Epizootiology of blood parasite infections in passerine birds from central New Jersey

CARL E. KIRKPATRICK' Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia,

PA 19104. U.S. A.

AND

HANNAH B. SUTHERS Department of Biology, Princeton University, Princeton, NJ 08544, U.S.A.

Received November 25, 1987

KIRKPATRICK, C. E., and SUTHERS, H. B. 1988. Epizootiology of blood parasite infections in passerine birds from central New Jersey. Can. J. Zool. 66: 2374-2382.

One or more genera of hematozoa were found in blood smears of 204 (29.3%) of 697 birds representing 59 species. Para- sites included Leucocytozoon spp. (1 6.6 % prevalence), Haemoproteus spp. (9.9 %), Plasmodium spp. (3.0%), Trypano- soma spp. (4.2%), and microfilariae of filariid nematodes (1.0%). Of 353 birds (48 species) tested for trypanosomiasis by blood culture, 141 (39.9%) were found to be positive. Several new host-parasite associations were identified. Prevalences were lowest in the summer and highest in the spring. During the summer, hatching-year birds were found to be infected less frequently than were older birds. Ground-feeding birds were infected with Leucocytozoon spp. at a significantly higher rate than were birds that characteristically feed above ground. Although the vertical stratum preferred for foraging was signifi- cantly associated with that of nesting, no significant association between preferred nesting stratum and prevalence of Leucocy- tozoon spp. infection among locally breeding birds was found. The results of this study, when compared with those of others, indicate that the epizootiology of avian hematozoan infections may vary greatly according to the locality studied. An examina- tion of the effects of blood sampling (whether by nail clipping or jugular venipuncture) on the birds revealed no significantly adverse effect of sampling on bird survival.

KIRKPATRICK, C. E., et SUTHERS, H. B. 1988. Epizootiology of blood parasite infections in passerine birds from central New Jersey. Can. J. Zool. 66 : 2374-2382.

Un ou plusieurs genres d'himatozoaires ont it6 trouvis dans les frottis sanguins de 204 sur 697 (29,3 %) oiseaux examinis appartenant a 59 espkces. Parmi les parasites, il faut mentionner Leucocytozoon spp. (friquence de 16,6 %), Haemoproteus spp. (9,9%), Plasmodium spp. (3,0%), Trypanosoma spp. (4,2%) et des microfilaires de nimatodes filariidis (1,0%). La culture des cellules sanguines de 353 oiseaux (48 espkces) a mis en ividence des trypanosomiases chez 141 d'entre eux (39,9%). Plusieurs associations h6te-parasite nouvelles ont i t6 constaties. La friquence des divers parasites s'est avirie minimale en it6 et maximale au printemps. Durant l ' i t i , les oiseaux de l'annie itaient infectis moins friquemment que les oiseaux plus figis. Les oiseaux qui se nourrissent sur le sol avaient des taux d'infection de Leucocytozoon spp. significative- ment plus ilevis que les oiseaux qui se nourrissent giniralement dans la vegetation. Bien que la strate verticale prifirie pour la recherche de nourriture soit significativement reliie a la strate prifirie pour la nidation, il n'y avait pas de corrilation significative entre la strate prifirie pour la nidation et la friquence des infections de Leucocytozoon spp. chez les oiseaux qui nichaient localement. La comparaison entre ces risultats et ceux d'autres etudes indique que l'ipizootiologie des infections d'oiseaux causies par les himatozoaires peut varier considirablement d'une localiti a une autre. Des observations ont permis de constater que le prilkvement du sang (par rognage de la griffe ou par ponction de la veine jugulaire) reste sans effet sur la survie des oiseaux.

[Traduit par la revue]

Introduction and examination of blood smears for the detection of circulat-

Although many surveys have been published on the occur- ing tr~panosomes~ and (iv) to the effect of

rence of the helminthic and protozoan blood parasites borne by sampling on bird survival. In addition, we describe several

arthropod vectors (66hematozoa-) of wild birds in North newly recognized host-parasite associations.

~mer i ca , relatively few were conducted in New Jersey (Greiner et al. 1975; Jochen 1966; Kirkpatrick and Lauer 1985; Williams and Bennett 1978). Moreover, there is little information on the hematozoa of passerine birds found in inland regions of the state, because previous surveys were conducted predominantly in Atlantic coastal regions (Greiner et al. 1975; Jochen 1966; Williams and Bennett 1978).

This study was undertaken with the following objectives: (i) to determine the prevalences of various hematozoa in pas- serine birds; (ii) to discover factors, both intrinsic and extrin- sic to the hosts, associated with increased or decreased risks of parasitism; (iii) to assess the relative merits of blood culture

'Author to whom reprint requests should be sent at the following address: Department of Veterinary Pathobiology, University of Illi- nois, 2001 South Lincoln Avenue, Urbana, IL 61801, U.S.A.

Materials and methods Birds

Passerine birds were captured in mist nets near the village of Hope- well, Mercer County, New Jersey (40°24'N, 74"46'W), from Sep- tember 1984 through May 1986. Habitats available on the study site include deciduous woodland and overgrown fields. Captured birds were marked with serially numbered U.S. Fish and Wildlife Service metal leg bands, and they were aged and sexed by external character- istics (Canadian Wildlife Service 1980). Body mass of birds was mea- sured to the nearest 0. l g with a Pesola spring scale. The systematic classification and nomenclature of the birds, as used in this report, follows the American Ornithologists' Union (1983). The classifica- tion of the birds according to feeding habit was described previously (Suthers 1985). Nesting habit classification is derived from Harrison (1975). Recaptured birds were identified by the numbered leg bands; they were not reexamined for parasites.

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KIRKPATRICK AND SUTHERS 2375

Blood sampling Blood was obtained for thin smears either by clipping a nail or by

jugular venipuncture. Hemostasis of clipped nails was accomplished by application of a saturated solution of ferric subsulfate. Venipunc- ture was performed using disposable sterile tuberculin syringes fitted with either 27- or 30-gauge needles. In the field, smears were dried rapidly with the aid of an aerosol duster. After fixation in absolute methanol (30-60 s) in the laboratory, smears were immersed in commercially prepared Giemsa stain concentrate (azure B type, Harleco, EM Diagnostic Systems, Gibbstown, NJ 08027 U.S.A.) diluted in 50 volumes of phosphate-buffered (pH 6.8) water for 25 min. Infection prevalences were determined by scanning blood smears (covered with cover slips) under 4 0 0 ~ magnification for at least 10 min. Parasitemias (i.e., concentrations of parasites in the blood, expressed as a percentage of erythrocytes) were estimated by actually counting at least 1000 erythrocytes in blood smears. Cultures for trypanosomes were made from approximately 30 pL of blood aseptically drawn by jugular venipuncture and injected into 2-mL rubber-stoppered, plain glass vials containing 1 mL sterile Sch-20-C medium. See Kirkpatrick and Lauer (1985) for details on these methods, including the formulation of Sch-20-C medium. Blood cul- tures that did not exhibit trypanosomes by day 15, as determined by phase-contrast microscopic examination, were considered negative.

Statistical analyses Assessment of factors potentially influencing the risk of infection

was performed using either the X2 test or Fisher's exact test and, to control for identified confounding variables, by the X2 test of Mantel and Haenszel (1959). McNemar's X2 test (Fleiss 1981) was used to evaluate paired data on methods for detecting Trypanosoma spp. infections. The method for calculating sensitivity and specificity of diagnostic tests for trypanosomiasis was according to Mausner and Bahn (1974). The effect of parasitization (i.e., whether or not birds were infected) on host body mass was tested by Wilcoxon's rank-sum test. Values of P < 0.05 were considered significant.

Voucher specimens Representative blood smears have been deposited in the Interna-

tional Reference Centre for Avian Hematozoa (IRCAH), Memorial University, St. John's, Newfoundland, Canada A1B 3x9. These slides have been assigned IRCAH accession numbers 97847-97870 and 98002-98018.

Results Parasite prevalences and new host records

Migrants predominated during April and May as well as from late August through October of each year. Breeding occurred at the study site during June and July, with the first locally hatched birds captured in late June.

In total, 697 birds, representing 59 species of 19 families, were tested for circulating blood parasites by examination of blood smears (Table 1). Of the 59 avian species examined, 37 (63%) were found to be parasitized, although sample sizes of 10 or more were obtained for only 17 species. Of all of the birds sampled, 204 (29.3 %) were found to be infected with one or more genera of hematozoa.

Leucocytozoon spp. were the most prevalent and microfilariae the least prevalent of the parasites studied. Leucocytozoon rnajoris, L. fringillinarum, L. dubreuili, and L. sakharofi were identified in 52,48, 5 , and 5 birds, respectively. The fol- lowing are newly recorded hosts for L. majoris: Hylocichla mustelina , Catharus fiscescens, Vireo griseus, Carpodacus purpureus, and Pipilo erythrophthalmus. Newly recognized hosts for L. sakharoffii are Zonotrichia albicollis and C. fis- cescens .

Haemoproteus beckeri, H. vireonis, and H. hedymeles were identified in six Dumetella carolinensis, nine Vireo spp., and

one Pheucticus ludovicianus, respectively. Haemoproteus sp. in Wilsonia canadensis is a new host-parasite record. Most of the Haemoproteus spp. are in need of redescription; hence, specific identifications of most were not attempted.

Plasmodium vaughani was found in three birds, P. circum- flexum, in two birds, and P. elongatum, P. hexameriumltenue, and P. polare, in one bird each. Icteria virens, Pipilo ery- throphthalmus, and Dendroica magnolia are newly recognized hosts for P. polare, P. elongatum, and P. circumflexum, respectively. Plasmodium spp. could be identified only to the subgeneric level in certain cases, as follows: Haemamoeba (n = 4) and Novyella (n = 6).

In some cases, parasites could not be identified below the genus level because only immature stages were found, because too few parasites were detected, or because of unsettled taxo- nomic status. New host-parasite records are claimed based on their absence in a recent host-parasite checklist of the avian hematozoa (Bennett et al. 1982) and in a recent report of a large hematozoa survey of passerine birds in Vermont (Barnard and Bair 1986).

Parasitemias of Leucocytozoon spp. were mostly < 0.1 % (96% of infected birds). The highest Leucocytozoon spp. para- sitemias found were 0.2 %, viz. L. dubreuili in Catharus gutta- tus and L. fringillinarum in Geothlypis trichas. Of the 69 birds infected with Haemoproteus, 14 (20%) had parasitemias of 0.1 % or greater, ranging as high as 12.6 % for one Zonotrichia albicollis. Plasmodium spp. parasitemias were also mostly < 0.1 % (62 % of infected birds). The remaining Plasmodium spp. parasitemias ranged from 0.5 to 19.7 % ; the Icteria virens infected with P. polare, noted above, had the highest intensity. Parasitemias of all of the extracellular parasites (Trypano- soma spp. and microfilariae) were < 0.1 %.

Circulating trypanosomes were tested by blood culture in 353 birds representing 48 species of 19 families (Table 2). Thirty-five (73 %) of the 48 avian species examined were posi- tive for trypanosomiasis. Positive cultures were found for 14 1 (39.9%) of the birds sampled. Because the systematics of avian Trypanosomu spp. are largely unsettled (Baker 1976), and because descriptions of cultured forms of these parasites are generally lacking, the identification of the trypanosomes found here was not attempted below the genus level. Trypano- soma spp. infections were recorded for the first time in Vireo griseus, Vermivora pinus, and Parus bicolor.

Among the birds found to have been infected (whether by examination of blood smear or blood culture), we determined the prevalences of infection by one, two, or three genera of parasites. Table 3 shows that, except in the Muscicapidae and the Vireonidae, the majority of the birds were infected with a single parasite genus.

Comparison of diagnostic tests for avian trypanosomiasis Blood smears were examined from all but 9 of the 353 birds

tested by blood culture for circulating trypanosomes. Of the 344 birds examined for Trypanosoma spp. by both methods, 19 (5.5 %) were positive and 201 (58.4 %) were negative by both methods. Of the remaining birds, 122 (35.5 %) were posi- tive by culture only, whereas just 2 (0.6%) were positive by smear only; this difference is significant (McNemar's X 2 = 1 14.2 df = 1, P < Considering our blood culture test as the definitive diagnostic test for avian trypanosomiasis, the validity of the blood smear examination as a screening test was calculated. The sensitivity (i.e., the ability of the test to iden- tify correctly those birds infected with trypanosomes) of blood

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CAN. J . ZOOL. VOL. 66. 1988

TABLE 1 . Prevalence of blood parasites, as determined by examination of blood smears, in passerine birds from New Jersey, 1984 - 1986

No. of birds No. infected withb: Nesting Feeding stratuma stratuma Examined Infected (%) L H P T M

Ty rannidae Empidonax traillii

(willow flycatcher) A Corvidae

Cyanocitta cristata (blue jay) A

Paridae Parus bicolor

(tufted titmouse) A Muscicapidae

Regulus calendula (ruby-crowned kinglet) A

Catharus fuscescens (veery) G

Catharus ustulatus" (Swainson's thrush) A

Catharus guttatus" (hermit thrush) A

Hylocichla mustelina (wood thrush) A

Turdus migratorius (American robin) A

Mimidae Dumetella carolinensis

(gray catbird) A Bombycillidae

Bombycilla cedrorum (cedar waxwing) A

Vireonidae Vireo griseus

(white-eyed vireo) A Vireo olivaceus

(red-eyed vireo) A Emberizidae

Vermivora pinus (blue-winged warbler) G

Dendroica magnolia (magnolia warbler) A

Dendroica coronata (yellow-rumped warbler) A

Dendroica striata (blackpoll warbler) A

Mniotilta varia (black-and-white warbler) G

Setophaga ruticilla (American redstart) A

Seiurus aurocapillus (ovenbird) G

Geothlypis trichas (common yellowthroat) G

Wilsonia canadensis (Canada warbler) G

Icteria virens (yellow-breasted chat) A

Cardinalis cardinalis (northern cardinal) A

Pheucticus ludovicianus (rose-breasted grosbeak) A

Pipilo erythrophthalmus (rufous-sided towhee) G

Passerella iliaca (fox sparrow) G

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KIRKPATRICK AND SUTHERS

TABLE 1 (concluded)

No. of birds No. infected withb: Nesting Feeding stratuma stratuma Examined Infected (%) L H P T M

Melospiza melodia (song sparrow)

Melospiza lincolnii (Lincoln's sparrow)

Melospiza georgiana (swamp sparrow)

Zonotrichia albicollisc (white-throated sparrow)

Junco hyemalis ' (dark-eyed junco)

Molothrus ater (brown-headed cowbird)

Icterus galbula (northern oriole)

Fringillidae Carpodacus purpureus

(purple finch) Carpodacus mexicanus

(house finch) Carduelis tristis

(American goldfinch) Negative speciesd Grand total Percent infected

NOTE: Some birds had multiple parasite infections. aPreferred nesting and feeding strata: A, above ground; G, ground; 0 , occasional ground; V , variable (data from Suthers 1985; Harrison

1975). 'L, Leucocytozoon: H, Haemoproteus; P, Plasmodium; T , Trypanosoma; M , microfilariae of filariid nematodes. 'Nonresident species. Note, however, that not all individuals of known resident species necessarily remain at the study site throughout the

breeding season. d~pec ies examined but not found parasitized included the following (n, nesting stratum, feeding stratum): Picoides pubescens (downy

woodpecker) (5, A, 0 ) , Myiarchus crinitis (great crested flycatcher) ( I , A, A), Sayornis phoebe (eastern phoebe) (2, A, A), Contopus virens (eastern wood-pewee) ( I , A, A), Empidonarflaviventris (yellow-bellied flycatcher) (2, G, A), Spizella pusilla (field sparrow) (17, A, 0 ) , Passerina cyanea (indigo bunting) (2, A, A), Dendroica petechia (yellow warbler) (6, A, A), Dendroica caerulensis (black-throated blue warbler) ( I , A, A), Dendroicapensylvanica (chestnut-sided warbler) (1, A, A), Dendroica discolor (prairie warbler) (1, A, A), Seiurus nove- boracensis (northern waterthrush) (3 , G, G), Oporornis formosus (Kentucky warbler) ( I , G , G) , Mimus polyglottus (northern mockingbird) ( I , A, 0 ) , Toxostoma rufum (brown thrasher) ( 1 , V , G), Thryothorus ludovicianus (Carolina wren) (1. V , O), Troglodytes aedon (house wren) (9, A, 0 ) . Troglodytes troglodytes (winter wren) ( 3 . V , 0 ) , Parus atricapillus (black-capped chickadee) (2, A, A), Parus carolinensis (Caro- lina chickadee) (5, A, A), Regulus satrapa (golden-crowned kinglet) (5, A, A), and Polioptila caerulea (blue-gray gnatcatcher) (1, A, A).

smear examination was 13.5%, and its specificity (i.e., the ability of the test to identify correctly those birds not infected) was 99 % .

Host factors and their relationship to parasitism Age-classes were determined (Canadian Wildlife Service

1980) for 646 (93 %) of the birds examined for parasites by the blood smear technique and for 341 (97%) of the birds tested for trypanosomiasis by blood culture. All further references to trypanosomiasis prevalence pertain to blood culture data. Examinations of blood smears indicated that hematozoan prev- alence in hatching-year (HY) birds (i.e., those born in the calendar year) was significantly less than that in after-hatch- ing-year (AHY) birds (i.e., those born in a previous calendar year) during the summer (Fisher's exact test, df = 1, P = 0.047) (Table 4). Although the prevalence of trypanosomiasis was also less in HY birds than in AHY birds in summer, the difference was not significant (Table 5).

Host species were categorized according to the level (i.e., vertical stratum) at which they spend most of their time forag- ing (Suthers 1985) (Table 1). Because the "occasional ground- feeding" group was considered to be so heterogeneous (the proportion of time spent either above or on the ground during

feeding probably varies widely among species), statistical analysis was limited to comparisons between birds feeding on the ground and those feeding above ground. The ground- feeding group was significantly more likely than the group feeding above ground to be infected with at least one kind of parasite (x2 = 27.2, df = 1, P < lop6) (Table 6). Parasite genera were then analyzed individually to determine which was contributing to the observed difference in prevalence according to feeding habit. Only Leucocytozoon spp. infec- tions were found in ground-feeding birds to an extent signifi- cantly greater than that of the other group (x2 = 33.5, df = 1, P < (Table 6). Because of the unequal sample sizes, it is possible that a relatively large sample of a heavily infected host species could have overwhelmed smaller samples from lightly infected species. Therefore, we again tested the associ- ation of feeding habit and parasite prevalences using those host species in which at least 10 individuals were examined (Table 1). Thus, of 225 ground-feeding birds of six species, 93 (41 %) were infected with at least one kind of parasite, and 65 (29%) were infected with Leucocytozoon spp. Of 174 aboveground feeders (six species), 32 (18%) were infected overall, with 28 (9 %) infected with Leucocytozoon spp. As before, ground-feeding behavior was found to be a significant

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2378 CAN. J . ZOOL. VOL. 66, 1988

TABLE 2. Prevalence of trypanosomes in passerine birds from New Jersey, 1984- 1986, as assessed by blood culture

No. of birds

Examined Infected (%)

Ty rannidae Empidonax jlaviventris

Corvidae Cyanocitta cristata

Paridae Parus bicolor

Muscicapidae Polioptila caerulea Catharus fuscescens Catharus ustulatusa Hylocichla mustelina Turdus migratorius

Mimidae Dumetella carolinensis Toxostoma rufum

Bombycillidae Bombycilla cedrorum

Vireonidae Vireo griseus Vireo olivaceus

Emberizidae Vermivora pinus Dendroica petechia Dendroica magnolia Mniotilta varia Setophaga ruticilla Seiurus aurocapillus Geothlypis trichas Wilsonia canadensis Icteria virens Cardinalis cardinalis Pheucticus ludovicianus Pipilo erythrophthalmus Spizella pusilla Melospiza melodia Melospiza georgiana Zonotrichia albicollisa Junco hyemalisu Molothrus ater Icterus galbula

Fringillidae Carpodacus purpureus Carpodacus mexicanus Carduelis tristis

Negative speciesb Grand total

NOTE: See Table I for common names of birds. "Nonresident species. 'species examined but not found parasitized including the following (n):

Picoides pubescens (4), Myiarchus crinitus ( I ) , Sayornis phoebe (2), Empidonax trailli (4), Melospiza lincolnii ( I ) , Passerina cyanea (2), Den- droica coronata (2), Dendroicu striata ( I ) , Seiurus noveboracensis (3), Oporornis formosus ( I ), Troglodytes aedon (7), Parus carolinensis ( I ), and Catharus guttatus (1).

risk factor for hematozoan infection overall (x2 = 23.0, df = 1, P < and for Leucocytozoon spp. in particular (x2 = 8.3, df = 1, P < 0.01). Except for Seiurus aurocapillus, at least a third of the birds of each of ,the ground-feeding species were infected with at least one kind of hematozoan.

Similarly, prevalences of Trypanosoma spp. infections between the ground-feeding and above-ground-feeding groups

were compared (Table 7). However, no significant relation- ship of feeding habit to trypanosomiasis prevalence was revealed.

Birds were classified further according to the vertical stratum of preferred nesting location (Table 1). The nesting habits of some species were considered too variable to cate- gorize. Testing the association between nesting strata (i.e., ground or above ground) and prevalence of parasitism did not reveal significant associations for parasitism generally or for parasitism by Haemoproteus spp., Plasmodium spp., or Try- panosoma spp. in particular. In contrast, ground-nesting host species were found to be parasitized by Leucocytozoon spp. to a significantly greater extent than were those nesting above ground (x2 = 2 1 .O, df = 1, P < lop5). However, when the four species of birds not known to nest in New Jersey (Table 1) were excluded from the analysis, no significant association between nest location and Leucocytozoon spp. infection became evident. No significant association between nest location and Trypanosoma spp. infections was found, whether the four strictly migratory host species were included in the analysis or not.

Host sex was determined for 646 (93%) of the birds from which blood smears were made and for 343 (97 %) of the birds from which blood cultures for trypanosomes were made. No significant differences in parasite prevalence were found between the two sexes (data not shown).

The relatively large sample of Zonotrichia albicollis (Table 1) allowed us to test additional host factors that may have been associated with parasitism. Body mass, ordinal score for sub- cutaneous fat in the ventral thorax (0-3 scale), age, and sex were each tested individually (by Wilcoxon's rank-sum test for body mass and by X2 analysis for the other factors) for pos- sible associations with infection (based on examination of blood smears), but no significant associations were discovered.

Association of season with parasite prevalences Seasonal prevalences of blood parasites in birds are given in

Tables 4 and 5. Overall, prevalences of parasitism increased from summer to fall to spring (birds were not sampled during winter). These trends (tested by X2 analysis) were significant, according to both blood smears (x2 = 39.8, df = 2, P <

(Table 4) and blood cultures (x2 = 14.1, df = 2, P < (Table 5).

Adjustment of positive associations with parasite prevalences for confounding variables

Because more than one of the tested factors intrinsic and extrinsic to the hosts was found to be significantly associated with the prevalences of certain parasites, some of these factors were tested again and mutually controlled (Mantel and Haens- zel 1959). When feeding habit was adjusted for host age and season, the effect of feeding habit on parasite prevalences (as determined by examination of blood smears) remained signifi- cant (P < When the four species of birds not known to breed at the study site (Table 1) were excluded from the feeding habit analysis, significant associations between preva- lences of Leucocytozoon spp. (P < 0.01) and Plasmodium spp. (P < and ground-feeding behavior were found. Age remained significantly associated with parasitism when adjusted for feeding habit (P = 0.003). Since all spring- captured birds were considered to be the same age (i.e., AHY), the possible confounding effect of season on age- related parasite prevalence could not be tested. The association between preferred foraging habit and nesting strata was tested.

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TABLE 3. Frequencies of single and multiple blood parasitism, as determined by examina- tion of blood smears and blood culture, in 176 passerine birds in New Jersey, 1984- 1986

No. of individuals infected with":

No. of infected No. of infected 1 parasite 2 parasite 3 parasite Host family host species individuals genus genera genera

Tyrannidae Corvidae Paridae Muscicapidae Mimidae Bombycillidae Vireonidae Emberizidae Fringillidae

Total 3 8 176 1 13 (64) 49 (28) 14 (8)

NOTE: See Table 1 for parasite genera found. 'Percentage of infected individuals is given in parentheses.

TABLE 4. Prevalence of blood parasites in passerine birds in New Jersey, as determined by examina- tion of blood smears, according to age-class and season, 1984- 1986

No. infected withh: Age- No. classu examined L H P T M Any parasite'

Summer HY 113 7 5 1 3 0 13 (12) AHY 6 8 6 7 1 3 2 15 (22) Total 189 13 (7) 12 (6) 2 (1) 6 (1) 2 (3) 28 (15)

Fall HY 246 50 26 9 6 0 85 (35) AHY 60 7 6 0 1 0 13 (22) Total 348 60 (17) 34 (20) 9 (3) 8 (2) 0 (0) 103 (30)

Spring AHY 160 43(27) 23(14) lO(6) 15(9) 5 ( 3 ) 73 (76)

'HY, hatching year; AHY, after hatching year; total includes birds of indeterminate age. 'L, Leucocytozoon; H, Haemoproteus; P, Plasmodium; T, Trypanosoma; M, microfilariae of filariid nematodes. Values

given are number positive with percent positive in parentheses. 'Sums of percent positives for individual parasites exceed total percent positive because of multiple infections in some birds.

Omitting those species that showed either variable nest loca- tion or variable feeding behaviors (Table l), the feeding and nesting strata were found to be significantly associated for the 44 species tested (Fisher's exact test, df = 1, P = 0.001). Thus, ground feeders were more likely to be ground nesters and vice versa. Consequently, it became necessary to retest the association between foraging behavior and parasite preva- lences, controlling for the potential confounding effect of nest location behavior. When this was done, ground-feeding birds were still found to be infected with hematozoa in general (x2 = 41.9, and with Leucocytozoon spp. in particular (x2 = 30.2), significantly more frequently than were aboveground feeders (df = 1 and P < in both cases).

Eflects of blood sampling and method of sampling on birds Records were kept on birds banded during 1984 and 1985

and recaptured at the study site until the end of summer 1986.

TABLE 5. Prevalence of Trjpanosoma spp. infections in passerine birds in New Jersey, as determined by blood culture, according to age-class and season, 1984 - 1986

No. of birds

Age- classu Examined Infected (%) - -

Summer HY 105 28 (27) AHY 5 9 21 (36) Total 165 50 (30)

Fa1 1 HY 43 15 (35) AHY 12 5 (42) Total 59 24 (41)

Spring AHY 129 67 (52)

"HY, hatching year; AHY, after hatching year; total includes birds of indeterminate age.

The return rate of sampled birds was compared with that of unsampled birds of 22 representative species (Table 8). The Discussion difference in return rates between sampled (6.5%) and unsampled (9.7 %) birds was not significant. The effect of the In their analysis of the distribution of avian hematozoa in method of blood collection on rate of return was examined North America, Greiner et al. (1975) separated the continent next (Table 9). There was no significant difference between into seven topographic regions. ~ c c o r d i n ~ l ~ , New Jersey the return rates of nail-clipped birds (5.1 %) and those of birds occupies the southeastern corner of their region 5 (the Great subjected to venipuncture (4.4 %). Lakes and northeastern Appalachian - Laurentian region).

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TABLE 6. Prevalence of blood parasites, as determined by examination of blood smears, according to feeding habit of 204 infected birds in New Jersey, 1984- 1986

No. infected withu: No.

examined L H P T M Any parasiteb

Ground feeding 265 79(30) 28(11) 12(5) lO(4) 2 ( < 1 ) l l l ( 4 2 ) Occasional ground feeding 16 1 1 1 (7) 19 (1 2) 4 (3) 4 (3) 4 (3) 37 (23) Aboveground feeding 271 26(10) 22(8) 5 ( 2 ) 15(6) 1 ( < 1 ) 56 (21)

NOTE: Values given are number positive with percent positive in parentheses. "L, Leucocytozoon; H, Haemoproteus: P, Plasmodium; T , Trypanosoma: M , microfilariae of filariid nematodes. b ~ u m s of numbers and percent positives for individual parasite genera exceed total numbers and percent positives because of multiple

infections in some birds.

TABLE 7. Prevalence of Trypanosoma spp. infections, as determined by blood culture, according to feeding habit of 14.1 infected passerine birds in New Jersey, 1984- 1986

No. of birds

Examined Infected (%)

Ground feeding 115 49 (43) Occasional ground feeding 100 39 (39) Aboveground feeding 138 53 (38)

TABLE 8. Effect of blood sampling on rate of bird return

Bled

Returned Yes No Total

Yes 2 8 37 65 No 400 345 745 Total 428 382 8 10

NOTE: Data for 22 selected species of birds sampled in New Jersey during 1984 and 1985 and recaptured through summer 1986.

Reviewing the literature available through 1974, they found that the overall prevalence of hematozoa in region 5 birds was approximately 38 % , with 22 % infected with Leucocytozoon, 18 % with Haemoproteus, and 3 % with Plasmodium. In com- parison, we found a lower prevalence of avian hematozoa, except for that of Plasmodium, which was identical. However, the overall prevalence of avian hematozoa (29.3 %) was much higher in our study than in two previous studies of New Jersey birds that included a large proportion of passerines (5.4%, Jochen 1966; 9.4%, Williams and Bennett 1978). In those studies, birds were sampled predominantly in Atlantic coastal sites, whereas we sampled birds in the interior of the state. Furthermore, one of those studies examined birds only during the summer. Nevertheless, it appears that New Jersey birds are less likely to be found parasitized than are birds sampled elsewhere (based, in all cases, on examination of blood smears) in region 5 of Greiner et al. (1975) (see also Barnard and Bair 1986). The relative proportions of birds infected with the various genera of hematozoa followed those of other birds in region 5, with Leucocytozoon spp. found most frequently (Barnard and Bair 1986; Greiner et al. 1975; Williams and Bennett 197 8).

In this study, as in most others (reviewed by Greiner et al.

TABLE 9. Effect of blood sampling method on rate of bird return

Sampling methoda

Returned NC VP Total

Yes 14 16 30 No 262 345 607 Total 276 36 1 637

NOTE: Data for all birds sampled in New Jersey during 1984 and 1985 and recaptured through summer 1986.

"NC, nail clipping; VP, venipuncture.

1975), Trypanosoma spp. were the protozoan parasites found least frequently in smears of avian peripheral blood. However, in our study, when blood specimens were cultured especially to detect them, Trypanosoma spp. were found to be the most common of the hematozoa. A similar disparity between the results of blood smear and blood culture examinations for Try- panosoma spp. was found in our previous study of New Jersey raptors (Kirkpatrick and Lauer 1985). We are not aware of previous attempts to culture the blood of North American pas- serine~ for trypanosomes but, based on our results, it is likely that reports on Trypanosoma spp. prevalence in birds based on examination of blood smears have understated the actual prev- alence of these parasites. As a diagnostic screening test for avian trypanosomiasis, we found that blood smear examina- tion had high specificity, but poor sensitivity (i.e., there were too many false negatives). In chronic infections, Trypanosoma spp. trypomastigotes are more noticeable in the bone marrow than in the blood circulation (Baker 1976). Similarly, Plas- modium spp. are probably more common in birds than surveys relying on blood smears would reveal. Others have shown that subinoculation of blood into nonimmune hosts is a more sensi- tive test for avian malaria (Herman 1938; Herman et al. 1966).

According to examination of blood smears taken during summer, the proportion of HY birds infected with protozoa was about half that of AHY birds. The prepatent periods of all of the protozoan genera studied are short enough (i.e., < 3 weeks) that we should have detected most infections occurring in those birds fledged at or near the study site (Baker 1976; Fallis and Desser 1977; Fallis et al. 1974; Seed and Manwell 1977). Migratory birds are thought to acquire hema- tozoan infections on their breeding grounds (Greiner et al. 1975). In contrast, results of our study apparently suggest that birds captured at the New Jersey study site were more likely to have acquired the parasites elsewhere. However, in a 3-year study of passerines in New Brunswick, Bennett et al. (1975)

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KIRKPATRICK AND SUTHERS 238 1

found that climatic factors varied markedly from one year to the next and that these factors seemed to influence hematozoan transmission. Thus, it is possible that extending our study would have revealed as anomalous the finding that summer HY birds experienced relatively fewer infections.

During the southward migration in autumn we would expect the pool of local HY birds sampled to be diluted by birds fledged at localities north of the study site. The prevalence of hematozoan infection in fall HY birds exceeded that of sum- mer (i.e., local) HY birds. This result suggests that transmis- sion of hematozoa may, indeed, occur at more northerly breeding grounds. In support of this hypothesis, a recent study of Vermont birds (mostly passerines) indicated peak preva- lences of hematozoan infections during summer, with nearly identical prevalences in HY (7 1 .6 %) and AHY (7 1.7 %) age- groups (Barnard and Bair 1986). Thus, active transmission among birds of all hematozoan genera was inferred for the Vermont study area. We suggest, therefore, that transmission of hematozoa among birds may not necessarily occur predomi- nantly on the breeding ground, i.e., some breeding grounds may be more favorable for hematozoan transmission than others. Whether significant transmission occurs in a given locality or not may depend on the nature of the habitat, cli- mate, and other factors peculiar to a certain area that influence insect vector abundance and activity (Bennett et al. 1975).

All of the birds sampled in spring were considered to be adults, and it is this group that evidenced the greatest preva- lence of hematozoan infection overall. It is likely that these birds arrived infected (as opposed to acquiring their infections locally) and that the high hematozoan prevalence was due, in part, to relapses of latent infections. The relapse phenomenon has been described before, and it may be of adaptive signifi- cance to the parasites (Beaudoin et al. 1971; Bennett and Cameron 1974; Dorney and Todd 1960). Parasitemia relapses coincident with insect-vector emergence and availability of parasite-naive hosts (i.e., nestling birds) should facilitate para- site transmission.

Related to the idea that hematozoan transmission among birds occurs mainly on breeding grounds is the hypothesis that birds initially become infected as nestlings (Greiner et al. 1975). Furthermore, since many insect vectors actively seek blood meals at certain altitudes or vertical strata (reviewed in Greiner et al. 1975; van Riper et al. 1986), then the height of nest location favored by a particular avian species may influ- ence susceptibility to infection by insect-borne parasites. Greiner et al. (1975) tested the effect of host-species nesting preference on parasite prevalence, but the results were incon- clusive. We did not find a significant association between parasite prevalence and preferred nest location for avian spe- cies known to breed at our study site. At a given breeding site, avian species may need to be carefully examined separately to determine whether birds become infected as nestlings. For example, Herman (1938) found that red-winged blackbird (Agelaius phoeniceus) nestlings did not become infected with Plasmodium spp. at a Massachusetts breeding ground.

We also tested the idea of vertical stratification of vectors by comparing hematozoan infection prevalence with host forag- ing behavior. We found that ground-feeding birds were over three times more likely to be parasitized with Leucocyto- zoon spp. than were avian species that habitually feed above ground. This association remained significant when host spe- cies not breeding in New Jersey were excluded from the analy- sis as well as when the potentially confounding variable of

preferred nesting stratum was controlled statistically. This appears to suggest that, at least for the New Jersey study site, vectors of Leucocytozoon spp. (viz. ornithophilic simuliids (black flies); Fallis et al. 1974) for passerine birds may be more active at ground level. However, unlike mammalophilic black flies, most ornithophilic species studied are predomi- nantly crepuscular feeders. In addition, most woodland-inhab- iting black fly species seek avian hosts mainly above ground (Anderson and DeFoliart 196 1 ; Bennett 1960; Service 198 1 ; Wenk 1981). Thus, it does not appear likely that ground- feeding behavior of passerine birds, found by us to be strongly associated with increased risk of Leucocytozoon spp. infec- tion, is necessarily related directly to vector behavior. It should be noted that factors other than altitude influence the finding of hosts by black I-lies (Service 1981; Sutcliffe 1986; Wenk 1981). Moreover, it is possible that there may exist wide variation in host susceptibility to hematozoa. Another, unexamined behavioral factor that may influence the risk of acquiring hematozoan infections is the vertical stratum at which hosts prefer to roost at night. This behavior may be important for those hematozoa transmitted by vectors feeding at night.

It is possible that impaired function and increased destruc- tion of erythrocytes may result from infection of birds with the intracellular hematozoa (e. g . , Leucocytozoon , Haemoproteus, and Plasmodium). Asexual reproduction of these parasites in various tissues (e.g., lung, liver, spleen, kidney) may also have pathologic consequences. For example, Hayworth et a1 . (1987) showed that experimental infection of canaries with Plasmodium relictum resulted in impaired host oxygen assimi- lation and thermoregulation at times of peak parasitemia. However, except for certain host -parasite associations (e.g., L. simondi in juvenile waterfowl (Fallis et al. 1974) and intro- duced P. relictum in some native Hawaiian birds (van Riper et al. 1986)), there is scant documented evidence for signifi- cant morbidity or mortality due to these parasites among avian populations in the wild (Baker 1976; Fallis and Desser 1977; Fallis et al. 1974; Seed and Manwell 1977). Nevertheless, the results of recent studies (Hamilton and Zuk 1982; Read 1987) suggest that susceptibility of passerine birds to hematozoa is important in sexual selection. Specifically, positive correla- tions were found between hematozoan prevalence among vari- ous host species and male plumage brightness, male song complexity, and body mass. Thus, hematozoan infections may exert important pressures on avian hosts that are more subtle than overt disease. We looked for a correlation between preva- lence of blood parasitism and body mass within a particular host species (2. albicollis) without success.

Under laboratory conditions, Stangel (1986) found that the withdrawal of blood (in larger quantities than that reported here) from small birds did not seem to cause significant stress, as measured by mortality rate and changes in body mass. Studies conducted in galliform and columbiform birds have indicated that birds recover rapidly from hemorrhage, both in terms of plasma volume and erythrocyte numbers, relative to mammals (Schindler and Gildersleeve 1987). Japanese quail, for example, recover completely from 30% acute blood loss within 72 h. Under field conditions, we did not find any signif- icantly adverse effects of blood sampling on bird survival as measured by rates of return to the study site. Nor did we find a significant difference in bird survival based on the mode of blood sampling (i .e., nail clip vs. jugular venipuncture). Thus, our results support Stangel's (1986) view that, compared with

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the stresses of capture, confinement, and handling, stress attending the removal of small quantities of blood from small birds is relatively low.

Acknowledgements This study was supported, in part, by a grant from the U.S.

Department of Agriculture Formula Fund and by a grant from the IBM Corporation for computer equipment. We are grateful to the many students who assisted us in the field, particularly Pamela S. Hoppe, Virginia P. Trexler-Myren, and Victor Apanius. We thank Gordon F. Bennett, Scott K. Robinson, Ronald M. Weigel, Victor Apanius, Cynthia Lord, and Anna M. Lyles for reading the manuscript.

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