Source: UNIVERSITY OF NEBRASKA submitted to
MANNHEIMIA HAEMOLYTICA: CHARACTERIZATION OF ISOLATES ASSOCIATED WITH FATAL BRONCHOPNEUMONIA OF CATTLE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0212145
Grant No.
(N/A)
Project No.
NEB-39-146
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 1, 2007
Project End Date
Aug 31, 2012
Grant Year
(N/A)
Project Director
McVey, D. S.
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
VETERINARY BIOMEDICAL SCIENCE
Non Technical Summary
The bovine respiratory disease (BRD) complex has been and is the most important cause of economic losses for the cattle background. The costs of mortality, therapy and poor growth performance have contributed to these losses. Treatment costs alone average over $15.00 per head and labor and feed efficiency factors probably increase the actual costs. The total losses due to BRD approach $1 billion per year. Even though the aggregate scientific understanding of the host animal responses and interactions with the microbial agents has increased substantially, the effectiveness of immunization and antimicrobial therapy has not reduced the prevalence or severity of BRD. Through completion of the work described in this proposal, our contribution to the field will be a more complete understanding of how M. haemolytica responds to the inflammatory environment present in the infected lung in order to persist in the tissues. This is significant because it may lead to methods to identify and treat persistant strains of M. haemolytica and therefore successfully prevent or treat the pneumonia. The findings of the proposed research would be useful to develop therapeutic agents and treatment strategies that would inhibit the response mechanisms and render the bacteria more susceptible to other, conventional (and less costly) treatments.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113399109010%
3113399110025%
3113399118015%
3114010110010%
3153399110020%
3154010109020%
Goals / Objectives
My long-term goal is to contribute to the understanding of virulence mechanisms of bacterial pathogens with particular emphasis on bacterial pneumonia of cattle and sheep. An important first step of this long-term goal is to determine how M. haemolytica responds to and resists microbicidal and clearance mechanisms of the immune system. The central hypothesis of this first step is that M. haemolytica responds to the killing mechanisms of phagocytic cells by induction of resistant phenotypes capable of continued growth and persistence within the tissues. In order to address this goal, we must first complete a phenotypic characterization of virulent M. haemolytica isolates. The primary aim of this project outline is to characterize the biochemical and structural phenotypes of the persistant isolates. This first set of aims is the principle focus of this project. The working hypothesis for this primary aim is that isolates of M. haemolytica from lungs of cattle that suffered fatal bronchopneumonia are phenotypically resistant to multiple classes of microbicidal compounds (typical of killing mechanisms of phagocytic cells) and metabolically capable of utilizing diverse energy and structural substrates to maintain high rates of growth. An additional objective of this work will be the identification of markers of these population phenotypes.
Project Methods
1.Strains to be tested and characterized - The initial set of bacteria to be tested will include approximately 100 to 125 isolates of M. haemolytica obtained from bovine lung samples submitted to the Nebraska VDC. The isolates selected will be from lungs with fatal, fibrinopurulent bronchopneumonia, typical of severe, fatal cases of pneumonic mannheimiosis. 2. Microbial physiology and growth kinetics, generation times and growth kinetics will be determined for each isolate as described. Each isolate will be characterized by carbon source utilization, production of proteolytic enzymes as well as toxin and capsule expression, secretion and structure. 3. Antimicrobial Susceptibility Testing, at the time of original isolation and selection of parental strains, antimicrobial disc susceptibility testing methods will be used to estimate MICs. The standard minimum inhibition concentrations (MIC) of the antimicrobial compounds will be determined (as required) by the standard twofold broth (in bovine heart infusion broth, BHIB) dilution method for aerobic bacteria. Commercial reagents are available for this test method. Determination of mutant prevention concentrations (MPC) will be determined as described. Culture expansion will be performed by two-step culture, first on BAP and then in BHIB. Culture incubation will be at 37C. The MPC is the lowest dilution that completely inhibits the growth of the inoculum. The MPC determinations will be made in duplicate and geometric means will be recorded. The principle difference between the MIC and MPC is the number of bacteria used to inoculate each test medium. 4.Killing kinetics, both MIC and MPC are based on inhibition of bacterial growth. For some isolates with relatively high MPC values, the killing kinetics of the specified drug may be determined. 5. Limited proteomic analysis, limited proteomic analysis of the bacteria will be achieved by two methods. The first is by polyacrylamide gel electrophoresis of outer membrane preparation profiles and lipopolysaccharide profiles. This will be followed by characterization of total protein extracts by a combination of strong cation exchange chromatography and reverse phase liquid chromatography coupled to UV detection of proteins and peptides. 6. Characterization of efflux activity, efflux activity will be determined as resistance to a standard set of chemicals using the standard twofold broth dilution method These determinations will also be made in duplicate. Variation in pH or cation concentrations can be used to characterize efflux activity in to either resistance nodulation division (RND), major facilitator superfamily (MFS) or multidrug and toxic compound extrusion (MATE) superfamily. 7. Limited genomic analysis, the parental and derived mutant strains will be subjected to genomic analysis by restriction fragment length polymorphism. The genomic polymorphisms that are identified will be utilized with other findings to identify specific mutations in individual genes.

Progress 09/01/07 to 08/31/12

Outputs
OUTPUTS: D.S. McVey left the employment of the University of Nebraska-Lincoln in 2011 and the project was terminated. The most signficant outputs were reported in the 2010-11 report. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The project was terminated as the principle investigator left the employment of the University of Nebraska-Lincoln in 2011. The 2010/10 to 2011/09 progress report describes the outcomes and impacts of the project.

Publications

  • No publications reported this period


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Bovine Respiratory disease complex (BRDC) also known as "shipping fever" is responsible for major economic losses in the US and global cattle industry. The bacterium Mannheimia (M.) haemolytica is considered the predominant bacterial pathogen associated with acute pleuropneumonia of BRDC. There is a need to develop molecular assays for epidemiological and pathological studies involving M. haemolytica. Both Pulse-Field Gel Electrophoresis (PFGE) and Metabolic Fingerprinting assays were evaluated as potential methods for assessing phenotypic and genotypic variations within the major serotypes of M. haemolytica. The PFGE successfully separated M. haemolytica strains and did reveal significant similarities and significant differences among M. haemolytica isolates and was able to consistently segregate closely related isolates. Also, a subset of M. haemolytica resistant to multiple antibiotics did consistently cluster together in multiple dendogram analysis schemes. However, the results also demonstrate that the PFGE method requires more standardization and refinement to be used on a routine basis. Clinical isolates of M. haemolytica appear to have very conserved metabolic activity fingerprints. The antimicrobial sensitivity characteristics of this resistant M. haemolytica sub-clade were highly conserved. The nature of the resistance phenotype suggests that a set of common mutations are likely responsible for these properties. In conclusion, there is genetic variation among M. haemolytica clinical isolated and consistent clustering of a fingerprint associated with a highly resistant antimicrobial sensitivity phenotype. Continued work to standardize these methods for epidemiological studies and to understand the mechanism of antibiotic resistance is warranted. PARTICIPANTS: The project relied on cooperation of the Nebraska Veterinary Diagnostic Center, University of Nebraska Lincoln. Colaborations were established with the USDA ARS Meat Animal Reseach Center (MARC, Dr. Carol Chitko McKown) and with Dr. Michael Aply, Professor, Kansas State University. The project work with Dr. Apley involved use of Mannheimia haemolytica isoalte characterization tools as related to isolates from pneumonic lungs relative to clinical signs, disease lesions and antimicrobial resistance. This work will be submitted for publication early in 2012. This work was also completed with sponsorship and cooperation of Bayer, Inc. Animal Health. The work at the MARC will involve more complete genetic sequenceing of the bacteria characterized through this project thus far. TARGET AUDIENCES: cattle veterinarians beef producers animal health research scientists animal health industry PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The characterization of the Mannhemia haemolytic completed in this project has now lead to additional studies the will provide key phenotype markers for antimicrobial compound resistance and, eventually, for relative virulence. Publications are expected in 2012.

Publications

  • No publications reported this period


Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Bovine respiratory disease (BRD) is of major economic importance to the United States and the global cattle industry. The U.S. feedlot industry estimates an annual loss as high as one billion dollars due to the loss of production, increased labor expenses, drug costs and death because of BRD. Many affected animals develop a severe bronchopneumonia caused Mannheimia (M.) haemolytica. Antibiotics are therefore frequently used to treat animals with clinical BRD. There is interest and concern regarding increased isolation of antibiotic-resistant pathogens associated with this disease. In this study, 50 lungs, tracheas and tonsils from calves with acute or peracute bronchopneumonia were subjected to extensive diagnostic laboratory investigation to identify and quantitate bacterial pathogens present. The distributions of the bacteria within the pneumonic lung tissue were also investigated. Only one bacterial pathogen was isolated from 36% of cases examined and Mannheimia haemolytica was the most common pathogen isolated from all tissues. Approximately 50% of the lungs contained > 10e5 colony forming units (CFU) per gram of tissue and 12% contained > 10e8 cfu. Multi-drug resistant bacteria were isolated from some respiratory tissues but this was not associated with any specific anatomical location within the respiratory system or the density of the bacteria isolated. Viral and Mycoplasma spp. pathogens were detected in some tissues but there was no correlation to bacterial species or numbers isolated or to distribution. The isolates were further evaluated using two fingerprinting methods, metabolic fingerprinting and pulsed field gel electrophoresis (PFGE). A lambda ladder PFG Marker (New England BioLabs, Ipswich, MA) was used as a marker. The PFGE images were analyzed using TotalLab (Nonlinear Dynamics, Newcastle upon Tyne, UK) TL120v206f and TL120DMv2006 software. Interestingly a set of multi-drug resistant isolates cluster within a potential sub-clade of organisms indicating that the resistant phenotype may be expressed among a limited genotype of M. haemolytica. Metabolic fingerprints were obtained for all isolates using Biolog GN2 MicroPlates according to the manufactures directions (Biolog, Hayward, CA). The M. haemolytica isolates were harvested from chocolate agar plates incubated at 37C with 5% supplemental CO2. The GN2 plates were inoculated and then incubated at 37C with 5% CO2 for 18 to 24 hours. The plates were analyzed using Microlog@ software (Biolog, Hayward, CA). In this metabolic profile analysis, the same set of multi-drug resistant isolates was observed to be closely related. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Current isolates of M. haemolytica are increasingly resistant to several classes of antibiotics. While the PFGE results verified substantial variation among the clinical isolates of M. haemolytica, it is noteworthy that a group of closely related, multi-drug-resistant isolates do cluster as a probable sub-clade. This relatedness was also observed in the metabolic fingerprinting of the isolates. These absolutes came from multiple occurrences of BRD on multiple cattle feeding operations in three states. It will be important to learn the mechanisms of resistance expressed in these isolates as well as genetics of the antimicrobial resistance. While the isolation frequency of these multi-drug-resistant bacteria is not very high (less than 5%), it will be very important to monitor the antimicrobial sensitivity profile of these bacteria in the future. In the same manner, it will be important to understand clonal selection processes and the epidemiology of colonization and infection by resistant strains of M. haemolytica.

Publications

  • Griffin, D, MM Chengappa, J Kuszak, and DS McVey. 2010. Bacterial Pathogens of the Bovine Respiratory Disease Complex. Veterinary Clinics of North America: Bovine Respiratory Diseases. (In Press).
  • Kapil, S, and DS McVey; editors.2010. Veterinary Clinics of North America: Emerging, Reemerging, and Persistent Infectious Diseases of Cattle. 26(1). March 2010.
  • McVey, DS, and J. Shi. 2010. Vaccination strategies for emerging diseases of livestock. Veterinary Clinics of North America: Emerging, Reemerging, and Persistent Infectious Diseases of Cattle. 26 (1):173-183.


Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: The Bovine Respiratory Disease Complex (BRDC), also known as "shipping fever" is responsible for major economic losses in the US and global cattle industry. The bacterium Mannheimia (M.) haemolytica is considered the predominant bacterial pathogen associated with the acute pleuropneumonia of BRDC. There is a need to develop molecular assays for epidemiological and pathological studies involving M. haemolytica. Both Pulse-Field Gel Electrophoresis (PFGE) and Metabolic Fingerprinting assays were evaluated as potential methods for assessing phenotypic and genotypic variations within the major serotypes of M. haemolytica. The PFGE successfully separated M. haemolytica strains and did reveal significant similarities and significant differences among M. haemolytica isolates. Also, a subset of highly resistant M. haemolytica did consistently cluster together in multiple dendogram analysis schemes. However, the results also demonstrate that the PFGE method requires more standardization and refinement to be used on a routine basis. Clinical isolates of M. haemolytica appear to have very conserved metabolic activity fingerprints. The antimicrobial sensitivity characteristics of this resistant M. haemolytica sub-clade were highly conserved. The nature of the resistance phenotype suggests that a set of common mutations are likely responsible for these properties. In conclusion, there is genetic variation among M. haemolytica clinical isolates and consistent clustering of a fingerprint associated with a highly resistant antimicrobial sensitivity phenotype. Continued work to standardize these methods for epidemiological studies and to understand the mechanism of antibiotic resistance is warranted. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
These studies have identified potential genetic fingerprints of multiple antibiotic resistant Mannheimia haemolytica isolates. This in turn may be useful for the identification of the resistance mechanisms and the underlying genetic basis of disease. Also, these methods may be useful for epidemiological studies investigating the incidence of BRD and the sources of infection. It is clear form these data that Mannheimia haemolyticais a relatively diverse organism that is likely still undergoing evolutionary selection.

Publications

  • No publications reported this period


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: A poster and oral presentation were delivered at an international bovine medicine convention in late 2007. Further work will result in a paper, master's degree thesis and likely an additional poster in 2008/2009. PARTICIPANTS: David Scott McVey received the DVM degree from the University of Tennessee in 1980. He spent three years in a dairy practice in East Tennessee. He earned the PhD degree in Veterinary Microbiology from Texas A&M University in 1986. While at Texas A&M University he was awarded the Jack Delaplane Award for research excellence in livestock diseases. Dr. McVey joined the faculty at Kansas State University in 1986 as an Assistant Professor of Immunology. He was promoted to the rank of Associate Professor in 1992. While at Kansas State University, Dr. McVey directed the Clinical Immunology and Flow Cytometry service laboratories. He was board certified in the ACVM in 1989 (Immunology) and 1990 (Bacteriology). Dr. McVey was course coordinator for immunology and also taught significant portions of infectious disease courses to undergraduate, graduate and veterinary students. Dr. McVey was awarded the SmithKline Beecham Award for Research Excellence in 1992. In late 1995, Dr. McVey joined Rhone Merieux as a Production Animal Scientist working in developmental research in veterinary biological products and diagnostics. He was awarded the Veterinary Information Network Special Services Award in 1997. In January of 1998, he joined Pfizer Bioprocess Research as a Senior Research Investigator and his most recent position there was Director of Laboratory Sciences, Biologicals Development of Pfizer Animal Health in Lincoln, NE (through April 2006). Dr. McVey is now at the University of Nebraska, where he is Professor of Clinical Microbiology and Director of the Veterinary Diagnostic Center and supervises diagnostic bacteriology and teaches bacteriology in the University of Nebraska-Iowa State University joint program for veterinary medicine. Dr. McVey was recipient of the Distinguished Alumni Award from the University of Tennessee College of Veterinary Medicine in 2007. He also is a past President of the American College of Veterinary Microbiologists and has also a served on the Blue Ribbon Panel for Counter Measures for Terrorist Threats to Agriculture for the President of the United States (2003-2004). Dr. McVey is the author of over 40 research articles, book chapters and patents as well as numerous abstracts and reports. Jennifer Kuszak is a graduate student working on the molecular fingerprint analysis of M. haemolytica. Her project has provided an opportunity for study and research work in the laboratory. TARGET AUDIENCES: Veterinary diagnosticians, veterinary practitioners, research scientists and beef producers PROJECT MODIFICATIONS: As the project progressed, the need to develop and use more specific and powerful means to characterize and type the M. haemolytica was obvious. As a result we have developed methods for both metabolic and genetic fingerprinting of the organism. These techniques combined with antimicrobial sensitivity testing results provide a very powerful means to fingerprint, or "type," the organism.

Impacts
Lungs from 20 calves with acute and fatal pneumonia were subjected to quantitative and qualitative analysis. The calves from which these lungs were selected died prior to antimicrobial treatment. Tissue samples were processed through mesh screens for serial dilution to quantitate bacteria per gram of pneumonic tissue by serial dilution and subsequent plate counts of colony forming units. The most common bacterium isolated was Mannheimia (M.) haemolytica and it was present from 2 X 103 to 5 X 108 colony forming units per gram of affected lung tissue. The lung tissue at the periphery of the consolidated tissue was predominantly infected with M. haemolytica. However, Pasteurella (P.) multocida, P. trehalosi, Histophilus somni and other bacteria were isolated from 16 of 20 lungs, most often from the center of the consolidated tissue as well as from within the conductance zones. Multiple phenotypes of M. haemolytica were isolated from individual lungs. The predominant phenotype was mucoid, resistant to tetracyclines and expressed patterns of restricted carbon source utilization. In addition, a set of approximately 60 clinical isolates of M. haemolytica have been used to develop metabolic and pulsed field gel electrophoresis fingerprints. This work will enhance our ability to distinguish between strains and isolates of M. haemolytica. Continued recognition that the bacterial pneumonia of the BRDC is polymicrobial could have significant impact on therapeutic strategies as well as clinical outcomes. The total numbers of bacteria present in an infected lung are high, but consistent with other forms of bacterial pneumonias in other mammalian species. The polymicrobial and polyclonal nature of the infections may also increase the likelihood of systemic inflammatory distress syndromes. Also clear, is the density and diversity of the infections characterized are great enough in magnitude to reach the thresholds of spontaneous mutant generation. Antimicrobial therapeutic doses should ideally be above mutant prevention concentrations. Continued development of methods to assess antimicrobial resistance is required and complete clinical validation of these data will be required. It is also clear that molecular and metabolic fingerprinting provide a very useful set of tools for epidemiological-based evaluation of the role of Mannheimia haemolytica in the BRDC.

Publications

  • Polymicrobial Etiology of Bacterial Pneumonia Associated with the Bovine Respiratory Disease Complex. McVey, DS. 2007. Annual Meeting of the American Association of Bovine Practitioners, Vancouver, British Columbia