BUi_Final Presentation_SDZSP
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The document summarizes a Salmonella outbreak in lorikeets at the San Diego Zoo Safari Park from October 4-20, 2014. It provides details on the outbreak timeline and affected birds. It also reviews immunology concepts, describes how titers can be used to monitor the immune response and vaccine effectiveness, and outlines the park's future monitoring plan which includes monthly blood sampling and booster vaccines every six months.
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BUi_Final Presentation_SDZSP
- 1. Christine T. Bui, MBA Advisor: Dr. Meredith Clancy April 22, 2015 San Diego Zoo Safari Park
- 2. Recap of Salmonella outbreak Outbreak Protocol Immunology Review Titers Preparation Testing and Interpretation Future monitoring Overview
- 3. Outbreak Timeline: Oct 4 – Oct 20, 2014 Offending Pathogen: Salmonella ser. Typhimurium Lorikeet Statistics: Total # birds in L.L on Oct 1st = 71 birds No Clinical Signs = 56 birds Symptomatic but survived = 7 birds (+/- 3 birds) Died/Euthanized = 8 birds Outbreak Recap
- 4. Outbreak Protocol If Salmonella spp. confirmed in single bird Test 20% of flock Consider batch samples for PCR & Enterics Panel Monitor flock If S. Typhimurium is genotyped & 3-5 sick birds Close exhibit Split Lorikeets into groups and quarantine MDB diagnostics & treatment pending lab results Sample environment and food prep areas• Testing 10-15% of population monthly • Hygiene/Husbandry protocols
- 8. Used to determine Exposure to antigen Strength of immune response If a booster vaccination is needed If a previous vaccine helped the immune system against the antigen Abnormal results Autoimmune disease Failure of vaccine Immune deficiency Viral infection Titers
- 9. Obtain pre-vaccination blood sample Vaccination Booster in 2-3weeks Obtain post-vaccination blood sample in 2-3 weeks Preparing for Titers
- 10. Titer Testing Image reference: Kimball, John. Kimball’s Biology Pages
- 11. Initial Salmonella Titers 0 2 4 6 8 10 12 14 16 18 negative 1:10 1:20 1:40 1: 80 1: 160 1: 320 1: 640 1: 1280 NumberofLorikeets Initial Salmonella Titer Values Number of Lorikeets vs. Initial Salmonella Titers Sample collection date: 11 Dec ‘14- 25 Feb ‘15
- 12. Salmonella Titers 0 1 2 3 4 5 6 7 8 9 Log10 Lorikeet ID Initial Titer Titer at vxn
- 13. Salmonella Titers 0 20 40 60 80 100 120 140 813112 813258 814209 812306 813032 812307 813269 813031 812271 Log10 Lorikeet ID Initial Titer Titer at vxn
- 14. Future Projections Effective Vaccine Program Ineffective Vaccine Program 0 2 4 6 8 10 12 14 16 18 20 NumberofLorikeets Titer Values 0 2 4 6 8 10 12 14 16 NumberofLorikeets Titer Values
- 15. Obtain blood sample from 10% of flock every 1-2 months to follow titers Booster every 6 months Unless titers suggest otherwise Fecal PCR for any sick bird Future monitoring
- 16. Recap of Salmonella outbreak Outbreak Protocol Immunology Review Titers Preparation Testing and Interpretation Future monitoring Summary
- 17. Abdolvahab, Farzan and Robert M. Friendship. “A clinical field trial to evaluate the efficacy of vaccination in controlling Salmonella infection and the association of Salmonella-shedding and weight gain in pigs.” Canadian Journal of Veterinary Research 2010. 74:258-263 “Autogenous vaccines”. Hygieia Biological Laboratories 2014 http://www.hygieialabs.com/autogenous.html Boseret, Geraldine, L. Bertrand, JG Mainil, E Thiry, and C Saegerman. Zoonoses in pet birds: review and perspectives” Journal of Veterinary Research 2013, 44 (1):36-53 Gast, RK and CW Beard. “Serological detection of experimental Salmonella enteritidis infections in laying hens”. Avian Diseases. 1990 Jul- Sep;34(3):721-8. Henochowicz, Stuart. “Antibody Titers” Maryland. 9 April 2015http://www.nlm.nih.gov/medlineplus/ency/article/003333.htm House, JK, MM Ontiveros, NM Blackmer, EL Dueger, JB Fitchhorn, GR McArthur, and BP Smith. “Evaluation of an autogenous Salmonella bacterin and a modified live Salmonella serotype Choleraesuis vaccine on a commercial dairy farm.” American Journal of Veterinary Research 2001. Dec 62(12):1897-1902. Kehoe, Spencer. “Outbreak Protocol- Lorikeet Landing”. San Diego Zoo Safari Park. Oct 2014. Mastroeni, P, JA Chabalgoity, SJ Dunstan, DJ Maskell, and G Dougan. “Salmonella: Immune Responses and Vaccines”. The Veterinary Journal 2000. 161:132–164 McDonough, Patrick L, RH Jacobson, JF Timoney, A Mutalib, DC Kradel, Y Chang, S Shin, DH Lein, S Trock, K Wheeler. “Interpretations of Antibody Responses to Salmonella enterica Serotype Enteritidis gm Flagellin in Poultry Flocks Are Enhanced by a Kinetics-Based Enzyme- Linked Immunosorbent Assay.” Clinical and Diagnostic Laboratory Immunology 1998 Jul; 5(4): 550–555. McDonough, Patrick L and Sue Stehman. “Usefulness of Vaccination and Vaccine for Control of Salmonellosis”. New York State Cattle Health References
- 18. McGuirk, Sheila and Simon Peek. “Salmonellosis in Cattle: a Review”. American Association of Bovine Practitioners. 36th Annual Conference Proceedings. Ohio Sept 2003 Parham, Peter. The Immune System. Garland Science. 3rd Ed 2009. Randall, LP, DJ Eaves, SW Cooles, V Ricci, A Buckley, MJ Woodward, and LJV Piddock. “Fluoroquinolone treatment of experimental Salmonella enterica serovar Typhimurium DT104 infections in chickens selects for both gyrA mutations and changes in efflux pump gene expression.” J. Antimicrob. Chemother. August 2005. 56(2): 297-306. Randall, LP, SW Cooles, NC Coldham, KS Stapleton, LJV Piddock, MJ Woodward. “Modification of Enrofloxacin treatment regimens for poultry experimentally infected with Salmonella enterica serovar Typhimurium DT104 to minimize selection of resistance.” Antimicrob. Agents Chemother. December 2006. 50(12):4030-4037 Sareyyuopglu, B, AC Ok, Z Cantekin, AH Yardimci, M Akan, and A Akcay. “Polymerase chain reaction detection of Salmonella spp in fecal samples of pet birds.” Avian Diseases. Mar 2008. 52(1):163-167 Sheela, Ruby R, U Babu, J Mu, S Elankumaran, DA Bautista, RB Raybourne, RA Heckert, W Song. “Immune Responses against Salmonella enterica Serovar Enteritidis Infection in Virally Immunosuppressed Chickens.” Clinical and Diagnostic Laboratory Immunology. July 2003. 10(4): 670-679. Smith KE, F Anderson , C Medus, F Leano, and J Adams. “Outbreaks of Salmonellosis at elementary schools associated with dissection of owl pellets.” Vector Zoon Dis 2005. 5:133–136. Tran, Thi QL, S Quessy, A Letellier, A Desrosiers, M Boulianne. “Immune response following vaccination against Salmonella Enteritidis using 2 commercial bacterins in laying hens.” Canadian Journal of Veterinary Research. July 2010. 74 (3): 185-192 “Vaccine Titer Monitoring” Michigan State University. Diagnostic Center for Population and Animal Health. Jan 2015. http://www.animalhealth.msu.edu/ClientEducation/MKTG.CARD.VIROLOGY.001.PDF Ward, MP, JC Ramer, J Poudfoot, MM Garner, C Juan-Salles, and CC Wu. “Outbreak of Salmonellosis in a Zoologic Collection of Lorikeets and Lories (Trichoglossus, Lorius, and Eos spp.)“ Avian Diseases 47(2): 493-498 References Continued
- 19. Questions?
- 20. THANK YOU All Hospital Techs & Keepers & Lab folk, Docs, Collection Managers, Animal Care Team, and the many wonderful people and animals I’ve met along the way! Oh yeah! Who is this girl?
Editor's Notes
- Today I’m going to recap the Salmonella Outbreak the Safari Park had back in October and the outbreak protocol we’re instituting. I’m then going to review the immune system and the role vaccines play in order for everyone to understand the aims of the Salmonella vaccines program we’ve initiated. Now, in order to determine the effectiveness of our vaccine, I’ll discuss titers (how they’re prepared, how we test for them and how we interpret results). Finally, I’ll wrap up with future monitoring parameters we can institute.
- Back in October between the 4-20, we had a total of 8 birds that either died or were euthanized due to infection with Salmonella Typhimurium. Of the 71 birds we had in Lorikeet Landing, 7 of these birds were symptomatic showing clinical signs such as lethargy, fluffed, and dyspnea. 56 of the birds showed no outward clinical signs. Looking back at this outbreak, the overall morbidity rate was 21% and the mortality rate was 11%. Reference: Kehoe, Spencer. “Outbreak Protocol- Lorikeet Landing”. San Diego Zoo Safari Park. Oct 2014. LORIKEET STATISTICS--------------------------- Case Definition: Animals showing clinical signs during the month of October, 2014 Morbidity (Incidence Rate): 21-25% Morbidity 15-18 cases with clinical symptoms / 71 individuals at risk at start of October Mortality Measure (cause-specific): 11% Mortality 8 Salmonella deaths / 71 individuals at risk at start of October Case-Fatality Measure: 44-53% case-fatality 8 deaths / 15-18 cases of disease in October 2014
- As part of this outbreak, we came up with a protocol for future use in case of another outbreak. [read slide] The most important aspect of this protocol is to [animation] testing 10-15% of the population monthly and revising hygiene/husbandry protocols as needed. MDB Diagnostics for SDZSP: CBC/Chemistry, fecal cytology + gram stain in house, Fecal PCR, culture and sensitivity Reference: Kehoe, Spencer. “Outbreak Protocol- Lorikeet Landing”. San Diego Zoo Safari Park. Oct 2014. Randall, LP, DJ Eaves, SW Cooles, V Ricci, A Buckley, MJ Woodward, and LJV Piddock. “Fluoroquinolone treatment of experimental Salmonella enterica serovar Typhimurium DT104 infections in chickens selects for both gyrA mutations and changes in efflux pump gene expression.” J. Antimicrob. Chemother. August 2005. 56(2): 297-306. Randall, LP, SW Cooles, NC Coldham, KS Stapleton, LJV Piddock, MJ Woodward. “Modification of Enrofloxacin treatment regimens for poultry experimentally infected with Salmonella enterica serovar Typhimurium DT104 to minimize selection of resistance.” Antimicrob. Agents Chemother. December 2006. 50(12):4030-4037
- let’s do a quick review of immunology. The immune system is divided into two major components: the Innate system and Adaptive system. Innate is a non-specific response to antigen or pathogens and does not involve immune memory. (Things like our skin, surface barriers and local inflammatory response are example components of this system) Adaptive is responsible for pathogen and antigen specific responses and results in immune memory. (There is a lag time between exposure and maximal response) Adaptive system is further divided into: Humoral component: involves antibody production Cell mediated component: involves antigen specific T- cell production, phagocytosis, cytokine release and the creation of immune memory *[animation] This is where vaccines plays a role
- This image depicts what our adaptive immune system does in the face of a novel pathogen. When first exposed to an antigen/pathogen, it takes our immune system about a week to mount a response; after which the number of circulating T-cells and antibodies produced increase and then decrease over time as the inciting pathogen is no longer a cause of concern. We’ll have a certain low level of circulating antibodies that contribute to our immune memory. When the immune system is challenged a second time with the same pathogen, it’s able to amount a faster immune response. References: Parham, Peter. The Immune System. Garland Science. 3rd Ed 2009.
- Where vaccines come in to play is essentially they help keep antibody levels high ( and therefore immune memory is also high) such that when a second exposure event occurs, our immune system can respond as quickly as possible. Essentially what we’re trying to do with our Salmonella vaccine is create a stronger adaptive immunity in the flock, such that if we get another exposure to Typhimurium, our birds will be better protected in hopes that it will reduce severity of disease and decrease mortality rate. How to we measure antibody levels in the body? {slide} Reference: Parham, Peter. The Immune System. Garland Science. 3rd Ed 2009. McGuirk, Sheila and Simon Peek. “Salmonellosis in Cattle: a Review”. American Association of Bovine Practitioners. 36th Annual Conference Proceedings. Ohio Sept 2003
- This segways us into Titers. Titers measure the amount of antibodies in the blood to a specific pathogen. 1) They can be used to determine…. Normal titer values will vary on specific values for immunization. For example certain pathogens are well studied, such as canine distemper and parvovirus. We know from research that if a dog’s distemper titer is below 1:32, and a parvovirus titer is below 1:80, it’s recommended that the dog be boostered to keep immune memory and circulating antibodies high. 2) abnormal results can infer… References: Henochowicz, Stuart. “Antibody Titers.” Maryland 9 April 2015. http://www.nlm.nih.gov/medlineplus/ency/article/003333.htm “Vaccine Titer Monitoring.” Michigan State University. Diagnostic Center for Population and Animal Health. Jan 2015. http://www.animalhealth.msu.edu/ClientEducation/MKTG.CARD.VIROLOGY.001.PDF
- In order to prepare ourselves for assessing Titers, we have to accomplish several steps. 1) Obtained pre-vxn blood sample. This will help us determine our ‘normal’ titer 2) Vaccinate 3) Booster 2-3 wks: one study in chickens noted antibody titers peaking at 1-2 weeks post-inoculation and then steadily declined over time although they were still seropositive 10 weeks out. Another study noted antibody levels persisted 3-34 wks post vaccination +booster. 4) Obtain post vxn sample- and then compare it to pre-vxn for interpretation *Since we discovered a specific serovar was causing our outbreak, the best vaccine to give our flock was an autogenous one vs commercially available salmonella vaccines. Autogenous vaccines are made fr the serotype of bacteria isolated from the flock. Because the vaccine is highly specific for the serotype of bacteria, it can evoke a serotype specific antibody response that may provide more efficient or quicker defense against the pathogen. Other information: *fecal PCR, fecal culture Reference: “Autogenous vaccines”. Hygieia Biological Laboratories 2014 http://www.hygieialabs.com/autogenous.html Gast RK1, Beard CW. Serological detection of experimental Salmonella enteritidis infections in laying hens. Avian Dis. 1990 Jul-Sep;34(3):721-8. McDonough, Pat and Sue Stehman. “Usefulness of Vaccination and Vaccine for Control of Salmonellosis”. Ney York State Cattle Health Assurance Program. pp1-4 Tran, Thi QL, S Quessy, A Letellier, A Desrosiers, M Boulianne. “Immune response following vaccination against Salmonella Enteritidis using 2 commercial bacterins in laying hens.” Canadian Journal of Veterinary Research. July 2010. 74 (3): 185-192
- Titer- highest dilution in which antibodies are detected. The higher the dilution series, the more antibodies the bird has. [Explain how serial dilutions are done] set up multiple vials with 9mls of dilutant; add 1 ml of sample; mix, take 1ml of that sample and put into next vial…so on and so on. *reference image: Kimball, John. Kimball’s Biology Pages. “Titers”. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Titer.html 12 May 2011
- N= 65 Lorkieets; Negative n= 17; 1: 10 n=8; 1:20, 1:40 n=9; 1:80 n=8; 1:160, 1:320 n=6; 1:640, 1:1280 n=1 Dates of sample collection: 11 Dec 2014 through 25 Feb 2015. Interpretation: 26% of the birds initially had negative titers- no antibodies were detected so they were not exposed, or not exposed at a high enough level to amount an detectable immune response. 74% of the birds had detectable titers=> these birds had exposure to Salmonella. As I noted earlier, normal titer values will vary on specific values for immunization. We do not yet know what a ‘normal’ titer is for our flock. We need more data from post-vaccine samples and continued surveillance. With continued surveillance, we may some day also know what titer level is considered ‘protective’ for our flock. **** SAMPLE TITER VALUES IN CHICKENS*** 1:201:60, 1:60 cut off gave best K-ELISA sen/spe, >400 Ideally in an effective vaccine, you want a 4 fold increase in titer levels References: McDonough, Patrick L, RH Jacobson, JF Timoney, A Mutalib, DC Kradel, Y Chang, S Shin, DH Lein, S Trock, K Wheeler. “Interpretations of Antibody Responses to Salmonella enterica Serotype Enteritidis gm Flagellin in Poultry Flocks Are Enhanced by a Kinetics-Based Enzyme-Linked Immunosorbent Assay.” Clinical and Diagnostic Laboratory Immunology 1998 Jul; 5(4): 550–555. Sheela, Ruby R, U Babu, J Mu, S Elankumaran, DA Bautista, RB Raybourne, RA Heckert, W Song. “Immune Responses against Salmonella enterica Serovar Enteritidis Infection in Virally Immunosuppressed Chickens.” Clinical and Diagnostic Laboratory Immunology. July 2003. 10(4): 670-679. Tran, Thi QL, S Quessy, A Letellier, A Desrosiers, M Boulianne. “Immune response following vaccination against Salmonella Enteritidis using 2 commercial bacterins in laying hens.” Canadian Journal of Veterinary Research. July 2010. 74 (3): 185-192
- Interpretation: Yellow bar that is higher than initial titer signifies bird is amounting an immune response to salmonella. The 2 week post vaccine titers we’ll obtain is then what we can use to compare to the yellow bars. We’d like at least a 4 times increase in titers in order to obtain sero-conversion in the flock. Sero-conversion does not always correlate with acquired resistance to infection, but rather a measure of active immunity. it may help us in establishing normal or protective titers. Reference: Mastroeni, P, JA Chabalgoity, SJ Dunstan, DJ Maskell, and G Dougan. “Salmonella: Immune Responses and Vaccines”. The Veterinary Journal 2000. 161:132–164
- In an effective vaccination program, we’d want to see a trend shifting the titer graph towards the right; meaning more birds have higher antibody levels, better immune memory, and they’re better protected. If we concluded that for our flock titers >=1:80 is considered protective, then our vaccine would be 80% effective. And any bird we test whose titer is < 1:80 should be boostered. An ineffective vaccine may give us titers that look like (graph on right); Titers are shifted to the left, where more birds have negative to low titer values. (In this made up scenario 75% of birds have titers <1:80, leaving only 25% of the flock with antibody levels adequate for protective immunity). If our titer results suggest abnormal titer values, that could mean: we have an ineffective vaccine maybe our flock is more sensitive to Salmonella and are unable to amount an appropriate immune response The duration of effect of our vaccine is <2-3 weeks, therefore we’re missing peak titer values QUESTION: How can we tell if it’s our vaccine or birds? It’s difficult to determine. One study of autogenous salmonella vaccine had no effect on fecal shedding when compared to MLV vaccine in cattle Pig study showed vaccinated pigs decreased amount of shedding over entire production stage. Reference: House JK, Ontiveros MM, Blackmer NM, Dueger EL, Fitchhorn JB, McArthur GR, Smith BP. “Evaluation of an autogenous Salmonella bacterin and a modified live Salmonella serotype Choleraesuis vaccine on a commercial dairy farm.” Am J Vet Res. 2001 Dec;62(12):1897-902. Abdolvahab, Farzan and Robert M. Friendship. “A clinical field trial to evaluate the efficacy of vaccination in controlling Salmonella infection and the association of Salmonella-shedding and weight gain in pigs.” Canadian Journal of Veterinary Research 2010. 74:258-263
- ? Sensitivity/specificity of salmonella fecal PCR? It’ll be interesting to see the titers post vaccination and from continued monitoring.
- In Summary, I’ve briefly reviewed the Salmonella outbreak that occurred back in October 2014. I have reviewed components of the immune system and the role vaccines play in it. These reviews helped us to understand our thought process behind the creation of a Salmonella vaccine and how we’re using titers to monitor our flock.
- Salmonella titers in chickens