Poor systemic antibody response after vaccination of commercial broiler breeders with Mycoplasma gallisepticum vaccine ts-11 not associated with susceptibility to challenge

A live attenuated Mycoplasma gallisepticum vaccine, ts-11, has been used for control of M gallisepticum in several countries. The rapid serum agglutination test is usually used as an indicator of flock response to vaccination; however, in some flocks, the detected response may be weak or absent. We investigated whether the low level, or lack, of systemic antibodies in ts-11-vaccinated flocks is correlated with susceptibility to infection after challenge with a virulent M. gallisepticum strain. Birds from 2 separate ts-11-vaccinated commercial flocks with no, or weak, rapid serum agglutination responses (at 11 or 14 wk postvaccination) were randomly selected and subjected to aerosol challenge with either M gallisepticum strain Ap3AS or sterile mycoplasma broth. A group of nonvaccinated specific-pathogen-free chickens at similar age were also exposed to aerosolization with M. gallisepticum strain Ap3AS and used as positive controls. Postmortem examination of the birds, performed 2 wk after challenge, revealed no significant difference in microscopic tracheal lesions or mucosal thicknesses between the ts-11-vaccinated field birds irrespective of their aerosolization treatment. However, both microscopic tracheal lesions and tracheal mucosal thicknesses of nonvaccinated challenged birds were significantly greater than those of ts-11 vaccinates. Hence, broiler breeders vaccinated in the field showed significant protection against virulent M. gallisepticum challenge even when no serum antibody was detected by rapid serum agglutination test. These results reveal that seroconversion detected by rapid serum agglutination test after ts-11 vaccination is not a reliable predictor of protection against M. gallisepticum infection. The possible significance of local antibody response and cell-mediated immunity against M. gallisepticum infection is discussed.     

Characterization of a ts-1-like Mycoplasma galisepticum isolate from commercial broiler chickens

Several commercial broiler flocks in northeastern Georgia that were the progeny of the same parent flock (Flock 40) were diagnosed as Mycoplasma gallisepticum (MG) positive by serology, culture, and PCR. Flock 40 had been vaccinated with ts-11 live MG vaccine. Several isolates were obtained from the MG-positive broiler flocks, and these isolates were indistinguishable from the ts-11 vaccine strain by the molecular strain differentiation methods used. A pathogenicity study was performed to compare the virulence of one of the isolates, K6216D, to the ts-11 vaccine strain. K6216D elicited a significantly stronger antibody response and significantly increased colonization of the tracheas and air sacs. K6216D also elicited significantly greater air sac and tracheal lesions than the ts-11 vaccine strain at 10 and 21 days postinoculation (P < or = 0.05). This is the first report of a field case of the apparent reversion to virulence and vertical transmission of the ts-11 vaccine.     

The effects of ts-11 strain Mycoplasma gallisepticum vaccination in commercial layers on egg production and selected egg quality parameters

Live Mycoplasma gallisepticum (MG) vaccines have been USDA approved and licensed for use in commercial layer chickens since 1988; however, egg production and egg quality data exist only for the F strain of MG. Information pertinent to the effects of ts-11 MG on egg and eggshell quality parameters, as well as egg size distribution, is lacking. In this study, pullets were inoculated at 10 wk of age with ts-11 strain MG and placed in biological isolation units at 10 birds/unit. Hen-day egg production, eggshell strength, Haugh unit score, pimpling incidence, and blood/meat spot incidence were monitored and recorded in each trial through a 45-wk production cycle. Further, eggs from all treatments were collected daily, Monday-Thursday, and individually weighed. Results of this study indicate that no significant difference was observed between the treatments for the parameters measured or for egg size distribution. Therefore, these data should lessen producers' concerns pertaining to the impact of ts-11 strain MG on egg production, egg and eggshell quality parameters, and egg size distribution.     

Induction of humoral immune response and protective immunity in chickens against Salmonella enteritidis after a single dose of killed bacterium-loaded microspheres.

Formalin-inactivated Salmonella enteritidis phage type 4 strain 119/95 (SE) was encapsulated in biodegradable poly (DL-lactide co-glycolic acid) PLGA; (65:35) microspheres by a modified water-in-oil-in-water (w/o/w) double-emulsion solvent extraction/evaporation technique. These SE-loaded microspheres (SE-MS) were porous and spherical in shape with diameters of 0.4-10 microm and 20-80 microm in two preparations. SE-MS were subsequently used to vaccinate specific-pathogen-free chickens in a single dose in order to investigate the potency of a single-dose vaccination in inducing immune responses and protective immunity. In Experiment 1, 4-wk-old chickens that were vaccinated intramuscularly with 20-80-microm SE-MS generated long lasting (over 6 mo) and persistently high serum anti-SE immunoglobulin (Ig)G antibody response. In Experiment 2, 2-wk-old chickens were vaccinated orally with 0.4-10-microm or intramuscularly with 20-80-microm SE-MS and challenged with 10(9) colony-forming units of homologous SE strain at 6 wk postvaccination. When challenged intramuscularly, one each of the orally vaccinated (n = 10) and the intramuscularly vaccinated birds (n = 10) showed clinical signs and death, whereas all of the nonvaccinated control birds (n = 12) were sick and 11 of them were killed. When challenge was via oral route, 26.1% of cloacal swabs and 24.0% of organs (liver, spleen, and cecum) collected from orally vaccinated birds (n = 35) were positive for SE, comparable to 27.9% of feces and 18.7% of organs from the intramuscularly vaccinated birds (n = 35). These figures were significantly lower than those for nonvaccinated birds (n = 30) from which 59.3% of feces and 44.0% of organs tested SE positive (P < 0.05). The humoral immune response was also determined after vaccination with a single dose. The intramuscular vaccination elicited higher serum IgG response than oral administration, but the latter elicited a significant intestinal mucosal IgA antibody response. This is the first evidence that chickens vaccinated with killed SE-loaded PLGA microspheres, intramuscularly and orally in a single dose, developed systematic and local immune responses, thereby conferring protective immunity.     

Duration of immunity engendered by a single dose of a cold-adapted strain of Avian pneumovirus

The duration of immunity after a single dose of a cold-adapted strain of Avian pneumovirus (APV) was studied. Turkeys were vaccinated at 1 wk of age and challenged with virulent virus 3, 7, 10, and 14 wk later. Nonvaccinated groups were also challenged at the same times. No clinical signs were observed in the vaccinated birds after vaccination or after any challenge. No viral RNA was shed by the vaccinated birds after any challenge. The nonvaccinated birds shed viral RNA after all challenges. Avian pneumovirus-specific humoral antibodies were detected in the vaccinated birds until 14 wk after vaccination. The results of this preliminary study indicate that inoculation with a single dose of a cold-adapted strain of APV at 1 wk of age provides protection until 15 wk of age.     

The efficacy of three commercial Mycoplasma gallisepticum vaccines in laying hens

The efficacy of three commercial Mycoplasma gallisepticum (MG) immunizing agents-a bacterin, a recombinant fowlpox-MG vaccine, and a live F-strain vaccine-was compared in specific-pathogen-free hens in egg production. Three groups of 25 chickens were vaccinated with one of the vaccines at 10 wk of age and 25 birds were not vaccinated. At 25 wk of age (and approximately 50% egg production), 20 birds from each of the three vaccinated groups and 15 nonvaccinated controls were challenged with virulent R-strain via aerosol; the birds were necropsied and evaluated at 10 days post-challenge. The MG bacterin and live F-strain vaccinations were both protective and resulted in significant differences in air sac lesions, tracheal lesions, and ovarian regression compared to the nonvaccinated controls and the recombinant fowlpox-MG vaccine (P < or = 0.05). The evaluation of ovarian regression is a useful method of testing the efficacy of MG vaccines in laying hens.     

Minimization of pathologic changes in Ornithobacterium rhinotracheale infection in turkeys by temperature-sensitive mutant strain

The protection elicited by a temperature-sensitive (Ts) mutant of Ornithobacterium rhinotracheale (ORT) vaccine against challenge with pathogenic strain was investigated. In Experiment 1, specific serologic response to ORT was detected in 12%-19% of Ts-vaccinated birds at 3 wk postvaccination by either drinking water or oculo-nasal instillation. At 7 days postchallenge, 100% of Ts-vaccinated turkeys of all groups were able to respond with an ORT-specific antibody response, but the control group was not, suggesting the potential of Ts strain to evoke immune protection. The study also revealed a statistically significant ability of the Ts strain to protect vaccinated turkeys against gross lesions caused by the pathogenic strain of ORT in treated groups vs. control. In Experiment 2, seroconversion was detected by enzyme-linked immunosorbent assay in birds after they were given the Ts strain in drinking water in field conditions. The results of the field study showed mean scores of gross lesions of nonvaccinated/challenged groups to be up to seven times higher than those of the vaccinated/challenged group. In addition, reisolation rates and quantification of ORT colonies per gram of lung tissue were significantly lower for vaccinated/challenged than for nonvaccinated/challenged turkeys. In conclusion, results from laboratory and field experiments suggest that use of the Ts mutant strain of ORT as a live vaccine would be a suitable method to evoke protection against ORT infection in turkeys.     

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).     

Studies of the transmission routes of Ornithobacterium rhinotracheale and immunoprophylaxis to prevent infection in young meat turkeys

The importance and prevention of the horizontal as well as the vertical transmission of Ornithobacterium rhinotracheale were investigated. In our first experiment we observed that specific-pathogen-free broiler chickens that were placed in hatching incubators at a commercial turkey hatchery during hatch showed respiratory tract lesions at postmortem examination that were positive for O. rhinotracheale by bacteriology and immunohistology. It appeared that vertical transmission occurred and that horizontal transmission of O. rhinotracheale is possible. In a second experiment, the turkeys derived from vaccinated parents showed significantly fewer respiratory tract lesions at postmortem examination at 16 days of age than the birds derived from nonvaccinated parents. In a third experiment, all vaccinated young birds, regardless of the vaccination state of their parents, showed significantly fewer respiratory tract lesions at 6 wk of age. We concluded that vaccination of the breeders reduces vertical transmission and that vaccination of the progeny is needed to resist challenge at 6 wk of age.     

Comparison of different attenuation strategies in development of a Salmonella hadar vaccine

The purpose of this work was to develop a live, attenuated vaccine strain to protect chickens against colonization by group C Salmonella. We constructed two candidate vaccines: a deltacya deltacrp derivative and a deltaphoP derivative of Salmonella hadar. White Leghorn chickens were vaccinated at day of age and at 2 wk with one of the two strains. A nonvaccinated group served as a control. At 4 wk of age, all birds were challenged with wild-type S. hadar and necropsied 6 days later. Numbers of S. hadar in the ceca were determined. Enzyme-linked immunosorbent assay-derived serum immunoglobulin G responses against S. hadar lipopolysaccharide indicated that both strains induced a serum antibody response. The average optical density450 for birds vaccinated with the deltaphoP or deltacya deltacrp derivatives was 0.456 and 0.881, respectively. Although the deltacya deltacrp derivative induced higher levels of serum antibody, it did not provide an immune response protective against colonization by S. hadar. Conversely, birds vaccinated with the deltaphoP strain showed significant protection against S. hadar challenge. Seventy percent of the nonvaccinates, 60% of the deltacya deltacrp vaccinates, and 15% of deltaphoP vaccinates were positive for S. hadar in tissues. In a second experiment, birds were vaccinated with either the deltaphoP strain or buffer and challenged with a 10-fold higher dose than in the first experiment. After challenge, all of the birds in both groups were colonized. The geometric mean number of cecal S. hadar isolated from the control group was 1.0 x 10(6) colony-forming units (CFU)/g, and from the vaccinated group, this value was 32 CFU/g, indicating a four to five log reduction in colonization by the challenge strain.     

Cold-adapted strain of avian pneumovirus as a vaccine in one-day-old turkeys and the effect of inoculation routes

To determine the optimum route of vaccination, we inoculated 1-day-old turkeys with a cold-adapted strain of avian pneumovirus (APV) by oculonasal, oral, or aerosol route. Another two groups served as nonvaccinated-challenged and nonvaccinated-nonchallenged groups. Birds in all vaccinated and nonvaccinated-challenged groups were challenged with virulent APV 3 wk postvaccination. After challenge, no vaccinated bird developed clinical signs or virus shedding, whereas nonvaccinated-challenged birds developed clinical signs (clinical score = 11.2/bird) and shed virus from their choanal cleft. Birds in all three vaccinated groups seroconverted at 3 wk postvaccination. The nonvaccinated-nonchallenged group remained free of clinical signs and virus shedding and did not develop APV antibodies throughout the course of the study. These results suggest that this cold-adapted strain of APV is safe and effective in 1-day-old turkeys when given by any of the three routes.     

Immunogenicity and Efficacy of a Commercial Feline Leukemia Virus Vaccine

Twenty young adult specific pathogen-free cats were randomly divided into two groups of 10 animals each. One group was vaccinated with two doses of feline leukemia virus vaccine according to the manufacturer's recommendations. All 20 cats were challenge exposed oronasally (4 times over a 1-week period), beginning 3 weeks after immunization, with a virulent subgroup A strain of FeLV (CT600-FeLV). The severity of the FeLV infection was enhanced by treating the cats with methylprednisolone acetate at the time of the last FeLV exposure. Ten of 10nonvaccinated cats became persistently viremic compared with 0/10 of the vaccinates. ELISA antibodies to whole FeLV were present at high concentrations after immunization in all of the vaccinated cats, and there was no observable anamnestic antibody response after challenge exposure. ELISA antibodies to whole FeLV appeared at low concentrations in the serum of nonvaccinated cats after infection but disappeared as the viremia became permanently established. Virus neutralizing antibodies were detected in 3/10 vaccinates and 0/10 nonvaccinates immediately before FeLV challenge exposure, and in 8/10 vaccinates and 1/10 nonvaccinates 5 weeks later. Although vaccination did not consistently evoke virus neutralizing antibodies, it appeared to immunologically prime cats for a virus-neutralizing antibody response after infection. Active FeLV infection was detected in bone marrow cells taken 14 weeks after infection from 10/10 nonvaccinates and 0/10 vaccinates. Latent FeLV infection was not detected in bone marrow cells from any of the vaccinated cats 14 weeks after challenge exposure.     

Subclinical infectious bursal disease in an integrated broiler production operation.

A field study was designed to determine the prevalence of subclinical infectious bursal disease (IBD) in broiler chickens from a commercial poultry company. Bursae of Fabricius (BF) from two vaccinated and three nonvaccinated broiler flocks were evaluated histologically, and antibody profiles of these broiler and matched parent breeder flocks were established. Lesions of IBD, including lymphoid necrosis, stromal edema, and infiltrates of heterophils and macrophages, were first detected in BF at 24 days of age in both vaccinated and nonvaccinated chickens. At 41 days, all BF had lesions characteristic of IBD, including severe lymphoid depletion, proliferation of epithelial cells, and mild fibroplasia. Although mean maternal antibody levels (measured by enzyme-linked immunosorbent assay) in broilers were apparently protective through day 12, IBD antibodies decreased to nonprotective levels (below 1,000) by day 16 or 20. Titers began to increase by day 28 or 32 because of field exposure. Sentinel birds, placed with broiler flocks, also developed IBD antibody titers. Broiler breeders had low and nonuniform antibody titers. Prevalence of field IBD exposure was high, and existing vaccination programs were not effective.     

A comparison of the oral application and injection routes using the onderstepoort biological products fowl typhoid vaccine, its safety, efficacy and duration of protection in commercial laying hens

This study was undertaken to establish whether the Onderstepoort Biological Products Fowl Typhoid (OBPft) vaccine registered as an injectable vaccine was effective and safe when administered orally to commercial layers. Its efficacy and duration of protection were compared with application by intramuscular injection. Commercial brown layer hens were used as they were found to be highly susceptible to Salmonella gallinarum infections. In the vaccine safety trial birds were euthanased at timed intervals spanning 4 weeks postvaccination. Necropsies were performed and samples were taken and tested. No clinical signs or mortalities could be attributed to the OBPft vaccine nor could active shedding of the vaccine strain be detected. Slight pathological changes were noted with both routes of vaccination; however, these changes were transient, returning to normal within the observation period. The injected groups showed a better serological response with the rapid serum plate agglutination (RSPA) test than the orally vaccinated groups. In the duration of protection trial, birds were challenged at 3-8-week intervals post-vaccination. All unvaccinated birds died. Protection 8 and 16 weeks after vaccination was above 60 %,by 24 weeks after challenge, the vaccine protection was below 30 %. It was found that there was no significant difference (P < 0.05) in the protection offered by either the oral or injected route of vaccination with the OBPft vaccine.     

Differentiation of Mycoplasma gallisepticum vaccine strains ts-11 and 6/85 from commonly used Mycoplasma gallisepticum challenge strains by PCR

Mycoplasma gallisepticum (MG) is an important avian pathogen causing significant economic losses within the poultry industry. In an effort to develop tools to aid in MG research and diagnostics, we have compared sequences of the attenuated MG vaccine strain ts-11 to those of commonly used pathogenic challenge strains in search of a simple means of differentiation. Via gapA sequence alignments and comparisons, we have identified and designed primers facilitating strain differentiation. When applied to conventional polymerase chain reaction (PCR) assay at low annealing temperature, the primer sets allow for the differentiation of MG attenuated vaccine strains ts-11 as well as the attenuated MG vaccine strain 6/85 from the commonly utilized MG challenge strains R(low), R, and S6. Conventional PCR differentiation is based on the visualization of sole products with the attenuated MG strains ts-11 and 6/85 and the lack of the corresponding products from MG strains R(low), R, and S6. When applied to MG strain F, product visualization varies with the applied primer set. The differentiation of MG strains ts-11 and 6/85 from the pathogenic challenge strains was also accomplished via real-time analyses, however, the primer sets were not able to differentiate MG strains ts-11 and 6/85 from selected MG field isolates.     

Safety of temperature sensitive mutant Mycoplasma gallisepticum vaccine

A temperature sensitive (ts) vaccine strain designated ts-11 was selected after exposure of a low passage culture of the immunogenic Australian field isolate (strain 80083) of Mycoplasma gallisepticum to 100 mg/ml of N-methyl-N-nitro-N-nitrosoguanidine. Viable counts (assayed as colour changing units (CCU)/25 microliters) of a thawed stock culture of ts-11 were typically log10 3 to log10 5 higher when incubated at 33 degrees C (the permissive temperature) than duplicate viable counts incubated at 39.5 degrees C (the restrictive temperature). Doses of approximately 2 x 10(7) CCU of ts-11 caused no gross lesions or loss of egg production when inoculated into the air sacs of susceptible chickens and no clinical or pathological signs of sinusitis when inoculated into the infraorbital sinuses of susceptible turkey poults, whereas the parent strain 80083 was demonstrably pathogenic. However, 1 of 10 poults inoculated intra-abdominally with approximately 2 x 10(7) CCU of ts-11 did show signs of mild airsacculitis. Eight-week-old pullets were vaccinated by eye drop with up to 1.4 x 10(7) CCU of ts-11 and simultaneously subjected to several stressful management practices, without apparent ill effects. Administration by coarse aerosol of 5 ml of ts-11 vaccine/25 day-old broilers, with or without 25 doses of infectious bronchitis virus vaccine caused no obvious signs of respiratory disease. The non virulent ts phenotype was maintained after 3 passages of strain ts-11 in chickens. Chickens vaccinated 3 weeks previously with ts-11 or with strain 80083 were placed in contact with susceptible chickens for a period of 2 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of a Mycoplasma gallisepticum strain exhibiting reduced virulence for prevention and control of poultry mycoplasmosis.

Two experiments were conducted to evaluate the virulence and vaccination efficacy of a Mycoplasma gallisepticum (MG) isolate designated MG Intervet 6/85. Virulence of the strain was determined by evaluation of airsacculitis scores following aerosol exposure to the isolate before and after 10 sequential passes in either commercial broiler chickens or commercial turkeys. Two-week-old specific-pathogen-free chickens were vaccinated by aerosol exposure. The birds were challenged with the R' strain of MG at either 4 or 8 weeks post-vaccination. Efficacy was evaluated by airsacculitis scores determined 21 days after challenge. Ten repetitive back-passes of the isolate in chickens and turkeys did not substantially increase the virulence. Virulence for both chickens and turkeys was minimal, while protection elicited by aerosol vaccination in young chickens against virulent R' strain was significant (P less than or equal to 0.05) compared with unvaccinated controls.     

Use of mgc2-polymerase chain reaction-restriction fragment length polymorphism for rapid differentiation between field isolates and vaccine strains of Mycoplasma gallisepticum in Israel

Increasing use of Mycoplasma gallisepticum (MG) live vaccines has led to a need for a rapid test for differentiation of MG field strains from the live vaccine strains ts-11 and 6/85. We examined the differentiating potential of diagnostic polymerase chain reaction (PCR) primers targeted to the gene mgc2, encoding a cytadherence-related surface protein uniquely present in MG. The mgc2-PCR diagnostic primers are specific for MG in tests of all avian mycoplasmas or bacteria present in the chicken trachea and are sensitive enough to readily detect MG in tracheal swabs from field outbreaks. Differentiation of vaccine strain ts-11 was based on identification of restriction enzyme sites in the 300-base-pair (bp) mgc2-PCR amplicon present in ts-11 and missing in MG isolates from field outbreaks in Israel. Restriction sites for the enzymes HaeII and SfaN1 were identified in the amplified region in strain ts-11 and were not found in 28 field isolates of MG, comprising a representative cross section of all the MG isolates from the period 1997-2003. In practice, differential diagnosis of MG is achieved within 1 day of submission of tracheal swab samples by mgc2-PCR amplification and restriction of the amplicon with HaeII, giving a 270-bp fragment for ts-11 or no restriction for other MG strains tested. Application of the mgc2-PCR-restriction fragment length polymorphism (mgc2-PCR-RFLP) assay enabled differential diagnosis of both components of a mixture of ts-11 and non-ts-11 DNA, detecting the field strain in the presence of a large excess of ts-11. The test was successfully applied in vivo for monitoring vaccinates in a ts-11 vaccine trial. In principle, the test may also be used to identify the 6/85 vaccine strain, which yields a 237-bp product, readily differentiated from the approximately 300-bp PCR product of all other strains tested. Further testing of field isolates will be necessary to determine the applicability of this test in the United States and other countries.     

Vaccination of calves against Salmonella dublin with aromatic-dependent Salmonella typhimurium

Ten Holstein calves were divided into 2 groups. Five calves served as nonvaccinated controls, and 5 calves were vaccinated IM at 2 and 3 weeks of age with 10(9) aromatic-dependent (aro-) Salmonella typhimurium strain SL1479 containing O antigens 1, 4, 12. Serious adverse reactions to vaccination were not observed in the calves. Mean maximum rectal temperature increase in the vaccinated calves was 1.5 C. One calf had diarrhea and depressed appetite for 1 day after vaccination. At 5 weeks of age, all calves were challenge exposed orally with 1.5 X 10(11) virulent S dublin strain SL1367 (O antigens 9,12). After challenge-exposure inoculum was given, 1 of 5 vaccinated calves died and 4 of the 5 nonvaccinated calves died (P less than 0.05). Thus, some cross serotype protection against S dublin was induced by parenteral vaccination of calves with aro- S typhimurium strain SL1479, although protection was not complete.