Avian Hemosporidian Parasite Lineages in Four Species of Free-ranging Migratory Waterbirds from Mongolia, 2008

Plasmodium, Haemoproteus, and Leucocytozoon are Apicomplexan hemosporidian parasites that can infect birds and have the potential to cause clinical disease. Several authors have investigated blood parasites in Asian waterbirds (Manwell and Kuntz 1966; Ishtiaq et al. 2007; Yoshimura et al. 2014), but none have focused on hemosporidian parasites in migratory birds in Mongolia. Wetlands in the mountainous region of Khovsgol Aimag Mongolia serve as a breeding ground and stopover location for migratory birds (BirdLife International 2004). Our study areas included three lakes (Erhel Nuur, 49°57′33′′N, 99°54′08′′E; Tsangiyn Dalai Nuur, 49°14′00′′N, 99°03′00′′E; and Tsengel Nuur, 49°44′54′′N, 101°01′01′′E) and a wetland complex of rivers, ponds, and lakes (Darkhad Valley, 51°13′00′′N, 99°24′00′′E) where waterbirds are known to breed and congregate during migration. We screened migratory waterbirds in these wetlands for hemosporidian parasites and examined hatch year (HY) bird samples to determine if parasite transmission occurs locally.

We captured birds 17 July 2008 to 3 September 2008 using hand nets and a spotlight, either from the shore or from a boat at night (geese, cormorants, and shelducks), or during the day on the breeding grounds (gulls; Fig. 1 and Supplementary Table), as described by Gilbert et al. (2012). Blood samples were collected from 151 birds. Species sampled included the Bar-headed Goose (Anser indicus; 30 after hatch year [AHY] birds), Great Cormorant (Phalacrocorax carbo; 25 AHY, 28 HY), Mongolian Gull (Larus mongolicus; 15 AHY, 23 HY), and Ruddy Shelduck (Tadorna ferruginea; 0 AHY, 30 HY) (see Supplementary Table). All birds appeared healthy when captured and were released after sampling.

Blood was drawn from the jugular vein and approximately 200 μL of blood from each bird was transferred on to an individual Whatman 903 Protein Saver Card (GE Healthcare, Piscataway Township, New Jersey, USA), air-dried, and stored at room temperature. For hemosporidian parasite PCR testing, DNA was extracted from a 6-mm filter paper punch using the QIAmp® DNA Mini Kit (Qiagen, Valencia, California, USA). Nested PCR amplification of a 478-base pair (bp) portion of the hemosporidian parasite cytochrome b gene was performed with HotStarTaq™ Master Mix (Qiagen) using previously described methods (Hellgren et al. 2004). Negative (no template) and positive controls for Plasmodium, Haemoproteus, and Leucocytozoon were run with each reaction. As an internal amplification control, all DNA extracts were tested for amplification of a 457-bp product of the avian host cytochrome b gene (Townzen et al. 2008). All DNA extracts yielded positive amplification of the internal amplification control. The PCR products of the expected molecular weight from the hemosporidian parasite cytochrome b amplification were purified using ExoSAP-IT® (Affymetrix, Santa Clara, California, USA) and directly sequenced in the forward and reverse directions (Genewiz Inc., South Plainfield, New Jersey, USA). All sequences were trimmed of their primers to 476 bp, and sequences were subjected to BLAST analysis using GenBank, National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and the MalAvi database (Bensch et al. 2009). Final sequences were deposited in GenBank under accessions KJ577823–KJ577838 and KU361371. Blood smears were prepared from fresh whole blood immediately after collection. Once dry, the slides were fixed in methanol and stained with a Romanowsky-type stain within 12 h of collection. Samples PCR-positive for hemosporidian parasites, with the exception of one slide that was missing (MN08 B-0333B, Great Cormorant), were independently examined by two certified wildlife pathologists, microscopically, for hemosporidian parasites. Pathologists independently scanned each of the 16 blood films at 100×, 200×, and 400× and performed focused parasite screening for 3 to 5 min at 630× (Leica DM 2500 microscope, Leica Microsystems Inc., Buffalo Grove, Illinois, USA). Additionally, one pathologist reviewed 100 fields of each blood film under 1,000× oil immersion (Nikon Eclipse 55i, Nikon Instruments Inc., Melville, New York, USA; Valkiūnas 2005). Cytologic preparations were of good quality with well-spread erythrocytes and insignificant cell lysis. The slide staining varied mildly, with regional basophilia or pallor affecting ≤50% of the slide in approximately 25% of cases, but there was adequate cytoplasmic and nuclear differentiation in all slides, and morphologic details of all cell types were apparent.

Apicomplexan hemosporidian parasites were identified in 17 of 151 samples by PCR (11%, 95% Adjusted Wald confidence interval: 7.1–17.4%). The percentages of each avian species that were positive were as follows: AHY Bar-headed Goose (7%, 2/30), HY Great Cormorant (11%, 3/28), AHY Great Cormorant (20%, 5/25), HY Mongolian Gull (4 %, 1/23), AHY Mongolian Gull (7%, 1/15), and HY Ruddy Shelduck (17%, 5/30). No HY or AHY Bar-headed Geese were tested. No organisms were identified microscopically in any of the PCR-positive slides. The absence of hemosporidian parasites on blood smears may reflect low levels of infection. Alternatively, infection could be limited to exoerythrocytic stages (prepatent period) or parasitism of deep vascular or organ sites (latent stage), as has been suggested in penguins (Cranfield et al. 1994); or these lineages could undergo abortive development in these birds, resulting in an absence of gametocytes (Valkiūnas et al. 2014).

Six Plasmodium lineages were identified in six birds (Table 1). One of the sequences, from a HY Mongolian Gull (A860), was 100% identical to a P_SGS1 lineage, identified from a Hawfinch (Coccothraustes coccothraustes) in Russia, which was morphologically and genetically characterized as Plasmodium relictum (Palinauskas et al. 2007). The sequence from an AHY Great Cormorant (B280) was 100% identical to a P_SW2 lineage previously found in a Common Grasshopper Warbler (Locustella naevia) from Russia (Ilgūnas et al. 2013). The sequences from samples A-0812 and A-0712 (HY Ruddy Shelduck), and B-0263 (AHY Great Cormorant) were submitted to the MalAvi database as new lineages: P-TADFER01, P-TADFER02, and P-PHACAR01.

One Haemoproteus lineage was recovered from seven birds: one AHY Bar-headed Goose, three HY Ruddy Shelduck, and two HY and an AHY Great Cormorant; it did not match morphologically characterized species from the MalAvi in GenBank. This lineage was submitted to the MalAvi database as ANSIND01.

Three Leucocytozoon lineages were recovered from four birds. Two of the samples, an AHY Bar-headed Goose (A235) and an AHY Great Cormorant (B298), were 100% identical to TUSW04 lineage found in Tundra Swans (Cygnus columbianus) from Alaska (GenBank JQ314223; Ramey et al. 2012). The second Leucocytozoon lineage from an AHY Mongolian Gull (A706) was 100% identical to CIAE02 lineage (GenBank EF607287) found in a Western Marsh Harrier (Circus aeruginosus) in Germany (Krone et al. 2008). The third Leucocytozoon lineage from an HY Great Cormorant (B-0256) did not match a known sequence and was submitted to the MalAvi database as PHACAR02.

In conclusion, we identified 10 lineages of hemosporidian parasites in migratory waterbirds that breed in Mongolia, and a hemosporidian parasite prevalence of 11%, in apparently healthy AHY and HY birds. Hemosporidian parasites in adult birds could represent various stages of infection and, because organisms were not identified in blood films, further characterization was not possible. All four bird species included in this study follow distinct migratory routes from their boreal breeding grounds in the wetlands of Mongolia to wintering areas in the milder climates of the Indian subcontinent, Southeast Asia, China, or the Korean Peninsula. For example, the Bar-headed Goose overwinters from the Tibetan Plateau south through much of the Indian Subcontinent (Newman et al. 2012). The wintering distribution of Mongolian Great Cormorants is poorly known but could include areas of the Indian Subcontinent, Southeast Asia, and China (Orta et al. 2016). Mongolian Gulls migrate to coastal China and the Korean Peninsula (Brazil 2009). Mongolian breeding populations of Ruddy Shelducks migrate to eastern and northeastern India, Myanmar, and possibly areas of China (Prosser et al. 2009; Newman et al. 2012; Carboneras and Kirwan 2016). While adult birds could have become infected at any point along the migration route, infection in HY birds would have occurred at the breeding grounds. Our results expand the known range of hemosporidian parasite lineages and provide important baseline data that contribute to a better understanding of the geographic range and types of hemosporidian parasites that infect waterbirds along their migratory routes and at their breeding grounds in northern Mongolia.

We thank the Wildlife Conservation Society's Wildlife Health and Health Policy Program for providing samples. This project was funded by donors supporting the Wildlife Conservation Society Zoological and Wildlife Health and Health Policy Programs, and funding was provided by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and the Department of Health and Human Services under contract HHSN266200700007C.

Supplementary material for this article is online at http://dx.doi.org/10.7589/2015-11-311.