Journal of Wildlife Diseases, 41(2), 2005, pp. 342±353 q Wildlife Disease Association 2005 CHARACTERIZATION OF CANARYPOX-LIKE VIRUSES INFECTING ENDEMIC BIRDS IN THE GALAÂ PAGOS ISLANDS Teresa Thiel,1 Noah K. Whiteman,1 Ana TirapeÂ,2,3 Maria Ines Baquero,2,4 Virna CedenÄo,2,3,4 Tim Walsh,5,6,7 Gustavo JimeÂnez UzcaÂtegui,6 and Patricia G. Parker1,5 1 Department of Biology, University of Missouri±St. Louis, Missouri 63121, USA 2 Laboratory of Fabricio Valverde, GalaÂpagos National Park, Isla Santa Cruz, GalaÂpagos, Ecuador 3 Concepto Azul, Guayaquil, Ecuador 4 Biotechnology Program, University of Guayaquil, Guayaquil, Ecuador 5 Charles Darwin Research Station, Puerto Ayora, Isla Santa Cruz, GalaÂpagos, Ecuador 6 Current address: Washington State University, Washington Animal Disease Diagnostic Laboratory, Pullman, Washington 99165-2037, USA 7 Corresponding author (email: [email protected]) ABSTRACT: The presence of avian pox in endemic birds in the GalaÂpagos Islands has led to concern that the health of these birds may be threatened by avipoxvirus introduction by domestic birds. We describe here a simple polymerase chain reaction±based method for identi®cation and discrimination of avipoxvirus strains similar to the fowlpox or canarypox viruses. This method, in conjunction with DNA sequencing of two polymerase chain reaction±ampli®ed loci totaling about 800 bp, was used to identify two avipoxvirus strains, Gal1 and Gal2, in pox lesions from yellow warblers (Dendroica petechia), ®nches (Geospiza spp.), and GalaÂpagos mockingbirds (Nesomimus parvulus) from the inhabited islands of Santa Cruz and Isabela. Both strains were found in all three passerine taxa, and sequences from both strains were less than 5% different from each other and from canarypox virus. In contrast, chickens in GalaÂpagos were infected with a virus that appears to be identical in sequence to the characterized fowlpox virus and about 30% dif- ferent from the canarypox/GalaÂpagos group viruses in the regions sequenced. These results in- dicate the presence of canarypox-like viruses in endemic passerine birds that are distinct from the fowlpox virus infecting chickens on GalaÂpagos. Alignment of the sequence of a 5.9-kb region of the genome revealed that sequence identities among Gal1, Gal2, and canarypox viruses were clustered in discrete regions. This indicates that recombination between poxvirus strains in com- bination with mutation led to the canarypox-like viruses that are now prevalent in the GalaÂpagos. Key words: Avian pox, avipoxvirus, canary pox, fowlpox, GalaÂpagos. INTRODUCTION in Hawaii (Wikelski et al., 2004), there is The GalaÂpagos Islands are volcanic in increasing concern about the introduction origin (Christie et al., 1992; White et al., of avian diseases that could result in ex- 1993) and located on the equator almost tinctions of GalaÂpagos avifauna. The ap- 1,000 km west of mainland Ecuador in pearance of avian pox±like lesions in both South America. Their isolation and relative domestic chickens and endemic birds on desolation delayed permanent colonization GalaÂpagos has heightened these concerns. by humans, and biodiversity remains most- Avian pox is a mild to severe disease of ly intact, with only about 5% of species birds that has been reported worldwide in having been lost (Gibbs et al., 1999); this approximately 60 avian species represent- includes none of the 28 breeding land bird ing 20 families. Avian pox is caused by species, 26 of which are endemic. Al- DNA viruses of the family Poxviridae, ge- though 90% of the archipelago was set nus Avipoxvirus, and transmission can oc- aside as a national park in 1959, the resi- cur through introduction into a break in dent human population, along with tour- the skin or, more commonly, when vec- ism, has grown rapidly, and exotics are tored by a biting insect. Disease is most continually being introduced despite in- commonly characterized by cutaneous creasing efforts to exclude them. Because proliferative lesions that harden to thick pathogens can have especially severe ef- scabs, but a diptheritic or wet form with fects when introduced to previously isolat- mucosal lesions within the digestive and ed avian populations, as well documented upper-respiratory tracts can occur (Ger- 342 THIEL ET AL.ÐCANARYPOX IN THE GALAÂ PAGOS 343 lach, 1999). The cutaneous form is most a single strain of virus (Ball, 1987); how- commonly observed in passerine birds ever, recombination between coinfecting (Gerlach, 1999). In GalaÂpagos, the order viruses may also contribute to the wide di- Passeriformes is represented by eight fam- versity observed between avipoxviruses. ilies with 28 species (Castro and Phillips, The dynamics of multihost pathogens in 1996). Several of these species are severely natural populations is important to under- threatened including the mangrove ®nch standing general patterns of rapid evolu- (Cactospiza heliobates; total population tion of viruses and their effect on natural approximately 100 individuals), the Flo- populations (Woolhouse et al., 2001; reana mockingbird (Nesomimus trifascia- Cleaveland et al., 2002). In GalaÂpagos, tus; approximately 200 individuals), the pox-like symptoms have been described in EspanÄ ola mockingbird (Nesomimus mac- several species of endemic birds, including donaldi; approximately 2,500 individuals), GalaÂpagos mockingbirds (N. parvulus par- the medium tree ®nch (Camarhynchus vulus), GalaÂpagos doves, yellow warblers pauper), and the large tree ®nch (Camar- (D. petechia), and some GalaÂpagos ®nches hynchus psittacula). The diptheritic form (Geospiza spp.) (Jimenez, 2003). Most of the disease is observed most frequently data on the effects of avian pox are from in Psittaciformes, Phasianiformes, and sev- the mockingbirds. eral Columbiformes (Gerlach, 1999). Col- During the 1982±83 El NinÄ o events, umbiformes present on GalaÂpagos include 56% of mockingbirds displaying pox-like a single endemic species, the GalaÂpagos lesions died on Genovesa, compared with dove (Zenaida galapagoensis), and the in- 39% of asymptomatic individuals (Curry troduced rock pigeon (Columba livia). and Grant, 1989). In that study, signi®- Thirteen strains of avipoxvirus have cantly more adults were affected than ju- been identi®ed worldwide, and strains vary veniles, partly because the epidemic peak- in virulence and host speci®city. Avipoxvi- ed before peak hatching. Prevalence in ruses from endemic forest birds in Hawaii GalaÂpagos mockingbirds was higher in (Apapane [Himatione sanguinea] and Ha- nestlings and juveniles than in adults on waiian crow [Corvus hawaiiensis]) include the island of Santa Cruz; a higher resight- two strains that differ signi®cantly from ing rate for young birds without symptoms fowlpox virus by restriction fragment than those with lesions indicated higher length polymorphism genetic analysis (Tri- mortality for infected birds (Vargas, 1987). pathy et al., 2000); their pathogenicity was mild in chickens. Protective immunity also Pox-like lesions were also observed among is strain related, and in vaccine trials, birds mockingbirds on Champion (an islet off are often unprotected if challenged with Floreana) during the 1982±83 El NinÄo different strains (Winter®eld and Reed, (Grant et al., 2000). 1985; Sarma and Sharma, 1988; Saini et The ®rst objective of this study was to al., 1990a, b). This indicates that signi®- develop a simple, speci®c diagnostic test cant antigenic differentiation may exist for avian pox that could be adapted for use among strains, and in part, this may result in the GalaÂpagos, where traditional virus from the rapid evolution of avipoxviruses propagation techniques are not economi- by recombination between strains. Repli- cally feasible. The second objective was to cation in avipoxviruses and other viruses in characterize the avipoxviruses that are in- Poxviridae involve genomic intermediates fecting native birds. The availability of comprising many tandem repeats of the large published regions of sequence for entire genome (Moyer and Graves, 1981). the fowlpox and canarypox viruses pro- Recombination, which occurs at extraor- vides the foundation for development of dinarily high frequencies in these viruses, rapid polymerase chain reaction±based is an essential part of replication involving methods for detection of these viruses. 344 JOURNAL OF WILDLIFE DISEASES, VOL. 41, NO. 2, APRIL 2005 METHODS DNA from both viruses (CA3-2F CTAATAGA- Field methods TACTAACGGAGAAG; CA3-2R TTAAATAA- AGAAATGTAAAGAC). Between May and July of 2002 and 2003, wild birds were captured with mistnets near the PCR ampli®cation and sequencing Charles Darwin Research Station on the island PCR ampli®cation with primer set CA3-2 or of Santa Cruz, Galapagos, Ecuador. In January m 2003 and July 2003, birds were captured on the CAX was performed in 50- l volumes of 67 island of Isabela, Galapagos Ecuador. Samples mM Tris-HCl (pH 8.8), 16 mM (NH4)2SO4, 0.01% Tween0, 1.5 mM MgCl2, 2.0 mM each of cutaneous lesions were removed using sterile m scalpels; these were transferred to a plastic vial dNTP, 0.01 mg bovine serum albumen, 0.6 M for each primer, 1 U Taq polymerase (Bioline, and frozen in liquid nitrogen for transport. In m some instances, samples were suspended in Randolph, Massachusetts, USA), and 2 l ex- 95% ethanol and frozen. Any bleeding related tracted DNA (concentration unknown). DNA to sample collection was stopped by applying isolated from fowlpox virus from a chicken in mild pressure with sterile cotton. the United States (kindly provided by D.N. Tri- pathy) was used as a positive control. A touch- DNA extraction down PCR program was used, beginning with an annealing temperature of 50 C and decreas- Samples were frozen in liquid nitrogen, pul- ing 0.5 C every cycle for 14 cycles to a ®nal verized to powder, and incubated at 65 C for annealing temperature of 43 C for an additional at least 6 hr in 250 ml Longmire's lysis buffer 25 cycles. Denaturation was at 94 C, and ex- (0.1 M Tris-HCl, pH 8.0, 0.1 M EDTA, 10 mM tension was 72 C, with a 45-sec hold at each NaCl, 0.5% SDS) with Proteinase-K (®nal con- temperature in the cycle.