Ferroptosis is a newly discovered form of cell death due to iron-dependent lipid peroxidation. In animal breeding, many environmental factors could lead to oxidative stress, which in turn reduce animal immunity and production performance. Polysaccharide of Atractylodes macrocephala Koidz (PAMK) has antioxidation, immunomodulatory, and inflammatory modulating effects. For investigating the effect of PAMK on splenic ferroptosis in gosling caused by lipopolysaccharide (LPS), 40 one-day-old Magang goslings were randomly divided into 4 groups (CON group, LPS group, PAMK group, and LPS+PAMK group). The protein expression of the ferroptosis marker Glutathione Peroxidase 4 (GPX4), the relative mRNA expression of ferroptosis-related genes and cytokines, and the oxidative stress and iron content of spleen tissues were examined. The correlation between ferroptosis and inflammatory factors was further analyzed by principal component analysis. The results showed that, compared with CON group, LPS caused alterations in the expression of the ferroptosis pathway genes and cytokines, which could upregulate levels of ferroptosis and inflammation. However, after treated with PAMK, the inflammation and ferroptosis was alleviated. Meanwhile, PAMK restored the expression and distribution of GPX4. In addition, PAMK alleviated the oxidative stress caused by LPS and reduced the iron content in spleen. Principal component analysis showed that cytokines were more closely related to antioxidant indexes. The CON, PAMK and LPS+PAMK groups had similar effects on the four components, with the LPS and PAMK groups showing the furthest difference in results. The result indicated that PAMK could reduce the level of oxidative stress and inflammatory cytokines in spleen of gosling caused by LPS, and jointly alleviate ferroptosis by regulating genes related to the ferroptosis pathway.

Polysaccharide of Atractylodes macrocephala Koidz (PAMK) has been well recognized as an immune enhancer that can promote lymphocyte proliferation and activate immune cells. The purpose of this study was to evaluate the effects of PAMK on humoral and cellular immune functions in immunosuppressed geese. Geese of the Control group were provided with normal feed, the PAMK group was provided with 400 mg·(kg body weight)⁻¹ PAMK, the cyclophosphamide (CTX) group was injected with 40 mg·(kg body weight)⁻¹ cyclophosphamide, while the CTX+PAMK group received the combination of PAMK and CTX. Spleen development and percentages of leukocytes in peripheral blood were examined. Principal component analysis was conducted to analyze correlations among humoral and cellular immune indicators. The results showed that PAMK alleviated the damage to the spleen, the decrease in T- and B-cell proliferation, the imbalance of leukocytes, and the disturbances of humoral and cellular immunity caused by CTX. Principal component analysis revealed that the relevance of humoral-immunity-related indicators was greater, and the CTX+PAMK group manifested the largest difference from the CTX group but was close to the Control group. In conclusion, PAMK alleviates the immunosuppression caused by CTX in geese, and the protective effect on humoral immunity is more obvious and stable.

Polysaccharide of Atractylodes macrocephala Koidz (PAMK) has been reported to have beneficial effects on regulation of immune responses in mammals and poultry. Nonetheless, the immunoregulatory mechanism of action of PAMK remains unclear. The Toll-like receptor 4 (TLR4) signaling cascade has been proved as a classic polysaccharide-regulated pathway. The aim of this study was to explore the effects of PAMK on the TLR4 signaling pathway in the regulation of spleen function in mice. Ninety-six 5-week-old BALB/c female mice were randomly allocated into four groups with three replicates per group and eight mice per replicate in a single-factor completely randomized experimental design. The control group was fed a basic diet (PAMK free); the other three groups were fed 100, 200, or 400 mg/kg PAMK for 28 days. The spleen index, concentrations of cytokines, and mRNA and protein expression levels of genes related to TLR4 signaling were determined in spleen tissue. Compared with the control group, the spleen index significantly increased in all treatment groups. Concentrations of interleukin 2 (IL-2), IL-4, interferon γ (IFN- γ ), and tumor necrosis factor α (TNF- α ) in the medium-PAMK group also increased significantly. PAMK in the medium-PAMK group significantly increased both mRNA and protein expression of TLR4, myeloid differentiation factor 88 (MyD88), TNFR-associated factor 6 (TRAF6), TRAF3, and nuclear factor kappa B (NF- κ B) in the spleen. In conclusion, PAMK may increase immune-response capacity of the spleen in mice via TLR4–MyD88–NF- κ B signaling.

Major viruses, including duck-origin avian influenza virus, duck-origin Newcastle disease virus, novel duck parvovirus, duck hepatitis A virus, duck Tembusu virus, fowl adenovirus, and duck enteritis virus, pose great harm to ducks and cause enormous economic losses to duck industry. This study aims to establish a multiplex polymerase chain reaction (m-PCR) method for simultaneous detection of these seven viruses. Specific primers were designed and synthesized according to the conserved region of seven viral gene sequences. Then, seven recombinant plasmids, as the positive controls, were reconstructed in this study. Within the study, D-optimal design was adopted to optimize PCR parameters. The optimum parameters for m-PCR were annealing temperature at 57 °C, Mg2+ concentration at 4 mM, Taq DNA polymerase concentration at 0.05 U/μL, and dNTP concentration at 0.32 mM. With these optimal parameters, the m-PCR method produced neither cross-reactions among these seven viruses nor nonspecific reactions with other common waterfowl pathogens. The detection limit of m-PCR for each virus was 1 × 104 viral DNA copies/μL. In addition, the m-PCR method could detect a combination of several random viruses in co-infection analysis. Finally, the m-PCR method was successfully applied to clinical samples, and the detection results were consistent with uniplex PCR. Given its rapidity, specificity, sensitivity, and convenience, the established m-PCR method is feasible for simultaneous detection of seven duck-infecting viruses and can be applied to clinical diagnosis of viral infection in ducks.

As companion animals, felines play an important role in human's family life, and their healthcare has attracted great attention. Viruses such as feline calicivirus (FCV), feline herpesvirus 1 (FHV-1), and feline parvovirus virus (FPV) are the most common pathogens that cause severe infectious disease in baby cats. Thus, preclinical detection and intervention of these three viruses is an effective means to prevent diseases and minimize their danger condition. In this study, a triplex TaqMan quantitative real-time polymerase chain reaction (qRT-PCR) assay was developed to detect these three viruses simultaneously. The detection limit of FPV, FCV, and FHV-1 was 5 × 10¹copies/assay, which exhibited higher sensitivity (about 10- to100-fold) than conventional PCR. The coefficients of variation (CVs) of the intra-assay variability were lower than 1.86%, and that of inter-assay variability were lower than 3.19%, indicating the excellent repeatability and reproducibility of the triplex assay. Additionally, the assay showed good specificity. Finally, samples from 48 cats were analyzed using the established assay and commercial kits. As a result, the total positive rates for these viruses were 70.83 or 62.5%, respectively, which demonstrated that the developed qRT-PCR assay was more accurate than the commercial kits and could be used in clinical diagnosis.