The impact of mutation and gene conversion on the local diversification of antigen genes in African trypanosomes

Patterns of genetic diversity in parasite antigen gene families hold important information about their potential to generate antigenic variation within and between hosts. The evolution of such gene families is typically driven by gene duplication, followed by point mutation and gene conversion. There is great interest in estimating the rates of these processes from molecular sequences for understanding the evolution of the pathogen and its significance for infection processes. In this study, a series of models are constructed to investigate hypotheses about the nucleotide diversity patterns between closely related gene sequences from the antigen gene archive of the African trypanosome, the protozoan parasite causative of human sleeping sickness in Equatorial Africa. We use a hidden Markov model approach to identify two scales of diversification: clustering of sequence mismatches, a putative indicator of gene conversion events with other lower-identity donor genes in the archive, and at a sparser scale, isolated mismatches, likely arising from independent point mutations. In addition to quantifying the respective probabilities of occurrence of these two processes, our approach yields estimates for the gene conversion tract length distribution and the average diversity contributed locally by conversion events. Model fitting is conducted using a Bayesian framework. We find that diversifying gene conversion events with lower-identity partners occur at least five times less frequently than point mutations on variant surface glycoprotein (VSG) pairs, and the average imported conversion tract is between 14 and 25 nucleotides long. However, because of the high diversity introduced by gene conversion, the two processes have almost equal impact on the per-nucleotide rate of sequence diversification between VSG subfamily members. We are able to disentangle the most likely locations of point mutations and conversions on each aligned gene pair

Comparison of antimicrobial resistance phenotypes and genotypes in enterotoxigenic Escherichia coli isolated from Australian and Vietnamese pigs

This study aimed to compare the antibiogram phenotype and carriage of antimicrobial resistance genes (ARGs) of 97 porcine multidrug-resistant (MDR) enterotoxigenic Escherichia coli (ETEC) isolates obtained from Vietnam and 117 porcine MDR-ETEC obtained from Australia, two countries with different antimicrobial regulation systems. An antimicrobial resistance index (ARI) was calculated to quantify their potential significance to public health. Both Vietnamese and Australian isolates had moderate to high levels of resistance to commonly used antibiotics (ampicillin, tetracycline and sulphonamides). None of the Australian isolates were resistant to fluoroquinolones or third-generation cephalosporins and none possessed associated plasmid-mediated ARGs. However, 23.1% of Australian isolates were resistant to gentamicin owing to ARGs associated with apramycin or neomycin resistance [e.g. aac(3)-IV] that impart cross-resistance to gentamicin. Whilst Vietnamese isolates carried aminoglycoside ARGs, 44.4% of commercial pig isolates were resistant to gentamicin in comparison with 0% of village pig isolates. The plasmid-mediated fluoroquinolone ARG qnrB was commonly detected in Vietnamese isolates (52.3% commercial, 44.1% village), but phenotypic resistance was low (3.2% and 11.8%, respectively). The mean ARI for Vietnamese isolates (26.0) was significantly different (P < 0.001) from the mean ARI for Australian isolates (19.8), primarily reflecting fluoroquinolone resistance in the former collection. This comparison suggests the effectiveness of regulations that slow the dissemination of 'critical' resistance by restricting the availability of important classes of antimicrobials

Description of a New Form of Naked-eyed Medusa (Thaumantias achroa), with Histological Details

n/

Flukes in Man.

n/

Spatio-temporal Models of Lymphangiogenesis in Wound Healing

Several studies suggest that one possible cause of impaired wound healing is failed or insufficient lymphangiogenesis, that is the formation of new lymphatic capillaries. Although many mathematical models have been developed to describe the formation of blood capillaries (angiogenesis), very few have been proposed for the regeneration of the lymphatic network. Lymphangiogenesis is a markedly different process from angiogenesis, occurring at different times and in response to different chemical stimuli. Two main hypotheses have been proposed: 1) lymphatic capillaries sprout from existing interrupted ones at the edge of the wound in analogy to the blood angiogenesis case; 2) lymphatic endothelial cells first pool in the wound region following the lymph flow and then, once sufficiently populated, start to form a network. Here we present two PDE models describing lymphangiogenesis according to these two different hypotheses. Further, we include the effect of advection due to interstitial flow and lymph flow coming from open capillaries. The variables represent different cell densities and growth factor concentrations, and where possible the parameters are estimated from biological data. The models are then solved numerically and the results are compared with the available biological literature.Comment: 29 pages, 9 Figures, 6 Tables (39 figure files in total

Class I major histocompatibility complexes loaded by a periodate trigger

Class I major histocompatibility complexes (MHCs) present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. The unstable nature of unliganded MHC necessitates the production of recombinant class I complexes through in vitro refolding reactions in the presence of an added excess of peptides. This strategy is not amenable to high-throughput production of vast collections of class I complexes. To address this issue, we recently designed photocaged MHC ligands that can be cleaved by a UV light trigger in the MHC bound state under conditions that do not affect the integrity of the MHC structure. The results obtained with photocaged MHC ligands demonstrate that conditional MHC ligands can form a generally applicable concept for the creation of defined peptide−MHCs. However, the use of UV exposure to mediate ligand exchange is unsuited for a number of applications, due to the lack of UV penetration through cell culture systems and due to the transfer of heat upon UV irradiation, which can induce evaporation. To overcome these limitations, here, we provide proof-of-concept for the generation of defined peptide−MHCs by chemical trigger-induced ligand exchange. The crystal structure of the MHC with the novel chemosensitive ligand showcases that the ligand occupies the expected binding site, in a conformation where the hydroxyl groups should be reactive to periodate. We proceed to validate this technology by producing peptide−MHCs that can be used for T cell detection. The methodology that we describe here should allow loading of MHCs with defined peptides in cell culture devices, thereby permitting antigen-specific T cell expansion and purification for cell therapy. In addition, this technology will be useful to develop miniaturized assay systems for performing high-throughput screens for natural and unnatural MHC ligands

Identification and HLA-Tetramer-Validation of Human CD4(+) and CD8(+) T Cell Responses against HCMV Proteins IE1 and IE2

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4+ and CD8+ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8+ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4+ and CD8+ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4+ and CD8+ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4+ and 44 CD8+ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy

Holograms to Focus Arbitrary Ultrasonic Fields through the Skull

[EN] We report 3D-printed acoustic holographic lenses for the formation of ultrasonic fields of complex spatial distribution inside the skull. Using holographic lenses, we experimentally, numerically and theoretically produce acoustic beams whose spatial distribution matches target structures of the central nervous system. In particular, we produce three types of targets of increasing complexity. First, a set of points are selected at the center of both right and left human hippocampi. Experiments using a skull phantom and 3D printed acoustic holographic lenses show that the corresponding bi-focal lens simultaneously focuses acoustic energy at the target foci, with good agreement between theory and simulations. Second, an arbitrary curve is set as the target inside the skull phantom. Using time-reversal methods the holographic beam bends following the target path, in a similar way as self-bending beams do in free space. Finally, the right human hippocampus is selected as a target volume. The focus of the corresponding holographic lens overlaps with the target volume in excellent agreement between theory in free-media, and experiments and simulations including the skull phantom. The precise control of focused ultrasound into the central nervous system is mainly limited due to the strong phase aberrations produced by refraction and attenuation of the skull. Using the present method, the ultrasonic beam can be focused not only at a single point but overlapping one or various target structures simultaneously using low-cost 3D-printed acoustic holographic lens. The results open new paths to spread incoming biomedical ultrasound applications including blood-brain barrier opening and neuromodulation.This work is supported by the Spanish Ministry of Economy and Innovation (MINECO) through Project No. TEC2016-80976-R. N.J. and S.J. acknowledge financial support from Generalitat Valenciana through Grants No. APOSTD/2017/042, No. ACIF/2017/045, and No. GV/2018/11. F.C. acknowledges financial support from Agencia Valenciana de la Innovacio through Grant No. INNCON00/18/9 and European Regional Development Fund (Grant No. IDIFEDER/2018/022).Jiménez-Gambín, S.; Jimenez, N.; Benlloch Baviera, JM.; Camarena Femenia, F. (2019). Holograms to Focus Arbitrary Ultrasonic Fields through the Skull. Physical Review Applied. 12(1):014016-1-014016-14. https://doi.org/10.1103/PhysRevApplied.12.014016S014016-1014016-14121GABOR, D. (1948). A New Microscopic Principle. Nature, 161(4098), 777-778. doi:10.1038/161777a0Microscopy by reconstructed wave-fronts. (1949). Proceedings of the Royal Society of London. Series A. Mathematical and Physical Sciences, 197(1051), 454-487. doi:10.1098/rspa.1949.0075Leith, E. N., & Upatnieks, J. (1962). Reconstructed Wavefronts and Communication Theory*. Journal of the Optical Society of America, 52(10), 1123. doi:10.1364/josa.52.001123Ni, X., Kildishev, A. V., & Shalaev, V. M. (2013). Metasurface holograms for visible light. Nature Communications, 4(1). doi:10.1038/ncomms3807Huang, L., Chen, X., Mühlenbernd, H., Zhang, H., Chen, S., Bai, B., … Zhang, S. (2013). Three-dimensional optical holography using a plasmonic metasurface. Nature Communications, 4(1). doi:10.1038/ncomms3808Ma, G., & Sheng, P. (2016). Acoustic metamaterials: From local resonances to broad horizons. Science Advances, 2(2), e1501595. doi:10.1126/sciadv.1501595Cummer, S. A., Christensen, J., & Alù, A. (2016). Controlling sound with acoustic metamaterials. Nature Reviews Materials, 1(3). doi:10.1038/natrevmats.2016.1Liu, Z. (2000). Locally Resonant Sonic Materials. Science, 289(5485), 1734-1736. doi:10.1126/science.289.5485.1734Fang, N., Xi, D., Xu, J., Ambati, M., Srituravanich, W., Sun, C., & Zhang, X. (2006). Ultrasonic metamaterials with negative modulus. Nature Materials, 5(6), 452-456. doi:10.1038/nmat1644Yang, M., Ma, G., Yang, Z., & Sheng, P. (2013). Coupled Membranes with Doubly Negative Mass Density and Bulk Modulus. Physical Review Letters, 110(13). doi:10.1103/physrevlett.110.134301Li, Y., Liang, B., Gu, Z., Zou, X., & Cheng, J. (2013). Reflected wavefront manipulation based on ultrathin planar acoustic metasurfaces. Scientific Reports, 3(1). doi:10.1038/srep02546Xie, Y., Wang, W., Chen, H., Konneker, A., Popa, B.-I., & Cummer, S. A. (2014). Wavefront modulation and subwavelength diffractive acoustics with an acoustic metasurface. Nature Communications, 5(1). doi:10.1038/ncomms6553Jiménez, N., Cox, T. J., Romero-García, V., & Groby, J.-P. (2017). Metadiffusers: Deep-subwavelength sound diffusers. Scientific Reports, 7(1). doi:10.1038/s41598-017-05710-5Jiménez, N., Romero-García, V., Pagneux, V., & Groby, J.-P. (2017). Rainbow-trapping absorbers: Broadband, perfect and asymmetric sound absorption by subwavelength panels for transmission problems. Scientific Reports, 7(1). doi:10.1038/s41598-017-13706-4Qi, S., Li, Y., & Assouar, B. (2017). Acoustic Focusing and Energy Confinement Based on Multilateral Metasurfaces. Physical Review Applied, 7(5). doi:10.1103/physrevapplied.7.054006Bok, E., Park, J. J., Choi, H., Han, C. K., Wright, O. B., & Lee, S. H. (2018). Metasurface for Water-to-Air Sound Transmission. Physical Review Letters, 120(4). doi:10.1103/physrevlett.120.044302Li, Y., Jiang, X., Liang, B., Cheng, J., & Zhang, L. (2015). Metascreen-Based Acoustic Passive Phased Array. Physical Review Applied, 4(2). doi:10.1103/physrevapplied.4.024003Li, Y., & Assouar, M. B. (2015). Three-dimensional collimated self-accelerating beam through acoustic metascreen. Scientific Reports, 5(1). doi:10.1038/srep17612Kaina, N., Lemoult, F., Fink, M., & Lerosey, G. (2015). Negative refractive index and acoustic superlens from multiple scattering in single negative metamaterials. Nature, 525(7567), 77-81. doi:10.1038/nature14678Li, J., Fok, L., Yin, X., Bartal, G., & Zhang, X. (2009). Experimental demonstration of an acoustic magnifying hyperlens. Nature Materials, 8(12), 931-934. doi:10.1038/nmat2561Melde, K., Mark, A. G., Qiu, T., & Fischer, P. (2016). Holograms for acoustics. Nature, 537(7621), 518-522. doi:10.1038/nature19755Xie, Y., Shen, C., Wang, W., Li, J., Suo, D., Popa, B.-I., … Cummer, S. A. (2016). Acoustic Holographic Rendering with Two-dimensional Metamaterial-based Passive Phased Array. Scientific Reports, 6(1). doi:10.1038/srep35437Zhu, Y., Hu, J., Fan, X., Yang, J., Liang, B., Zhu, X., & Cheng, J. (2018). Fine manipulation of sound via lossy metamaterials with independent and arbitrary reflection amplitude and phase. Nature Communications, 9(1). doi:10.1038/s41467-018-04103-0Memoli, G., Caleap, M., Asakawa, M., Sahoo, D. R., Drinkwater, B. W., & Subramanian, S. (2017). Metamaterial bricks and quantization of meta-surfaces. Nature Communications, 8(1). doi:10.1038/ncomms14608Brown, M. D., Cox, B. T., & Treeby, B. E. (2017). Design of multi-frequency acoustic kinoforms. Applied Physics Letters, 111(24), 244101. doi:10.1063/1.5004040Hertzberg, Y., & Navon, G. (2011). Bypassing absorbing objects in focused ultrasound using computer generated holographic technique. Medical Physics, 38(12), 6407-6415. doi:10.1118/1.3651464Zhang, P., Li, T., Zhu, J., Zhu, X., Yang, S., Wang, Y., … Zhang, X. (2014). Generation of acoustic self-bending and bottle beams by phase engineering. Nature Communications, 5(1). doi:10.1038/ncomms5316Marzo, A., Seah, S. A., Drinkwater, B. W., Sahoo, D. R., Long, B., & Subramanian, S. (2015). Holographic acoustic elements for manipulation of levitated objects. Nature Communications, 6(1). doi:10.1038/ncomms9661Ter Haar, >Gail, & Coussios, C. (2007). High intensity focused ultrasound: Physical principles and devices. International Journal of Hyperthermia, 23(2), 89-104. doi:10.1080/02656730601186138Gélat, P., ter Haar, G., & Saffari, N. (2014). A comparison of methods for focusing the field of a HIFU array transducer through human ribs. Physics in Medicine and Biology, 59(12), 3139-3171. doi:10.1088/0031-9155/59/12/3139Fry, F. J., & Barger, J. E. (1978). Acoustical properties of the human skull. The Journal of the Acoustical Society of America, 63(5), 1576-1590. doi:10.1121/1.381852Thomas, J.-L., & Fink, M. A. (1996). Ultrasonic beam focusing through tissue inhomogeneities with a time reversal mirror: application to transskull therapy. IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control, 43(6), 1122-1129. doi:10.1109/58.542055Hynynen, K., & Jolesz, F. A. (1998). Demonstration of Potential Noninvasive Ultrasound Brain Therapy Through an Intact Skull. Ultrasound in Medicine & Biology, 24(2), 275-283. doi:10.1016/s0301-5629(97)00269-xSun, J., & Hynynen, K. (1998). Focusing of therapeutic ultrasound through a human skull: A numerical study. The Journal of the Acoustical Society of America, 104(3), 1705-1715. doi:10.1121/1.424383Aubry, J.-F., Tanter, M., Pernot, M., Thomas, J.-L., & Fink, M. (2003). Experimental demonstration of noninvasive transskull adaptive focusing based on prior computed tomography scans. The Journal of the Acoustical Society of America, 113(1), 84-93. doi:10.1121/1.1529663Tanter, M., Thomas, J.-L., & Fink, M. (1998). Focusing and steering through absorbing and aberrating layers: Application to ultrasonic propagation through the skull. The Journal of the Acoustical Society of America, 103(5), 2403-2410. doi:10.1121/1.422759Hertzberg, Y., Volovick, A., Zur, Y., Medan, Y., Vitek, S., & Navon, G. (2010). Ultrasound focusing using magnetic resonance acoustic radiation force imaging: Application to ultrasound transcranial therapy. Medical Physics, 37(6Part1), 2934-2942. doi:10.1118/1.3395553Jolesz, F. A. (Ed.). (2014). Intraoperative Imaging and Image-Guided Therapy. doi:10.1007/978-1-4614-7657-3Shen, C., Xu, J., Fang, N. X., & Jing, Y. (2014). Anisotropic Complementary Acoustic Metamaterial for Canceling out Aberrating Layers. Physical Review X, 4(4). doi:10.1103/physrevx.4.041033Maimbourg, G., Houdouin, A., Deffieux, T., Tanter, M., & Aubry, J.-F. (2018). 3D-printed adaptive acoustic lens as a disruptive technology for transcranial ultrasound therapy using single-element transducers. Physics in Medicine & Biology, 63(2), 025026. doi:10.1088/1361-6560/aaa037Ferri, M., Bravo, J. M., Redondo, J., & Sánchez-Pérez, J. V. (2019). Enhanced Numerical Method for the Design of 3-D-Printed Holographic Acoustic Lenses for Aberration Correction of Single-Element Transcranial Focused Ultrasound. Ultrasound in Medicine & Biology, 45(3), 867-884. doi:10.1016/j.ultrasmedbio.2018.10.022Hynynen, K., McDannold, N., Vykhodtseva, N., & Jolesz, F. A. (2001). Noninvasive MR Imaging–guided Focal Opening of the Blood-Brain Barrier in Rabbits. Radiology, 220(3), 640-646. doi:10.1148/radiol.2202001804Tyler, W. J., Tufail, Y., Finsterwald, M., Tauchmann, M. L., Olson, E. J., & Majestic, C. (2008). Remote Excitation of Neuronal Circuits Using Low-Intensity, Low-Frequency Ultrasound. PLoS ONE, 3(10), e3511. doi:10.1371/journal.pone.0003511Schneider, U., Pedroni, E., & Lomax, A. (1996). The calibration of CT Hounsfield units for radiotherapy treatment planning. Physics in Medicine and Biology, 41(1), 111-124. doi:10.1088/0031-9155/41/1/009Mast, T. D. (2000). Empirical relationships between acoustic parameters in human soft tissues. Acoustics Research Letters Online, 1(2), 37-42. doi:10.1121/1.1336896Mazziotta, J. C., Toga, A. W., Evans, A., Fox, P., & Lancaster, J. (1995). A Probabilistic Atlas of the Human Brain: Theory and Rationale for Its Development. NeuroImage, 2(2), 89-101. doi:10.1006/nimg.1995.1012Yushkevich, P. A., Piven, J., Hazlett, H. C., Smith, R. G., Ho, S., Gee, J. C., & Gerig, G. (2006). User-guided 3D active contour segmentation of anatomical structures: Significantly improved efficiency and reliability. NeuroImage, 31(3), 1116-1128. doi:10.1016/j.neuroimage.2006.01.015Treeby, B. E., & Cox, B. T. (2010). Modeling power law absorption and dispersion for acoustic propagation using the fractional Laplacian. The Journal of the Acoustical Society of America, 127(5), 2741-2748. doi:10.1121/1.3377056Treeby, B. E., Jaros, J., Rendell, A. P., & Cox, B. T. (2012). Modeling nonlinear ultrasound propagation in heterogeneous media with power law absorption using a k-space pseudospectral method. The Journal of the Acoustical Society of America, 131(6), 4324-4336. doi:10.1121/1.4712021Jiménez, N., Camarena, F., Redondo, J., Sánchez-Morcillo, V., Hou, Y., & Konofagou, E. E. (2016). Time-Domain Simulation of Ultrasound Propagation in a Tissue-Like Medium Based on the Resolution of the Nonlinear Acoustic Constitutive Relations. Acta Acustica united with Acustica, 102(5), 876-892. doi:10.3813/aaa.919002Jiménez, N., Romero-García, V., Pagneux, V., & Groby, J.-P. (2017). Quasiperfect absorption by subwavelength acoustic panels in transmission using accumulation of resonances due to slow sound. Physical Review B, 95(1). doi:10.1103/physrevb.95.014205Tsang, P. W. M., & Poon, T.-C. (2013). Novel method for converting digital Fresnel hologram to phase-only hologram based on bidirectional error diffusion. Optics Express, 21(20), 23680. doi:10.1364/oe.21.023680Lirette, R., & Mobley, J. (2017). Focal zone characteristics of stepped Fresnel and axicon acoustic lenses. doi:10.1121/2.0000703Gatto, M., Memoli, G., Shaw, A., Sadhoo, N., Gelat, P., & Harris, R. A. (2012). Three-Dimensional Printing (3DP) of neonatal head phantom for ultrasound: Thermocouple embedding and simulation of bone. Medical Engineering & Physics, 34(7), 929-937. doi:10.1016/j.medengphy.2011.10.012Robertson, J., Martin, E., Cox, B., & Treeby, B. E. (2017). Sensitivity of simulated transcranial ultrasound fields to acoustic medium property maps. Physics in Medicine and Biology, 62(7), 2559-2580. doi:10.1088/1361-6560/aa5e98Hill, C. R., Bamber, J. C., & ter Haar, G. R. (Eds.). (2004). Physical Principles of Medical Ultrasonics. doi:10.1002/0470093978O’Neil, H. T. (1949). Theory of Focusing Radiators. The Journal of the Acoustical Society of America, 21(5), 516-526. doi:10.1121/1.1906542Chen, D.-C., Zhu, X.-F., Wei, Q., Wu, D.-J., & Liu, X.-J. (2018). Broadband acoustic focusing by Airy-like beams based on acoustic metasurfaces. Journal of Applied Physics, 123(4), 044503. doi:10.1063/1.501070

Veterinary Students’ Knowledge and Perceptions About Antimicrobial Stewardship and Biosecurity—A National Survey

A better understanding of veterinary students’ perceptions, attitudes, and knowledge about antimicrobial stewardship and biosecurity could facilitate more effective education of future veterinarians about these important issues. A multicenter cross-sectional study was performed by administering a questionnaire to veterinary students expected to graduate in 2017 or 2018 in all Australian veterinary schools. Four hundred and seventy-six of 1246 students (38%) completed the survey. Many students were unaware of the high importance of some veterinary drugs to human medicine, specifically enrofloxacin and cefovecin (59% and 47% of responses, respectively). Fewer than 10% of students would use appropriate personal protective equipment in scenarios suggestive of Q fever or psittacosis. Students expected to graduate in 2018 were more likely to select culture and susceptibility testing in companion animal cases (OR 1.89, 95% CI 1.33–2.69, p < 0.001), and were more likely to appropriately avoid antimicrobials in large animal cases (OR 1.75, 95% CI 1.26–2.44, p = 0.001) than those expected to graduate in 2017. However, 2018 graduates were less likely to correctly identify the importance rating of veterinary antimicrobials for human health (OR 0.48, 95% CI 0.34–0.67, p < 0.001) than 2017 graduates. Students reported having a good knowledge of antimicrobial resistance, and combating resistance, but only 34% thought pharmacology teaching was adequate and only 20% said that teaching in lectures matched clinical teaching. Efforts need to be made to harmonize preclinical and clinical teaching, and greater emphasis is needed on appropriate biosecurity and antimicrobial stewardship