Dynamics of parasitemia of malaria parasites in a naturally and experimentally infected migratory songbird, the great reed warbler Acrocephalus arundinaceus

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Experimental Parasitology 119 (2008) 99–110 www.elsevier.com/locate/yexpr

Dynamics of parasitemia of malaria parasites in a naturally and experimentally infected migratory songbird, the great reed warbler Acrocephalus arundinaceus Pavel Zehtindjiev a, Mihaela Ilieva a, Helena Westerdahl b, Bengt Hansson b, Gediminas Valki unas c, Staffan Bensch b,* a

Institute of Zoology, Bulgarian Academy of Sciences, Boulevard, Tzar Osvoboditel 1, 1000 Sofia, Bulgaria b Department of Ecology, Lund University, Ecology Building, SE-223 62 Lund, Sweden c Institute of Ecology, Vilnius University, Vilnius-21 LT-08412, Lithuania Received 17 April 2007; received in revised form 19 December 2007; accepted 30 December 2007 Available online 13 January 2008

Abstract Little is known about the development of infection of malaria parasites of the genus Plasmodium in wild birds. We used qPCR, targeting specific mitochondrial lineages of Plasmodium ashfordi (GRW2) and Plasmodium relictum (GRW4), to monitor changes in intensities of parasitemia in captive great reed warblers Acrocephalus arundinaceus from summer to spring. The study involved both naturally infected adults and experimentally infected juveniles. The experiment demonstrated that P. ashfordi and P. relictum lineages differ substantially in several life-history traits (e.g. prepatent period and dynamics of parasitemia) and that individual hosts show substantial differences in responses to these infections. The intensity of parasitemia of lineages in mixed infections co-varied positively, suggesting a control mechanism by the host that is general across the parasite lineages. The intensity of parasitemia for individual hosts was highly repeatable suggesting variation between the host individuals in their genetic or acquired control of the infections. In future studies, care must be taken to avoid mixed infections in wild caught donors, and when possible use mosquitoes for the experiments as inoculation of infectious blood ignores important initial stages of the contact between the bird and the parasite. Ó 2008 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Avian malaria; Plasmodium; Acrocephalus arundinaceus; Mixed infections; qPCR

1. Introduction The most virulent of the four malaria parasites of humans, Plasmodium falciparum, causes many millions of deaths annually (Sachs and Malaney, 2002). At a first glance, it therefore seems a paradox that most bearers of malaria parasites appear fully asymptomatic in field studies carried out in endemic malarial regions (Bruce et al., 2000). Absence of apparent fitness costs of malaria infections is not unique to field studies of P. falciparum and humans, but have frequently been reported in other host–parasite systems (Schall, 2002; Valki unas, 2005). *

Corresponding author. E-mail address: staff[email protected] (S. Bensch).

0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.12.018

The solution to this paradox is probably that the vast majority of infected host individuals included in field studies are carrying chronic infections. At this stage of infection, parasitemia is light as a result of an efficient host immune system that keeps the parasite population under control. The host individuals that for any reasons are unable to cope with the infection (acute primary or relapse infections), die either directly from the disease, or become eliminated by predators. Remaining in the population is therefore mainly two kinds of healthy individuals; those that never have been infected and those that have recovered and can cope with the infection. The proportions of these groups in the host population depend on the rate of parasite transmission, virulence and clearance of infections (Frank, 1996).

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Avian malaria parasites of the genus Plasmodium consist of approximately 40 described morphological species (Valki unas, 2005), however, molecular analyses of parasite mitochondrial lineages suggest that there are many more biological species because similar molecularly lineages often have distinctly different host distributions and an apparent absence of recombination (Ricklefs and Fallon, 2002; Bensch et al., 2004; Beadell et al., 2006). The majority of morphologically or molecularly identified species of avian malaria parasites are known mainly from observations of their blood stages, either from a diagnostic morphology or a DNA-sequence. Both the traditional microscopic based and the more recent molecular methods have been used extensively to investigate malaria prevalence in natural populations (Peirce, 1981; Scheuerlein and Ricklefs, 2004; Valki unas, 2005; Ricklefs et al., 2005; Pe´rez-Tris and Bensch, 2005a). Population and experimental studies have confirmed the prediction that infections of Plasmodium spp. (and species of the related genus Haemoproteus) in field studies, where light chronic infections prevail, is rarely associated with detectable host fitness costs (Weatherhead, 1990; Woodworth et al., 2005; Valki unas et al., 2006b; Bensch et al., 2007). A few studies have even observed a positive association between being infected and measures of host fitness (Westerdahl et al., 2005; Kilpatrick et al., 2006). Such observations suggest that the chronically infected individuals consist of a relatively more fit subset after parasites have selected against low performing phenotypes, or that mild infections increase the capacity of the immune system to prevent or control other, more debilitating infections. Heavy primary infections, with intensities of parasitemia above 10%, are well known from the many infection experiments carried out during the last century (van Riper et al., 1994; Valki unas, 2005) and are also common in wildlife but are underestimated when using common methods of catching birds such as mist-netting (Valki unas, 2005). Numerous studies of natural bird populations hence fail to address several important aspects of host–parasite interactions. For many species of malaria parasites and other haemosporidians, we thus lack information about their biology and ecology including e.g. time and place of transmission, prepatent periods, dynamics of parasitemia and the occurrence in internal organs by tissue stages of the parasites during primary infections and most importantly, virulence, defined as the amount of damage the parasite is causing its hosts (Frank, 1996). In a long term study of a Swedish population of great reed warblers Acrocephalus arundinaceus, previous studies have found about 20 mitochondrial lineages of blood haemosporidian parasites of the genera Plasmodium and Haemoproteus, most of which qualify as distinct species based on host distribution and/or apparent absence of recombination (Bensch et al., 2000, 2007; Waldenstro¨m et al., 2004). These studies have been carried out in the species breeding quarter in northern Europe, however, evidence has accumulated that transmission of most of the lineages occurs only in sub-Saharan Africa at the birds winter quar-

ters of this long-distant migrant (Waldenstro¨m et al., 2002; Bensch et al., 2007; Hellgren et al., 2007). Although individual birds have been studied over several breeding seasons, this longitudinal study has ignored the most crucial part of the contact between the host and the parasites, i.e. when naive birds are infected for the first time at the wintering grounds. To enable us to make detailed studies of the host–parasite interactions during primary infections, we decided to carry out infection experiments on captive great reed warblers and to run the experiment for several months until the stage of chronic infections was reached. For the infection experiments we chose to study the two most common lineages of malaria parasites of the genus Plasmodium (GRW2 and GRW4) naturally infecting great reed warblers. Among breeding great reed warblers, prevalence of the lineage GRW2 was of 5.8% (Bensch et al., 2007) and it is the single known lineage of Plasmodium ashfordi (Valki unas et al., 2007). The few identified alternative hosts for this lineage include four species of warblers (Hellgren et al., 2007) and house martin Delichon urbica (A. Marzal in preparation). In contrast, the lineage GRW4 was much more prevalent (15.5%) among breeding great reed warblers (Bensch et al., 2007) and is a member of a much more lineage rich clade of parasites within the morphospecies Plasmodium relictum (Valki unas et al., 2007). The lineage GRW4 is also much more of a host generalist, having been identified in more than 30 species from 13 different bird families (Beadell et al., 2006; Hellgren et al., 2007). Also worth noting is that GRW4 is the Plasmodium lineage introduced to Hawaii less than 100 years ago where it seems to have contributed to the decline and extinction of several endemic bird species during the 20th century (van Riper et al., 1986; Beadell et al., 2006). Accurate detection of Plasmodium spp. is difficult to achieve during light chronic infections, which are frequently overlooked in blood films (Jarvi et al., 2003; Waldenstro¨m et al., 2004). At chronic infections, Plasmodium spp. parasitemia might be as low as 1/1,000,000 infected erythrocytes and even less, so accurate determination of malaria infections by microscopy might take several hours for each sample and it requires high qualification expertise (Iezhova et al., 2005). To efficiently measure intensity of parasitemia, we therefore quantified the amount of parasite DNA with quantitative PCR (qPCR) which has been demonstrated to be both fast and accurate (Bell and RanfordCartwright, 2002). The main aim of the infection experiment carried out during this study was to investigate the dynamics of parasitemia in juvenile birds during their first encounter with malaria parasites; we did that by inoculating infected blood from adult great reed warblers to uninfected juveniles of the same species. For each bird, we measured the intensity of parasitemia repeatedly throughout the experiment in order to determine the length of the prepatent period of infection and the level of parasitemia at its peak, and during the following chronic stage of infections. This allowed

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us to investigate if there were general life-history differences between P. relictum and P. ashfordi, which belong to different subgenera of malaria parasites, Haemamoeba and Novyella, respectively (Valki unas, 2005). Because the birds were not kept in mosquito proof cages, we also monitored the level of natural transmission of other malaria lineages, which were not included in the inoculation experiments. These analyses demonstrated that we also had uncontrolled natural transmission of two other Plasmodium lineages among the captive birds; this provided us with the opportunity to investigate some patterns of mixed infections, i.e. if their parasitemia develops independently or in positive or negative associations with experimental infections. We set up the following predictions. First, we tested the well established premise (e.g. van Riper et al., 1986; Valki unas, 2005) that the naive birds, experimentally inoculated with either P. ashfordi (lineages GRW2) or P. relictum (GRW4), should develop higher parasitemia than seen in naturally infected adult birds, because at primary infections the birds should become sicker than during chronic stage of the infections. Second, we predicted that GRW2 should pose a higher fitness cost to great reed warblers and therefore develop higher levels of parasitemia. This prediction is based on the observations that GRW2 infections are associated with the diversity of the host’s MHC genes (Westerdahl et al., 2005), that GRW2 is a rarer parasite (prevalence 5.8%) than is GRW4 (15.5%), and on the assumption that the intensity of parasitemia is correlated to virulence as previously seen in other malaria parasite–host systems (Mackinnon and Read, 2004). Third, to find out whether the infections affected the performance of the birds, we monitored several indicators of condition (subcutaneous fat level, body mass, body temperature and moult progression) with the prediction that infections should reduce the body mass, delay moult and affect body temperature. In parallel to the infection experiment, we also followed the dynamics of secondary parasitemia in naturally infected adult great reed warblers that had chronic infections already when captured and that we held in captivity throughout the winter when they normally are at their African winter quarter (Bensch et al., 2007). According to the adaptive transmission hypothesis (Hasselquist et al., 2007), we predicted to find increasing parasitemia relative to initial levels during the period November–January when the sub-Saharan wintering areas are wet, competent vectors should be abundant and transmission conditions optimal.

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Table 1 Prevalence and identity of blood parasites in 52 adult great reed warblers captured at the Kalimok station in Bulgaria during June 2005 Lineage

Species

Number of birds

GenBank#

GRW1 ACSTE1 GRW3 GRW5 GRW2 GRW4 SGS1 RTSR1 Not determined Uninfected

Haemoproteus payevskyi Haemoproteus sp. Haemoproteus sp. Haemoproteus sp. Plasmodium ashfordi Plasmodium relictum Plasmodium relictum Plasmodium sp. Plasmodium or Haemoproteus

18 1 1 4 2* 8 2** 1 1 14

AF254964 EF032811 AF254965 DQ368346 AF254962 AF254975 AF495571 AF495568

* **

One of these was later shown to have mixed infections with GRW4. One of these was later shown to have mixed infections with GRW11.

for presence of avian malaria parasites using molecular methods (see below). The molecular results obtain in mid July demonstrated the presence of eight different lineages of Haemosporidians of which four were Plasmodium sp. (Table 1). We selected nine birds naturally infected with P. ashfordi (lineage GRW2, two birds), and P. relictum (GRW4, five birds and SGS1, two birds) to be kept as donors of infection for the coming experiments. These infected adult birds were also used to monitor dynamics of secondary parasitemia of these two malaria parasites during this study. The remaining birds were released at the capture site. In late August, 25 juvenile great reed warblers were captured around the station and placed in captivity for later use in the infection experiments. 2.2. Conditions for keeping the birds All birds during the experiment were kept in a large aviary in open air conditions from June to November and in an indoor aviary from December to April. The sizes of both aviaries were around 20 square meters. The cages were supplied with suitable vegetation from the typical habitat of the species. During the winter period, the indoor aviary was provided with bio light in a 12 h photoperiod and temperatures between 15 and 25 °C to provide conditions more similar to those in African winter quarters. The food provided for the birds in the aviaries included water, meal worms, larva of flies and a mix of eggs based substance enriched by vitamins and minerals. In selected intervals, birds were caught in mist nets inside the aviary when blood samples and measurements were taken.

2. Methods 2.3. Handling of donor and experimental birds 2.1. Study site and experimental setup The study was carried out at the Kalimok station, NE Bulgaria (44°010 N, 26°260 E) between June 2005 and May 2006. In June 2005, we captured 52 adult great reed warblers in the reed beds surrounding the Kalimok station. The captured birds were placed in captivity and screened

The captive birds were inspected on average every tenth day throughout the study to monitor parasitemia, the visible fat reserves estimated on a nine-class scale (Kaiser, 1993), body mass (to the nearest 0.1 g), body temperature (with a digital thermometer inserted in the cloacae) and progress of moult of flight feathers (Ginn and Melville,

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1983). We collected blood samples in SET-buffer (about 20 ll blood in 500 ll buffer; 0.15 M NaCl, 0.05 M Tris, 0.001 M EDTA, pH 8.0) for molecular analyses and blood films were prepared on glass slides for microscopic examination. Juveniles were examined more frequently (every 3– 7 days) during the month following the inoculation. 2.4. Microscopic analyses Blood films were air-dried, fixed in absolute methanol, and stained with Giemsa. A minimum of 100 fields were examined both at low (250) and high (1000) magnification. In order to validate the qPCR method for estimating intensities of parasitemia, we selected three samples from each of 10 individuals (five individuals with GRW2 and five with GRW4) for extensive microscopic smear counts. Three persons (PZ, MI and DD) calculated the intensity of parasitemia from these smears according to Godfrey et al., 1987) by counting the number of parasites per 10,000 erythrocytes examined. In the statistical analyses, 0 counts of infection were given the score of 0.001% as this is the next order of magnitude below the detection limit (0.01%) when examining 10,000 erythrocytes. 2.5. Experimental infections For inoculations we obtained 0.2 ml of blood from the brachial vein of infected donor birds, mixed this with 0.8 ml of 3.7% solution of trisodium citrate dehydrate (C6N5Na3O72H2O) and immediately (within 5 min) injected 0.3 ml of the mix in the pectoral muscle of experimental juvenile birds. The first infection experiment was carried out on 21 September 2005 (Table 2) when blood from two adults, one carrying the lineage GRW2 (individTable 2 Details about the two infection experiments with Plasmodium ashfordi (lineage GRW2) and P. relictum (GRW4) Adult donors (intensity of parasitemia) Exp 1 (21 September) GRW2 #968 (0.29%)a,b

GRW4

#042 (0.012%)b

Exp 2 (3 December) GRW2 #043 (0.022%)

GRW4

a

#039 (0.0001%)

Juveniles (other lineages present/survived the winter or date of death) #221 (GRW4/27 October) #228 (GRW11/survived) #232 (SGS1/19 November) #424 (-/19 January) #428 (-/survived) #429 (-/23 January) #214 #227 #299 #413 #414 #426

(GRW11/11 February) (SGS1/survived) (-/survived)) (-/survived) (-/8 December) (SGS1/survived)

Also GRW4 at intensity of parasitemia of 0.0025%. Not sampled 21September. Intensity of parasitemia is the geometric mean from 31 August and 3 October. b

ual #968) and one GRW4 (#042), were inoculated to six juvenile birds (three with GRW2 and three with GRW4). A control bird was inoculated with the same amount of an uninfected blood and buffer mixture. The second experiment was carried out on 3 December 2005 (Table 2) when six uninfected juveniles were inoculated with infected blood from two other donors (#043 for GRW2 and #039 for GRW4, again in a 3 + 3 experimental birds design). In both experiments, the intensities of parasitemia in the donor birds were substantially higher for GRW2 than for GRW4 (Table 2). Microscopic inspections of blood smears showed that asexual (infectious) stages (trophozoites and meronts) dominated over sexual (not infectious) stages (gametocytes) in all four donors at the time for the inoculation experiments. 2.6. Molecular analyses of malaria parasites DNA was extracted using standard phenol/chloroform methods (Sambrook et al., 1989) and diluted to a concentration of 25 ng/ll for standard PCR and 1 ng/ll for quantification of parasitemia using real-time qPCR. We determined presence and absence of circulating parasites and identification of parasite lineages using a nested PCR-approach and direct sequencing as in Waldenstro¨m et al. (2004). Each bird was typed 2–4 times (from samples taken at different dates) by sequencing to confirm its molecular lineages (e.g. GRW2, GRW4, etc.). We used an Mx3000 real-time PCR instrument (Stratagene) with SYBR-green based detection and separate runs for P. relictum and P. ashfordi, with lineage specific primers for GRW2 (GRW2/8F 50 -CAAATTTTAACTGGTGT CTTATTAGCC, GRW2/9R 50 -AAAGCACCATCCG CTCCATAA-30 ) and GRW4 (GRW4/11F 50 -ATTAG CAGAACAAAGAAACTTAACA-30 , GRW4/11R 50 -CA TAGAATGAACATATAAACCAG-30 ). These primers target a region of 101 bp (GRW2) and 105 bp (GRW4) including primers. The fragments are located outside the region of the cytochrome b used for identification (Waldenstro¨m et al., 2004). We used an UDG/dUTP containing mix (Platinum SYBR Green qPCR SuperMix-UDG, Invitrogen) and pre-incubation at 50 °C at 2 min to avoid crosscontamination of PCR products between batches and included multiple negative controls in each experiment. Each reaction of a total of 25 ll included 5 ng of DNA, 3 mM (GRW1 and GRW4) or 5 mM (GRW2) MgCl2, 12.5 ll of SuperMix, 0.1 lM ROX, and 0.4 lM (GRW4) or 0.2 lM (GRW2) of each primer. The temperature profile, after the initial incubation at 50 °C at 2 min and 95 °C at 2 min, consisted of 42 cycles of 95 °C at 15 s, 55 °C at 30 s and 72 °C at 30 s. Each sample was run in duplicates and scored as average values. To produce standard curves we started with DNA samples of birds that had levels of parasitemia assessed from smears, and made a series of a six step 5 dilutions by adding DNA from an uninfected nestling great reed warbler in proportions 4:1. We discarded and reran experiments producing standard

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curves that were steeper than 3.8, as this is indicative of inefficient amplifications and errors in the estimated intensities of parasitemia. Typically, Ct-values (the first cycle number when there is a detectable signal above background) ranged between 23 and 37 in most experiments. Before calculating relative infection levels, the individual dissociation curves were manually inspected to confirm specific amplifications. For each individual, we used qPCR to analyze samples collected approximately every 5 day during the first month following the inoculation and every 10 day during the following months and those birds surviving until May 2006 were thus analyzed up to 27 times. 2.7. Statistical analyses We 10log transformed all parasitemia estimates before statistical analyses. Mean intensity of parasitemia values are reported as geometric means. For calculation of mean intensity of parasitemia, we only included days when parasites were recorded in the blood (non-zero values of intensity of parasitemia). To estimate the time for experimental birds to reach a substantial level of infection, we chose a level of 0.04% infected erythrocytes based on the observation that this was the mean intensity of parasitemia among donors for the lineage with the highest parasitemia (GRW2) throughout the experiment. When calculating mean intensity of parasitemia for the experimental infections, we only included days after parasitemia reached 0.04%. Individual repeatabilities of intensity of parasitemia were calculated for each lineage by ANOVA (Lessells and Boag, 1987). 3. Results 3.1. Intensity of parasitemia The intensity of parasitemia estimated from smears (mean from three independent counts) and qPCR (Fig. 1) was strongly and significantly correlated (r = 0.846, P < 0.001). Worth noting is that the correlation coefficients between the repeated smear counts undertaken by the three persons were similar in magnitude (r = 0.736–0.865) to when their mean values were compared with the qPCR estimates (Fig. 1). This suggests that qPCR is at least as accurate as smear counts for estimating intensities of parasitemia. The smear counts seemed on average to result in lower estimates of intensities of parasitemia. 3.2. Details of the experiment All six juveniles inoculated with P. ashfordi (GRW2) infected blood were confirmed by sequence analysis to develop the same infection (Fig. 2). One of the six juveniles inoculated with P. relictum (GRW4) infected blood died 5 days post infection before parasitemia became patent. The remaining five birds were confirmed by sequencing to develop GRW4 infections (Fig. 3). In addition, a GRW4

Fig. 1. The correlation between the intensity of parasitemia as estimated from smear counts and qPCR. Filled circles show data for GRW2 and open circles for GRW4. The dotted line is the expected 1:1 ratio. Because smear counts based on 10,000 erythrocytes cannot detect intensities of parasitemia <0.01%, zero-counts were given the value of 0.001 in the statistical analyzes.

infection was also confirmed in one of the juveniles (#221) included in the experiment with the lineage GRW2. As this bird was inoculated with blood from the adult #968 which by microscopy and PCR also was confirmed to carry simultaneous infections of GRW2 and GRW4, experimental transmission is the most likely explanation to the mixed infection. None of the other birds, including the control bird inoculated with uninfected blood, developed GRW2 or GRW4 infections. The facilities for keeping birds in captivity at Kalimok were not protected from mosquitoes, which are common at the study site. Since the target lineages of the experiment (GRW2 and GRW4) appear to have transmission in Africa only (Bensch et al., 2007), we reckoned that contact with local mosquitoes should not interfere with the experiment. The results from the present study support that the infection of birds with lineages GRW2 and GRW4 was only accomplished by inoculation of infected blood. However, the lineages SGS1 and GRW11 were apparently naturally transmitted with mosquitoes between the birds within the captive population (Table 2). From samples taken at the time of capture in August, all the juveniles were confirmed parasite negative by PCR, but three individuals developed SGS1 and two GRW11 infections during the time in captivity. Four of these mixed infections involved juveniles infected with GRW2 and one juvenile infected with GRW4. However, because the qPCR protocol cannot distinguish between GRW4 and SGS1/GRW11 and that the latter two lineages might be at a lower intensity of parasitemia, it is likely that we have overlooked some concomitant SGS1/GRW11 infections in the GRW4 experimental birds. Several of the experimentally infected birds died during the period between September 2005 and May 2006 (Table 2). The mortality did not differ significantly between birds infected with GRW2 (3 of 6), GRW4 (3 of 6) or among

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Fig. 2. Dynamics of parasitemia of Plasmodium ashfordi (lineage GRW2, red) and P. relictum (combined data for lineages GRW4/SGS1/GRW11, blue) in six juvenile great reed warblers experimentally inoculated with infected blood from the adult donor #968 on 21 September (a, d and e) and donor #043 on 3 December (b, c and f). The birds on the left panel (a, b, c) survived until the monitoring of parasitemia was terminated (shaded grey area) whereas the birds on the right panel died 35–70 days post infection. The intensity of parasitemia (10log scale) was measured with qPCR (see Section 2). Three digit numbers indicate individuals as listed in Table 2. The birds #214 and #227 showed non-zero values on day 0 as a result of uncontrolled transmission of SGS1 and GRW11. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

the adults (5 of 9). It appears however that there are multiple causes to the mortality. In addition to the uncontrolled and local transmission of the lineages SGS1 and GRW11 among the captured birds, analyses of blood smears confirmed presence of Isospora (synonym Atoxoplasma) infections in several of the birds. No adults but all juveniles except individuals #221 and #299 developed apparent Isospora infections. In some birds Isospora infections reached high intensity of parasitemia (up to 24% of leucocytes in bird #429 on the day of its death) and might have contributed to the observed mortality.

In the naturally infected adults, the infection of Plasmodium lineages GRW2, GRW4 and SGS1 did not show any evidence of increased parasitemia during the winter. The adult #968 showed an example of strong relapse infection of GRW2 already in the middle of July (maximum intensity of parasitemia 37.6%), but except for a smaller relapse infection in October (2.1%) the level of parasitemia was kept around 0.1% throughout the winter. Though some of the adults had days without detectable parasitemia both by microscopy and PCR, none of them cleared the infec-

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Fig. 3. Dynamics of parasitemia of Plasmodium relictum (combined data for the lineages GRW4/SGS1/GRW11) in juvenile great reed warblers experimentally inoculated with infected blood from (a) donor #042 on 21 September and (b) donor #039 on 3 December. Three digit numbers indicate individuals as listed in Table 2. Two birds (#424 and #429) died before the monitoring of parasitemia was terminated (shaded area).

tions during this experiment, and thus kept the infections for up to 300 days. 3.3. Differences in intensity of parasitemia between individual hosts and parasite lineages Combining adults and experimental birds, GRW2 showed higher mean and maximum intensity of parasitemia than both SGS1 and GRW4 (Table 3; GRW2 vs. SGS1, mean t12 = 3.65, P = 0.003, max, t12 = 1.94, P = 0.08; GRW2 vs. GRW4, mean, t18 = 2.23, P = 0.039, max, t18 = 2.09, P = 0.05). No difference in mean or maximum parasitemia was observed between SGS1 and GRW4 (mean parasitemia, t16 = 1.63, P = 0.12, max parasitemia, t16 = 0.84, P = 0.40). The lineage GRW4 reached significantly higher mean (t9 = 2.99, P = 0.015) and maxi-

mum (t10 = 2.31, P = 0.044) parasitemia in experimental juveniles than in naturally infected adults. The highest GRW2 parasitemia (85% estimated from smears, 65% by qPCR) was seen in a juvenile (experimental) bird, but there was no statistical difference compared with adults (mean parasitemia, t6 = 0.86, P = 0.42; max parasitemia, t6 = 0.02, P = 0.98). Following experimental infections, GRW4 was detected in the blood on average 12.5 days before the first detection of GRW2 (Table 2). A parasitemia of 0.04% was reached between 16 and 20 days post infection for GRW4, significantly earlier than the 33–101 days observed for GRW2 (Table 3). Although the intensity of parasitemia is changing over time, the individuals seemed to keep the variation around an individual specific level. This conclusion was based on

Table 3 Mean and maximum intensity of parasitemia of three Plasmodium spp. lineages in adult donor and experimental juvenile great reed warblers

Donors n Mean parasitemia in % (range) Maximum parasitemia % (range) Experimental juveniles n Time to first detection in days (range) Time to reach 0.04% parasitemia in days (range) Mean parasitemia in %

Maximum parasitemia in % (range)

GRW2 (A)

GRW4 (B)

SGS1 (C)

t-Test

2 0.046 (0.006–0.324) 1.179 (0.037–37.6)

6 0.0024 (0.0006–0.011) 0.0555 (0.005–0.360)

2 0.0008 (0.0007–0.0009) 0.0064 (0.0032–0.0132)

tA,B = 2.36, tB,C = 1.30, tA,B = 1.53, tB,C = 1.80,

6 25.1 (12–33) 49.0 (33–101) 0.0357a (0.0005–0.433)

6 12.6 (12–15) 17.6 (16–20) 0.0152a (0.0040–0.047)

4 —

tA,B = 3.35, P = 0.009

—

tA,B = 2.67, P = 0.025

0.0029 (0.0005–0.0074)

1.111 (0.023–65.47b)

0.209 (0.0268–1.296)

0.195 (0.025–1.227)

tA,B = 2.62, tA,C = 1.83, tB,C = 2.47, tA,B = 1.02, tA,C = 1.07, tB,C = 0.55,

For experimental juveniles, the number of days from inoculation to when parasitemia first reached the intensity of 0.04% are given a Including days after reaching a parasitemia of 0.04%. b Maximum intensity of parasitemia from smear analysis was 85%.

P = 0.05 P = 0.24 P = 0.17 P = 0.12

P = 0.55 P = 0.11 P = 0.043 P = 0.32 P = 0.31 P = 0.59

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Table 4 Repeatability of the intensity of parasitemia in great reed warblers infected with lineages of Plasmodium ashfordi (GRW2) and P. relictum (GRW4, SGS1) Parasite lineage

Repeatability

df

F

P

GRW2 GRW4 SGS1

0.534 0.472 0.165

7, 77 9, 151 5, 69

12.03 15.20 3.41

<0.0001 <0.0001 =0.008

repeatability analyses showing significant repeatabilities across individuals for all the three Plasmodium lineages ranging from R = 0.26 in SGS1 to R = 0.53 in GRW2 (Table 4). 3.4. Mixed infections Six of the individuals carrying P. ashfordi (GRW2) were simultaneously infected with P. relictum (GRW4, n = 2 or SGS1, n = 4). Once the lineage GRW2 had reached a peak of parasitemia, it consistently held higher parasitemia than the P. relictum lineages (Fig. 2) and the parasitemia of two species appeared to develop in parallel. For three of the birds with mixed infections, we had more than 5 simultaneous estimates of the parasitemia for both parasite species (Fig. 4) and in each of these cases there was a strong positive correlation (r = 0.390–0.773; P = 0.017–0.052). We then calculated the mean parasitemia for both species in the six birds showing simultaneous infections between GRW2 and GRW4/SGS1. In all cases, the parasitemia of GRW2 was higher by approximately one order of magnitude (Figs. 4 and 5). The levels of mean intensity of parasitemia of GRW2 and GRW4/SGS1 appeared to be positively correlated but the association was not significant (r = 0.63, P = 0.16). 3.5. Cost of infections Three birds infected with P. ashfordi (GRW2) died 35– 70 days post infection; the death occurred after approximately 10 days of marked increase of intensity of parasitemia (Fig. 2). The samples taken the same day or the day before death showed relatively high intensities of parasitemia (0.2%, 4.2% and 6.7%). For two of the birds, this was their highest score of intensity and for one bird the second highest recorded score during the experiment. Two birds infected with P. relictum (GRW4) died during this experiment (Fig. 3); the death occurred 119 (individual #424) and 124 (individual #429) days post infection. The death occurred approximately 10 days after decrease (0.0003% on the day it died) or increase of parasitemia (0.14% on the day it died). Body mass of all infected juvenile birds increased during this experiment. Peak body mass of the juvenile birds was recorded in late October or early November, when the body mass had increased between 40% and 79% relative the mass at capture in late August. Such increase of body

Fig. 4. The correlation between the intensity of parasitemia of Plasmodium ashfordi (lineage GRW2) and P. relictum (combined lineages for GRW4/SGS1) in two juveniles (a: #228; b: #227) and one adult great reed warbler (c: #968).

mass is expected as the birds are preparing for the autumn migration to tropical Africa. When comparing the birds in the first experiment, that were infected in September during the period when they were building up fat stores for migration, with the birds in the second experiment that were infected after reaching peak body mass, there was no difference in peak body mass or time to reach peak body mass. Detailed analyses are difficult due to small sample sizes. Moult (starting date and extent), fat levels and body temperature did not show any consistent differences between experimental groups or correlations with intensity of parasitemia (data not shown but can be obtained from the authors on request).

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Fig. 5. The relationship between mean intensity of parasitemia of Plasmodium ashfordi (lineage GRW2) and P. relictum (GRW4 grey circles, SGS1 black circles) in great reed warblers with mixed infections (r = 0.63, P = 0.16). Below the stippled line is the intensity of parasitemia higher in P. ashfordi than in P. relictum.

4. Discussion 4.1. Comparisons between qPCR and smear counts Overall, there was a good agreement between intensities of parasitemia estimated by qPCR and smear counts for both P. relictum and P. ashfordi. The three independent smear counts were not more closely correlated to each other than was their mean values to the qPCR estimates, supporting that parasitemia is measured at least as accurately with qPCR as with the traditional microscopic method. Why the smear counts on average resulted in lower estimates of parasitemia than qPCR is not clear, but a possibility is that young and developing parasites are frequently missed when doing smear counts. Smear counts are based on direct counting of infected erythrocytes and the error will hence increase as the parasite becomes rare relative uninfected erythrocytes. The qPCR method has an error of about ±1 cycle when determining the Ct-values, from which the parasitemia (in %) can be estimated if the sample is run with a calibrated standard curve. One cycle difference represents a factor of 2 around the estimate so if assuming that the true value of parasitemia is 10%, the observations for the qPCR estimates are expected to fall between 5% and 20%. At high intensities of parasitemia, smear counts are clearly expected to be more accurate than qPCR, however, at lower intensities when smear counts are based on one or a few infected cells seen per 10,000 erythrocytes, the qPCR is expected to be more accurate. The problem with doing smear counts at low intensities is illustrated by the seven samples in Fig. 1 for which none of the three persons detected any parasites. 4.2. Comparisons between malaria lineages We found several differences in life-history traits between the lineages of the studied species of malaria par-

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asites. The prepatent period was shorter, and the intensity of parasitemia at the peak and during chronic infection was lower in P. relictum (GRW4) than in P. ashfordi (GRW2). It is worth noting that the locally transmitted lineages SGS1 and GRW11 had dynamics of parasitemia more similar to GRW4 which agrees with them also being members of the P. relictum clade (Valki unas et al., 2007). In a recent study of blackbirds Turdus merula it was shown with qPCR that two Plasmodium lineages (named TM1 and TM2) differed in the intensity of parasitemia by 2–3 orders of magnitude (Bentz et al., 2006). Unfortunately, these parasites were not identified morphologically so it is not known to which subgenus they belong. The species P. relictum belongs to the subgenus Haemamoeba whereas P. ashfordi belongs to the Novyella (Valki unas, 2005). Malaria parasites belonging to these subgenera markedly differ in their development in avian hosts. Well investigated species of Haemamoeba, such as P. relictum, P. gallinaceum, P. cathemerium and others are characterized by rapid increase of primary parasitemia with well-pronounced peak of the parasitemia, following by rapid crisis and long-lasting low chronic parasitemia (Garnham, 1966; Valki unas, 2005), which is in accordance with the present study. Life cycles are unknown or poorly studied for the great majority of Novyella species, which are frequently considered to be relatively benign to their avian hosts. Well investigated Novyella species, such as P. vaughani and P. rouxi are characterized by slowly developing primary parasitemia without well-pronounced peak of parasitemia and long-lasting chronic parasitemia (Mohammed, 1958; Valki unas, 2005; Iezhova et al., 2005). From this point of view, extremely high experimental primary parasitemia (up to 85%) and high mortality of birds with experimental P. ashfordi infection is unexpected and a unique finding for species of Novyella; that warrants further investigation, including comparison of mono-infections in experimental birds and investigation of tissue studies. Although intensive sampling has been conducted, no cases of European transmission of GRW4 of P. relictum or GRW2 of P. ashfordi have been confirmed. Great reed warblers are therefore most likely obtaining these parasites while being in their Sub-Saharan winter quarters (Waldenstro¨m et al., 2002; Bensch et al., 2007). This interpretation is supported by the results from this study. Although uncontrolled Plasmodium spp. transmission occurred among the captive birds, it only involved the lineages SGS1 and GRW11, two lineages of P. relictum for which European transmission has previously been established (Bonneaud et al., 2006; Hellgren et al., 2007). Because of the winter transmission of GRW2 and GRW4, and that parasites should time the peak parasitemia when transmission chances are as highest (i.e. when competent vectors are present), we expected to observe relapse infections (increase in parasitemia) in adult birds during the winter. Although relapse infections were seen in some of the adult birds, these did not coincide with the winter season. It should

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be noted that the failure to find ‘‘winter relapse infections” in this experiment does not preclude that this happens when the birds are migrating to Africa. For the lineage GRW4 of P. relictum, we found that naive birds developed higher parasitemia than seen in the naturally infected adult birds during chronic infection, a pattern commonly recorded in avian malarial infection experiments (Atkinson et al., 2000; Woodworth et al., 2005; Iezhova et al., 2005). This pattern was however not statistically confirmed for the lineage GRW2 of P. ashfordi. The two naturally infected donors of GRW2 showed remarkably high levels of parasitemia in captivity (maximum 37.6%). Hence, the absence of the predicted difference between primary and chronic intensity of parasitemia for GRW2 is not due to low parasitemia in naive birds, but that the level of parasitemia in chronically infected great reed warblers for this malaria species sometimes is high. Our data show that development of P. ashfordi in great reed warblers is different from currently investigated species of Novyella (Mohammed, 1958; Valki unas, 2005; Iezhova et al., 2005), which, however, might be due to the effect of mixed infections and warrants further investigation. The lineage GRW2 had a significantly longer prepatent period than GRW4, it reached much higher peak of parasitemia in the infected birds than did GRW4 and the rate of decrease of its parasitemia was slower. From studies of rodent and human malaria it is known that the height of peak parasitemia and the parasitemia clearance time are useful indirect measures of parasite virulence (Mackinnon and Read, 2004) indicating that GRW2 is a more virulent parasite than GRW4 is for great reed warblers. It is probable that P. ashfordi (GRW2) is one of the most pathogenic species of the subgenus Novyella (Iezhova et al., 2005; Valki unas, 2005). In support of this, the mortality of the GRW2 infected birds appeared to coincide with rapidly increasing parasitemia. However, due to low sample sizes and mortality from other reasons, we could not establish that GRW2 infection results in higher mortality than GRW4 infection. Neither did the other measured correlates to fitness give any clear indications of difference in virulence between the lineages. We found no apparent differences in how body temperature, in birds shown to be reduced during peak parasitemia (Hayworth et al., 1987), was affected by the two parasites. Similarly, body mass and timing and speed of moult appeared not to be affected differently by the two parasites. It should be noted however that since the birds were provided with abundant and easily available food, the cost of infections in terms of food intake can have been much milder than in the wild (Valki unas, 2005). Interestingly, we found significant repeatabilities in the level of the intensity of parasitemia across individuals for each of the investigated lineages. This result suggests that individual birds keep the infections at individual distinct levels. Whether such levels are set by the hosts’ immune system (genetic or acquired), the growth rate (virulence)

of the parasite population, or both is however not known. One possibility is that the level of parasitemia is reflecting the individual’s tolerance against the infection; those hosts with higher chronic parasitemia are more tolerant against the parasite as seen in several studies of humans (Boutlis et al., 2006). 4.3. Mixed infections Due to (i) the uncontrolled natural transmission of the P. relictum lineages SGS1 and GRW11 at our study site and (ii) the presence of mixed infection of the lineages GRW2 and GRW4 in one donor bird, some of the experimental juvenile birds were infected by more than one lineage. By combining information from microscopy (Valki unas et al., 2007), DNA sequencing and qPCR analyses, five of the six birds inoculated with GRW2 were also found to be infected by different P. relictum lineages. In contrast, we only confirmed one of six birds inoculated with GRW4 to be co-infected with SGS1 or GRW11. We do not think that this difference is resulting from GRW2 infected birds being more prone to pick up other lineages but rather, that the difference is more likely to be a methodological artifact. Our qPCR protocol cannot differentiate between GRW4 and SGS1/GRW11. With direct sequencing it should be possible to record mixed infections by looking for double base callings in the electropherograms as long as the lineages in simultaneous infections have similar intensity of parasitemia (Pe´rez-Tris and Bensch, 2005b). However, our data suggests that SGS1 and GRW11 occur at a much lower parasitemia than GRW4, and may therefore not be detected by sequencing samples for concomitantly infected birds (Valki unas et al., 2006a). The four different species of human malaria parasites often occur in mixed infections and sometimes all four can be found in a single hosts (Richie, 1988). When occurring together, their temporal dynamic appears to be negatively correlated (Bruce et al., 2000), e.g. when P. falciparum has high parasitemia, Plasmodium vivax usually has low and vice versa, indicating that they are differentially controlled by the human immune system. In contrast, we found evidence of a positive correlation between the intensity of parasitemia of the lineages in mixed infections in great reed warblers, both when looking within and between individuals. Such a pattern of parallel change of parasitemia in different species of parasites suggests that the host is controlling the infections indiscriminately, e.g. by nitric oxide production, rather than through a mechanism that relies on genotype-specific immunity (Bruce and Day, 2002; Boutlis et al., 2006). However, previous studies of naturally malaria infected great reed warblers suggest that the MHC (Major Histocompatibility Complex) genotype of the bird is important for the outcome of the GRW2 infections, also pointing towards the actions of more parasite specific immune responses (Westerdahl et al., 2005).

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4.4. Conclusion The two studied species of avian malaria parasites are distinctly different in several life-history traits, characteristics that should have consequences for the interactions with their hosts. Moreover, different individual hosts seem to respond differently to the same malaria species. The remarkably high diversity of avian malaria parasites (Pe´rez-Tris et al., 2007) and, consequently, the even higher number of potential parasite–host species interactions, suggests that the selective force by the malaria infections on bird populations will be both complex and important to understand when studying population dynamics and fitness of phenotypes. Though most studies of avian malaria parasites hitherto have focused on prevalence data among chronically infected individuals, we hope that the present study will stimulate to more detailed studies of the biology of the parasites. An important parameter to establish is the fitness consequences associated with primary infections, which are most dangerous for avian hosts (Valki unas, 2005) and might be easiest studied under controlled experimental conditions. The experience from this first study shows that serious precautions must be taken to avoid mixed infections in wild captured donors and uncontrolled transmission by mosquitoes. If possible, experiments using competent vectors are preferred since the first stage in natural infections involves sporozoites to which the immune system may respond differently than to already established merozoites. It is important to stress that accurate measures of fitness can only be obtained in natural free ranging populations. To understand the consequences of malaria and other parasitic infections in wildlife, it will be instrumental to combine experimental research and field studies. Acknowledgments The work was supported by grants from the Swedish Research Council (VR), the Swedish Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS), Carl Tryggers Stiftelse, Schybergs stiftelse, the Royal Swedish Academy of Sciences and Crafoordska Stiftelsen. The partial support of the Lithuanian State Science and Studies Foundation to G.V. is gratefully acknowledged. The experiment was carried out with the permission form the Bulgarian Ministry of Environment and Waters (#28/02.06.2005). We thank Dimitar Dimitrov (DD) for help with smear counts and Olof Hellgren for constructive comments on the manuscript. References Atkinson, C.T., Dusek, R.J., Woods, K.L., Iko, W.M., 2000. Pathogenicity of avian malaria in experimentally infected Hawaii amakihi. Journal of Wildlife Diseases 36, 197–204. Beadell, J.S., Ishtiaq, F., Covas, R., Melo, M., Warren, B.H., Atkinson, C.T., Bensch, S., Graves, G.R., Jhala, Y.V., Peirce, M.A., Rahmani, A.R., Fonseca, D.M., Fleischer, R.C., 2006. Global phylogeographic

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