BACKGROUND: Chicken coccidiosis is a protozoan disease that leads to considerable economic losses in the poultry industry. Live oocyst vaccination is currently the most effective measure for the prevention of coccidiosis. However, it provides limited protection with several drawbacks, such as poor immunological protection and potential reversion to virulence. Therefore, the development of effective and safe vaccines against chicken coccidiosis is still urgently needed.
METHODS: In this study, a novel oral vaccine against Eimeria tenella was developed by constructing a recombinant Lactobacillus plantarum (NC8) strain expressing the E. tenella RON2 protein. We administered recombinant L. plantarum orally at 3, 4 and 5 days of age and again at 17, 18 and 19 days of age. Meanwhile, each chick in the commercial vaccine group was immunized with 3 × 102 live oocysts of coccidia. A total of 5 × 104 sporulated oocysts of E. tenella were inoculated in each chicken at 30 days. Then, the immunoprotection effect was evaluated after E. tenella infection.
RESULTS: The results showed that the proportion of CD4+ and CD8+ T cells, the proliferative ability of spleen lymphocytes, inflammatory cytokine levels and specific antibody titers of chicks immunized with recombinant L. plantarum were significantly increased (P < 0.05). The relative body weight gains were increased and the number of oocysts per gram (OPG) was decreased after E. tenella challenge. Moreover, the lesion scores and histopathological cecum sections showed that recombinant L. plantarum can significantly relieve pathological damage in the cecum. The ACI was 170.89 in the recombinant L. plantarum group, which was higher than the 150.14 in the commercial vaccine group.
CONCLUSIONS: These above results indicate that L. plantarum expressing RON2 improved humoral and cellular immunity and enhanced immunoprotection against E. tenella. The protective efficacy was superior to that of vaccination with the commercial live oocyst vaccine. This study suggests that recombinant L. plantarum expressing the RON2 protein provides a promising strategy for vaccine development against coccidiosis.

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.

Toxoplasma gondii is an intracellular protozoan parasite that infects all nucleated cells except the red blood cells. Currently, nucleic acid vaccines are being widely investigated in Toxoplasma gondii control, and several nucleic acid vaccine candidate antigens have shown good protection in various studies. The aim of this study was to construct a nucleic acid vaccine with Toxoplasma gondii SRS29C as the target gene. We explored the nucleic acid vaccine with Toxoplasma surface protein SRS29C and the combined gene of SRS29C and SAG1 and evaluated its immunoprotective effect against Toxoplasma gondii. To amplify the gene fragment and clone it to the expression vector, the recombinant plasmid pEGFP-SRS29C was constructed by PCR. Eukaryotic cells were transfected with the plasmid, and the expression of the target protein was assessed using the Western blot method. The level of serum IgG was determined via ELISA, and the splenic lymphocyte proliferation ability was detected using the CCK-8 method. The percentages of CD4+ and CD8+ T cells were measured by flow cytometry. Mice were immunised three times with single-gene nucleic acid vaccine and combination vaccine. Splenic lymphocytokine expression was determined using ELISA kits. The mice\'s survival time was monitored and recorded during an in vivo insect assault experiment, and the vaccine\'s protective power was assessed. The outcomes showed that PCR-amplification of an SRS29C gene fragment was successful. The 4,733-bp vector fragment and the 1,119-bp target segment were both recognised by double digestion. Additionally, after transfection of the recombinant plasmid pEGFP-SRS29C, Western blot examination of the extracted protein revealed the presence of a target protein strip at 66 kDa. The test results demonstrated that the IgG content in the serum of the pEGFP-SRS29C group and the co-immunization group was significantly higher than that of the PBS group and the empty vector group. The IgG potency induced by the co-immunization group was higher than that of the pEGFP-SRS29C group and the pEGFP-SAG1 group, the number of splenic lymphocyte proliferation number was higher than that of the PBS group and the empty vector group. The CD4+/CD8+ T ratio was higher than that of the PBS group and the empty vector group. The expression of IFN-γ and TNF-α in the splenocytes of the pEGFP-SRS29C group and the combined immunisation group was significantly higher following antigen stimulation. In the worm attack experiments, mice in the PBS and empty vector groups perished within 9 days of the worm attack, whereas mice in the pEGFP-SRS29C group survived for 18 days, mice in the pEGFP-SAG1 group survived for 21 days, and mice in the co-immunization group survived for 24 days. This demonstrates that the constructed Toxoplasma gondii nucleic acid vaccine pEGFP-SRS29C and the combined gene vaccine can induce mice to develop certain humoral and cellular immune responses, and enhance their ability to resist Toxoplasma gondii infection.

Toxoplasmosis is one of the most dangerous zoonotic diseases, causing serious economic losses worldwide due to abortion and reproductive problems. Vaccination is the best way to prevent disease; thus, it is imperative to develop a candidate vaccine for toxoplasmosis. BAG1 and ROP8 have the potential to become vaccine candidates. In this study, rTgBAG1, rTgROP8, and rTgBAG1-rTgROP8 were used to evaluate the immune effect of vaccines in each group by detecting the humoral and cellular immune response levels of BABL/c mice after immunization and the ability to resist acute and chronic infection with Toxoplasma gondii (T. gondii). We divided the mice into vaccine groups with different proteins, and the mice were immunized on days 0, 14, and 28. The protective effects of different proteins against T. gondii were analysed by measuring the cytokines, serum antibodies, splenocyte proliferation assay results, survival time, and number and diameter of brain cysts of mice after infection. The vaccine groups exhibited substantially higher IgG, IgG1, and IgG2a levels and effectively stimulated lymphocyte proliferation. The levels of IFN-γ and IL-2 in the vaccine group were significantly increased. The survival time of the mice in each vaccine group was prolonged and the diameter of the cysts in the vaccine group was smaller; rTgBAG1-rTgROP8 had a better protection. Our study showed that the rTgBAG1, rTgROP8, and rTgBAG1-rTgROP8 recombinant protein vaccines are partial but effective approaches against acute or chronic T. gondii infection. They are potential candidates for a toxoplasmosis vaccine.

Eimeria tenella is the most pathogenic and harmful intestinal parasitic protozoan. Recombinant DNA vaccines open options for promising strategies for preventing avian coccidiosis, replacing chemical drugs and live oocyst vaccines. Two important antigenic proteins, EtAMA3 (also known as SporoAMA1) and EtRON2L2, act together to promote the invasion of E. tenella sporozoites. In this study, a recombinant DNA vaccine, designated pcDNA3.1(+)-AR, was constructed based on EtAMA3DII, EtRON2L2D3, and EtRON2L2D4. Chickens were intramuscularly immunized with different doses (25, 50, or 100 μg) of pcDNA3.1(+)-AR to evaluate its immunoprotective effects in vivo. The chickens in the 50 μg and 100 μg groups had higher cytokine concentrations (interleukin 2, interferon-gamma, and interleukin 10), and lesion scores (81.9% and 67.57%, respectively) and relative oocyst production (47% and 19%, respectively) reduced compared with the unchallenged group, indicating partial protection against E. tenella. These results suggest that pcDNA3.1(+)-AR is a promising vaccine candidate against avian coccidiosis.

BACKGROUND: Rabbit coccidiosis is a parasitism caused by either one or multiple co-infections of Eimeria species. Among them, Eimeria intestinalis is the primary pathogen responsible for diarrhea, growth retardation, and potential mortality in rabbits. Concerns regarding drug resistance and drug residues have led to the development of recombinant subunit vaccines targeting Eimeria species as a promising preventive measure. The aim of this study was to assess the immunoprotective efficacy of recombinant subunit vaccines comprising EiROP25 and EiROP30 (rhoptry proteins (ROPs)) against E. intestinalis infection in rabbits.
METHODS: Cloning, prokaryotic expression, and protein purification were performed to obtain EiROP25 and EiROP30. Five groups of fifty 35-day-old Eimeria-free rabbits were created (unchallenged control group, challenged control group, vector protein control group, rEiROP25 group, and rEiROP30 group), with 10 rabbits in each group. Rabbits in the rEiROP25 and rEiROP30 groups were immunized with the recombinant proteins (100 μg per rabbit) for primary and booster immunization (100 μg per rabbit) at a two-week intervals, and challenged with 7 × 104 oocysts per rabbit after an additional two-week interval. Two weeks after the challenge, the rabbits were euthanized for analysis. Weekly collections of rabbit sera were made to measure changes in specific IgG and cytokine level. Clinical symptoms and pathological changes after challenge were observed and recorded. At the conclusion of the animal experiment, lesion scores, the relative weight increase ratio, the oocyst reduction rate, and the anticoccidial index were computed.
RESULTS: Rabbits immunized with rEiROP25 and rEiROP30 exhibited relative weight gain ratios of 56.57% and 72.36%, respectively. Oocysts decreased by 78.14% and 84.06% for the rEiROP25 and rEiROP30 groups, respectively. The anticoccidial indexes were 140 and 155. Furthermore, there was a noticeable drop in intestinal lesions. After the primary immunization with rEiROP25 and rEiROP30, a week later, there was a notable rise in specific IgG levels, which remained elevated for two weeks following challenge (P < 0.05). Interleukin (IL)-2 levels increased markedly in the rEiROP25 group, whereas IL-2, interferon gamma (IFN-γ), and IL-4 levels increased substantially in the rEiROP30 group (P < 0.05).
CONCLUSIONS: Immunization of rabbits indicated that both rEiROP25 and rEiROP30 are capable of inducing an increase in specific antibody levels. rEiROP25 triggered a Th1-type immune protection response, while rEiROP30 elicited a Th1/Th2 mixed response. EiROP25 and EiROP30 can generate a moderate level of immune protection, with better efficacy observed for EiROP30. This study provides valuable insights for the promotion of recombinant subunit vaccines targeting rabbit E. intestinalis infection.

Toxoplasma gondii (T. gondii) is one of the most common pathogenic protozoa in the world, and causes toxoplasmosis, which in varying degrees causes significant economic losses and poses a serious public health challenge globally. To date, the development of an effective vaccine for human toxoplasmosis remains a challenge. Given that T.gondii calcium-dependent protein kinase 3 (CDPK3), dense granule protein 35 (GRA35) and rhoptry organelle protein 46 (ROP46) play key roles during Toxoplasma gondii invasion of host cells, we developed a protein vaccine cocktail including these proteins and validated its protective efficacy. The specific protective effects of vaccine on mice were analyzed by measuring serum antibodies, cytokines, splenocyte proliferation, the percentage of CD4+ and CD8+ T-lymphocytes, survival rate, and parasite cyst burden. The results showed that mice vaccinated with a three-protein cocktail produced the highest levels of immune protein antibodies to IgG, and high levels of IFN-γ, IL-2, IL-4, and IL-10 compared to other mice vaccinated with two proteins. In addition, CD4+ and CD8+ T cell percentages were significantly elevated. Compared to the control groups, mice vaccinated with the three-protein cocktail survived significantly longer after acute infection with T. gondii and had significantly fewer cysts after chronic infection. These results demonstrated that a cocktail vaccine of TgCDPK3, TgGRA35, and TgROP46 can effectively induce cellular and humoral immune responses with good protective effects in mice, indicating its potential as vaccine candidates for toxoplasmosis.

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.

Toxoplasma gondii can cause congenital infection and abortion in humans and warm-blooded animals. T. gondii dense granule proteins, GRA35, GRA42, and GRA43, play a critical role in the establishment of chronic infection. However, their potential to induce protective immunity against T. gondii infection remains unexplored.
This study aimed to test the efficacy of a DNA vaccine encompassing GRA35, GRA42, and GRA43 in inducing protective immunity against the highly virulent T. gondii RH strain (type I) and the brain cyst-forming PRU strain (type II).
The eukaryotic plasmids pVAX-GRA35, pVAX-GRA42, and pVAX-GRA43 were constructed and formulated into two- or three-gene cocktail DNA vaccines. The indirect immunofluorescence assay (IFA) was used to analyze their expression and immunogenicity. Mice were immunized with a single-gene, two-genes, or multicomponent eukaryotic plasmid, intramuscularly. We assessed antibody levels, cytotoxic T-cell (CTL) responses, cytokines, and lymphocyte surface markers by using flow cytometry. Additionally, mouse survival and cyst numbers in the brain of mice challenged 1 to 2 months postvaccination were determined.
Specific humoral and cellular immune responses were elicited in mice immunized with single-, two-, or three-gene cocktail DNA vaccine, as indicated by significant increases in serum antibody concentrations of total IgG, IgG2a/IgG1 ratio, cytokine levels (IFN-γ, IL-2, IL-12, IL-4, and IL-10), lymphocyte proliferation, lymphocyte populations (CD4+ and CD8+ T lymphocytes), CTL activities, and survival, as well as decreased brain cysts, in comparison with control mice. Moreover, compared with pVAX-GRA35 + pVAX-GRA42, pVAX-GRA42 + pVAX-GRA43, or pVAX-GRA35 + pVAX-GRA43, multicomponent DNA vaccine with three genes (pVAX-GRA35 + pVAX-GRA42 + pVAX-GRA43) induced the higher humoral and cellular immune responses, including serum antibody concentrations, cytokine levels, lymphocyte proliferation, lymphocyte populations, CTL activities and survival, resulting in prolonged survival time and reduced brain cyst loads. Furthermore, mice immunized with pVAX-GRA35 + pVAX-GRA42, pVAX-GRA42 + pVAX-GRA43, or pVAX-GRA35 + pVAX-GRA43 showed greater Th1 immune responses and protective efficacy than the single-gene-vaccinated groups.
These results demonstrate that TgGRA35, TgGRA42, or TgGRA43 are vaccine candidates against T. gondii infection, and the three-gene DNA vaccine cocktail conferred the strongest protection against T. gondii infection.

Avian coccidiosis caused by Eimeria is a serious parasitic disease that poses a threat to the poultry industry. Currently, prevention and treatment mainly rely on the administration of anticoccidials and live oocyst vaccines. However, the prevalence of drug resistance and the inherent limitations of live vaccines have driven the development of novel vaccines. In this study, the surface protein (Et-SAG14), a previously annotated rhoptry protein (Eten5-B), and a gametocyte phosphoglucomutase (Et-PGM1) were characterized and the vaccine potential of the recombinant proteins were evaluated. Et-SAG14 was dispersed in the form of particles in the sporozoite and merozoite stages, whereas Et-PGM1 was distributed in the apical part of the sporozoite and merozoite stages. The previously annotated rhoptry Eten5-B was found not to be located in the rhoptry but distributed in the cytoplasm of sporozoites and merozoites. Immunization with rEten5-B significantly elevated host interferon gamma (IFN-γ) and interleukin 10 (IL-10) transcript levels and exhibited moderate anticoccidial effects with an anticoccidial index (ACI) of 161. Unexpectedly, both recombinant Et-SAG14 and Et-PGM1 immunization significantly reduced host IFN-γ and IL-10 transcription levels, and did not show protection against E. tenella challenge (ACI < 80). These results suggest that the rEten5-B protein can trigger immune protection against E. tenella and may be a potential and effective subunit vaccine for the control of coccidiosis in poultry.