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.

Scrodentoids H and I, a Pair of Natural Epimerides from Scrophularia dentata, Inhibit Inflammation through JNK-STAT3 Axis in THP-1 Cells.

BACKGROUND: Scrophularia dentata is an important medicinal plant and used for the treatment of exanthema and fever in Traditional Tibetan Medicine. Scrodentoids H and I (SHI), a pair of epimerides of C19-norditerpenoids isolated from Scrophularia dentata, could transfer to each other in room temperature and were firstly reported in our previous work. Here, we first reported the anti-inflammatory effects of SHI on LPS-induced inflammation. PURPOSE: To evaluate the anti-inflammatory property of SHI, we investigated the effects of SHI on LPS-activated THP-1 cells. METHODS: THP-1 human macrophages were pretreated with SHI and stimulated with LPS. Proinflammatory cytokines IL-1ß and IL-6 were measured by RT-PCR and enzyme-linked immunosorbent assays (ELISA). The mechanism of action involving phosphorylation of ERK, JNK, P38, and STAT3 was measured by western Blot. The NF-κB promoter activity was evaluated by Dual-Luciferase Reporter Assay System in TNF-α stimulated 293T cells. RESULTS: SHI dose-dependently reduced the production of proinflammatory cytokines IL-1ß and IL-6. The ability of SHI to reduce production of cytokines is associated with phosphorylation depress of JNK and STAT3 rather than p38, ERK, and NF-κB promoter. CONCLUSIONS: Our experimental results indicated that anti-inflammatory effects of SHI exhibit attenuation of LPS-induced inflammation and inhibit activation through JNK/STAT3 pathway in macrophages. These results suggest that SHI might have a potential in treating inflammatory disease.

Scrodentoid A Inhibits Mast Cell-Mediated Allergic Response by Blocking the Lyn-FcεRIß Interaction.

Background: Mast cells are considered an attractive therapeutic target for treating allergic diseases, and the Lyn-FcεRIß interaction is essential for mast cell activation. This study investigated the antiallergic effect of scrodentoid A (SA) on mast cells and mast cell-mediated anaphylaxis. Methods: For in vitro experiments, mast cells were treated with SA. Cell proliferation was tested using the XTT assay. The mRNA expression of various cytokines and chemokines was measured using qPCR. The levels of histamine, eicosanoids (PGD2, LTC4), and cytokines were measured using enzyme immunoassay kits. Signaling was investigated using Western blotting and immunoprecipitation. For in vivo experiments, the antiallergic activity of SA was evaluated using two mouse models of passive anaphylaxis as passive cutaneous and systemic anaphylaxis. The mechanism was investigated through immunohistochemistry and immunofluorescence. Results: SA considerably inhibited immunoglobulin (Ig) E-mediated mast cell activation, including ß-hexosaminidase release, mRNA and protein expression of various cytokines, and PGD2 and LTC4 release. Oral administration of SA effectively and dose-dependently suppressed mast cell-mediated passive cutaneous and systemic anaphylaxis. SA significantly attenuated the activation of Lyn, Syk, LAT, PLCγ, JNK, Erk1/2, and Ca2+ mobilization without Fyn, Akt, and P38 activation by blocking the Lyn-FcεRIß interaction. Conclusions: SA suppresses mast cell-mediated allergic response by blocking the Lyn-FcεRIß interaction in vitro and in vivo. SA may be a promising therapeutic agent for allergic and other mast cell-related diseases.