1. Investigation of Chronic Disease in a Captive Mara Colony Mexico

2. Index Case July 21, 2001 July 21, 2001 2 year old captive born male mara (Dolichotis patagonum) died shortly after presenting with weakness and anorexia 2 year old captive born male mara (Dolichotis patagonum) died shortly after presenting with weakness and anorexia

3. Index Case-con’t Animal had disseminated fungal infection affecting mostly the liver, adrenal and lymph nodes Animal had disseminated fungal infection affecting mostly the liver, adrenal and lymph nodes Cytology evaluation of the lymph node, adrenal and liver revealed numerous cytoplasmic yeast-like organisms Cytology evaluation of the lymph node, adrenal and liver revealed numerous cytoplasmic yeast-like organisms 2-4 um in diameter 2-4 um in diameter Clear halo surrounding them Clear halo surrounding them In the cytoplasm of phagocytic cells and adrenocortical cells In the cytoplasm of phagocytic cells and adrenocortical cells Gomori’s methenamine silver stain positive Gomori’s methenamine silver stain positive Histoplasma capsulatum var capsulatum was isolated from spleen Histoplasma capsulatum var capsulatum was isolated from spleen

4. Case Confirmation Gross necropsy findings Gross necropsy findings Histological findings Histological findings Other testing Other testing Special stains Special stains Transmission electron microscopy Transmission electron microscopy Organism isolation Organism isolation Exoantigen test Exoantigen test Immunohistochemistry Immunohistochemistry

5. Establishing Endemic Levels Intradermal testing Intradermal testing Common test in humans and equine Common test in humans and equine Test location on abdomen Test location on abdomen 18/29 animals negative at 72 hours 18/29 animals negative at 72 hours No clinical, hematolgical, radiographic or immunologic evidence of histoplasmosis in these animals No clinical, hematolgical, radiographic or immunologic evidence of histoplasmosis in these animals No prior cases of histoplasmosis in this collection No prior cases of histoplasmosis in this collection

6. Is There An outbreak? There is no evidence of histoplasmosis previous to these cases in the entire collection There is no evidence of histoplasmosis previous to these cases in the entire collection Is there the probability that more animals in the mara colony could have been exposed ot the disease? Is there the probability that more animals in the mara colony could have been exposed ot the disease? Maras use dens dug in the ground, each den is used several times by different females Maras use dens dug in the ground, each den is used several times by different females The intradermal testing, that is the only practical way to detect positive animals, was negative. The intradermal testing, that is the only practical way to detect positive animals, was negative.

7. Descriptive Epidemiology Herd history Herd history Herd history Herd history Environmental factors Environmental factors Environmental factors Environmental factors

8. Herd History Mara colony at park for >20 years Mara colony at park for >20 years 1997: 5 animals from Argentina added to group 1997: 5 animals from Argentina added to group 1 male, 4 females 1 male, 4 females Reproduction good since introduction (66 births) Reproduction good since introduction (66 births) During time of the index case During time of the index case Herd composed of 29 animals Herd composed of 29 animals

9. Environmental Factors Diet Diet Water ad libitum Water ad libitum Oranges, carrots, chopped alfalfa and rabbit pellets Oranges, carrots, chopped alfalfa and rabbit pellets Enclosure Enclosure 120m 2 120m 2 Fenced with 1.20m high wire mesh and bamboo on one side Fenced with 1.20m high wire mesh and bamboo on one side Sandy substrate Sandy substrate Often see squirrels (Spermophilus variegatus), Mice (Mus musculus) and various birds inside the enclosure. Often see squirrels (Spermophilus variegatus), Mice (Mus musculus) and various birds inside the enclosure. Often see opossum (Didelphis virginiana), ringtail cat (Bassariscus astutus) and stray cats (Felis domesticus) inside the park. Often see opossum (Didelphis virginiana), ringtail cat (Bassariscus astutus) and stray cats (Felis domesticus) inside the park. Several wild colonies of insectivorous bats (Tadarida brasiliensis) Several wild colonies of insectivorous bats (Tadarida brasiliensis)

10. Subsequent Cases April 25, 2002 April 25, 2002 Died after presenting with progressive weight loss Died after presenting with progressive weight loss Severe granulomatous typhlocolitis and moderate granulomatous gastrohepatic lymphadenitis Severe granulomatous typhlocolitis and moderate granulomatous gastrohepatic lymphadenitis Similar organisms in the cytoplasm of macrophages Similar organisms in the cytoplasm of macrophages

11. Case #2 Gross necropsy findings Gross necropsy findings Thickening of the large intestinal mucosa Thickening of the large intestinal mucosa Watery to mucoid small intestinal contents Watery to mucoid small intestinal contents Histopathology Histopathology Severe, chronic grnaulomatous and fibrosing hepatitis and adrenalitis with foci of necrosuppurative inflammation Severe, chronic grnaulomatous and fibrosing hepatitis and adrenalitis with foci of necrosuppurative inflammation Granulomatous typhlocolitis Granulomatous typhlocolitis Adrenocortical cells had abundant similar organisms in their cytoplasm with no associated lesion Adrenocortical cells had abundant similar organisms in their cytoplasm with no associated lesion Histoplasma capsulatum var. capsulatum was isolated from the liver and adrenal gland. Histoplasma capsulatum var. capsulatum was isolated from the liver and adrenal gland.

12. Is This An Outbreak? Retrospective review of 54 animals revealed no other lesions compatible with Histoplasmosis Retrospective review of 54 animals revealed no other lesions compatible with Histoplasmosis Previously non-tested animals were tested with high-dose antigen testing Previously non-tested animals were tested with high-dose antigen testing All animals negative All animals negative Investigate immune status of animals and contamination status of environment Investigate immune status of animals and contamination status of environment

13. Immune Status and Environment No previous evidence of histoplasmosis in this mara colony No previous evidence of histoplasmosis in this mara colony High probability that more animals in the colony could have been exposed to disease High probability that more animals in the colony could have been exposed to disease Intradermal testing is the only practical way to test these animals Intradermal testing is the only practical way to test these animals questionable accuracy questionable accuracy Immunological status of the colony would contribute to presence of disseminated histoplasmosis in more animals Immunological status of the colony would contribute to presence of disseminated histoplasmosis in more animals

14. Hypotheses Unusual susceptibility of this species to the disease Unusual susceptibility of this species to the disease Low immunological status of the group Low immunological status of the group Predisposing them to disease Predisposing them to disease High environmental contamination High environmental contamination

15. Investigate Hypothesis Review literature for similar cases Review literature for similar cases Review literature for similar cases Review literature for similar cases Environmental Investigation Environmental Investigation Environmental Investigation Environmental Investigation

16. Literature Review No reports of disseminated histoplasmosis in maras or any rodent species No reports of disseminated histoplasmosis in maras or any rodent species Report of a localized mucocutaneous histoplasmosis in a rabbit Report of a localized mucocutaneous histoplasmosis in a rabbit Reports of histoplasmosis affecting the skin and lymph nodes of Badgers in Europe Reports of histoplasmosis affecting the skin and lymph nodes of Badgers in Europe

17. Environmental Investigation Environmental sampling of mara areas Environmental sampling of mara areas No growth No growth Mice inoculated with extracts of environmental samples DID seroconvert Mice inoculated with extracts of environmental samples DID seroconvert Demonstrates presence of organism in the enclosure Demonstrates presence of organism in the enclosure 2 bat colonies in the park were sampled 2 bat colonies in the park were sampled No culture growth No culture growth NO seroconversion in mice NO seroconversion in mice

18. Environmental Investigation Climatic conditions of park does not promote the proliferation of the fungus Climatic conditions of park does not promote the proliferation of the fungus Sandy substrate, low humidity, lack of bird or bat droppings Sandy substrate, low humidity, lack of bird or bat droppings But indirect evidence of fungal presence was found But indirect evidence of fungal presence was found Bats play an important role in fungal dissemination Bats play an important role in fungal dissemination Abundance of insectivorous bats in park Abundance of insectivorous bats in park Other carnivorous wild animals in the park could disseminate the fungus Other carnivorous wild animals in the park could disseminate the fungus Serology is pending Serology is pending

19. Treatment and Intervention Disinfect Disinfect Foot baths with phenolic base products Foot baths with phenolic base products Dedicated tools to affected areas Dedicated tools to affected areas 80cm of dirt removed from enclosure and replaced 80cm of dirt removed from enclosure and replaced Disinfection of holding area Disinfection of holding area Communicate to staff Communicate to staff Provided with masks Provided with masks Review of personal hygiene Review of personal hygiene Quarantine Quarantine All maras moved to quarantine All maras moved to quarantine

20. Long Term Control of immune status of the colony Control of immune status of the colony Review sex ratio and demographics Review sex ratio and demographics Review genetics Review genetics Monitor wild bat colonies within the park Monitor wild bat colonies within the park

21. References Chavez-Tapia D.B., R. Vargas-Yañes, G. Rodríguez-Arellanes, G. R. Peña-Sandoval, J. J. Flores-Estrada, M Reyes-Mo0ntes, M. L. Taylor. 1998. El murciélago como reservorio y responsable de la dispersión de Histoplasma capsulatum en la naturaleza. Rev. Inst. Nal. Enf. Resp. Mex. 11(3):187-191 Chavez-Tapia D.B., R. Vargas-Yañes, G. Rodríguez-Arellanes, G. R. Peña-Sandoval, J. J. Flores-Estrada, M Reyes-Mo0ntes, M. L. Taylor. 1998. El murciélago como reservorio y responsable de la dispersión de Histoplasma capsulatum en la naturaleza. Rev. Inst. Nal. Enf. Resp. Mex. 11(3):187-191 Kapustin N. Kanitz C. and Muech T. 1999. Identification of a retrovirus in Bennett`s (Macropus rufrogriseus frutica) and Dama (Tammar) (Macrophus eugenni) Wallabies. Proc. Am. As. Zoo Vet. Pp; 262 Kapustin N. Kanitz C. and Muech T. 1999. Identification of a retrovirus in Bennett`s (Macropus rufrogriseus frutica) and Dama (Tammar) (Macrophus eugenni) Wallabies. Proc. Am. As. Zoo Vet. Pp; 262 Morita T., M. Kishimoto, A. Shimada, Y. Matsumoto and J. Shindo. 2001. Disseminated histoplasmosis in a Sea Otter (Enhydra lutris). J. Comp. Path. 125: 219-223. Morita T., M. Kishimoto, A. Shimada, Y. Matsumoto and J. Shindo. 2001. Disseminated histoplasmosis in a Sea Otter (Enhydra lutris). J. Comp. Path. 125: 219-223. Rezabek G. B., J. M. Donahue, R. C. Giles, M. B. Petrites-Murphy, K. B. Poonacha, J. R. Rooney, B, J, Smith, T. W. Swerczek and R. R. Tramontin. 1993. Histoplasmosis in Horses., J. Comp Path. 109:47-55. Rezabek G. B., J. M. Donahue, R. C. Giles, M. B. Petrites-Murphy, K. B. Poonacha, J. R. Rooney, B, J, Smith, T. W. Swerczek and R. R. Tramontin. 1993. Histoplasmosis in Horses., J. Comp Path. 109:47-55. Rosas-Rosas A., C. J. Salles, G. Rodríguez-Arellanes, M. L. Taylos, M. M. Garner. 2004. Disseminated Histoplasma capsulatum var. capsulatum infection in a captive mara ( Dolichotis patagonum) In Press…… Rosas-Rosas A., C. J. Salles, G. Rodríguez-Arellanes, M. L. Taylos, M. M. Garner. 2004. Disseminated Histoplasma capsulatum var. capsulatum infection in a captive mara ( Dolichotis patagonum) In Press…… Taylor M. L., C. B. Chavez-Tapia, R. Vargas-Yañez, G. Rodríguez-Arellanes, G.R Peña- Sandoval, C Toriello, A. Perez and M. R. Reyez-Montes. 1999. Environmental conditions Favouring Bat infection with histoplasma capsulatum in Mexican shelters. Am J. Trop. Med. Hyg. 61(6):914-919. Taylor M. L., C. B. Chavez-Tapia, R. Vargas-Yañez, G. Rodríguez-Arellanes, G.R Peña- Sandoval, C Toriello, A. Perez and M. R. Reyez-Montes. 1999. Environmental conditions Favouring Bat infection with histoplasma capsulatum in Mexican shelters. Am J. Trop. Med. Hyg. 61(6):914-919. Taylos M.L, Reyes-Montes M. Chavez-Tapia C. B. Curiel-Quesada E. Duarte-Escalante E. Rodríguez-Arellanes G. Peña-Sandoval G. R., and Valenzuela-Tovar F. 2000. Ecology and molecular epidemiology findings of Histoplasma capsulatum, in Mexico. Res. Adv. In Microbiology. 1:29-35. Taylos M.L, Reyes-Montes M. Chavez-Tapia C. B. Curiel-Quesada E. Duarte-Escalante E. Rodríguez-Arellanes G. Peña-Sandoval G. R., and Valenzuela-Tovar F. 2000. Ecology and molecular epidemiology findings of Histoplasma capsulatum, in Mexico. Res. Adv. In Microbiology. 1:29-35.