A farmer in Hanjiang District, Yangzhou City, Jiangsu Province, raised more than 500 60-day-old SH chickens. The chickens were free ranged in the backyard during the day and fed with rice and grass (Fig. 1A), and they entered the hen house freely at night. On 12 July 2015, a disease began to occur in the chicken flock, with symptoms that included crouched heads, drooping wings and the excretion of watery yellow-green diarrhea (Fig. 1B). In total, 15–20 chickens died daily. On 22 July 2015, after more than 200 chickens had already died, the owner visited the Animal Disease Diagnosis Center of the College of Veterinary Medicine, Yangzhou University, and there were still nearly 30 sick chickens. After a consultation and field investigation, the flock was vaccinated against infectious diseases, such as H5-subtype bird flu, Newcastle disease, infectious bronchitis and bursal disease, in accordance with local immunization procedures. Albendazole (Tongren Pharmaceutical Co. Ltd., Shanghai, China) was orally administered once to 45-day-old chickens at a dose of 20 mg/kg of body weight to expel parasites, and diclazuril (Baoling Animal Health Product Co. Ltd., Tianjin, China) was added to the drinking water at a dose of 0.5 ppm per chicken to prevent coccidiosis. Doxycycline (Tongren Pharmaceutical Co. Ltd., Shanghai, China) was used to treat the remaining sick chickens at a dose of 25 mg/kg of body weight, twice daily for 5 days, with no obvious effects.

Four dead chickens were dissected, and the pathological changes were similar. The livers were enlarged and ulcerated lesions with circular depressions had formed on the surface. The lesions were light yellow or yellowish green in the center, the peripheral edges were uplifted, and they had a unique crater-like shape (Fig. 1C). The ceca were swollen (Fig. 1D) and contained casein-like emboli. After the removal of the contents, necrosis and ulcers of the ceca were seen. No significant changes were found in other organs. No trematode, cestode, nematode or coccidial oocysts were found in the contents of the small intestines and ceca by conventional parasitic examinations.

Appropriate amounts of liver and cecal tissues were cut and observed in the hematoxylin and eosin stained tissue sections. Then, the sections were mounted with neutral balsam for microscopic examination. A large number of circular eosinophilic histomonad-like parasites appeared in the liver and cecal tissues (Fig. 2).

Approximately 1 g of necrotic tissues from diseased chicken livers were aseptically removed and placed into a sterile 1.5 ml Eppendorf tubes. The tissues were smashed and inoculated into 10 ml medium that was preheated to 40°C, and then incubated anaerobically in an incubator at 40°C. The medium consisted of 9 ml of M199 medium (Gibco, Grand Island, New York, USA), 1 ml of horse serum (Gibco), and 11 mg of rice flour (Sigma, St. Louis, Missouri, USA), and was inoculated with a ring of cecal bacteria grown on a Columbia blood agar plate. The cecal bacteria were obtained by inoculating Colombia blood agar plate with the cecal contents of healthy chickens and storing them overnight at 37°C. A microscopic examination after 36 hr revealed that the isolated parasites were round or oval. There were rice flour particles in the cells that were refractive, 10–20 μm in size, with a pendulum-like movement that was slow and weak (Fig. 3). Primers were designed targeting the H. meleagridis 18S rRNA gene (P1: 5′-GAAAGCATCTATCAAGTGGAA-3′ and P2: 5′-GATCTTTTCAA ATTAGCTTTAAA-3′) [7]. The primers were synthesized by Sangon Biotech (Shanghai, China). The parasite DNA was extracted using an EasyPure Genomic DNA Kit (TransGen Biotec, Beijing, China) and was identified by PCR. The sequencing of the 3 PCR products (each performed 3 times) showed 100% identity levels and that a specific fragment of 576 bp was amplified, named H. meleagridis SH (GenBank Accession No. MH449665). The 18S rRNA sequences were added to an existing database of 10 other parabasalid sequences, including strains isolated from different hosts (turkey and chicken) and regions (France, Australia and America), as well as 2 outgroup sequences. Accession numbers for the sequences used in the phylogenetic analyses are indicated in Fig. 4. A maximum likelihood analysis was carried out with MEGA. The sequence of H. meleagridis SH was 100% homologous to the 18S rRNA sequence of H. meleagridis (JX963670) in GenBank. Combining the morphological observations and phylogenetic analysis, the isolated parasites were identified as H. meleagridis.

The cloning was carried out using the limiting dilution method of monoclonal antibodies. The cultured parasites reaching their growth peak were diluted to ~10³ cells/ml with preheated medium, and 1–2 μl of the medium was drawn onto cover slips to observe the parasites under a microscope. Single parasites were labeled and transferred to Eppendorf tubes containing 1 ml of medium and anaerobically cultured in an incubator at 40°C. After 3–4 days of cultivation, the culture broth containing propagated parasites was inoculated into fresh basal medium at a 1:10 ratio for the propagation of the parasites. The cloned parasite strain was named HM-JSYZ-C and cryopreserved.

Five 14-day-old SH chickens (purchased from the Institute of Poultry, Chinese Academy of Agricultural Sciences, Jiangsu, China) were inoculated with the HM-JSYZ-C parasite strain through cloacal infection. These chickens were raised in an isolated environment after hatching and fed a drug-free feed formula with free access to water. Animal care and all experiments were performed according to the rules of the Animal Experiment Ethics Committee of Yangzhou University. The inoculum used was 10⁵ parasites/chicken. Another 5 chickens were inoculated with parasite-free medium as a negative control. After infection, morbidity and clinical symptoms were observed daily, and the chickens were killed after 15 days to observe cecal and liver lesions. The symptoms after inoculation with the HM-JSYZ-C strain in the chickens included depression and diarrhea. Two chickens died within 15 days, and the mortality reached 40%. In the infected group, crater-like circular ulcerative necrotic areas appeared on the liver surface of dead and surviving chickens, the cecal wall was thickened and intestinal emboli formed in the ceca (Fig. 5A). Chickens in the negative control group did not get sick or die, and no significant macroscopic lesions were observed in their livers and ceca (Fig. 5B).