Avian hemosporidian parasites from northern California oak woodland and chaparral habitats

During spring-summer 2003-2004, the avian community was surveyed for hemosporidian parasites in an oak (Quercus spp.) and madrone (Arbutus spp.) woodland bordering grassland and chaparral habitats at a site in northern California, a geographic location and in habitat types not previously sampled for these parasites. Of 324 birds from 46 species (21 families) sampled (including four species not previously examined for hemosporidians), 126 (39%) were infected with parasites identified as species of one or more of the genera Plasmodium (3% of birds sampled), Haemoproteus (30%), and Leucocytozoon (11%). Species of parasite were identified by morphology in stained blood smears and were consistent with one species of Plasmodium, 11 species of Haemoproteus, and four species of Leucocytozoon. We document the presence of one of the parasite genera in seven new host species and discovered 12 new parasite species-host species associations. Hatching-year birds were found infected with parasites of all three genera. Prevalence of parasites for each genus differed significantly for the entire sample, and prevalence of parasites for the most common genus, Haemoproteus, differed significantly among bird families. Among families with substantial sample sizes, the Vireonidae (63%) and Emberizidae (70%) were most often infected with Haemoproteus spp. No evidence for parasite between-genus interaction, either positive or negative, was found. Overall prevalence of hemosporidians at the northern California sites and predominance of Haemoproteus spp. was similar to that reported in most other surveys for the USA, Canada, and the Caribbean islands.     

Paucity of hematozoa in Colombian birds

Sixty-four birds of 43 species were caught at six localities in Colombia during the dry season in March 1998 and investigated for hematozoa by microscopic examination of stained blood films. Haemoproteus coatneyi, Plasmodium vaughani, Leucocytozoon sp., and microfilariae were identified. The overall prevalence of infection was 8%. Prevalences of infection for Haemoproteus spp., Plasmodium spp., Leucocytozoon spp., and microfilariae were 3%, 2%, 2%, and 3%, respectively. All hemosporidian infections encountered were of low intensity (< 1% of infected erythrocytes). The low prevalences and intensities of hemosporidian parasites in this study are in accord with other records from the Neotropics.     

Blood parasites of birds in Cameroon

Birds from south-central Cameroon, western Africa, were surveyed for blood parasites from August to October 1986. Of 331 birds examined, representing 65 species of 15 families and 6 orders (mostly passerines), 55 (17%) were found to be infected with 1 or more genera of hemotropic parasites. These included: Haemoproteus spp. (11% prevalence), Leucocytozoon spp. (3%), Plasmodium spp. (2%), Trypanosoma spp. (1%), and microfilariae of filariid nematodes (1%). Several new host-parasite associations were identified.     

Further observations on the blood parasites of birds in Uganda

Birds from three National Parks (Bwindi Impenetrable, Kibale, and Queen Elizabeth) in western Uganda were surveyed during the dry season in July 2003 and investigated for hematozoa by microscopic examination of stained blood films. Of 307 birds examined, representing 68 species of 15 families and four orders, 61.9% were found to be infected with blood parasites. Species of Haemoproteus (15.3% prevalence), Plasmodium (20.5%), Leucocytozoon (40.1%), Trypanosoma (11.4%), Hepatozoon (2.6%), Atoxoplasma (0.3%), and microfilariae (3.9%) were recorded. Except for Haemoproteus spp. infections, the overall prevalence of hematozoa belonging to all genera was significantly higher in this study than was previously reported in Uganda. Thirty-six species of birds were examined for blood parasites for the first time and 112 new host-parasite associations were identified. Eighty-one were at the generic and 31 at the specific level of the hematozoa. Hepatozoon and Atoxoplasma spp. were detected for the first time in Uganda.     

Presence of Plasmodium and Haemoproteus in breeding prothonotary warblers (Protonotaria citrea: Parulidae): temporal and spatial trends in infection prevalence

Prothonotary warbler (Protonotaria citrea) has shown a long-term decline in abundance in the United States. As a long-range migrant, these warblers are exposed to parasites in both tropical and temperate regions. The focus of this study was to use molecular techniques to examine the temporal prevalence patterns of heamosopridian parasites Plasmodium and Haemoproteus in breeding prothonotary warblers. The prevalence (presence or absence) of Plasmodium and Haemoproteus species was assayed using primer sets for the cytochrome b gene of the mitochondrial DNA. Blood samples were obtained from 187 adult prothonotary warblers collected at 3 central Virginia, U.S.A., breeding sites. The relationship between haemosporidian parasite infections and reproductive success also was examined. We found that 71% of captured prothonotary warblers were infected with haemosporidian parasites, specifically, with 36% prevalence for Haemoproteus spp. and 44% prevalence for Plasmodium spp., during the 2008 breeding season; for both parasites, prevalence increased throughout the season. We found significant variation in haemosporidian parasite prevalence across the breeding season that was strongly site specific. Conversely, we found no significant effects of haemosporidian parasite infections on the reproductive success of prothonotary warblers. This is in sharp contrast to recent reports suggesting considerable effects of these parasites on the reproductive success of wild birds.     

Nested cytochrome b polymerase chain reaction diagnostics underestimate mixed infections of avian blood haemosporidian parasites: microscopy is still essential

Numerous polymerase chain reaction (PCR)-based methods have been developed and used increasingly to screen vertebrate blood samples for the diagnosis of haemosporidian blood parasites (Sporozoa, Haemosporida), but a rigorous evaluation of the sensitivity of these methods for detecting mixed infections of different haemosporidian species belonging to the same and different genera and subgenera is lacking. This study links the information obtained by nested cytochrome b PCR and traditional microscopy in determining mixed haemosporidian infections in naturally infected birds. Samples from 83 individual passerine birds with single infections of Haemoproteus or Plasmodium spp., as determined by mitochondrial DNA amplification, also were investigated by microscopic examination of stained blood films. Thirty-six samples (43%) were found to harbor mixed Haemoproteus, or Plasmodium spp. infections, or both. Thus, the PCR assays alone underestimate the occurrence of mixed infections of haemosporidian parasites in naturally infected birds. To determine the true species composition of the haemosporidians in each individual host, PCR diagnostics need to be improved. Specific primers for Haemoproteus spp. and Plasmodium spp. should be developed. Ideally, a combination of the approaches of both microscopy and PCR-based methods is recommended for this purpose.     

Haemoproteus spp. and Leukocytozoon spp. in a captive raptor population

Raptors are commonly infected with two blood parasites of the family Haemoproteidae, Haemoproteus spp. and Leukocytozoon spp. To determine if age or length of time in captivity influence prevalence of Haemoproteus spp. and Leukocytozoon spp. infection in captive raptors, blood samples were collected from 55 birds from April 1999 to May 2000. Blood smears were examined for parasitemia and influence of age and length of time in captivity at the time of sample collection were compared. We found juvenile and adult birds were more likely to be infected with Leukocytozoon spp. than were nestlings (P = 0.006) and birds present for > 365 days were more likely to be infected with Haemoproteus spp. and/or Leukocytozoon spp. than were birds captive for < 365 days.     

Hematozoa in endemic subspecies of common kestrel in the Cape Verde Islands

We examined 130 Common Kestrel (Falco tinnunculus) representing two endemic subspecies and nine resident island populations on the Cape Verde archipelago between 1996 and 1999 to study diversity, prevalence, and intensity of hematozoa. Hematozoan diversity was very low; we detected only Plasmodium fallax, a species that is rarely found in Falconoformes, and, possibly, Haemoproteus brachiatus. Moreover, prevalence of Plasmodium fallax was low (1.5%) with a mean intensity of infection of 0.05 protozoa/10(-3) erythrocytes. Only one bird (0.8%) was infected with a gametocyte that was most likely Haemoproteus brachiatus; the intensity in this infected bird was 1.5 protozoa/10(-3) erythrocytes. A single parasite or two parasites were observed in blood smears in four additional birds, but identification to genus was not possible. This is the first record of blood parasites in birds on the Cape Verde Archipelago. The low prevalence of these parasites might be because of arid and less-favorable conditions for the pathogen's vectors. The sedentary nature and high level of isolation of the island kestrel populations are also factors that could decrease the probability of infection.     

Additional observations on blood parasites of birds in Costa Rica

Birds from the Area de Conservacion Guanacaste in northwestern Costa Rica were surveyed for blood parasites in June 2001 and December 2001-January 2002. Of 354 birds examined, representing 141 species of 35 families and 15 orders, 44 (12.4%) were infected with blood parasites. Species of Haemoproteus (4.8% prevalence), Plasmodium (0.6%), Leucocytozoon (0.3%), Trypanosoma (2.0%), and microfilariae (7.6%) were recorded. Twelve species of birds in this survey were examined for blood parasites for the first time. Several new host-parasite associations were identified.     

A comparison of the blood parasites in three subspecies of the yellow wagtail Motacilla flava

One-hundred and eighty yellow wagtails Motacilla flava belonging to 3 subspecies (Motacilla flava feldegg, Motacilla flava flava, Motacilla flava thunbergi) were caught during the spring migration in south Kazakhstan and investigated by microscopic examination of stained blood smears. Haemoproteus anthi, Haemoproteus motacillae, Leucocytozoon fringillinarum, Leucocytozoon majoris, Plasmodium relictum, Plasmodium polare, Atoxoplasma sp., Trypanosoma sp., and microfilariae were identified. The overall prevalence of infection was 47.8%. Prevalences of Haemoproteus spp. (27.2%), Plasmodium spp. (25.0%), Leucocytozoon spp. (8.9%), Atoxoplasma spp. (4.4%), Trypanosoma spp. (1.1%), and microfilariae (0.6%) were recorded. No differences were discernible in parasite fauna or intensities of infection between males and females or between different subspecies. However, prevalence of infection of Haemoproteus, Leucocytozoon, and Plasmodium spp. was different in different host subspecies. These differences can be explained by differences in geographical location of breeding areas of these birds.     

A comparative analysis of microscopy and PCR-based detection methods for blood parasites

We compared information obtained by both microscopy and nested mitochondrial cytochrome b PCR in determining prevalence of haemosporidian infections in naturally infected birds. Blood samples from 472 birds of 11 species belonging to 7 families and 4 orders were collected in Europe, Africa and North America. Skilled investigators investigated them using the PCR-based screening and microscopic examination of stained blood films. The overall prevalence of haemosporidian infections, which was determined combining results of both these methods, was 60%. Both methods slightly underestimated the overall prevalence of infection, which was 54.2% after the PCR diagnostics and 53.6% after microscopic examination. Importantly, both these tools showed the same trends of prevalence of Haemoproteus spp. (21% by PCR and 22% by microscopy), Plasmodium spp. (17% and 22%) and Leucocytozoon spp. (30% and 25%) in the same sample, testifying that microscopy is a reliable tool in determining patterns of distribution of blood haemosporidian parasites in naturally infected birds. We encourage using optical microscopy in studies of blood parasites in parallel to the now widely employed molecular methods. Microscopy is relatively inexpensive and provides valuable information about directions how molecular methods can be further improved and most effectively applied, especially in the field studies of parasites. Importantly, blood films, which are used for microscopic examination, should be of good quality; they should be examined properly by skilled investigators. In spite of relatively long duration of microscopy of each sample, such examination provides opportunities for simultaneous determination and verification of taxonomically different parasites. Presently, different PCR protocols must be used for the detection of parasites belonging to different genera; this is expensive and time-consuming.     

Blood parasites of some birds from Ghana

A total of 135 birds of 26 species in 13 families was examined for blood parasites; 43 birds (31.9%) of 13 species were infected; species of the Ploceidae were the most heavily infected. Species of Haemoproteus occurred most commonly 29 birds) while Leucocytozoon and Plasmodium species were virtually absent. There was no significant difference in the prevalence of hematozoa in birds from the mature rainforest and those in a savannah-urban setting.     

The sensitivity of microscopy and PCR-based detection methods affecting estimates of prevalence of blood parasites in birds

Inferences about the evolution of host-parasitic relationships are often made based on the prevalence of avian malaria, which is usually estimated in a large sample of birds using either microscopic or molecular screening of blood samples. However, different techniques often have variable accuracy; thus, screening methodology can raise issues about statistical bias if method sensitivity varies systematically across parasites or hosts. To examine this possibility, published information was collected on the prevalence of species in 4 genera of avian blood parasites ( Plasmodium, Haemoproteus, Leucocytozoon, and Trypanosoma) from various sources that used different tools. The data were tested to determine if the application of different methods provided different estimates for the same hosts. In these comparisons between the main methodologies, the PCR-based molecular methods were generally found to provide higher estimates for Plasmodium spp. prevalence than microscopic tools, while there was no significant tendency for such a trend in species of Haemoproteus and Leucocytozoon. When analyzing intraspecific variance of prevalence within molecular studies, some studies provided consistently higher estimates for Haemoproteus spp. prevalence than others, indicating that differences between studies can affect detected estimates. Within microscopic studies, surveys that examined more microscopic fields were more likely to report higher prevalence for Plasmodium spp. than those relying on fewer microscopic fields. Consequently, studies making comparisons across parasite genera and/or host species from different sources need to consider several types of bias originating from variation in method sensitivity.     

Prevalence of avian haemosporidian parasites and their host fidelity in the central Philippine islands

We examined the prevalence and host fidelity of avian haemosporidian parasites belonging to the genera Haemoproteus, Leucocytozoon and Plasmodium in the central Philippine islands by sampling 23 bird families (42 species). Using species-specific PCR assays of the mitochondrial cytochrome b gene (471base pairs, bp), we detected infections in 91 of the 215 screened individuals (42%). We also discriminated between single and multiple infections. Thirty-one infected individuals harbored a single Haemoproteus lineage (14%), 18 a single Leucocytozoon lineage (8%) and 12 a single Plasmodium lineage (6%). Of the 215 screened birds, 30 (14%) presented different types of multiple infections. Intrageneric mixed infections were generally more common (18 Haemoproteus/Haemoproteus, 3 Leucocytozoon/Leucocytozoon, and 1 Plasmodium/Plasmodium) than intergeneric mixed infections (7 Haemoproteus/Leucocytozoon and 1 Haemoproteus/Leucocytozoon/Plasmodium). We recovered 81 unique haemosporidian mitochondrial haplotypes. These clustered in three strongly supported monophyletic clades that correspond to the three haemosporidian genera. Related lineages of Haemoproteus and Leucocytozoon were more likely to derive from the same host family than predicted by chance; however, this was not the case for Plasmodium. These results indicate that switches between host families are more likely to occur in Plasmodium. We conclude that Haemoproteus has undergone a recent diversification across well-supported host-family specific clades, while Leucocytozoon shows a longer association with its host(s). This study supports previous evidence of a higher prevalence and stronger host-family specificity of Haemoproteus and Leucocytozoon compared to Plasmodium.     

High prevalence and diversity of blood parasites of passerine birds in Southern Turkmenistan

Thirty nine specimens of passerine birds belonging to 19 species and eight families were investigated by blood smear technique in four localities of Southern Turkmenistan in 3-18 August 1991. The overall prevalence of infection was 59%. Protists from the orders Haemosporida (genera Haemoproteus, Plasmodium, Leucocytozoon), Kinetoplastida (Trypanosoma), and Adeleida (Hepatozoon), as well as Microfilaria were found. Haemoproteids (the prevalence of infection is 44%), leucocytozoids (23%), malarial parasites (13%) and trypanosomes (13%) were most frequently recorded. Only low chronic infections (< 1% of infected cells for the great majority of intracellular parasites, and a few trypanosomes and Microfilaria in each blood smear) were seen. Haemoproteus belopolskyi, H. balmorali, H. dolniki, H. magnus, H. minutus, H. fringillae, H. majoris, Leucocytozoon dubreuili, and Trypanosoma avium were recorded for the first time in Turkmenistan. The former five above-mentioned species of haemoproteids are new records for the fauna of Middle Asia. Gametocytes of leucocytozoids in fusiform host cells were found for the first time in passerine birds in the Holarctic. The host is Parus bokharensis. Due to the wide distribution and the opportunity to collect a large parasitological material using harmless for hosts methods, bird haemosporidian parasites can be used as convenient models for ecological and evolutionary biology studies in South Turkmenistan. The heavily infected Orphean Warbler Sylvia hortensis is an especially convenient host for such purposes.     

Prevalence and diversity patterns of avian blood parasites in degraded African rainforest habitats

Land use changes including deforestation, road construction and agricultural encroachments have been linked to the increased prevalence of several infectious diseases. In order to better understand how deforestation affects the prevalence of vector-borne infectious diseases in wildlife, nine paired sites were sampled (disturbed vs. undisturbed habitats) in Southern Cameroon. We studied the diversity, prevalence and distribution of avian malaria parasites ( Plasmodium spp.) and other related haemosporidians (species of Haemoproteus and Leucocytozoon ) from these sites in two widespread species of African rainforest birds, the yellow-whiskered greenbul ( Andropadus latirostris , Pycnonotidae) and the olive sunbird ( Cyanomitra olivacea , Nectariniidae). Twenty-six mitochondrial cytochrome b lineages were identified: 20 Plasmodium lineages and 6 Haemoproteus lineages. These lineages showed no geographic specificity, nor significant differences in lineage diversity between habitat types. However, we found that the prevalence of Leucocytozoon and Haemoproteus infections were significantly higher in undisturbed than in deforested habitats ( Leucocytozoon spp. 50.3% vs. 35.8%, Haemoproteus spp. 16.3% vs. 10.8%). We also found higher prevalence for all haemosporidian parasites in C. olivacea than in A. latirostris species (70.2% vs. 58.2%). Interestingly, we found one morphospecies of Plasmodium in C. olivacea , as represented by a clade of related lineages, showed increased prevalence at disturbed sites, while another showed a decrease, testifying to different patterns of transmission, even among closely related lineages of avian malaria, in relation to deforestation. Our work demonstrates that anthropogenic habitat change can affect host–parasite systems and result in opposing trends in prevalence of haemosporidian parasites in wild bird populations.     

Prevalence and differential host-specificity of two avian blood parasite genera in the Australo-Papuan region

The degree to which widespread avian blood parasites in the genera Plasmodium and Haemoproteus pose a threat to novel hosts depends in part on the degree to which they are constrained to a particular host or host family. We examined the host distribution and host-specificity of these parasites in birds from two relatively understudied and isolated locations: Australia and Papua New Guinea. Using polymerase chain reaction (PCR), we detected infection in 69 of 105 species, representing 44% of individuals surveyed (n = 428). Across host families, prevalence of Haemoproteus ranged from 13% (Acanthizidae) to 56% (Petroicidae) while prevalence of Plasmodium ranged from 3% (Petroicidae) to 47% (Ptilonorhynchidae). We recovered 78 unique mitochondrial lineages from 155 sequences. Related lineages of Haemoproteus were more likely to derive from the same host family than predicted by chance at shallow (average LogDet genetic distance = 0, n = 12, P = 0.001) and greater depths (average distance = 0.014, n = 11, P < 0.001) within the parasite phylogeny. Within two major Haemoproteus subclades identified in a maximum likelihood phylogeny, host-specificity was evident up to parasite genetic distances of 0.029 and 0.007 based on logistic regression. We found no significant host relationship among lineages of Plasmodium by any method of analysis. These results support previous evidence of strong host-family specificity in Haemoproteus and suggest that lineages of Plasmodium are more likely to form evolutionarily-stable associations with novel hosts.     

Haematozoa infections in a Great Tit Parus major population in Central Portugal: relationships with breeding effort and health

Blood parasites may act as modulators of their hosts' ecology, life histories and fitness. We studied the prevalence of Plasmodium sp., Haemoproteus sp. and Leucocytozoon sp. and their effects on morphological, biochemical and haematological variables and on breeding effort of Great Tits Parus major . Total prevalence (percentage of individuals infected by any parasite) ranged from 7.7% to 61.1%. There was an overall positive association in prevalence between the three haematozoan parasites. No effect of sex or age on infection status was observed. Negative impacts of infection on physiological condition depended largely on year and/or season and included effects on body condition index, plasma protein and haemoglobin index. There were also indications that parasite infection increased immune response and stress levels and activated antioxidant defence mechanisms. Males with higher fledging success had a higher probability of Haemoproteus infection, and females laying heavier eggs had a higher probability of Plasmodium infection. However, clutch size was negatively associated with the probability of infection by Leucocytozoon and Haemoproteus . Surprisingly, males raising second broods had a lower prevalence of both Haemoproteus and Leucocytozoon . Only 5.7% of first-brood nestlings were infected, but those in infected nestboxes had a lower heterophil/lymphocyte ratio. This study confirms the pathogenicity of blood parasites to the host by demonstrating negative effects of infection on both physiology and breeding performance.     

Hematozoa of raptors from southern New Jersey and adjacent areas

Blood smears from 259 birds of 12 species, representing four families of raptors, from New Jersey, Pennsylvania, Delaware, and Virginia were examined for blood parasites. Infected birds constituted 59.1% of the total. Birds were infected with one or more of the following genera of protozoa: Leucocytozoon (43.2%); Haemoproteus (21.6%); Plasmodium (1.2%); and Trypanosoma (1.2%). Blood culture of 142 raptors of 11 species for Trypanosoma revealed a prevalence of 41.5%. Plasmodium circumflexum is reported for the first time in Accipiter striatus, and Trypanosoma sp. in Buteo jamaicensis.     

Molecular characterization of malarial parasites in captive passerine birds

Seven of 28 passerine birds that died in captivity were positive for malarial parasites by polymerase chain reaction targeting the mitochondrial cytochrome b (cytB) and apicoplast ribosomal RNA (rRNA) genes. Each bird was infected with a single parasite lineage having a unique genotype. Apicoplast rRNA sequences were present both in Haemoproteus spp. and Plasmodium spp. and had typically high adenosine + thymidine content. Phylogenies for cytB and apicoplast rRNA sequences were largely congruent and supported previous studies that suggest that Plasmodium-Haemoproteus spp. underwent synchronous speciation with their avian hosts, interrupted by sporadic episodes of host switching. Apicoplast phylogeny further indicated that Haemoproteus spp. are ancestral to Plasmodium spp. All the 7 infected passerine birds had histologic lesions of malaria, and malarial parasites may have contributed to the death of at least 4 animals. These findings provide new genetic data on passerine hematozoa, including initial sequences of apicoplast DNA, and emphasize the relevance of parasite prevalence, evolutionary relationships, and host switching to modern management and husbandry practices of captive birds.