Transcriptomic analysis uncovers a biphasic response to precocious Eimeria acervulina infection in chicken duodenal tissue.

Live anticoccidial vaccines, either formulated with unattenuated or attenuated Eimeria parasites, are powerful stimulators of chicken intestinal immunity. Little is known about the dynamics of gene expression and the corresponding biological processes of chicken responses against infection with precocious line (PL) of Eimeria parasites. In the present study, we performed a time-series transcriptomic analysis of chicken duodenum across 15 time points from 6 to 156 hours post-infection (p.i.) with PL of E. acervulina. A high-quality profile showing two distinct changes in chicken duodenum mRNA expression was generated during the infection of Eimeria. Early response revealed that activation of the chicken immune response was detectable from 6 h.p.i., prominent genes triggered during the initiation of asexual and sexual parasite growth encompass immune regulatory effects, such as interferon gamma (IFN-γ), interferon regulatory factor 1 (IRF1), and interleukin-10 (IL10). The late response was identified significantly associating with maintaining cellular structure and activating lipid metabolic pathways. These analyses provide a detailed depiction of the biological response landscape in chickens infected by the PL of E. acervulina, contributing significant insights for the investigation of the host-parasite interactions and the management of parasitic diseases.

Transcriptomic Insights into the Developmental Dynamics of Eimeria acervulina: A Comparative Study of a Precocious Line and the Wild Type.

Coccidiosis, a parasitic disease caused by single or multiple Eimeria species, leads to significant economic losses in the poultry industry. The Eimeria life cycle includes schizogony, gametogony, and sporogony. To investigate the dynamics of gene expression and regulatory networks during the development of Eimeria acervulina, we employed time-course transcriptomics to rigorously compare the gene expression patterns between a precocious line (PL) and the wild type (WT) of E. acervulina. The results revealed that the PL enters into gametogony 12 h earlier than the WT, and both the PL and WT exhibited distinct clustering patterns during the development phase. A weighted gene co-expression network analysis (WGCNA) identified genes specifically expressed at four distinct developmental stages, schizogony, gametogony, sporulated oocysts, and unsporulated oocysts, clarifying the key biological processes at each stage. This study used global transcriptome profiling to elucidate molecular variations throughout the E. acervulina life cycle, providing critical insights into molecular characterization and valuable resources for investigating other apicomplexan parasites of public health importance.

Oral delivery of Eimeria acervulina transfected sequentially with two copies of the VP2 gene induces immunity against infectious bursal disease virus in chickens.

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.

Developing efficient strategies for localizing the enhanced yellow fluorescent protein subcellularly in transgenic Eimeria parasites.

Eimeria species serve as promising eukaryotic vaccine vectors. And that the location of heterologous antigens in the subcellular components of genetically modified Eimeria may determine the magnitude and type of immune responses. Therefore, our study aimed to target a heterologous fluorescent protein to the cell surface or microneme, two locations where are more effective in inducing protective immunity, of Eimeria tenella and E. acervulina sporozoites. We used an enhanced yellow fluorescent protein (EYFP) as a tagging biomarker, fusing variously with some localization or whole sequences of compartmental proteins for targeting. After acquiring stable transgenic Eimeria populations, we observed EYFP expressing in expected locations with certain strategies. That is, EYFP successfully localized to the surface when it was fused between signal peptides and mature products of surface antigen 1 (SAG1). Furthermore, EYFP was efficiently targeted to the apical end, an optimal location for secretory organelle known as the microneme, when fused to the C terminus of microneme protein 2. Unexpectedly, EYFP exhibited dominantly in the apical end with only weak expression on the surface of the transgenic sporozoites when the parasites were transfected with plasmid with EYFP fused between signal peptides and mature products of E. tenella SAG 13. These strategies worked in both E. tenella and E. acervulina, laying a solid foundation for studying E. tenella and E. acervulina-based live vaccines that can be further tailored to the inclusion of cargo immunogens from other pathogens.

Microneme-located VP2 in Eimeria acervulina elicits effective protective immunity against infectious bursal disease virus.

Using transgenic Eimeria spp. to deliver exogenous antigens is a viable option for developing multivalent live vaccines. Previous research revealed that the location of antigen expression in recombinant Eimeria dictates the magnitude and type of immune responses. In this study, we constructed genetically modified Eimeria acervulina that expressed VP2 protein, a protective antigen from infectious bursal disease virus (IBDV), on the surface or in the microneme of sporozoites. After vaccination, VP2-specific antibody was readily detected in specific pathogen-free chickens receiving transgenic E. acervulina parasites expressing VP2 in microneme, but animals vaccinated with which expressing VP2 on surface failed to produce detectable antibody after two times immunizations. Moreover, the bursal lesion of microneme-located VP2 transgenic E. acervulina immunized chickens was less severe compared with un-immunized animals after IBDV challenge infection. Therefore, genetically modified E. acervulina that express IBDV-derived VP2 in micronemes are effective in inducing specific antibody responses against VP2, while parasites that have VP2 expression on cell surface are not suitable. Thus, the use of Eimeria parasites as vaccine vectors needs to consider the proper targeting of exogenous immunogens. Our results have implications for the design of other vector vaccines.

Anammox activity improved significantly by the cross-fed NO from ammonia-oxidizing bacteria and denitrifying bacteria to anammox bacteria.

Nitric oxide (NO) has been suggested as an obligate intermediate in anaerobic ammonium oxidation (anammox), nitrification and denitrification. At the same time, ammonia-oxidizing bacteria (AOB) and denitrifying bacteria (DNB) are always existed in anammox flora, so what is the role of NO produced from AOB and DNB? Could it accelerate nitrogen removal via the anammox pathway with NO as an electron acceptor? To investigate this hypothesis, nitrogen transforming of an anammox biofilter was analyzed, functional gene expression of anammox bacteria (AnAOB), AOB and DNB were compared, and NO source was verified. For anammox biofilter, anammox contributed to 91.3 % nitrogen removal with only 14.4 % of AnAOB being enriched, while DNB was dominant. Meta-omics analysis and batch test results indicated that AOB could provide NO to AnAOB, and DNB also produced NO via up-regulating nirS/K and down-regulating nor. The activation of the anammox pathway of NH4++NO→N2 caused the downregulation of nirS and nxr in Ca. Kuenenia stuttgartiensis. Additionally, changes in nitrogen transforming pathways affected the electron generation and transport, limiting the carbon metabolism of AnAOB. This study provided new insights into improving nitrogen removal of the anammox system.

Evaluation of the effect of the indoor environment on the physiological responses of early-gestation sows in a commercial house in China.

Objective: The 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. Methods: A 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. Results: The 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. Conclusion: This 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.

Tissue-resident, memory CD8+ T cells are effective in clearing intestinal Eimeria falciformis reinfection in mice.

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.

Antibiotic Changes Host Susceptibility to Eimeria falciformis Infection Associated with Alteration of Gut Microbiota.

Eimeria falciformis is a murine-infecting coccidium that mainly infects the cecum and colon where it coexists with a large number of endogenous bacteria. Here, we found that mice treated with a broad-spectrum antibiotic cocktail including ampicillin, neomycin, metronidazole, and vancomycin had less oocyst production and milder pathological consequences after E. falciformis infection than mice without antibiotics, regardless of the inoculation doses. Furthermore, we showed that antibiotic treatment reduced parasitic invasion and prolonged asexual stage during E. falciformis infection, which may result in alleviating the infection. Interestingly, when further defining different antibiotic combinations for E. falciformis infection, it was shown that mice treated with ampicillin plus vancomycin had substantially attenuated E. falciformis infections as measured by cecal parasite counts and histopathological features. In contrast, treatment with metronidazole plus neomycin was beneficial to E. falciformis infection. Analyses of gut microbiota revealed various changes in bacterial composition and diversity following antibiotic treatments that were associated with host susceptibility to E. falciformis infection. Together, these findings suggest that gut microbiota may regulate the course and pathogenicity of E. falciformis infection, while the mechanisms need to be further investigated, especially for the development of coccidial vaccines for use in farm animals.

Establishment of Recombinant Eimeria acervulina Expressing Multi-Copies M2e Derived from Avian Influenza Virus H9N2.

The potential of Eimeria parasites as live vaccine vectors has been reported with successful genetic manipulation on several species like E. tenella, E. mitis and E. necatrix. Among seven Eimeria species infecting chickens, E. acervulina is a highly prevalent, moderately pathogenic species. Thus, it is valuable for the study of transfection and for use as a potential as vaccine vector. In this study, a plasmid containing expression cassette with enhanced yellow fluorescent protein (EYFP), red fluorescent protein (RFP) and 12 copies of extracellular domain of H9N2 avian influenza virus M2 (M2e) protein was used for the transfection. Nucleofected sporozoites were inoculated into birds through wing vein. Recombinant E. acervulina oocysts with 0.1% EYFP+ and RFP+ populations were collected from the feces of the inoculated birds. The fluorescent rate of transgenic parasites reached over 95% after nine successive propagations with a pyrimethamine selection in vivo and fluorescent-activated cell sorting (FACS) of progeny oocysts. The expression of M2e in the transgenic parasites (EaM2e) was confirmed by Western blot and its cytoplasm localization in sporozoites was displayed by an indirect immunofluorescent assay (IFA). Meanwhile, we found that the fecundity of EaM2e was equivalent to that of wild type E. acervulina (EaWT). Taken together, the stable transfection of E. acervulina was successfully established. Future studies will focus on whether transgenic E. acervulina can serve as a live vaccine vector.

Morphological and Phylogenetic Analysis of Eustrongylides sp. and Gnathostoma spinigerum Parasitizing the Asian Swamp Eel Monopterusalbus in China.

Nematode infections transmitted to humans by the consumption of wild or cultured eels are increasingly being reported. In the present study, 120 Asian swamp eel, Monopterus albus (Zuiew), individuals collected from China were examined for parasite infections, and 78 larval nematodes were isolated. Morphological and molecular characteristics, including sequence and phylogenetic analysis of the internal transcribed spacer (ITS) and cytochrome c oxidase subunit I (COI) gene regions, were employed to identify these nematodes at the lowest taxonomic level possible. Asian swamp eel was infected with two zoonotic parasite taxa: Gnathostoma spinigerum advanced third-stage larvae, with 6.67% prevalence and mean intensity = 1.25, and Eustrongylides sp. fourth-stage larvae, with 26.67% prevalence and mean intensity = 2.13. These findings evidence the need to enhance public hygiene and food safety awareness toward eel consumption.

Internal daughter formation of Toxoplasma gondii tachyzoites is coordinated by transcription factor TgAP2IX-5.

Toxoplasma gondii rapidly propagates through endodyogeny of tachyzoites, a process in which daughter parasites divide within the cell of the mother parasite. Recent studies have revealed that transcription factors with AP2-domain participate in the process of cell division in T. gondii. However, the concise regulation of the division cycles by AP2 proteins is poorly understood. In this study, we evaluated the effect of the transcription factor TgAP2IX-5 on the daughter cell formation in T. gondii. TgAP2IX-5 is a nuclear protein and is highly expressed during the S phase of the cell cycle of tachyzoites. TgAP2IX-5-disrupted strain showed a severe defect in replication and completely blocked lytic parasite growth. Following 3-indoleacetic acid treatment or without treatment of AP2IX-5-AID-3HA tagged strain for 30 min, 1 and 2 hr, the differentially expressed genes were 8, 54 and 202, respectively. Among these genes, the significantly downregulated ones were AP2 proteins, inner membrane complex (IMC) proteins and SAG-related proteins. Interestingly, loss of TgAP2IX-5 leads to a defect in internal daughter IMC formation and abnormalities in the morphology of organelles during cell division. Together, our study suggests that TgAP2IX-5 is crucial in regulating IMC formation of daughter cells in T. gondii.

Efficient Single-Gene and Gene Family Editing in the Apicomplexan Parasite Eimeria tenella Using CRISPR-Cas9.

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.

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites.

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second-generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.

Selection and characterization of a precocious line of Eimeria media.

Coccidiosis, caused by the infection of Eimeria parasites, is one of the most common diseases in domestic rabbits. Live anticoccidial vaccine formulated with attenuated precocious lines of pathogenic eimerian parasites is expected to be valuable for the control of rabbit coccidiosis as a similar strategy to produce anticoccidial vaccines against chicken coccidiosis has being used for several decades. Eimeria media, moderate pathogenic, is widespread in China. Therefore, attenuated anticoccidial vaccines against rabbit coccidiosis should contain vaccine strain(s) of E. media. In this study, a precocious line of E. media (Empre) was selected by collecting and propagating the early excreted oocysts with 16 successive generations. The prepatent period of Empre reduced from 108 h of its parental strain (Emwt) to 70 h. The fecundity of Empre was about 1/10 to 1/3 lower than that of Emwt. Each sporocyst of Empre sporulated oocyst contained only one large refractile body instead of two smaller ones seen in the parental strain. When vaccinated with 1 × 103 or 1 × 104 precocious line oocysts, the rabbits were completely protected against homologous challenge with the parental strain 14 days post challenge by terms of body weight gain and oocyst output counting, indicating the efficacy of Empre. Meanwhile, all immunized rabbits showed no clinical sign post immunization, indicating the safety of Empre. For co-immunization, 1 × 103Empre oocysts and 5 × 102 oocysts of a precocious line of E. intestinalis (EIP8) were inoculated to each rabbit in a trial. No diarrhea or mortality was found after vaccination, and the weight gains of the vaccinated group were similar to that of unvaccinated-unchallenged control (UUC) group, while the weight gains of the vaccinated group were similar to that of unvaccinated-unchallenged control (UUC) group (P > 0.05), but significantly higher than that of UCC group (P < 0.01) after challenge, indicating it is safe and effective when using co-immunization. These results together show that Empre, as a precocious line, is a good candidate of precocious line of E. media for anticoccidial vaccine development.

Co-immunization with two recombinant Eimeria tenella lines expressing immunoprotective antigens of E. maxima elicits enhanced protection against E. maxima infection.

BACKGROUND: Live anticoccidial vaccines have been a tremendous success for disease prevention. The establishment of the reverse genetic manipulation platform has enabled the development of Eimeria parasites, the live anticoccidial vaccine strains, as vaccine vectors. In our previous study, recombinant E. tenella expressing a single immunodominant antigen of E. maxima (Et-EmIMP1) was able to protect chickens against challenge infection with E. maxima. This promising result encouraged us to further explore strategies to improve the protection efficacy of recombinant Eimeria and develop it as a vaccine vector. RESULTS: We constructed a novel recombinant Eimeria line expressing apical membrane antigen 1 of E. maxima (Et-EmAMA1) and then immunized chickens with Et-EmAMA1 and/or Et-EmIMP1. We found that the E. maxima soluble antigen-specific cell-mediated immunity was much stronger in the birds that were co-immunized with Et-EmAMA1 and Et-EmIMP1 than in those that were immunized with Et-EmAMA1 or Et-EmIMP1 alone. The oocyst production after E. maxima infection was significantly reduced in the recombinant Eimeria-immunized birds compared with the wild-type-immunized and naïve birds. The oocyst production in the birds co-immunized with Et-EmAMA1 and Et-EmIMP1 was consistently the lowest among the treatment groups after E. maxima infection. CONCLUSIONS: These results demonstrated that Eimeria is an effective vaccine vector that can carry and deliver heterologous Eimeria antigens to the host immune system and trigger specific immune responses. Our results also suggested that increasing the number of recombinant Eimeria lines is an effective approach to enhance protective immunity against infections with heterologous pathogens.

Stable transfection of Eimeria necatrix through nucleofection of second generation merozoites.

Eimeria spp., the causative agents of coccidiosis, are the most common protozoan pathogens of chickens. Infection with these parasites can result in poor development or death of animals leading to a devastating economic impact on poultry production. The establishment of transfection protocols for genetic manipulation of Eimeria species and stable expression of genes would help advance the biology of these parasites as well as establish these organisms as novel vaccine delivery vehicles. Here, we report the selection of the first stable transgenic E. necatrix population, EnHA1, consitutively expressing the EYFP reporter following transfection of the 2nd generation merozoites with a linear DNA fragment harboring the EYFP reporter gene, the HA1 gene from the avian influenza virus H9N2 and the TgDHFR-TS selectable marker, which confers resistance to pyrimethamine. Transfected merozoites were inoculated into chickens via the cloacal route, and feces from 18 h to 72 h post inoculation were collected and subjected to subsequent serial passages, FACS sorting and pyrimethamine selection. A gradual increase in the number of EYFP-expressing sporulated oocysts was noticed with more than 90% EYFP + oocysts obtained after five passages. Immunofluorescence assay confirmed successful expression of the HA1 antigen in the EnHA1 population. The ability to genetically manipulate E. necatrix merozoites and express heterologous genes in this parasite will pave the way for possible use of this organism as a vaccine-delivery vehicle.

Comparative transcriptome analysis of Eimeria maxima (Apicomplexa: Eimeriidae) suggests DNA replication activities correlating with its fecundity.

BACKGROUND: Chicken coccidiosis, caused by the infection of Eimeria species, leads to important economic losses to the poultry industry. Vaccination with attenuated live parasites seems to be the best way to control this disease. Attenuated eimerian parasites with shortened prepatent times show great changes in intracellular development compared to their parent strains but the mechanisms involved in these biological differences are still unclear. RESULTS: In this study, we obtained a precocious line of E. maxima by sequential selection of 22 generations of early shed oocysts in chickens and performed a comparative transcriptome analysis of three different developmental stages of the precocious line and its parent strain using Illumina high-throughput sequencing. Our E. maxima precocious line showed decreased pathogenicity, reduced fecundity and a greatly shorted prepatent time of only 98 h. We found that typical gene changes in the stage development from unsporulated to sporulated oocyst and from sporulated oocyst to merozoite were marked by upregulated organelle genes and protein translation related genes, respectively. Additionally, major differences between the precocious line and its parent strain were detected in the merozoite stage, characterized by downregulated genes involved in protein cleavage and DNA replication activities. CONCLUSIONS: Our study generated and characterized an E. maxima precocious line, illustrating gene expression landscapes during parasite development by transcriptome analysis. We also show that the suppressed DNA replication progress in the merozoite stage in the precocious line may result in its reduced fecundity. These results provide the basis for a better understanding of the mechanism of precocity in Eimeria species, which can be useful in studies in early gametocytogenesis in apicomplexan parasites.

Eimeria tenella infection perturbs the chicken gut microbiota from the onset of oocyst shedding.

Coccidiosis is a serious threat to the poultry industry, resulting in substantial economic losses worldwide. The effective development of alternative treatments for coccidiosis that does not involve chemotherapy drugs and does not result in antibiotic resistance relies on gaining a clearer understanding of the interaction between host intestinal microbiota and enteric coccidia. Here, we established an Eimeria tenella infection model in chickens and subsequently monitored the changes in the overall intestinal microbiome using 16S rRNA gene sequencing. We found that the gut (i.e. fecal) microbiota of infected chicken differed from that of uninfected naïve animals. Levels of non-pathogenic bacteria, including Lactobacillus and Faecalibacterium declined, whereas those of pathogenic bacteria, including Clostridium, Lysinibacillus, and Escherichia, increased over time in response to E. tenella infection. Similar dynamic changes of the fecal microbiota were observed in both Arbor Acres broilers and White Leghorn chickens, indicating that the perturbation of the microbiota was directly induced by E. tenella infection. Our findings could be used to further elucidate the serious damage to host health caused by coccidia infection, leading to the development of new effective treatment options for coccidiosis.

Influence of Eimeria falciformis Infection on Gut Microbiota and Metabolic Pathways in Mice.

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.