Genetic comparison of the Mycoplasma gallisepticum 6/85 vaccine strain and 6/85-like field isolates.

Mycoplasma gallisepticum infection in poultry leads to disease and pathology that can reduce producer profits. Live attenuated vaccines are available that can limit or completely prevent the effects of infection. Field isolates that are genetically related to the attenuated vaccine strains have been isolated, raising the question of whether the attenuation of the vaccine strains is limited and can lead the strains to revert to more virulent forms. The 6/85 live attenuated vaccine is derived from a field isolate collected in the United States. Analysis of the genome of sequenced M. gallisepticum strains revealed a cluster of 10 6/85-like strains that group with the 6/85 vaccine strain. Four genomic regions were identified that allowed for strain differentiation. The genetic differences between strains points toward nine of the ten strains most likely being sister strains to the 6/85 vaccine strain. Insufficient differences are present in the tenth strain to make a definitive conclusion. These results suggest that most if not all strains similar to the live attenuated vaccine strain are field isolates of the parent strain used to derive the live attenuated vaccine.

Whole-Genome Sequencing of Three M. gallisepticum Isolates Similar to the 6/85 Vaccine Strain.

Effective control of Mycoplasma gallisepticum infection is accomplished through vaccination with live attenuated vaccines. However, virulent M. gallisepticum strains with genetic markers consistent with those of vaccine strains were found in infected flocks. We report here the complete genetic sequences of three isolates that are genetically similar to the 6/85 vaccine strain.

Complete Genome Sequences of Three Mycoplasma gallisepticum 6/85-like Isolates.

Control of Mycoplasma gallisepticum infection can be accomplished through vaccination. However, virulent field strains with genetic markers identical to vaccine strains have been identified. Here, we report the sequencing of three field isolates with genetic markers identical to the M. gallisepticum 6/85 vaccine strain.

Effects of the In Ovo Vaccination of the ts-11 Strain of Mycoplasma gallisepticum in Layer Embryos and Posthatch Chicks.

The transmission of the ts-11 strain of Mycoplasma gallisepticum (MG) vaccine (ts-11MGV) between incubated eggs and between hatchlings that was administrated via in ovo injection, and its subsequent effects on their posthatch performance were evaluated. Marek's disease diluent alone (sham-injected) or containing either 3.63 × 101, 102, 103, or 104 cfu of ts-11MGV was manually in ovo-injected into the amnion on 18 days of incubation. Egg residue analysis, percentage incubational egg weight loss, hatchability of viable injected eggs, and hatchling body weight (BW) were assessed. Selected hatchlings from each treatment replicate group were swabbed in the choanal cleft for MG DNA detection. Female chick live performance was also assessed through 21 days of posthatch age. Unexposed control sentinel chicks were allocated to each treatment replicate group to assess horizontal transmission. Birds were later swabbed and bled respectively, for detection of MG DNA and IgM production at 21 days posthatch. In all birds, no MG DNA was detected and SPA tests for IgM were negative. Among all variables, only 0 to 21 day BW gain was significantly affected by treatment and was lower in the 3.63 × 104 ts-11 MGV treatment in comparison to all the other treatments. Because ts-11MGV does not exhibit vertical or horizontal transmission capabilities under commercial conditions, it may not be a good candidate for in ovo injection.

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.

Complete Genome Sequences of Two Vaccine Strains and One Field Isolate of Mycoplasma gallisepticum.

Mycoplasma gallisepticum infection of poultry can cause significant losses for poultry producers. Live attenuated M. gallisepticum vaccines mitigate the losses caused by infection, although the antigens that lead to immune protection have not been identified. Here, we report the sequencing of two vaccine strains and one field strain.

Complete Genome Sequences of Two Mycoplasma gallisepticum F-Strain Variants.

Mycoplasma gallisepticum pathology in poultry is preventable by vaccination with live M. gallisepticum vaccines. Research has suggested possible differences in host response between F-strain-based vaccines. The genomes of the AviPro vaccine and F99 parent strains were sequenced for comparison with the already sequenced F-strain vaccine.

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.

DNA microarray technology used for studying foodborne pathogens and microbial habitats: minireview.

Microarray analysis is an emerging technology that has the potential to become a leading trend in bacterial identification in food and feed improvement. The technology uses fluorescent-labeled probes amplified from bacterial samples that are then hybridized to thousands of DNA sequences immobilized on chemically modified glass slides. The whole gene or open reading frame(s) is represented by a polymerase chain reaction fragment of double-strand DNA, approximately 1000 base pair (bp) or 20-70 bp single-strand oligonucleotides. The technology can be used to identity bacteria and to study gene expression in complex microbial populations, such as those found in food and gastrointestinal tracts. Data generated by microarray analysis can be potentially used to improve the safety of our food supply as well as ensure the efficiency of animal feed conversion to human food, e.g., in meat and milk production by ruminants. This minireview addresses the use of microarray technology in bacterial identification and gene expression in different microbial systems and in habitats containing mixed populations of bacteria.

Flax-retting by polygalacturonase-containing enzyme mixtures and effects on fiber properties.

Enzyme-retting of flax was accomplished via individual treatment with four polygalacturonase (PGase) containing solutions of various fungal sources and the resulting fibers were characterized. The retting solutions were equilibrated to contain 2.19 U of PGase activity as determined via a dinitrosalicylic acid (DNS) reducing sugar assay. As compared with the buffer control, treatment with the various enzyme solutions increased the yield of fine fibers. Treatment with Aspergillus niger PGase resulted in a 62% increase in fine fiber yield as compared with the buffer control and fiber strength did not statistically differ (P

Cloning of the O-acetylserine lyase gene from the ruminal bacterium Selenomonas ruminantium HD4.

The gene coding for O-acetylserine lyase (OASL) was cloned from a Selenomonas ruminantium HD4 Lambda ZAP II genomic library by degenerative probe hybridization and complementation. Sequence analysis revealed a 933 bp ORF with a G + C content of 53%. The ORF had significant homology with enzymes involved in cysteine biosynthesis. A CuraBLASTN homology search showed that the ORF shared 59% nucleotide identity with the cysK of Bacillus subtilis. The deduced amino acid sequence exhibited high (>70%) similarity with the CysK of B. subtilis and other cysteine synthesis proteins from Mycobacterium tuberculosis, Mycobacterium leprae, and Spinacia oleracea. Further analysis predicted that the gene product was a member of the pyridoxal phosphate enzyme family and of cytoplasmic origin. Phylogenetic analysis clustered the S. ruminantium gene product with the OASLa isoform of B. subtilis and the OASLb isoforms of Streptococcus suis, Escherichia coli, and Campylobacter jejuni. The OASL of S. ruminantium HD4 was also able to complement the cysM cysK double mutations in Escherichia coli NK3 and allow for growth on minimal media that contained either sulfate or thiosulfate as the sole source of sulfur. These results suggest that the gene functions as a cysM in S. ruminantium HD4. In conclusion, this research describes the cloning and expression of an O-acetylserine lyase gene from the predominant ruminal anaerobe S. ruminantium HD4. To our knowledge, this is the first report characterizing genes involved in sulfur metabolism from the genus Selenomonas.

Cloning of the L-lactate dehydrogenase gene from the ruminal bacterium Selenomonas ruminantium HD4.

A clone from a Selenomonas ruminantium HD4 Lambda ZAP II genomic library was isolated by its ability to complement the anaerobic growth deficiency of an Escherichia coli (pfl, ldh) double mutant. The 1.0-kb insert from the clone was sequenced and revealed a single open reading frame (ORF, 957-bp) which was preceded by a putative Shine-Dalgarno (SD) sequence (AGGGGG). The potential SD sequence corresponded to 3' 16S rRNA sequences of various Selenomonas strains. The ORF was predicted to encode a protein of 318 amino acids with a calculated molecular mass of 34,975 Da and an isoelectric point of 5.54. In addition, the ORF contained 51 mol % G + C and this is consistent with the average G + C content (54%) of the S. ruminantium chromosome. The cloned S. ruminantium gene exhibited 59% nucleotide identity and 61% deduced amino acid similarity with L-lactate dehydrogenases (L-LDH) of Pediococcus acidilactici and Bacillus megaterium, respectively. Incorporation of the cloned S. ruminantium gene into E. coli DC1368 (pfl, ldh) restored anaerobic growth on glucose and L-LDH activity was detected in cell extracts. Because lactate accumulation within the rumen can be detrimental to animal performance, characterizing the gene(s) involved in lactate production by predominant ruminal bacteria will lead to a better understanding of lactate metabolism within the rumen.