Enteritis of Early Weaned Pigs : II. Pathogenesis

Strains of hemolytic E. coli are implicated in edema disease and enteritis of swine. Immunological experiments were conducted to determine the specific role played by hemolytic E. coli in the etiology of these diseases. When cell-free extracts prepared from a frequently isolated E. coli — 0139:K82(B) were injected 48 hours apart into a healthy pig, symptoms of edema disease were produced on both occasions. Similar symptoms were produced when this extract was injected into a colostrum-deprived pig raised in isolation.

The Schultz-Dale reaction revealed no difference between the contractions of the ilea of sensitized and non-sensitized guinea-pigs. In vitro treatment of a single non-sensitized guinea-pig uterus with extracts of five different strains of hemolytic E. coli produced sharp contractions in every trial. A similar treatment with extracts of four non-hemolytic E. coli strains also stimulated the non-sensitized guinea-pig uterus but the magnitude of the contractions was much less. These studies indicated that the cell-free extracts of hemolytic E. coli produced a marked nonspecific toxic reaction.

     

Serological Identification of Escherichia coli Isolated From Cats and Dogs

In a group of 112 cats, examined during an outbreak of diarrhea, 104 (92.8%) yielded Escherichia coli with identifiable O groups as compared to 12 (21.8%) of 55 control cats apparently free of intestinal ailments. E. coli group O6 occurred in 49.1% of the cats with diarrhea, and persisted in this group of cats at about the same rate when examined for a period of ten months. E. coli group O6 was not isolated from the control group.

Specimens were also collected from dogs with various clinical signs. E. coli O4 and O6 strains appear to be important potential pathogens for canine species and were found to spread from one dog to another when in close contact. E. coli group O4 was identified in ten and O6 in three of 14 dogs examined. All isolates in which the O group could be identified were hemolytic.

     

Limitations of the Infant Mouse Test for Escherichia coli Heat Stable Enterotoxin

A study was undertaken to evaluate the response of different test systems to preparations of heat-stable enterotoxin (ST) derived from Eschericihia coli strains recovered from diarrheal disease of humans, pigs and calves. Sterile broth culture supernatants of enterotoxigenic strains of E. coli were heated at 65°C for 30 minutes and tested for the presence of heat-stable enterotoxin. Three test systems, namely, ligated intestine of weaned pigs, ligated intestine of rabbits and the infant mouse test were used in attempts to detect ST in the culture supernatants. Two patterns of reaction were observed in response to ST-containing preparations: either the preparation elicited a response in the three tests or the preparation elicited a reaction only in the ligated pig intestine. A response in all three tests were observed for 5/5 human ST-producing E. coli, 5/5 bovine enterotoxigenic E. coli, 5/5 “atypical” porcine enterotoxigenic E. coli, 3/3 St⁺LT^- porcine E. coli of serogroup O138:K81 and 4/24 LT⁺ST⁺ porcine E. coli. A response only in the ligated pig intestine was obtained with 5/5 ST⁺LT^- porcine E. coli belonging to serogroups other than O138:K81 and to 20/24 ST⁺LT⁺ E. coli from pigs. The results are consistent with the view that there are two kinds of ST, one of which (ST1) reacts in all three tests and the other (ST2) which reacts only in the ligated pig intestine. The findings underscore the limitations of the infant mouse test as a means of detecting ST in porcine isolates of E. coli, since the test fails to detect ST produced by a large number of these E. coli strains. There appeared to be a relationship between kind(s) of ST produced and the animal species from which the producing organism was recovered.     

Immunity to Escherichia coli in Pigs: Anti-enterotoxins in Colostrum and Serum from Vaccinated and Non-vaccinated Sows

Colostrum from non-vaccinated sows did not contain naturally occurring antibodies to heat labile Escherichia coli enterotoxin. Vaccination of sows by either the intramuscular or intramammary routes with a live formalinized E. coli vaccine resulted in the production of colostrum capable of neutralizing the heat labile toxin. Intramammary vaccination resulted in the production of colostrum which significantly reduced the enterotoxigenic effects of the vaccine strain of E. coli organisms but not that of a heterologous strain.

Vaccination of the sows resulted in the production of serum antibodies to heat labile enterotoxin. Antibodies to heat stable enterotoxin were not demonstrable in the colostrum of either non-vaccinated or vaccinated sows.

     

Distribution of Porcine Fecal Coliflora Throughout a Barn

Three hundred and seventy strains of fecal Escherichia coli were isolated from pigs in one barn and 475, 539 and 490 strains were isolated at each of three successive samplings in another barn. The majority of the E. coli isolates obtained at any one sampling belonged to a small number of E. coli types. Three repeated samplings in one barn indicated that the dominant E. coli types harboured by pigs in this barn were constantly changing. The results also suggested that, within a particular barn, a successive batch of pigs could experience the same sequence of E. coli types. Colicin production appeared to be associated with dominant strains and the proportion of colicin producers in different investigations ranged from 36 to 68%.     

Studies of Escherichia Coli in Gnotobiotic Pigs: V. Evaluation of the Effects of Orål and Parenteral Administration of Immune Serum

The effects of orally and/or parenterally administered immune serums were evaluated in 36 gnotobiotic pigs infected with an enteropathogenic strain of Escherichia coli. Pigs were euthanatized at predetermined time intervals between 6 and 48 hours prostinfection. The results were evaluated on the basis of: 1) clinical observations, 2) necropsy observations, 3) counts of viable E. coli in segments of the small intestine, 4) attempts to isolate E. coli from the heart, liver, and bile, 5) microscopic examination of fixed intestinal sections to determine the location of E. coli and morphologic evidence of the host response, and 6) determination of the pH of the various portions portions of the gastrointestinal tract.     

Hemotological and Febrile Responses of Rabbits to Listerial Hemolysins

The effects of an infusion of partially purified hemolysin (9-125) from Listeria monocytogenes were studied in intact rebbits. A similar preparation from trypticase soy broth (TSB) was employed for control purposes. Alterations of several blood constituents and febrile responses resulting from both of these products were noted. After administration of hemolysin into the blood stream, profound leukopenia, marked reduction of platelets, and changes in the blood constituents were recorded. The data comparing the effect of both hemolysin and TSB are presented.

Both groups exibited pyrogenic responses. The test animals had elevated temperatures for up to 24 hours while the control group had temperature almost in normal range within eight hours of injection. Pyrogenic responses and changes in the constituents of peripheral blood suggest that the hemolysins possess cytotoxic activity and thus may play a role in the pathogenesis of Listeria monocytogenes in the disease process.

     

Rapid purification of a 110-kilodalton hemolysin of Actinobacillus pleuropneumoniae by monoclonal antibody-affinity chromatography.

An efficient, single-step method for purification of the 110-kilodalton (kDa) hemolysin of Actinobacillus pleuropneumoniae was developed. An immunoaffinity column was made by cross-linking murine monoclonal antibody 8C2 to the 110-kDa hemolysin of A pleuropneumoniae strain J45 serotype 5 to protein A-agarose beads. Purified hemolysin with high hemolytic activity was obtained after washing the column with phosphate-buffered saline solution, and eluting the hemolysin with 50 mM diethylamine, pH 11.0. The same column was also used to purify the hemolysin from A pleuropneumoniae strain 4074 serotype 1. The purification procedure could be completed within 5 hours, and almost 50% of the total hemolytic activity and hemolysin protein was recovered in pure form.     

Defences of the bovine mammary gland against infection and prospects for their enhancement

The equine alternative complement pathway has been partially characterized and compared to the equine classical activation pathway. A dose-dependent lysis of RbRBC was observed with peak lytic values noted within 10 minutes at 37°C when rabbit red blood cells (RbRBC) were used as an alternative pathway activator. Sheep red blood cells (SRBC) sensitized with rabbit hemolysin or partially purified equine IgM antibodies were equally sensitive to lysis. Dilution of the commercial hemolysin by $\text{1}\text{5}$ reduced lysis from 90% to 38% in the presence of constant cell numbers. Hemolysis of SRBC peaked at 10 minutes and the majority of lysis occurred within 10 minutes. Dilution of equine sera by as little as $\text{1}\text{5}$ decreased hemolytic activity for SRBC to 21.5% from greater than 90% with undiluted sera. The alternative pathway protein, equine factor B, was tested using RbRBC and monitored by its differential susceptibility to heat treatment at 50°C. This treatment led to almost complete inactivation after a 15-minute incubation. An apparent heat-dependent decay of certain classical pathway components was also observed after 50°C treatment. This sensitivity was indicated by a reduction in the lytic activity for sensitized SRBC. Treatment for 15 minutes at 56°C with either RbRBC or SRBC was sufficient to abolish hemolytic activity in all equine sera tested. Chelation of cations with 0.04 M EDTA blocked expression of alternative and classical pathway activation; however, chelation of Ca⁺⁺ ions with 10 mM EGTA containing 1 mM Mg⁺⁺ ions permitted lysis of the RbRBC but not the SRBC. A dose-related Mg⁺⁺-ion dependence for RbRBC hemolytic activity was observed as the concentration of Mg⁺⁺ was increased to 1.0 mM. In addition, our results obtained with pre-colostral foal serum strongly suggest that natural antibody to RbRBC was of little importance in the lysis observed with these cells. These results also show that the equine alternative pathway activation may require Ca⁺⁺ ions. If Ca⁺⁺ ions are required, the equine alternative pathway is quite different from any other mammalian complement system so far described. Our results suggest that the alternative pathway of activation is of major importance in the equine complement system. Confirmation of this hypothesis requires both purification of the components involved as well as further characterization.     

Studies of Escherichia Coli in Gnotobiotic Pigs: IV. Comparison of Enteropathogenic and Nonenteropathogenic Strains

Four gnotobiotic pigs were infected with an enteropathogenic strain of Escherichia coli, and 4 were infected with a nonenteropathogenic strain of E. coli. Pigs killed in pairs at 6, 12, 24, and 48 hours PI. Four pigs were maintained as germfree controls. The discussions were based on the results of 1) clinical observations, 2) necropsy observations, 3) counts of viable E. coli in segments of the small intestine, 4) attempts to isolate E. coli from the heart, liver, and bile, 5) microscopic examination of fixed intestinal sections to determine the location of E. coli and morphologic evidence of the host response, and (6) determinations of the pH of the contents of the various portions of the gastrointestinal tract.

No diarrhea, fluid accumulation, or impairment of the digestive capacity were noted in the pigs infected with the nonenteropathogenic strain of E. coli. The number of viable E. coli detected in the respective segments of the homogenized small intestine was similar in pigs infected with either strain.

Diarrhea occurred continuously starting 18 hours PI in the pigs infected with the enteropathogenic strain and killed 24 or 48 hours PI. The pH of the contents of the cecum and colon became markedly more alkaline simultaneously with the increase in the heterogeneity and fluid content of the cecum and colon and thus appeared to correlate well with the onset of the clinical diarrhea. No enteritis was detected grossly or microscopically.

The characteristics that determine the enteropathogenicity of a strain of E. coli could not be defined from the results, but it was noted that the host response appeared to be quite similar to that of infant rabbits experimentally infected with Vibrio cholera.

     

A Comparative Study of the Rabbit and Pig Gut Loop Systems for the Assay of Escherichia coli Enterotoxin

A comparison was made between segments of pig and rabbit small intestine in their response to heat-labile (LT) and heat-stable (ST) preparations from porcine enteropathogenic Escherichia coli. Either whole cell lysates or dialysed broth culture supernatants were used as sources of LT and soft agar culture fluids as a source of ST. Whole cell lysates of all thirteen LT-producing E. coli strains tested regularly elicited fluid accumulation in rabbit gut loops. Whole cell lysates of certain E. coli strains considered to be nonenteropathogenic in pigs could also elicit a positive response in rabbit gut loops. When graded doses of LT were tested in pig and rabbit gut loops, the rabbit was more sensitive and is therefore considered preferable to the pig for quantitation of LT. In the rabbit, upper (jejunal) and lower (ileal) small intestine were compared for their response to LT and it was found that ileal loops were twice as sensitive but more prone to false positive reactions. When soft agar culture fluids of several enteropathogenic E. coli strains were tested in the rabbit, the response was inconsistent, and it was concluded that the rabbit is unsuitable for the assay of the heat-stable enterotoxin.     

The Significance of Proliferation and Enterotoxin Production by Escherichia coli in the Intestine of Gnotobiotic Pigs

The significance of enterotoxin production and proliferative ability of Escherichia coli in the intestinal tract as related to porcine enteric colibacillosis was studied in 68 gnotobiotic pigs.

The animals were monocontaminated at seven to ten days of age with eight selected strains of E. coli. The strains were two naturally occurring porcine enteropathogens — P155 (0149:K91;K88a,c:H10) and P307 (08:K87;K88a,b:H19), two nonenteropathogenic strains — P104 (0139:K82:H1) and F11 (018-ab:K?:H14), and four enterotoxigenic derivatives of the above strains — P104(P155), P104(P307), F11(P155) and F11(P307). The response of the animals was evaluated on the basis of clinical observations and necropsy lesions 22 hours after exposure to the organisms. E.coli counts were determined at seven different levels of the intestinal tract. Cell free extracts of the intestinal contents were examined for enterotoxic activity by the ligated pig intestine loop test.

All of the strains possessing the enterotoxin plasmid produced enterotoxin in the pig's intestine and were capable of causing diarrhea. The nonenteropathogenic E. coli failed to do so. The strains possessing the P155 enterotoxin plasmid were more virulent than the corresponding derivatives with the P307 enterotoxin plasmid. Strains P155, P307 and P104(P155) proliferated in the upper small intestine at a greater rate and were more virulent than the other strains. The numbers attained in the upper small intestine by the other enterotoxigenic derivatives were comparable to those of their nonenteropathogenic parent strains.

It was considered that enterotoxin produced by E. coli was the essential factor for causing a diarrheic response in gnotobiotic pigs. The virulence of each of the tested strains appeared to be governed by the degree of enterotoxicity associated with a particular enterotoxin plasmid, the numbers attained by these organisms in the upper small intestine, (but not in the lower small intestine or in the colon), and by other undetermined factors.

     

Preliminary Observations on the Isolation of Escherichia coli Bacteriophages from the Intestinal Contents of Chickens

Eschericia coli bacteriophages were isolated from the intestines of chickens. These phages had different lytic patterns, and were propagated in nutrient broth containing 0.4 gm calcium chloride/litre. The agar layer technique was used to determine the routine test dilution (RTD) and plaque morphology. The phages differed in their 1) morphology, 2) RTD values, and their ability to lyse E. coli strains from various animals. All phages isolated lysed human K12 E. coli strains, whereas only two phages lysed the chicken E. coli strain. Phages isolated lysed E. coli from chicken, bovine, ovine, equine, and human, but not from porcine, canine and other avian species.     

Studies of Escherichia coli in Gnotobiotic Pigs VI. Effects of Feeding Bacteria-Free Filtrates of Broth Cultures

Nine gnotobiotic pigs derived from one gilt were fed bacteria-free filtrates prepared from: 1) cultures of an enteropathogenic strain of Escherichia coli 09:K·:NM (Strain 340), 2) cultures of a nonenteropathogenic strain of E. coli 08.K·.H16 (Strain CDC-1466-56), and 3) uninoculated culture medium.

Diarrhea was observed initially two to four hours after feeding the filtrate prepared from the enteropathogenic E. coli. The duration of diarrhea was five to ten hours. No diarrhea was observed after feeding filtrate prepared from uninoculated medium or cultures of nonenteropathogenic E. coli.

The pH values of the feces increased with the onset of diarrhea and decreased to normal after diarrhea stopped.

No histopathological lesions were found.

     

Comparative Studies on Corynebacter1Um Pyogenes Toxin Formation in Monocultures and Mixed Cultures: The Demonstration of a Stimulating Effect of Peptococcus Indolicus and Stuart-Schwan Cocci

The growth and the toxin (i.e. hemolysin) producing capacity of Corynebacterium pyogenes were studied in monocultures and in co-cultures with 1 or more of the organisms frequently accompanying it in summer mastitis in cattle (Peptococcus indolicus, Stuart-Schwan cocci, Bacteroides melaninogenicus subsp. levii, Fusobacterium necrophorum and Streptococcus dysgalactiae) or with organisms seldom associated with summer mastitis (Streptococcus uberis, Streptococcus agalactiae, non-toxic staphylococci and Escherichia coli).Pc. indolicus, and to some extent also Stuart-Schwan cocci, stimulated the growth as well as the hemolysin producing capacity of Gb. pyogenes (Table 1) while Str. dysgalactiae, Str. uberis, Str. agalactiae, E. coli and the majority of the staphylococci reduced these activities. Most F. necrophorum strains stimulated the growth, but not the hemolytic activity. With B. melaninogenicus the results were inconclusive.The effect of Pc. indolicus appeared to be associated with the production of a filterable factor (Tables 2 and 3).Mouse toxicity and hemolytic activity of culture filtrates were closely correlated (Table 4).     

Pathogenicity of Vibrio Coli for Swine II. Experimental Infection of Conventional Pigs with Vibrio Coli

The pathogenicity of V. coli for conventional swine was studied by inoculating pigs with cultures of V. coli and V. coli infected gut of gnotobiotic pigs. Thus, six conventional pigs were inoculated with strains of V. coli freshly isolated from infected gnotobiotic pigs. The cultures were grown in simulated sows milk, and added to the feed. Two other groups, of three pigs each, were infected by administration of minced intestine from two gnotobiotic pigs, heavily infected with the organism. Vibrio was isolated from all pigs, including five of the six controls, but larger numbers were isolated from the inoculated groups, especially from those fed macerated gut. Clinical signs of disease were not observed.     

Peptococcus (S. Micrococcus) indolicus. The demonstration of two varieties of hemolysin forming strains.

On the basis of biochemical and serological criteria 2 hemolysin forming varieties of peptococci were identified as Peptococcus indolicus.Of a total of 16 hemolytic strains examined 9 originated from the vagina of clinically healthy cows, 4 from mastitis secretions from dry cows, 2 from the interdigital skin of clinically healthy sows, and 1 from a subcutaneous abscess in a pig.Two strains were designated a-hemolytic and 14 β-hemolytic.On blood agar plates colonies of the α-hemolytic variety were surrounded by narrow zones of almost complete hemolysis, while colonies of the β-hemolytic variety were surrounded by broad zones of incomplete hemolysis. The hemolysins were termed α- and β-hemolysin, respectively.The β-hemolysin, but not the α-hemolysin, could be demonstrated in cultures grown in liquid media. The β-hemolysin was found to be filtrable, relatively thermoresistant, and non-dermonecrotic.By gel diffusion analyses the 2 α-hemolytic strains were referred to Serotype C. Ten of the β-hemolytic strains belonged to Serotype C, 2 to Type B, 1 to Type D, and 1 to Type E.     

Studies on the Serological and Immunological Response of Chickens to Endotoxin and Endotoxoid

Chicks two, three and four weeks of age respond well serologically to endotoxin given intravenously, with the older chicks giving a better response. The induced immunity against live organism challenge produced by homologous endotoxin from Escherichia coli or Salmonella typhimurium is specific between these two organisms, however, cross-immunity occurs between E. coli 045 and E. coli 0109 and between Salmonella enteritidis and S. typhimcrium and further, endotoxins from different organisms may be combined to provide immunity against the homologous organisms. Endotoxoid administered to chicks rendered them more susceptible to live organism challenge for five days, following which some immunity developed.     

Hemolytic interactions of Dermatophilus congolensis.

The strains of Dermatophilus congolensis grew on blood agar with washed sheep erythrocytes with marked total hemolysis. In testing for hemolytic interactions they gave a significant synergistic effect of a characteristic shape with Rhodococcus equi and Streptococcus agalactiae, whereas with Staphylococcus aureus producing beta hemolysin and with Staphylococcus aureus producing delta hemolysin a simultaneous synergistic as well as antagonistic effect were observed. First of all a conspicuous inhibition of in the beta hemolysin zone began and then the hemolytic effect of D. congolensis was enhanced. A similar double reaction was also observed with Listeria ivanovii. With delta hemolysin there was an inhibition of the hemolytic effect of D. congolensis and at the same time a synergistic effect could be observed. Also D. congolensis gave a weak synergistic effect with Micrococcus lylae and Listeria monocytogenes, and a further weak antagonistic effect with alpha hemolysin of Staphylococcus aureus, Staphylococcus hyicus, Staphylococcus chromogenes and Micrococcus luteus. No interaction of D. congolensis was established with Corynebacterium pseudotuberculosis.