Polyerositis and Arthritis Due to Escherichia coli in Gnotobiotic Pigs

Forty gnotobiotic pigs from six litters were exposed orally to Escherichia coli 083:K·:NM at 69 to 148 hours of age, while 17 pigs from the same litters served as unexposed controls. Clinical signs of infection included fever, anorexia, diarrhea, lameness, and reluctance to move.

Eighty-four percent of the exposed pigs in four litters died, while only 13% in two litters died. Gross and microscopic lesions included serofibrinous to fibrinopurulent polyserositis in 96% of the exposed pigs in four litters and 33% of the exposed pigs in two litters. A few pigs had gross and/or microscopic lesions of arthritis. Escherichia coli was routinely isolated from the serous and synovial cavities of infected pigs.

Anti-hog cholera serum administered orally as a colostrum substitute gave partial protection against E. coli infection.

     

Studies of Escherichia Coli in Gnotobiotic Pigs: II. The Immune Response*

The immune response of orally infected and parenterally vaccinated (E. COLI 08:K.:H21) gnotobiotic pigs was studied by bactericidal and indirect hemagglutination test methods. The bacterial agglutination test proved to be of no value in this study.

The results demonstrated that antibody was detectable within 8 days following oral infection, but the titers remained very low until after the pigs were vaccinated intravenously. The titers of the sera were markedly increased following intravenous vaccination.

There was no detectable absorption of antibody from the gut of gnotobiotic pigs fed immune serum at 4 to 6 days of age and no detectable difference between the immune response of these pigs and those not fed serum.

     

Studies of Escherichia coli in Gnotobiotic Pigs: I. Experimental Reproduction of Colibacillosis*

The successful experimental production of colibacillosis in young gnotobiotic pigs by the oral administration of E. COLI (08:K.:H21 was accomplished. Colibacillosis was not produced in gnotobiotic pigs infected with either of the 2 other strains of E.COLI employed.

Necropsy observations were presented and discussed. Emphasis was placed on the absence of grossly visible enteritis in pigs that died in the acute phase of the disease. The gross appearance of the intestinal tract changed some hours after death to resemble that associated with enteritis.

Bacteremia was detected in the gnotobiotic pigs orally infected with each of the 3 strains of E. COLI, and the special characteristics of the colostrum-deprived pig were stressed as important factors in these findings.

     

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 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.

     

Studies of Escherichia coli in Gnotobiotic Pigs: III. Evaluation of Orally Administered Specific Antisera*

The protective effect of orally administered immune (E. COLI 08:K.:H21) serum was demonstrated in experimentally infected gnotobiotic pigs. The temporary protective effect of the immune serum and the correlation of protection with IN VITRO antibody determinations were discussed. The results indicated that the protective action of the immune serum is apparently not dependent upon the complement-antibody bactericidal system. It was suggested that the most plausible mechanism of action of the immune serum was the inactivation of endotoxin in the lumen of the intestine.     

Experimental Atrophic Rhinitis in Gnotobiotic Pigs

Twenty-nine caesarian derived colostrum deprived germfree pigs were reared in isolators in groups of three to four per isolator. At seven days of age each group was inoculated intranasally with one of four strains of Bordetella bronchiseptica (designated B, J, L and 55B), or Pseudomonas aeruginosa or a mucoid strain of Escherichia coli, all previously isolated from nasal mucus of pigs affected with clinical atrophic rhinitis. Another group was inoculated simultaneously with B. bronchiseptica B and Pasteurella multocida. The animals were observed for clinical signs of atrophic rhinitis and monitored bacteriologically at weekly intervals for seven weeks. Then they were bled for serology and killed and their respiratory organs examined for gross and histopathological lesions.

All of the pigs inoculated with the Bordetellae had inflammation of the nasal mucosa and developed positive serum antibody titers against all four of the Bordetella strains used in this study. Strain J caused sneezing and turbinate atrophy in three of four pigs. One of the three pigs inoculated with strain L died in ten days from bronchopneumonia and pericarditis and had turbinate atrophy. Strains B and B55 caused no turbinate atrophy, but two out of three pigs inoculated with both B. bronchiseptica B and P. multocida had turbinate atrophy. No nasal lesions were observed in the pigs inoculated with E. coli or P. aeruginosa or in the noninoculated germfree controls.

The results indicate a variation in the ability of different strains of B. bronchiseptica to cause turbinate atrophy in pigs and demonstrate that nasal infections by these organisms stimulate serum antibody response. Presence of P. multocida appears to increase the severity of the lesions. As the E. coli and Pseudomonas failed to produce atrophic rhinitis, they are probably of no significance as primary etiological agents in the atrophic rhinitis syndrome in swine.

     

Duration of Mycoplasma hyopneumoniae Infection in Gnotobiotic Pigs

Sixteen gnotobiotic pigs raised in flexible plastic isolators (four pigs per isolator) were inoculated with a culture of Mycoplasma hyopneumoniae. One pig was killed and underwent necropsy at weekly intervals for the following 16 weeks. Macroscopic lesions were observed in the lungs of 13 of 16 pigs and microscopic lesions were found in 14 of 16 pigs. Mycoplasma hyopneumoniae was cultured from the trachea or lungs from 10 of the 16 pigs. Scanning electron microscope studies showed areas of damage to the cilia, collections, of leucocytes and mucus, and mycoplasma in the trachea as well as the bronchi. These conditions were found in all the pigs seen at necropsy from nine to 16 weeks postinoculation and there was no evidence of noticeable regression or recovery during this 16 week period.     

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.

     

Experimental Colibacillosis in Gnotobiotic Baby Pigs II. Pathology

Sixty-two neonatal gnotobiotic pigs were used in three experiments to determine the lesions produced by two closely related strains of Escherichia coli O138:K81:NM (of Michigan origin) and O138:K81 (of Minnesota origin). Exposure was by subcutaneous injection of bacterial culture into the umbilical stump or by oral inoculation.

Gross signs common to monocontaminated pigs included distention of the flaccid small and large intestines with fluid contents. Edema was prominent in various tissues of most pigs exposed via the umbilical stump but not in those exposed orally.

Histological lesions were predominantly in the gastrointestinal tract and were variable. At one extreme acute hemorrhagic enteritis was present in two pigs, while at the other extreme in a few pigs it was difficult to distinguish tissues of infected pigs from those of noninfected germfree pigs. Significant histological lesions common to monocontaminates included mild inflammatory reaction, hydropic degeneration of the intestinal epithelium, evidence of interference with normal function of the villus-draining mechanisms, and vascular changes generally indicated by edema.

The findings suggest that interference with normal absorption of nutrients plays at least some role in the pathogenesis of colibacillosis in young gnotobiotic pigs.

     

Immunity to Escherichia coli in Pigs. The Role of Milk in Protective Immunity to E. coli Enteritis

Milk whey from immunized sows increased the survival time of gnotobiotic piglets infected with Escherichia coli. The survival time of infected piglets fed milk whey from non-vaccinated sow's was the same as that of similar pigs fed condensed cow milk.

The significance of milk antibodies in immune protection against E. coli enteritis is discussed and compared with that of absorbed colostral antibodies. From calculations presented it would appear that seven day old piglets receive approximately 1 g of gamma globulin daily from milk and that this is equal to the piglets total serum gamma globulin content. After seven days of age the gamma globulin content of piglet serum falls, whereas that of milk remains constant; milk is, therefore, potentially a major source of immunoglobulins with protective activity against E. coli associated enteritides.

     

Experimental Colibacillosis in Gnotobiotic Baby Pigs I. Microbiological and Clinical Aspects

Sixty-two gnotobiotic pigs were used in three experiments to determine the means whereby two related strains of Escherichia coli colonized the intestinal tract. Pigs were exposed by a method simulating neonatal contamination of the umbilical stump. Bacteremia was produced within one and one half hours, and by 24 hours the infection was generally well established in the gastrointestinal tract. By 48 hours after exposure, the bacteremia had subsided so that only an occasional isolation from organs other than the gastrointestinal tract was made.

Oral exposure of one litter of germfree pigs produced heavy colonization of the entire gastrointestinal tract within four hours. Evidence of intermittent bacteremia was present in pigs of this litter. Diarrhea appeared earlier when pigs were exposed orally than when they were exposed by way of the umbilical stump.

     

Effect of Bacteriophage in Enterotoxigenic Escherichia coli (ETEC) Infected Pigs

We evaluated effect of enterotoxigenic Escherichia coli (ETEC) specific lytic phage CJ12 in ETEC infected pigs. Phage was mixed with feed at a ratio of 1:1,000 (0.1%). One week after initially providing phage mixed feed, pigs were challenged orally with 10(11) CFU of ETEC and body weight, diarrhea score, bacterial CFU and phage PFU in the feces were measured. Pigs of phage treated groups C (10(6) PFU/g) and D (10(8) PFU/g) showed more resistance to diarrhea due to ETEC infection compared to positive control group B on the third day after the initial challenge. Moreover, during the quantitation of ETEC in feces, both groups C and D showed approximately 63.92 and 60.73% reduced ETEC compared to positive control group B. Phages were successfully isolated from feces in both groups C and D during the experiment without any adverse effects, suggesting the possibility of using CJ12 as a feed additive.