A single-cell pedigree analysis of alternative stochastic lymphocyte fates.

In contrast to most stimulated lymphocytes, B cells exposed to Toll-like receptor 9 ligands are nonself-adherent, allowing individual cells and families to be followed in vitro for up to 5 days. These B cells undergo phases typical of an adaptive response, dividing up to 6 times before losing the impetus for further growth and division and eventually dying by apoptosis. Using long-term microscopic imaging, accurate histories of individual lymphocyte fates were collected. Quantitative analysis of family relationships revealed that times to divide of siblings were strongly related but these correlations were progressively lost through consecutive divisions. A weaker, but significant, correlation was also found for death times among siblings. Division cessation is characterized by a loss of cell growth and the division in which this occurs is strongly inherited from the original founder cell and is related to the size this cell reaches before its first division. Thus, simple division-based dilution of factors synthesized during the first division may control the maximum division reached by stimulated cells. The stochastic distributions of times to divide, times to die, and divisions reached are also measured. Together, these results highlight the internal cellular mechanisms that control immune responses and provide a foundation for the development of new mathematical models that are correct at both single-cell and population levels.

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.

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.

Identification of Mycoplasma synoviae immunogenic surface proteins and their potential use as antigens in the enzyme-linked immunosorbent assay.

Specific immunogenic proteins of Mycoplasma synoviae (MS) strain WVU-1853 were purified and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western transfer using anti-MS, anti-M. gallisepticum, and non-immune chicken sera. A cluster of prominent immunoreactive proteins with molecular masses from 46 to 52 kDa (p46-52) and less reactive single proteins of approximately 22 and 92 kDa were shown to partition into the detergent phase of Triton X-114 MS lysates, suggesting that these amphiphilic polypeptides are integral membrane proteins. Monoclonal antibodies, produced by immunizing mice with MS whole cell proteins and shown to bind species-specific determinants, reacted strongly with p46-52 and less intensely with the 22-kDa protein, but they did not react with the 92-kDa protein. A protein fraction extracted from the Triton X-114 detergent phase and further purified by ion-exchange chromatography was found to be highly enriched in p46-52 and was used as antigen in a prototype enzyme-linked immunosorbent assay (ELISA) to detect MS antibodies. Seventy serum samples were taken variously from specific-pathogen-free chickens experimentally infected with MS or MG and from commercial broiler breeder chickens vaccinated for MS and MG. All samples were tested by both rapid serum agglutination and a prototype ELISA described herein; the two tests were strongly correlated (r = 0.776). The results indicate that the ELISA antigen used--the immunodominant cell surface proteins in the p46-52 cluster--appears to be a good candidate for use in the development of improved rapid diagnostics of MS infections in birds.

A proposed formula for calculating energy needs of children with cerebral palsy.

This study compared two methods of calculating the energy needs of children with CP: the traditional method, using RDA for chronological age; and the Krick method, which calculates the BMR and includes factors for muscle tone, movement or level of activity, and energy requirements for normal and catch-up growth. 30 tube-fed children, aged between nine months and 18 years, who were inpatients for longer than one week at the Kennedy Institute, were reviewed. 14 were female. They were weighed at admission and discharge to evaluate the rate of growth; calorie prescriptions at discharge were based on the clinical course. The Krick method was found to be a more potent predictor of the discharge prescription than the RDA method.