Search Results (16)

Chicken coccidiosis is a parasitic disease caused mainly by Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix, with most cases presenting as mixed infections. Currently, although a subunit vaccine (CoxAbic) targeting Eimeria maxima via maternal immunization is commercially available, no genetically engineered multivalent subunit vaccine exists against mixed infections caused by these four Eimeria species simultaneously. Therefore, we developed a tetravalent recombinant subunit vaccine (designated TEIN) by fusing key antigen genes (TA4, 3-1E, IMP1, NA4) from these four Eimeria species and expressing the construct in Pichia pastoris. A total of 500 chickens were randomly allocated into 25 experimental subgroups (n = 20 each), consisting of five groups (control, challenged, adjuvant, pPIC9K, and TEIN) and five challenge conditions (infection with Eimeria tenella, Eimeria acervulina, Eimeria maxima, Eimeria necatrix, or a mixture of four species). Immunization was performed via leg intramuscular injection at 14 and 21 days of age. At 28 days of age, all chickens except the controls were orally challenged with 1 × 10⁴ sporulated oocysts. Statistical analysis was performed using one-way or two-way ANOVA as appropriate. Results showed that chickens vaccinated with the TEIN subunit vaccine exhibited significantly elevated serum levels of IgY, IL-2, IL-10, and IFN-γ, as well as an increased splenic lymphocyte CD4⁺/CD8⁺ ratio. The anticoccidial indices (ACI) against Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix, and their mixed infection reached 174.82, 174.58, 174.41, 180.61, and 175.95, respectively. Moreover, no significant differences were observed in hematological parameters, serum biochemical markers, or histopathological findings between the vaccinated and control groups. These results demonstrate the vaccine’s potential as a promising candidate for controlling mixed coccidial infections. Full article

Avian coccidiosis is an intestinal disease caused by Eimeria spp. infection. A deeper understanding of the interaction between host gut microbiota and the Eimeria parasite is crucial for developing alternative therapies to control avian coccidiosis. Here, we used full-length sequencing of 16S ribosomal RNA amplicons to compare changes in the gut microbiota of chickens infected with Eimeria tenella, Eimeria maxima, and Eimeria necatrix, aiming to identify both species-specific and common alterations in gut microbiota at 4 and 10 days post-infection. The result revealed that infection with all three Eimeria species led to a decrease in the abundance of the microbial genera Limosilactobacillus, Streptococcus, Alistipes, Lactobacillus and Phocaeicola, while the abundance of Bacteroides, Escherichia and Ligilactobacillus increased. Escherichia and Enterococcus were most abundant in the jejunum of the E. necatrix-infected group and in the cecum of the E. tenella-infected group, whereas Megamonas abundance was highest in the E. maxima-infected group. LEfSe analysis showed that infection with all three Eimeria species significantly reduced the abundance of 13 bacterial species, including Acetilactobacillus jinshanensis, Bacteroides ndongoniae, Barnesiella viscericola, Christensenella minuta, Enterocloster clostridioformis, Gemella haemolysans_A, Granulicatella adiacens, Lawsonibacter sp000177015, Limosilactobacillus reuteri, Limosilactobacillus reuteri_D, Limosilactobacillus vaginalis_A, Limosilactobacillus caviae, Limosilactobacillus vaginalis. Infection with E. tenella significantly increased the abundance of five bacterial species, including Bacteroides fragilis, Enterococcus cecorum, Helicobacter pylori, Methylovirgula ligni, and Phocaeicola sp900066445. Infection with E. maxima significantly increased the abundance of seven bacterial species, including Clostridioides difficile, Faecalibacterium prausnitzii, Mediterraneibacter torques, Muribaculum intestinale, Mediterraneibacter massiliensis, Phascolarctobacterium faecium, and Phocaeicola plebeius. Infection with E. necatrix significantly increased the abundance of seven bacterial species, including Alistipes sp900290115, Anaerotignum faecicola, Bacteroides fragilis_A, Escherichia coli, Harryflintia acetispora, Pseudoclostridium thermosuccinogenes, and Tidjanibacter inops_A. The results showed that Eimeria infection causes significant species- and time-dependent changes in the gut microbiota of chickens. These findings enhance our understanding of coccidiosis pathogenesis and offer potential targets for developing probiotics. Full article

Chicken coccidiosis is a major parasitic disease in poultry that causes substantial economic losses worldwide. The accurate and simultaneous diagnosis of mixed infections with multiple Eimeria species remains challenging using conventional methods. In this study, we developed a multiplex TaqMan-MGB qPCR assay targeting the ITS1 region for simultaneous detection and quantification of four major pathogenic species: E. acervulina, E. necatrix, E. maxima, and E. tenella. The assay exhibited high specificity without cross-reactivity and achieved a sensitivity 100-fold greater than conventional PCR, with detection limits ranging from 10¹ to 10² copies/μL. Standard curves demonstrated strong linearity (R² ≥ 0.95) and amplification efficiency (95–113%). Repeatability was robust, with intra- and inter-assay CVs below 2%. Applied to 165 clinical samples collected from poultry farms in Fujian Province, China (2022–2024), the assay detected an overall Eimeria infection rate of 93.3%, higher than conventional PCR (89.7%). Epidemiological analysis revealed E. tenella (78.8%, [130/165]) and E. necatrix (78.8%, [130/165]) were dominant, while mixed infections were frequent (83.3%, [136/154]), including dual (45.5%, [70/154]), triple (31.2%, [48/154]), and quadruple (11.7%, [18/154]) infections. This multiplex TaqMan-MGB qPCR provides a rapid, sensitive, and quantitative tool for detecting multiple Eimeria species, greatly improving diagnostic efficiency and supporting clinical diagnosis, surveillance, and control strategies. Full article

SSB proteins play essential roles in DNA replication, recombination, and repair in bacteria, archaea, and eukarya. This study investigates the transcript levels, identification, expression and purification, subcellular localization, and immune protective potential of the SSB-like proteins of Eimeria necatrix (EnSSB), exploring its role in the development of E. necatrix and its potential as a candidate antigen for a subunit vaccine against avian coccidiosis. The level of EnSSB gene transcription was highest in unsporulated oocysts (UO), followed by gametocytes (GAM) (p < 0.05). The gene consisted of an open reading frame of 1488 nucleotides encoding a protein of 495 amino acid residues with a predicted molecular weight of 53.31 kDa. EnSSB contained a SSB domain with a conserved OB (oligonucleotide/oligosaccharide binding) fold. The molecular mass of the native protein, as determined by Western blot analysis, was ~58 kDa in second-generation merozoites (MZ-2) and UO. In addition to the 58 kDa band, four other bands (~98 kDa, ~82 kDa, ~36 kDa and ~28 kDa) were detected in GAM. No bands were detected in MZ-3. Indirect immunofluorescence and immuno-electron microscopy localized EnSSB in the cytoplasm of macrogametocytes but not in wall-forming bodies and oocyst wall. Animal challenge experiments demonstrated that rEnSSB elicited robust IgY responses, increased splenic T lymphocytes and body weight gain, reduced intestinal lesion scores and oocyst shedding, and presented anticoccidial index (ACI) more than 160. These findings not only offer a foundation for understanding the role of EnSSB protein in regulating the development of E. necatrix, but also present a potential protective antigen of E. necatrix for the development of a subunit vaccine against avian coccidiosis. Full article

Coccidiosis, caused by Eimeria spp., significantly reduces poultry productivity and causes major economic losses. Traditional control methods are limited by drug resistance and high production costs. Recent genomic and bioinformatic advances have enabled the identification of novel antigens, making recombinant subunit vaccines a promising next-generation strategy by eliciting robust cellular and humoral immune responses. This study investigates the E. necatrix gametocyte protein 56 (EnGAM56) as a potential candidate for recombinant subunit vaccines. The full-length E. necatrix gametocyte gam56 gene (Engam56-F) was amplified, expressed in vitro, and characterized via SDS-PAGE and Western blot. Immunofluorescence assays revealed that EnGAM56-F is specifically localized in gametocytes and unsporulated oocysts. Chickens immunized with recombinant proteins (rEnGAM56-F and rEnGAM56-T) were evaluated for immunoprotection against E. necatrix infection through lesion scores, weight gain, oocyst production, anticoccidial index (ACI), and antibody and cytokine levels. The synergistic effects were evaluated by employing various combinations of recombinant proteins, including rEtGAM22, rEtGAM56-T, and rEtGAM59. Results showed that EnGAM56-F encodes a 468-amino acid protein with distinct tyrosine-serine-rich and proline-methionine-rich regions. rEnGAM56-F was specifically recognized by both anti-6 × His tag antibodies and convalescent serum from chickens infected with E. necatrix. Both rEnGAM56-F and rEnGAM56-T provided immune protection, with rEnGAM56-T showing superior efficacy. The combination of rEnGAM (22 + 59 + 56-T) yielded the strongest immune response, followed by rEnGAM (22 + 56-T). These findings highlight the potential of EnGAM56 as a candidate for recombinant subunit anticoccidial vaccines. Full article

Coccidian parasites possess complex life cycles involving asexual proliferation followed by sexual development, producing oocysts that are transmitted from host to host through feces, guaranteeing disease transmission. Eimeria necatrix is a highly pathogenic coccidian causing high mortality in birds. This study examined ultrastructural changes occurring during the third merogony, microgametogenesis, and macrogametogenesis of E. necatrix. The third-generation meront contained eight merozoites, each with coccidian-specific features like conoid, rhoptries, micronemes, and dense granules. Microgametes had a nucleus, mitochondrion, two flagella, and a basal apparatus. Macrogametes surrounded by two membranes (M1 and M2), contained organelles like WFB1, WFB2, endoplasmic reticulum, mitochondria, and tubular structures. Oocyst wall formation began with M2 separating from M1 and forming a loose veil around the organism. The WFB1 fused together to form the outer layer of the oocyst wall between M1 and M2, while M4 formed beneath M1. The WFB2 fused with the M4 to discharge its contents external to M4, which fused together to form the inner layer of the oocyst wall. Immunogold electron microscopy co-localization result showed that EnGAM22 localized to WFB1 and the outer wall, while EnGAM59 localized to WFB2 and the inner wall, suggesting they are key structural components of the oocyst wall. Full article

Family poultry production systems (FPPSs) in Chile and Argentina produce high-quality and nutritious food. However, little is known about chicken coccidiosis in these production systems. This work aimed to determine the Eimeria sp. positivity rate, circulating species, general farm management, and knowledge of the disease in FPPSs by gaining access to peri-urban markets in these two countries. The overall Eimeria sp. positivity rate, determined in 88 fecal samples, was 85.1%. Oocysts per gram in Argentinean meat-producing FPPSs were significantly higher than in Argentinean and Chilean egg-producing FPPSs. Multiplex-PCR based on seven Eimeria spp. Sequence-Characterized Amplified Region (SCAR) markers showed a great diversity of circulating species, with E. mitis (70.3%), E. acervulina (62.2%), and E. tenella (59.5%), followed by E. maxima (43.2%), E. praecox (32.4%), E. necatrix (18.9%), and E. brunetti (5.4%). Low awareness among family producers and low implementation of control measures were found. Importantly, pathogenic species were found even in asymptomatic chickens, which represents a potential chicken health threat. Furthermore, the administration of sulfonamides to broilers with clinical signs poses a risk of environmental contamination. This is the first comprehensive cross-sectional study showing that Eimeria sp. is a persistent parasite in peri-urban FPPSs in Argentina and Chile. Full article

Zearalenone (ZEN) is a toxic secondary metabolite produced by the Fusarium fungi, which widely contaminates grains, food, and feed, causing health hazards for humans and animals. Therefore, it is essential to find effective ZEN detoxification methods. Enzymatic degradation of ZEN is believed to be an eco-friendly detoxification strategy, specifically thermostable ZEN degradation enzymes are needed in the food and feed industry. In this study, a novel ZEN lactone hydrolase ZHRnZ from Rosellinia necatrix was discovered using bioinformatic and molecular docking technology. The recombinant ZHRnZ showed the best activity at pH 9.0 and 45 °C with more than 90% degradation for ZEN, α-zearalenol (α-ZOL), β-zearalenol (β-ZOL) and α-zearalanol (α-ZAL) after incubation for 15 min. We obtained 10 mutants with improved thermostability by single point mutation technology. Among them, mutants E122Q and E122R showed the best performance, which retained more than 30% of their initial activity at 50 °C for 2 min, and approximately 10% of their initial activity at 60 °C for 1 min. The enzymatic kinetic study showed that the catalytic efficiency of E122R was 1.3 times higher than that of the wild-type (WT). Comprehensive consideration suggests that mutant E122R is a promising hydrolase to detoxify ZEN in food and feed. Full article

The codon usage bias (CUB) of genes encoded by different species’ genomes varies greatly. The analysis of codon usage patterns enriches our comprehension of genetic and evolutionary characteristics across diverse species. In this study, we performed a genome-wide analysis of CUB and its influencing factors in six sequenced Eimeria species that cause coccidiosis in poultry: Eimeria acervulina, Eimeria necatrix, Eimeria brunetti, Eimeria tenella, Eimeria praecox, and Eimeria maxima. The GC content of protein-coding genes varies between 52.67% and 58.24% among the six Eimeria species. The distribution trend of GC content at different codon positions follows GC1 > GC3 > GC2. Most high-frequency codons tend to end with C/G, except in E. maxima. Additionally, there is a positive correlation between GC3 content and GC3s/C3s, but a significantly negative correlation with A3s. Analysis of the ENC-Plot, neutrality plot, and PR2-bias plot suggests that selection pressure has a stronger influence than mutational pressure on CUB in the six Eimeria genomes. Finally, we identified from 11 to 15 optimal codons, with GCA, CAG, and AGC being the most commonly used optimal codons across these species. This study offers a thorough exploration of the relationships between CUB and selection pressures within the protein-coding genes of Eimeria species. Genetic evolution in these species appears to be influenced by mutations and selection pressures. Additionally, the findings shed light on unique characteristics and evolutionary traits specific to the six Eimeria species. Full article

Coccidiosis in chickens is a parasitic disease of economic importance for the poultry industry. In Ecuador, there is limited information regarding the prevalence of Eimeria spp. on commercial broiler farms. Therefore, a total of 155 poultry farms in the provinces of Pichincha and Santo Domingo de los Tsáchilas were surveyed. The analysis of fresh fecal samples was conducted to determine the parasitic load of six of the seven chicken Eimeria species (excluding E. mitis) through multiplex PCR. Additionally, an epidemiological survey was performed to assess the risk factors associated with the infection using a multivariable logistic regression model. All samples tested positive for the presence of Eimeria spp., despite the farmers having implemented prophylactic measures, and no clinical coccidiosis cases were recorded. The parasitic load varied between 25 and 69,900 oocyst per gram. The species prevalence was as follows: Eimeria spp. 100%, E. maxima 80.4%, E. acervulina 70.6%, E. praecox 55.4%, E. tenella 53.6%, E. necatrix 52.2%, and E. brunetti 30.8%. The main species combination was E. cervuline, E. maxima, E. necatrix, and E. praecox (23.90%), followed by E. tenella, as a unique species (10.69%), and then E. acervulina, E. maxima, and E. praecox (8.81%). It was observed that farms operated by independent producers had a higher amount of Eimeria spp. and higher probability of the presence of E. brunetti, E. necatrix, E. praecox, and E. tenella. Poultry houses located below 1300 m above sea level were associated with a higher parasitic load and the presence of E. brunetti. Birds younger than 35 days of age and from open-sided poultry houses (with rudimentary environmental control) had a higher probability of presenting E. maxima. Drinking water from wells increased the risk of E. praecox presence. Research aimed at designing control strategies to improve health management on poultry farms in the region would help minimize the impact of coccidiosis. Full article

Coccidiosis in chickens is one of the major problems in the poultry industry, caused by protozoan parasites of the genus Eimeria. The current study used morphological and molecular characteristics to identify Eimeria spp. infecting domestic chickens (Gallus gallus) in the Riyadh region of Saudi Arabia. In this study, 120 domestic poultry were examined and 30 were found to be infected with oocysts of Eimeria spp. (25%). According to the morphology of the recorded oocysts, five species were found. Eimeria necatrix was the first species discovered, and it was distinguished by oblong, ovoid-shaped oocysts with double-layered walls that measured 20 (23–23) and 17 (16–20) μm. The second species was Eimeria maxima, which had oval- to egg-shaped oocysts with double-layered walls and measurements of 28 (26–29) and 23 (20–24) μm. The third species was Eimeria tenella, characterized by oval-shaped oocysts with double-layered walls and measurements of 21 (20–24) × 17 (16–20) μm. Eimeria praecox was the fourth species that was characterized by spherical-shaped oocysts with single-layered walls and measurements of 21 (19–23) × 20 (19–20) μm. Eimeria acervulina was the last species to have oval-shaped oocysts with double-layered walls and measurements of 20 (18–25) and 17 (14–20) μm. The percentages of infection with Eimeria species were as follows: E. tenella, 10.84%; E. necatrix, 5.84%; E. acervulina, 4.16%; E. maxima, 2.5%; and E. praecox, 1.66%. Nested PCR based on the amplification of internal transcribed spacer I (ITS-I) regions confirmed the presence of the five Eimeria species in the examined fecal samples with their specific amplicon sizes: E. necatrix (383 bp), E. maxima (145 bp), E. tenella (278 bp), E. praecopx (116 bp), and E. acervulina (321 bp). Full article

Poultry necrotic enteritis (NE) is a complex and multifactorial disease caused by Clostridium perfringens types. Earlier, the disease was prevented and/or controlled through the addition of in-feed antibiotics and antimicrobial growth promoters (AGPs). The ban on the use of these agents as feed additives has been a major reason for re-emergence of this disease leading to huge economic losses to the world poultry industry. Understanding the pathogenesis of NE by developing an effective experimental model remains challenging and lacks consistency owing to the involvement of several critical factors involved in causing lesions of disease in the field. In this study, locally characterized C. perfringens types, i.e., ACP (toxinotype A), and GCP (toxinotype G), obtained from NE outbreaks on commercial farms in China (2020–2022), were used to experimentally induce NE in Specific-Pathogen-Free (SPF) chicks. The lesion scores observed on day 20 were 1.9 ± 1.10 (GCP strain) and 1.5 ± 1.08 (ACP strain), and both had significant difference as compared to the control group. The inclusion of fishmeal in addition to oral clostridial dose, i.e., fishmeal (day 7 onward) + Clostridia (7.5 × 10⁸ cfu/mL consecutively for 04 days) induced a lesion score of 2.0 ± 1.15 in respective groups. Use of coccidia (Eimeria necatrix) on day 9 followed by clostridia challenge enhanced the lesion scores to 2.5 ± 1.08 and 2.2 ± 1.23 for type G and type A strains, respectively. When both predisposing factors (coccidia + fish meal) were given together, i.e., fishmeal (day 7 onward) and coccidia (day 9) along with clostridia, the lesion scores were 3.2 ± 1.22 (GCP + coccidia + fish meal) and 3.0 ± 1.15 (ACP + coccidia + fish meal). These results were significantly different from group 1 (ACP) and 2 (GCP), in which only C. perfringens was used to induce NE. The clinical signs as well as histopathological lesions in experimentally induced groups were found similar as reported in the literature. The two type G strains identified in this study were also used for susceptibility testing against various drugs. Both strains were found to be resistant to amikacin, doxycycline, metronidazole, neomycin, nystatin, polymyxin B, streptomycin, and tetracycline. Variable susceptibility was seen against ceftriaxone, florfenicol, gentamicin, and kanamycin drugs. Amoxicillin, ampicillin, cefotaxime, ciprofloxacin, enrofloxacin, ofloxacin, and penicillin were effective drugs based upon their low level of resistance and therefore they might be preferred over other antimicrobial agents for proper treatment/prophylaxis of NE infections. Further studies are needed to study the pathogenesis of NE in detail in experimentally induced models along with continuous monitoring of the resistance pattern of C. perfringens strains in the field. Full article

Avian Eimeria species vary in their replication location, fecundity, and pathogenicity. They are required to complete the development within the limited space of host intestines, and some synergistic or antagonistic effects occur among different Eimeria species. This study evaluated the impact of Eimeria mitis on the outcome of Eimeria necatrix or Eimeria tenella challenge infection. The severity of E. mitis/E. necatrix and E. mitis/E. tenella mixed infections were quantified by growth performance evaluation, survival rate analysis, lesion scoring, blood stool scoring, and oocyst output counting. The presence of E. mitis exacerbated the outcome of co-infection with E. tenella, causing high mortality, intestinal lesion score, and oocyst production. However, E. mitis/E. tenella co-infection had little impact on the body weight gain compared to individual E. tenella infection. In addition, the presence of E. mitis appeared not to enhance the pathogenicity of E. necatrix, although it tends to inhibit the growth of challenged birds and facilitate oocyst output and mortality in an E. mitis/E. necatrix co-infection model. Collectively, the results suggested a synergistic relationship between E. mitis and E. tenella/E. necatrix when sharing the same host. The presence of E. mitis contributed to disease pathology induced by E. tenella and might also advance the impact of E. necatrix in co-infections. These observations indicate the importance of accounting for differences in the relationships among different Eimeria species when using mixed infection models. Full article

Trichoderma is the most commonly used fungal biocontrol agent throughout the world. In the present study, various Trichoderma isolates were isolated from different vegetable fields. In the isolated microflora, the colony edges varied from wavy to smooth. The mycelial forms were predominantly floccose with hyaline color and conidiophores among all the strains were highly branched. Based on morphological attributes, all the isolates were identified as Trichoderma harzianum. The molecular identification using multilocus sequencing ITS, rpb2 and tef1α, genes further confirmed the morphological identification. The average chitinase activity varied from 1.13 units/mL to 3.38 units/mL among the various isolates, which increased linearly with temperature from 15 to 30 °C. There was an amplified production in the chitinase production in the presence of Mg⁺ and Ca²⁺ and Na⁺ metal ions, but the presence of certain ions was found to cause the down-regulated chitinase activity, i.e., Zn²⁺, Hg²⁺, Fe²⁺, Ag⁺ and K⁺. All the chitinase producing Trichoderma isolates inhibited the growth of tested pathogens viz., Dematophora necatrix, Fusarium solani, Fusarium oxysporum and Pythium aphanidermatum at 25% culture-free filtrate concentration under in vitro conditions. Also, under in vivo conditions, the lowest wilt incidence and highest disease control on Fusarium oxysporum was observed in isolate BT4 with mean wilt incidence and disease control of 21% and 48%, respectively. The Trichoderma harzianum identified in this study will be further used in formulation development for the management of diseases under field conditions. Full article

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. Full article