Vaccination of turkeys with cell-free culture filtrate of Pasteurella multocida: effects of dilution, iron chelation, and heterologous challenge.

Two experiments were done to further define cell-free culture filtrate (CCF) from Pasteurella multocida and its endotoxin content in protecting turkeys against challenge. In the first experiment, the greater-than-30,000-molecular-weight fraction of P. multocida strain R44/6 (serotype 3/4/9/12) CCF was used in 10-fold dilutions given by air-sac inoculation or aerosol to vaccinate turkeys, which were subsequently challenged with either homologous (P-1059, serotype 3) or heterologous (X-73, serotype 1) strains. Endotoxin content of the CCF fraction was high. Compared with positive controls given either live Clemson University vaccine or a commercial bacterin, homologous protection was provided by undiluted CCF and 1:10 dilutions of CCF, but there was no heterologous protection. In the second experiment, CCF of strain R44/6 in regular and iron-limiting media and CCF of strain FC127B (serotype 1/4) were used alone or in combination to vaccinate turkeys, which were challenged as in the first experiment. Homologous but not heterologous protection occurred, even though growth of strain R44/6 in iron-limiting media reduced endotoxin content of CCF by approximately 93%. These results indicate that endotoxin levels of less than 10% but greater than 1% of those in CCF from regular media are sufficient to induce protection in turkeys against homologous challenge but that CCF from either regular or iron-limiting medium does not provide protection against heterologous challenge.     

Evaluation of Pasteurella multocida mutants of low virulence. I. Development and pathogenicity

Mutagenesis of the Clemson University (CU) vaccine strain of Pasteurella multocida with N-methyl-N-nitro-N-nitrosoguanidine resulted in temperature-sensitive mutants that grew at 37 C but not at 42 C. Seven such mutants were evaluated for immunogenicity in turkeys. From these seven, only two, PM#1 and PM#3, provided turkeys with a level of protection against challenge with a virulent serotype 3 P. multocida strain (P-1059) comparable to the protection provided by the CU strain. Intravenous (IV) inoculation of PM#1, PM#3, or CU was used to assess differences in virulence. PM#1 and PM#3 resulted in lower rates of mortality and lameness than the CU strain. Histopathological evaluation of spleens 24, 48, and 72 hours after IV inoculation demonstrated that the CU strain induced significantly more fibrinoid necrosis of the spleen than either PM#1 or PM#3.     

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.     

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.     

Onset and duration of immunity and minimum dosage with CU cholera vaccine in turkeys via drinking water

Growing turkeys were partly protected against fowl cholera 4 days after vaccination with the live Clemson University (CU) strain of Pasteurella multocida administered in drinking water, and they were highly protected from 1 to 4 weeks after vaccination. The commercially available lyophilized vaccine and the freshly cultured vaccine of the CU strain did not differ in the level of immunity induced. Immunity was relatively high in turkeys vaccinated with 1:2 and 1:4 dilutions of the recommended dosage (4 X 10(8) P. multocida) but was significantly (P less than 0.05) lower in turkeys vaccinated with a 1:8 dilution of the recommended dosage. Immunity continued for 13 weeks after the last vaccination in turkeys vaccinated twice 3 weeks apart, but it persisted for only 8 weeks in those vaccinated only once.     

Pasteurella multocida infection in the domestic rabbit: immunization with a streptomycin-dependent mutant.

Fourteen Pasteurella multocida-free rabbits were inoculated intranasally with a streptomycin-dependent mutant of P. multocida serotype 12:A. Vaccinations with approximately 10(8) colony forming units were done on days 0, 14 and 28. Two weeks later the animals were separated into groups, which included 12 rabbits divided into two control groups of six unvaccinated Pasteurella-free animals. Seven vaccinated rabbits were challenged intranasally with the homologous virulent parent strain and the other seven vaccinates were challenged with a virulent strain of serotype 3:A. Rabbits were necropsied two weeks later. The vaccinated group challenged with the parent strain showed a more rapid nasal clearance of the organism than the vaccinated group challenged with the heterologous strain. However, the number of positive cultures of P. multocida recovered from tissues post-challenge were similar in vaccinated and control animals. In a significant number of animals, vaccination with serotype 12:A induced detectable antibody production to somatic antigens of both 12:A and heterologous strain 3:A.     

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.     

DNA fingerprinting of plasmid-containing serotype A: 3,4 Pasteurella multocida isolated from cases of fowl cholera in chickens and turkeys

The live, attenuated vaccine strains of Pasteurella multocida have been hypothesized to be responsible for homologous serotype outbreaks of fowl cholera on farms that use the commercial vaccines. We have further hypothesized that the naturally occurring Clemson University (CU) vaccine strain may be transformed to virulence by the acquisition of plasmid DNA. To test this hypothesis, we obtained seven homologous serotype (A:3,4) P. multocida isolates, all plasmid bearing, that were cultured from fowl cholera cases in vaccinated flocks and compared the isolates with the CU reference vaccine by molecular methods. Restriction fragment length polymorphisms (RFLPs) were detected by DNA/DNA hybridization with labeled probes specific for the cya, aroA, and rrn genes of P. multocida. The RFLPs obtained from BglII-digested genomic DNA probed with cya demonstrated no differences among the isolates. Although three isolates probed with aroA showed a RFLP identical to the vaccine strain, five isolates were distinctly different. Isolates probed with rrn grouped into three different restriction patterns that were dissimilar from that of the vaccine strain. Therefore, we have shown that these fowl cholera isolates are different from the CU vaccine strain and that these outbreaks were not vaccine related.     

Antigenic properties of four serotypes of Pasteurella multocida determined by enzyme-linked immunosorbent assay

Broiler minibreeder hens were used to produce monovalent antisera to bacterins prepared from serotypes 1, 3, 4, and 3 X 4 cross (CU strain) of P. multocida and to a polyvalent fowl cholera bacterin containing serotypes 1, 3, and 4. Antiserum to the CU strain (live vaccine) was also produced. Monovalent enzyme-linked immunosorbent assay (ELISA) plate antigens were prepared by separately sonicating each of the strains. Polyvalent plate antigen (Poly 3) was prepared by combining, in equal amounts after sonication, antigens from serotypes 1, 3, and 4. Each antiserum was assayed against its homologous ELISA plate antigen and against all other heterologous plate antigens, including Poly 3. The strongest reactions, as indicated by the highest absorbance values, were observed in homologous ELISAs. The CU strain may be the best monovalent ELISA plate antigen for detecting antibodies formed in response to a commercial polyvalent bacterin and to vaccinations with the live CU strain. Overall, monovalent serotype 1 (strain X-73) antiserum did not react well with any other heterologous ELISA plate antigen, whereas monovalent antisera of serotypes 4 (strain P-1662) and 3 X 4 (CU strain) reacted equally strongly with monovalent serotype 4 ELISA plate antigen. Background binding of negative serum was significantly lower (P less than 0.05) when using CU plate antigen than when using any of the other plate antigens.     

The pathogenesis of Pasteurella multocida serotype A:3,4 infection in turkeys: a comparison of two vaccine strains and a field isolate

The pathogenesis of avian pasteurellosis caused by two vaccine strains, M-9 and Clemson University (CU), and a highly virulent field isolate, 86-1913, of Pasteurella multocida (serotype A:3,4) was studied in 7-week-old turkeys inoculated by an oculo-nasal-oral technique. Turkeys inoculated with strain CU and isolate 86-1913 developed severe progressive bacteremia that began at 4 hours postinoculation (PI) and peaked at 16-20 hours PI. Turkeys inoculated with strain CU and isolate 86-1913 had significantly higher concentrations of bacteria in blood and tissues, and greater histologic lesion scores for necrosis, heterophil infiltrates, and intralesional bacteria than turkeys inoculated with strain M-9. Immunohistochemical staining specific for P. multocida demonstrated numerous extracellular bacteria in tissues from turkeys inoculated with strain CU and isolate 86-1913. The mortality for turkeys inoculated with isolate 86-1913 was significantly higher than for turkeys receiving the two vaccine strains.     

Prevention of experimental haemorrhagic septicaemia with a live vaccine

Pasteurella multocida serotype B:3,4 isolated from a fallow deer in England was used as a vaccine to prevent haemorrhagic septicaemia. The deer strain was less virulent for calves than typical serotype B:2 of haemorrhagic septicaemia strains. It elicited antibodies in cattle that protected mice against serotype B:2 infection. The live deer vaccine containing 2 X 10(7) viable organisms per dose was used to immunise calves. Six months after vaccination, five of six calves were protected against serotype B:2 challenge. Two calves challenged nine months after vaccination survived the same challenge. The live vaccine was more efficacious than an alum precipitated vaccine in protecting calves against B:2 challenge.     

Safety and efficacy of two live Pasteurella multocida aro-A mutant vaccines in chickens

Two auxotrophic aro-A mutants of Pasteurella multocida designated PMP1 (serotype 1) and PMP3 (serotype 3) were tested as vaccine candidates to protect chickens against fowl cholera. A reliable intratracheal challenge method was established that resulted in > or = 75% mortality in both specific-pathogen-free chickens and commercial broiler breeders 24 hr after challenge. Dose protection studies indicated that at least 10(6) colony-forming units (CFU) of PMP1 and 10(8) CFU of PMP3 were required to provide complete protection against challenge in all birds. Although high doses of 10(9) CFU of the vaccine strains produced some endotoxinlike reactions, lower but protective dose levels produced no clinical sign or lesion in any chicken. Both vaccine strains provided cross-protection with a heterologous challenge strain PM206 (serotype 4). Future studies will examine the duration of protective immunity induced by the two vaccine candidates, PMP1 and PMP3, and cross-protection against other serovars.     

DNA fingerprinting for differentiation of field isolates from reference vaccine strains of Pasteurella multocida in turkeys

The genomes from field isolates of Pasteurella multocida in turkeys and those of P multocida reference CU and M9 vaccine strains were analyzed and compared after cleavage with restriction endonucleases. The electrophoretic profiles obtained with DNA fragments from field isolates and vaccine strains of the same serotype were characteristic and reproducible. These features indicated the existence of differences among the isolates of the same serotype that cannot currently be detected, using available serotyping methods. However, several field isolates had electrophoretic profiles similar to those of either CU or M9 vaccine strain. It was concluded that restriction endonuclease analysis of DNA genomes from P multocida isolated from turkeys provides the information for differentiation of field isolates from vaccine strains of the same serotype.     

Transmission of Pasteurella multocida on California turkey premises in 1988-89.

Restriction endonuclease analysis (REA) of whole-cell DNA was used to determine possible sources of Pasteurella multocida for each outbreak of fowl cholera occurring in turkey flocks in eight commercial poultry companies in California from October 1988 to September 1989. Over this period, 179 isolates of P. multocida were obtained from dead turkeys in 80 meat and breeder flocks on 43 premises. P. multocida was isolated from wildlife on five premises. Isolates were characterized by subspecies, serotype, presence of plasmid DNA, and REA type. In 52 (65%) flocks, all isolates of P. multocida had the same REA pattern as the M9 live vaccine strain following digestion of DNA with the restriction enzyme SmaI. Field strains of P. multocida were obtained from 27 (34%) flocks, and one flock (1%) yielded both M9 and a field strain of the organism. REA of field strains of P. multocida revealed 17 different SmaI REA types. Based on matching SmaI REA types, potential sources of P. multocida were identified for 15 of the 28 flocks infected with field strains of the organism, and transmission between turkey premises was a possibility in only seven flocks.     

Comparison of live avirulent M-9, Minnesota, and CU fowl cholera vaccines

The M-9 and Minnesota (MN) avirulent Pasteurella multocida vaccines were evaluated and compared with the Clemson University (CU) vaccine, which had been shown to be highly effective in preventing fowl cholera in turkeys. Neither the M-9 nor the MN vaccine given in the drinking water was as effective as the CU vaccine in protecting turkeys against challenge with virulent P. multocida. When grown in brain-heart infusion (BHI) agar as recommended, the M-9 was not as efficacious as when it was grown in BHI broth. The M-9 was as effective as the CU vaccine only when grown in BHI broth and given at 10 times the standard dosage. Injection of the M-9 vaccine into the air spaces of the head at a site near the caudal rim of the ear after one vaccination in the drinking water was not as effective for hyperimmunizing potential breeders as was the CU vaccine injected at the same site. A microtiter agglutination test demonstrated a significant (P less than 0.05) correlation between the level of anti-P. multocida antibody found 1 week after vaccination and survival after challenge with virulent P. multocida.     

Serum resistance as an indicator of virulence of Pasteurella multocida for turkeys

Wildlife isolates of Pasteurella multocida, whose virulence for turkeys had previously been determined by intravenous inoculation, were characterized regarding their ability to survive incubation in fresh non-immune turkey serum. The relative virulence of the isolates was significantly associated with their ability to resist the bactericidal power of the serum as determined by standard plate counts following incubation. Organisms with a high survival value were more virulent; those with a low survival value were less virulent. A statistical model was specified and was successfully used to predict relative virulence of the P. multocida isolates. This method of assaying serum resistance was rapid, repeatable, and practical and could be performed with minimal laboratory equipment. Also studied was the serum resistance of seven serotype 3, 4 isolates obtained from the lungs of M9-vaccinated turkeys from seven flocks experiencing increased mortality due to fowl cholera. These isolates were shown to be identical to the M9 vaccine by restriction endonuclease analysis of chromosomal DNA. Six of the seven isolates had higher serum survival values than the original M9 vaccine.     

Molecular epidemiology of Pasteurella multocida in turkeys.

Pasteurella multocida isolated from turkeys during an outbreak of fowl cholera was characterized by serotype and heterogeneity of genes encoding rRNA (ribotype) to investigate the epidemiology of the organism. Isolates were collected between October 1985 and July 1986. The M9 or Clemson University fowl cholera vaccine-like strain was detected in 17% of the flocks with fowl cholera. One particular strain, isolated only from breeder flocks, was recovered from 7 of the 10 breeder flocks examined in this study. Intracompany transmission appeared to be common, implying a failure in biosecurity. Circumstantial evidence indicated that in the field; the incubation period of P multocida in a turkey flock may be between 2 to 7 weeks. Wildlife did not appear to be an important reservoir of P multocida for turkeys during this study period. Ribotyping results tended to discount several of the possible interflock transmissions, as suggested by examination of serotyping results alone; however, serotyping in combination with ribotyping proved helpful in understanding the epidemiology of P multocida in turkeys.     

Lysates of turkey-grown Pasteurella multocida: effects of solubilizing agents on the immunologic properties of membrane vesicles

Membrane vesicles from lysed suspensions of turkey-grown Pasteurella multocida were treated with various solubilizing agents to release protein that may contain cross-protection factor. Potassium thiocyanate, NaOH-glycine, lithium diiodosalicylate, guanidine hydrochloride, n-butanol, dimethyl sulfoxide, Triton X-100, and sodium lauryl sarcosinate were each tested as solubilizing agents. Vaccines made from combining solubilized membrane vesicles with complete lysate supernatant fluid produced various degrees of protection against challenge exposure with a heterologous serotype of P multocida in turkeys. Only vaccines prepared from membranes that were solubilized with potassium thiocyanate and sodium lauryl sarcosinate protected as well as complete lysate from turkey-grown P multocida. The amount of protein in each vaccine did not relate to protection. Distinct chemical differences were observed between lysates prepared from turkey-grown P multocida and lysates prepared from 41 C broth-grown P multocida. The external morphology of P multocida, after treatment with lysozyme and EDTA, was similar whether grown in broth or in turkeys.     

Protection of chickens by ribosomal vaccines from Pasteurella multocida: dependence on homologous lipopolysaccharide

Chickens were protected against fowl cholera by ribosomal vaccines prepared from noncapsulated Pasteurella multocida. Passive hemagglutination (PHA) titers to lipopolysaccharide (LPS) and the degree of protection conferred by ribosomal vaccines were diminished or abolished when ribosomes were chromatographed on an immunoadsorbent column. Addition of subimmunogenic amounts of serotype 1 (homologous) LPS to highly purified ribosomes resulted in vaccines that protected against challenge exposure and produced PHA titers to homologous LPS. Addition of serotype 5 LPS to highly purified ribosomes did not protect chickens against challenge exposure with serotype 1 P multocida, but produced PHA titers to serotype 5 LPS. Combinations of serotype 1 ribosomal RNA and serotype 1 (homologous) LPS did not protect chickens or produce PHA titers to LPS. Purified ribosomes from Brucella abortus, Aspergillus fumigatus, and chicken liver were combined with LPS from P multocida and were evaluated as vaccines. Brucella abortus and A fumigatus ribosomes combined with LPS protected chickens as well as did bacterin made from whole cells of P multocida. Chicken liver ribosomes combined with LPS did not provide protection. To determine whether a protein carrier would substitute for ribosomes, methylated bovine albumin (MBA) was combined with LPS and evaluated as a vaccine. A serologic response to LPS was induced by MBA-LPS vaccine, but the vaccine offered no better protection than when LPS was used alone as vaccine. Ribosome-LPS vaccines produced serologic responses to LPS that were at least 5-fold greater than those produced by MBA-LPS vaccine.     

Attenutated live fowl cholera vaccine. III. Laboratory and field vaccination trials in turkeys and chickens

Administered via the drinking water, M-3-G, an attenuated strain of Pasteurella multocida of serotype 1, was found to immunize turkeys and chickens against fowl cholera. Immunity was tested by challenging birds intramuscularly, by palatine cleft swab, or orally after 3 vaccinations. No reactions to vaccination were noted in 390 turkeys in 12 laboratory trials, nor in 20,245 vaccinated in field trials. Chickens showed no vaccination reactions, and immunity was elicited by challenge in a laboratory trial and in face of natural outbreaks in the field, where 11,600 chickens were vaccinated. No vaccination reactions were noted, although most birds involved in the trials were carrying Mycoplasma spp. Immunity was found to last about 10 weeks after the last vaccination. The immunizing properties of M-3-G are compared with the CU strain.