Comparative proteomic analysis across the developmental stages of the Eimeria tenella.

Eimeria tenella is the main pathogen responsible for coccidiosis in chickens. The life cycle of E. tenella is, arguably, the least complex of all Coccidia, with only one host. However, it presents different developmental stages, either in the environment or in the host and either intracellular or extracellular. Its signaling and metabolic pathways change with its different developmental stages. Until now, little is known about the developmental regulation and transformation mechanisms of its life cycle. In this study, protein profiles from the five developmental stages, including unsporulated oocysts (USO), partially sporulated (7 h) oocysts (SO7h), sporulated oocysts (SO), sporozoites (S) and second-generation merozoites (M2), were harvested using the label-free quantitative proteomics approach. Then the differentially expressed proteins (DEPs) for these stages were identified. A total of 314, 432, 689, and 665 DEPs were identified from the comparison of SO7h vs USO, SO vs SO7h, S vs SO, and M2 vs S, respectively. By conducting weighted gene coexpression network analysis (WGCNA), six modules were dissected. Proteins in blue and brown modules were calculated to be significantly positively correlated with the E. tenella developmental stages of sporozoites (S) and second-generation merozoites (M2), respectively. In addition, hub proteins with high intra-module degree were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway enrichment analyses revealed that hub proteins in blue modules were involved in electron transport chain and oxidative phosphorylation. Hub proteins in the brown module were involved in RNA splicing. These findings provide new clues and ideas to enhance our fundamental understanding of the molecular mechanisms underlying parasite development.

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.

Study on the alleviative effect of Lactobacillus plantarum on Eimeria falciformis infection.

Coccidia of the genus Eimeria are specialized intracellular parasitic protozoa that cause severe coccidiosis when they infect their hosts. Animals infected with Eimeria develop clinical symptoms, such as anorexia, diarrhea, and hematochezia, which can even cause death. Although the current preferred regimen for the treatment of coccidiosis is antibiotics, this treatment strategy is limited by the ban on antibiotics and the growing problem of drug resistance. Therefore, the exploration of alternative methods for controlling coccidiosis has attracted much attention. Lactobacillus plantarum has been shown to have many beneficial effects. In this study, L. plantarum M2 was used as a research object to investigate the effect of L. plantarum on intestinal inflammation induced by infection with Eimeria falciformis in mice by detecting indicators, such as oocyst output, serum cytokines, and the intestinal microbiota. Compared with that in the infection group, the percent weight loss of the mice that were administered with L. plantarum M2 was significantly reduced (P < 0.05). Supplemented L. plantarum M2 and probiotics combined with diclazuril can reduce the total oocyst output significantly (P < 0.05, P < 0.001). L. plantarum M2 had outstanding performance in maintaining intestinal barrier function, and the levels of the mucin MUC1 and the tight junction protein E-cadherin were significantly elevated (P < 0.01, P < 0.05). Studies have shown that probiotic supplementation can alleviate adverse reactions after infection and significantly improve intestinal barrier function. In addition, probiotics combined with diclazuril could optimize the partial efficacy of diclazuril, which not only enhanced the effect of antibiotics but also alleviated their adverse effects. This study expands the application of probiotics, provides new ideas for alternative strategies for coccidia control, and suggests a basis for related research on lactobacilli antagonizing intracellular pathogen infection.IMPORTANCECoccidia of the genus Eimeria are specialized intracellular parasitic protozoa, and the current preferred regimen for the treatment of coccidiosis is antibiotics. However, due to antibiotic bans and drug resistance, the exploration of alternative methods for controlling coccidiosis has attracted much attention. In this work, we focused on Lactobacillus plantarum M2 and found that probiotic supplementation can alleviate adverse reactions after infection and improve intestinal barrier function. This study proposes the possibility of using lactic acid bacteria to control coccidiosis, and its potential mechanism needs further exploration.

Host protein EPCAM interacting with EtMIC8-EGF is essential for attachment and invasion of Eimeria tenella in chickens.

The five epidermal growth factor-like domains (EGF) of Eimeria tenella microneme protein 8 (EtMIC8) (EtMIC8-EGF) plays a vital role in host cell attachment and invasion. These processes require interactions between parasite proteins and receptors on the surface of host cells. In this study, five chicken membrane proteins potentially interacting with EtMIC8-EGF were identified using the GST pull-down assay and mass spectrometry analysis, and only chicken (Gallus gallus) epithelial cell adhesion molecule (EPCAM) could bind to EtMIC8-EGF. EPCAM-specific antibody and recombinant EPCAM protein (rEPCAM) inhibited the EtMIC8-EGF binding to host cells in a concentration-dependent manner. Furthermore, the rEPCAM protein showed a binding activity to sporozoites in vitro, and a significant reduction of E. tenella invasion in DF-1 cells was further observed after pre-incubation of sporozoites with rEPCAM. The specific anti-EPCAM antibody further significantly decreased weight loss, lesion score and oocyst output during E. tenella infection, displaying partial inhibition of E. tenella infection. These results indicate that chicken EPCAM is an important EtMIC8-interacting host protein involved in E. tenella-host cell adhesion and invasion. The findings will contribute to a better understanding of the role of adhesion-associated microneme proteins in E. tenella.

Expression of Toll-like receptors and host defence peptides in the cecum of chicken challenged with Eimeria tenella.

Chicken coccidiosis, caused by Eimeria protozoa, affects poultry farming. Toll-like receptors (TLRs) and host defence peptides (HDPs) help host innate immune responses to eliminate invading pathogens, but their roles in Eimeria tenella infection remain poorly understood. Herein, 14-day-old chickens were treated orally with 50,000 E. tenella oocysts and the cecum was dissected at different timepoints. mRNA expression of 10 chicken TLRs (chTLRs) and five HDPs was measured by quantitative real-time PCR. chTLR7 and chTLR15 were upregulated significantly at 3 h post-infection while other chTLRs were downregulated (p < .05). chTLR1a, chTLR1b, chTLR2b and chTLR4 peaked at 36 h post-infection, chTLR3, chTLR5 and chTLR15 peaked at 72 h post-infection and chTLR21 expression was highest among chTLRs, peaking at 48 h post-infection (p < 0.05). For HDPs, cathelicidin (CATH) 1 to 3 and B1 peaked at 48 h post-infection, liver-expressed antimicrobial peptide 2 peaked at 96 h post-infection, and CATH 2 expression was highest among HDPs. CATH2 and CATH3 were markedly upregulated at 3 h post-infection (p < .05). The results provide insight into innate immune molecules during E. tenella infection in chicken, and indicate that innate immune responses may mediate resistance to chicken coccidiosis.

Morphological and molecular characterization of Eimeria magna infecting local rabbit (Oryctolagus cuniculus) in Alkarg City, Saudi Arabia.

Coccidiosis is one of the most prevalent diseases found in local rabbits (Oryctolagus cuniculus), which is caused by the Eimeria. The study aimed to more reliably identify Eimeria species (Eimeria magna) infecting Local Rabbits in Alkarg City, Saudi Arabia, based the method on the molecular properties and morphological and molecular biological techniques. Sub-spheroidal oocysts measuring 21-27 × 12-16 (24 × 14.4) µm (20 n) and with a length/width (L/W) ratio of 0.9-1.1 (1.0) were identified by microscopic analysis of a fecal sample. Oocysts feature a bi-layered wall that is 1.0-1.2 (1.1) µm thick. About two-thirds of the wall's thickness is made up of a smooth outer layer. A polar granule is present, but neither a micropyle nor an oocyst residuum is present. The ovoidal sporozoites measure 15-18 × 8-11 (16.5 × 9.5) µm, have an L/W ratio of 1.6-1.8 (1.7), and take up around 21% of the oocyst's total surface. The mean size of the sub-Stieda body is 1.4 × 2.3 µm, while the average size of the Stieda body is 0.9 × 1.8 µm. The para-Stieda body is lacking. Sporocyst residuum appears membrane-bound and has an uneven form made up of several granules. With two refractile bodies below the striations and pronounced striations at the more pointed end, sporozoites are vermiform, measuring an average of 11.6 × 4.0 µm. The results of the sequencing for the 18S rDNA gene confirmed the species of Eimeria parasites found in the host (rabbits). The current parasite species is closely related to the previously described and deposited E. magna and deeply embedded in the genus Eimeria (family Eimeriidae). According to the findings, single oocyst molecular identification of Eimeria may be accomplished through consistent use of the morphological and molecular results. It is possible to draw the conclusion that the current research supplies relevant facts that help assess the potential infection and future control measures against rabbit coccidiosis to reduce the financial losses that can be incurred by the rabbit industry in Saudi Arabia.

Eimeria falciformis extracellular vesicles differentially express host cell lncRNAs.

Long noncoding RNAs (lncRNAs) are regulatory transcripts during protozoan infections in the host intestinal epithelial cells (IECs). Apicomplexan Eimeria falciformis sporozoite extracellular vesicles (EVs) contain virulence factors that modulate host IECs pro-inflammatory genes and immune responses. In this study, E. falciformis sporozoites were made to interact with inactivated host cells, and the parasite EVs were separated from total secretome by ultracentrifugation and purified on density gradient medium. Dose-dependent bio-activity of E. falciformis EVs was investigated by RNA sequencing, functional annotation and quantitative PCR. It was found that E. falciformis EVs induced mRNA, circRNA, and lncRNA expressions in mouse IECs. Of 38, 217 lncRNAs assembled, 157 and 152 were upwardly and downwardly expressed respectively. Differentially expressed lncRNAs were associated with cytokines, pyroptosis, and immune signaling pathways including FoxO, NF-κB, MAPK, and TGF-ß. In essence, E. falciformis EVs altered host cell RNA expressions during the interaction with host IECs. Also, differentially expressed lncRNAs are potential diagnostic transcripts during Eimeria infections.

Phylogenetic analysis of Eimeria tenella isolates from chicken of sub-tropical mountains of Meghalaya, India.

BACKGROUND: Coccidiosis is the most common and pathogenic intestinal disease caused by different species of Eimeria is chicken. In this study, we describe the prevalence, molecular diagnosis and evolutionary insight of Eimeria tenella in chicken of Meghalaya's sub-tropical mountainous area. METHODS AND RESULTS: Faecal samples (337 no.) and dead chicks (298 no.) were collected every month from January to July' 2023 from poultry farms (4nos.) in and around Umiam, Ri-Bhoi, Meghalaya. The chicks were categorized into different age groups viz. < 3, 3-6 and > 6 weeks. Samples were examined by flotation techniques and post-mortem. The oocysts were sporulated in 2.5% potassium dichromate solution. Eimeria tenella's 18 S rRNA gene genomic DNA was extracted, amplified, and sequenced. Fecal sample and postmortem examinations revealed 24.04% and 33.22% infections of Eimeria sp., respectively. Oocyst per gram (OPG) was recorded highest and lowest in July (26,500) and February (9800), respectively. Amplification of the 18 S rRNA small subunit gene (SSU) by Polymerase Chain Reaction (PCR) revealed a 1790 bp band size. The amplicon was sequenced and deposited in the NCBI database. BLAST analyses of the SSU rRNA gene of E. tenella, Umiam, Meghalaya isolate (OR458392.1) revealed sequence similarities of more than 99% with SSU rRNA gene sequences available in the NCBI database. Pair wise alignment exhibited nucleotide homology ranging from 71.59 to 100.0% with the maximum sequence homology (100.0%) shared with the E. tenella isolate from Turkey (HQ680474.1) and the lowest homology of 95.6% with UK (HG994972.1). Umiam isolate were found to have 97.08% and 100.0% nucleotide similarities with E. tenella from both the UK (AF026388.1) and the USA (U40264.1), respectively. However, nucleotide similarities of 98.24%, 85.33%, 84.75% and 81.35% were observed with E. tenella strain Bangalore (JX312808.1), E. tenella isolate Kerala-1 (JX093898.1), E. tenella isolate Kerala-3 (JX093900.1) and E. tenella isolate Kerala-2 (JX093899.1), respectively. Phylogenetic analysis of SSU rRNA sequences of E. tenella Umiam, Meghalaya isolate with cognate sequences throughout the world revealed these sequences are distinct but at the same time share a close phylogenetic relationship with Indian isolates from Bangalore and Andhra Pradesh. In addition, the distant phylogenetic relationship was observed with cognate gene sequences of United States of America, Canada, China. CONCLUSION: Phylogenetic analysis of SSU rRNA sequences of E. tenella Umiam, Meghalaya isolate with cognate sequences throughout the world revealed these sequences are distinct but at the same time share a close phylogenetic relationship with Indian isolates from Bangalore and Andhra Pradesh. Distant phylogenetic relationship was observed with cognate gene sequences of United States of America, Canada, China.

The financial cost of coccidiosis in Algerian chicken production: a major challenge for the poultry sector.

Coccidiosis, caused by parasites of the genus Eimeria, is a significant economic burden to the poultry industry. In this study, we conducted a comprehensive analysis to evaluate the financial losses associated with Eimeria infection in chickens in Algeria, relying on data provided by key stakeholders in the Algerian poultry industry to assess sub-clinical as well as clinical impact. We employed the updated 2020 version of a model established to estimate the cost of coccidiosis in chickens, taking into consideration specific cultural and technical aspects of poultry farming in Algeria. The findings predict economic losses due to coccidiosis in chickens of approximately £86.7 million in Algeria for the year 2022, representing £0.30 per chicken raised. The majority of the cost was attributed to morbidity (74.9%), emphasizing the substantial economic impact of reduced productivity including decreased bodyweight gain and increased feed conversion ratio. Costs associated with control measures made up 20.5% of the total calculated cost, with 4.6% of the cost related to mortality. These figures provide a clear indication of the scope and economic impact of Eimeria infection of chickens in Algeria, illustrating the impact of practices common across North Africa. They underscore the ongoing requirement for effective preventive and control measures to reduce these financial losses while improving productivity and welfare, ensuring the economic sustainability of the Algerian poultry industry.

Age-dependent development trends (models) of intestinal significant microbiota species and Eimeria oocysts in coccidia-challenged broiler chickens as affected by dietary encapsulated organic acids and anticoccidial drugs.

ABSTRACTThe study was conducted to investigate the effect of dietary encapsulated organic acids (EOAs) and anticoccidials on the age-dependent development trend of intestinal Lactobacillus, E. coli, coliforms, and Eimeria in Eimeria spp.-infected broiler chickens from reused litter. In total, 525 mixed-sex 1-day-old broiler chickens were used in an uninfected/un-supplemented control plus a 2 (no EOA or 0.1% EOA) × 3 (no anticoccidial, 0.05% maduramicin, and 0.02% diclazuril) factorial arrangement of treatments as a completely randomized design with five replicates of 15 chickens. Results indicated that the cubic model is the best model for explaining the development trends of the intestinal microbial population in uninfected and infected chickens (affected by the EOAs and anticoccidials). Based on the cubic models, the microbial populations had development trends with a decreasing slope from 1-day-old until the early or middle finisher period. EOAs and anticoccidials, especially their simultaneous usage, improved (P < 0.05) the linear and cubic models' slope (affected negatively by Eimeria infection). A polynomial model (order = 6) was determined as the best model for explaining the EOAs and anticoccidial effects on the trend of intestinal Eimeria oocysts in infected chickens. The infection peak (which happened at 25 days) was reduced by EOAs and anticoccidials, especially their simultaneous usage. In conclusion, cubic and polynomial (order = 6) regressions are the best models fitted for explaining the microbiota and Eimeria oocysts trends, respectively. EOAs and anticoccidials, especially their simultaneous usage, had beneficial effects on the microbiota and Eimeria development trends and gastrointestinal health in coccidia-infected broiler chickens. RESEARCH HIGHLIGHTSCubic regression is the best model for explaining intestinal microbiota development.Polynomial regression is the best model for intestinal Eimeria oocysts development.Age-development trends are affected by dietary encapsulated organic acids and anticoccidials.

Prevalence, geographic distribution and risk factors of Eimeria species on commercial broiler farms in Guangdong, China.

BACKGROUND: Coccidiosis is one of the most frequently reported diseases in chickens, causing a significant economic impact on the poultry industry. However, there have been no previous studies evaluating the prevalence of this disease in broiler farms in Guangdong province. Therefore, this study aims to conduct an epidemiological investigation into the occurrence of Eimeria species and associated risk factors in intensive management conditions across four regions in Guangdong province, China. A total of 394 fecal samples were collected from 89 broiler farms in Guangdong province. The prevalence of Eimeria species infection was determined using PCR, and the occurrence of Clostridium perfringens type A was assessed using quantitative real-time PCR. RESULTS: The results showed an overall prevalence of 98.88% (88/89) at the farm level and 87.06% (343/394) at the flock level. All seven Eimeria species were identified, with E. acervulina (72.53%; 64/89), E. tenella (68.54%; 61/89), and E. mitis (66.29%; 59/89) at the farm level, and E. acervulina (36.55%; 144/394), E. mitis (35.28%; 139/394), and E. tenella (34.01%; 134/394) at the flock level. The predominant species combination observed was a co-infection of all seven Eimeria species (6.74%; 6/89), followed by a combination of E. acervulina, E. tenella, E. mitis, E. necatrix, E. brunetti, and E. maxima (5.62%, 5/89). A combination of E. acervulina, E. tenella, E. mitis, E. necatrix, E. brunetti, and E. praecox (4.49%; 4/89) was also observed at the farm level. Furthermore, the study identified several potential risk factors associated with the prevalence of Eimeria species, including farm location, chicken age, drinking water source, control strategy, and the presence of C. perfringens type A were identified as potential risk factors associated with prevalence of Eimeria species. Univariate and multivariate analyses revealed a significant association between E. necatrix infection and both grower chickens (OR = 10.86; 95% CI: 1.92-61.36; p < 0.05) and adult chickens (OR = 24.97; 95% CI: 4.29-145.15; p < 0.001) compared to starter chickens at the farm level. Additionally, farms that used groundwater (OR = 0.27; 95% CI: 0.08-0.94; p < 0.05) were less likely to have E. maxima compared to those that used running water. At the flock level, the prevalence of E. tenella was significantly higher in the Pearl River Delta (OR = 2.48; 95% CI: 1.0-6.15; p = 0.05) compared to eastern Guangdong. Interestingly, flocks with indigenous birds were less likely to have E. brunetti (OR = 0.48; 95% CI: 0.26-0.89; p < 0.05) compared to flocks with indigenous crossbred birds. Furthermore, flocks that used anticoccidial drugs (OR = 0.09; 95% CI: 0.03-0.31; p < 0.001) or a combination of vaccines and anticoccidial drugs (OR = 0.06; 95% CI: 0.01-0.25; p < 0.001) were less likely to be positive for E. tenella compared to flocks that only used vaccines. Finally, flocks with C. perfringens type A infection were significantly more likely to have E. necatrix (OR = 3.26; 95% CI: 1.96-5.43; p < 0.001), E. tenella (OR = 2.14; 95% CI: 1.36-3.36; p < 0.001), E. brunetti (OR = 2.48; 95% CI: 1.45-4.23; p < 0.001), and E. acervulina (OR = 2.62; 95% CI: 1.69-4.06; p < 0.001) compared to flocks without C. perfringens type A. CONCLUSIONS: This study conducted an investigation on the prevalence, distribution, and risk factors associated with Eimeria species infection in broiler chickens in Guangdong. The farm-level prevalence of Eimeria species was higher than the previous prevalence figures for other areas and countries. E. brunetti was identified at higher prevalence in Guangdong than previously survived prevalence in different regions in China. Farm location, chicken age, drinking water source, control strategy, and the presence of C. perfringens type A were considered as potential risk factors associated with prevalence of Eimeria species. It is imperative to underscore the necessity for further surveys to delve deeper into the occurrence of Eimeria species under intensive management conditions for different flock purposes.

Efficacy of liver free and Chitosan against Eimeria tenella in chickens.

Eimeria spp. are the pathogen that causes coccidiosis, a significant disease that affects intensively reared livestock, especially poultry. Anticoccidial feed additives, chemicals, and ionophores have routinely been employed to reduce Eimeria infections in broiler production. Therefore, the shift to antibiotic-free and organic farming necessitates novel coccidiosis preventive strategies. The present study evaluated the effects of potential feed additives, liver free and chitosan, against Eimeria tenella infection in White Leghorn broiler female chickens. One hundred sixty-five 1-day-old White Leghorn broiler female chicks were divided into 11 groups (15 female chicks per group), including the positive control group (G1), the negative control group (G2), a chitosan-treated group (G3), a chitosan-treated-infected group (G4), the liver free-treated group (G5), the liver free-treated-infected group (G6), the liver free-and-chitosan-treated group (G7), the liver free-and-chitosan-infected group (G8), the therapeutic liver free-and-chitosan-treated-infected group (G9), the sulfaquinoxaline-treated group (G10), and the sulfaquinoxaline-treated-infected group (G11). Chitosan was fed to the chicks in G3 and G4 as a preventative measure at a dose of 250 mg/kg. The G5 and G6 groups received 1.5 mg/kg of Liverfree. The G7 and G8 groups received chitosan and Liverfree. The G10 and G11 groups were administered 2 g/L of sulfaquinoxaline. From the moment the chicks arrived at Foshan University (one-day-old chicks) until the completion of the experiment, all medications were given to them as a preventative measure. G8 did; however, receive chitosan and liver free as therapeutic supplements at 7 dpi. The current study showed that the combination of liver free and chitosan can achieve better prophylactic and therapeutic effects than either alone. In E. tenella challenged chickens, G8 and G9 chickens showed reduced oocyst shedding and lesion score, improved growth performance (body weight, body weight gain, feed intake, feed conversion ratio, and mortality rate), and cecal histology. The current study demonstrates that combining liver free and chitosan has superior preventive and therapeutic benefits than either alone, and they could also be used as alternative anticoccidial agents.

Cryptosporidium spp. and Eimeria spp. (Apicomplexa: Eimeriorina) of freshwater Cyprinid fish species in the Kura River basin in Azerbaijan territory.

This study aims to determine the prevalence of Cryptosporidium and Eimeria spp. oocysts in fish specimens in the river Kura. It was conducted during the 2021-2022 at two sites: Mingachevir reservoir in central Azerbaijan and in Neftchala district where the river finally enters the Caspian Sea through a delta of the Kura River estuary. The diagnosis of oocysts was performed microscopically. Fine smears from the intestine epithelial layers stained by Ziehl-Neelsen for Cryptosporidium oocysts. To identify Eimeria oocysts, each fish's faecal material and intestinal scrapings were examined directly under a light microscope in wet samples on glass slides with a coverslip. Results revealed a prevalence of Cryptosporidium and Eimeria species infections in fish hosts from both territories Rutilus caspicus, Alburnus filippi, Abramis brama orientalis and Carassius gibelio. Of 170 investigated fish specimens, 8.8% (15/170) were infected with Cryptosporidium species oocysts. Eimeria species oocysts were identified in 20.6% (35/170). The presence of Cryptosporidium and Eimeria infections in fish specimens are natural infections. However, their presence in fish species may be attributed to the age of the fish species and water pollution. This is the first report regarding the prevalence of Cryptosporidium oocysts in fish species in Azerbaijan.

Eimeria tenella pyrroline -5-carboxylate reductase is a secreted protein and involved in host cell invasion.

Chicken coccidiosis, which caused by Eimeria spp, is a parasitic protozoal disease. At present, control measures of this disease depend mainly on anticoccidial drugs and live vaccines. But these control strategies have drawbacks such as drug resistance and limitations in live vaccines production. Therefore, novel control approaches are urgently need to study to control this disease effectively. In this study, the function and characteristics of the pyrroline-5-carboxylate reductase of Eimeria tenella (EtPYCR) protein were preliminary analyzed. The transcription and translation level were analyzed by using qPCR and Western blot. The results showed that the mRNA transcription and translation levels of EtPYCR were higher in unsporulated oocysts (UO) and second generation merozoites (Mrz) than that in sporulated oocysts (SO) and sporozoites. Enzyme activity showed that the enzyme activity of EtPYCR was also higher in the UO and Mrz than that in the SO and sporozoites. Immunofluorescence localization showed EtPYCR was mainly located on the top of sporozoites and the whole cytoplasm and surface of Mrz. The secretion assay indicated that EtPYCR was secretion protein, but not from micronemes. Invasion inhibition assay showed that rabbit anti-rEtPYCR polyclonal antibodies can effectively inhibit sporozoite invasion of DF-1 cells. These results showed that EtPYCR possess several important roles that separate and distinct from its conversion 1-pyrroline-5-carboxylate (P5C) into proline and maybe involved in the host cell invasion and development of parasites in host cells.

Evaluation of the immunoprotective effect of the recombinant Eimeria intestinalis rhoptry protein 25 and rhoptry protein 30 on New Zealand rabbits.

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). CONCLUSION: 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.

Parasitological and molecular investigation of consequences of raw meat feeding (BARF) in dogs and cats: implications for other pets living nearby.

Considering the original nature of carnivorism among dogs and cats, nowadays these pets are increasingly fed BARF (Biologically Appropriate Raw Food/Bone And Raw Food) diet to improve their health conditions. However, this diet may also carry health risks, such as vitamin or mineral deficiencies, and infection with pathogens including parasites. In our study, fecal samples of 89 pet animals kept on BARF diet were subjected to coprological examination followed by molecular analyses. Six of them shed eggs of Dicrocoelium dendriticum. This result was confirmed with PCR and sequencing, and in one case, the DNA of Fasciola hepatica was also demonstrated. In addition, oocysts of Cystoisospora canis, a Cystoisospora ohioensis-like sp. and Eimeria stiedai, as well as sporocysts of a Sarcocystis sp. were also detected. All samples were negative for Neospora caninum and Toxoplasma gondii. In conclusion, no evidence was found for the infection of BARF-fed dogs and cats with parasites that are usually associated with this diet and considered as clinico-pathological risk factors for these pets themselves (e.g., N. caninum, T. gondii). However, fluke eggs (probably originating from ruminants) and oocysts of E. stiedai (from rabbit liver in the food) were demonstrated as pseudoparasites. These species are usually not considered among parasite-associated risks of BARF-feeding, implying that other animals living near BARF-fed pets are neglected in this context. However, where intermediate hosts of D. dendriticum occur in urban areas, BARF-feeding may indirectly affect later other dogs and cats. It was also shown here that BARF-feeding may contribute to the contamination of the environment with E. stiedai oocysts, thus increasing the risks of biliary coccidiosis in nearby living pet rabbits that would otherwise not have access to oocysts of E. stiedai.

Biological characteristics of a precocious line of Eimeria tenella.

The Eimeria tenella Yulin strain (EtYL), which is sensitive to most anti-coccidial drugs, was isolated in the Yulin area of Guangxi, China. Then, Eimeria tenella Yulin precocious line (pEtYL), a precocious line with a prepatent period of 108 h, was obtained through early selection. The biological characteristics of pEtYL, including its morphology, purity, oocyst excretion curve, reproductive capacity, pathogenicity, immunogenicity, and preservation time, were comprehensively analyzed. The results showed that the isolated precocious line of E. tenella exhibited high purity, relatively weak pathogenicity, and good immunogenicity and can be used as a live vaccine line for chicken coccidiosis.

Transcriptomic responses of rabbits to infections by precocious line and wild-type Eimeria media: revealing molecular signatures and pathway differences in liver and duodenum during the peak and terminal phases of oocyst production.

Eimeria media is a principal pathogen responsible for rabbit coccidiosis, targeting the rabbit's intestinal epithelial cells. This parasitism damages the intestinal mucosal barrier, initiating a systemic immune and inflammatory response that jeopardizes the sustainable growth of rabbit farming. To understand the implications of infection on the host's immune and metabolic responses, we employed RNA-Seq to analyze RNA from the liver and duodenum tissues of post-infected rabbits infected with both the precocious line and wild-type strain of E.media. Comprehensive transcriptomic analysis revealed that the two parasites exhibit divergent transcriptomic imprints on host tissues. While the precocious line predominantly modulates immune-centric pathways with significant differential gene enrichment, wild-type strain favors pathways that affect metabolism. In addition, our study pinpointed a set of genes that undergo significant modifications in response to these effects. These revelations grant a fresh avenue to probe deeper into the symbiotic intricacies of the E.media and its rabbit host.

Synthesis and evaluation of febrifugine derivatives as anticoccidial agents.

A series of new febrifugine derivatives with a 4(3H)-quinazolinone scaffold were synthesized and evaluated for their anticoccidial activity both in vitro and in vivo. The targets' in vitro activity against Eimeria tenella was studied using quantitative real-time reverse transcription polymerase chain reaction and Madin-Darby bovine kidney cells. Most of these compounds demonstrated anticoccidial efficacy, with inhibition ratios ranging from 3.3% to 85.7%. Specifically, compounds 33 and 34 showed significant inhibitory effects on the proliferation of E. tenella and exhibited lower cytotoxicity compared to febrifugine. The IC50 values of compounds 33 and 34 were 3.48 and 1.79 µM, respectively, while the CC50 values were >100 µM for both compounds. Furthermore, in a study involving 14-day-old chickens infected with 5 × 104 sporulated oocysts, treatment with five selected compounds (22, 24, 28, 33, and 34), which exhibited in vitro inhibition rate of over 50% at 100 µM, at a dose of 40 mg/kg in daily feed for 8 consecutive days showed that compound 34 possessed moderate in vivo activity against coccidiosis, with an anticoccidial index of 164. Structure-activity relationship studies suggested that spirocyclic piperidine may be a preferable substructure to maintain high effectiveness in inhibiting Eimeria spp., when the side chain 1-(3-hydroxypiperidin-2-yl)propan-2-one was replaced.

Changes in the physiological, hematological and parasitological parameters of Dorper sheep in relation to residual feed intake and confinement environment.

The selection of animals with greater feed efficiency has unknown parasitological and physiological parameters when confined in full sun or shade. Thus, we aimed to assess the effect of residual feed intake (RFI) and the confinement environment on the hematological, parasitological and physiological parameters in male Dorper sheep (n = 60; 30 with positive RFI and 30 with negative RFI) distributed in 2 confinement environments (full sun and shade), in a 2 × 2 factorial scheme, with 15 animals for treatment. Animals kept in the shade showed an increase (P < 0.05) of erythrocytes, hemoglobin and albumin. Animals kept in full sun showed higherOocysts gamma glutamyltransferase, direct bilirubin, transaminase aspartate and respiratory rate (P<0.05). There was a higher incidence of Eimeria spp oocysts in RFI negative animals (P<0.05). Positive RFI animals increased respiratory rate (P<0.05). The RFI did not influence the blood parameters of Dorper sheep, however, it had an effect on respiratory rate and presence of Eimeria spp. oocysts. Thermal environment promoted changes in blood parameters and the physiological parameters of Dorper sheep.