Progress 10/01/05 to 09/30/10
Outputs
OUTPUTS: This project has focused on a small ruminant model for Transmissible Spongiform Encephalopathies (TSE) using white-tailed deer and goats, with the development of a diagnostic test the ultimate objective. During the present reporting period, this has resulted in five scientific presentations at regional, national and international meetings. Sequencing of the genome of Spiroplasma contributed to the goals of this project. Culture/isolation techniques were optimized to culture Spiroplasma spp. The goal of this project was to expand the capacity of the model system to look at TSEs and other future regulatory/emerging pathogenic diseases of domestic livestock and/or wildlife species. Focusing on TSE, the project has made significant contributions to the understanding of the link between Spiroplasma spp and TSE. PARTICIPANTS: Philip Elzer (PI), Dearl Sanders, Fred Enright, Frank Bastian, William Todd, Dennis French and Ron Thune. Others included: Will Forbes,Joel Walker, Sue Hagius, Stephen Buco, Charles Boudreaux, Christie Landry, Hilari French, Patrick Cutbirth, Diana Babin, Tamara Chouljenko, Svetlana Oard and Russ Freeland. TARGET AUDIENCES: Target audiences include the TSE scientific community and local and state deer producers as well as international mycoplasma research workers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Different inoculation routes (intracranial, intravascular, intradermal) in a neonatal caprine host were investigated. Daily blood samples for the first seven days post-inoculation and weekly thereafter were put into M1D culture and incubated at 30C. At six weeks, the animals were sacrificed and tissues were harvested for histopathology, PCR, and culture to further isolate the organism. S. mirum was isolated from whole blood culture in 58% of the total animal group (IC-0%, IV-100%, ID-50%) and from brain cultures in 11% of the total animal group (IC-0%, IV-25%, ID-0%). Further investigation will help elucidate S. mirum's potential role in TSE. Three different species of Sprioplasma were used to study the effects of disinfectants (chemical/physical) on growth. Stock cultures of each species were mixed with dilutions of halogens, alcohols, detergents, chlorhexidine, hydrogen peroxide, and aldehydes or different levels of physical control (heat and UV light) to test resistance to environmental conditions. The organisms were exposed to various dilutions of the disinfectants and then inoculated into fresh culture at different time points. Overall, spiroplasmas were found to be susceptible to halogen dilutions greater than 1%, alcohol dilutions greater than 50%, detergent dilutions greater than 0.05%, aldehyde dilutions greater than 0.1%, and to hydrogen peroxide dilutions greater than 0.1%. Similar growth patterns were found at all-time points tested. Spiroplasmas were also susceptible to heat at 56C and UV light if exposed for longer than 1 hour. An antibiotic profile was created using a standard veterinary MIC susceptibility plate using SMCA. SMCA was found to be susceptible to only clindamycin and tylosin tartrate of the antibiotics tested. SMCA was also found to be resistant to complement. Overall, the spiroplasmosis animal models have proven to be valuable in the understanding the organism and its role in the disease process. In summary the association of Spiroplasma spp. with TSE-like syndromes has been furthered through novel culture techniques and animal modeling.
Publications
- Bastian, F.O., C. M. Boudreaux, S.D. Hagius,M. S. Bulgin, S.J. Sorensen-Melson, F.M. Enright, and P.H. Elzer (2010). Spiroplasma Found in the Eyes of Scrapie Affected Sheep. 135th ANA, San Francisco, CA, September 2010, Poster M134.
- Freeland, R., H. French, C. Boudreaux, S. Hagius, F.Bastian, and P.Elzer. (2010). Characterization of acute effects caused by Spiroplasma mirum in goats via three different inoculation routes. Phi Zeta Research Day, Baton Rouge, LA, September 29, 2010.
- Bastian, F., C. Boudreaux, S. Hagius, M. Bulgin, S. Sorensen-Melson, F. Enright, and P. Elzer. (2010). Spiroplasma found in the eyes of Scrapie sheep research flock. Phi Zeta Research Day, Baton Rouge, LA, September 29, 2010.
- French, H. and P. Elzer. (2010). Sensitivity profile of Spiroplasma mirum and Spiroplasma melliferum to disinfection. Phi Zeta Research Day, Baton Rouge, LA, September 29, 2010.
- Landry, C.A., F.O. Bastian, and R.L. Thune. (2010). Complete Genome Sequence of Spiroplasma mirum Suckling Mouse Cataract Agent. Phi Zeta Research Day, Baton Rouge, LA, September 29, 2010.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: This project has continued in the development of a small ruminant model for Transmissible Spongiform Encephalopathies (TSE) using white-tailed deer and goats. This has resulted in seven scientific presentations at regional and national meetings as well as for producer groups. A new isolation technique was developed to culture Spiroplasma spp. Extramural funding supported this project by sequencing the genome of Spiroplasma spp. PARTICIPANTS: Philip Elzer, Dearl Sanders, Fred Enright, Frank Bastian, William Todd and Ron Thune. Others included: Will Forbes,Joel Walker, Sue Hagius, Stephen Buco, Charles Boudreaux, Christie Landry, and Hilari French. Several undergraduates are also involved. TARGET AUDIENCES: Target audiences include the TSE scientific community and local and state deer producers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In this study, we examined eyes from deer and sheep inoculated intracranially with the suckling mouse cataract (SMCA) strain of Spiroplasma mirum and showed severe retinopathy akin to retinal lesions in naturally occurring scrapie in sheep. Astrogliosis was seen in the retinal neuronal layer. Spiroplasma antigen was found by immunocytochemistry concentrated in the eyes, especially in the vitreous humor and corneal endothelia. This led to our culturing a novel Spiroplasma sp. from eyes obtained from a scrapie sheep research flock at the University of Idaho into cell-free media and bovine corneal endothelial (BCE) cells. The scrapie spiroplasma isolates were identified by dark field and electron microscopy, and molecular studies. There was no immune cross-reactivity between scrapie spiroplasma isolates and SMCA. Understanding the susceptibility of ophthalmic tissues to spiroplasma infection may clarify its role in the pathogenesis of TSE.
Publications
- Bastian, F., W. Todd, C. Boudreaux, F. Enright. Tubulofilamentous Structures in TSE Represent Morphological Markers of Spiroplasmosis. 85th Annual Meeting of the American Association of Neuropathologists, June 11, 2009, San Antonio, TX. Poster 86.
- Bastian, F., W. Todd, C. Boudreaux, F. Enright. Tubulofilamentous Structures in TSE Represent Morphological Markers of Spiroplasmosis. J Neuropathol Exp Neurol, May 2009, 68(5):574.
- Cutbirth. P., H.M. French, C.M. Boudreaux, F.O. Bastian and P.H. Elzer. (2009). The use of embryonated egg for the isolation and culture of Spiroplasma spp. Merck-Merial National Veterinary Scholar Symposium. Raleigh, NC, July 2009.
- Bastian, F.O., C.M. Boudreaux, S.D. Hagius, M.S. Bulgin, S. J. Sorensen-Melson, F.M. Enright, and P.H. Elzer. (2009). Spiroplasma found in eyes from Scrapie sheep research flock. Microbial Pathogenesis and Host Response, Cold Spring Harbor Laboratory, September 8-12,2009.
- French, H.M., S.D Hagius, C.M. Boudreaux, F.M. Enright, F.O. Bastian and P.H. Elzer. (2009). Embryonated egg fatalities due to Spiroplasma mirum. Phi Zeta Research Day, Baton Rouge, LA, September 23, 2009.
- Cutbirth. P., H.M. French, C.M. Boudreaux, F.O. Bastian and P.H. Elzer. (2009). The use of embryonated egg for the isolation and culture of Spiroplasma spp. Phi Zeta Research Day, Baton Rouge, LA, September 23, 2009.
- Bastian, F.O., C.M. Boudreaux, S.D. Hagius, M.S. Bulgin, S. J. Sorensen-Melson, F.M. Enright, and P.H. Elzer. (2009). Spiroplasma found in scrapie sheep eyes is a clue to the cause of transmissible spongiform encephalopathies. Phi Zeta Research Day, Baton Rouge, LA, September 23, 2009.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: This project has continued to characterize a small ruminant model for Transmissible Spongiform Encephalopathies (TSE) using white-tailed deer and goats. This has resulted in several scientific presentations at local, regional and national meetings as well as to producer groups. The preliminary data has enabled the researchers to successfully solicit extramural funding from two sources. PARTICIPANTS: Philip Elzer, PI; Co PIs: Dearl Sanders, Fred Enright, Dennis French, Bill Todd, Frank Bastian, Ronald Thune. The PI and Co PIs designed and implemented the project. Other participants included: Will Forbes (deer husbandry and maintenance of control animals); Joel Walker, Sue Hagius, Daniel Langlois, Stephen Buco (deer husbandry, maintenance of inoculated animals and necropsy, laboratory assistance); Mitch Boudreaux, Denise Fernandez (laboratory and molecular procedures);Hilari French (veterinary/graduate student - serology, necropsy and histology). Organizations involved: LSU AgCenter Department of Veterinary Science and the Bob R. Jones Idlewild Research Station; LSU School of Veterinary Medicine; National Science Foundation. TARGET AUDIENCES: Target audiences include the TSE scientific community and deer producers. PROJECT MODIFICATIONS: Tissue culture and egg inoculation have been added to the laboratory procedures utilized to detect the presence of spiroplasma in tissues.
Impacts
Four-month old farm-raised white-tailed deer and neonatal goats were inoculated intracranially with Spiroplasma mirum (SMCA). One of four deer developed clinical signs of TSE after 7 months incubation at which time both experimental and control deer were necropsied. All deer brains were examined histologically to determine the location and nature of any lesions. The control brains showed no abnormalities; the injected deer exhibited neuronal degeneration, spongiform changes, and astrogliosis in the same topographic distribution as naturally occurring TSE. This successful replication of the disease validates this model for further investigation of the TSE pathogenesis and emphasizes the role of spiroplasmosis as a cause of this disease. The caprine model is being monitored both clinically and serologically for spiroplasmosis. The ruminant models should lead to the elucidation of the immunological and pathological processes involved in the natural host with TSE.
Publications
- Bastian, F.O., W.J. Todd, F.M. Enright, H.M. French, and P.H. Elzer. (2008). Experimental spiroplasmosis as a TSE model. 102nd American Society for Microbiology, Boston, MA, June 2008.
- Babin, D, S. Oard, J. Oard and P. Elzer. (2008). Determination of a caprine humoral antibody response against cholera toxin B expressed in a transgenic plant vaccine. Phi Zeta Research Day, Baton Rouge, LA, September 24, 2008.
- Bastian, F.O., W. J. Todd, H. M. French, C. M. Boudreaux, F. M. Enright, and P. H. Elzer. (2008). Spiroplasma experimentally induce astrogliosis and retinopathy in ruminants characteristic of transmissible spongiform encephalopathies. Experimental Biology 2008, San Diego, CA, April 5-9, 2008, FASEB J. 22: 173.5.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: This project has lead to the development of a small ruminant model for Transmissible Spongiform Encephalopathies (TSE) using white-tailed deer. This has resulted in one publication and numerous scientific presentations at regional and national meetings as well as producer groups. The preliminary data has enhanced the ability of the researchers to solicit extramural funding.
PARTICIPANTS: Philip Elzer, PI; Co PIs: Dearl Sanders, Fred Enright, Dennis French, Frank Bastian. The PI and Co PIs designed and implemented the project. Other participants included: Will Forbes (deer husbandry and maintenance of control animals); Joel Walker, Sue Hagius, Chad Towns, Natalie Cooper, April Ooley and Stephen Buco (deer husbandry, maintenance of inoculated animals and necropsy); Hilari French (veterinary/graduate student - necropsy and histology). Organizations involved: LSU AgCenter Department of Veterinary Science and the Bob R. Jones Idlewild Research Station; LSU School of Veterinary Medicine.
TARGET AUDIENCES: Target audiences include the TSE scientific community and deer producers.
PROJECT MODIFICATIONS: The first experiment was performed using neonatal animals; all future experiments will use weaned animals. This modification was necessary to ensure fitness of the animals as neonates present husbandry issues.
Impacts
Farm raised white-tailed deer inoculated intracranially with Spiroplasma mirum (SMCA) developed signs of TSE after 1.5 to 5.5 months incubation. Three of four deer developed clinical signs of neurological deterioration, while the controls showed no abnormalities. All deer brains were examined at necropsy to determine the location and nature of any lesions. All control brains were normal; and four of four SMCA inoculated deer brains revealed spongiform encephalopathy in the cerebral cortex, cerebellar cortex and the brain stem. The impact of these findings emphasizes the importance of the deer model's role in providing information about TSE pathogenesis, which thus far is a negative association between diseased brains with Spiroplasma and abnormal prions.
Publications
- Bastian, F.O., D.E. Sanders, W.A. Forbes, S.D. Hagius, J.V. Walker, W.G. Henk, F.M. Enright, and P.H. Elzer. (2007). Spiroplasma spp. from TSE brains or ticks induce spongiform encephalopathy in ruminants. J Med Microbiol 56:1235-1242.
- Bastian, F.O., D.E. Sanders, W.A. Forbes, S.D. Hagius, J.V. Walker, W.G. Henk, F.M. Enright, and P.H. Elzer. (2007). Spiroplasma spp. Isolated from Rabbit Ticks or TSE-affected Brains Induce Spongiform Encephalopathy in Ruminants. AANP, Washington, DC, April 2007.
- French, HM., PH. Elzer, FM. Enright and FO. Bastian. (2007). TSE neurological triad in experimental Spiroplasma mirum infection in white-tailed deer. Phi Zeta Research Day, Baton Rouge, LA, September 26, 2007.
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Progress 01/01/06 to 12/31/06
Outputs
Neonatal goats and sheep were inoculated via intracranial injections in the right hemisphere between the fontanels using a 21-gauge needle with Spiroplasma in an attempt to induce Transmissible Spongiform Encephalopathies (TSE). Control groups received injections of media. Animals were euthanatized one year post inoculation since no clinical signs developed. Upon necropsy, numerous histological lesions similar to TSE like lesions were observed in the brains of the Spiroplasma injected animals compared to none in the control animals. White tailed-deer have currently been injected with Spiroplasma in an attempt to induce TSE like symptoms and or lesions.
Impacts
The goal of this project is to expand the capacity of the model system to look at TSE and other future regulatory/emerging pathogenic diseases of domestic livestock and/or wildlife species. The results of this project will lead to the development of a ruminant model and diagnostic tests to assist in the control and or eradication of these diseases.
Publications
- No publications reported this period
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