1. Large Scale Immune Profiling of Infected Humans & Goats Reveals Differential Recognition of Brucella melitensis Antigens Video by Kathryn Dullard 17150258

2. Brucellosis Zoonotic infectious disease endemic in regions especially in Peru and the Middle East The reservoirs of Brucella melitensis: goats and sheep Brucellosis typically begins with a flu-like illness Inflammation of the liver and spleen, &gastrointestinal or respiratory symptoms can occur. Commonly used screening tests Problematic for differentiate between vaccination & infection in goats Problematic in differentiating acute, chronic and relapsing forms of brucellosis in humans living in endemic regions

3. Hypothesis The immune response to B.melitensis infection of the natural reservoir host (goat) & the accidental host (human) is similar despite potentially different routes of infection. Current Knowledge of Protein Antigens Relatively small number of immunogenic Brucella abortus protein – recognized by cattle, sheep & mice. Limited studies on recognition of Brucella melitensis in goats & sheep. No individual antigen has proven to be of sufficient diagnostic utility

4. Materials & Methods Current serological assays to diagnose human brucellosis are based on detection of agglutinating anti-LPS antibodies. Protein microarray A set of 1406 Open Reading Frames from B. melitensis, 1009 antigens selected based on the prediction of protein localization sites, and 397 Open Reading Frames were randomly selected. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. Data analysis Serodominant antigens = antigens with mean signal intensity > mean + 2 standard deviations above the negative controls.

5. B. melitensis Serum Samples Human sera: 42 serums = Blood Culture +, Rose Bengal + 18 serums = Blood Culture -, Rose Bengal + 18 serums = Blood Culture -, Rose Bengal – 57 serums = Control Goat sera: Positive and negative (uninfected) controls from a previously conducted vaccine safety study 15 serum samples = specific pathogen-free goat flock as an additional negative control Dose & Route of Inoculum: Humans infected by ingestion of infected goat’s milk products containing a small inoculum the goats were infected with a high dose (107 CFU) of B. melitensis strain 16M via the conjunctiva.

6. Goat Serodiagnostic Antigens 49 serodominant antigens were identified among 1406 antigens tested. 18 of those antigens were serodiagnostic The remaining 31 cross reactive antigens reacted similarly among all goats samples Human Serodiagnostic Antigens 33 serodominant identified from the 1406 tested 13 of those antigens were serodiagnostic 20 cross reactive antigens reacted similarly amoung all human samples Validation of serodiagnostic accuracy was conducted with immunostrips

7. Comparing Antigenic Proteins among Humans & Goats 2 common serodiagnostic antigens between humans and goats Top Antigen = Bp26 protein 26KD periplasmic immunogenic protein Immunodominant antigen in infected cattle, sheep, goats & humans 2 nd Antigen = Protease DO (HtrA) Antigens useful for brucellosis diagnosis 11 exclusively for humans 16 exclusively for goats 8 common cross reactive antigens 12 exclusively for humans 23 exclusively for goats

8. The natural reservoir host (goat) & the accidental host (human) have fundamentally different immune responses against B. melitensis

9. Discussion Identified several novel serodiagnostic antigens specific for human infection; Useful for the practical development of diagnostics Current assays – Identification of antibodies to LPS Brucella LPS is cross-reavtive with several other species; E. coli, Y. enterocolitica, F. tularensis Identification of diagnostic protein antigens may facilitate the development of more specific serodiagnostic assays Serodiagnostic Antigens for Humans & Goats

10. Serodiagnostic Antigens Specific for Humans Identified several novel serodiagnostic antigens specific for human infection; Useful for the practical development of diagnostics Current assays – Identification of antibodies to LPS Brucella LPS is cross-reavtive with several other species; E. coli, Y. enterocolitica, F. tularensis Identification of diagnostic protein antigens may facilitate the development of more specific serodiagnostic assays

11. Serodiagnostic Antigens Specific for Goats

12. Conclusion Seroreactive & metabolic pathways expressed during infection in these hosts. B. melitensis utilization of peptides and amino acids for growth during infection. Immune responses may be related to species further comment on this possibility is limited by the lack of available data. Both dose and route of inoculum may have contributed to the differential antigen recognition Humans are assumed to have been infected by ingestion of infected goat’s milk products containing a small inoculum the goats were infected with a high dose (107 CFU) of B. melitensis strain 16M via the conjunctiva. Protein expression differences by different strains of B. melitensis unlikely but must be considered as well.