Journal of Wildlife Diseases, 46(3), 2010, pp. 718–723 # Wildlife Disease Association 2010

TESTING FOR SALMONELLA SPP. IN RELEASED PARROTS, WILD PARROTS, AND DOMESTIC FOWL IN LOWLAND PERU

Oscar Butron1,3 and Donald J. Brightsmith2,3,4 1 Facultad de Veterinaria y Zoote´cnica, Universidad Peruana Cayetano Heredia, Lima, Peru 2 Schubot Exotic Bird Health Center, Texas A&M University, College Station, Texas 77845-4467, USA 3 Authors contributed equally to this work 4 Corresponding author (email: [email protected])

ABSTRACT: Wild animal populations face threats from pathogens from both intentionally released captive animals and domestic animals that accompany human settlements. From December 2004 through August 2005, we studied free living macaws and parrots in the Tambopata National Reserve in the Peruvian Amazon and semicaptive domestic fowl in human settlements adjacent to the reserve. In 1992–1993, large macaws (Aras spp.) that were serologically positive for Salmonella Pullorum were released into this reserve, which hosts dense populations of free-living parrots and macaws. We collected cloacal swabs from 64 birds and cultured for Salmonella spp. via standard laboratory methods. All 35 psittacines tested were culture negative for Salmonella spp., while 31% of 29 domestic fowl were culture positive. Our findings suggest that the domestic fowl that accompany human settlement in this region carry and shed Salmonella spp. that could threaten wild bird populations in and around the reserve. Key words: Ara, conservation, macaw, parrot, pathogen pollution, reintroduction, Salmo- nella spp.

INTRODUCTION low pathogenicity in other avian species (D’Aoust and Prescott, 2007). This sug- Wildlife populations, including rare and gests that large populations of free-living endangered species, can suffer mass parrots in the Tambopata National Re- mortality and possibly extinction from serve may have been exposed to Salmo- disease outbreaks (Friend et al., 2001; nella Pullorum. Additionally, thousands of Lips et al., 2008). Many recent outbreaks parrots consume soil at a large river-edge are the result of ‘‘pathogen pollution,’’ in cliff or ‘‘clay lick’’ located within 1 km of which pathogens are inadvertently spread the release site (Brightsmith, 2004). These to immunologically naı¨ve populations by parrots feed and defecate in extremely human activities (Daszak et al., 2000). high densities on this lick providing ample Release of captive raised or translocated opportunity for fecal-oral transmission of animals is a valuable wildlife management Salmonella and other pathogens. technique. However, releases risk intro- Released birds are not the only poten- ducing pathogens to wild populations tial sources of pathogens faced by wild (Cunningham, 1996). In the early 1990s, populations (Daszak et al., 2000). Each 32 large macaws (Scarlet Macaw, Ara year, millions of live domestic fowl are macao; Red-and-green Macaw, A. chlor- sold and transported worldwide (USDA opterus; and Blue-and-yellow Macaw, A. Foreign Agricultural Service, 2005). Some ararauna) were hand-reared and released of these fowl end up in backyard flocks into the Tambopata National Reserve in adjacent to natural forest and protected the lowlands of southeastern Peru (Ny- areas where they come in contact with cander et al., 1995; Brightsmith et al., wild species (Roelke-Parker et al., 1996; 2005). Serologic testing in 1994 revealed Chen et al., 2005; Hernandez-Divers et that most of the released birds were al., 2006). The purpose of this study was to antibody positive for Salmonella serotype determine if Salmonella spp. were present Pullorum but the wild birds were negative in free-living parrots in the Tambopata (Karesh et al., 1997). Salmonella Pullorum National Reserve and in domestic fowl in is a common pathogen of poultry but has local communities adjacent to the reserve.

718 BUTRON AND BRIGHTSMITH—PARROTS, FOWL AND SALMONELLA IN PERU 719

MATERIALS AND METHODS We sampled chicks from Scarlet and Red-and- green Macaw nests. We accessed the nests Study sites using single rope climbing techniques (Perry This study was conducted in and adjacent to and Williams, 1981). Chicks were placed in a the Tambopata National Reserve (275,000 ha), bucket and lowered to the ground for sam- in the Department of Madre de Dios in the pling. Chicks were all sampled at 2–3 wk of lowlands of southeastern Peru. The main age. We also sampled adult macaws that had study site was Tambopata Research Center been hand-raised and released at Tambopata (TRC, 13u89170S, 69u369290W, 250 m eleva- Research Center during 1992–1993. To do tion, 3,200 mm of rain per year), which lies at this, we captured macaws by hand as they the interface of tropical moist and subtropical foraged in the lodge (Brightsmith et al., 2005). wet forest (Tosi, 1960; Brightsmith, 2004). The To determine if Salmonella was present in center is surrounded by mature floodplain domestic fowl (chickens, ducks, and turkeys) in forest, successional floodplain forest, Mauritia the communities along the border of the flexuosa (Arecaceae) palm swamp, and upland Tambopata National Reserve, we located small forest (Foster et al., 1994). The center is a farms with backyard domestic fowl, obtained tourist lodge owned and operated by Rain- permission from farm owners, and captured forest Expeditions, which has supported parrot domestic fowl by hand. The small farms research since 1989 (Brightsmith, 2008). At sampled contained a total of about 170 domestic the time of this study, the lodge was located birds, of which about 20% were sampled. The about 300 m from a large ‘‘clay lick,’’ a 500-m- number sampled was limited by the ability to long, 25- to 30-m high bank of exposed soil quickly capture the free-ranging animals and along the western bank of the upper Tambo- the willingness of the owners to have their pata River, where over 1,000 psittacine birds animals pursued and handled. All Psittaciformes of up to 17 species gather daily to consume soil and domestic fowl were manually restrained for (Brightsmith, 2004). cloacal swab collection. All samples were We also studied domestic fowl from five collected from August to December 2005. communities along the Tambopata River near the northern border of the Tambopata National Analyses Reserve. The communities studied were: Balti- Upon collection, all cloacal swabs were more (12u499590S, 69u259590W), Condenado placed in Stuart transport medium (Becton (12u52910S, 69u25910W), Sachavacayoc (12u519 Dickinson [BD], Franklin Lakes, New Jersey, 00S, 69u22910W), La Torre (12u469590S, 69u USA) and selenite broth (BD), and refrigerated 9 0 9 0 9 19 1 W), and Infierno (12u43 59 ,69u13 at 4–8 C for up to 3 days until the samples were 0 59 W). These small communities consist mainly transported to the laboratory for analysis. of families with subsistence and small-scale Presence of Salmonella spp. was determined commercial agriculture operations, who sup- by culture in the Laboratory of Microbiology plement their income with hunting, gold and Immunology of the Faculad de Veterinaria mining, timber harvest, and collection of y Zootecnia of the Universidad Peruana Caye- nontimber forest products. Most families main- tano Heredia. Samples were pre-enriched in tain a few domestic fowl (chickens [Gallus peptone water (Merck, Whitehouse Station, gallus], ducks [genera Anus and Cairina], or New Jersey, USA) for 24 hr and enriched in turkeys [Meleagris gallopavo]) and some also selenite cystine broth (BD) and tetrathionate keep parrots as pets. broth (Merck) for 24 hr then plated on xylose– lysine–desoxycholate agar (DIFCO, BD) and Capture Salmonella–Shigella agar (DIFCO) and incu- To determine if Salmonella spp. were pre- bated at 37 C for 24 hr. Presumptive positive sent in the wild parrots, we captured birds at growths (dark bacterial colonies) were grown the clay lick adjacent to TRC using a blind on tryptic soy agar (Merck) and isolates from surrounded by five snare traps. The traps them were inoculated on Kligler triple sugar consisted of a long pole with two thin branches iron agar (TSI, Merck) and lysine-decarboxyl- covered with nylon nooses at the top. When ase agar (LDA, DIFCO) and incubated at 42 C parrots perched on these branches their feet for 24 hr. Positive isolation of Salmonella spp. became ensnared in the nooses. When a parrot was determined by the slope reverting to red, became entrapped, the pole was lowered to indicating a lactose-negative reaction, a yellow the ground and the bird removed for sampling. butt indicator of a glucose-positive reaction, a To determine if Salmonella spp. were purple reaction throughout the LDA, and a present in the macaw population around blackening of the Kligler’s agar superimposed TRC, we sampled both chicks and adult birds. on the previous reactions due to the production 720 JOURNAL OF WILDLIFE DISEASES, VOL. 46, NO. 3, JULY 2010

of hydrogen sulfide (H2S), as the characteristic TABLE 1. Testing for Salmonella spp. in released reactions caused by most Salmonella spp. macaws and wild parrots in the Tambopata National species in Kligler’s agar (Vadillo et al., 2002). Reserve, Peru and domestic fowl in human settlements adjacent to the reserve. Species sampled are included in the Methods section. RESULTS Positive Positive Cloacal swabs from 35 individual psit- Sampled (n) (%)95% CI tacine birds from the area around TRC were cultured for Salmonella spp. These Released macaws 5 0 0 NA Scarlet Macaw included four Scarlet Macaws and one chicks 5 0 0 NA Red-and-green Macaw that were hand Wild adult parrots 25 0 0 NA raised and released in 1993–1994 (Karesh Total parrots 35 0 0 NA et al., 1997). One of these Scarlet Macaws Poultry 22 6 27 7–47% Ducks 7 3 43 3–93% was sampled twice (3 mo apart). In Total fowl 29 9 31 13–9% addition, we cultured cloacal swabs from five chicks from four different nests of wild Scarlet Macaws and 25 wild adult our ability to recovery Salmonella spp. and psittacines captured at the clay lick, the methods used only detect individuals including one Scarlet Macaw, three Blue- that are shedding bacteria at the time of and-yellow Macaws (Ara ararauna), one sample collection. It is possible that some Red-bellied Macaw (Orthopsittaca mani- of these birds were infected and were lata), eight Blue-headed Parrot (Pionus shedding Salmonella spp. intermittently. menstruus), 11 Mealy Parrots (Amazona Given that the released macaws, includ- farinosa), and one Yellow-crowned Parrot ing those antibody positive for Salmonella (A. ochrocephala). None of these birds Pullorum, have used the clay lick for many cultured positive for Salmonella. Of the years, we were concerned that they may domestic fowl from the local communities, have infected other birds with Salmonella 31% (95% CI513–49%, n529) cultured spp., leading to endemic salmonellosis positive for Salmonella spp. Of the chick- among these wild parrot populations. ens, 27% (95% CI57–47%, n 5 22) were Among wild birds, salmonellosis outbreaks positive, and of the ducks 43% (95% CI 5 can occur via fecal-oral transmission when 3–93%, n57) were positive (Table 1). large numbers of birds congregate (Tizard, 2004). Salmonella spp. can remain viable DISCUSSION for up to 200 days in soil, and some birds None of the hand-raised macaws tested can become subclinical carriers and inter- were culture positive for Salmonella spp., mittently shed bacteria (Gerlach, 1994). suggesting that the population of released Mortalities of wild parrots due to salmo- macaws may not be currently shedding nellosis have not been reported, but Salmonella spp. None of the wild birds antibodies against S. Pullorum have been trapped at the clay lick tested positive for found in wild and captive Blue-fronted Salmonella spp., suggesting that shedding Amazons (Amazona aestiva) in Bolivia of Salmonella spp. is not common among (Deem et al., 2005). In addition, Salmo- the thousands of birds that use this clay nella spp. are known to infect a variety of lick (Brightsmith, 2004). However, addi- psittaciformes in captivity (Panigrahy et tional testing in this region is needed as al., 1979; Shima and Osborn, 1989) and only 24 of the .1,000 birds that use this outbreaks of different intensity have been site could be sampled due to the difficulty reported in aviaries (Shima and Osborn, of trapping wild psittacines. In addition, 1989; Ward et al., 2003). hold time, transport time, storage condi- The risk of introducing pathogens to tions, and media type may have affected native populations is cited as the major BUTRON AND BRIGHTSMITH—PARROTS, FOWL AND SALMONELLA IN PERU 721 reason to avoid reintroduction of psitta- The relationships between human and cine birds (Derrickson and Snyder, 1992; ecosystem health are complex. Many Snyder et al., 1996). In 1994, the majority pathogens introduced to frontier areas of the hand-raised macaws tested at TRC can spread from domestic animals that (61% of 13) had antibody titers suggestive accompany human settlement (Roelke- of exposure to Salmonella Pullorum (Kar- Parker et al., 1996; Hernandez-Divers et esh et al., 1997), while none of the eight al., 2006). In areas where newly arriving wild macaws tested had positive antibody domestic animals and their pathogens are titers. This suggests exposure of the channeled through a few commercial released birds to Salmonella Pullorum, a markets before being dispersed to rural known poultry pathogen, during the rear- communities, improved sanitary controls ing process. However, the epidemiological at markets could reduce the risk of implications of these antibody findings are pathogen pollution for millions of hectares ambiguous. Positive antibody titers do not of rural and wild lands. Improved markets necessarily imply ongoing Salmonella spp. coupled with education to improve the infection because in some cases antibodies sanitary conditions of backyard fowl could can still be detected long after the bacteria provide multiple benefits, including im- has cleared and fecal shedding has ceased proved human health through reduced (Gast, 1997). As Salmonella spp. infections exposure to zoonotic diseases, improved are often transitory, it is possible that the welfare of local people through reduced released birds were exposed to the path- livestock loss to disease, and protection of ogen during hand rearing and cleared the wildlife from the introduction of new or bacteria from their system before release emerging pathogens. As a result, conser- into the wild. In addition, lack of known vation practitioners should collaborate sensitivity and specificity of Salmonella with agricultural extension agents and Pullorum serologic testing in wild birds public health officials to encourage local results in unknown validity (D’Aoust and authorities to increase biosecurity and Prescott, 2007). health monitoring in live animal markets In the communities adjacent to the and educate animal owners about good Tambopata National Reserve, over 30% of sanitary practices. Additional attention to the backyard domestic fowl were culture live animal markets may also provide other positive for Salmonella spp. These results benefits such as reduced illegal wild confirm that the domestic fowl that accom- animal trade, improved control of zoonotic pany human settlement carry pathogens diseases, and identification and control of that could pose a risk to wildlife populations new and emerging diseases (Karesh et al., (Daszak et al., 2000; Hernandez-Divers et 2005). Given the economic cost and health al., 2006). This finding highlights the need implications of diseases linked directly to for additional investigation regarding other the live animal trade like SARS and avian pathogens these domestic fowl may harbor. influenza (Bell et al., 2004; Kilpatrick et The risk of pathogen spread is exacerbated al., 2006), increased disease surveillance of as people in these communities also keep live animal markets should be cost effec- wildlife as pets (Deem et al., 2005). During tive and provide multiple benefits at the this study, we observed pet Cobalt-winged local, national, and global levels. Parakeets (Brotogeris cyanoptera) and a ACKNOWLEDGMENTS Spix’s Guan (Penelope jacquacu) kept along- side domestic fowl. Because pets are often Thanks to I. Tizard and the staff of allowed to roam freely and frequently Tambopata Research Center for support of this project. Funding was provided by the escape, pathogens could be spread from Schubot Exotic Bird Health Center at Texas domestic fowl to wild native bird popula- A&M University and Rainforest Expeditions. tions via free-roaming pets. This research was carried out under permis- 722 JOURNAL OF WILDLIFE DISEASES, VOL. 46, NO. 3, JULY 2010

sion granted by the Peruvian government’s REYNEL,A.KRATTER,P.K.DONAHUE, AND L. J. Instituto Nacional de Recursos Naturales BARKLEY. 1994. The Tambopata-Candamo Re- (INRENA) and by the leaders of the local served Zone of southeastern Peru: A biological communities involved. Protocols used in this assessment. Conservation International, Wash- research were approved by the Duke Univer- ington, DC, 184 pp. sity Institutional Animal Care and Use Com- FRIEND, M., R. G. MCLEAN, AND F. J. DEIN. 2001. mittee. This manuscript was improved by the Disease emergence in birds: Challenges for the comments of P. Mendoza, I. Tizard, J. J. twenty-first century. Auk 118: 290–303. Heatley, and two anonymous reviewers. GAST, R. K. 1997. Paratyphoid infections. In Diseases of poultry. 10th Edition, B. W. Calnek, H. J. LITERATURE CITED Barnes, C. W. Beard, L. R. McDougald and Y. M. Saif (eds.). Iowa State University Press, BELL, D., S. ROBERTON, AND P. HUNTER. 2004. Animal Ames, Iowa, pp. 97–121. origins of SARS coronavirus: Possible links with GERLACH, H. 1994. Bacteria. In Avian medicine: the international trade in small carnivores. Principles and application, B. Ritchie, G. J. Philosophical Transactions of the Royal Society Harrison and L. R. Harrison (eds.). Wingers, of London. Biological Sciences 359: 1107–1114. Lake Worth, Florida, pp. 949–983. RIGHTSMITH B , D. J. 2004. Effects of weather on avian HERNANDEZ-DIVERS, S. M., P. VILLEGAS,F.PRIETO,J. geophagy in Tambopata, Peru. Wilson Bulletin C. UNDA,N.STEDMAN,B.RITCHIE,R.CARROLL, 116: 134–145. AND S. J. HERNANDEZ-DIVERS. 2006. A survey of ———. 2008. Rainforest Expeditions and Earth- selected avian pathogens of backyard poultry in watch as funding partners for Macaw (Ara spp.) northwestern Ecuador. Journal of Avian Medi- research in southeastern Peru. Neotropical cine and Surgery 20: 147–158. Ornithology 19(Suppl): 173–181. KARESH, W. B., A. D. CAMPO,W.E.BRASELTON,H. ———, J. HILBURN,A.DEL CAMPO,J.BOYD,M. PUCHE, AND R. A. COOK. 1997. Health evaluation FRISIUS,R.FRISIUS,D.JANIK, AND F. GUILLE´ N. of free-ranging and hand-reared macaws (Ara 2005. The use of hand-raised Psittacines for spp.) in Peru. Journal of Zoo and Wildlife reintroduction: A case study of scarlet macaws Medicine 28: 368–377. (Ara macao) in Peru and Costa Rica. Biological ———, R. A. COOK, AND E. L. BENNETT. 2005. Conservation 121: 465–472. Wildlife trade and global disease emergence. CHEN, H., G. J. D. SMITH,S.Y.ZHANG,K.QIN,J. Emerging Infectious Diseases 11: 1000–1002. WANG,R.G.WEBSTER,J.S.M.PEIRIS, AND Y. KILPATRICK, A. M., A. A. CHMURA,D.W.GIBBONS,R. GUAN. 2005. H5N1 virus outbreak in migratory C. FLEISCHER,P.P.MARRA, AND P. DASZAK. 2006. waterfowl. Nature 436: 191–192. Predicting the global spread of H5N1 avian CUNNINGHAM, A. A. 1996. Disease risks of wildlife influenza. Proceedings of the National Academy translocations. Conservation Biology 10: 349– of Sciences of the United States of America 103: 353. 19368–19373. D’AOUST, P. Y., AND J. F. PRESCOTT. 2007. Salmonel- LIPS, K. R., J. DIFFENDORFER,J.R.MENDELSON, III, losis. In Infectious diseases of wild birds, N. J. AND M. W. SEARS. 2008. Riding the wave: Thomas, D. B. Hunter and C. T. Atkinson (eds.). Reconciling the roles of disease and climate Blackwell, Ames, Iowa, pp. 270–288. change in amphibian declines. Public Library of DASZAK, P., A. A. CUNNINGHAM, AND A. D. HYATT. Science Biology 6: e72. 2000. Emerging infectious diseases of wildlife— NYCANDER, E., D. H. BLANCO,K.M.HOLLE,A.D. Threats to biodiversity and human health. CAMPO,C.A.MUNN,J.I.MOSCOSO, AND D. G. Science 287: 443–449. RICALDE. 1995. Manu and Tambopata: Nesting DEEM, S. L., A. J. NOSS,R.L.CUE´ LLAR, AND W. B. success and techniques for increasing reproduc- KARESH. 2005. Health evaluation of free-ranging tion in wild macaws in southeastern Peru. In The and captive Blue-fronted Amazon parrots (Ama- large macaws: Their care, breeding and conser- zona aestiva) in the Gran Chaco, Bolivia. Journal vation, J. Abramson, B. L. Spear and J. B. of Zoo and Wildlife Medicine 36: 598–605. Thomsen (eds.). Raintree Publications, Ft. DERRICKSON, S. R., AND N. F. R. SNYDER. 1992. Bragg, California, pp. 423–443. Potentials and limits of captive breeding in PANIGRAHY, B., J. E. GRIMES,M.I.RIDEOUT,R.B. parrot conservation. In New World parrots in SIMPSON, AND L. C. BRUMBLES. 1979. Zoonotic crisis, N. F. R. Snyder and S. R. Beissinger diseases in psittacine birds: apparent increased (eds.). Smithsonian, Washington, DC, pp. 133– occurrence of chlamydiosis (psittacosis), salmo- 164. nellosis, and giardiasis. Journal of the American FOSTER, R. B., T. PARKER, A, A. H. GENTRY,L.H. Veterinary Medical Association 175: 359–361. EMMONS,A.CHICCHO´ N,T.SCHULENBERG,L. PERRY, D. R., AND J. WILLIAMS. 1981. The tropical RODRI´GUEZ,G.LARNAS,H.ORTEGA,J.ICOCHEA, rain forest canopy: A method providing total W. WUST,M.ROMO,C.J.ALBAN,O.PHILLIPS,C. access. Biotropica 13: 283–285. BUTRON AND BRIGHTSMITH—PARROTS, FOWL AND SALMONELLA IN PERU 723

ROELKE-PARKER, M. E., L. MUNSON,C.PACKER,R. Memoria explicativa sobre el mapa ecolo´gico del KOCK,S.CLEAVELAND,M.CARPENTER,J. Peru´ . Instituto Interamericano de las Ciencias O’BRIEN,A.POSPLSCHLL,R.HOFMANN-LEHMANN, Agricolas de la Organizacio´n de los Estados H. LUTZ,G.L.M.MWAMENGELE,M.N.MGASA, Americanos, Lima, Peru, 271 pp. G. A. MACHANGE,B.A.SUMMERS, AND M. J. G. USDA FOREIGN AGRICULTURAL SERVICE. 2005. Import APPEL. 1996. A canine distemper virus epidemic and export data by commodity, www.fas.usda. in Serengeti lions (Panthera leo). Nature 379: gov/dlp/traderuns/2005/05-12/december2005. 441–445. html. Accessed May 2008. SHIMA, A. L., AND K. G. OSBORN. 1989. An epornitic VADILLO, S., S. PI´RIZ, AND E. MATEOS. 2002. Manual of Salmonella typhimurium in a collection of de Microbiologia Veterinaria. McGraw-Hill, lories and lorikeets. Journal of Zoo and Wildlife Madrid, 853 pp. Medicine 20: 373–376. WARD, M. P., J. C. RAMER,J.PROUDFOOT,M.M. SNYDER, N. F. R., S. R. DERRICKSON,S.R.BEISSINGER, GARNER,C.JUAN-SALLE´ S, AND C. C. WU. 2003. J. W. WILEY,T.B.SMITH,W.D.TOONE, AND B. Outbreak of Salmonellosis in a zoologic collec- MILLER. 1996. Limitations of captive breeding in tion of lorikeets and lories (Trichoglossus, endangered species recovery. Conservation Bi- Lorius, and Eos spp.). Avian Diseases 47: 493– ology 10: 338–348. 498. TIZARD, I. 2004. Salmonellosis in wild birds. Seminars in Avian and Exotic Pet Medicine 13: 50–66. Received for publication 29 May 2008. TOSI, J. A. 1960. Zonas de vida natural en el Peru´ . Accepted 30 December 2009.