Molecular and Serological Detection of Toxoplasma gondii in South China Tiger

Yingying Wang, Jianyi Yu and Lichen Zhou*

Shanghai Zoo, 2381 Hongqiao Road, Changning District, Shanghai, China, 200335

ABSTRACT

Toxoplasmosis is one of the most important zoonotic diseases with serious health risks for wild animals. The South China tiger, the most endangered tiger subspecies endemic to China, remain only a little more than 200 today, was susceptibly infected with Toxoplasma gondii. In this study, we used polymerase chain reaction to detect DNA, and enzyme-linked immunosorbent assay to test for T. gondii antibodies. Antibodies (S/P>0.31) to T. gondii were found in 20 (26.32%) of the 76 tigers, while all blood samples tested through nested PCR were negative. This is the first investigation of T. gondii infection in South China tiger.

Article Information

Received 24 September 2021

Revised 02 October 2021

Accepted 15 October 2021

Available online 22 September 2022

(early access)

Published 21 July 2023

Authors’ Contribution

LZ and YW designed the experiment. YW and JY collected the samples. YW performed the majority of the experiments and wrote the paper.

Key words

Toxoplasmosis, The South China tiger, PCR, ELISA

DOI: https://dx.doi.org/10.17582/journal.pjz/20210924080958

* Corresponding author: [email protected]

0030-9923/2023/0004-1997 $ 9.00/0

Copyright 2023 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Toxoplasmosis is one of the most common parasitic infections in humans and in domesticated and wild animals all over the world. This disease causes severe neurologic, ocular, and systemic diseases inneonates and those with weakened immune systems (Dubey and Beattie, 1988; Dubey and Su, 2009). Toxoplasma infection poses serious health concerns for wildlife. In the past, outbreaks were predominately sporadic and self-limited. Recently, however, major outbreaks of the disease have led to irretrievable losses in wildlife. Toxoplasmosis represents a potential risk to biodiversity, modifying the behavior and structure of animal populations while driving some species to complete extinction (Daszak et al., 2000; Williams et al., 2002). Toxoplasmosis is caused when Toxoplasma gondii parasitizes in cells, infecting various parts of the host’s body and damaging tissues and organs. The condition eventually leads to a decline in the host’s immune function and an increased prevalence.

The Felidae family plays a prominent role in the epidemiology of T. gondii infection because felids release millions of oocysts in a short period of time, often in one to two weeks through their feces. This causes pollution in soil, food, and water. T. gondii oocystsin feline feces contaminate surroundings and cause sporulation in terrestrial and aquatic environments.

There are many stray cats in the zoos in China.

In addition, the living space of captive wild animals is limited, and there is more daily contact with each other, so captive wild animals in zoos are at high risk of becoming infected and spreading toxoplasmosis. Beijing, Shanghai, Chengdu, Fuzhou, and other Chinese cities have various levels of captive wild animals infected by Toxoplasma gondii. Even the Siberian tiger breeding base in Heilongjiang and the giant panda breeding base in Chengdu have been infected by T. gondii (Yang, 2020).

The South China tiger, the most endangered tiger subspecies endemic to China, has long been extinct in the wild. It has been classified as Critically Endangered (CR) by the World Conservation Union (IUCN). There remain only a little more than 200 of this species today, and all are in captivity. Tigers become can become infected by T. gondii through ingestion of bradyzoites in raw meat that contains tissue cysts, and oocysts on rare occasion. Infection can also occur by consuming food or water contaminated with oocysts shed by stray cats, or through vertical transmission of tachyzoites from mother to fetus.

With the rise of urban zoos, human populations and captive wildlife are interacting more frequently. Because it is difficult to recognize specific clinical symptoms or representative signs of zoonosis in wild animals, continuous surveys of the prevalence of toxoplasma infection in wild animals are important from a public health perspective (Cutler et al., 2010). On the other hand, little is known about diseases in the South China tiger, and strategies are lacking that control and prevent infectious diseases in this critically endangered species (Daszak et al., 2000; King et al., 2004). This short communication describes a study to evaluate toxoplasma infection in captive South China tigers in China.

Materials and methods

Seventy blood samples of captive South China tigers were collected from nine zoos (Shanghai, Hangzhou, Chengdu, Chongqing, Guangzhou, Meihuashan, Linyi, Suzhou, Nanchang) in China (Fig. 1). Sera were separated by centrifugation at 2,000 g for 5 min. The sera were stored at -20°C for toxoplasma antibodies detection, and the blood samples were processed for genomic DNA extraction using a Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions.

T. gondii was detected by Nested PCR targeting B1 DNA around the XhoI and PmlI restriction sites, according to a previously described method (Grigg and Boothroyd, 2001). PCR primers used for amplification are as follows: Pml/S1, 59-TGTTCTGTCCTATCGCAACG (positions 128 to147); Pml/S2, 59-TCTTCCCAGACGTGGATTTC (positions 152 to 171); Pml/AS1, 59-ACGGATGCAGTTCCTTTCTG (positions 688 to 707); and Pml/AS2, 59-CTCGACAATACGCTGCTTGA (positions 663 to 682), yielding a 504bp product. PCR was carried out in the final volume of 20μL containing 10μLPCR mix (2×) (Takara Dalian, China), 0.4 μL of each forward and reverse primer (10μmol/L), 2μL DNA (200 ng/mL) and 7.2 μL DNase/RNase-free water. The amplified PCR products were separated by electrophoresis in 1.5% agarose gels.

Serum antibodies against T. gondii were screened using the EVL Toxo test ELISA kit (European Veterinary Laboratory, Woerden, The Netherlands), according to the manufacturer’s recommendations. The serum samples and controls were diluted to 1:500 and tested in duplicate. The optical density (OD) was measured at 450 nm with an ELISA plate reader (Thermo Fisher, Waltham, MA, USA). The S/P (samples/positive control) ratio for each sample was calculated according to the formula: S/P = (OD450of the sample - OD450 of negative control)/ (OD450ofpositive control - OD450 of negative control). Samples with S/P ratio lower than 0.31 were considered negative for T. gondii antibodies. If the S/P ratios were greater than or equal to 0.31, the samples were considered positive.

The prevalence of T. gondii infection in South China tigers of different sexes, ages, regions, and sampling times was analyzed using the Chi Square Test in SPSS (version 18.0, SPSS Inc. Chicago, IL, USA). A probability (p) value of< 0.05 was considered statistically significant.

Results and discussion

The present study is the first epidemiological investigation of T. gondii infection in South China tiger in China. Antibodies (S/P>0.31) to T. gondii were found in 20(26.32%) of the 76 tigers, sampled from all nine sampling regions: Shanghai, Hangzhou, Chengdu, Chongqing, Guangzhou, Meihuashan, Nanchang, Suzhou and Linyi. The seropositive rates in Chongqing (66.7%) and Chengdu (50.0%) were significantly higher than those in the other sampling regions. The difference in the frequency of S/P ratio on T. gondii infection in male (X =0.26, SD=0.16) and female (X =0.22, SD=0.13) tigers was not significant. The antibody-positive rate of sub-adult tigers (80.0%) was higher compared with young (16.0%) and adult tigers (26.1%). Sampling time had an impact on the results the positive rate of serum samples was much higher in October and December than in other months (January, February, April, July, November). Variance in test results on the basis of region, age, or time of the sample for the detected parasite showed significant differences. Epidemiological survey results for the detection of T. gondii are available in Table I.

The prevalence of T. gondii in the Chongqing and Chengdu tigers was noticeably higher compared to tigers in other places in China. This may be related to sporulation and survival of coccidial oocysts in the environment due to temperature and humidity.

We found that the offspring of antibody-positive female tigers were also antibody-positive. This is consistent with the conclusion of Sharma et al. (2019). T. gondii infection can transmit tachyzoites from mother to fetus through vertical transmission (Sharma et al., 2019).

Table I. General characteristics and prevalence of Toxoplasma gondiiin South China tigers in Eastern China (n=76).

T. gondii can infect a variety of mammals. Though felines are the primary hosts for the parasite, other animals feed on the egg sacs of felines and become infected (Dubey, 2008; Munhoz et al., 2017). At present, captive African lions (Panthera leo), Lynxes (Lynx canadensis), and Siberian tigers (Panthera tigris altaica) are susceptible to T. gondii (Chen et al., 2014). Evidence of infection mostly discovered through serological detection has been found for 31 of the world’s 39 felid species (Dubey, 2016).

It is necessary to prevent and control the spread of T. gondii during daily feeding. First, we should control the number of stray cats, optimize the layout of zoos, and reduce contact between stray cats and South China tigers. Additional measures will include strengthening disinfection management, interrupting the transmission route of T. gondii, and ensuring the health and safety of infected South China tigers by quarantining them even after treatment, which in turn will guard the health of other wild animals in the zoo. Preventing cross infection and repeat infection in zoos is crucial in managing this dangerous condition.

In our study, all blood samples tested through nested PCR were negative for the presence of T. gondii. Lack of positive results from nested PCR in seropositive tigers was most likely related to T. gondii infection in tigers in the past. Moreover, the difference between the results may derivefrom the fact that T. gondii is temporarily present in blood, and it is therefore possible that it is not readily detected by nested PCR.

Acknowledgements

The authors would like to thank the staff of Shanghai Zoo, Hangzhou Zoo, Chengdu Zoo, Chongqing Zoo, Guangzhou Zoo, Longyan Meihuashan Nature Reserve, Nanchang Zoo, Suzhou Zoo and Linyi Zoo, for assistance in handling the tigers.

Funding

This research was supported by he Special Fund for Scientific Research of Shanghai Landscaping & City Appearance Administrative Bureau (GZ200404); the Opening Foundation of Beijing Key Laboratory of Captive Wildlife Technologies in Beijing Zoo (ZDK202102); and the Science and Technology Project of Shanghai Zoo (SZ170305; SZ190204; SZ160206).

Statement of conflict of interest

The authors have declared no conflicts of interest.

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