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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.

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Differentiation of field isolates of Pasteurella multocida serotype 3,4 from live vaccine strain by genotypic characterization.

Fifty-five serotype 3,4 isolates of Pasteurella multocida, isolated from turkeys dead from fowl cholera, were characterized (fingerprinted) genotypically for comparison with the serotype 3,4 live fowl cholera vaccine principally used in turkeys in California. Twenty-three isolates were obtained from turkeys vaccinated with the M9 live vaccine, and 32 additional isolates were from turkeys not vaccinated for fowl cholera. Methods of characterization included restriction endonuclease analysis of chromosomal DNA and ribotyping, a technique for highlighting restriction site heterogeneity of highly conserved ribosomal RNA genes and associated sequences using a radiolabeled rRNA probe. Eight different genotypes or ribotypes were detected in these isolates by the above methods. Of 23 isolates from M9-vaccinated turkeys flocks, 19 were the same ribotype as M9. Thirty of 32 isolates recovered from unvaccinated turkeys were different ribotypes from M9. The remaining two isolates resembled M9 and were recovered from two different flocks placed in succession on a turkey farm where a flock placed previously had been vaccinated with M9, suggesting interflock transmission. Ribotyping and restriction endonuclease analysis appear to be useful tools to aid in the determination of the role that the live vaccine plays in fowl cholera epidemiology.

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Dermal necrosis caused by Pasteurella multocida infection in turkeys.

Severe dermal necrosis caused by Pasteurella multocida Serotype 1 was diagnosed in three dressed turkey carcasses and two live turkeys from a commercial flock. The dressed carcasses were among several condemned at a processing plant. The isolate, P. multocida Serotype 1, produced progressive dermal necrosis when experimentally inoculated into injured skin of turkeys. The organism was reisolated from the dermal lesions. The turkey houses were found to be infested by mice; the skin injury and infection with P. multocida probably originated from mouse bites.

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[Typing and virulence testing of Pasteurella haemolytica field strains].

The greater part of 145 typed Pasteurella haemolytica strains from calf could be attributed to Type A 1. Hence, in the context of virulence testing, this is the most important type at present for calf. Clearly manifest pneumonia was caused in calf by other strains of Types A 2, A 6, A 12, and T 10 which were also tested for their virulence parameters. The same applied to a number of strains which had so far been characterised merely as T or A/T strains.

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Screening pig herds for toxigenic Pasteurella multocida and turbinate damage in a health scheme for atrophic rhinitis.

The Pig Health Control Association launched a health scheme for atrophic rhinitis in 1978. For several years pig herds were monitored by scoring the degree of turbinate damage and by clinical inspections. When laboratory facilities became available for detecting toxigenic Pasteurella multocida, nasal swabs were taken from pigs in Association herds during 1988 and 1989 to determine whether the organism was present. Sows were screened routinely and in the worst affected herds, sucklers and weaners were also swabbed. In 12 of 19 herds with consistently low snout scores toxigenic P multocida were not isolated, and in 15 herds which developed higher snout scores with time toxigenic P multocida were also not found. Eleven herds had never been listed by the Association, either because their snout scores were consistently high or because they had received importations of stock from herds with high snout scores; of six of these herds with the most persistently high snout scores five showed varying degrees of the clinical signs of atrophic rhinitis, but none of the six showed evidence of infection with toxigenic P multocida, and the organism was not found in the other five herds in the group. There seems to be an overlap between the clinical and gross pathological signs of atrophic rhinitis seen in some herds not infected with toxigenic P multocida and the mild and spasmodic signs of atrophic rhinitis seen in some herds which are substantially infected with the organism.

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Serum antibody response to carbohydrate antigens of Pasteurella haemolytica serotype 1: relation to experimentally induced bovine pneumonic pasteurellosis.

The antibody responses to the capsular carbohydrate (CC) purified from Pasteurella haemolytica serotype 1 were determined by an ELISA, using 135 sera from 6 calves vaccinated with phosphate-buffered saline solution, formalin-killed P haemolytica bacterins, live P haemolytica, or an extract of P haemolytica referred to as carbohydrate-protein subunit (CPS). Calves vaccinated with live P haemolytica, bacterins, or CPS developed serum antibodies to CC. Bacterins containing Freund incomplete adjuvant or Freund complete adjuvant induced higher antibody responses than did bacterins containing aluminum hydroxide. In 4 of 6 experiments, high antibody responses to CC were significantly (P less than 0.05) correlated with resistance to transthoracic challenge exposure with P haemolytica. When calves were challenge exposed with a dose of P haemolytica that was 4.5 times greater than the standard challenge exposure dose or when calves that had been vaccinated with CPS were challenge exposed, antibody responses did not significantly (P greater than 0.05) correlate with resistance to challenge exposure. The amount of serum antibodies to CPS increased significantly (P less than 0.05) when calves were vaccinated with live or killed P haemolytica or with CPS, compared with that in calves given saline solution. In 5 of 6 experiments, correlation between high antibody responses and resistance to challenge exposure was significant (P less than 0.05). The correlation between those variables was not significant (P less than 0.07) for CPS-vaccinated calves. In the ELISA, treatment of CPS with sodium m-periodate, to oxidize periodate-sensitive carbohydrate epitopes, failed to markedly alter the antibody response to CPS.(ABSTRACT TRUNCATED AT 250 WORDS)

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Clinical and microbiologic findings in lambs inoculated with Pasteurella haemolytica after infection with ovine adenovirus type 6.

Colostrum-deprived lambs (10 to 20 days old) were inoculated with either ovine adenovirus type 6 (OAV-6; n = 6), Pasteurella haemolytica type A1 (n = 6), or OAV-6 followed by P haemolytica 5 days later (n = 10). Another group (n = 3) served as sham-inoculated controls. Lambs inoculated with OAV-6 or P haemolytica developed mild and moderate respiratory tract disease of 6 and 3 days' duration, respectively. Lambs inoculated with virus and bacteria developed clinical signs of respiratory tract disease of greater intensity and duration (9 days) than with either agent alone. Within 3 hours of bacterial inoculation, all lambs that received P haemolytica were anorectic, listless, and febrile, and had hyperpnea and dyspnea. Ovine adenovirus type 6 was isolated from all virus-inoculated lambs. Although P haemolytica was not recovered from all bacteria-inoculated lambs, it was recovered for a longer period in the group that received both agents. Antibody to OAV-6 was detected in virus-inoculated lambs as early as day 6 after inoculation. The control lambs remained clinically normal and neither virus nor bacteria were recovered at necropsy.

Adenoviridae↗

Infection of the middle nasal meatus of calves with Pasteurella haemolytica serotype 1.

Eight healthy nonstressed calves were inoculated with Pasteurella haemolytica serotype 1, by instilling a broth culture into the middle nasal meatus of the left nostril. The inoculated left nostrils shed P haemolytica from the ventral nasal meatus at a steady rate for a mean of 7 days, whereas the uninoculated right nostrils of the same calves shed P haemolytica sporadically and in lower concentrations. The duration, frequency, and concentration of P haemolytica shed from the inoculated nostrils was significantly (P less than 0.05) greater than from the nostrils of other healthy calves that had been exposed by instilling the culture into the ventral nasal meatus of both nostrils in a previous study. The concentration of antibodies (IgG, IgA, and IgM) to P haemolytica increased significantly (P less than 0.05) in serum and nasal secretions after exposure. Four weeks after initial P haemolytica exposure, calves were exposed to infectious bovine rhinotracheitis virus and became clinically ill. Four calves were induced to shed P haemolytica from both nostrils by the virus infection; thus, they were harboring the bacterium and were susceptible to active recolonization. Four calves were not induced to shed P haemolytica. The apparent reason was not that they were resistant to active colonization, but that they were no longer harboring the bacterium, because they became active shedders after they were reinfected with P haemolytica.

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Induction of pulmonary antibodies to Pasteurella haemolytica following intraduodenal stimulation of the gut-associated lymphatic tissue in cattle.

The induction of pulmonary antibodies to a bacterial antigen following intraduodenal (D) stimulation of the gut-associated lymphatic tissue (GALT) was investigated. Six calves were divided into two groups of three calves each. The GALT-primed calves received an ID dose of live Pasteurella haemolytica A1 followed by a subcutaneous (SC) dose of killed P. haemolytica. The sham-primed calves received an ID dose of phosphate-buffered saline solution (PBSS) followed by a SC dose of killed bacteria. Serum and pulmonary lavage fluids were collected weekly from each calf and assayed for titers of leukotoxin neutralizing antibodies (LNA), as well as IgG and IgA (lavage fluids only) to P. haemolytica. The GALT-primed calves responded to the ID stimulation by bacteria with increased serum IgG. The sham-primed calves had no change in antibody titers following ID stimulation. The GALT-primed calves had increased serum IgG, lavage IgG and IgA and increased LNA titers in both lavage fluids and serum following the SC dose of killed bacteria. The sham-primed calves demonstrated only an increase in serum IgG following the SC inoculation. A challenge study to evaluate if antibodies induced by GALT stimulation could reduce pulmonary lesions was performed using six calves divided into two groups. One group received an ID dose of P. haemolytica followed two weeks later by a SC dose of killed P. haemolytica. The sham vaccinated calves received an ID dose of PBSS followed in two weeks by a SC dose of killed bacterin. Calves were challenged by an intrapulmonary dose of live P. haemolytica A1 eleven days after the SC inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

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Pasteurella anatipestifer infections in California turkey flocks: circumstantial evidence of a mosquito vector.

Outbreaks of Pasteurella anatipestifer infections in California turkey flocks were investigated and found to have a seasonal distribution, with a peak incidence in fall, coinciding with peak Culex mosquito populations. An experiment was conducted to test the hypothesis that mosquitoes may serve as vectors for P. anatipestifer infections in turkeys. Four 7-week-old turkey poults were exposed for 7 days to mosquitoes captured from turkey barns during a field outbreak of P. anatipestifer serotype 1 infection. One turkey developed serum antibodies to serotype 1, detectable by enzyme-linked immunosorbant assay, and was resistant to an intravenous inoculation of P. anatipestifer serotype 1 at 4 weeks postexposure. Giemsa-stained blood smears from this bird and from three 7-week-old turkeys inoculated intravenously with P. anatipestifer revealed the presence of rod-shaped bacteria in or on the surface of host erythrocytes. No such rod-shaped bodies were found on erythrocytes of an uninoculated control turkey.

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Pasteurella multocida infection in Japanese quail (Coturnix coturnix japonica).

Three flocks of Japanese quail, approximately 75,000 birds each, experienced acute high mortality beginning at 24 to 28 days of age. Gross lesions were absent or were composed of either multifocal small pale areas on livers and spleens or lungs slightly darker in color than normal. Histopathology revealed multifocal splenic and hepatic necrosis and interstitial pneumonia. Pasteurella multocida, serotype 3,4, was isolated from affected tissues. The quail were successfully treated with chlortetracycline, and the organism was apparently eliminated from the premises by thorough cleaning, disinfection, and insect and rodent control. Experimental studies showed Japanese quail to be highly susceptible to disease caused by the P. multocida isolated from the affected flocks.

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Immunoperoxidase evaluation of pneumonic lesions induced by Pasteurella multocida in calves.

To evaluate the relationship between pneumonic lesions and distribution of bacteria, lungs from calves inoculated with Pasteurella multocida were examined histologically by use of immunoperoxidase technique. Pneumonic lesions fundamentally consisted of broncho-pneumonia with fibrinopurulent pleuritis. The lesions were confirmed to be associated with inoculated P multocida, using the immunoperoxidase technique. The P multocida antigen was detected not only in the bacterial clusters in the lesions, but also in the cytoplasm of infiltrating neutrophils and macrophages. Further, immunoelectron microscopy indicated that the inoculated bacteria generally were phagocytosed and digested by neutrophils.

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Characterization of Pasteurella haemolytica strains isolated from goats in Hungary.

Thirty-five Pasteurella haemolytica strains were isolated in Hungary from goat carcasses sent for postmortem examination from two farms with large goat flocks. All strains belonged to biotype A and with the exception of one strain of serotype A8 they belonged to serotype A2. No untypable strains were found by the indirect haemagglutination test.

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Antibodies to Pasteurella haemolytica somatic antigens in two models of the bovine respiratory disease complex.

Serum samples obtained from feeder calves before and after entry into the market system (days 0 to 7) were assayed for antibodies to Pasteurella hamolytica biotype A, serotype 1 capsular polysaccharide (CPS) and lipopolysaccharide/outer membrane protein (LPSp) by isotype in a kinetic-augmented, antigen-capture ELISA. These test results, plus indirect hemagglutination (IHA) antibody titers, and hemolysin-in-gel test (HIGT) findings were compared with clinical performance data during the initial 4 weeks in the feedlot (receiving period). High concentrations of HIGT antibody, at the point of initial assembly of feeder calves at weaning and during the subsequent 7-day marketing period, were associated with freedom from bovine respiratory disease (BRD) during the receiving period. High or rapidly increasing concentrations of anti-CPS IgG1 during the marketing period were also associated with less BRD. However, high concentrations of anti-LPSp IgG1 during the marketing period were associated with increased BRD during the receiving period. There was no correlation between the concentrations of antibody determined by IHA tests early in the marketing period and freedom from BRD during the receiving period. High concentrations of antibody determined by this test at entry into the feedlot (day 7) were associated with a high incidence of BRD. Calves vaccinated with a P haemolytica bacterin had significantly (P less than 0.05) higher HIGT values and concentrations of anti-LPSp IgG1 and IHA antibody than did nonvaccinated calves on entry into the feedlot (day 7). Vaccination appeared to have little effect on the amount of anti-CPS IgG1. Of all the tests used to quantitate antibody, the HIGT correlated best with clinical performance.(ABSTRACT TRUNCATED AT 250 WORDS)

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Effect of Pasteurella haemolytica (A1) capsular polysaccharide on sheep lung in vivo and on pulmonary surfactant in vitro.

Capsular polysaccharide (CP) of Pasteurella haemolytica (type A1) was first deposited by fiberoptic bronchoscopy into the lungs of sheep to examine lesions and changes in bronchoalveolar lavage cell populations and, later, was mixed with pulmonary surfactant to investigate alterations in physical properties or surface tension. At 22 hours after deposition, minimal lesions were seen in the lungs only at and contiguous to the site of CP deposition in 2 of 4 sheep. Microscopically, alveoli and interlobular septa were filled with edema fluid. Terminal airways and alveoli contained a moderate amount of neutrophils that varied between sheep. Significant differences in number or type of bronchoalveolar lavage cells were not observed in the weekly lavages between each group or among sheep within each group, either before or after deposition of CP or physiologic saline solution. After 6 hours of incubation at 37 C, CP-surfactant mixtures were examined with a surface tensiometer and centrifuged in sucrose gradients. The CP bound to surfactant, resulting in formation of a precipitate with a surface tension of 31.6 +/- 0.1 dynes/cm and a density of 1.07 to 1.08 g/ml. Lipopolysaccharide of P haemolytica, used as a control, also bound to surfactant, resulting in a complex with a surface tension of 57.7 +/- 0.4 dynes/cm and a density of 1.06 to 1.10 g/ml. Surfactant alone had a surface tension of 32.6 +/- 0.2 dynes/cm and density of 1.05 to 1.06 g/ml. The CP appears by itself not to be a direct major factor in the lung damage that develops in cases of pneumonic pasteurellosis.(ABSTRACT TRUNCATED AT 250 WORDS)

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Blood bactericidal assay (Pasteurella haemolytica) comparison of morbidity in marketed feeder calves.

An in vitro bactericidal assay that used bovine heparinized blood was investigated for its usefulness in detecting differences in the bactericidal immunity of calves against Pasteurella haemolytica serotype 1 (Ph1). Greater than 90% of killing occurred within 30 minutes. The substitution of fetal calf serum for autologous calf plasma caused loss of bactericidal activity of the blood. Decomplemented calf serum also was low in bactericidal activity. The blood bactericidal assay appears to be opsonin antibody-dependent and complement-dependent. The coefficient of variation (CV) that can be expected with this assay was established by use of a group of 8 calves; within-day CV maximum was 0.9, and between-day CV maximum was 2.1. The blood bactericidal assay was used to evaluate 30 calves under typical market stress from 4 farms in eastern Tennessee. All calves had decreased bactericidal activity, as they moved into a feedyard in Texas. The bactericidal activity was reduced among sick calves, based on the severity of clinical signs. Morbidity was highest during the first 14 days in the feedlot. During this period, healthy calves had a decreased bactericidal index (BI) of 4 points, and calves with clinical signs of bovine respiratory tract disease for 3 days had a decreased BI of 8 points. The average reduction in the BI of calves with clinical signs of bovine respiratory tract disease for 6 or more days was 14 points.

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Field trial of a live streptomycin dependent Pasteurella multocida serotype A:12 vaccine in rabbits.

A live, streptomycin dependent, Pasteurella multocida (SDPM) serotype A:12 vaccine was evaluated for preventing pasteurellosis in two commercial rabbitries. Rabbits were inoculated intranasally at 5 weeks old with either 0.25 ml of vaccine containing 10(8) colony forming units/ml or 0.25 ml of diluent (control). A proportion of rabbits received a second intranasal inoculation 1 month later. Partial protection against P. multocida infection was observed 1 and 2 months after inoculation in rabbits given only one dose of vaccine. The incidence of clinical signs of pasteurellosis was similar in vaccinated and nonvaccinated market-age rabbits inoculated 4 to 6 weeks previously. In does maintained in the breeding colony, P. multocida infection and upper respiratory disease occurred more frequently in vaccinated than nonvaccinated rabbits. Humoral antibody responses (IgA, IgM, IgG) followed longitudinally were similar in vaccinated and nonvaccinated does. Hence, the SDPM vaccine was not efficacious in controlling P. multocida infection at these two rabbitries.

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Characterization of outer membrane protein-enriched extracts from Pasteurella multocida isolated from turkeys.

Outer membrane protein (OMP)-enriched extracts of avian strains of Pasteurella multocida were examined by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Culture medium did not have a significant effect on the OMP profiles of strains of P multocida examined; however, in vivo propagation had an appreciable effect on the OMP profile composition of the reference strain P-1059. Such bacteria, expressed several additional OMP in the 27-kD, 48-kD, 56-kD, 60-kD, 80-kD, and 94-kD molecular mass regions. These OMP were not detected in the electrophorogram of strain P-1059 grown in vitro. The OMP profiles of reference strains of the 16 serotypes of P multocida did not identify any serotype-specific protein markers. Field strains of serotype A:3 had variation in OMP profiles and did not express OMP that all were identical to that expressed by the reference strain P-1059. The live attenuated CU and M9 bacterial vaccine strains expressed strain-specific OMP markers of 48-kD and 45-kD molecular masses, respectively. These strain-specific OMP markers may be used to differentiate these strains from virulent field strains that are of the same serotype and isolated from turkeys that have succumbed to pasteurellosis as a result of vaccine-related reactions or breakdown in immunity.

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