mg is considered positive, and in that case the cultures would be considered positive for STa production. The low level of ST production may be associated with the method of culture. It has been suggested that STa production by E; 9911 strains isolated from animals other than humans may not be maximal in trypticase soy broth (Scotland gt 31;, 1985). Alderete and Robertson (1977) have indicated that 184 the production of ST is relatively low in aerated cultures and have suggested that a synthetic medium, casamino acids-yeast extract medium, be used. Also, the ability of E; 9911 strains to secrete STa toxin is occasionally lost on subculturing 19 yitrg (Burgess gt 31;, 1978; Dean gt 31;, 1972). Since cGMP was only slightly elevated in this study and the 987P-positive L 9911 broth culture suckling mouse test results were in the equivocal range the 987P- strain of ETEC used in the study may be an STb producer. If so results from this chapter, lack of elevation of cGMP, and Chapter 2, lack of adhesion of 987P-positive E; 9911 to brush borders would correlate well with the results of Broes gt 31;, 1988. Broes gt 31; reported that a 8Tb producing, 987P-positive strain of E; 9911 colonized the neonatal pigs' small intestine to a moderate extent but did not adhere to the intestinal epithelium. 185 SUMMARY This study was used to determine if enterotoxin production in gnotobiotic pigs challenged with 987P- positive E; 9911 could be affected by the oral administration of 987P MCA. The cGMP content of the pig ileal mucosa was determined using a cGMP radioimmunoassay. No statistically significant difference in cGMP values was present between the treated and control animals in the two litters. Values ranged from 2.0 to 3.7 pmole/mg protein with an average value of 2.7 for the control animals and 3.0 for the treated animals. Intestinal content filtrates from the piglets were used in a suckling mouse assay for the detection of STa in luminal contents. All intestinal content filtrates were negative for STa in the assay. Based on the results of this study, at the time of euthanasia, substantial amounts of enterotoxin were not present in the piglets' small intestines. Diarrhea had abated by the end of the two day observation period, so an accurate assessment of enterotoxin production in this study is difficult. BIBLIOGRAPHY Abraham, S.N., Hasty, D.L., Simpson, W.A. and Beachey, E.H.:Antiadhesive properties of a quaternary structure-specific hybridoma antibody against type 1 fimbriae on Egghgtigm 9911. J. Exp. Med. 158:1114- 1128, 1983. Acres, S.D., Isaacson, R.E., Babiuk, L.A. and Kapitany, R.A.: Immunization of calves against enterotoxigenic colibacillosis by vaccinating dams with purified K99 antigen and. whole cell bacterins. Infect. Immun. 25:121-126, 1979. Acres, S.D., Isaacson, R.E., Khachatourians, G., Babiuk, L. and Kapitany R.A.: Vaccination of cows with purified K99 antigen, K99+ anucleated live E; 9911 (ALEC), and whole cell bacterins containing enterotoxigenic E; 9911 for prevention of enterotoxigenic colibacillosis of calves. In Breseeding§_2f the Essend Internatignal fixmeesium 9n Ng993t31 121313133, Saskatoon, Saskatchewan, Canada, pp. 443-455, 1978. Aimoto, S., Takao, T., Shimonishi, 8., Hara, 8., Takeda, T., Takeda, Y. and Miwatani, T.: Amino-acid sequence of a heat-stable enterotoxin produced by enterotoxigenic Egghgtignig 9911. Eur. J. Biochem. 257-263, 1982. Alderete, J.F. and Robertson, D.C.: Purification and chemical characterization of the heat-stable enterotoxin produced by porcine strains of enterotoxigenic Egghgtignn 9911. Infect. Immun. 19:1021-1030, 1978. Altmann. K., Pyliotis, N.A. and Mukkur, T.K.S.: A new method for the extraction and purification of K99 pili from enterotoxigenic Egghgtighig 9911 and their characterization. Biochem. J. 201:505-513, 1982. Aly, R. and Shinefield, H.R.: Eggtgt131 Intgtfgtgggg. CRC Press, Boca Raton, FL, 1982. 186 187 Anderson, M.J., Whitehead, J.S. and Kim, Y.S.: Interaction of Egghgzign13 49911 K88 antigen with porcine intestinal brush border' membranes. Infect. Immun. 29:897-901, 1980. Aning, K.G. and Thomlinson, J.R.: Adhesion factor distinct from K88, K99, F41, 987P, CFAI and CFAII in porcine Eggngtignig 9911. Vet. Rec. 112:251, 1983. Anonymous: Porcimune. Pitman-MOore Inc., Washington Crossing, NJ, 1981 (a technical report). Anonymous: Magic bullet for baby pigs. Anim. Nutr. Health 38:22-26, 1983. Anonymous: Bar-Guard-99 pamphlet. Anchor Animal Health, 1987. ‘ Arbuckle, J.B.R.: The location of Egghgtigh13 9911 in the pig intestine. J. Med. Microbiol. 3:333-340, 1970. Arbuckle, J.B.R.: Enteropathogenic Egghgrighig 9911 on the intestinal mucopolysaccharide layer of pigs. Vet. Path. 104:93-97, 1971. Arbuckle, J .B.R.: Observations on the association of pathogenic Egghg;19913,,9911 ‘with. small intestinal villi of pigs. Res. Vet. Sci. 20:233-236, 1976. Awad-Masalmeh, M., Moon, H.W., Runnels, P.L. and Schneider, R. A.: Pilus production, hemagglutination, and adhesion by porcine strains of enterotoxigenic Egghgtignig 9911 lacking K88, K99, and 987P antige- ns. Infect. Immun. 35: 305- -313, 1982. Bertschinger, H. U., Moon, H. W. and Whipp, S. C. : Association of E_99g:19913, 9911, with the small intestinal epithelium I. Comparison of enteropathogenic and nonenteropathogenic porcine strains in pigs. Infect. Immun. 5:595-605, 1972. Bienstock, J.: The local immune response. Amer. J. Vet. Res. 36:488-491, 1975. Bijlsma, I.G.W., de Nijs, A., van der Meer, C. and Frik, J. F.: Different pig phenotypes affect adherence of Eggngt19913 9911 to jejunal brush borders by K88ab, K88ac or K88ad antigen. Infect. Immun. 37:891-894., 1982. 188 Blood, D.C., Radostits, O.M. and Henderson, J.A.: Balliere, London, 1983. Bourne, F.J.: Humoral immunity in the pig. Vet. Rec. 98:499-501, 1976. Brinton, C.C., Jr.: Non-flagellar appendages of bacteria. Nature 183:782-786, 1959. Brinton, C.C. , Jr. : The structure, function, synthesis and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram negative bacteria. Trans. N.Y. Acad. Sci. 27:1003-10054, 1965 Brinton, C.C, Jr.: Contributions of pili to the specificity of the bacterial surface and a unitary hypothesis of conjugal infectious heredity. In 1mg 59.921.219.12: 91 $29.11 5112:9999- Edited by B D- Davis and L. Warren, Prentice-Hall, Englewood Cliffs, NJ, pp. 37-70, 1967. Brinton, C.C., Jr.: The piliation phase syndrome and the uses of purified pili in disease control. In Wings 9.: the 211mm 19.1.9; W Magma, Atlanta, GA, pp. 33-7o,1973. Brinton, C.C., Jr., Fusco, P., Wood, S.W., Jayappa, J.G., Goodnow, R.A. and Strayer, J.: A complete vaccine for neonatal swine colibacillosis and the prevalence of Egghgnignn 9.911 on swine isolates. Vet. Med. Small Anim. Clin. 78:962-966, 1983. Broes, A., Fairbrother, J.M., Lariviere, S., Jacques, M. and Johnson, W.M.: Virulence properties of enterotoxigenic Egghgzignig 9911 08:KX105 strains isolated from, diarrheic piglets. Infect. Immun. 56:241-246, 1988. Bruner, D.S. and Gillespie, J.H.: 11393919 Infegtieus Dim 2f m1; Animals. University Press, Cornell, Ithaca, NY, p. 139, 1973. Bullen, J. J., Roger, H. J. and Leigh, L.: Iron-binding proteins in milk and resistance of mm 9911 infection in infants. Brit. Med. J. 1: 69- 75, 1972. 189 Burgess, M.N., Bywater, R.J., Cowley, C.M., Mullan, N.A. and Newsome, P.M. : Biological evaluation of a methanol-soluble, heat-stable E9999319913 9911 enterotoxin in infant mice, pigs, rabbits, calves. Infect. Immun. 21:526-531, 1978. Burgess, M.N., Cowley, C.M., Melling, J., Mullan, N.A. and Newsome, P.M. : Assay of the heat-labile enterotoxin of Egghgtignig 9911 in infant rabbits. J. Med. Microbiol. 12:291-302, 1979. Burns, D.L., Moss, J. and vaughn, M.: Choleragen- stimulated release of guanyl nucleotide from turkey erythrocyte membranes. J. Biol. Chem. 257:32-34, 1982. Burrows, M.R., Sellwood, R. and Gibbons, R.A.: Haemagglutinating and adhesive properties associated with the K99 antigen of bovine strains of Egghgt1gn13 9911. J. Gen. Microbiol. 96:269-275, 1976 Cassel, D. and. Selinger, z.: Mechanism. of adenylate cyclase activation by cholera toxin: Inhibition of GTP hydrolysis at the regulatory site. Proc. Natl. Acad. Sci. U.S.A. 74:3307-3311, 1974. Chan, S.K. and Gianella, R.A.: Amino acid sequence of heat stable enterotoxin produced by Eggngt19913 9911 pathogenic for man. J. Biol. Chem. 256:7744-7746, 1981. Chandra, R.K;: Prospective studies of the effect of breast feeding on incidence of infection and allergy. Acta Paediatr. Scand. 68:691-694, 1979. Chanter, N.: Structural and functional differences of the anionic and cationic antigens in K99 extracts of Eggngt19913 9911 B41. J. Gen. Microbiol. 126:1585-1589, 1982. Christie, B.R. and Waxler, G.L.: Experimental colibacillosis in gnotobiotic baby pigs. II. Pathology. Can. J. Comp. Med. 37:271-280, 1973. Clements. J.D. and Finkelstein, R.A.: Immunological cross-reactivity between a heat-labile enterotoxin(s) of 292922121119 29111 and subunits of £2112 29919292 enterotoxin. Infect. Immun. 21:1036-1039, 1978a. 190 Clements, J .D. and Finkelstein, R.A.: Demonstration of shared and unique immunological determinants in enterotoxins from 1119219 9119.123: and Menuhin 9911. Infect. Immun. 22:709-713, 1978b. Clements, J.D. and Finkelstein, R.A.: Isolation and characterization of homogeneous heat-labile enterotoxins with high specific activity from 9911 cultures. Infect. Immun. 4:760-769, 1979. Cohen, M.B., Guarino, A., Shukla, R. and Giannella, R.A.: Age-related differences in receptors for Egghgt1gn13 9911 heat-stable enterotoxin in the small and large intestine of children. Gastroenterology 94:367-373, 1988. Contrepois, M., Dubourguier, H.C., Parodi, A.L., Girardeau, J.F. and Ollier, J.L.: Septicemic mm 9911 and experimental infection of calves. Vet. Microbiol. 12:109-118, 1986. Cunningham, A.S.: Morbidity in breast-fed and artificially fed infants. J. Pediatr. 95:685-689, 1979. Curtis, J. and Bourne, F.J.: Immunoglobulin quantitation in sow serum, colostrum and milk and the serum of young pigs. Biochim. et Biophys. Acta 236:319-332, 1971. Dafni, z. and Robbins, J. B.: Purification of heat labile enterotoxin from Egghgt19h13 9911 078: H11 by affinity chromatography with antiserum to 219:19 9991gt3g toxin. J. Infect. Dis. 133: 8138-8141, 1976. Dallas, W.S.: Conformity between heat-labile toxin genes from human and porcine enterotoxigenic W 9911. Infect. Immun. 40:647-652, 1983. Dallas, W.S. and Falkow, 8.: Amino acid sequence homology between cholera toxin and Egghgtignig 9911 heat-labile toxin. Nature 288: 499-501, 1980. Dean, A.G., Ching, Y.-C., Williams, R.A. and Harden, L.B.: Test for “9991191113 9911 enterotoxin using infant mice: Application in a study of diarrhea in children in Honolulu. J. Infect. Dis. 125:407-411, 1972. 191 Dean, E.A. and Isaacson, R.E.: In y1tr9 adhesion of piliated Egghgt19913 9911 to small intestinal villous epithelial cells from rabbits and the identification of a soluble 987P receptor-containing fraction. Infect. Immun. 36:1192-1198, 1982. Dean, E.A. and Isaacson, R.E.: Location and distribution of a receptor for the 987P pilus of Egghgt1gh13 9911 in small intestines. Infect. Immun. 47:345-348, 1985a. Dean, E.A. and Isaacson, R.E.: Purification and characterization of a receptor for the 987P pilus of Egghgr1gh13 9911. Infect. Immun. 47:98-105, 1985b. de Graaf, F.K., Klemm, P. and Gaastra, W.: Purification, characterization and partial covalent structure of Eggngt1gn13 9911 adhesive antigen K99. Infect Immun. 33:877-883, 1980. de Graaf, F.K., Krenn, B.E. and Klaasen, P.: Organization and expression of genes involved in the biosynthesis of K99 fimbriae. Infect. Immun. 43:508-514, 1984. de Graaf, F.K. and Roorda, I.: Production,purification, and characterization of the fimbrial adhesive antigen F41 isolated from calf enteropathogenic W 9911 strain B41M. Infect. Immun. 36:751-758, 1982. de Jonge, H.R.: The localization of guanylate cyclase in rat small intestinal epithelium. FEBS Lett. 53:237- 242, 1975. de Jonge, H.R.: Cyclic GMP-dependent protein kinase in intestinal brush borders. Adv. Cyclic Nucleotide Res. 14:315-333, 1981. de Jonge, H.R.: The mechanism of action of Egghg119h13 9911 heat-stable enterotoxin. Biochem. Soc. Trans. 12:180-184, 1984. de M01, P., Hemelhof, W., Retore, P., Takeda, T., Miwatani, T., Takeda, Y. and Butzler, J.P.: A competitive immunosorbent assay for the detection of heat stable enterotoxin of Egghgt19913 9911. J. Med. Microbiol. 20:69-74, 1985. de M01, P., Van Wijnendaele, F., Hemelhof, W. and Corazza, Y.: Possible field test for ST-producing Egghgt1gn13 9911. Lancet 1:524-525, 1983. 192 Deneke, C. P., McGowan, K., Thorne, G. M. and Gorbach, S.L.: Attachment of human and pig (K88) enterotoxigenic Eggngzign13 9911 strains to either human or porcine small intestinal cells. Infect. Immun. 45: 522-524, 1984. DePinho, R.A., Feldman, L.B. and Scharff, M.D.: Tailor-made monoclonal antibodies. Ann. Intern. Med. 104:225-233, 1986. Dixon, J.M.S.: The fate of bacteria in the small intestine. J. Pathol. Bacterial. 79:131-139, 1960. Dobrescu, L. and Huygelen, C. : Protection of piglets against E; 9911 enteritis by immunization of the sow with. a vaccine containing' heat-labile enterotoxin (LT). Zbl. Vet. Med. 323:79-88, 1976. Donta, S.T., Moon, H.W. and Whipp, S.C.: Detection of heat-labile Egghgt1gh13 9911 enterotoxin with the use of adrenal cells in tissue culture. Science 183:334-336, 1974. Donta, S.T. and Viner, J.P.: Inhibition of the steroidogenic effects of cholera and heat-labile Eggngt1gn13, 9911, enterotoxins by’ GMl ganglioside: Evidence for a similar receptor site for the two toxins. Infect. Immun. 11: 982-985, 1975. Dorner, F., Jaksche, H. and Stockl, W.: enterotoxin: Purification, partial characterization, and immunological observations. J3 Infect. Dis. 133:8142-8156, 1976. Dorner, F., Mayer, P. and Leskova, R.: Immunity to c ' 9911 in piglets: The role of colostral antibodies directed against heat-labile enterotoxin in experimental neonatal diarrhea. Zbl. Vet. Med. 27:207-221, 1980. Dougan, G., Dowd, G. and Kehoe, M.: Organization of K88ac encoded polypeptides in the L 9911 cell envelope: Use of minicells and membrane protein mutants for studying assembly. J. Bacteriol. 153:364-370, 1983. Drees, D.T. and Waxler, G.L.: Enteric colibacillosis in gnotobiotic swine: A fluorescence microscopic study. Am. J. Vet. Res. 31:1147-1157,1970. 193 Dreyfus, L.A., Frantz, J.C. and Robertson, D.C.: Chemical properties of heat-stable enterotoxins produced by enterotoxigenic Eggngflgnig 9911 of different host origins. Infect. Immun. 42:539-548, 1983. Dreyfus, L.A., Jasa-Friedmann, L. and Robertson, D.C.: Characterization of the mechanism of action of Eggngt1gn13 9911 heat-stable enterotoxin. Infect. Immun. 443493-501, 1984. Dubos, R.: Staphylocci and infection and immunity. Am. J. Dis. Child. 1053643-645, 1963. Duguid, J.P., Clegg, S.and Wilson, M.I.: The fimbrial and non-fimbrial haemagglutinins of Egghgt19913 9911. J. Med. Microbiol. 12:213-227, 1979. Duguid, J.P. and Old, D.C.: Adhesive properties of Enterobacteriaceae. In Eagtgt131 mgrgngg. Edited by E.H. Beachey, Chapman and Hall, London, pp. 184-217, 1980. Duguid, J.P., Smith, I.W., Dempster, G. and Edmunds, P.N.: Non-flagellar filamentous appendages ("fimbriae”) and haemagglutinating activity in Bacterium coli. J. Path. Bact. 703335-348, 1955. Evans. D.G. and Evans, D.J., Jr.: New surface associated heat-labile colonization factor antigen (CFA/II) produced by' enterotoxigenic Egghgt19913_ 9911 of serogroups 06 and 08. Infect. Immun. 21:638-647, 1978. Evans, D.G., Evans, D.J., Jr., Pierce, N.F.: Differences in the response of rabbit small intestine to heat- labile and heat-stable enterotoxins of 3999119313 9911. Infect. Immun. 7:873-880, 1973. Evans, D.G., Silver, R.P., Evans, D.J.,Jr., Chase, D.G. and Gorbach, S.L.: Plasmid-controlled colonization factor associated with virulence in Egghgt1gn13 9911 enterotoxigenic for humans. Infect. Immun. 123656-667, 1975. Evans, D.J., Jr., Chen, L.C., Curlin, G.T. and Evans, D.G.: Stimulation of adenyl cyclase by W 9911 enterotoxin. Nature 236: 137-138, 1972. Evans, D.J., Jr. and Evans, D.G.: Direct serological assay for the heat-labile enterotoxin of Eggngt1gn13 9911, using passive immune hemolysis. Infect. Immun. 163604-609, 1977. 194 Evans, D.J., Jr., Evans, D.G. and Dupont, H.L.: Hemagglutination patterns of enterotoxigenic and enteropathogenic Ecchczichic ccii determined with human, bovine, chicken and guinea pig erythrocytes in the presence and absence of mannose. Infect. Immun. 233336-346, 1979. Evans, D.J.,Jru, Evans, D.G., Richardson, S.H. and Gorbach, S.L.: Purification of the polymixin-released, heat-labile enterotoxin of ccii. J} Infect. Dis. 133:897-8102, 1976. Evans, P.A., Newby, T.J., Stokes, C.R., Patel, D. and Bourne, F.J.: Antibody response of the lactating sow to oral immunization with Ecchcrichic ccii. Scand. J. Immunol. 113419-429, 1980. Ey, P. L., Prowse, S. J. and Jenkins, C. R.: Isolation of pure IgGl, IgG;a and 1932b immunoglobulins from mouse serum using protein. A-sepharose. Immunochemistry 15. 429-436, 1978. Fachon-Kalweit, 8., Elder, B.L. and Fives-Taylor, P.: Antibodies that bind to fimbriae block adhesion of Streptococcus sanguis to saliva-coated hydroxyapatite. Infect. Immun. 483617-624, 1985. Paris, A., Lindahl, M. and Wadstrém, T.: GMZ-like glycoconjugate as possible erythrocyte receptor for the CFA/I and K99 haemagglutinins of enterotoxigenic Ecchcnichig ccii. FEMS MUic:rol>ic>l. Leattz. 73 265- 269, 1980. Field, M.: Secretion of the small intestine. In Ehycicicgy 2f the gastrointestinal Enact. Edited by L-Ro Johnson, vol. 2., Raven Press, New York, pp. 963- 982, 1981. Field, M., Graf. L.B., Jr., Laird, W.J. and Smith, P.L.: Heat-stable enterotoxin of Ecchcrichig ccii: in XLLIQ effects on guanylate cyclase activity, cyclic GMP concentration, and ion transport in small intestine. Proc. Natl. Acad. Sci. U.S.A. 75:2800-2804, 1978. Finkelstein, R.A. , Larue, 14.x. , Johnston, D.W. , Vasil, H.L., Cho, G.J. and Jones, J.R.: Isolation and properties of heat labile enterotoxin from enterotoxigenic fischcrichia ccii. J. Infect. Dis. 13338120-8137, 1976. 195 Finkelstein, R.A. and Yang, 2: Rapid test for identification of heat-labile enterotoxin-producing Ecchcgichig ccii colonies. J. Clin. Microbiol. 18323- 28, 1983. Frantz, J. C., Dhatnagar, P.M., Brown, A.L., Garret, L.K. and Hughes, J. L.: Investigation of a synthetic Ecchccichic ccii: Heat-stable enterotoxin as an immunogen for swine and cattle. Infect. Immun. 55:1077-1084, 1987. Frantz, J.C., Jase-Friedman, L. and Robertson, D.C.: Binding of Ecchczichic ccii heat-stable enterotoxin to rat intestinal cells and brush border membranes. Infect. Immun. 433622-630, 1984. Frantz, J. C. and Mellencamp, M.W.: Production and testing of mm 291.1 (Mb) toxoid- In W mm mm mum on Neonatal Digits: University of Saskatchewan, Saskatoon, Saskatchewan, Canada, pp. 500-517, 1984. Frantz, J.C. and Robertson, D.C.: Immunological properties of Ecchccichic ccii heat-stable enterotoxins: Development of a radioimmunoassay specific for heat-stable enterotoxins with suckling mouse activity. Infect. Immun. 333193-198, 1981. Freter, R.: Coproantibody and bacterial antagonism as protective factors in experimental enteric cholera. J. Exp. Med. 1043419-426, 1956. Freter, R.: Mechanism of association of bacteria with mucosal surfaces. In achccich and Microozganicm Echhcgchici_y. Edited by K. Elliot, Ciba Foundation Symposium 80, Pitman Medical, Turnbridge Wells, United Kingdom, pp. 36-55, 1981. Foged, N.T., Klemm, P., Elling, F., Jorsal, S.E. and Zeuthen, J. 3 Monoclonal antibodies to K88ab, K88ac and K88ad fimbriae from enterotoxigenic Ecchcrichic ccii. Microbial Pathogenesis 1:57-69, 1986. Forstner, J. F.: Intestinal mucins in health and disease. Digestion 173234-263, 1978. Fubara, E.S. and Freter, R.: Protection against enteric bacterial infection by secretory IgA. J. Immunol., 1163395-403, 1973. 196 Furer, E., Cryz, S.J., Dorner, F., Nicolet, J., Wanner, M. and Germanier, R.: Protection against colibacillosis in neonatal pigs by immunization of dams with procholeragenoid. Infect. Immun. 353887-894, 1982. Furer, E., Cryz, S.J. and Germanier, R.: Protection of piglets against neonatal colibacillosis based on antitaxic immunity. Dev. Biol. Stand. 533161-167, 1983a. Furer, E., Cryz, S.J. Jr., Dorner, F. and Germanier, R.: Protection of neonatal piglets against colibacillosis by immunization of dams with procholeragenoid. 1h Mlle—en“ in Eeeeereh ef Chelere and Related W. Edited by S. Kuwahara and N .F. Pierce, Martinus Nijhaff Publishers, Boston, pp. 29-33, 1983b. Fusco, P. To, A., To, S. and Brinton,C., Jr.: The purification and characterization of four types of Eseheriehie 9.9.1.1 pili and the Specificity of E... eeli pili far immunity, colonization and adhesion. In 2mm Lie-Jam centerenee en Chelere. Atlanta. Georgia, pp. 60-70, 1978. Gaastra, W. and de Graaf, F.K.: Host-specific fimbrial adhesins af noninvasive enterotoxigenic Escherichia ccii strains. Microbial. Rev. 423129-161, 1982. Gaastra, W., Klemm, P. and de Graaf, F.K.: The nucleotide sequence of the K88ad protein subunit of porcine enterotoxigenic Eschczichia ccii. FEMS Microbial. Lett. 183177-183, 1983. Gaastra, W., Klemm, P., Walker, J.M. and de Graaf, F.K.: K88 fimbrial proteins amino and carbaxyl terminal sequences of intact proteins and cyanogen bromide fragments. FEMS Microbial. Lett. 6315-18, 1979. Gaastra, W., Maai, F.R., Stuitje, A.R. and de Graaf, F.K.: The nucleotide sequence of the gene encoding the K88ab protein subunit of porcine enterotoxigenic Escherichia ccii. FEMS Microbial. Lett. 12:41-46, 1981. Geary, S.J., Marchlewicz, E.A. and Finkelstein, R.A.: Comparison of heat-labile enterotoxins from porcine and human strains of Ecchczichic ccii. Infect. Immun. 363215-220, 1982. 197 Georges, H.C., Wachsmuth, I.K., Birkness, R.A., Moseley, 8.1. and Georges, A.J.3 Genetic probes for enterotoxigenic Ecchcxichic ccii, isolated farm childhood diarrhea in the Central African Republic. J. Clin. Microbial. 183199-202, 1983. Gerrard, J.W.3 Breast feeding: Second thoughts. Pediatrics 543757-764, 1974. Getty, R.: Epidural anesthesia in the hog - its technique and applications. In Scientifie Breeeeeinge 1.9.9111 Annual Meeting am. pp- 83-98. 1963. Gianella, R.A.: Suckling mouse model for detection of heat-stable ficchc;ichic_ ccii_ enterotoxin: Characteristics of the model. Infect. Immun. 14:95-99, 1976. Gianella, R.A., Drake, K.W. and Luttrell, M.: Development of a radioimmunoassay for Ecchczichig heat-stable enterotoxin: Comparison with the suckling mouse biaassay. Infect. Immun. 333186-192, 1981. Gianella, R.A., Luttrell, M. and Thompson, M.: Binding of Ecchczichic ccii heat-stable enterotoxin to receptors on rat intestinal cells. Amer. J. Physiol. 2453G492-G498, 1983. Gibbons, R.A., Jones, G.W. and Sellwood, R.: An attempt to identify the intestinal receptor for the K88 adhesin by means of a haemagglutinatian inhibition test using glycoproteins and fractions from saw colostrum. J. Gen. Microbial. 863228-240, 1975 . Gibbons, R.A., Sellwood, R., Burrows, M. and Hunter, P.A.: Inheritance of resistance to neonatal E; ccii diarrhoea in the pig: Examination of the genetic system. Thear. Appl. Genet. 51:65-70, 1977. Gibbons, R.J. and van Houte, J.: Selective bacterial adherence to oral epithelial cells and its role as an ecological determinant. Infect. Immun. 33567-573, 1971. Gill, D.M., Clements, J.D., Robertson, D.C. and Finkelstein, R.A.: Subunit number and arrangement of Ecchczichig ccii heat-labile enterotoxin. Infect. Immun. 333677-682, 1981. 198 Gill, D.M., Evans, D.J., Jr. and Evans, D.G.: Mechanism of activation of adenylate cyclase in yiczc by palymixin-released, heat-labile enterotoxin af ccii. J. Infect. Dis. 13338103, 1976. Girardeau, J.P.: A new in xitzc technique for attachment to intestinal villi using enteropathogenic Egchcrichic ccii. Ann. Microbial. 131B331-37, 1980. Glass, R.I., Svennerholm, A.-M., Stall, B.J., Kahn, M.R., Belayet, K.M., Hassain, M.I.H. and Holmgren J.: Protection against cholera in breast-fed children by antibodies in breast milk. New Engl. J. Med. 30831389-1392, 1983. Godinc. J-W-: Eeneclenal Antibediees. Principle: and 21ccciccc. 2nd ed., Orlando, Academic Press, FA, 1986. Galderman, L., Kaplan, B. and Rubinstein, E.3 Ecchczichic ccii adherence to the intestine of mice. Israel J. Med. Sci. 213410-414, 1985. Goldman, A.S. and Smith, C.W.: Host resistance factors in human milk. J. Pediatr. 8231082-1090, 1973. Gray, J.D.A. and Shiner, M.: Influence of gastric pH on gastric and jejunal flora. Gut 83574-581, 1967. Greenberg, H. , McAuliffe, V. , Valdesusa, J. , Wyatt, R. , Flares, J. , Kalica, A. , Hashina, Y. and Singh, N. 3 Seralagical analysis of the subgroup protein of ratavirus, using monoclonal antibodies. Infect. Immun. 39391-99, 1983a. Greenberg, R.N., Dunn, J.A., Guerrant, R.L.: Reduction of the secretory response to Ecchcnichic chi enterotoxin by thial and disulfide compounds. Infect. Immun. 413174-180, 1983b. Greenberg, R.N. , Guerrant, R.L. , Chang, B. , Robertson, D.C. and Murad, F.: Inhibition of Egchcnichic ccii heat-stable enterotoxin effects on intestinal guanylate cyclase and fluid secretion by guinacrine. Biochem. Pharmacol. 3132005-2009, 1982a. Greenberg, R.N., Murad, F., Chang, 8., Robertson, D.C. and Guerrant, R.L.: Inhibition of Ecchczichic ccii heat- stable enterotoxin by indamethacin and chlarpramazine. Infect. Immun. 293908-913, 1980. 199 Greenberg, R.N. , Murad, F. and Guerrant, R.L.: Lanthanum chloride inhibition of the secretory response to ccii heat-stable enterotoxin. Infect. Immun. 353483-488, 1982b. Greenberg, H.B., Sack, D.A., Rodriguez, W., Sack, R.B., Wyatt, R.G. and Kalica, A.R.: Micratiter solid-phase radioimmunoassay for detection of Ecchczichic heat-labile enterotoxin. Infect. Immun. 173541-545, 1977. Grimes, S.D., Waxler, G.L., Newman, J.P.: Adhesion of K99-positive Egchcgichig ccii to intestinal brush borders of pigs. Am. J. Vet. Res. 473385-388, 1986. Gross, R.J. and Rowe, B.: Ecchczichic c_a_l_i diarrhoea. J. Hyg. Camb. 95:531-550, 1985. Guadalini, S. Raa, M.C., Smith, P.L. and Field, M.: cGMP modulation of ileal ion transport: in 11:19 effects of Bcchczichic ccii heat-stable enterotoxin. Am. J. Physiol. 2433G36-G41, 1982. Guerrant, R.I., Bruntan, L.L., Schnaitman, Y.C. , Rebhun, L. I. and Gilman, L.G.: Cyclic adenosine monophosphate and alteration of Chinese hamster ovary morphology: A rapid and sensitive in 113.129 assay for Vibria Cholera and Ecchcnichig ccii. Infect. Immun. 103320-327, 1974. Guerrant, R.L., Hughes , J. M., Chang, B., Robertson, D. C. and Murad, F. . Activation of intestinal guanylate cyclase by heat-stable enterotoxin of W ccii: Studies of tissue specificity, potential receptors, and intermediates. J. Infect. Dis. 1423220-228, 1980. Guerrant, R. L., Moore, R.A., Kirschenfeld, P.M. and Sande, M.A.: Role of taxigenic and invasive bacteria in acute diarrhea of childhood. New Engl. J. Med. 2933567-573, 1975. Guerina, N.G., Kessler, T.W., Guerina, V.J., Neutra, M.R., Clegg, H.W., Langermann, S., Scannapieca, E.A., and Galdmann, D.A.: The role of pili and capsule in the pathogenesis of neonatal infections with Ecchczichig ccii K1. J. Infect. Dis. 1483395-405, 1983. 200 Guinee, PuA.M. and Jensen, W.H.: Behavior of Ecchccichic ccii K antigens K88ab, K88ac, and K88ad in immunaelectrapharesis, double diffusion, and hemagglutination. Infect. Immun. 233700-705, 1979a. Guinee, P.A.M. and Jansen, W.H.: Detection of enterotoxigenicity and attachment factors in ccii strains of human, porcine and bovine origin: A comparative study. Zbl. Bakt. Hyg. J. Abt. Orig. A 2433245-257, 1979b. Guinee, P.A.M., Veldkamp, M.J. and Jansen, W.H.: Improved Minca medium for the detection of K99 antigen in calf enterotoxigenic strains of Ecchczichic ccii. Infect. Immun. 153676-678, 1977. Gyles, C.L.: Immunological study of the heat-labile enterotoxins of Eccherichia ccli and yihric chclerae. Infect. Immun. 93564-570, 1974a. Gyles, C.L.: Relationships among heat-labile enterotoxins of Escherichia cell and yihric chcleree. J- Infect- Dis. 129:277-283, 1974b. Gyles, C.L. and Barnum, D.A.: A heat-labile enterotoxin from strains of Ecchcnichic ccii enteropathogenic for pigs. J. Infect. Dis. 120:419-426, 1969. Hadad, J .J . and Gyles, C.L.: Scanning and transmission electron microscopic study of the small intestine of colostrum-fed calves infected with selected strains of Ecchczichia ccii. Am. J. Vet. Res. 43:41-49, 19828. Hadad, J.J. and Gyles, C.L.: The role of K antigens of enteropathogenic Ecchczichig ccii in colonization of the small intestine of calves. Can. J. Comp. Med. 46:21-26, 1982. Hamilton, D.L., Johnson, M.R., Farsyth, G.W., Rae, W. E. and Nielsen, N. 0.: The effect of cholera toxin and heat labile and. heat stable Ecchccichic, ccii on cyclic AMP concentrations in small intestinal mucosa of pig and rabbit. Can. J. Comp. Med. 423327-331,1978. Hanssan, G.C., Karlssan, K.-A., Larson, G., Stramberg, N. and Thurin, J .: Carbohydrate-specific adhesion of bacteria to thin-layer chramatagrams: A rationalized approach to the study of host cell glycolipid receptors. Anal. Biochem. 146:158-163, 1985. 201 Hewlett, E.L., Guerrant, R.L., Evans, D.J., Jr. and Greenaugh, W.B.: Toxins af yihgig chclcrcc and Ecchccichig ccii stimulate adenyl cyclase in rat fat cells. Nature 149:371-373, 1974. Hohmann, A., Wilson, M.R.3 Adherence of emterapathagenic Ecchgichic coli to intestinal epithelium in xi 9. Infect. Immun. 123866-880, 1975. Holmgren, J.: Actions of cholera toxin and the prevention of cholera. Nature 292:413-417, 1981. Holmgren, J.: Toxins affecting intestinal transport processeS- In Len limiehce cf Escherichia celi- Edited by M. Sussman, Academic Press, London, pp. 177-191, 1985. Holmgren, J., Fredman, P., Lindblad, M., Svennerholm, A.M. and Svennerholm, L.: Rabbit intestinal glycoprotein receptor for Escherichia coli heat-labile enterotoxin lacks affinity for cholera toxin. Infect. Immun. 383424-433, 1982. Holmgren, J., Svennerholm, A.-M. and Lindblad, M.: Receptor-like glycocampaunds in human milk that inhibit classical E1 T or 219119 chcicncc cell adherence (hemagglutination). Infect. Immun. 39:147- 154, 1983. Honda, T., Akhtar, 0., Glass, R.I. and Galam Kibriya, A.K.M.: A simple assay to detect Ecc_hc:ichi_a ccii producing heat-labile enterotoxin: Results of a field study of the Biken test in Bangladesh. Lancet 113609-610, 1981a. Honda, T., Taga, S., Takeda, Y. and Miwatani, T.: Modified Elek test for the detection of heat-labile enterotoxin af enterotoxigenic L ccli. J. Clin. Microbiol. 1331-5, 1981b. Honda, T., Tsuji, T., Takeda, Y. and Miwatani, T.: Immunological nanidentity of heat-labile enterotoxins from human and porcine enterotoxigenic We ccii. Infect. Immun. 34: 337- 340, 1981c. Haskins, L.S.: Degradation of mucus glycoproteins in the gastrointestinal tract. In Thc glycccahjhgct c. Edited. by' M.I. Horowitz and. W. Pigman, vol. II, Academic Press, New York, pp. 235-253, 1978. 202 Haughten, R.A., Engert, R.F., Ostreshi, J.M., Hoffman, S.R. and Klipstein, E.A.: A completely synthetic toxoid vaccine containing Ecchczichic ccii heat- stable toxin and antigenic determinants of heat- labile toxin B subunit. Infect. Immun. 483735-740, 1985. Hughes, J.M., Murad, F., Chang, B. and Guerrant, R.L.: Role of cyclic GMP in the action of heat-stable enterotoxin of Ecchc;ichic_ ccii. Nature 2713755-756,1978. Isaacson RE: K99 surface antigen of Egchczichig ccii: Purification. and. partial characterization. Infect. Immun. 15:272-279, 1977. Isaacson, R.E.: K99 surface antigen of Ecchczichic ccii: Antigenic characterization. Infect. Immun. 223555-559,1978. Isaacson, R.E., Calmenera, J. and Richter, P.: Ecchczichic ccii K99 pili are composed of one subunit species. FEMS Microbial. Lett. 123229-232, 1981. Isaacson, R.E., Dean, E.A., Morgan, R.L. and Moon, H.W.3 Immunization of suckling pigs against enterotoxigenic Ecchccichic_ ccii induced diarrheal disease by vaccinating dams with purified K99 or 987P pili: Antibody production in response to vaccination. Infect. Immun. 293824-826, 1980. Isaacson, R,E., Fusco, P.C., Brinton, C.C. and Moon, H.W.3 In xicrc adhesion of Escherichia ccli to porcine small intestinal epithelial cells: Pili as adhesive factors. Infect. Immun. 213392-397, 1978. Isaacson, R.E., Nagy, B. and Moon ,H.W.: Colonization of porcine small intestine by Ecchczichia ccii: Colonization and adhesion factors of pig enteropathogens that lack K88. J. Infect. Dis. 135: 531-539, 1977. Isaacson, R.E. and Richter, P.: Ecchcflchic ccli 987P pilus: Purification and partial characterization. J. Bacteriol. 1463784-789, 1981. Jason, J.M., Nierburg, P. and Marks, J.S.: Mortality and infectious disease associated with infant-feeding practices in developing countries. Pediatrics 7438702-8727 , 1984 . 203 Jayappa, H.G., Goodnow, R.A. and Geary, S.J.: Role of Ecchcgichia ccii type 1 pilus in colonization of porcine ileum and its protective nature as a vaccine in controlling colibacillosis. Infect. Immun. 483350-354, 1985. Jayappa, H.G., Strayer, J.G. and Goodnow, R.A.: Experimental infection and field trial evaluation of a ‘multiple-pilus phase-cloned bacterin for the simultaneous control of neonatal colibacillosis and mastitis in swine. In Encccedihgc, Fourth Ia_t___ichalernat' _Slmcccicm ch Diarrhea. University of Saskatchewan, Saskatoon, Saskatchewan, Canada, pp. 518-540, 1983. Jayappa, H., Strayer, J.G. and Goodnow, R.A.: Efficacy of Egghecichig ccii bacterin containing K88, K99, 987P and type 1 pili antigens in controlling natural and laboratory induced colibacillosis in neonatal pigs. In Proceegihgs of hhc__§hh Inhernggia hgi Pig ycterinagy §cciecy angcess, Ghent, Belgium, p. 86, 1984. Jensen,C.0.: Q,0, Jehsch'§ §cicc§c§_ Ecpers, i8§§-19Q§, val.1, Ejaar Munksgaard, Copenhagen, 290, 1948. Cited by Kohler (1972). Jones, G.W.: The attachment of bacteria to the surfaces of animal cells. In Miczchici Ihcccaccichc. Edited by J.L. Reissig, Chapman and Hall, London, pp. 139- 176, 1977. Jones, G.W. and Isaacson, R.E.: Prateinaceous bacterial adhesins and their receptors. Crit. Rev. Microbiol. 10:229-260, 1983. Jones, G. W. and Rutter, J. M.: Role of the K88 antigen in the pathogenesis of neonatal diarrhea caused by Ecchccichig ccii in piglets. Infect. Immun. 63 918- 927, 1972. Jones, G. W. and. Rutter, J. M.: The association of K88 antigen with haemagglutinating activity in porcine strains of Ecchczichic ccii. J. Gen. Microbiol. 843135-144,1974. Jubb, K.V.F., Kennedy, P.C. and Palmer, N.: Bcchcicgy c; mugs—tic Mic, 3rd ed., vol. 2, Academic Press, Inc, New York, pp. 128-130, 1985. 204 Kallenius, G., Korhonen, T.K., Vaisanen-Rhen, V. and Svenson, S.B.3 Adherence assays. In Enhczchccccgici fincficcc Angigcnc. Edited by T.K. Karhonen, E. A. Dawes and P.H. Makela, Elsevier Science Publishers B.V., Amsterdam, pp. 321-332, 1985. Kantor, H.S., Tao, P. and Wisdom, C.: Action of .ccii_ enterotoxin: Adenylate cyclase behavior of intestinal epithelial cells in culture. Infect. Immun. 931003-1010, 1974. Kapitany, R.A., Farsyth, G.W., Scoot, A., McKenzie, S.F. and Worthington, R.W.: Isolation and partial characterization of two different heat-stable enterotoxins produced by bovine and porcine strains of enterotoxigenic Eschcnichic ccii. Infect. Immun. 263173-177, 1979a. Kapitany, RJA., Scoot, A., Farsyth, G.W., McKenzie, S.L. and Worthington, R.W. 3 Evidence for two heat-stable enterotoxins produced by enterotoxigenic Ecchczichic ccii. Infect. Immun. 243965-966, 1979b. Kapitany, R.A, Scoot, A., Farsyth, G.W. and Worthington, R.W.: Purification and comparison of the heat stable enterotoxins produced by bovine and porcine strains of Escherichia _lico In Exceeedims cf the secch .Ihterhaticnai. ENIHEHEUHn :en HQQDQLQI. Inauunusi. Saskatoon, Saskatchewan, Canada, pp. 107-118, 1978. Kawai, Y. and Maratami, M.: Intestinal enzyme activities in germfree, conventional and gnotobiotic rats associated. with indigenous :microorganisms, Infect. Immun. 193777-778, 1978. Kearns, M. and Gibbons, R.A.: The possible nature of the pig intestinal receptor for the K88 antigen of Ecchczichig ccii. FEMS Microbiol. Lett. 63165-168, 1979. Kehoe, M., Sellwood, R., Shipley, P. and Dougan, G.: Genetic analysis of K88-mediated adhesion of enterotoxigenic Ecchczichig ccii. Nature 291:122-126, 1981. Kennedy, D.J., Greenberg, R.N., Dunn, J.A., Abernathy, R., Ryers, J.S. and Guerrant, R.L.: Effects of Escherichia coli heat-stable enterotoxin STb on intestines of mice, rabbits and piglets. Infect. Immun. 463639-643, 1984. 205 Kim, Y.B.: Immunology - introductory remarks. In Bcccn; Admces in Germfree W. Edited by Sneaki. s., Ozawa, A. and Hashimota, K., Toikai University Press, Tokyo, pp. 483-491, 1981. Klemm, P. 3 The complete amino-acid sequence of the K88 antigen, a fimbrial protein from Ecchcnichia ccli. Eur. J. Biochem. 1173617-627, 1981. Klemm, P.: Fimbrial adhesins of Eschcnichia ccli. Rev. Infect. Dis. 7. 321-340, 1985. Klemm, P. and. Mikkelsen, L.: Prediction of antigenic determinants and secondary structures of the K88 and CFAI fimbrial proteins from enteropathogenic Ecchczichic ccii. Infect. Immun. 38. 41-45, 1982. Klipstein, F.A., Engert, R.F. and Clements, J.D.: Development of a vaccine of cross-linked heat-stable and heat-labile enterotoxins that protects against ccii producing either enterotoxin. Infect. Immun. 373550-557,1982. Klipstein, F.A., Engert, R.F., Clements, J.D. and Houghten, R.A.: Protection against human and porcine enterotoxigenic strains of Ecchczichic ccii in rats immunized with a cross-linked toxoid vaccine. Infect. Immun. 403924-929, 1983a. Klipstein, F.A., Engert, R.F., Clements, J.D. and Houghten, R.A.: Vaccine for enterotoxigenic Ecchccichic ccii based on synthetic heat-stable toxin cross-linked to the B subunit of heat-labile toxin. J.Infect. Dis. 147,3318-326, 1983b. Klipstein, F.A., Engert, R.F., Houghten, R.A. and Rowe, B.: Enzyme-linked immunosorbent assay for Ecchczichic ccii heat-stable enterotoxin. J. Clin. Microbiol. 193798-803, 1984. Knoop, F.C. and Abbey, D.M.3 Effect of chemical and pharmacological agents on the secretory activity induced by Ecchczichic ccii heat-stable enterotoxins. Can. J. Microbiol. 273754-758, 1981. Knoop, F.C. and Thomas, D.D.3 Stimulation of calcium uptake and cyclic GMP synthesis in rat basophilic leukemia cell by Ecchcpichic ccii heat-stable enterotoxin. Infect. Immun. 413971-977, 1983. 206 Knutton. 8., Lloyd, D. R., Candy, D. C. A. and MCNeish, A.S.: Ultrastructural study of adhesion of enterotoxigenic Ecchczichic ccii to erythrocytes and human intestinal epithelial cells. Infect. Immun. 443519-527,1984. Kohler, E.M.3 Studies of Ecchcxichig ccii in gnotobiotic pigs. V. Evaluation of the effects of oral and parenteral administration of immune serum. Can. J. Comp. Med. and Vet. Sci. 313283-289, 1967. Kohler, E.M.3 Protection of pigs against neonatal enteric colibacillosis with colostrum and milk from orally vaccinated sows. Am. J. Vet. Res. 353331-338, 1974. Kohler, E.M.3 Neonatal enteric colibacillosis of pigs and current research on immunization. J. Amer. Vet. Med. Assoc. 1733588-591, 1978. Kohler, E.M. and Bohl, E.H.3 Studies of Ecchcxichig ccii in gnotobiotic piglets. III. Evaluation of orally administered specific antisera. Can. J. Comp. Med. 303233-237, 1966. Kohler, E.M., Cross, R.F. and Bohl, E.H.3 Protection against neonatal enteric colibacillosis in pigs suckling orally vaccinated sows. Am. J. Vet. Res. 363757-764, 1975. Kohler, G. and Milstein, C.3 Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497, 1975. Korhonen, T.K., Rhen, M., vaisanen-Rhen, V. and Pere, A.: In mhhclsay cf the hacterial cell Enyelcpe- Edited by D.E.S. Stewart-Tull and M. Davies, Wiley, Chichester, pp.319-354, 1985. Kovar, H.G., Serula, M.K., Marks, J.S. and Fraser, D.W.3 Review of the epidemiologic evidence for an association between infant feeding and infant health. Pediatrics 7438615-638, 1984. Kunkel, S.L. and Robertson, D.C.: Purification and chemical characterization of the heat-labile enterotoxin produced by enterotoxigenic Ecchczichia ccii. Infect. Immun. 253586-596, 1979. Kwan, C.N. and Wishnow, R.M.3 Ecchczichih enterotoxin-induced steroidogenesis in cultured adrenal tumor cells. Infect. Immun. 103146-151, 1974. 207 Lallier, R., Bernard, F., Gendreau, M., Lazure, C., Seidan, N.G., Chretien, M. and St.-Pierre, S.A.3 Isolation and purification of Ecchcrichig heat-stable enterotoxin of porcine origin. Anal. Biochem. 127:267-275, 1982. Larsen, S.A. and Homer, D.R.3 Relation of breast versus bottle feeding to hospitalization for gastroenteritis in a middle-class U.S. population. J. Pediatr. 923417-418, 1978. Laux, DC., McSweegan, E.F. and Cohen, P.S.3 Adhesion of enterotoxigenic Ecchczichih ccii to immobilized intestinal mucosal preparations: A model for adhesion to mucosal surface components. J. Microbiol. Methods 2327-39, 1984. Laux, D.C. , McSweegan, E.F. , Williams, T.J. , Waldowski, E.A. and Cohen, P.S.3 Identification and characterization of mouse small intestine ‘mucosal receptors for Ecchcxichic ccii K-12 (K88ab). Infect. Immun. 52:18-25, 1986. Lazure, C., Sedah, N.G., Chretien, M., Lattier, R. and St. Pierre, 8.: Primary structure determination of Egchccichia ccii heat stable enterotoxin of porcine origin. Can. J. Biochem. Cell. Biol. 613287-292, 1983. Lee, C.H., Moseley, S.L., Moon, H.W., Whipp, S.C., Gyles, C.L. and So, M.: Characterization of the gene encoding heat-stable toxin II and preliminary molecular epidemiological studies of enterotoxigenic Ecchccichic ccii heat-stable toxin II producers. Infect. Immun. 423264-268,1983. Levine, M.M.3 Ecchczichi§_,ccii that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorraghic and enteroadherent. J. Infect. Dis. 1553377-389, 1987. Levine, M.M., Black, R.E., Brinton, C.C., Clements, M.L., Fusco, P., Hughes, T.P., O'Donnell, 8., Robins- Browne, R., Woods, 8. and Young, C.R.3 Reactogenicity, immunogenicity and efficacy studies of Ecchcrichig ccii type 1 pili parenteral vaccine in man. Scand. J. Infect. Dis. 33:83-95, 1982. 208 Lindahl, M., Paris, A. and Wadstrom, T.: A GM2-like receptor for CFA/I and K99. In Aficchmcnh cf ° ta Gastrsuintestihai Scrface- Edited by E. Boedeker, CRC Press, Boca Raton, pp. 189-198, 1984. Lindahl, M. and Wadstrom, T.: K99 surface haemagglutinin of enterotoxigenic L c_o_1i recognize terminal N-acetylgalactosamine and sialic acid residues of glycophorin and other complex glycoconjugates. Vet. Microbiol. 93249-257, 1984. Lindhalm, L., Holmgren, J ., Wikstrom, M., Karlsson, U., Andersson, K. and Lycke, N.: Monoclonal antibodies to cholera toxin with special reference to cross-reactions with Ecchcxichic ccii heat-stable enterotoxin. Infect. Immun. 403570-576, 1983. Linggood, M.A. and Porter, P.: Antibody induced elimination of the plasmid controlled K88 adhesion factor from a porcine 'enteropathogen. Immunol. 353125-127, 1978. Lockwood, D.E. and Robertson, D.C.: Development of a competitive enzyme-linked immunosorbent assay (ELISA) for Ecchcrichic ccii heat-stable enterotoxin (STa) . J. Immunol. Methods. 753295-307, 1984. Lowry, O.H. , Rosebrough, N.J . , Farr, A. L. and Randall , R.J . 3 Protein measurement with the fol in phenol reagent. J. Biol. Chem. 1933265-275, 1951. Malick, L.E. and Wilson, R.B.3 Modified thiocarbohydrazide procedure for scanning electron microscopy: Routine use for normal, pathological, or experimental tissues. Stain Technology 503265-269 , 1975. Majumdar, A.S. and Ghose, A.C.3 Protective effects of antichalerae antibodies in human colostrum. Infect. Immun. 363962-965, 1982. Mainil, J.G., Sadowski, P.L., Tarsio, M. and Moon, H.W.3 In 1119 emergence of enterotoxigenic Ecchchichic ccii variants lacking genes for K99 fimbriae and heat- stable enterotoxin. Infect. Immun. 5533111-3116, 1987. Mantle, M., Mantle, D. and Allen, A.: Polymeric structure of pig small-intestinal mucus glycoprotein. Biochem. J. 1953 277-285. 1981. 209 Marshall, T. and Allen, A.: The isolation and characterization of the high-molecular weight glycoprotein from pig colonic mucus. Biochem J. 1733569-578, 1978. Mashiter, K., Mashiter, G.D., Haugher, R.L. and Field, J.B.3 Effect of cholera and E; ccii enterotoxins on cyclic adenosine. 3',5'-monophosphate levels and intermediary metabolism in the thyroid. Endocrinol. 923541-549, 1973. Masson, P.L., Heremans, J.F. and Dive, C.H.3 An iron- binding protein common to many external secretions. Clin. Chin. Acta 143735-739, 1966 Miler, I., Cerna, J., Travnicek, J., Rejnek, J. and Kruml, J .3 The role of immune pig colostrum, serum, and immunoglobulins IgG, IgM, and IgA in local intestinal immunity against enterotoxic strains of ccii 055 in germfree piglets. Folia Microbiol 203433-438, 1975. Miniats, O.P, Mitchell, L. and Barnum, D.: Response of gnotobiotic pigs to Ecchcnichic ccii. Can. J. Comp. Med. 343269-276, 1970. Mooi, F.R. and de Graaf, F.K.: Isolation and characterization of K88 antigens. FEMS iMicrobiol. Lett. 5317-20, 1979. Mooi, F.R., de Graaf, F.K. and van Embden, J.D.A.3 Cloning, mapping and expression of the genetic determinant that encodes for the K88ab antigen. Nucleic Acids Res. 63849-865, 1979. Mooi, F.R.., Harms, N., Bakker, D. and de Graaf, F.K.: Organization and expression of genes involved in the production of the K88ab antigen. Infect. Immun. 3231155-1163, 1981. Mooi, F.R., van Buren, M., Koopman, G., Roosendaal, B. and de Graaf, F.K.: K88ab gene of Ecchcgichic ccii encodes a fimbria-like protein distinct from the K88ab fimbrial adhesin. J3 Bacterial. 1593482-487, 1984. Mooi, F.R., Wijfjes, A. and de Graaf, F.K.: Identification and characterization of precursors in the biosynthesis of the K88ab fimbria of ‘Ecchczichic ccii. J. Bacteriol. 154341-49, 1983. 210 Mooi, F.R., Wouter, C., Wijfjes, A. and de Graaf, F.K.: Construction and characterization of mutants impaired in the biosynthesis of- the K88ab antigen. J. Bacteriol. 1503512-521, 1982. Moon, R.W. 3 Pathogenesis of enteric diseases caused by Ecchczichic ccii. Adv. Vet. Sci. Comp. Med. 183179- 211, 1974.' Moon, H.W.3 Mechanism in the pathogenesis of diarrhea: A review, J. Am. Vet. Med. Assoc. 1723443-448, 1978a. Moon, H.W.3 Pili as protective antigens in vaccines for the control of enterotoxigenic Ecchczichic ccii infections. In Ercceedinesi secend International firmnesinn. en. Hennatal. Diarrhea. University of Saskatchewan, Saskatoon, Saskatchewan, Canada, pp. 393-405, 1978b. Moon, H.W.3 Luminal and mucosal factors of small intestine affecting pathogenic colonization. In ficccccczy Diarrhcg. Edited by M. Field, J.S. Fordtran and 8.6. Schultz, American Physiological Society, Bethesda, MD, pp. 127-139, 1980. Moon, H.W.3 Protection against enteric colibacillosis in pigs suckling orally vaccinated dams: Evidence for pili as protective antigens. Am. J. Vet. Res. 423173-177, 1981. Moon, H.W.3 Intestine. In Qcii Echhcicgy, edited by N.F. Cheville, 2nd ed., Iowa State University Press. Ames, Iowa, pp. 503-529, 1983. Moon, H.W., Baetz, A.L. and Giannella, R.A.: Immunization of swine with heat stable Ec,.ccii_ enterotoxin coupled to a carrier protein does not protect suckling pigs against an E; ccii strain that produces heat stable enterotoxin. Infect. Immun. 393990-992, 1983. Moon, H.W., Isaacson, R.E. and Pohlenz, J.: Mechanism of association of enteropathogenic Ecchczichic ccii with intestinal epithelium. Am. J. Clin. Nutr. 323119- 127, 1979. Moon, H.W., Kohler, E.M., Schneider, R.A. and Whipp, S.C. Prevalence of pilus antigens, enterotoxin types and enteropathogenicity among K88-negative enterotoxigenic Bcchczichia ccii from neonatal pigs. Infect. Immun. 273222-230, 1980. 211 Moon, H.W., Nagy, B., Isaacson, R.E. and. prskov, I.3 Occurrence of K99 antigen on Ecchccichig isolated from pigs and colonization of pig ileum by K99+ enterotoxigenic E_,_ coii from calves and pigs. Infect. Immun. 153614-620, 1977. Moon, H.W. and Runnels, P.L.: Trials with somatic (O) and capsular (K) polysaccharide antigens of enterotoxigenic Ecchczichic ccli as protective antigens in vaccines for swine. In W cf the hearth Externaticnal Sympcsism en hecnatal 11mm, Saskatoon, Saskatchewan, Canada, pp. 558-569, 1984. Moon, H.W. and Whipp, S.C.: Development of resistance with age by swine intestine to effects of enteropathogenic Ecchcgichic SL911... J. Infect. Dis. 1223220-223, 1970. Moon, H. W. and Whipp, S. C. . Systems for testing the enteropathogenicity of Egchcnichic coli. Ann. N. Y. Acad. Sci. 1763197-211, 1971. Moon, H.W., Whipp, S.C. and Baetz, A.L.: Comparative effects of enterotoxins from Eschczichic ccii and yihzic cmciczg on rabbit and swine small intestine. Lab. Invest. 253 133-140, 1971. Moon, H.W., Whipp, S.C. and Skarvedt, S.M.: Etiologic diagnosis of diarrheal diseases of calves: Frequency and methods for detecting enterotoxin and K99 antigen production by Eschcnichig ccii. Am. J. Vet. Res. 3731025-1029, 1976. Morgan, R.L., Isaacson, R.E., Moon, H.W., Brinton, C.C. and To, C.C.: Immunization of suckling pigs against enterotoxigenic Escherichia ccii-induced diarrheal disease by vaccinating dams with purified 987 or K99 pili: Protection correlates with pilus homology of vaccine and challenge. Infect. Immun. 223771-777, 1978. Morilla, A., Rico, J., Estrada, A., Martell, M. and Rosales, C.3 Nonspecific stimulation of the immune system of baby pigs. 1. Use of blood serum 2. Use of oil adjuvantS- In Ereceecincs cf the 3th Internaticnal Bic Yeterinary congress. Ghent. Belgium, p. 215, 1984. 212 Morris, J. A., Sojka, W. J. and Ready, R.A.: Serological comparison of the Eschczichia ccii prototype strains for the F(Y) and Att 25 adhesins implicated in neonatal diarrhoea in calves. Res. Vet. Sci. 38:246- 247, 1985. Morris, J.A., Stevens, A.E. and Sajka, W.J .3 Preliminary characterization of cell-free K99 antigen isolated from Ecchcnichic ccii B41. J. Gen. Microbiol. 993353-357, 1977. Morris, J.A., Stevens, A.E. and Sojka, W.J.: Anionic and cationic components of the K99 surface antigen from ccii B41. J. Gen. Microbiol. 1073 173- 175, 1978a. Morris, J.A., Stevens, A.E. and Sojka, W.J.: The isoelectric point of cell free K99 antigen exhibiting hemagglutinating properties. Infect. Immun. 1931097- 1098, 1978b. Morris, J.A., Thorns, C., Scott, A.C., Sojka, W.J. and Wells, G.A.: Adhesion ih yim and in viyc associated with an adhesive antigen (F41) produced by K99 mutant of the reference strain Ecchcnichic ccli B41. Infect. Immun. 3631146-1153, 1982. Morris, J.A., Thorns, C.J. and Sojka, W.J.3 Evidence for two adhesive antigens on the K99 reference strain Ecchczichic ccii B41. J. Gen. Microbiol. 1183107-113, 1980a. Morris, J. A., Thorns, C. J., Wells, G. A. H., Scott, A. C. and Sojka, W. J.: The production of F41 fimbriae by piglet strains of enterotoxigenic Ecchczichic ccii that lack K88, K99 and 987P fimbriae. J. Gen. Microbial. 12932753-2759, 1983. Morris, J. A., Wray, C. and Sojka, W.J.: Passive protection of lambs against enteropathogenic Ecchccichic ccii; Role of antibodies in serum and colostrum of dams vaccinated with K99 antigen. J. Med. Microbiol. 133265-271, 1980b. Moseley, S. L., Dougan, G., Schneider, R. A. and Moon, H. W.: Cloning of chromosomal DNA encoding the F41 adhesin of enterotoxigenic Egchcnchic ccii and genetic homology between adhesins F41 and K88. Infect. Immun. 1673799-804, 1986. 213 Moseley, S.L., Hardy, J.W., Huq, M.I., Echeveria, P. and Falkow, 8.3 Isolation and nucleotide sequence determination of a gene encoding a heat-stable enterotoxin of Ecchczichig ccii. Infect. Immun. 3931167-1174, 1983. Moseley, S.L., Hug, I., Alim, A.R.M.A., So, M., Samadpour- Motalebi, M. and Falkow, 8.3 Detection of enterotoxigenic Eschcrichia ccii by DNA colony hybridization. J. Infect. Dis. 1423892-898, 1980. Moss, 'J., Garrison, S., Fishman, P.H. and Richardson, S.H.: Gangliosides sensitize unresponsive fibroblasts to Ecchcnichih coli heat-labile enterotoxin. J. Clin. Invest. 643381-384, 1979. Moss, J. and Richardson, S.H. 3 Activation of adenylate cyclase by heat-labile Egchczichih ccii enterotoxin. Evidence for ADP-ribosyltransferase activity similar to that of chaleragen. J. Clin. Invest. 623281-285, 1978. Moss, J. and Vaughn, N.: Mechanism of activation of adenylate cyclase by choleragen and E_,_ ccii heat- labile enterotoxin. In W him, eds. Field, M., Fordtran, J.S. and Schultz, S.G., American Physiological Society, Bethesda, MD, pp.107-126, 1980. Mouricout, M. and Julian, R.: Inhibition of mannose-resistant haemagglutination of sheep erythrocytes by enterotoxigenic Ecchccichig ccii in the presence of plasma glycoprotein glycans. FEMS Microbiol. Lett. 373145-149, 1986. Mouricout, M., Petit, J.P. and Julien, R.: Modes d'action d'agents inhibiteurs de l'adhesion de Egchcrichic ccii eterotoxinogenes (ECET) aux glycoproteines de la muqueuse intestinale bovine. Rev. Inst. Pasteur, Lyon 193161-168, 1986. Mouricout, M.A. and Julien, R.A.: Pilus-mediated binding of bovine enterotoxigenic Ecchcnichia ccii to calf small intestinal mucins. Infect. Immun. 5531216-1223, 1987. Murray, M.: Local immunity and its role in vaccination. Vet. Rec. 933500-504, 1973 214 Mushin, R., Ford, F.C.P. and Hughes, J.C.: Aspects of colonization of the mouse intestine by coliform bacteria. J. Med. Microbiol. 33573-583, 1970. Myers, L.L.3 Passive protection of calves against enteric colibacillosis by vaccination of the dams with capsular and fimbrial K antigens of E_,_ ccii. In Mines cf_the seccnd IntematienaLSynnesinm en nicthcc, University of Saskatchewan, Saskatoon, Saskatchewan, pp. 427-437, 1978. Myers, L.L. and Guinee, P.A.M.: Occurrence and characteristics of ETEC isolated from calves with diarrhea. Infect. Immun. 1331117-1119, 1976. Myers, L.L., Newman, F.S., Wilson, R.A. and Catlin, J.E.3 Passive immunization of calves against experimentally induced enteric colibacillosis by vaccination of dams. Am. J. Vet. Res. 34:29-33, 1973. Nagy, L.K., Mackenzie, T. and Painter, K.R.3 Protection of the nursing pig against experimentally induced enteric colibacillosis by vaccination of dam with fimbrial antigens of E,_ ccii (K88, K99 and 987P). Vet. Rec. 1173408-413, 1985. Nagy, B., Moon, H. W. and Isaacson, R. B.: Colonization of porcine small intestine by Ecchczichia cc1i3Ileal colonization and adhesion by pig enteropathogens that lack K88 antigen and by some acapsular mutants. Infect. Immun. 13:1214-1220, 1976. Nagy, B., Moon, H.W. and Isaacson, R.E.: Colonization of porcine intestine by enterotoxigenic Ecchczichic ccii: selection of pdliated forms in yiyci adhesion of piliated forms to epithelial cells in yihzc, and incidence of a pilus antigen among porcine enteropathogenic EL ccii. Infect. Immun. 163344-352, 1977. Nagy, B., Moon, H.W., Isaacson, R.E., To, C.C. and Brinton, C.C.: Immunization of suckling pigs against enteric enterotoxigenic Ecchcxichic ccii_infection by vaccinating dams with purified pili. Infect. Immun. 213269-274, 1978. Nakazawa, M., Haritani, M., Sugimoto, C. and Kashiwazaki, M.: Colonization of enterotoxigenic Ecchccichic ccii exhibiting mannose-sensitive hemagglutination to the small intestine of piglets. Microbiol. Immunol. 303485-489, 1986. 215 Newby, T.J. and Stokes, C.R.3 The intestinal immune system and oral vaccination. Vet. Immunol. and Immunopath. 6367-105, 1984. Newsome, P.M., Burgess, M.N., Bywater, R.J., Cowley, C.M. and Mullan, N.A.: Studies of the biological activities and mechanism of action of heat stable E,_ ccli enterotoxins. In_2rcceecincs_ef the second en. , University of Saskatchewan, Saskatoon, Saskatchewan, Canada, pp. 119-135, 1978. Newsome, P.M., Burgess, M.N. and Mullan, N.A.: Effect of Ecchcrichig ccii heat-stable enterotoxin on cyclic GMP levels in mouse intestine. Infect. Immun. 223290-291, 1978. Nielsen, N.O., Moon,H.W. and Roe, W.E.3 Enteric colibacillosis in swine. J. Am. Vet. Med. Assoc. 15331590-1606, 1968. ¢rskov, F., prskov, H., Smith, H.W. and Sojka, W.J.3 The establishment of K99, a thermolabile transmissible W ccii K antigen, previously call "Kco", possessed by calf and lamb enteropathogenic strains. Acta. Path. Microbiol. Scand. Sect. B 83:31-36, 1975. ¢rskov, I. and prskav, F.: Episome-carried surface antigen K88 of Ecchcrichic ,ccii I. Transmission of the determinant of the K88 antigen and influence on the transfer of chromosomal markers. J. Bacteriol. 91:69-75, 1966. ¢rskov, I. and prskov, F.: Serology of machia ccii fimbriae. Prog. Allergy 33380-105, 1983. prskov, I., firskov, F., Sojka, W.J. and Leach, J.M.: Simultaneous occurrence of E_,_ ccii B and L antigens in strains from diseased swine. Acta Pathol. Microbiol. Scand. 53: 404-422, 1961. ¢rskov, I., ¢rskov, F., Sojka, W.J. and Wittig, W.: K antigens K88ab(L) and K88ac(L) in E. coli A new O antigen: 0147 and a new K antigen 3 K89(B). Acta Path. et Microbiol. Scand. 623439-447, 1964. Ottow, J .C.G.3 Ecology, physiology and genetics of fimbriae and pili. Ann. Rev. Microbiol. 29379-108, 1975. 216 Parry, S.H.and Porter, P.: Immunological aspects of cell membrane adhesion demonstrated by porcine enteropathogenic Ecchcxichic,ccii. Immunol. 34:41-49, 1978. Picken, R.N., Mazaitis, A.J., Maas, W.K., Rey, M. and Heyneker, H.3 Nucleotide sequence of the gene for heat-stable enterotoxin II of Ecchccichic ccii. Infect. Immun. 423269-275, 1983. Poh1,P., Lintermans, P., van Muylem, K. and Schotte, M.: Enterotoxigenic Ecchczichic .ccii from Icalves that synthesize an adhesive antigen different from K99. Annales de Medicine Veterinaire 126, 569-571, 1982. Porter, P.: Porcine colostral IgA and IgM antibodies to ccii and their intestinal absorption by the neonatal pig. Immunol. 173617-626, 1969a. Porter, P.: Transfer of immunoglobulins IgG, IgA and IgM to lacteal secretions in the parturient saw and their absorption by the neonatal piglet. Biochim. et Biophys. Acta 181:381-392, 1969b. Porter, P., Noakes, D.E. and Allen, W.D.3 Intestinal Secretion of immunoglobulins and antibodies to E_._ ccii in the pig. Immunol. 183909-920, 1970a. Porter, P., Noakes, D.E. and Allen, W.D.3 Secretory IgA and antibodies to E_,_ ccii in porcine colostra and milks and their significance in the alimentary tract of young pigs. Immunol. 183245-257, 1970b. Powell, D.A., Hu, P.C., Wilson, M., Collier, A.M. and Baseman, J.B.3 Infect. Immun. 133959-966, 1976. Rao, M.C., Guandalini, S., Smith, P.L. and Field, M.: Mode of action of heat-stable enterotoxin: Tissue and subcellular specificities and role of cyclic GMP. Biochim. Biophys. Acta 632335-46, 1980 Rapacz, J. and Hasler-Rapacz, J.: Polymorphism and inheritance of swine small intestinal receptors mediating adhesion of three serological variants of ccii-producing K88 pilus antigen. Animal Genetics 173305-321, 1986. Raybould, T.J.G., Wilson, P.J., McDougal, L.J. and Watts, T.C.3 A porcine-murine hybridoma that secretes porcine monoclonal antibody of defined specificity. Am. J. Vet. Res. 4631768-1769,1985. 217 Rejnek, J., Travnicek, J., Kastka, J., Sterzl, J. and Lanc, A.: Study of the effect of antibodies in the intestinal tract of germ-free baby pigs. Folia Microbiol. 13:36-42, 1968. Ronnberg, B. , Wadstrom, T. and Jernvall, H.3 Structure of a heat stable enterotoxin produced by a human strain of Ecchcnichia ccii. FEBS Lett. 1553183-186, 1983. Roosendaal, B., Gaastra, W. and de Graaf, F.K.: The nucleotide sequence of the gene encoding the K99 subunit of enterotoxigenic Ecchcrichia ccii. FEMS Microbiol. Lett. 223253-258, 1984. Runnels, P.L., Moon, H.W. and Schneider, R.A.: Development of resistance with host age to adhesion of K994- Eachczichig ccii to isolated intestinal epithelial cells. Infect. Immun. 283298-300, 1980. Rutter, J.M.: Ecchcnichic ccli infections in piglets: Pathogenesis, virulence and vaccination. Vet. Rec. 963171-175 , 1975 . Rutter, J.M., Burrows, M.R., Sellwood, R. and Gibbons, R.A.: A genetic basis for resistance to enteric disease caused by Ecchczichic ccii. Nature 2573135- 136, 1973. Rutter, J.M. and Jones, G.W.: Protection against enteric diseases caused by Ecchcnichic cgii: A model for vaccination with a virulence determinant? Nature 2423531-532, 1973. Rutter, J.M. and Anderson, J .C.3 Experimental neonatal diarrhoea caused by an enteropathogenic strain of W ccii in piglets: A study of the disease and the effect of vaccinating the dam. J. Med. Microbiol . 53197-210 , 1972 . Rutter, J.M., Jones, G.W., Brown, G.T.H., Burrows, M.R. and Luther, P.D.3 Antibacterial activity in colostrum and milk associated with protection of piglets against enteric disease caused by K88-positive Ecchczichia ccii. Infect. Immun. 133667-676, 1976. Sack, D.A. and Sack, B.: Test for enterotoxigenic Ecchccichic ccii using Y1 adrenal cells in miniculture. Infect. Immun. 113334-336, 1975. 218 Sadowski, P.L.: Protection of pigs from fatal colibacillosis through the use of ”a" and "c" monoclonal antibodies to the K88 pilus. In My; fifthnnnnalheetincefthecenferencecfnesearch flczhczc, Chicago, Illinois, p. 52, 1984 (abstract). Sadowski, P.L., Acres, S.D. and Sherman, D.M.3 Monoclonal antibody for the protection of neonatal pigs and calves from toxic diarrhea. In R113 _Bjclcgy cf H31 Lifeiciences W in Eistechnclmz. Basic Edited by A. Hollander, A. I. Laskin and P. Rogers, vol. 25, Plenum Press, New York, pp. 93-99, 1983. Sadowski, P.L., Koebelbeck, V., Marshall, R.F., Kuecker, R.D. and Spronk, G.D.3 A 987P specific monoclonal antibody with the ability to reduce mortality due to colibacillosis in neonatal pigs. In W Guidc and Mst a Becki 1st International Imcicgy Winn. University of Guelph, Guelph, Ontario, Canada, p. 141, 1986 (abstract). Saeed, A.M.K., Sriranganathan, N., Cosand, W. and Burger, D.3 Purification and characterization of heat-stable enterotoxin from bovine enterotoxigenic Ecchcnichig ccii. Infect. Immun. 403701-707, 1983. Salit, LE. and Gotschlich, E.C.3 Hemagglutination by purified type 1 Ecchcrichic ccli pili. J. Exp. Med. 14631169-1181, 1977. Sandow, M.J. and Whitehead, R. 3 Progress Report: The paneth cell. Gut 203420-431, 1979. Saunders, C.N., Stevens, A.J., Spence, LB. and Sojka, W.,J.3 Ecchczigic ccli infection: Reproduction of the disease in "Pathogen-free" piglets. Res. Vet. Sci., 43347-357, 1963. Savage, D.C., Siegel, J.E., Snellen, J.E., et al: Transit time of epithelial cells in the small intestines of germfree mice and ex-germfree mice associated with indigenous microorganisms. Appl. Environ. Microbiol. 423996-1001, 1981. Schacter, M. and Williams, D.3 Biosynthesis of mucus glycoproteins. In Encns in Health and Disease. Edited by E.M. Chantler, LB. Elder and M. Epstein, vol. II, Plenum Press, New York, pp. 3-15, 1982. 219. Schifferli, D.M., Abraham, S.N. and Beachey, E.H.3 Use of monoclonal antibodies to probe subunit- and polymer-specific epitopes of 987P fimbriae of Ecchcxichic ccii. Infect. Immun. 553923-930, 1987. Schlink, G.T.3 Protection against F41 colibacillosis with a combination K88, K989, 987P and F41 subunit vaccine. In sixty:Sixth Annual Meeting conference cf Research Eerkers__in Animal__nisease. Chicago, Illinois, p. 28, 1985 (abstract). Schneider, R.A. and To, S.C.M.3 Enterotoxigenic ccii strains that express K88 and 987P pilus antigens. Infect. Immun. 363417-418, 1982. Scoot, A., Owen, B.D. and Agar, J.L.: Influence of orally administered porcine immunoglobulins on survival and performance of newborn colostrum-deprived pigs. J. Anim. Sci. 3531201-1205, 1972. Scotland, S.M., Gross, R.J. and Rowe, B.: Laboratory tests for enterotoxin production, enteroinvasion and adhesion in diarrhoeagenic Efichczichic ccii. In Thc Yirnlence of Escherichia celi- Edited by H- Sussman, Academic Press, London, pp. 395-405, 1985. Sellwood, R.: Ecchccichig ccii diarrhoea in pigs with or without the K88 receptor. Vet. Rec. 1053228-230, 1979. Sellwood, R.: Genetic susceptibility to intestinal infection - animal models. In gcncticc inc Hetercceneity ef.srmmrn1 - Discrcers- Edited by J.I. Rotter, I.M. Samloff and D.L. Rimoin, Academic Press, New York, pp. 535-549, 1980a. Sellwood, R.: The interaction of the K88 antigen with porcine intestinal epithelial cell brush. borders. Biochem. et Biophys. Acta 6323326-335, 1980b. Sellwood, R3 An intestinal receptor for the K88 antigen of porcine enterotoxigenic Ecchcxichic ccii. In Attachment of crcanisns tn the cat nacc_a- Edited by E.C. Boedecker, CRC Press, Inc., Boca Raton, FL, CRC Press, Inc., pp. 167-175, 1984a. Sellwood, R.: Inhibition of adhesion of Ecchccichig K88 antigen by mammary secretions of susceptible and resistant sows. Vet. Microbiol. 93477-486, 1984b. 220 Sellwood, R. Gibbons, R.A., Jones, G.W. and Rutter, J.M.: Adhesion of enteropathogenic Ecchcgichic ccii to pig intestinal brush borders: The existence of two pig phenotypes. J. Med. Microbiol. 83405-411, 1975. Sellwood, R. and Kearns, M.: Inherited resistance to Ecchczichig ccii diarrhea in pigs: The genetics and nature of the intestinal receptor. In EIQDEIQIE cf Kncxiecge in the W. Edited by Ed)- Janowitz and D.B. Sachar, Projects in Health, Inc., Upper Monclair, New Jersey, pp. 113-122, 1979. Seriwatana, J., Echeverria, P., Escamilla, J., Glass, R., Huq, I., Rockhill, R. and Stall, B.J.: Identification cf enterotoxigenic Ecchczichig ccii in patients with diarrhea in Asia with three enterotoxin gene probes. Infect. Immun. 423152-155, 1983. Shedlofsky, S. and Freter, R.: Synergism between ecological and immunological control mechanisms of intestinal flora. J. Infect. Dis. 1293296-303, 1974. Sherman, D.M., Acres, S.D., Sadowski, P.L., Springer, J.A., Bray, B., Raybould, T.J.G. and Muscoplat, C.C.: Protection of calves against fatal enteric colibacillosis by orally administered Ecchcrichic ccii K99-specific monoclonal antibody. Infect Immun. 423653-658, 1983. Sherman, D.M. and Markham, R.J .F.3 Current and future applications of monoclonal antibodies against bacteria in veterinary medicine. In Mchccichgi Eachcrig. Edited by A.J. Macario and E.C. de Macario, vol. III, Academic Press, Inc., New York, pp. 295-340, 1986. Shimizu, M., Sakano, T., Yamamoto, J. and Kitajma, K.3 Incidence and some characteristics of fimbriae FY and 31A of Ecchczichia ccii isolates from calves with diarrhea in Japan. Microbiol. Immunol. 313417-426, 1987. Shipley, P.L., Gyles, C.L. and Falkow, 8.3 Characterization of plasmids that encode for the K88 colonization antigen. Infect. Immun. 203559-566,1978. Shrank, 6.0. and Verwey, W.F.3 Distribution of cholera organisms in experimental yihzic chcicrcc infections: Proposed mechanism of pathogenesis and antibacterial immunity. Infect. Immun. 133195-203, 1976. 221 Silverblatt, F.J. and Cohen, L.S.3 Antipili antibody affords protection against experimental ascending pyelonephritis. J. Clin. Invest. 643333-336, 1979. Silverblatt, F.J., Weinstein, R. and Rene, P.: Protection against experimental pyelonephritis by antibody to pili. Scand. J. Infect. Dis. Suppl. 33:79-82, 1982. Skerman, F. J., Formal, S. B. and Falkow, S.3 Plasmid associated enterotoxin production in a strain of Ecchczichia ccii isolated from humans. Infect. Immun. 53622-624,1972. Slomiany, B.L. and Slomiany, A.: Lipids of mucous secretions of the alimentary tract. In achcchmch; cf Qreanisns to the cat Enccsa- Edited by EC- Boedecker, CRC Press, Inc., Boca Raton, FL, pp. 24-31, 1984. Smit, H., Gaastra, W., Kamerling, J.P., Vliegenthart, J.F.G. and de Graaf, F.K.: Isolation and structural characterization of the equine erythrocytes receptor for enterotoxigenic Ecchczichic ,ccii_ K99 fimbrial adhesin. Infect. Immun. 463578-584, 1984. Smith, H.W.3 The nature of the protective effect of antisera against Ecchczichig ccii diarrhoea in piglets. J. Med. Microbiol. 53345-352, 1972. Smith, H.W. and Gyles, C.L.: The relationship between two apparently different enterotoxins produced by enteropathogenic strains of E. coli of porcine origin. J. Med. Microbiol. 33387-401, 1970. Smith, H. W. and Halls, 8.3 . Observations by the ligated intestinal segment and oral inoculation methods on Eachcrichig ccii infections in pigs, calves, lambs and rabbits. J. Path. Bact. 933499-529,1967. Smith. H.W. and Halls, 8.: The transmissible nature of the genetic factor in (Ecchcrichic (ccii that controls enterotoxin production. J. Gen. Microbiol. 523319-334, 1968. Smith, H.W. and Huggins, M.B.3 The influence of plasmid- determined and other characteristics of enteropathogenic Ecchczichig ccii on their ability to proliferate in the alimentary tracts of piglets, calves and lambs. J. Med. Microbiol. 113471-492, 1978. 222 Smith, H.W. and Jones, J.E.T.: Observations on the alimentary tract and its bacterial flora in healthy and diseased pigs. J. Path. and Bact. 863387-412, 1963. Smith, H.W. and Linggood, M.A.: Observations on the pathogenic properties of K88, Hly, and Ent plasmids of Egchczichia ccii with particular reference to porcine diarrhea, J. Med. Microbiol. 43467-485, 1971a. Smith, H.W. and Linggood, M.A.: The effect of antisera in protecting pigs against experimental W diarrhoea and edema disease. J. Med. Microbiol. 43487-493, 1971b. Smith, H.W. and Linggood, M.A.: Further observations on Ecchcrichic ccli enterotoxins with particular regard to those produced by atypical piglet strains and by calf and lamb strains: the transmissible nature of these enterotoxins and of a K antigen possessed by calf and lamb strains. J. Med. Microbiol. 53243-250, 1972. So, M., Boyer, H.W., Betlach, M. and Falkow, 8.3 Molecular cloning of an Eschccichic ccii plasmid determinant that encodes for the production of heat-stable enterotoxin. J. Bacteriol. 1283463-472, 1976. So, M. and McCarthy, B.J .3 Nucleotide sequence of the bacterial transposan Tn 1681 encoding a heat-stable toxin and its identification in enterotoxigenic Ecchci'ichia ccii strains. Proc. Natl. Acad. Sci. USA 77:4011-4015, 1980. Soderlind, D. and Mollby, R.: Enterotoxins, O-groups and K88 antigen in Eschczichic ccli from neonatal pigs with and without diarrhea. Infect. Immun. 24:611- 616, 1979. Sojka, W.J.3 Ecchcrichic ccii in domestic animals and poultry. Commonwealth Agricultural Bureau, Weybridge, England, 1965. Sojka, W.J., Wray, C. and Morris, J.B.3 Passive protection of lambs against experimental enteric colibacillosis by colostral transfer of antibodies from K99-vaccinated ewes. J. Med. Microbiol. 113493- 499, 1978. 223 Spicer, E.K, Kavanaugh, W M., Dallas, W.S., Falkow, S., Konigsberg, W. H. and Schafer, D. B.: Sequence homologies between A subunits of Ecchczichichii and 11.th chcicrcc enterotoxins. Proc. Natl. Acad. Sci. U.S.A. 78. 50- 54, 1981. Spicer, E.K. and Noble, J.A.: Ecchczichia ccii heat- labile enterotoxin. Nucleotide sequence of the A subunit gene. J. Biol. Chem. 25735716 5721, 1982. Staley, T.E., Jones, E.W. and Carley, L.D.3 Attachment and penetration of Ecchczichic ccii into intestinal epithelium of the ileum in newborn pigs. Amer. J. Pathol. 563371-392, 1969. Staley, T.E. and Wilson, I.B.3 Recovery of intestinal membrane binding sites for K88 E; ccii from p1g mucosal organ cultures. Fed. Proc. 323507, 1983 (abstract). Staples, S.J., Asher, S.E. and Giannella, R.A.: Purification and characterization of heat stable enterotoxin produced by a strain of E; ccii pathogenic to man. J. Biol. Chem. 25534716-4721, 1980. Steel, R.G.D. and Torrie, J H.. . 3 Principles of Statistical A Eicnetrical £IQ£§§EI§§ Apnrcachr Edited by C- Napier and J.W. Maisel, McGraw-Hill, New York 1980. Stirm, S., prskov, F., firskov, I. and Birch-Anderson, A.: Episome-carried surface antigen K88 of E__chc1ichic ccii III. Morphology. J. 1967a. Bacterial . 93 3 740-748 , Stirm, 8., ¢rskov, F., prskov, I. and Mansa, B.: Episome-carried surface antigen K88 of Ecchcrichic ccii II. Isolation and Bacterial. chemical analysis. J. 933731-739, 1967b. Stoliar, O.A., Kaniecki-Green, E., Pelley, R.P., Klaus, M.H. and Carpenter, C.C.J .3 Secretary IgA against enterotoxins in breast milk. Lancet 131258-1261, 1976. Strombeck, D R. and Harold, 0.: Binding of cholera toxin to mucins and inhibition by gastric mucin. Infect. Immun. 1031266-1272, 1974. 224 Suarez, S., Paniagua, C., Alvarez, M. and. Rubia, P.: Features of enterotoxigenic Ecchccichic ccii strains of porcine origin that express K88 and 987P fimbrial antigens. Vet. Microbiol. 13:65-68, 1987. Svennerholm, A.-M. and Holmgren, J.: Identification of Ecchczichig ccii heat-labile enterotoxin by means of a ganglioside immuonosorbent assay (GMl-ELISA) procedure. Curr. Microbial. 1319-23, 1978. Svendsen, J. and Wilson, M.R.: Immunity to Ecchcrichia ccii in pigs: Effect of feeding colostrum or serum from intramammarily or intramuscularly vaccinated saws to Eschczjchic ccii-infected gnotobiotic pigs. Am. J. Vet. Res. 32. 899- 904, 1971. Takao, T., Hitouji, T., Aimoto, S., Shimonishi, Y., Hara, 8., Takeda, T. and Miwatani, T.: Amino-acid sequence of a heat-stable enterotoxin isolated from enterotoxigenic Ecchc:ichic_ ccii isolated from patients. FEBS Lett. 15231-5, 1983. Takeda, Y., Honda, T., Sima, H., Tsuji, T. and Miwatani, TL: Analysis of antigenic determinants in cholera enterotoxin and heat-labile enterotoxins from human and porcine enterotoxigenic Ecchczichic ccii. Infect. Immun. 41:50-53, 1983. Thomas, L. V., Cravioto, A., Scotland, 8. M. and Rowe, B.: New fimbrial antigen type (E8775) that may represent a colonization factor in enterotoxigenic Ecchczichig ccii in humans. Infect. Immun. 3531119-1124,1982. Thomas, D.D. and Knoop, F.C.: Effect of heat-stable enterotoxin of Ecchczichic ccii on cultured mammalian cells. J. Infect. Dis. 1473450-459, 1983. Thompson, M.R., Brandwein, H., Labine-Racke, M. and Giannella, R.A. 3 Simple and reliable enzyme-l inked immunosorbent assay with monoclonal antibodies for detection of Ecchcxichic coli heat-stable enterotoxins. J. Clin. Microbial. 20:59-64, 1984. Thompson, M. R., Luttrell, M., Overmann, G. and Gianella, R. A.: Revised amino acid sequence for a heat-stable enterotoxin produced by an Ecchcrichia ccii strain (18D) that is pathogenic for man. Infect. Immunol. 473834-836, 1985. 225 To, S.C.: Prevention of colibacillosis in neonatal swine with a 4-pilus E. coli bacterin. Mod. Vet. Pract. 65:39-41, 1984a. To, S.C.M.3 F41 antigen among porcine enterotoxigenic ficchccichig ccii strains lacking K88, K99, and 987? pili. Infect. Immun. 433549-554, 1984b. To, S.C.M., Moon, H.W. and Runnels, P.L.: Type 1 pili (F1) of porcine enterotoxigenic Ecchcrichia ccii: Vaccine trial and tests for production in the small intestine during disease. Infect. Immun. 4331-5, 1984. Tsuji, T., Taga, 8., Honda, T., Takeda, Y. and Miwatani, T.: Molecular heterogeneity of heat-labile enterotoxins from human and porcine enterotoxigenic Ecchczichic ccii. Infect. Immun. 383444-448, 1982. Tzipori, M., Withers, M. and Hayes, J.: Attachment of E. coli-bearing K88 antigen to equine brush- border membranes. vet. Microbial. 93561-570, 1984. van der Waaij, D., Berghuis-de Vries, J.M. and Lekkerkerk, J.E.C.3 Colonization resistance of the digestive tract in conventional and antibiotic-treated mice. J. Hyg. 693405-411, 1971. van Doorn, J, Oudega, B., Mooi, F.R. and de Graaf, F.K.: Subcellular localization of polypeptides involved in the biosynthesis of K88ab fimbriae. FEMS Lett. 13399-104, 1982. Wadstrom, T., Smyth, C.J., Faris, A., Johnson, P. and Freer, J.H.: Hydrophobic adsorptive and hemagglutinating properties of enterotoxigenic Bcchcrichic ccii with different colonizing factors: K88, K99 and colonization factor antigens and adherence factors. In Ezcceedihgg, ficcchc . 2n. lusunxtal. Ddsuuflnsa. University of Saskatchewan, Saskatoon, Saskatchewan, Canada, pp. 29-55, 1979. Waldman, S.A., O'Hanley, P., Falkow, S., Schoolnik, G. and Murad, F.: A simple, sensitive, and specific assay for the heat-stable enterotoxin of Ecchczichig ccii. J. Infect. Dis. 149383-89, 1984. Waxler, G.L., Schmidt, D.A. and Whitehair, C.R.3 Techniques for rearing gnotobiotic pigs. Am. J. Vet. Res. 273300-307, 1966. 226 Weikel, C.S., Nellans, H.W. and Guerrant, R.L.: In yiyc and in yim effects of a novel enterotoxin, STb, produced by Ecchczichic coli. J. Infect. Dis. 1533893-900, 1986a. Weikel, C.S., Tiemens, R.M., Moseley, S.L., Hug, I.M. and Guerrant, R.L.: Species specificity and lack of production of 8Tb enterotoxin by strains isolated from humans with diarrheal illness. Infect. Immun. 523323-325, 1986b. Weinstein, R. and Silverblatt, F.J.: Antibacterial mechanisms of antibody to mannose-sensitive pili of Ecchcgichig ccii. J. Infect. Dis. 147:882-889, 1983. Weiser, M.M.3 The synthesis of intestinal glycoproteins with special reference to vitamin D-dependent processes, attachment of microorganisms, and membrane shedding- In Attachment of mm to the an; Muccsa. Edited by E.C. Boedecker, CRC Press, Inc., Boca Raton, FL, pp 89-98, 1984. Welsh, J.M. and May, J.T.3 Antiinfective properties of breast milk. J. Pediatr. 9431-9, 1979. Wensink, J., Gankema, H., Jansen, W.K., Guinee, P.A.M. and Witholt, 8.: Isolation of the membranes of an enterotoxigenic strain of Ecchcnighic ccii and distribution of enterotoxin activity in different subcellular fractions. Biochim. Biophys. Acta. 5143128-136, 1978. Westerman, R.B., Mills, R.W., Phillips, R.M., Fortner, G.W. and Greenwood, J.M.: Predominance of the ac variant in K88-positive Eschcrichic ccii isolates from swine. J. Clin. Microbiol. 263149-150, 1988. White, F., Wenham, G., Sharman, G.A.M., Jones, A.S., Rattray, E.A.S. and McDonald, 1. 3 Stomach function in relation to a scours syndrome in the piglet. Brit. J. Nutr. 233847, 1969. Wilson, I.B., Staley, T.E., Bush, L.J. and Gilliland, S.E.: Recovery of intestinal membrane binding sites for K88 L ccii from pig mucosal organ cultures. Molecular and Cellular Biochemistry 62:57-65, 1984. Wilson, M.R.: Immunity to Ecchccichic ccli in pigs: Efficacy of a live formalized vaccine under field conditions. Br. Vet. J. 130: 599-605, 1974. 227 Wilson, M.R.: Enteric colibacillosis. In Discgcgc cf fixing. Edited by A.D. Leman, B. Straw, B., R.D. Glock, W.C. Mengeling, R.M.C. Penny and E. Schall, 6th ed., Iowa State University Pres, Ames, IA, pp. 520-528, 1986. Wilson, M.R.: The influence of preparturient intramammary vaccination on bovine mammary secretions. Antibody activity and protective value against ccii enteric infections. Immunol. 233947-955, 1972. Wilson, M.R. and Hohmann, A.W.3 Immunity to Ecchcrichic ccii, in pigs: Adhesion of enteropathogenic Escherichia chi to isolated intestinal epithelial cells. Infect. Immun. 103776-782, 1974. Wilson, M.R. and Svendsen, J .3 Immunity to Ecchczichic ccii in pigs. The role of milk in protective immunity to E_,_ ccli enteritis. Can. J. Comp. Med. 353239-243, 1971. Walk, W., Svennerholm, A.-M. and Holmgren, J.: Isolation of Ecchcgichic ccii heat-labile enterotoxin by affinity chromatography: Characterization of subunits. Curr. Microbial. 33339-344, 1980. Wostman, E.S. and Bruckner-Kardoss, E.3 Functional characteristics of gnotobiotic rodents. In M W in Mfrs: Research- Edited by 8- Sasaki. A. Ozawa, and K. Hashimoto, Tokai University Press, Tokyo, pp. 321-325, 1981. Yamamoto, T. and Yokota, T.: Sequence of heat-labile enterotoxin of Ecchczichig ccii pathogenic for humans. J. Bacteriol. 1553728-733, 1983. Yano, T,, Leite, D.S., de Camargo, I.J.B. and de Castro, A.E.P.3 A probable new adhesive factor (F42) produced by enterotoxigenic Egchccichic ccli isolated from pigs. Microbiol. Immunol. 303495-508, 1986. Yolken, R.M., Greenberg, H.B., Merson, M.R., Sack, R.B. and Kapikian, A.z.3 Enzyme-linked immunosorbent assay for detection of Eschcnichic ccli heat-labile enterotoxin. J. Clin. Microbiol. 63439-444, 1977. Young, L.S.3 Monoclonal antibodies: technology and application to gram-negative infections. Infection 13 (suppl. 2)38224-8229, 1985. 228 Zenzer, T.V. and Metzger, J .F.3 Comparison of the action of fifichcrichic ccli enterotoxin on the thymocyte adenylate cyclase-cyclic adenosine monophosphate system to that of cholera toxin and prostaglandin E1. Infect. Immun. 103503-509, 1974. VITA The author was born in Tuskegee Institute, Alabama on February 24, 1958. Her primary and secondary education was completed in several schools located throughout the United States and Germany. She graduated from Tuskegee Institute's School of Veterinary Medicine in 1982. In the summer of 1982, the author entered a graduate program in the Department of Pathology at Michigan State University. She completed a Master's Degree in 1984 and then entered the Department of Pathology's Doctoral Program. The author has accepted an assistant professor position in. Michigan State University's Department of Pathology. 229