DIAGNOSIS AND EPIZOOTIOLOGY OF EMERGING DISEASES OF WILDLIFE, LIVESTOCK, AND POULTRY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010852
• Project No.: MICL02465
• Project Start Date: Nov 1, 2016
• Project End Date: Oct 31, 2021

Project Director
Bolin, ST.

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
Pathobiology & Diagnostic Investigation

Non Technical Summary
Emerging diseases of domestic or free ranging ranging animals are caused frequently by infectious agents that may be bacteria, viruses, fungi, or parasite. Many of those agents are zoonotic and are of importance to human health as well as animal health. While emerging zoonotic agents are of obvious concern for all sectors of the society, emerging infectious agents associated with animals, but not humans, can have devastating effects on animal populations and, consequently, affect the human food supply, recreational activities, sporting events, and local economies. A recent example occurred in 2015, when the poultry industry of the Midwestern United States experienced the worst outbreak of avian influenza that ever occurred in North America. The causal virus was new to the United States and its introduction to the country resulted in the loss of over 50 million turkeys and chickens that were killed by the virus, or were destroyed to control spread of the virus. This project will investigate three emerging or re-emergin diseases of economic importance. The goals will be to develop a laboratory model for assessing survival of Mycobacterium bovis in ensiled forage, conduct a multicounty surveillance for Pseudogymnoascus destructans (P. destructans, the fungus that causes white nose syndrome) in big brown bats in Michigan, initiate and complete studies for prevalence of hemoplasmas in tissue specimens submitted for routine diagnostic testing (tissues not submitted as suspect cases of hemoplasma infection) for cattle, deer, sheep, and bison. The hemoplasmas are insidious bacteria that colonize the blood causing anemia, ill thrift, and an assortment of other disease conditions that negatively impact production of meat, milk, or wool; and those may have reservoirs in wildlife.

Animal Health Component

100%

Research Effort Categories

Basic

20%

Applied

60%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4099	1100	70%
311	4099	1102	30%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1100 - Bacteriology; 1102 - Mycology;

Keywords

bovine tuberculosis

white nose syndrome

hemoplasma

Goals / Objectives
Goal 1. Develop a laboratory model for assessing survival of Mycobacterium in ensiled forage. Successful completion of this objective will result in a reproducible model for ensiling feedstuffs contaminated with Mycobacterium bovis (M. bovis), sampling the ensiled feed for viable M. bovis using culture, detection of non-cultivable and dormant M. bovis using fluorescent markers only expressed in a live organism, and detection of genetic material from M. bovis in ensiled feed using polymerase chain reaction. The detection of non-cultivable and dormant M. bovis will require creation of a recombinant organism capable of expressing a fluorescent signal if alive and dormant. This has been done with laboratory adapted strains of mycobacteria; however, we will try to do this with a field strain of mycobacteria from Michigan.Goal 2. Conduct a multicounty surveillance for Pseudogymnoascus destructans (P. destructans, the fungus that causes white nose syndrome) in big brown bats in Michigan. Successful completion of this objective will provide information on the prevalence of P. destructans in big brown bats. We also will compare the use of rapid detection of the fungus using ultraviolet light with presence of histologic lesions and with results of PCR assays that detect the fungus. This work will require collaboration with the Michigan Departments of Natural Resources (MDNR) and Community Health (MDCH). The bats will be submitted by the public to the MDCH for rabies evaluation. All bats negative for rabies (big brown bats and other species of bats) are transported to the MDNR Disease Research Laboratory for examination for white nose syndrome using observation of lesions and ultraviolet light scanning. Samples from all bats, regardless of lesions or fluorescent signal, will be submitted for PCR assay.Goal 3. Initiate and complete studies for prevalence of hemoplasmas in tissue specimens submitted for routine diagnostic testing (tissues not submitted as suspect cases of hemoplasma infection) for cattle, deer, sheep, and bison. Hemoplasma infections of canine and feline are well documented and subject to routine testing. There is a lack of information on the hemoplasmas of livestock and free ranging animals, especially in North America. The prevalence of those organisms is unknown. The hemoplasmas cannot be cultured in vitro, and would escape detection by traditional laboratory testing methods for infectious agents. Convenience samples of tissue from the aforementioned species will be tested for presence of hemoplasmas using PCR assays targeting their genetic material. A sub-objective will be to determine the species hemoplasma detected by PCR through nucleic acid sequencing. This part of the objective will include use of diagnostic specimens submitted as suspicious of hemoplasma infection. Successful completion of this project will provide useful information on prevalence on hemoplasmas in large animals and their association with disease.

Project Methods
Objective A. Develop a laboratory model for assessing survival of Mycobacterium in ensiled forage. Mycobacterium bovis strain MDCH # 358258 will be used in this study. Strain MDCH # 358258 was originally isolated from a white-tailed deer in Northeast MI and is identical to the strain found in cattle in that region. Bacteria will be propagated to mid-log growth phase. The bacteria will be diluted to a final concentration of at least 1.0 x 106 CFU/ml. Exactly 500 grams of forage (a mixed grass) will be placed into multiple 8" x 12" nylon/polyethylene 4-mil high-performance film vacuum pouch (Bunzl Koch Supplies, St. Louis, MO). The forage will be inoculated with 5ml of M. bovis in culture medium and mixed thoroughly to ensure equal distribution of the bacteria. Air will be evacuated and the bags sealed using a chamber vacuum packager (MVS-31X Vacuum Packager, Minipack America, Orange, CA). Sealed bags will be held at 20°C ± 2°C. That temperature is equivalent to the average temperature for the region from June 1- September 1. At designated intervals, replicate bags will be opened and placed into sterile blender jars with an appropriate volume of enrichment broth and pH indicator. Samples will be blended for 30 to 60 seconds, decontaminated using a NaOH/HCl digest method, and M. bovis will be concentrated using centrifugation. Bacterial pellets will be suspended in 2 ml of residual digest liquid and inoculated on both solid and liquid media. Media will be monitored weekly for growth. A standard curve will be used with a real-time qPCR assay targeting the bacterial insertion sequence 6110 to quantify mycobacteria in a residual sample volume. To identify dormant bacteria, the M. bovis used for inoculation of the forage will be transformed with the pMV306::hsp60-fluc firefly luciferase reporter plasmid that will stably incorporate into the M. bovis genome. Samples of processed silage (see above) will be tested for reporter function as determined using the Bright-Glo luciferase reagent (Promega) and measuring luminescence using a Perkin Elmer Espire ultra-sensitive luminometer. As a positive and negative control for live/dead analysis, we will use heat killed and actively growing M. bovis cells. At each testing interval, a sample of non-inoculated control silage will be sent for fermentation profile. This analysis will done to verify the fermentation process was completed and will include measurements of dry matter, lactic acid, acetic acid, lactic/acetic acid ratio, propionic acid, butyric acid, iso-butyric acid, total acids, crude protein and pH.Objective B. Conduct a multicounty surveillance for Pseudogymnoascus destructans (P. destructans, the fungus that causes white nose syndrome) in big brown bats in Michigan. Over 1,000 bats that have been tested free of rabies and were collected over a one year period in counties within the Lower Peninsula of Michigan will be examined for presence of Pseudogymnoascus destructans. That is approximately the number of bats tested for rabies each year and it is anticipated that most of those will be big brown bats. However, there should be representatives of some of the other 8 species bats found in Michigan. The muzzle, wing, and tail membranes of each bat will be examined for orange-yellow fluorescence using an ultraviolet (UV) light. Suspect areas of fluorescence will be excised and submitted for histopathology and PCR assay for detection of characteristic lesions and fungal DNA. Similar samples of tissue will be submitted for PCR assay from bats not showing fluorescence. Hence, approximately 1,000 PCR assays will be performed on presumptive positive and presumptive negative bats to allow an assessment of sensitivity and specificity of the PCR assay. To prevent cross-contamination of samples from bats, each bat will be handled with latex gloves and examined over a paper plate, suspect tissue will be excised with a disposable razor blade, and a toothpick will be used place the tissue samples into labeled 1.5 mL micro-centrifuge tubes. The latex gloves, paper plate, disposable razor blade and toothpick will be discarded after each bat is processed. Extraction of DNA from fungus can be problematic; however, we have found the sodium dodecyl sulfate digestion method is effective with tissues from bats. Briefly, a 5mm x 5 mm piece of wing or facial skin in a 1.5 micro-centrifuge tube will be incubated in 60 uL of a digestion buffer and proteinase K for 2 hours at 56 C. The digestion buffer consists of 50mM trisaminomethane, pH 8.5, 1mM ethylenediaminetetraacetic acid, and 0.5% sodium dodecyl sulfate. This will be followed by incubation at 100 C for 10 minutes. Finally, 20 uL of polysorbate 20 will be added to each tube to neutralize the PCR inhibitory effect of sodium dodecyl sulfate. The crude DNA from this extraction process will be used in PCR assays following methods described by Lorch et al. (ref 34, Literature Review) to amplify a 625 base pair segment of DNA incorporating the 5.8S rDNA and the flanking internal transcribed spacers from P. destructans. All PCR amplicons will be subjected to nucleic acid sequencing and in silico analysis to verify source of the DNA. The results of the study will be submitted to the Michigan Department of Natural Resources Wildlife Disease Research Division for epidemiologic analysis, as this group maintains records of location found, species, and condition of bats submitted for rabies testing.Objective C. Initiate and complete studies for prevalence of hemoplasmas in tissue specimens submitted for routine diagnostic testing (tissues not submitted as suspect cases of hemoplasma infection) for cattle, deer, sheep, and bison. The Diagnostic Center for Population and Animal Health at Michigan State University performs over 700 field, gross, or complete post mortem examinations on farm animals each year. In addition, over 20,000 blood samples are submitted each year from "healthy" cattle for routine disease surveillance or analysis of vitamin concentration in blood. Diagnostic samples submitted to the Diagnostic Center from recent disease outbreaks in cattle from Michigan and Wisconsin, bison from Texas, and various species of cervids from Canada that have tested positive hemoplasmas have been stored frozen for further analysis. Thus, a large number of convenience samples, from animals with clinical signs of disease or animals that presumably are healthy, are available for testing for hemoplasmas in blood or splenic tissue. Testing for hemoplasmas will be done using PCR primers targeting the 16S rRNA gene, the RNase P RNA Gene, or the intergenic spacer region between the 16S and 23S rRNA genes. Those PCR assays are done routinely at the Diagnostic Center. The choice of those gene targets allows comparison of nucleic acid sequence from amplicons of positive PCR assays with similar sequences deposited in public data bases. The derivation of nucleic acid sequences will be done by the Research Technology Support Facility at Michigan State University, the derived sequences will be trimmed and edited using Sequencher software (Gene Codes Corporation, Ann Arbor, MI). The nucleotide data base at the National Center for Biotechnology Information will be used for comparison of nucleic acid sequences. Other freeware data bases will be used for phylogenetic analyses. As part of this objective, a collaborative project has been initiated with private veterinary practitioners in Wisconsin, and with academic veterinary clinicians, clinical pathologists, and epidemiologists at the University of Wisconsin, to perform an extended study of hemoplasmas in one or two well managed dairy herds known to be positive for hemoplasmas. This will allow access to production records from before and after treatment is initiated or management practices have been implemented to control hemoplasma.

Progress 11/01/16 to 10/31/21

Outputs
Target Audience:Wildlife biologists and environmentalists, regulatory agencies for agriculture, regulatory agencies for fisheries and wildlife, cattle producers, and veterinarians that support the cattle industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The 5 year project involved 3 veterinary students, 2 of which were credited with publications and have continued on for post veterinary graduate education in pathology or epidemiology. How have the results been disseminated to communities of interest?The reuslts of the goals have been puplished in peer reviewed journals, oral presentations of the results have been given at national meetings, and to interested regional commodity meetings. Goal 3 has been expanded with additional funding and will involve a graduate student. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Goal 1 was completed in 2019. A field strain of Mycobacterium bovis that was isolate from a deer in Michigan was used to inoculate samples of corn, grass and mixed grass silage. Survival of the Mycobacterium was assessed using bacterial culture at regular intervals after inoculation of the forages and before insiling, after the ensiling process was initiated, and during storage periods of the silage up to 4 months after inoculation. Isolation of the Mycobacterium was not possible a few days after the ensiling process started; however presence of Mycobacterial DNA was possible until the end of the test period. The above experiment was repeated using fluorescently labeled Mycobacterium bovis that would show fluorescence only if the bacterium was alive. This approach failed, as the sensitivity of detection of the fluoescen label in silage was insufficient. One publication came from this project. Goal 2 was completed. This project involved a summer student from Michigan State University and close collaboration with the Michigan Department on Natural Resources (MDNR). Conveience samples from over 1,000 bats submitted to the Michigan Department of Health and Human Serviices for rabies testing. Bats negative for rabies were tested for Pseudogymnoascus destructans, the causal fungus of white nose syndrome. Most of the bats tested were big brown bats with a few little brown bats. The big brown bats tested negative for the fungus,as most bats in Michigan are big brown bats, this was a positive finding. Visual observation for fungal disease, examination off bat wings and nose for fungal fluoescence, histopatholgy of bat tissues, and PCR were used as diagnostic procedures. Fungal disease was detected in little brown bats by all diagnostic procedures. One publication came from this project. The collaboration with the MDNR continued with a separated project on Eastern Equine Encephalities virus in ruffed grouse that lead to a publication in 2021. Goal 3. This project has received additonal funding from the Michigan Alliance for Animal Agriculture and will continue with different objectives. The original goal has been completed. The project involved 2 veterinary students, one each from Michigan State University and from the University of Wisconsin. The project included 80 dairy farms in Wisconsin or Michigan and 30 cattle from each farm. Hemoplasmas were detected in samples from all farms with Mycoplasma hemobos slightly more prevalent than Mycoplasma wenyonii. The testing include serologic detection of antibody against bovine leukemia virus and those results indicated the in-herd prevalence of that virus was about half that of the hemoplamas. One of the students presented the findings a national meeting and was the primary author on a peer reviewed publication in 2020.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Eastern Equine Encephalitis Virus in Visibly Affected Ruffed Grouse (Bonasa umbellus) in Michigan, Minnesota, and Wisconsin, USA. Anderson M, Melotti J, Dinh E, Fitzgerald SD, Cooley TM, Bolin S, Elsmo EJ, Businga N, W�nschmann A, Carstensen M.J Wildl Dis. 2021 Apr 1;57(2):453-456. doi: 10.7589/JWD-D-20-00113.PMID: 33822164 Detection of pathogens in blood or feces of adult horses with enteric disease and association with outcome of colitis. Kopper JJ, Willette JA, Kogan CJ, Seguin A, Bolin SR, Schott HC 2nd.J Vet Intern Med. 2021 Sep;35(5):2465-2472. doi: 10.1111/jvim.16238. Epub 2021 Aug 12.PMID: 34382708

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Wildlife biologists, regulatory agenicies for agriculture or for fisheries and wildlife, dairy producers and dairy veterinarians Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Goal 2 provided for training of a veterinary student in diagnostic methods, study design, epidemiological methods, and scientific writing. Goal 3 has involved two veterinary students and has provided on farm training in sample collection, acquisition of herd history, and recognition of dairy farm management practices. Goal 4 will provide training opportunity for a postdoctoral fellow and a graduate student. How have the results been disseminated to communities of interest?Results from Goal 2 have been published in a wildlife disease journal. Those results also have been communicated at scientific meetings. Early results from Goal 1 were published in 2019 and yearly reports have been presented to Michigan cattle producers. Some findings from Goal 3 were presented at the annual meeting of American Association of Bovine Practitioners in 2019. A manuscript has been prepared and I ready for submission to a peer reviewed journal. Goal 4 has been delayed by an unexpected equipment failure and by the COVID 19 pandemic. However, work on this goal has restarted recently and initial pilot studies have been successful. Progress reports have been submitted to the funding source. What do you plan to do during the next reporting period to accomplish the goals?Funding for Goal 1 has run out so work on this goal has stopped. Goal 2 is completed. Field work for Goal 3 is done and a manuscript will be submitted in the next reporting period. Goal 4 has funding for another year and will be the focus of work for the next reporting period.

Impacts
What was accomplished under these goals? Goal 1. A genetically modified M. bovis was created that has a fluorescent reporter, which is only expressed in a live bacterium. The sensitivity of detection for this reporter was approximately 10,000 viable, but dormant organisms. Culture conditions for isolation of viable M. bovis were modified to increase the sensitivity for bacterial isolation and extend the time M. bovis could be isolated from ensiled feed. A new approach for exposure of M. bovis to temperature, pH, and chemical changes that occur during the ensiling process was tested and found to facilitate recovery of M. bovis from ensiled feeds. A pilot study was done using the M. bovis with fluorescent reporter, and the improved recovery and culture methods. The results of the pilot study were encouraging and full-scale experiment was initiated. The findings from the study included improved methods for recovery of cultivable M. bovis were able to extend the time for detection of live M. bovis embedded in ensiled forages; however, the fluorescent reporter inserted into M. bovis did not enhance detection of live organism. Study design may have influenced the latter finding. Goal 2. This goal has been completed and a manuscript has been published detailing the methods used and the results from testing over 1,000 bats. Further activity on this goal is not anticipated, as funding sources for additional work have not been secured. Goal 3. Extramural funding was secured and a project on prevalence of hemoplasma in dairy cattle was initiated in June of 2018. The project involves a 2-state surveillance of approximately 100 herds containing numbers of cattle ranging from hundreds to thousands. The objective is to determine the within herd and regional herd prevalence of hemoplasmas in dairy cattle. The project includes a producer survey of management practices that may revel risk factors for infection of dairy cattle with hemoplasmas. To date, over 80 herds and 2,000 cattle have been tested for infection with hemoplasmas. A major finding of the project is bovine hemoplasmas are widespread in dairy cattle in both Wisconsin and Michigan. Epidemiological analyses have been done and a manuscript has been prepared. Submission of the manuscript to a peer reviewed journal is expected in October of 2020. Goal 4. Generate an optimized system to detect misfolded proteins directly from lymph nodes, intestinal lymphoid tissues (rectal anal junction or ileum Peyer's patches), white blood cells, or the brain of deer affected by chronic wasting disease. This is a new goal the was initiated because extramural funding has been obtained. To accomplish this goal, necessary equipment was purchased and received. Training in use of the equipment is scheduled and seed prion is being produced. The project was scheduled to begin in November 2019. The equipment purchased to perform the RT-QuIC procedure for detection of chronic wasting disease in deer was not adequate and had to be replaced. Adequate equipment is now in place and initial pilot studies indicate the RT-QuIC procedure is working as expected. The COVID pandemic has prevented travel to the Rocky Mountain Laboratories in Hamilton Montana for training in production of seed prion for use in the RT-QuIC procedure. Communications with the colleagues at that facility are ongoing and alternative plans for training are being developed.

Publications

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Targeted audiences included wildlife professionals, dairy and beef producers, veterinarians, animal disease regulatory officials, laboratory diagnosticians, and public health professionals. Changes/Problems:A additional Goal 4 is requested to be added to the project. The plane for this goal is to generate an optimized system to detect misfolded proteins directly from lymph nodes, intestinal lymphoid tissues (rectal anal junction or ileum Peyer's patches), white blood cells, or the brain of deer affected by chronic wasting disease. We will use both the USDA approved ELISA and immunohistochemical methods that are performed routinely at the Michigan State University Veterinary Diagnostic Laboratory on thousands of samples yearly as gold standards for positive and negative results. The availability of that large resource of defined samples will facilitate investigation of the RT-QuIC technology with modifications designed to enhance detection of chronic wasting disease misfolded proteins. Potential diagnostic tissues include lymph nodes, intestinal lymphoid tissues (rectal anal junction or ileum Peyer's patches), white blood cells, or the brain of deer. What opportunities for training and professional development has the project provided?A veterinary student that worked on goal 3 presented findings from the project at a national meeting. Goal 4 is new and will provide opportunity for graduate student participation. How have the results been disseminated to communities of interest?Results from Goal 2 have been published in a wildlife disease journal. Those results also have been communicated at scientific meetings. Early results from Goal 1 were published in 2019 and yearly reports have been presented to Michigan cattle producers. Some findings from Goal 3 will be presented at the annual meeting of American Association of Bovine Practitioners in 2019. Goal 4 is new and progress on the goal should occur during the next reporting period. What do you plan to do during the next reporting period to accomplish the goals?Funding for Goal 1 has run out so work on this goal has stopped. Goal 2 is completed. Field work for Goal 3 is done. Epidemiological analyses for Goal 3 remain to be done. Goal 4 has funding for 2 years and will be the focus of work for the next reporting period.

Impacts
What was accomplished under these goals? Goal 1. A genetically modified M. bovis was created that has a fluorescent reporter, which is only expressed in a live bacterium. The sensitivity of detection for this reporter was approximately 10,000 viable, but dormant organisms. Culture conditions for isolation of viable M. bovis were modified to increase the sensitivity for bacterial isolation and extend the time M. bovis could be isolated from ensiled feed. A new approach for exposure of M. bovis to temperature, pH, and chemical changes that occur during the ensiling process was tested and found to facilitate recovery of M. bovis from ensiled feeds. A pilot study was done using the M. bovis with fluorescent reporter, and the improved recovery and culture methods. The results of the pilot study were encouraging and full-scale experiment was initiated. The findings from the study included improved methods for recovery of cultivable M. bovis were able to extend the time for detection of live M. bovis embedded in ensiled forages; however, the fluorescent reporter inserted into M. bovis did not enhance detection of live organism. Study design may have influenced the latter finding. Goal 2. This goal has been completed and a manuscript has been published detailing the methods used and the results from testing over 1,000 bats. Further activity on this goal is not anticipated, as funding sources for additional work have not been secured. Goal 3. Extramural funding was secured and a project on prevalence of hemoplasma in dairy cattle was initiated in June of 2018. The project involves a 2-state surveillance of approximately 100 herds containing numbers of cattle ranging from hundreds to thousands. The objective is to determine the within herd and regional herd prevalence of hemoplasmas in dairy cattle. The project includes a producer survey of management practices that may revel risk factors for infection of dairy cattle with hemoplasmas. To date, over 80 herds and 2,000 cattle have been tested for infection with hemoplasmas. A major finding of the project is bovine hemoplasmas are widespread in dairy cattle in both Wisconsin and Michigan. Epidemiological analyses are ongoing.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: M. Mazaki-Tovi, S. R. Bolin & P. A. Schenck. Adipokines secretion in feline primary adipose tissue culture in response to dietary fatty acids. BMC Veterinary Research 15, Article 324, 2019. Daniel L. Grooms DVM, PhD; Steven R. Bolin DVM, PhD; Jessica L. Plastow BS; Ailam Lim PhD; Joseph Hattey BS; Phillip T. Durst MS; Steven R. Rust PhD; Michael S. Allen PhD; Daniel D. Buskirk PhD; Richard W. Smith DVM. Survival of Mycobacterium bovis during forage ensiling. American Journal of Veterinary Research 80:87-94, 2019.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Targeted audiences included wildlife professionals, dairy and beef producers, veterinarians, animal disease regulatory officials, and public health professionals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Goal 2 provided for training of a veterinary student in diagnostic methods, study design, epidemiological methods, and scientific writing. Goal 3 has involved two veterinary students and has provided on farm training in sample collection, acquisition of herd history, and recognition of dairy farm management practices. How have the results been disseminated to communities of interest?Results from Goal 2 have been published in a wildlife disease journal. Those results also have been communicated at scientific meetings. Activity in Goals 1 and 3 began recently and formal reports have not been made; however, the motivation behind Goal 1 and some of the project details have been communicated to local stakeholder groups. What do you plan to do during the next reporting period to accomplish the goals?Goal 1 will receive additional effort during the next reporting period to complete laboratory testing. If extramural funding permits, another experiment with M. bovis in ensiled feed will be done to provide additional data needed for publication of results. On-farm collection of samples for Goal 3 has started and this will continue into 2019. The number of samples to be tested is large, which will necessitate banking samples for testing during the next year and delay data analysis until mid-2019.

Impacts
What was accomplished under these goals? Goal 1. A genetically modified M. bovis was created that has a fluorescent reporter, which is only expressed in a live bacterium. The sensitivity of detection for this reporter was approximately 10,000 viable, but dormant organisms. Culture conditions for isolation of viable M. bovis were modified to increase the sensitivity for bacterial isolation and extend the time M. bovis could be isolated from ensiled feed. A new approach for exposure of M. bovis to temperature, pH, and chemical changes that occur during the ensiling process was tested and found to facilitate recovery of M. bois from ensiled feeds. A pilot study was done using the M. bovis with fluorescent reporter, and the improved recovery and culture methods. The results of the pilot study were encouraging and full-scale experiment was initiated. Goal 2. This goal has been completed and a manuscript has been published detailing the methods used and the results from testing over 1,000 bats. Further activity on this goal is not anticipated, as funding sources for additional work have not been secured. Goal 3. Extramural funding was secured and a project on prevalence of hemoplasma in dairy cattle was initiated in June of 2018. The project involves a two state surveillance of approximately 100 herds containing numbers of cattle ranging from hundreds to thousands. The objective is to determine the within herd and regional herd prevalence of hemoplasmas in dairy cattle. The project includes a producer survey of management practices that may revel risk factors for infection of dairy cattle with hemoplasmas. To date, over 80 herds and 2,000 cattle have been tested for infection with hemoplasmas. The project will continue into 2019.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Pablo Estima-Silva P, Marcolongo-Pereira C, , Lemos dos Santos B, Coelho ACB, Amaral LA, Lim A, Bolin SR, Schild AL. Equine multinodular pulmonary fibrosis in southern Brazil: pathology and differential diagnosis. Pesq. Vet. Bras. 37(11):1247-1252, 2017 DOI: 10.1590/S0100-736X2017001100009 Zarski LM, High EA, Rahul K.Nelli RK, Bolin SR, Williams KJ, Hussey G. Development and application of a quantitative PCR assay to study equine herpesvirus 5 invasion and replication in equine tissues in vitro and in vivo. Journal of Virological Methods, 248: 44-53, 2017.

Progress 11/01/16 to 09/30/17

Outputs
Target Audience:Targeted audiences included wildlife professionals, dairy and beef producers, veterinarians, animal disease regulatory officials, and public health professionals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Goal 2 was used for training of a veterinary student in diagnostic methods, study design, epiemiological methods, and scientific writing. Goal 3 will involve a vetrinary studend and will provide on farm training in sample collection, acquisition of herd history, and recognition of dairy farm management practices. How have the results been disseminated to communities of interest?Results from Goal 2 have been published in a wildlife journal. Those results also have been communicated at scientific imeetings. Acitivity in Goals 1 and 2 were started recently and no reports have been made. What do you plan to do during the next reporting period to accomplish the goals?Goal 1 will recieve the most effort during the next reporting period. The laboratory portions of the study shoull be completed during the next year. Data analysis for that study will begin, but it is not likely that the analysis will be completed during the next reportin period. On farm sample collection for Goal 3 will be started in the next reporting period. The laboratory portions of the study will follow sample collection.

Impacts
What was accomplished under these goals? Goals 1 and 2 were designed to impact regulatory programs for the control of bovine tuberculosis and white nose syndrome in bats. Specifically, Goal 1 uses state of the art procedures to assess possible survival of Mycobacterium bovis in fermented foods. The immediate focus in on fermented forages for cattle, but the methods developed could be used on fermented foods for humans. The study desinged for Goal 2 will impact wildlife programs that monitor bat populations for white nose because the focus is on common species of bats do not use caves or abanded mines as an hibernacula. Multiple procedures for detection of white nose syndrome were compared that ranged from inexpensive and simple to perform to procedures that would be done in diagnostic facility. Goal 3 will not be initiated until 2018, but its impact will be on diary producers and veterinarians that service the dairy industry. Specifically, this goal is designed to provide informaton on the distribution of blood borne mycoplasma in diary cattle, the effects of that organism on dairy production, and risk factors for dissemniation of the organism among dairy cattle and diary farms.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Lila M.Zarski, Emily A.High, Rahul K.Nelli, Steven R.Bolin, Kurt J.Williams, GiselaHussey. Development and application of a quantitative PCR assay to study equine herpesvirus 5 invasion and replication in equine tissues in vitro and in vivo. Journal of Virological Methods, Volume 248, October 2017, Pages 44-53. Samantha L. Darling, Ailam Lim, Julie R. Melotti, Daniel J. O'Brien, Steven R. Bolin. Prevalence and Distribution of Pseudogymnoascus Destructans in Michigan Bats Submitted for Rabies Surveillance. Journal of Wildlife Diseases 53(3):482-490. 2017 Colleen S. Bruning-Fann, Suelee Robbe-Austerman, John B. Kaneene, Bruce V. Thomsen, John D. Tilden Jr, Jean S. Ray, Richard W. Smith, Scott D. Fitzgerald, Steven R. Bolin, Daniel J. O'Brien, Thomas P. Mullaney, Tod P. Stuber, James J. Averill, David Marks. Use of whole-genome sequencing and evaluation of the apparent sensitivity and specificity of antemortem tuberculosis tests in the investigation of an unusual outbreak of Mycobacterium bovis infection in a Michigan dairy herd. Journal 0f the American Veterinary Medical Association 251(2):206-216, 2017