SEROPREVALENCE OF NINE LEPTOSPIRA INTERROGANS SEROVARS IN WILD CARNIVORES, UNGULATES, AND PRIMATES FROM A ZOO POPULATION IN A METROPOLITAN REGION OF CHILE Author(s): Eduardo Moreno-Beas, D.V.M., Pedro Abalos, D.V.M., M.Sc., and Ezequiel Hidalgo-Hermoso, D.V.M. Source: Journal of Zoo and Wildlife Medicine, 46(4):774-778. Published By: American Association of Zoo Veterinarians https://doi.org/10.1638/2014-0139.1 URL: http://www.bioone.org/doi/full/10.1638/2014-0139.1
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Journal of Zoo and Wildlife Medicine 46(4): 774–778, 2015 Copyright 2015 by American Association of Zoo Veterinarians
SEROPREVALENCE OF NINE LEPTOSPIRA INTERROGANS SEROVARS IN WILD CARNIVORES, UNGULATES, AND PRIMATES FROM A ZOO POPULATION IN A METROPOLITAN REGION OF CHILE Eduardo Moreno-Beas, D.V.M., Pedro Abalos, D.V.M., M.Sc., and Ezequiel Hidalgo-Hermoso, D.V.M.
Abstract: Serum samples from 130 individuals representing 42 species of carnivores, ungulates, and primates from a population of captive mammals in Metropolitan Region in Chile were tested for antibodies against nine serovars of Leptospira interrogans using the microscopic agglutination test. Ten percent of the animals were seropositive to one or more serovars. Seroprevalence was significantly higher in ungulates (20.4%) compared to carnivores (3.8%) and primates (3.4%). There were no significant differences in seroprevalence among sex and age ranges. The most frequent serovar detected was Autumnalis, present in 53.4% of antibody-positive animals. Most positive animals had titers of 1 : 200, except for a maned wolf (Chrysocyon brachyurus) with titers of 1 : 400 against serovar Hardjo. To the authors’ knowledge, this is the first report of Leptospira exposure detected in native endangered pudu (Pudu puda) and the first confirmation of exposure to L. interrogans in captive wild mammals in Chile. Leptospirosis should be considered as a differential diagnosis in future disease presentation for hepatitis or abortions in captive mammals in Chile. Key words: Chile, Leptospira interrogans, seroprevalence, zoo mammals.
INTRODUCTION Leptospirosis is an infectious disease caused by the spirochete Leptospira interrogans. All mammals, including aquatic species, can be considered susceptible to infections involving L. interrogans, with mammals serving as carriers of this bacteria, which is found worldwide, with the exception of Antarctica.1 Wild animals are susceptible to infection with a variety of Leptospira serovars and can serve as incidental hosts.14 The type of infection the animal acquires depends on the endemism of the serovar acting on the species involved, and the transmission of the bacteria depends on many factors, including climate, population density, and the degree of contact between the reservoir and the accidental host.1,6,11 Susceptible animals acquire the infection by direct or indirect contact with the urine or tissues of infected animals, leading to a chronic infection of the renal tubules. The prevalence of chronic excretion in the urine increases with the age of the animal.11 These bacteria do not survive well in acidic urine but remain viable in alkaline urine,
From the Conservation and Research Department, Parque Zoolo´gico Buin Zoo, Panamericana Sur Km 32, Buin, Chile (Moreno-Beas, Hidalgo-Hermoso); and the Departamento de Medicina Preventiva Animal, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Av. Santa Rosa 11735, La Pintana, Santiago, Chile (Moreno-Beas, Abalos). Correspondence should be directed to Dr. Moreno-Beas (
[email protected]).
making herbivores and animals with alkaline urine relatively more important as disseminators.1 Today leptospirosis is considered a reemerging zoonosis, causing outbreaks in humans worldwide and becoming an agent of major public health importance.17,23 Seropositivity to serovars of L. interrogans in wild free-living animals has been described in the literature but is reported to a lesser extent in captive animals.4,14 Free-living animals are usually positive to serotypes common in their native areas.24 In captivity, animals of different ecological origins and different epidemiologic conditions are forced to live in close proximity to each other, and this facilitates exposure and infection to several serovars of L. interrogans.14,25 Under these conditions, the possibility of contact with animal reservoirs, such as free-living rodents, increases, creating opportunities for exposure to Leptospira and other infectious agents.10,13,25 The purpose of this study was to determine the seroprevalence of nine serovars of L. interrogans in wild captive mammals in Chile, which serve as the causal agent of a reemerging zoonosis present in Chile and therefore a threat to zoo animal collections and humans.
MATERIALS AND METHODS One hundred thirty blood samples were collected from carnivores (52), ungulates (49), and primates (29) from a zoo population in Metro-
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Table 1. Chile.
The species distribution of tested captive animals from a zoo population in Metropolitan Region,
Taxonomic group
Carnivores
Primates
Ungulates
Species
Tested (No.)
Titers 1 : 100 (No.)
Alopex lagopus Canis lupus Caracal caracal Chrysocyon brachyurus Galactis cuja Genetta genetta Leopardus pardalis Lycaon pictus Mephitis mephitis Panthera leo Panthera onca Panthera tigris Potos flavus Procyon lotor Puma concolor Suricata suricata Ursus arctos Ursus malayanus Vulpes vulpes Alouatta caraya Ateles paniscus Cebus albifrons Cebus apella Cercopithecus aethiops Colobus guereza Lagothrix lagotricha Lemur catta Papio hamadryas Camelus bactrianus Cervus elaphus Dama dama Gacella Thomsoni Thomsoni Giraffa camelopardalis Lama glama Lama guanicoe Lama pacos Ovis aries Pudu puda Tapirus terrestris Tragelaphus angasii Tragelaphus spekii
1 1 1 6 1 6 4 2 2 3 1 3 2 5 2 2 1 3 6 4 1 1 7 1 3 1 5 6 2 4 6 5 3 4 3 5 5 7 3 1 1
0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 2 1 0 2 3 0 0 1
politan Region in Chile. Samples from clinically healthy animals of both sexes and different ages (Table 1) were included. The animals were not vaccinated against leptospirosis. The serum samples were separated by centrifugation at 1,400 g for 15 min and were stored at 208C until analysis. Serum samples were tested using the microscopic agglutination test (MAT) to serovars Pomona, Grippotiphosa, Copenhageni, Hardjo, Canicola, Bratislava, Autumnalis, Tarassovi, and Bataviae, grown in Ellinghausen–McCullough– Johnson–Harris liquid medium in the Bacterio-
logical Laboratory of the Agricultural and Livestock Service (Route 68, Km 12, Lo Aguirre, Regio´n Metropolitana, Santiago, Chile). The positive antibody titer criteria provided by to the World Organization for Animal Health (OIE; 1 : 100, end point titer) were used.20 The analysis considered the following variables: sex, taxonomic group (carnivores, ungulates, and primates), and age ranges (,6 mo, 6–18 mo, 18–36 mo, and .36 mo) according to medical records. Age records were unavailable for 12 animals. As a result of the small sample sizes, the association of
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Table 2. Chile.
JOURNAL OF ZOO AND WILDLIFE MEDICINE
Antibody-positive animals to L. interrogans serovars in a zoo population in Metropolitan Region,
Taxonomic group
Carnivore Carnivore Primate Ungulate Ungulate Ungulate Ungulate Ungulate Ungulate Ungulate Ungulate Ungulate Ungulate a
Species
Sex
Age range (mo)
Titersa
Chrysocyon brachyurus Leopardus pardalis Papio hamadryas Camelus bactrianus Tragelaphus spekii Lama guanicoe Lama glama Lama glama Pudu puda Pudu puda Pudu puda Ovis aries Ovis aries
male male male male male male female male female female female female female
6–18 .36 .36 .36 18–36 .36 .36 .36 .36 .36 .36 .36 .36
Co ¼ 1 : 200 H ¼ 1 : 400 Ca ¼ 1 : 200 A ¼ 1 : 100 A ¼ 1 : 100 Br ¼ 1 : 100 P ¼ 1 : 100 H ¼ 1 : 100 A ¼ 1 : 200 A ¼ 1 : 100 A ¼ 1 : 100 G ¼ 1 : 100 Br ¼ 1 : 100 A ¼ 1 : 100 A ¼ 1 : 200 Ca ¼ 1 : 200
Co indicates Copenhageni; H, Hardjo; Ca, Canicola; A, Autumnalis; Br, Bratislava; P, Pomona; G, Grippotyphosa.
variables was analyzed with the Fisher exact test, with an a of P , 0.05 considered statistically significant.
RESULTS Of the 42 mammal species involved in the study, nine (21.4%) had antibody-positive reactions to MAT. Of the 130 samples examined, 13 (10%) had titers of 1 : 100 against one or more of the serovars investigated. The pudu (Pudu puda), with three individuals, was the species with the most seropositive individuals. All seropositive animals reacted to only one serovar, except for one guanaco (Lama guanicoe) with a reaction against the serovars Hardjo and Autumnalis and one maned wolf (Chrysocyon brachyurus) with antibody titers against Copenhageni, Hardjo, and Canicola (Table 2). There were no significant differences between the percentages of females (8.6%) and males (11.7%) that had positive titers. Age was not statistically related to titers of 1 : 100, despite the fact that 84.6% of positive animals were more than 36 mo of age. Seropositivity was significantly higher (P , 0.05) in ungulates (20.4%) compared to carnivores (3.8%) and primates (3.4%). Of the seven serovars detected in this study, the most frequent was Autumnalis (53.4%), followed by serovars Bratislava, Canicola, and Hardjo present in 15.4% of reactions each, and then followed by Pomona, Copenhageni, and Grippotyphosa in 7.7% of reactions each.
DISCUSSION The lack of previous studies on the seroprevalence of L. interrogans in wild mammals in captivity in Chile and the lack of recent data about the
infection status of domestic animals in rural regions make it difficult to compare results taking into account local factors such as environment, vector species, disseminators, and predominant serovars. For the purposes of comparison, it is important to note that this study used an endpoint titer of 1 : 100, as recommended by the OIE, while other studies12,20 commonly use titers that are lower than this. For example, the prevalence obtained in the present study was less than the 25% predicted and determined in a zoo in Korea, where the survey10 used an endpoint titer of 1 : 25, potentially overestimating seroprevalence. The number of serovars included in the present study was lower than in other studies10,13–15; therefore, it is likely that there were some animals that were seropositive for serovars not tested in the present study and not showing an antigenic cross reaction with the serovars used, thereby underestimating the seroprevalence. Diagnosis of leptospirosis is difficult, depending on laboratory assays such as the MAT, the indirect hemagglutination assay, or enzyme-linked immunosorbent assay, or one can detect Leptospira in tissues or fluids by culture, dark-field microscopy, immunostaining, or polymerase chain reaction (PCR).1 PCR assays are increasingly used for the detection of Leptospira, but this analysis is not a routine diagnostic method for their lack validation.20 MAT was used for Leptospira screening in this study as it is currently considered the gold standard, has a low cost, is available in Chile, and has the ability to detect prevalence of infections.20 Despite the fact that seropositivity was significantly higher in ungulates (20.4%), it is not possible to determine whether these taxa play a role in the epidemiology of L. interrogans in this
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MORENO-BEAS ET AL.—SEROPREVALENCE OF LEPTOSPIRA IN ZOO MAMMALS
Table 3.
Review of antibody-positive animals to L. interrogans kept in zoos between 1975 and 2007. Serovarsb
Taxonomic group
n
a
Ca
P
G
Carnivora 49 12 6 4 Ungulata 73 5 14 6 Primate 7 1 0 4 Marsupialia 0 0 0 0 Lagomorphia 2 0 0 0 Rodentia 0 0 0 0 Seropositive mammals 129 18 20 14 Prevalence (%) 98.5 14.0 15.5 10.9
Co
I
Ma
H
Br
Ba
A
Bt
Pa
Py
Hb
5 0 0 0 0 0 5 3.9
17 17 0 0 0 0 34 26.4
2 0 0 0 0 0 2 1.6
0 0 0 0 0 0 0 0
0 1 0 0 0 0 1 0.8
0 0 0 0 0 0 0 0
1 2 0 0 0 0 3 2.3
0 0 0 0 0 0 0 0
0 8 7 1 3 2 0 0 1 0 0 0 0 0 0 0 0 0 1 11 10 0.8 8.5 7.8
T
W
0 1 0 0 0 0 1 0.8
2 0 0 0 0 0 2 1.6
Sample size. Ca indicates Canicola; P, Pomona; G, Grippotyphosa; Co, Copenhageni; I, Icterohaemorrhagiae; Ma, Mankarso; H, Hardjo; Br, Bratislava; Ba, Ballum; A, Autumnalis; Bt, Bataviae; Pa, Panama; Py, Pyrogenes; Hb, Hebdomalis; T, Tarassovi; W, Wolffi. a
b
captive population because MAT only demonstrates an exposure of the animal to the bacteria. Studies based on bacterial isolation from urine or renal carriage status are needed to identify the exact role of these animals in the epidemiology of the disease, and in particular to ascertain if they can act as reservoirs.1,8 Although it was determined that the seroprevalence of L. interrogans in this collection was low, the evidence of exposure to the agent should be considered for a differential diagnosis in future disease presentation in the park. Veterinarians should suspect leptospirosis in animals with signs of renal or hepatic failure, uveitis, pulmonary hemorrhage, acute febrile illness, or reproductive failure.1,24 It is important to note the high seroprevalence of the serovar Autumnalis, for which rodents have been identified as reservoirs in other countries.21 Based on these results, it is essential to carry out frequent surveys in captive and free-living wild mammals, as well as domestic animals, to determine if this serovar has taken on greater importance in Chile, whether rodents are acting as disseminators of Autumnalis, or if the high seroprevalence reflects a situation unique to the zoo. Mammals may have been readily exposed to leptospirosis via rodent vectors, in spite of rigorous attempts at rodent control within the confines of the zoo.16 Results may be indicative of the presence of a serovar belonging to the serogroup Autumnalis and not specifically to the serovar Autumnalis, because MAT provides a general impression about present serogroups in the population without specifically identifying the infecting serovar in all cases.2,12 Furthermore, the other serovars included in these results match the serovars present in reports3–5,7,9,10,13,15,18,19,22 from 1975 to 2007, in which 129 of 131 mammals kept
in zoos in the United States, Brazil, Mexico, Korea, and Canada were reported as antibodypositive to the serovars Icterohaemorrhagiae (26.4%) (antigenically associated with Copenhageni11), Pomona (15.5%), Canicola (13.9%), and Grippotyphosa (10.8%), without considering Autumnalis, Bratislava, or Hardjo (Table 3). The present study represents the first report on the occurrence of Leptospira antibodies in native endangered pudu and in Chilean captive wild mammals. Further research is necessary to evaluate the role of ungulates in the epidemiology of the infection. It is imperative that the control of pest species in this collection be improved and that the exposure to Leptospira in rodents be evaluated, as these rodents may be serving as the principal vector of the bacteria. Acknowledgments: The authors kindly thank the Buin Zoo personnel for their help in sample collection and storage. Thanks are also extended to Patricia Avalos, Maria Cristina Madariaga, and Alexza Pezoa for performing the Leptospira serology at the Agricultural and Livestock Service.
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