The cellular immune response in the tracheal mucosa to Mycoplasma gallisepticum in vaccinated and unvaccinated chickens in the acute and chronic stages of disease.

Mycoplasma gallisepticum causes a lymphoproliferative response in the tracheal mucosa of infected birds. The studies reported here aimed to determine, using immunohistochemical and immunofluorescent staining, which lymphocyte subsets were infiltrating the mucosa during the acute and chronic phases of disease and to determine whether these subsets differed in birds that had been vaccinated with strain ts-11. In vaccinates there was no detectable infiltration of T or B lymphocytes between 1 and 6 weeks after infection with a virulent strain. Unvaccinated birds had an initial influx of CD8+TCR- lymphocytes at 1 week, with the numbers decreasing over the next 5 weeks. CD8+TCR+ cells increased over the 6 weeks. The proportion of CD4+TCRalphabeta2+ cells also increased, whilst there was an increase in CD4+TCRalphabeta1+ cells at 3 weeks and then a decrease by 6 weeks. B lymphocytes were not detected until 3 weeks after infection, and their appearance coincided with a decrease in the concentration of mycoplasma DNA detectable in the trachea. The formation of clusters of CD8+TCR- lymphocytes was a prominent feature of the early response, while at 3 and 6 weeks after infection clusters of B cells became prominent, although in some cases they surrounded a cluster of CD8+ cells. These observations suggest a primary role for local antibody mediated responses in controlling M. gallisepticum infection, but also show that there are probably significant natural killer and cytotoxic T cell responses to infection, although the efficacy of these in controlling infection was not able to be determined.

Age related differences in the immune response to vaccination and infection with Mycoplasma gallisepticum.

Several studies have suggested that there are age related differences in the responses of chickens to vaccination and infection with Mycoplasma gallisepticum, but there have not been any systematic comparisons of the responses of young birds to vaccination with those of birds the same age to infection. The aim of the studies described here was to examine the immune responses of chickens between 1 and 6 weeks of age to vaccination and to infection with M. gallisepticum. Birds under 4 weeks of age developed significantly more severe clinical signs after infection with virulent M. gallisepticum than birds that were 4 or 6-weeks old at the time of infection, with greater concentrations of M. gallisepticum in the trachea. Chickens younger than 4 weeks old at the age of vaccination were protected from clinical signs of disease after challenge and from development of more severe lesions in the trachea and air sacs, although this protective immunity was significantly less effective in controlling the replication of M. gallisepticum in the trachea. Examination of the subsets of lymphocytes infiltrating the tracheal mucosa did not detect any age related differences. Thus the ts-11 vaccine has been shown to be safe in birds between 1 and 4 weeks of age and to be effective in preventing development of severe disease in them after challenge, even though birds of the same age were more susceptible to development of disease when infected with virulent M. gallisepticum.

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.

Induction of enzootic pneumonia in pigs by the administration of an aerosol of in vitro-cultured Mycoplasma hyopneumoniae.

The aim of this study was to establish whether enzootic pneumonia could be induced reliably in piglets by administering an aerosolised culture of Mycoplasma hyopneumoniae. Groups of five M hyopneumoniaefree Landrace x Large White piglets weaned at 11 to 14 days of age were exposed to aerosols of in vitro cultures of a virulent strain of M hyopneumoniae. In three separate trials, 14 of 15 pigs exposed to the bacteria developed pneumonia, but pigs exposed to the culture medium alone did not develop the disease. Lung pathology, both gross and histological, indicated acute disease. Ten of the pigs were tested for seroconversion by Western blot and they were all positive. The growth rates of the infected pigs were significantly reduced and the water consumption of the infected groups was also depressed. M hyopneumoniae was recovered from eight of the 15 infected pigs.

Automated, fluorescence-based approach for the specific diagnosis of chicken coccidiosis.

We have established a fluorescence-based electrophoretic approach for the specific identification of all seven currently recognised species of Eimeria infecting chickens. The second internal transcribed spacer (ITS-2) of ribosomal DNA is amplified by polymerase chain reaction (PCR) from any of the seven species using a single set of oligonucleotide primers (of which the reverse one is fluorescently labelled). The amplicons are heat-denatured, subjected to denaturing polyacrylamide gel electrophoresis in a 377 DNA sequencer (ABI). The chromatograms produced are stored electronically and then analysed using GeneScan 3.1 software. Using control DNA samples representing monospecific lines of Eimeria, regions in the chromatograms have been defined for the specific identification of each of the seven species, although some variation in the chromatograms (reflecting population variation) was detectable within two species. Electrophoretic reading and analysis is carried out automatically using a computer imaging system, thus making it a time- and cost-effective approach. It is well suited for high-throughput diagnostic screening of oocyst samples and should find applicability as a tool for prevalence studies, monitoring of coccidiosis outbreaks and the quality control of vaccines.

Rapid PCR detection of Salmonella in horse faecal samples.

A rapid polymerase chain reaction (PCR) assay was developed for detecting Salmonella in faeces of horses and assessed on samples from horses admitted to a veterinary hospital. Direct detection was achieved by amplification of part of ompC after extraction of DNA from faeces using a spin column method to reduce the amount of inhibitory substances in samples. An internal positive control was included to detect false negative results. While the sensitivity of the PCR assay was less than culture when assessed on faeces inoculated with Salmonella, its sensitivity on faecal samples obtained from horses was much greater than culture. Salmonella DNA was detected in 40% of faecal samples using the PCR assay while Salmonella were cultured from only 2% of the samples. The PCR assay has potential for use in either routine diagnosis or for detection of the carrier status in animals.

Molecular epidemiology of Salmonella Heidelberg in an equine hospital.

From 1992 to 1997, multi-drug resistant (MDR) Salmonella Heidelberg isolates were cultured from a number of horses hospitalised in a veterinary hospital in Victoria, Australia. To examine the relationships between the cases, 28 isolates from the hospital were compared by pulsed field gel electrophoresis (PFGE), IS200 element profiles, antimicrobial resistance patterns, plasmid profiles and phage typing. The PFGE patterns following digestion with XbaI and BlnI restriction endonucleases showed that the isolates from the veterinary hospital originated from a common source. These isolates also had indistinguishable IS200 profiles. However, PFGE was more discriminatory than IS200 profiles. All the veterinary hospital isolates and one independent isolate had the same antimicrobial resistance pattern and had at least one plasmid in common. Localisation of antimicrobial resistance genes indicated that the veterinary hospital isolates had more than one plasmid carrying resistance genes and that the genes encoding sulphathiazole and trimethoprim resistance were not on these plasmids. Phage typing was ineffective as 22 of the 28 isolates were untypeable. In conclusion, the combination of different methods used for epidemiological studies suggested that a single strain of MDR S. Heidelberg was isolated from horses admitted to the hospital for 6 years and caused salmonellosis in susceptible horses within that period with no apparent correlation between the antimicrobials used and retention of its MDR phenotype.

Single-strand restriction fragment length polymorphism analysis of the second internal transcribed spacer (ribosomal DNA) for six species of Eimeria from chickens in Australia.

Species of Eimeria from chickens from Australia were characterised using a polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) approach. The ribosomal DNA region spanning the second internal transcribed spacer (ITS-2) was amplified from genomic DNA by PCR, digested separately with three restriction endonucleases (CfoI, Sau3AI and TaqI) and the fragments separated by denaturing gel electrophoresis. The PCR products amplified from the six species varied from approximately 70 to 620 bp on agarose gels, with differences in size and number of bands among species, but no apparent variation within a species. The PCR-RFLP analysis of ITS-2 amplicons on denaturing gels gave characteristic profiles for individual species (except for minor variation in profiles within some species). The results indicate that ITS-2 contains useful genetic markers for the identification of six Eimeria species occurring in Australia.

Lymphocytic infiltration in the chicken trachea in response to Mycoplasma gallisepticum infection.

A prominent feature of disease induced by Mycoplasma gallisepticum is a lymphoproliferative response in the respiratory tract. Although this is also seen in other mycoplasma infections, including Mycoplasma pneumoniae, the phenotype of the lymphocytes infiltrating the respiratory tract has not been determined. In this study, the numbers and distribution of lymphocytes in the tracheas of chickens infected with a virulent strain of M. gallisepticum were examined. Three groups of chickens were experimentally infected with M. gallisepticum and three unchallenged groups were used as controls. One infected and one control group were culled at 1, 2 and 3 weeks post infection. Tracheas were removed and examined for the presence and number of T cells carrying CD4, CD8, TCRgamma7, TCRalphabeta1 or TCRalphabeta2 markers. There was no significant difference in the number of CD8+ cells in the upper, middle and lower trachea. High numbers of both CD4+ and CD8+ cells were found with variable numbers of TCRalphabeta1+ and TCRalphabeta2+, but no TCRgammadelta+, cells throughout the time course. The distribution of CD4 cells was dispersed, while the CD8+ cells were clustered in follicular-like arrangements. No difference was detected in the distribution of TCRalphabeta1+ and TCRalphabeta2+ cells. The titre of mycoplasma genomes in the trachea decreased significantly from 1 to 2 weeks, while the mucosal thickness of the trachea increased significantly from 1 to 2 weeks then decreased from 2 to 3 weeks, indicating resolution of the lesions following control of infection. This study is the first to examine the phenotypes of T lymphocytes infiltrating the respiratory tract during mycoplasma infections. The findings suggest involvement of specific stimulation of CD8+ cells, particularly in the acute phase of disease.

Colonization of the respiratory tract by a virulent strain of avian Escherichia coli requires carriage of a conjugative plasmid.

The E3 strain of E. coli was isolated in an outbreak of respiratory disease in broiler chickens, and experimental aerosol exposure of chickens to this strain induced disease similar to that seen in the field. In order to establish whether the virulent phenotype of this strain was associated with carriage of particular plasmids, four plasmid-cured derivatives, each lacking two or more of the plasmids carried by the wild-type strain, were assessed for virulence. Virulence was found to be associated with one large plasmid, pVM01. Plasmid pVM01 was marked by introduction of the transposon TnphoA, carrying kanamycin resistance, and was then cloned by transformation of E. coli strain DH5alpha. The cloned plasmid was then reintroduced by conjugation into an avirulent plasmid-cured derivative of strain E3 which lacked pVM01. The conjugant was shown to be as virulent as the wild-type strain E3, establishing that this plasmid is required for virulence following aerosol exposure. This virulence plasmid conferred expression of a hydroxamate siderophore, but not colicins, on both strain E3 and strain DH5alpha. Carriage of this plasmid was required for strain E3 to colonize the respiratory tracts of chickens but was not necessary for colonization of the gastrointestinal tract. However, the virulence plasmid did not confer virulence, or the capacity to colonize the respiratory tract, on strain DH5alpha. Thus, these studies have established that infection of chickens with E. coli strain E3 by the respiratory route is dependent on carriage of a conjugative virulence plasmid, which confers the capacity to colonize specifically the respiratory tract and which also carries genes for expression of a hydroxymate siderophore. These findings will facilitate identification of the specific genes required for virulence in these pathogens.

A novel mechanism for control of antigenic variation in the haemagglutinin gene family of mycoplasma synoviae.

High-frequency phase and antigenic variation of homologous lipoprotein haemagglutinins has been seen in both the major avian mycoplasma pathogens, Mycoplasma synoviae and Mycoplasma gallisepticum. The expression and, hence, antigenic variation of the pMGA gene family (encoding these lipoproteins in M. gallisepticum) is controlled by variation in the length of a trinucleotide repeat motif 5' to the promoter of each gene. However, such a mechanism was not detected in preliminary observations on M. synoviae. Thus, the basis for control of variation in the vlhA gene family (which encodes the homologous haemagglutinin in M. synoviae) was investigated to enable comparison with its homologue in M. gallisepticum and with other lipoprotein gene families in mycoplasmas. The start point of transcription was identified 119 bp upstream of the initiation codon, but features associated with control of transcription in other mycoplasma lipoprotein genes were not seen. Comparison of three copies of vlhA revealed considerable sequence divergence at the 3' end of the gene, but conservation of the 5' end. Southern blot analysis of M. synoviae genomic DNA revealed that the promoter region and part of the conserved 5' coding sequence occurred as a single copy, whereas the remainder of the coding sequence occurred as multiple copies. A 9.7 kb fragment of the genome was found to contain eight tandemly repeated regions partially homologous to vlhA, all lacking the putative promoter region and the single-copy 5' end of vlhA, but extending over one of four distinct overlapping regions of the 3' coding sequence. Examination of sequential clones of M. synoviae established that unidirectional recombination occurs between the pseudogenes and the expressed vlhA, with duplication of pseudogene sequence and loss of the corresponding region previously seen in the expressed gene. Expression of the 5' end of two variants of the vlhA gene showed that they differed in their reaction with monoclonal antibodies specific for this region. These data suggest that the control of vlhA antigenic variation in M. synoviae is achieved by multiple gene conversion events using a repertoire of coding sequences to generate a chimeric expressed gene, with the greatest potential for variation generated in the region encoding the haemagglutinin. Thus, completely distinct mechanisms have been adopted to control antigenic variation in homologous gene families.

High-resolution electrophoretic procedures for the identification of five Eimeria species from chickens, and detection of population variation.

To overcome limitations of conventional approaches for the identification of Eimeria species of chickens, we have established high resolution electrophoretic procedures using genetic markers in ribosomal DNA. The first and second internal transcribed spacer (ITS-1 and ITS-2) regions of ribosomal DNA were amplified by polymerase chain reaction (PCR) from genomic DNA samples representing five species of Eimeria (E. acervulina, E. brunetti, E. maxima, E. necatrix and E. tenella), denatured and then subjected to denaturing polyacrylamide gel electrophoresis (D-PAGE) or single-strand conformation polymorphism (SSCP) analysis. Differences in D-PAGE profiles for both the ITS-1 and ITS-2 fragments (combined with an apparent lack of variation within individual species) enabled the unequivocal identification of the five species, and SSCP allowed the detection of population variation between some isolates representing E. acervulina, which remained undetected by D-PAGE. The establishment of these approaches has important implications for controlling the purity of laboratory lines of Eimeria, for diagnosis and for studying the epidemiology of coccidiosis.

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

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

Indirect enzyme-linked immunosorbent assay for detection of immunoglobulin G reactive with a recombinant protein expressed from the gene encoding the 116-kilodalton protein of Mycoplasma pneumoniae.

Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactivity with patient sera and the most antigenic was further assessed for its serodiagnostic potential by indirect enzyme-linked immunosorbent assay (ELISA). The ELISA based on the recombinant protein was equivalent in sensitivity to the commercial test (Serodia Myco II; Fujirebio Inc.) to which it was compared. Southern and Western blotting data suggested that the recombinant protein derived from the 116-kDa protein of M. pneumoniae could provide a species-specific diagnostic tool, although further assessment is required.

Multigene families encoding the major hemagglutinins in phylogenetically distinct mycoplasmas.

Mycoplasma synoviae has two major membrane antigens, MSPA and MSPB, both of which are phase variable and which may be coordinately involved in adhesion of the organism to erythrocytes. A single gene (vlhA) from M. synoviae was characterized, and polypeptides were expressed from nonoverlapping 5' and 3' regions in Escherichia coli. The expression product of the vlhA 5' region reacted with specific reagents against MSPB, while that of the 3' region reacted with specific reagents against MSPA. Analysis of the predicted amino acid sequence showed a characteristic signal peptidase II cleavage site, and the presence of the acylation site was confirmed by identification of a lipid-associated membrane protein, similar in molecular mass to MSPB, in [3H]palmitate-labelled membrane proteins. Further sequence analysis of the vlhA gene revealed a high identity with the Mycoplasma gallisepticum pMGA1.7 gene, a member of a large translated family. The vlhA gene was shown to hybridize to multiple restriction fragments of the M. synoviae genome, suggesting that it was also a member of a multigene family. These findings indicate that coordinate phase variation of the two major surface antigens of M. synoviae WVU may be due to their expression from the same gene and that homologous gene families encode the major hemagglutinins of two phylogenetically distinct mycoplasmas. The presence of homologous multigene families in such phylogenetically distinct species, but not in the genomes of more closely related species, suggests that the families may have been transferred horizontally.

Expression of two members of the pMGA gene family of Mycoplasma gallisepticum oscillates and is influenced by pMGA-specific antibodies.

Certain monoclonal antibodies and polyclonal antisera directed to pMGA, the major protein of Mycoplasma gallisepticum, were tested for the ability to influence the surface phenotype of the cell population which resulted from their inclusion in growth medium. The polyclonal antiserum and one monoclonal antibody (MAb 66) resulted in an alteration of surface phenotype; specifically, populations of cells grown either on plates or in broth cultures which contained these reagents ceased the expression of pMGA and instead expressed an antigenically unrelated new polypeptide (p82). Upon the removal of antibody, the progeny of these cells regained pMGA expression and produced antigenically sectored colonies. The basis of this switch between pMGA+ and pMGA- states was shown to be transcriptional. The p82 polypeptide, the expression of which resulted from growth of cells in antibodies, was another member of the pMGA gene family and was located just downstream from the pMGA gene normally expressed by the M. gallisepticum cells used. Collectively the results of this work suggest that this organism has evolved an unusual means of altering the antigenic composition of its surface in response to antibodies or to other environmental cues.

Production of temperature-sensitive clones of Mycoplasma synoviae for evaluation as live vaccines.

An Australian field isolate of Mycoplasma synoviae (MS), 89079/7NS, was exposed to the mutagen N-nitro-N'-methyl-N-nitrosoguanidine. Fifteen clones from the exposed culture were characterized for temperature sensitivity. Four clones labelled B, D, G, and H were temperature sensitive and were further characterized for their ability to colonize chickens and elicit an immune response. Serum antibody responses to MS were detected 3 wk after infection, by eyedrop, in 10 of 10 birds inoculated with 86079/7NS and clones B and G and in 9 of 10 birds inoculated with clone H. No MS antibody response was observed in any bird inoculated with clone D. MS was recovered from the upper trachea of 10 of 10 birds inoculated with clones B, G, and H at 2 wk after infection. No MS was isolated from birds inoculated with clone D. Clone H, designated MS-H, was selected as a potential vaccine candidate.

Efficacy of a temperature-sensitive Mycoplasma synoviae live vaccine.

A temperature-sensitive clone of Mycoplasma synoviae (MS), MS-H, derived from the chemical mutagenesis of the Australian field isolate 86079/7NS, was investigated for efficacy as a live vaccine. Titers of MS-H vaccine ranged between 1.16 x 10(8) and 8.4 x 10(8) color changing units/ml when incubated at 33 C and were consistently > or = 10(2) lower when incubated at 39.5 C. Laboratory-produced MS-H vaccine protected 8 out of 10 specific-pathogen-free Webster white leghorn chickens against a combined experimental challenge of aerosols of the virulent wild MS strain 88064/FP and T-strain infectious bronchitis vaccine administered intratracheally. Laboratory and commercially produced vaccines were compared for efficacy against thoracic air sac challenge, with both achieving similar levels of air sac protection. Air sac lesion incidences of five air sacs with lesions out of 32 and four out of 32 were seen in groups vaccinated with laboratory and commercial vaccines, respectively, compared with 13 out of 20 in nonvaccinated specific-pathogen-free hybrid white leghorn chickens. An attempt to determine the dose response of the commercial vaccine was conducted with 0.5, 2, and 4 times the standard dose. Air sac lesion incidences of 16 air sacs with lesions out of 32 in the 0.5 times, 11 out of 32 in the 2 times, and 1 out of 32 in the 4 times dose groups were observed.

Safety of a temperature-sensitive clone of Mycoplasma synoviae as a live vaccine.

A temperature-sensitive (ts+) clone derived from the Australian Mycoplasma synoviae (MS) field isolate 86079/7NS was produced by chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and assessed for safety as a live vaccine. This clone, designated MS-H, was assessed for pathogenicity in three different models with air sac lesions as the criterion. No air sac lesions were observed when MS-H was administered to specific-pathogen-free hybrid white leghorn (HWL) chickens by eyedrop at 10 times the normal dose or directly into the thoracic air sacs or as an aerosol administered to specific-pathogen-free Webster white leghorn chickens with concurrent intratracheal T-strain infectious bronchitis virus (IBV). MS-H did not revert to virulence or lose the ts+ phenotype when passaged through five in vivo and 10 in vitro passages. No adverse effects were seen when HWL chickens were vaccinated concurrently with MS-H and combinations of Mycoplasma gallisepticum ts-11 vaccine, IBV vaccine, and infectious laryngotracheitis virus vaccine. Lateral transmission of MS-H was found to occur when vaccinated HWL chickens were mixed with unvaccinated chickens 2 wk after vaccination. At 1 wk after mixing, one out of two unvaccinated chickens had seroconverted to MS and was culture positive for MS. At 2 wk after mixing, both contact chickens were positive for MS by culture and serology.

Field evaluation of the safety and efficacy of a temperature-sensitive Mycoplasma synoviae live vaccine.

Mycoplasma synoviae (MS) strain MS-H was used in three separate commercial flocks for large-scale evaluation of the safety and efficacy of the vaccine under commercial conditions. MS-H successfully colonized meat and layer-breeders vaccinated by eyedrop and persisted for up to 55 wk after vaccination. Restriction fragment length polymorphism analysis showed that MS-H was the only strain isolated from two vaccinated flocks. In a third flock, challenge with a wild-type MS occurred, and this strain was isolated from both vaccinated and unvaccinated birds. Vertical transmission of MS-H was investigated by culturing pipped embryos and testing broiler progeny for MS antibody at processing (56 days old). No evidence of vertical transmission was detected. Lateral transmission of MS-H strain from vaccinated to unvaccinated birds occurred in one of the commercial flocks. Forty-one of 50 isolates of MS-H obtained from vaccinated flocks maintained their temperature-sensitive phenotype, but nine isolates showed a nontemperature-sensitive phenotype.