Leptospirosis in a Mexican Grey Wolf (Canis lupus baileyi): Survey for Leptospira Infection in Captive and Local Free-Ranging Wildlife and Feral Animals at Africam Safari, Puebla, México
American Association of Zoo Veterinarians Conference 2001
Carles Juan-Sallés1; Alberto Parás1; Luis García-Feria1; Michael M. Garner2; José A. Ramos-Vara3; Miguel Ángel Luna4; Osvaldo Martínez1; Alejandra Hernández1
1Animal Health Department, Africam Safari Zoo, (Col. Azcárate), Puebla, México; 2Northwest ZooPath, Snohomish, WA, USA; 3Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, MO, USA; 4CENID Microbiología, Cuajimalpa, D.F., México


A 3.5-year-old, captive-born, male Mexican grey wolf (Canis lupus baileyi) housed in a facility with two other Mexican wolves was reported to have voided dark urine; urinalysis revealed hematuria, hemoglobinuria, and bilirubinuria. The animal was anorexic. Complete blood count (CBC) and serum biochemistries revealed leukocytosis, mostly due to absolute neutrophilia, azotemia, hyperbilirubinemia, and elevated aspartate transferase (AST), alanine transferase (ALT), and alkaline phosphatase (ALK); plasma was icteric. Treatment was initiated with oral doxycycline, and changed to intramuscular oxytetracycline and fluid therapy 3 days later. The animal’s condition progressively deteriorated and it died 6 days after presentation. This wolf had been vaccinated for leptospirosis (Nobivac Lepto, Intervet) once yearly since its birth (twice in 1998). At necropsy, body weight was 20 kg; gross findings consisted of depleted fat stores, icterus, ascites, reddened enlarged lymph nodes, hemorrhages in the intestinal serosa and endocardium, and reddened kidneys and gastric mucosa. Histopathology revealed moderate to severe, chronic lymphocytic interstitial nephritis with severe, acute, diffuse tubular necrosis and hemoglobin casts within tubules. Warthin-Starry stain on renal tissue revealed low numbers of organisms morphologically compatible with spirochetes in the lumina of tubules. There was severe intracanalicular cholestasis in the liver. Immunohistochemistry for leptospiral antigen on renal tissue using previously described procedures6 was negative. Serologic testing for 15 Leptospira interrogans serovars performed on serum collected 3 days after presentation at two different laboratories revealed positive titers (1:200) to L. icterohaemorrhagiae and borderline titers (1:50) to L. canicola. The two cage mates remained clinically unaffected, but hemogram revealed hemoconcentration and leukocytosis in one animal (blood urea nitrogen and creatinine were within normal limits in this wolf). A few days later the leukocytosis had resolved and serum biochemistry for hepatic and renal function was normal. One month later, this animal had hyperbilirubinemia. Serologic testing in the two cage mates revealed positive titers (1:100 or 1:200) to both L. canicola and L. wolffi at the time of death of the cage mate. The wolf with leukocytosis was seronegative for L. wolffi 1 month later. This animal died of unrelated causes 3 months later. Histopathology revealed mild multifocal plasmacytic interstitial nephritis that was negative for tubular spirochetes with Warthin-Starry stain.

The clinical, pathologic, and histochemical findings in the deceased Mexican grey wolf are characteristic of leptospirosis. This animal had previously been vaccinated against L. canicola and L. icterohaemorrhagiae, the two serovars for which borderline and positive titers were found respectively at the time of clinical presentation. Therefore, these titers were most likely vaccine-induced and the serovar involved in this case undetectable with the serovar panels used. The lack of seroconversion to L. wolffi (not included in the vaccine) in the two cagemates makes the interpretation of positive titers to this serovar difficult.

A preliminary serosurvey for leptospirosis has been performed at two different laboratories using the microscopic agglutination test for 11, 12, or 15 Leptospira interrogans serovars in captive carnivores (felids, hyenas, bears, and red pandas) with banked sera, in trapped wildlife (Didelphis virginiana, Spermophilus mexicanus, and Bassariscus astutus), and in feral domestic animals (dogs, and cats). All captive carnivores except the Mexican grey wolves were tested serologically prior to vaccination for leptospirosis. Additionally, urine from trapped wildlife and feral domestic animals was tested for leptospiruria by dark-field microscopy. Overall, this serosurvey has revealed seropositivity for 10 different serovars. All tested D. virginiana and B. astutus were negative; however, leptospiruria associated with chronic tubulointerstitial nephritis was found in a D. virginiana. Dark-field microscopy of all other urine samples was negative with the possible exception of a cat. Four of five S. mexicanus had borderline titers to L. pyrogenes and/or L. grippotyphosa. Two dogs had borderline or positive titers to multiple serovars (8 between the two dogs) and a third dog not tested serologically had chronic plasmacytic interstitial nephritis; the vaccination history for these dogs is unknown. Leptospira immunohistochemistry in three selected opossums (including that affected with nephritis) and this last dog was negative.

Seropositivity to seven serovars has been found in captive felids included in this survey with titers up to 1:800; in contrast, only one of five feral domestic cats had borderline titers to L. pomona. Ten of 10 wild cats from the conventional zoo (CZ) were seropositive to one or more serovars in contrast to 1/10 positive captive felids from the wild animal park (WAP). Six of 10 seropositive felids at the CZ had positive titers both to Leptospira hardjo, L. tarassovi, and L. wolffi. Leptospiremia had been diagnosed in October 1996 in four P. onca by dark-field microscopy, and two had abnormalities in the urinalysis (hematuria, bilirubinuria, proteinuria, pyuria, and/or positivity for urobilinogen) and normal CBC and biochemistry panels for renal and hepatic function. A single hyena and black bear accounted for most of the positive and borderline titers found in these species. The only captive carnivore species serologically negative was the red panda.

These preliminary findings suggest that leptospirosis may be an important infectious disease at the primary authors facility as at other zoologic institutions.1,3,4 These findings also highlight not only the problems with the diagnosis and control of leptospirosis in zoo animals, mostly due to the existence of more than 200 pathogenic serovars (and, thus, the limited value of serologic testing, immunohistochemistry, and vaccination programs)3-5 but also the difficulties in continuously controlling the direct and indirect interactions of zoo animals with free-ranging wildlife and feral domestic animals. The use of a more sensitive diagnostic test, such as the PCR assay, may help to better characterize the epizootiology of leptospirosis in zoos and thus prevent it.2

Literature Cited

1.  Gamble KC. Multi-species leptospirosis in an urban zoo: exhibit modification impact on a wildlife point source. In: Proceedings of the Joint Conference of the American Association of Zoo Veterinarians/International Association of Aquatic Animal Medicine. 2000:153–154.

2.  Ghneim G. Development and validation of a polymerase chain reaction (PCR) assay to detect leptospiruria in California sea lions (Zalophus californianus). In: Proceedings of the Joint Conference of the American Association of Zoo Veterinarians/International Association of Aquatic Animal Medicine. 2000:506.

3.  McNamara T, Linn M, Calle P, Cook R, Karesh W, Raphael B. Leptospirosis: an under-reported disease in zoo animals? In: Proceedings of the American Association of Zoo Veterinarians. 1997:248–251.

4.  Parás AL. Estudio serológico de brucelosis y leptospirosis en una población de venado cola blanca (Odocoileus virginianus) albergada en el Zoológico de Chapultepec. (Tesis de Licenciatura). México, D.F., Fac. Med. Vet. y Zoot., U.N.A.M. 1990.

5.  Quinn PJ, Carter ME, Markey BK, Carter GR. Clinical Veterinary Microbiology. London, UK: Wolfe Publishing; 1994:292–299.

6.  Ramos-Vara JA, Beissenherz ME. Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed, paraffin-embedded tissues: experience with 63 markers. J Vet Diagn Invest. 2000;12:07–311.


Speaker Information
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Carles Juan-Sallés, DVM
Department of Pathology
Africam Safari
Puebla, México

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