Anaplasma phagocytophilum-Occupied Vacuole Interactions with the Host Cell Cytoskeleton
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cultivation of Uninfected and A. phagocytophilum Infected Host Cell Lines
2.2. Antibodies
2.3. Immunofluorescence Assays and Microscopy
2.4. Infection Assays
2.5. Western Blot Analyses
2.6. qRT-PCR
2.7. UBC9 siRNA Knockdown
2.8. Assessment of the Effect of WFA on A. phagocytophilum Infection
2.9. Statistical Analyses
3. Results
3.1. Intermediate Filaments and Microtubules Assemble around the ApV
3.2. SUMO-2/3 but Not SUMO-1 Colocalizes with Vimentin Assembled around the ApV
3.3. Keratin Filaments that Assemble around the ApV Colocalize with SUMO-2/3 Moieties
3.4. Vimentin Expression and the Relative Abundance of Insoluble Vimentin Are Increased in A. phagocytophilum Infected Cells
3.5. SUMOylation Is Critical for Vimentin Assembly at the ApV
3.6. Active Bacterial Protein Synthesis Is Not Necessary for Vimentin Assembly and SUMO-2/3 Localization at the ApV
3.7. Pharmacologic Inhibition Soluble Vimentin Reduces the A. phagocytophilum Load
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
ApV | Anaplasma phagocytophilum-occupied vacuole |
LSCM | Laser-scanning confocal microscopy |
WFA | Withaferin A |
SCV | Salmonella-containing vacuole |
Erk1/2 | Extracellular signal-related kinases 1 and 2 |
MAPK | Mitogen-associated protein kinases |
References
- Chen, S.M.; Dumler, J.S.; Bakken, J.S.; Walker, D.H. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J. Clin. Microbiol. 1994, 32, 589–595. [Google Scholar] [PubMed]
- Dumler, J.S.; Choi, K.S.; Garcia-Garcia, J.C.; Barat, N.S.; Scorpio, D.G.; Garyu, J.W.; Grab, D.J.; Bakken, J.S. Human granulocytic anaplasmosis and Anaplasma phagocytophilum. Emerg. Infect. Dis. 2005, 11, 1828–1834. [Google Scholar] [CrossRef] [PubMed]
- Stuen, S.; Granquist, E.G.; Silaghi, C. Anaplasma phagocytophilum—A widespread multi-host pathogen with highly adaptive strategies. Front. Cell. Infect. Microbiol. 2013, 3, 31. [Google Scholar] [CrossRef] [PubMed]
- Truchan, H.K.; Seidman, D.; Carlyon, J.A. Breaking in and grabbing a meal: Anaplasma phagocytophilum cellular invasion, nutrient acquisition, and promising tools for their study. Microbes Infect. 2013, 15, 1017–1025. [Google Scholar] [CrossRef] [PubMed]
- Rikihisa, Y. Mechanisms of obligatory intracellular infection with Anaplasma phagocytophilum. Clin. Microbiol. Rev. 2011, 24, 469–489. [Google Scholar] [CrossRef] [PubMed]
- Bakken, J.S.; Dumler, J.S.; Chen, S.M.; Eckman, M.R.; Van Etta, L.L.; Walker, D.H. Human granulocytic ehrlichiosis in the upper Midwest United States. A new species emerging? JAMA 1994, 272, 212–218. [Google Scholar] [CrossRef] [PubMed]
- Bakken, J.S.; Dumler, S. Human granulocytic anaplasmosis. Infect. Dis. Clin. N. Am. 2008, 22, 433–448. [Google Scholar] [CrossRef] [PubMed]
- CDC. Annual Cases of Anaplasmosis in the United States. Available online: http://www.cdc.gov/anaplasmosis/stats/index.html (accessed on 3 January 2016).
- Dumler, J.S. The biological basis of severe outcomes in Anaplasma phagocytophilum infection. FEMS Immunol. Med. Microbiol. 2012, 64, 13–20. [Google Scholar] [CrossRef] [PubMed]
- Goodman, J.L.; Nelson, C.; Vitale, B.; Madigan, J.E.; Dumler, J.S.; Kurtti, T.J.; Munderloh, U.G. Direct cultivation of the causative agent of human granulocytic ehrlichiosis. N. Engl. J. Med. 1996, 334, 209–215. [Google Scholar] [CrossRef] [PubMed]
- Munderloh, U.G.; Lynch, M.J.; Herron, M.J.; Palmer, A.T.; Kurtti, T.J.; Nelson, R.D.; Goodman, J.L. Infection of endothelial cells with Anaplasma marginale and A. phagocytophilum. Vet. Microbiol. 2004, 101, 53–64. [Google Scholar] [CrossRef] [PubMed]
- Ojogun, N.; Kahlon, A.; Ragland, S.A.; Troese, M.J.; Mastronunzio, J.E.; Walker, N.J.; Viebrock, L.; Thomas, R.J.; Borjesson, D.L.; Fikrig, E.; et al. Anaplasma phagocytophilum outer membrane protein A interacts with sialylated glycoproteins to promote infection of mammalian host cells. Infect. Immun. 2012, 80, 3748–3760. [Google Scholar] [CrossRef] [PubMed]
- Seidman, D.; Hebert, K.S.; Truchan, H.K.; Miller, D.P.; Tegels, B.K.; Marconi, R.T.; Carlyon, J.A. Essential domains of Anaplasma phagocytophilum invasins utilized to infect mammalian host cells. PLoS Pathog. 2015, 11, e1004669. [Google Scholar] [CrossRef] [PubMed]
- Beyer, A.R.; Truchan, H.K.; May, L.J.; Walker, N.J.; Borjesson, D.L.; Carlyon, J.A. The Anaplasma phagocytophilum effector AmpA hijacks host cell SUMOylation. Cell. Microbiol. 2015, 17, 504–519. [Google Scholar] [CrossRef] [PubMed]
- Truchan, H.K.; VieBrock, L.; Cockburn, C.L.; Ojogun, N.; Griffin, B.P.; Wijesinghe, D.S.; Chalfant, C.E.; Carlyon, J.A. Anaplasma phagocytophilum Rab10-dependent parasitism of the trans-Golgi network is critical for completion of the infection cycle. Cell. Microbiol. 2016, 18, 260–281. [Google Scholar] [CrossRef] [PubMed]
- Truchan, H.K.; Cockburn, C.L.; Hebert, K.S.; Magunda, F.; Noh, S.M.; Carlyon, J.A. The pathogen-occupied vacuoles of Anaplasma phagocytophilum and Anaplasma marginale interact with the endoplasmic reticulum. Front. Cell. Infect. Microbiol. 2016, 6, 22. [Google Scholar] [CrossRef] [PubMed]
- Sukumaran, B.; Mastronunzio, J.E.; Narasimhan, S.; Fankhauser, S.; Uchil, P.D.; Levy, R.; Graham, M.; Colpitts, T.M.; Lesser, C.F.; Fikrig, E. Anaplasma phagocytophilum AptA modulates Erk1/2 signalling. Cell. Microbiol. 2011, 13, 47–61. [Google Scholar] [CrossRef] [PubMed]
- Niu, H.; Yamaguchi, M.; Rikihisa, Y. Subversion of cellular autophagy by Anaplasma phagocytophilum. Cell. Microbiol. 2008, 10, 593–605. [Google Scholar] [CrossRef] [PubMed]
- Wickstead, B.; Gull, K. The evolution of the cytoskeleton. J. Cell Biol. 2011, 194, 513–525. [Google Scholar] [CrossRef] [PubMed]
- Kumar, Y.; Valdivia, R.H. Actin and intermediate filaments stabilize the Chlamydia trachomatis vacuole by forming dynamic structural scaffolds. Cell Host Microbe 2008, 4, 159–169. [Google Scholar] [CrossRef] [PubMed]
- Fletcher, D.A.; Mullins, R.D. Cell mechanics and the cytoskeleton. Nature 2010, 463, 485–492. [Google Scholar] [CrossRef] [PubMed]
- Kim, S.; Coulombe, P.A. Intermediate filament scaffolds fulfill mechanical, organizational, and signaling functions in the cytoplasm. Genes Dev. 2007, 21, 1581–1597. [Google Scholar] [CrossRef] [PubMed]
- Pall, T.; Pink, A.; Kasak, L.; Turkina, M.; Anderson, W.; Valkna, A.; Kogerman, P. Soluble CD44 interacts with intermediate filament protein vimentin on endothelial cell surface. PLoS ONE 2011, 6, e29305. [Google Scholar] [CrossRef] [PubMed]
- Hirata, Y.; Hosaka, T.; Iwata, T.; Le, C.T.; Jambaldorj, B.; Teshigawara, K.; Harada, N.; Sakaue, H.; Sakai, T.; Yoshimoto, K.; et al. Vimentin binds IRAP and is involved in GLUT4 vesicle trafficking. Biochem. Biophys. Res. Commun. 2011, 405, 96–101. [Google Scholar] [CrossRef] [PubMed]
- Haglund, C.M.; Welch, M.D. Pathogens and polymers: Microbe-host interactions illuminate the cytoskeleton. J. Cell Biol. 2011, 195, 7–17. [Google Scholar] [CrossRef] [PubMed]
- Thomas, V.; Fikrig, E. Anaplasma phagocytophilum specifically induces tyrosine phosphorylation of ROCK1 during infection. Cell. Microbiol. 2007, 9, 1730–1737. [Google Scholar] [CrossRef] [PubMed]
- Kaminsky, R.; Denison, C.; Bening-Abu-Shach, U.; Chisholm, A.D.; Gygi, S.P.; Broday, L. SUMO regulates the assembly and function of a cytoplasmic intermediate filament protein in C. elegans. Dev. Cell 2009, 17, 724–735. [Google Scholar] [CrossRef] [PubMed]
- Wilkinson, K.A.; Henley, J.M. Mechanisms, regulation and consequences of protein SUMOylation. Biochem. J. 2010, 428, 133–145. [Google Scholar] [CrossRef] [PubMed]
- Snider, N.T.; Omary, M.B. Post-translational modifications of intermediate filament proteins: Mechanisms and functions. Nat. Rev. Mol. Cell Biol. 2014, 15, 163–177. [Google Scholar] [CrossRef] [PubMed]
- Matic, I.; van Hagen, M.; Schimmel, J.; Macek, B.; Ogg, S.C.; Tatham, M.H.; Hay, R.T.; Lamond, A.I.; Mann, M.; Vertegaal, A.C. In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy. Mol. Cell. Proteom. 2008, 7, 132–144. [Google Scholar] [CrossRef] [PubMed]
- Geiss-Friedlander, R.; Melchior, F. Concepts in sumoylation: A decade on. Nat. Rev. Mol. Cell Biol. 2007, 8, 947–956. [Google Scholar] [CrossRef] [PubMed]
- Snider, N.T.; Weerasinghe, S.V.; Iniguez-Lluhi, J.A.; Herrmann, H.; Omary, M.B. Keratin hypersumoylation alters filament dynamics and is a marker for human liver disease and keratin mutation. J. Biol. Chem. 2011, 286, 2273–2284. [Google Scholar] [CrossRef] [PubMed]
- Huang, B.; Troese, M.J.; Howe, D.; Ye, S.; Sims, J.T.; Heinzen, R.A.; Borjesson, D.L.; Carlyon, J.A. Anaplasma phagocytophilum APH_0032 is expressed late during infection and localizes to the pathogen-occupied vacuolar membrane. Microb. Pathog. 2010, 49, 273–284. [Google Scholar] [CrossRef] [PubMed]
- IJdo, J.W.; Wu, C.; Magnarelli, L.A.; Fikrig, E. Serodiagnosis of human granulocytic ehrlichiosis by a recombinant HGE-44-based enzyme-linked immunosorbent assay. J. Clin. Microbiol. 1999, 37, 3540–3544. [Google Scholar] [PubMed]
- Huang, B.; Troese, M.J.; Ye, S.; Sims, J.T.; Galloway, N.L.; Borjesson, D.L.; Carlyon, J.A. Anaplasma phagocytophilum APH_1387 is expressed throughout bacterial intracellular development and localizes to the pathogen-occupied vacuolar membrane. Infect. Immun. 2010, 78, 1864–1873. [Google Scholar] [CrossRef] [PubMed]
- Troese, M.J.; Carlyon, J.A. Anaplasma phagocytophilum dense-cored organisms mediate cellular adherence through recognition of human P-selectin glycoprotein ligand 1. Infect. Immun. 2009, 77, 4018–4027. [Google Scholar] [CrossRef] [PubMed]
- Troese, M.J.; Kahlon, A.; Ragland, S.A.; Ottens, A.K.; Ojogun, N.; Nelson, K.T.; Walker, N.J.; Borjesson, D.L.; Carlyon, J.A. Proteomic analysis of Anaplasma phagocytophilum during infection of human myeloid cells identifies a protein that is pronouncedly upregulated on the infectious dense-cored cell. Infect. Immun. 2011, 79, 4696–4707. [Google Scholar] [CrossRef] [PubMed]
- Kahlon, A.; Ojogun, N.; Ragland, S.A.; Seidman, D.; Troese, M.J.; Ottens, A.K.; Mastronunzio, J.E.; Truchan, H.K.; Walker, N.J.; Borjesson, D.L.; et al. Anaplasma phagocytophilum Asp14 is an invasin that interacts with mammalian host cells via its C terminus to facilitate infection. Infect. Immun. 2013, 81, 65–79. [Google Scholar] [CrossRef] [PubMed]
- Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
- Salmon, M.; Zehner, Z.E. The transcriptional repressor ZBP-89 and the lack of Sp1/Sp3, c-Jun and Stat3 are important for the down-regulation of the vimentin gene during C2C12 myogenesis. Differentiation 2009, 77, 492–504. [Google Scholar] [CrossRef] [PubMed]
- Toivola, D.M.; Boor, P.; Alam, C.; Strnad, P. Keratins in health and disease. Curr. Opin. Cell Biol. 2015, 32, 73–81. [Google Scholar] [CrossRef] [PubMed]
- Romano, R.C.; Carter, J.M.; Folpe, A.L. Aberrant intermediate filament and synaptophysin expression is a frequent event in malignant melanoma: An immunohistochemical study of 73 cases. Mod. Pathol. 2015, 28, 1033–1042. [Google Scholar] [CrossRef] [PubMed]
- Perez-Sala, D.; Oeste, C.L.; Martinez, A.E.; Carrasco, M.J.; Garzon, B.; Canada, F.J. Vimentin filament organization and stress sensing depend on its single cysteine residue and zinc binding. Nat. Commun. 2015, 6, 7287. [Google Scholar] [CrossRef] [PubMed]
- Komura, K.; Ise, H.; Akaike, T. Dynamic behaviors of vimentin induced by interaction with GlcNAc molecules. Glycobiology 2012, 22, 1741–1759. [Google Scholar] [CrossRef] [PubMed]
- Murray, M.E.; Mendez, M.G.; Janmey, P.A. Substrate stiffness regulates solubility of cellular vimentin. Mol. Biol. Cell 2014, 25, 87–94. [Google Scholar] [CrossRef] [PubMed]
- Niu, H.; Xiong, Q.; Yamamoto, A.; Hayashi-Nishino, M.; Rikihisa, Y. Autophagosomes induced by a bacterial Beclin 1 binding protein facilitate obligatory intracellular infection. Proc. Natl. Acad. Sci. USA 2012, 109, 20800–20807. [Google Scholar] [CrossRef] [PubMed]
- Grin, B.; Mahammad, S.; Wedig, T.; Cleland, M.M.; Tsai, L.; Herrmann, H.; Goldman, R.D. Withaferin a alters intermediate filament organization, cell shape and behavior. PLoS ONE 2012, 7, e39065. [Google Scholar] [CrossRef] [PubMed]
- Guignot, J.; Servin, A.L. Maintenance of the Salmonella-containing vacuole in the juxtanuclear area: A role for intermediate filaments. Microb. Pathog. 2008, 45, 415–422. [Google Scholar] [CrossRef] [PubMed]
- Wasylnka, J.A.; Bakowski, M.A.; Szeto, J.; Ohlson, M.B.; Trimble, W.S.; Miller, S.I.; Brumell, J.H. Role for myosin II in regulating positioning of Salmonella-containing vacuoles and intracellular replication. Infect. Immun. 2008, 76, 2722–2735. [Google Scholar] [CrossRef] [PubMed]
- Szeto, J.; Namolovan, A.; Osborne, S.E.; Coombes, B.K.; Brumell, J.H. Salmonella-containing vacuoles display centrifugal movement associated with cell-to-cell transfer in epithelial cells. Infect. Immun. 2009, 77, 996–1007. [Google Scholar] [CrossRef] [PubMed]
- Man, S.M.; Ekpenyong, A.; Tourlomousis, P.; Achouri, S.; Cammarota, E.; Hughes, K.; Rizzo, A.; Ng, G.; Wright, J.A.; Cicuta, P.; et al. Actin polymerization as a key innate immune effector mechanism to control Salmonella infection. Proc. Natl. Acad. Sci. USA 2014, 111, 17588–17593. [Google Scholar] [CrossRef] [PubMed]
- Mostowy, S.; Cossart, P. Septins: The fourth component of the cytoskeleton. Nat. Rev. Mol. Cell Biol. 2012, 13, 183–194. [Google Scholar] [CrossRef] [PubMed]
- Mor-Vaknin, N.; Legendre, M.; Yu, Y.; Serezani, C.H.; Garg, S.K.; Jatzek, A.; Swanson, M.D.; Gonzalez-Hernandez, M.J.; Teitz-Tennenbaum, S.; Punturieri, A.; et al. Murine colitis is mediated by vimentin. Sci. Rep. 2013, 3, 1045. [Google Scholar] [CrossRef] [PubMed]
- Carlyon, J.A.; Abdel-Latif, D.; Pypaert, M.; Lacy, P.; Fikrig, E. Anaplasma phagocytophilum utilizes multiple host evasion mechanisms to thwart NADPH oxidase-mediated killing during neutrophil infection. Infect. Immun. 2004, 72, 4772–4783. [Google Scholar] [CrossRef] [PubMed]
- Thomas, V.; Samanta, S.; Wu, C.; Berliner, N.; Fikrig, E. Anaplasma phagocytophilum modulates gp91phox gene expression through altered interferon regulatory factor 1 and PU.1 levels and binding of CCAAT displacement protein. Infect. Immun. 2005, 73, 208–218. [Google Scholar] [CrossRef] [PubMed]
- Mott, J.; Rikihisa, Y. Human granulocytic ehrlichiosis agent inhibits superoxide anion generation by human neutrophils. Infect. Immun. 2000, 68, 6697–6703. [Google Scholar] [CrossRef] [PubMed]
- Wang, T.; Malawista, S.E.; Pal, U.; Grey, M.; Meek, J.; Akkoyunlu, M.; Thomas, V.; Fikrig, E. Superoxide anion production during Anaplasma phagocytophila infection. J. Infect. Dis. 2002, 186, 274–280. [Google Scholar] [CrossRef] [PubMed]
- Carlyon, J.A.; Chan, W.T.; Galan, J.; Roos, D.; Fikrig, E. Repression of rac2 mRNA expression by Anaplasma phagocytophila is essential to the inhibition of superoxide production and bacterial proliferation. J. Immunol. 2002, 169, 7009–7018. [Google Scholar] [CrossRef] [PubMed]
- Rennoll-Bankert, K.E.; Garcia-Garcia, J.C.; Sinclair, S.H.; Dumler, J.S. Chromatin-bound bacterial effector ankyrin A recruits histone deacetylase 1 and modifies host gene expression. Cell. Microbiol. 2015, 17, 1640–1652. [Google Scholar] [CrossRef] [PubMed]
- Garcia-Garcia, J.C.; Rennoll-Bankert, K.E.; Pelly, S.; Milstone, A.M.; Dumler, J.S. Silencing of host cell CYBB gene expression by the nuclear effector AnkA of the intracellular pathogen Anaplasma phagocytophilum. Infect. Immun. 2009, 77, 2385–2391. [Google Scholar] [CrossRef] [PubMed]
- IJdo, J.W.; Mueller, A.C. Neutrophil NADPH oxidase is reduced at the Anaplasma phagocytophilum phagosome. Infect. Immun. 2004, 72, 5392–5401. [Google Scholar] [CrossRef] [PubMed]
- Choi, K.S.; Dumler, J.S. Early induction and late abrogation of respiratory burst in A. phagocytophilum-infected neutrophils. Ann. N. Y. Acad. Sci. 2003, 990, 488–493. [Google Scholar] [CrossRef] [PubMed]
- Banerjee, R.; Anguita, J.; Roos, D.; Fikrig, E. Cutting edge: Infection by the agent of human granulocytic ehrlichiosis prevents the respiratory burst by down-regulating gp91phox. J. Immunol. 2000, 164, 3946–3949. [Google Scholar] [CrossRef] [PubMed]
- Walker, D. Sumoylation: Wrestling with filaments. Nat. Rev. Mol. Cell Biol. 2010, 11, 3. [Google Scholar] [CrossRef] [PubMed]
- Andreou, A.M.; Tavernarakis, N. SUMOylation and cell signalling. Biotechnol. J. 2009, 4, 1740–1752. [Google Scholar] [CrossRef] [PubMed]
- Soellner, P.; Quinlan, R.A.; Franke, W.W. Identification of a distinct soluble subunit of an intermediate filament protein: Tetrameric vimentin from living cells. Proc. Natl. Acad. Sci. USA 1985, 82, 7929–7933. [Google Scholar] [CrossRef] [PubMed]
- Zhang, Y.Q.; Sarge, K.D. Sumoylation regulates lamin A function and is lost in lamin A mutants associated with familial cardiomyopathies. J. Cell Biol. 2008, 182, 35–39. [Google Scholar] [CrossRef] [PubMed]
- Ribet, D.; Hamon, M.; Gouin, E.; Nahori, M.A.; Impens, F.; Neyret-Kahn, H.; Gevaert, K.; Vandekerckhove, J.; Dejean, A.; Cossart, P. Listeria monocytogenes impairs SUMOylation for efficient infection. Nature 2010, 464, 1192–1195. [Google Scholar] [CrossRef] [PubMed]
- Bartetzko, V.; Sonnewald, S.; Vogel, F.; Hartner, K.; Stadler, R.; Hammes, U.Z.; Bornke, F. The Xanthomonas campestris pv. vesicatoria type III effector protein XopJ inhibits protein secretion: Evidence for interference with cell wall-associated defense responses. Mol. Plant Microbe Interact. 2009, 22, 655–664. [Google Scholar] [CrossRef] [PubMed]
- Orth, K.; Xu, Z.; Mudgett, M.B.; Bao, Z.Q.; Palmer, L.E.; Bliska, J.B.; Mangel, W.F.; Staskawicz, B.; Dixon, J.E. Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like protein protease. Science 2000, 290, 1594–1597. [Google Scholar] [CrossRef] [PubMed]
- Dunphy, P.S.; Luo, T.; McBride, J.W. Ehrlichia chaffeensis exploits host SUMOylation pathways to mediate effector-host interactions and promote intracellular survival. Infect. Immun. 2014, 82, 4154–4168. [Google Scholar] [CrossRef] [PubMed]
© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Truchan, H.K.; Cockburn, C.L.; May, L.J.; VieBrock, L.; Carlyon, J.A. Anaplasma phagocytophilum-Occupied Vacuole Interactions with the Host Cell Cytoskeleton. Vet. Sci. 2016, 3, 25. https://doi.org/10.3390/vetsci3030025
Truchan HK, Cockburn CL, May LJ, VieBrock L, Carlyon JA. Anaplasma phagocytophilum-Occupied Vacuole Interactions with the Host Cell Cytoskeleton. Veterinary Sciences. 2016; 3(3):25. https://doi.org/10.3390/vetsci3030025
Chicago/Turabian StyleTruchan, Hilary K., Chelsea L. Cockburn, Levi J. May, Lauren VieBrock, and Jason A. Carlyon. 2016. "Anaplasma phagocytophilum-Occupied Vacuole Interactions with the Host Cell Cytoskeleton" Veterinary Sciences 3, no. 3: 25. https://doi.org/10.3390/vetsci3030025