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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Ruminant Diseases and Immunology Research » Research » Research Project #432020

Research Project: Identification of Disease Mechanisms and Control Strategies for Bacterial Respiratory Pathogens in Ruminants

Location: Ruminant Diseases and Immunology Research

2017 Annual Report

Objective 1. Define the virulence determinants and mechanisms involved with the principal bacteria associated with bovine respiratory disease, including identifying microbial mechanisms used by commensal bacteria to become pathogens, and identifying the mechanisms of bacterial colonization in target hosts. Subobjective 1.1: Identify microbial mechanisms used by commensal bacteria to become pathogens. Subobjective 1.2: Identify the mechanisms of bacterial colonization of the host. Objective 2. Determine the host-pathogen interactions associated with respiratory infections, including developing animal disease models to study respiratory disease complex, identifying the host factors that drive the early innate immune response to bacterial respiratory infections, and characterizing functional genomics of the host associated with respiratory infection. Subobjective 2.1. Continue the development of animal disease models to study respiratory disease complex. Subobjective 2.2. Identify the host factors that drive the early innate immune response to bacterial infection. Subobjective 2.3. Characterize functional genomics of the host associated with respiratory infection. Objective 3: Develop intervention strategies to reduce antibiotic use, including developing vaccines that will induce early mucosal immunity in young animals, and developing and evaluating immune-modulators to prevent and/or treat respiratory disease. Subobjective 3.1. Develop vaccines that induce early mucosal immunity in young animals. Subobjective 3.2. Develop and evaluate immune modulators to prevent and/or treat respiratory disease.

Binding of bacteria to mucosal surfaces, and evasion of host innate and adaptive immunity are critical to successful colonization and maintenance of infection. Identification of key molecular players in these interactions should enable potentially effective intervention strategies. We will utilize a coordinated and multipronged approach to characterize molecular mechanisms promoting respiratory bacterial pathogen colonization, adherence, and persistence in cattle. We plan to use experimental ruminant models and specific mutants to describe molecular mechanisms enabling bacteria to colonize, adhere and grow in the respiratory tract, and to examine the influences of primary viral infection on secondary bacterial infections. While much knowledge has been gained regarding the individual pathogens involved in BRDC, less is known concerning co-infections involving bacterial and viral respiratory pathogens. Given the expertise of our research team, and specific etiologic agent prevalence in the field, we will focus on BVDV and BRSV as the viral pathogens, and Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and Mycoplasma bovis as the bacterial agents. We plan to continue the development of reproducible models of viral predisposition to bacterial disease and to characterize the host and infectious agents’ response using a comprehensive approach. Bacterial genes or gene products identified in these studies will be used, based on their importance in colonization, for developing and testing novel vaccines. Furthermore, we will examine the potential of immunomodulators to enhance the host response to infection with respiratory pathogens. The overriding goal of this plan is to develop preventative measures that aid in the reduction or elimination of BRDC in beef and dairy cattle. Reductions in BRDC will be of substantial economic benefit to cattle producers. However, as specific bacterial pathogens involved in BRDC are significant causes of morbidity and mortality in wild ruminant populations, there are aspects of this research plan that include those species. For example, bighorn sheep suffer severe die-offs as a result of respiratory disease and it is considered the major factor impacting the long-term sustainability of bighorn sheep populations. Moreover, M. bovis has additionally emerged in North American bison, causing substantial economic losses to producers and threatening the stability of heritage herds. Therefore, strategies to reduce respiratory disease in wild ruminant populations will be of substantial value to the public interest in sustaining wildlife populations, as well as reduce economic losses to bison producers.

Progress Report
In support of Objective 1 we have acquired nearly 200 Mycoplasma (M.) bovis isolates from bison and cattle suffering from Mycoplasma-related disease as well as from healthy animals. All isolates have been cloned and properly stored to support future studies. In addition, we completed our genetic marker analysis known as multilocus sequence typing (MLST) of these and additional Mycoplasma isolates and have detected novel M. bovis sequence types among both bison and cattle isolates. In support of this objective, recent clinical tissue samples were obtained from over 200 healthy bison across 12 geographic locations in the United States and Canada from which 4 different species of Mycoplasma were cultured. These isolates were cloned and added to our collection of approximately 2000 historical samples dating back to 1988 from over 30 different herds of bison in the United States and Canada. These samples are supporting our research efforts into understanding the virulence factors of this pathogen that differ between cattle and bison. These findings are being used to guide development of more effective vaccines. In support of Objective 2 relevant bovine genes, such as boToll-like receptor (boTLR)2 and boTLR4, have been cloned into mammalian expression vectors. A human cell line, which does not normally express these proteins has been transfected with the respective vectors containing the genes to generate boTLR expressing cell lines. Once all stable cell lines are established, relevant studies utilizing these cell lines will be conducted. In two co-infection experiments, calves were challenged with M. bovis and/or Bovine Viral Diarrhea Virus (BVDV). The objective of these studies is to determine if there is synergy between these two organisms which leads to enhanced respiratory disease. Tissue samples were collected at necropsy and stored for further analyses. To date, RNA has been extracted from most of these tissues. In support of Objective 3 a potential new vaccine for Bovine Respiratory Syncytial Virus (BRSV) and human Respiratory Syncytial Virus (RSV) has been tested by ARS researchers at Ames, Iowa, in collaboration with researchers at Rutgers University and Mount Sinai Medical School. Studies published using the vaccine in mice have shown the vaccine to provide protection. Preliminary vaccination and challenge trials in calves have now been conducted and the vaccine was found to provide incomplete protection. Thus, results of these studies in cattle have provided researchers with data that indicates that dosage of the vaccine needs to be re-evaluated for larger animal species. With this in mind, ARS researchers at Ames, Iowa this year conducted a new trial using a 5-fold higher dose of the vaccine and administered two doses three weeks apart. In the first trial, vaccinated calves were found to have reduced lung lesions following virulent challenge compared to non-vaccinated calves. The most effective BVDV vaccines currently available commercially are not recommended for use in pregnant or stressed animals. An alternative, yet effective BVDV vaccine, is therefore needed for use in such animals. ARS researchers at Ames, Iowa, have previously developed a Mannheimia (M.) haemolytica vaccine strain with reduced ability to cause disease due an alteration in the toxin gene yet continues to express a protective portion of the toxin. When delivered to calves, the vaccine elicited immune responses which were highly protective against the disease. Regions of the protective BVDV proteins were identified and optimized for expression in Mannheimia and the DNA fragment was commercially synthesized. This gene (DNA) fragment will be subsequently introduced into the M. haemolytica vaccine strain to express BVDV protective molecules. It is expected that such a vaccine strain, when delivered orally, will elicit both protective immune responses against BVDV. Many bacteria, including Histophilus (H.) somni, employ elaborate systems to procure molecules from host tissues and incorporate these molecules onto their membranes. Such measures are known to protect the bacteria against killing by host defense mechanisms. ARS researchers at Ames, Iowa have constructed a H. somni mutant which cannot uptake a specific molecule. The mutant and the H. somni parent were assessed for removal of this molecule from the culture media. The parent strain, unlike the mutant, was shown to deplete this molecule from culture media. These findings demonstrated that the H. somnis uptake mutant behaved as anticipated and likely is reduced in its ability to cause disease due to an inability to the specific molecule for incorporation onto its outer membrane. A second proposed mutation will be inserted into this H. somni mutant for the express purpose of constructing a safe vaccine candidate of H. somni possessing two important mutations that will reduce the ability of H. somni to cause disease. Pneumonia, caused principally, by M. haemolytica and Mycoplasma (M.) ovipneumonia, is thought to be responsible for the dramatic decline of bighorn populations throughout the western United States. ARS researchers at Ames, Iowa have constructed a M. haemolytica vaccine candidate that also expresses and secretes relevant M. ovipneumoniae proteins designed to elicit potentially protective antibodies. The vaccine strains described here when administered orally will establish colonization in nasal passages and tonsils to produce specific immune responses. This live vaccine is intended to reduce the incidence of pneumonia in bighorn sheep due to M. haemolytica and M. ovipneumonia. Moreover the vaccine can be dispensed via baited foods to enable mass application to free ranging animals. In addition, the M. ovipneumoniae molecules secreted by the M. haemolytica vaccine candidate were expressed in escherichia (E.) coli.

1. Killing of respiratory bacteria by host white blood cell-derived small proteins. Host white blood cell-derived proteins can kill respiratory organisms, like bacteria associated with Bovine Respiratory Disease Complex (BRDC), an important disease in cattle. ARS researchers at Ames, Iowa have compared the ability of certain small proteins produced by bovine white blood cells to kill bacteria associated with BRDC. Of the four proteins examined, one was found to be very effective in killing Mycoplasma (M.) haemolytica, Pasteurella (P.) multocida and H. somni. This protein was selected for further studies to examine the killing of H. somni in more detail, as this had not been done previously. This protein was able to kill by damaging the bacterial membrane. It will be important to determine whether this protein could be used as a treatment to reduce the severity of BRDC in cattle.

2. Estimated prevalence of Mycoplasma (M.) bovis in bison herds. A collaborative study, between ARS researchers at Ames, Iowa and researchers in the United States and Canada, designed to estimate the prevalence of M. bovis in Canadian bison herds with or without past history of M. bovis-associated disease and to determine potential risk factors for infection was completed. Based on the presence of antibody against M. bovis in bison herds, on average, 12% of individual bison and 79% of the herds had been exposed to this pathogen; the proportion of positive animals ranged from 11-41% and 3-9% for herds with or without history of M. bovis-associated disease, respectively. Eight of 11 herds with no history of M. bovis-associated disease were positive for mycoplasma antibodies, which suggests that either bison can be subclinically infected or mycoplasma infection may be underdiagnosed. Risk factors associated with a high level of positivity include type of operation (highest in feedlot bison versus cow-calf herds) and age (for females, highest in those over 30 months old). These data provide insights needed for the development of appropriate prevention and control measures for mycoplasma-related disease in bison herds. As mycoplasma-related disease in bison herds has resulted in significant morbidity and mortality over the last several years, such measures would prevent or reduce these losses.

3. Comparison of toxin-binding protein expression on Bighorn sheep and domestic sheep white blood cells. Bighorn sheep are more susceptible to pneumonia caused by Mannheimia haemolytica than domestic sheep, with a toxin produced by this bacterium associated with the severity of the disease. ARS researchers at Ames, Iowa and collaborators compared expression of the toxin binding proteins on neutrophils between bighorn and domestic sheep. Contrary to the hypothesis, domestic sheep neutrophils expressed more toxin binding protein on neutrophils than those of bighorn sheep, yet domestic sheep neutrophils were less sensitive to the toxin than bighorn sheep neutrophils. These data provide additional insight in regards to the mechanisms of interaction between the bacterial toxin and host cells. Such information will be important in the development of methods to alter this interaction and reduce disease severity in bighorn sheep.

Review Publications
Linz, B., Ivanov, Y., Preston, A., Brinkac, L., Parkhill, J., Kim, M., Harris, S.R., Goodfield, L.L., Fry, N.K., Gorring, A.R., Nicholson, T.L., Register, K.B., Losada, L., Harvill, E.T. 2016. Acquisition and loss of virulence-associated factors during genome evolution and speciation in three clades of Bordetella species. Biomed Central (BMC) Genomics. 17:767. doi: 10.1186/s12864-016-3112-5.
Ivanov, Y., Linz, B., Register, K.B., Newman, J., Taylor, D., Boschert, K., Le Guyon, S., Wilson, E., Brinkac, L., Sanka, R., Greco, S.C., Klender, P.M., Losada, L., Harvill, E. 2016. Identification and taxonomic characterization of Bordetella pseudohinzii spp. nov. isolated from laboratory-raised mice. International Journal of Systematic and Evolutionary Microbiology. 66:5452-5459. doi: 10.1099/ijsem.0.001540.
Nelson, C.D., Lippolis, J.D., Reinhardt, T.A., Sacco, R.E., Powell, T.L., Drewnoski, M.E., O'Neill, M., Beitz, D.C., Weiss, W.P. 2016. Vitamin D status of dairy cattle: Outcomes of current practices in the dairy industry. Journal of Dairy Science. 99:10150-10160.
Walsh, D.P., Cassirer, F.E., Bonds, M.D., Brown, D.R., Edwards, W.H., Weiser, G.C., Drew, M.L., Briggs, R.E., Fox, K.A., Miller, M.W., Shanthalingam, S., Srikumaran, S., Besser, T.E. 2016. Concordance in diagnostic testing for respiratory pathogens of Bighorn Sheep. Wildlife Society Bulletin. 40(4):634-642. doi: 10.1002/wsb.721.
Dassanayake, R.P., Shanthalingam, S., Liu, W., Casas, E., Srikumaran, S. 2017. Differential susceptibility of bighorn sheep (Ovis Canadensis) and domestic sheep (Ovis Aries) neutrophils to Mannheimia Haemolytica Leukotoxin is not due to differential expression of cell surface CD18. Journal of Wildlife Diseases. 53(3):625-629. doi: 10.7589/2016-11-244.
Casas, E., Kehrli Jr., M.E. 2016. A review of selected genes with known effects on performance and health of cattle. Frontiers in Veterinary Science. 3(113). doi: 10.3389/fvets.2016.00113.
Bras, A.L., Suleman, M., Woodbury, M., Register, K.B., Barkema, H., Perez-Casal, J., Windeyer, M. 2017. A serologic survey of Mycoplasma spp. in farmed bison (Bison bison) herds in western Canada. Journal of Veterinary Diagnostic Investigation. 29(4):513-521. doi: 10.1177/1040638717710057.
Klima, C.L., Zaheer, R., Briggs, R.E., McAllister, T.A. 2017. A multiplex PCR assay for molecular capsular serotyping of Mannheimia haemolytica serotypes 1, 2, and 6. Journal of Microbiological Methods. 139(2017):155-160. doi. 10.1016/j.mimet.2017.05.010.
Madsen-Bouterse, S.A., Schneider, D.A., Zhuang, D., Dassanayake, R.P., Balachandran, A., Mitchell, G.B., O'Rourke, K.I. 2016. Primary transmission of chronic wasting disease versus scrapie prions from small ruminants to transgenic mice expressing ovine and cervid prion protein. Journal of General Virology. 97(9):2451-2460.