Neutrophil mediated and IgA dependent antibacterial immunity against enteropathogenic Escherichia coli in the porcine intestinal mucosa.

The role of polymorphonuclear neutrophils (PMN) in the antibacterial immunity against enteropathogenic Escherichia coli (EEC) 0:149 in the porcine intestine was studied using intestinal Thiry-Vella loop (T-V loop) as a model. Intraluminal immunizations of T-V loops resulted in elevated levels of immunoglobulin A (IgA) anti-EEC 0:149 antibody in the loop secretions, an infiltration of PMN in the lumen of the loops and an increase in the concentrations of lactoferrin (LF), lysozyme (LY), cationic proteins (CP), and a specific bactericidal response in the immunized loops. PMN were observed by electron microscopy (EM) to be actively phagocytic in the lumen of the immune loops. EM observations of loop fluids as well as the abrogating effect of iron on the in vivo bactericidal response strongly suggest that the pMN played an important role in the bactericidal response in the loops against EEC. In addition to phagocytosis by PMN and subsequent intracellular killing, disintegration of PMN in the lumen of the loops and extracellular killing of EEC by the antibacterial products of PMN such as LF, LY and CP, with and/or without synergistic effect of IgA antibodies, also contribute to the bactericidal response of the immunized loops.

Effects of immune colostrum on the expression of a K88 plasmid encoded determinant: role of plasmid stability and influence of phenotypic expression of K88 fimbriae.

Passaging of the K88-positive Escherichia coli strain CN6913 through synthetic medium containing immune colostrum gave rise to large numbers of K88-negative CN6913 variants. These K88-negative variants had all lost a single large plasmid known to encode the K88 genetic determinant. Four other large plasmids harboured by this strain were unaffected. Viable K88-positive and K88-negative variants of CN6913 accumulated at a similar rate in synthetic medium and in medium containing non-immune colostrum. In the presence of immune colostrum, viable cells of the K88-negative variant accumulated faster and to a greater extent in cultures than the K88-positive variant if incubated at 37 degrees C, which favours the phenotypic expression of K88. However, when similar cultures were incubated at 18 degrees C, a temperature known to inhibit phenotypic expression of K88, the accumulation of viable cells of the two variants was strictly comparable in all media and no loss of plasmid or increase in K88-negative variants was observed. Cells containing a pBR322-based K88-encoding recombinant plasmid were also eliminated by immune colostrum whereas cells containing pBR322 were not. Plasmids encoding the K99 antigen were not readily eliminated from strains passaged through medium containing immune colostrum. K99-negative variants that were detected still harboured the K99-encoding plasmid.

Protection of the nursing pig against experimentally induced enteric colibacillosis by vaccination of dam with fimbrial antigens of E coli (K88, K99 and 987P).

Pregnant gilts were vaccinated with two doses of alhydrogel adsorbed fimbrial antigens of Escherichia coli (K88ab, K88ac, K99 and 987P) supplemented with beta toxoid of Clostridium perfringens type C. Their piglets, and piglets of nonvaccinated gilts, were subsequently orogastrically challenged with one or other of the four fimbrial types of enteropathogenic E coli. Some of the vaccinated animals were reinjected with a single dose of the vaccine during second gestation and their piglets, and piglets of non-vaccinated sows, were challenged the same way as were litters of gilts. Blood serum and colostra were examined for antibodies to the four fimbrial antigens of E coli and for antitoxin to beta toxin of C perfringens type C. It was found that: (1) a highly significant reduction in mortality and morbidity was achieved in vaccinated litters against all four challenge strains of E coli; (2) excretion of K88ab and K88ac but not of K99 and 987P challenge strains was significantly reduced; (3) revaccination of sows by a single dose of the vaccine during second gestation conferred complete protection against mortality and highly significant protection against morbidity; (4) no correlation was noted between colostral or seroagglutinins to fimbrial antigens of E coli and mortality rates in litters challenged with homologous fimbrial types of E coli, but good correlation was found between colostral precipitins to K88 antigens and mortality rates in litters; (5) antitoxin value in 97 per cent of colostrum of vaccinated sows was 10 iu equivalent of C perfringens type C toxin or more per ml of colostrum.

Evaluation of procholeragenoid against experimental colibacillosis in piglets of vaccinated dams.

A toxoid prepared from the toxin of Vibrio cholera was adjuvanted with aluminium hydroxide and used for immunisation of pregnant gilts. Litters of these and of non-vaccinates were experimentally challenged with Escherichia coli producing either heat labile and heat stable (LT and ST) enterotoxins or ST enterotoxin only. Both the challenge strains of E coli produced high rates of mortality (64 and 68 per cent) and morbidity (80 and 100 per cent) in litters of non-vaccinated dams. Statistically highly significant protection against the LT/ST enterotoxin producing strain of E coli was obtained accompanied by the absence of colonisation of the small intestine by the pathogen. No protection against the ST enterotoxin producing strain was found. It is suggested that this vaccine would not confer passive protection to piglets against K99 and 987-positive E coli which usually produce ST enterotoxin only.

Purification, characterization and immunological properties of the serotype-specific capsular polysaccharide of Pasteurella haemolytica (serotype A1) organisms.

The serotype-specific capsular polysaccharide from two strains of Pasteurella haemolytica serotype A1 organisms was purified and characterized by chemical analysis and NMR spectroscopy. The polymer has the structure----3)-O-(2-acetamido-2-deoxy-4-O-acetyl-beta-D-mannopyranos yluronic acid)-(1----4)-O-(2-acetamido-2-deoxy-beta-D-mannopyranose)-(1----. The polysaccharide was immunogenic (able to evoke production of antibodies) for sheep but not for rabbits. Immuno electron-microscopy studies using the Protein A-gold technique showed the polysaccharide to be peripherally located on the bacterial surface. Reduction, oxidation and de-O-acetylation of the polymer did not appear to alter its immunological precipitability with specific antiserum, but all three treatments destroyed its ability to adhere to sheep erythrocytes at neutral pH. De-N-acetylation of the polymer destroyed both immunological precipitability and erythrocyte adherence.

Multi-adhesin vaccines for the protection of the neonatal piglet against "E. coli" infections.

It has been shown that the initial stages of enteric infection with E. coli organisms involve attachment of the organism to the intestinal villi. Attachment is mediated by specific protein adhesins on the surface of the organism such as K88ab, K88ac, K99 and 987P (1). Purified preparations of each of these adhesins have been used for the vaccination of pregnant gilts and been shown to confer passive protection to their piglets against homologous but not heterologous challenge with E. coli (2, 3). In this study gilts have been vaccinated with multi-adhesin vaccines combining these components (K88, K99, 987P) and litters born to them were challenged together with litters of non-vaccinated gilts, with the appropriate E. coli strains. It was shown that vaccines confer substantial protection in terms of mortality, reduction in diarrhoea and excretion of the organisms.

Passive immunity in calf diarrhea: vaccination with K99 antigen of enterotoxigenic Escherichia coli and rotavirus.

Twenty-four pregnant cows were vaccinated intramuscularly with K99 extract from enterotoxigenic Escherichia coli and inactivated rotavirus as follows: six cows were injected with 2 ml of oil-adjuvanted vaccine; six cows were injected with 0.5 ml of oil-adjuvanted vaccine; six cows were injected with 4 ml of aluminum hydroxide-adjuvanted vaccine twice with a four-week interval; and six cows were unvaccinated as controls. Calves born to these cows were challenged with enterotoxigenic E. coli at 6 to 18 h after birth. Serum and milk antibodies to K99 and rotavirus in cows vaccinated with either dose of oil vaccine were significantly increased until at least 28 days after calving. In cows vaccinated with alhydrogel vaccine, there was a significant K99 antibody increase in serum and in colostrum but not in milk and a significant rotavirus antibody increase only in colostrum. Five of six calves born to unvaccinated cows developed enterotoxic colibacillosis after challenge, and all excreted the challenge strain of enterotoxigenic E. coli. None of the 18 calves in the three vaccinated groups developed clinical colibacillosis, and fecal excretion of the challenge organism was reduced. A combined enterotoxigenic E. coli-rotavirus vaccine may prove useful in preventing some outbreaks of calf diarrhea.

Scanning electronmicroscopy of the jejunum in enteritis necroticans.

Intestinal tissue resected at laparotomy from patients in Papua New Guinea at various clinical stages of enteritis necroticans, locally known as pig-bel, has been examined under the scanning electronmicroscope. Evidence obtained from parallel studies of experimental infection in pigs is presented. Progressive destruction of the intestinal mucosa was seen during the course of the disease in man. Numerous filamentous rods morphologically consistent with the appearance of Clostridium perfringens type C, were seen to be attached the affected areas of gut and were associated with the necrotic tissue. The mechanism of pathogenicity includes a stage of attachment to the surfaces of jejunal villi, local multiplication, and the production of beta toxin which may be protected from tryptic digestion by the inadequacy of pancreatic protease production in susceptible subjects and by the ingestion of a trypsin inhibitor. The association of the condition with pork feasting is discussed.

Duration of anti-adhesive and bactericidal activities of milk from vaccinated sows on Escherichia coli O149 in the digestive tract of piglets during the nursing period.

Piglets were exposed orogastrically to Escherichia coli to enable study of the duration of anti-adhesive and bactericidal activities of milk of sows vaccinated with a K88 enriched E coli vaccine. There was a marked increase in the number of the challenge strain in the digestive tract of weaned piglets of all ages (between 888 and 2144 per cent). In contrast, there was a decrease in their number (75 per cent) in the day-old colostrum-fed piglets. When the piglets were two weeks old milk was still capable of reducing the rate of proliferation of the pathogen but at five weeks it proliferated at equal rates in the digestive tract of both suckling and weaned litter-mates. The rate of adhesion of the K88 positive E coli to the small intestine of colostrum deprived piglets was high (5 x 108/g). Rate of adhesion fell gradually in weaned piglets from 5.4 x 107/g at two weeks to 2.0 x 106/g at four to five weeks of age. In contrast, resistance of the small intestine of suckling pigs to adhesion by K88-positive E coli remained relatively stable through the five week period of nursing bacterial counts ranging from 5 x 104/g to 3 x 104/g of tissue.

The effect of colostrum or past colibacillosis on the adhesion of Escherichia coli to the small intestine of the pig.

Nursing litters of vaccinated (K88 antigen) and non-vaccinated gilts as well as weaned piglets, which had recovered from natural colibacillosis, were exposed to Escherichia coli O149: K91(B), K88ac(L) orogastrically and 3-4 h after exposure a proportion of each group was killed for adhesion studies; others were kept for clinical observations. From killed piglets duodenal contents were collected and after washing of the duodenum by standardised techniques it was homogenised. Viable counts of E coli O149 in these specimens were carried out and compared. Colicounts in the duodenal contents and in the homogenate of washed duodenum of naturally infected piglets were also compared. There was less than tenfold difference in counts of K88-positive E coli between duodenal contents and washed duodenum of piglets which were (A) naturally infected, (B) colostrum deprived and experimentally infected, or (C) were from colostrum fed groups where those litter mates which were kept for clinical observation died of colibacillosis. There was 10(3)-10(5)-fold difference in E coli O149 counts between comparable specimens from piglets which (a) recently recovered from K88-positive E coli infection at the time of oral challenge or (b) were from colostrum fed groups where those litter mates which were kept for clinical observation survived oral challenge. It is concluded that adhesion of K88-positive E coli to the epithelium of the anterior small intestine of the pig is a feature of natural as well as experimental colibacillosis. Such adhesion may be prevented by (i) earlier natural infection of K88-positive E coli or (ii) by the ingestion of colostrum of K88 antigen vaccinated dams.

In vitro studies on the antimicrobial effects of colostrum and milk from vaccinated and unvaccinated pigs on Escherichia coli.

Unsupplemented porcine colostrum and milk exhibited a powerful bactericidal effect for porcine strains of E coli incubated in vitro at 37 degrees C. This activity was independent of complement but was susceptible to acid pH, to the presence of soluble iron and to the selective immunoprecipitation of IgG, IgA and IgM. Manifestation of bactericidal activity required bacteria in an active state of metabolism and the length of incubation was an important factor in demonstrating the quality of the anticoli activity, ie, proliferation-inhibitory, bacteriostatic or bactericidal. Whey obtained by acid precipitation or by the application of rennin was devoid of bactericidal activity but was capable of slowing down proliferation of E coli. There was no correlation between lysozyme and anticoli activity although the complete removal of lysozyme by adsorption on to bentonite reduced bactericidal titres. With very few exceptions the highest bactericidal titres were recorded for colostrum, but even 28 days post partum about one half of 22 undiluted milk samples exhibired bactericidal activity.

Protection of cattle against experimental haemorrhagic septicaemia by the capsular antigens of Pasteurella multocida, types B and E.

Aluminum hydroxide adjuvant vaccines containing endotoxin-free capsular antigens of Pasteurella multocida, types B and E, were administered to cattle. Dose dependent serological responses were observed which were similar for both antigens. The immunised cattle were subjected to intravenous challenge by a virulent type E strain. All animals which received the highest vaccine dose survived and all unimmunised control animals died and a vaccine dose-response relationship was obtained. The results of passive mouse protection and indirect haemagglutination tests (type E) on the sera of immunised cattle corresponded with the degree of protection against challenge of the cattle.

Isolation of a protective, non-toxic capsular antigen from Pasteurella multocida, types B and E.

Separation of the capsular antigen and endotoxin from saline extracts of Pasteurella multocida type B was achieved by fractional precipitation from aqueous solution by addition of polar organic solvents. Biological tests for the presence of endotoxin showed that it was absent from capsular antigen preparations so obtained. Properties of the capsular antigen suggested that it was a high molecular weight acidic polysaccharide. The solvent fractionation method was found to be equally applicable to separation of capsular antigen and endotoxin of P multocida type E. The type B capsular antigen in the presence of aluminium hydroxide gel adjuvant, was poorly immunogenic in rabbits. In cattle, however, a dose-dependent serological response was obtained as demonstrated by the mouse passive protection test.