Protective immunity to infectious bronchitis in broilers vaccinated against Marek's disease either in ovo or at hatch and against infectious bronchitis at hatch.

Two experiments were conducted using commercial broiler chickens to determine if Marek's disease (MD) vaccines HVT/SB-1 and HVT plus CVI-988 given either in ovo or at hatch adversely affected the efficacy of infectious bronchitis (IB) vaccines (Ark and Mass serotypes) given by eyedrop on the day of hatch. Nonvaccinated negative controls and controls that received only IB vaccines were included in each study. Birds were challenged with either infectious bronchitis virus (IBV) Mass-41 or IBV Ark-99 on either day 26 or 27 of age. Protection was assessed 5 days post-IBV challenged by virus isolation from the trachea. The day of hatch mean antibody titer to IBV was 12,668 +/- 4704 and 2503 +/- 3243 by enzyme-linked immunosorbent assay in experiments 1 and 2, respectively. In each study, nonvaccinated controls had a significantly higher (P < or = 0.05) incidence (88%-100%) of IBV challenge virus isolation than did controls vaccinated for IB but not for MD. Analysis of data from both studies showed that protection to IB in groups that received only IB vaccines at hatch ranged from 55.0% to 77.3%, whereas protection to IB in groups receiving both MD and IB vaccines ranged from 50.0% to 95.5%. In both experiments and within IBV challenge serotype, broilers given MD vaccines (in ovo or at hatch) and IB vaccines at hatch had protection rates to IBV challenges that were not significantly less (P < or = 0.05) than IB protection rates of groups that received only IB vaccines at hatch. Analysis of these data shows that administration of high-titered MD vaccines either in ovo or at hatch did not affect the efficacy of an IB vaccination (serotypes Ark and Mass) given by eyedrop at hatch.

Efficacy of a novel infectious bursal disease virus immune complex vaccine in broiler chickens.

Two experiments were conducted to test the efficacy of a novel infectious bursal disease virus (IBDV) vaccine in broiler chickens with maternal IBDV immunity. The IBDV vaccine was formulated by mixing IBDV strain 2512 with bursal disease antibodies (BDA) to produce the IBDV-BDA complex vaccine. In Expt. I, 1-day-old Cobb x Cobb broiler chickens were vaccinated subcutaneously with either IBDV-BDA or commercial live intermediate IBDV vaccine (vaccine A) or were left unvaccinated. In Expt. 2, the vaccine A group was not included; instead, IBDV strain 2512 was included. Chickens were maintained in isolation houses. On day 28 (Expt. 1) and day 32 (Expt. 2) of age, chickens from each group were challenged with a standard USDA IBDV (STC strain) challenge. Challenged and unchallenged chickens were evaluated for their bursa/body weight ratios and antibody titers 3 days post-challenge. Bursae collected from Expt. 2 were examined histologically to evaluate bursal lesions and confirm gross examination. None of the unvaccinated chickens was protected against the challenge virus as evidenced by the presence of acute bursal lesions (edema/hemorrhage). All chickens receiving the IBDV-BDA complex or the IBDV strain 2512 (Expt. 2) were protected from the challenge virus as evidenced by no acute bursal lesions. Additionally, chickens receiving the IBDV-BDA complex vaccine or the IBDV strain 2512 had antibody titers to IBDV, indication the presence of an active immune response. In Expt. 1, chickens vaccinated with vaccine A and challenged had bursal lesions similar to those observed in the unvaccinated, challenged chickens. These chickens also showed no indication of active immunity against the virus. These results suggest that the 1-day-of-age-administered IBDV-BDA complex vaccine can induce active immunity and protection against a standard IBDV challenge in the face of variable levels of maternal IBDV immunity.

Antibody responses of chickens to inoculation with Mycoplasma gallisepticum membrane proteins in immunostimulating complexes.

Membrane proteins of Mycoplasma gallisepticum (MG) strain R were extracted with the detergent Mega-10 and incorporated into immunostimulating complexes (ISCOMs). A membrane protein of approximately 64 kD (p64) molecular weight was a major component of MG ISCOMs. Six-week-old specific-pathogen-free leghorn chickens were inoculated by various routes (subcutaneous; combined intranasal and eyedrop; and combined subcutaneous, intranasal, and eyedrop) with 10 micrograms MG proteins in ISCOMs, or inoculated subcutaneously with 10 micrograms MG proteins in Freund's adjuvant. Subcutaneous inoculation of MG ISCOMs, or MG Freund's adjuvant resulted in higher sero-positive rates (detected by enzyme-linked immunosorbent assay) in serum and respiratory tract washings, compared to combined routes of MG ISCOM inoculation. In chickens inoculated subcutaneously with MG ISCOMs antibodies were first detected at 31 days postinoculation (PI) and the sero-positive rate peaked at 56 days PI. Sero-positive rates started to decline at day 64 PI. In the Freund's adjuvant group, MG antibodies were first detected at day 21 PI, and the sero-positive rate peaked at day 39 PI and did not decline. MG antibodies were detected by ELISA in upper respiratory tract and tracheal washes from chickens inoculated subcutaneously with MG ISCOMs and MG Freund's adjuvant. Immunoblots to MG strain R whole cell proteins showed that respiratory tract washings and sera from chickens inoculated subcutaneously with MG ISCOMs contained immunoglobulins to MG proteins, with a prominent reaction to p64.

Protective immune response to Mycoplasma gallisepticum demonstrated in respiratory-tract washings from M. gallisepticum-infected chickens.

Chickens inoculated with Mycoplasma gallisepticum (MG) produced IgA, IgM, and IgG detectable in washings from the upper respiratory tract (URTW; nasal sinuses and turbinates) and lower respiratory tract (LRTW; trachea, lungs, and air sacs). URTW and LRTW from infected chickens had significant protective effects in a MG-inoculated tracheal-ring-organ-culture system. Protective effects in vitro correlated positively with total MG-specific immunoglobulin titer, but not IgA titer, as determined by enzyme-linked immunosorbent assay. URTW and LRTW from infected chickens inhibited attachment of MG to tracheal-ring-organ cultures in a dose-dependent manner. This suggests that chickens produce a protective immune response to MG that locates in the respiratory tract and that attachment inhibition may be responsible for this protective effect.

Inhibition of Mycoplasma gallisepticum growth and attachment to chick tracheal rings by antibodies to a 64-kilodalton membrane protein of M. gallisepticum.

A Mycoplasma gallisepticum (MG) strain R protein of 64 kilodaltons (p64) was partially digested from the surface of the bacterium by trypsin. Monospecific polyclonal anti-p64 IgG inhibited attachment of MG to chick tracheal rings by as much as 69%. However, trypsin treatment of viable MG cells did not reduce attachment to tracheal rings or hemagglutination titer. Anti-p64 IgG inhibited growth of MG strain R in broth and on solid media, inhibited the uptake of radiolabeled thymidine, but did not inhibit hemagglutination. Anti-p64 IgG inhibited growth of eight MG strains on solid medium. The degree of growth inhibition varied widely depending on the strain and correlated positively with the reported virulence of the MG strains with one exception (A5969). An IgG monoclonal antibody to p64 (MyG 001) inhibited growth of MG strain R on solid and in broth media. The strong attachment-inhibition activity of anti-p64 IgG may result from its growth-inhibiting activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of MG strains suggested that p64 is expressed in higher amounts in vitro in virulent strains (R, S6) than in strains of low virulence (F, M876, K503, K703, K730). P64 should be used to immunize chickens to determine if it can stimulate a growth and attachment-inhibiting response in the respiratory tract.

Clinical Mycoplasma gallisepticum infection in multiplier breeder and meat turkeys caused by F strain: identification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, restriction endonuclease analysis, and the polymerase chain reaction.

In February 1991, a flock of North Carolina multiplier breeder turkeys experienced respiratory signs, sinusitis, airsacculitis, and increased mortality. Mycoplasma gallisepticum (MG) was isolated, and appropriate control measures were initiated. Ultimately, this outbreak involved several breeder flocks of an integrated turkey production company before the last infected flock was identified in May 1991. During this time, MG was also isolated from a flock of commercial layer-type chickens raised as pullets in close proximity to the index turkey flock. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and restriction endonuclease analysis indicated that these isolates were identical to each other and to examples of the vaccinal F strain. Additionally, MG isolates from the affected turkey breeder and layer flocks were identified as MG F strain by use of an F strain-specific DNA probe and polymerase chain reaction. A separate outbreak of MG disease in several meat-turkey flocks of a Midwest producer/processor yielded isolates identified as F strain by the polymerase chain reaction. These studies demonstrated: 1) the utility of newer technologies for disease outbreak investigations; and 2) the potential of MG F strain to cause disease in breeder and meat turkeys under field conditions.

Efficacy of danofloxacin and tylosin in the control of mycoplasmosis in chicks infected with tylosin-susceptible or tylosin-resistant field isolates of Mycoplasma gallisepticum.

The efficacy of danofloxacin was compared with that of tylosin in the control of induced mycoplasmosis. In three experiments, disease was induced in broiler chicks by intrapulmonary injection of field isolates of Mycoplasma gallisepticum (MG) originating from Brazil, a different isolate being used for each experiment. Starting the day after inoculation, groups of chicks were medicated for 3 days via the drinking water with danofloxacin (50 ppm) or tylosin (500 ppm) or were left as unmedicated controls. Chicks were observed for 21 days. A severe mycoplasmosis was induced in unmedicated birds, characterized by mortality, depression, and respiratory signs. Danofloxacin was highly efficacious in controlling this infection in all three experiments, whereas tylosin was efficacious in only one. This difference could be related directly to the reduced in vitro susceptibility to tylosin displayed by the two isolates of MG. In the two experiments where isolates were tylosin-resistant, danofloxacin was significantly (P < or = 0.05) superior to tylosin in reducing mortality, maintaining weight gain, and reducing the prevalence of air-sac lesions, isolation of MG, and seroconversion.

A comparison of danofloxacin and tylosin in the control of induced Mycoplasma gallisepticum infection in broiler chicks.

Experimental mycoplasmosis was induced in 1-day-old chicks by intrapulmonary inoculation of Mycoplasma gallisepticum (MG). This method of infection proved to be useful for evaluating the efficacy of antimicrobial medication, by measuring mortality, weight gain, pathological responses, frequency of reisolation of MG, and seroconversion. Using this model, the efficacies of danofloxacin (a novel fluoroquinolone) and tylosin were compared for two MG isolates, a reference isolate (the R-strain) and a field isolate from California. Danofloxacin administered in the water at 50 ppm for 3 days was equivalent to tylosin at 500 ppm for 3 days in the degree of control of mortality and maintenance of weight gain. Danofloxacin was superior to tylosin in preventing air-sac lesions, reducing the frequency of reisolation of MG, and preventing seroconversion in surviving birds.

Comparison of Mycoplasma synoviae isolates by immunoblotting.

SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot profiles using antisera from M. synoviae F10-2AS-inoculated chickens detected heterogeneity among 10 M. synoviae isolates. This variation was primarily seen in immunoblots as a difference in the molecular size of immunoreactive proteins below 50 kDa and corresponded to an area of variability seen in SDS-PAGE. No two isolates had the same immunoblot profile below 50 kDa. Hyperimmune antiserum to a M. synoviae F10-2AS protein of 41 kDa (p41) reacted with protein(s) ranging from 41 to 48 kDa in nine of 10 isolates and strongly with one of 87 kDa in one isolate. The immunoreactivity of antiserum from inoculated chickens and the hyperimmune antiserum to p41 varied from intense to weak with the 10 M. synoviae isolates. All of the highly immunogenic proteins of M. synoviae F10-2AS, including p41, partitioned into the Triton X-114 detergent phase, indicating that they are amphiphilic in nature and integral membrane proteins. The data suggest that M. synoviae is capable of varying the molecular size of a highly immunogenic integral membrane protein.

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.

Breeder turkey hens seropositive and culture-negative for Mycoplasma synoviae.

Four flocks of clinically normal turkey breeder hens were shown to have suspect and positive Mycoplasma synoviae (MS) hemagglutination-inhibition (HI), enzyme-linked immunosorbent assay, and, in some cases, serum plate agglutination serology in the absence of MS isolation. In all cases, HI serology for Mycoplasma gallisepticum (MG) and M. meleagridis was negative. Acholeplasma laidlawii was isolated from some hens in each of these MS-seropositive culture-negative flocks. Immunoblotting was used to help determine if this positive MS serology was a result of cross-reactive antibodies to A. laidlawii or to some other Mycoplasma species. When sera from two of the flocks were reacted with MS antigen in immunoblotting, a strong and characteristic MS immunoblot profile was seen. Immunoblotting gave no evidence of a strong antibody response to A. laidlawii, M. iowae, or MG. This suggests the presence (or earlier presence) of MS in these flocks that is difficult to isolate by routine methods. Furthermore, this work shows that immunoblotting can be an important tool in the diagnosis of poultry diseases.

An outbreak of Mycoplasma synoviae infection in North Carolina turkeys: comparison of isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and restriction endonuclease analysis.

Mycoplasma synoviae (MS) isolates made in 1988-89 from turkey flocks in North Carolina, Missouri, and Ontario, Canada, were compared with each other and MS reference strains (WVU-1853, F10-2AS, Neb-3S, and K1968) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of cell proteins and restriction endonuclease analysis (REA) of DNA. SDS-PAGE and REA indicated considerable homology among MS reference strains and recent field isolates. However, sufficient differences were resolved to identify the MS reference strains as different from each other and the field isolates, and to classify seven of nine recent field isolates as a cluster of nearly identical strains. The results suggest that flocks infected with members of the cluster were epizootiologically associated, possibly by a common or point source of infection.

Vaccination of turkeys with cell-free culture filtrate of Pasteurella multocida: effects of ultrafiltration and endotoxin removal.

Cell-free culture filtrate (CCF) of Pasteurella multocida strain R44/6 (serotype 3/4/9/12) was fractionated by ultrafiltration into fractions of less than 10,000, greater than 10,000, greater than 30,000, and 10,000 to 30,000 molecular weight (MW). The less-than-10,000-MW fraction contained little endotoxin comparable to bacteriologic medium; the 10,000-to-30,000-MW fraction had a moderate amount of endotoxin, whereas the greater-than-10,000- and greater-than-30,000-MW fractions contained high levels of endotoxin. Following ultrafiltration, each fraction, except the less-than-10,000-MW fraction, was divided into two equal parts, and endotoxin was removed from one part. Turkeys were vaccinated with the various MW fractions of CCF, with and without endotoxin, via the air sacs at 6 and 9 weeks of age and compared with negative controls given bacteriologic medium and positive controls vaccinated with a commercial bacterin. Before oral challenge with strain P-1059 (serotype 3) at 12 weeks of age, antibody titers were detected only in positive control turkeys. Protection against challenge, as measured by post-challenge mortality and body-weight gain, was provided by the greater-than-10,000-, greater-than-30,000-, and 10,000-to-30,000-MW fractions containing endotoxin and the commercial bacterin. Turkeys that had been vaccinated with bacteriologic medium and the four different fractions without endotoxin were not protected. Results indicated that endotoxin in CCF of P. multocida is critical in protecting turkeys from pasteurellosis.

Humoral immune response of turkeys to strain S6 and a variant Mycoplasma gallisepticum studied by immunoblotting.

Two putative variant Mycoplasma gallisepticum (MG) strains (M876 and M35), originally isolated from commercial turkeys, were compared with eight well-characterized MG strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE protein profiles indicated that the variant strains were correctly classified as MG based on homologous patterns in species-specific regions of the electrophoretic profiles. However, differences in protein profiles also indicated that variant strains M876 and M35 were different from each other and the other MG strains tested. Immunoblotting was used to assess the humoral immune response of turkeys to infection with the S6 reference strain or M876 variant strain of MG. Immunoblots using antisera to M876 showed that seroconversion to this isolate was slower, and to fewer MG proteins when compared with immunoblots using antisera to S6. Immunoblot analyses further indicated that pooled antisera from turkeys inoculated with either S6 or M876 reacted with each of 10 MG strains tested. However, pooled S6 antisera reacted with greater intensity and with more MG proteins than did pooled M876 antisera. The species-specific immunodominant proteins with the greatest potential for use as antigens in serologic tests appeared to be those of 64 (p64) and 56 (p56) kilodaltons molecular mass.

Comparison of Mycoplasma gallisepticum strains and identification of immunogenic integral membrane proteins with Triton X-114 by immunoblotting.

Pooled chicken antisera from 33 and 77 days post Mycoplasma gallisepticum strain R contact-exposure reacted with cell proteins of 19 M. gallisepticum strains. These pooled antisera reacted with more proteins and with greater intensity to reference strains (R, PG31, S6, and A5969) and nine field strains than they did with six other field strains including three (703, 503, and 730) that have been described as serological variants. Following extraction with Triton X-114 the majority of immunogenic M. gallisepticum proteins partitioned exclusively or primarily into the detergent phase indicating that they are integral membrane proteins. This included three immunogenic species-specific proteins (p64, p56 and p26). M. gallisepticum p56 was detected, by immunoblotting, in 18 of 19 strains suggesting that it could serve as an antigen for serological tests. P26 was evident in 13 of 19 strains. Hyperimmune antiserum to p64 reacted with a 64 kDa protein in 19 M. gallisepticum strains, but did not react with seven other avian Mycoplasma spp. There was no evidence found supporting the view that p64 is the hemagglutinin of M. gallisepticum.

Response of broiler chickens to fowl cholera vaccination at 1 to 6 weeks of age.

Broiler chickens, in groups of 10, received a single vaccination by the stick-wing route at 1, 2, 3, 4, 5, 6, or 11 weeks of age with live Clemson University strain of Pasteurella multocida. Twenty non-vaccinates kept in isolation served as controls. Cholera serum antibody titers in all chickens were determined by enzyme-linked immunosorbent assay at weekly intervals. Chickens vaccinated once at 1, 2, 3, 4, 5, and 6 weeks, respectively, attained 25.2%, 28.7%, 34.7%, 46.2%, 51.8%, and 64.6% of the titers of those vaccinated once at 11 weeks of age. Variation in antibody response was greatest in chickens vaccinated at 1 or 2 weeks of age. Additionally, chickens vaccinated at 1 or 2 weeks of age showed the longest response time (5 weeks) to reach maximum antibody titers after a single vaccination. When the original vaccinates were revaccinated at 11 weeks of age, all showed a secondary response equal to or greater than that seen in chickens vaccinated once at 11 weeks of age. Age of the chickens at the time of vaccination and antibody titer were positively correlated (r = 0.997). Overall antibody responses to vaccination were higher and much more uniform as birds increased in age.

The humoral immune response of chickens to Mycoplasma gallisepticum and Mycoplasma synoviae studied by immunoblotting.

The humoral immune response over time of White Leghorn chickens experimentally infected with Mycoplasma gallisepticum or M. synoviae by an aerosol inoculation or a contact exposure were compared by immunoblotting. The response of chickens infected with M. gallisepticum were similar with respect to proteins recognized and intensity of response, regardless of mode of infection. On the other hand, chickens infected by aerosolization of M. synoviae responded to more proteins and with greater intensity than did M. synoviae contact-exposed birds. Chickens infected with M. gallisepticum responded with antibodies to over 20 proteins, while chickens infected with M. synoviae responded with antibodies to 12 proteins. Field sera from chickens naturally infected on commercial poultry farms with M. gallisepticum or M. synoviae were analyzed by immunoblotting and were found to react with a number of mycoplasma proteins. However, no correlation was seen when comparing intensity of immunoblot staining and hemagglutination-inhibition titer of the field sera. The experimental antisera were used to identify species-specific proteins of M. gallisepticum and M. synoviae. Six immunogenic species-specific proteins of M. gallisepticum with relative molecular masses of 82 (p82), 65-63 (p64), 56 (p56), 35 (p35), 26 (p26), and 24 (p24) kilodaltons (kDa) were identified. Two species-specific proteins of M. synoviae with relative molecular masses of 53 (p53) and 22 (p22) kDa were identified. Additionally, a highly immunogenic 41 (p41) kDa protein of M. synoviae was identified. Species-specific proteins identified in these mycoplasmas and the 41 kDa protein of M. synoviae were purified by preparative SDS-PAGE in amounts sufficient for further characterization and for use in serodiagnostic tests.

Evaluation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis purified proteins of Mycoplasma gallisepticum and M. synoviae as antigens in a dot-enzyme-linked immunosorbent assay.

Selected immunogenic proteins of Mycoplasma gallisepticum (MG) strain R and M. synoviae (MS) isolate F10-2AS were purified from sodium dodecyl sulfate-polyacrylamide gels. Purified MG proteins of 65 to 63 (p64) kilodaltons (kDa), and 26 and 24 (p26/24) kDa, and purified MS proteins of 53 (p53) kDa, 41 (p41) kDa, and 22 (p22) kDa were evaluated as potential antigens for an enzyme-linked immunosorbent assay (ELISA). Chicken antisera to MG, MS, or oil-emulsion vaccines were used to evaluate these purified proteins as antigens in a dot-ELISA. MG antigen p64 detected antibodies 3 days after the serum plate agglutination (SPA) test and 7 days before the hemagglutination-inhibition (HI) test. Antigen p64 detected antibodies to 12 MG isolates, and in sera from field outbreaks of MG. No cross-reactions with MS-positive antisera were seen with antigen p64. MG antigen p26/24 did not perform as well as p64. MS antigen p41 detected antibodies 5 days after the SPA test and at least 11 days before the HI test, and in sera from field outbreaks of MS. However, some MG-positive antisera reacted with p41. MS antigens p53 and p22 did not perform well.

Further studies of Mycoplasma gallisepticum serum plate agglutination antigen grown in medium with artificial liposomes substituting for serum.

Frey's medium supplemented with artificial liposomes substituting for serum was evaluated for Mycoplasma gallisepticum (MG) serum plate agglutination (SPA) antigen. Antigens prepared in batch (static) culture were compared with antigens grown in a fermenter. All batch-grown MG liposome antigens were highly sensitive, specific, and resulted in a greater yield compared with fermenter-grown liposome antigens. Compared with antigens prepared in Frey's medium with 12% swine serum (regular FMS) or with commercial SPA antigens, liposome antigens had a higher degree of specificity; however, they were similar in sensitivity and antigen yield. The only growth parameter to affect the yield per liter of batch-grown liposome antigen was the concentration of liposomes in the growth medium. The reduced yield and sensitivity of antigens grown in a fermenter may have been due to autoclaving the medium instead of sterilizing by filtration. There was no obvious difference between patterns of serum-medium-grown, liposome-medium-grown, or commercial SPA antigens upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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.