Monoclonal Antibodies to S and N SARS-CoV-2 Proteins as Probes to Assess Structural and Antigenic Properties of Coronaviruses.

Antibodies targeting the spike (S) and nucleocapsid (N) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential tools. In addition to important roles in the treatment and diagnosis of infection, the availability of high-quality specific antibodies for the S and N proteins is essential to facilitate basic research of virus replication and in the characterization of mutations responsible for variants of concern. We have developed panels of mouse and rabbit monoclonal antibodies (mAbs) to the SARS-CoV-2 spike receptor-binding domain (S-RBD) and N protein for functional and antigenic analyses. The mAbs to the S-RBD were tested for neutralization of native SARS-CoV-2, with several exhibiting neutralizing activity. The panels of mAbs to the N protein were assessed for cross-reactivity with the SARS-CoV and Middle East respiratory syndrome (MERS)-CoV N proteins and could be subdivided into sets that showed unique specificity for SARS-CoV-2 N protein, cross-reactivity between SARS-CoV-2 and SARS-CoV N proteins only, or cross-reactivity to all three coronavirus N proteins tested. Partial mapping of N-reactive mAbs were conducted using truncated fragments of the SARS-CoV-2 N protein and revealed near complete coverage of the N protein. Collectively, these sets of mouse and rabbit monoclonal antibodies can be used to examine structure/function studies for N proteins and to define the surface location of virus neutralizing epitopes on the RBD of the S protein.

Identification of Sero-Diagnostic Antigens for the Early Diagnosis of Johne's Disease using MAP Protein Microarrays.

Considerable effort has been directed toward controlling Johne's disease (JD), a chronic granulomatous intestinal inflammatory disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) in cattle and other ruminants. However, progress in controlling the spread of MAP infection has been impeded by the lack of reliable diagnostic tests that can identify animals early in the infection process and help break the transmission chain. To identify reliable antigens for early diagnosis of MAP infection, we constructed a MAP protein array with 868 purified recombinant MAP proteins, and screened a total of 180 well-characterized serum samples from cows assigned to 4 groups based on previous serological and fecal test results: negative low exposure (NL, n = 30); negative high exposure (NH, n = 30); fecal-positive, ELISA-negative (F + E-, n = 60); and both fecal- and ELISA-positive (F + E+, n = 60). The analyses identified a total of 49 candidate antigens in the NH, F + E-, and F + E+ with reactivity compared with the NL group (p < 0.01), a majority of which have not been previously identified. While some of the antigens were identified as reactive in only one of the groups, others showed reactivity in multiple groups, including NH (n = 28), F + E- (n = 26), and F + E+ (n = 17) groups. Using combinations of top reactive antigens in each group, the results reveal sensitivities of 60.0%, 73.3%, and 81.7% in the NH, F + E-, and F + E+, respectively at 90% specificity, suggesting that early detection of infection in animals may be possible and enable better opportunities to reduce within herd transmission that may be otherwise missed by traditional serological assays that are biased towards more heavily infected animals. Together, the results suggest that several of the novel candidate antigens identified in this study, particularly those that were reactive in the NH and F + E- groups, have potential utility for the early sero-diagnosis of MAP infection.

A defined antigen skin test for the diagnosis of bovine tuberculosis.

Bovine tuberculosis (bTB) is a major zoonotic disease of cattle that is endemic in much of the world, limiting livestock productivity and representing a global public health threat. Because the standard tuberculin skin test precludes implementation of Bacille Calmette-Guérin (BCG) vaccine-based control programs, we here developed and evaluated a novel peptide-based defined antigen skin test (DST) to diagnose bTB and to differentiate infected from vaccinated animals (DIVA). The results, in laboratory assays and in experimentally or naturally infected animals, demonstrate that the peptide-based DST provides DIVA capability and equal or superior performance over the extant standard tuberculin surveillance test. Together with the ease of chemical synthesis, quality control, and lower burden for regulatory approval compared with recombinant antigens, the results of our studies show that the DST considerably improves a century-old standard and enables the development and implementation of critically needed surveillance and vaccination programs to accelerate bTB control.

Conditional Function of Autoaggregative Protein Cah and Common cah Mutations in Shiga Toxin-Producing Escherichia coli.

Cah is a calcium-binding autotransporter protein involved in autoaggregation and biofilm formation. Although cah is widespread in Shiga toxin-producing Escherichia coli (STEC), we detected mutations in cah at a frequency of 31.3% in this pathogen. In STEC O157:H7 supershedder strain SS17, a large deletion results in a smaller coding sequence, encoding a protein lacking the C-terminal 71 amino acids compared with Cah in STEC O157:H7 strain EDL933. We examined the function of Cah in biofilm formation and host colonization to better understand the selective pressures for cah mutations. EDL933-Cah played a conditional role in biofilm formation in vitro: it enhanced E. coli DH5α biofilm formation on glass surfaces under agitated culture conditions that prevented autoaggregation but inhibited biofilm formation under hydrostatic conditions that facilitated autoaggregation. This function appeared to be strain dependent since Cah-mediated biofilm formation was diminished when an EDL933 cah gene was expressed in SS17. Deletion of cah in EDL933 enhanced bacterial attachment to spinach leaves and altered the adherence pattern of EDL933 to bovine recto-anal junction squamous epithelial (RSE) cells. In contrast, in trans expression of EDL933 cah in SS17 increased its attachment to leaf surfaces, and in DH5α, it enhanced its adherence to RSE cells. Hence, the ecological function of Cah appears to be modulated by environmental conditions and other bacterial strain-specific properties. Considering the prevalence of cah in STEC and its role in attachment and biofilm formation, cah mutations might be selected in ecological niches in which inactivation of Cah would result in an increased fitness in STEC during colonization of plants or animal hosts.IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) harbors genes encoding diverse adhesins, and many of these are known to play an important role in bacterial attachment and host colonization. We demonstrated here that the autotransporter protein Cah confers on E. coli DH5α cells a strong autoaggregative phenotype that is inversely correlated with its ability to form biofilms and plays a strain-specific role in plant and animal colonization by STEC. Although cah is widespread in the STEC population, we detected a mutation rate of 31.3% in cah, which is similar to that reported for rpoS and fimH The formation of cell aggregates due to increased bacterium-to-bacterium interactions may be disadvantageous to bacterial populations under conditions that favor a planktonic state in STEC. Therefore, a loss-of-function mutation in cah is likely a selective trait in STEC when autoaggregative properties become detrimental to bacterial cells and may contribute to the adaptability of STEC to fluctuating environments.

Comparative analysis of super-shedder strains of Escherichia coli O157:H7 reveals distinctive genomic features and a strongly aggregative adherent phenotype on bovine rectoanal junction squamous epithelial cells.

Shiga toxin-producing Escherichia coli O157:H7 (O157) are significant foodborne pathogens and pose a serious threat to public health worldwide. The major reservoirs of O157 are asymptomatic cattle which harbor the organism in the terminal recto-anal junction (RAJ). Some colonized animals, referred to as "super-shedders" (SS), are known to shed O157 in exceptionally large numbers (>104 CFU/g of feces). Recent studies suggest that SS cattle play a major role in the prevalence and transmission of O157, but little is known about the molecular mechanisms associated with super-shedding. Whole genome sequence analysis of an SS O157 strain (SS17) revealed a genome of 5,523,849 bp chromosome with 5,430 open reading frames and two plasmids, pO157 and pSS17, of 94,645 bp and 37,446 bp, respectively. Comparative analyses showed that SS17 is clustered with spinach-associated O157 outbreak strains, and belongs to the lineage I/II, clade 8, D group, and genotype 1, a subgroup of O157 with predicted hyper-virulence. A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence-related loci. Phenotypic analyses revealed a distinctive and strongly adherent aggregative phenotype of SS17 on bovine RAJ stratified squamous epithelial (RSE) cells that was conserved amongst other SS isolates. Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism. Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells.

Complete nucleotide sequence of pRS218, a large virulence plasmid, that augments pathogenic potential of meningitis-associated Escherichia coli strain RS218.

BACKGROUND: Escherichia coli is the most predominant Gram-negative bacterial pathogen associated with neonatal meningitis. Previous studies indicated that the prototypic neonatal meningitis E. coli (NMEC) strain RS218 (O18:K1:H7) harbors one large plasmid. Objectives of the present study were to analyze the complete nucleotide sequence of this large plasmid (pRS218) and its contribution to NMEC pathogenesis using in vitro and in vivo models of neonatal meningitis. RESULTS: The plasmid is 114,231 bp in size, belongs to the incompatibility group FIB/IIA (IncFIB/IIA), and contains a genetic load region that encodes several virulence and fitness traits such as enterotoxicity, iron acquisition and copper tolerance. The nucleotide sequence of pRS218 showed a 41- 46% similarity to other neonatal meningitis-causing E. coli (NMEC) plasmids and remarkable nucleotide sequence similarity (up to 100%) to large virulence plasmids of E. coli associated with acute cystitis. Some genes located on pRS218 were overly represented by NMEC strains compared to fecal E. coli isolated from healthy individuals. The plasmid-cured strain was significantly attenuated relative to the RS218 wild-type strain as determined in vitro by invasion potential to human cerebral microvascular endothelial cells and in vivo by mortalities, histopathological lesions in the brain tissue, and bacterial recovery from the cerebrospinal fluid of infected rat pups. CONCLUSIONS: The pRS218 is an IncFIB/IIA plasmid which shares a remarkable nucleotide sequence similarity to large plasmids of E. coli associated with cystitis. Both in vitro and in vivo experiments indicated that pRS218 plays an important role in NMEC pathogenesis.

Screening of Mycobacterium avium subsp. paratuberculosis mutants for attenuation in a bovine monocyte-derived macrophage model.

Vaccination remains a major tool for prevention and progression of Johne's disease, a chronic enteritis of ruminants worldwide. Currently there is only one licensed vaccine within the United States and two vaccines licensed internationally against Johne's disease. All licensed vaccines reduce fecal shedding of Mycobacterium avium subsp. paratuberculosis (MAP) and delay disease progression. However, there are no available vaccines that prevent disease onset. A joint effort by the Johne's Disease Integrated Program (JDIP), a USDA-funded consortium, and USDA-APHIS/VS sought to identify transposon insertion mutant strains as vaccine candidates in part of a three phase study. The focus of the Phase I study was to evaluate MAP mutant attenuation in a well-defined in vitro bovine monocyte-derived macrophage (MDM) model. Attenuation was determined by colony forming unit (CFUs) counts and slope estimates. Based on CFU counts alone, the MDM model did not identify any mutant that significantly differed from the wild-type control, MAP K-10. Slope estimates using mixed models approach identified six mutants as being attenuated. These were enrolled in protection studies involving murine and baby goat vaccination-challenge models. MDM based approach identified trends in attenuation but this did not correlate with protection in a natural host model. These results suggest the need for alternative strategies for Johne's disease vaccine candidate screening and evaluation.

Transcriptional analysis of diverse strains Mycobacterium avium subspecies paratuberculosis in primary bovine monocyte derived macrophages.

In this study we analyzed the macrophage-induced gene expression of three diverse genotypes of Mycobacterium avium subsp. paratuberculosis (MAP). Using selective capture of transcribed sequences (SCOTS) on three genotypically diverse MAP isolates from cattle, human, and sheep exposed to primary bovine monocyte derived macrophages for 48 h and 120 h we created and sequenced six cDNA libraries. Sequence annotations revealed that the cattle isolate up-regulated 27 and 241 genes; the human isolate up-regulated 22 and 53 genes, and the sheep isolate up-regulated 35 and 358 genes, at the two time points respectively. Thirteen to thirty-three percent of the genes identified did not have any annotated function. Despite variations in the genes identified, the patterns of expression fell into overlapping cellular functions as inferred by pathway analysis. For example, 10-12% of the genes expressed by all three strains at each time point were associated with cell-wall biosynthesis. All three strains of MAP studied up-regulated genes in pathways that combat oxidative stress, metabolic and nutritional starvation, and cell survival. Taken together, this comparative transcriptional analysis suggests that diverse MAP genotypes respond with similar modus operandi for survival in the host.

The evidence for Mycobacterium paratuberculosis in Crohn's disease.

PURPOSE OF REVIEW: Though long hypothesized, the putative link between Mycobacterium avium paratuberculosis and Crohn's disease remains neither confirmed nor refuted. This article reviews published contributions that directly or indirectly address this question. RECENT FINDINGS: Epidemiologic studies, looking for M. avium paratuberculosis DNA in Crohn's tissue, show a strong association between the agent and this disease. Supporting data, however, are presently inconclusive on a causal role. Genetic studies provide indirect support for a role of mycobacteria in Crohn's disease, by identifying susceptibility genes that encode proteins implicated in innate immunity to intracellular bacteria. Clinical trial data support at least a short-term benefit for antimycobacterial therapy in Crohn's disease, but the microbial specificity of this response is presently unknown. SUMMARY: There appears to be a strong association between M. avium paratuberculosis and Crohn's disease, but the causality of this association is unknown. Consequently, the therapeutic implications of this association require further study. A number of critical questions about the biology of M. avium paratuberculosis remain unanswered. Data from studies of this organism, and its interaction with the immune system, can help address proposed reasons for or against a role of M. avium paratuberculosis in the etiology of Crohn's disease.

Association of germ-line polymorphisms in the feline p53 gene with genetic predisposition to vaccine-associated feline sarcoma.

A case-control study was conducted in order to investigate the association of polymorphisms in the genomic sequence of the feline p53 gene with a predisposition to vaccine-associated feline sarcoma (VAFS). In the study, 50 domestic short hair cats with a confirmed histopathologic diagnosis of VAFS were matched to disease-free controls (1:2) by age, sex, and breed. Cats from both the diseased (case) and control groups were also negative for feline leukemia virus and feline immunodeficiency virus. Germ-line DNA was prepared from blood samples from cats in both groups and analyzed for sequence variation at 8 polymorphic sites in the p53 gene. A strong association was found between VAFS and the presence of specific nucleotides at 2 of the polymorphic sites. The strongest association was observed for a single-base insertion in intron 7 of the gene with an odds ratio of 8.99 (95% confidence interval = 3.42-23.57, P < 0.0001). The results of the study indicate that analysis of the presence or absence of the identified genetic markers in apparently healthy disease-free cats may help in predicting which individual animals are at greater risk of developing the disease.

Rapid expression of Mycobacterium avium subsp. paratuberculosis recombinant proteins for antigen discovery.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease, a chronic granulomatous enteritis of ruminants and other species. Detection of infection in animals is hampered by the lack of sensitive and specific diagnostic assays. We describe here an approach that utilizes translationally active PCR fragments for the rapid in vitro transcription and translation of recombinant proteins for antigen discovery in M. avium subsp. paratuberculosis. The investigations showed that the MAP1272c protein selectively reacts with sera from Johne's disease-positive cattle and represents an antigen of potential utility in M. avium subsp. paratuberculosis immunodiagnostics.

Comparative transcriptional analysis of human macrophages exposed to animal and human isolates of Mycobacterium avium subspecies paratuberculosis with diverse genotypes.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in animals and has been hypothesized to be associated with Crohn's disease in humans. Recently, M. avium subsp. paratuberculosis isolates recovered from Crohn's disease patients were shown to have limited diversity, implying the existence of human disease-associated genotypes and strain sharing with animals (A. H. Ghadiali et al., J. Clin. Microbiol. 42:5345-5348, 2004). To explore whether these genotypic differences or similarities among human and animal isolates translated to functionally significant attributes such as variance in host preference and/or difference in magnitude of infections, we performed a global scale analysis of M. avium subsp. paratuberculosis isolates that were representative of different genotypes and host species using DNA microarrays. Genome-wide characterization of the transcriptional changes was carried out using a human monocytic cell line (THP-1 cells) in response to different genotypes of M. avium subsp. paratuberculosis isolates recovered from various hosts. We identified several differentially expressed genes during early intracellular infection, including those involved in common canonical pathways such as NF-kappaB, interleukin-6 (IL-6), mitogen-activated protein kinase/extracellular signal-regulated kinase, and Jun N-terminal protein kinase signaling, as well as genes involved in T helper type 1 (Th1) responses (such as CCL5 ligand) and those that encode several proinflammatory cytokines and chemokine receptors. The cattle and human isolates of M. avium subsp. paratuberculosis, regardless of their short sequence repeat (SSR) genotype, induced similar global gene expression patterns in THP-1 cells. They differentially regulated genes necessary for cell survival without causing major alterations in proinflammatory genes. In contrast, the sheep isolates representing diverse SSR genotypes closely resembled the global gene expression pattern of an M. avium subsp. avium isolate, and they significantly up-regulated proinflammatory genes related to IL-6, T-cell receptor, B-cell receptor, and death receptor signaling within THP-1 cells. Additionally, we demonstrated consistency among infecting genotypes of M. avium subsp. paratuberculosis isolated from diverse hosts [cattle (n=2), human (n=3), sheep (n=2), and bison (n=1)] in quantitative reverse transcription-PCR analysis of seven differentially expressed genes. While the levels of expression induced by the bison isolate were different compared with cattle or human isolates, they followed the common anti-inflammatory, antiapoptotic trend. Our data suggest that the macrophage responses to M. avium subsp. paratuberculosis isolates from cattle and human sources, regardless of genotype, follow a common theme of anti-inflammatory responses, an attribute likely associated with successful infection and persistence. However, these expression patterns differ significantly from those in THP-1 cells infected with sheep isolates of M. avium subsp. paratuberculosis or the M. avium subsp. avium isolate. These data provide a transcriptional basis for a variety of pathophysiological changes observed during early stages of infection by different strains of M. avium subsp. paratuberculosis, a first step in understanding trait-allele association in this economically important disease.

Current understanding of the genetic diversity of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease (or paratuberculosis). Paratuberculosis is a chronic gastroenteritis mainly affecting cattle, sheep and other ruminants. MAP is also of concern due to the heretofore unresolved issue of its possible role in Crohn's disease in humans. We present here a review of MAP (i) mobile genetic elements; (ii) repetitive elements; (iii) single nucleotide polymorphisms; and (iv) whole-genome comparisons to study the molecular epidemiology of MAP. A summary of the findings to date is presented, and the discriminatory power, advantage and disadvantages of each of the methods are compared and discussed.

Cytokine responses of bovine macrophages to diverse clinical Mycobacterium avium subspecies paratuberculosis strains.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease (JD) persistently infects and survives within the host macrophages. While it is established that substantial genotypic variation exists among MAP, evidence for the correlates that associate specific MAP genotypes with clinical or sub-clinical disease phenotypes is presently unknown. Thus we studied strain differences in intracellular MAP survival and host responses in a bovine monocyte derived macrophage (MDM) system. RESULTS: Intracellular survival studies showed that a bovine MAP isolate (B1018) and a human MAP isolate (Hu6) persisted in relatively higher numbers when compared with a sheep MAP isolate (S7565) at 24-hr, 48-hr and 96-hr post infection (PI). MDMs stimulated with B1018 up-regulated IL-10 at the transcript level and down-regulated TNFalpha at the protein and transcript levels compared with stimulations by the S7565 and Hu6. MDMs infected with Hu6 showed a down regulatory pattern of IL-10 and TNFalpha compared to stimulations by S7565. Cells stimulated with B1018 and Hu6 had low levels of matrix metalloprotease-3 (MMP3) and high levels of tissue inhibitor of metalloprotease-1 (TIMP1) at 96-hr PI relative to MDMs stimulated by S7565. CONCLUSION: Taken together, results suggest that the bovine (B1018) and the human (Hu6) MAP isolates lead to anti-inflammatory and anti-invasive pathways in the macrophage environment whereas the sheep (S7565) MAP isolate induces a pro-inflammatory pathway. Thus the infecting strain genotype may play a role in polarizing the host immune responses and dictate the clinicopathological outcomes in this economically important disease.

Three novel herpesviruses associated with stomatitis in Sudan plated lizards (Gerrhosaurus major) and a black-lined plated lizard (Gerrhosaurus nigrolineatus).

Glossal stomatitis was observed in a Sudan plated lizard (Gerrhosaurus major) with severe dyspnea. On necropsy, intranuclear inclusion bodies were seen in the periglottal lingual epithelium. Labial stomatitis was seen in a second Sudan plated lizard and a black-lined plated lizard (G. nigrolineatus). Degenerate polymerase chain reaction (PCR) primers targeting a conserved region of herpesvirus DNA-dependent DNA polymerase gene were used to amplify products from lesions from each lizard. Nucleotide sequencing of the PCR products showed that the sequence from each lizard was unique. Phylogenetic and comparative sequence analyses suggest that these viruses are novel members of the subfamily Alphaherpesvirinae, and they are here termed gerrhosaurid herpesviruses 1-3. Results of our analyses suggest that the genus Gerrhosaurus can be infected by these novel herpesviruses.

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: evidence for limited strain diversity, strain sharing, and identification of unique targets for diagnosis.

The objectives of this study were to understand the molecular diversity of animal and human strains of Mycobacterium avium subsp. paratuberculosis isolated in the United States and to identify M. avium subsp. paratuberculosis-specific diagnostic molecular markers to aid in disease detection, prevention, and control. Multiplex PCR of IS900 integration loci (MPIL) and amplified fragment length polymorphism (AFLP) analyses were used to fingerprint M. avium subsp. paratuberculosis isolates recovered from animals (n = 203) and patients with Crohn's disease (n = 7) from diverse geographic localities. Six hundred bacterial cultures, including M. avium subsp. paratuberculosis (n = 303), non-M. avium subsp. paratuberculosis mycobacteria (n = 129), and other nonmycobacterial species (n = 168), were analyzed to evaluate the specificity of two IS900 integration loci and a newly described M. avium subsp. paratuberculosis-specific sequence (locus 251) as potential targets for the diagnosis of M. avium subsp. paratuberculosis. MPIL fingerprint analysis revealed that 78% of bovine origin M. avium subsp. paratuberculosis isolates clustered together into a major node, whereas isolates from human and ovine sources showed greater genetic diversity. MPIL analysis also showed that the M. avium subsp. paratuberculosis isolates from ovine and bovine sources from the same state were more closely associated than were isolates from different geographic regions, suggesting that some of the strains are shared between these ruminant species. AFLP fingerprinting revealed a similar pattern, with most isolates from bovine sources clustering into two major nodes, while those recovered from sheep or humans were clustered on distinct branches. Overall, this study identified a high degree of genetic similarity between M. avium subsp. paratuberculosis strains recovered from cows regardless of geographic origin. Further, the results of our analyses reveal a relatively higher degree of genetic heterogeneity among M. avium subsp. paratuberculosis isolates recovered from human and ovine sources.

Genomic homogeneity between Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis belies their divergent growth rates.

BACKGROUND: Mycobacterium avium subspecies avium (M. avium) is frequently encountered in the environment, but also causes infections in animals and immunocompromised patients. In contrast, Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow-growing organism that is the causative agent of Johne's disease in cattle and chronic granulomatous infections in a variety of other ruminant hosts. Yet we show that despite their divergent phenotypes and the diseases they present, the genomes of M. avium and M. paratuberculosis share greater than 97% nucleotide identity over large (25 kb) genomic regions analyzed in this study. RESULTS: To characterize genome similarity between these two subspecies as well as attempt to understand their different growth rates, we designed oligonucleotide primers from M. avium sequence to amplify 15 minimally overlapping fragments of M. paratuberculosis genomic DNA encompassing the chromosomal origin of replication. These strategies resulted in the successful amplification and sequencing of a contiguous 11-kb fragment containing the putative Mycobacterium paratuberculosis origin of replication (oriC). This fragment contained 11 predicted open reading frames that showed a conserved gene order in the oriC locus when compared with several other Gram-positive bacteria. In addition, a GC skew analysis identified the origin of chromosomal replication which lies between the genes dnaA and dnaN. The presence of multiple DnaA boxes and the ATP-binding site in dnaA were also found in M. paratuberculosis. The strong nucleotide identity of M. avium and M. paratuberculosis in the region surrounding the origin of chromosomal replication led us to compare other areas of these genomes. A DNA homology matrix of 2 million nucleotides from each genome revealed strong synteny with only a few sequences present in one genome but absent in the other. Finally, the 16s rRNA gene from these two subspecies is 100% identical. CONCLUSIONS: We present for the first time, a description of the oriC region in M. paratuberculosis. In addition, genomic comparisons between these two mycobacterial subspecies suggest that differences in the oriC region may not be significant enough to account for the diverse bacterial replication rates. Finally, the few genetic differences present outside the origin of chromosomal replication in each genome may be responsible for the diverse growth rates or phenotypes observed between the avium and paratuberculosis subspecies.

Transcriptional response of Pasteurella multocida to defined iron sources.

Pasteurella multocida was grown in iron-free chemically defined medium supplemented with hemoglobin, transferrin, ferritin, and ferric citrate as iron sources. Whole-genome DNA microarrays were used to monitor global gene expression over seven time points after the addition of the defined iron source to the medium. This resulted in a set of data containing over 338,000 gene expression observations. On average, 12% of P. multocida genes were differentially expressed under any single condition. A majority of these genes encoded P. multocida proteins that were involved in either transport and binding or were annotated as hypothetical proteins. Several trends are evident when the data from different iron sources are compared. In general, only two genes (ptsN and sapD) were expressed at elevated levels under all of the conditions tested. The results also show that genes with increased expression in the presence of hemoglobin did not respond to transferrin or ferritin as an iron source. Correspondingly, genes with increased expression in the transferrin and ferritin experiments were expressed at reduced levels when hemoglobin was supplied as the sole iron source. Finally, the data show that genes that were most responsive to the presence of ferric citrate did not follow a trend similar to that of the other iron sources, suggesting that different pathways respond to inorganic or organic sources of iron in P. multocida. Taken together, our results demonstrate that unique subsets of P. multocida genes are expressed in response to different iron sources and that many of these genes have yet to be functionally characterized.

Evaluation of in vitro chemosensitivity of vaccine-associated feline sarcoma cell lines to vincristine and paclitaxel.

OBJECTIVE: To determine the in vitro sensitivity of 4 vaccine-associated feline sarcoma (VAFS) cell lines to the chemotherapeutic agents vincristine and paclitaxel. SAMPLE POPULATION: Cell lines derived from 4 VAFS specimens. PROCEDURES: Cell lines were cultured in vitro and individually exposed to various concentrations of vincristine and paclitaxel. Survival was estimated after 24 and 72 hours of exposure to each drug, and the drug concentrations that resulted in 50 and 90% reduction in number of viable cells (IC50 and IC90, respectively) were calculated. RESULTS: Both vincristine and paclitaxel had significant dose-dependent effects on the viability of the VAFS cell lines. After 72 hours of drug exposure, the IC50 and IC90 of vincristine for the 4 cell lines were between 0.005 to 0.039 microg/ml and 0.045 to 1.027 microg/ml, respectively. The IC50 and IC90 values for paclitaxel were between 0.037 to 0.092 microg/ml and 2.450 to 15.413 microg/ml, respectively. CONCLUSIONS: Results of pharmacokinetic studies on vincristine and paclitaxel in other species suggest that concentrations greater than the IC50 values may be possible for both drugs in feline patients as well. The drug concentrations at which viable cell numbers were reduced by 90% may also be attained in vivo for some cases, but detailed information is needed regarding the distribution, concentration, duration of availability, and toxicity of various drugs in cats. Carefully chosen combinations of antineoplastic agents need to be screened to identify treatment protocols that may be further evaluated clinically for the treatment of VAFS.