Immune response of pigs to parenteral vaccination with an aromatic-dependent mutant of Salmonella typhimurium.

Cellular and humoral immune responses to parenteral vaccination with an aromatic-defined (aroA) Salmonella typhimurium and to oral challenge with the S. typhimurium parent strain were examined in pigs. The effectiveness of aroA S. typhimurium vaccination for prevention of clinical disease following challenge was also evaluated. A split litter model was utilized and analysis of variance was by least squares. The statistical model accounted for the effects of vaccination and litter. Parenteral vaccination of pigs with the aroA mutant induced a significant O-polysaccharide (O-ps) specific lymphocyte blastogenic response as well as a significant antibody response to O-ps, lipopolysaccharide and killed bacteria. The aroA strain was avirulent in pigs, was not shed in the feces and significantly reduced the severity of diarrhea following oral challenge.     

Sequential titration of bovine lung and serum antibodies after parenteral or pulmonary inoculation with Pasteurella haemolytica

Calves were vaccinated by intrabronchial or subcutaneous injection of formalinized Pasteurella haemolytica. Antibody in serum, nasal washings, and bronchoalveolar washings was titrated sequentially before and after calves were vaccinated and then challenge exposed with live homologous bacteria. Bronchoalveolar washings were collected by fiberoptics bronchoscopy, and antibody was titrated by indirect (antiglobulin) bacterial agglutination. Responsiveness to vaccination was related in initial serum antibody concentrations. Calves with serum antibody titers of 1:20 or more were nonresponsive, whereas with few exceptions, calves having titers of less than 1:20 responded to vaccination. Results indicated that serum and lung antibody were induced by subcutaneous or by intrabronchial inoculation of formalinized P haemolytica. By either route of immunization, serum antibody was more persistent than was lung antibody, and pulmonary challenge exposure with live P haemolytica did not alter existing titers.     

Bovine Vibriosis: The Distribution and Specificity of Antibodies Induced by Vaccination and Infection and the Immunofluorescent Localization of the Organism in infected Heifers

Two groups of three Holstein heifers were immunized respectively with Vibrio fetus venerealis and Vibrio fetus intestinalis incorporated in Freund's complete adjuvant. Both serum and vaginal mucus agglutination titers increased following immunization. Vaginal mucus samples were more frequently positive when the homologous cells were used as antigen in the agglutination test.

Ten non-immunized heifers were inoculated with another strain of V. fetus venerealis and slaughtered at periods of 30 to 40 and 60 to 70 days post-inoculation (DPI). Agglutinating antibodies were present in the vaginal mucus of some infected individuals by five weeks post-inoculation. In the course of the experiment 11 vaginal mucus samples were obtained which agglutinated heated cells of the infecting strain; one aggglutinated whole cells. Precipitins toward homologous antigens could not be demonstrated in vaginal mucus but four of six samples tested precipitated a heat stable extract from an intestinal strain of the same O-serotype. Bacterial antigen was detected by immunofluorescence on the surface, as well as within and beneath the epithelium at all levels of the reproductive tract regardless of time of slaughter. Lesions in infected animals consisted of focal and diffuse lymphocytosis, plasmacytosis, and epithelial vacuolation. Diffuse neutrophilic infiltration of the oviducts was observed.

Agglutinins appeared in the serum of each of nine heifers immunized with whole cells of same venereal strain. Group mean serum titers for whole and heated cells were 1/28,000 and 1/1,300 respectively. Vaginal mucus samples agglutinated whole cells in 48% of tests while 6.3% reacted with heated cells. Serum, but not vaginal mucus, of immunized animals precipitated soluble antigens of the immunizing strain. The immunizing strain of V. fetus did not infect the reproductive tract of any of six immunized heifers upon challenge.

     

Effects of antigen and recombinant porcine cytokines on pig dendritic cell cytokine expression in vitro

To evaluate variables influencing in vitro immune response induction, pig monocyte-derived DCs (moDCs) were treated with putative type-1 and type-2 antigens (Ags, killed Mycobacterium tuberculosis (Mtb) and hen egg white lysozyme (HEWL)) and recombinant porcine cytokines (IL-6, IL-10, IL-12, IFN-gamma and TNF-alpha). Responses were measured as moDC cytokine mRNA expression. Treatment of moDCs with HEWL increased IL-13 but not IL-12, IFN-gamma or IL-10 mRNA, suggesting a DC2 phenotype. Addition of TNF-alpha, IFN-gamma or IL-12 to HEWL-treated moDCs increased IL-12p35 and reduced IL-13 mRNA; suggesting a DC1 phenotype. Mtb increased moDC IL-12p35, IFN-gamma and to a lesser extent IL-13 mRNA. This DC1 bias was enhanced by TNF-alpha, IFN-gamma or IL-12, which increased IL-12p35 and to a lesser extent IL-10 mRNA but reduced IL-13 mRNA. Addition of IL-10 to Mtb-pulsed moDCs reduced IL-12p35, IFN-gamma and IL-13, but increased IL-10 mRNA, suggesting diversion from DC1 to DC2. Thus porcine moDCs treated with Ag and/or cytokines alter moDC cytokine expression confirming their likely ability to initiate and steer acquired immune response.     

The effect of hen-egg antibodies on Clostridium perfringens colonization in the gastrointestinal tract of broiler chickens

We evaluated the ability of hen-egg antibodies (HEA) to reduce intestinal colonization by Clostridium perfringens in broiler chickens. Antibodies against C. perfringens or cholera toxin (negative control) were obtained from the eggs of laying hens hyperimmunized using a C. perfringens bacterin or cholera toxin. Eggs were collected, pooled, and egg antibodies were concentrated by polyethylene-glycol precipitation. An initial experiment was conducted to determine the in vivo activity of the administered antibody along the length of the intestine. Thereafter, two feeding trials were performed to assess the efficacy of feed amended with the egg antibodies in reducing the level of colonization of C. perfringens in challenged birds. Antibody activity declined from proximal to distal regions of the intestine but remained detectable in the cecum. In the first experiment there was no significant reduction in the number of C. perfringens in the birds fed the diet amended with the anti-C. perfringens egg antibody, compared to the birds that received the anti-cholera toxin egg antibody (n = 10), at any of the sampling times. In the second experiment there was a significant decrease in C. perfringens intestinal populations 72 h after treatment (n = 15) as assessed by culture-based enumeration, but there was no decrease as measured by quantitative PCR based on the C. perfringens phospholipase C gene. Intestinal-lesion scores were higher in the birds that received the anti-C. perfringens HEA. Our work suggests that administration of HEA did not reduce the level of C. perfringens intestinal colonization and conversely might exacerbate necrotic enteritis.     

Multidrug resistance and distribution of Salmonella serovars in slaughtered pigs

The present study was undertaken to estimate the occurrence and distribution of multidrug resistance (MDR) among Salmonella serovars isolated from slaughtered pigs at Debre Zeit, Ethiopia. A total of 501 different samples were examined of which 42 (41.6%) of 101 mesenteric lymph nodes, 22 (21.8%) of 101 tongues, 17 (16.8%) of 101 caecal contents, 11 (11.1%) of 99 livers and two (2%) of 99 muscle (diaphragm and abdomen) samples were Salmonella positive. Of the 94 Salmonella isolates representing 15 different serovars, 69 (73.4%) were multidrug resistant (resistance to two or more antimicrobials). Among the Salmonella serovars a high level of MDR was observed in S. Hadar, S. Kentucky, S. Blockley and S. Enteritidis mainly to tetracycline (88.6%), streptomycin (82.9%), nitrofurantoin (74.3%), nalidixic acid and ciprofloxacin (42.9% each), sulfisoxazole (21.1%) and spectinomycin (20%). The pattern of MDR varied from two to eight antimicrobials among the resistant Salmonella serovars. The common profiles of resistance among the MDR serovars were the combined resistance to nitrofurantoin, streptomycin and tetracycline (R type NitStrTet, 51.4%), ciprofloxacin, nalidixic acid and nitrofurantoin (R type CipNalNit, 10%), ciprofloxacin, nalidixic acid, spectinomycin, streptomycin, sulfisoxazole and tetracycline (R type CipNalSptStrSulTet, 14.3%) and to ciprofloxacin, kanamycin, nalidixic acid, neomycin, nitrofurantoin, streptomycin and tetracycline (R type CipKanNalNeoNitStrTet, 10%). Results of the present study indicate the widespread occurrence and distribution of MDR Salmonella serovars in slaughtered pigs which could be a potential source of human MDR Salmonella infections.     

Immune response phenotype induced by controlled immunization of neonatal pigs varies in type 1:type 2 bias

Immune response (IR) of pigs varies by litter and by individual such that ratios of type-1 and type-2 IR differ. Estimates of heritability for antibody and cell-mediated IR suggest that genotype and the environment contribute approximately 20% and 80% to this variation. It is hypothesized that the IR phenotype of outbred neonatal pigs is immature and variable progressing with age from type-2 bias to a more balanced phenotype. To test this, pigs were IR phenotyped by a standardized protocol using two intramuscular injections of the combined type-1 and type-2 antigens (Ag) Candida albicans (CA) and hen egg white lysozyme (HEWL). Immune response was measured by wheal and flare reaction to HEWL and double skin fold thickness (DSFT) response to each Ag injected intradermally at 35 days of age. Blood was collected at 14 and 35 days of age to measure immunoglobulin IgG(1), IgG(2) and IgE isotype-relatedness of antibody (Ab) to CA and HEWL. Comparison was made between two different groups of pigs (A) and (B), from the same herd tested separately at an interval of two and a half years. An unexpected group difference in IR bias was observed. Bias in IR was not consistently toward type-2. Increase in DSFT to CA, an indicator of type-1 IR, was greater in A while frequency of wheal and flare to injection of HEWL, a type-2 IR correlate, was greater in B. Frequency of individuals with positive serum Ab activity to both Ags was greater in B than A for most isotypes. Ratios of Ab activity by type-1 and 2 isotypes and DSFT to type-1 and 2 Ags indicate diminished type-1 relative to type-2 biased IR response in B. We conclude that in normal neonatal pigs under standard husbandry IR bias is not invariably toward type-2. Phenotype varied between groups in type-1:type-2 bias with implications for protective and immunopathogenic IR. While the etiology was not pursued it is possible that unidentified environmental variables may have induced this change in IR phenotype.     

Construction, characterization, and evaluation of the vaccine potential of three genetically defined mutants of avian pathogenic Escherichia coli

The delta galE, delta purA, and delta aroA derivatives of avian septicemic Escherichia coli EC99 strain (O78 serogroup) were constructed with a suicide vector containing the pir-dependent R6K replicon and the sacB gene of Bacillus subtilis. The resultant isogenic mutants were stable and lacked approximately 45%, 36%, and 52% of the genes for galE, purA, and aroA, respectively. The delta purA and delta aroA mutants did not grow on minimal medium, whereas the delta galE mutant grew on minimal medium but was sensitive to galactose-induced lysis. The reversion frequencies of all three mutants were <10(-12). The mutants were highly attenuated for virulence as determined by administration of approximately 10(7) colony-forming units of each mutant to 1-day-old chicks by the subcutaneous route. Chickens were vaccinated with the mutants by spray (droplet size approximately 20 microm) at 1 and 14 days of age to determine safety, immunogenicity, and efficacy. The mutants were found to be safe. Seven days after a second vaccination, immunoglobulin (Ig)Y antibodies to E. coli in serum and air sac washings were detected by enzyme-linked immunosorbent assay. In both serum and air sac washings, IgY antibodies were significantly higher in chickens vaccinated with the mutants as compared with phosphate-buffered saline-treated controls but were significantly lower compared with chickens that were vaccinated with the parent strain. In serum, but not in air sac washings, IgY antibodies were significantly lower in chickens vaccinated with the mutants compared with the parent strain. The vaccinated chickens were given infectious bronchitis virus intranasally at 17 days of age and were challenged with homologous (EC99 strain) or heterologous (O2 serogroup) E. coli 4 days later. Chickens that received wild-type EC99 strain or its mutant derivatives were protected from homologous but not from heterologous challenge. This study indicates that the delta galE, delta purA, and delta aroA mutants are safe and moderately immunogenic but the protection conferred by the mutants is serogroup specific.