Molecular detection and genetic diversity of Leucocytozoon sabrazesi in chickens in Thailand

Abstract

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.

Figure 1

Gametocytes of L. sabrazesi in chicken’s cells identified from Geimsa stained blood smear. The high magnification micrographs showed the elongated macrogametocytes (A), elongated microgametocytes (B) and rounded microgametocyte (the left side) and rounded and elongated macrogametocytes (the right side) (C). The arrows present parasites’ nuclei of macrogametocytes. The arrowheads indicate parasites’ nuclei of microgametocytes. The dashed arrows demonstrate hosts’ nuclei.

Figure 2

A Maximum Likelihood (ML) phylogenetic tree of L. sabrazesi cytb gene sequences in this study (blodface) and those taken from GenBank. The numbers on each node correspond to the bootstrap analysis of 1000 replicates (percentage more than 50% were listed). The GenBank assession numbers, the country and the parasite species name of the sequences are exhibited. Two gene sequences of Haemoproteus sp. were employed as the outer groups. The scale bar exhibits the number of substitutions per site.

Figure 3

TCS network of haplotypes based on the L. sabrazesi cytb gene sequences detected in Thailand (A) and other countries (B).

Figure 4

Nucleic and amino acid entropy plots obtained from L. sabrazesi cytb gene sequences. Entropy plot of nucleic acid sequences from Thailand (A) as well as Thailand and other countries (B). Entropy plot of amino acid sequences from Thailand (C) as well as Thailand and other countries (D).

Figure 5

Geographical location of northern, western and southern regions where chicken blood samples are collected and examined. Legends indicate the distribution of Leucocytozoon sabrazesi Thailand strains discriminated in chickens from Mae Suai district (MS) in Chiang Rai province (CR), Sai Yok district (SY) in Kanchanaburi province (KB) and Srinagarindra (SD), Khuan Khanun (KK), Kong Ra (KR) and Mueang Phatthalung (MP) districts in Phatthalung province (PL).