Public Practice Opportunities for Veterinary Students to Enhance Veterinary Public Health Education.

Veterinarians have a long history of contributing to animal and human health; simultaneously, the veterinary medical profession has held the tenet of protecting public health. Veterinary education has shifted with societal needs over time and currently has curricula at US colleges of veterinary medicine (CVMs) largely focused on clinical practice and basic sciences. The focus of many veterinary curricula produces a veterinarian who meets the needs of the US pet owner. A void often exists in the knowledge and understanding of new veterinary graduates in the field of public practice and, in particular, public health. Students need to be able to find other learning environments and opportunities that help bridge this void. This article captures possible opportunities as best practices. Advising US veterinary students interested in public health and public health policy while considering these opportunities will help to enhance the likely experiences students have during their formal veterinary education. While no list of opportunities can be inclusive of all possibilities, the experiences listed here provide a solid foundation of options for students to include in the individualized aspects of their veterinary education.

Signalling inhibitors against Mycobacterium tuberculosis--early days of a new therapeutic concept in tuberculosis.

Tuberculosis causes nearly two million deaths per year world-wide. In addition multidrug-resistant mycobacterial strains rapidly emerge so novel therapeutic approaches are needed. Recently, several promising mycobacterial target molecules were identified, which are involved in bacterial or host cell signalling e.g. the serine/threonine protein kinases, PknB and PknG, NAD kinase and the NAD synthetase. Here we describe some early efforts in the development of novel signal transduction inhibitory anti-mycobacterial drugs using a multiple target approach, with special emphasis on the kinase inhibitory field. Initially, we are using the Nested Chemical Library (NCL) technology and pharmacophore modelling. A hit-finding library, consisting of approximately 19000 small molecules with a bias for prototypic kinase inhibitors from our NCL library and commercial sources was virtually screened against these validated target molecules. Protein structures for the virtual screening were taken from the published three dimensional crystal structures of the enzymes. The hits from the virtual screening were subsequently tested in enzymatic assay systems. Potent hits were then tested for biological activity in macrophages, infected with mycobacteria. The final goal of this exercise is not only to identify potent anti-mycobacterial substances, but also a common pharmacophore for the mycobacterial target PknG in combination with PknB, NAD kinase and/or NAD synthetase. This common pharmacophore still needs to be a unique pharmacophore for the mycobacterial target proteins over human off-targets. Such a pharmacophore might then drive the optimization of a completely new profile of an antibiotic agent with activity against latent mycobacteria and resistance mycobacterial strains.

Modeling the relationship between food animal health and human foodborne illness.

To achieve further reductions in foodborne illness levels in humans, effective pre-harvest interventions are needed. The health status of food animals that are destined to enter the human food supply chain may be an important, although often overlooked, factor in predicting the risk of human foodborne infections. The health status of food animals can potentially influence foodborne pathogen levels in three ways. First, diseased animals may shed higher levels of foodborne pathogens. Second, animals that require further handling in the processing plant to remove affected parts may lead to increased microbial contamination and cross-contamination. Finally, certain animal illnesses may lead to a higher probability of mistakes in the processing plant, such as gastrointestinal ruptures, which would lead to increased microbial contamination and cross-contamination. Consequently, interventions that reduce the incidence of food animal illnesses might also help reduce bacterial contamination on meat, thereby reducing human illness. Some of these interventions, however, might also present a risk to human health. For example, the use of antibiotics in food animals can reduce rates of animal illness but can also select for antibiotic-resistant bacteria which can threaten human treatment options. In this study, we present a mathematical model to evaluate human health risks from foodborne pathogens associated with changes in animal illness. The model is designed so that potential human health risks and benefits from interventions such as the continued use of antibiotics in animal agriculture can be evaluated simultaneously. We applied the model to a hypothetical example of Campylobacter from chicken. In general, the model suggests that very minor perturbations in microbial loads on meat products could have relatively large impacts on human health, and consequently, small improvements in food animal health might result in significant reductions in human illness.

Stakeholder position paper: economist's perspectives on antibiotic use in animals.

Economics seeks to understand and explain the allocation of resources within society and to suggest appropriate policy to achieve goals such as optimal efficiency. Relating antimicrobial use and antibiotic resistance is a complex task and requires very clear and careful analyses of many relationships. To address these relationships, antibiotic use data must be linked with other basic data. Ideally, a complex relational database needs to be developed. These must include data on: antimicrobial usage, farm-level animal productivity, basic disease, basic farm management, consumer response, and antimicrobial resistance at multiple levels (farm, during process, on a variety of both animal, vegetable and grain-derived food products, from humans, from other animal species, and from the environment) among other data. Such a complex relational database can help with the attribution and risk assessment process. Also these linked data are needed in order to develop the economic production models and other economic models, as well as control for confounding so that there is both adequate and appropriate statistical control. Antimicrobial usage data may become economically important for reasons unrelated to animal productivity and animal health. With expanding global trade of animals and animal products, there have been changes in restrictions and regulations associated with the movement of products. Future trade opportunities may be linked to antimicrobial usage. The European Union banned the use of antimicrobials for growth-promoting purposes based on perceived risk and on public opinion. Monitoring antimicrobial use, disease occurrence, and resistance patterns following changes in usage that are a result of either policy changes or response to new information will help in the understanding of the complex relationships in microbial and disease ecology. There is a lot of evidence substantiating the productivity and profitability gains to producers from the use of antimicrobials. There is also evidence for the potential gains to consumers, both from an economic perspective as well as from a health perspective. Economists can help identify the implications of these relationships both at a micro- and macro-economic level; both relative to producer and to consumer welfare. We are at a pivotal time in history with sufficient analytical expertise and tools to address this complex issue from a scientific perspective. Linking various agencies so there can be coordination of data collection and data sharing is needed to successfully address this topic.

Consortia and centers of excellence: issues and answers.

As the demand for food-supply veterinarians changes, while the level of expertise necessary in this field markedly increases, there is a need to examine alternative modalities for delivering food-supply veterinary education. It seems clear that not all veterinary schools in the United States can sustain optimally sized facilities for the broad-based training in all species of food animals that the current and future food-supply veterinarian needs. An alternative model is for select schools to establish consortial centers of excellence in specific food-animal species, to which students from other schools can go for optimum final-year education. This alternative mode of food supply-veterinary medical education is discussed here.

Influence of Salmonella in pigs preharvest and during pork processing on human health costs and risks from pork.

Salmonellosis in humans is a costly disease traditionally assumed to be associated with exposure to contaminated food. We have developed a farm-to-fork model that allows estimation of the human health costs and risks associated with Salmonella in pork. This analysis focuses on the stages of the pork production chain up to the point of producing a chilled pork carcass. The model predicts the number of human cases of salmonellosis associated with pork (mean, 99,430; 90% confidence interval, 20,970 to 245,560) and the corresponding social costs (mean, $81.53 million; 90% confidence interval, $18.75 million to $197.44 million). Sensitivity and scenario analyses suggest that changes in Salmonella status during processing are more important for human health risk and have a higher benefit:cost ratio when compared with on-farm strategies for Salmonella control. Specifically, benefit:cost ratios are less than 1 (indicating they are not likely to be profitable from a social economic perspective) for the on-farm strategies of vaccination and meal feeding, whereas rinsing carcasses at various temperatures with and without sanitizer all have benefit:cost ratios greater than 1 (indicating they are profitable from a social economic perspective). This type of modeling is useful for evaluation of the relative cost effectiveness of interventions at different points in the food chain when allocating limited food safety dollars and is best used for examining trends and alternative strategies rather than for providing definitive dollar value estimates of risk. The dollar value estimates must be considered in the context of the wide confidence intervals.

Associations between on-farm and slaughter plant detection of Salmonella in market-weight pigs.

Thirty swine production units in the midwestern United States were studied to assess the relationship of herd-level prevalence of Salmonella on the farm prior to slaughter versus at slaughter. Fecal samples were collected from 30 pigs on each farm within 48 h of slaughter, and 30 ileocecal lymph node samples were collected in the same shipment cohort at slaughter. Samples were cultured by conventional methods, and Salmonella identity was confirmed by serotyping. Overall, 11.7% (n = 105) of the fecal samples and 14.9% (n = 133) of the ileocecal lymph node samples were positive for Salmonella. Seventeen of the farms (56.7%) had one or more positive fecal samples, and 24 (80.0%) had one or more positive ileocecal lymph node samples. Twenty-four recognized serotypes and three additional distinct antigenic types were identified. Among all isolates, 56.5% had serotypes that were duplicated both on the farm and at slaughter for a particular cohort, whereas the remaining samples lacked a duplicate serotype in the other sample type. There was a positive correlation in the prevalence of Salmonella between fecal samples and ileocecal lymph node samples (Spearman's p = 0.75; 95% confidence interval [CI], 0.62 to 0.89). Linear regression analysis was used to identify two farms that biased the regression estimates. Excluding these farms, 62% of the variance in farm slaughter Salmonella prevalence was accounted for by on-farm prevalence. The analyses suggest that the prevalence of Salmonella spp. at slaughter can be predicted from preslaughter on-farm sampling and vice versa.

Effects of Mycoplasma hyopneumoniae vaccine on pigs naturally infected with M. hyopneumoniae and porcine reproductive and respiratory syndrome virus.

The effects of a single-dose Mycoplasma hyopneumoniae vaccine was studied in growing pigs. Each of 24 vaccinated cohorts of approximately 1200 pigs reared in separate barns was matched by time, farm site, and sex with unvaccinated cohorts. Pigs were naturally exposed to M. hyopneumoniae and porcine reproductive respiratory syndrome virus (PRRSv). Daily weight gain was 42 g per pig per day higher and mortality rate was 15.2/1000 pigs lower for vaccinated cohorts. Age at PRRSv onset was associated with mortality rate, but did not modify vaccine effects. M. hyopneumoniae vaccination was effective in promoting growth in spite of concurrent PRRSv infection.

Models of antimicrobial resistance and foodborne illness: examining assumptions and practical applications.

Antimicrobial resistance is an issue of increasing global concern. Several investigators have suggested that antibiotic use in food-producing animals is a major contributor to the increasing incidence of antimicrobial-resistant organisms causing illness in humans (F. J. Angulo, K. R. Johnson, R. V. Tauxe, and M. L. Cohen, Microb. Drug Res. 6:77-83, 2000; P. D. Fey, T. J. Safranek, M. E. Rupp, E. F. Dunne, R. Efrain, P. C. Iwen, P. A. Bradford, F. J. Angulo, and S. H. Hinrichs, N. Engl. J. Med. 342:1242-1249, 2000; S. A. McEwen and P. J. Fedorka-Cray, Commun. Infect. Dis. 34(Suppl. 3):S93-S106, 2002; D. L. Smith, A. D. Harris, J. A. Johnson, E. K. Silbergeld, and J. G. Morris, Jr., Proc. Natl. Acad. Sci. USA 99:6434-6439, 2002; D. G. White, S. Zhao, R. Sudler, S. Ayers, S. Friedman, S. Chen, P. F. McDermott, D. D. Wagner, and J. Meng, N. Engl. J. Med. 345:1147-1154, 2001; W. Witte, Science 279:996, 1998). In this paper, we discuss this and other assumptions relevant to a quantitative risk assessment model for salmonellosis in humans. We also discuss other important aspects of modeling food safety and food-associated antimicrobial resistance risk to humans. We suggest that the role of food-producing animals in the origin and transmission of antimicrobial resistance and "foodborne" pathogens has been overestimated and overemphasized in the scientific literature; consequently, nonfoodborne transmission, including pet-associated human cases, has been underemphasized. Much evidence exists for the potential contribution to infectious disease that may be of human or pet origin (that may contact humans through food but not be of a food origin). Risk analyses that do not acknowledge the potential for these sources of cross-contamination will understate the contribution that origin has in the realm of foodborne and food-associated diseases (e.g., Salmonella) and the resulting uncertainty levels in the food system, thus leading to biased inferences. We emphasize the importance of evaluating both the foodborne and nonfoodborne transmission risk for salmonellosis and outline the basics of an analytical modeling approach in food safety with examples to illustrate strengths and limitations in the modeling. Examples illustrate, on a simplistic level, how varying assumptions and other inputs can influence the output of food-associated quantitative risk models.

A protein kinase inhibitor as an antimycobacterial agent.

The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) was found to inhibit the growth of two different mycobacterial strains, the slow-growing Mycobacterium bovis Bacille Calmette Guerin (BCG) and the fast-growing saprophyte Mycobacterium smegmatis mc2 155, in a dose-dependent manner. While screening for the effect of kinase inhibitors on mycobacterial growth, millimolar concentrations of H7 induced a 40% decrease in the growth of M. bovis BCG when measured as a function of oxidative phosphorylation. This H7-induced decrease in growth was shown to involve a 2-log fold decrease in the viable counts of M. smegmatis within a 48-h period and a 50% reduction in the number of BCG viable counts within a 10-day period. Micromolar concentrations of H7 compound induced a significant decrease in the activity of the Mycobacterium tuberculosis protein serine/threonine kinase (PSTK) PknB. The inhibition of mycobacterial growth as well as the inhibition of a representative M. tuberculosis protein serine/threonine kinase PknB suggests that conventional PSTK inhibitors can be used to study the role that the mycobacterial PSTK family plays in controlling bacterial growth.

Monitoring promoter activity and protein localization in Mycobacterium spp. using green fluorescent protein.

Two green fluorescent protein (Gfp) fusion vectors were constructed for use in Mycobacterium spp. The first plasmid facilitates quantification of mycobacterial promoter activity. The second vector permits construction of translational fusions of mycobacterial proteins to Gfp in order to study subcellular localization including protein secretion. Using this translational fusion construct, we verify that a Gfp fusion to the putative secreted M. tuberculosis protein ChoD is translocated to the extracellular milieu when cloned and expressed in Mycobacterium smegmatis.

N-Acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis.

Mycothiol is a novel thiol produced only by actinomycetes and is the major low-molecular-weight thiol in mycobacteria. Mycothiol was previously shown to be synthesized from 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside by ligation with cysteine followed by acetylation. A novel mycothiol-dependent detoxification enzyme, mycothiol conjugate amidase, was recently identified in Mycobacterium smegmatis and shown to have a homolog, Rv1082, in Mycobacterium tuberculosis. In the present study we found that a protein encoded by the M. tuberculosis open reading frame Rv1170, a homolog of Rv1082, possesses weak mycothiol conjugate amidase activity but shows substantial deacetylation activity with 1-D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins), a hypothetical mycothiol biosynthetic precursor. The availability of this protein enabled us to develop an assay for GlcNAc-Ins, which was used to demonstrate that GlcNAc-Ins is present in M. smegmatis at a level about twice that of mycothiol. It was shown that GlcNAc-Ins is absent in mycothiol-deficient mutant strain 49 of M. smegmatis and that this strain can concentrate GlcNAc-Ins from the medium and convert it to mycothiol. This demonstrates that GlcNAc-Ins is a key intermediate in the pathway of mycothiol biosynthesis. Assignment of Rv1170 as the gene coding the deacetylase in the M. tuberculosis genome represents the first identification of a gene of the mycothiol biosynthesis pathway. The presence of a large cellular pool of substrate for this enzyme suggests that it may be important in regulating mycothiol biosynthesis.

Cholesterol is accumulated by mycobacteria but its degradation is limited to non-pathogenic fast-growing mycobacteria.

In this report we show that fast-growing non-pathogenic mycobacteria degrade cholesterol from liquid media, and are able to grow on cholesterol as a sole carbon source. In contrast, slow-growing mycobacteria, including pathogenic Mycobacterium tuberculosis and bacillus Calmette-Guérin (BCG), do not degrade and use cholesterol as a carbon source. Nevertheless, pathogenic mycobacteria are able to uptake, modify, and accumulate cholesterol from liquid growth media, and form a zone of clearance around a colony when plated on solid media containing cholesterol. These data suggest that cholesterol may have a role in mycobacterial infection other than its use as carbon source.

DNA-Based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III.

Bacteria of the Burkholderia cepacia complex consist of five discrete genomic species, including genomovars I and III and three new species: Burkholderia multivorans (formerly genomovar II), Burkholderia stabilis (formerly genomovar IV), and Burkholderia vietnamiensis (formerly genomovar V). Strains of all five genomovars are capable of causing opportunistic human infection, and microbiological identification of these closely related species is difficult. The 16S rRNA gene (16S rDNA) and recA gene of these bacteria were examined in order to develop rapid tests for genomovar identification. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA revealed sequence polymorphisms capable of identifying B. multivorans and B. vietnamiensis but insufficient to discriminate strains of B. cepacia genomovars I and III and B. stabilis. RFLP analysis of PCR-amplified recA demonstrated sufficient nucleotide sequence variation to enable separation of strains of all five B. cepacia complex genomovars. Complete recA nucleotide sequences were obtained for 20 strains representative of the diversity of the B. cepacia complex. Construction of a recA phylogenetic tree identified six distinct clusters (recA groups): B. multivorans, B. vietnamiensis, B. stabilis, genomovar I, and the subdivision of genomovar III isolates into two recA groups, III-A and III-B. Alignment of recA sequences enabled the design of PCR primers for the specific detection of each of the six latter recA groups. The recA gene was found on the largest chromosome within the genome of B. cepacia complex strains and, in contrast to the findings of a previous study, only a single copy of the gene was present. In conclusion, analysis of the recA gene of the B. cepacia complex provides a rapid and robust nucleotide sequence-based approach to identify and classify this taxonomically complex group of opportunistic pathogens.

A novel mycothiol-dependent detoxification pathway in mycobacteria involving mycothiol S-conjugate amidase.

Mycothiol, 1-D-myo-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy-alpha-D-glucopyranoside (MSH), is composed of N-acetylcysteine (AcCys) amide linked to 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins) and is the major thiol produced by most actinomycetes. When Mycobacterium smegmatis was treated with the alkylating agent monobromobimane (mBBr), the cellular mycothiol was converted to its bimane derivative (MSmB). The latter was rapidly cleaved to produce GlcN-Ins and the bimane derivative of N-acetylcysteine (AcCySmB), a mercapturic acid that was rapidly exported from the cells into the medium. The other product of cleavage, GlcN-Ins, was retained in the cell and utilized in the resynthesis of mycothiol. The mycothiol S-conjugate amidase (amidase) responsible for cleaving MSmB was purified to homogeneity from M. smegmatis. A value of K(m) = 95 +/- 8 microM and a value of k(cat) = 8 s(-)(1) was determined for the amidase with MSmB as substrate. Activity with 100 microM mycothiol or with the monobromobimane derivative of 1-D-myo-inosityl-2-(L-cysteinyl)amido-2-deoxy-alpha-D-glucopyra nos ide (CySmB-GlcN-Ins) or of 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-(alpha, beta)-D-glucopyranoside (AcCySmB-GlcN) was at least 10(3) lower than with 100 microM MSmB, demonstrating that the amidase is highly specific for S-conjugates of mycothiol. Conjugates of mycothiol with the antibiotic cerulenin, N-ethylmaleimide, 3-(N-maleimidopropionyl)-biocytin, and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin also exhibited significant activity. The sequence of the amino-terminal 20 residues was determined, and an open reading frame (Rv1082) coding for 288 residues having an identical predicted amino-terminal amino acid sequence was identified in the Mycobacterium tuberculosis genome. The Rv1082 gene (mca) from M. tuberculosis was cloned and expressed in Escherichia coli, and the expressed protein was shown to have substrate specificity similar to the amidase from M. smegmatis. These results indicate that mycothiol and mycothiol S-conjugate amidase play an important role in the detoxification of alkylating agents and antibiotics.

The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis.

In bacteria, extracellular signals are generally transduced into cellular responses via a two-component system. However, genome sequence data have now revealed the presence of 'eukaryotic-like' protein kinases and phosphatases. Mycobacterium tuberculosis appears to be unique among bacteria in that its genome contains 11 members of a newly identified protein kinase family. These M. tuberculosis eukaryotic-like protein kinases could be key regulators of metabolic processes, including transcription, cell development and interactions with host cells.

Expression and characterization of the Mycobacterium tuberculosis serine/threonine protein kinase PknB.

PknB is a member of the newly discovered eukaryotic-like protein serine/threonine kinase (PSTK) family of proteins. The pknB gene was cloned and expressed in Escherichia coli. The active recombinant protein was purified and shown to be reactive with antiphosphoserine antibodies, as well as with antibodies to the phosphorylated eukaryotic Ser/Thr kinases mitogen-activated protein kinase kinase 3 and 6, P38, and Creb. In vitro kinase assays demonstrated that PknB is a functional kinase that is autophosphorylated on serine/threonine residues and is also able to phosphorylate the peptide substrate myelin basic protein. Analysis of pknB expression in Mycobacterium tuberculosis indicates the presence of pknB mRNA in (i) organisms grown in vitro in bacteriological media, (ii) a murine macrophage in vitro infection model, and (iii) in vivo alveolar macrophages from a patient with tuberculosis.

Species identification and phylogenetic relationships based on partial HSP60 gene sequences within the genus Staphylococcus.

The phylogenetic relationships among 36 validly described species or subspecies within the genus Staphylococcus were investigated by cloning and sequencing their 60 kDa heat-shock protein (HSP60) genes using a set of universal degenerate HSP60 PCR primers. The cloned partial HSP60 DNA sequences from nine Staphylococcus aureus strains were highly conserved (97-100% DNA sequence similarity; mean 98%), indicating that the HSP60 gene of multiple isolates within the same species have little microheterogeneity. At the subspecies level, DNA sequence similarity among members of S. aureus, Staphylococcus schleiferi, Staphylococcus cohnii and Staphylococcus capitis ranged from 91 to 98%. At the interspecies level, sequence similarity among 23 distinct species of staphylococci ranged from 74 to 93% (mean 82%). By comparison, the highest sequence similarity of Bacillus subtilis and Escherichia coli with members within the genus Staphylococcus was only 70 and 59%, respectively. Importantly, phylogenetic analysis based on the neighbour-joining distance method revealed remarkable concordance between the tree derived from partial HSP60 gene sequences and that based on genomic DNA-DNA hybridization, while 16S rRNA gene sequences correlated less well. The results demonstrate that DNA sequences from the highly conserved and ubiquitous HSP60 gene offer a convenient and accurate tool for species-specific identification and phylogenetic analysis of staphylococci.