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.     

Detection of antibodies to Mycoplasma gallisepticum vaccine ts-11 by an autologous pMGA enzyme-linked immunosorbent assay

Mycoplasma gallisepticum is a poultry pathogen that causes respiratory disease and loss of egg production worldwide. A live attenuated 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. With the use of specific monoclonal antibodies against M. gallisepticum strain S6 pMGA in immunoaffinity purification, the major membrane antigen of ts-11 was purified. An indirect enzyme-linked immunosorbent assay (ELISA) was developed with the purified antigen, and its potential for detection of antibodies induced after ts-11 vaccination was compared with an indirect ELISA with M. gallisepticum strain S6 pMGA. In the presence of high levels of ts-11-induced antibodies, both antigens detected similar numbers of positive sera. However, when lower levels of antibodies were present, ts-11 pMGA showed a higher sensitivity than S6 pMGA. Further examination of ts-11 pMGA with Mycoplasma synoviae-infected chicken sera revealed that ts-11 pMGA is specific for M. gallisepticum antibodies. With a panel of sera from ts-11-vaccinated or non-ts-11-vaccinated field chickens, the ts-11 pMGA ELISA was found to be more sensitive than the commercial rapid serum agglutination test in detecting antibodies to ts-11 vaccine. The results from this study suggest that the major membrane antigen of M. gallisepticum may have slightly different antigenic profiles in different strains, thereby necessitating the use of autologous antigens in serodiagnostic assays to increase sensitivity of the tests for mycoplasma antibodies. Thus, the low level of antibody response after ts-11 vaccination is, at least partially, due to the low ability of the current diagnostic antigens to bind ts-11 antibodies.     

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.     

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.     

A chronicle of serologic response in commercial layer chickens to vaccination with commercial F strain Mycoplasma gallisepticum vaccine

Vaccination of multi-age layer operations, wherein one million plus commercial layer chickens are housed, has been spurious until the development of a self-propelled, constant-speed spray vaccinator. Still, even with its use, live Mycoplasma gallisepticum (MG) vaccinations have been questionable in terms of seroconversion. Using the vaccinator as a research tool over the past 5 yr, factors have been elucidated which impact seroconversion to one live MG vaccine in particular, the F strain of MG (FMG). These factors include the type of nozzle used to spray the vaccine, the temperature of the water used to rehydrate and administer the vaccine, and the pH and osmolarity of the fluid used to apply the vaccine. In the present study, one farm was monitored for its seroconversion rates over 4 1/2 yr, during which time the FMG vaccination protocol was amended as factors were identified that enhanced seroconversion rates. The results of this study showed that implementation and inclusion of the optimized factors into the vaccination protocol for FMG enhanced seroconversion rates because they went from an initial 50%-55% positive seroconversion rate to a consistent 100% positive seroconversion rate over the 56-mo study period.     

Safety of Mycoplasma gallisepticum vaccine strain 6/85 after backpassage in turkeys

The objective of this research was to evaluate the safety of the 6/85 strain vaccine strain of Mycoplasma gallisepticum in turkeys by backpassing the vaccine strain up to 10 times by contact infection in turkeys and challenging turkeys with the resulting backpassaged strain. The vaccine strain, however, did not spread to in-contact turkeys, and it was necessary to reisolate the organism before challenging turkeys for the next passage. The challenge strain, therefore, was one that had been backpassaged four times in turkeys, with a total in vivo time in turkeys of 66 days. The backpassaged 6/85 vaccine strain was no different in pathogenicity than the original vaccine strain, except that at 10 days postchallenge, it was isolated in higher numbers from air sacs. Both the original 6/85 vaccine strain and the backpassaged strain were apathogenic in turkeys, except for a slightly increased diameter of the tracheal mucosa at 10 days postchallenge; at 20 days postchallenge the tracheal mucosal thickness was no different from that of controls.     

Influence of F strain Mycoplasma gallisepticum infection on response of commercial layers to heat exposure

Commercial layers were inoculated with F strain Mycoplasma gallisepticum (MG) and housed in either conventional chicken houses or the lower-stress environment of biological isolation units. At the end of 2 weeks, all treatment groups were placed in environmental chambers and subjected to 4 hr of heat stress (40 C with a dew point of 21 C). Rectal temperature, an indicator of response to high heat, was monitored. Rectal temperatures of F strain MG-inoculated hens housed in the conventional chicken house environment were significantly higher than those of uninoculated controls, whereas rectal temperatures of hens held in isolation units were comparable to those of their uninoculated controls.     

Safety and efficacy of the avirulent Mycoplasma gallisepticum strain K5054 as a live vaccine in poultry

A Mycoplasma gallisepticum (MG) isolate from an atypically mild outbreak in turkey breeders was found to be similar to house finch isolates by DNA analyses. A preliminary study in turkeys showed that this isolate (K5054) caused very mild lesions and protected turkeys against subsequent challenge with a virulent MG strain. In this study, K5054 was further evaluated as a potential vaccine strain in commercial layer-type chickens and turkeys. The safety of K5054 was evaluated by aerosol challenge followed by evaluation of gross and histopathologic lesions as well as serologic reactions and isolation of MG from the trachea and air sacs. Infection of chickens (trial 1) and turkeys (trial 2) with K5054 resulted in little evidence of MG lesions. There was weak seroconversion, and K5054 was consistently reisolated from the tracheas of chickens and turkeys. The efficacy of K5054 as a vaccine was evaluated by aerosol challenge of vaccinated chickens (trial 3) and turkeys (trial 4) with virulent R strain. There was evidence of protection from lesions associated with MG.     

Evaluation of protection against Mycoplasma gallisepticum infection in chickens vaccinated with the F strain of M. gallisepticum

The effect of vaccination with the F strain of Mycoplasma gallisepticum (MG) on protection against challenge with a tylosin-resistant strain of MG was evaluated. White leghorn chickens vaccinated via eyedrop at 6 weeks of age were subsequently challenged with various dilutions of the tylosin-resistant MG strain, as were unvaccinated controls. Three days later, tracheal swabs were collected and cultured in medium with and without tylosin to distinguish between the vaccine and challenge strains. The mean infectious dose of the challenge strains was 3.8 log10 higher in the vaccinated group than in the controls, and the vaccinated group harbored fewer challenge organisms in the trachea. These findings suggest that the F strain of MG induces protection against infection with field strains of MG and that long-term vaccination with the F strain in multiple-age layer farms may result in replacement of field MG strains by the F strain.     

The serological response of young dogs to the Flury LEP strain of rabies virus vaccine

The serological response of puppies from Nigeria to live Flury low egg passage (LEP) rabies vaccine was determined. Two sets of puppies were used: one set from rabies-vaccinated bitches and another set from non-vaccinated bitches. Puppies were vaccinated intramuscularly with Flury LEP strain rabies vaccine and serially bled from the 4th week to the 30th week. Serum rabies virus neutralizing antibodies (VNA) were measured by a modified rapid fluorescent focus inhibition test (RFFIT). Puppies from non-vaccinated bitches responded well to vaccination after the 4th week and through to the 10th week of age, showing a progressive increase in VNA. In contrast, puppies from vaccinated bitches responded well to rabies vaccination only at 10 weeks of age, although detectable maternal rabies VNA and rabies anti-ribonucleoprotein (RNP) antibodies had decreased by 6 weeks post partum.     

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.     

Effects of increased dosages of the Mycoplasma gallisepticum vaccine MYCOVAC-L in layer chickens subsequently challenged with virulent M. gallisepticum: egg production and serologic response

Ten-week-old Hy-Line Commercial W-36 pullets were spray-vaccinated with MYCOVAC-L at the manufacturer's recommended dosage (1x) or at 15 times that rate (15x). At 22 or 45 wk of age, subsets of 1x- and 15x-vaccinated pullets were challenged with the virulent Mycoplasma gallisepticum (MG) strain Rlow. Percent hen-day egg production was determined through week 55. Analyses for treatment effects on overall (22-56 wk) percent hen-day egg production revealed no significant differences between nonchallenged 1x and nonchallenged 15x MYCOVAC-L treatments. Among 1x MYCOVAC-L-vaccinated groups, Rlow challenge at 45 wk corresponded to significantly (P < or = 0.01) lower overall egg production compared with the unchallenged 1x-vaccinated control (70.88% vs. 79.15%, respectively). Conversely, at the 15x MYCOVAC-L dosage level, overall egg production was not significantly affected by virulent MG challenge at 45 wk compared with its unchallenged counterpart (84.09% vs. 81.03%, respectively) and could indicate increased protection from virulent MG challenge. Serologic monitoring indicated the virulent MG challenge was consistently (100%) associated with seroconversion. Comparisons among the nonchallenged experimental treatments indicated that vaccinations at the 15x MYCOVAC-L dosage rate were associated with a greater seroconversion rate at weeks 21, 27, and 44, but not at week 50.     

Serologic response of roosters to gradient dosage levels of a commercially available live F strain-derived Mycoplasma gallisepticum vaccine over time

Spray application is a commonly used, time- and labor-efficient means to deliver live Mycoplasma gallisepticum (MG) vaccine to laying hens in commercial production facilities. The dosage of vaccine received by spray-vaccinated birds can vary due to variation in the spray plume and the vaccine suspension droplet trajectory. In this study, a total of 48 Hy-Line W-36 males were placed individually in isolation units following eye-drop application of gradient levels (1 x, 10(-1) x, 10(-2) x, 10(-3) x, 10(-4) x, 10(-5) x, 10(-6) x, and unvaccinated control) of the MG vaccine. The determined titer associated with a 1 x dose was 2 x 10(6) colony-forming units/dose. Serologic response was assessed weekly following vaccination via serum plate agglutination (SPA) for weeks one through seven postvaccination (p.v.). In addition, immunologic response was assessed at 5, 6, and 7 wk p.v. via MG enzyme-linked immunosorbent assay (ELISA). As indicated by SPA analyses, a 1 x dose of vaccine resulted in 100% seroconversion, and dose levels of 10(-1) x and 10(-2) x resulted in 75% and 37.5% seroconversion, respectively, at 6 wk p.v. The MG ELISA results at 6 wk p.v. demonstrated immunologic responses in 100%, 57.1%, and 28.6% of the 1 x, 10(-1) x, and 10(-2) x dosed birds, respectively. The lower dosage levels of 10(-3) x, 10(-4) x, 10(-5) x, and 10(-6) x did not elicit a response from any bird at 6 wk p.v. Utilizing the SPA data, a logistic regression model was used to determine the relationship between dosage level and seroconversion rate (R2 = 0.999 with a standard error of prediction of 1.6%). The model predicted a required effective dosage of 0.26 x for 90% seroconversion at 6 wk p.v. under test conditions.     

Factors affecting the serological response of dogs and cats to rabies vaccination

After being vaccinated against rabies some cats and dogs fail to show an antibody titre adequate to meet the requirements of the UK Pet Travel Scheme. To investigate this problem, the data derived from 16,073 serum samples submitted to the Veterinary Laboratories Agency for serological testing between 1999 and 2002, 1002 samples submitted to BioBest during March and April 2001, and 1264 samples associated with one make of vaccine submitted to BioBest between June 2001 and January 2003, were analysed. The probability of antibody titre failing to reach at least 0.5 iu/ml was analysed by logistic regression as a function of the choice of vaccine, the interval between vaccination and sampling, the sex and age of the animal, and its country of origin. In dogs, all these factors, except sex, had highly significant (P < 0.001) effects on the test failure rate, and in cats all the factors had a significant effect (P < 0.05).     

An ultrastructural study on the interaction of Mycoplasma gallisepticum with the chicken tracheal epithelium

Seven-day-old chickens wee intratracheally inoculated with Mycoplasma gallisepticum. The tracheas collected 6 and 14 days after chickens were inoculated were subjected to titration of mycoplasma and examination by light and electron microscopy. The mycoplasma organisms grew well; 10(7) to 10(8) color-changing units in a milligram of tissue were determined. Tracheal lesions occurred in close association with the presence of mycoplasmas and were characterized by degeneration of the epithelial cells and inflammatory cellular infiltration of the mucosa. Mycoplasmas were predominantly found extracellularly and only rarely in phagocytic vacuoles of the epithelial cells. Although the mycoplasmas exhibited considerable pleomorphism in size and shape, most of them were oval or round, and the largest diameters were between 300 and 700 mn. Elongated and irregular forms were also observed, particularly in those mycoplasmas adhering to the epithelial cells. The organism had a limiting unit membrane, the fibrillar nuclear area, the peripheral cytoplasmic area containing numerous ribosomes, and a terminal bleb structure. Mycoplasmas attached to the epithelial cells by their blebs close to the host cell membrane. At the attachment site, neither fusion of the membranes of the mycoplasma and host cell nor injury to the host cell membrane could be demonstrated. Nevertheless, seemingly, the intimate association between the adhering mycoplasmas and the epithelial cells might be an important factor in pathogenesis of the disease.     

Use of Brucella abortus vaccine strain RB51 in pregnant cows after calfhood vaccination with strain 19 in Argentina

One hundred and seven pregnant cows, which had been calfhood vaccinated with Brucella abortus strain 19 (S-19) were revaccinated with either S-19 or strain RB51 (S-RB51). All S-19-revaccinated animals seroconverted, while none of the RB51-revaccinated animals seroconverted. Two out of 25 (8%) S-19-revaccinated animals aborted, while none of the 57 RB51-revaccinated group aborted. Four of the S-19-revaccinated animals shed S-19 in the milk for at least 7 days, while only 1 cow shed S-RB51 for at least 3 days (but <7 days) post-parturition. Revaccination of strain 19 calfhood-vaccinated, pregnant cattle with S-RB51 appears to be a safe procedure with no diagnostically negative consequences.     

Identification of Ehrlichia ruminantium (Gardel strain) IFN-gamma inducing proteins after vaccination with a killed vaccine

IFN-γ is considered as a key factor in protection against heartwater of ruminants, caused by the obligate intracellular bacterium Ehrlichia ruminantium. In this study, a better definition of the molecular masses of IFN-γ inducing proteins of the Gardel strain of E. ruminantium was obtained by the use of continuous flow electrophoresis (CFE) and sensitized polyclonal lymphocytes. Out of 15 E. ruminantium CFE fractions tested within the 14–39 kDa region, eight were commonly reacted to by all goats. Interestingly, half of these fractions fall within the 23–29 kDa region, shown previously to contain polymorphic B-cell epitopes. Thus, the results suggest that this region also contains T-cell epitopes potentially involved in protection. Also, several proteins were found to be more immunogenic than the serologically immunodominant MAP1 protein. Finally, high activity within the 15–19 kDa region was observed, which confirms previous work done with CD4+ T-cell lines obtained from cattle immunized with a South African strain of E. ruminantium. The proteins falling within the molecular weight ranges defined in this study may have potential as vaccine antigens.