Immunity to Escherichia coli in pigs: Antibody Secretion by the Mammary Gland after Intramammary or Intramuscular Vaccination with an E. coli Vaccine

Indirect hemagglutinating antibody titres in individual gland samples of colostrum and milk from 13 sows were measured. Five of the sows were vaccinated via a mammary gland and five by the intramuscular route with a live formalinised Escherichia coli vaccine and three remained as non-vaccinated controls.

Antibody titres were higher in colostral and milk whey from the vaccinated sows than from non-vaccinated groups. The inoculated gland in the group of sows given vaccine by the intramammary route secreted milk containing markedly more antibodies to the vaccine E. coli strain than did the non-vaccinated glands. Milk from the vaccinated gland did not contain higher titres to heterologous E. coli O antigens than milk from non-vaccinated glands. Serum titres were the same or higher than the titres in colostrum from non-vaccinated glands.

     

Escherichia coli Agglutinins in Cow Serum, Colostrum and the Nursing Calf

Immunochemical properties of Escherichia coli O antibodies present in bovine serum and colostrum were investigated. Dam and calf serum samples plus colostral whey samples were fractionated by gel filtration, and the 7S and 19S fractions isolated. Antibody activity against the O antigens of four recognized E. coli bovine pathogens was determined by the indirect hemagglutination test on the whole serum and colostral whey samples and the 7S and 19S fractions thereof. Mercaptoethanol reduction was used to chemically study the immunochemistry of the E. coli O antibodies.

The E. coli O antibodies in dam serum were entirely 19S macroglobulins and appeared to be IgM immunoglobulins. The antibodies in colostrum and calf serum were both 7S and 19S globulins. Reasons for believing these 7S antibodies may be IgG, and the 19S antibodies IgA, immunoglobulins are presented.

     

Immunity to Escherichia coli in pigs: Serum Gamma Globulin Levels, Indirect Hemagglutinating Antibody Titres and Bactericidal Activity Against E. coli in pigs up to five Weeks of Age

Serum gamma globulin levels, indirect hemagglutinating antibody titres and bactericidal activity against the 0149:K91;K88ac:H10 Serotype of Escherichia coli were determined in pigs up to five weeks of age from vaccinated and non-vaccinated sows.

Gamma globulin levels at two days of age were approximately twice adult levels, by three weeks of age they were one quarter of adult levels and remained so until five weeks of age. Indirect hemagglutinating antibody activity was highest at two days of age, fell until three weeks of age and then rose. Little or no indirect hemagglutinating antibody activity was detected in sera taken at two days of age from pigs from non-vaccinated sows. Only three of 26 two day old pigs had demonstrable bactericidal activity; by three weeks of age 16 of 26 had bactericidal activity. Serum from piglets of vaccinated sows had no more bactericidal activity than did sera from non-vaccinated sows.

     

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

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

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

     

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.

     

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 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: 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.

     

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.     

Autolytic Changes in the Digestive System of Germfree, Escherichia coli Monocontaminated, and Conventional Baby Pigs

Tissues from three groups of pigs (germfree, Escherichia coli monocontaminated, and healthy conventional pigs) were collected at intervals between 24 minutes and 7 hours 12 minutes after death. Histological differences between the three groups were present in the alimentary mucosa and were most striking in the ileum and colon.

Autolytic change was detected only in the digestive system and the sequence of autolytic events was similar in all groups. The time of onset and rate of progress differed markedly between groups and between segments of the alimentary canal. In the conventional group, autolysis started quickly and progressed rapidly. In the germfree group it was later in onset and slower in progress. The E. coli infected group was intermediate. The initial visible change occurred in the small intestine.

The morphological differences and autolytic changes were described.

     

日粮添加枯草芽胞杆菌对大肠埃希菌感染仔猪免疫功能的影响

本试验旨在研究饲粮中添加枯草芽胞杆菌对大肠埃希菌K88感染仔猪血清免疫功能的影响。选用平均体重6.8kg±0.5kg的去势"杜×长×大"三元杂交公猪8头,随机分为2组,每头猪单栏饲养,对照组饲喂基础饲粮,试验组饲喂基础饲粮+0.1%枯草芽胞杆菌,每头猪灌服致病性大肠埃希菌K88菌液(1×1011 CFU)。采用ELISA方法检测血清中细胞因子和猪瘟抗体水平。结果表明,采食添加枯草芽胞杆菌的处理组仔猪比对照组仔猪血清IL-10水平提高(P0.01),血清IL-8和TNF-α水平降低(P0.01),猪瘟抗体阻断率提高(P0.05)。结果提示,仔猪采食含枯草芽胞杆菌的饲粮时,能够调节机体免疫功能,缓解致病性大肠埃希菌K88感染造成的炎症反应。研究工作为枯草芽胞杆菌的推广应用提供了试验依据。     

Development of Post‐weaning Diarrhoea in Piglets. Relation to Presence of Escherichia coli Strains and Rotavirus

Weaning of piglets complicated with an exposure to pathogenic strains of Escherichia coli was scrutinized in two sets. The first set comprised 20 animals representing two litters and the second set included 30 animals from five litters. The piglets were either left as controls or exposed to one or three pathogenic strains of E. coli. Aiming to simulate a natural exposure the challenge strains were spread on the floor of the pens at weaning. In addition the pigs experienced several non‐infectious stress factors commonly occurring at that occasion. Some groups were given adrenocorticotropic hormone (ACTH), aiming to simulate a stressful weaning. The balance and the composition of the faecal coliform populations, measured by a metabolic fingerprinting method, was disturbed among all animals following weaning. This disturbance was more pronounced and lasted longer among piglets exposed to pathogenic strains of E. coli. All piglets exposed to pathogenic E. coli shed these strains in faeces. Diarrhoea was induced in the groups exposed to E. coli, but not among the control animals. Pigs not treated with ACTH and subjected to a single pathogenic strain of E. coli became infected but did not develop diarrhoea unless if coinciding with shed of rotavirus. Control pigs excreting rotavirus had no diarrhoea. Diarrhoea was most frequent in the groups exposed to three pathogenic strains of E. coli, and in these groups diarrhoea was seen in the absence of rotavirus. ACTH administration amplified the clinical signs. The litter of origin influenced the development of post‐weaning diarrhoea.     

Protection Against Neonatal Escherichia coli Diarrhoea in Pigs by Vaccination of Sows with a New Vaccine that Contains Purified Enterotoxic E. coli Virulence Factors F4ac,F4ab,F5 and F6 Fimbrial Antigens and Heat‐Labile E. coli Enterotoxin (LT) Toxoid

The efficacy of a new vaccine against neonatal Escherichia coli diarrhoea in piglets containing purified F4ab, F4ac, F5 and F6 fimbriae and detoxified heat‐labile toxin (LT) was tested in challenge experiments by the method described by the European Pharmacopoeia (3rd edn, EDQM, Council of Europe, Strasbourg, France). A group of 11 young sows from a herd without E. coli problems was vaccinated 6–8 and 2–4 weeks prior to expected farrowing and another group of nine young sows were non‐vaccinated controls. Escherichia coli antibody titres were determined in serum samples taken from the sows before first vaccination and before farrowing and in colostrum samples. The newborn piglets were allowed to suckle colostrum from their mother immediately after birth. The piglets were marked with individually numbered ear tags. Approximately 12 h after birth, 118 piglets from vaccinated sows and 79 piglets from non‐vaccinated control sows were challenged by oral instillation of 5 ml of a freshly prepared culture of one of the challenge strains [O8:K87:F4ab (LT+) or O149:K91:F4ac (LT+) or O9:K30:F5 or O9:K103:F6 respectively]. The challenge cultures contained as a mean 6.8 × 10⁹ CFU/ml. After challenge the piglets were observed for 7 days and mortality and morbidity were recorded. Vaccinated sows developed significant levels of antibody titres in colostrum and serum. Control sows stayed at a low/seronegative level. The protective efficacy was excellent because 66.7–87.5% of the piglets from vaccinated sows remained without clinical signs after challenge. Only 0.0–28.0% of the piglets from non‐vaccinated sows remained healthy and more than 47.1% of the piglets in this group died after challenge. It is concluded that the new vaccine is very effective in protection of piglets against neonatal E. coli diarrhoea.     

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.

     

Determination of antibiotic resistance pattern and virulence genes in Escherichia coli isolated from bovine with subclinical mastitis in southwest of Iran

The aims of the present study were to investigate the prevalence of some virulence genes and also determine the antimicrobial resistance pattern of E. coli isolated from bovine with subclinical mastitis. The milk of 502 cows was collected from 8 dairy herds in the southwest of Iran. Conventional biochemical tests were used for identification of E. coli at the species level. Antimicrobial susceptibility patterns of E. coli isolates were determined by disc agar diffusion method and polymerase chain reaction (PCR) was used for detection of seven virulence genes including f17A, afaE-8, afaD-8, eaeA, cnf1, cnf2, and iucD. Seventy (13.94%) isolates of E. coli were identified in 502 milk samples. The highest rate of resistance was observed against tetracycline (18.6%), while none of the isolates were resistant to streptomycin. Eight (11.5%) out of 70 E. coli isolates carried at least one of the virulence genes. The afaD-8 was the most prevalent gene detected in 5 (7.1%) isolates. The afaE-8, iucD, and eaeA were detected in 3, 3, and 2 isolates respectively. Low prevalence of virulence factors may be indicating that most of the E. coli isolates originated from the commensal flora of cows and enter to the udders via environment contamination with feces.

     

Effect of Enteropathogenic Escherichia coli Infection on the Transmembrane Potential of the Swine Intestine

Transmural potential difference and short-circuit current were measured on membranes of swine small intestine as an index of transport activity for sodium ion. The measurements were made in vitro on membranes prepared from control and E. coli 0138-infected intestinal loops from weanling pigs. Measurements were made on duodenal and upper, middle and lower jejunal segments.

No differences were noted in the transmembrane potential difference and the short-circuit current between the control and the experimentally infected loops.

     

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 Immunization of Sows with Inactivated Transmissible Gastroenteritis (TGE) Virus

Four sows were immunized with inactivated TGE virus. The virus was either propagated in pig kidney cell cultures, or obtained from the intestines of experimentally infected gnotobiotic pigs, and inactivated by treatment with β-propiolactone. The inactivated virus preparations were administered to sows by intramammary inoculation. Two sows received two inoculations; the other two sows received three inoculations of inactivated virus. The antibody responses in the serum and milk were determined and piglets nursing the sows were experimentally challenged with virulent TGE virus. Three inoculations of the virus preparation stimulated much higher levels of serum and milk antibody than did two inoculations. A schedule of three inoculations of sows with inactivated TGE virus was effective in stimulating protection against TGE for piglets nursing these sows.     

Analysis of milk-derived isolates of E. coli indicating drug resistance in central Ethiopia

Mastitis is one of the most important diseases in dairy cows throughout the world and is responsible for significant economic losses to the dairy industry. This study was performed to characterize the genetic basis of drug resistance in Escherichia coli isolated from cases of clinical and sub-clinical bovine mastitis. A total of 224 California mastitis test (CMT)-positive milk samples were collected from December 2015 to April 2016 to characterize the phenotypic and genetic basis of antimicrobial resistance in E. coli isolated from raw milk from dairy farms found in Burayu, Sebeta, and Holeta areas of Ethiopia. The prevalence of E. coli was 7.1% (16) and both phenotypic and molecular techniques were used to identify E. coli antimicrobial susceptibility trait. The most commonly observed phenotypic resistance was against ampicillin (68.7%), sulphamethazole-trimethoprim (50%), and streptomycin (25%). Multidrug resistance phenotypes were found in 11 of 16 (68.7%) of E. coli isolates. Tetracycline (tet (A)) and chloramphenicol (cml (A)) genes were the most predominant encoding resistance genes identified (50%) each, followed by gentamycin resistance encoding gene (aac (3)-IV) (37.5%). Overall, 11 (68.7%) of the isolates had multidrug resistance genes responsible to two or more classes of antibiotics. The most common pattern detected was cml (A) and tet (A) together 37.5% followed by aac (3)-IV and tet (A) 25%. The current study indicated that raw milk could be regarded as critical source of antibiotic-resistant pathogenic E. coli.

     

Serological Identification of Escherichia coli Isolated From Cats and Dogs

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

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