Toxoplasmosis, a globally prevalent zoonosis caused by Toxoplasma gondii (T. gondii), poses major threats to both human and animal health, leading to reproductive losses in livestock and severe disease in immunocompromised individuals. Although enzyme-linked immunosorbent assay (ELISA) and PCR are widely used for diagnosis and surveillance, they may be limited by turnaround time, laboratory instrumentation, and, in the case of serological assays, the need for species-specific reagents. To enable rapid, equipment-minimal detection applicable to multiple host species, we developed a point-of-care (POC) double-antigen sandwich colloidal gold immunochromatographic assay (GICA) based on recombinant surface antigen 2 (rSAG2) of T. gondii. In this assay, rSAG2 served both as the capture antigen immobilized on the test line and as the colloidal gold-conjugated detection probe. Key parameters, including conjugation pH, antigen loading, and buffer composition, were systematically optimized. The resulting strip showed a detection limit of a 1:40 serum dilution, and no cross-reactivity was observed with sera positive for 23 common pathogens from different host species. Good repeatability and storage stability for 4 months at 4°C were also observed. For field evaluation, 409 clinical serum samples from six animal groups (100 chickens, 68 dogs, 30 cats, 81 pigs, 80 yaks, and 50 sheep) were tested, and seropositivity rates ranged from 2.9% to 31.3% across the sampled groups. In a subset of chicken (n = 46) and dog (n = 44) sera tested in parallel with the corresponding commercial ELISA kits, the rSAG2-GICA showed good preliminary agreement, with overall agreement rates of 91.3% and 97.7%, respectively. Collectively, this rSAG2-based GICA shows potential as a rapid and practical tool for on-site serological screening of T. gondii antibodies across multiple host species and may support epidemiological surveillance in diverse animal populations.

Avian coccidiosis caused by Eimeria species threatens the global poultry industry. Current control measures rely mainly on anticoccidial drugs and live vaccines, but drug resistance and safety concerns drive the need for novel vaccines such as subunit vaccines. Among Eimeria species, Eimeria necatrix is one of the most pathogenic. Its life cycle involves sporozoites invading the jejunum and undergoing two generations of schizogony; the released second-generation merozoites then migrate specifically to the cecum for further development—a step essential for completing the life cycle. Previously, we identified EnMIC as the key molecule mediating this cecum-specific invasion, and its essential adhesive domain EnMAR2. In the present study, we evaluated the immunoprotective potential of EnMIC and EnMAR2. Recombinant subunit vaccines of EnMIC and EnMAR2 were administered to chickens. Humoral and cellular immune responses were assessed by measuring serum IgY, intestinal IgA, T-cell subsets (CD4⁺/CD8⁺), and cytokine transcripts (IL-2, IFN-γ, IL-4) in the spleen and cecal tonsils. After challenge with E. necatrix, protective effects were evaluated by survival rate, weight gain, lesion index, oocyst output, and the anticoccidial index (ACI). Both vaccines significantly elevated serum IgY and intestinal IgA levels. They also increased splenic IL‑2, IFN‑γ, and IL‑4 mRNA levels, while downregulating these cytokines in cecal tonsils. Furthermore, the rEnMIC group showed an increased proportion of CD4⁺ T cells in both the spleen and cecal tonsils. In the challenge trial, both the rEnMIC and rEnMAR2 groups exhibited significantly better weight gain, lower lesion index, and greater oocyst reduction than the non‑immunized infected group. Their ACI values were 168.92 and 166.60, respectively. These results demonstrate that EnMIC and EnMAR2, as key mediators of E. necatrix site-specific cecal invasion, can induce both humoral and cellular immune responses when delivered as recombinant protein vaccines. The vaccines effectively alleviated pathological damage, reduced oocyst output, and improved growth performance. Therefore, EnMIC and EnMAR2 represent promising novel vaccine candidates against E. necatrix infection in chickens.

Rhizosphere microorganisms play crucial roles in regulating crop secondary metabolism and stress adaptation, yet the mechanisms by which they coordinately enhance both 2-acetyl-1-pyrroline (2-AP) and stress resistance in fragrant rice remain unclear. Here, we report a newly isolated a Rhizopus strain TYR1 (CGMCC NO: 42,112) from fragrant rice rhizosphere. Through field trials, pot experiments, hydroponics, and plate assays combined with transcriptomics, qRT-PCR, metabolite quantification, and enzyme activity analysis, we demonstrate that TYR1 specifically colonizes rice roots and simultaneously increases grain 2-AP content by 38.04

Root-knot nematodes secrete effectors into plant cells to facilitate parasitism. A candidate effector, MiMSP8, of Meloidogyne incognita has been shown to localise within the nucleus when transiently expressed in Nicotiana benthamiana leaf cells, but its role is still unknown. We demonstrate that the MiMSP8 protein is expressed in the dorsal gland of M. incognita juveniles and it can also be detected within giant cells induced by nematodes. Silencing of MiMSP8 impairs nematode parasitism while overexpression of MiMSP8 increases susceptibility to nematode infection. MiMSP8 interacts with SlU2AF35, the small subunit of the U2 snRNP auxiliary factor (U2AF) in tomato. This interaction competes with SlU2AF65 for binding to SlU2AF35, thereby disrupting the formation of heterodimers of the key splicing factor U2AF. Overexpression of MiMSP8 in tomato hairy roots leads to genome-wide alternative splicing changes involved in multiple biological processes. MiMSP8 interferes with the binding between SlU2AF35 and pre-mRNAs of a subset of genes. Silencing SlU2AF35 results in abnormal gene splicing in plants and increases their sensitivity to nematode parasitism. Collectively, our findings reveal that M. incognita deploys a nuclear localised effector to target a key component of the spliceosome and disrupt the splicing of plant pre-mRNA to promote parasitism.

Toll-like receptor (TLR)-mediated recognition of specific ligands is critical for initiating innate immunity against Eimeria infections. However, the avian-specific TLR15 has unclear immunological functions, including its specific ligands and role in anti-coccidia immunity. In this study, an aspartyl protease from Eimeria tenella (EteASP) was identified as a potential TLR15 ligand by co-immunoprecipitation. Two distinct TLR15-expressing innate immune cell populations, macrophages and dendritic cells, were employed as models to investigate the immune responses triggered by recombinant EteASP protein (rEteASP). In macrophages, rEteASP induced robust immune activation, associated with the stimulation of both NF-κB and MAPK pathways. This activation correlated with increased production of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), nitric oxide, and reactive oxygen species, alongside enhanced phagocytosis. Furthermore, rEteASP promoted classical dendritic cell activation in chicken bone marrow-derived dendritic cells, marked by stellate morphology and upregulated CD11c and MHC II surface expression. The activation patterns in chBMDCs mirrored those in macrophages, characterized by increased production of immune mediators. A mixed lymphocyte reaction (MLR) further demonstrated that rEteASP-activated dendritic cells efficiently triggered naive T cell activation, driving a shift towards balanced Th1/Th2-type cellular immune response. Collectively, these findings establish EteASP as a potent immunomodulator capable of activating key innate immune effector functions in macrophages and dendritic cells, and bridging to adaptive immunity. Our data implicate TLR15 as a potential receptor contributing to these responses, broadening the investigation of TLR15 ligands in protozoa and offering novel insights into Eimeria-host immune interactions. However, the potential involvement of additional pattern recognition receptors in EteASP recognition and the definitive role of TLR15 requires further investigation.

The root-knot nematode Meloidogyne incognita is an obligate biotrophic pathogen that causes extensive losses to agriculture worldwide. Effectors secreted by the parasite play an essential role during nematode infection through suppressing plant innate immunity. Here, we identify and characterize a M. incognita effector designated as MiV86, which is secreted into plant cells and positively regulates nematode parasitism. We show that MiV86 interacts with RING finger protein 217 (NbRNF217), an RBR-type E3 ubiquitin ligase of Nicotiana benthamiana, which negatively regulates plant immunity in an enzymatic activity-dependent manner. Moreover, we demonstrate that NbRNF217 targets and ubiquitinates the helper nucleotide-binding leucine-rich repeat receptor protein NRC4, resulting in its relocation and degradation through the 26S proteasome and endosomal/vacuolar pathways. NbRNF217 regulates its homeostasis through self-catalyzed ubiquitination or external ubiquitination modifications, and we show that MiV86 inhibits the ubiquitination of NbRNF217 in planta without affecting its activity, thereby promoting the degradation of NRC4, which also contributes to the resistance of N. benthamiana against M. incognita. Our findings reveal a mechanism by which a nematode effector hijacks an E3 ubiquitin ligase to attenuate NRC4-mediated plant immunity, facilitating nematode parasitism.

Meloidogyne incognita is one of the most widely distributed plant-parasitic nematodes and causes severe economic losses annually. The parasite produces effector proteins that play essential roles in successful parasitism. Here, we identified one such effector named MiCE108, which is exclusively expressed within the nematode subventral esophageal gland cells and is upregulated in the early parasitic stage of M. incognita. A yeast signal sequence trap assay showed that MiCE108 contains a functional signal peptide for secretion. Virus-induced gene silencing of MiCE108 impaired the parasitism of M. incognita in Nicotiana benthamiana. The ectopic expression of MiCE108 in Arabidopsis suppressed the deposition of callose, the generation of reactive oxygen species, and the expression of marker genes for bacterial flagellin epitope flg22-triggered immunity, resulting in increased susceptibility to M. incognita, Botrytis cinerea, and Pseudomonas syringae pv. tomato (Pst) DC3000. The MiCE108 protein physically associates with the plant defense protease RD21A and promotes its degradation via the endosomal-dependent pathway, or 26S proteasome. Consistent with this, knockout of RD21A compromises the innate immunity of Arabidopsis and increases its susceptibility to a broad range of pathogens, including M. incognita, strongly indicating a role in defense against this nematode. Together, our data suggest that M. incognita deploys the effector MiCE108 to target Arabidopsis cysteine protease RD21A and affect its stability, thereby suppressing plant innate immunity and facilitating parasitism.

Clinical avian coccidiosis is typically caused by coinfection with several Eimeria species. Recombinant protein and DNA vaccines have shown promise in controlling coccidiosis. On this basis, DNA vaccines that encode multiple epitopes from different Eimeria species may provide broad protection against coinfections. In this study, we designed a fusion gene fragment, 14EGT, that contained concentrated T-cell epitopes from four common antigens of Eimeria species (14-3-3, elongation factor 2, glyceraldehyde-3-phosphate dehydrogenase, and transhydrogenase). The multiepitope DNA vaccine pVAX1-14EGT and recombinant protein vaccine pET-32a-14EGT (r14EGT) were then created based on the 14EGT fragment. Subsequently, cellular and humoral immune responses were measured in vaccinated chickens. Vaccination-challenge trials were also conducted, where the birds were vaccinated with the 14EGT preparations and later exposed to single or multiple Eimeria species to evaluate the protective efficacy of the vaccines. According to the results, vaccination with 14EGT preparations effectively increased the proportions of CD4 + and CD8 + T cells and the levels of Th1 and Th2 hallmark cytokines. The levels of serum IgG antibodies were also significantly increased. Animal vaccination trials revealed alleviated enteric lesions, weight loss, and oocyst output compared to those of the control groups. The preparations were found to be moderately effective against single Eimeria species, with the anticoccidial index (ACI) ranging from 160 to 180. However, after challenge with multiple Eimeria species, the protection provided by the 14EGT preparations was not satisfactory, with ACI values of 142.18 and 146.41. Collectively, the results suggest that a multiepitope vaccine that encodes the T-cell epitopes of common antigens derived from Eimeria parasites could be a potential and effective strategy to control avian coccidiosis.