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

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.

Conventional diagnostic method for Leucocytozoon sp.infection is direct microscopic observation of the circulating gametocytes in Giemsa-stained blood smear.In addition, detection of parasites DNA or RNA using polymerase chain reaction (PCR) method utilizing primers derived from parasites mitochondrial cytochrome b (cytb) gene could be more common reliable and widely used in diagnosing the infections particular in laboratory for high sensitivity and specificity even when blood smears are negative in case of low parasitemia or early stage of infection in animals 9,12,[20][21][22] .Little is known about the prevalence of Leucocytozoon sabrazesi in Thailand.The aim of this study was to further investigate the occurrence and genetic diversity of this parasite in chickens from three regions (northern, western and southern) of Thailand.Likewise, haplotype diversity and entropy analysis among the isolated sequences discriminated in this study and those from other countries are investigated.These results could provide the information on the genetic diversity structure of this parasite's population for further development of the immunodiagnostic methods and vaccine strategies.

Occurrence of L. sabrazesi infection in chicken blood samples
Of the 313 chicken blood samples were examined under light microscope to observe the intra-erythrocytic L. sabrazesi gametocytes in blood smears (Fig. 1).Two hundred and fifty-two blood samples out of 313 (80.51%) were positive as investigated by the PCR analysis targeting cytb gene of L. sabrazesi (Table 1).The representative PCR products of parasite examined were 248 bp.The highest occurrence of L. sabrazesi infection (116/127; 91.34%) was found in Chiang Rai province, followed by Kanchanaburi province (72/86; 83.72%) and Phatthalung province (64/100; 64%) as shown in Table 1.The association between the occurence of L. sabrazesi infection and the factors, i.e., age (4, 4-12 and > 12 months), management system (backyard in households, free-range farms and non-evaporative cooling houses) and water sources (pond in the farm and river) nearby were considered to be statistically significant (p < 0.05), while the significant different was not found counting upon the chicken's breed (native and layer), gender (male and female), type of feeds (commercial and natural) and insect control system (Table 2).

Phylogenetic and similarity analysis of L. sabrazesi cytb gene sequences
In this study, the phylogenetic tree based on the alignment of the 15 Thailand sequences of L. sabrazesi cytb gene with 6 other sequences taken from the GenBank were classified as 2 clades.Clade 1 was divided into two subclades (subclade 1-1 and subclade 1-2).The sequences assigned to subclade 1-1 exhibited the genetic variability of L. sabrazesi cytb gene sequences from Chiang Rai and Kanchanaburi provinces of Thailand together with sequences from Malaysia, Thailand and Myanmar obtained from the GenBank.Subclade 1-2 was composed of two sequences from Kanchanaburi province and showed phylogenetic proximity.Five sequences from Phatthalung province of Thailand comprised clade 2 and also showed phylogenetic proximity.The cytb gene sequences among L. sabrazesi were highly conserved when compared with other strains as outer groups (Fig. 2).In addition, the similarity ranged between 89.5 and 100% for Thailand cytb sequences.The similarity of subclade 1-1 and subclade 1-2 was in the range of 93.3-99.5% and 94.3-100% among the Thailand L. sabrazesi sequences, respectively, while the similarity of the sequences within 2nd clade ranged between 89.5 to 100% (Table 3).The nucleic acid substitution rate in cytb sequences among L. sabrazesi was estimated under the Tamura and Nei 23 mode as shown in Table 4.

Haplotype diversity
The TCS Network tool was employed to construct the haplotype network of L. sabrazesi cytb gene sequences.Haplotype diversity based on cytb gene found in Chiang Rai, Kanchanaburi and Phatthalung provinces of Thailand was diverse when compared to worldwide gene sequences (Fig. 3).In Thailand, a total of 13 different haplotypes were analyzed including the 5 sequences (haplotype #1 to #3 and #8), 5 sequences (haplotype #4 to #7) and 5 sequences (haplotype #9 to #13) taken from chickens in Kanchanaburi, Phatthalung and Chiang Rai provinces, respectively.Bearing in mind, haplotype #4 was composed of 2 identical sequences (PT1 and PT2) and haplotype #8 was also comprised of SY1 and SY5 sequences (Fig. 3A and Table 5).In addition, 18 haplotypes indicated in TCS network exhibited that haplotypes #1 to #8, #12, #14 to #18 were detected in chicken in three provinces of Thailand.The rest of haplotypes was detected in other countries worldwide (Fig. 3B and Table 5).

Discussion
Leucocytozoonosis is an important disease causing clinical infection in chickens in many areas of the world, based on widespread distribution of the vector simuliid flies or culicoides midges.There are many conventional methods for detection of Leucocytozoon infection in chickens, such as clinical signs, hematological findings, microscopic examination of mature gametocytes in blood smears and serological tests.However, these methods require expertise since parasites are often missed when parasitemia are significantly low.Accurate diagnosis is needed to develop for appropriate treatment, transmission control and disease management.Thus, molecular assay, i.e., nested PCR, is a sensitive diagnostic method and used for detection and characterization of parasite DNA strains after gametocytes disappeared in host's blood circulation.
Up to now, the detection of Leucocytozoon infection in chickens has only been reported in some areas of Thailand 18,19 .In addition, there is an obvious lack of relevant information on leucocytozoonosis isolated in chickens in Thailand.Our study is the first investigation that demonstrated a molecular occurrence of L. sabrazesi infection in chickens throughout Chiang Rai, Kanchanaburi and Phattalung provinces of Thailand.
In this study, our findings revealed a high molecular occurrence of L. sabrazesi in chicken blood samples in three regions (northern, western and southern) of Thailand.The molecular detection exhibited that of the animal sampled, 80.51% (252/313) were positive for L. sabrazesi based on the cytb gene (Table 1).In our work, the cytb gene has been used as a good marker for providing sufficient variation to establish phylogeographic patterns on the large scale and useful for epidemiological approaches of leucocytozoonosis 24,25 .The occurrence of L. sabrazesi infections was highest in chickens aged < 4 months (95%) compared to chickens aged 4-12 months (90.11%) and > 12 months (54.95%), which is in line with the previous studies by Naqvi et al. 26 , who showed the www.nature.com/scientificreports/highest prevalence in chickens aged less than 6 months (24 weeks) was 78.8%.From our findings, it is possible that the intensive infection could be consistent with free-range farm system showing the higher statistically significant infection than other systems.Likewise, the climate condition, such as rainfall, are often involved with the occurrence of L. sabrazesi infection due to the longer survival of the parasite vectors (black flies and biting midges) 27 .Moreover, western and southern regions of Thailand have a long rainy season (about 3-4 months);   www.nature.com/scientificreports/this tropical area is a significant influence on the risk of L. sabrazesi infection and also seems to be a predisposing factor for the development of the parasite vectors which can survive in humid environment 9,26,[28][29][30] .
Although the genetic diversity of Leucocytozoon sp.based on the sequences of cytb gene has been investigated in several countries 8,12,14,16,22,25,31 , little is known about the genetic diversity and the phylogeny of L. sabrazesi Thailand strain.In this study, the cytb gene in chicken population sampled in the northern, western and southern areas of Thailand was employed to determine the genetic diversity of L. sabrazesi in these regions.The phylogenetic analysis of chicken L. sabrazesi cytb gene Thailand isolate showed two clades together with the sequences from Malaysia and Myanmar.Our results exhibited that the genetic diversity observed in a phylogram was confirmed by the high similarity value for L. sabrazesi cytb gene (89.5-100%).This finding indicated high conserved sequences and phylogenetic proximity of cytb gene circulating in both Thailand and other countries.
In this work, the cytb gene sequence in blood samples of chickens in the northern, western and southern parts of Thailand were analyzed.Our results showed that the L. sabrazesi population was low diverse in Thailand, with the presence of probably more than one haplotype.The genotype of this gene was identified in haplotype networks.It was carried out with sequences detected in this study together with other sequences obtained from GenBank database that found in Malaysia and Myanmar.In this study, there has been mild different of morphological traits when compare the sequences from Chiang Rai haplotype to other haplotypes.Because Chiang Rai sequence has more prevalence and some chickens show pale comb or skinny correlate to amount of gametocytes in the blood smear.The severity of high prevalence is probably associated with areas where parasite's vectors are abundant.The more prevalent haplotype is haplotype#12, which is 0-4 month age chickens.This indicated that there was some genetic diversity of cytb gene observed in the different haplotype networks in Thailand and other countries.Furthermore, the cytb sequence shared genetic traits with all sequences as ascertained previously from Malaysia and Myanmar.This finding exhibited that the genetic diversity among L. sabrazesi populations varied in accordance with the geographical area.
Regarding the analysis of L. sabrazesi nucleic acid sequences, our results approved the polymorphism with 26 entropy peaks reaching up to 1.21 (sequence within Thailand) and 1.04 (sequence worldwide).In addition, our results showed 5 entropy peaks of amino acid sequences reaching up to 0.97 (sequence within Thailand) and 0.84 (sequence worldwide).These indicated that different genotypes may involve being a genetic diversity of L. sabrazesi distribution in Thailand.

Conclusions
This study is the first report indicating a molecular occurrence and genetic diversity of L. sabrazesi in chicken blood samples in Thailand.Our findings showed that L. sabrazesi cytb gene is genetically conserved in Thailand and other countries.These could help to ameliorate the understanding of phylogeny and genetic diversity among cytb gene of L. sabrazesi Thailand strain.Therefore, the periodical assessment of the occurrence of leucocytozoonosis is necessitate to control the affectivity of the effective treatment and prevention throughout the country to reduce the infection of chicken vector-borne parasites.

Study locations and sample sizes
The present study was conducted in three regions of Thailand (Fig. 5).Chicken blood samples were collected in Mae Suai district (19°39′24″N, 99°32′30″E) of Chiang Rai province in northern region, in Sai Yok district (14°6′56″N, 99°8′40″E) of Kanchanaburi province in western region, and in four districts including Srinagarindra (7°34′24″N, 99°56′30″E), Khuan Khanun (7°44′6″N, 100°0′36″E), Kong Ra (7°24′12″N, 99°57′0″E) and Mueang Phatthalung (7°37′6″N, 100°4′24″E) of Phatthalung province in southern region.Total 313 blood samples were randomly collected from the chickens raised in both backyard in households, free-range farms and nonevaporative cooling houses during December 2019 and October 2020.The sample sizes were calculated using the Table 4.The nucleic acid substitution rate in L. sabrazesi cytb sequences as detected in chickens in Thailand and other countries.Each entry is the probability of substitution from one base (row) to another base (column).Rates of different transitional substitutions are shown in bold and those of transversional substitutions are shown in italics.The maximum Log likelihood for this computation was − 554.346 and − 588.782 for the sequences within Thailand and worldwide, respectively.www.nature.com/scientificreports/formula based on an equation, n = t 2 × p(1 − p)/m 2 , inserting the following values: the prevalence of L. sabrazesi infection among chickens in Thailand (p) 18,19 , a 95% confidence level (t) and 5% margin of error (m).

Collection of blood samples and microscopic examination
Between 0.3 and 2 ml of blood was collected via either the brachial wing vein or medial metatarsal vein of each chicken.They were kept into the centrifuge tube containing lithium heparin to preclude coagulation, and stored www.nature.com/scientificreports/ at − 80 °C until further used.In addition, some thin fresh blood smears were prepared for each chicken on glass slides, air-dried, fixed in 100% methanol for 1 min, and then stained with Giemsa stain (SIGMA-ALDISH, Germany) for 40 min.

DNA extraction
Genomic DNA of L. sabrazesi was extracted from all collected blood samples of chickens using Tissue DNA Extraction Kit (OMEGA, bio-tek, USA) following the protocol of Watthanadirek et al. 32 and Junsiri et al. 33 with some modifications.Briefly, 20 µl of blood samples were mixed with 25 µl of OB Protease solution and 250 µl of BL buffer and incubated at 70 °C for 1 min.After adding with 250 µl of 100% ethanol, samples were transferred to HIBIND DNA Mini Column.Finally, DNA samples were eluted in 50 µl Milli-Q water and kept at − 20 °C until used.The concentration and purity of DNA were defined with NANODROP 2000 Spectrophotometers (THERMO SCIENTIFIC) at 260/280 and 260/230 ratios.

Amplification and detection of L. sabrazesi DNA
The specific primer pairs designed from L. sabrazesi cytb sequence submitted in GenBank database under accession numbers AB299369.1,were utilized to amplify DNA fragments of the cytb gene.L. sabrazesi cytb gene was amplified by nested PCR using 2 pairs of specific primers.In the first step of amplification, the primers, namely LsF1 (5′-CAT ATA TTA AGA GAA TTA TGGAG-3′) and LsR1 (5′-ATA AAA TGY TAA GAA ATA CCA TTC -3′) were used.In the second step, the primers, namely LsF2 (5′-TAA TCA CAT GGG TTT GTG GA-3′) and LsR2 (5′-GCT TTG GGC TAA GAA TAA TACC-3′) were also used.The expected size of amplification products was 248 bp.PCR reaction mixtures consisting of 50 ng DNA template, 0.2 µM each of the primers, 0.125 mM of each deoxynucleoside triphosphate (dNTPs), 3 mM MgCl 2 , 0.25 U Tag DNA polymerase (NEW ENGLAND BIOLABS, UK), 1× standard Tag reaction buffer and nuclease free water, were put through in a thermal cycle (BIO-RAD, USA) with the following condition: 40 cycles of denaturation at 94 °C for 1 min (1st step) and for 20 s (2nd step), annealing at 50 °C for 1 min (1st step) and 53 °C for 20 s (2nd step), extension at 68 °C for 1 min (1st step) and for 30 s (2nd step) as well as a final extension at 68 °C for 5 min (1st and 2nd steps).PCR product was observed by 1.2% agarose gel stained with Fluorostain DNA Fluorescent Staining Dye (SMOBIO, Taiwan) and viewed under ultraviolet (UV) transilluminator.A 100 bp Plus DNA Ladder (THERMO FISHER SCIENTIFIC, USA) was used as standard for defining the molecular mass of PCR products.
Cloning of the mitochondrial cytb gene from L. sabrazesi DNA L. sabrazesi cytb gene was cloned into vector with following specific primers: LsF 5′-CAC CTA ATC ACA TGG GTT TGT GGA -3′ and LsR 5′-GCT TTG GGC TAA GAA TAA TACC-3′.The 4 nucleotides (CACC) were added at 5′ end of forward primer with the overhang sequence (GTGG) in pET100/D-TOPO ® vector (INVITROGEN, USA) to enable directional cloning.The PCR reaction was conducted with the protocols as described in previous section.PCR products were purified using ULTRACLEAN 15 DNA Purification Kit (MO BIO LABORATORIES, USA) following the manufacturer's instructions for cloning.The 20 ng of the blunt-end PCR products were inserted in the pET100/D-TOPO vector (INVITROGEN LIFE TECHNOLOGIES, USA).Then 3 µl of the cloning reactions were transformed into chemically competent Escherichia coli cells (INVITROGEN, USA).Subsequently, 200 µl of transformed bacterial culture was spread on the Luria Bertani (LB) agar plates containing 100 µg ampicillin and incubated for overnight at 37 ºC.The positive clones were selected and grown in LB medium containing ampicillin for overnight.Finally, the recombinant plasmids (pET100-cytb) were extracted from the competent cells using AXYPREP Plasmid Miniprep Kit (AXYGEN BIOSCIENCE, USA) following the manufacturer's instructions and analyzed for correctly sized inserts by agarose gel electrophoresis 32,33 .

Sequencing analysis
Purified PCR products were confirmed by Sanger method of DNA sequencing.All DNA sequences were analyzed by BLAST (The National Center for Biotechnology Information, NCBI, http:// www.ncbi.nlm.nih.gov/ BLAST).All sequences were deposited in GenBank, accession numbers are provided in Table 6.

Phylogenetic sequence analysis
Leucocytozoon sabrazesi cytb gene sequences were used for sequence alignment and phylogenetic analysis as shown in Table 6 and two gene sequences of Haemoproteus sp. were employed as the outer groups (accession

Figure 1 .
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 .
Figure2.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 .
Figure 3. TCS network of haplotypes based on the L. sabrazesi cytb gene sequences detected in Thailand (A) and other countries (B).

Figure 4 .
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 .
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).

Table 1 .
Summary of L. sabrazesi infection in chickens from Chiang Rai, Kanchanaburi and Phatthalung provinces analyzed by PCR assay.

Table 2 .
Factors associated with L. sabrazesi infection of chickens in Chiang Rai, Kanchanaburi and Phatthalung provinces.χ 2 = Chi-square; df = degree of freedom.

Table 3 .
Similarity of the L. sabrazesi cytb gene sequences as detected in chicken sampled in Thailand.

Table 5 .
Polymorphism and genetic diversity of L. sabrazesi cytb gene sequences as detected in chickens in Thailand and other countries.N: number of analyzed sequences; VS: number of variable sites; GC: G × C content; h: number of haplotypes; Dh: diversity of haplotypes; SD: standard deviation; π: nucleotide diversity (per site); K: average number of nucleotide differences.

Table 6 .
The L. sabrazesi cytb nucleotide sequences amplified in Thailand strains were deposited in GenBank database.