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. 2022:29:22.
doi: 10.1051/parasite/2022022. Epub 2022 Apr 27.

Molecular genetic diversity and bioinformatic analysis of Leucocytozoon sabrazesi based on the mitochondrial genes cytb, coxI and coxIII and co-infection of Plasmodium spp

Pornpiroon Nooroong  1 , Amaya Watthanadirek  1 , Sutthida Minsakorn  1 , Napassorn Poolsawat  1 , Witchuta Junsiri  1 , Nitipon Srionrod  1 , Siriphan Sangchuai  2 , Runglawan Chawengkirttikul  3 , Panat Anuracpreeda  1
Affiliations

Molecular genetic diversity and bioinformatic analysis of Leucocytozoon sabrazesi based on the mitochondrial genes cytb, coxI and coxIII and co-infection of Plasmodium spp

Pornpiroon Nooroong et al. Parasite. 2022.

Abstract
in English, French

Leucocytozoon sabrazesi is an intracellular haemoprotozoan parasite responsible for leucocytozoonosis, which is transmitted by insect vectors and affects chickens in tropical and subtropical areas in many countries. It causes huge economic losses due to decreased meat and egg production. In the present study, we used nested PCR to determine the genetic diversity of L. sabrazesi based on the cytb, coxI, coxIII and concatenated genes in chickens in Thailand. In addition, we found co-infections between L. sabrazesi and Plasmodium spp. (P. gallinaceum or P. juxtanucleare) in chickens that were not identified by microscopic examination of blood smears. The phylogenetic analysis indicated that L. sabrazesi cytb and coxIII genes were conserved with similarity ranging from 99.9 to 100% and 98 to 100%, respectively whereas the coxI gene was diverse, with similarities ranging from 97 to 100%. These findings ascertained the nucleotide analysis of the cytb, coxI, coxIII and concatenated sequences in which 4, 8, 10 and 9 haplotypes were found, respectively. In addition, it was found that the large number of synonymous substitutions and conservative amino acid replacements in these mitochondrial genes occurred by non-synonymous substitution. The evolutionary analysis of the Ka/Ks ratio supported purifying selection and the negative values of both Fu's Fs and Tajima's D indicate selective sweep especially for the coxI gene. The entropy and Simplot analysis showed that the genetic variation in populations of Plasmodium spp. was higher than in Leucocytozoon. Hence, the nucleotide sequences of three mitochondrial genes could reflect the evolutionary analysis and geographic distribution of this protozoan population that switches hosts during its life cycle.

Title: Diversité génétique moléculaire et analyse bioinformatique de Leucocytozoon sabrazesi basée sur les gènes mitochondriaux cytb, coxI et coxIII et la co-infection avec Plasmodium spp.

Abstract: Leucocytozoon sabrazesi est le parasite hémoprotozoaire intracellulaire responsable de la leucocytozoonose, qui est transmise par des insectes vecteurs et affecte les poulets dans les zones tropicales et subtropicales de nombreux pays. Il provoque d’énormes pertes économiques en raison de la diminution de la production de viande et d’œufs. Dans la présente étude, nous avons utilisé la PCR nichée pour déterminer la diversité génétique de L. sabrazesi sur la base des gènes cytb, coxI, coxIII et concaténés chez des poulets en Thaïlande. De plus, nous avons trouvé des co-infections entre L. sabrazesi et Plasmodium spp. (P. gallinaceum ou P. juxtanucleare) chez des poulets, qui n’ont pas été identifiées par l’examen microscopique de frottis sanguins. L’analyse phylogénétique a indiqué que les gènes cytb et coxIII de L. sabrazesi étaient conservés avec une similarité allant respectivement de 99,9 à 100 % et de 98 à 100 %, alors que le gène coxI était diversifié, avec des similarités allant de 97 à 100 %. Ces découvertes ont confirmé l’analyse des nucléotides des séquences cytb, coxI, coxIII et concaténées dans lesquelles 4, 8, 10 et 9 haplotypes ont été trouvés, respectivement. De plus, il a été constaté que le grand nombre de substitutions synonymes et de remplacements conservateurs d’acides aminés dans ces gènes mitochondriaux se produisaient par substitution non synonyme. L’analyse évolutive du rapport Ka/Ks a soutenu la sélection purificatrice et les valeurs négatives des Fs de Fu et D de Tajima indiquent un balayage sélectif, en particulier pour le gène coxI. L’entropie et l’analyse Simplot ont montré que la variation génétique de la population de Plasmodium spp. était plus élevée que pour Leucocytozoon. Par conséquent, les séquences nucléotidiques de trois gènes mitochondriaux pourraient refléter l’analyse évolutive et la répartition géographique de cette population de protozoaires qui changent d’hôte au cours de leur cycle de vie.

Keywords: Chickens; Co-infection; Genetic diversity; Leucocytozoon sabrazesi; Mitochondrial genes; Plasmodium spp.; Thailand.

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Figures

Fig. 1
Fig. 1
Phylogenetic tree of the cytb gene sequences in this study (bold face) and those taken from GenBank. The boostrap values calculated from NJ, ML, MP and BA are labeled on each branch.
Fig. 2
Fig. 2
Phylogenetic tree of the coxI gene sequences in this study (bold face) and those obtained from GenBank. The boostrap values calculated from NJ, ML, MP and BA are labeled on each branch.
Fig. 3
Fig. 3
Phylogenetic tree of the coxIII gene sequences in this study (bold face) and those taken from GenBank.The boostrap values calculated from NJ, ML, MP and BA are labeled on each branch.
Fig. 4
Fig. 4
Phylogenetic tree of the concatenated gene sequences in this study (bold face) and those obtained from GenBank. The boostrap values calculated from NJ, ML, MP and BA are labeled on each branch.
Fig. 5
Fig. 5
Entropy analysis of L. sabrazesi cytb, coxI and coxIII gene sequences. Entropy plot of multiple nucleic acid sequence alignment of the cytb (A), coxI (B) and coxIII (C) genes. The red peaks indicate the high variation at each position of the nucleic acid sequences. Entropy plot of multiple amino acid sequence alignment of CYTb (D), COXI (E) and COXIII (F). The red peaks indicate the high variation at each position of amino acid sequences.
Fig. 6
Fig. 6
Nucleic acid substitution rate and base composition of cytb, coxI, coxIII and concatenated sequences among Leucocytozoon spp. and Plasmodium spp. Tables showing the transition and transversion from nucleotide substitution in cytb (A), coxI (B.), coxIII (C) and concatenated (D) genes. Graph incidating the synonymous and non-synonymous substitutions of cytb (E), coxI (F), coxIII (G) and concatenated (H) genes of Leucocytozoon spp. and Plasmodium spp.
Fig. 7
Fig. 7
TCS network of haplotypes based on Leucocytozoon spp. and Plasmodium spp. cytb gene sequences (A) detected in Thailand and other countries. The number of bars on lines between a haplotype and another represent the number of nucleotide mutation (B).
Fig. 8
Fig. 8
TCS network of haplotypes based on Leucocytozoon spp. and Plasmodium spp. coxI gene sequences (A) detected in Thailand and other countries. The number of bars on lines between a haplotype and another represent the number of nucleotide mutation (B).
Fig. 9
Fig. 9
TCS network of haplotypes based on Leucocytozoon spp. and Plasmodium spp. coxIII gene sequences (A) detected in Thailand and other countries. The number of bars on lines between a haplotype and another represent the number of nucleotide mutation (B).
Fig. 10
Fig. 10
TCS network of haplotypes based on Leucocytozoon spp. and Plasmodium spp. concatenated gene sequences (A) detected in Thailand and other countries. The number of bars on lines between a haplotype and another represent the number of nucleotide mutation (B).
Supplementary Figure 1.
Supplementary Figure 1.
Alignment of nucleic acid sequences of the cytb gene among L. sabrazesi and Plasmodium spp. The highest similarity of nucleotide positions is represented with dark blue color, while white color represents the least similarity of each nucleic acid position (A). Multiple amino acid sequence alignment of CYTb protein among L. sabrazesi and Plasmodium spp. The highest similarity of physicochemical properties (BLOSUM score 62) of each amino acid position is represented with blue color, while white color represents the least similarity of each amino acid position (B).
Supplementary Figure 2:
Supplementary Figure 2:
Alignment of nucleic acid sequences of coxI gene among L. sabrazesi and Plasmodium sp.p The highest similarity of nucleotide positions is represented with dark blue color, while white color represents the least similarity of each nucleic acid position (A). Multiple amino acid sequence alignment of COXI protein among L. sabrazesi and Plasmodium spp. The highest similarity of physicochemical properties (BLOSUM score 62) of each amino acid position is represented with blue color, while white color represents the least similarity of each amino acid position (B).
Supplementary Figure 3:
Supplementary Figure 3:
Alignment of nucleic acid sequences of coxIII gene among L. sabrazesi and Plasmodium sp. The highest similarity of nucleotide positions is representd with dark blue color, while white color represents the least similarity of each nucleic acid position(A). Multiple amino acid sequence alignment of COXIII protein among L. sabrazesi and Plasmodium spp. The highest similarity of physicochemical properties (BLOSUM score 62) of each amino acid position is represented with blue color, while white color represents the least similarity of each amino acid position (B).
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