Peer-Reviewed Publications

Little research has focused on biotechnology for animal nutrition. We wanted to investigate the possibility of simplifying a modern biotechnology for practical application to broiler research. A study was conducted to evaluate plasma protein status as affected by dietary lysine using Matrix-assisted-laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). Broiler chicks were fed either a diet deficient (1.00%) or adequate (1.22%) in lysine from 0 to 17 d of age. Body weight was determined at 17 d of age. Upon validation of different growth conditions, blood sample representative from each experimental unit was taken and pooled by treatment. Blood plasma was then submitted to MALDI-TOF MS analyses. Resultant spectra, representative of the smallest-mass subset (<25 kDa) of the proteins in each sample was collected. Despite the limited mass range, the data sets were extremely complex; each set of data had almost a quarter of a million datapoints per sample. Plasma from the chicks fed the lysine adequate diet appeared to have a higher number of highly stringent and differentially expressed protein peaks when compared to the underfed lysine chicks. There appears to be a tendency for increased variability as proteins of higher nominal mass are expressed in MALDI-TOF MS, based on calculation of coefficient of variation (CV). However, such CV is not surprising to any equipment or laboratory assay, and appears to be of low impact to this procedure (<0.1% CV). Results from this study exemplify the feasibility of MALDI-TOF MS for biomarker identification of nutritional status.

Pasteurella multocida is an important animal pathogen that may also infect humans through animal bites and scratches. After comparison of transcriptional regulator gene sequences from the P. multocida genome with other DNA sequences at GenBank, we identified two genes (i.e., Pm0762 and Pm1231) uniquely present in P. multocida. By using oligonucleotide primers (Pm0762F/R and Pm1231F/R) designed from these genes in PCR, it was found that specific DNA products of expected sizes were obtained with genomic DNA from P. multocida only, but not from other bacteria. These results indicated that the putative transcriptional regulator genes Pm0762 and Pm1231 are species-specific, and that the PCR methods targeting these genes provide a useful means of rapidly and precisely identifying P. multocida from other bacteria. Further elucidation of the roles and functions of these putative transcriptional regulator genes (Pm0762 and Pm1231) and their protein products may help provide valuable insight into the molecular mechanism of P. multocida virulence and pathogenicity.

Listeria ivanovii is a Gram-positive bacterial pathogen that is capable of causing abortions and stillbirths in farm animals, particularly sheep and cattle. In terms of morphological, biochemical and molecular characteristics, L. ivanovii resembles other Listeria species such as L. monocytogenes, a pathogen of both man and animals. In this study, through comparative analysis of genomic DNA from the six Listeria species, a L. ivanovii specific clone (liv22-228) containing a 946 bp insert was isolated. This clone contained the 5' ends of two divergently transcribed L. ivanovii genes and an intergenic spacer region, similar in organization to homologous regions from the L. innocua and L. monocytogenes genomes. Regions of low homology in the clone were identified by comparing to the L. innocua and L. monocytogenes genomes, and oligonucleotide primers (liv22-228F and liv22-228R) were designed. These primers amplified a 463 bp band from genomic DNA of L. ivanovii strains only, but not from other Listeria species or common bacteria. Thus, PCR employing L. ivanovii specific primers (liv22-228F and liv22-228R) provides a useful and straightforward method for rapid and precise determination of L. ivanovii.

The importance of angiosperms to sustaining humanity by providing a wide range of 'ecosystem services' warrants increased exploration of their genomic diversity. The nearly completed sequences for two species representing the major angiosperm subclasses, specifically the dicot Arabidopsis thaliana and the monocot Oryza sativa, provide a foundation for comparative analysis across the angiosperms. The angiosperms also exemplify some challenges to be faced as genomics makes new inroads into describing biotic diversity, in particular polyploidy (genome-wide chromatin duplication), and much larger genome sizes than have been studied to date.

The entire chicken genome sequence will be available by the time this review is in press. Chickens will be the first production animal species to enter the "postgenomic era." This fundamental structural genomics achievement allows, for the first time, complete functional genomics approaches for understanding the molecular basis of chicken normo- and pathophysiology. The functional genomics paradigm, which contrasts with classical functional genetic investigations of one gene (or few) in isolation, is the systematic holistic genetic analyses of biological systems in defined contexts. Context-dependent gene interactions are the fundamental mechanics of all life. Functional genomics uses high-throughput large-scale experimental methods combined with statistical and computational analyses. Projects with expressed sequence tags in chickens have already allowed the creation of cDNA microarrays for large-scale context-dependant mRNA analysis (transcriptomics). However, proteins are the functional units of almost all biological processes, and protein expression very often bears no correlation to mRNA expression. Proteomics, a discipline within functional genomics, is the context-defined analysis of complete complements of proteins. Proteomics bridges the "sequence-to-phenotype gap;" it complements structural and other functional genomics approaches. Proteomics requires high capital investment but has ubiquitous biological applications. Although currently the fastest-growing human biomedical discipline, new paradigms may need to be established for production animal proteomics research. The prospective promise and potential pitfalls of using proteomics approaches to improve poultry pathogen control will be specifically highlighted. The first stage of our recently established proteomics program is global protein profiling to identify differentially expressed proteins in the context of the commercially important pathogens. Our trials and tribulations in establishing our proteomics program, as well some of our initial data to understand chicken immune system function, will be discussed.

Listeria monocytogenes is an opportunistic bacterial pathogen that has accounted for an important portion of human foodborne diseases worldwide. In this study, through comparative analysis of L. innocua and L. monocytogenes genomic sequences, we selected a L. monocytogenes specific gene (lmo0733) that has the potential for specific detection of L. monocytogenes. Using PCR primers (lmo0733F and lmo0733R) derived from this gene, a specific fragment of 453 bp was amplified only from genomic DNA of L. monocytogenes strains. PCR products from other Listeria species as well as other Gram-positive and -negative species were not detectable, confirming the specificity of this assay. Thus, the PCR test employing primers lmo0733F and lmo0733R represents an additional tool in the diagnostic arsenal for rapid, sensitive and specific detection and identification of human infections due to L. monocytogenes.

Proteomics is the study of the entire protein compliment of an organism. The blood plasma is the only tissue in which an organism's entire proteome may be potentially represented. First results toward mapping the broiler plasma proteome are presented here. Blood was taken from eight 18 day-old representative commercial broiler chickens. Plasma was isolated from each sample and pooled. For initial sample fractioning a 0.4 µl aliquot of the pooled plasma was run on one dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Based on relative amounts of protein, the gel was divided into three fractions. The proteins were in-gel digested with trypsin. Two-dimensional liquid chromatography in-line with electrospray ionization tandem mass spectrometry was then used for "shot-gun" qualitative plasma proteomics. The resulting tandem mass spectra were then searched against the non-redundant chicken protein database. Generally accepted high stringency statistical criteria for protein identification were used. Eighty-four chicken proteins were identified. Our work demonstrates the future potential for plasma proteomics for identifying biomarkers of disease and production in chickens.

Several heterologous expression systems were tested for their ability to express a unique maize cysteine proteinase Mir1. A baculovirus-based expression system using Trichoplusia ni larvae as host resulted in the expression of Mir1 that was correctly processed and exhibited proteinase activity. Expression in Escherichia coli resulted in accumulation of Mir1, but it had limited solubility and enzymatic activity. Large quantities of Mir1 were produced when Pichia pastoris was used as the host, but the enzyme was insoluble and inactive.

The cereal species, of central importance to our diet, began to diverge 50-70 million years ago. For the past few thousand years, these species have undergone largely parallel selection regimes associated with domestication and improvement. The rice genome sequence provides a platform for organizing information about diverse cereals, and together with genetic maps and sequence samples from other cereals is yielding new insights into both the shared and the independent dimensions of cereal evolution. New data and population-based approaches are identifying genes that have been involved in cereal improvement. Reduced-representation sequencing promises to accelerate gene discovery in many large-genome cereals, and to better link the under-explored genomes of `orphan' cereals with state-of-the-art knowledge.

Listeria monocytogenes is an opportunistic bacterial pathogen that is an important cause of human food-borne illness worldwide. However, L. monocytogenes strains demonstrate considerable variation in pathogenic potential. In this report, virulent and avirulent L. monocytogenes isolates were compared by using a comparative screening strategy. Two clones were identified that contained DNA that was only present in virulent L. monocytogenes strains. PCR primers were designed for three genes from these clones and for five other selected L. monocytogenes genes. All eight primer sets predominantly detected virulent L. monocytogenes isolates, as determined by a mouse virulence assay; one of the putative internalin genes, lmo2821, was detected in all strains that were considered to be virulent. Primers from these eight genes were then tested by PCR against a larger panel of bacterial strains; each of the genes was detected predominantly in clinical or food L. monocytogenes isolates, rather than environmental isolates. The findings from this study suggest that virulent L. monocytogenes strains may possess genes that are not present in avirulent isolates, which could serve as markers for PCR assessment of L. monocytogenes virulence.

The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that resembles the lid subcomplex of proteasomes. Through its ability to regulate specific proteasome-mediated protein degradation events, CSN controls multiple aspects of development. Here, we report the cloning and characterization of AtCSN2, the last uncharacterized CSN subunit from Arabidopsis. We show that the AtCSN2 gene corresponds to the previously identified FUS12 locus and that AtCSN2 copurifies with CSN, confirming that AtCSN2 is an integral component of CSN. AtCSN2 is not only able to interact with the SCF(TIR1) subunit AtCUL1, which is partially responsible for the regulatory interaction between CSN and SCF(TIR1), but also interacts with AtCUL3, suggesting that CSN is able to regulate the activity of other cullin-based E3 ligases through conserved interactions. Phylogenetic analysis indicated that the duplication and subsequent divergence events that led to the genes that encode CSN and lid subunits occurred before the divergence of unicellular and multicellular eukaryotic organisms and that the CSN subunits were more conserved than the lid subunits during evolution. Comparative analyses of the subunit interaction of CSN revealed a set of conserved subunit contacts and resulted in a model of CSN subunit topology, some aspects of which were substantiated by in vivo cross-link tests.

Cot-based cloning and sequencing (CBCS), a synthesis of Cot analysis, DNA cloning and high-throughput sequencing, promises to accelerate the study of eukaryotic genomes. In particular, CBCS will (1) permit efficient gene discovery in species with substantial quantities of repetitive DNA, (2) allow the sequence complexity (i.e. all the unique sequence information) of large genomes to be elucidated at a fraction of the cost of shotgun sequencing, and (3) enhance genome sequencing efforts by facilitating capture of low-copy sequences not secured by EST sequencing. CBCS should accelerate comparative genomics research, especially in large genomes such as those of many crops.

The effects of F-strain Mycoplasma gallisepticum (FMG) inoculation during the pullet period on the subsequent performance and egg characteristics of commercial Single Combed White Leghorn hens were evaluated. In two trials, BW, feed consumption, egg production (EP), egg weight, egg size class, relative eggshell water vapor conductance, and relative percentages of eggshell, yolk and albumen weights were determined through approximately 60 wk of age. In each trial, pullets at 12 wk of age were randomly assigned to negative pressure biological isolation units. Birds in one-half of the total units were inoculated with FMG, and the other half were sham-inoculated with sterile media. In both trials, onset of lay was delayed approximately 1 wk in layers inoculated with FMG. Control birds that had not been previously inoculated with FMG laid their first egg at 18 wk of age, while birds that had been previously inoculated with FMG laid their first egg at 19 wk of age. In Trial 1, FMG-inoculated hens laid significantly fewer total eggs, which became apparent at each week after Week 42. In Trial 2, a numerical decrease in total EP occurred, and the percentage of undersized eggs laid by FMG-inoculated birds was significantly lower at 19 wk of age but was higher at 20 and 21 wk when compared to controls. Mortality was not significantly different between the treatments in either trial. These data demonstrate that when birds are housed in isolation facilities and inoculated with FMG at 12 wk of age, onset of lay is delayed. These data also suggest that FMG may lead to delays in undersize EP and decreases in total EP. However, because significant FMG effects on these parameters were observed in only one trial, additional studies may be necessary to verify these effects.

Plants frequently respond to herbivorous insect attack by synthesizing defense proteins that deter insect feeding and prevent additional herbivory. Maize (Zea mays L.) lines, resistant to feeding by a number of lepidopteran species, rapidly mobilize a unique 33-kDa cysteine protease in response to caterpillar feeding. The accumulation of the 33-kDa cysteine protease in the maize mid-whorl was correlated with a significant reduction in caterpillar growth that resulted from impaired nutrient utilization. Black Mexican Sweetcorn callus transformed with mir1, the gene encoding the 33-kDa cysteine protease, expressed the protease and growth of caterpillars reared on the transgenic callus was reduced 60-80%. Scanning electron microscopy was used to examine the effect of plant material expressing the 33-kDa cysteine protease on the structure of the caterpillar peritrophic matrix. Because the peritrophic matrix surrounds the food bolus, assists in digestive processes, and protects the caterpillar midgut from physical and chemical damage, disruption of peritrophic matrix may reduce caterpillar growth. The results indicated that the peritrophic matrix was severely damaged when caterpillars fed on resistant maize plants or transgenic Black Mexican Sweetcorn. The accumulation of the 33-kDa cysteine protease in response to caterpillar feeding, and its ability to damage the insect peritrophic matrix, represents an unusual host-plant resistance mechanism that may have applications in agricultural biotechnology.