Morphological and molecular characteristics of Plasmodium juxtanucleare in layer chicken from three districts of Yogyakarta, Indonesia

Abstract

Background and aim: Blood parasite infections in poultry, such as Plasmodium, are a serious threat to the poultry industry due to their potential to cause economic losses. To date, there has been inadequate research on the morphological and molecular detection of the different Plasmodium species that infect poultry in Indonesia. Therefore, this study aimed to analyze the morphological and molecular characteristics of Plasmodium spp. and the several predisposing factors for Plasmodium infection in layer chickens from three districts of Yogyakarta, Indonesia.

Materials and methods: One hundred and five blood samples from layer chickens were collected from 13 farms located in three districts of Yogyakarta (Sleman, Bantul, and Kulon Progo) between September and November 2022. Blood samples were subjected to microscopic and polymerase chain reaction (PCR) analyses. Sequencing was performed using basic local alignment search tools to identify the nucleotide structure of cytochrome b. Phylogenetic analysis of Plasmodium was performed using the MEGA-X software.

Results: Microscopic examination revealed that 17/105 positives (16.19%) were positive for blood parasite infection. Trophozoites, erythrocytic meronts, and microgametocytes of Plasmodium were found in blood samples. Based on the morphological examination, the species found in the samples was close to Plasmodium juxtanucleare. Polymerase chain reaction examination revealed that 21/60 samples were positive for Plasmodium (35%). The Plasmodium species identified from the sequenced samples were proven to be P. juxtanucleare. The P. juxtanucleare from Thailand was closely related to samples (99.64%-100%) with a genetic distance of 0%-1%. In addition, age, population, and cage type were not significantly associated with Plasmodium infection.

Conclusion: Based on microscopic and PCR examinations, the Plasmodium species found in the three districts of Yogyakarta was P. juxtanucleare. The genetic distance between samples from the three districts of Yogyakarta was closely related (0%-1%) to P. juxtanucleare from Thailand and Japan. There was no correlation between Plasmodium infection and age, cage type, or population.

Keywords: avian malaria; cytochrome b gene; layer chicken; polymerase chain reaction.

Figure-1

Trophozoites phase of Plasmodium juxtanucleare (A); Microgametocyte (B); Erythrocytic meront (C).

Figure-2

Results of PCR on agarose gel with the percentage of parasitemia from each positive band. PCR negative results (Lanes 1 and 3); PCR-positive results (Lanes 2 and 4—10); positive control (P) from sample proved by sequencing; negative control (N).

Figure-3

Phylogenetic tree gene cytochrome b of samples, Plasmodium spp., Plasmodium relictum, Plasmodium juxtanucleare, and Plasmodium gallinaceum based on nucleotide sequence using Neighbor-Joining method along with Kimura 2 parameter. Sleman (SL); Bantul (BA); Kulon Progo (WA).

Figure-4

Phylogenetic tree gene cytochrome b of samples, Plasmodium spp., Plasmodium relictum, Plasmodium juxtanucleare, and Plasmodium gallinaceum based on protein sequence using Neighbor-Joining method along with Kimura 2 parameter. Sleman (SL); Bantul (BA); Kulon Progo (WA).