In August 2019, five adult black swans from a wetland park of Beijing died within a week in succession. Before they died, no obvious symptoms were observed except mild diarrhea.
The fresh carcasses were sent to the National Research Center for Wildlife Borne Diseases for postmortem and histopathological examination. At routinely pathological investigation, the ceca were swollen and the mucosa were hemorrhages and anabrosis (Fig. 1a). A large number of moving trophozoites were observed by microscopic examination. The livers were enlarged and accompanied by the color turned dark red and the edge was blunt (Fig. 1b). No visible lesions were found in other organs. Histopathological examination showed that cecal hemorrhage, intestinal villi edema, disordered arrangement, epithelial cells exfoliated, and many parasites were found in lamina propria (Fig. 2a). Vacuolar degeneration of hepatocytes and interlobular bile duct hyperplasia were observed in the liver tissues. A large number of mononuclear inflammatory cells infiltrated between the liver lobules, and the fibrous tissue proliferated moderately (Fig. 2b).
Histological sections from the livers and ceca of the birds were further processed for in situ hybridization (ISH) using the described probe specific for T. gallinarum and H. meleagridis [12, 13]. The positive signals with the T. gallinarum probe were found in the caeca (Fig. 2c) but not in the livers (Fig. 2d). The result of ISH in the caeca and livers showed no signal with the H. meleagridis probe.
Using two trichomonad primer sets, TFR1/R2 and 18S-F/R, the ITS and 18S rRNA region of the isolates were successfully amplified with specific single band size of approximately 350 bp and 600 bp in the gel [14, 15] (Fig. 3), respectively. Notably, the PCR products were subcloned into T-vectors before sequencing to ensure that the specific sequences be successfully sequenced. Both sequences were clustered with the reference sequences of T. gallinarum download from GenBank database under phylogenetic analyses (Fig. 4a, b).
Other potential pathogens, such as Coccidia spp., Blastocystis spp. and hepatitis E virus were negative using the method previously reported [16,17,18].
Taken together, after eliminating potential pathogens, such as H. meleagridis, Coccidia, Blastocystis spp., hepatitis E virus as well as pathogenic bacteria, the presence of T. gallinarum was eventually confirmed by microscopic examination, histopathology, specific PCR amplification and ISH. Therefore, the death of the black swan was likely to be caused by T. gallinarum.