Coccidiosis, caused by different species of Eimeria parasites, is an economically important disease of poultry and livestock worldwide. Here we report previously unknown alterations in the gut microbes and metabolism of BALB/c mice infected with Eimeria falciformis . Specifically, we observed a significant shift in the abundance of cecal bacteria and disrupted metabolism in parasitized animals. The relative abundances of Lachnospiraceae bacterium NK4A136, Ruminiclostridium , Alistipes , and Lactobacillus declined in response to E. falciformis infection, whereas Escherichia , Shigella , Helicobacter , Klebsiella , and Bacteroides were increased. Carbohydrate and amino acid metabolites in the serum samples of infected mice were significantly altered compared to naïve controls. Levels of amino acids, including asparagine, histidine, l -cysteine, tryptophan, lysine, glycine, serine, alanine, proline, ornithine, methionine, and valine, decreased on day 7 postinfection before returning to baseline on day 14. In addition, increased levels of indolelactate and mannitol and a reduced amount of oxalic acid indicated impaired carbon metabolism upon parasitic infection. These data demonstrate that intestinal coccidial infection perturbs the microbiota and disrupts carbon and nitrogen metabolism.

The “self-cleaving” 2A sequence of picornavirus, which mediates ribosome-skipping events, enables the generation of two or more separate peptide products from one mRNA containing one or more “self-cleaving” 2A sequences. In this study, we introduced a single 2A sequence of porcine teschovirus-1 (P2A) linked to two fluorescent protein genes, the enhanced yellow fluorescent protein (EYFP) gene and the red fluorescent protein (RFP) gene, in a single cassette into transgenic Eimeria tenella (EtER). As expected, we obtained two separated protein molecules rather than a fused protein, although the two molecules were translated from the same mRNA carrying a single “self-cleaving” 2A sequence. Importantly, RFP led by a secretion signal was secreted into parasitophorous vacuoles, while EYFP localized mainly to the nucleus of EtER. Our results demonstrate that the “self-cleaving” 2A sequence actively mediated cleavage of polyproteins in the apicomplexan parasite E. tenella.

Coccidiosis is one of the most serious diseases of livestock and birds in the world. Vaccination with live-parasite anticoccidial vaccines with genetic manipulation improving the immunogenicity of vaccine strains would be the best means for controlling coccidiosis in breeder and layer stocks, even in fast-growing broilers.

We investigated the efficacy of a single bacterium strain, Bacillus subtilis (B. subtilis) YW1, on the performance, morphology, cecal microbiota, and intestinal barrier function of laying hens. A total of 216 28-week-old Hy-line Brown laying hens were divided into three dietary treatment groups, with six replicates of 12 birds each for 4 weeks. The control group (Ctr) was fed a basal diet and the treatment groups, T1 and T2, were fed a basal diet supplemented with B. subtilis at a dose rate of 5 × 10⁸ CFU/kg and 2.5 × 10⁹ CFU/kg, respectively. Dietary supplementation with B. subtilis did not significantly affect overall egg production in both groups, with no obvious changes in average egg weight and intestine morphology. B. subtilis administration also improved the physical barrier function of the intestine by inducing significantly greater expression levels of the tight junction protein occludin in T1 (p = 0.07) and T2 (p < 0.05). Further, supplementation with B. subtilis effectively modulated the cecal microbiota, increasing the relative level of beneficial bacteria at the genus level (e.g., Bifidobacterium p < 0.05, Lactobacillus p = 0.298, Bacillus p = 0.550) and decreasing the level of potential pathogens (e.g., Fusobacterium p < 0.05, Staphylococcus p < 0.05, Campylobacter p = 0.298). Overall, B. subtilis YW1 supplementation cannot significantly improve the egg production; however, it modulated the cecal microbiota towards a healthier pattern and promoted the mRNA expression of the tight junction protein occludin in laying hens, making B. subtilis YW1 a good probiotic candidate for application in the poultry industry, and further expanding the resources of strains of animal probiotics.

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.