Direct evidence of cheetah (Acinonyx jubatus) as intermediate host of Toxoplasma gondii through isolation of viable strains.

Toxoplasma gondii causes lifelong infection in most definitive and intermediate hosts. Clinical cases of toxoplasmosis in captive cheetahs have been reported. However, there are few reports of viable T. gondii strains isolated from cheetahs. Here, T. gondii infection was investigated using molecular and serological assays in cheetahs from China. Modified agglutination test (MAT) (cut-off: 1:25) indicated that all six examined cheetahs (n = 6) showed T. gondii antibodies. Toxoplasma gondii DNA was detected in three out of five cheetahs. Two viable T. gondii strains were isolated from the striated muscles of two cheetahs using mice bioassay. They were designated as TgCheetahCHn1 and TgCheetahCHn2. Genetic characterization of DNA derived from tachyzoites was performed using RFLP-PCR of 10 markers. Toxoplasma gondii TgCheetahCHn1 is ToxoDB PCR-RFLP genotype #319, and the alleles of ROP18/ROP5 types were 3/7. TgCheetahCHn2 is ToxoDB genotype #9, and the alleles of ROP18/ROP5 were 3/6. The average survival time of TgCheetahCHn1-infected Swiss mice was 22 ± 1 days (n = 23), and the mice did not have detectable T. gondii-specific antibodies until 117 ± 30 days post-inoculation (n = 8), therefore, TgCheetahCHn1 had intermediate virulence. TgCheetahCHn2 was avirulent for Swiss mice. Few brain tissue cysts (0-50) were observed in the mice inoculated with TgCheetahCHn1 or TgCheetahCHn2. The results provide direct evidence of cheetah as intermediate host of T. gondii.

Isolation and Characterization of a Viable Toxoplasma gondii from Captive Caracal (Caracal caracal).

Toxoplasma gondii is a widespread protozoan parasite that infects most warm-blooded animals, and felids can serve as intermediate and definitive hosts. Pathological diagnosis and serological and etiological investigations were conducted on a captive caracal (Caracal caracal) carcass collected from China in 2022. Pathological diagnosis revealed that cardiac insufficiency, pulmonary edema, hepatic failure, and renal insufficiency were the causes of the caracal's death. A modified agglutination test (cut-off: 1:25) revealed that IgG antibodies against T. gondii were present in the myocardium juice (1:1600), ascitic fluid (1:3200), and hydropericardium (1:800). A viable T. gondii (TgCaracalCHn2) strain was isolated from the tissue samples (heart, brain, spleen, and skeletal muscle) of this caracal using a mouse bioassay. The genotype of TgCaracalCHn2 was ToxoDB#5 (Type II variant), as determined via RFLP-PCR. The strain was avirulent in Swiss mice and matched the prediction of ROP18 and ROP5 gene alleles of TgCaracalCHn2 (2/2). Mild tissue cysts (203 ± 265) were observed in mice brains after inoculation with TgCaracalCHn2 tachyzoites. ToxoDB#5 is the dominant genotype in North American wildlife, and this is the first documented isolation of T. gondii ToxoDB#5 from China. This indicates that caracal plays an important role in the transmission of this T. gondii genotype.

Isolation and Genetic Characterization of Toxoplasma gondii from a Patas Monkey (Erythrocebus patas) in China.

Many cases of Toxoplasma gondii infection have been reported worldwide in non-human primates (NHPs), especially in captive New World monkeys. However, few studies on toxoplasmosis in Old World monkeys have been conducted. In this study, serological and molecular biological analyses were carried out to look for T. gondii antibodies and T. gondii infection in 13 NHPs from China. T. gondii infection was confirmed in 8 NHP cases. T. gondii antibodies were detected in 1/5 New World monkeys and in 4/7 Old World monkeys. T. gondii DNA was detected in 3/5 New World monkeys and 5/7 Old World monkeys. The one ring-tailed lemur was negative for both antibodies and DNA of T. gondii. The most common clinical manifestations of T. gondii infection were malaise, poor appetite, emaciation, and foamy nasal discharge. The most common histopathological findings were interstitial pneumonia, necrotic hepatitis, necrotizing myocarditis, lymphadenitis, and necrotic splenitis. One viable T. gondii strain was successfully isolated from the myocardium of a patas monkey (Erythrocebus patas) by bioassay in mice. T. gondii tachyzoites were obtained from cell cultures and were designated as TgMonkeyCHn2. The genotype of this strain belongs to ToxoDB genotype #9, and the allele of ROP18/ROP5 gene was 3/6. TgMonkeyCHn2 tachyzoites were avirulent in Swiss mice. To our knowledge, this is the first report of fatal toxoplasmosis in a patas monkey. T. gondii infection in patas monkeys may indicate environmental contamination by oocysts. The patas monkey is a new host record for T. gondii.

Epidemiology and isolation of viable Toxoplasma gondii strain from macropods.

Wallabies and other marsupials are highly susceptible to Toxoplasma gondii. In this study, 26 macropod samples were collected (8 red kangaroos, 4 Parma wallabies, 8 red-necked wallabies, 5 albino red-necked wallabies and 1 Eastern grey kangaroo), including tissue (n = 9) and serum (n = 17) samples. According to the modified agglutination test (MAT) results (cutoff 1:25), 50% (95% Cl: 32.06-67.94%) of the macropods had T. gondii antibodies. Among them, species, survival state, and sampling date were risk factors for T. gondii susceptibility (P < 0.05). T. gondii DNA was detected in two (cases #14 and #15) of the nine cases obtained from macropod tissues. One viable T. gondii strain (TgRooCHn4) was isolated from an albino red-necked wallaby (Macropus rufogriseus, case #14) via bioassay in mice. TgRooCHn4 belongs to ToxoDB genotype #3, using the 10 multilocus PCR-RFLP markers. The ROP18 and ROP5 gene allele types of TgRooCHn4 were 2/2, which was predicted to be non-lethal to mice. The virulence of TgRooCHn4 tachyzoites was avirulent in mice. Most macropods sampled from Hernan province in 2021 and 2022 were positive with T. gondii infection. A flood occurred in July 2021 in Zhengzhou from Henan province may promote the transmission of T. gondii oocysts. To our knowledge, this is the first T. gondii strain isolated from albino red-necked wallaby. However, further investigation is required to enhance our understanding of the transmission and prevention of toxoplasmosis in sensitive zoo animals.

Dietary yucca extract and Clostridium butyricum promote growth performance of weaned rabbits by improving nutrient digestibility, intestinal development, and microbial composition.

Yucca has abundant amounts of polyphenolics, steroidal saponins, and resveratrol and its extract can be used as a feed additive in the animal husbandry, which might contribute to the improvement in the growth and productivity in rabbit production. Hence, the current study aimed to examine the effects of yucca extract alone and in combination with Clostridium butyricum (C. butyricum) on growth performance, nutrient digestibility, muscle quality, and intestinal development of weaned rabbits. A total of 400 40-day-old male rabbits were randomly divided into 4 treatment groups for 40 days: (1) basal diet group, (2) basal diet contained 300 mg/kg of yucca extract, (3) basal diet supplemented with 0.4 × 1010 colony-forming units (CFU)/kg of C. butyricum, and (4) the blend of 0.4 × 1010/kg CFU of C. butyricum and 300 mg/kg of yucca extract. The supplementation of yucca extract or C. butyricum increased body weight (BW) of rabbits depending on the age, the combined addition of yucca extract and C. butyricum significantly increased BW, weight gain, and feed intake, companying with increased the digestibility of crud protein, fiber, phosphorous, and calcium as compared to control diet (P < 0.05). Furthermore, yucca extract and C. butyricum treatment alone and in combination notably increased the villus high and the ratio of villus high to crypt depth of rabbits (P < 0.05). The combined supplementation of yucca extract and C. butyricum altered the intestinal microbiota of rabbits, as demonstrated by increased the abundance of beneficial bacteria Ruminococcaceae and decreased the proportion of pathogenic bacteria such as Pseudomonadaceae and S24-7. In addition, the rabbits fed the diet with yucca extract and the blend of yucca extract and C. butyricum had significantly increased pH45min, decreased pressing loss, drip loss, and shears force when compared with rabbits received control diet (P < 0.05). Diet with C. butyricum or its mixture with yucca extract increased the fat content of meat, while the combined addition of yucca extract and C. butyricum declined the content of fiber in meat (P < 0.05). Collectively, the combined use of yucca extract and C. butyricum showed better results on growth performance and meat quality, which might be closely associated with the improved intestinal development and cecal microflora of the rabbits.

Additional evidence of tigers (Panthera tigris altaica) as intermediate hosts for Toxoplasma gondii through the isolation of viable strains.

Toxoplasmosis is one of the most common zoonotic diseases in the world. Felines excrete Toxoplasma gondii oocysts, which play a key role in the transmission of this protozoon. Pathological diagnoses were performed on four carcasses of captive tigers collected from 2019 to 2021 in China, and T. gondii was surveyed using serology, molecular analysis, and aetiology. Striated muscle samples of the tigers (n = 4) were bioassayed in mice. DNA derived from T. gondii tachyzoites was isolated and characterized using PCR-RFLP. The pathological diagnoses revealed that ageing, declined immune function, liver, and kidney failures caused the deaths in the tigers examined. A modified agglutination test (cut-off: 1:25) revealed that IgG antibodies to T. gondii were 100% (4/4) in the captive tigers. Two viable T. gondii strains (TgTigerCHn3 and TgTigerCHn4) were isolated from tiger striated muscles and seeded on the Vero cell culture for further propagation. The genotypes of TgTigerCHn3 and TgTigerCHn4 were ToxoDB#20 and ToxoDB#2, respectively. The two strains were avirulent for Swiss mice, which matched the ROP18 and ROP5 gene alleles of TgtigerCHn3 (3/4) and TgtigerCHn4 (3/3). Few brain tissue cysts (0-213) were observed in the mice after inoculation with TgTigerCHn3 and TgTigerCHn4. This is the first documented isolation of T. gondii ToxoDB#20 and ToxoDB#2 from tigers. The results provide additional direct evidence of tiger as intermediate hosts for T. gondii. Tigers in the zoos may potentially transmit T. gondii to other animals and humans.

Comparative Immunological Response and Pathobiology of Mice Inoculated with Toxoplasma gondii Isolated from Different Hosts.

Toxoplasma gondii is an obligate intracellular parasite that has a worldwide distribution and can infect almost all warm-blood animals. Serological tests are the main detection methods for T. gondii infection in animals and humans. Little is known of biological behavior, antibody responses, and virulence of T. gondii strains in mice from China. Here we document antibody responses, tissue cyst burden, and mouse virulence of T. gondii strains isolated from different hosts in China. All T. gondii strains formed tissue cysts in the brains of mice and positively correlated with the T. gondii antibody titer (R2 = 0.3345). These results should aid in the diagnosis and characterization of T. gondii isolates.