Fluorescent microspheres as a positive indicator in an intratracheal infection model.

Animal models testing the ability of vaccines and therapeutic agents to prevent pathology from induced respiratory infection are an important means of testing and validating the vaccines and therapeutic agents. However, the lack of induced pathology in test subjects could be either indicative of protection or a problem with the animal model system. This work describes the improvement of a chicken model system of intratracheal infection using fluorescent microspheres as a positive indicator of infection. It was shown that fluorescent microspheres and Mycoplasma gallisepticum bacteria both dispersed to the same areas of the chicken respiratory system and that the microspheres remained detectable in the chicken lung tissue for at least 7 days following infection. The microspheres used are detectable using a black light, allowing for visualization during necropsy. Using the updated model system, three live M. gallisepticum vaccines were tested both for their ability to elicit a humoral immune response following vaccination, and for their ability to protect from air sac lesion pathology at two different time points following vaccination. Results showed the protective effects of the different M. gallisepticum vaccines prevented the induction of pathology, consistent with previous results. The presence of the fluorescent microspheres provided a positive method of identifying the properly infected chickens and a means of differentiating failed experimental infections so that those samples could be removed, resulting in improved consistency in infection results.

The impact of vaccination route on Mycoplasma gallisepticum vaccine efficacy.

Mycoplasma gallisepticum infection can lead to major financial losses for poultry producers. Control of M. gallisepticum infection in the layer industry is generally obtained through vaccination due to the nature of the multi-aged flocks in the facilities. Live vaccines can provide significant protection from the pathogenic effects of M. gallisepticum infection. However, differing management practices, including vaccination procedures, can lead to significant variations in the efficacy of the same vaccine. The site of vaccine deposition has been shown to be one important factor significantly influencing the vaccination outcome. Previous research has shown that vaccine applied to the eyes or sprayed on the head is significantly more effective than when sprayed on the body. Vaccine application to the eyes, through the nares (nasal), and 2 routes through the oral cavity were studied to further characterize the most efficient route for delivery. Results of this work demonstrate that eye drop vaccination is significantly more effective than nasal vaccination, and vaccine delivered through the oral cavity has a negligible contribution to overall vaccination outcome.

Proteomics inference of genes involved in host adaptation of Mycoplasma gallinarum.

Different from most other host-specific mycoplasmas, Mycoplasma gallinarum has been isolated from various hosts, such as poultry, pig, cattle, and sheep. The wide distribution among different hosts, the low pathogenesis, and the weak host immunological responses suggest this mycoplasma has a unique host adaptation mechanism. In this study, we applied two-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (2D LC-MS/MS) to characterize the protein profiling of M. gallinarum. Our results suggest that M. gallinarum possesses homologs of cytadhesin proteins found in other mycoplasmas lacking an organized tip organelle. Our results showed that there are possibly multiple aminopeptidase gene homologs present in M. gallinarum, which might be involved in nutrient acquisition of M. gallinarum. The information present here would be useful for future studies to identify genes responsible for the colonization and host adaptation properties of M. gallinarum.

Induction of a mycoplasma gallisepticum pMGA gene in the chicken tracheal ring organ culture model.

Genetic and molecular methods to investigate the pathogenesis of the poultry respiratory pathogen Mycoplasma gallisepticum are quite limited. Therefore, the objective of this study was to design and evaluate a functional genomics approach to identify M. gallisepticum genes involved in colonization of the poultry respiratory tract. To serve as a transcriptional reporter, a promoterless lacZ gene from Escherichia coli was cloned into the Tn4001 transposon. The transposon was used to randomly mutagenize the chromosome of the M. gallisepticum S6 strain, and a bank of 1386 transposon mutants containing lacZ fusions to mycoplasma chromosomal DNA was assembled. Each mycoplasma clone containing the lacZ reporter was independently screened in the chicken tracheal ring organ culture (TROC) model system for increased production of beta-galactosidase. A twofold or greater increase in beta-galactosidase was consistently observed for eight mutants. In one of the mutants, the transposon was inserted in a pMGA gene encoding a cell surface adhesin involved in hemagglutination. Therefore, these data indicate that screening of a M. gallisepticum transposon reporter bank with a chicken TROC model is useful for the identification of genes induced during poultry colonization and virulence.