Molecular survey and genetic diversity of Plasmodium sp. infesting domestic poultry in northeastern Thailand

A total of 116 blood samples were collected from backyard birds in two provinces (Maha Sarakham and Nongkhai) in the northeastern part of Thailand (Fig. 1). Fifty-one blood samples were from Nongkhai (30 from ducks, 10 from turkeys and 11 from geese) and sixty-five were from Maha Sarakham (30 from ducks, 26 from turkeys and 9 from geese). Samples were taken from March to September 2022 of approximately 0.1–0.5 mL of blood drawn from the brachial vein into anticoagulant ethylenediaminetetraacetic acid tubes. These were stored on ice during transport to the laboratory at the Faculty of Veterinary Sciences of Mahasarakham University. The blood samples were screened for haemosporidian parasite infection by a thin-blood-smear technique and measurement of pack cell volume (PCV). The remaining blood was stored at −20°C until DNA extraction. All animal handling and blood collection steps were performed by veterinarians and access to all backyard birds was approved by their owners. All blood samples were randomly collected from symptomatic birds that their owners selected for blood testing. In addition, samples were collected from asymptomatic (healthy) animals.

Fig. 1.

Blood samples were examined for haemosporidian parasite infection under a light microscope by thin-blood-smear technique. The blood smear was prepared by dropping blood onto a slide, smearing it with a spreader, air-drying it for approximately 10 s and fixing it with 100% methanol for 5 min. Subsequently, the blood films were stained with 10% Giemsa solution for 15 min and the parasites were detected in monolayer fields under a light microscope (Nikon, Tokyo, Japan).

The percentage of total blood volume which was red blood cells, or the PCV value, was assessed from the height of the erythrocyte column in a microhaematocrit tube after centrifugation. Blood was directly placed into the heparinised microhaematocrit tube and centrifuged at 10,000 rpm for 3 min (Hettich Zentrifugen, Tuttlingen, Germany). The ratio of the volume of packed red blood cells to the total blood volume was measured and expressed as a percentage. The PCVs (%) of the infected and uninfected groups were compared using the independent samples t-test. A P-value less than 0.05 was considered statistically significant.

Approximately 20 μL of each whole blood sample was mixed homogenously with 180 μL of 1× phosphate-buffered saline. DNA was extracted from 200 μL of the mixed solutions following the protocol with the GF-1 Blood DNA Extraction Kit (Vivatis, Shah Alam, Malaysia) and stored at −20°C.

A nested-PCR method using primers targeting a partial mitochondrial cyt b gene was performed. For the external nested PCR, the primer pair of HaemNFI (5′-CATATATTAAGAGAAITATGGAG-3′) and HaemNR3 (5′-ATAGAAAGATAAGAAATACCATTC-3′) which can amplify the DNA of Plasmodium, Haemoproteus and Leucocytozoon was used (10). For the internal nested PCR, the primers HaemF (5′-ATGGTGCTTTCGATATATGCATG-3′) and HaemR2 (5′-GCATTATCTGGATGTGATAATGGT-3′) were used to amplify the DNA from Plasmodium and Haemoproteus (2), and HaemFL (5′-ATGGTGTTTTAGATACTTACATT-3′) and HaemR2L (5′-CATTATCTGGATGAGATAATGGIGC-3′) were used to amplify the Leucocytozoon DNA (10).

Both amplification steps of the nested-PCR reaction were performed in a 25 μL reaction volume consisting of 1 U of Taq polymerase (Thermo Scientific, Waltham, MA, USA), 1.5 mM of MgSO₄, 0.2 mM of dNTPs, 1× PCR buffer, 0.4 μM of each primer and template DNA (5 μL of extracted DNA for the first PCR and 2 μL of PCR product from the first amplification for the second PCR). The PCR conditions comprised 35 cycles of denaturation for 45 s at 95°C, annealing for 45 s at 50°C for the first step and at 50°C (Leucocytozoon) and 53°C (Plasmodium and Haemoproteus) for the second steps, extension for 90 s at 72°C and a final extension at 72°C for 5 min. DNA from fighting cocks previously tested for Leucocytozoon and Plasmodium/ Haemoproteus infections served as positive controls. The PCR master mixes containing only the primers with no DNA template served as negative controls. The nested PCR generated approximately 480 base pairs (bp), which were subsequently identified by 1% agarose gels stained with ViSafe Red Gel Stain (Vivantis) and visualised under ultraviolet light on a gel documentation system.

All positive samples containing the partial cyt b gene were purified and sequenced directly at a commercial sequencing company (ATGC Co., Pathum Thani, Thailand). The sequences obtained were multiply aligned using ClustalW in the BioEdit program with final adjustment performed manually, and compared for similarity with sequences deposited in the GenBank database using the BLAST program hosted by NCBI (https://www.ncbi.nlm.nih.gov/). The haplotype diversity of Plasmodium sp. was calculated in the DnaSP6 program (25).

The sequences of the partial cyt b gene of each Plasmodium haplotype in this study were deposited in GenBank (accession Nos OR341178–OR341184). A total of 40 Plasmodium sequences (7 haplotypes from this study and 33 related sequences from the MalAvi and GenBank databases) were analysed using the maximum-likelihood method in MEGA X (13) for construction of a phylogenetic tree, with sequences of two Haemoproteus sp. used as an outgroup. Bootstrap analysis with 1,000 replications was used to estimate the confidence of the branching patterns of the trees.

In total, 116 blood smears from backyard poultry were examined under the microscope for haemosporidian parasite infections. Plasmodium sp. was observed in ducks and turkeys in Nongkhai and Maha Sarakham provinces (Fig. 2). However, Leucocytozoon sp. and Haemoproteus sp. were not found in all backyard poultry blood smears. In Nongkhai, the prevalence of Plasmodium spp. was 3.3% in ducks and 10% in turkeys. In Maha Sarakham, the prevalence of Plasmodium spp. was 6.7% in ducks and 26.9% in turkeys (Table 1). All positive samples identified by light microscopy were successfully amplified by nested PCR.

Fig. 2.

The molecular approach using the nested PCR method detected an additional 26 positive samples from microscopically negative samples. Microscopic examination indicated that no DNA fragments of Leucocytozoon sp. or Haemoproteus sp. were amplified. In Nongkhai and Maha Sarakham provinces, the molecular prevalence of Plasmodium spp. in ducks was 13.3% and 23.3%, and in turkeys was 50% and 76.9%, respectively. The prevalence of Plasmodium spp. in geese was 9.1% in Nongkhai. In summary, the overall prevalence of Plasmodium spp. was 31.9% (95% confidence interval from 23.4 to 40.4), with the highest prevalence observed in turkeys at 25/36 (69.4%), lower prevalence in ducks at 11/60 (18.3%) and the lowest in geese at 1/20 (5%).

The obtained sequences of the partial mitochondrial cyt b gene of Plasmodium parasites in this study were approximately 446-bp-long fragments (416–470 bp). All 37 positive PCR products were sequenced and seven haplotypes were identified. Among 37 sequences, 32, 4 and 1 sequences were identified as Plasmodium sp., P. gallinaceum and P. juxtanucleare, respectively.

There were 32 sequences of Plasmodium sp. which represented four lineages (accession Nos OR341178–OR341180 and OR341184), 4 sequences of P. gallinaceum which represented two lineages (accession Nos OR341181 and OR341183), and 1 sequence of P. juxtanucleare which represented one lineage (accession No. OR341182). In addition, two lineages of Plasmodium sp. found in this study had not been previously described (Table 2).

The ACCBAD01 lineage (accession No. OR341180) identified in ducks and turkeys was found to be identical (100% similarity) to Plasmodium sp. in a Tyto alba (barn owl) (accession No. MK390829.1) and an Accipiter badius (shikra) (accession No. JN639001.1), both from Thailand. The ORW1 lineage (accession No. OR341179) identified in ducks and turkeys had 100% similarity to Plasmodium sp. in a Gyps bengalensis (white-rumped vulture) from India (accession No. EF552403.1) and a Pycnonotus sinensis (light-vented bulbul) from China (accession No. KJ145050.1). The GALLUS01 lineage (accession No. OR341181) identified in turkeys was closely related (96.63-100% similarity) to P. gallinaceum in a Gallus gallus (chicken) from Thailand (accession No. LC506179.1). Under accession No. OR341182, the GALLUS02 lineage identified in turkeys was 99.56% identical to P. juxtanucleare in a Gallus gallus spadiceus (Burmese red junglefowl) from Thailand (accession No. KU248845.1) and another from Malaysia (accession No. KT290918.1).

A duck was the source of the ANAPLA lineage (accession No. OR341178), which was highly (99.78%) similar to Plasmodium sp. in a Tyto alba (accession No. MK390829.1) and an Accipiter badius (accession No. JN639001.1), both from Thailand. The MELGAL lineage (accession No. OR341183) identified in a turkey was a very close match (99.77% similarity) to P. gallinaceum in a Gallus gallus from Thailand (accession No. LC506179.1). Finally, the lineage designated FANTAIL01 (accession No. OR341184) identified in geese was identical (100% similarity) to Plasmodium sp. in a Rhipidura rufifrons (rufous fantail) from Australia (accession No. AY714196.1) and Milvus spp. (kites) from Spain (accession No. HF543648.1).

Phylogenetic analysis showed that the Plasmodium spp. could be divided into three main clades – Clade A, Clade B and Clade C. Most of the Plasmodium examples isolated from this backyard bird population belonged to Clades A and B, while Clade C contained a sequence of Plasmodium sp. from geese. Clade A consisted of the ORW1 lineage detected in ducks and turkeys and the GALLUS02 lineage detected in turkeys. Clade B contained the ACCBAD01 lineage detected in ducks and turkeys, the GALLUS01 lineage detected in turkeys, the ANAPLA lineage detected in ducks and the MELGAL lineage detected in turkeys (Fig. 3). The results showed that P. gallinaceum and P. juxtanucleare were found only in turkeys, while unidentified Plasmodium was found in turkeys, ducks and geese. However, the unidentified Plasmodium in geese was genetically different from the unidentified Plasmodium in ducks and turkeys.

Fig. 3.

Concerning PCV values, the mean averages in each animal species were in the normal range. Although the infected groups showed a lower trend of PCV, the results revealed no statistical difference between the infected and uninfected groups (Table 3). Moreover, there were no clinical signs in any poultry infected with Plasmodium spp.