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Abstract 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

Abstract Background The gastrointestinal epithelium plays an important role in directing recognition by the immune system, and epithelial cells provide the host's front line of defense against microorganisms. However, it is difficult to cultivate avian intestinal epithelial cells in vitro for lengthy periods, and the lack of available cell lines limits the research on avian intestinal diseases and nutritional regulation. Chicken coccidiosis is a serious intestinal disease that causes significant economic losses in the poultry industry. In vitro, some cell line models are beneficial for the development of Eimeria species; however, only partial reproduction can be achieved. Therefore, we sought to develop a new model with both the natural host and epithelial cell phenotypes. Methods In this study, we use the SV40 large T antigen (SV40T) gene to generate an immortalized cell line. Single-cell screening technology was used to sort positive cell clusters with epithelial characteristics for passage. Polymerase chain reaction (PCR) identification, immunofluorescence detection, and bulk RNA sequencing analysis and validation were used to check the expression of epithelial cell markers and characterize the avian intestinal epithelial cell line (AIEC). AIECs were infected with sporozoites, and their ability to support the in vitro endogenous development of Eimeria tenella was assessed. Results This novel AIEC consistently expressed intestinal epithelial markers. Transcriptome assays revealed the upregulation of genes associated with proliferation and downregulation of genes associated with apoptosis. We sought to compare E. tenella infection between an existing fibroblast cell line (DF-1) and several passages of AIEC and found that the invasion efficiency was significantly increased relative to that of chicken fibroblast cell lines. Conclusions An AIEC will serve as a better in vitro research model, especially in the study of Eimeria species development and the mechanisms of parasite–host interactions. Using AIEC helps us understand the involvement of intestinal epithelial cells in the digestive tract and the immune defense of the chickens, which will contribute to the epithelial innate defense against microbial infection in the gastrointestinal tract. Graphical Abstract

Avian coccidiosis, a parasitic disease prevalent in poultry, is caused by Eimeria species and leads to significant economic losses. The use of attenuated live oocyst vaccines has been adopted as an alternative to the use of anticoccidial drugs. However, the accurate detection and differentiation of vaccine strains from virulent ones remain challenging. Therefore, this study presents a novel TaqMan polymerase chain reaction (PCR) detection method that offers enhanced sensitivity, specificity, and reproducibility compared with traditional PCR techniques. Through whole-genome resequencing and bioinformatics analysis, we identified a molecular marker gene, Em_marker6, with a unique 21-base pair deletion specific to the Eimeria maxima attenuated vaccine strain. Optimized primers and probes targeting this marker enabled rapid quantification cycle value achievement and high fluorescence intensity. The standard curve’s slope of −3.540 and correlation coefficient of 0.9971 confirmed precise quantification capabilities. The TaqMan PCR method detected as few as 30 plasmid DNA copies and 50 oocysts per reaction, outperforming traditional PCR techniques by an order of magnitude. No cross-reactivity was observed with other E. maxima wide-type strains or common intestinal pathogens, ensuring the exclusive detection of the E. maxima EMPY vaccine strain. Weekly testing over 3 weeks demonstrated minimal variability, indicating robust consistency in the method’s application. Testing on 61 clinical samples revealed a 57.38% positivity rate for E. maxima species and 13.11% for the vaccine strain. The Em_marker6 gene exhibited genetic stability across multiple generations, confirming the detection method’s robust stability for the attenuated E. maxima vaccine strain. This study significantly advances the field of avian coccidiosis research and control by providing a valuable tool for monitoring vaccine purity and preventing inadvertent infections in vaccinated flocks, aligning with global efforts to curb antibiotic use in animal feed.

Trichomonas gallinae is a protozoa that parasitizes the upper gastrointestinal and respiratory tracts of various animals and birds, including Columbidae, Passeriformes, and Falconiformes. Polymerase chain reaction-based T. gallinae ITS1/5.8S/ITS2 gene typing yields inconsistent results owing to methodological differences. To standardize the statistical analysis of T. gallinae genotype distributions, this study employed MEGA-X software with the Tamamura 3-parameter (T92) + G model in the neighbor-joining method, with 2,000 bootstrap replicates, to calculate a systematic evolutionary tree. The resulting tree comprised 12 branches, ITS-OBT-Tg-1 to ITS-OBT-Tgl, with similar phylogenetic relationships. Relevant literature review yielded T. gallinae prevalence data in Columbidae. Statistical analysis was conducted from two perspectives: non-biological and biological factors, using chi-square tests and ordered logistic regression analysis. T. gallinae positivity rates differed significantly across diverse regions (χ2 = 4,609.9, P = 0.000, df = 4) and at various times (χ2 = 2,810.8, P = 0.000, df = 3). However, temperature and precipitation did not significantly affect T. gallinae positivity rates. Additionally, T. gallinae positivity rates differed significantly among diverse hosts (χ2 = 2,958.6, P = 0.000, df = 14) and by host age (χ2 = 478.5, P = 0.000, df = 2) and sex (χ2 = 96.00, P = 0.000, df = 1). This comprehensive analysis aimed to control T. gallinae transmission, reduce economic and species resource losses, and provide a foundation for future related research.

Coccidiosis is a costly intestinal disease of chickens caused by Eimeria species. This infection is associated with high mortality, reduced feed efficiency, and slowed body weight gain. The diagnosis and control of coccidiosis becomes challenging due to the fact that chickens can be infected by seven different Eimeria species and often occur mixed-species co-infections. Grasping the epidemiology of Eimeria species is crucial to estimate the efficiency of poultry management. This study aimed to explore the distribution of Eimeria species in broiler chickens in China after administering live anticoccidial vaccines. A total of 634 samples were obtained, and the survey results showed that the prevalence of Eimeria was 86.12% (546/634), and the most common species were E. acervulina (65.62%), E. necatrix (50.95%), E. mitis (50.79%), E. tenella (48.42%), and E. praecox (41.80%). Most samples indicated mixed-species infections (an average of 3.29 species per positive sample). Notably, 63.98% of samples contain 3 to 5 Eimeria species within a single fecal sample. The most prevalent combinations were E. acervulina–E. tenella (38.96%) and E. acervulina–E. necatrix (37.22%). Statistical analysis showed that flocks vaccinated with trivalent vaccines were significantly positive for E. necatrix in grower chickens (OR = 3.30, p

Avian trichomoniasis, caused by the protozoan parasite Trichomonas gallinae, is a prevalent and economically significant disease in pigeons. This study investigated the drug resistance of T. gallinae isolates in Guangdong Province, China. The results revealed that 25.3% (20/79) of the isolates were resistant to one or more of the four nitroimidazole drugs tested, namely, metronidazole, dimetridazole, secnidazole, and tinidazole. Secnidazole elicited the highest resistance rate (19.0%; 15/79), followed by tinidazole (17.7%; 14/79), metronidazole (17.7%; 14/79), and dimetridazole (13.9%; 11/79). An enormous majority of the resistant isolates (70.0%; 14/20) exhibited resistance to multiple drugs. Additionally, the resistance rate was significantly higher in isolates from birds aged < 30 days (53.3%; 8/15) than in those from older birds (23.1%; 12/52). Moreover, no drug resistance was detected in female pigeons. The genotype of the isolated strain was also associated with drug resistance. Specifically, 50.0% (15/30) of ITS-B genotypes exhibited resistance to drugs, while only 10.2% (5/49) of ITS-A genotypes demonstrated resistance. This study also found the growth characteristics of different Trichomonas isolates to be influenced by their genotypes and initial inoculum concentrations. These findings underscore the urgent need for effective measures to control and prevent drug-resistant T. gallinae infections in pigeons, thus ensuring the stable development of the pigeon industry.

Parasitic protozoa are single-celled organisms that have adapted to live in cells of humans and animals. The protozoan parasites include Leishmania spp., Trypanosoma spp., Plasmodium spp., Toxoplasma gondii, Cryptosporidium spp., and Eimeria spp., which can cause protozoal diseases that seriously endanger human and animal health and cause huge economic losses in the livestock industry. Parasite development and reproduction after invasion of the host require a large number of purine nucleotides, and the corresponding purine bases are catalyzed by purine phosphoribosyl transferase to generate the corresponding purine nucleotides. Purine phosphoribosyltransferases (PPRT) are important metabolic enzymes involved in the ribophosphorylation of purine bases and are present in many protozoan parasites. Adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) belong to PPRT. APRT and HGXPRT convert adenine, hypoxanthine, guanine, and xanthine into adenosine-5'-monophosphate (AMP), inosine-5 '-monophosphate (IMP), guanosine-5'-monophosphate (GMP), and xanthosine- 5'-monophosphate (XMP), respectively. Purine nucleotides are involved in many functions as components of DNA and RNA, as enzyme cofactors in metabolic pathways, as sources of energy in protozoan parasites. Purine phosphoribosyltransferases are key enzyme in the purine salvage pathway of parasitic protozoa, which is significantly different from the de novo synthesis pathway of the host. In recent years, purine phosphoribosyltransferases have become the interesting research hotspot as an antiparasitic drug candidates target, with major progress in the screening of compounds against protozoan parasites. This review focuses on the basic characteristics, biological functions, inhibitors screening and application of purine phosphoribosyltransferases in parasitic protozoa of Leishmania spp., Trypanosoma spp., Plasmodium spp., and Toxoplasma gondii, so as to provide reference for the research of drug targets and the screening of new inhibitors against parasitic protozoa.

Abstract 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.

Marine symbiotic and epiphyte microorganisms are sources of bioactive or structurally novel natural products. Metabolic blockade-based genome mining has been proven to be an effective strategy to accelerate the discovery of natural products from both terrestrial and marine microorganisms. Here, the metabolic blockade-based genome mining strategy was applied to the discovery of other metabolites in a sea anemone-associated Streptomyces sp. S1502. We constructed a mutant Streptomyces sp. S1502/Δstp1 that switched to producing the atypical angucyclines WS-5995 A–E, among which WS-5995 E is a new compound. A biosynthetic gene cluster (wsm) of the angucyclines was identified through gene knock-out and heterologous expression studies. The biosynthetic pathways of WS-5995 A–E were proposed, the roles of some tailoring and regulatory genes were investigated, and the biological activities of WS-5995 A–E were evaluated. WS-5995 A has significant anti-Eimeria tenell activity with an IC50 value of 2.21 μM. The production of antibacterial streptopyrroles and anticoccidial WS-5995 A–E may play a protective role in the mutual relationship between Streptomyces sp. S1502 and its host.

This is a paper in the field of metallurgical engineering. In this paper, natural clay mineral serpentine was subjected to mechanochemical activation (MA) for the highly-efficient removal of Ni (Ⅱ) in wastewater and simultaneous recovery of metal nickel as secondary resources. The effectiveness of serpentine for precipitation and recovery of Ni was investigated, with the process mechanism analyzed. The results showed MA of serpentine led to the disordering of the stable crystal structure of serpentine, thereby resulting in the release of large amounts of Mg2+ ions and hydroxyl group in aqueous solution, and the resultant interaction between Ni (Ⅱ) and OH-, with the formation of amorphous Ni(OH)2 as sediments. The maximum processing capacity of activated serpentine for Ni (Ⅱ) reached up to 226.04 mg/g, meanwhile the final Ni (Ⅱ) concentration met up with the national discharging standard, indicating the feasibility of remediation of heavy metal contaminations by a very low dosage of activated serpentine. Concluded from these results, mechanochemical activation can be expected to boost the reactivity of natural serpentine, thereby improving the utilization efficiency of low-cost serpentine for the clean-up of environment pollutions and recovery of metal resources, which is conducive to promoting the green development of mineral-resource-environment cross-fields.

Abstract Background Necrotic enteritis (NE) is an infectious intestinal disease caused by Clostridium perfringens (C. perfringens) that is now re-emerging and causing concern within the poultry industry. Previously, the supplementation of antibiotics in feed was the most popular control strategy against C. perfringens. However, with the ban on supplementing growth-promoting antibiotics in livestock feed, alternatives to antibiotics will be essential in order to control necrotic enteritis. A possible alternative to antibiotics could be the medium or long chain fatty acids (MCFA or LCFA) as these are able to destroy cell membranes which in turn results in the death of bacteria. In this study, the in vitro antimicrobial activity of different combinations with microencapsulated caprylic acid (C8: 0), capric acid (C10: 0), lauric acid (C12: 0) and myristic acid (C14: 0) against C. perfringens and in vivo control the NE-inducing C. perfringens in broiler chicken were analyzed. Results The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) assay results revealed that three different combinations of medium/long chain fatty acids varied in antimicrobial activities against C. perfringens type A strain (CVCC52, quality control), C. perfringens type A strain (C8-1), C. perfringens type G strain (D25) and C. perfringens type G strain (MZ1). Specifically, combination of C12: 0 and C14: 0 (C12–14) showed the highest antimicrobial activity against the four strains of C. perfringens (MIC ≤ 12.5 μg/mL, MBC = 50 μg/mL), followed by the combination of C10: 0 and C12: 0 (C10–12) (MIC, MBC ≤ 50 μg/mL). The in vivo study, 189 of 818-crossbred chickens that were fed a wheat-based diet and randomly divided into nine groups, with six treatment groups supplemented with either a high dose (1 g/kg) or low dose (0.5 g/kg) of three combinations respectively. The remaining three groups comsisted of a positive group supplement with avilamycin (0.01 g/kg), an infected control and an uninfected control. All chickens were challenged with C. perfringens from day 14 to day 17, except those in the uninfected control group. On day 20, the duodenum and jejunum necrotic lesions scores were calculated and the results showed that there was significant decrease in the C12–C14 high dose group (1.43 ± 0.23, 0.48 ± 0.13) and the C10–12 high dose group (1.52 ± 0.19, 0.48 ± 0.11) compared to the infected group (2.86 ± 0.21, 1.20 ± 0.28). Conclusions This finding indicated that dietary microencapsulated C12–C14 and C10–C12 could inhibit the growth of C. perfringens in chickens, which proves is viability to serve as an alternative to antibiotics used for necrotic enteritis caused by C. perfringens.

Eimeria tenella (E. tenella) is a protozoal parasite that can cause severe cecal lesions and death in chickens, seriously harming the chicken industry. Conventional control strategies mainly rely on anticoccidial drugs. However, the emerging problems of anticoccidial resistance and drug residues necessitate exploring potential drug targets for developing new anticoccidial drugs. Fructose-1,6-bisphosphate aldolase (ALD) is an essential enzyme for parasite energy metabolism that has been considered a potential drug target. In this study, we analyzed the molecular and biochemical properties of E. tenella ALD2 (EtALD2). EtALD2 mRNA expression was highest in second-generation merozoites, whereas the protein level was highest in unsporulated oocysts. Indirect immunofluorescence showed that EtALD2 was mainly distributed in sporozoite’ cytoplasm. The natural product inhibitor (morin) was screened by computer-aided drug screening. Enzyme kinetic and inhibition kinetic assays showed that morin had a good inhibitory effect on EtALD2 activity (IC50 = 10.37 μM, Ki = 48.97 μM). In vitro inhibition assay demonstrated that morin had an inhibitory effect on E. tenella development, with an IC50 value of 3.98 μM and drug selection index of 177.49. In vivo, morin significantly improved cecal lesions (p

Abstract Background Cryptosporidium is one of the most prevalent parasites infecting both birds and mammals. To examine the prevalence of Cryptosporidium species and evaluate the public health significance of domestic chickens in Guangdong Province, southern China, we analyzed 1001 fecal samples from 43 intensive broiler chicken farms across six distinct geographical regions. Methods Individual DNA samples were subjected to nested PCR-based amplification and sequencing of the small subunit of the nuclear ribosomal RNA gene (SSU rRNA). Analysis of the 60 kDa glycoprotein gene (gp60) was performed to characterize the subtypes of C. meleagridis. Results The overall prevalence of Cryptosporidium was 13.2% (95% CI 11.1–15.3) (24 of 43 farms), with C. meleagridis (7.8%), C. baileyi (4.8%) and mixed infections (0.6%). Using the gp60 gene, three subtype families, IIIb, IIIe and IIIg, were identified, including six subtypes: one novel (IIIgA25G3R1a) and five previously reported (IIIbA23G1R1c, IIIbA24G1R1, IIIbA21G1R1a, IIIeA17G2R1 and IIIeA26G2R1). Within these subtypes, five known subtypes were genetically identical to those identified in humans. Conclusions This is the first report of C. meleagridis in chickens from Guangdong. The frequent occurrence of C. meleagridis in domestic chickens and the common C. meleagridis subtypes identified in both humans and chickens is of public health significance. Our study indicates that broiler chickens represent a potential zoonotic risk for the transmission of Cryptosporidium in this region. Graphical Abstract

Enterocytozoon bieneusi is a common pathogen in a broad range of vertebrate hosts. To assess the prevalence and genotypes of E. bieneusi in farmed masked palm civet (Paguma larvata), 537 fecal samples from seven provinces in China were tested by nested PCR of the polymorphic internal transcribed spacer (ITS) region. Among of all the samples, 60.0% (325/531) were positive for E. bieneusi, with the highest prevalence in Hebei province (85.0%). Sequence analysis revealed the presence of nine E. bieneusi genotypes, including four known genotypes (SHR1, PL2, PL4, CHG19) and five novel genotypes (PL12 to PL16). Genotypes SHR1 and PL2 were the most common genotypes in seven provinces. Phylogenetic analysis showed that three genotypes (CHG19, PL4 and PL16) were distributed to Group 1, and six genotypes (SHR1, PL2, PL12, PL13, PL14 and PL15) formed a novel clade, which was named group 12. Findings highlight the need to conduct additional research to elucidate the epidemiology of E. bieneusi in farmed masked palm civet.

Abstract Background Poultry necrotic enteritis (NE) is an economically important disease caused by C. perfringens. The disease causing ability of this bacterium is linked with the production of a wide variety of toxins. Among them, necrotic enteritis B-like (NetB) toxin is reported to be involved in the pathogenesis of NE; in addition there is some circumstantial evidence that tpeL toxin may enhance virulence, but this is yet to be definitely shown. The situation becomes more complicated in the presence of a number of predisposing factors like co-infection with coccidia, type of diet and use of high protein diet. These co-factors alter the intestinal environment, thereby favoring the production of more toxins, leading to a more severe disease. The objective of this study was to develop a successful animal model that would induce clinical signs and lesions of NE using C. perfringens type G strains obtained from field outbreaks. A separate trial was simultaneously considered to establish the role of dietary factor with coccidial co-infection in NE. Results The results have shown that use of net-B positive C. perfringens without predisposing factors induce moderate to severe NE (Av. Lesion score 1.79 ± 1.50). In a separate trial, addition of fish meal to a feed of C. perfringens challenged birds produced higher number of NE cases (Av. Lesion score 2.17 ± 1.28). However, use of less virulent E. necatrix strain along with fish meal in conjunction with net-B positive strain did not alter the severity of NE lesions in specific pathogen free chicken (Av. Lesion score 2.21 ± 1.13). Conclusions This study suggests that virulent C. perfringens type G strains can induce NE lesions in the absence of other predisposing factors. Birds in the clostridia challenged group showed moderate to severe NE lesions. Use of less virulent coccidia strain contributed to a lesser extent in increasing the severity of disease. Maize based diet along with fishmeal (1:1) increased the severity of lesions but statistically it was non-significant. The NE lesions in all experimental groups were found to be present more frequently in the duodenum. In this way, this study provided an effective model for in vivo production of NE in poultry birds.

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.

Coccidiosis is an intestinal disease of poultry that endangers animal health and causes economic loss to poultry production. For decades, the control of the coccidiosis relies mainly on anticoccidial drugs and live vaccines. However, problems such as widespread of drug resistance, public health pressure after mass administration of anticoccidial drugs, and the risk of expansion of coccidia after administration of live avian coccidiosis vaccine make new anticoccidial alternatives urgent for health development of avian industry. A number of new vaccines have emerged recently, including recombinant vaccines, DNA vaccines and genetically engineered live vaccines. Additionally, various adjuvants, such as DNA adjuvants, recombinant adjuvants, plant-derived adjuvants, probiotics and prebiotics have been evaluated in animal trials. In this article, an overview of the host immune response to avian coccidia is presented, and current vaccine and adjuvant control strategies are outlined as well. It is hoped that this review will contribute to a deeper understanding of avian coccidiosis prevention and control strategies and lay a theoretical foundation in the field.

Avian coccidiosis is an important intestinal protozoan disease that has caused major economic losses to the poultry industry. Clostridium butyricum can not only maintain the stability of the intestinal barrier, but can also improve the production performance of broiler chickens. We studied the effects of feeding C. butyricum alone, administration of coccidiosis vaccine alone, and the combined administration of C. butyricum and coccidiosis vaccine on body weight gain, feed consumption, and feed conversion ratio of broilers. Meanwhile, intestinal contents of 8- and 15-day-old broilers were collected, and their intestinal microbiome was characterized by high-throughput sequencing of the V3–V4 region of 16S rDNA. We analyzed the oocysts per gram values and lesion scores in the C. butyricum alone group, in a group challenged with the coccidiosis-causing parasite, Eimeria, and in groups simultaneously challenged Eimeria and pretreated with C. butyricum, the coccidiosis vaccine, or combined C. butyricum and coccidiosis vaccine. Intestinal tissue samples were collected from 32-day-old broilers for microbiome analysis. Our results showed that combination of C. butyricum with coccidiosis vaccine significantly improved the performance of broiler chickens and also significantly reduced the oocysts per gram value and intestinal lesions caused by Eimeria sp. infection. Furthermore, C. butyricum and coccidiosis vaccine administered alone or in combination significantly increased the relative abundance of the immune biomarker genus Barnesiella. The significant increase in the abundance of the Clostridia_UCG.014, Eubacterium coprostanoligenes group and Bacteroides was a key factor in controlling Eimeria sp. infection.

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.

Abstract Background Enterocytozoon bieneusi, a common opportunistic fungal pathogen, has a wide range of hosts. Limited epidemiological data on E. bieneusi intestinal infections in companion animals (dogs and cats) in China exists. In this study, fecal samples (651 from dogs and 389 from cats) in Guangzhou city, Guangdong Province, China, were collected, and the ribosomal internal transcribed (ITS) spacer region from the DNA extracted from them was Polymerase Chain Reaction (PCR)-amplified and sequenced. Results Based on the sequencing data, E. bieneusi was identified in the fecal samples collected from 149 (22.9%) and 79 (20.3%) dogs and cats. Of the factors investigated, poor living conditions appeared to be the major risk factor for contracting the pathogen. Eleven E. bieneusi genotypes, six known (PtEb IX, GD1, D, CD9, EbpC, I) and five novel (designated here as GD2– GD6), were found in dogs. Eight genotypes, six known (PtEb IX, GD1, D, CD9, EbpC, Type IV) and two novel (GD2 and GC1), were identified in cats. Genotype PtEb IX was most common in both dogs and cats, followed by genotype GD1. Conclusions Although PtEb IX was the most common E. bieneusi genotype in dogs, this is the first report of this genotype dominating in cats. The same genotype distribution of the pathogen between the two different companion animals species in the same geographic area indicates that inter-species transmission is probable. The widespread existence of zoonotic E. bieneusi genotypes (D, EbpC, Type IV) in companion animals indicates that they are potential sources of environmental contamination and infections in humans.

Background/Aims: Lung cancer continues to be the leading cause of cancer related deaths worldwide due to its high incidence, malignant behavior and lack of major advancements in treatment strategy. The occurrence and development of lung cancer is closely related to inflammation. Thus, we conducted the present study to investigate the effects of IL-35 (Interleukin 35), a newly identified anti-inflammatory factor, on non-small cell lung cancer (NSCLC), which accounts for about 85% of all lung cancers. Methods: We first evaluated the IL-35 expression in 384 pairs of NSCLC samples and their adjacent normal mucosa by realtime PCR, ELISA (Enzyme-linked immunoassay) and tissue microarrays. Then the role of IL-35 on patient survival rates, cancer progression and their sensitivity to chemotherapy drugs were assessed. Results: IL-35 was barely expressed in the NSCLC tissues but highly expressed in the adjacent normal tissues. The down-regulation of IL-35 was significantly correlated with the results of American Joint Committee on Cancer stage, differentiation and it was also shown to be an independent prognostic indicator of disease-free survival and overall survival for patients with NSCLC. Overexpression of IL-35 in NSCLC cells suppressed cell migration, invasion, proliferation, colony formation through suppressing β-catenin. IL-35 inhibited NSCLC formation in the mice model and sensitize the cancer cells to chemotherapy drugs. Conclusion: Our results showed that IL-35 plays an inhibitory role in NSCLC development and function as a novel prognostic indicator and a potential therapeutic target.

Endoplasmic reticulum (ER) is a kind of organelle with multiple functions including protein synthesis, modification and folding, calcium storage, and lipid synthesis. Under stress conditions, ER homeostasis is disrupted, which is defined as ER stress (ERS). The accumulation of unfolded proteins in the ER triggers a stable signaling network named unfolded protein response (UPR). Hydrogen sulfide is an important signal molecule regulating various physiological and pathological processes. Recent studies have shown that H2S plays an important role in many diseases by affecting ERS, but its mechanism, especially the signaling pathways, is not fully understood. Therefore, in this review, we summarize the recent studies about the signaling pathways involved in the effects of H2S on ERS in diseases to provide theoretical reference for the related in-depth researches.

Abstract Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.

The gastrointestinal epithelium guides the immune system to differentiate between commensal and pathogenic microbiota, which relies on intimate links with the type I IFN signal pathway. Epithelial cells along the epithelium provide the front line of host defense against pathogen infection in the gastrointestinal tract. Increasing evidence supports the regulatory potential of long noncoding RNAs (lncRNAs) in immune defense but their role in regulating intestinal epithelial antimicrobial responses is still unclear. Cryptosporidium, a protozoan parasite that infects intestinal epithelial cells, is an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children in developing countries. Recent advances in Cryptosporidium research have revealed a strong type I IFN response in infected intestinal epithelial cells. We previously identified a panel of host cell lncRNAs that are upregulated in murine intestinal epithelial cells following microbial challenge. One of these lncRNAs, NR_033736, is upregulated in intestinal epithelial cells following Cryptosporidium infection and displays a significant suppressive effect on type I IFN-controlled gene transcription in infected host cells. NR_033736 can be assembled into the ISGF3 complex and suppresses type I IFN-mediated gene transcription. Interestingly, upregulation of NR_033736 itself is triggered by the type I IFN signaling. Moreover, NR_033736 modulates epithelial anti-Cryptosporidium defense. Our data suggest that upregulation of NR_033736 provides negative feedback regulation of type I IFN signaling through suppression of type I IFN-controlled gene transcription, and consequently, contributing to fine-tuning of epithelial innate defense against microbial infection.

Coal and gas outbursts mostly occur during mining at geostructural belts. Pre-drainage coalbed methane using hydraulic fracturing is one of the methods to prevent outbursts. However, the coal in geostructural belts is to be soft and crushed with special mechanical properties and pore structure. To explore the feasibility of hydraulic fracturing in geostructural belts, a field investigation on enhanced coalbed methane using hydraulic fracturing with vertical well was conducted at the Yangquan Coalfield, China. This case puts forward a method for the location selection of vertical well in geostructural belts. In addition, a triple-control technology for hydraulic fracturing, which is characterized by pressure control, flow control and sand ratio control of fracturing fluid, is presented. The results show that the average gas production and maximum gas drainage capacity of the test well were 5.67 and 12.88 times than those of the regular well, respectively, achieving good drainage effects.

Clostridium perfringens poses a serious threat to small ruminants by causing moderate to severe enterotoxaemia. Due to its ability to produce a wide arsenal of toxins, it is ranked among the most prevalent and important pathogens in livestock. This study focused on the molecular characterization of different Clostridium perfringens types along with their antimicrobial resistance profile. An overall higher prevalence of C. perfringens (46.1%) was detected based on mPCR among sheep and goats (healthy and diseased) in the Punjab province, Pakistan. The majority of the isolates were characterized as type A (82%), followed by type D (18%). Among the isolates from diseased sheep and goats, 27% were positive for cpa, 49% for cpa and cpb2, 9% for cpa and etx, 15% for cpa, cpb2 and etx. In the case of isolates from healthy sheep and goats, 59% were positive for cpa, 34% for cpb2 and cpa, 4% for cpa and etx, and 3% for cpa, cpb2 and etx. The prevalence of the beta2 toxin gene in the diseased sheep and goat population was 64% as compared to 37% in healthy animals. All 184 isolates (100%) were sensitive to rifampin and ceftiofur; the majority (57%) was sensitive to teicoplanin, chloramphenicol, amoxicillin, linezolid and enrofloxacin. A lower proportion of isolates (43%) were sensitive to ciprofloxacin and only 14% were susceptible to erythromycin. The findings of this study highlight the higher prevalence of C. perfringens in small ruminants and indicate that detailed pathogenesis studies are necessary to understand the explicit role of various toxins in causing enteric infections in sheep and goats including how they might be exploited to develop vaccines against these diseases.

Cryptosporidium parvum is a zoonotic intracellular protozoan responsible for the diarrheal illness cryptosporidiosis in humans and animals. Although a number of zoite surface proteins are known to be expressed during, and believed to be involved in, attachment and invasion of host cells, the molecular mechanisms by which C. parvum invades the host epithelial cells are not well understood. In the present study, we investigated the gene expression patterns, protein localization in developmental stages in culture, and in vitro neutralization characteristics of Cpgp40/15 and Cpgp40. Indirect immunofluorescence assay showed that Cpgp40/15 is associated with the parasitophorous vacuole membrane (PVM) during intracellular development. Both anti-gp40/15 and anti-gp40 antibodies demonstrated the ability to neutralize C. parvum infection in vitro. Further studies are needed to fully understand the specific role and functional mechanism of Cpgp40/15 (or gp40/15 complex) in the invasion of the host or in the PVM and to determine the feasibility of gp40/15 as a vaccine candidate for cryptosporidiosis in vivo.