An Attempt to Experimentally Produce Edema Disease in Swine by Oral Administration of Escherichia Coli Serotype 0139:K82:H1

Diarrhea, reduced appetite, and nervousness were observed following oral exposure of six pigs to freeze-thaw extract and living culture of E. coli strain 0139:K82:H1.

The most significance gross change was the appearance of subserosal edema of the mesentry, especially of the mesocolon.

Histological findings included perivascular edema of the brain and reticulum cells as well as lymphocytic aggregates in the renal cortex simulating early neoplasia. Edema of the submucosa of gallbladder, the duodenum, the spiral colon and the fundic stomach as well as the lymph nodes was also observed.

The syndrome which was produced resembled, in many aspects, edema disease although some complicating factors were ob-

     

The Pathophysiological Effects of Moraxella bovis Toxins on Cattle, Mice and Guinea Pigs

In three experiments, cattle, mice and guinea pigs were inoculated with viable cultures of Moraxella bovis or fractions of this organism. Fractions were obtained by disruption of cells with a fractionator at 20,000 pounds per square inch, and separating the cell wall and cell sap fractions by differential centrifugation. Cell sap fractions were further separated by ultra-centrifugation, heating and precipitation with (NH₄)₂ SO₄. Different fractions induced different pathophysiological manifestations. The cell wall fractions caused localized lesions (necrosis) at the site of injection, and emphysema and congestion of the lungs. Cell sap fractions induced a “shock syndrome,” as well as hemorrhage and inflammation of the intestines, hemorrhage and congestion of lymph nodes, liver, adrenal and spleen. Cell sap also induced conjunctivitis in mice and guinea pigs, and periocular edema, myosis, ocular pruritus and lacrimation in cattle.

The authors suggest that M. bovis probably produces endotoxins and exotoxins as well as possibly a specific oculopathic substance, but more definitive work is needed to confirm this. They caution that consideration of these toxins should be made in any application of M. bovis for vaccines or other immunological studies.

     

Glasser's Disease of Swine Produced by the Intratracheal Inoculation of Haemophilus suis

The intracheal inoculation of pigs with Haemophilus suis led to the production of Glasser's disease at every attempt without significant pulmonary involvement. Isolation of this organism from the experimental animals was possible only in the acute phase of the disease.

The indirect fluorescent antibody technique when applied to frozen sections of tissues obtained from the experimentally infected pigs at autopsy, revealed a few rod forms but mostly “round bodies” of H. suis in animals from which the organism was isolated, and “round bodies” only in the pigs from which the organism was not isolated.

Attention is drawn to the similarities between the lesions caused by H. suis and Mycoplasma hyorhinis, and to the confusion which may result therefrom. It is stressed that the laboratory diagnosis of these two diseases is complicated by the fact that both agents may not be isolated on the media commonly used in diagnostic laboratories. Both organisms necessitate the use of special media where the clinical and autopsy results indicate polyserositis and arthritis.

     

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.

     

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.

     

Experimental Edema Disease of Swine (E. Coli ENTEROTOXEMIA) I. Detection and Preparation of an Active Principle

Freeze-thaw lysates prepared from strains of Escherichia coli belonging to serogroups O138, O139, and O141 contained a principle (edema disease principle) which induced edema disease in swine. All freeze-thaw lysates contained endotoxic activity that tended to obscure the edema disease syndrome and methods were developed to reduce such activity. Freeze-thaw lysates prepared from E. coli O139 induced the most characteristic edema disease syndrome. Partially purified edema disease principle prepared from O139 freeze-thaw lysates by sequential precipitation with ammonium sulphate and streptomycin sulphate had increased specific activity with markedly reduced endotoxic activity. This material was insoluble at acidic pH but readily soluble at alkaline pH. The effective molecular weight of edema disease principle, based on retention and filtration properties of diaflo membranes, appeared to be greater than 50,000 and less than 100,000. The biological activity of edema disease principle was thermolabile. Sodium deoxycholate treatment of edema disease principle further reduced endotoxic activity. A thermolabile, ammonium sulphate precipitable material was prepared from E. coli O139 that induced a predictable syndrome which resembled edema disease clinically and pathologically following intravenous inoculation in pigs.     

Comparison of Pathogenicity of Various Isolates of Bordetella Bronchiseptica in Young Pigs

Eight groups of 12-to 24-hour-old pigs were procured from a respiratory disease-free herd of swine and reared in isolation using a box-rearing procedure. They were inoculated intranasally at 3 days of age with different isolates of Bordetella bronchiseptica.

It was found at necropsy 4 weeks post-inoculation that 4 isolates of swine origin, an isolate of rabbit origin and an isolate of cat origin caused mild to moderate turbinate atrophy in 22 of 24 pigs. An isolate of rat origin caused mild turbinate atrophy in 1 of 4 pigs and an isolate of dog origin caused no turbinate atrophy. Pneumonia was present in most of the pigs inoculated with the swine, cat and rabbit isolates.

Bordetella bronchiseptica was recovered in heavy growth from the nasal and tracheal exudate collected at necropsy from pigs inoculated with the 4 isolates of swine origin and the isolate of cat origin. Fewer organisms were isolated from nasal exudate collected from pigs inoculated with the rat, dog and rabbit isolates.

     

Observations on the Etiological Agent of Plasmacytosis of Mink: Evidence Suggesting a Relationship of the Agent to the Granules of a Mycobacterium Species

A hypothesis based on a possible connection between the granules produced by a species of Mycobacterium and the agent causing Plasmacytosis in mink is suggested. The presence of these granules in the identical tissues of mink from which a virus had previously been isolated, is noted. Granules with the ability to produce a “germ tube” with acid-fast staining characteristics were found to be present in these tissues. Preliminary cytological studies have shown these granules to be similar to those described by Much. When tissues containing the granules were injected into guinea pigs, rabbits and chickens and these were later tested with avian tuberculin, positive skin reactions occurred. A disease was reproduced in chickens which simulated avian leucosis. In guinea pigs a disease was reproduced which resembled Plasmacytosis in mink with some histological differences. Rabbits appeared to be refractory to infection with the dosage and route of inoculation used. The results obtained from bacteriological studies, tissue culture, animal inoculation, as well as observations made on the cytological properties of the granules, are described and discussed.     

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.

     

Virus Pneumonia of Pigs; Attempts at Propagation of the Causative Agent in Cell Cultures and Chicken Embryos

Two strains of the agent of virus pneumonia, were tested for the ability to propagate in 12 types of cell cultures and in chicken embryos. The 5 primary cell cultures used were: swine kidney, lung, bone marrow, testicle, and chicken embryo kidney; and the 7 serial passage cell cultures were: swine kidney, kidney-tumor, testicle, bone-marrow, bovine kidney, and human cervical carcinoma (HeLa). The agent of virus pneumonia was propagated in primary swine kidney and in HeLa cell cultures as shown by the production of typical gross and microscopic lesions in pigs inoculated with cell future fluids. Third passage cell culture fluids, produced typical gross lesions in pigs, but fourth passage cell culture fluids produced only microscopic lesions, and no lesions were produced by sixth and eleventh passage fluids. Control pigs receiving fluids from uninoculated cell cultures remained free of gross or microscopic lesions, as did uninoculated controls. Cytopathic effects were not detected in any of the inoculated cell cultures and no cellular changes were detected by staining with Giemsa stain or acridine orange.

Neither lesions nor deaths occurred in chicken embryos inoculated with both strains of virus pneumonia virus. Pneumonia was not produced in pigs inoculated with suspensions from second chicken embryo passage of the 2 strains inoculated by the chorioallantioic sac, the amniotic sac, and the yolk sac routes.

Identical gross and microscopic lesions were produced in pigs inoculated with either pneumonic lung suspensions or with virulent cell culture fluids. Gross lesions consisted of areas of light to reddish-purple consolidation usually limited to the anterior, cardiac, and intermediate lobes of the lungs. Pleuritis and pericarditis were never present in experimentally produced virus pneumonia. The microscopic lesions were characterized by: 1. perivascular and peribronchiolar lymphoid infiltration and hyperplasia, 2. alveolar interstitial thickening and infiltration, and 3. alveolar exudates consisting of alveolar cells, lymphocytes, plasma cells, and neutrophiles.

     

Seasonal Fluctuations of Nematode Populations in Breeding Ewes and Lambs

The seasonal changes in nematode populations of a flock of sheep in the Montreal area were determined using serial fecal egg counts, fecal culture of larvae and necropsy worm counts. It was found that Ostertagia spp.,Nematodirus spp., Trichostrongylus agei, Trichostrongylus spp. and Chabertia ovina over-wintered on pasture and could initiate patent infections the following spring. The development of populations of H. contortus was typical of that seen with most of the other species and was characterized by the following series of events. In early winter when the study was started with stabled pregnant ewes, most of the populations were immature and the egg counts were low and remained so throughout the entire winter. However, in the spring, following lambing, large numbers of adult worms were seen with a consequent decrease in immatures and a sudden increase in egg counts. When the ewes and lambs were pastured together, the egg counts in ewes dropped consequent to “self-cure”, the “spring-rise” providing the major source of overwhelming infections for lambs with deaths by the end of July. As the season progressed larvae taken in by both ewes and lambs did not mature, and by early fall, most of the worm population consisted of immature forms. It appeared that H. contortus could not have more than two generations in ewes or lambs in a single grazing season.     

Experimental Infection of Pigs with ‘<Emphasis Type="Italic">Candidatus</Emphasis> Helicobacter suis’

‘Candidatus Helicobacter suis’ is a spiral-shaped bacterium that colonizes the stomach of more than 60% of slaughter pigs. The role of ‘Candidatus Helicobacter suis’ in gastric disease of pigs is still unclear. Experimental studies in pigs are lacking because this bacterium is unculturable until now. An inoculation protocol using ‘Candidatus Helicobacter suis’ infected mouse stomach homogenate was used to reproduce the infection in pigs. Control animals were inoculated using negative mouse stomach homogenate. Pigs were inoculated three times with one-week intervals and euthanized 6 weeks post inoculation. Tissue samples were taken from different mucosal stomach regions to detect ‘Candidatus Helicobacter suis’ by PCR and urease test. Mucosal inflammation was evaluated on formalin-fixed tissue samples. Lesions in the pars oesophagea were scored macroscopically. Infection was succesful in all challenged animals, with the antrum and the fundus being predominantly positive. Infection was associated with infiltration of lymphocytes and plasma cells in the antral mucosa, evolving to follicular gastritis. No apparent inflammation of the fundic stomach region was detected in the infected animals. A clear link between ‘Candidatus Helicobacter suis’ and pars oesophageal lesions could not be found.     

Enzootic Pneumonia of Pigs: Identification of a Causative Mycoplasma in Infected Pigs and in Cultures by Immunofluorescent Staining

Immunofluorescent staining has been used to identify Mycoplasma hyopneumoniae in smears of broth cultures, in infected pig testicle cell cultures, and in frozen cut sections of pneumonic lungs from field and experimentally produced cases of enzootic pneumonia. In the pneumonic pig lung, fluorescent staining was limited to the surface of the bronchial and bronchiolar epithelium and to the contained exudate. In a series of trials using experimentally infected pigs fluorescence was not detected until 25 days post-infection and was regularly seen in pigs killed thereafter. Porcine immune globulin precipitated from the serum of experimentally infected pigs and conjugated with fluorescein isothiocyanate was reactive and specific for the detection of M. hyopneumoniae. Immune globulin conjugates prepared from the serum of hyperimmunized rabbits were reactive but in some cases produced a faint non-specific staining of frozen tissue sections. No such non-specific reactions were noted on stained culture smears or cell cultures.

Fluorescence was not seen in known positive preparations stained with non-immune pig globulin conjugates or in preparations from uninoculated cell cultures or pigs, stained with non-immune or immune globulin conjugates.

Mycoplasma hyorhinis was detected by immunofluorescent staining with homologous conjugates, in smears of broth cultures and in tissue sections from pigs with polyserositis.

Immunofluorescent staining was found to be species specific and useful for the early species identification of mycoplasma isolated from pigs.

     

Swine Enzootic Pneumonia: Age Susceptibility and Treatment Schemata

Pigs were found to be susceptible to Mycoplasma hyopneumoniae-induced swine enzootic pneumonia (SEP) when four hours old. Chlortetracycline was incapable of preventing transmission of SEP from infected pigs on the drug to susceptible, untreated pigs. When continuous medication was started at one or two weeks postinoculation, chlortetracycline partially or completely inhibited formation of SEP lesions but did not clear M. hyopneumoniae from inoculated pigs. Chlortetracycline administered orally was capable of completely suppressing the formation of SEP lesions in inoculated pigs if given prophylactically and if milk was withheld for several hours after each treatment; lesion suppression was incomplete if milk was given ad libitum. In either case treated animals remained infected with M. hyopneumoniae.     

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.

     

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.     

Pasteurella haemolytica of Cattle: Serotype, Production of Bete-galactosidase and Antibacterial Sensitivity

Two hundred and three isolates of Pasteurella haemolytica from cattle were studied. They originated from the nasal cavity of cattle in housed herds; the nasal cavity and pneumonic lungs of experimental feedlot calves and from pneumonic bovine lungs submitted for bacteriological diagnosis.

To determine whether a single characteristic or combination of characteristics might be a feature of isolates collected from animals with pneumonic pasteurellosis (Shipping Fever), the following tests were made. Cultures were serotyped by indirect haemagglutination; the ability to produce beta-galactosidase was examined in the ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) test and antibacterial sensitivity tests were done. None of these factors could be directly related to the role of P. haemolytica in “Shipping Fever”.

     

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.

     

A Survey of the Alimentary Tract of Cattle for Clostridium Perfringens

Contents of the rumen, abomasum, ileum, and colon of 100 fattened cattle were examined for the presence of Cl. Perfringens. Liquid medium iniculated with each sample of gut content was tested for the presence of toxins of Cl. Perfringens.

Identification of Cl. Perfringens was based on atmospheric requirements for growth, colonial morphology, and stormy fermentation in litmus milk. Identification of toxins was based on neutralization tests in guinea pigs and mice.

Cl. Perfringens was isolated from 202 of 399 samples. In 105 additional cultures, colonies characteristic of Cl. Perfringens were present but could not be isolated in pure culture.

Cl. Perfringens type D toxin was identified in only one culture, which was inoculated with ileum contents. Type A toxin was identified in eight of the 24 samples from the one lot of samples in which no type A antitoxin was used. There were no identifications of toxigenic types B, C, or E.

The results indicate that an isolation from necropsy specimens of untyped Cl. Perfringens or type A Cl. Perfringens is in itself of little significance. The infrequency of occurrence of the other toxigenic types in this survey of healthy cattle indicates that recovery of these types from necropsy specimens may be of more significance in determining the cause of death.

     

Studies on the Susceptibility of Water Buffaloes to Leptospira

Eight mature farming type, Taiwan, water buffaloes were inoculate with L. australis A while six received L. canicola. Before inoculation all animals were negative to the microscopic-agglutination test (agglutinationlysis test) using the above species as antigen.

No sign of clinical leptospirosis was observed although four animals developed temperatures.

Cultures made from buffalo blood, kidneys and urine and from blood of guinea pigs inoculated with kidney emulsion and urine from the inoculated buffalo were all negative for leptospiral organisms.

Blood samples drawn from the water buffalo 2, 3 and 4 weeks post inoculation were negative to the microscopic-agglutination test except for one animal. Blood from the animal taken two weeks post-inoculation was positive at 1:100 dilution with L. australis A antigen but that taken at 3 and 4 weeks was negative.