Abstract

Leptospirosis, the disease caused by the spirochete Leptospira interrogans, is a zoonosis with worldwide distribution. Leptospira interrogans is known to infect and cause disease in a broad range of mammalian species.⁴ A great deal of work and research has been done on this agent in domestic animals. The development of a vaccine against the five most common serovars (L. pomona, L. hardjo, L. icterohaemorrhagiae/copenhageni, L. grippotyphosa, L. canicola) has greatly decreased the incidence of this disease problem in vaccinated populations. As a result, although aware of it, many veterinarians may have little practice experience with this pathogen.

Classically, after being shed by a carrier host into the environment, organisms gain entry into the susceptible host via mucocutaneous or percutaneous routes. The spirochetes multiply and are spread hematogenously and localize in the liver, kidney, and uterus. In the acute form of the disease, particularly with certain serovars (e.g., L. canicola) that can produce a hemolysin, hemolytic anemia and icterus are prominent gross manifestations of the infection. Acute hepatic injury from bacterial localization within hepatocytes can cause elevation of liver enzymes and hyperbilirubinemia. Following the hepatic phase of the disease, organisms localize in the kidney, leading to focal to diffuse tubulonephritis. In addition to hepatic and renal involvement, organisms may also localize to the lungs, heart, and brain with subsequent inflammation in these tissues. Chronically infected tissues are frequently infiltrated by lymphocytes, plasma cells, and fibrous connective tissue.² Subclinical infections of adults can result in abortions or neonatal losses and have a significant impact on reproduction programs.

Diagnosis of Leptospira exposure is ideally achieved through the collection of paired serum samples and the demonstration of a rise in titer against a specific serovar. Examination of urine by phase contrast microscopy and visualization of the spirochetes, or evaluation of formalin-fixed urine by electron microscopy are other methods of diagnosis. Fluorescent antibody (FA) testing of fresh urine or fresh or frozen tissue collected at the time of necropsy will confirm the presence of leptospiral antigen. Cultivation of leptospires is problematic due to the fragile and fastidious nature of the organism. Of all diagnostic methods, it is the one with the lowest yield.

As is true of many other diseases, the diagnosis of leptospirosis in zoo animals presents a serious challenge. While serologic testing is always the preferred method of diagnosis, reluctance to immobilize zoo species for repeated blood sampling at fixed time intervals often makes it impossible to obtain paired samples to demonstrate a rise in titer. In addition, zoo animals may die with no antecedent clinical workup or blood samples temporally related to the time of death. Vaccination of animals in the absence of prior baseline serology can further complicate interpretation of serologic results. In the absence of serologic data, diagnosis often rests on histopathologic evaluation of tissues. In cases where animals are in an autolyzed condition, the success of culture attempts is decreased, and histopathologic interpretation of changes in hepatocytes and renal tubular epithelium is more difficult. Silver stains may be used on fixed tissue sections to detect leptospires, but this technique too is fraught with complications. Immunohistochemical identification of leptospires exists but is not yet widely available. Financial limitations may impede the ability of an institution to routinely submit fresh tissues for FA as a screening method. Physical lack of freezers may make it impossible to preserve tissue samples at -70°C for FA testing if indicated by histopathology. Most important of all, in addition to the previously described handicaps, leptospiral infections in non-domestic species may not “fit” classic descriptions of the disease. Atypical presentations of the disease may lead both the clinician and pathologist to overlook this diagnosis and underestimate the impact this disease may be having on a zoological collection.

At the Wildlife Conservation Society, the loss of a red panda (Ailurus fulgens) resulted in a heightened awareness of this disease threat and also raised two very important issues. First, although it was impossible to do serologic testing on the panda that died acutely with massive pulmonary hemorrhage and renal tubular necrosis and hepatocellular dissociation, silver positive organisms were demonstrated on histologic sections, and FA was also positive on liver and kidney. Serology performed on the exposed cagemate was initially reported as negative by the diagnostic laboratory. Since it was possible the serovar infecting the red panda was not necessarily the same as the five serovars seen and tested for in domestic species, serologic testing for an additional 12 serovars was performed. A rise in titer against L. autumnalis, a serovar most commonly associated with rodents, was demonstrated. Given their location within a large urban park, the red pandas could readily have been exposed to leptospirosis via rodent vectors, in spite of rigorous attempts at rodent (mice/rats) control within the confines of the zoo.

This case raised concerns as to how many cases might previously have gone undetected. Up until that time, all Leptospira serologic testing had been limited to the five common serovars. As a result of this case, all 17 serovars are routinely tested for now. However, the limitations of even the extended panel must be recognized, given the fact that over 200 pathogenic serovars have been described and can potentially be transmitted by any mammal.

The detection of an “unusual” serovar raised a second issue. The red panda had not been vaccinated against leptospirosis due to the risk of using commercially available multivalent canine vaccines and inducing canine distemper.¹ However, even if a leptospire bacterin had been used, it might not have provided any protection against the specific serovar causing disease in this individual, as L. autumnalis was not included in any commercial bacterin. This prompted discussions as to the logic of vaccination programs currently used in zoological collections.

The Department of Pathology began a retrospective study and reviewed 10 years of pathology records for known or suspected cases of leptospirosis. Tissues from incoming “sudden death cases” were submitted for FA, and culture, and silver stains were routinely performed on tissue sections. Twenty cases involving 16 species of exotic mammals (Table 1), housed both in indoor and outdoor exhibits, were confirmed by FA testing of fresh tissues collected at necropsy or those available in the frozen archive library.

Table 1. Species in which Leptospira infection was confirmed between 1986–1996

Animal

Red panda (Ailurus fulgens)
Roosevelt elk fetus (Cervus elaphus roosevelti)
Flying squirrel (Glaucomys sabrinus)
Pere David’s deer fetus (Elaphurus davidianus)
Indian rhinoceros fetus (Rhinoceros unicornis)
Barasingha fetus (Cervus duvaucelii)
Sambar deer neonate (Cervus unicolor)
Sun squirrel (Heliosciurus gambianus) (n=4)
Senegal bushbaby (Galago senegalensis)
Degu (Octodon degus)
Pygmy goat (Capra hircus)
Okapi (Okapia johnstoni)
Sugar glider (Petaurus breviceps)
Slender-horned gazelle (Gazella leptoceros)
Wild mouse (Mus musculus)
Eld’s deer (Cervus eldi thamin)
Wild muskrat (Ondatra zibethicus)

Several cases (red panda, Senegal bushbaby, degu, sugar glider) presented with acute hemolytic crisis very similar to the syndrome reported in domestic animals.⁴ Of greater interest, however, were those cases that exhibited no detectable gross lesions at necropsy, as was the case with most of the hoofstock neonates. “Poor-doer” calves are common necropsy submissions in zoological collections. Failure to nurse, hypothermia, and maternal neglect are often contributory factors leading to death. In the absence of specific gross findings, unless the prosector has a high index of suspicion for leptospirosis, it is possible that appropriate diagnostic tests may not be performed. As the leptospiral status of the dam is often unknown, failure to pursue the diagnosis in neonates may result in under-reporting of the disease as a whole. This can have serious implications for breeding herds and captive propagation programs.

The loss of four sun squirrels (Heliosciurus gambianus) challenged another “classic” concept of leptospirosis. Rodents have generally been reported as carrier species and are considered one of the main sources of infection for other mammals.³ And yet, all sun squirrels died with massive peracute leptospiremia and tissue necrosis. This particular exotic rodent species, at least, proved to be extremely susceptible to the disease.

These preliminary studies on leptospirosis in zoo animals have raised more questions than answers and challenged many basic assumptions about the disease. Certainly, all institutions should already have stringent pest control programs in place. Protecting food and water stations from contamination is standard procedure in zoos. However, from a realistic point of view, no matter how intensive a zoos’ rodent/feral mammal control program might be, it is unlikely that introduction of this disease will be prevented. The problem is probably far more widespread than is currently suspected. For, even if zoos can prevent accidental exposure of collection animals by indigenous wildlife, there is still the risk of acquiring infection from subclinical carriers already within the herd. More aggressive screening of all incoming quarantined animals may help detect carriers prior to release. A combination of urinalysis, vaginal swabs, and paired serologic tests is the best hope of diagnosing this disease. Concerns over the risks of handling or immobilizations necessary to perform these procedures need to be weighed against the potential risk of losing animals to the disease.

The importance of serologic testing cannot be over-emphasized. Without it, while the other tests will confirm the presence of leptospirosis in general, only culture or a rise in titer will help pinpoint the actual serovar involved. This information will be necessary before vaccination programs can be evaluated. Given the number of pathogenic serovars that might potentially be introduced to a zoo collection via feral mammals or in-house carriers, do currently available vaccines actually provide protection against disease? If so, for how long? There is evidence that exotic species do not respond to vaccination in a uniform manner (Dr. Bolin, personal communication). That being the case, it is obvious there will be a need for standardized clinical vaccine trails in key species before this question can be answered. Until then, it would be best not to be lulled into a false sense of security by an animal’s vaccination status. The identification and treatment of this disease problem within zoological collections will not be as straightforward as it might be in domestic species. But, given the importance of the individuals found in zoos and the impact fetal death or decreased reproductive performance may have on the future of certain species, additional studies are warranted.

Acknowledgments

We would like to thank and acknowledge Dr. McDonough and the microbiology staff of the diagnostic laboratory of the New York State College of Veterinary Medicine for their help and guidance. Special thanks to Mr. Alfred Ngbokoli for technical assistance in preparation of routine and silver impregnation studies of histologic specimens.

Literature Cited

1.  Bush, M., R.J. Montali, D. Brownstein, A.E. James, and M.J. Appel. 1976. Vaccine-induced canine distemper in a lesser panda (red panda). J. Am. Vet. Med. Assoc. 169(9):959–960.

2.  Greene, C.E., E.B. Shotts. 1990. Leptospirosis. In: Greene, C.E. (ed.). Infectious Diseases of the Dog and Cat. W.B. Saunders, Philadelphia, PA. Pp. 498–507.

3.  Thiermann, A.B. 1981. The Norway rat as a selective chronic carrier of Leptospira icterohaemorrhagiae. J. Wildl. Dis. 17(1):39–43.

4.  Torten, M. and R.B. Marshall. 1994. Leptospirosis. In: Beran, G.W. (ed.). Handbook of Zoonoses, Section A: Bacterial, Rickettsial, Chlamydial, and Mycotic. CRC Press, Boca Raton, FL. Pp. 245–264.