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.

Pathogenicity differences of multiple isolates of Aspergillus fumigatus in turkeys.

Sixteen Aspergillus fumigatus isolates of environmental, mammalian, and avian origin were used to assess: 1) intra-air-sac inoculation as a viable challenge alternative to aerosol exposure, and 2) isolate variability in pathogenicity. Development of lesions, antibody response in survivors, mortality, and weight gains were assessed. Turkey poults were challenged with equal numbers of viable conidia. Total number of conidia given per experimental group varied markedly and did not influence mortality. Antibody response as measured by the enzyme-linked immunosorbent assay and agar gel immunodiffusion test was erratic, although most poults with high antibody scores had marked lesions and low weight. Lesions were characterized by necrogranulomatous pneumonia and airsacculitis with marked visceral involvement. The source of the isolate was not a factor in mortality, although this was biased by the high numbers of isolates from birds with aspergillosis. The single environmental isolate produced no mortality.

Fowl cholera epornitic: antigenic characterization and virulence of selected Pasteurella multocida isolates.

An epornitic of fowl cholera involving turkey flocks of several farms within a 15-mile radius in Utah was studied. Pasteurella multocida strains isolated from birds in affected flocks were antigenically characterized as A:1, A:3, and B:4, based on capsular sero-grouping and somatic serotyping results. Experimental exposure of poults with each of two strains representing the rarely reported capsular group B indicated that both were virulent.

Pasteurella multocida colonization and invasion in experimentally exposed turkey poults.

Turkey poults were examined microbiologically to detect colonization and invasion following exposure to selected strains of Pasteurella multocida. A variety of host tissues was examined 24 hours or less after exposure via the upper respiratory tract. Recovery of P. multocida from the dorsal pharynx was used as an indicator of colonization; recovery from selected internal organs was used as an indicator of invasion. The frequency of colonization and invasion by capsulated and uncapsulated forms of an avirulent live-vaccine strain was low. A virulent strain readily colonized and invaded. Colonization occurred in less than 6 hours after exposure, and invasion occurred between 6 and 12 hours after exposure.

Virulence and toxigenicity of capsular serogroup D Pasteurella multocida strains isolated from avian hosts.

Five capsular serogroup D strains of Pasteurella multocida isolated from avian hosts were examined for virulence and toxigenicity. Virulence was based on development of lethal infections or lesions following intramuscular exposure of turkey poults. The four strains isolated from turkeys varied from slightly to moderately virulent; the strain isolated from a chicken was avirulent. Poults exposed by intra-airsac inoculation with relatively few organisms of the more virulent of the strains had a high mortality rate; however, intranasal exposure of poults with this strain did not cause clinical disease or establish infections. All strains from turkeys were toxigenic, producing heat-labile toxins that killed poults when administered intraperitoneally and caused focal dermal lesions when administered intradermally. Using these criteria, the strain from a chicken was not toxigenic. The demonstration of virulence, particularly the high mortality in poults exposed via air sacs, indicates avian capsular serogroup D strains are a potential cause of fowl cholera.

Somatic serotypes of Pasteurella multocida strains isolated from avian hosts (1976-1988).

During the period of 1976-88, 733 strains of Pasteurella multocida isolated from avian hosts were submitted to the National Animal Disease Center for somatic serotyping. The serotypes were determined and are presented in a summarized form in this report. The most common serotypes were 3 (29%); 1 (18%); 3,4 (12%); and 3,4,12 (9%). The 733 strains had been isolated from 25 species of avian hosts; 400 (55%) were from turkeys. The most common serotypes of strains from turkeys were 3 (38%); 3,4 (18%); 3,4,12 (11%); and 4 (4%).

An avidin-biotin enhanced dot-immunobinding assay for the detection of Mycoplasma gallisepticum and M. synoviae serum antibodies in chickens.

A dot-immunobinding assay was enhanced by the incorporation of avidin and biotin reagents into the test system (DAB assay). This assay was used to detect serum antibodies to Mycoplasma gallisepticum (MG) and M. synoviae (MS) from chickens. Serum samples were tested by rapid serum plate (RSP), hemagglutination-inhibition (HI), and DAB assay methods. These results were compared. The DAB assay was at least 20 times more sensitive in detecting antibodies for MS and at least 75 times more sensitive in detecting antibodies for MG than the HI test. The DAB assay was as specific as the HI test. The DAB assay was also more sensitive and specific than the RSP test. Some cross-reactions occurred when low dilutions of high-titer sera were used in the DAB assay. Parameters for determining negative, suspicious, and positive samples were established. The DAB assay for MG and MS may have several applications, including use as a screening test and a confirmatory test.

Virulence and infectivity of A: 14 strains of pasteurella multocida for turkey poults.

Strains of Pasteurella multocida, capsular serogroup A and somatic serotype 14, (A: 14) isolated from turkeys which had succumbed to fowl cholera were examined for virulence and infectivity. Groups of 2-week-old poults were injected intramuscularly with one of three strains. Slight to moderate virulence, based on mortality, was noted. Poults that survived exposure were frequently affected with exudative airsacculitis. Intramuscular, intra-air sac and intranasal exposure of 6-week-old poults with the most virulent strain, as determined in the first experiment, caused the death of one of five intramuscularly-exposed poults. No other clinical evidence was observed. At post mortem examination, marked exudative airsacculitis occurred in all intra-air sac exposed poults, both in inoculated and other air sacs. Pneumonia was also observed in these poults but only affected lungs adjacent to inoculated air sacs. Some poults became carriers of P. multocida.

Solubilization of membrane-associated cross-protection factor(s) of Pasteurella multocida.

Pasteurella multocida harvested from the blood of turkeys dying of experimental fowl cholera were purified by centrifugation and lysed. The soluble and membrane-associated components of the bacteria were separated by centrifugation. Nonionic (octylglucoside) and zwitterionic (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; CHAPS) detergents were tested for their abilities to solubilize the cross-protection factor(s) (CPF) from the membrane-associated component. Protection studies in turkeys showed that optimum solubilization was by 1.0% octylglucoside and 0.5% CHAPS. Antibodies from turkeys made against solubilized membrane-associated CPF passively cross-protected poults against challenge. Ion exchange chromatography of detergent-solubilized CPF resulted in elution of two protein-containing peaks, each of which conferred active immune protection.

Virulence of avian capsular serogroup B Pasteurella multocida for turkey poults.

Two strains of capsular serogroup B Pasteurella multocida isolated from avian hosts (swan and turkey) were evaluated for virulence based on lethality for turkey poults. Groups of poults were exposed intramuscularly to various concentrations of organisms of each strain. Both strains were virulent. The strain isolated from a turkey was highly virulent: all exposed poults died in less than 24 hours, including those exposed to only 79 organisms. This highly virulent strain was neither highly invasive nor highly infective: intrapharyngeal exposure with 7.9 x 10(6) organisms resulted in death of only one of five poults, and attempts to isolate the organism from pharyngeal mucosae and livers of surviving poults were unsuccessful. The high degree of virulence of a B capsular group strain isolated from a turkey indicates a disease-producing potential for members of this uncommon serogroup of P. multocida.

Airsacculitis in turkeys exposed to Mycoplasma synoviae membranes.

In studies to investigate the pathogenesis of mycoplasmal airsacculitis, exudative lesions were produced in turkeys by intra-air-sac inoculation with Mycoplasma synoviae cell membranes and viable organisms. Membrane inocula containing 5 mg of protein produced more severe lesions than inocula containing either 2.5 mg or 1 mg protein. Turkeys exposed to 5 mg of membrane protein developed moderately severe airsacculitis; those exposed to viable organisms developed markedly severe airsacculitis. Microscopic examinations revealed that membrane-induced lesions were generally similar to those resulting from infection but were less severe. At the termination of the study, 8 days after exposure, M. synoviae was isolated from respiratory tract tissues of all turkeys exposed to live organisms, but it was not isolated from any of those exposed to membranes or from unexposed control turkeys. Antibody against M. synoviae was demonstrated with the tube agglutination test in sera from turkeys exposed to membranes and those exposed to live organisms, but it was not demonstrated in sera from unexposed control turkeys.

Capsular groups of Pasteurella multocida isolated from avian hosts.

Avian strains of Pasteurella multocida representing a variety of host types, geographic locations, and somatic serotypes were examined to provide information on the distribution of capsular serogroups. Of the 246 strains studied, 166 were capsular group A, 4 were group B, 4 were group D, and 14 were group F; 58 strains were non-encapsulated and consequently not serogroupable. This is the first report of serogroup B P. multocida from avian hosts in the United States. The 188 serogroupable strains represented 12 variations in somatic serotypes and were isolated from 11 types of avian hosts representing 25 states of the United States, Bangladesh, Singapore, and Canada.

Effects of Pasteurella multocida endotoxins on turkey poults.

Studies of turkey poult responses to Pasteurella multocida endotoxins indicated that lipopolysaccharide (LPS) preparations from two highly pathogenic strains and free endotoxin from one of these strains were all similar in lethal toxicity. Lethal intravenous doses were generally high, 1 mg or more for 1-week-old poults (13.3 mg/kg). The toxic effects of LPS were not increased by repeated administration of small hourly doses. For both forms of endotoxin, the relationship between dose and response was considered erratic. Attempts to increase the susceptibility of poults to LPS by administering a liver-damaging substance (galactosamine) or a histamine-releasing substance (compound 48/80) or by performing surgical bursectomy were not effective. The LPS did not provoke a dermal Shwartzman reaction, even though doses used were 10 times those that produced a characteristic reaction in a rabbit.

Serogroup F, a new capsule serogroup of Pasteurella multocida.

Four capsule serogroups (A, B, D, and E) have been described by using passive hemagglutination tests. Serogroups A and D predominant in pasteurelloses of avian species. A new capsule serogroup of Pasteurella multocida has been isolated from turkeys in Arkansas, California, Indiana, Iowa, Missouri, New Jersey, and Virginia. Strains belonging to the new serogroup were somatic serotype 1, 3, 7, or 12, and they varied in virulence for mice and poults. Antisera made in rabbits passively protected mice against challenge with the same serogroup regardless of somatic serotype.

The effects of heat-labile Bordetella avium toxin on turkey poults.

Intraperitoneal exposure of turkey poults to various concentrations of Bordetella avium sonicate containing heat-labile toxin indicated a high degree of toxicity: poults died after exposure to sonicate containing as little as 6.32 micrograms of protein. The toxic effects were dose-related: poults that received sonicate containing 158 micrograms of protein died in 4 to 6 1/2 hours, those that received 31.6 and 6.32 micrograms of protein died in 25 to 30 hours, and those that received 1.2 micrograms survived the 6-day course of the study and were apparently unaffected. Histologic examination of poults that received lethal doses of the toxin revealed degeneration and necrosis of parenchymal cells of the liver and pancreas as well as hyperemia, hemorrhage, and necrosis of the mucosa of the small intestine. No lesions were observed in poults that received the sublethal dose of toxin or in unexposed poults. Repeated intranasal exposure of poults to the sonicate did not produce clinical signs of disease or gross lesions.

Turkey coryza: selected tests for detection and neutralization of Bordetella avium heat-labile toxin.

Bordetella avium heat-labile toxin (HLT) was lethal for poults, mice, and embryonating chicken eggs. It produced hemorrhagic lesions in turkey and guinea pig skin. Antiserum made in turkeys neutralized the lethality of the toxin and its ability to produce hemorrhagic skin lesions. Further, antiserum against HLT of an Ohio strain neutralized lethality of HLT of strains from Iowa, North Carolina, and West Germany. The antiserum did not neutralize lethality of HLT from B. bronchiseptica. Bordetella avium HLT was not ciliostatic for turkey tracheal-ring cultures and did not stimulate adenyl cyclase activity using mouse adrenal cell cultures.

Fowl cholera: influence of Bordetella avium on vaccinal immunity of turkeys to Pasteurella multocida.

Turkeys exposed to Bordetella avium were vaccinated against fowl cholera with live Pasteurella multocida vaccine. Previous exposure to B. avium resulted in impairment of systemic immunity conferred by the vaccine: 86% of the vaccinated turkeys exposed to B. avium at 1 day old developed lesions or died of fowl cholera after challenge at 15 weeks old with virulent P. multocida. Of vaccinated turkeys not previously exposed to B. avium, only 26% had lesions or died of fowl cholera.