Pathology and molecular characterization of Leucocytozoon caulleryi from backyard chickens in Khon Kaen Province, Thailand

Abstract Aim: The aim of this study was to characterize Leucocytozoon caulleryi from backyard chickens in Khon Kaen Province, Thailand. Materials and Methods: Tissue samples were collected from backyard chickens suspected to have leucocytozoonosis and subjected to histopathology examination. The BLAST Tool at NCBI GenBank (Basic Local Alignment Research Tools) (http://www.ncbi.nlm.nih.gov/BLAST) was used to identify the nucleotide sequence of the partial cytochrome c oxidase subunit I (cox I) gene. A Phylogenetic tree for analysis of L. caulleryi was constructed by using MEGA 7.0 software (https://www.megasoftware.net/). Results: The necropsy results revealed the subcutaneous hemorrhages of pectoral muscles, multifocal hemorrhages of the thymus and pectoral muscles, hemorrhage of the proventriculus and peritoneal cavity, and megaloschizonts of the pancreas and intestine, including subcapsular hemorrhages of the liver. Microscopic examination revealed numerous megaloschizonts of Leucocytozoon spp. in the pectoral muscles, intestine, pancreas, and thymus. Molecular analysis of the partial cox I gene showed that the causal agent was closely related to L. caulleryi reported in Japan. Conclusion: From these results, L. caulleryi was determined to be the causal agent of leucocytozoonosis and was closely associated with L. caulleryi reported in Japan.

Infected birds exhibit acute clinical signs such as internal bleeding, anemia, lethargic, diarrhea, pallor, and experience mortality and reduced egg production [6,7]. The schizogonies and sporozoites stages are transmitted through the salivary glands of an insect vector, and developed to the first generation of schizonts in various organs following invasion of the vascular endothelium. The second generation of megaloschizonts was identified using histopathological procedures. At the gametogony stage, merozoites are released from megaloschizonts and transformed into gametocytes in erythrocytes [8]. Therefore, leucocytozoonosis can be diagnosed by microscopic examination, and identified from stained blood or infected tissue sections, or using molecular assays [9,10]. In general, infection with Leucocytozoon spp. is identified using polymerase chain reaction (PCR), which is more sensitive than microscopic examination because the DNA of the parasite can be detected even when gametocytes are not found in thin blood smears [11]. There are no drugs available to treat avian leucocytozoonosis. As previously reported, treatment with pyrimethamine combined with sulfadimethoxine was found to be partially effective, while primaquine was active against Leucocytozoon spp. gametocytes [11]. Vaccination was carried out with the second generation schizonts and oil-adjuvant recombinant R7 that presented effective results in laboratory and field trials [12,13].
The L. caulleryi mitochondrial genome encodes three protein-coding genes, cytochrome b (cyt b), Available at www.veterinaryworld.org/Vol.14/October-2021/8.pdf cytochrome c oxidase subunits I (cox I) and III (cox III), and large subunit rRNA genes [14]. The cyt b and cox I genes have been extensively used for the detection of avian L. caulleryi [15,16]. The cox I gene is a highly conserved mitochondrially encoded protein and so is frequently used for species determination and phylogenetical analysis studies [17].
The objectives of this research were to identify the species of Leucocytozoon and study the phylogeny of Leucocytozoon calleryi from backyard chickens in Thailand. Gross and microscopic examination and molecular techniques were used to characterize chicken leucocytozoonosis cases.

Ethical approval
The study was approved by the Institutional Animal Care and Use Committee of Khon Kaen University, based on the Ethics of Animal Experimentation of National Research Council of Thailand, No: IACUC-KKU-55/64.

Study period, location, and sampling
In June 2018, five 12-to-18-day-old carcasses of backyard chickens from Khon Kaen Province in Thailand were subjected for necropsy at the Veterinary Diagnostic Laboratory, Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen, Thailand. These birds were submitted from a flock with a history of depression, subcutaneous hemorrhages, and mortality. The mortality was 100% within 1 week.

Gross histopathology
Tissue samples from lung, heart, liver, thymus, pectoral muscle, proventriculus, gizzard, intestine, and pancreas were collected from infected chicken carcasses. The tissues were fixed with 10% neutral buffered formalin solution and embedded in paraffin sections that were stained with hematoxylin and eosin for histopathological examination.

DNA extraction and PCR assays
Genomic DNA was extracted from tissue of the infected chicken using NucleoSpin Tissue, Mini kits for DNA (Macherey-Nagel, Düren, Germany), following the manufacturer's instructions. PCR reactions were performed with a pair of primers, as described previously, to amplify the 588 bp mitochondrial cox I gene [18]. The PCR reaction was carried out in a DNA thermal cycler (Major Cycler, Major Science, Taiwan) using DreamTaq Green PCR Master Mix (2X) (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Briefly, 3 μL of the DNA template was added to 22 μL of PCR reaction mixture consisting of 12.5 μL of DreamTaq Green PCR Master Mix solution (Thermo Fisher Scientific, Waltham, MA, USA), 0.5 μL each of 10 μM forward and reverse primers, and 8.5 μL of nuclease-free water. Thermal cycling was performed with an optimized profile of initial denaturation at 95°C for 5 min, followed by 40 cycles at 95°C for 30 s, 55°C for 30 s, and 68°C for 60 s, and a final extension at 68°C for 10 min. The PCR products were analyzed using electrophoresis in 1.2% agarose gels containing RedSafe Nucleic Acid Staining Solution (JH Science, Kirkland, WA, USA). The gels were run at 100 volts for 30 min and visualized using ultraviolet light in a Gel Doc™ XR+ Gel Documentation System (Bio-Rad, Hercules, CA, USA). The PCR products of the PCR-positive samples were reamplified for gel purification using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions, and subjected to direct sequencing by First BASE Laboratories Sdn Bhd, Seri Kembangan, Selangor, Malaysia.

Gross histopathology
Gross pathology of the birds showed subcutaneous hemorrhages of the wings and legs, including multifocal hemorrhages in the pectoral and thigh muscles, thymus and bursa of Fabricius, with friable hepatic lobes and splenomegaly. Two birds were found to have subcapsular liver hemorrhages. Other lesions included petechiae of the lung, heart, proventriculus, gizzard, pancreas, liver, and kidney in some birds (Figure-1). The pectoral muscles, intestine, pancreas, and thymus carried numerous Leucocytozoon megaloschizonts and merozoites (Figure-2).

Phylogenetic analysis
A total of 588 bp of PCR products were successfully amplified (Figure-3) and all five DNA sequences were identical to the cox I gene of L. caulleryi. The obtained sequence was deposited in GenBank with the accession number MK903018. Sequence analysis showed 100% nucleotide sequence homology with the reference strain, and complete identity with the L. caulleryi mitochondrial DNA genome from Japan (GenBank accession no. AB302215.1) (Figure-4). Available at www.veterinaryworld.org/Vol.14/October-2021/8.pdf

Discussion
The objective of this study was to characterize L. caulleryi infecting backyard chickens using gross histopathology and molecular techniques. Gross lesions were found in various organs examined, including subcutaneous hemorrhages of pectoral muscles, legs and wings, subcapsular liver hemorrhage, and hemorrhages in pectoral and thigh muscles and the thymus.
These lesions were similar to those reported from a previous study of broiler breeders in South Korea, which found subcutaneous hemorrhages in the legs and wings, thigh and pectoral muscles, thymus, heart,   pancreas, and kidneys [2]. Leucocytozoonosis cases in South Korea present with gross lesions of the friable hepatic lobes, splenomegaly, and degenerated ovaries and oviducts in layer chickens from commercial farms [19]. These lesions cause high mortality, causing economic losses to the poultry industry.
L. caulleryi frequently causes lethal hemorrhagic disease in chickens [6], and anemia due to the destruction of gametocytes, erythrocytes, and the vascular endothelium. Five birds in this report died with clinical signs, including depression and subcutaneous hemorrhage. The clinical signs of protozoal disease and mortality result from anemia produced by antierythrocytic factors, as large numbers of gametocytes block pulmonary capillaries or parasites invade the vessels and vascular endothelium of important organs such as the brain and heart. The parasites form megaloschizonts that block the vessels, resulting in multifocal necrosis [8]. L. caulleryi infection in layer chickens causes mortality and reduced egg production [20,21]. In Thailand, there have been reports of the prevalence of blood parasites, including L. caulleryi, in backyard chickens and fighting cocks [22,23].
Microscopic histopathological examination is used to provide definitive diagnoses of L. caulleryi infections in chickens [24,25]. Histopathological studies of L. caulleryi in this study revealed numerous megaloschizonts in organs such as the pancreas, intestine, lung, and pectoral muscle. Petechiae were found in muscle tissue and megalozchizonts in spleen, liver, lung, and heart [26].
Molecular analysis of a cox I gene indicated that L. caulleryi in this study was phylogenetically identical to those previously reported in Japan, where avian leucocytozoonosis is endemic [3,14]. In this study, DNA was directly extracted from the tissue samples.
Other studies have separated the gametocytes of L. caulleryi from whole blood using flow cytometry, and the gametocytes might be used for further genetic analyzes [27]. These results indicated that molecular techniques were useful for the diagnosis of diseases and the development of intervention strategies for avian leucocytozoonosis.
The arthropod vectors and their population density were considered for disease control and the eradication in vectors of backyard and free-range chickens. C. arakawa has been identified in chickens positive for Leucocytozoon spp. reared in open houses [5]. The prevalence of this protozoal disease frequently increases during the spring season due to arthropod vectors such as black flies (Simulium spp.) and biting midges (Culicoides spp.) [28]. Study of Culicoides spp. have been reported in several countries in Asia with climates suitable for the survival and reproduction of insect vectors [29]. The prevalence of leucocytozoonosis in chickens increases in late summer or early in the rainy season because this period facilitates Culicoides arakawae distribution throughout Asian countries. In Thailand, the prevalence of infection with blood parasites in backyard chickens was highest in the summer season, even higher than the rates of infection in the rainy season [22]. Simulium asakoae complex and Simulium chumpornense have been reported to be possible vectors of Leucocytozoon in poultry and wild birds in Thailand [29]. Thus, the occurrence of avian leucocytozoonosis is related to the distribution of C. arakawae [5], and disease control in chickens requires the elimination of insect vectors from the environment [30].

Conclusion
Chickens that died suddenly showed clinical signs of depression and pallor, and gross lesions, Available at www.veterinaryworld.org/Vol.14/October-2021/8.pdf including subcutaneous hemorrhages, multifocal hemorrhages of various organs. L. caulleryi was determined to be the causal agent and was very similar to the strain reported in Japan. Histopathological examination and molecular analysis are valuable tools for the detailed investigation of avian leucocytozoonosis.

Authors' Contributions
SJ, TP, and SJ: Designed the work. SJ and TP: Collected the samples. SJ: Carried out the histopathology laboratory experiment and drafted the manuscript. All authors read and approved the final manuscript.