Protective efficacy of Eimeria maxima EmLPL and EmTregIM-1 against homologous challenge in chickens.

Chicken coccidiosis has inflicted significant economic losses upon the poultry industry. The primary strategies for preventing and controlling chicken coccidiosis include anticoccidial drugs and vaccination. However, these approaches face limitations, such as drug residues and resistance associated with anticoccidial drugs, and safety concerns related to live vaccines. Consequently, the urgent development of innovative vaccines, such as subunit vaccines, is imperative. In previous study, we screened 2 candidate antigens: Eimeria maxima lysophospholipase (EmLPL) and E. maxima regulatory T cell inducing molecule 1 (EmTregIM-1). To investigate the immune protective effect of the 2 candidate antigens against Eimeria maxima (E. maxima) infection, we constructed recombinant plasmids, namely pET-28a-EmLPL and pET-28a-EmTregIM-1, proceeded to induce the expression of recombinant proteins of EmLPL (rEmLPL) and EmTregIM-1 (rEmTregIM-1). The immunogenic properties of these proteins were confirmed through western blot analysis. Targeting EmLPL and EmTregIM-1, we developed subunit vaccines and encapsulated them in PLGA nanoparticles, resulting in nano-vaccines: PLGA-rEmLPL and PLGA-rEmTregIM-1. The efficacy of these vaccines was assessed through animal protection experiments. The results demonstrated that rEmLPL and rEmTregIM-1 were successfully recognized by anti-E. maxima chicken sera and His-conjugated mouse monoclonal antibodies. Immunization with both subunit and nano-vaccines containing EmLPL and EmTregIM-1 markedly mitigated weight loss and reduced oocyst shedding in chickens infected with E. maxima. Furthermore, the anticoccidial indexes (ACI) for both rEmLPL and PLGA-rEmLPL exceeded 160, whereas those for rEmTregIM-1 and PLGA-rEmTregIM-1 were above 120 but did not reach 160, indicating superior protective efficacy of the rEmLPL and PLGA-rEmLPL formulations. By contrast, the protection afforded by rEmTregIM-1 and PLGA-rEmTregIM-1 was comparatively lower. Thus, EmLPL is identified as a promising candidate antigen for vaccine development against E. maxima infection.

Inhibitory effect of Eimeria maxima IFN-γ inhibitory molecules on the immune function of T cell subsets in chickens.

It has been reported that infection of chicken coccidian could inhibit the production of Th1 cytokine IFN-γ, thereby evading clearance by the host immune system. The present study aimed to have a further investigation into the effects of Eimeria maxima IFN-γ inhibitory molecules (EmHPSP-2 and EmHPSP-3) on the immune function of chicken peripheral blood mononuclear cells (PBMC) and various T cell subsets. First, separated PBMC or sorted T cell subsets were used for incubation with recombinant proteins of EmHPSP-2 (rEmHPSP-2) and EmHPSP-3 (rEmHPSP-3). Subsequently, the effects of rEmHPSP-2 and rEmHPSP-3 on proliferative capacity, nitric oxide (NO) release and mRNA levels of cytokines of the above cells were detected. The sorting purity of CD8+, CD4+ CD25-, CD4+, and CD4+ CD25+ T cells was 93.01, 88.88, 87.04, and 81.26%, respectively. The NO release of PBMC was significantly inhibited by rEmHPSP-2 and rEmHPSP-3. The proliferation of PBMC and CD4+ T cells was significantly inhibited by rEmHPSP-2 and rEmHPSP-3, whereas CD8+, CD4+ CD25-, and CD4+ CD25+ T cells was significantly promoted by the 2 proteins. The 2 proteins significantly downregulated interferon-gamma (IFN-γ) mRNA level, upregulated the transcriptional levels of interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-ß1) in PBMC. IFN-γ and IL-2 transcriptional levels were markedly inhibited in CD8+ T cells. IFN-γ transcriptional level was significantly inhibited, but IL-4 was promoted by rEmHPSP-2 and rEmHPSP-3 in CD4+ CD25- T cells. Meanwhile, the inhibitory effects of rEmHPSP-2 and rEmHPSP-3 on the transcriptional levels of IFN-γ and IL-2 were more obvious in CD4+ T cells containing CD25+ cells compared with the CD25+ cells depletion group. It was found that IL-10, TGF-ß1, and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) mRNA levels were significantly upregulated upon stimulation of chicken CD4+ CD25+ T cells by proteins. This study is not only of great significance to clarify the immune evasion mechanism of chicken coccidia, but also provides candidate antigen molecules for development of a novel vaccine against chicken coccidiosis.

Differential changes in cerebellar transmitter content and expression of calcium binding proteins and transcription factors in mouse administered with amphetamine.

Cerebellar mechanisms were explored underlying the effects of amphetamine (Amph) on the brain, by monitoring primarily the neurochemistry of the cerebellum. Adult mice received repeated intermittent injections of d-Amph, 5mg/kg or saline, twice daily for three days and once on day 4. As revealed by the biochemical analysis, the levels of GABA content were increased by 68-93% in the cerebellar vermis and hemisphere of mice at 4h after the first (acute) or the last (repeated) Amph injections, though the glutamate content was unaltered, compared to the respective saline-treated controls. By contrast, at 4h post-repeated Amph, in the vermis, the level of norepinephrine was approximate 38% lower than the corresponding control and 5-hydroxytryptamine (5-HT) resembled the control, whereas in the hemisphere, the norepinephrine content was similar to control and 5-HT 66% higher, implying cerebellar lobe-dependent changes. However, in both lobes, at 4h after the acute and repeated Amph exposures, changes of the transmitter content were correlated with reductions of 50-64% in the levels of the phosphorylated (p) MAP kinase (K) expression and 39-55% in the calbindin-D28k (CB) of the Purkinje cell somata, and increases of 289-556% in pCREB, 373-594% c-FOS, and 51-76% calretinin of the granular layer, as shown by immunohistochemical analysis. The up-regulated GABA content in the vermis and hemisphere may be associated with the decreased expression of Purkinje somatal CB and pMAPK, implicating a relation between the Ca(2+) of Purkinje cells and the level of GABA. Furthermore, the prominent increases of the granular layer pCREB, c-FOS and calretinin may influence the activity of Purkinje cells, which are known to be modulated by the granule cells. The data indicate that the Amph exposure selectively alters specific transmitters in the cerebellar lobes and modifies the cellular expression of distinct signaling proteins in the cerebellar layers.