The burden of gastrointestinal parasites in zoo animals has serious implications for their welfare and the health of veterinarians and visitors. Zhuyuwan Zoo is located in the eastern suburb of Yangzhou city in eastern China, in which over 40 species of zoo animals are kept. In order to understand the infection status of GI parasites in Zhuyuwan Zoo, a total of 104 fresh fecal samples collected randomly from birds (n = 19), primates (n = 19), and non-primate mammals (n = 66) were analyzed using the saturated saline flotation technique and nylon sifter elutriation and sieving method for eggs/oocysts, respectively. Two Ascaris species were molecularly characterized. The results showed that the overall prevalence of parasitic infection was 42.3% (44/104). The parasitic infection rate in birds, primates, and non-primate mammals were 26.3% (5/19), 31.6% (6/19), and 50.0% (33/66), respectively. A total of 11 species of parasites were identified, namely, Trichostrongylidae, Capillaria sp., Trichuris spp., Strongyloides spp., Amidostomum sp., Toxascaris leonina, Baylisascaris transfuga, Parascaris equorum, Paramphistomum spp., Fasciola spp., and Eimeria spp. Paramphistomum spp. eggs were first detected from the captive Père David’s deer, and Fasciola spp. eggs were first reported from sika deer in zoo in China. A sequence analysis of ITS-2 and cox1 showed that the eggs isolated from the African lion (Panthera leo Linnaeus, 1758) were T. leonina, and the eggs from the brown bear (Ursus arctos Linnaeus, 1758) were B. transfuga. The public health threat posed by these potential zoonotic parasitic agents requires attention. These results lay a theoretical foundation for prevention and control of wild animal parasitic diseases at zoos in China.

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.

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.

Background: Eimeria necatrix , a member of the Apicomplexa phylum, is one of the most pathogenic parasites, causing high mortality in chickens. Microneme proteins (MICs) play essential roles in host cell recognition and invasion by apicomplexan parasites and are also attractive candidates for vaccine development. However, comprehensive studies on E. necatrix MICs remain limited. Methods: Eimeria necatrix MIC3 gene (En MIC3 ) was amplified and expressed in Escherichia coli . The recombinant protein (rEnMIC3) was characterized via SDS-PAGE and Western blot. The antigenicity of rEnMIC3 and its localization in sporozoites (SZ) and second-generation merozoites (MZ-2) of E. necatrix were determined by Western blot and indirect immunofluorescence analyses (IFAs). The dynamic expression of EnMIC3 across different developmental stages and its impact on sporozoite invasion of host cells were analyzed. The immune protection provided by rEnMIC3 was evaluated in chickens using weight gain, lesion scores, oocyst production, anticoccidial index (ACI), and antibody levels. Results: The open reading frame of En MIC3 was 798 bp, encoding a 265-amino acid protein with a predicted molecular weight of 28.50 kDa. EnMIC3 contained a signal peptide and a single epidermal growth factor (EGF)-like domain. The rEnMIC3 with an approximate molecular weight of 36 kDa could be specifically recognized by convalescent sera from chickens infected with E. necatrix . The molecular mass of the native protein was approximately 35 kDa, and it localizes to the apical region in SZ but exhibits a cytoplasmic distribution in MZ-2. EnMIC3 mRNA was expressed at significantly higher levels in SZ than in MZ-2, whereas protein expression displayed an inverse pattern. Anti-rEnMIC3 polyclonal antibodies inhibited sporozoite invasion of DF-1 cells in a dose-dependent manner. Vaccination with rEnMIC3 conferred effective protection against E. necatrix challenge, with the high-dose group (200 µg) achieving the highest ACI value (171.32) and markedly elevated serum antibody levels. Conclusions: These findings not only offer a foundation for understanding the role of EnMIC3 protein in the host invasion of E. necatrix but also present a potential protective antigen of E. necatrix for the development of a subunit vaccine against avian coccidiosis.