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Chicken coccidiosis caused by Eimeria spp. can occur on almost all poultry farms, causing huge economic losses to the industry. Genetically manipulated Eimeria parasites as a vaccine vector to deliver viral antigens have been reported. In our preliminary study, transgenic E. acervulina expressing a VP2 gene (Ea-VP2) of the infectious bursal disease virus (IBDV) demonstrated partial protection against IBDV infection. To enhance immune responses, we aimed to increase the VP2 gene copy number in transgenic E. acervulina. In this study, we used a novel plasmid vector carrying a VP2 gene fused with three flag tags and a red fluorescent reporter gene (mCherry). The vector was introduced into Ea-VP2 sporozoites through nucleofection, leading to the generation of Ea-2VP2. Subsequent analysis revealed a notable escalation in the fluorescent rate, increasing from 0.11 to 95.1% following four consecutive passages facilitated by fluorescent-activated cell sorting. Verification via PCR, Western blot, and immunofluorescence confirmed the successful construction of the Ea-2VP2 population. Despite lower fecundity compared to wild-type E. acervulina, Ea-2VP2 maintained immunogenicity. Our research effectively created a transgenic E. acervulina strain transfected sequentially with two copies of the VP2 gene from IBDV. This modification resulted in an increased humoral immune response after primary immunization in chickens. Additionally, it demonstrated a degree of protection within the bursa against IBDV infection. Future studies will focus on further enhancing immune response levels.

ObjectiveThe environment influences the sow's health and physiology during gestation. This study was conducted to evaluate indoor environmental parameters and physiological responses of early-gestation sows and investigate the possible methods for assessing the thermal environment in commercial houses.MethodsA total of 20 early-gestation sows (commercial purebred Yorkshire) with an average body weight of 193.20 ± 3.62 kg were used for this study in winter, spring, summer, and autumn. The indoor environment parameters comprising dry-bulb temperature (Tdb), relative humidity (RH), and carbon dioxide (CO2) were recorded in 30-min intervals. Physiological parameters including heart rate (HR) and respiration rate (RR) of sows were also measured every 30 min. Wet-bulb temperature (Twb) was calculated using Tdb, RH and atmospheric pressure was recorded at a nearby weather station.ResultsThe average indoor Tdb and RH were 12.98 ± 2.03°C and 80.4 ± 6.4% in winter, 18.98 ± 2.68°C and 74.4 ± 9.0% in spring, 27.49 ± 2.05°C and 90.6 ± 6.4% in summer, and 17.10 ± 2.72°C and 64.5 ± 10.9% in autumn. A higher average concentration of CO2 was observed in winter (1,493 ± 578 mg/m3) than in spring (1,299 ± 489 mg/m3), autumn (1,269 ± 229 mg/m3), and summer (702 ± 128 mg/m3). Compared with the HR and RR in the optimum environment, high RH in the house led to a significant decrease in both HR and RR (P < 0.05). In addition, a significant decline in HR was also obtained at high temperatures (P < 0.05). A temperature humidity index (THI), THI = 0.82 × Tdb + 0.18 × Twb, was determined for early-gestation sows, and the THI thresholds were 25.6 for HR. The variation in THI in summer showed that heat stress still occurred under the pad-fan cooling system.ConclusionThis study demonstrated the critical significance of considering physiological responses of early-gestation sows in commercial houses and THI thresholds. We recommend that much more cooling measures should be taken for early-gestation sows in summer.

Eimeria, a cousin of malarial parasites, causes coccidiosis that results in huge losses in the poultry industry. Although live coccidiosis vaccines have been developed and used widely for the successful control of the disease, the mechanism underlying protective immunity remains largely unknown. Using Eimeria falciformis as a model parasite, we observed that tissue-resident memory CD8+ T (Trm) cells accumulated in cecal lamina propria following E. falciformis infection in mice, especially after reinfection. In convalescent mice challenged with a second infection, E. falciformis burden diminished within 48-72 h. Deep-sequencing revealed that CD8+ Trm cells were characterized by rapid up-regulation of effector genes encoding pro-inflammatory cytokines and cytotoxic effector molecules. While FTY720 (Fingolimod) treatment prevented the trafficking of CD8+ T cells in peripheral circulation and exacerbated primary E. falciformis infection, such treatment had no impact on the expansion of CD8+ Trm cells in convalescent mice receiving secondary infection. Adoptive transfer of cecal CD8+ Trm cells conferred immune protection in naïve mice, indicating that these cells provide direct and effective protection against infection. Overall, our findings not only explain a protective mechanism of live oocyst-based anti-Eimeria vaccines but also provide a valuable correlate for assessing vaccines against other protozoan diseases.

Eimeria species are pathogenic protozoa with a wide range of hosts and the cause of poultry coccidiosis, which results in huge economic losses to the poultry industry. These parasites encode a genome of ∼8000 genes that control a highly coordinated life cycle of asexual replication and sexual differentiation, transmission, and virulence. However, the function and physiological importance of the large majority of these genes remain unknown mostly due to the lack of tools for systematic analysis of gene functions. Here, we report the first application of CRISPR-Cas9 gene editing technology in Eimeria tenella for analysis of gene function at a single gene level as well as for systematic functional analysis of an entire gene family. Using a transgenic line constitutively expressing Cas9, we demonstrated successful and efficient loss of function through non-homologous end joining as well as guided homologous recombination. Application of this approach to the study of the localization of EtGRA9 revealed that the gene encodes a secreted protein whose cellular distribution varied during the life cycle. Systematic disruption of the ApiAp2 transcription factor gene family using this approach revealed that 23 of the 33 factors expressed by this parasite are essential for development and survival in the host. Our data thus establish CRISPR-Cas9 as a powerful technology for gene editing in Eimeria and will set the stage for systematic functional analysis of its genome to understand its biology and pathogenesis, and will make it possible to identify and validate new targets for coccidiosis therapy.

Vaccine delivery is critical in antigen discovery and vaccine efficacy and safety. The diversity of infectious diseases in humans and livestock has required the development of varied delivery vehicles to target different pathogens. In livestock animals, previous strategies for the development of coccidiosis vaccines have encountered several hurdles, limiting the development of multiple species vaccine formulations. Here, we describe a novel vaccine delivery system using transgenic Eimeria tenella expressing immunodominant antigens of Eimeria maxima. In this delivery system, the immune mapped protein 1 of E. maxima (EmIMP1) was delivered by the closely related species of E. tenella to the host immune system during the whole endogenous life cycle. The overexpression of the exogenous antigen did not interfere with the reproduction and immunogenicity of transgenic Eimeria. After immunization with the transgenic parasite, we detected EmIMP1’s and E. maxima oocyst antigens’ specific humoral and cellular immune responses. In particular, we observed partial protection of chickens immunized with transgenic E. tenella against subsequent E. maxima infections. Our results demonstrate that the transgenic Eimeria parasite is an ideal coccidia antigen delivery vehicle and represents a new type of coccidiosis vaccines. In addition, this model could potentially be used in the development of malaria live sporozoite vaccines, in which antigens from different strains can be expressed in the vaccine strain.