Enzootic Pneumonia in Pigs: Propagation of a Causative Mycoplasma in Cell Cultures and in Artificial Medium

Three strains of a new species of mycoplasma were recovered from pneumonic pig lungs, known free of Mycoplasma hyorhinis, by prolonged incubation in pig testicle cell cultures. The three strains produced a characteristic cytopathic effect in the cell cultures. A highly enriched meat-infusion-broth medium was evolved and permitted regular propagation of these organisms. Pneumonia could consistently be produced by intratracheal inoculation of pigs with the mycoplasma propagated in the enriched broth medium or in cell cultures. The mycoplasma were recovered from the lungs of experimentally infected pigs by inoculation into the broth medium. Comparative studies of the pneumonia producing mycoplasma and of M. hyorhinis were carried out in cell cultures, broth media, and in pigs infected experimentally by different routes. The morphological characteristics of the mycoplasma, grown in the different media, are described and illustrated.     

Enzootic Pneumonia of Pigs: Complement-Fixation Tests for the Detection of Mycoplasma Antibodies in the Serum of Immunized Rabbits and Infected Swine

The direct, the modified direct and the indirect complement-fixation tests were investigated as methods for the detection of antibodies for the enzootic pneumonia mycoplasma and for Mycoplasma hyorhinis in the serum of infected pigs and of immunized rabbits.

Only the modified direct complement-fixation test in which the guinea-pig complement is supplemented with fresh, normal unheated calf serum was suitable for the detection of mycoplasma antibodies in sera of infected swine. Based on the close correlation between the production of typical lung lesions in experimentally infected pigs and the appearance of significant serum antibody titres, the modified direct complement-fixation test provides for the first time a sensitive, specific in vitro method for the detection of enzootic pneumonia in the live pig. This test also permitted the in vitro differentiation of the mycoplasma causing enzootic pneumonia from M. hyorhinis which causes polyserositis.

Antibodies in the sera of rabbits were demonstrable by the ordinary direct complement-fixation test. However, in contast to the observation made with swine sera, only a slight quantitative antigenic difference between the enzootic pneumonia mycoplasma and M. hyorhinis was seen when the tests were performed with rabbit serum antibodiies.

     

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.

     

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.

     

Immunofluorescent Studies of Bovine Para-Influenza 3 Virus I. Cell Cultures and Experimentally Infected Hamsters

Immunofluorescent techniques were used to follow the replication of the SF-4 strain of bovine para-influenza 3 (PI-3) virus in Madin-Darby Bovine Kidney (MDBK), secondary bovine kidney (BK), Embryonic Bovine Trachea (EBTr) cell cultures and experimentally infected hamsters. The virus replicated equally well in MDBK and secondary BK cell cultures but less successfully in EBTr cultures. Nuclear fluorescence was not observed in the cell systems studied.

Infection was limited to the epithelial lining of the upper respiratory tract, trachea and bronchi of experimentally infected hamsters. Virus was most easily identified in smears prepared from turbinate mucosa suggesting a potential diagnostic technique for use in identifying PI-3 infected animals.

     

Diagnosis of Transmissible Gastroenteritis in Pigs by Means of Immunofluorescence

The fluorescent antibody (FA) test for the diagnosis of field outbreaks of transmissible gastroenteritis (TGE) in baby pigs was compared to other available means including: virus isolation by inoculation of test pigs, intestinal lesions especially villous atrophy, and clinical observations.

Immunofluorescent tests were done on frozen sections of the small intestine and it was possible to make a specific diagnosis within two hours after collecting samples. The results obtained with the FA test compared favorably with virus isolation from infected tissues. It was considered a more advantageous procedure as long as infected pigs were in a relatively early phase of the disease. Because of the variability of the lesions as related to the stage of infection, pathologic diagnoses were less satisfactory. Field diagnoses made on the basis of clinical signs were least reliable.

     

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.

     

The Effect of Medicated Feed on the Nasal Microflora and Weight Gain of Pigs

Antimicrobial agents were added to the feed of swine for three weeks to determine the interrelationships of potentially pathogenic agents in the nasal tract, turbinate atrophy and weight gains.

Bordetella bronchiseptica was not isolated from the groups fed the combination of chlortetracycline, penicillin and sulfamethazine. B. bronchiseptica was found in some pigs after the feeding trail, but this organism was not significantly associated with turbinate atrophy at the time of slaughter.

Mycoplasma hyorhinis was not found in the nasal passages of the pigs that received feed containing high concentration chlortetracycline but was found in pigs that received other diets. Hemophilus suis was not significantly reduced by any of the treatments used.

The organisms studied in the pigs were not isolated from the personnel handling the pigs.

     

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.

     

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.

     

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.     

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 on Transmissible Gastroenteritis of Swine I. The Isolation and Identification of a Cytopathogenic Virus of Transmissible Gastroenteritis in Primary Swine Kidney Cell Cultures

Two virus isolates from transmissible gastroenteritis (TGE) of swine were adapted to grow in primary swine kidney cells. Growth of the virus was indicated by the resistance of the infected cells to the cytopathic effect of a virus diarrhea virus of cattle, and by the development of large round cells on the cell sheet.

Evidence that these virus isolates were TGE was obtained by the development of signs of the disease followed by death of exposed SPF pigs, or the resistance of the recovered pigs to further signs of disease when they were exposed to virulent TGE contained in virus bearing intestinal tissue.

The in vitro and in vivo serum neutralization tests, along with staining of infected cells by fluorescein conjugated TGE antiserum, gave further indication of the specific nature of the virus growing in the cell cultures.

     

Detection of Actinobacillus pleuropneumoniae Infection in Pigs

It is difficult to control the spread of porcine haemophilus pleuropneumonia caused by Actinobacillus pleuropneumoniae because there is no sensitive and specific way to accurately determine whether or not a pig herd is infected. This paper reports bacteriological and serological techniques used to detect A. pleuropneumoniae infection in pigs from a herd with endemic disease.

The bacteria were isolated from the anterior nasal mucosa of grower pigs, but not from younger or older pigs. Bacteriological culture of several tissues from the respiratory tract showed that nine of ten young finishing pigs were infected, but culture of lung tissue from slaughtered hogs detected infection in only 39 of 288 (13.5%). Both cooler storage temperature and use of selective medium prolonged the time that lung tissue could be stored and the organism still recovered. An enzyme-linked immunosorbent assay detected serotype-specific antibodies in serum of infected pigs.

     

Differentiation Among Strains of Goat Mycoplasma by Incident Light Immunofluorescence

Antigenic differentiation between strains of goat mycoplasma was studied by direct fluorescent antibody reactions employing incident (vertical) ultraviolet light. Agar colonies of the mycoplasma grown in petri dishes were fixed by alcohol in situ, and stained with conjugated globulin before examination with ultraviolet light.

The fluorescent antibody (FA) conjugate against Vom strain of Mycoplasma mycoides var. capri was Vom strain-specific, no cross reaction with Mexico, Connecticut, or Maryland strains. Similarly, the Mexico strain conjugate was specific for colonies of Mexico, and did not cross with the Vom, strain. Additionally, the conjugate of the PG-2 strain of Mycoplasma agalactiae, which was specific for the colonies of PG-2 was refractory for the strain #99 of M. agalactiae.

It was therefore possible to utilize an immunofluorescent technique (incident ultraviolet light) to demonstrate differences among strains of M. mycoides var. capri and M. agalactiae.

     

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.

     

Study of the Practicability of Various Diagnostic Methods in the Demonstration of Swine Fever Virus of High and Low Virulence in Organs of Experimentally Infected Pigs

Inoculation experiments were performed with two German strains of swine fever virus. In Experiment I, ten pigs of Danish Landrace were inoculated with a strongly virulent strain, while in Experiment II ten pigs were inoculated with weakly virulent virus.The animals were killed at varying times after inoculation and organs taken out for examination by means of fluorescent antibodies (FA), by the complement fixation test (CFT), and by the agar gel diffusion test (AGT). Cryostat sections of tonsils, spleen and lymph nodes were examined by FA staining. Tissue suspensions from the same organs were inoculated into primary pig kidney tissue cultures, which were also stained with FA. Antigen produced from spleen tissue was used in the CFT and pieces of pancreas tissue in the AGT.The strongly virulent virus could be demonstrated easily by FA in all the inoculated pigs, both by direct staining of cryostat sections and by staining of inoculated tissue cultures. The CFT and AGT were positive when the tested organs originated from animals killed at a more advanced stage of the disease.While the weakly virulent virus could be demonstrated by FA staining of tissues from eight of the ten pigs in Experiment II, virus was found in none of these animals by the FA tissue culture method. The CFT was positive in one case and the AGT in three cases.In both experiments it was found that FA staining of cryostat sections of tonsils was a particularly suitable method for the demonstration of virus.The results are discussed and compared with recent German and American studies.     

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: 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.

     

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.