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.     

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.     

Prolactin and colibacillus-induced fluid transport in ligated intestinal segments.

Two experiments, using the ligated intestinal segment technique, were conducted to determine whether the pituitary hormone prolactin (PRL) could reduce Escherichia coli-induced fluid loss into the small intestine of 2- to 3-week-old pigs. Inoculation of 10(6) to 10(8) enteropathogenic E coli organisms into ligated jejunal segments caused a significant accumulation of luminal fluid within 12 hours. In the first experiment, intraluminal inoculation with 0.5 mg of ovine PRL along with the bacteria did not have any effect on fluid accumulation. Systemic IV treatment of the animals with 1.0 mg of ovine PRL at 3-hour intervals, beginning either immediately after or 9 to 10 hours before intestinal ligation, did not significantly (P less than 0.05) reduce fluid accumulation as compared with control animals. In the second experiment, IM administration of 100 microgram of thyrotropin-releasing hormone (TRH) at 3-hour intervals, beginning 6 hours before intestinal ligation, significantly (P less than 0.05) increased circulating PRL concentrations, as measured by radioimmunoassay. However, TRH treatment did not reduce the accumulation of luminal fluid in E coli-inoculated segments.     

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.

     

Thermostable factor(s) in soya producing a net excess of secretion in the ligated gut test in pigs

The ligated gut test (LGT) is the standard method for the examination of Escherichia coli strains for enterotoxin production in pigs. As solid pig feed has been associated with diarrhea, soya products (the main protein source for piglets) were investigated with the same test as E. coli strains. After injection of different soya products into ligated segments of the small intestine fluid accumulation was observed, indicating a net excess of secretion. The factor in soya products responsible for this effect was found to be thermostable, as its effect was unaltered after heating at 120 degrees C during an hour. No indications of a possible allergic phenomenon accounting for the fluid accumulation were found. From the results of this study it is concluded that soyabean products can produce results in the LGT similar to those produced by enterotoxigenic E. coli strains.     

Thermostable factor(s) in soya producing a net excess of secretion in the ligated gut test in pigs

Summary The ligated gut test (LGT) is the standard method for the examination ofEscherichia coli strains for enterotoxin production in pigs. As solid pig feed has been associated with diarrhea, soya products (the main protein source for piglets) were investigated with the same test asE. coli strains.After injection of different soya products into ligated segments of the small intestine fluid accumulation was observed, indicating a net excess of secretion. The factor in soya products responsible for this effect was found to be thermostable, as its effect was unaltered after heating at 120°C during an hour. No indications of a possible allergic phenomenon accounting for the fluid accumulation were found.From the results of this study it is concluded that soyabean products can produce results in the LGT similar to those produced by enterotoxigenicE. coli strains.     

Studies of the pathogenesis of enteric E. coli infections in weaned pigs. The significance of the milk of the dam in preventing the disease.

Milk from sows whose progeny developed post weaning E. coli diarrhoea (PWD milk) and from sows which were immunized by intramuscular vaccination using a homologous strain of E. coli (immune milk) were tested in ligated segments of pig intestine. The results showed that PWD milk neutralized the enterotoxigenic, fluid accumulating capacity of the lysate of the disease-causing E. coli pathogen. A similar effect was seen by using immune milk (Table I). Neither PWD milk nor immune milk contained sufficient antibacterial activity to neutralize the fluid accumulating capacity of live cultures of E. coli O149:K91, while such activity was contained in immune serum. It is concluded that milk from sows whose progeny developed PWD contains antibodies capable of neutralizing the enterotoxigenic effects of the homologous E. coli organisms. It is suggested that the presence in milk from these sows of antibody-mediated activity against enteropathogenic E. coli organisms may be instrumental in preventing the disease in the progeny during the suckling period and consequently, when this protective milk supply stops at weaning, the disease may develop in susceptible animals, mainly because their own production of specific E. coli antibodies is insufficient to prevent PWD.     

The prevalence of enterotoxigenic Escherichia coli in the feces of calves with diarrhea.

A study was undertaken to determine the prevalence of enterotoxigenicity among Escherichia coli isolated from calves with diarrhea and from a control group of normal calves. The test organisms consisted of 200 E. coli recovered from scouring calves less than two weeks of age and 100 E. coli from normal calves. The enterotoxigenicity of the cultures was evaluated by three methods, namely, injection of ligated segments of piglet intestine, injection of ligated segments of calf intestine and oral inoculation of suckling mice. Live cutures of all the test organisms were used for the ligated intestine studies whereas sterile broth culture supernatants were used in the suckling mouse tests. Of the isolates from scouring calves, 36% were enterotoxigenic in the piglet intestine and 28% in the calf intestine. Amongst the isolates from normal calves, none was enterotoxigenic in the piglet intestine and one was enterotoxigenic in the calf test system. The ligated piglet intestine was considered unsuitable for determining the enterotoxigenicity of bovine E. coli, whereas the ligated calf intestine test was satisfactory and correlated completely with the suckling mouse test. The enterotoxigenic E. coli of bovine origin produced an enterotoxin that resembled the heat stable enterotoxin of typical porcine enteropathogenic E. coli.     

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.

     

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.     

Synergistic effects of Miconazole and Polymyxin B on microbial pathogens

The therapeutic value of antibiotics depends on the susceptibility of the infecting microorganism and the pharmacological profile of the drugs. To assess the value of an antibiotic combination of polymyxin B and miconazole this study examined the in vitro synergistic potential of the two drugs on Gram-negative and Gram-positive bacteria and yeast. Antifungal and antibacterial activity was tested by minimum inhibitory concentration (MIC) of broth macrodilution and urea broth microdilution, by fluorescence microscopy and flow cytometry. Synergism was calculated using the fractional inhibitory concentration index (FICi). With Staphylococcus intermedius as target we found up to an eightfold reduction of the individual MICs when both drugs were combined. However, the FICi was 0.63 suggesting no real interaction between the two drugs. With Escherichia coli, Pseudomonas aeruginosa, and Malassezia pachydermatis as targets the antimicrobial drug combination reduced the MICs of polymyxin B and miconazole from fourfold to hundredfold resulting in FICi between 0.06 and 0.5 which defines a synergistic action. Thus, if polymyxin B and miconazole are combined their effect is greater than the sum of the effects observed with polymyxin B and miconazole independently, revealing bactericidal and fungicidal synergism. Our results indicate a strong therapeutic value for the combination of these antimicrobial agents against Gram-negative bacteria and yeast and a weaker value against Gram positive bacteria for clinical situations where these pathogens are involved.     

Response of Gnotobiotic Pigs to Escherichia coli

In a study of the response of gnotobiotic pigs to coliform infections, 45 one-week-old germfree pigs were divided into five groups and each group was inoculated orally with a different strain of Escherichia coli. Three of these were enteropathogenic swine strains, P307[08:K87(B), K88 a,b (L):H19]; P570 [0138:K81]; P568[0141:K85a,b(B), K88a,b(L):H4], one was a virulent human strain, H224, [026:K60(B6)], and one was a non-enteropathogenic swine strain, P581[OX13:K68]. It was attempted to protect a portion of the pigs with orally administered specific antisera and sera from non-immunized specific pathogenfree (SPF) pigs. Observations were made on the clinical response, bacterial counts of feces and intestinal contents, gross pathological changes, distribution of the organisms in organs and serum hemagglutinin titers.

Infection with E. coli P307 resulted in diarrhea, dehydration and death, unless the pig was protected with specific antiserum. The pigs infected with E. coli P570 had a transient diarrhea but retained their appetites and recovered. Those infected with the other three strains remained healthy throughout. No circulating hemagglutinating antibody against the test strains of E. coli could be detected in any of the pigs seven days or earlier post-inoculation.

Relationship could not be established between the numbers of viable E. coli in the feces and the presence of clinical colibacillosis. Orally administered specific antiserum afforded protection against strain P307, but did not reduce the number of E. coli in the gut or alter their distribution in the internal organs. This suggested that the protective effect of specific antibody in the intestine was due to its action on a metabolite (enterotoxin) produced by E. coli P307 rather than the organism itself.

     

Virulence of wild and mutant strains of Salmonella typhimurium in ligated intestinal segments of calves, pigs, and rabbits

A ligated intestine model in calves, pigs, and rabbits was tested for its value as an indicator of virulence of potential vaccine strains of Salmonella typhimurium. A wild virulent strain (3860C), a laboratory strain LT2, and mutants of these 2 strains were evaluated. Inoculation of calf intestinal segments with strain 3860C revealed that fluid responses were greatest in the proximal portion of the small intestine and that doses greater than 10(7) organisms were required to produce fluid responses and mucosal damage. Immunoperoxidase-stained sections of intestine revealed that a large dose of Salmonella organisms was required before mucosal invasion could be detected. Aromatic (aroA), galactose epimerase (galE), and diaminopimelic acid (dap) mutants of strain 3860C all resulted in much less fluid response, mucosal invasion, and mucosal damage compared with those by the parent organism. Strain LT2 induced such weak responses that it was not possible to evaluate reductions in virulence of its mutants. In 6-week-old pigs, there was no fluid response to any strains; however, in 1-week-old pigs, there was fluid response to the wild strain and some of its mutants. In adult rabbits, fluid responses were not observed, except when the wild strain was inoculated in the proximal portion of the small intestine. The calf and 1-week-old pig models appeared to be best suited for assessment of virulence of mutant strains of S typhimurium.     

Concentrations of energy substrates in oviduct fluid in unilaterally ovariectomised pigs

Nichol et al (1992, Journal of Reproduction and Fertility, 96, 699–707) identified a pre- to post-ovulatory decrease (approx 1mM) in the amount of glucose in pig oviduct fluid. The present studies investigated whether the decrease was due to metabolism by embroyos and/or oviduct tissues, and also whether there was a local influence of the ovary on the oviduct fluid content of energy substrates. Unilaterally ovariectomised pigs were used, in which, through compensation, oviducts that contained twice the normal number of embryos could be compared with oviducts which contained no embryos. Following unilateral ovariectomy and after two oestrous cycles of normal duration, surgery was performed 88 hours after the beginning of standing heat to obtain oviduct fluid samples, just before embryonic entry into the uterus. Luminal fluid samples from the ampulla and ampullary-isthmic junction from oviducts with and without an adjacent ovary were assayed for glucose, pyruvate and lactate concentrations. No significant differences were found between the glucose, pyruvate and lactate concentrations in fluids from the ampulla or ampullaryisthmic junction from oviducts containing embryos compared with absence of embryos (P> 0·05). Therefore, the post-ovulatory decrease was not due to the presence of embryos or to a local effect of the ipsilateral ovary. Consequently, pig oviduct fluid concentrations of glucose, lactate and pyruvate are seemingly regulated by systemic mechanisms.     

Evaluation of various methods for the detection of enteropathogenic Escherichia coli in diarrheic calves.

Evaluation of the Escherichia coli population in the small intestine of diarrheic calves was performed by estimating the number of colony-forming units and the observation of direct gram-stained smears of mucosal scrapings. Enterotoxigenicity of the isolates was determined by the infant mouse test and the ligated intestinal segment in calves. The influence of various broth culture media on the production of enterotoxin was also studied. Serologic characterization of the E coli isolates was performed. A total of 190 cases of diarrhea in bovine neonates was studied and it was found that enteropathogenic E coi was not responsible for more than 20% of the cases. A practical approach for the recognition of enteropathogenic E coli in a routine diagnostic laboratory is proposed.     

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.

     

The role of K antigens of enteropathogenic Escherichia coli in colonization of the small intestine of calves.

The colonizing and proliferating abilities of enterotoxigenic acapsular or K99- mutants of bovine enteropathogenic Escherichia coli strains were compared with those of their capsulated and K99+ parent strains in the small intestine of infected colostrum-fed calves. Calves infected with the enteropathogenic E. coli parent strains developed profuse diarrhea and severe dehydration. None of the calves which received the acapsular mutant developed diarrhea and one of three calves inoculated with the K99- enterotoxigenic mutant developed moderate diarrhea. The parent enteropathogenic E. coli strains colonized the middle and lower small intestine; in these areas a layer of specific immunofluorescence against the enteropathogenic E. coli covered most villi and 80% of the organisms were associated with the intestinal wall. The acapsular mutant strain failed to colonize the small intestine and fluorescent bacteria were not observed in any area of the small intestine. The K99- mutant proliferated to a lesser extent than did the K99+ parent strain in all areas of the small intestine but moderately colonized the lower small intestine where fluorescent bacteria were observed to cover parts of the intestinal villi.     

Minimal Inhibitory Concentrations of Twenty Antimicrobial Agents to Animal Pathogens

Minimal inhibitory concentration (M.I.C.) values as determined by an agar-plate-dilution method for 60 bacterial isolates, consisting of Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus of animal origin against 20 antimicrobial drugs are presented. Of all the drugs, gentamicin had the best in vitro antibacterial activity in terms of M.I.C. when considering all the species of organisms together, while spectinomycin had the least activity.

An inoculum replicator was a convenient tool in carrying out the agar-plate-dilution method.

A comparison of the M.I.C. values of 42 isolates of S. typhimurium with the results obtained by low level method and the Bauer-Kir-by method showed that with few exceptions, there is a general agreement.

     

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.     

The effects of intestinal Escherichia coli 263, intravenous infusion of Escherichia coli 263 culture filtrate and iron dextran supplementation on iron metabolism in the young pig

An experiment using 32 pigs in a 2(3) factorial arrangement of treatments was used to determine the effects on the (1) level of iron dextran supplementation, (2) iv infusion of an Escherichia coli 263 culture filtrate and (3) presence of E. coli 263 in a ligated intestinal segment, on the ability of the young pig to limit systemic Fe availability. Iron dextran was administered im 3 d postpartum. Culture filtrate was infused iv, E. coli were injected into ligated intestines and blood sampling was started at 14 d postpartum. Blood was taken every 2 h for 22 h, after which pigs were euthanized and livers, spleens and kidneys were removed. Pigs receiving 400 mg of iron dextran (HiFe) exhibited greater serum Fe (SFe) and lower total Fe-binding capacity (TIBC) than pigs injected with 100 mg Fe (LoFe). The effects of the E. coli culture filtrate infusion appeared to be associated with endotoxin-induced circulatory shock. The presence of E. coli in the intestine increased TIBC in LoFe pigs, but not in HiFe pigs. The increase in TIBC coincided with the time of maximal fluid secretion into the intestine. Intestinal E. coli also caused an increase in liver Fe content, particularly in HiFe pigs. These data suggest that intestinal E. coli can cause a shift of Fe from the plasma to the reticuloendothelial system, and pigs receiving high supplemental dosages of Fe are less able to limit the availability of Fe to microorganisms.