Fatal Toxoplasma gondii infection in the giant panda

Toxoplasma gondii can infect nearly all warm-blooded animals. We report an acute fatal T. gondii infection in the endangered giant panda (Ailuropoda melanoleuca) in a zoo in China, characterized by acute gastroenteritis and respiratory symptoms. T. gondii infection was confirmed by immunological and molecular methods. Multilocus nested PCR-RFLP revealed clonal type I at the SAG1 and c29-2 loci, clonal type II at the SAG2, BTUB, GRA6, c22-8, and L358 loci, and clonal type III at the alternative SAG2 and SAG3 loci, thus, a potential new genotype of T. gondii in the giant panda. Other possible pathogens were not detected. To our knowledge, this is the first report of clinical toxoplasmosis in a giant panda.


Introduction
Toxoplasmosis, caused by the obligate intracellular protozoan Toxoplasma gondii, is an important zoonosis worldwide. It is a major public health concern, mainly because of congenital disease, infection of immunocompromised patients, and an emerging severe form of acquired toxoplasmosis in immunocompetent patients [1]. The lifecycle of T. gondii includes sexual multiplication within cats and asexual multiplication within nearly all warm-blooded animals, including humans [6]. Humans and animals become infected by eating undercooked or raw meat containing cysts, or by ingesting food or water contaminated with sporulated oocysts.
The giant panda (Ailuropoda melanoleuca) is an emblematic endangered species and regarded as a national treasure and ''living fossil'' in China [16]. Its population is estimated at approximately 1600 in the wild, and the captive population is more than 300 [7,13]. The health of giant pandas has attracted global attention. Here, we report an acute fatal T. gondii infection in the giant panda in China.

Case presentation
In February 2014, a seven-year-old giant panda named Jin Yi was found dead at Zhengzhou Zoo, Henan Province, China. The panda did not eat at noon on February 7. On February 8, the panda was found lying in the room with head buried in the abdomen. Treatment measures included intramuscular administration of cephalosporin and intravenous infusion of glucose. The animal had difficulty breathing overnight and was found dead in the morning of February 9.
A complete necropsy was conducted. Severe pathologic lesions were found, localized to the gastrointestinal tract and lungs. The gastrointestinal tract contained little or no ingesta, had multifocal mucosal hemorrhage, and dry, hard-packed digesta in the duodenum. Lungs were congested and chyme blocked the respiratory tract. Histologically, macrophages containing T. gondii tachyzoites were seen in the alveoli (Fig. 1). Other lesions included congestion in the intestinal lamina propria and submucosa, gastric epithelial necrosis, and sloughing.
Serum and tissue samples were collected for examination of potential pathogens that may cause hemorrhagic gastroenteritis. The animal had an antibody titer for T. gondii of 200 by the modified agglutination test [5]. T. gondii DNA was detected in the liver, spleen, lungs, kidneys, and small and large intestines by nested PCR targeting the B1 gene [12]. The immunofluorescence assay (IFA) revealed T. gondii tachyzoites present in the lung and small intestine tissues (Fig. 2), suggesting acute orally acquired toxoplasmosis in the giant panda, probably occurring 7-10 days before signs.
The positive DNA samples were directly typed by multilocus nested PCR-RFLP (Mn-PCR-RFLP) using 10 genetic markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico), and the reference strains, including GT1, PTG, CTG, MAS, TgCgCa1, TgCatBr5, TgCatBr40, TgCatBr64, and TgRsCr1, were used as positive controls. The results revealed clonal type I at the SAG1 and c29-2 loci, clonal type II at the SAG2, BTUB, GRA6, c22-8, and L358 loci, and clonal type III at the alternative SAG2 and SAG3 loci, showing a potential new atypical genotype of T. gondii in the giant panda. Other potential pathogens, including viruses and bacteria that cause acute gastroenteritis, or respiratory disease, were not detected. These results demonstrated that the giant panda died from acute toxoplasmosis due to a T. gondii strain of an atypical genotype.

Discussion
Toxoplasma gondii is considered to be one of the most successful eukaryotic pathogens, based on the number of host species and percentage of animals infected worldwide. The consequences of infection with T. gondii are associated with the host species and parasite genotypes. Primary infections in adults are mostly asymptomatic, but severe, acute, disseminated toxoplasmosis can occur in immunocompetent hosts when infected with some isolates [11]. Many T. gondii genotypes identified in animals and humans show high genetic diversity of T. gondii in China [10]. In addition to the atypical ToxoDB#9, there are several other atypical T. gondii genotypes identified in animals and humans in China 1 [2,17,14]. Atypical T. gondii strains have been shown to cause severe clinical disease in immunocompetent hosts [4,9].
Despite its taxonomic classification as a carnivore, the giant panda has a diet that is primarily herbivorous, almost exclusively bamboo. The panda still retains decidedly ursine teeth and will eat meat when available [15]. In addition to bamboo, the captive panda is given some formulated biscuits or other dietary supplements. There are a number of stray cats and small rodents in the zoo, and these animals can freely roam in the habitat of the giant panda. The infection may be obtained by consuming food or water contaminated with sporulated oocysts, or by ingestion of rodents infected with T. gondii [3]. Treatment   of the disease should include pyrimethamine plus sulfadiazine. Avoiding consumption of raw or undercooked meat is the main measure recommended to prevent T. gondii infection [8].