Serologic Survey for Leptospira Spp. In Captive Neotropical Felids in Foz Do Iguaçu, Paraná, Brazil

SEROLOGIC SURVEY FOR LEPTOSPIRA SPP. IN CAPTIVE NEOTROPICAL FELIDS IN FOZ DO IGUAÇU, PARANÁ, BRAZIL Author(s) :Leila Sabrina Ullmann, D.V.M., Juliano L. Hoffmann, D.V.M., M.Sc., Wanderlei de Moraes, D.V.M., Zalmir S. Cubas, D.V.M., M.Sc., Leonilda Correia dos Santos, B.Sc, M.Sc., Rodrigo Costa da Silva, D.V.M., Ph.D., Nei Moreira, D.V.M., Ph.D., Ana Marcia Sa Guimaraes, D.V.M., M.Sc., Lucilene Granuzzio Camossi, D.V.M., M.Sc., Helio Langoni, D.V.M., Ph.D., and Alexander W. Biondo, D.V.M., Ph.D. Source: Journal of Zoo and Wildlife Medicine, 43(2):223-228. 2012. Published By: American Association of Zoo Veterinarians DOI: http://dx.doi.org/10.1638/2010-0091.1 URL: http://www.bioone.org/doi/full/10.1638/2010-0091.1

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Journal of Zoo and Wildlife Medicine 43(2): 223–228, 2012 Copyright 2012 by American Association of Zoo Veterinarians

SEROLOGIC SURVEY FOR LEPTOSPIRA SPP. IN CAPTIVE ´ , BRAZIL NEOTROPICAL FELIDS IN FOZ DO IGUACU, ¸ PARANA Leila Sabrina Ullmann, D.V.M., Juliano L. Hoffmann, D.V.M., M.Sc., Wanderlei de Moraes, D.V.M., Zalmir S. Cubas, D.V.M., M.Sc., Leonilda Correia dos Santos, B.Sc, M.Sc., Rodrigo Costa da Silva, D.V.M., Ph.D., Nei Moreira, D.V.M., Ph.D., Ana Marcia Sa Guimaraes, D.V.M., M.Sc., Lucilene Granuzzio Camossi, D.V.M., M.Sc., Helio Langoni, D.V.M., Ph.D., and Alexander W. Biondo, D.V.M., Ph.D.

Abstract: Leptospirosis is a bacterial zoonosis of worldwide distribution and is endemic in tropical countries, where rodents and other wild mammals are abundant and may act as reservoirs. Leptospirosis has become a concern in captive wild animals, due mostly to their exposure to contaminated urine or environment. Although domestic cats (Felis catus) have been reported refractory to leptospirosis, serology and disease in captive wild felids is still unclear. In this study 57 adult, clinically healthy felids, including 1 Geoffroy’s cat (Leopardus geoffroyi), 3 jaguarundis (Puma yagouaroundi), 17 margays (Leopardus wiedii), 22 little spotted cats (Leopardus tigrinus), and 14 ocelots (Leopardus pardalis) kept in captivity at the Sanctuary at the Itaipu Binacional ¸ hydroelectric power plant (Bela Vista Biological Sanctuary), Foz do Iguacu City, Parana´ State, Brazil, were serologically surveyed for the presence of antibodies against 28 serovars of Leptospira spp. by microagglutination test (MAT). Two animals (3.5%) were seropositive: one male ocelot to the serovar Cynopteri (titer 100) and one female margay to Autumnalis (100) and Butembo (200). The captive-born, 5-yr-old ocelot had been solitary housed in an individual cage. The approximately 21-yr-old wild-caught margay was also kept individually. None of the tested animals showed signs of leptospirosis. During a study conducted 4 yr previously in the same facility, this particular margay also tested positive for the same two serovars, among others. The present study indicates that the felids tested for Leptospira spp. by MAT were exposed to serovars, but did not demonstrate clinical signs of disease. Comparison with a previous study suggests that serovar titers may vary over time and that leptospirosis dynamics remains unclear in wild felids. Key words: Captive wild cats, leptospirosis, microagglutination test, serology.

INTRODUCTION Ten of 36 known Felidae species are distributed throughout the Neotropics. Eight of them occur in the Brazilian territory, namely ocelots (Leopardus pardalis), margays (Leopardus wiedii), littlespotted cats (Leopardus tigrinus), Geoffroy’s cats (Leopardus geoffroyi), pampas cats (Leopardus colocolo), jaguarundis (Puma yagouaroundi), cougars (Puma concolor), and jaguars (Panthera onFrom the Department of Veterinary Hygiene and Public Health, School of Veterinary Medicine and Animal Science, Sa˜o Paulo State University, Botucatu, Sa˜o Paulo 18618-000, Brazil (Ullmann, Hoffmann, da Silva, Camossi, Langoni); the Sanctuary at the Itaipu Binacional Hydroelectric Power Plant (Bela Vista Biological Sanctuary–BVBS), Foz do Iguac¸u, Parana´ 85866900, Brazil (Moraes, Cubas, Santos); the College of Veterinary Medicine, Campus Palotina, Federal University of Parana´ , Palotina, Parana´ 85950-000, Brazil (Moreira); the School of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907, USA (Guimaraes); and the Department of Veterinary Medicine, Federal University of Parana´, Curitiba, Parana´ 80035050, Brazil (Biondo). Correspondence should be directed to Dr. Biondo ([email protected]).

ca).28 Studies in free-ranging feline populations are relatively difficult owing to the secretive nature of these animals. In captivity, sample collection is less complex, thus allowing researchers to explore the epidemiology of infectious diseases in exotic species.23 Despite biosecurity measures in most zoos, captive animals still can be exposed to infectious agents, many of which are potentially zoonotic.8,22,27 Leptospirosis is an emerging zoonotic disease of worldwide distribution. It is caused by spirochetes of the genus Leptospira, which includes saprophytic and pathogenic species.17 The latter are classified into more than 200 serovars based on the microagglutination test (MAT). Leptospira serotyping has been recognized as an essential tool for clinical and epidemiologic research, and may indicate the host reservoirs involved in disease transmission.13 Leptospira serovars may have host preference, and often signs of the disease are mild or absent in reservoir hosts as they become adapted to the pathogen.6 Wild animals are exposed to a wide variety of Leptospira serovars and often present with titers to serovars common to their native areas. The

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interaction between infectious agent and host may change in captivity because animals of different ecologic areas may share a common environment, thus exposing animals to different serovars not found in their native habitat.19 Moreover, captivity may facilitate the exposure of felids to rodents, raccoons, opossums, feral cats, and stray dogs, which are considered important reservoirs of leptospires.3,6 Few surveys have been performed on leptospirosis in domestic and wild felids (Table 1), mostly because they are considered refractory to the disease and serology varies by locality, species, and institution studied. Domestic cats (Felis catus) develop seropositivity to serovars Pomona and Ballum with concurrent leptospiruria after 8 wk of experimental infection.7 Natural infection in cats with the serovar Grippothyphosa and the development of leptospiruria has been observed.2 Cats with leptospirosis may present with clinical disease, with elderly cats that have contact with dogs more likely to be infected.24 Serologic reports of Leptospira spp. in wild felids include American bobcats (Felis rufus),11,26 cougars,10,12,18,19 ocelots,10,19 little spotted cats,10 jaguarundis, margays, and jaguars10,19 (Table 1). Suggestive cases of acute leptospirosis were confirmed in a free-ranging cougar in the U.S.,12 and in a captive cougar in Brazil that presented with clinical signs of hematuria, bilirubinuria, and death associated with chronic interstitial nephritis.19 A previous study was conducted in 2002 at the Bela Vista Biological Sanctuary (BVBS) and a nearby zoo that evaluated jaguars, cougars, ocelots, margays, little-spotted cats, and jaguarundis (Biondo, unpubl. data). In this study, 28 out of 61 animals (45.9%) were seropositive for Leptospira as evaluated by using MAT.10 The high prevalence of antibodies against Leptospira spp. in captive wild cats and the epidemiology of the disease were not fully understood. The objective of the present study was to evaluate for a serologic response to Leptospira spp. in a zoologic collection of 57 felids of five species, in a facility known to have a rodent infestation. Of the 57 felids, 32 had been previously tested and 25 had never been tested.

MATERIALS AND METHODS The present study is part of a larger multiinstitutional research project that involves researchers from two campuses of the Federal University of Parana´, Sa˜o Paulo State University, and BVBS.

Animals and blood collection A total of 57 clinically healthy adult cats, including 1 Geoffroy’s cat (L. geoffroyi), 3 jaguarundis (P. yagouaroundi), 17 margays (L. wiedii), 22 little-spotted cats (L. tigrinus), and 14 ocelots (L. pardalis), kept in captivity in a breeding center in ¸ the BVBS, city of Foz do Iguacu, Parana´ State, Brazil, were sampled in 3 days in November 2006 for this study. Twenty-three of these animals were captive born and 34 were wild caught, with a gender distribution of 27 males and 30 females. All animals were maintained in individual enclosures in close proximity to one another. All were fed with raw meat and water was replaced two times a day. Animals were captured with nets and then chemically restrained under different protocols with ketamine hydrochloride (10.7–28.1 mg/kg), xylazine hydrochloride (0.67–1.86 mg/kg) and/or diazepam (0.27–1.10 mg/kg), and atropine sulfate (0.01–0.09 mg/kg) for sample collection. Blood samples were obtained by venipuncture of the jugular or femoral vein, transferred to a serum separator tube, and centrifuged at 1,600 g for 10 min. Sera were then stored at 208C until analysis. Serologic analysis Serologic analysis was conducted at the Laboratory of Zoonosis Research, School of Veterinary Medicine and Animal Science, Sa˜o Paulo State University, Campus of Botucatu, Brazil, using MAT. Samples were tested against 28 serovars kept in EMJH medium, cultured weekly: Australis, Bratislava, Autumnalis, Bataviae, Canicola, Djasiman, Sentot, Hebdomadis, Icterohaemorrhagiae, Copenhageni, Pomona, Pyrogenes, Hardjo (Prajitno), Castellonis, Wolffi, Andamana, Patoc, Mini (Ctg), Szwajisak, Hardjo (Bovis), Javanica, Cynopteri, Grippotyphosa, Butembo, Panama, Tarassovi, Shermani, and Whitcombi. These are the most frequently found serovars and serogroups of leptospires in Brazil. Serum was initially diluted to 1:50 with phosphate buffer solution pH 7.4 and tested against serovars listed. A sample was considered a reagent sample when more than 50% of leptospires agglutinated, and a titer of 100 was considered as cutoff. Negative and positive controls were used when performing the test.6

RESULTS AND DISCUSSION Overall, 2/57 (3.5%) animals were seropositive for Leptospira spp., which contrasts with the 12/32

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Table 1. Serologic survey of Leptospira spp. in domestic and wild felids, where fractions represent seropositive animals/total number of animals surveyed. Animals

Species

Serovars

Reference

Domestic cats Experimental infection Domestic cat (13/14) Domestic cat (9/10) Natural infection Domestic cat (1/8) Domestic cat (1/2) Domestic cat (29/200)

Felis catus F. catus

Pomona and Ballum Icterohaemorrhagiae and Canicola

7 16

F. catus F. catus F. catus

Grippothyphosa Pomona Shermani, Pomona, Icterohaemorrhagiae, and Grippothyphosa Pomona Hardjo, Autumnalis, and Icteroharmorrhagiae Icteroharmorrhagiae and Patoc Rachmati, Bratislava, Ballum, Bataviae, Salinen, Panama, and Canicola Pyrogenes, Bataviae, Autumnalis, and Icterohaemorrhagiae

2 20 28

Grippotyphosa and Pomona Suggestive, but not confirmed Pomona Pomona Pomona Pomona Icterohaemorrhagiae Pomona and Icterohaemorrhagiae Pomona Hardjo Castellonis and Hardjo Grippotyphosa and Bratislava Patoc, Butembo, and Grippotyphosa Patoc, Butembo, Andamana, and Autumnalis Patoc Icterohaemorrhagiae and Andamana Icterohaemorrhagiae and Canicola Copenhageni, Grippotyphosa, Pomona, Icterohaemorrhagiae, Patoc, and Andamana Copenhageni, Grippotyphosa, Canicola, and Icterohaemorrhagiae Patoc, Grippotyphosa, Pomona, Andamana, and Icterohaemorrhagiae Icterohaemorrhagiae Grippotyphosa and Icterohaemorrhagiae Sentot Cynopteri Autumnalis and Butembo

11 12 18 18 18 18 4 4 4 4 10 10 10 10

Domestic cat (22/172) Domestic cat (8/87)

F. catus F. catus

Domestic cat (9/200) Domestic cat (33/99)

F. catus F. catus

Domestic cat (7/31)

F. catus

15 1 14 24 25

Wild felids American bobcat (2/8) Cougar (1/1) Cougar (1/2) Jaguarundi (1/2) Margay (1/3) Jaguar (1/2) Cougar (1/4) Jaguar (8/11) Serval (2/2) Jaguarundi (1/21) Jaguar (2/4) Cougar (2/3) Ocelot (5/9) Margay (9/15)

Felis rufus Puma concolor P. concolor Puma yagouaroundi Leopardus wiedii Panthera onca P. concolor P. onca Leptailurus serval P. yagouaroundi P. onca P. concolor Leopardus pardalis L. wiedii

Little-spotted cat (10/27) Ocelot (2/4) Cougar (2/3)3 Jaguar (17/97)

Leopardus tigrinus L. pardalis P. concolor P. onca

Cougar (9/70)

P. concolor

Ocelot (15/73)

L. pardalis

Little-spotted cat (2/41) Jaguarundi (3/53) Jaguarundi (1/4) Ocelot (1/14) Margay (1/17)

L. tigrinus P. yagouaroundi P. yagouaroundi L. pardalis L. wiedii

(37.5%) seropositivity reported in a previous survey 4 yr earlier11 and is similar to very recent findings from a zoo facility of Sa˜o Paulo State where only 1/35 (2.86%) animals (a jaguarundi)

10 5 5 9

9 9 9 9 29 Present study Present study

was found seropositive for the serovar Sentot from nine wild felid species analyzed.29 In the present study, the first seropositive animal was a solitary, 5-yr-old, captive-born male ocelot that

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was positive for the serovar Cynopteri (100), classified into the Cynopteri serogroup. The second animal was a wild-born solitary female margay that had lived at the institution for 18 yr and was seropositive for serovars Autumnalis (100) and Butembo (200), classified into the same serogroup, Autumnalis. Because the latter serovars are part of the same serogroup and the animal presented low titers, the possibility of cross-reactivity cannot be excluded. Although no comprehensive survey has been previously performed in wildlife animals in Parana state, serovars Cynopteri, Autumnalis, and Butembo have been found in wildlife species throughout Brazil and may indicate exposure to similar potential sources of infection.3,4,5,9,10,29 In the previous study, the female margay had shown positive titers to serovars Autumnalis (3,200), Butembo (200), Pomona (1,600), Icterohaemorrhagiae (800), and Copenhageni (100).10 Because the original titer for Autumnalis was very high, current serologic results for this female margay may indicate persistent antibodies. The Butembo titer may be due to a cross-reaction, elevated antibody levels from previous exposure, or new contact with the agent.6 Because no previous report has been found on titer persistence in either domestic or wild felids, causes for changes in antibody levels over time can be speculated. Recently, a tapir specimen from the Sorocaba Zoo had titer 1,600 to serovars Icterohaemorrhagiae and Pomona, and after 2.5 yr titer to the same serovars was 400. No other tapirs that share the same enclosure had titers, which may indicate persistence of antibody levels.29 The most prevalent serovar found in the previous survey was Patoc, which accounted for 7/12 (58.33%) seropositive samples, with titers varying from 100 to 800. Patoc is considered an environmental nonpathogenic serovar. Previous research indicates that Leopardus sp. might play a role as Patoc reservoirs in the studied region, as most of the animals were naturally found in the area.10 However, Patoc was not found in the present study, suggesting that the Leptospira serovars present in the area may have changed over time, or that Leopardus sp. were not reservoirs and thus do not shed the organism into the environment. When only the animals that were seropositive to pathogenic serovars (n ¼ 5) are considered, the comparison between both surveys can be conducted: 3.5% in the present study compared to 15.6% (5/32) in the study by Guerra-Neto et al.10 The difference in seroprevalence may be ex-

plained by the lower sample size of the present study, persistence of titers, improvement in the health status of the animal population, or reduction in the rodent populations in the facility. In general, there are no standard laboratory protocols, particularly for serologic techniques, for leptospirosis, thereby potentially leading to inadequate coordination of methods and interpretation.6 This may explain the differences in reading and interpreting techniques in different laboratories by different technicians. According to the BVBS veterinarians, there were no changes in animal management, water supply, nutrition, enclosure cleaning, pest control, or drug therapy during the period between both surveys that could explain the difference seen between the two studies. Because seroprevalence in the present study was very low and no clinical signs were observed, no further leptospirosis monitoring or intervention, such as changes in husbandry or handling of felids or enhanced rodent population control, was implemented. Leptospirosis titer maintenance is not fully established in felids; however, the removal of an infection source may be the most likely cause of the lower seroprevalence found in the second survey. Comparing the studies, in the first study, 61 felids were tested, and of the 57 tested in the second study, 32 overlapped with the first study, including 3 jaguarondis, 4 ocelots, 11 margays, and 14 little-spotted cats. Out of these 32 felids, 12 (including the margay seropositive in the present study) had titers to one or more serovars in the first study; however, none demonstrated clinical signs of leptospirosis. Some seropositive animals from the first study were transferred to another facility and were not included in the present one. Studies based on bacterial isolation from urine are needed in order to identify the exact role of these animals in the epidemiology of the disease, especially to ascertain if they can act as reservoirs, as suggested by Guerra-Neto et al.11 for felids of the Leopardus genus. Based on the findings of the present study and other recent reports in wild cats,21 detection of serovars not transmitted by rodents should be further investigated to fully establish the role of felids in the Leptospira spp. life cycle. Acknowledgments: Funding for Leila Ullmann’s research fellowship was provided by the State of Sa˜o Paulo Research Foundation (FAPESP). Studies were conducted in partnership

ULLMANN ET AL.—LEPTOSPIRA SPP. SURVEY IN NEOTROPICAL FELIDS OF BRAZIL

with Itaipu Binacional, Foz do Iguacu; the Federal Parana´ University, Curitiba; and the Sa˜o Paulo State University (UNESP), Brazil. We kindly thank the help given by the Bela Vista Sanctuary and Itaipu Binacional personal for the sample collection and storage. The result of this survey does not reflect the opinion of Itaipu.

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29. Ullmann, L. S. 2011. Inque´rito sorolo´gico, molecular e fatores de risco para leptospirose em mamı´feros cativos, papel dos animais sinantro´picos presentes no local e aspectos de sau´de pu´blica. M.Sc. ¸ Dissertaca˜o, Univ. Estadual Paulista, Botucatu, Sa˜o Paulo, Brazil. Received for publication 27 May 2010