Source: MONTANA STATE UNIVERSITY submitted to NRP
ESTABLISHMENT OF A SPECIFIC PATHOGEN-FREE RESEARCH FLOCK OF DOMESTIC SHEEP FOR MYCOPLASMA OVIPNEUMONIAE PATHOGENESIS, INFECTION AND PREVENTION STUDIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1019305
Grant No.
2019-67016-29907
Cumulative Award Amt.
$194,000.00
Proposal No.
2018-06895
Multistate No.
(N/A)
Project Start Date
May 1, 2020
Project End Date
Apr 30, 2022
Grant Year
2020
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Microbiology and Immunology
Non Technical Summary
This is a FASE-EPSCoR seed award focused onMycoplasma ovipneumoniae (M. ovi) infection in sheep.M. ovi is a bacterium that infects the upper airways of domestic sheep, causing mild respiratory disease and production losses due to reduced weight gains, but that causes severe and often fatal lung infections when transmitted to wild Bighorn sheep populations.There is a considerable interest from sheep farmers, wildlife conservationists and the general public to better understand the infectionrisks that domestic sheep may pose to Bighorn sheep health in the Northwestern United States, including Montana. We seek to establish a research program at Montana State University in order to develop new preventive, treatment or management measures to eliminate subclinical infection of domestic sheep with M. ovi. and other upper respiratory tract pathogens. We hypothesize that preventing M. ovi. infection in domestic sheep will both prevent production losses associated with M. ovi. infection in domestic sheep and greatly reduce the risk for Bighorn sheep infection with this organism. The goal of this project is toestablish a specific pathogen-free (SPF) research herd of domestic sheep that harbors neither M. ovi. nor other bacteria that are considered to be co-factors for disease development. Specifically, we will use a combination of supervised lambing and colostrum-free motherless rearing followed by isolation of the flock from other ruminants in order to derive pathogen-free domestic sheep that are immunologically naïve to M. ovi. The SPF herd will be screened regularly for M. ovi. and other respiratory pathogens to confirm disease-free status, and ewes will be re-bred to produce animals for experimentation. This herd will be a key tool for future studies in M. ovi. infection, immunity, transmission and disease pathogenesis, all of which remain underinvestigated. The proposed work addresses USDA priorities relevant to program area priority code A1221, Animal Health and Disease.
Animal Health Component
0%
Research Effort Categories
Basic
20%
Applied
0%
Developmental
80%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31136101100100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3610 - Sheep, live animal;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
In order to understand the pathogenesis of bacterial pneumonia in domestic sheep and bighorn sheep and to develop and test novel preventive and therapeutic approaches, we seek to develop a specific pathogen-free (SPF) research herd of domestic sheep for experimental infections. We will use supervised lambing to eliminate any postnatal contact between the lambs and the ewes, motherless, colostrum-free rearing and segregation of the resulting flock from other sheep and ruminants. This approach will prevent colonization of the lambs by opportunistic bacterial pathogens and viruses that may be present in the ewes' respiratory tract or transmitted through the colostrum or through contact with skin, feces and respiratory fluids. The SPF research flock generated with this method will be regularly monitored for bacterial and viral colonization and will then be used for infection and challenge experiments in future studies on sheep pneumonia. By re-breeding the offspring from year 1, we plan to have a herd of approximately 30 SPF animals available after 1.5 years, so that 20 animals will be available for experimental work while the remaining sheep will be bred for herd maintenance.The proposed project will involve the following activities:Acquisition of pregnant ewes for synchronized lambing.Supervised lambing and separation of lambs from ewes.Motherless, colostrum-free rearing of lambs in our ABSL-2 facilityTesting of nasal swabs, bronchoalveolar lavage, tissues, sera for pathogens including M. ovi.Re-breeding of SPF ewe lambs with SPF ram lambs to increase flock size.
Project Methods
We here will derive a SPF sheep herd by taking lambs away from their mothers at birth, depriving them of sheep colostrum and hand-rearing them away from other sheep and ruminants.Selection of Pathogens to Eliminate: In addition to eliminating M. ovi. from the flock, we also seek to eliminate Mannheimia haemolytica, Bibersteinia trehalosi and Parainfluenza 3 virus (PI3), which are considered potential co-factors in M. ovi. pathogenesis. All organisms have been detected in the lungs of pneumonic bighorn sheep and have been shown to induce pneumonia upon experimental infection. M. haemolytica, B. trehalosi and PI3 also are associated with pulmonary lesions in domestic sheep. We will perform diagnostic testing for these organisms to confirm SPF status. Notably, our supervised lambing/motherless rearing and segregation regime will likely lead to the exclusion of additional pathogens and commensals from the SPF flock.Ewe Breeding and Housing: Fifteen pregnant mixed-breed ewes (Rambouillet, Suffolk, Targhee, Columbia, and/or Hampshire) aged 4 - 5 year old will be purchased several weeks before the projected lambing date from a local sheep farmer. Pregnancy will be confirmed using abdominal ultrasound. Presence or absence of M. ovi. is not an inclusion/ exclusion criterion for our study. However, all ewes will be tested for M. ovi., M. haemolytica, B. trehalosi and PI 3 at the beginning of the experiment using nasal swab and serum samples. Prior to lambing, ewes will be fed hay from the Johnson Family Livestock Facility farm, grain, and an appropriate vitamin/mineral supplement.Supervised Lambing: Pregnant ewes will be kept in a paddock with a covered shelter prior to lambing. Lambing dates and litter sizes will be estimated by transabdominal ultrasound screening. During the anticipated lambing period, ewes will be monitored around the clock for signs of imminent delivery. Ewes developing signs of labor will be separated from the flock and moved into a designated lambing pen for assisted lambing. Lambs will be manually delivered onto a clean plastic sheet and will then be transferred into a separate, heated rearing area within the Johnson Family Livestock Facility ABSL-2 laboratory, where they will undergo cleaning, umbilical disinfection and a general health check by a designated team of animal care staff.Motherless Rearing: Lambs will be housed in a heated animal room inside the Johnson Family Livestock Facility in groups of 5 - 10 animals, with siblings and lambs of similar ages housed together. During the first 24 h, we will bottle-feed the lambs with 200 mL/kg bodyweight of bovine colostrum. The colostrum will be obtained from a local dairy farm, tested for the absence of M. ovi, M. haemolytica and other Pasteurellacea, stored at -20?C and thawed as needed. We will then train lambs to use bucket feeders and raise them on a commercial lamb milk replacer dietfor 5 weeks. Meal frequency will be decreased over time from 6 meals/day on days 1 - 5 to 4 meals/day on days 6 - 10 and 3 meals/day thereafter, as previously described. Water, high quality hay and lamb pellets will be offered ad libitum. Animal care personnel responsible for the lambs will not have any contact with other non-SPF sheep. Lamb health will be scored daily by animal care personnel, as described previously, and animals that develop signs of disease will be isolated and treated by our contracted large animal veterinarian as needed. Lamb body weights will be recorded once per week.Weaning: We will wean lambs at the age of 5 weeks, when animals have reached a body weight of 20 - 25 kg. Following weaning, we will keep the lambs inside the ABSL-2 laboratory for another 10 - 14 days to minimize stress. When lambs have adapted to a solid diet at around 7 - 8 weeks of age, we will move all animals to an outdoor paddock with secure double fencing that has never been used for sheep housing. All other non-SPF sheep will be removed from the outdoor paddock areas prior to moving the SPF lambs to the outdoor paddock. Herd Maintenance and Re-breeding: At 4 - 5 months of age, ram lambs will be evaluated, and 2 - 3 rams with excellent body condition and weight gains will be selected for maintenance breeding. Of the remaining males, 4 - 5 animals will be euthanized, and tissues (lung, trachea, nasal tissue, mediastinal lymph nodes, thymus, spleen, liver) will be collected for pathogen detection. If additional rams are available, they will be used for preliminary infection experiments. All healthy ewe lambs will be kept at the facility and bred with the SPF rams at 8 - 10 months of age to produce additional sheep for our research flock. We will collect nasal swab samples from all animals every 3 months and BALF every 6 months to test for Mycoplasma ovipneumoniae, Mannheimia haemolytica, Bibersteinia trehalosiand parainfluenza 3 virus.Sheep that develop signs of disease will be separated from the other animals and treated as needed. Should any mortalities occur, cause of death will be determined by necropsy at the Montana Veterinary Diagnostic Laboratory.Diagnostic Testing for Respiratory Pathogens: Regular monitoring of the sheep herd for maintenance of the SPF status is a major component of our pilot project. Samples will be analyzed for Mycoplasma ovipneumoniae, Mannheimia haemolytica, Bibersteinia trehalosiand parainfluenza 3 virus using PCR, ELISA or bacterial culture, as appropriate. We will collect nasal swabs, bronchoalveolar lavage fluid (BALF) and serum samples from all sheep in the study at regular intervals. BALF will be obtained under local anesthesia from animals aged 4 months and older, using a flexible fiber-optic endoscope. Blood samples for serology will be obtained by venipuncture of the jugular vein. In addition, 4 - 5 ram lambs from year 1 will be euthanized for collection of the following tissues: Nasal tissue, tracheal mucosa, lung parenchyma, mediastinal lymph nodes, thymus, spleen and liver. Placental samples collected from the non-SPF ewes will also be tested for M. ovi. We will also screen all feed supplies and water sources for possible contamination with any of the pathogens.Samples will be analyzed at the Washington Animal Disease Diagnostic Laboratory for M. ovi. infection by PCR and bacterial culture, and for the presence of M. ovi.-specific serum antibodies by ELISA. Serological analysis for PI3 infection will be performed at the Montana Veterinary Diagnostic Laboratory. The presence of M. haemolytica will be determined in our laboratory by PCR specific for M. haemolytica O-sialoglycoprotein endopeptidase, as previously described. Likewise, we will use quantitative PCR to identify B. trehalosi based on the expression of B. trehalosi manganese-dependent superoxide dismutase, as described by Dassanayake, et al. (2013). Samples that test positive for either M. haemolytica or B. trehalosi will be analyzed by PCR for the expression of the leukotoxin gene, which is associated with respiratory pathology.

Progress 05/01/20 to 04/30/22

Outputs
Target Audience:The target audiences for our study are scientists interested in sheep infectious disease and immunology, veterinarians, sheep farmers and individuals involved in wildlife conservation. To share our data with scientists and veterinarians, we have presented our work at the 101st and the 102nd Conference of Research Workers in Animal Diseases (CRWAD) and have published three manuscripts in peer reviewed journals, two of them with Dr. Bimczok assenior author. The PI also presented her project as part of Montana State University's MAES seminar series to a Montana State University audience. Two graduate student also have given oral presentations about our work at an internal research seminar series. To share our data with other stakeholders in the sheep industry, we have participated in a local workshop series on M. ovipneumoniae mitigation strategies for domestic and bighorn sheep that included microbiology and animal science faculty from MSU, MSU's Extension service, representatives from the MT Department of Fish, Wildlife and Parks and a representative from the Wild sheep Foundation. This workshop series has led to the development of a collaborative research proposal submitted to the Fish Wildlife and Parks Service. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training in large animal husbandry and experimentation and in immunology and infectious disease to one Master's student in the Bimczok lab, Ms. Thea Johnson, who graduated with an M.S. degree Microbiology and Immunology in May 2021. The project has also provided training in infectious disease and microbiology to a Ph.D. student, Mr. B. Tegner Jacobson. In addition, our team provided key resources and expertise to a Master's student in Dr. A. Rynda-Apple's lab, Ms. Ema Robinson, who graduated in May 2022. All graduate students have had the opportunity to present their data at national research conferences (CRWAD). In addition, six MSU undergraduate students contributed to various aspects of our project on either the animal husbandry side of the project or on the laboratory side, thereby gaining valuable experience in ewe and neonatal lamb care or in research methods. How have the results been disseminated to communities of interest?To share our data with scientists and veterinarians, we have presented our work at the virtual 101st Conference of Research Workers in Animal Diseases (CRWAD) as a recorded poster presentation. Three posters associated with our work were presented at the 102nd CRWAD in 2021. We also have published three peer-reviewed manuscripts in Veterinary Microbiology, the Veterinary Record, and Viruses. To share our data with other stakeholders in the sheep industry, we have participated in a local workshop series on M. ovipneumoniae mitigation strategies for domestic and bighorn sheep that included microbiology and animal science faculty from MSU, MSU's Extension service, representatives from the MT Department of Fish, Wildlife and Parks and a representative from the Wild Sheep Foundation. Resources generated with our project have been shared with multiple collaborators, including Dr. Mark Jutila, Department of Microbiology and Cell Biology, Montana State University; Dr. Agnieszka Rynda-Apple, Department of Microbiology and Cell Biology, Montana State University; Working Dogs for Conservation (wd4c.org), Missoula, MT; and Dr. Martin Ganter, Veterinary University of Hannover, Germany. These collaborative efforts have further enhanced M. ovipneumoniae research efforts nationally and internationally. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? With the funding from this grant, we have successfully derived a research flock of specific pathogen-free domestic sheep that is free from key respiratory pathogens involved in M. ovipneumoniae pneumonia, including M. ovipneumoniae, Pasteurella multocida, Mannheimia haemolytica, Bibersteinia trehalosi and parainfluenza-3 virus (PI3). We used supervised lambing and artificial rearing with colostrum replacer and milk replacer to prevent transmission of bacteria from ewes to the newborn lambs. With this approach, a flock consisting of 12 males and 18 females was initiallyderived, with an above-average survival rate of 93.7%. In this newly generated SPF flock, we studied whether a commercial colostrum replacer that contained dried bovine serum as an antibody source would provide adequate immunological protection to newborn lambs. Immunoglobulin G (IgG) uptake was analyzed by ELISA, and the persistence of antigen-specific antibodies was analyzed by parainfluenza 3 virus (PI-3) neutralization assay. Serum antibody ELISA performed on days 1 and 14 revealed IgG levels of 17.9 ± 2.8 and 27.5 ± 2.5 mg/ml, respectively. PI-3 antibodies derived from the colostrum replacer were present for 86.3 ± 10.6 days. We concluded that antibodies derived from bovine serum protein delivered to lambs via a commercial colostrum replacer are readily absorbed and persist for months, suggesting that these products may offer adequate protection. These data have been published in the journal "Veterinary Record". All animals were initially housed in heated animal rooms inside our large animal BSL-2 facility. After weaning at approximately 6 weeks of age, eight lambs were selected for a pilot infection study and were kept inside the BSL-2 facility, whereas the other animals were moved to an outside paddock. In subsequent years, our SPF flock has been housed in outside paddocks. Animals are then moved into the BSL-2 facility for infection experiments as needed. Repeated testing of the whole SPF flock over the past two years year has confirmed that the sheep did not spontaneously acquire M. ovipneumoniae, PI-3 or Pasteurellaceae in the outdoor paddocks. Sentinel sheep that were euthanized did not display any macroscopic or microscopic abnormalities of the respiratory tract. All sheep also are regularly tested for the presence of intestinal parasites by analysis of fecal samples and treated as necessary. To maintain our flock and produce additional SPF lambs, we bred the 16 SPF ewe lambs that were born in 2020 with two of the ram lambs in December 2020 and January 2021. The ewes were estrus-synchronized, and two rams were introduced for a total of three cycles. Fourteen new lambs were born in May 2021 and were confirmed to be free ofM. ovipneumoniaeand Pasteurellaceae at 4-6 weeks of age. The SPF ewes were again bred in November and December 2021, and 22 lambs were born in April and early May 2022. A similar breeding strategy will be used in the coming years to maintain the SPF flock and to generate lambs for experimental infections. In order to understand whether M. ovipneumoniae alone induces respiratory disease in lambs with no previous exposure to or immunity against M. ovipneumoniae, we performed an M. ovipneumoniae challenge experiment in eight of the SPF lambs in August 2020, and blood, serum, bronchoalveolar lavage (BAL) and nasal swabs were collected. We inoculated four 4-month-old, specific-pathogen-free lambs intranasally, orally and conjunctivally with fresh nasal wash fluids from M. ovipneumoniae-infected sheep. The lambs were monitored for M. ovipneumoniae colonization, M. ovipneumoniae-specific antibodies, clinical signs, and cellular and molecular correlates of lung inflammation for eight weeks. In order to determine whether asymptomatic M. ovipneumoniae infection could be successfully cleared by antibiotic treatment, all lambs then were treated with gamithromycin and observed for an additional four weeks. M. ovipneumoniae inoculation resulted in stable colonization of the upper respiratory tract in all M. ovipneumoniae-inoculated, but in none of the four mock-infected control lambs. All M. ovipneumoniae-infected lambs developed a robust antibody response to M. ovipneumoniae within 2 weeks. However, we did not observe significant signs of respiratory disease, evidence of lung damage or inflammation in any of the infected lambs. Interestingly, treatment with gamithromycin, which blocked growth of the M. ovipneumoniae in vitro, failed to reduce M. ovipneumoniae colonization. These observations indicate that, in the absence of co-infections, M. ovipneumoniae caused asymptomatic colonization of the upper respiratory tract that was resistant to clearance by the host immune response and by gamithromycin treatment. These data have been published in "Veterinary Microbiology". The 14 lambs born in 2021 were used in a collaborative study with Dr. Rynda-Apple to investigate whether co-infection with Influenza D virus (IDV) would alter clinical progression of M. ovi infection in sheep. Lambs were inoculated intranasally with M. ovipneumoniae or mock infection, followed after four weeks by infection with IDV. Pathogen shedding was tracked, and immunological responses were evaluated by measuring acute phase response and IDV-neutralizing antibody titers. While lamb health statuses remained subclinical, M. ovipneumoniae-exposed lambs had significantly elevated body temperatures during IDV infection compared to M. ovipneumoniae-naïve, IDV-infected lambs. Moreover, we found a positive correlation between prior M. ovipneumoniae burden, early-infection IDV shedding, and IDV-neutralizing antibody response. Our findings suggest that IDV infection may not induce clinical symptoms in domestic sheep, but previous M. ovipneumoniae exposure may promote mild IDV-associated inflammation. These findings were recently published in "Viruses".

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Impact of Mycoplasma ovipneumoniae on the phagocytic ability of alveolar macrophages. Jacobson, Brian T.; Johnson, Thea; Adams, Noah; Jutila, Mark; Rynda-Apple, Agnieszka; Bimczok, Diane CRWAD, Chicago, IL, December 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Use of virus-like protein cages to reduce Mycoplasma ovipneumoniae infection in domestic sheep. Rynda-Apple, Agnieszka; Robinson, Emily; Bimczok, Diane; Jutila, Mark; Jacobson, Tegner; Luu, Abby; Johnson, Thea; Schulein, Clyde; Jones, Kerri. CRWAD, Chicago, IL, December 2021?
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Isolation of potential lytic bacteriophage for Mycoplasma ovipneumoniae (M. ovi). Jutila, Mark; Jackiw, Larissa; Jacobson, B. Tegner; Hedges, Jodi; Aspelin, Klara; Brumfield, Susan; Young, Mark; Bimczok, Diane; Wiedenheft, Blake. CRWAD, Chicago, IL, December 2021
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Experimental infection of specific-pathogen-free domestic lambs with Mycoplasma ovipneumoniae causes asymptomatic colonization of the upper airways that is resistant to antibiotic treatment. Johnson T, Jones K, Jacobson BT, Schearer J, Adams N, Thornton I, Mosdal C, Jones S, Jutila M, Rynda-Apple A, Besser T, Bimczok D. Vet Microbiol. 2022 Feb;265:109334. doi: 10.1016/j.vetmic.2022.109334. Epub 2022 Jan 10. PMID: 35033769
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Transfer and persistence of bovine immunoglobulins in lambs fed a colostrum replacer. Johnson T, Jacobson BT, Jones K, Mosdal C, Jones S, Vitkovic M, Kruppenbacher S, Sebrell A, Bimczok D. Vet Rec. 2022 Jul 16:e1974. doi: 10.1002/vetr.1974. Online ahead of print. PMID: 35841612
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Pathophysiology of Influenza D Virus Infection in Specific-Pathogen-Free Lambs with or without Prior Mycoplasma ovipneumoniae Exposure. Ema Robinson, Clyde Schulein, B. Tegner Jacobson, Kerri Jones, Jonathon Sago, Victor Huber, Mark Jutila, Diane Bimczok, Agnieszka Rynda-Apple. Viruses 2022, 14(7), 1422; https://doi.org/10.3390/v14071422
  • Type: Theses/Dissertations Status: Published Year Published: 2021 Citation: Experimental infection of specific pathogen-free domestic lambs with Mycoplasma ovipneumoniae. Johnson, Thea Haviland (Montana State University - Bozeman, College of Agriculture, 2021)
  • Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2022 Citation: Friends or Foes: The Role of Influenza D Viruses in Respiratory Coinfections. Robinson, Ema (Montana State University - Bozeman, College of Agriculture, 2022)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Experimental Infection of Specific Pathogen Free Domestic Lambs with Mycoplasma ovipneumoniae. Thea Johnson, Kerri Jones, Agnieszka Rynda-Apple, Thomas Besser, Mark Jutila, Diane Bimczok. CRWAD virtual, December 2020


Progress 05/01/20 to 04/30/21

Outputs
Target Audience:The target audiences for our study are scientists interested in sheep infectious disease and immunology, veterinarians, sheep farmers and individuals involved in wildlife conservation. To share our data with scientists and veterinarians, we have presented our work at the 101st Conference of Research Workers in Animal Diseases (CRWAD) and have written two manuscripts. One manuscript on our pilot M. ovipneumoniae infection study has been resubmitted to "Veterinary Microbiology" after revisions. A second manuscript on the use of bovine serum-based colostrum replacer will be submitted in the next few weeks. To share our data with other stakeholders in the sheep industry, we have participated in a local workshop on M. ovipneumoniae mitigation strategies for domestic and bighorn sheep that included microbiology and animal science faculty from MSU, MSU's Extension service, representatives from the MT Department of Fish, Wildlife and Parks and a representative from the Wild Sheep Foundation. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training in large animal husbandry and experimentation and in immunology and infectious disease to one Master of Science student in the Bimczok lab, Ms. Thea Johnson, who graduated with an M.S. degree Microbiology and Immunology in May of this year. The project also provided training in infectious disease and microbiology to a Ph.D. student, Mr. B. Tegner Jacobson, during his lab rotation. Following his laboratory rotations, Mr. Jacobson decided to join the Bimczok laboratory and will continue to work on our M. ovipneumoniae project over the next several years. In addition, five MSU undergraduate students contributed to various aspects of our project on either the animal husbandry side of the project or on the laboratory side, thereby gaining valuable experience in ewe and neonatal lamb care or in research methods. How have the results been disseminated to communities of interest?To share our data with scientists and veterinarians, we have presented our work at the 101st Conference of Research Workers in Animal Diseases (CRWAD) and have written two manuscripts. One manuscript on our pilot M. ovipneumoniae study has been resubmitted to "Veterinary Microbiology" after revisions. A second manuscript on the use of bovine serum-based colostrum replacer will be submitted in the next few weeks. To share our data with other stakeholders in the sheep industry, we have participated in a local workshop on M. ovipneumoniae mitigation strategies for domestic and bighorn sheep that included microbiology and animal science faculty from MSU, MSU's Extension service, representatives from the MT Department of Fish, Wildlife and Parks and a representative from the Wild sheep Foundation. What do you plan to do during the next reporting period to accomplish the goals?Work on this award began in the fall of 2019 through a pre-award funding mechanism. We have now successfully established an M. ovipneumoniae- and Pasteurellacea-free flock of domestic sheep, we have demonstrated the absence of relevant respiratory pathogens, and we have bred the ewe lambs from the original SPF flock to produce a new generation of SPF lambs. In addition, we have already performed a pilot infection experiment with M. ovipneumoniae. Therefore, the original goals for our project have been successfully met. Going forward, we will continue to perform bi-monthly testing for M. ovipneumoniae, Pasteurellaceae and parainfluenza virus to confirm the maintenance of the SPF status of the flock. We also have applied for follow up funding from the USDA so that we can utilize our SPF flock and infection model to explore the pathogenesis of M. ovipneumoniae-associated respiratory disease in more detail.

Impacts
What was accomplished under these goals? With the funding from this grant, we have successfully derived a research flock of specific pathogen-free domestic sheep that is free from key respiratory pathogens involved in sheeppneumonia, including M. ovipneumoniae, Pasteurella multocida, Mannheimia haemolytica, Bibersteinia trehalosi and parainfluenza-3 virus (PI3). We used supervised lambing and artificial rearing with colostrum replacer and milk replacer to prevent transmission of bacteria from ewes to the newborn lambs. With this approach, a flock consisting of 12 males and 18 females was successfully derived, with an above-average survival rate of 93.7%. All animals were initially housed in heated animal rooms inside our large animal BSL-2 facility. After weaning at approximately 6 weeks of age, eight lambs were selected for a pilot infection study and were kept inside the BSL-2 facility, whereas the other animals were moved to an outside paddock. Repeated testing over the past year has confirmed that the sheep did not spontaneously acquire M. ovipneumoniae or Pasteurellaceae in the outdoor paddocks. Serum antibodies to parainfluenza 3 were detected 2 weeks after birth, but declined over time and were undetectable at 6 months, consistent with passive transfer of antibodies from the milk replacer, which contained bovine serum, rather than active infection of the SPF lambs. Going forward, bi-monthly testing for M. ovipneumoniae, Pasteurellaceae and parainfluenza virus will be conducted to demonstrate continuing SPF status of the flock. All sheep also are regularly tested for the presence of intestinal parasites by analysis of fecal samples and treated as necessary. To maintain our flock and produce additional SPF lambs, we bred the 16 SPF ewe lambs that were born in 2020 with two of the ram lambs in December 2020 and January 2021. The ewes were estrus-synchronized, and two rams were introduced for a total of three cycles. Fourteen new lambs were born in May 2021 and were confirmed to be free of M. ovipneumoniae and Pasteurellaceae at 4-6 weeks of age. A similar breeding strategy will be used in the coming years. In order to understand whether M. ovipneumoniae alone induces respiratory disease in lambs with no previous exposure to or immunity against M. ovipneumoniae, we performed an M. ovipneumoniae challenge experiment in eight of the SPF lambs in August 2020, and blood, serum, bronchoalveolar lavage (BAL) and nasal swabs were collected. Four SPF lambs aged 15-16 weeks were selected for experimental infection with M. ovipneumoniae, and four additional lambs, matched for age and sex, were selected as a control group. The SPF lambs were inoculated with ceftiofur-treated nasal wash fluids from lambs with clinical symptoms of respiratory disease that had tested positive for M. ovipneumoniae by nasal swab PCR. Microbiological analysis of the pooled nasal wash fluid confirmed that both M. haemolytica and B. trehalosi were present before ceftiofur treatment but were undetectable after the treatment. We then inoculated sheep with 50 mL of the treated nasal wash fluid (M. ovipneumoniae group) or PBS (control group) by infusing liquid into the nostrils, the oral cavity and the conjunctival sacs. After inoculation, the M. ovipneumoniae group and the control group were housed in separate rooms of the JFLF to prevent aerosol transmission of M. ovipneumoniae and were monitored for 12 weeks. All lambs in the M. ovipneumoniae group, but none of the lambs in the control group, showed positive PCR results for M. ovipneumoniae at two weeks post infection (p.i.). M. ovipneumoniae levels peaked at 2-4 weeks, declined at 6 weeks and then plateaued. Colonization with M. ovipneumoniae was confirmed by successful culture of viable mycoplasma in nasal swab samples from two of the experimental lambs at 4 weeks post inoculation. All infected lambs developed a strong M. ovipneumoniae-specific antibody response that peaked at 4 weeks p.i. and remained high throughout the experimental period, whereas no significant M. ovipneumoniae-reactive antibodies were detected in the control group. Interestingly, no significant increase in clinical symptoms was seen in the M. ovipneumoniae-infected compared to the uninfected lambs throughout the 12-week experiment. Average daily gains measured between days 20 and 55 of the experiment also did not differ significantly between M. ovipneumoniae-infected and control lambs. Previous studies had shown increased numbers of AMs in BAL samples from M. ovipneumoniae pneumonia. However, our analyses of BAL fluids for lung damage (lactate dehydrogenase assay), total cell counts or inflammatory cell recruitment also revealed no significant differences between the M. ovipneumoniae-infected and the control animals. These data indicate that M. ovipneumoniae alone does not cause respiratory disease in domestic SPF-lambs under controlled laboratory conditions. In order to determine whether asymptomatic M. ovipneumoniae infection could be successfully cleared by antibiotic treatment, all lambs were injected with two doses of gamithromycin (Zactran®) eight weeks after challenge with M. ovipneumoniae. Although the M. ovipneumoniae from the nasal wash inoculum were susceptible to gamithromycin in vitro, gamithromycin neither eliminated nor significantly decreased the level of M. ovipneumoniae infection as determined by qPCR in any of the lambs either at 2 or at 4 weeks after the treatment. These findings show that additional studies are needed to design appropriate strategies to eliminate asymptomatic carriers.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: T. Johnson, K. Jones, B.T. Jacobson, J. Schearer, N. Adams, I. Thornton, C. Mosdal, S. Jones, M. Jutila, A. Rynda-Apple, T. Besser, D. Bimczok Experimental infection of specific-pathogen-free domestic lambs with Mycoplasma ovipneumoniae causes asymptomatic colonization of the upper airways that is resistant to antibiotic treatment
  • Type: Theses/Dissertations Status: Published Year Published: 2021 Citation: T.H. Johnson, Experimental infection of specific pathogen-free domestic lambs with Mycoplasma ovipneumoniae. Montana State University M.S. thesis, 2021
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: T. Johnson, K. Jones, A. Rynda-Apple, T. Besser, M. Jutila, D. Bimczok. Experimental Infection of Specific-Pathogen-Free Domestic Lambs with Mycoplasma ovipneumoniae Does not Cause Clinical Disease. 101st Conference of Research Workers in Animal Diseases, Dec. 5-8, 2020