Molecular characterization of chicken infectious anaemia virus (CIAV) in China during 2020-2021.

Chicken infectious anaemia virus (CIAV) has been identified as the causative agent of chicken infectious anaemia (CIA), causing huge economic losses to the poultry industry globally. In this study, a total of 573 clinical samples were collected from 197 broiler farms in 17 provinces of China during 2020-2021. Among them, 375 samples (375/573, 65.4%) were positive for CIAV by real-time PCR. The positive rate of CIAV detection between different regions of China ranged from 46.67% (North China) to 81.25% (Central China). The nucleotide sequences of the VP1 gene were obtained for 91 CIAV strains, whole genome sequencing was successful for 72 out of 91 strains. Phylogenetic analysis based on the VP1 gene revealed that 91 CIAV strains currently circulating in China belong to three genotypes (II, IIIa and IIIb), and most of the CIAV strains belong to genotype IIIa. Phylogenetic analysis of the whole genome showed that 71 CIAV strains belong to genotype IIIa, and one strain belongs to genotype II. Sequence analysis showed several amino acid substitutions in both the VP1, VP2 and VP3 proteins. Our results enhance the understanding of the molecular characterization of CIAV infection in China.RESEARCH HIGHLIGHTS A molecular systematic survey of CIAV in China during 2020-2021.CIAV genotype IIIa is the predominant genotype in China.

Prevalence and diversity of Trichomonas gallinae in meat pigeons (Columba livia) in Guangdong Province, People's Republic of China.

Pigeon farming for meat has developed into an important economic industry in most countries, especially in China. Trichomoniasis, caused by the protozoan parasite Trichomonas gallinae, is a worldwide disease in pigeons. However, studies of the prevalence and distribution of T. gallinae lineages in domestic pigeons in southern China are limited. In this study, a total of 636 pigeon throat swabs samples from four regions in Guangdong Province were screened for T. gallinae by in vitro culture assays and microscopy. The results revealed an overall prevalence of T. gallinae infection in southern China of 26.6% (169/636). There were significant differences in the infection rate of T. gallinae between the four regions (χ2 = 117.948, df = 4, P = 0.000), with up to 44.6% in the Pearl River Delta region. The infection rate of young pigeons was as high as 70.8%. The rDNA sequences (18S rRNA/ITS1-5.8S rRNA-ITS2) of 153 positive samples were amplified and sequenced. Results identified 58.2% (89/153) overall as ITS-A (18S-VI) (also known as ITS-OBT-Tg-1) and 41.8% (64/153) as ITS-B (18S-IV) (also known as ITS-OBT-Tg-2). Thus, ITS-A (18S-VI) was the dominant T. gallinae genotype in southern China, especially in young pigeon (97.0%, 32/33). In conclusion, a high prevalence of T. gallinae infection in domestic pigeons was identified in southern China, particularly in the Pearl River Delta region. The ITS-A (18S-VI) was the dominant genotype highly pathogenic, which may weaken the immune system of pigeons, and cause a negative impact on the development of the pigeon industry in China.

Identification and Characterization of a Novel Apical Membrane Antigen 3 in Eimeria tenella.

Eimeria tenella is an obligate intracellular parasite in the phylum Apicomplexa. As described for other members of Apicomplexa, apical membrane antigen 1 (AMA1) has been shown to be critical for sporozoite invasion of host cells by E. tenella. Recently, an E. tenella paralogue of AMA1 (EtAMA1), dubbed sporoAMA1 (EtAMA3), was identified in proteomic and transcriptomic analyses of E. tenella, but not further characterized. Here, we show that EtAMA3 is a type I integral membrane protein that has 24% -38% identity with other EtAMAs. EtAMA3 has the same pattern of Cys residues in domains I and II of AMA1 orthologs from apicomplexan parasites, but high variance in domain III, with all six invariant Cys residues absent. EtAMA3 expression was developmentally regulated at the mRNA and protein levels. EtAMA3 protein was detected in sporulated oocysts and sporozoites, but not in the unsporulated oocysts or second-generation merozoites. EtAMA3 is secreted by micronemes and is primarily localized to the apical end of sporozoites during host-cell invasion. Additionally, pretreatment of sporozoites with rEtAMA3-specific antibodies substantially impeded their invasion into host cells. These results suggest EtAMA3 is a sporozoite-specific protein that is involved in host-cell sporozoite invasion.

Molecular characterization and functional analysis of Eimeria tenella citrate synthase.

Chicken coccidiosis, caused by an obligate intracellular protozoan parasite of the genus Eimeria, is a major parasitic disease in the intensively reared poultry industry. Due to the widespread use of anticoccidial drugs, resistance has become an inevitable problem. In our previous study, Eimeria tenella citrate synthase (EtCS) was found to be up-expressed in two drug-resistant strains (diclazuril-resistant and maduramycin-resistant strains) compared to drug-sensitive strain by RNA sequence. In this study, we cloned and expressed EtCS and obtain its polyclonal antibodies. Quantitative real-time polymerase chain (qPCR) reactions and Western blots were used to analyze the transcription and translation levels of EtCS in sensitive and three drug-resistant strains. Compared with the sensitive strain, the transcription of EtCS was both significantly upregulated in diclazuril-resistant and maduramycin-resistant strains, but was not significantly different in salinomycin-resistant strain. No significant difference was seen in translation level in the three drug-resistant strains. Indirect immunofluorescence indicated that EtCS was mainly located in the cytoplasm of sporozoites except for posterior refractile bodies and in the cytoplasm and surface of merozoites. Anti-rEtCS antibody has inhibitory effects on E. tenella sporozoite invasion of DF-1 cells and the inhibition rate is more than 83%. Binding of the protein to chicken macrophage (HD11) cells was confirmed by immunofluorescence assays. When macrophages were treated with rEtCS, secretion of nitric oxide and cell proliferation of the macrophages were substantially reduced. These results showed that EtCS may be related to host cell invasion of E. tenella and involve in the development of E.tenella resistance to some drugs.

Molecular characterization of serine/threonine protein phosphatase of Eimeria tenella.

Avian coccidiosis is a widespread and economically significant poultry disease caused by several Eimeria species, including Eimeria tenella. Previously, E. tenella serine/threonine protein phosphatase (EtSTP) was found to be differentially expressed in drug-sensitive (DS) and drug-resistant strains using RNA-seq. In the present study, we found that transcription and translation levels of EtSTP were higher in diclazuril-resistant (DZR) strains and maduramicin-resistant (MRR) strains than in DS strains using quantitative real-time PCR (qPCR) and Western blotting. Enzyme activity results indicated that the catalytic activity of EtSTP was higher in the two drug-resistant strains than in DS strains. Western blot and qPCR analysis also showed that expression levels of EtSTP were higher in unsporulated oocysts (UO) and second-generation merozoites (SM). Indirect immunofluorescence localization showed that EtSTP was located in most areas of the parasite with the exception of refractile bodies, and fluorescence intensity was enhanced during development. In vitro inhibition experiments showed that the ability of sporozoites (SZ) to invade cells was significantly decreased after treatment with anti-rEtSTP antibody. These results indicated that EtSTP acted mainly during the developmental and reproductive stages of the parasite and may be related to the resistance of coccidia to external drug pressure.

Molecular characterization of 60S ribosomal protein L12 of E. tenella.

This study analyzed the large-subunit (60S) ribosomal protein L12 of Eimeria tenella (Et60s-RPL12). A full-length cDNA was cloned, and the recombinant protein was expressed in E. coli BL21 and inoculated in rabbits to produce the polyclonal antibody. Quantitative real-time polymerase chain reaction and western blotting were used to analyze the transcription levels of Et60s-RPL12 and translation levels in different developmental stages of E. tenella. The results showed that the mRNA transcription level of Et60s-RPL12 was highest in second-generation merozoites, whereas the translation level was highest in unsporulated oocysts. Indirect immunofluorescence showed that Et60s-RPL12 was localized to the anterior region and surface of sporozoites, except for the two refractile bodies. As the invasion of DF-1 cells progressed, fluorescence intensity was increased, and Et60s-RPL12 was localized to the parasitophorous vacuole membrane (PVM). The secretion assay results using staurosporine indicated that this protein was secreted, but not from micronemes. The role of Et60s-RPL12 in invasion was evaluated in vitro. The results of the invasion assay showed that polyclonal antibody inhibited host cell invasion by the parasite, which reached about 12%. However, the rate of invasion was not correlated with the concentration of IgG.

Effects of a complex probiotic preparation, Fengqiang Shengtai and coccidiosis vaccine on the performance and intestinal microbiota of broilers challenged with Eimeria spp.

BACKGROUND: Coccidiosis, a prominent intestinal protozoan disease, carries significant economic implications for the poultry industry. The aim of this study was to evaluate the effects of Fengqiang Shengtai (BLES), a probiotics product, and coccidiosis vaccine in modulating the intestinal microbiome and providing insight into mitigating the occurrence and management of avian coccidiosis. METHODS: Broilers included in the study were divided into four pre-treatment groups: the Pre-Con group (commercial diet), Pre-BLES group (BLES supplement), Pre-Vac group (coccidiosis vaccination) and Pre-Vac-BLES group (combined vaccination and BLES). Body weight gain, feed consumption and feed conversion ratio were monitored from age 25 to 55 days. Cecum contents were collected at 8 and 15 days of age for comparative analysis of intestinal microbiomes. In the Pre-BLES and Pre-Vac-BLES groups, probiotics were administered at a dose of 0.01 g per chicken between ages 3 to 6 days and 10-13 days. At 3 days of age, chickens in the Pre-Vac and Pre-Vac-BLES groups were vaccinated with 1700 sporulated oocysts of the live coccidiosis vaccine per chicken. At the age of 25 days, Eimeria spp. challenge experiments were performed based on the aforementioned immunization strategy, and the oocysts per gram (OPG) in the feces, intestinal lesion score and intestinal pathological characteristics were evaluated. Specifically, 30 chickens were randomly selected from each group and orally administered 34,000 sporulated oocysts of Eimeria spp. per chicken, re-defined as Eimeria group, BLES-Eimeria group, Vac-Eimeria group and Vac-BLES-Eimeria group, respectively. Additionally, 30 chickens were randomly selected from the Pre-Con group and included as negative control without Eimeria spp. CHALLENGE: Intestinal microbiota was sequenced and analyzed when the broilers were 32 days old. RESULTS: A significant improvement was observed in body weight gain of the broilers in the Pre-BLES and Pre-Vac-BLES group at 45 days of age. Analysis of the intestinal microbiota revealed a positive correlation between the experimental groups receiving BLES and coccidiosis vaccines at 8 and 15 days of age with the Enterococcus genus and Lachnospiraceae NK4A136 group, respectively. In addition to the reduced lesion score and OPG values, the combination of coccidiosis vaccine and BLES also reduced the intestinal epithelial abscission induced by coccidiosis vaccines. The results of intestinal microbial function prediction demonstrated that N-glycan biosynthesis and ferroptosis were the prominent signal pathways in the Vac-BLES-Eimeria group. CONCLUSIONS: Taken together, the results of the present study suggest that supplementation of BLES with coccidiosis vaccine represents a promising strategy for improving growth performance, alleviating clinical manifestations and inducing favorable alterations to the intestinal microbiota in broiler chickens affected by coccidiosis.

Effects of Bacillus subtilis and coccidiosis vaccine on growth indices and intestinal microbiota of broilers.

Avian coccidiosis is the most serious parasitic disease in the poultry industry. Therefore, the aim of the current study was to explore the effects of B. subtilis and live coccidiosis vaccine alone or in combination on the production performance and anticoccidiosis, as well as the dynamic changes of intestinal microbial community. Nine hundred ninty Mahuang chickens were randomized into 4 preimmune groups including control group, coccidiosis vaccine immunization group; B. subtilis administration group and a group that was administrated a combination of live coccidiosis vaccine and B. subtilis group. Intestinal mucosal scraps collected from all these experimental groups at the age of 8 d and 15 d for microbial community 16S rRNA gene sequencing. At the age of 25 d, 30 broilers from each preimmune group were randomly assigned to a subgroup infected with Eimeria spp. and renamed as CI, V-CI BS-CI, and VBS-CI group. The production performance was monitored at the age of 25 d, 35 d, 45 d, and 55 d for the rest broilers from each pre-immune group. Otherwise, in the Eimeria spp. challenge stage, intestinal mucosal scraps collected for microbial community sequencing, while duodenum, jejunum, and cecum collected for pathological examination after sacrifice at the age of 32 d. In addition, the oocysts per gram of feces (OPG) and intestinal lesion score of broilers after Eimeria spp. challenge were also counted. Overall, the probiotics and coccidiosis vaccine resulted in the significantly improvement of the production performance. Otherwise, the intestinal lesion score and OPG after Eimeria spp. infection was significantly decreased in the VBS-CI group (P < 0.05). Moreover, these protective effects may also be closely related to genus such as Romboutsia, Blautia, and Butyricococcus, as well as microbiota functions like the quorum sensing pathway. According to these results, a combination of B. subtilis and coccidiosis vaccines can improve performance and provide additional protection against Eimeria spp. infection.

Molecular characterization and analysis of the ATPase ASNA1 homolog gene of Eimeria tenella in a drug sensitive strain and drug resistant strains.

The widespread use of drugs has exacerbated the resistance of Eimeria tenalla to anti-coccidial drugs. Using RNA-seq, we previously found the ATPase ASNA1 homolog of E. tenella (EtASNA1) was differentially expressed in resistant strains and drug sensitive (DS) strain. In our study, we used western blotting and quantitative real-time PCR (qRT-PCR) to analyze the translational and transcriptional levels of EtASNA1 in a diclazuril-resistant (DZR) strain, maduramicin-resistant (MRR) strain, salinomycin-resistant (SMR) strain, and DS strain and found EtASNA1 was highly expressed in three drug-resistant strains. The qRT-PCR and western blotting results also showed that the expression levels of EtASNA1 increased with increasing drug concentration, and the transcription levels of the DZR strains isolated from the field were higher than those of the DS strain. In addition, we used in vivo and in vitro tests to analyze the changes of EtASNA1 expression after DZR, MRR, and DS strain infections in chickens, and in vitro inoculation of DF-1 cells in the presence of drugs. The addition of drugs caused expression to be upregulated. The results of qRT-PCR and western blotting also showed that the expression levels of EtASNA1 in second-generation merozoites (SM) and unsporulated oocysts (UO) were significantly higher than those in the other two developmental stages. The immunofluorescence localization of EtASNA1 indicated that the protein was distributed throughout the sporozoites (SZ) and SM, except for the refractile bodies of SZ. In vitro inhibition experiments showed that anti-EtASNA1 antibody incubation significantly inhibited SZ invasion of DF-1 cells. The above results showed that EtASNA1 may be related to host cell invasion of E. tenella and may be involved in the development of E. tenella resistance to some drugs.

Further investigation of the characteristics and biological function of Eimeria tenella apical membrane antigen 1.

Apical membrane antigen 1 (AMA1) is a type I integral membrane protein that is highly conserved in apicomplexan parasites. Previous studies have shown that Eimeria tenella AMA1 (EtAMA1) is critical for sporozoite invasion of host cells. Here, we show that EtAMA1 is a microneme protein secreted by sporozoites, confirming previous results. Individual and combined treatment with antibodies of EtAMA1 and its interacting proteins, E. tenella rhoptry neck protein 2 (EtRON2) and Eimeria-specific protein (EtESP), elicited significant anti-invasion effects on the parasite in a concentration-dependent manner. The overexpression of EtAMA1 in DF-1 cells showed a significant increase of sporozoite invasion. Isobaric tags for relative and absolute quantitation (iTRAQ) coupled with LC-MS/MS were used to screen differentially expressed proteins (DEPs) in DF-1 cells transiently transfected with EtAMA1. In total, 3953 distinct nonredundant proteins were identified and 163 of these were found to be differentially expressed, including 91 upregulated proteins and 72 downregulated proteins. The DEPs were mainly localized within the cytoplasm and were involved in protein binding and poly(A)-RNA binding. KEEG analyses suggested that the key pathways that the DEPs belonged to included melanogenesis, spliceosomes, tight junctions, and the FoxO and MAPK signaling pathways. The data in this study not only provide a comprehensive dataset for the overall protein changes caused by EtAMA1 expression, but also shed light on EtAMA1's potential molecular mechanisms during Eimeria infections.

TITLE: Étude approfondie des caractéristiques et de la fonction biologique de l'antigène 1 de la membrane apicale d'Eimeria tenella. ABSTRACT: L'antigène 1 de la membrane apicale (AMA1) est une protéine membranaire intégrale de type I hautement conservée chez les parasites Apicomplexa. Des études antérieures ont montré que l'AMA1 d'Eimeria tenella (EtAMA1) était importante pour l'invasion des cellules hôtes par les sporozoïtes. Nous montrons ici qu'EtAMA1 est une protéine des micronèmes sécrétée par les sporozoïtes, confirmant les résultats précédents. Un traitement individuel et combiné avec des anticorps d'EtAMA1 et de ses protéines en interaction, la protéine 2 du cou des rhoptries d'E. tenella (EtRON2) et la protéine spécifique d'Eimeria (EtESP), a provoqué des effets anti-invasion significatifs et dépendants de la concentration sur le parasite. La surexpression d'EtAMA1 dans les cellules DF-1 a montré une augmentation significative de l'invasion par les sporozoïtes. Des marqueurs isobares pour la quantification relative et absolue (iTRAQ) couplés à LC-MS/MS ont été utilisés pour cribler des protéines exprimées différentiellement (PED) dans des cellules DF-1 transfectées de manière transitoire avec EtAMA1. Au total, 3953 protéines non redondantes distinctes ont été identifiées et 163 d'entre elles se sont révélées exprimées de manière différentielle, dont 91 régulées à la hausse et 72 régulées à la baisse. Les PED étaient principalement localisées dans le cytoplasme et étaient impliquées dans la liaison aux protéines et la liaison au poly (A)-ARN. Les analyses de KEEG ont suggéré que les voies clés auxquelles appartenaient les PED comprenaient la mélanogenèse, les épissosomes, les jonctions étroites et les voies de signalisation FoxO et MAPK. Les données de cette étude fournissent non seulement un ensemble de données complet pour les modifications globales des protéines causées par l'expression d'EtAMA1, mais mettent également en lumière les mécanismes moléculaires potentiels d'EtAMA1 pendant les infections par Eimeria.

Eimeria tenella Translation Initiation Factor eIF-5A That Interacts With Calcium-Dependent Protein Kinase 4 Is Involved in Host Cell Invasion.

Eimeria tenella is an apicomplexan, parasitic protozoan known to infect poultry worldwide. An important calcium-dependent protein kinase (CDPK) has been identified in plants, green algae, ciliates and apicomplexan, such as E. tenella. CDPKs are effector molecules involved in calcium signaling pathways, which control important physiological processes such as gliding motility, reproduction, and host cell invasion. Given that CDPKs are not found in the host, studying the functions of CDPKs in E. tenella may serve as a basis for developing new therapeutic drugs and vaccines. To assess the function of CDPK4 in E. tenella (EtCDPK4), a putative interactor, translation initiation factor eIF-5A (EteIF-5A), was screened by both co-immunoprecipitation (co-IP) and His pull-down assays followed by mass spectrometry. The interaction between EteIF-5A and EtCDPK4 was determined by bimolecular fluorescence complementation (BiFC), GST pull-down, and co-IP. The molecular characteristics of EteIF-5A were then analyzed. Quantitative real-time polymerase chain reaction and western blotting were used to determine the transcription and protein levels of EteIF-5A in the different developmental stages of E. tenella. The results showed that the transcription level of EteIF-5A mRNA was highest in second-generation merozoites, and the protein expression level was highest in unsporulated oocysts. Indirect immunofluorescence showed that the EteIF-5A protein was found throughout the cytoplasm of sporozoites, but not in the refractile body. As the invasion of DF-1 cells progressed, EteIF-5A fluorescence intensity increased in trophozoites, decreased in immature schizonts, and increased in mature schizonts. The secretion assay results, analyzed by western blotting, indicated that EteIF-5A was a secreted protein but not from micronemes. The results of invasion inhibition assays showed that rabbit anti-rEteIF-5A polyclonal antibodies effectively inhibited cell invasion by sporozoites, with an inhibition rate of 48%.

Eimeria tenella Eimeria-specific protein that interacts with apical membrane antigen 1 (EtAMA1) is involved in host cell invasion.

BACKGROUND: Avian coccidiosis is a widespread, economically significant disease of poultry, caused by several Eimeria species. These parasites have complex and diverse life-cycles that require invasion of their host cells. This is mediated by various proteins secreted from apical secretory organelles. Apical membrane antigen 1 (AMA1), which is released from micronemes and is conserved across all apicomplexans, plays a central role in the host cell invasion. In a previous study, some putative EtAMA1-interacting proteins of E. tenella were screened. In this study, we characterized one putative EtAMA1-interacting protein, E. tenella Eimeria -specific protein (EtEsp). METHODS: Bimolecular fluorescence complementation (BiFC) and glutathione S-transferase (GST) fusion protein pull-down (GST pull-down) were used to confirm the interaction between EtAMA1 and EtEsp in vivo and in vitro. The expression of EtEsp was analyzed in different developmental stages of E. tenella with quantitative PCR and western blotting. The secretion of EtEsp protein was tested with staurosporine when sporozoites were incubated in complete medium at 41 °C. The localization of EtEsp was analyzed with an immunofluorescence assay (IFA). An in vitro invasion inhibition assay was conducted to assess the ability of antibodies against EtEsp to inhibit cell invasion by E. tenella sporozoites. RESULTS: The interaction between EtAMA1 and EtEsp was confirmed with BiFC and by GST pull-down. Our results show that EtEsp is differentially expressed during distinct phases of the parasite life-cycle. IFA showed that the EtEsp protein is mainly distributed on the parasite surface, and that the expression of this protein increases during the development of the parasite in the host cells. Using staurosporine, we showed that EtEsp is a secreted protein, but not from micronemes. In inhibition tests, a polyclonal anti-rEtEsp antibody attenuated the capacity of E. tenella to invade host cells. CONCLUSION: In this study, we show that EtEsp interacts with EtAMA1 and that the protein is secreted protein, but not from micronemes. The protein participates in sporozoite invasion of host cells and is maybe involved in the growth of the parasite. These data have implications for the use of EtAMA1 or EtAMA1-interacting proteins as targets in intervention strategies against avian coccidiosis.

Which is the safer method of labor induction for oligohydramnios women? Transcervical double balloon catheter or dinoprostone vaginal insert.

OBJECTIVE: To compare the effectiveness and safety of two cervical ripening methods in term primiparous women with unfavorable cervices and oligohydramnios. METHODS: Women (126 cases) with oligohydramnios [amniotic fluid index (AFI) ≤5 cm] and a low Bishop Score (≤6) were assigned randomly to use double balloon catheter (mechanical method group, 67 cases) or dinoprostone 10 mg controlled-release vaginal insert (pharmacological method group, 59 cases) for induction of labor. The study's primary outcome was caesarean section rate (CSR). The secondary outcome measures included maternal and neonatal morbidity, an incremental changes in Bishop Score, and intrapartum interventions. RESULTS: There was no significant difference between the mechanical method group and the pharmacological method group in CSR and change in Bishop Score. Tacysystole, non-reassuring fetal heart patterns, and cases of newborn umbilical-cord arterial blood pH<7.1 were significantly lower with the mechanical method compared with the pharmacological method (p < 0.05). More patients needed additional intervention in the mechanical method group. CONCLUSIONS: Both methods resulted in a similar CSR. Double balloon catheter resulted in fewer labor complications, but more frequent use of oxytocin and amniotomy. Compared with dinoprostone vaginal insert, double balloon catheter use may be less problematic in women with oligohydramnios.