1254

MALARIA PREVALENCE AND WHITE-BLOOD-CELL RESPONSE TO INFECTION IN A TROPICAL AND IN A TEMPERATE THRUSH

Robert E. Ricklefs1 and Kimberly S. Sheldon2

Department of Biology, University of Missouri-St. Louis, 8001 Natural Bridge Road, St. Louis, Missouri 63121, USA

Abstract.—To evaluate the possibilities and limitations of using white-blood-cell (WBC) counts to characterize investment in immune-system function, we compared the prevalence and intensity of malaria infections and concentrations of WBCs in the American Robin (Turdus migratorius) in Michigan and Missouri and in the Clay-colored Robin (T. grayi) in central Panama. We ascertained infection by polymerase chain reaction (PCR) screening and quantifi ed infection intensity and WBC concen-trations by visual inspection of blood smears. Because few parasites were observed on smears, we assumed that most cases of malaria represented chronic, rather than acute, infections. Prevalence of haemosporidian infection (Panama 41%, Missouri 57%, Michigan 63%) did not diff er signifi cantly between locations. However, most infections in Panama were undetected on blood smears, whereas more than half were apparent on smears in Michigan and Missouri. Among WBCs, lymphocytes were the most abundant type, followed by heterophils; eosinophils and lympho-cytes were more common in the North American sample than in Panama, and their numbers in Michigan were signifi cantly higher in infected than in noninfected individuals. Tropical T. grayi apparently maintained infections at lower intensities but appeared to accomplish this in spite of lower abundances of immune-system cells. Furthermore, analyses of blood smears of Turdus spp. from elsewhere in the tropics revealed a wide range of prevalence, which suggests that either the regional presence of haemosporidians or the ability of hosts to control infections cannot be learned from limited samples. Additional species surveyed in Michigan and Panama revealed no consistent pa ern in either infection intensity or WBC concentrations. Infection and response appear to be highly idiosyncratic. In agreement with other authors, we caution that blood parameters are diffi cult both to interpret and to sam-ple adequately in tests of regional or other eff ects. Received 15 January 2006, accepted 7 November 2006.

Keywords: avian malaria, blood parasites, immune response, lymphocyte, Plasmodium, Turdus.

Prevalencia de Malaria y Respuesta de Glóbulos Blancos a la Infección en un Zorzal de la Zona Tropical y Uno de la Zona Templada

Res men.—Para evaluar las posibilidades y limitaciones del uso de conteos de glóbulos blancos para caracterizar la inversión en la función del sistema inmune, comparamos la prevalencia e intensidad de infecciones de malaria y las concentraciones de glóbulos blancos (GB) en Turdus migratorius en Michigan y Missouri, y en T. grayi en la región central de Panamá. Determinamos la infección mediante exploración con PCR y cuantifi camos la intensidad de la infección y las

1 E-mail: ricklefs@umsl.edu2 Present address: Department of Biology, Box 351800, University of Washington, Sea le, Washington 98195, USA.

The Auk 124(4):1254–1266, 2007© The American Ornithologists’ Union, 2007. Printed in USA.

Malaria Infections in Thrushes (Turdus)October 2007] 1255

Haemosporidian pathogens of birds, which include the Apicomplexa genera Leucocytozoon, Plasmodium, and Haemoproteus, provide an excellent research model for host–parasite interactions. Haemosporidian infections are characterized by high prevalence (Valkiūnas 2005), potential pathological eff ects (Atkinson et al. 1988, 1995; cf. Dufva and Allander 1989, McConkey et al. 1996, Ots and Hõrak 1998, Garvin et al. 2003b), and demonstrated stimu-lation of immune responses (Massey et al. 1996; Ots and Hõrak 1996, 1998; Ots et al. 1998; Apanius et al. 2000; Garvin et al. 2003b). It is not surprising, therefore, that biologists have used data on haemosporidian blood parasites to assemble comparative information on immune-system responses (Martin et al. 2001, Tella et al. 2002). However, interpretation of simple measures of disease status and immune-system response depends on understanding variation in such measures. In particular, static samples of disease prevalence and white-blood-cell (WBC) counts cannot resolve the etiology of the disease or distinguish between acute (crisis) and chronic infections (van Riper et al. 1986, Atkinson and van Riper 1991, Atkinson et al. 1995).

We undertook the present study of haemo-sporidian parasites and WBC counts to assess some components of this variation. We focused

particularly on comparisons of temperate and tropical songbirds. Several studies have sug-gested that haemosporidian infections are generally less prevalent in the tropics than in temperate regions (Ricklefs 1992, Valkiūnas et al. 2003). “Parasite prevalence” refers to the propor-tion of a host population that is infected, which is traditionally measured by visual inspection of blood smears (Godfrey et al. 1987, Valkiūnas 2005). However, prevalence data have not been compared broadly with adequate control for host taxonomy. Moreover, prevalence might be con-founded by immune response to the extent that it refl ects the ability of host individuals to control chronic infections below the level of detection by visual examination of blood smears.

Li le is known about the relative respon-siveness of the immune system in birds and its eff ectiveness in combating haemosporidian infec-tions. Infections by Plasmodium elicit immune responses in birds, as shown by the presence of antibodies and the eventual control of infec-tions (e.g., Atkinson et al. 2001a, b). In a survey of haemo sporidian infections in six species of passerine birds in the Lesser Antilles, infected individuals exhibited higher WBC counts, par-ticularly lymphocyte, than those whose blood smears were parasite-free (Apanius et al. 2000). Other studies have also noted elevated WBC

concentraciones de GB a través de la inspección visual de frotis sanguíneos. Debido a que pocos parásitos fueron observados en los frotis, suponemos que la mayoría de los casos de malaria representaron infecciones crónicas, no agudas. La prevalencia de infección de hemosporidios (Panamá 41%, Missouri 57%, Michigan 63%) no fue signifi cativamente diferente entre localidades, pero la mayoría de las infecciones en Panamá no se detectaron en los frotis sanguíneos, mientras que en Michigan y Missouri más de la mitad fueron aparentes en los frotis. Entre los GB, los linfocitos fueron el tipo más abundante, seguidos por los heterófi los. Los eosinófi los y linfocitos fueron más comunes en la muestra de Norte América que en la de Panamá y sus números en Michigan fueron signifi cativamente más altos en individuos infectados que en los no infectados. La especie tropical T. grayi aparentemente mantuvo infecciones a intensidades más bajas, pero parecería lograr esto a pesar de tener menor abundancia de células del sistema inmune. Además, los análisis de frotis sanguíneos para Turdus spp. de otras áreas tropicales revelan un amplio rango de prevalencia, sugiriendo que la presencia regional de hemosporidios o la habilidad de los hospederos para controlar las infecciones no pueden ser determinadas con base en un número limitado de muestras. Otras especies estudiadas en Michigan y Panamá no revelaron un patrón consistente en cuanto a la intensidad de infección o a las concentraciones de GB. La infección y la respuesta parecen ser altamente idiosincráticas. De acuerdo con lo documentado por otros autores, advertimos que los parámetros sanguíneos son difíciles de interpretar y de muestrear adecuadamente en estudios sobre los efectos regionales o de otros tipos.

Ricklefs and Sheldon1256 [Auk, Vol. 124

counts associated with haemosporidian infection (Massey et al. 1996; Ots and Hõrak 1996, 1998; Ots et al. 1998; Garvin et al. 2003b).

Here, we assess haemosporidian parasite prev-alence and immune-system response to infection in two species of thrush, the American Robin (Turdus migratorius) in Michigan and Missouri and the Clay-colored Robin (T. grayi) in the Republic of Panama. Available information on haemosporidian prevalence in T. migratorius and T. grayi reveals lower incidence of infection in tropical compared with temperate populations. Our objective was to determine whether para-site prevalence assessed by polymerase chain reaction (PCR), infection intensity quantifi ed on blood smears, and immune responses, indicated by WBC counts for infected and uninfected indi-viduals (reviewed in Ots and Hõrak 1998, Ots et al. 1998), diff er between the species. We also surveyed samples of additional songbird species in the same localities to determine the generality with respect to region of the results for Turdus.

Materials and Methods

Turdus grayi is a 74-g (Dunning 1993) Neo-tropical thrush inhabiting open or semi-open landscapes from southern Mexico (Oaxaca and Nuevo León) to Colombia (American Ornithologists’ Union [AOU] 1998). The 77-g (Dunning 1993) Nearctic T. migratorius breeds in temperate regions from Alaska and northern Canada, through most of the United States, to southern Mexico (Oaxaca and Veracruz) at higher elevations. In the northern part of the range, T. migratorius are migratory, with popula-tions shi ing southward in autumn (AOU 1998).

Field studies.—We conducted studies on T. grayi during the breeding and early postbreed-ing season (March–August 2003) at Gamboa, central Panama (9.1°N, 79.7°W, 60 m elevation). We investigated T. migratorius during the tem-perate breeding and early postbreeding season (May–August 2003) at the Kellogg Biological Station in Hickory Corners, southern Michigan (42.4°N, 85.4°W, 290 m elevation), and during part of the nonbreeding season (October 2003) at the Anheuser Busch Wildlife Conservation Area in St. Charles County, near St. Louis, Missouri (38.8°N, 90.5°W, 145 m elevation). Individuals of other species were caught at the same time in Panama and Michigan. All studies were conducted under appropriate permits from

national and state governments in accordance with protocols approved by the Institutional Animal Care and Use Commi ee (IACUC) of the University of Missouri-St. Louis.

Blood sampling.—Birds were captured using mist nets, and blood samples were acquired in heparinized microcapillary tubes a er the brachial vein of the wing was punctured with a 27-gauge syringe needle. Thin smears were pre-pared on individually labeled microscope slides using one or two drops of blood (Godfrey et al. 1987). Smears were air-dried, fi xed in absolute methanol, and stained with Giemsa. Five to 10 µL of additional blood was stored in lysis buff er. For each slide, randomly chosen fi elds totaling ∼10,000 erythrocytes (red blood cells [RBCs]) were inspected under oil-immersion objective using 400× magnifi cation by a single observer (K.S.S.), who was unaware of the infec-tion status as determined by PCR screening (see below). Leukocytes were identifi ed as lym-phocytes, monocytes, eosinophils, heterophils, basophils, and thrombocytes (Hawkey and Denne 1989) and were quantifi ed as number of cells per 104 RBCs. We quantifi ed parasitized RBCs (haemosporidian gametocytes, primarily) in the same way. Numbers of RBCs were esti-mated by comparing microscope fi elds with a series of 11 photographs with accurate counts of RBCs over a range of densities (736–2,710 RBCs per fi eld) used to quantify WBCs and parasites.

Molecular assays.—DNA extraction and screen-ing by PCR amplifi cation of a 154-nucleotide segment of parasite RNA-coding mitochon-drial DNA followed Fallon et al. (2003b). We a empted to amplify and sequence a segment of the cytochrome-b gene for all samples that tested positive in PCR screening using methods outlined in Fallon et al. (2004, 2005).

Statistics.—Heterogeneity in numbers of infected and non-infected individuals in diff er-ent samples was tested by likelihood-ratio chi-square values for contingency tables (G-tests; Sokal and Rohlf 1995). Diff erences in numbers of parasites and WBCs of various classes between samples were compared by nonparametric one-way analyses of variance (ANOVA) using the Kruskal-Wallis statistic (Siegel 1956). Nested analyses of variance of parasite prevalence were based on taxonomic levels in Sibley and Monroe (1990). All analyses were done using SAS (SAS Institute, Cary, North Carolina) so ware proce-dures FREQ, CATMOD, and NPAR1WAY.

Malaria Infections in Thrushes (Turdus)October 2007] 1257

Results

Parasite prevalence.—The idea that haemo-sporidian prevalence in bird populations is lower in the tropics than at temperate latitudes is based primarily on compilations of smear data for North America (Greiner et al. 1975) and the Neotropics (White et al. 1978, Sousa and Herman 1982). A broader survey of such data for Turdus including Europe, Southeast Asia, and Africa, showed that tropical–temper-ate diff erences are not consistent (Table 1). For example, prevalence of Haemoproteus in two temperate-zone samples (North America and Europe) was 6.4% and 18.2%, respectively, com-pared with 0–19.1% in various tropical samples; for Plasmodium, the prevalence in the temperate-zone samples (7.6% and 10.4%) also was brack-eted by the tropical samples (0–34.8%).

Parasite prevalence in this study, judged by visual inspection of fi elds containing ∼10,000 RBCs, was 1 of 47 individual T. grayi in Panama, 6 of 19 T. migratorius in Michigan, and 7 of 21 T. migratorius in Missouri. The prevalence in T. grayi (2.1%) did not diff er signifi cantly from the combined prevalence for Haemoproteus and Plasmodium of 5.4% (χ2 = 0.70, P = 0.40; White et al. 1978) and 10.9% (χ2 = 3.20, P = 0.074; Sousa and Herman 1982) reported in published stud-ies (Table 1). Prevalence in the Michigan (33%)

and Missouri (32%) samples of T. migratorius did not exceed signifi cantly the value of 26% summed over earlier North American studies (Michigan, χ2 = 0.30, P = 0.59; Missouri, χ2 = 0.55, P = 0.46; Table 1).

Screening by PCR detected all the infected individuals identifi ed from blood smears, as well as an additional 18 infections out of 46 screened individuals in Panama, an additional 6 of 19 in Michigan, and an additional 4 of 19 in Missouri; DNA samples were not obtained for one individual in Panama and two in Missouri. Thus, PCR was more eff ective than micros-copy (G = 24.3, P < 10–6; Freed and Cann 2006). According to PCR screening, the prevalence of malaria parasites in our samples was 41% in Panama, 57% in Missouri, and 63% in Michigan. Prevalence did not diff er between the Missouri and Michigan samples (G = 0.03, P = 0.85); the temperate-zone samples combined had margin-ally higher prevalence than the Panama sample (G = 3.0, P = 0.08). Panama samples were obtained during the dry season (March) and the wet season (July). Parasite prevalence determined by PCR was 16 of 37 (43%) in the dry season and 3 of 9 (33%) in the wet season (G = 0.3, P = 0.60). Juvenile individuals were not sampled in Michigan, and hatch-year (HY) and a er-hatch-year (AHY) individuals were not distinguished in Missouri. The dry-season

Table 1. Published prevalence of infections by the haemosporidian parasites Leucocytozoon (LEU), Haemoproteus (HAE), and Plasmodium (PLA) in Turdus migratorius, T. grayi, and other species of Turdus in several regions, based on visual inspection of blood smears.

Percentage of prevalence

Species a Location b Sample Infected LEU HAE PLANew World

T. migratorius North America (5) 1,323 75.3 56.8 18.2 7.6T. grayi Neotropics (6) 55 9.1 0.0 3.6 1.8T. grayi Panama (7) 55 34.5 21.8 10.9 0.0Turdus spp. (1) Neotropics (6) 422 17.3 0.5 5.2 4.7

Old WorldTurdus spp. (2) Europe (8) 940 30.5 14.1 6.4 10.4Turdus spp. (3) Tropical Africa (8) 24 41.7 20.8 0.0 20.8Turdus spp. (4) Tropical Southeast Asia (9) 440 46.8 1.1 19.1 34.8Turdus spp. (4) SE Asia (excluding T. obscurus) (9) 180 25.0 1.7 7.8 15.0

a Species: (1) 14 species (T. albicollis, T. amaurochalinus, T. chiguanco, T. fumigatus, T. fuscater, T. grayi, T. ignobilis, T. leucomelas, T. nudigenis, T. olivater, T. rufi torques, T. rufi ventris, T. rufopalliatus, T. seranus); (2) 6 species (T. iliacus, T. merula, T. philmelos, T. pilaris, T. torquatus, T. viscivorus); (3) 2 species (T. libonyana and T. olivaceus); (4) 9 species (T. unicolor, T. cardis, T. boulboul, T. merula, T. chrysolanus, T. celaenops, T. obscurus, T. rufi collis, T. naumanni).

b Sources: (5) Greiner et al. 1975; (6) White et al. 1978; (7) Sousa and Herman 1982; (8) Peirce 1981, Benne et al. 1974, Benne and Herman 1976, Wink and Benne 1976, Benne et al. 1977, Peirce 1984; (9) McClure et al. 1978.

Ricklefs and Sheldon1258 [Auk, Vol. 124

sample in Panama did not include HY birds; however, the wet-season sample included four HY and fi ve AHY individuals; the three infec-tions were detected in AHY birds.

Identity of parasite lineages.—We sequenced up to 350 nucleotides of the cytochrome-b gene of 10 parasites in Panama, 10 in Missouri, and 14 in Michigan. Thus, sequencing success was 34 of 42 positive screens (81%). We identifi ed four lin-eages of haemosporidian parasites in T. migrato-rius in North America, three of which could be assigned to the genus Plasmodium on the basis of their phylogenetic relationship to known lin-eages of Plasmodium (Bensch et al. 2000, Perkins and Schall 2002, Ricklefs and Fallon 2002), and one to Haemoproteus, represented by a single infection in our sample. Panamanian T. grayi infections yielded three lineages of Plasmodium.

Infection intensity.—Infection intensity (in -fected cells per 104 RBCs) was low overall, and we assume that these values represented chronic rather than acute infections. However, more par-asites were counted on blood smears taken from infected individuals (identifi ed by PCR screen-ing) in Missouri and Michigan than in Panama. In Michigan, we counted no infected cells in smears from six infected T. migratorius; the remaining six infected individuals had 1, 2, 3, 4, 6, and 11 parasites per 104 RBCs. In the Missouri sample, four infected individuals lacked visible parasites on smears. Among seven additional PCR-posi-tive blood samples, parasite intensities were 1, 1, 6, 6, 7, 19, and 29 per 104 RBCs. The Michigan and Missouri samples did not diff er signifi cantly (Kruskal-Wallis, χ2 = 0.93, P = 0.33). The highest value belonged to the single Haemoproteus infec-tion in the sample. By contrast, among 19 PCR-positive Panamanian T. grayi, visual inspection revealed only a single parasitized cell on a single smear. This distribution diff ered signifi cantly from the combined temperate-zone samples (χ2 = 12.4, P = 0.0004). The average frequency of parasites in the blood of infected birds was 0.06 parasites per 104 RBCs in Panama, 2.25 in Michigan, and 6.27 in Missouri (4.00 excluding the one Haemoproteus infection).

White blood cells.—Numbers of diff erent types of white blood cell per 10,000 RBCs in each sampling locality, and for infected and unin-fected individual thrushes, are shown in Table 2. Three diff erent pa erns are evident in the WBC counts (Fig. 1). First, numbers of thrombocytes and basophils did not diff er among Panama, Ta

le 2

. Cou

nts

of w

hite

blo

od c

ells

per

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eryt

hroc

ytes

on

smea

rs f

rom

uni

nfec

ted

bird

s an

d in

divi

dual

s in

fect

ed w

ith a

vian

mal

aria

in

Mic

higa

n an

d M

isso

uri (

T. m

igra

tori

us) a

nd P

anam

a (T

. gra

yi).

M

ichi

gan

Mis

sour

i Pa

nam

a

U

ninf

ecte

d In

fect

ed

Uni

nfec

ted

Infe

cted

U

ninf

ecte

d In

fect

ed

(n =

7)

(n =

12)

(n

= 8

) (n

= 1

1)

(n =

27)

(n

= 1

9)

Vari

able

M

ean

SD

Mea

n SD

M

ean

SD

Mea

n SD

M

ean

SD

Mea

n SD

HET

6.

57

4.79

6.

83

8.22

15

.00

13.1

8 8.

18

8.32

2.

85

3.58

2.

34

3.79

EOS

4.00

6.

40

8.92

5.

79

5.38

4.

78

3.00

3.

10

1.85

2.

85

2.16

2.

17BA

S 0.

14

0.38

0.

42

0.51

1.

75

2.25

0.

91

1.22

0.

33

0.96

0.

47

0.90

LYM

32

.43

26.7

4 70

.75

28.5

1 49

.50

59.5

4 45

.18

32.0

2 18

.93

13.7

8 28

.26

19.3

6M

ON

4.

86

2.19

5.

58

4.93

0.

63

0.74

1.

27

1.42

3.

11

3.03

3.

53

4.25

THR

43.4

3 24

.55

52.1

7 26

.47

40.6

3 29

.27

52.5

6 66

.89

60.3

0 42

.78

55.2

1 28

.16

PAR

A

0.00

0.

00

2.25

3.

39

0.00

0.

00

6.27

9.

45

0.00

0.

00

0.05

0.

23A

bbre

viat

ions

: HET

= h

eter

ophi

ls, E

OS

= eo

sino

phils

, BA

S =

baso

phils

, LY

M =

lym

phoc

ytes

, MO

N =

mon

ocyt

es, T

HR

= th

rom

bocy

tes,

and

PA

RA

= m

alar

ia p

aras

ites.

Malaria Infections in Thrushes (Turdus)October 2007] 1259

Missouri, and Michigan (Kruskal-Wallis, throm-bocytes, χ2 = 4.4, P = 0.11; basophils, χ2 = 4.9; P = 0.087) or between parasitized (χ2 = 1.4, P = 0.24) and nonparasitized individuals (χ2 = 0.0, P = 0.99). Second, heterophils and monocytes diff ered, in complementary fashion, between localities; heterophils were more common in blood smears in Michigan and, especially, Missouri, than in Panama (heterophils, χ2 = 21.9, P < 0.0001; monocytes, χ2 = 18.3, P < 0.0001), but their numbers did not respond to parasitism in either locality (heterophils, χ2 = 0.3, P = 0.56; monocytes, χ2 = 0.0, P = 0.86). Third, lymphocytes

and eosinophils, although diff ering in frequency between locations (lymphocytes, χ2 = 18.2, P = 0.0001; eosinophils, χ2 = 12.4, P = 0.0021) also were signifi cantly elevated in infected individu-als in Michigan (lymphocytes, χ2 = 6.9, P = 0.009; eosinophils, χ2 = 3.5, P = 0.062).

Comparison with species other than Turdus.—To determine whether the results obtained for Turdus in Panama and Michigan could be generalized to a broader sample of bird spe-cies, we compared counts of WBCs on smears for 50 individuals of 18 species in Panama and 36 individuals of 11 species in Michigan

Fig. 1. Numbers of white blood cells in uninfected and infected individuals of Turdus migratorius in Michigan and Missouri and T. grayi in Panama. Bars represent standard errors, but most distribu-tions were highly positively skewed and significance tests were based on nonparametric rank-order statistics.

Ricklefs and Sheldon1260 [Auk, Vol. 124

(Table 3). Smears were chosen to provide an approximately even number of noninfected and infected (PCR-screened) individuals; infected samples numbered 22 in Panama and 16 in Michigan. Except for signifi cantly elevated levels of thrombocytes in the Panamanian birds (Kruskal-Wallis, χ2 = 5.7, P = 0.017), counts of the other cells did not diff er between locations. Among PCR-positive individuals, infection intensity did not diff er signifi cantly between Panama and Michigan (χ2 = 0.3, P = 0.58). Except for a tendency of heterophils to be elevated in infected birds in Michigan (χ2 = 3.8, P = 0.050) and in Panama (χ2 = 6.3, P = 0.012), infection status had no eff ect on WBC counts.

Discussion

Application of polymerase-chain-reaction assays and what smear data reveal.—Recent studies using PCR and DNA sequencing to quantify haemo-sporidian parasite prevalence and to identify parasite lineages have shown that visual inspection of smears generally underestimates prevalence, sometimes substantially (Feldman et al. 1995; Perkins et al. 1998; Bensch et al.

2000; Jarvi et al. 2002, 2003; Richard et al. 2002; Waldenström et al. 2002; Fallon et al. 2003b; Freed and Cann 2003, 2006). In our compari-son between tropical and temperate species of Turdus, PCR revealed a substantially higher prevalence than visual screening and increased tropical prevalence (40%) to a level that did not diff er signifi cantly from the temperate sample (63%). If the lower intensities of infection in T. grayi caused some infections to be missed by PCR (Hellgren et al. 2004), the gap in prevalence would be even narrower. The sensitivity of our PCR screening appears to be about one parasite per 105 RBCs (Fallon et al. 2003b), though read-ing smears is at least an order of magnitude less sensitive. Thus, prevalence from visual inspec-tion of smears likely reveals more about the intensity of infections than about the proportion of individuals infected (Atkinson et al. 2001b). In addition, neither smears nor PCR screening of DNA extracted from blood can recognize infections that become quiescent in the liver or other internal tissues (Atkinson and van Riper 1991, Atkinson et al. 2001a, Valkiūnas 2005).

Diff erent intensities of infections in Panama and Michigan could refl ect either properties

Table 3. Cell counts, for taxa other than Turdus, of passerine birds from Michigan and Panama.

Michigan Panama

Uninfected Infected Uninfected Infected (n = 20) (n = 16) (n = 28) (n = 22)

Variable Mean SD Mean SD Mean SD Mean SDHET 3.55 3.72 6.00 4.44 3.93 5.33 7.32 5.77EOS 0.60 1.10 2.19 4.86 1.21 3.61 1.14 3.23BAS 0.10 0.31 0.19 0.54 0.07 0.38 0.36 1.04LYM 17.25 14.36 16.44 12.87 18.07 13.31 21.55 14.95MON 0.30 0.66 0.75 1.39 1.32 1.76 0.68 1.13THR 28.95 22.73 39.56 24.59 43.21 23.35 66.14 65.66PARA 0.45 a 1.61 6.88 13.70 0.04* 0.19 3.82 12.25

a Two Michigan individuals that tested negative with PCR had parasite intensities of 2 × 10–4 and 7 × 10–4; one Panama individual that tested negative had a parasite count of 1 × 10–4.

Note: Sample includes 86 passerine birds (number infected in parentheses) captured in Michigan and Panama. Michigan: Baltimore Oriole (Icterus galbula) = 3 (3), Common Grackle (Quiscalus quiscula) = 1 (1), Common Yellowthroat (Geothlypis trichas) = 2 (1), Field Sparrow (Spizella pusilla) = 5 (0), Gray Catbird (Dumetella carolinensis) = 7 (4), House Wren (Troglodytes aedon) = 6 (2), Myrtle Warbler (Dendroica coronata) = 1 (1), Rose-breasted Grosbeak (Pheucticus ludovicianus) = 2 (2), Song Sparrow (Melospiza melodia) = 8 (1), and Yellow Warbler (D. petechia) = 1 (1). Panama: Blue-gray Tanager (Thraupis episcopus) = 2 (1), Black-tailed Flycatcher (Myiobius atricaudus) = 1 (0), Buff -throated Saltator (Saltator maximus) = 6 (0), Buff -throated Woodcreeper (Xiphorhynchus gu atus) = 2 (2), Buff -breasted Wren (Thryothorus leucotis) = 8 (0), Crimson-backed Tanager (Ramphocelus carbo) = 6 (4), Golden-collared Manakin (Manacus vitellinus) = 6 (2), Northern Waterthrush (Seiurus novaboracensis) = 1 (1), Red-capped Manakin (Pipra mentalis) = 1 (1), Rufous-and-white Wren (T. rufalbus) = 1 (1), Western Slaty Antshrike (Thamnophilus atrinucha) = 1 (1), Song Wren (Cyphorhinus phaeocephalus) = 3 (3), Spo ed Antbird (Hylophylax naevioides) = 1 (0), Variable Seedeater (Sporophila americana) = 6 (1), White-breasted Woodwren (Henicorhina leucosticta) = 1 (1), White-bellied Antbird (Myrmeciza longipes) = 2 (2), White-shouldered Tanager (Tachyphonus luctuosus) = 1 (1), and Yellow-green Vireo (Vireo fl avoviridis) = 1 (1).

Malaria Infections in Thrushes (Turdus)October 2007] 1261

of the parasites or properties of their hosts. In particular, Plasmodium infections are gener-ally maintained at much lower intensities than Haemoproteus infections (S. M. Fallon and R. E. Ricklefs unpubl. data). Thus, it is necessary to identify the lineage of haemosporidian parasite when comparing infection intensity and WBC counts. In the present study, each population of thrushes was infected, with one exception, by several lineages of Plasmodium; infection intensity within areas was unrelated to parasite lineage; and the lineages from the tropical and temperate areas were interspersed phylogeneti-cally (data not shown). Thus, infection intensity in this comparison likely refl ects the ability of hosts to control chronic infections.

If this were the case, it would be tempting to conclude that tropical T. grayi can resist or control disease organisms be er than temperate-zone T. migratorius. Although this conclusion might be valid for malaria infections in these two populations, it need not apply to other parasites or pathogens, and it likely does not refl ect gen-eral diff erences between tropical and temperate-zone populations. The surveys summarized in Table 1 of parasite prevalence in blood smears represent varied investigators, methods of sam-pling, sample sizes, and seasons of study, but nonetheless indicate that tropical populations of Turdus spp. in Africa and southeast Asia can harbor either high prevalences or high intensities or both of malaria parasite infections. Thus, even though Neotropical Turdus spp. tend to have, on average, low malaria prevalence, this is certainly not true of all regions in the tropics, and diff erent species of hosts within regions may vary widely in parasite prevalence.

At the taxonomic level of family, average prevalence of avian haemosporidian infec-tion judged from blood smears does exhibit signifi cant variation (Greiner et al. 1975). This variation has been related to other a ributes of the hosts, including development time (Ricklefs 1992). A nested hierarchical analysis of covariance of prevalence within Neotropical species also showed signifi cant correlations between the prevalence of Plasmodium and Haemoproteus at the levels of host families within Passeriformes and genera within fami-lies. Correlations between the parasite genera were not evident among host species within genera, where most of the variation in preva-lence of both parasites is concentrated (data

not shown; see also Scheuerlein and Ricklefs 2004). Thus, higher taxonomic groups appear to diff er either in their general susceptibility or resistance to both genera of parasites. However, most of the variation among species is idiosyn-cratically related to such factors as sampling error, variation in prevalence of diff erent genera of parasites owing to particular environmental conditions aff ecting vectors, and variation in the resistance of host species to diff erent lineages of parasites.

Prevalence of malaria observed in blood smears o en has a pronounced cycle in sea-sonal environments, declining through the fall and winter and then re-emerging from latent stages in the spring to coincide with the onset of the breeding season (Applegate 1970, 1971; Benne and Cameron 1974; Klei and DeGiusti 1975; Atkinson et al. 1988; Stacey et al. 1990; Hatchwell et al. 2000; Garvin et al. 2003a; Schrader et al. 2003). By contrast, we have noted li le seasonal variation in prevalence or lineage type in samples from one location in southwestern Puerto Rico that has highly seasonal precipitation (Fallon et al. 2004). The Panama and Michigan samples both spanned the breeding seasons of the local populations of Turdus. A third sample obtained near St. Louis, Missouri, during September and October, well a er the end of the breeding season and into the molt period (Pyle 1997), had similar prevalence of avian malaria compared with the breeding-season sample from Michigan (57% vs. 63%). Mosquitoes were still active when these birds were sampled, and so these infections may have been acquired late in the summer. The infec-tions were from the same lineage of Plasmodium that was most common in Michigan earlier in the summer. The relatively high prevalence in the autumn birds from Missouri could refl ect the greater ability of PCR screening to detect declining infections; but in fact, the infection intensities of these birds were higher, on aver-age, than those of the early-summer sample from Michigan.

Diff erences in white-blood-cell counts between areas.—Comparing samples from Michigan and Panama, which were obtained during the same part of the annual cycle, overall levels of WBCs in apparently uninfected birds were greater in the Michigan populations, except for basophils and monocytes, which were similar. Diff erences in WBC levels could refl ect either

Ricklefs and Sheldon1262 [Auk, Vol. 124

“baseline” levels of immune-system cells in the peripheral blood or diff erences in general health. Whether high levels of WBCs indicate a high constitutive position in anticipation of infection, a strong immune response to ongoing infections, or a relatively ineff ective immune system requiring the recruitment of large numbers of WBCs can be determined only by experimental studies. However, responses to naturally occurring infections, such as malaria, may be informative.

Among samples of Turdus, lymphocytes increased dramatically in birds with detect-able parasites in peripheral blood, particularly in Michigan. Eosinophils also increased in response to infection, but the eff ect was seen only in the Michigan birds, where counts of eosinophils about doubled. Eosinophils are known to increase in response to infec-tion, particularly by macroparasites, in other systems (Roi et al. 1989, Janes et al. 1994). Because lymphocytes are responsible for anti-body production (Rose et al. 1979, Janes et al. 1994), it is not surprising that they are also recruited in the peripheral blood. Considering that the WBC response was stronger in Michigan than in Panama and that parasite intensity was greater in Michigan, the absence of a WBC response in the late-summer sample from Missouri, in which parasite intensity was even higher, provides an interesting contrast for further investigation.

Comparisons with non-Turdus passerine birds.—We examined a variety of other species in both Michigan and Panama to determine whether the diff erences in infection intensity and WBC response between the tropical and temperate locality observed in Turdus were generalizable to other species. This second sample supported the pa ern observed in Turdus only in the posi-tive (but nonsignifi cant) response of eosinophil levels to malaria infection in Michigan and the somewhat higher parasitemias (also nonsig-nifi cant) observed in Michigan. Levels of most types of WBCs were lower in this sample than in the Turdus samples. In contrast to the results for Turdus, heterophils rather than eosinophils responded signifi cantly to malaria infection in Michigan and in Panama. Heterophils form a major component of the initial response of the immune system to infection (Dietert et al. 1996); they clear bacteria and other infectious agents by phagocytosis and are important components

of the infl ammation response (Lane 1996). Heterophil levels are also, apparently, associ-ated with stress (Post et al. 2003a, b).

An increase in heterophil levels in response to malaria infection was also reported for a variety of passerine birds in the Lesser Antilles (Apanius et al. 2000). These infections involved primarily Haemoproteus rather than Plasmodium (Fallon et al. 2003a), and this is the case for many of the non-Turdus species in Panama (R. E. Ricklefs unpubl. data). Ots and Hõrak (1998) noted that Haemoproteus infections in Great Tits (Parus major) increased lymphocyte counts but not the number of heterophils. Further sampling will be required to determine how the WBC response depends on the type of malaria parasite.

Conclusions.—In comparative studies, it is important to include both PCR screening and visible inspection of blood smears in analy-ses of chronic haemosporidian infections in birds. Polymerase chain reaction gives a more accurate assessment of parasite prevalence, whereas enumerating infections on blood smears provides information about the abil-ity of host individuals to control infections in relation to the virulence of the parasite. Most variation in parasite prevalence appears as dif-ferences among species in the same genus of host, which indicates the highly idiosyncratic nature of parasite infections. Although infec-tion prevalence in host populations may vary over time and space (Bensch and Åkesson 2003, Fallon et al. 2004), these values can be relatively stable within a host population over periods of up to a decade (Fallon et al. 2004, Ricklefs et al. 2005). Thus, a large component of variation in prevalence includes the outcomes of coevolu-tionary interactions between host and parasite populations (Apanius et al. 2000, Fallon et al. 2003a). The role of vector abundance in mediat-ing variation in prevalence has not been studied in a broadly comparative context (Super and van Riper 1995). Host–parasite coevolution-ary interactions are suffi ciently labile that the prevalence of one or a few parasites in one or a few host species cannot reveal the general dis-ease environment or the outcome of life-history evolution. Indeed, the infl uence of such factors is apparent primarily at higher taxonomic lev-els, where data averaged over many species and genera provide more specifi c, taxon-dependent pa erns (e.g., Ricklefs 1992).

Malaria Infections in Thrushes (Turdus)October 2007] 1263

White blood cells are one group of immune-system components, including phagocytotic cells and producers of specifi c antibodies, and these cells respond to infection by haemospo-ridian parasites in some populations of birds. Counts in uninfected individuals may indi-cate constitutive “background” levels of these cells or the activity of the immune system in responding to other types of infections. In either case, it is clear from the data on birds infected with Plasmodium that diff erent populations of hosts respond in diff erent ways, or may not respond at all with respect to concentrations of WBCs. The immune system has many diff erent pathways of response (Adamo 2004, Matson et al. 2005, Tieleman et al. 2005), and each compo-nent provides a diff erent insight into the overall capacity of the immune response of any given individual. If existing data on parasite preva-lence are a guide, however, general pa erns in the status of the immune system in wild populations of birds will require information on large samples of species. Narrower compari-sons are likely to refl ect the special relationships between particular host and parasite popula-tions. Ultimately, experimental infection fol-lowed by monitoring the etiology of the disease in individual birds will provide the most direct and useful information.

Ackno ledgments

This study was supported by National Science Foundation grants DEB-0089226 and IBN-0212587. Logistical support was provided by the Smithsonian Tropical Research Institute in Panama and by the Kellogg Biological Station in Michigan. We thank B. Swanson and J. Gray for help with PCR and sequencing. Associate Editor K. P. Johnson and several reviewers made constructive comments that greatly improved the manuscript.

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Associate Editor: K. P. Johnson