2. Lecuona, Luis_REC

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Concomitant Administration of GonaCon ™ and Rabies Vaccine in Female Dogs ( Canis familiaris ) in Mexico Luis Lecuona , DVM Agricultural Specialist in Animal Health USDA/APHIS/IS-WS Mexico Luis.Lecuona@aphis.usda.gov ACC&D’s 5 th International Symposium on Non-surgical Contraceptive Methods for Pet Population Control Portland, Oregon, USA. June 20 th – 22 nd , 2013

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Dog Rabies Vaccination and Dog Rabies Cases in Mexico Dog Rabies Vaccination and Human Rabies Cases in Mexico Impact of the Rabies Program in Mexico Lab Studied and Positive Samples in Mexico Rabies PEP in Mexico

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Stabilization of the Canine Population Dog and cat spaying and neutering Voluntary donation Euthanasia To reduce the risk of attacks on humans To Create a Culture of Responsible Owner

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North America Rabies Management Plan Signed at the 8 th NARMP Meeting October , 2008. Atlanta, GA

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To evaluate the safety of updated GonaCon TM formula in Mexico. To evaluate progesterone levels and the titers of antibodies against gonadotropin releasing hormone ( GnRH ). To document any effects of toxicity in domestic female dogs in Mexico. To see that simultaneous applications of GonaCon TM and the parenteral rabies vaccination used in Mexico does not limit the immune response to both treatments. To evaluate the risk of possible local adverse reactions on the application area of updated GonaCon TM formula. OBJECTIVES The number and kind of adverse local reactions is less and in a minor intensity that the reported in previous studies. The new GonaCon TM reduces the progesterone levels and develop antibodies titers against gonadotropin releasing hormone ( GnRH ). GonaCon TM has less adverse effects of toxicity in domestic female dogs in Mexico compared with previous versions. The simultaneous applications of GonaCon TM and the parenteral rabies vaccination in Female dogs in Mexico do not limit the immune response for both products. The updated GonaCon TM formula is not generate local adverse reactions that could suggest discontinuing its use in Mexico . HYPOTHESIS GonaCon TM Study in Mexico

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PROVEZA’s Shelter Facilities. Villa de Tezontepec , Hgo GonaCon TM Study in Mexico

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GonaCon TM Study in Mexico Selection of female dogs for the study (Sep – Nov/2010). Integration of groups – GonaCon TM importation (Oct/10 – Jan/11). Field trial: 3 groups: 61 study days: Daily and weekly clinical evaluation: Sampling procedures on D0, D31 y D61 (Jan - Mar/11). Laboratory Analysis (CBC, BCP, VNA, THR, GnRH ), (Jan - Jul/11) Macro and Microscopic findings evaluation (Mar - Sep/11). Preliminary report integration (Sep - Oct/11). Final Report (Dec/11) Study Phases Group A: Just Rabies Vaccine (RPM). Group B: Just GonaCon TM (LPM). Group C: Rabies Vaccine (RPM) + Gonacon TM (LPM).

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GonaCon TM Study in Mexico Conjugated to a large mollusk hemocyanin protein and emulsified with the adjuvant AdjuVac TM A 0.5 ml dose 500 µg of the GnRH conjugate 21µg of inactivated Mycobacterium avium (Adjuvant) Gentamicin (Life Technologies, Carlsbad, CA) Fungizone (Life Technologies, Carlsbad, CA ) Formulation produced in a clean room to inhibit bacterial and fungal activity. Pre-loaded in 3ml Air- Tite luer -lock syringes (Air- Tite Products, Virginia Beach, VA, USA) Formulation

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Field Trial (61 Days Period) GonaCon TM Study in Mexico

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GonaCon TM Study in Mexico Field Trial (61 Days Period)

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GonaCon TM Study in Mexico 6 female dogs (in houses). Just rabies vaccine. 1 dog with serosanguinous discharge at D58. No Physical alterations, prostration, lesion or limping. 2 female dogs finished the study pregnant. Weight average: D0 - 21.22 kg. ± 10.54: D61 - 21.25 kg ± 10.86. Group A 7 Female dogs (Confined). Just GonaCon TM . 1 st Week - 4 dogs with pain and/or hyperthermia in LPM. 1 dog had puppies during the selection period (11/10); 2 more had puppies during the 1 st week of the study (01/11). 1 dog with a small spot in the application site of GonaCon TM at D16. All female dogs conclude the study with muscle atrophy on the LPM. No prostration or limping. Weight average: D0 - 15.29 kg ± 5.26: D61 - 13.86 kg ± 3.44. Group B 7 Female dogs (Confined). Rabies Vaccine + GonaCon TM . 1 st Week - 2 dogs with hyperthermia in LPM. 1 dog with serosanguinous discharge on D16* and other in D59. 1 dog with a small spot in the application site of GonaCon TM at D16. All female dogs conclude the study with muscle atrophy on the LPM. No prostration or limping. Weight average: D0: 15.74 kg ± 4.62: D61 - 15.93 kg ± 4.14. Group C *Alive at D94, with a new serosanguinous discharge Field Observations (61 Days Period)

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GonaCon TM Study in Mexico Field Trial (61 Days Period)

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GonaCon TM Study in Mexico Blood Sampling Procedures (5 Samples set for each dog/ day)

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Lab Findings Erythroblastopenia Leukocytosis Leukopenia Thrombocytopenia. Polycythemia GonaCon TM Study in Mexico Cell Blood Count (CBC) (Hematocrit, Hemoglobin, Mean Corpuscular Value, Mean Corpuscular Hemoglobin, Leucocytes & Platelets) Group A Group B Group C D0 2 1 1 6 0 D31 3 0 1 1 0 D61 0 0 0 2 2 D0 1 0 7 7 0 D31 0 0 2 1 1 D61 0 0 1 4 3 D0 0 1 0 6 1 D31 0 0 2 0 0 D61 0 0 1 3 2

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GonaCon TM Study in Mexico Blood Chemistry Parameters (BCP) (Glucose, Urea, Creatinine , Uric Acid, Cholesterol & Triglycerides) Lab Findings Hypoglycemia Hyperazotemia Hypercholesterolemia Hypocholesterolemia Hypouremia Group A Group B Group C D0 4 6 1 0 0 D31 1 6 0 0 0 D61 0 5 1 0 0 D0 1 5 0 0 0 D31 1 6 0 2 0 D61 0 7 0 0 0 D0 3 7 0 0 0 D31 0 7 0 2 0 D61 0 7 0 0 2

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GonaCon TM Study in Mexico Progesterone (THR = ng /ml)

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GonaCon TM Study in Mexico Progesterone (THR = ng /ml)

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GonaCon TM Study in Mexico Rabies Antibodies Titers (VNA – FAVN*) *Maximum possible value 13.8 UI/ml

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*Maximum possible value 13.8 UI/ml GonaCon TM Study in Mexico Rabies Antibodies Titers (VNA – FAVN*)

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GonaCon TM Study in Mexico Antibodies Titers Against Gonadotropin Releasing Hormone (GnRH – D x 1,000)

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GonaCon TM Study in Mexico Antibodies Titers Against Gonadotropin Releasing Hormone (GnRH – D x 1,000)

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Progesterone At Day 0, THR levels in Groups A and B were lower than Group C (F=1.64, P=0.222). At Day 31 THR levels in Groups B and C were significantly lower than Group A (F=1.11, P=0.0351), which did not receive GonaCon ™. At Day 61, THR levels in Groups B and C were equal and lower than Group A (F=2.22, P=0.137 ). GonaCon TM Study in Mexico Statistical Findings Rabies VNA titers All dogs were reported to have been rabies vaccinated; dogs in Group B were not revaccinated against rabies. At Day 0, rabies VNA titers in Groups A and C were similar, but lower than Group B (F=2.46, P=0.116). At Day 31, rabies VNA titers in Group B were lower than Group A. Also, Group C titers were lower than Group B (F=3.01, P=0.076). At Day 61, rabies VNA titers in Groups B and C were lower than Group A (F=3.92, P=0.040) . Anti- GnRH antibody titers GonaCon TM was never used in these dogs before this study. At Day 0, no anti- GnRH immune response was detected. At Day 31, anti- GnRH titers among dogs in Groups B and C were similar, but higher than Group A (F=18.19, P=0.0001). The same results were observed on Day 61 (F=10.27, P=0.001) .

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GonaCon TM Study in Mexico Macroscopic Findings Left cardiac hypertrophy (4). Valvular endocarditis (2). Controls Group B Group C Several gastrointestinal parasites diseases in different clinical status: Dipilidium , Strongiloides , Ascaridiasis , Ancylostomiasis (Mucous enteritis in several sections of the intestine). Left cardiac hiyertrophy (5). Left cardiac hypertrophy (2). Slight to severe diffuse hepatosis (1). Chronic perihepatitis (3). Slight to severe diffuse hepatosis (4). Chronic perihepatitis (2). Slight diffuse hepatosis (1). Chronic Perihepatitis (1). Peyer’s patch hyperplasia (1). Peyer’s patch hyperplasia (5). Peyer’s patch hyperplasia (2). Slight to severe chronic interstitial nephritis (3). Nephrosis (1). Slight to severe chronic interstitial nephritis (2). Nephrosis (2). Slight to severe chronic interstitial nephritis (4). Studies in 6 animals. Studies in 5 of 7 animals. Studies in 6 of 7 animals.

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Studies in 6 dogs. Controls Group B Group C Studies in 5 of 7 dogs. Studies in 6 of 7 dogs. Ovary: Increased number of atresic follicles. Important reduction of second, preantrium and third follicles. Basophilic aspect into the tunica ambuginea . Muscle: 10 samples. Group B. No apparent findings(3) * . Moderate and focal chronic granulomatous myositis (1). Diffuse coagulative necrosis (1). Group C. Slight focal chronic granulomatous myositis (2). Slight to severe multifocal necrosis (3). * Samples from application site GonaCon TM Study in Mexico Preliminary Microscopic Findings Ovary: Not apparent findings. Several phases of follicle development and reduced number of atresic follicles. Muscle: Not apparent findings. Hypophysis : Not apparent findings. Hypophysis : Slight diffuse congestion and edema (4). Coagulative necrosis ( Basophílic Cells), More acidophilic cells (1). Hypophysis : Slight diffuse congestion (4). Difuse edema (3). Coagulative necrosis (Basophilic cells), More acidophilic cell (4).

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GonaCon TM Study in Mexico Microscopic Findings Hypophysis ( Pituitary Gland ) Diffuse congestion and edema Normal Coagulative necrosis

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GonaCon TM Study in Mexico Microscopic Findings Ovary Reduction of second, preantrium and third follicles Normal

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GonaCon TM Study in Mexico Microscopic Findings Muscle Normal Severe multifocal necrosis Chronic granulomatous myositis Diffuse Coagulative necrosis

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Less and minor intensity of adverse local reactions than in previous formulation used. GonaCon TM reduces progesterone levels and develop antibodies titers against GnRH . GonaCon TM had not generated adverse effects of toxicity. Simultaneous application of GonaCon TM and rabies vaccine did not limited the immune response. GonaCon TM Study in Mexico Conclusions and Recommendations

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GonaCon TM generated NOT measured muscle atrophy. Observed chronic granulomatose myositis and the diffuse coagulative necrosis were not limping source. No dejection, ulceration or paralysis were observed. A new study in owned female dogs during a long period (2 years) could be useful to measure the real possibilities to use the product in Mexico . GonaCon TM Study in Mexico Conclusions and Recommendations

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All dogs were clinically evaluated and reported in several stages of metabolic alterations (hepatic and renal function). It looks like these metabolic problems are not related with the study but is needed more analysis about this condition . A full report of macro and microscopic findings in the collected organs and tissues will be useful. All animals into the study had previously received rabies vaccine. We have no chances to get non vaccinated dogs in the field. GonaCon TM Study in Mexico Special Considerations – Lessions Learned

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The FAVN test were adjusted for the top of 13.8 UI/ml. It is needed to compare FAVN and RIFFT titers to better understand rabies antibodies responses. On the last days was detected the muscle atrophy. We had not a previously established strategy to make systematic measurements, but observations were recorded. During the study 2 female dogs into the Group B, and 1 into the Group C, that were pregnant and had puppies in that time are kept alive and they are clinically healthy. GonaCon TM Study in Mexico Special Considerations – Lessions Learned

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Dr. Fernando Vargas Pino MVZ. Verónica Gutiérrez Cedillo Undrerdirection of Zoonosis . DGAPP/CENAPRECE/SALUD Lic . Pedro Luis Noble Monterrubio Dra. Ana María Tavares Jiménez MVZ Erick José Canales Vargas MVZ Luis René Gress Ortega MVZ José Manuel Méndez García Health Services in Hidalgo State MVZ. Patricia Beatriz García Reyna MVZ. Juan Ocampo López Academic Area of Veterinary Medicine and Zootechnics Animal and Plant Science Institute/UAEH Charles E. Rupprecht, PhD Rabies Section CDC, Atlanta, GA. EUA Scott Bender, DVM Navajo Nation Veterinarian Program. Navajo Nation Department of Agriculture Lowell A. Miller, PhD Kathleen A. Fagerstone, PhD NWRC/USDA/APHIS/WS Dennis Slate, PhD Luis Lecuona, DVM USDA/APHIS/WS GonaCon TM Study in Mexico Authors and Institutions

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GonaCon TM Study in Mexico REPORT UNDER PUBLICATION PROCESS 5th International Symposium on Non-Surgical Contraceptive Methods of Pet Population Control

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Thank You! Luis Lecuona , DVM Agricultural Specialist in Animal Health USDA/APHIS/IS-WS Mexico Luis.Lecuona@aphis.usda.gov

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