ABSTRACT
Toxoplasma gondii oocysts in the environment are a threat to humans and animals. This study is aimed to evaluate the prevalence of T. gondii in white spoonbills and isolate viable T. gondii from white spoonbills. In 28.6% (2/7) of white spoonbills, T. gondii antibodies were found in heart juice by the modified agglutination test (cut-off: 1:4). T. gondii DNA was detected in tissues of 42.9% (3/7) white spoonbills. One viable T. gondii strain, named TgSpoonbillCHn1, was isolated from the myocardium of a white spoonbill by bioassay in mice. DNA extracted from TgSpoonbillCHn1 tachyzoites was characterized by PCR-restriction fragment length polymorphism with ten markers and the virulence genes ROP5 and ROP18. The results revealed that it was ToxoDB#2 (Type III). The ROP18/ROP5 genotype combination predicts that this strain is avirulent for mice, which is supported by the infection experiments in mice. This is the first report of the isolation of viable T. gondii strain from white spoonbil. The prevalence of T. gondii in white spoonbills may be indicative of environmental contamination of oocysts. This report provides direct evidence of white spoonbill as an intermediate host of T. gondii.
KEYWORDS: T. gondii, Platalea leucorodia, isolation, genotype, virulence
Dear editor: Toxoplasma gondii is an obligate intracellular parasite that has a worldwide distribution and can infect almost all warm-blood animals, including birds [1]. However, T. gondii behaves differently in different hosts. Birds are considered resistant to clinical toxoplasmosis. The serological response for T. gondii infection in birds was erratic and transient, the serological results of T. gondii should be interpreted with caution [2,3]. White spoonbill (Platalea leucorodia), as a large wading bird, they obtain food from water, sludge, or the ground. Their food includes small aquatic creatures, insects, crustaceans, tiny fish, and loach. The infection status of T. gondii in white spoonbill is an excellent indicator to monitor the environment for T. gondii oocysts contamination. However, there is still a lack of direct evidence that T. gondii can infect spoonbill.
In this study, from September 2018 to June 2020, eleven adult white spoonbills died of bloody mass stools in a zoo (34°46′ N, 113°39′ E, Henan, China) (Table 1, Figure S1). These birds were fed fish and loaches, also foraged from the artificial lake. Fresh tissue samples from seven white spoonbills were collected. These samples were submitted to the Laboratory of Veterinary Pathology of Henan Agricultural University (Zhengzhou, China) for pathological diagnosis and also allow us to investigate T. gondii infection in white spoonbills.
Table 1.
Background data and isolation of Toxoplasma gondii from white spoonbills (Platalea leucorodia).
| Samples ID | Received date | Sex, age | Clinical signs | Pathological findings | MAT titersa | PCRb | Mice bioassay c |
|---|---|---|---|---|---|---|---|
| Case#1 | Sep 27, 2018 | Female adult | Loose stools with fresh blood | nd | nd | nd | nd |
| Case#2 | Sep 30, 2018 | Female adult | Loose stools with fresh blood | nd | nd | nd | nd |
| Case#3 | Oct 3, 2018 | Female adult | Salivate, depressed, anorexia | Hydropericardium, liver congestion | <1:2 | + | 0/4 |
| Case#4 | Oct 3, 2018 | Female adult | Depressed, anorexia | Myocardial hemorrhage, glandular stomach mucosal hemorrhage, liver congestion | 1:16 | + | 1/5, 4/5, 5/5, 5/5, 2/2, 3/3, 3/3, 5/5 |
| Case#5 | Oct 5, 2018 | Female adult | Depressed, anorexia | Myocardial hemorrhage | nd | nd | nd |
| Case#6 | Oct 5, 2018 | Female adult | Salivate, depressed, anorexia | Myocardial hemorrhage | nd | nd | nd |
| Case#7 | Oct 7, 2018 | Male, adult | Salivate, loose and black stools, depressed, anorexia | Hydropericardium, endocardial hemorrhage | 1:16 | - | nd |
| Case#8 | Oct 7, 2018 | Female adult | Salivate, depressed, anorexia | Splenomegaly, liver congestion, kidney hemorrhage | <1:2 | - | nd |
| Case#9 | Oct 11, 2018 | Female adult | Loose stools with fresh blood, salivate, depressed, anorexia | Pulmonary congestion, kidney hemorrhage | <1:2 | - | nd |
| Case#10 | Nov 11, 2018 | Female adult | Loose and black stools, depressed, anorexia | Enlarged liver and kidney | <1:2 | - | nd |
| Case#11 | Sep 13, 2019 | Female adult | Loose and black stools, depressed, anorexia | Heart coronary fat hemorrhage, liver congestion | <1:2 | + | nd |
Titration: 1:2–1:8192.
Primer were TOX5/TOX8.
No. of positive mice/No. of inoculated mice.
nd: not done.
The degree of fat storage and muscle development in these white spoonbills were well. Bloody stools (6/11), salivate (4/11), hydropericardium (2/9), myocardium or heart coronary fat hemorrhage (5/9), white pulp necrosis (4/7) were observed by gross and microscopic examination (Figure S1 B–E). Pathological diagnosis showed that adenovirus infection was the major cause of death in these white spoonbills. T. gondii parasites were not found in tissue sections of all seven white spoonbills.
T. gondii antibodies were identified by the modified agglutination test (MAT) (cut off = 1:4) [4]. Our survey indicated that 28.6% (2/7, case#4 and case#7) (95% CI, 7.56%–64.76%) of the white spoonbills were seropositive for T. gondii, with titres of 1:16 (Table 1). The current understanding of the specificity and sensitivity of the serological diagnosis of T. gondii infection in birds is limited. Viable T. gondii were isolated from 6 of 1025 chickens with MAT titre of <1:5 by bioassay in cats [5]. Cabezón reported 6.2% T. gondii antibodies in the Eurasian spoonbill (5/81) by MAT, with titres greater than or equal to 1:25 [6]. Work found T. gondii infection in one species of Pelecaniformes, a red-footed booby (Sula sula), and parasites were confirmed by immunohistochemical (IHC) staining in the heart and cerebrum [7]. However, the serum antibody reaction to T. gondii was not reported.
PCR assays for T. gondii were performed using primer Tox-8 and Tox-5 [8]. T. gondii DNA was detected in tissues of 42.9% (3/7) (95% CI, 15.75%–75.02%) white spoonbills. T. gondii DNA was detected in myocardium digestive juices of white spoonbills (case#3, case#4), and in lung and heart of white spoonbill case#11.
The myocardium (20 g) of white spoonbill case#3 and case#4 was bioassayed in mice individually [1]. For the TOX#25-4 group (case#4), mouse (M#796) had seroconversion antibodies for T. gondii, and cysts (n = 120 in the whole brain) were found in the brain at 43 DPI (Figure S1 F). T. gondii cysts were verified by IHC (Figure S1 G). The T. gondii from the mouse brain was propagated in cell culture successfully (15 DPI) and designated as TgSpoonbillCHn1. DNA samples extracted from T. gondii tachyzoites in cell cultures were characterized by PCR-RFLP [9,10]. Its genetic typing was ToxoDB#2 (type III). ToxoDB#2 is widely distributed worldwide [11]. ToxoDB#2 T. gondii strains were previously found in cats [12] and sheep [13] from central China, indicating that, except for ToxoDB#9, ToxoDB#2 is one of the major endemic genotypes in China. Until now, there were more than one hundred viable T. gondii strains isolated from avian species (excluding chickens) worldwide, most from Anseriformes, Columbiformes and Accipitriformes [2,3]. However, only one T. gondii strain isolated from Pelecaniformes [14]. Genetic diversity of T. gondii isolates from birds follows the global patterns, with ToxoDB #1, #3, and #2 being dominant in Africa and Europe [3]. 22 DNA extracted from tissues of birds from China were genotyped, they were ToxoDB #10, #1, #3 and #9 [3]. TgSpoonbillCHn1 was the first genotyped strain from viable T. gondii isolate in birds from China.
The virulence of TgSpoonbillCHn1 was evaluated in Swiss mice. The ROP18/ROP5 genotype combination (3/3) suggests this strain is avirulence for mice, which matched with the mouse virulence evaluation in this study (Table S1).
The source of T. gondii infection for these white spoonbills is less clear. Vertical transmission of T. gondii is sporadic in chicken; however, this is unclear in other species of birds, including white spoonbill. These white spoonbills were hatched and grew in a zoological park. White spoonbills may have acquired T. gondii through the ingestion of oocysts from felids feces or mechanical transport hosts (fish and loaches in the feed). This indicated that T. gondii oocysts contaminate the habitat environment (water, soil) of white spoonbills. The high seropositive of T. gondii antibodies in captive tigers (80%) from central China support this hypothesis [15].
This is the first report on the isolation of T. gondii from white spoonbill, providing direct evidence that white spoonbill is an intermediate host of T. gondii. White spoonbills could serve as a good sentinel animal for T. gondii contamination in the environment.
Supplementary Material
Acknowledgments
We would thank Ruijing Su and Dong hui (Henan Agricultural University, Zhengzhou, China) for genotyping and collecting samples. We thank Chunlei Su (University of Tennessee, Knoxville, USA) for his help in preparation of this manuscript. This study was financed by the Natural Science Foundation of Henan Province, China (202300410214).
Funding Statement
This work was supported by Natural Science Foundation of Henan Province, China [grant number 202300410214].
Disclosure statement
No potential conflict of interest was reported by the author(s).
References
- 1.Dubey JP. Toxoplasmosis of animals and humans. Boca Raton (FL: ): CRC Press, Taylor & Francis Group; 2010, p. 1–313. [Google Scholar]
- 2.Dubey JP. A review of toxoplasmosis in wild birds. Vet Parasitol. 2002;106:121–153. [DOI] [PubMed] [Google Scholar]
- 3.Dubey JP, Murata FHA, Cerqueira-Cézar CK, et al. Epidemiologic significance of Toxoplasma gondii infections in turkeys, ducks, ratites, and other wild birds: 2009-2020. Parasitology. 2020:1–30. DOI: 10.1017/S0031182020001961. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Dubey JP, Desmonts G.. Serological responses of equids fed Toxoplasma gondii oocysts. Equine Vet J. 1987;19:337–339. [DOI] [PubMed] [Google Scholar]
- 5.Dubey JP, Laurin E, Kwok OCH.. Validation of the modified agglutination test for detection of Toxoplasma gondii in free-range chickens by using cat and mouse bioassay. Parasitology. 2016;143:314–319. [DOI] [PubMed] [Google Scholar]
- 6.Cabezón O, García-Bocanegra I, Molina-López R, et al. Seropositivity and risk factors associated with Toxoplasma gondii infection in wild birds from Spain. PLoS One. 2011;6(12):e29549. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Work TM, Massey JG, Lindsay D, et al. Toxoplasmosis in three species of native and introduced Hawaiian birds. J Parasitol. 2002;88(5):1040–1042. [DOI] [PubMed] [Google Scholar]
- 8.Reischl U, Bretagne S, Krüger D, et al. Comparison of two DNA targets for the diagnosis of toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes. BMC Infect Dis. 2003;3:7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Su C, Shwab EK, Zhou P, et al. Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii. Parasitology. 2010;137:1–11. [DOI] [PubMed] [Google Scholar]
- 10.Shwab EK, Jiang T, Pena HF, et al. The ROP18 and ROP5 gene allele types are highly predictive of virulence in mice across globally distributed strains of Toxoplasma gondii. Int J Parasitol. 2016;46:141–146. [DOI] [PubMed] [Google Scholar]
- 11.Shwab EK, Saraf P, Zhu XQ, et al. Human impact on the diversity and virulence of the ubiquitous zoonotic parasite Toxoplasma gondii. P Natl Acad Sci USA. 2018;115:E6956–E6963. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Yang Y, Ying Y, Verma SK, et al. Isolation and genetic characterization of viable Toxoplasma gondii from tissues and feces of cats from the central region of China. Vet Parasitol. 2015;211:283–288. [DOI] [PubMed] [Google Scholar]
- 13.Jiang N, Su R, Jian F, et al. Toxoplasma gondii in lambs of China: heart juice serology, isolation and genotyping. Int J Food Microbiol. 2020;322:108563. [DOI] [PubMed] [Google Scholar]
- 14.Silva MA, Pena HFJ, Soares HS, et al. Isolation and genetic characterization of Toxoplasma gondii from free-ranging and captive birds and mammals in Pernambuco state, Brazil. Revista Brasileira de Parasitologia Veterinária. 2018;27:481–487. [DOI] [PubMed] [Google Scholar]
- 15.Yang Y, Dong H, Su R, et al. Direct evidence of an extra-intestinal cycle of Toxoplasma gondii in tigers (Panthera Tigris) by isolation of viable strains. Emerg Microbes Infec. 2019;8:1550–1552. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.