Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy.

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.

Marinobacter shengliensis subsp. alexandrii Subsp. Nov., Isolated from Cultivable Phycosphere Microbiota of Highly Toxic Dinoflagellate Alexandrium catenella LZT09 and Description of Marinobacter shengliensis Subsp. shengliensis Subsp. Nov.

Phycosphere hosts the boundary of unique holobionts harboring dynamic algae-bacteria interactions. During our investigating the microbial consortia composition of phycosphere microbiota (PM) derived from diverse harmful algal blooms (HAB) dinoflagellates, a novel rod-shaped, motile and faint yellow-pigmented bacterium, designated as strain LZ-6 T, was isolated from HAB Alexandrium catenella LZT09 which produces high levels paralytic shellfish poisoning toxins. Phylogenetic analysis based on 16S rRNA gene and two housekeeping genes, rpoA and pheS sequences showed that the novel isolate shared the highest gene similarity with Marinobacter shengliensis CGMCC 1.12758 T (99.6%) with the similarity values of 99.6%, 99.9% and 98.5%, respectively. Further phylogenomic calculations of average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values between strains LZ-6 T and the type strain of M. shengliensis were 95.9%, 96.4% and 68.5%, respectively. However, combined phenotypic and chemotaxonomic characterizations revealed that the new isolate was obviously different from the type strain of M. shengliensis. The obtained taxonomic evidences supported that strain LZ-6 T represents a novel subspecies of M. shengliensis, for which the name is proposed, Marinobacter shengliensis subsp. alexandrii subsp. nov. with the type strain LZ-6 T (= CCTCC AB 2018388TT = KCTC 72197 T). This proposal automatically creates Marinobacter shengliensis subsp. shengliensis for which the type strain is SL013A34A2T (= LMG 27740 T = CGMCC 1.12758 T).

Autophagy induced by monensin serves as a mechanism for programmed death in Eimeria tenella.

Monensin (Mon), the first ionophoric antibiotic has widely been used for the treatment and prevention of coccidiosis in poultry until recently, however, at present; its efficacy has been compromised with the emergence of many Mon-resistant strains. Knowledge of the mode of the action of anti-parasitic agents is as important as for other antimicrobials, especially for discovery and long term use of the existing drugs. However, little is known about anti-parasitic drug: monensin's, mechanism of action and physiological alteration in Eimeria tenella. In this study, we explored Mon effects on the viability of Mon-Sensitive GZ (MonS-GZ) and Mon-Resistant GZ (MonR-GZ) Eimeria tenella strains using trypan blue staining and investigated Mon-induced autophagy using Western blotting, indirect immunofluorescence assay, and transmission electron microscopy. The results showed that monensin leads to programmed death of E. tenella parasites by inducing autophagy as a mechanism of anticoccidial action. Mon-induced autophagy was indicated by the decreased sporozoites survival rate, ATG8 over expression and localization, and intracellular vacuolar structures and autophagosomes formation in MonS-GZ strain while in MonR-GZ strains autophagy pathway was not triggered. The autophagy inhibitor 3-methyladenine (3-MA) effectively blocked programmed cell death and saved the MonS-GZ sporozoites. These findings indicated that autophagy serves as a potentially important mechanism of E. tenella cell death in response to Mon and disruption of the autophagy pathway may lead to emergence of drug resistance against this anti-parasitic drug.

Occurrence and genotypes of Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. in household, shelter, breeding, and pet market dogs in Guangzhou, southern China.

Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. are common intestinal protozoans that infect humans and animals worldwide. A survey that assessed the prevalence, molecular characteristics, and zoonotic potential of these pathogens was conducted on a variety of dogs in Guangzhou, southern China. A total of 651 canine stool samples from household (n = 199), shelter (n = 149), breeding (n = 237), and pet market dogs (n = 66) were collected from eight districts in Guangzhou. Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. were detected by PCR amplification of the SSU rRNA gene. Giardia duodenalis-positive specimens were further assigned into assemblages using the glutamate dehydrogenase gene. Cryptosporidium spp., G. duodenalis, and Blastocystis sp. were found in 21 (3.2%), 20 (3.1%), and 35 (5.4%) samples, respectively. The overall prevalence of shelter dogs (40.28%, 60/149) was significantly higher than that of household (3.0%, 6/199), breeding (2.1%, 5/237), and pet market dogs (7.5%, 5/66) (χ2 = 154.72, df = 3, P < 0.001). Deworming in the past 12 months had a strong protective effect on the risk of contracting parasite infections (P < 0.001). No significant differences were detected between age or sex groups (P > 0.05). Dog-specific C. canis (n = 19) and zoonotic C. parvum (n = 2) were the only two Cryptosporidium species. Sequence analysis revealed the presence of three G. duodenalis assemblages: dog-specific assemblages D (n = 14) and C (n = 5), and cat-specific F (n = 1). Zoonotic Blastocystis ST3 (n = 28) was the dominant subtype, followed by ST1 (n = 6) and ST10 (n = 1). To our knowledge, this is the first large-scale investigation on the occurrence and molecular characteristics of Blastocystis sp. in dogs in China. Our results indicated that the dogs seemed to play a negligible role as reservoirs for Cryptosporidium spp. and G. duodenalis transmission to humans, but they are potential novel suitable hosts of Blastocystis sp. A strict sentinel surveillance system of dogs should be established to minimise the zoonotic risk of spreading blastocystosis among humans and dogs.

Melatonin Alleviates Neuroinflammation and Metabolic Disorder in DSS-Induced Depression Rats.

There is a bidirectional relationship between inflammatory bowel disease (IBD) and depression/anxiety. Emerging evidences indicate that the liver may be involved in microbiota-gut-brain axis. This experiment focused on the role of melatonin in regulating the gut microbiota and explores its mechanism on dextran sulphate sodium- (DSS-) induced neuroinflammation and liver injury. Long-term DSS-treatment increased lipopolysaccharide (LPS), proinflammation cytokines IL-1ß and TNF-α, and gut leak in rats, breaking blood-brain barrier and overactivated astrocytes and microglia. Ultimately, the rats showed depression-like behavior, including reduction of sucrose preference and central time in open field test and elevation of immobility time in a forced swimming test. Oral administration with melatonin alleviated neuroinflammation and depression-like behaviors. However, melatonin supplementation did not decrease the level of LPS but increase short-chain fatty acid (SCFA) production to protect DSS-induced neuroinflammation. Additionally, western blotting analysis suggested that signaling pathways farnesoid X receptor-fibroblast growth factor 15 (FXR-FGF 15) in gut and apoptosis signal-regulating kinase 1 (ASK1) in the liver overactivated in DSS-treated rats, indicating liver metabolic disorder. Supplementation with melatonin markedly inhibited the activation of these two signaling pathways and its downstream p38. As for the gut microbiota, we found that immune response- and SCFA production-related microbiota, like Lactobacillus and Clostridium significantly increased, while bile salt hydrolase activity-related microbiota, like Streptococcus and Enterococcus, significantly decreased after melatonin supplementation. These altered microbiota were consistent with the alleviation of neuroinflammation and metabolic disorder. Taken together, our findings suggest melatonin contributes to reshape gut microbiota and improves inflammatory processes in the hippocampus (HPC) and metabolic disorders in the liver of DSS rats.

Effects of Probiotics on Depressive or Anxiety Variables in Healthy Participants Under Stress Conditions or With a Depressive or Anxiety Diagnosis: A Meta-Analysis of Randomized Controlled Trials.

Background: Probiotics have been associated with the treatment of depression and anxiety. However, the results reported in the literature have been inconsistent, and no meta-analysis specifically reported probiotics used on participants with varying levels of emotional state. Methods: This meta-analysis aimed to study the effectiveness of probiotics on anxious or depressive symptomatology for participants under stress conditions or with a depressive or anxiety disorder diagnosis. Medline, PubMed, EMBASE, and the Cochrane Library were searched through December 2019 for randomized controlled trials (RCTs). The primary outcomes were depression and anxiety scores. Main inclusion criteria: RCTs of probiotics for participants with a mood or emotional disorder diagnosis or under stress situations; and all participants were adults (age ≥16 years); Assessed by the modified Jadad assessment scale found seven high-quality studies and three low-quality studies. Results: Ten clinical trials (n = 685 total participants) were included based on the inclusion and exclusion criteria. All studies were assessed as low or moderate risk of bias. The meta-analysis showed that probiotics could significantly reduce the depression scale for patients with anxiety and depression, and healthy participants under stress. However, there was no significant difference between the probiotics and placebo groups in the reduction of patient anxiety scores, even if they are depressive or anxious patients or healthy participants under stress. Subgroup analysis revealed that probiotics had significant effect on depressive symptoms just in patients with depression, and no significant change in anxiety in patients, and no improvement in participant performance under stress. Conclusions: Probiotics could alleviate depressive symptoms in patients with a depression diagnosis or depression scores also in anxiety disorder diagnosis, and suggesting that probiotics may be adjunct therapies for mood or emotional disorders. Therefore, it is essential that probiotics could be more involved in the treatment of patients with depression in the future. The evidence of probiotics successfully treating depression is still insufficient, and more high-quality studies on patients with depression are still needed.

The effect of autophagy on the survival and invasive activity of Eimeria tenella sporozoites.

Autophagy is a cellular process that is vital for the maintenance of homeostasis in eukaryotic cells. Currently, autophagy-related genes (atgs) in the Eimeria tenella genome database have been reported, but very little is known about the effects of autophagy on the survival and invasive activity of this protozoan. In this study, we investigated the autophagy in E. tenella sporozoites under starvation and autophagy-modulators treatments and evaluated the autophagy influence on cellular adenosine triphosphate (ATP) levels, the survival rate and the invasive activity of the sporozoites. The results showed that the autophagy could be induced in the sporozoites by starvation or inducer rapamycin (RP), but it could be inhibited by 3-methyladenine (3-MA) treatment. The sporozoites after starvation and RP-treatment displayed punctate signals of EtATG8 and formed autophagosomes. The survival rate of the sporozoites under starvation was significantly lower than that in the control group, whereas the ATP levels in sporozoite were far greater than those in the control. The quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) showed that the invasive activity of the sporozoites was up- and down-regulated by RP and 3-MA induction, respectively. Our results indicate that autophagy has effects on the survival and invasive activity of E. tenella sporozoites, which may provide new insights into anti-coccidial drugs.

Molecular epidemiology of Cryptosporidium spp. in dairy cattle in Guangdong Province, South China.

To determine the prevalence of Cryptosporidium in dairy cattle in Guangdong Province, South China, 1440 fecal samples were collected from 10 farms and screened for Cryptosporidium with PCR. The overall prevalence of Cryptosporidium was 4.38% (63/1440), and the infection rates in preweaned calves, postweaned calves, heifers and adults were 6.4% (19/297), 6.19% (33/533), 1.48% (4/271) and 2.06% (7/339), respectively. Three Cryptosporidium species, Cryptosporidium andersoni (n = 33), Cryptosporidium bovis (n = 22) and Cryptosporidium ryanae (n = 8) were detected by DNA sequence analysis of the 63 positive samples, and C. andersoni was identified as the most common species on the dairy cattle farms. In preweaned calves, C. bovis was the most prevalent species (9/19, 47.4%). In contrast, C. andersoni was the predominant species (19/33, 57.6%) in postweaned calves and the only species found in heifers and adults. The zoonotic species Cryptosporidium parvum was not detected in this study. Twenty-four C. andersoni isolates were successfully classified into three multilocus sequence typing (MLST) subtypes. MLST subtype A4,A4,A4,A1 was the predominant subtype, and MLST subtype A2,A5,A2,A1, previously found in sheep, was detected in cattle for the first time. A linkage disequilibrium analysis showed that the C. andersoni isolates had a clonal genetic population structure. However, further molecular studies are required to better understand the epidemiology of Cryptosporidium in Guangdong.

Genetic characteristics and geographic segregation of Giardia duodenalis in dairy cattle from Guangdong Province, southern China.

Giardia duodenalis is a gastrointestinal parasitic protozoan with important veterinary and public health implications worldwide. In this study, a total of 1440 fecal specimens were collected from dairy calves in Guangdong Province, China. The overall prevalence of G. duodenalis was 2.2% (31/1440) by polymerase chain reaction (PCR). The prevalence was 8.4% and 1.1% in pre- and post-weaned calves, respectively. Both assemblages A (6.5%, 2/31) and E (93.5%, 29/31) were found based on sequence analysis of the SSU rRNA gene. All G. duodenalis-positive samples were assayed with PCR followed by sequencing the beta giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes, and 20, 23, and 18 sequences were obtained, respectively. Multilocus genotyping yielded 10 assemblage E multilocus genotypes (MLGs). All assemblage E MLGs were genetically distinct from those obtained in cattle from Henan, Xinjiang, Shanghai, and Shaanxi in China. This finding was also supported by FST, Nm, and median-joining network analysis. Results of this study indicated that G. duodenalis was found in low prevalence in dairy calves in Guangdong Province, southern China; nevertheless, the infected calves might represent a public health risk in this region. Additionally, the differences in assemblage E MLGs from the five analyzed regions revealed significant geographic segregation of G. duodenalis in China.