REFINEMENT OF PERITONITIS MODEL IN COMME

• Sponsoring Institution: Cooperating Schools of Veterinary Medicine
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 0208748
• Project Start Date: Sep 7, 2006
• Project End Date: Dec 31, 2009
• Program Code: [(N/A)]- (N/A)

Recipient Organization
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES,IA 50011

Performing Department
VETERINARY MEDICINE

Non Technical Summary
Peritonitis of laying hens has been the single most important cause of morbidity, mortality, and financial loss from disease for commercial egg producers during the last decade. The egg production industry is desperately seeking a solution to this problem. The authors of this proposal feel that the potential for future funding from national funding agencies is very high and that data from this project would make future proposals to those agencies much more competitive.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3210	1100	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3210 - Egg-type chicken, live animal;

Field Of Science
1100 - Bacteriology;

Keywords

e. coli

p. trehalose

peritonitis

aluminum hydroxide adjuvant

titration

necropsied

bacterin

immunization

Goals / Objectives
a.Determine the lowest dose of P. trehalose and E. coli that will consistently produce lesions of peritonitis in laying hens. This dosage will be used in future work to determine if vaccination will prevent mortality and lesions associated with peritonitis. b.Determine if two doses of a bacterin against P. trehalose will protect against peritonitis in laying hens. An aluminum hydroxide adjuvant will be used. c.Determine if two doses of a bacterin containing both P. trehalose and E. coli will protect against peritonitis in laying hens. An aluminum hydroxide adjuvant will be used.

Project Methods
We intend to undertake a series of titration experiments to determine the lowest dose required for each of these two pathogens which will consistently create peritonitis in laying hens. Once the lowest dosages have been determined, we will immunize chickens with two injections of a killed P. trehalose vaccine prior to challenge with P. trehalose and E. coli. Third, we will immunize chickens with two injections of a bacterin containing killed P. trehalose and E. coli prior to challenge with P. trehalose and E. coli. For each titration experiment, the oviduct of two control chickens will be inoculated with sterile media on Day 1. Also on Day 1, the oviduct of 12 adult chickens (10 principals + 2 controls) in egg production will be inoculated with P. trehalose at 11:00 am. On Day 2, the oviduct of 12 chickens (10 principals + 2 controls) will be inoculated with E. coli. Survivors will be necropsied on Day 5. Titration experiments willbe done to determine the lowest dose of P. trehalose and E. coli that will consistently create peritonitis. On Day 1, 8 chickens will be injected intramuscularly with an experimental bacterin containing killed P. trehalose bacteria plus an aluminum hydroxide adjuvant. On Day 14, the same 8 chickens will be inoculated with a second dose of the bacterin. On Day 28, the oviduct of 16 adult chickens (8 vaccinates + 8 nonvaccinates) in egg production will be inoculated with P. trehalose (dosage x determined from Experiments 1 - 4) at 11:00 am. On Day 29, the oviduct of 16 chickens (8 vaccinates + 8 nonvaccinates) will be inoculated with E. coli (dosage y - lowest effective dosage determined from experiments. On Day 32, all surviving chickens will be euthanized with carbon dioxide and evaluated for lesions of peritonitis. Also,the same procedure will be used for immunization with a bacterin containing both P. trehalose and E. coli.

Progress 09/07/06 to 12/31/09

Outputs
OUTPUTS: 1. Loss of pathogenic G. anatis isolate. Early in 2006, we successfully reproduced peritonitis in 60% of laying hens by inoculating a combination of G. anatis and E. coli into the oviduct. While project researchers were attending the AVMA convention in July 2006, the electrical supply to our laboratory was lost for a period of time leading to elevated temperatures in freezers and death of our G. anatis isolates. 2. Non-pathogenic isolates of G. anatis do not produce disease. During January 2007, a new strain of G. anatis was obtained and used in four experimental trials in an attempt to re-create the peritonitis lesions seen during the 2006 studies. None of the trials with the new isolate were successful and we have concluded that this new isolate of G. anatis was a nonpathogen. 3. Search for pathogenic isolates of G. anatis. We collected a significant number of isolates of G. anatis from the field and subjected these isolates to an embryo lethality assay (ELA) previously described for E. coli. On the basis of this assay, G. anatis isolates could be divided into pathogens of high, intermediate, and low virulence. Also, we looked at the genes of many of the new G. anatis isolates and found that the virulence genes roughly corresponds to the ELA results. Based upon the ELA and virulence gene assays, we selected additional G. anatis isolates to determine if these isolates will induce peritonitis lesions similar to those observed in field outbreaks. 4. In November 2007, we attempted to prove that G. anatis is a pathogen for laying hens by intravenous inoculation of a G. anatis strain previously shown to be a pathogen by Danish researchers. The Danish isolate was lethal to embryos and contained appropriate virulence genes. We inoculated 9 chickens with 100 bacteria, 9 chickens with 10,000 bacteria, 10 chickens with 1,000,000 bacteria, and 10 chickens with 100,000,000 bacteria. None of the laying hens inoculated developed clinical illness or lesions of peritonits. PARTICIPANTS: Principal Investigator: Darrell Trampel, DVM, PhD College of Veterinary Medicine Iowa State University [e-mail] [phone] (Telephone) [phone] (FAX) Co-Investigator: Lisa K. Nolan, DVM, PhD Chair, Department of Vet. Microbiology & Preventive Medicine College of Veterinary Medicine Iowa State University [e-mail] [phone] (Telephone) [phone] (FAX) TARGET AUDIENCES: Egg producers Poultry veterinarians Veterinary diagnosticians

Impacts
Peritonitis associated with E. coli causes significant morbidity and mortality in many commercial egg production facilities. Bacteria are always associated with lesions of peritonitis in laying hens. E. coli is almost always present and Gallibacterium anatis is often isolated as well. Results of this research would indicate that peritonitis in laying hens can result from an ascending bacterial infection of the oviduct. In field outbreaks, both E. coli and G. anatis probably reach the peritoneal cavity by that route. However, G. anatis probably plays only a secondary role in the development of peritionitis in laying hens. E. coli appears to be the primary pathogen.

Publications

• No publications reported this period