Abstract

Johne's disease is an infectious disease of ruminants caused by Mycobacterium avium sp. paratuberculosis (MAP). The organism primarily affects the small intestines resulting in poor digestion, diarrhea, and chronic weight loss. Transmission is by fecal contamination of feed and water, in milk and colostrum, in utero,¹³ and via semen.⁹ Once infected, an animal can harbor the organism for months to years before testing positive or demonstrating clinical signs.

In recent years Johne's disease has been diagnosed in captive non-domestic hoofstock^3,10-12,15 and in free-ranging wildlife.⁸ The Johne's Testing Center at the University of Wisconsin reports isolating the organism from 9.1% of U.S. zoos submitting fecal samples in the last 2 yr.¹⁰ Diagnosis is complicated in non-domestic hoofstock by the fact that serologic assays have not been validated in these species. Factors which complicate control of the disease in a zoo setting include infrequent handling of animals, mixed species displays, dealing with rare and valuable animals, budget constraints, inter-zoo animal exchange, and public relations issues.¹⁰

Case 1

A fecal sample was collected on a 10-yr-old male addax (Addax nasomaculatus) on 19 February 1998. It was submitted to the Johne's Testing Center at the University of Wisconsin, Madison, Wisconsin for radiometric culture. This was part of a routine surveillance program for Johne's disease at Busch Gardens, Tampa, Florida. An acid-fast organism was cultured^2,4,5,7,14 and identified by DNA probe^2,7,14,16 as MAP. Johne's disease had not been diagnosed in the addax herd since routine surveillance began in 1992 and this male had been separated from the herd for the last 5 yr. During this period of separation, eight samples were submitted previous to the 19 February 1998 sample. Seven of these cultures were negative and the other culture was contaminated.

This animal was isolated and 10 more fecal samples submitted for MAP culture over the next 7 mo. All samples were negative on culture. On 1 September 1998 this animal was immobilized for electroejaculation. Two semen samples and one fecal sample were submitted for MAP culture. One semen sample was contaminated and the other semen sample and fecal sample were negative on culture.

A fecal sample was submitted to the state lab for a MAP DNA probe at the same time and was found to be negative. Serum was submitted at this time for a Johne's disease enzyme linked immunosorbent assay (ELISA)^1,2,4 and found to be negative.

On 30 September 1998 this animal was immobilized for a right flank laparotomy and samples taken from the liver, mesenteric lymph node, and a section of the ileocecal junction, and submitted for histopathology and MAP culture. All samples were culture negative. No acid-fast organisms were seen and changes consistent with Johne's disease were not detected on histopathology.

Two more fecal samples were submitted for MAP culture on 31 October 1998 and 7 March 1998 and were found to be negative. Unable to confirm Johne's disease but having serious concern for potential exposure of "clean" females and three "clean" herds of other species, a management decision was made to humanely euthanatize this animal in April 1999. Twenty-one tissue samples were submitted to the Johne's Testing Center for culture. Eight of these tissues were positive on culture for acid-fast organisms that were identified as MAP by DNA probe (Table 1). No organisms were seen by acid-fast stain of histopathologic tissues samples. A mild lymphoplasmacytic and eosinophilic mucosal infiltrate of the ileocecal valve was identified. There were mildly acid-fast positive granular material within macrophages in the lymph node, suggestive of ceroid.

Suggested criteria for diagnosis of Johne's disease in an individual animal include positive fecal culture or tissue culture and at least one of the following: clinical signs of wasting compatible with Johne's disease, histology compatible with Johne's disease, or a Johne's disease management unit history of infection within the last year.⁶ Antemortem diagnostic procedures and history did not support or confirm that this animal was infected with MAP. Management elected euthanasia because of minimal genetic value to the Species Survival Plan, minimal display value, and decided that the risk of spreading Johne's disease outweighed any potential benefits of holding the animal or surplusing it. A decision that proved to be the correct one in the end.

Case 2

Johne's disease was diagnosed in a herd of springbok (Antidorcas marsupialis) at Busch Gardens, Tampa, Florida. The herd male was isolated to an off-display holding area. Fecal samples were collected and sent to the Johne's Testing Center for MAP culture. Eleven samples were submitted between 12 November 1993 and 1 April 1998. Six of these were negative on culture, three were contaminated, and two were positive for acid-fast organisms. The sample collected 16 January 1995 cultured positive for acid-fast organisms, but due to a heavy growth of contaminating bacteria the lab was unable to recover it for speciation by DNA probe. The sample collected on 1 April 1998 was positive for acid-fast organisms and confirmed as MAP by DNA probe. Eight more fecals were collected between 1 April 1998 and 22 August 1998 and submitted for MAP culture. Six of these samples were negative and one was contaminated. The sample collected 13 August 1998 was positive for acid-fast organisms but the DNA probe determined that these organisms were not MAP.

Given the herd history and one positive culture for MAP a decision was made to euthanatize the herd male. The animal was immobilized on 31 August 1998 for euthanasia. Prior to euthanasia the animal was electroejaculated and seven semen samples were submitted for MAP culture. Gastrointestinal and reproductive tissue samples were collected and submitted for culture (Table 2). Acid-fast organisms were cultured from the mesenteric lymph node, ileocecal junction lymph node, ileocecal valve, ileum, and testicle. DNA probes confirmed that the organisms cultured from the mesenteric lymph node and testicle were MAP. The other three acid-fast cultures were not probed for confirmation. Culture of MAP from the testicle of this animal suggests that transmission in semen is a potential concern in this species and should be considered in other exotic ruminant species.

Table 1. Tissue culture results from necropsy of a male addax euthanatized for suspected mycobacterium infection.

Table 2. Tissue/semen culture results from a male springbok euthanatized due to suspected mycobacterium infection.

References

1.  Burnside DM, BO Rowley. 1994. Evaluation of an enzyme-linked immunosorbent assay for diagnosis of paratuberculosis in goats. Am. J. Vet. Res. 55: 465.

2.  Collins MT. 1995. New developments in the diagnosis of Johne's disease in domestic and zoological small ruminants. Proc. Am. Assoc. Zoo Vet./Wildl. Dis. Assoc./Am. Assoc. Wildl. Vet. Joint Conf. Pp. 144-150.

3.  Collins MT, JE Oosterhuis. 1993. Diagnosis and control of paratuberculosis in exotic hoofed stock. Proc. Am. Assoc. Zoo Vet. Pp. 386-387.

4.  Collins MT, SE Balzer, DG Teubert. 1995. Temporal study to evaluate the serum antibody ELISA, gamma interferon test kit, and radiometric fecal culture for diagnosis of paratuberculosis in naturally infected adult dairy cattle. Proc. Fourth Inter. Coll. Paratuberculosis. Pp. 54-60.

5.  Collins MT, KB Kenefick, DC Sockett. 1990. Enhanced radiometric detection of Mycobacterium paratuberculosis using filter concentrated fecal specimens. J. Clin. Microbiol. 28:2514-2519.

6.  Cook RA. 1998. Overview and general considerations. Proc. Workshop on the Diagnosis, Prevention, and Control of Johne's Disease in Non-Domestic Hoofstock. Pp. 37-53.

7.  Cousins DV, RJ Evans, BR Francis. 1995. Use of BACTEC radiometric culture method and polymerase chain reaction for rapid screening of faeces and tissues for Mycobacterium paratuberculosis. Aust. Vet. J. 72:458-462.

8.  Jessup DA, ES Williams. 1999. Paratuberculosis in free-ranging wildlife in North America. In:Fowler, M.E. and E.R. Miller (eds.). Zoo and Wild Animal Medicine, 4th ed. W.B. Saunders Company, Philadelphia, Pennsylvania. Pp. 616-620.

9.  Larsen AB, OHV Stalheim, DE Hughes. 1981. Mycobacterium paratuberculosis in the semen and genital organs of a semen-donor bull. J. Am. Vet. Med. Assoc. 179:169.

10. Manning EJB. 1998. Johne's disease in non-domestic hoofstock: Issues and overview. Proc. Workshop on the Diagnosis, Prevention, and Control of Johne's Disease in Non-Domestic Hoofstock. Pp. 7-11.

11. Manning EJB, MT Collins. 1999. Paratuberculosis in zoo animals. In:Fowler ME, ER Miller (eds.). Zoo and Wild Animal Medicine, 4th ed. W.B. Saunders Company, Philadelphia, Pennsylvania. Pp. 612-616.

12. Manning EJB, MT Collins. 1998. Johne's Disease in zoo animals. Proc. Am. Assoc. Zoo Vet./Am. Assoc. Wildl. Vet. Pp.128-130.

13. Seitz SE, LE Heider, WD Hueston,. 1989. Bovine fetal infection with Mycobacterium paratuberculosis. J. Am. Vet. Med. Assoc. 194:1423-1426.

14. Sockett DC, DJ Carr, MT Collins. 1992. Evaluation of conventional and radiometric fecal culture and commercial DNA probe for diagnosis of Mycobacterium paratuberculosis infections in cattle. Can. J. Vet. Res. 56:148-153..

15. Thoen CO, WD Richards, JL Jarnagin. 1977. Mycobacteria isolated from exotic animals. J. Am. Vet. Med. Assoc. 170:987-990.

16. Vary PH, PR Anderson, E Green. 1990. Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne's Disease. J. Clin. Microbiol. 28:933-937.