Fowl cholera vaccination of growing turkeys with CU strain via routes other than oral

Turkeys developed a high level of protective immunity and serum anti-Pasteurella multocida antibody when vaccinated with the Clemson University (CU) strain of P. multocida via the following routes: wing-web puncture with the recommended dosage or 1:10 dilution of this dosage, subcutaneous, crop injection, cloacal perfusion of the bursa of Fabricius, and intratracheal. The development of immunity after vaccination via the wing-web puncture and subcutaneous routes was dose-dependent: the lower dosages induced less serum antibody and protective immunity. Immunity was greatest 3 weeks after vaccination via wing-web puncture, although immunity was evident as early as 4 days after vaccination. A high level of antibody and protective immunity with no adverse reactions was induced by vaccinating twice, either by the drinking water followed by a similar dosage parenterally or by a parenteral route followed by the drinking water; these regimens were similar to one vaccination in the drinking water. Vaccination via wing-web puncture and possibly subcutaneous injection and bursal perfusion could be used for flocks with a history of a high mortality after vaccination with the CU vaccine in the drinking water and suspected of being immunosuppressed.     

Vaccination of turkey breeder hens and toms for fowl cholera with CU strain

Unvaccinated laying breeder hens and semen-producing toms were susceptible to the CU strain of Pasteurella multocida and highly susceptible to a virulent strain of P. multocida. Laying breeders vaccinated with CU strain when environmental temperatures were low ceased egg production during the first week after vaccination and had 29% mortality, whereas those vaccinated when temperatures were moderate had only a 25% decrease in egg production and 17% mortality. Comparable nonlaying breeders vaccinated during moderate temperatures did not die. Although few semen-producing toms died postvaccination and the quantity and quality of semen was not affected, 21.7% developed torticollis. Laying breeders were protected against CU vaccine and challenge with virulent P. multocida if vaccinated every 4 weeks beginning when 7 weeks old. Potential breeders vaccinated before laying with combinations of 3 vaccinations via drinking water, wing-web puncture, or inoculation into the air spaces of the head through the auditory tube were protected against challenge after the onset of laying. However, vaccination via wing-web puncture at 25 weeks of age resulted in abscesses that failed to resolve. The combination of vaccinations most effective in protecting laying breeders was vaccination in the drinking water at 7 and 11 weeks and inoculation into the air spaces of the head at 15 weeks.     

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.     

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.     

Effects of bursectomy, irradiation, and cyclophosphamide on turkeys vaccinated with CU cholera strain

Turkeys surgically bursectomized, irradiated, and/or injected with cyclophosphamide at 1 day were vaccinated with the live Clemson University (CU) strain of Pasteurella multocida. Bursectomized turkeys vaccinated via drinking water or wing-web puncture at 7 weeks and challenged at 11 weeks had a significantly (P less than 0.05) lower survival rate after challenge than unbursectomized controls. Bursectomized and unbursectomized turkeys vaccinated via drinking water at 7 weeks, revaccinated via the auditory tube at 11 weeks, and challenged at 15 weeks had similar survival rates. The vaccinated bursectomized turkeys had significantly (P less than 0.05) lower levels of serum anti-P. multocida antibody than vaccinated unbursectomized controls. Radiation had no immunosuppressive effect. The immunosuppressive effect of cyclophosphamide was dosage-dependent. Bursectomy and injection of cyclophosphamide in the same turkey were complementary. It was concluded that in young turkeys, the development of immunity to the avirulent CU vaccine is highly dependent upon the bursa of Fabricius, but that as they grow older the bursa is of less importance, particularly if they were vaccinated via a parenteral route, such as in the air spaces of the head.     

Relationship between anti-Pasteurella multocida antibody titers after CU vaccination and survival after challenge

The relationship between serum anti-Pasteurella multocida antibodies and survival rates after challenge was determined in turkeys vaccinated one or more times with the live avirulent Clemson University (CU) vaccine and then challenged with a virulent isolate (9481) of P. multocida in the drinking water. A microtiter agglutination test for assaying anti-P. multocida serum antibodies demonstrated a highly significant (P less than 0.001) correlation between the serum antibody titer 1 week after the initial or single vaccination and the survival rate after challenge, and a significant (P less than 0.01) correlation between the antibody titer immediately before challenge and the survival rate after challenge. A highly significant (P less than 0.0001) correlation was also observed between the antibody titer before vaccination and the survival rate after challenge. This relationship was considered the result of an anamnestic response by the CU vaccine to a previous sensitization by antigens of other microbial organisms, probably in the intestine and similar antigenically to P. multocida. In contrast, a significant (P less than 0.05) but negative correlation was seen between the antibody titer 1 week after challenge and the survival rate. This relationship was thought to be the result of a marked stimulation of the antibody titer by the systemic infection of P. multocida that subsequently killed the turkeys.     

Studies on Pasteurella multocida. IV. Immunofluorescence detection of the organisms in spleen and lungs of turkeys vaccinated with live oral vaccines

Oral vaccination of turkeys with live avirulent strains of P. multocida (M-2283 and CU strain) resulted in the local as well as systemic dissemination of the organisms. The persistence of P. multocida in the lungs and splenic tissues of these vaccinated turkeys was demonstrated by the indirect immunofluorescence technique. All of the tissues examined up to the first week post vaccination were P. multocida positive. Cryostat sections of lungs from birds vaccinated with the avirulent strain M-2283 were negative at 2 weeks post vaccination, while the spleen continued to be positive up to the third week post vaccination. In contrast to the group of turkeys vaccinated with strain M-2283, lung and spleen cryostat sections from turkeys vaccinated with strain CU remained positive up to the fourth week post vaccination. Tissues examined thereafter were negative for the presence of P. multocida from both groups.The major immune mechanism in the defense against fowl cholera is still unknown. If local immunity is primarily responsible, the CU strain may be the better vaccine strain as it persists in the lungs for 2 weeks longer than the M-2283 strain. However, if systemic immunity is chiefly responsible for immunity, both strains could protect equally, since they persist in the spleen for approximately the same period of time.     

Fowl cholera immunity induced by various vaccines in broiler minibreeder chickens determined by enzyme-linked immunosorbent assay

Broiler minibreeder hens were vaccinated for protection against fowl cholera at 12 and 21 weeks of age using several vaccination schemes, which included a live Pasteurella multocida (CU strain) vaccine, two commercial polyvalent fowl cholera oil-based bacterins, and two experimentally prepared polyvalent oil-based bacterins. Some treatment groups received only live or killed vaccines, whereas others received a live vaccine at 12 weeks followed by a killed product at 21 weeks. At 42 weeks of age, all birds that received the live CU vaccine twice or once followed by a bacterin survived challenge. Birds that received killed vaccines only were significantly less protected but still showed a respectable survival rate of 86%. All unvaccinated controls died within 72 hr after challenge. At 72 weeks of age, overall protection was lower than that at 42 weeks, regardless of vaccination treatment. Antibody titers were usually higher in birds that received bacterins than in those receiving live vaccines, yet overall protection was still greater in those birds that received the live cholera vaccine twice.     

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.     

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.     

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.     

Effects of adjuvant-augmented germling vaccines in turkey poults challenged with Aspergillus fumigatus

Turkey poults were vaccinated with combinations of two different germling preparations and three adjuvants (N-acetylmuranyl-L-alanyl-D-isoglutamine, Pasteurella multocida lipopolysaccharide [LPS], and avridine) at 1 and 2 weeks of age, and their immunity was challenged by sublethal exposure to aerosols of Aspergillus fumigatus conidia at 1 month of age. Fewer turkeys in the groups given vaccines prepared from germlings grown on Dorset's and Henley's medium (D&H) had organisms in lung tissue at 2 weeks after challenge exposure as compared with those vaccinated with germling grown on neopeptone dialysate (Neo). The LPS of P. multocida appeared to be the most efficacious of the adjuvants in the D&H vaccine group, as A. fumigatus was isolated from only one of eight turkeys in this group; the number of organisms per gram of lung tissue was low compared with other vaccine groups at 2 weeks after challenge exposure; and poults given D&H vaccine with LPS as adjuvant had less-severe lung lesions than other groups. These differences in lung lesions were more marked at 2 weeks than at 8 weeks after challenge exposure. The only difference among other parameters in the vaccinated turkeys was lower heterophil counts in the turkeys given D&H-prepared vaccines than in unvaccinated controls. This was probably due to less-severe infections resulting from protective effects of these vaccines.     

Attenuated live fowl cholera vaccine. I. Development of vaccine strain M3G of Pasteurella multocida

A live cholera vaccine was developed from a virulent avian septicemia strain of Pasteurella multocida serotype 1. The virulent parental strain was mutagenized with N-methyl-N'-nitro-N-nitroso guanidine. Mutants were selected that had either smaller colonies at 37 C or temperature sensitivity for growth at 41 C. Four small-colony mutants and 2 temperature-sensitive mutants were studied. All the mutants were avirulent for turkeys. Sixteen days after turkeys were vaccinated with each mutant, both the vaccinates and unvaccinated controls were challenge-exposed to virulent P. multocida of the homologous serotype and the heterologous serotype 3. Two of the small-colony mutant strains protected against both homologous and heterologous challenge. Suggested for a live cholera vaccine is P. multocida M3G, a small-colony-forming mutant, innocuous for both mice and turkeys and stable against reversion.     

Immunization of turkey breeder hens against fowl cholera by combined oral and wing-web administration of attenuated (CU) pasteurella multocida

Turkey breeder candidates were exposed to attenuated Pasteurella multocida (Clemson University strain) via both mouth (one or three times) and wing-web stick (one or two times). Significant protection lasting to 25-30 weeks post-vaccination was conferred under such immunization programs. The best protection with the fewest adverse effects of vaccination was established when orally vaccinated turkeys were subsequently vaccinated via wing-web at 20 and 25 weeks of age. High doses of attenuated P. multocida via wing-web produced lameness (synovitis and osteomyelitis) and severe wing lesions in growing turkeys.     

Evaluation of Pasteurella multocida mutants of low virulence. II. Immunologic response of turkeys

Vaccination of turkeys by administering Pasteurella multocida mutant PM#1 or PM#3, either by the oculo-nasal-oral method or in the drinking water, induced a level of protection comparable to vaccination with the Clemson University (CU) strain or the M-9 vaccine. The level of protection was not altered when PM#1, PM#3, or the CU strain was grown in brain-heart infusion (BHI) broth or BHI agar. Under extremely severe challenges, the CU strain provided a greater level of protection than PM#1, PM#3, or the M-9 vaccine. It also was apparent from this study that the less-virulent mutant organisms and the M-9 vaccine require a higher concentration of organism per vaccine dose than the CU strain to provide similar protection.     

Comparison of vaccination protocols of broiler breeder hens for Pasteurella multocida utilizing enzyme-linked immunosorbent assay and virulent challenge

A commercial enzyme-linked immunosorbent assay (ELISA) to detect serological response to vaccination and virulent challenge with type 1 (X-73) Pasteurella multocida was used to determine the best vaccination protocol for broiler breeders against fowl cholera. Birds vaccinated twice, at 10 and 19 weeks of age, with the avirulent Clemson University (CU) strain both times, with a commercial bacterin first and the CU strain second, or with the CU strain first and bacterin second had the highest survival rates (98-100%) following challenge at 25 weeks. The two groups that received the CU strain and bacterin also produced the highest mean ELISA antibody titers (greater than 10,000). Birds vaccinated once, at 10 weeks, with the CU strain had the same survival rate as birds vaccinated twice with bacterin (90 and 91%). Under the conditions of this experiment, an ELISA titer greater than or equal to 1000 resulted in at least a 92% survival rate after virulent challenge (23% survival in nonvaccinates).     

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.     

Efficacy and duration of immunity of a combined equine influenza and equine herpesvirus vaccine against challenge with an American-like equine influenza virus (A/equi-2/Kentucky/95)

It has been recommended that modern equine influenza vaccines should contain an A/equi-1 strain and A/equi-2 strains of the American and European-like subtype. We describe here the efficacy of a modern updated inactivated equine influenza-herpesvirus combination vaccine against challenge with a recent American-like isolate of equine influenza (A/equine-2/Kentucky/95 (H3N8). The vaccine contains inactivated Influenza strains A-equine-1/Prague'56, A-equine-2/Newmarket-1/'93 (American lineage) and A-equine-2/ Newmarket-2/93 (Eurasian lineage) and inactivated EHV-1 strain RacH and EHV-4 strain V2252. It is adjuvanted with alhydrogel and an immunostim. Horses were vaccinated at the start of the study and 4 weeks later. Four, six and eight weeks after the first vaccination high anti-influenza antibody titres were found in vaccinated horses, whereas at the start of the study all horses were seronegative. After the challenge, carried out at 8 weeks after the first vaccination, nasal swabs were taken, rectal temperatures were measured and clinical signs were monitored for 14 days. In contrast to unvaccinated control horses, vaccinated animals shed hardly any virus after challenge, and the appearance of clinical signs of influenza such as nasal discharge, coughing and fever were reduced in the vaccinated animals. Based on these observations, it was concluded that the vaccine protected against clinical signs of influenza and, more importantly, against virus excretion induced by an American-like challenge virus strain. In a second experiment the duration of the immunity induced by this vaccine was assessed serologically. Horses were vaccinated at the start of the study and 6 and 32 weeks later. Anti-influenza antibody titres were determined in bloodsamples taken at the first vaccination, and 2, 6, 8, 14, 19, 28, 32, 37, 41, 45 and 58 weeks after the first vaccination. Vaccinated horses had high anti-influenza antibody titres, above the level for clinical protection against influenza, against all strains present in the vaccine until 26 weeks after the third vaccination.     

Fowl cholera immunity in broiler breeder chickens determined by the enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies in broiler breeders vaccinated (wing web) with the CU fowl cholera vaccine. Birds were bled weekly from 9 to 26 weeks, every other week from 26 to 40 weeks, and every 4 weeks from 40 to 56 weeks of age. Overall mean ELISA antibody titers (9 to 56 weeks) reported as log10 values and survivability of the vaccinates after virulent challenge were as follows: unvaccinated--5.75, 48%; birds vaccinated once at 8 weeks--5.91, 78%; birds vaccinated twice at 8 and 14 weeks--6.11, 100%; birds vaccinated thrice at 8, 14, and 20 weeks--6.23, 100%; birds vaccinated twice at 8 and 20 weeks--6.12, 100%; and birds vaccinated twice at 8 and 20 weeks (plus fowl pox at 8 weeks)--6.08, 95%. Survivability in the vaccinates after virulent challenge with strain X-73 Pasteurella multocida was 100% in birds with ELISA antibody titers (log10) greater than 6.02. Under the conditions of this experiment, birds vaccinated two or three times between 8 and 20 weeks proved to be sufficiently immune at 56 weeks of age to withstand a virulent fowl cholera challenge. Birds not vaccinated or vaccinated only once at 8 weeks were not sufficiently immunized to withstand virulent challenge.     

Safety, efficacy and cross-protectivity of a live intranasal aerosol haemorrhagic septicaemia vaccine

The safety, efficacy and cross-protectivity of a live intranasal aerosol haemorrhagic septicaemia vaccine containing Pasteurella multocida serotype B:3,4 were tested in young cattle and buffaloes in Myanmar, where more than 1.5 million animals had been inoculated with this vaccine between 1989 and 1999. A recommended dose of 2 x 10(7) viable organisms was used for the efficacy test. The administration of 100 times the recommended dose to 50 cattle and 39 buffalo calves was innocuous. Seven months after they were vaccinated, three of three buffaloes were protected and 12 months after they were vaccinated, three of four buffaloes were protected against a subcutaneous challenge with serotype B:2 which killed three of three unvaccinated buffaloes. Twelve months after they were vaccinated, eight of eight cattle survived a serotype B:2 challenge, which killed four of four unvaccinated controls. The vaccinated cattle had developed serum antibodies detectable by the passive mouse protection test. Indirect haemagglutination tests on sera taken from cattle 10 days and five weeks after they were vaccinated showed high titres of antibodies. The serum of vaccinated cattle cross-protected passively immunised mice against infection with P. multocida serotypes E:2, F:3,4 and A:3,4.