Ornithological Monographs Volume (2006), No. 60, 98-111 © The American Ornithologists' Union, 2006. Printed in USA. CHAPTER 7 AVIAN HEMATOZOA IN SOUTH AMERICA: A COMPARISON OF TEMPERATE AND TROPICAL ZONES KATE L. DURRANT/'* JON S. BEADELL/ FARAH ISHTIAQ/ GARY R. GRAVES/ STORRS L. OLSON/ EBEN GERING/ M. A. PEIRCE/ CHRISTOPHER M. MILENSKY/ BRIAN K. SCHMIDT/ CHRISTINA GEBHARD/ AND ROBERT C. FLEISCHER^ ^Genetics Program, National Zoological Park, 3001 Connecticut Avenue NW, Washington, D.C. 20008, USA; ^Division of Birds, Smithsonian Institution, National Museum of Natural History, Washington, D.C. 20013, USA; and ^MP International Consultancy, Bexhill-on-Sea, East Sussex, United Kingdom ABSTRACT.•We used screening techniques based on polymerase chain reaction (PCR) to explore the avian hematozoan parasites (Plasmodium spp. and Haemoproteus spp.) of two previously uninvestigated regions of continental South America. Comparisons of tropical- zone Guyana and temperate-zone Uruguay revealed that overall prevalence of Plasmodium and Haemoproteus species detected in a diverse sampling of potential hosts was significantly higher in Guyana. The difference in prevalence between the two geographic zones appears to be attributable to ecological differences rather than taxonomic sampling artifacts. Diversity of hematozoan haplotypes was also higher in Guyana. We found no relationship between hema- tozoan haplotype and host family sampled within or between regions. We found very few Plasmodium and no Haemoproteus haplotypes shared between the two regions, and evidence of geographic structuring of hematozoan haplotypes between the two regions. We suggest that a lack of hematozoan haplotype transmission between the two regions may be attributable to the migratory patterns of each region's avian hosts. Received 11 April 2005, accepted 21 November 2005. RESUMEN.•Usamos técnicas de investigación basadas en reacción en cadena de polímeros (RCP) para explorar hemoparásitos avícolas (Plasmodium spp. y Haemoproteus spp.) en dos regiones no investigadas de Sudamérica. Las comparaciones de la zona tropical de Guyana y de la zona templada de Uruguay revelaron que la frecuencia general de especies de Plasmodium y Haemoproteus encontrados en una muestra diversa de hospederos potenciales fue significativamente más alta en Guyana. La diferencia en frecuencia entre las dos zonas geográficas aparentemente se debe a diferencias ecológicas que debido al muestreo taxonómico. La diversidad de hematozoos haplotipicos fue también más alta en Guyana. No encontramos una relación entre hematozoos haplotipicos y familias de hospederos muestreados dentro o entre las regiones. Encontramos solo algunos cuantos haplotipos de Plasmodium en común entre las dos regiones, pero no se encontraron haplotipos de Haemoproteus, ni evidencia de una estructuración geográfica de haplotipos de hematozoos entre las dos regiones. Por lo que sugerimos que la ausencia de transmisión de haplotipos de hematozoos entre las dos regiones puede ser atribuida a los patrones de emigración, para cada región, de las aves hospederas. HEMATOZOAN PARASITES (Plasmodium spp. important role in this difference by influencing and Haemoproteus spp.) are commonly found the density of vectors or potential hosts or the in blood smears from birds on every conti- ease of transmission. Comparison of hemato- nent except Antarctica (Bennett et al. 1993). zoan parasites of temperate and tropical zones Hematozoan prevalence may differ between may reveal differences related to climatic fac- geographic locations, and climate may play an tors. For example, Ricklefs (1992), surveying results from analyses based on blood smears, found a 2.6x greater infection rate in temperate "E-mail: [email protected] than in tropical zones. Temperate and tropical 98 AVIAN HEMATOZOA IN SOUTH AMERICA 99 regions may differ in the types of disease vec- haplotypes between the two zones to determine tors that occur, or in the distribution and pat- the potential for transmission between regions. terns of movement of potential hosts during migration (Ricklefs et al. 2005). METHODS Application of genetic techniques based on polymerase chain reaction (PCR) for screening avian hematozoan infections has resulted in a Sample collection and processing.•Tissue samples were obtained from birds collected during U.S. number of recent papers on regional surveys National Museum of Natural History (USNM, concentrating on a few areas of the globe: Africa Smithsonian Institution) collecting expeditions to (Waldenström et al. 2002), North America (Fallón Guyana (1994-2000) and Uruguay (2002-2003). et al. 2003, Ricklefs et al. 2005), and the Australo- Voucher specimens for tissues are in the USNM collec- Papuan region (Beadell et al. 2004). Thus far, the tion (numbers available on request). Samples in each South American continent has not been surveyed region were subsets of the total number of species col- using these PCR-based screening techniques. lected and were chosen to reflect a variety of potential The new screening techniques are also uncov- hosts for avian hematozoa, mostly passerines but also ering a wide array of phylogenetically varied representatives of other groups, such as doves, par- lineages of avian hematozoa (Perkins and Schall rots, and waterfowl. Taxonomy follows that used on the Handbook of the Birds of the World Internet Bird 2002). It has been proposed that the many newly Collection website (see Acknowledgments). Samples discovered hematozoan haplotypes may corre- came from many locations and habitat types in both spond to new species (Perkins 2000, Bensch et regions (Fig. 1). Geographic coordinates of each local- al. 2004). Haplotypes of hematozoa are often ity are given in Table 1. Samples from tropical-zone shared between geographic regions, carried by Guyana were collected during the dry season {n = 184) migratory birds as they travel between winter- or toward the end of the wet season (n = 11). Samples ing and breeding grounds (Waldenström et al. from temperate-zone Uruguay were collected during 2002, Ricklefs et al. 2005). Migratory movement August-September 2002 and June-July 2003. is generally between a tropical-zone winter- Some birds collected in Uruguay had thin blood ing ground and a temperate-zone breeding smears prepared from a small volume of peripheral blood for hematozoan detection (n = 141). These sam- ground. Therefore, birds that are found in two ples were fixed in 95% ethanol and stained with Giemsa disjunct geographic and ecological regions may as described in Garnham (1966). Blood smears were be expected to share hematozoa with common scanned under a microscope for 10 min at 400x mag- haplotypes if conditions for transmission are nification. In this time limit, -20,000 erythrocytes could met in each region. be examined, the estimate for passerines being 455 Avian hematozoa were previously classified erythrocytes per field at this magnification (Gering and partially on the basis of the host taxon, and some Atkinson 2004). Intense infections may have as many species were believed to be host specific, par- as one parasitemia per 2,000 erythrocytes (Godfrey et ticularly in the genus Haemoproteus (Atkinson al. 1987), whereas extremely mild infections may have and Van Riper 1991). If this holds, host species <1 parasitemia per 50,000 erythrocytes (Jarvi et al. 2002); our method falls between the two, striving for that occur in each of two separate geographic maximum efficiency. We noted the presence or absence and ecological regions may be expected to share of parasitemia for each individual, but not the intensity phylogenetically similar hematozoan parasites. of infection. Each hematozoan detected was digitally Alternatively, hematozoan haplotypes found in photographed using a Nikon Coolpix 4500 camera and many different host species may indicate host identified from these photos by M. Peirce to genus and switching, or a greater range of host sharing species where possible using hematozoan morphologi- than previously expected. cal characteristics and host taxonomy. A subset of the Our aims were: (1) to estimate avian hemato- samples was examined both by blood smear analysis zoan prevalence within and between two climatic and PCR-based screening techniques (n = 85). zones, temperate and tropical, on the South We extracted host and parasite DNA from the tis- sue samples using the manufacturer's protocols sup- American continent, using PCR-based screening plied with DNeasy kits (Qiagen, Valencia, California). methods; (2) to determine the phylogeny of the We tested the quality of each DNA extraction by hematozoan haplotypes found in the two regions; amplifying a small fragment of avian cytochrome-b (3) to explore host specificity of hematozoan hap- (cyt b) DNA using primers cytb-2RC and cytb-wow lotypes within and between each region; and (4) (268 base pairs [bp]) (Dumbacher et al. 2003). This to examine the degree of sharing of hematozoan amplification was successful in all cases. 100 ORNITHOLOGICAL MONOGRAPHS NO. 60 ^¿y~N N W^T^-E jn=27 lOOmlkï C '^ \ Ifll kms / ^ V f n=e ^^^ / C^ \/' ¡f^ ^ N^ /1 *n=33 ^ n=lO F G » / Uruguay ....n=is. v \ ^. •^'' '' '-^ n=38^V • • j'. "y .V I 'j-.^ 75 \u- ^ • J) .-^••i.f'r-'^ • J >*/ \fs(^r\ '-'^A.'^"¿ à''" : n=39 /- / ! % V, fc "=46 M / '•n=20 I n=S5 \\ mj \ \ ^ N \ ""••-,. ^X*"^ n='2 N FIG. 1. Map showing prevalence at each sampling location for (A) tropical-zone Guyana and (B) temperate- zone Uruguay. Numbers in gray boxes are sample sizes too low for prevalence charts at certain