Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
PATHOGENESIS OF MORAXELLA BOVOCULI SP. NOV IN BOVINE OCULAR INFECTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0211472
Grant No.
(N/A)
Project No.
CA-V-VME-4013-AH-301
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Apr 15, 2007
Project End Date
Apr 14, 2012
Grant Year
(N/A)
Project Director
Angelos, JO.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
MEDICINE & EPIDEMOLOGY
Non Technical Summary
Moraxella bovoculi sp. nov is a novel species of Moraxella that has been isolated from eyes of calves with infectious bovine keratoconjunctivitis (IBK; pinkeye); it is not known if M. bovoculi sp. nov can actually cause IBK. This project examines whether M. bovoculi sp. nov when inoculated into the eyes of calves will induce IBK and therefore attempts to see whether Kochs postulates can be established with respect to M. bovoculi sp. nov and IBK.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113310106015%
3113310110017%
3113410106017%
3113410110017%
3114010106017%
3114010110017%
Goals / Objectives
We hypothesize that M. bovoculi sp. nov will cause corneal ulceration typical of IBK in calves and that we will be able to reculture M. bovoculi sp. nov from eyes of calves that develop corneal ulcers, thereby establishing Kochs postulates for M. bovoculi sp. nov and IBK. Proposed Activities: For this study, 3 specific objectives are proposed: #1: Inoculate eyes of calves that have been: a) ultraviolet sunlamp irradiated or b) non-irradiated with 1x1010 cfu of M. bovoculi sp. nov isolate #237. #2: examine these calves daily for up to 14 days to determine if any calves develop corneal ulcers. #3: identify any bacteria isolated from ulcerated eyes.
Project Methods
Healthy weaned dairy calves (n=20) will be purchased through the UC Animal Resources Services and housed at the animal vivarium on campus. The ocular secretions from both eyes of each calf will be cultured for 3 consecutive days following arrival to ensure the animals are free of ocular infection with M. bovis or M. bovoculi sp. nov. Ocular secretions will be streaked onto 5% bovine blood agar plates and incubated at 35C for 24 hr prior to examination for colonies with morphology typical of M. bovis and/or M. bovoculi sp. nov. We already have established methods in our laboratory to positively identify M. bovis and M. bovoculi sp. nov using biochemical and molecular (PCR amplification of the 16S-23S interspacer region) criteria. Any calves harboring either organism will be removed from the study. Beginning on day 4, both eyes of 8 calves will be irradiated with a sunlamp at a distance of 60 cm for 10 minutes and then inoculated by instilling 1 X 1010 colony forming units of β-hemolytic M. bovoculi sp. nov isolate 237 suspended in 1 mL of trypticase soy broth (TSB) drop wise onto the corneal surface. We have previously utilized this method for experimentally inducing IBK. The irradiation and experimental infections will be repeated on days 5 and 6. The eyes of 8 additional calves will not receive corneal irradiation, however, these calves will infected as described above on days 4, 5, and 6. Two calves will be irradiated following the same protocol on days 4, 5, and 6, however, these calves will be sham infected with 1 ml of sterile TSB containing heat killed (52 C, 1 hour) M. bovoculi cultures on days 4, 5, and 6. From days 5 through 18, both eyes of each calf will be examined daily according to standard methods for ocular examinations that we have previously employed in previous studies of IBK. Ocular secretions will be collected and cultured daily from any calves that develop corneal opacities; confirmation of corneal ulcers will be made by examining for the presence of fluorescein dye uptake. The presence of M. bovoculi sp. nov will be determined on the basis of molecular criteria (see above). Any calves that develop corneal ulcers with a diameter greater than 1 cm or corneal perforations will be humanely euthanized and necropsied. In that case, the eyes will be harvested and examined histologically for corneal pathology.

Progress 04/15/07 to 04/14/12

Outputs
OUTPUTS: The most significant outputs and dissemination activities for the entire life of this project include: 1)journal publications related to experimental studies with Moraxella bovoculi vaccination and antibiotic sensitivity; 2)lay publications in California Cattleman Magazine related to Moraxella bovis and Moraxella bovoculi as these organisms relate to prevention of cattle pinkeye; and 3)presentations given by Dr. Angelos to cattle producer groups in California describing prevention of cattle pinkeye and of the role that Moraxella bovoculi may play in pinkeye prevention. PARTICIPANTS: John Angelos - principal investigator/project director John Maas - UC Veterinary Cooperative Extension; collaborated with PI in developing pinkeye related articles for publication in stakeholder publications. Dave Coons - laboratory manager; assisted in laboratory work related to the project during its lifetime Louise Ball - laboratory manager prior to Dave Coons; assisted in laboratory work related to the project during its lifetime John Hess - assisted in vaccine trials related to this project over its lifetime Veronica Villaneuva - assisted in challenge studies related to this project over its lifetime Judy Edman - assisted in vaccine trials related to this project over its lifetime TARGET AUDIENCES: Target audiences included cattle producers throughout California that were reached via publications in California Cattleman Magazine as well as through local cattle producer talks that Dr. Angelos participated in during the lifetime of this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project has led to a change in knowledge as it relates to Moraxella bovoculi. During the lifetime of this project we learned that Moraxella bovoculi does not cause corneal ulcerations when exposed to calf corneas. Initially we believed that this novel organism that we first reported on in 2007 was another cause of pinkeye (infectious bovine keratoconjunctivitis; IBK) in cattle along with Moraxella bovis, however, experimental challenge of calves with Moraxella bovoculi only demonstrated development of conjunctivitis in infected calves. We also learned that vaccination of calves with recombinant Moraxella bovoculi cytotoxin did not protect calves against IBK. This further supported the notion that Moraxella bovoculi is most likely not a primary pathogen in causing IBK in cattle. The fact that Moraxella bovoculi did not cause corneal ulcers in our studies has also led to a change in actions in that it has changed how we think of Moraxella bovoculi in terms of its role in causing IBK. Most likely this organism behaves as a risk factor for IBK much like mycoplasma and infectious bovine rhinotracheitis virus infection acting as risk factors for IBK in calves. Our studies with Moraxella bovoculi also led to a change in actions in that we have begun (based on our research with Moraxella bovoculi) looking for additional Moraxella spp. antigens to use in recombinant Moraxella bovis vaccines to prevent pinkeye in cattle.

Publications

  • No publications reported this period


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: During this reporting period we continued our work with the recombinant M. bovis porin protein. In late 2011 we were successful in demonstrating expression of this protein in outer membrane preparations of Moraxella bovis and Moraxella bovoculi. While we were not able to clearly show agglutination of whole M. bovis or M. bovoculi with antiserum against this porin protein, we have at this time evidence to pursue use of this protein in a vaccine to determine if this antigen, when combined with our previously tested recombinant M. bovis cytotoxin, will protect calves against naturally occurring infectious bovine keratoconjunctivitis (IBK). PARTICIPANTS: John Angelos - principal investigator/project director John Maas - UC Veterinary Cooperative Extension; collaborated with PI in developing pinkeye related articles for publication in stakeholder publications. Dave Coons - laboratory manager TARGET AUDIENCES: Dr. Angelos presented 7 lectures to cattle stakeholder groups throughout northern California in 2011: February 24, Pinkeye: New Epidemics of an Old Disease, Annual Cattle Health Meeting, Siskiyou County Cattlemen, Montague, CA. February 25, Pinkeye: New Epidemics of an Old Disease, Winter Animal Health Meeting, Glenn County Cattlemen, Willows, CA. February 25, Pinkeye: New Epidemics of an Old Disease, Winter Animal Health Meeting, Shasta County Cattlemen, Cottonwood, CA. February 26, Pinkeye: New Epidemics of an Old Disease, Winter Animal Health Meeting, Humboldt County Cattlemen, Eureka, CA. May 7, Pinkeye Update, Beef Quality Assurance Program, Cole Facility, UC Davis, Davis, CA. August 25, Pinkeye Identification, Treatment, and Prevention, Yolo County Cattlemen and Wool Growers Association 19th Annual Fall Tour Educational Meeting, Esparto, CA. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The results we have obtained from our laboratory testing for presence of M. bovis porin on outer membrane preparations has given justification to pursue future testing of this antigen in recombinant subunit vaccines to prevent IBK.

Publications

  • May 2011: Angelos JA and Maas J. 2011. Protection Against Pinkeye: Wet Winter Warns of Pinkeye Outbreak. California Cattleman Magazine; pp. 28-30.
  • 2011: Angelos JA, Gohary K, Ball LM, and Hess JF. 2011. Randomized controlled field trial to assess efficacy of a Moraxella bovis pilin-cytotoxin-Moraxella bovoculi cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis. AJVR In Press (accepted Oct 13, 2011)
  • 2011: Angelos JA, Ball LM, and Byrne BA. 2011. Minimum inhibitory concentrations of selected antimicrobial agents for Moraxella bovoculi associated with infectious bovine keratoconjunctivitis. J Vet Diagn Invest, 23(3): 552-555.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: During this reporting period (calendar year 2010) a process for purification of the recombinant M. bovis porin protein (this protein was previously identified in M. bovoculi and M. bovis and is ~72% identical between the two Moraxella species) was developed. The method for purification was scaled up and this protein was submitted to an outside vendor for development of rabbit antisera to the protein. Initial attempts to demonstrate inhibition of growth of M. bovis and M. bovoculi in the presence of rabbit antisera against porin were met with inconclusive results. A variety of methods were attempted to identify if rabbit antisera to this protein could agglutinate whole M. bovis and M. bovoculi or latex beads coated with M. bovis or M. bovoculi proteins. The initial results suggest that agglutination could not be demonstrated. During this time, two additional beta barrel outer membrane proteins of M. bovis were also cloned and expressed in E. coli. These were: 1) an iron repressible outer membrane protein (IrpA); and 2) a transferrin receptor protein (TrpA). PARTICIPANTS: John Angelos - principal investigator/project director Dave Coons - laboratory manager; DNA sequencing/PCR/cloning; expressing recombinant proteins John Maas - UC Veterinary Cooperative Extension ; collaborated with PI in developing pinkeye related articles for publication in stakeholder publications. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Results of the overall project (Pathogenesis of M. bovoculi) obtained to date and its previous outputs have aided in the overall understanding/knowledge base of M. bovoculi and its role in pathogenesis of IBK that have helped the investigator in guiding stakeholders in approaches to prevention of infectious bovine keratoconjunctivitis (pinkeye) and recommendations on how to deal with M. bovoculi infections in herds. Such understanding is conveyed to stakeholders via publications in stakeholder media (California Cattleman Magazine). Based on our current understanding of M. bovoculi, it is most likely acting as another risk factor for pinkeye in cattle similar to flies, dust, UV radiation, Mycoplasma, and infectious bovine rhinotracheitis virus (IBR virus); this is largely concluded on the results of the challenge trial that we conducted early in the course of this overall project as well as from anecdotal information conveyed to the principal investigator (J. Angelos) during interactions with private veterinarians as well as local cattle producers at cattle producer meetings.

Publications

  • Galvao KN and Angelos JA. 2010; Ulcerative blepharitis and conjunctivitis in adult dairy cows and association with Moraxella bovoculi. Canadian Veterinary Journal, 51(4): 400-2.
  • Angelos JA. 2010; Infectious Keratoconjunctivitis (Pinkeye, Infectious ophthalmia) In: Kahn CM, (ed), The Merck Veterinary Manual, 10th Edition, Merck & Co., Inc., Whitehouse Station, NJ. 470-472.
  • Angelos JA. 2010; Moraxella In Gyles CL, Prescott JF, Songer JG, and Thoen CO, (eds), Pathogenesis of Bacterial Infections in Animals, 4th Edn., Wiley-Blackwell, Ames. 469-481.
  • Angelos JA. 2010; Moraxella bovoculi and infectious bovine keratoconjunctivitis: cause or coincidence Veterinary Clinics of North America: Food Animal Practice, Vol. 26(1), Elsevier (Saunders), Philadelphia. pp. 73-8.
  • Angelos JA and Maas J. April 2010; Planning Your Prevention Program: Don't turn blind-eye to pinkeye California Cattleman Magazine; pp 23-25.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: During this funding period we successfully cloned and sequenced a full lenth porin gene (POR) from Moraxella bovis and ~90% of a homologous porin gene from Moraxella bovoculi. For the portion of POR that has been cloned into M. bovoculi (~90% of gene thus far), there is ~72% identity with M. bovis POR. The POR gene from M. bovis was cloned into an expression vector and a recombinant POR subunit from M. bovis has been expressed in E. coli. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Beef and Dairy cattle industries; bovine pinkeye vaccine manufacturers; cattle producers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Both M. bovis and M. bovoculi express porin genes that are >70% identical over the identified region of the gene.

Publications

  • No publications reported this period


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: During this past year we have focused on additional pathogenic factors of M. bovoculi besides pili. We identified proteins in outer membrane preparations that were related to siderophore receptors and porins of other bacteria. The genes encoding these proteins were identified in M. bovis and we are currently involved in identifying, cloning, and sequencing related proteins in M. bovoculi. PARTICIPANTS: John Angelos - principal investigator/project director Louise Ball - laboratory supervisor; isolated cell surface proteins Dave Coons - laboratory supervisor following Louise Ball; DNA sequencing/PCR/cloning TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We originally were trying to identify pilin of M. bovoculi, however, repeated attempts to isolate pilin related proteins were not successful. This failure led to an alternative method to isolate cell surface proteins (immunoprecipitation and mass spectrometric analysis) from M. bovoculi. We subsequently identified putative siderophore receptor and porin proteins from M. bovoculi as well as M. bovis.

Publications

  • No publications reported this period


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: During October and November of 2007, a challenge trial was conducted at CLAS on the UC Davis campus to determine whether Koch's postulates could be established for Moraxella bovoculi and infectious bovine keratoconjunctivitis (IBK; "pinkeye"). Calves were purchased from 2 organic dairies; these calves were approximately 3 weeks of age at the start of the trial. Both eyes and both nares of each of 15 calves were cultured daily for 3 days; in addition, the eyes and nares were cultured for Mycoplasma. Calves that were found to be free of Moraxella/mycoplasma species in their eyes were assigned to control (2 calves) or challenge (4 calves) groups. The calves were then exposed to ultraviolet sunlamps for 10 minutes per day for 3 consecutive days. Following exposure on each day, both eyes of calves in the control group were instilled with 1 ml of a killed suspension of Moraxella bovoculi isolate 237; challenge group calves received 1 ml of a live culture of Moraxella bovoculi isolate 237 instilled into each eye. The eyes of all calves were examined each day for 14 days after the last challenge (last day of ultraviolet exposure) for the development of corneal ulcers. During this time, eyes were cultured every other day. PARTICIPANTS: John Angelos - principal investigator/project director; collected ocular swabs; examined calf eyes daily Louise Ball - laboratory supervisor; performed microbiologic analyses of bacterial cultures collected from eyes; maintained project data spread sheet Veronica Villanueva - laboratory assistant; assisted in holding calves for ocular and nasal swab collection and serum collection; assisted in holding calves for the ultraviolet exposure to sunlamps. TARGET AUDIENCES: Bovine veterinarians, researchers, diagnostic microbiology laboratories, and veterinary biologics companies are the target audiences. PROJECT MODIFICATIONS: The sunlamps will need to be revised that were used in this study in order to determine whether the previously developed model for causing IBK in calves with Moraxella bovis does or does not work for Moraxella bovoculi. In addition, a different more virulent field strain of Moraxella bovoculi will be used in the future.

Impacts
Results of this study suggest that challenge with Moraxella bovoculi isolate 237 caused conjunctivitis in calves. The ultraviolet output of the sunlamps that were used to cause corneal irritation (a previously documented model of causing corneal ulceration in calves with Moraxella bovis) is suspected to be less than optimal, and therefore this study will need to be revised and repeated with new sunlamps. As of this writing, the ability of Moraxella bovoculi to cause corneal ulceration in calves still needs to be determined.

Publications

  • Angelos JA, Ball LM. Differentiation of Moraxella bovoculi sp. nov. from other coccoid moraxellae by the use of polymerase chain reaction and restriction endonuclease analysis of amplified DNA. J Vet Diagn Invest. 2007 Sep;19(5):532-4.
  • Angelos JA, Ball LM, Hess JF. Identification and characterization of complete RTX operons in Moraxella bovoculi and Moraxella ovis. Vet Microbiol. 2007 Nov 15;125(1-2):73-9.
  • Angelos JA, Ball LM. Relatedness of cytotoxins from geographically diverse isolates of Moraxella bovis. Vet Microbiol. 2007 Oct 6;124(3-4):382-6.
  • Angelos JA, Spinks PQ, Ball LM, George LW. Moraxella bovoculi sp. nov., isolated from calves with infectious bovine keratoconjunctivitis. Int J Syst Evol Microbiol. 2007 Apr;57(Pt 4):789-95.