First Evidence of Feline Herpesvirus, Calicivirus, Parvovirus, and Ehrlichia Exposure in Brazilian Free-Ranging Felids

Filoni, C; Catão-Dias, J L; Bay, G; Durigon, E L; Jorge, R S P; Lutz, H; Hofmann-Lehmann, R (2006). First evidence of feline herpesvirus, calicivirus, parvovirus, and Ehrlichia exposure in Brazilian free-ranging felids. Journal of Wildlife Diseases, 42(2):470-7. Postprint available at: http://www.zora.uzh.ch

University of Zurich Posted at the Zurich Open Repository and Archive, University of Zurich. Zurich Open Repository and Archive http://www.zora.uzh.ch

Originally published at: Journal of Wildlife Diseases 2006, 42(2):470-7. Winterthurerstr. 190 CH-8057 Zurich http://www.zora.uzh.ch

Year: 2006

First evidence of feline herpesvirus, calicivirus, parvovirus, and Ehrlichia exposure in Brazilian free-ranging felids

Filoni, C; Catão-Dias, J L; Bay, G; Durigon, E L; Jorge, R S P; Lutz, H; Hofmann-Lehmann, R

Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch

Originally published at: Journal of Wildlife Diseases 2006, 42(2):470-7. First evidence of feline herpesvirus, calicivirus, parvovirus, and Ehrlichia exposure in Brazilian free-ranging felids

Abstract

Serum samples from 18 pumas (Puma concolor), one ocelot (Leopardus pardalis), and two little spotted cats (Leopardus tigrinus) collected from free-ranging animals in Brazil between 1998 and 2004 were tested by indirect immunofluorescence (IFA) for antibodies to feline herpesvirus 1 (FHV 1), calicivirus (FCV), coronavirus (FCoV), parvo-virus (FPV), Ehrlichia canis, Anaplasma pha-gocytophilum, and Bartonella henselae. Serum samples also were tested, by Western blot and ELISA, for feline leukemia virus (FeLV) specific antibodies and antigen, respectively, by Western blot for antibodies to feline immunodeficiency virus (FIV), and by indirect ELISA for antibodies to puma lentivirus (PLV). Antibodies to FHV 1, FCV, FCoV, FPV, FeLV, FIV, PLV or related viruses, and to B. henselae were detected. Furthermore, high-titered antibodies to E. canis or a closely related agent were detected in a puma for the first time. Journal of Wildlife Diseases, 42(2), 2006, pp. 470–477 # Wildlife Disease Association 2006

First Evidence of Feline Herpesvirus, Calicivirus, Parvovirus, and Ehrlichia Exposure in Brazilian Free-ranging Felids

Claudia Filoni,1,6 Jose´ Luiz Cataõ-Dias,1,2 Gert Bay,3 Edison Luiz Durigon,4 Rodrigo Silva Pinto Jorge,5 Hans Lutz,3 and Regina Hofmann-Lehmann31Departamento de Patologia, Faculdade de Medicina Veterina´ria e Zootecnia, Universidade de Saõ Paulo, Avenida Prof. Dr. Orlando Marques de Paiva 87, 05508-270, Saõ Paulo, SP, Brazil; 2 Fundaçaõ Parque Zoolo´gico de Saõ Paulo, Avenida Miguel Ste´fano 4241, 04301-905, Saõ Paulo, SP, Brazil; 3 Clinical Laboratory, Vetsuisse Faculty, University of Zurich, Wintherthurerstrasse 260, CH-8057 Zurich, Switzerland; 4 Laborato´rio de Virologia Cl´ınica e Molecular, Departamento de Microbiologia, Universidade de Saõ Paulo, Instituto de Cieˆncias Biome´dicas, Edif´ıcio Biome´dicas II, Avenida Prof. Lineu Prestes, 1374, 05508-900, Saõ Paulo, SP, Brazil; 5 Departamento de Medicina Veterina´ria Preventiva e Sau´de Animal, Faculdade de Medicina Veterina´ria e Zootecnia, Universidade de Saõ Paulo, Avenida Prof. Dr. Orlando Marques de Paiva 87, 05508-270, Saõ Paulo, SP, Brazil; 6 Corresponding author (email: [email protected])

ABSTRACT: Serum samples from 18 pumas Data regarding viral infections in neo- (Puma concolor), one ocelot (Leopardus parda- tropic Brazilian felids are sparse and have lis), and two little spotted cats (Leopardus concentrated primarily on feline retro- tigrinus) collected from free-ranging animals in Brazil between 1998 and 2004 were tested by viruses. A feline immunodeficiency virus indirect immunofluorescence (IFA) for anti- (FIV) pol gene from a Brazilian zoo puma bodies to feline herpesvirus 1 (FHV 1), (Puma concolor) has been sequenced calicivirus (FCV), coronavirus (FCoV), parvo- (Carpenter et al., 1996) and antibodies to virus (FPV), Ehrlichia canis, Anaplasma pha- FIV have been reported from a Brazilian gocytophilum, and Bartonella henselae. Serum samples also were tested, by Western blot and free-ranging puma (Brown et al., 1993). ELISA, for feline leukemia virus (FeLV) Additionally, FIV provirus has been re- specific antibodies and antigen, respectively, portedly detected in Brazilian jaguars by Western blot for antibodies to feline (Panthera onca), pumas, jaguarundis (Her- immunodeficiency virus (FIV), and by indirect pailurus yagouarondi), ocelots (Leopardus ELISA for antibodies to puma lentivirus (PLV). Antibodies to FHV 1, FCV, FCoV, FPV, FeLV, pardalis), margays (Leopardus wiedii), FIV, PLV or related viruses, and to B. henselae pampas cat (Oncifelis colocolo), and little were detected. Furthermore, high-titered anti- spotted cats (Leopardus tigrinus) (Leal bodies to E. canis or a closely related agent and Ravazzolo, 1998). Although evidence were detected in a puma for the first time. of exposure to feline leukemia virus Key words: Anaplasma, Bartonella, Ehrli- chia, feline viruses, free-ranging felids, Leo- (FeLV) and FIV was not detected in pardus tigrinus, Leopardus pardalis, Puma a captive small neotropic felid population concolor, serology. from Sa˜o Paulo state in Brazil (Filoni et al., 2003), three captive neotropic felids Infections of nondomestic felids with were found to be FeLV viremic (one viruses that are common in domestic margay and two pampas cats) in a survey carnivores have been reported for numer- conducted in North American zoos (Ken- ous species worldwide (Mochizuki et al., nedy-Stoskopf, 1999). Captive jaguars, 1990; Olmsted et al., 1992; Paul-Murphy pumas, margays, a pampas cat, and et al., 1994; Hofmann-Lehmann et al., a free-ranging ocelot in Brazil were found 1996; Daniels et al., 1999; Leutenegger et FeLV positive on indirect immunofluores- al., 1999a; Fromont et al., 2000; Ostrowski cence assays (IFA) (Schmitt et al., 2003). et al., 2003). Although the implications of In Brazil, antibodies to feline corona- these infections on wild felid conservation virus (FCoV) were reported in captive are usually difficult to assess, it is broadly jaguars, pumas, margays, a pampas cat, accepted that monitoring these infections and a free-ranging ocelot (Schmitt et al., is an important component for the man- 2003). To the best of our knowledge, there agement of endangered populations (Mur- are no published reports of FHV 1, FCV, ray et al., 1999; Daszak et al., 2000). and FPV infections in wild felids in Brazil.

470 SHORT COMMUNICATIONS 471

Studies have demonstrated the exposure captured with the use of trained dogs (for of a variety of free-ranging and captive large felids) or live traps (small felids) and felids to Bartonella henselae, including immobilized with the use of darts and neotropic felids from North American a combination of tiletamine HCl and zoos (Kelly et al., 1998; Yamamoto et al., zolazepam HCl (TelazolH, Fort Dodge, 1998; Rotstein et al., 2000; Molia et al., Fort Dodge, Iowa, USA). At the time of 2004; Pretorius et al., 2004). An extensive sample collection, a complete physical serosurvey of pumas reported seropositive examination was performed; animals were animals throughout most of the geograph- radio-instrumented and were released at ical range of the species (Chomel et al., the place of capture for further monitor- 2004b); the overall seroprevalence in ing. One of the samples (serum from heart South American pumas was found to be clot) was obtained from a necropsy per- 22.4%. To our knowledge, there is no formed on the little spotted cat from previous report of Ehrlichia canis and Ubatuba, Sa˜o Paulo (Table 1, Fig. 1). Anaplasma phagocytophilum infections in Antibody titers to FHV 1, FCV, FCoV, captive or free-ranging neotropic Brazilian and FPV were determined in 21 serum felids. samples by IFA as described (Hofmann- The aim of this study was to serologi- Lehmann et al., 1996). All sera were cally survey free-ranging felids from Brazil screened at a dilution of 1:20. For FHV to determine the extent of exposure to 1, FCV, and FPV, positive and question- selected viruses (FHV 1, FCV, FCoV, able results were titrated in a two-fold FPV, FeLV, FIV, and the puma lentivirus, serial dilution starting at 1:20 until end- PLV) and hemoparasites (E. canis, A. point. For FCoV, positive samples were phagocytophilum, and B. henselae). titrated using 1:25, 1:100, 1:400, and TheGenomeResourceBankfrom 1:1600 dilutions. National Research Center for Carnivores Exposure to E. canis and A. phagocyto- Conservation—CENAP, unity of National philum or closely related agents was Environmental Agency—IBAMA in Brazil evaluated by IFA with the use of 1:80 provided serum samples, which were dilutions of the serum samples and Mega transported to Switzerland in full compli- Screen Fluoehrlichia c. slides (MegaCor ance with specific federal permits, like Diagnostik GmbH, 6912 Ho¨rbranz, Aus- Convention on International Trade in tria) or E. equi slides (VMRD, Inc. Endangered Species—CITES (Permit Pulman, Washington, USA). Serum sam- Numbers 0112928BR and 1562/04), Ge- ples from 20 animals were assayed for netic Heritage Management Council— antibodies to B. henselae by IFA (Glaus et CGEN and Agriculture Ministry (CSI al., 1997). All sera were screened at 1530/2004), on dry ice as diagnostic dilutions of 1:64 and 1:128. Titers of $64 specimens packed in compliance with were considered positive. Positive serum IATA Packing Instruction 650. Samples samples were titrated until endpoint by were stored at CENAP at 280 C or in twofoldserialdilutions. liquid nitrogen. For quality control of the IFA slides, Blood samples were collected from 18 aliquots of the cell cultures (140 ml) or free-ranging pumas, one ocelot, and two scrapings from the slides were tested for little spotted cats between 1998 and 2004 presence of unwanted antigens. Samples from four biomes (Amazon Forest, Atlan- were incubated at 40 C for 10 min in tic Forest, Cerrado, and Pantanal) that 300 ml lysis buffer and total nucleic acid included 13 municipalities in the states of (TNA) was extracted with the use of the Rondoˆ nia, Acre, Mato Grosso, Mato MagNA Pure LC instrument (Roche, Grosso do Sul, Parana´,Sa˜o Paulo e Rio Rotkreuz, Switzerland). The TNA samples de Janeiro (Fig. 1, Table 1). Animals were were analyzed by real-time TaqMan poly- 472 JOURNAL OF WILDLIFE DISEASES, VOL. 42, NO. 2, APRIL 2006

FIGURE 1. Map depicting the geographic areas within Brazil from which samples from 21 free-ranging felids were collected.

merase chain reaction (PCR) or reverse FCV (Kummrow et al., 2005). No un- transcriptase (RT) PCR on an ABI Prism wanted agents were detected in any 7700 Sequence Detection System (Ap- antigen preparations. plied Biosystems, Foster City, California, All sera were examined for the presence USA) for the presence of the agents of of antibodies to FeLV by Western blot interest: FIV (Leutenegger et al., 1999b), (Hofmann-Lehmann et al., 1995). Sam- FeLV (Hofmann-Lehmann et al., 2001), ples containing antibodies to gp70, p58, FCoV (Gut et al., 1999), FHV 1 (Vogtlin et p27, p15, and p12 were judged positive. al., 2002), FPV (Meli et al., 2004), and Additionally, the serum samples were TABLE 1. Serologic test results for free-ranging Puma concolor , Felis pardalis , and Felis tigrinis in Brazil.

Serologic results c Indirect fluorescent antibody test Western blot Ehrlichia Bartonella ELISA Indirect Biome Location a Species b n FHV-1 FCV FCoV FPV canis henselae FeLV FIV PLV

Amazon Forest VR P. concolor (3,4,5) 3 2 N d N3 N 3 N N N RA L. pardalis (18) 1 1 1 1 1 N 1 N N N Pantanal BM P. concolor (1,2,8,9,11, 19) 6 N 2 N N 1 5 e 11 2 CM P. concolor (15,16) 2 N N N 2 N 1 N N N Cerrado BaM P. concolor (7) 1 N N N N N 1 N N N JM P. concolor (12,13) 2 N N N 2 N 2 N N N PM P. concolor (14) 1 1 N N N N 1 N N N Atlantic Forest PP P. concolor (6) 1 1 1 N N N 1 N N N SS P. concolor (10) 1 N N N N N 1 1 N N lS P. concolor (17) 1 1 N N 1 N 1 N N N UP L. tigrinus (20) 1 N 1 N 1 N 1 N N N NR L. tigrinus (21) 1 N 1 N N N 1 N N N TOTAL 21 6 6 1 10 1 19 2 1 2 Antibody titer 20–160 20–40 100 40–640 20,480 64–1024 (range) HR OMNCTOS473 COMMUNICATIONS SHORT a VR 5 Vilhena/ Rondo ˆ nia (12 u379S, 60 u079W); RA 5 Rio Branco/Acre (09 u529S, 67 u529W); BM 5 Baraõ de Melgaço/ Mato Grosso (16 u079S, 55 u529W); CM 5 Corumba´/ Mato Grosso do Sul (19 u039S, 57 u419W); BaM 5 Bataguassu/Mato Grosso do Sul (21 u419S, 52 u269W); JM 5 Jardim/Mato Grosso do Sul 21 u269S, 56 u119W; PM 5 Ponta Pora˜/Mato Grosso do Sul (22u339S, 55 u419W); PP 5 Parque Nacional do Iguac ¸u /Parana´ (25u339S, 54 u339W); SS 5 Serra da Cantareira/Sa õ Paulo (23 u339S, 46 u419W); lS 5 Ilha Solteira/Sa õ Paulo (20 u269S, 51u189W); UP 5 Ubatuba/Saõ Paulo (23 u269S, 45 u039W); Nova Friburgo/Rio de Janeiro (22 u189S42u339W). b Individual animal identification number. c Number of animals positive. All animals tested negative for antibodies to Anaplasma phagocytophium on IFA; all animals tested negative on Direct Fe LV p27 ELISA. d N 5 negative. e No material was available from one puma for the B. henselae IFA. 474 JOURNAL OF WILDLIFE DISEASES, VOL. 42, NO. 2, APRIL 2006 tested for the presence of FeLV p27 as persist in the environment and this may a measure for viremia by a sandwich explain the single FCoV antibody positive ELISA (Lutz et al., 1983). One sample animal that was detected in this study. tested positive. However, upon retesting However, even though FHV 1 and FCV in the presence of mouse serum, it was do not persist well in the environment and determined that this represented a false require close contact to be transmitted, positive; the positive reaction resulted seropositive results to both of these viruses from cross-linking of the monoclonal were relatively common (29%) in this mouse anti-FeLV antibodies by feline survey. This situation is similar to that of antimouse antibodies and not by binding free-ranging lions in Africa, where high of the FeLV antigen. Antibodies to FIV antibody prevalence rates to FHV 1 have were detected by Western blot (Lutz et been reported (Hofmann-Lehmann et al., al., 1980, 1988). Samples with antibodies 1996). to p24 and p15 were considered positive. The finding of antibodies to retroviruses Antibodies reactive to a specific puma addresses the need for additional moni- lentivirus peptide were detected by an toring. Two animals had antibodies to indirect ELISA (Van Vuuren et al., 2003). FeLV, but FeLV antigen was not detected. Results of the serosurvey are summa- It is possible that these animals had rized in Table 1. This is the first reported undergone regressive FeLV infection and serologic evidence that FHV 1, FCV, FPV, mounted specific immune responses. The and the obligate intracellular bacterium E. finding of seropositive animals nonethe- canis (or antigenically related agents) are less may be of concern, because FeLV is present among free-ranging felids in potentially pathogenic (Jessup et al., 1993; Brazil. Antibodies to FCoV in a free- Fromont et al., 2000; Sleeman et al., 2001) ranging ocelot, antibodies to FeLV, FIV, and domestic cats (Felis catus) have been and PLV (or related lentiviruses) in free- implicated as a source of FeLV infection ranging pumas, and antibodies to B. to nondomestic felids (Jessup et al., 1993; henselae in all three feline species also Sleeman et al., 2001). One puma was were detected. Antibody prevalence rates found to be FIV seropositive. This animal based on all 21 animals were as follows: B. and a second puma also had antibodies to henselae (95%); FPV (48%); FHV 1 and PLV (Table 1). To determine the nature FCV (29%); FeLV and PLV (10%); and and origin of these viruses, sequence FCoV, FIV, and E. canis (5%). Antibodies analysis would be necessary; however, to A. phagocytophilum were not detect- material suitable for these analyses was able. unavailable. Our results indicate that co-infection Antibodies to E. canis were detected in with the studied viral pathogens is not one puma. Although the titer was high, common in the Brazilian neotropic felids: interpretation of this single positive is most animals (76%) were seropositive for difficult. It could represent evidence of only one or two of the selected viruses. An an E. canis infection or antibodies could exception was one Amazonic ocelot sero- have resulted from an infection with positive for four of the selected viruses. a related Ehrlichia species, such as E. The high prevalence of antibodies to chaffeensis or E. ewingii. Ticks are com- parvoviruses (48%) may relate to the mon in the area (Baraõ de Melgaço, Mato environmental stability of these nonenve- Grosso) where this animal was sampled loped viruses; free-ranging wild felids may and numerous tick species including be exposed in areas contaminated by Amblyomma ovale, A. parvum, A. cajen- feces, even if they are solitary, widely nense, Boophilus microplus, and Ixodes dispersed, and at low density (Barker and aragaoi have been reported from pumas Parish, 2001). In contrast, FCoV do not (Labruna et al., 2005). SHORT COMMUNICATIONS 475

A very high prevalence of antibodies to samples as well as to all the researchers B. henselae (95%), which causes cat that collected them during their field scratch disease in humans (Chomel et al., projects throughout the country. We 2004a) was detected. Brazilian free-rang- especially thank Rose Lilian Gasparini ing felids may be a reservoir for B. Morato and Ota´vio Borges Maia for having henselae, and zoo guidelines and wildlife always been promptly helpful in conduct- management programs in Brazil should ing all legal paperwork requested, and further emphasize recommendations for Ronaldo Gonçalves Morato and Roge´rio safe handling of animals. Bartonella hen- Cunha de Paula for reporting sample selae is mainly transmitted via cat fleas origins and help in map figure prepara- (Ctenocephalides felis) in domestic cats tion. We thank Dr. M. Meli, Dr. V. (Chomel et al., 1996) and is potentially Cattori, Y. Ahmed, E. Go¨nczi, T. Meili transmissible among domestic cats and Prodan, B. Weibel, and N. Tschopp, wild felids (Chomel et al., 2004b). In Clinical Laboratory, Vetsuisse Faculty, addition to fleas, ticks from the genus University of Zurich, for excellent techni- Ixodes have been suggested as potential cal assistance. Laboratory work was per- vectors for Bartonella spp. (Schouls et al., formed using the logistics of the Centre 1999; Chang et al., 2001; Chomel et al., for Clinical Studies at the Vetsuisse 2004a). Free-ranging felids are considered Faculty, University of Zurich. We are also as hosts for C. felis (Linardi and Guimar- indebted to Fundaçaõ Parque Zoolo´gico a˜es, 2000) and ticks such as Ixodes aragaoi de Saõ Paulo, Coordenaçaõ de Aperfei- have been collected and identified para- çoamento de Pessoal de N´ıvel Superior— sitizing a puma (Labruna et al., 2005). CAPES, Pro´-Reitoria de Po´s-Graduaçaõ According to the physical examinations from Universidade de Saõ Paulo and the at the moment of sampling there were no International Relations Office, University signs of the infections or related diseases of Zurich, Switzerland. Regina Hofmann- in the animals. The single necropsied Lehmann is the recipient of a professor- little spotted cat was in very good body ship by the Swiss National Science Foun- condition with the exception of heavy dation (PP00B-102866). This study was parasitosis, including Dirofilaria immitis. conducted by Claudia Filoni as partial At present the significance of these fulfillment of the requirements for a doc- pathogens to the overall health of these torate degree at Departamento de Patolo- wild felid populations is unknown. Since gia, Faculdade de Medicina Veterina´ria e the infectious agents investigated in the Zootecnia, Universidade de Saõ Paulo. present study are common domestic carnivore’s pathogens, it is not possible LITERATURE CITED to determine whether these agents have BARKER,I.K.,AND C. R. PARISH. 2001. Parvovirus been historically associated with neotrop- infections. In Infectious diseases of wild animals, ic felid populations or have been in- 3rd Edition, E. S. Williams and I. A. Barker troduced by interactions with domestic (eds.). Iowa State University Press, Iowa, carnivores. Isolation and molecular char- pp. 131–146. BROWN, E. W., S. MITHTHAPALA, AND S. J. O’BRIEN. acterization of these pathogens, both in 1993. Prevalence of exposure to feline immuno- domestic and wild carnivores, would be deficiency virus in exotic felid species. Journal of helpful to answer this question and may Zoo and Wildlife Medicine 24: 357–364. provide important baseline data to de- CARPENTER, M. A., E. W. BROWN,M.CULVER,W.E. velop effective programs aimed at in- JOHNSON,J.PECON-SLATTERY,D.BROUSSET, AND S. J. O’BRIEN. 1996. Genetic and phylogenetic fectious disease prevention and mitiga- divergence of feline immunodeficiency virus in tion. the puma (Puma concolor). Journal of Virology The authors express their sincere ap- 70: 6682–6693. preciation to CENAP–IBAMA for the CHANG, C. C., B. B. CHOMEL,R.W.KASTEN,V. 476 JOURNAL OF WILDLIFE DISEASES, VOL. 42, NO. 2, APRIL 2006

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