Anti-inflammatory and immunoregulatory effects of colistin sulphate on human PBMCs.

In previous studies, CST has been identified as having an immunostimulatory effect on Caenorhabditis elegans and macrophage of rats. Here, we further investigated its immunomodulatory effects on human peripheral blood mononuclear cells (PBMCs). LPS-stimulated PBMCs inflammatory model was established. Flow cytometry was applied to measure phagocytosis of PBMCs. Cytokine mRNA and protein expression levels of LPS-stimulated PBMCs with or without CST were measured by qRT-PCR and ELISA. The transcriptomic profile of CST-treated PBMCs was investigated by RNA-sequencing. Gene Ontology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) were applied to find potential signalling pathways. PBMCs showed a significant increase in phagocytic activity at 6 h after being incubated with CST at the concentration of 10 µg/mL. In the presence of LPS, CST maintained and promoted the expression of TNF-α and chemokine CCL24. The content of pro-inflammatory cytokines, such as IL-1ß, IL-6 and IFN-γ, which were released from LPS-stimulated PBMCs, was reduced by CST at 6 h. Anti-inflammatory cytokines, such as IL-4, IL-13 and TGF-ß1, were significantly increased by CST at 24 h. A total of 277 differentially expressed immune-related genes (DEIRGs) were detected and cytokine-cytokine receptor interaction was highly enriched. CST presented obvious anti-inflammatory and immunoregulatory effects in LPS-induced PBMCs inflammatory model not only by improving the ability of PBMCs to clear pathogens but also by decreasing pro-inflammatory cytokines and increasing anti-inflammatory cytokines. And the mechanism may be related to cytokine-cytokine receptor interaction.

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.

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.

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.

Towards Innovative Design and Application of Recombinant Eimeria as a Vaccine Vector.

Efficient delivery of antigenic cargo to trigger protective immune responses is critical to the success of vaccination. Genetically engineered microorganisms, including virus, bacteria, and protozoa, can be modified to carry and deliver heterologous antigens to the host immune system. The biological vectors can induce a broad range of immune responses and enhance heterologous antigen-specific immunological outcomes. The protozoan genus Eimeria is widespread in domestic animals, causing serious coccidiosis. Eimeria parasites with strong immunogenicity are potent coccidiosis vaccine candidates and offer a valuable model of live vaccines against infectious diseases in animals. Eimeria parasites can also function as a vaccine vector. Herein, we review recent advances in design and application of recombinant Eimeria as a vaccine vector, which has been a topic of ongoing research in our laboratory. By recapitulating the establishment of an Eimeria transfection platform and its application, it will help lay the foundation for the future development of effective parasite-based vaccine delivery vectors and beyond.

Genetic modification of the protozoan Eimeria tenella using the CRISPR/Cas9 system.

Eimeria tenella has emerged as valuable model organism for studying the biology and immunology of protozoan parasites with the establishment of the reverse genetic manipulation platform. In this report, we described the application of CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (endonuclease) system for efficient genetic editing in E. tenella, and showed that the CRISPR/Cas9 system mediates site-specific double-strand DNA breaks with a single guide RNA. Using this system, we successfully tagged the endogenous microneme protein 2 (EtMic2) by inserting the red fluorescent protein into the C-terminal of EtMic2. Our results extended the utility of the CRISPR/Cas9-mediated genetic modification system to E. tenella, and opened a new avenue for targeted investigation of gene functions in apicomplexan parasites.

Identification of candidate antigens by 2-DE Immunoblotting for diagnosis of Toxoplasma gondii infection in chickens and rabbits.

Toxoplasmosis, caused by apicomplexan parasite Toxoplasma gondii, is a common food-borne disease in humans. Undercooked meat is a potential source of T. gondii infection. As meat of chicken or rabbit is consumed worldwide, tools such as ELISA for the detection of infection of this parasite in rabbits and chickens are much-needed. To search diagnostic antigens of T. gondii special for rabbits and chickens, we conducted two dimensional electrophoresis (2-DE), Western blotting and mass spectrometry (MS) with T. gondii tachyzoite proteins. When probed with rabbit or chicken anti-T. gondii sera, about 60 positive spots among over 500 visible protein spots were detected. In subsequent mass spectrometric analysis, microneme 4 (MIC4) and a putative rhoptry protein are of diagnositic value among the 13 spots selectively picked from the equivalent gel. This study encourages further validation of these candidate antigens for the development of immunologic tools for the detection of T. gondii infection in chickens and rabbits.

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.

Selection and identification of a precocious line of Eimeria intestinalis with enlarged oocysts and deletion of one generation of schizogony.

Rabbit coccidiosis is a common parasitic disease and responsible for enormous economic losses in the rabbit industry. Eimeria intestinalis, one of the highly pathogenic and common Eimeria species infecting rabbits, is considered as an indispensable species for the development of live oocyst vaccines against rabbit coccidiosis. In this study, we report the successful selection of a precocious line (EIP8) from a wild-type strain of E. intestinalis (WT) by successively collecting and propagating the early excreted progeny oocysts. The EIP8 line had a prepatent period of only 132 h compared to 204 h for the WT. Oocysts of EIP8 were notably different from those produced by the WT strain by their significantly larger size (mean length: 29.3 vs 27.6 µm and mean width 20.5 vs 19.8 µm). Examination of tissue sections prepared from EIP8-infected rabbits revealed that this precocious line undergoes only two generations of schizogony before differentiating into gametocytes by 120 h post-infection. In contrast, WT parasites undergo three generations of schizogony and gametocytes are present by 168 h post-infection. EIP8 multiplication capacity reduced by more than 35-fold and a concomitant decrease in pathogenicity was detected. Interestingly, immunization with 103 or 104 EIP8 oocysts provided sufficient protection against homologous challenge with wild-type parasites, as body weight gain of immunized and challenged rabbits was similar to that of untreated animals, as well as more than 90% reduction of oocyst output was detected in immunized and challenged animals when compared to unimmunized and challenged animals. Together, these results show that the EIP8 precocious line of E. intestinalis is an attenuated immunogenic strain and a suitable candidate for the development of a live vaccine against rabbit coccidiosis.

The production of short chain fatty acid and colonic development in weaning piglets.

Weaning process widely affects the small intestinal structure and function in piglets, while the responses of large intestine to weaning stress are still obscure. The purpose of this study was to determine the developmental changes (i.e., short chain fatty acids (SCFAs) concentrations, growth parameters, crypt-related indices and antioxidant capacity) in colon of piglet during weaning. Forty piglets were weaned at day 21 and euthanized to collect colonic tissues and digesta samples on day 0, 1, 3, 7 and 14 post-weaning (n = 8). Piglet growth performance was improved (p < .001) on day 7 and 14 post-weaning. The concentrations of acetate, propionate, butyrate, valerate, isobutyrate, isovalerate and total SCFAs were higher (p < .001) during the late post-weaning period. The mRNA abundances of SCFAs transporters were greater (p < .001) on day 7 and 14. The absolute and relative weights, absolute length and perimeter of colon were greater (p < .001) on day 7 and 14. Similarly, post-weaning increases (p < .001) in colonic crypt depth and Ki67 positive cells numbers per crypt were observed during the same period. Colonic crypt fission indices decreased (p < .01), while total crypt numbers increased (p < .001) on day 14 after weaning. Moreover, total SCFAs concentration was significantly associated with colonic growth parameters and Ki67 cells/crypt (p < .001). In addition, catalase content was decreased on day 3, 7, and 14, whereas, the concentrations of total superoxide dismutase (T-SOD) and manganese-containing superoxide dismutase (MnSOD) were higher (p < .05) on day 1 and 3 post-weaning. These results showed that weaning process has a significant effect on colonic growth and development, which might be associated with the change of SCFAs concentrations in colon.

Transgenic Eimeria tenella Expressing Profilin of Eimeria maxima Elicits Enhanced Protective Immunity and Alters Gut Microbiome of Chickens.

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. Profilin from apicomplexan parasites is the first molecularly defined ligand for Toll-like receptor 11 (TLR11) and TLR12 in mice and is a potential molecular adjuvant. Here, we constructed a transgenic Eimeria tenella line (Et-EmPro) expressing the profilin of Eimeria maxima, the most immunogenic species of chicken coccidia, and evaluated the adjuvant effects of EmPro on the immunogenicity of E. tenella We found that immunization with the transgenic Eimeria parasites, compared with the wild type, elicited greater parasite antigen-specific cell-mediated immunity, characterized by increased numbers of interferon gamma (IFN-γ)-secreting lymphocytes. The transgenic parasite also induced better protective immunity against E. tenella challenge than the wild type. In addition, the diversity of the fecal microbiome of the birds immunized with the transgenic parasite differed from that of the microbiome of the wild-type-immunized birds, indicating interactions of Eimeria with the gut microbiome of chickens. Our results showing enhanced immunogenicity of E. tenella by use of EmPro as a molecular adjuvant derived from the most immunogenic affinis species represent a large step forward in the development of the next generation of coccidiosis vaccines using Eimeria as a vaccine platform expressing molecular adjuvants and potentially other pathogen antigens against not only coccidiosis but also other infectious diseases.

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.

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.

An optimized DNA extraction method for molecular identification of coccidian species.

Molecular identification of Eimeria parasites infecting poultry and livestock has been commonly used for more than 20 years. An important step of the molecular identification technique is the rupturing of the oocyst wall for DNA extraction. Previously, DNA extraction methods included pre-treatment with sodium hypochlorite and osmotic shock with saturated salt solution. Here, we present a modification of this technique for a more sensitive and efficient identification of Eimeria spp. in field samples. The disruption extent of the oocyst walls, yield of DNA extraction, and identification of species-specific DNA sequences by PCR were used to evaluate this optimized method. Incubation of oocysts in sodium hypochlorite for 1.5 h at 4 °C followed by treatment with a saturated salt solution for 1 h at 55 °C broke up the walls of most Eimeria tenella oocysts, as well as other coccidian species of chicken and rabbit, such as Eimeria intestinalis and even Cryptosporidium cuniculus. Notably, polymerase chain reaction (PCR) amplification of the intervening transcribed sequence 1 (ITS-1) was successfully performed with genomic DNA extracted from just 50 oocysts using this optimized method. Our findings will greatly promote the development of molecular diagnosis methods of coccidiosis and simplify coccidian species identification and categorization as well as infection prevalence, providing a significant advancement in the development of techniques for coccidiosis control and prevention.

"Self-cleaving" 2A peptide from porcine teschovirus-1 mediates cleavage of dual fluorescent proteins in transgenic Eimeria tenella.

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.

Calcium-dependent microneme protein discharge and in vitro egress of Eimeria tenella sporozoites.

Egress is a vital step in the endogenous development of apicomplexan parasites, as it assures the parasites exit from consumed host cells and entry into fresh ones. However, little information has previously been reported on this step of Eimeria spp. In this study, we investigated in vitro egress of Eimeria tenella sporozoites triggered by acetaldehyde. We found that addition of exogenous acetaldehyde induces egress of sporozoites from primary chicken kidney cells (PCKs) and stimulate secretion of E. tenella microneme 2 protein (EtMic 2). Moreover, by using cellular calcium inhibitors, we further proved that these processes were dependent on the intracellular calcium of the parasites. Our findings provide clues to the study of interaction between eimerian parasites and their hosts.

A Portable Laser Photoacoustic Methane Sensor Based on FPGA.

A portable laser photoacoustic sensor for methane (CH4) detection based on a field-programmable gate array (FPGA) is reported. A tunable distributed feedback (DFB) diode laser in the 1654 nm wavelength range is used as an excitation source. The photoacoustic signal processing was implemented by a FPGA device. A small resonant photoacoustic cell is designed. The minimum detection limit (1σ) of 10 ppm for methane is demonstrated.

Acquired adenoma of the nonpigmented ciliary epithelium: analysis of five cases.

PURPOSE: Adenoma of the non-pigmented ciliary epithelium (ANPCE) is extremely rare. The aim of this study is to present our experience in treating this rare entity and to determine the long-term surgical outcomes of local tumor resection (partial iridocyclectomy with lamellar sclerouvectomy) in five cases of ANPCE in China. PATIENTS AND METHODS: The medical data of four women and one man ranging in age from 28 to 46 years (median, 38 years) with ANPCE were reviewed retrospectively at the Zhongshan Ophthalmic Center of Sun Yat-sen University, China. All patients had received local tumor resection and the diagnosis was confirmed by histopathological examination. RESULTS: All five cases experienced blurred vision. The right eye was affected in four cases and the left in one. Slit-lamp biomicroscopy revealed a nodular, non-pigmented, gray-white mass with a slightly irregular surface. Tumor invasion through the peripheral iris was observed in two patients, two patients had multiple iris or ciliary cysts, and one patient had a partially bloodstained cornea. All tumors transmitted light readily. Ultrasound biomicroscopy showed a middle- or hyper-echoic solid mass in the ciliary body. MRI examination showed the tumor hyperintense to vitreous on T1WI and hypointense to vitreous on T2WI. Local resection was performed in all cases. After a median of 5.0 years of follow-up, no tumor recurrence was present in any of the cases and all involved eyes were saved. The visual acuity improved seven lines in three cases, decreased one line in one case, and vision was lost in one case. CONCLUSIONS: ANPCE often presents in adults as gray-white in color, with an irregular and sometimes multilobulated surface. The tumor transmits light well. Local resection of the mass generally provides the patient with useful vision. Recurrence after surgical removal is unlikely.

Early detection of Toxoplasma gondii-infected cats by interferon-gamma release assay.

Felines, the only definitive hosts that shed the environmentally-durable oocysts, are the key in the transmission of Toxoplasma gondii to all warm-blooded animals. They seroconvert as late as the third week and begin to shed oocysts as early as 3-8 days after being fed tissue cysts. Early detection of Toxoplasma-infected cats is crucial to evaluate Toxoplasma-contaminated environment and potential risks to public health. Moreover, it is fundamental for Toxoplasma infection control. Interferon-gamma release assay (IGRA) is a blood-based test assessing the presence of IFN-γ released by the T-lymphocytes directed against specific antigens, which is an ideal assay for early detection of Toxoplasma-infected cats. Here, cats were orally infected with the tissue cysts and blood was collected for toxoplasmic antigen stimulation, and the released IFN-γ was measured by ELISA. Results showed that Toxoplasma-infection was detected by IGRA as early as 4 days post-infection (dpi); while serum Toxoplasma IgM and IgG were detected by ELISA at 10 dpi and 14 dpi, respectively. Our findings demonstrated that IGRA-positive and ELISA-negative samples revealed an early Toxoplasma infection in cats, indicating a new strategy for the early diagnosis of Toxoplasma infection by combining IGRA and ELISA. Therefore, IGRA could emerge as a reliable diagnostic tool for the exploration of cat toxoplasmosis prevalence and its potential risks to public health.

Ethanol and isopropanol trigger rapid egress of intracellular Eimeria tenella sporozoites.

Egress from host cells is a vital step of the intracellular life cycle of apicomplexan parasites such as Toxoplasma gondii. This phenomenon has attracted attentions from many research groups. Previous studies have shown that ethanol could stimulate the release of microneme proteins by elevating intracellular Ca(2+) concentration of T. gondii, resulting in the parasite egress from host cells. However, little information about egress is known on Eimeria species, the causative agent of coccidiosis in poultry and livestock. In this report, we studied the effect of ethanol and isopropanol on the egress of eimerian parasites. Eimeria tenella sporozoites cultured in primary chicken kidney cells were treated with ethanol and isopropanol, then the egressed parasites were analyzed. Ethanol and isopropanol could induce the rapid egress of E. tenella sporozoites from host cells. No substantial damage was found in parasite-egressed host cells. Compared to the freshly isolated sporozoites, the re-invading ability and reproductivity of the egressed parasites significantly decreased by 43.4 and 44.1 % individually. We also found that fewer sporozoites egressed from host cells when the parasites developed for a longer time before the alcohol treatment. These results demonstrate an in vitro egress mode different from that of T. gondii, facilitating the deciphering of the mechanisms of egress of eimerian parasites.