Epidemiologic investigation of Riemerella anatipestifer in a commercial duck company by serotyping and DNA fingerprinting.

A commercial duck company that raises approximately two million Pekin ducks per year experienced an outbreak of Riemerella anatipestifer (RA) on nine farms over a 1-yr period. Owing to concerns that the bacteria was being spread from farm to farm, an investigation using serotyping and DNA fingerprinting was performed. The results revealed that there were three different strains of RA involved in the outbreak. One strain was spread from one farm to six other farms, while another strain from the same farm was spread to two other farms. These findings add additional proof of the value of DNA fingerprinting in disease outbreak investigations and further support the importance of implementing biosecurity protocols to stop the spread of disease-causing organisms.

Induction of Fos-like immunoreactivity in the rat brain following Pasteurella multocida endotoxin administration.

The functional neuroanatomy of the immune system link to the CNS was investigated by assessing neuronal activity with Fos immunohistochemistry following systemic lipopolysaccharide (LPS) administration. Two hours after LPS robust Fos-like immunoreactivity (Fos-IR) was observed in several nuclear groups in the brain including the paraventricular and supraoptic nuclei of the hypothalamus, central nucleus of the amygdala, and nucleus of the solitary tract. A similar but diminished pattern of Fos-IR was present at 6 hours and was absent 24 hours after LPS administration. Investigation of the functional neuroanatomy of the acute phase reaction could prove to be critical in enhancing the ability of individuals to combat insults such as tissue damage and inflammation. The central nervous system (CNS), particularly the hypothalamus, is intimately involved in the coordination of the various aspects of the acute phase reaction (reviewed in 1). Understanding the functional neuroanatomy by which the brain responds to immune system challenges would greatly augment the ability to control the deleterious and enhance the beneficial aspects of the acute phase reaction. In this study we have used lipopolysaccharide (LPS or endotoxin) administration as an experimental model to study immune system activation. LPS is a complex glycolipid and a component of the outer membrane of most Gram-negative bacteria (2). Administration of LPS has been demonstrated to induce the secretion of several proteins including interleukin-1 (IL-1), tumor necrosis factor (TNF), and interleukin-6 (IL-6; reviewed in 3). Further, it has been hypothesized that LPS induction of IL-1 and TNF is the key event in the pathogenesis of Gram-negative bacterial septic shock syndrome (2). Many recent studies have utilized immunohistochemistry for Fos, the product of the immediate early gene c-fos, as a marker of neuronal activation. Fos is a nuclear-binding protein that is expressed at increased levels in activated neurons (4). Although the exact function of Fos in the CNS is still unknown, it is thought that Fos is transcribed after cellular stimulation as a means to convert a stimulus into long-term genetic action (for reviews see 5,6). This study investigated the activation of the CNS by peripherally administered LPS isolated from the bacterium Pasteurella multocida. As a marker of neuronal activation, immunohistochemistry for the Fos protein was performed and image analysis was utilized to quantify the Fos induction in the CNS.

Immunoelectrophoresis employing avian antisera for the detection and quantitation of Pasteurella multocida antigens.

Immunoelectrophoresis with various buffer systems at high and low pH was examined for suitability to detect and quantitate Pasteurella multocida antigens with turkey or chicken anti-P. multocida sera. Counterimmunoelectrophoresis was used to develop a buffer system for one-dimensional, two-dimensional, and rocket immunoelectrophoresis. The effects of pH, buffer, and molarity on resolution of immunoprecipitates were determined; 0.05 M sodium acetate-acetic acid buffer at pH 5.6 was the most suitable buffer. This buffer could be used in counterimmunoelectrophoresis with turkey or chicken sera to detect minute amounts of P. multocida protein antigens (4.3 ng/test) or lipopolysaccharide (3.12 micrograms/test). One-dimensional immunoelectrophoresis with the acetate buffer system required treatment of the gels with a 17% NaCl solution to induce immunoprecipitation of P. multocida lipopolysaccharide. Other techniques using the acetate buffer system did not require the high salt treatment. In two-dimensional immunoelectrophoresis, antisera migrated in the second dimension at pH 8.6, but did not migrate at pH 5.6. Rocket immunoelectrophoresis with the acetate buffer system was effective for quantitating P. multocida antigens.

Typing of Pasteurella multocida from haemorrhagic septicaemia in Danish fallow deer (Dama dama).

Isolates of Pasteurella multocida ssp. multocida (n = 31) from a Danish population of fallow deer which succumbed to haemorrhagic septicaemia during 1992 1993 and isolates from the palatine tonsils of apparently healthy fallow deer from the same area (n=6) were typed and compared with P. multocida from other sources. Plasmids were net observed in the fallow deer strains and one unique pattern was observed by ribotyping using HindIII and by pulsed-field gel electrophoresis using SanlI as restriction endonuclease. All Danish fallow deer isolates belonged to serotype B:3,4. On restriction endonuclease analysis using HhaI as restriction endonuclease, all had a profile identical to that of a fallow deer isolate from the United Kingdom: profile 0033 of Wilson et al. On restriction endonuclease analysis using HpaII as restriction endonuclease, the Danish fallow deer isolates had a unique profile, designated 0062, which differed slightly from that of a fallow deer isolate from the United Kingdom. P. multocida from other animal species were genotypically different from the fallow deer isolates. It is concluded that a specific clone of P. multocida was responsible for the outbreak of haemorrhagic septicaemia among Danish fallow deer. A carrier rate of 27% was demonstrated among apparently normal animals from the same population.

Protection of Pasteurella multocida dermonecrotic toxin-challenged rats by toxoid-induced antibody.

Two different doses of glutaraldehyde-treated Pasteurella multocida dermonecrotic toxin (PMDT) were used to immunize rats. Rats developed serum IgG antibodies specific for native PMDT, and IgG titers increased with dose and number of toxoid immunizations. Survival rates in both active immunization and passive serum neutralization experiments were dependent on dose of toxoid vaccination and serum levels of anti-PMDT IgG. Vaccination with toxoid prevented weight loss but not leukocytosis and increased complement titers in toxin-challenged rats. Toxoid, itself, induced minimal leukocytosis but no alterations in complement titers or weight gain.

Influence of chondroitinase on indirect hemagglutination titers and phagocytosis of Pasteurella multocida serogroups A, D and F.

Capsules of Pasteurella multocida serogroups A, D and F contain mucopolysaccharides which block antigenic determinants and prevent phagocytosis. In this study, capsules of serogroup A, D and F strains of P. multocida were depolymerized by enzyme treatment. Capsule depolymerization of serogroup D and F strains with chondroitinase increased indirect hemagglutination (IHA) test titers and enhanced phagocytosis by swine neutrophils. Capsule depolymerization of serogroup A strains with hyaluronidase increased IHA titers, but depolymerization with chondroitinase did not. When serogroup A strains were treated with a combination of chondroitinase and hyaluronidase, IHA test titers were lower than titers of the same strains treated with hyaluronidase alone. Combined enzyme treatment of serogroup D strains resulted in IHA test titers similar to those of chondroitinase treatment alone.

Immunity induced in rats vaccinated with toxoid prepared from heat-labile toxin produced by Pasteurella multocida serogroup D.

Rats were vaccinated with a toxoid (D-toxoid) prepared from purified heat-labile toxin (D-toxin) produced by Pasteurella multocida serogroup D. Vaccination of rats with D-toxoid prevented death and other effects of D-toxin (hepatic necrosis, development of elevated leukocyte counts, lymphopenia, neutrophilia, and elevated complement titers) that occurred in phosphate buffered saline (PBS)-vaccinated control rats.

Replacement of sodium chloride with dextran or polyethylene glycol for immunoprecipitation of lipopolysaccharide with antibodies in chicken or turkey sera.

Dextran or polyethylene glycol could replace sodium chloride in agarose gels for inducing immunoprecipitation of Pasteurella multocida lipopolysaccharides with antibodies in chicken or turkey sera. Resolution of immunoprecipitates was best when 3% concentrations of either dextran or polyethylene glycol were used. Higher concentrations increased opacity of the gels. Nonspecific precipitation of serum or gamma-globulin fractions in gels was caused by the electrophoresis buffer, dextran, and polyethylene glycol. Dialysis of serum or gamma-globulin fractions against the electrophoresis buffer and soaking gels in buffers of pH greater than 7.0 that contained 3% polyethylene glycol reduced nonspecific precipitation. Incorporation of dextran or polyethylene glycol into gels enhanced immunoprecipitation in rocket immunoelectrophoresis but resulted in slower mobility of antigen.

Serum complement activity and serum enzymes in rats after a subcutaneous injection of toxin prepared from Pasteurella multocida type D.

Toxin produced by Pasteurella multocida type D was investigated for its effect on serum complement and serum biochemistry in rats. Rats were given a sublethal single subcutaneous injection of D toxin equivalent to 0.2 microgram/kg of body weight. Serum obtained 1, 3, 5 and 7 days post-treatment was tested for complement activity, total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). Serum complement titers were significantly elevated (P less than 0.05) at all times after injection of toxin compared to rats injected with diluent and tested at the same intervals. Bilirubin was decreased but both control and D toxin-treated rats had low concentrations of bilirubin in their sera. The other biochemical constituents measured had no consistent pattern that would indicate liver damage in the rats.

Use of rats to compare atrophic rhinitis vaccines for protection against effects of heat-labile protein toxin produced by Pasteurella multocida serogroup D.

Four bacterin-toxoid and three bacterin commercial vaccines against atrophic rhinitis were tested in rats for their capacity to immunize against the lethal and systemic effects of purified heat-labile protein toxin (D-toxin) produced by Pasteurella multocida serogroup D. Only one bacterin-toxoid vaccine stimulated sufficient immunity to prevent the death of all rats challenged with D-toxin. None of the vaccines prevented weight loss, leukocytosis or increases in serum complement titers in rats challenged with D-toxin. Rats provide an inexpensive animal model for testing the capacity of vaccines to generate antitoxic immunity against the lethal and systemic effects of D-toxin.

The effect of formalin-killing of Pasteurella multocida on the antigenicity and extractability of its lipopolysaccharide.

The extraction of lipopolysaccharides (LPS) from formalin-killed (FK) Pasteurella multocida strain X-73 and from cells not exposed to formalin (NF) were compared by the Westphal and phenol-chloroform-petroleum ether (PCP) extraction procedures. The LPS was determined by: (1) serologic analyses with antiserum specific for LPS; (2) analyses for toxicity; and (3) chemical analyses for components expected to be in LPS (such as hexoses, heptoses, amino sugars, 3-deoxyoctulosonic acid, and fatty acids). Strain X-73, the strain most virulent for chickens, was markedly affected by formalin killing. Unlike many strains, which readily yield LPS into the aqueous phase when extracted with phenol at 68 degrees by the Westphal procedure, strain X-73 did so only with FK and not with NF cells. With the NF cells, LPS was extracted by EDTA from the precipitate obtained during the Westphal procedure. With the PCP procedure, LPS was extracted readily from NF cells, but not from FK cells. The change in extractability of LPS as a result of formalin-killing was the same for both the encapsulated form of X-73 and a nonencapsulated variant derived from it. Although formalin-killing affected the extractability of LPS, no antigenic differences could be detected by immunodiffusion. However, the chick-embryo toxicity of LPS extracted from NF cells was greater than that of LPS from FK cells.

Studies of the pathogenic avian Haemophili.

The biochemical and physiological characteristics of 35 strains of avian haemophili from 7 countries were examined. All strains required V-factor but not X-factor for growth on artificial media. They produced acid in phenol-red broth containing fructose, glucose, and mannose. Acid production from other carbohydrates was variable or did not occur. Thirty-two strains were pathogenic to chickens. Pathogenicity varied with method of exposure. Hyaluronic acid was found in 9 strains. Hemagglutination of human or chicken erythrocytes was inhibited by its presence. Antimicrobial sensitivity patterns showed all strains to be sensitive to chloromycetin, erythromycin, furoxone, gentamicin, nalidixic acid, neomycin, novobiocin, spectinomycin, and tetracycline.

Partial purification of cross-protection factor(s) from Pasteurella multocida.

Membrane-associated cross-protection factor(s) (CPF) of in vivo-grown Pasteurella multocida were solubilized by detergent and partially purified by sequential chromatography on ion exchange, gel filtration, and hydroxyapatite columns. The CPF activity was determined by challenge of turkeys vaccinated with different chromatography fractions and challenge of poults passively immunized with serum from the same vaccinated turkeys. Analyses of different chromatography fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots suggested that proteins in the range of 59-65 kDa and a protein of 39-kDa molecular mass were associated with cross-protection. Although CPF activity was found to be associated with protein-containing material, treatment with periodate diminished cross-protection, indicating that carbohydrate determinants may also contribute to immune cross-protection.

Purification of a cross-protective antigen from Pasteurella multocida grown in vitro and in vivo.

A peptone-based medium was formulated to grow Pasteurella multocida in vitro, which expressed an antigen that induces cross protection in turkeys against different serotypes. Vaccines of various chromatographic fractions obtained from P. multocida grown in the medium induced active immune cross protection in turkeys, and sera from these turkeys passively cross protected naïve poults. An antigen of approximately 39 kD molecular size was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroelution from hydroxyapatite chromatographic fractions of both in vivo- and in vitro-grown P. multocida. The purified antigen from either source induced active immune cross protection but no passive protection in one of two experiments. Increasing the dose of vaccine resulted in both active and passive immune cross protection in the second experiment.

Cross-protection factor(s) of Pasteurella multocida: passive immunization of turkeys against fowl cholera caused by different serotypes.

An antiserum cross-protective against different serotypes of Pasteurella multocida was made in turkeys by inoculating them with killed serotype 3 organisms grown in vivo and then exposing them to live serotype 3 organisms. In passive-immunization studies, the antiserum protected young turkeys against the homologous and heterologous serotypes 1, 4, 5, 9, and 12. In addition, the antiserum protected against P. multocida of a heterologous capsule serogroup, serogroup F. A globulin and two IgG fractions purified from the antiserum protected against heterologous challenge with serotype 1. Turkey-grown P. multocida were chemically lysed and separated into soluble and insoluble components to make immunoadsorbents. Antibodies from the cross-protective antiserum isolated by the immunoadsorbents passively protected young turkeys against heterologous serotype I challenge.

Comparisons of serologic responses of white Leghorn and New Hampshire red chickens to purified lipopolysaccharides of Pasteurella multocida.

White leghorn and New Hampshire red chickens were inoculated with purified lipopolysaccharides of 14 serotypes of Pasteurella multocida to determine their ability to produce serotype-specific antisera for somatic antigen typing. Specific antisera were made by both breeds of chicken to lipopolysaccharides of serotypes 1, 3, 4, 6, 8, and 16. No specific antisera were made against lipopolysaccharides of serotypes 2, 5, 7, 12, and 14. Lipopolysaccharides of serotypes 10 and 11 failed to stimulate antibody production. White leghorns were more responsive than New Hampshire red chickens. White leghorn antisera had higher titers to lipopolysaccharides in passive hemagglutination tests and produced more intense precipitin reactions with heat-stable antigens in the gel-diffusion-precipitin test.

Turkey coryza: toxin production by Bordetella avium.

Twelve strains of Bordetella avium representing isolates from turkeys in the United States, the Federal Republic of Germany, and the Republic of South Africa were tested for toxin production. Sterile filtered sonicates from 9 of 12 strains contained a toxin that was lethal for 7-to-10-day-old poults. Mice were also susceptible to the lethal effects of the toxin. No differences in susceptibility to the toxin were found between Beltsville small white and broad-breasted white poults. The toxin was solubilized by sonication and inactivated by heating at 56 C for 30 min. Treatment with formalin or proteolytic enzymes inactivated the toxin, indicating that it is probably a protein. The evidence suggests that the toxin is involved in the pathogenesis of turkey coryza.

Pasteurella multocida virulence factors: selection of fowl cholera-inducing and non-inducing strains.

Relatively little information is available on Pasteurella multocida virulence factors involved in producing fowl cholera. Because of the complex nature of bacterial pathogenesis, the recommended approach for ascertaining these factors is to compare biological attributes of high- and low-virulence strains. To permit use of this approach for fowl cholera, P. multocida strains of high and low virulence were identified. Turkey poults were exposed intrapharyngeally and intravenously (IV) to two antigenically and biochemically similar strains. Based on mortality, strain P-1059 was highly virulent and strain P-1062 was avirulent. Microbiological examination indicated that only the virulent strain infected the pharyngeal mucosa of intrapharyngeally exposed poults and survived and multiplied in IV-exposed poults. These findings indicate strain differences in those virulence factors concerned with the colonization and multiplication stages of disease development.

Fowl cholera: protection against Pasteurella multocida by ribosome-lipopolysaccharide vaccine.

Ribosomal protein from Aspergillus fumigatus substituted for intact ribosomes in potentiating the immunogenicity of Pasteurella multocida lipopolysaccharide. Ribosomal protein behaved as a carrier for the lipopolysaccharide. The basic protein methylated albumin, but not protamine sulfate, substituted for ribosomal protein as a carrier for lipopolysaccharide. Synthetic single- and double-stranded polynucleotides did not function as an adjuvant to potentiate the immunogenicity of lipopolysaccharide or methylated albumin-lipopolysaccharide complexes. Double-stranded polynucleotide (poly A:poly U), added as an adjuvant for methylated albumin-lipopolysaccharide vaccine, produced sera with lowered passive hemagglutination antibodies to lipopolysaccharide, but it did not influence protection against challenge with P. multocida. No differences in protection were observed between different lines of specific-pathogen-free white leghorn chickens given ribosome-lipopolysaccharide vaccine. Humoral protection, demonstrated by passive-protection tests, was induced by ribosome-lipopolysaccharide vaccine. Cell-mediated immunity was not detected by delayed-type hypersensitivity skin test reactions.

Bacterin-induced protection of turkeys against fowl cholera following infection with Bordetella avium.

Groups of Beltsville small white turkeys, passively immunized and not passively immunized against Bordetella avium, were challenged with live B. avium at 2 days of age. Birds not passively immunized developed severe bordetellosis with early onset, whereas passively immunized birds developed mild bordetellosis with late onset. Following convalescence, birds with and without exposure to B. avium were vaccinated against fowl cholera with a water-in-oil bacterin. The birds were given a homologous challenge with serotype A: 3 Pasteurella multocida. Although no difference in protection against fowl cholera was seen between vaccinated birds that were previously infected with B. avium and those that were not, survivability was better in birds given two doses rather than 1 dose of bacterin.