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Leucocytozoon sabrazesi is the intracellular protozoa of leucocytozoonosis, which is transmitted by the insect vectors and affects chickens in most subtropical and tropical regions of the globe, except South America, and causing enormous economic losses due to decreasing meat yield and egg production. In this study, L. sabrazesi gametocytes have been observed in the blood smears, and molecular methods have been used to analyse the occurrence and genetic diversity of L. sabrazesi in blood samples from 313 chickens raised in northern, western and southern parts of Thailand. The nested polymerase chain reaction (nested PCR) assay based on the cytb gene revealed that 80.51% (252/313) chickens were positive of L. sabrazesi. The phylogenetic analysis indicated that L. sabrazesi cytb gene is conserved in Thailand, showed 2 clades and 2 subclades with similarity ranged from 89.5 to 100%. The diversity analysis showed 13 and 18 haplotypes of the sequences from Thailand and from other countries, respectively. The entropy analyses of nucleic acid sequences showed 26 high entropy peaks with values ranging from 0.24493 to 1.21056, while those of amino acid sequences exhibited 5 high entropy peaks with values ranging from 0.39267 to 0.97012. The results; therefore, indicate a high molecular occurrence of L. sabrazesi in chicken blood samples with the associated factors that is statistically significant (p < 0.05). Hence, our results could be used to improve the immunodiagnostic methods and to find appropriate preventive control strategies or vaccination programs against leucocytozoonosis in order to mitigate or eliminate the harmful impact of this infection on chicken industry.

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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.

Babesia bovis and B. bigemina are the most common tick-borne parasites that cause bovine babesiosis which effects livestock production, leading to economic losses in tropical and subtropical areas of the world. The aims of this study were to determine the molecular detection, genetic diversity and antigenicity prediction of B. bovis based on spherical body protein 2 (sbp-2) gene and B. bigemina based on rhoptry-associated protein 1a (rap-1a) gene in cattle in Thailand. By PCR assay, the molecular detection of B. bovis and B. bigemina infection revealed levels of 2.58% (4/155) and 5.80% (9/155), respectively. The phylograms showed that B. bovis sbp-2 and B. bigemina rap-1a sequences displayed 5 and 3 clades with similarity ranging between 85.53 to 100% and 98.28 to 100%, respectively, when compared within Thailand strain. Diversity analysis of sbp-2 and rap-1a sequences showed 18 and 4 haplotypes, respectively. The entropy analysis illustrated 104 and 7 polymorphic sites of sbp-2 and rap-1a nucleic acid sequences, respectively, while those of sbp-2 and rap-1a amino acid sequences showed 46 and 4 high entropy peaks, respectively. Motifs analysis exhibited the distribution and conservation among sbp-2 and rap-1a sequences. The continuous and discontinuous B-cell epitopes have also been evaluated in this work. Therefore, our findings may be used to ameliorate the understanding inputs of molecular phylogeny, genetic diversity and antigenicity of B. bovis and B. bigemina Thailand stains.

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