Parasitology Research (2024) 123:252

https://doi.org/10.1007/s00436-024-08260-8

RESEARCH

Avian haemosporidians of the genera Plasmodium and Haemoproteus

from resident and Neotropical migrant birds in Colombia
Maria Camila Hernández‑Ospina1 · Diego Chitan‑Guerrero1 · Johnathan Alvarez‑Londoño1,2 ·
Mauricio Bohada‑Murillo2,3 · Estefani T. Martínez‑Sánchez1,4 · Fredy A. Rivera‑Páez1 ·
Gabriel J. Castaño‑Villa5

Received: 29 March 2024 / Accepted: 31 May 2024

© The Author(s) 2024

Abstract
Avian haemosporidians of the genera Plasmodium and Haemoproteus are a group of widely distributed blood parasites
that can negatively affect the fitness of their hosts. Colombia contains the greatest diversity of birds on the planet, but
knowledge about the associations between haemosporidian and its avifauna is scarce and fragmented. We collected blood
samples from 255 birds (203 residents and 52 neotropical migrants) belonging to 27 families and 108 species. The study
was conducted in six localities in the inter-Andean valleys of the Cauca and Magdalena rivers. Parasites of the genera
Plasmodium and Haemoproteus were identified in the samples by morphological and molecular analysis of a fragment
of the mitochondrial gene cyt b. Among the samples, 9.3% (n = 24) were positive for Plasmodium or Haemoproteus.
Co-infection with Plasmodium and Haemoproteus was found in Red-eyed Vireo. Seventeen haemosporidian lineages
were identified, five of which were reported for the first time in resident birds (Common Ground Dove, Checker-throated
Stipplethroat, Tropical Kingbird, Pale-breasted Thrush, and Ruddy-breasted Seedeater) and one in the Summer Tanager
(neotropical migrant). The research results confirm the wide diversity of haemosporidian present in tropical lowlands
and the possible role of neotropical migratory birds in dissemination on haemosporidian along their migratory routes.

Keywords Avian malaria · Blood · Farmlands · Hummingbirds · Secondary forest · Thraupidae

Introduction (Ricklefs and Fallon 2002; Valkiūnas 2005; Lacorte et al.

2013; Ellis et al. 2020). Birds are susceptible to infection by
Haemosporidian (Apicomplexa: Haemosporida) are a group mosquito-borne Plasmodium genera (Culicidae) and Haemo-
of hemoparasites that infect a wide range of vertebrate hosts proteus (subgenera Parahaemoproteus and Haemoproteus),
globally, including reptiles, amphibians, mammals, and birds transmitted by Ceratopogonidae and Hippoboscidae flies,

Section Editor: Nawal Hijjawi

* Fredy A. Rivera‑Páez 3
Grupo de Investigación en Ecosistemas Tropicales,
[email protected] Departamento de Ciencias Biológicas, Facultad de Ciencias
Exactas y Naturales, Universidad de Caldas, Calle 65
1
Grupo de Investigación en Genética, Biodiversidad y Manejo No. 26‑10 A.A 275, Manizales, Caldas, Colombia
de Ecosistemas—GEBIOME, Departamento de Ciencias 4
Biológicas, Facultad de Ciencias Exactas y Naturales, Doctorado en Ciencias‑Biología, Facultad de Ciencias
Universidad de Caldas, Calle 65 No. 26‑10 A.A 275, Exactas y Naturales, Universidad de Caldas, Calle 65
Manizales, Caldas, Colombia No. 26‑10 A.A 275, Manizales, Caldas, Colombia
5
2
Maestría en Ciencias Biológicas, Facultad de Ciencias Grupo de Investigación en Genética, Biodiversidad y
Exactas y Naturales, Universidad de Caldas, Calle 65 Manejo de Ecosistemas—GEBIOME, Departamento
No. 26‑10 A.A 275, Manizales, Caldas, Colombia de Desarrollo Rural y Recursos Naturales, Facultad de
Ciencias Agropecuarias, Universidad de Caldas, Carrera 35
No. 62‑160 A.A 275, Manizales, Caldas, Colombia

Vol.:(0123456789)
252 Page 2 of 13 Parasitology Research (2024) 123:252

respectively (Valkiūnas 2005; Santiago-Alarcon et al. 2012; Materials and methods

Toscani Field et al. 2018). Haemosporidians of the genera
Plasmodium and Haemoproteus have complex life cycles Study area
with sexual reproductive stages in arthropod vectors and
asexual reproduction in their avian hosts (Valkiūnas 2005). The study was conducted in six localities within the depart-
However, asexual reproduction may be disrupted in a non- ment of Caldas, Colombia (Latitude: 4.07194, Longi-
competent avian host, and the mature forms of the para- tude: − 75.95722). The localities are situated in the inter-
site (gametocytes), which are infectious to the vector, are Andean valleys of the Cauca (Municipality of Palestina) and
not generated (i.e., abortive infection) (Valkiūnas 2005; Magdalena rivers (Municipalities of La Dorada, Samaná
Valkiūnas and Iezhova 2017). Infections caused by hae- and Victoria) (Fig. 1). The six localities are found within
mosporidian can have adverse effects on birds’ reproductive an elevation range of 126 and 1050 m a.s.l. They experi-
success and survival, due to the damage caused in tissues ence a bimodal rainfall pattern (average annual precipita-
and organs such as the spleen, liver, and brain, and conse- tion ranging from 2000 to 4000 mm). The highest rainfall
quently, affect their populations (Palinauskas et al. 2008, occurs between March and June and between September
2015; Risely et al. 2018). and December. The average temperature ranges between
Over the past few decades, the implementation of 20 and 27 °C (Martínez-Sánchez et al. 2018). The sampled
morphological and molecular analyses has allowed the localities in the inter-Andean valley of the Magdalena River
identification of a wide diversity of avian haemosporid- are farms dedicated to the production of avocado (Persea
ian morphospecies and lineages in the Neotropical region americana Mill.) or cocoa (Theobroma cacao L.), or buffalo
(Bensch et al. 2009; González et al. 2015; Fecchio et al. rearing (Bubalus bubalis L.) and two secondary forests. The
2018; 2019; Ellis et al. 2020). In the neotropics, there have two locations that were sampled in the inter-Andean valley
been reports of resident and migratory birds infected by the of the Cauca River correspond to two farms that produce
genera Plasmodium and Haemoproteus, which could play a cocoa (T. cacao) or citrus (Citrus spp.) production (Table 1).
significant role in the dispersal of haemosporidian through-
out the Americas (Valkiūnas 2005; DeBrock et al. 2021;
Lotta-Arévalo et al. 2023). In Colombia, 140 neotropical
Collection and processing of bird blood samples
migratory bird species are recorded during the spring and
Wild birds were captured from September 2021 to February
fall migrations (Echeverry-Galvis et al. 2022). Infection
2022 using six mist nets (12 × 2.5 m × 36 mm) at each loca-
by Plasmodium (Haemamoeba) cathemerium, Haemopro-
tion for five consecutive days, with a total sampling intensity
teus (Parahaemoproteus) vireonis, or Haemoproteus (Para-
of 1980 h/net. The nets were operated between 06:00 and
haemoproteus) coatneyi has been detected in birds of the
17:00 h. Captured birds were marked with a small cut on the
Vireonidae, Turdidae, or Cardinalidae families (González
first rectrix to avoid recounts and were subsequently released
et al. 2015; Pulgarín-R et al. 2019). In Colombia, there is
at the location where they were captured (Martínez-Sánchez
a limited and fragmented knowledge about haemosporid-
et al. 2018). The bird taxonomy followed the nomenclature of
ians and their interaction with wild birds, with studies
Remsen et al. (2023). Residency status (neotropical migratory
mainly conducted in localities of the Caribbean, Orinoco,
or resident) was determined according to Echeverry-Galvis
and High Andean regions (González et al. 2015; Alvarez-
et al. (2022). A blood sample (approximately 20 to 50 μl) was
Londoño et al. 2022; Lotta-Arévalo et al. 2023). This lack
obtained from each captured bird through brachial vein punc-
of information hinders the understanding of birds’ role in
ture using 25G and 27G gauge needles (Busi et al. 2020). To
the epidemiology of Plasmodium and Haemoproteus.
obtain the hummingbird blood sample, a small cut was made
In this context, and considering that the regions encom-
near the fingernail (Owen 2011). The samples were used to
passing the inter-Andean valleys of the Cauca and Magda-
create blood smears (at least two per bird), fixed in absolute
lena rivers are include in the migration routes of at least 83
methanol for 5 min, and stained with 5% GIEMSA solution,
species of neotropical migratory birds (Fierro-Calderón and
pH 7.2 for 45 min (Alvarez-Londoño et al. 2022). The remain-
Eusse 2010; Naranjo et al. 2012), this region could serve as
ing blood was also deposited on FTA cards (Flinders Technol-
a “transmission zone” where resident-migratory bird spe-
ogy Associates) for subsequent molecular analysis.
cies and haemosporidians of South and North American
origin converge (Moens and Pérez-Tris 2016; Fecchio et al.
2018). This research aimed to determine the diversity and
Morphological analysis
prevalence of Plasmodium and Haemoproteus in neotropi-
For morphological identification of haemosporidians,
cal resident and migratory wild birds at six locations in the
blood smears were analyzed using a light microscope,
inter-Andean valleys of the Cauca and Magdalena rivers.
Parasitology Research (2024) 123:252 Page 3 of 13 252

Fig. 1  Study area. A Map of Colombia depicting department of Cal- River Valley, (3) Corregimiento Berlín, (4) Finca El Edén, (5) Haci-
das (black). B Sampled localities in Cauca River Valley, (1) Granja enda Alcaparrosa, and (6) Charca de Guarinocito
Luker and (2) Granja Montelindo; sampled localities in Magdalena

Table 1  Descriptions of the localities in the department of Caldas where birds were sampled, Colombia. The localities correspond to the num-
bers presented in Fig. 1

Municipality Locality Locality Latitude Longitude Elevation (m Habitat type

number above sea
level)

Palestina Granja Luker 1 5.0735 − 75.682 1050 Cocoa agroforestry systems (Theobroma cacao L.)
Granja Montelindo 2 5.0753 − 75.6730 1010 Monocrop (Citrus nobilis Lour.)
Samaná Corregimiento Berlín 3 5.6005 − 74.9473 791 Secondary forest
Victoria Finca El Edén 4 5.3348 − 74.9137 1000 Mixed-cropping (Persea americana M. and Theo-
broma cacao L.)
La Dorada Hacienda Alcaparrosa 5 5.5756 − 74.6683 126 Grazing pasture with weeds and dispersed tree
Charca de Guarinocito 6 5.3353 − 74.7232 208 Secondary forest

specifically an Olympus BX43 optical microscope, with Molecular analysis

40 × and 100 × objectives; approximately 100–150 fields
were visualized per objective (Valkiūnas 2005). The para- DNA extraction from blood samples stored on FTA cards
sites were identified using specialized taxonomic guides was performed using the Wizard Genomic DNA Purifica-
(Valkiūnas 2005; Valkiūnas and Iezhova 2018; 2022). tion kit (Promega Corporation, Madison, USA) following
Images of the parasites were captured using an Olympus the manufacturer’s instructions. To detect Plasmodium and
DP28 digital camera and edited with Olympus CellSens Haemoproteus DNA, nested Polymerase Chain Reaction
Standard v3.22.11 software. (PCR) testing was conducted on a fragment of a cytochrome
252 Page 4 of 13 Parasitology Research (2024) 123:252

b (cyt b) gene. The initial PCR utilized primers AE064/ between the two runs below 0.01 and graphically using
AE066 to amplify a 1109 bp fragment for all three gen- Tracer (Rambaut and Drummond 2007). A total of 25% of
era of haemosporidians (Plasmodium, Haemoproteus, and the trees were discarded as run periods, and the remaining
Leucocytozoon) (Pacheco et al. 2018). One microliter of the trees were used to create a consensus tree applying a 50%
initial PCR was utilized for the second PCR, utilizing the majority rule. The phylogeny was visualized using FigTree
HaemF/HaemR2 primers that amplify a 480 bp fragment v1.3.1 (Rambaut 2013). All obtained sequences were
for Plasmodium and Haemoproteus (Hellgren et al. 2004). deposited in MalAvi and GenBank. The percent prevalence
To determine Plasmodium DNA, a nested PCR was con- of Plasmodium or Haemoproteus in birds was determined
ducted using the initial PCR amplicon and primers AE983/ using the following equation [(number of infected individu-
AE985 which amplify a 580 bp fragment (Pacheco et al. als / number of individuals examined) × 100] (Bush et al.
2018). Similarly, a nested PCR was performed using the ini- 1997). Molecularly positive samples that did not present
tial PCR amplicon and primers AE980/AE982 to determine forms of the parasite in the blood were not included in the
Haemoproteus DNA, resulting in amplification of a 346 bp prevalence estimation (Alvarez-Londoño et al. 2022).
fragment (Pacheco et al. 2018). Samples positive by PCR
with primers AE983/AE985 and primers AE980/AE982
were considered coinfections. All PCR reactions were con- Results
ducted with both a negative control (­ H2Odd) and positive
control (Haemoproteus columbae) (Alvarez-Londoño et al. A total of 255 birds belonging to 108 species and 27 fami-
2022). To visualize PCR products, horizontal electropho- lies (79% resident, 20% neotropical migrants, and 1%
resis was carried out using 1% agarose gels with 1X TBE introduced) were examined (Tables 2 and 3). Twelve per-
buffer, stained with SYBR SAFE (Thermo Fisher Scientific, cent of the bird species are neotropical migrants of Tyran-
Waltham, USA), and visualized in UV photodocumenter. nidae, Vireonidae, Turdidae, Parulidae, and Cardinalidae
Positive samples for HaemF/HaemR2 were sent to Macrogen (Tables 2 and 3). The 68.5% of the examined birds were
(Seoul, South Korea) for purification and Sanger sequenc- captured in agricultural areas, while 31.5% were found
ing. The obtained sequences were edited in the Geneious in secondary forests. Parasites from the genera Plasmo-
Prime program (2023.0.4. https://​www.​genei​ous.​com/) and dium and Haemoproteus were identified in 20 passerine
aligned in the MEGA11 software (Tamura et al. 2021). To and non-passerine species (Table 2). The prevalence of
identify Plasmodium and Haemoproteus genetic lineages, Plasmodium was 4.3% (11/255), while the prevalence of
we compared our sequences with sequences deposited in Haemoproteus was 3.5% (9/255). Seventy-one percent of
the public databases MalAvi (http://1​ 30.2​ 35.2​ 44.9​ 2/M
​ alavi/, the haemosporidian-infected birds were residents, while
Bensch et al. 2009) and GenBank (https://b​ last.n​ cbi.n​ lm.n​ ih.​ 29% were neotropical migrants. Neotropical migratory birds
gov/​Blast.​cgi). Sequences that had at least one nucleotide Red-eyed Vireo (Vireo olivaceus), Gray-cheeked Thrush
difference with the MalAvi sequences were considered new (Catharus minimus), Swainson’s Thrush (Catharus ustula-
lineages and were named according to the nomenclature pro- tus), and Northern Waterthrush (Parkesia noveboracensis)
posed by Bensch et al. (2009). were found infected by haemosporidians. These birds were
Phylogenetic reconstruction was conducted using Bayes- captured during the fall migration (between October and
ian inference on the identified lineages of Plasmodium and November 2021) (Table 2). At the same time, the Summer
Haemoproteus. The alignments included our sequences and Tanager (Piranga rubra) and Scarlet Tanager (Piranga oli-
sequences of the cyt b gene obtained from the MalAvi data- vacea) were captured in winter (February 2022) (Table 2).
base which were linked to morphospecies, and a sequence Morphologically, Plasmodium (Novyella) unalis infection
of Leucocytozoon buteonis BUTREG01 [DQ177264] was (trophozoites, meronts, and gametocytes) was identified
used as an outgroup. The total length of the alignments was in the Pale-breasted Thrush (Turdus leucomelas) (Fig. 2)
480 bp. The General Time Reversible model with invariant and Haemoproteus (Haemoproteus) paramultipigmentatus
sites and gamma distribution (GTR + I + G) was selected infection in the Common Ground Dove (Columbina pas-
according to the corrected Akaike information criterion serina), Haemoproteus (Parahaemoproteus) witti in the
using jModeltest v.2.1.6 (Darriba et al. 2012). Bayesian anal- Rufous-tailed Hummingbird (Amazilia tzacatl), Haemo-
ysis was carried out using MrBayes v3.2.7a (Ronquist and proteus (Parahaemoproteus) nucleocentralis in the Ruddy-
Huelsenbeck 2003) via the CIPRESS Science Gateway v3.3 breasted Seedeater (Sporophila minuta), and Haemoproteus
(Miller et al. 2010). Two independent Markov and Monte (Parahaemoproteus) erythrogravidus in the Blue-necked
Carlo chains (MCMC) were used simultaneously for a total Tanager (Stilpnia cyanicollis) in the Magdalena Valley
of 15 million generations, with four chains sampled every (Fig. 3). Haemoproteus (Parahaemoproteus) tyranni was
1000 generations. Convergence was evaluated by calculating detected in a Tropical Kingbird (Tyrannus melancholicus)
the mean and standard deviation of the frequencies divided in the Cauca River Valley (Table 2; Fig. 3).
Parasitology Research (2024) 123:252 Page 5 of 13 252

Table 2  Avian hosts positive with Plasmodium spp. and Haemopro- where birds were sampled in the department of Caldas, Colombia.
teus spp. in the department of Caldas. The GenBank accession codes The localities correspond to the numbers presented in Fig. 1
in bold correspond to the sequences obtained in this study. Localities
Order/family Host species English name No. infected birds/no. exam- Parasite species and lineages from Number of birds by locality [no. bird
(Scientific name) ined birds (% prevalence) MalAvi (% similarity) infected]

1 2 3 4 5 6

Columbiformes/
Columbidae Common ground dove (Columbina 1/2 (50) Haemoproteus (H.) paramultipig- 2[1]
passerina) mentatus COLPAS11*
Ruddy ground dove (Columbina 2/4 (50) Haemoproteus sp.b 4[2]
talpacoti)
Apodiformes/
Trochilidae Rufous-tailed hummingbird 1/2 (50) Haemoproteus (P.) witti EUP- 2[1]
(Amazilia tzacatl) MAC01 (100%)
Passeriformes/
Thamnophilidae Checker-throated stipplethroath 1/4 (25) Plasmodium sp. EPIFUL01* 4[1]
(Epinecrophylla fulviventris)
Formicariidae Black-faced antthrush (Formicarius 1/1 (100) Plasmodium sp.S 1[1]
analis)
Tyrannidae Tropical kingbird (Tyrannus 2/2 (100) Haemoproteus (P.) 2[2]
melancholicus) tyranni MYMAC03 (100%)
Tropical kingbird (Tyrannus Haemoproteus (P.) tyranni TYR-
melancholicus) MEL03*
Vireonidae Red-eyed vireo (Vireo olivaceus)ˆ 1/1 (100) Plasmodium sp. VIOLI03 (100%) 1[1]
Haemoproteus sp.S
Troglodytidae House wren (Troglodytes aedon) 1/3 (33.3) Plasmodium sp. SETAUD23 1[1] 2
(100%)
Turdidae Gray-cheeked thrush (Catharus 1/4 (25) Plasmodium (H.) matutinum 1 1[1] 2
minimus)ˆ LINN1 (100%)
Swainson’s thrush (Catharus 1/22 (4.54) Plasmodium sp. BT7 (100%) 2 7 7 5[1] 1
ustulatus)ˆ
Pale-breasted thrush (Turdus 1/1 (100) Plasmodium unalis TULEU09* 1[1]
leucomelas)
Passerellidae Orange-billed sparrow (Arremon 1/1 (100) Haemoproteus sp. ATLBRU01 1[1]
aurantiirostris) (100%)
Parulidae Northern waterthrush (Parkesia 1/4 (25) Plasmodium sp.a, b 2[1] 1 1
noveboracensis)ˆ
Cardinalidae Summer tanager (Piranga rubra)ˆ 1/1 (100) Plasmodium sp. PIRUB04* 1[1]
Scarlet tanager (Piranga 1/1 (100) Plasmodium sp. TACTHA01a 1[1]
olivacea)ˆ (100%)
Sooty ant-tanager (Habia gutturalis) 3/5 (60) Plasmodium sp. EMBHER01 5[3]
(100%)
Thraupidae Ruddy-breasted seedeater 1/6 (14) Haemoproteus (P.) nucleocentra- 6[1]
(Sporophila minuta) lis SPOMIN01*
Bananaquit (Coereba flaveola) 1/3 (33.3) Plasmodium sp. PADOM09 1[1] 1 1
(100%)
Blue-necked tanager (Stilpnia 1/1 (100) Haemoproteus (P.) erythrogravi- 1[1]
cyanicollis) dus TANCYA01 (100%)
Blue-gray tanager (Thraupis 1/4 (25) Plasmodium sp. PADOM09 4[1]
episcopus) (100%)

*New haemosporidian lineages reported in the study

ˆNeotropical migrant bird
S
Presence of blood forms, PCR negative
a
Molecularly positive samples that did not present forms of the parasite in the blood
b
Unidentified lineage (low quality sequence)
252 Page 6 of 13 Parasitology Research (2024) 123:252

Table 3  Non-infected bird species by haemosporidian parasites caught in the department of Caldas—Colombia (the localities correspond to the
numbers presented in Fig. 1)
Order Family Bird species No. of exam- Number of birds by locality
ined bird
1 2 3 4 5 6

Columbiformes Columbidae Leptotila verreauxi 2 1 1

Apodiformes Trochilidae Glaucis hirsutus 1 1
Threnetes ruckeri 1 1
Phaethornis striigularis 1 1
Phaethornis anthophilus 2 2
Phaethornis guy 2 2
Chlorostilbon gibsoni 1 1
Chalybura buffonii 6 1 5
Polyerata amabilis 1 1
Charadriiformes Charadriidae Vanellus chilensis 1 1
Coraciformes Momotidae Hylomanes momotula 1 1
Momotus subrufescens 2 2
Alcedinidae Chloroceryle aenea 2 1 1
Galbuliformes Bucconidae Notharchus tectus 2 2
Hypnelus ruficollis 1 1
Piciformes Ramphastidae Pteroglossus torquatus 1 1
Picidae Melanerpes rubricapillus 2 1 1
Colaptes punctigula 1 1
Psittaciformes Psittacidae Forpus conspicillatus 3 1 2
Passeriformes Thamnophilidae Cercomacra nigricans 2 2
Sipia palliata 1 1
Hafferia immaculata 2 2
Gymnopithys bicolor 3 3
Furnariidae Dendrocincla fuliginosa 8 3 4 1
Glyphorynchus spirurus 1 1
Xiphorhynchus triangularis 2 2
Lepidocolaptes souleyetii 1 1
Furnarius leucopus 4 3 1
Certhiaxis cinnamomeus 1 1
Synallaxis albescens 1 1
Pipridae Lepidothrix coronata 3 3
Manacus manacus 5 4 1
Machaeropterus striolatus 3 3
Ceratopipra erythrocephala 1 1
Tityridae Pachyramphus cinnamomeus 1 1
Tyrannidae Mionectes oleagineus 7 1 2 4
Leptopogon amaurocephalus 3 3
Tolmomyias sulphurescens 1 1
Oncostoma olivaceum 2 2
Poecilotriccus sylvia 2 2
Todirostrum cinereum 3 1 2
Zimmerius chrysops 1 1
Camptostoma obsoletum 1 1
Elaenia flavogaster 1 1
Elaenia chiriquensis 1 1
Tyrannulus elatus 1 1
Phyllomyias griseiceps 1 1
Parasitology Research (2024) 123:252 Page 7 of 13 252

Table 3  (continued)
Order Family Bird species No. of exam- Number of birds by locality
ined bird
1 2 3 4 5 6

Legatus leucophaius 1 1
Pitangus sulphuratus 1 1
Phaeomyias murina 1 1
Myiodynastes maculatus 1 1
Myiozetetes cayanensis 3 1 1 1
Myiozetetes similis 1 1
Myiarchus panamensis 1 1
Empidonax virescens* 1 1
Empidonax traillii* 1 1
Contopus cinereus 1 1
Vireonidae Vireo leucophrys 1 1
Hirundinidae Stelgidopteryx ruficollis 2 1 1
Troglodytidae Microcerculus marginatus 1 1
Turdidae Turdus grayi 1 1
Turdus ignobilis 2 2
Estrildidae Lonchura malacca+ 1 1
Fringillidae Euphonia laniirostris 1 1
Icteridae Icterus nigrogularis 1 1
Parulidae Leiothlypis peregrina* 1 1
Geothlypis philadelphia* 1 1
Setophaga castanea* 7 6 1
Setophaga ruticilla* 1 1
Setophaga petechia* 7 5 2
Basileuterus rufifrons 1 1
Mitrospingidae Mitrospingus cassinii 1 1
Cardinalidae Cyanoloxia brissonii 2 2
Thraupidae Sicalis flaveola 6 3 3
Volatinia jacarina 12 2 2 8
Eucometis penicillata 3 2 1
Ramphocelus dimidiatus 5 5
Dacnis lineata 1 1
Dacnis cayana 1 1
Sporophila crassirostris 1 1
Sporophila intermedia 3 1 2
Sporophila nigricollis 5 1 3 1
Sporophila schistacea 2 1 1
Saltator maximus 1 1
Saltator striatipectus 1 1
Stilpnia vitriolina 1 1
Tangara inornata 1 1
Thraupis palmarum 1 1
Total 184 35 20 50 49 23 7

*Neotropical migrant bird

+
Introduced bird

Molecularly, 17 haemosporidian lineages were determined were recorded in the Red-eyed Vireo (VIOLI03), the House
(10 Plasmodium and seven Haemoproteus). Lineages that were Wren (Troglodytes aedon) (SETAUD23), the Gray-cheeked
previously reported in the MalAvi database of Plasmodium Thrush (LINN1), the Swainson’s Thrush (BT7), the Scarlet
252 Page 8 of 13 Parasitology Research (2024) 123:252

Fig. 2  Parasites of the genera Plasmodium found in the study. a breasted Thrush. g–h Macrogametocyte of Plasmodium unalis
Erythrocytic meront of Plasmodium sp. (VIOLI03) in the Red-eyed (TULEU09) in the Pale-breasted Thrush. i Trophozoite of Plasmo-
Vireo. b Macrogametocyte of Plasmodium sp. (SETAUD23) in the dium sp. (PIRUB04) in the Summer Tanager. j Macrogametocyte of
House Wren. c Trophozoite of Plasmodium (H.) matutinum (LINN1) Plasmodium sp. (EMBHER01) in the Sooty Ant-Tanager. k Young
in the Gray-cheeked Thrush. d Trophozoite of Plasmodium una- erythrocytic meront of Plasmodium sp. (PADOM09) in the Blue-gray
lis (TULEU09) in the Pale-breasted Thrush. e Erythrocytic meront Tanager. Short black arrow pigment granules. Black long arrow mer-
of Plasmodium unalis (TULEU09) in the Pale-breasted Thrush. f ozoite. Black arrowhead parasite nucleus. White long arrow vacuole.
Microgametocyte of Plasmodium unalis (TULEU09) in the Pale- Scale bar = 10 μm

Tanager (TACTHA01), the Sooty Ant-Tanager (Habia gut- for the cyt b gene in this study are [OR654036–OR654050,
turalis) (EMBHER01), the Bananaquit (Coereba flaveola) OR767283–OR767285, OR805347]. Likewise, the sequences
(PADOM09), and the Blue-gray Tanager (Thraupis episco- of the new lineages were deposited in the MalAvi database.
pus) (PADOM09). However, Haemoproteus lineages were However, no parasitic forms of Plasmodium were found
detected in the Rufous-tailed Hummingbird (EUPMAC01), in blood smears of the Northern Waterthrush, the Scarlet
the Tropical Kingbird (MYMAC03), the Orange-billed Spar- Tanager, and the Gray-headed Tanager (Eucometis peni-
row (Arremon aurantiirostris) (ATLBRU01), and the Blue- cillata) (Table 2). Only one individual of the Red-eyed
necked Tanager (TANCYA01) (Table 2; Fig. 4). Some of these Vireo was co-infected with Plasmodium sp. (VIOLI03) and
lineages were documented for the first time in Colombia within Haemoproteus (P.) sp. (Table 2). The blood smears obtained
resident or neotropical migratory birds (SETAUD23, TAC- in this study are located in the Genetics Laboratory of the
THA01, VIOLI03, EMBHER01, EUPMAC01, ATLBRU01, Universidad de Caldas.
TANCYA01, and LINN1). Three new Plasmodium lineages
were detected, PIRUB04 infecting the Summer Tanager,
TULEU09 infecting the Pale-breasted Thrush, and EPIFUL01 Discussion
infecting the Checker-throated Stipplethroat (Epinecrophylla
fulviventris). Additionally, three new Haemoproteus line- The prevalence of Plasmodium spp. or Haemoproteus spp.
ages were found, COLPAS11 infecting the Common Ground was similar to that established in other localities in Colombia
Dove, TYRMEL03 infecting the Tropical Kingbird, and (Rodríguez and Matta 2001; Valkiūnas et al. 2003; Basto
SPOMIN01 infecting the Ruddy-breasted Seedeater (Table 2; et al. 2006; Alvarez-Londoño et al. 2022). It has been sug-
Fig. 4). Notably, the new H. (P.) nucleocentralis lineage gested that the prevalence of these haemosporidians in birds
SPOMIN01 was closely related (posterior probability = 1) to seems to be determined by habitat characteristics (e.g., lentic
the TANDES01 lineage, with an evolutionary distance of 2.4% water bodies, ponds, or forest type) that support the devel-
(Table S1, Fig. 4). The GenBank accession codes obtained opment, abundance, and diversity of their vectors or by
Parasitology Research (2024) 123:252 Page 9 of 13 252

Fig. 3  Parasites of the genera

Haemoproteus found in the
study. a–d Macrogametocytes
of H. (H.) paramultipigmentatus
in the Common Ground Dove.
e, f, h Microgametocytes of H.
(P.) witti in the Rufous-tailed
Hummingbird. g Macroga-
metocyte of H. (P.) witti in
Rufous-tailed Hummingbird. i–l
Macrogametocytes of H. (P.)
tyranni in the Tropical King-
bird. m, p Macrogametocytes
of H. (P.) nucleocentralis in the
Ruddy-breasted Seedeater. n,
o Microgametocytes of H. (P.)
nucleocentralis in the Ruddy-
breasted Seedeater. q, s, t Mac-
rogametocytes of H. (P.) eryth-
rogravidus in the Blue-necked
Tanager. r Microgametocyte of
H. (P.) erythrogravidus in the
Blue-necked Tanager. Black
arrowhead parasite nucleus.
Short black arrow pigment
granules. White arrowhead pro-
trusions of infected erythrocyte
envelope. Scale bar = 10 μm

ecophysiological traits of their hosts (González et al. 2015; previously been recorded only in other countries of the
Alvarez-Londoño et al. 2022). Particularly, one of the loca- Americas, such as the USA, Guyana, Peru, or Brazil, in
tions with the highest number of Plasmodium spp. infected both passerine and non-passerine birds (Beadell et al.
birds was the Granja Luker. On this farm, pools are formed 2006; Durrant et al. 2006; Bensch et al. 2009; Fecchio et al.
as a result of the crop drainage network, and there are bro- 2019). Likewise, we report the presence of P. (H.) matuti-
meliads attached to trees that serve as habitat for mosquitoes num LINN1 in Colombia infected to a migratory Gray-
of the genus Wyeomyia, recognized vectors of avian Plasmo- cheeked Thrush. This Plasmodium morphospecies has
dium (Bensch et al. 2009; Morcillo et al. 2023). previously been recorded in North America, Europe, Asia,
Seventy percent of the Plasmodium lineages reported and Australia, infecting birds of various orders including
in our study have previously been detected in America, Gruiformes, Charadriiformes, Strigiformes, or Passeri-
Europe, Asia, or Australia (Bensch et al. 2009; Fig. 4). formes (Bensch et al. 2009; Fig. 4). In particular, P. (H.)
However, some of these Plasmodium lineages were docu- matutinum has been observed in birds on the east coast of
mented for the first time in Colombia within resident or the USA (states of Michigan and New York) (Bensch et al.
neotropical migratory birds. In relation to this, we con- 2009). These localities are part of the migration routes
sider it as an extension of the geographic distribution range of the Gray-cheeked Thrush from their breeding grounds
across the Americas for the SETAUD23 and TACTHA01 in Asia (northeastern Siberia) and North America (from
lineages of Plasmodium sp. detected in House Wren and Alaska to the East Coast) (Udvardy 1994). This suggests
Scarlet Tanager, respectively. Both lineages have been pre- that neotropical migratory birds may play a key role in the
viously reported in North America in neotropical migratory dispersal of parasites of the genus Plasmodium between
birds wintering in Colombia (Bensch et al. 2009; Fig. 4). temperate and tropical regions of the Americas (Hellgren
We recorded the geographic expansion in the South Ameri- et al. 2007; Fecchio et al. 2018). We reported six new line-
can distribution of the VIOLI03 and EMBHER01 lineages ages of haemosporidians infecting resident and migratory
of Plasmodium sp., as well as the EUPMAC01 lineages birds, of which three lineages of Haemoproteus (COL-
of H. (P.) witti, ATLBRU01 of Haemoproteus sp., TAN- PAS11, TYRMEL03, and SPOMIN01) and one lineage of
CYA01 of H. (P.) erythrogravidus. These lineages had Plasmodium (TULEU09) were related to a morphospecies
252 Page 10 of 13 Parasitology Research (2024) 123:252

Fig. 4  Bayesian phylogeny of Haemoproteus spp. and Plasmodium found. Lineages and sequences reported in this study are shown in
spp. based on 117 lineages of cytochrome b gene. Morphologi- bold. The sequence of Leucocytozoon buteonis BUTREG01 was used
cal species names followed by MalAvi lineage, GenBank accession as an outgroup. The scale bar represents the number of nucleotide
number within square brackets, and the country where the sequences substitutions per site. Nodal support values indicate posterior prob-
are reported. The colored rectangles correspond to the geographic abilities
regions where the lineages from the MalAvi database have been

through morphological analysis. The TULEU09 lineage of infect the Rufous-collared Sparrow (Zonotrichia capen-
P. (N.) unalis recorded in the Pale-breasted Thrush appears sis) in Ecuador (Fig. 4). Moreover, the new COLPAS11
to be related to the TFUS06 lineage of P. (N.) unalis that lineage of H. (H.) paramultipigmentatus was detected
has been previously documented in the Great Thrush (Tur- in Common-ground Dove in the Magdalena River Val-
dus fuscater) in Colombia, as well as four other species of ley. This lineage grouped with the COLPAS03 and COL-
the genus Turdus in the Atlantic forest of Brazil (Turdus BUC01 lineages belonging to the morphospecies H. (H.)
rufiventris, T. leucomelas, Turdus albicollis, and Turdus paramultipigmentatus and Haemoproteus (Haemoproteus)
flavipes) (Mantilla et al. 2013; Tostes et al. 2018; Fig. 4). multipigmentatus, respectively (Fig. 4). These lineages are
In this regard, it has been suggested that there is a high closely associated with birds from the Columbidae fam-
degree of intraspecific polymorphism, and a wide diver- ily, with species distributed in Mexico, Ecuador, Brazil,
sity of host species are present within this parasite spe- Venezuela, and recently reported in Colombia (Valkiūnas
cies (Mantilla et al. 2013; Vanstreels et al. 2015; Tostes et al. 2010; Lotta-Arévalo et al. 2023). Another new lin-
et al. 2018). The newly discovered EPIFUL01 lineage of eage, TYRMEL03 of H. (P.) tyranni, was detected in a
Plasmodium sp. detected in the Checker-throated Stipple- Tropical Kingbird in the Cauca River Valley. This lineage
throat is part of a polytomy of diverse Plasmodium mor- was associated with the MYMAC03 lineage reported in
phospecies (Fig. 4). Therefore, this new lineage could be this study in another individual from the Tropical King-
another Plasmodium species, but further studies on this bird (Fig. 4). The MYMAC03 lineage had previously been
host are required to confirm. The new Plasmodium sp. lin- detected only through molecular methods in another mem-
eage PIRUB04 found in the Summer Tanager was related ber of the Tyrannidae family (Myiodynastes maculatus) in
to the lineages EMBHER01 in the Sooty-ant Tanager, Brazil (Ferreira et al. 2017). The morphospecies H. (P.)
SETAUD23 in the House Wren, TACTHA01 in the Scarlet tyranni does not have an associated lineage in the MalAvi
Tanager, and PADOM09 in the Bananaquit (evolutionary database; however, it has a significantly higher number of
distances of < 1.7%; Table S1). The latter lineages were records in North America (the USA and Canada), while
related to the Plasmodium (H.) cathemerium morphospe- in the rest of the Americas, it has only been previously
cies (ZONCAP15), which was previously documented to recorded in Panama, Brazil, and Venezuela, infecting
Parasitology Research (2024) 123:252 Page 11 of 13 252

mainly birds of the family Tyrannidae (e.g., Pitangus sul- Open Access This article is licensed under a Creative Commons Attri-
phuratus and T. melancholicus) (Valkiūnas 2005; Valera bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
et al. 2019; da Silva et al. 2022). Therefore, we consider as you give appropriate credit to the original author(s) and the source,
that the record of H. (P.) tyranni increases its distribution provide a link to the Creative Commons licence, and indicate if changes
range in South America and provides a lineage associated were made. The images or other third party material in this article are
with this morphospecies. The new lineage SPOMIN01 of included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
H. (P.) nucleocentralis was closely related to the lineage the article’s Creative Commons licence and your intended use is not
TANDES01 of the same morphospecies (detected only in permitted by statutory regulation or exceeds the permitted use, you will
Brazil). These two lineages have only been reported in two need to obtain permission directly from the copyright holder. To view a
species of the Thraupidae family (Sporophila minuta and copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Tangara desmaresti) (Anjos et al. 2021). This study con-
tributes to the knowledge of the diversity of Plasmodium
and Haemoproteus lineages and species present in neo- References
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