Tetratrichomonas gallinarum is parasitic protozoa with a wide host range. However, its lethal infection is rare reported.
Here, we described the first lethal cases of T. gallinarum infection in black swans in China. Five black swans died within a week in succession without obvious symptoms except mild diarrhea. At necropsy, severe lesions were observed in caeca with thickened caecal walls and hemorrhages in the mucosa. A large number of moving trophozoites were found in the contents of the cecum by microscopic examination. The livers were enlarged with multiple bleeding spots on the surface. Histopathology of the livers showed mononuclear cell infiltration and moderate hyperplasia of fibrous tissue. The histopathology of the cecum showed that the villi of the cecum were edematous. Finally, the presence of T. gallinarum was determined by specific PCR andin-situ hybridization assay. Additionally, common pathogens that can cause similar symptoms were excluded.
The death of the black swan was caused by T. gallinarum, suggesting that the parasite might be a new threat to the Cygnus birds.
Tetratrichomonas gallinarum is parasitic protozoa with a wide host range . Owing to sick birds are usually co-infected with other pathogens and artificially infected animals rarely develop symptoms, the pathogenicity of T. gallinarum is controversial [2,3,4]. Moreover, lesions caused by T. gallinarum in birds were sporadically reported in some countries, such as in chukar partridges, mockingbird, Waldrapp ibis and white pelican from America [5,6,7,8], in duck from Germany , in red-legged partridges from Great Britain , and in Layer chickens from the Netherlands . Here, we described the first fatal case of black swans (Cygnus atratus) associated with T. gallinarum infection in China, and the threat of the protozoa to Cygnus birds must be considered.
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.
Discussion and conclusions
Though T. gallinarum is commonly found gallinaceous and anseriform birds, it seldom causes diseases . The maturity of the immune system may be an important reason for the host to suffer from this parasite, as previous studies have found that most of the dead birds were juveniles or subadults [8, 9]. However, all the dead black swans in the present study were adult, thus the heterogeneity between T. gallinarum isolates might also be an important factor result in the differences in pathogenicity among hosts.
Studies conducted by Dimasuay and Rivera shown that T. gallinarum can be detected from healthy ducks (Anas platyrhynchos) , which suggested that the parasite might be commensal in some duck species. In the present study, some healthy ducks shared activity area with the black swans. Thus the T. gallinarum recovered from the black swans may be spillover from the ducks.
In conclusion, we described the first fatal case of black swans associated with T. gallinarum infection in China, suggesting that the protozoan might be a new threat to the Cygnus birds. A comprehensive epidemiological investigation of T. gallinarum in Cygnus birds is urgently needed in the future.
Availability of data and materials
The ITS and 18S nucleotide sequences of T. gallinarum generated in the present study have been deposited in GenBank database under the accession numbers MN448387and MN448388 as well as MN450306 and MN450307, respectively.
In situ hybridization
Internal transcribed spacer
Cepicka I, Hampl V, Kulda J, Flegr J. New evolutionary lineages, unexpected diversity, and host specificity in the parabasalid genus Tetratrichomonas. Mol Phylogenet Evol. 2006;39(2):542–51. https://doi.org/10.1016/j.ympev.2006.01.005.
Amin A, Liebhart D, Weissenbock H, Hess M. Experimental infection of turkeys and chickens with a clonal strain of Tetratrichomonas gallinarum induces a latent infection in the absence of clinical signs and lesions. J Comp Pathol. 2011;144(1):55–62. https://doi.org/10.1016/j.jcpa.2010.06.002.
Burns RE, Braun J, Armien AG, Rideout BA. Hepatitis and splenitis due to systemic tetratrichomoniasis in an American white pelican (Pelecanus erythrorhynchos). J Vet Diagn Investig. 2013;25(4):511–4. https://doi.org/10.1177/1040638713488368.
Richter B, Schulze C, Kammerling J, Mostegl M, Weissenbock H. First report of typhlitis/typhlohepatitis caused by Tetratrichomonas gallinarum in three duck species. Avian Pathol. 2010;39(6):499–503. https://doi.org/10.1080/03079457.2010.518137.
Liebhart D, Neale S, Garcia-Rueda C, Wood AM, Bilic I, Wernsdorf P, et al. A single strain of Tetratrichomonas gallinarum causes fatal typhlohepatitis in red-legged partridges (Alectoris rufa) to be distinguished from histomonosis. Avian Pathol. 2014;43(5):473–80. https://doi.org/10.1080/03079457.2014.959435.
Landman WJ, Molenaar RJ, Cian A, van der Heijden HM, Viscogliosi E. Granuloma disease in flocks of productive layers caused by Tetratrichomonas gallinarum. Avian Pathol. 2016;45(4):465–77. https://doi.org/10.1080/03079457.2016.1163325.
Richter B, Fragner K, Weissenböck H. Simultaneous detection of protozoa in the tissues of snakes by double in situ hybridization. Microsc Res Tech. 2008;71(4):257–9. https://doi.org/10.1002/jemt.20546.
Liebhart D, Weissenböck H, Hess M. In-situ hybridization for the detection and identification of Histomonas meleagridis in tissues. J Comp Pathol. 2006;135(4):237–42. https://doi.org/10.1016/j.jcpa.2006.08.002.
Bilic I, Jaskulska B, Souillard R, Liebhart D, Hess M. Multi-locus typing of Histomonas meleagridis isolates demonstrates the existence of two different genotypes. PLoS One. 2014;9(3):e92438. https://doi.org/10.1371/journal.pone.0092438.
Jarquin-Diaz VH, Balard A, Jost J, et al. Detection and quantification of house mouse Eimeria at the species level - challenges and solutions for the assessment of coccidia in wildlife. Int J Parasitol Parasites Wildl. 2019;10:29–40. https://doi.org/10.1016/j.ijppaw.2019.07.004.
Sun ZF, Larsen CT, Dunlop A, Huang FF, Pierson FW, Toth TE, et al. Genetic identification of avian hepatitis E virus (HEV) from healthy chicken flocks and characterization of the capsid gene of 14 avian HEV isolates from chickens with hepatitis-splenomegaly syndrome in different geographical regions of the United States. J Gen Virol. 2004;85(Pt 3):693–700. https://doi.org/10.1099/vir.0.19582-0.
We acknowledge Dr. Ping Wang specifically for his excellent PS technical assistance.
This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA19050204), Beijing Innovation onsortium of Agriculture Research System (BAIC04–2019); State Administration of Forestry and Grassland, China and Chinese Academy of Sciences (CZBZX-1).
Authors and Affiliations
National Research Center for Wildlife Borne Diseases, Institute of Zoology, Chinese Academy of Sciences, 1-5 Beichenxilu, Chaoyang District, Beijing, 100101, PR China
Shengyong Feng, Han Chang, Shuyi Han & Hongxuan He
College of Life Sciences, University of Chinese Academy of Sciences, Chaoyang District, Beijing, 100101, China
Shengyong Feng & Han Chang
Beijing General Station of Animal Husbandry, Chaoyang District, Beijing, 100107, China
Beijing Center for Animal Disease Control, Beijing, China
Shaanxi Institute of zoology, Xi’an, 710032, Shaanxi, China
Experimental design was done by HH and SF. Collection of samples was done by HC and YW. The experiments were done by HC SH, FL and QW. The manuscript was written by SF and revised by HH. All authors read and approved the final manuscript.
This study was approved by the Animal Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences. All samples were handled in accordance with good animal practices required by the Animal Ethics Procedures and Guidelines of the People’s Republic of China.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.