Melatonin alleviates lung injury in H1N1-infected mice by mast cell inactivation and cytokine storm suppression.

Influenza A virus (IAV) H1N1 infection is a constant threat to human health and it remains so due to the lack of an effective treatment. Since melatonin is a potent antioxidant and anti-inflammatory molecule with anti-viral action, in the present study we used melatonin to protect against H1N1 infection under in vitro and in vivo conditions. The death rate of the H1N1-infected mice was negatively associated with the nose and lung tissue local melatonin levels but not with serum melatonin concentrations. The H1N1-infected AANAT-/- melatonin-deficient mice had a significantly higher death rate than that of the WT mice and melatonin administration significantly reduced the death rate. All evidence confirmed the protective effects of melatonin against H1N1 infection. Further study identified that the mast cells were the primary targets of melatonin action, i.e., melatonin suppresses the mast cell activation caused by H1N1 infection. The molecular mechanisms involved melatonin down-regulation of gene expression for the HIF-1 pathway and inhibition of proinflammatory cytokine release from mast cells; this resulted in a reduction in the migration and activation of the macrophages and neutrophils in the lung tissue. This pathway was mediated by melatonin receptor 2 (MT2) since the MT2 specific antagonist 4P-PDOT significantly blocked the effects of melatonin on mast cell activation. Via targeting mast cells, melatonin suppressed apoptosis of alveolar epithelial cells and the lung injury caused by H1N1 infection. The findings provide a novel mechanism to protect against the H1N1-induced pulmonary injury, which may better facilitate the progress of new strategies to fight H1N1 infection or other IAV viral infections.

PI3K/AKT-mediated autophagy inhibition facilitates mast cell activation to enhance severe inflammatory lung injury in influenza A virus- and secondary Staphylococcus aureus-infected mice.

Influenza A virus infection causes considerable morbidity and mortality each year globally, and secondary bacterial infection further exacerbates the severity and fatality of the initial viral infection. Mast cells have substantial roles in protecting the respiratory tract mucosa, while their role in viral and bacterial co-infection remains unclear. The present study revealed that secondary Staphylococcus aureus infection significantly aggravated the activation of mast cells during the initial H1N1 infection both in vivo and in vitro, which was closely related to the increased inflammatory lung injury and mortality. Meanwhile, the secondary S. aureus infection suppressed autophagy and promoted inflammatory mediators released by mast cells through activating the PI3K/Akt signaling pathway. Blocking PI3K/Akt pathway by LY294002, an inhibitor of Akt phosphorylation, could rescue autophagy and inhibit the release of inflammatory mediators. Furthermore, based on the influenza A viral and secondary bacterial infected mice model, we showed that the combination of LY294002 and antiviral drug oseltamivir could effectively reduce the inflammatory damage and pro-inflammatory cytokines releasing in lungs, recovering body weight loss and improving the survival rate from the co-infections. In conclusion, secondary bacterial infection can inhibit autophagy and stimulate mast cell activation through the PI3K/Akt pathway, which might explain why secondary bacterial infection would cause severe and fatal consequences following an initial influenza A viral infection.

Toxoplasma gondii in lambs of China: Heart juice serology, isolation and genotyping.

Toxoplasmosis is one of the most common foodborne diseases in the world. The objective of this study was to determine Toxoplasma gondii infection in lambs from Henan province, China. A total of 166 lamb hearts were collected from 2017 to 2019. T. gondii infection was determined by the Modified Agglutination Test (MAT) using heart juice of lambs. 11 isolates (TgSheepCHn3 - TgSheepCHn13) were obtained from samples with MAT titers ≥1:100. The rate of T. gondii isolation increased with antibody titer against T. gondii (P < 0.05). No isolate was obtained from samples with titer 1:25 and 1:50, suggesting the cut-off titer for MAT is better set at 1:100. With cut-off value of 1:100, IgG antibodies to T. gondii were found in 25.3% (42/166) of the lambs by MAT. T. gondii parasite was not found in IHC and HE-stained tissue sections of lamb hearts (0/166). Sixty-seven heart tissues with ≥1:25 MAT titers were subjected to acid pepsin digestion and detected T. gondii by PCR. Only 7.5% (5/67) of DNA amplified products were found in heart tissues by the primer TOX5/TOX8. Brain tissue cysts were observed in all mice infected with the 11 isolates at day 60 post infection, suggesting these isolates are non-lethal to mice. PCR-RFLP analysis revealed that 7 isolates belonged to ToxoDB#2, 4 isolates belonged to ToxoDB#4. This is the first isolation of ToxoDB#2 and ToxoDB#4 from lambs in China. Interestingly, none of these isolates belongs to the ToxoDB#9 that is common in China. Our results suggest that the genetic diversity and population structure of T. gondii from China maybe more abundant and magical than previous speculation.

Toxoplasma gondii in four captive kangaroos (Macropus spp.) in China: Isolation of a strain of a new genotype from an eastern grey kangaroo (Macropus giganteus).

Marsupials are highly susceptible to Toxoplasma gondii infection. Here, we report T. gondii infection in four kangaroos from a zoo in China. Kangaroos were imported into China in 2000 and were since bred in zoo. In 2017-2018, four kangaroos died due to respiratory system disease or injury. The bodies were submitted to the laboratory to test for T. gondii infection. Antibodies to T. gondii were found in 75% (3/4) of the kangaroos via the modified agglutination test with the cut-off 1:25. Cysts were observed in the histopathological sections of tongue and diaphragm or squashes of fresh myocardium in two kangaroos. These cysts were confirmed as T. gondii by immunohistochemical staining and molecular biological analysis. One viable T. gondii strain was isolated from one kangaroo and designated as TgRooCHn1. DNA from T. gondii tachyzoites obtained from cell culture was characterized by 10 PCR-RFLP markers and the virulence genes ROP5 and ROP18. The genotype of this isolate did not match with any known genotypes; it was designated as ToxoDB#292. The virulence of TgRooCHn1 (104 tachyzoites) was non-lethal to mice, and it formed tissue cysts. To our knowledge, the present study is the first isolation of ToxoDB#292 strain from kangaroo. Improvemets for captive settings were initiated, including greater attention being paied to birds and stray cats, fed frozen meat for carnivores.

Evidence of red panda as an intermediate host of Toxoplasma gondii and Sarcocystis species.

Toxoplasma gondii has been found to infect almost all warm-blooded animals; however, some hosts lack direct evidence of T. gondii infection. The red panda (Ailurus fulgens) is an endangered species that mainly lives in temperate forests of South Asia. Here, T. gondii infection in red pandas from zoos in China were reported. Antibodies to T. gondii were found in 14.3% (2/14) of red pandas via the modified agglutination test (MAT) with a cut-off titer of 1:25. One viable T. gondii strain was isolated from tissues of red panda and designated as TgRedpandaCHn1. DNA from tachyzoites obtained from cell culture was characterized by PCR-RFLP with 10 markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) and virulence genes of ROP5 and ROP18. The results indicate that this isolate belonged to ToxoDB genotype #20. The ROP18/ROP5 genotype combination predicated that this strain is non-lethal to mice, which is supported by the infection in mice. T. gondii tissue cysts were readily formed and mice survived. Tissue cysts observed in the histopathological sections of the tongue and diaphragm of one red panda were speculated as sarcocysts, but not T. gondii base on morphological characteristics. To our knowledge, this study is the first to report on the isolation of T. gondii from red panda. Additionally, this report provides direct evidence of red panda as an intermediate host of T. gondii and Sarcocystis species.