Trichinella spiralis -induced immunomodulation signatures on gut microbiota and metabolic pathways in mice.

The hygiene hypothesis proposes that decreased exposure to infectious agents in developed countries may contribute to the development of allergic and autoimmune diseases. Trichinella spiralis, a parasitic roundworm, causes trichinellosis, also known as trichinosis, in humans. T. spiralis had many hosts, and almost any mammal could become infected. Adult worms lived in the small intestine, while the larvae lived in muscle cells of the same mammal. T. spiralis was a significant public health threat because it could cause severe illness and even death in humans who eat undercooked or raw meat containing the parasite. The complex interactions between gastrointestinal helminths, gut microbiota, and the host immune system present a challenge for researchers. Two groups of mice were infected with T. spiralis vs uninfected control, and the experiment was conducted over 60 days. The 16S rRNA gene sequences and untargeted LC/MS-based metabolomics of fecal and serum samples, respectively, from different stages of development of the Trichinella spiralis-mouse model, were examined in this study. Gut microbiota alterations and metabolic activity accompanied by parasite-induced immunomodulation were detected. The inflammation parameters of the duodenum (villus/crypt ratio, goblet cell number and size, and histological score) were involved in active inflammation and oxidative metabolite profiles. These profiles included increased biosynthesis of phenylalanine, tyrosine, and tryptophan while decreasing cholesterol metabolism and primary and secondary bile acid biosynthesis. These disrupted metabolisms adapted to infection stress during the enteral and parenteral phases and then return to homeostasis during the encapsulated phase. There was a shift from an abundance of Bacteroides in the parenteral phase to an abundance of probiotic Lactobacillus and Treg-associated-Clostridia in the encapsulated phase. Th2 immune response (IL-4/IL-5/IL-13), lamina propria Treg, and immune hyporesponsiveness metabolic pathways (decreased tropane, piperidine and pyridine alkaloid biosynthesis and biosynthesis of alkaloids derived from ornithine, lysine, and nicotinic acid) were all altered. These findings enhanced our understanding of gut microbiota and metabolic profiles of Trichinella -infected mice, which could be a driving force in parasite-shaping immune system maintenance.

Efficacy of liver free and Chitosan against Eimeria tenella in chickens.

Eimeria spp. are the pathogen that causes coccidiosis, a significant disease that affects intensively reared livestock, especially poultry. Anticoccidial feed additives, chemicals, and ionophores have routinely been employed to reduce Eimeria infections in broiler production. Therefore, the shift to antibiotic-free and organic farming necessitates novel coccidiosis preventive strategies. The present study evaluated the effects of potential feed additives, liver free and chitosan, against Eimeria tenella infection in White Leghorn broiler female chickens. One hundred sixty-five 1-day-old White Leghorn broiler female chicks were divided into 11 groups (15 female chicks per group), including the positive control group (G1), the negative control group (G2), a chitosan-treated group (G3), a chitosan-treated-infected group (G4), the liver free-treated group (G5), the liver free-treated-infected group (G6), the liver free-and-chitosan-treated group (G7), the liver free-and-chitosan-infected group (G8), the therapeutic liver free-and-chitosan-treated-infected group (G9), the sulfaquinoxaline-treated group (G10), and the sulfaquinoxaline-treated-infected group (G11). Chitosan was fed to the chicks in G3 and G4 as a preventative measure at a dose of 250 mg/kg. The G5 and G6 groups received 1.5 mg/kg of Liverfree. The G7 and G8 groups received chitosan and Liverfree. The G10 and G11 groups were administered 2 g/L of sulfaquinoxaline. From the moment the chicks arrived at Foshan University (one-day-old chicks) until the completion of the experiment, all medications were given to them as a preventative measure. G8 did; however, receive chitosan and liver free as therapeutic supplements at 7 dpi. The current study showed that the combination of liver free and chitosan can achieve better prophylactic and therapeutic effects than either alone. In E. tenella challenged chickens, G8 and G9 chickens showed reduced oocyst shedding and lesion score, improved growth performance (body weight, body weight gain, feed intake, feed conversion ratio, and mortality rate), and cecal histology. The current study demonstrates that combining liver free and chitosan has superior preventive and therapeutic benefits than either alone, and they could also be used as alternative anticoccidial agents.

Dietary chamomile flowers extract improved performance and mitigated aflatoxin B1 toxicity in rabbits.

Aflatoxin B1 (AFB1) is among the poisonous mycotoxins that contaminate food and feed. Limited studies are available on the efficacy of chamomile (Cha) against oxidative stress, liver damage and pro-inflammatory response induced by AFB1. The present study aims to evaluate the effects of Cha on the performance and protective effects against AFB1 in growing rabbits. The experimental rabbits were divided into four different groups, including Cha (70 mg kg day-1), AFB1 (AF; 30 µg kg day-1), AFB1+Cha (AFLCha) and control (CON). The results indicated that the AFB1 treatment had lower values of performance, and carcass parameters compared to the Cha and AFLCha treatments. Furthermore, the Cha and AFLCha groups had lower values of liver and kidney function activities compared to the AFB1 treatment. The higher values of antioxidant enzymes were observed in Cha and AFLCha treatments than in the AFB1 treatment. AFB1 treatments had higher levels of malondialdehyde and liver functions with lower levels of antioxidant enzymes (glutathione and superoxide dismutase) compared to Cha and CON groups. In conclusion, dietary Cha could mitigate the oxidative stress of AFB1-induced liver deterioration.

An ultrasensitive electrochemical sensor for detecting porcine epidemic diarrhea virus based on a Prussian blue-reduced graphene oxide modified glassy carbon electrode.

This study developed a novel, ultrasensitive sandwich-type electrochemical immunosensor for detecting the porcine epidemic diarrhea virus (PEDV). By electrochemical co-deposition of graphene and Prussian blue, a Prussian blue-reduced graphene oxide-modified glassy carbon electrode was made, further modified with PEDV-monoclonal antibodies (mAbs) to create a new PEDV immunosensor using the double antibody sandwich technique. The electrochemical characteristics of several modified electrodes were investigated using cyclic voltammetry (CV). We optimized the pH levels and scan rate. Additionally, we examined specificity, reproducibility, repeatability, accuracy, and stability. The study indicates that the immunosensor has good performance in the concentration range of 1 × 101.88 to 1 × 105.38 TCID50/mL of PEDV, with a detection limit of 1 × 101.93 TCID50/mL at a signal-to-noise ratio of 3σ. The composite membranes produced via co-deposition of graphene and Prussian blue effectively increased electron transport to the glassy carbon electrode, boosted response signals, and increased the sensitivity, specificity, and stability of the immunosensor. The immunosensor could accurately detect PEDV, with results comparable to real-time quantitative PCR. This technique was applied to PEDV detection and served as a model for developing additional immunosensors for detecting hazardous chemicals and pathogenic microbes.

Establishment of a method for detecting Trichinella spiralis in ovine muscle tissues using real-time fluorescence quantitative PCR.

Trichinellosis is caused by Trichinella spiralis, a meat-borne zoonotic disease transmitted to humans through the consumption of infected undercooked or raw meat. Surveillance using safe and precise diagnostic tools to diagnose T. spiralis in sheep is needed to assess the incidence and probability of transmission from sheep to humans. In this study, we developed a real-time PCR assay to detect T. spiralis DNA in ovine muscle samples that can be used as an alternative surveillance tool to ensure food safety using newly designed primers. The assay is specific for the Scfld4 gene of Trichinella (T1) and enables the detection of larvae in ovine muscle tissue samples with high sensitivity and specificity. Trichuris ovis, Oesophagostomum dentatum, Haemonchus contortus, and Bunostomum trigonocephalum showed no nonspecific amplification. The assay could detect Trichinella DNA concentrations as low as 0.0026 ng/µL, equivalent to 0.0064 larvae, indicating a high sensitivity for T. spiralis detection. We used this real-time PCR to detect 73 ovine muscle samples from an ovine abattoir, and five samples tested positive via real-time PCR but negative via microscopy. This assay may provide a more specific and sensitive method for rapidly detecting Trichinella larvae in ovine muscle tissues.

The phylogenetic position of Arhaphe deviatica within Hemipteran insects: A potential model species for eco-devo studies of symbiosis.

Insecta is known to be the most diverse group of species, exhibiting numerous forms of endosymbiotic associations. Molecular techniques have provided significant indicators for insect-microbe interactions. The present study aimed to register one of the true bugs of pentatomomorpha and clarify its taxonomic position through phylogenetic analysis of the partial 16S rRNA gene region. A maximum likelihood analysis retrieved a generally well-supported phylogeny based on Tamura 3-parameter model. Based on the partial mitochondrial 16S rRNA gene sequences, a phylogenetic study of suborder Heteroptera relationships within Hemipteras' order was constructed. Sequences of 221 bases of the 3' end of the gene from 28 species within 16 families were analyzed. This analysis and bootstrap confidence revealed two major clades comprising four suborders within Hemiptera, with a close relationship between Heteroptera + (Sternorrhyncha + (Auchenorrhycha + Coleorrhyncha)). Infraorder Pentatomomorpha is forming a sister group with a substantial bootstrap value to Cimicomorpha. Pyrrhocoroidea forms a sister relationship with Lygaeoidea + Coreoidea. There is a close relationship between Largidae and Pyrrhocoridae within Pyrrhocoroidea. The results show that the present species is firmly embedded in the genus Arhaphe with 94.35% sequence resemblance to its congeners. Besides, the recovered hemipteran species considered a potential model group for studying different symbionts. We propose both phylogenetic and ecological evolutionary developmental biology viewpoints for a more synthetic understanding of insect populations' molecular evolution.

Selecting Hub Genes and Predicting Target Genes of microRNAs in Tuberculosis via the Bioinformatics Analysis.

Tuberculosis (TB) is the world's most prevalently infectious disease. Molecular mechanisms behind tuberculosis remain unknown. microRNA (miRNA) is involved in a wide variety of diseases. To validate the significant genes and miRNAs in the current sample, two messenger RNA (mRNA) expression profile datasets and three miRNA expression profile datasets were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed (DE) genes (DEGs) and miRNAs (DE miRNAs) between healthy and TB patients were filtered out. Enrichment analysis was executed, and a protein-protein interaction (PPI) network was developed to understand the enrich pathways and hub genes of TB. Additionally, the target genes of miRNA were predicted and overlapping target genes were identified. We studied a total of 181 DEGs (135 downregulated and 46 upregulated genes) and two DE miRNAs (2 downregulated miRNAs) from two gene profile datasets and three miRNA profile datasets, respectively. 10 hub genes were defined based on high degree of connectivity. A PPI network's top module was constructed. The 23 DEGs identified have a significant relationship with miRNAs. 25 critically significant Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were discovered. The detailed study revealed that, in tuberculosis, the DE miRNA and DEGs form an interaction network. The identification of novel target genes and main pathways would aid with our understanding of miRNA's function in tuberculosis progression.

Naturally-derived targeted therapy for wound healing: Beyond classical strategies.

This review summarizes the four processes of wound healing in the human body (hemostasis, inflammatory, proliferation, and remodeling) and the most current research on the most important factors affecting cutaneous wound healing and the underlying cellular and/or molecular pathways. Local factors, including temperature, oxygenation, and infection, and systemic factors, such as age, diabetes, sex hormones, genetic components, autoimmune diseases, psychological stress, smoking and obesity are also addressed. A better understanding of the role of these factors in wound repair could result in the development of therapeutics that promote wound healing and resolve affected wounds. Additionally, natural products obtained from plants and animals are critical targets for the discovery of novel biologically significant pharmacophores, such as medicines and agrochemicals. This review outlines the most recent advances in naturally derived targeted treatment for wound healing. These are plant-derived natural products, insect-derived natural products, marine-derived natural products, nanomaterial-based wound-healing therapeutics (metal- and non-metal-based nanoparticles), and natural product-based nanomedicine to improve the future direction of wound healing. Natural products extracted from plants and animals have advanced significantly, particularly in the treatment of wound healing. As a result, the isolation and extraction of bioactive compounds from a variety of sources can continue to advance our understanding of wound healing. Undescribed bioactive compounds or unexplored formulations that could have a role in today's medicinal arsenal may be contained in the abundance of natural products and natural product derivatives.

Determination of lumefantrine as an effective drug against Toxoplasma gondii infection - in vitro and in vivo study.

Toxoplasma gondii is an obligate intracellular protozoan parasite, which can infect almost all warm-blooded animals, including humans, leading to toxoplasmosis. Currently, the effective treatment for human toxoplasmosis is the combination of sulphadiazine and pyrimethamine. However, both drugs have serious side-effects and toxicity in the host. Therefore, there is an urgent need for the discovery of new anti-T. gondii drugs with high potency and less or no side-effects. Our findings suggest that lumefantrine exerts activity against T. gondii by inhibiting its proliferation in Vero cells in vitro without being toxic to Vero cells (P ≤ 0.01). Lumefantrine prolonged mice infected with T. gondii from death for 3 days at the concentration of 50 µg L-1 than negative control (phosphate-buffered saline treated only), and reduced the parasite burden in mouse tissues in vivo (P ≤ 0.01; P ≤ 0.05). In addition, a significant increase in interferon gamma (IFN-γ) production was observed in high-dose lumefantrine-treated mice (P ≤ 0.01), whereas interleukin 10 (IL-10) and IL-4 levels increased in low-dose lumefantrine-treated mice (P ≤ 0.01). The results demonstrated that lumefantrine may be a promising agent to treat toxoplasmosis, and more experiments on the protective mechanism of lumefantrine should be undertaken in further studies.

Correction to: Oral inoculation of ultraviolet-irradiated Eimeria species oocysts protects chickens against coccidiosis.

The author name Salama Abohamra in the original published version of this article should have been Salama Abohamra Sayed Shany.

Soluble CXCL16 promotes TNF-α-induced apoptosis in DLBCL via the AMAD10-NF-κB regulatory feedback loop.

We had previously identified that the co-expression of transmembrane CXCL16 (TM-CXCL16) and its receptor CXCR6 is an independent risk factor for poor survival in patients with diffuse large B-cell lymphoma (DLBCL). However, the impact of the soluble form of CXCL16 (sCXCL16) on the pathogenesis of DLBCL remains unknown. In the present study, the synergistic effect of sCXCL16 and tumor necrosis factor α (TNF-α) on apoptosis in DLBCL cell lines (OCI-LY8 and OCI-LY10) was investigated in vitro. sCXCL16 reinforced TNF-α-mediated inhibition of DLBCL cell proliferation, as determined by the cell counting kit-8 assay. The results of annexin V staining showed that sCXCL16 enhanced TNF-α-induced apoptosis in OCI-LY8 and OCI-LY10 cells through a death receptor-caspase signaling pathway. The results of gene microarray suggested a significant upregulation of differentially expressed genes in the TNF signaling pathway. sCXCL16 increased the concentration of extracellular TNF-α by binding to CXCR6 to activate the nuclear factor-κB (NF-κB) signaling pathway. TNF-α also induced the secretion of sCXCL16 by increasing the expression of ADAM10, which is known to cleave TM-CXCL16 to yield sCXCL16. Moreover, bioinformatics analysis revealed that elevated TNF-α and ADAM10 expression levels in tumor tissues predicted better survival in patients with DLBCL. Thus, our study suggests that sCXCL16 enhances TNF-α-induced apoptosis of DLBCL cells, which may involve a positive feedback loop consisting of TNF-α, ADAM10, sCXCL16, and members of the NF-κB pathway. sCXCL16 and TNF-α may be used as prognostic markers in the clinic, and their combinational use is a promising approach in the context of DLBCL therapy.

Toxoplasma gondii oocyst-driven infection in pigs, chickens and humans in northeastern China.

BACKGROUND: Toxoplasma gondii, an intracellular apicomplexan protozoan parasite, can infect almost all warm-blooded animals. The aim of the present study was to investigate T. gondii oocyst-driven infection in pigs, chickens and humans in Jilin province, northeastern China. RESULTS: The serum samples of pigs, chickens and humans were sampled and tested by indirect enzyme-linked immunosorbent assays (ELISAs) using dense granule antigen GRA7, oocyst-specific protein OWP8, and sporozoite-specific protein CCp5A, respectively. Results showed a prevalence of 16.7% by GRA7-ELISA, and 12.2% by OWP8- and CCp5A-ELISA in pigs; 10.4% by GRA7-ELISA, 13.5% by OWP8-ELISA, and 9.4% by CCp5A-ELISA in chickens; and 14.2% by GRA7-ELISA, 3.6% by OWP8-ELISA, and 3.0% by CCp5A-ELISA in humans. No significant differences were observed between T. gondii seroprevalence in pigs and chickens among the three antigens-based ELISAs (P > 0.05). However, there were significant differences between T. gondii seroprevalence rates in humans (P < 0.05). These findings demonstrated a low prevalence of T. gondii oocyst-driven infection in humans, a medium prevalence in pigs, and a high prevalence in chickens. CONCLUSIONS: The present study demonstrated that different oocyst-driven infection rates in different animal species, which would help to design effective strategies to prevent T. gondii transmission. To our knowledge, this is the first study to differentiate T. gondii infective forms in pigs, chickens and humans in China.

Oral inoculation of ultraviolet-irradiated Eimeria species oocysts protects chickens against coccidiosis.

Prevention of coccidiosis is one of the best ways of controlling disease. Therefore, the present study was carried out to evaluate the protective effect of ultraviolet (UV)-irradiated sporulated oocysts of Eimeria species against coccidiosis in layer chickens. One hundred forty-four one-day-old layer chicks were randomly divided into 4 groups (n = 36), including non-immunized/non-challenged negative control group (NC group), non-immunized/challenged control group (NIC group), non-irradiated sporulated oocyst/challenged group (CA group), and UV-irradiated sporulated oocyst/challenged (UV group). At the age of 4 days, chickens in groups UV and CA were both orally inoculated with 1.0 × 104 UV-irradiated and non-irradiated sporulated oocysts of Eimeria species, respectively. Chickens in groups NIC and NC were served as positive and negative controls, respectively. Chickens in all groups were orally challenged with 7.5 × 104 sporulated oocysts of Eimeria species except the NC group at the age of 21 days. The results revealed that chicks receiving UV-irradiated sporulated oocysts had no signs of illness with minimal or no changes in the cecal integrity and a significantly lower oocyst shedding (OPG) than in the NIC group. Additionally, the cytokine gene expression profiles were evaluated. Expression levels of IL-2, IL-12, and IFN-γ were significantly higher in the spleen of chicks in the UV and CA groups than in the NC group post-challenge. As expected, treatment with irradiated oocysts resulted in a significant reduction in oocyst shedding and maintenance of cecal mucosal integrity. Furthermore, the body weight was higher in chickens inoculated with UV-irradiated oocysts than their non-irradiated counterparts. In conclusion, our results demonstrate that inoculation with UV-irradiated sporulated oocysts of Eimeria species can produce a substantial reduction in infection symptoms.

cGAS/STING/TBK1/IRF3 Signaling Pathway Activates BMDCs Maturation Following Mycobacterium bovis Infection.

Cyclic GMP-AMP synthase (cGAS) is an important cytosolic DNA sensor that plays a crucial role in triggering STING-dependent signal and inducing type I interferons (IFNs). cGAS is important for intracellular bacterial recognition and innate immune responses. However, the regulating effect of the cGAS pathway for bone marrow-derived dendritic cells (BMDCs) during Mycobacterium bovis (M. bovis) infection is still unknown. We hypothesized that the maturation and activation of BMDCs were modulated by the cGAS/STING/TBK1/IRF3 signaling pathway. In this study, we found that M. bovis promoted phenotypic maturation and functional activation of BMDCs via the cGAS signaling pathway, with the type I IFN and its receptor (IFNAR) contributing. Additionally, we showed that the type I IFN pathway promoted CD4⁺ T cells' proliferation with BMDC during M. bovis infection. Meanwhile, the related cytokines increased the expression involved in this signaling pathway. These data highlight the mechanism of the cGAS and type I IFN pathway in regulating the maturation and activation of BMDCs, emphasizing the important role of this signaling pathway and BMDCs against M. bovis. This study provides new insight into the interaction between cGAS and dendritic cells (DCs), which could be considered in the development of new drugs and vaccines against tuberculosis.

A Comparative Study of Biological Characteristics and Transcriptome Profiles of Mesenchymal Stem Cells from Different Canine Tissues.

Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy. Comparing the biological and transcriptome gene characteristics of MSCs from different sources provides an important basis for the screening of clinically used cells. The main purpose of this experiment was to establish methods for the isolation and culture of MSCs from five different canine sources, including adipose tissue, bone marrow, umbilical cord, amniotic membrane, and placenta, and compare biological and transcriptome characteristics of MSCs, in order to provide a basis for the clinical application of canine MSCs. MSCs were isolated from Chinese pastoral dogs, and the following experiments were performed: (1) the third, sixth, and ninth generations of cells were counted, respectively, and a growth curve was plotted to calculate the MSC population doubling time; (2) the expression of CD34 and CD44 surface markers was studied by immunofluorescence; (3) the third generation of cells were used for osteogenetic and adipogenic differentiation experiments; and (4) MSC transcriptome profiles were performed using RNA sequencing. All of the five types of MSCs showed fibroblast-like adherent growth. The cell surface expressed CD44 instead of CD34; the third-generation MSCs had the highest proliferative activity. The average population doubling time of adipose mesenchymal stem cells (AD-MSCs), placenta mesenchymal stem cells (P-MSCs), bone marrow mesenchymal stem cells (BM-MSCs), umbilical cord mesenchymal stem cells (UC-MSCs), and amniotic mesenchymal stem cells (AM-MSCs) were 15.8 h, 21.2 h, 26.2 h, 35 h, and 41.9 h, respectively. All five types of MSCs could be induced to differentiate into adipocytes and osteoblasts in vitro, with lipid droplets appearing after 8 days and bone formation occurring 5 days after AD-MSC induction. However, the multilineage differentiation for the remaining of MSCs was longer compared to that of the AD-MSCs. The MSC transcriptome profiles showed that AD-MSC and BM-MSCs had the highest homology, while P-MSCs were significantly different compared to the other four types of MSCs. All the isolated MSCs had the main biological characteristics of MSCs. AD-MSCs had the shortest time for proliferation, adipogenesis, and osteogenic differentiation.

Influence of Eimeria falciformis Infection on Gut Microbiota and Metabolic Pathways in Mice.

Coccidiosis, caused by different species of Eimeria parasites, is an economically important disease of poultry and livestock worldwide. Here we report previously unknown alterations in the gut microbes and metabolism of BALB/c mice infected with Eimeria falciformis Specifically, we observed a significant shift in the abundance of cecal bacteria and disrupted metabolism in parasitized animals. The relative abundances of Lachnospiraceae bacterium NK4A136, Ruminiclostridium, Alistipes, and Lactobacillus declined in response to E. falciformis infection, whereas Escherichia, Shigella, Helicobacter, Klebsiella, and Bacteroides were increased. Carbohydrate and amino acid metabolites in the serum samples of infected mice were significantly altered compared to naïve controls. Levels of amino acids, including asparagine, histidine, l-cysteine, tryptophan, lysine, glycine, serine, alanine, proline, ornithine, methionine, and valine, decreased on day 7 postinfection before returning to baseline on day 14. In addition, increased levels of indolelactate and mannitol and a reduced amount of oxalic acid indicated impaired carbon metabolism upon parasitic infection. These data demonstrate that intestinal coccidial infection perturbs the microbiota and disrupts carbon and nitrogen metabolism.

An ex vivo ruminal ovine model to study the immediate immune response in the context of bacterial lipopolysaccharide.

We have set up an ex vivo ovine ruminal model, which can mimic the multicellular process to explore the early steps in Salmonella typhimurium lipopolysaccharide (LPS) stimulation using RNA-seq technology. Ovine ruminal explants were collected for histological and transcriptional analysis and supernatants collected to quantitate lactate dehydrogenase (LDH) enzymes. A total of 8 and 523 genes were significantly over-expressed between LPS-treated and control tissues at 6 and 12 h, respectively. However, six and seven hundred and thirteen genes were substantially repressed between the aforementioned tissues, correspondingly. Key genes up-regulated in response to the addition of LPS were tumor necrosis factor (TNF), interlukin (IL)-1 beta(b), IL-6, IL-8, IL-17B, IL-19, MMP-1, MMP-3, and integrin alpha 2 (ITGA8, 9). This study shows for the first time that galectin-1 is up-regulated in an ex vivo ruminal segment model exposed to bacterial lipopolysaccharide following 6 h of incubation. The ruminal segment model has been shown to be a suitable tool to study the bacterial lipopolysaccharide effects on the ovine ruminal tissues prior to in vivo assessment.

An optimized DNA extraction method for molecular identification of coccidian species.

Molecular identification of Eimeria parasites infecting poultry and livestock has been commonly used for more than 20 years. An important step of the molecular identification technique is the rupturing of the oocyst wall for DNA extraction. Previously, DNA extraction methods included pre-treatment with sodium hypochlorite and osmotic shock with saturated salt solution. Here, we present a modification of this technique for a more sensitive and efficient identification of Eimeria spp. in field samples. The disruption extent of the oocyst walls, yield of DNA extraction, and identification of species-specific DNA sequences by PCR were used to evaluate this optimized method. Incubation of oocysts in sodium hypochlorite for 1.5 h at 4 °C followed by treatment with a saturated salt solution for 1 h at 55 °C broke up the walls of most Eimeria tenella oocysts, as well as other coccidian species of chicken and rabbit, such as Eimeria intestinalis and even Cryptosporidium cuniculus. Notably, polymerase chain reaction (PCR) amplification of the intervening transcribed sequence 1 (ITS-1) was successfully performed with genomic DNA extracted from just 50 oocysts using this optimized method. Our findings will greatly promote the development of molecular diagnosis methods of coccidiosis and simplify coccidian species identification and categorization as well as infection prevalence, providing a significant advancement in the development of techniques for coccidiosis control and prevention.

Mycoplasma gallisepticum MGA_0676 is a membrane-associated cytotoxic nuclease with a staphylococcal nuclease region essential for nuclear translocation and apoptosis induction in chicken cells.

Mycoplasma gallisepticum can infect a wide variety of birds including the commercial poultry. M. gallisepticum MGA_0676 is a putative lipoprotein, which is similar to bacterial thermostable nucleases. But the possible pathogenic effect of M. gallisepticum MGA_0676 has not been investigated so far. In the present study, we cloned the MGA_0676 gene after deletion of the amino-terminal signal sequence and mutagenesis of the Mycoplasma TGA tryptophan codons to TGG and expressed recombinant MGA_0676 protein in Escherichia coli. We identified and characterized MGA_0676 as a Ca(2+)-dependent cytotoxic nuclease of M. gallisepticum with a staphylococcal nuclease (SNc) region that displays the hallmarks of nucleases. Membrane protein immunoblot analysis and immunogold electron microscopy revealed that MGA_0676 locates on the membrane surface of M. gallisepticum. Furthermore, apoptosis assay using annexin V-FITC and propidium iodide (annexin V/PI) indicated that MGA_0676 played significant roles in apoptosis induction and pathological damages in chicken cells. Moreover, confocal microscopy showed that MGA_0676 localizes in the nuclei of host cells. Besides, after the SNc region was deleted, MGA_0676 lost its ability of nuclear localization, nuclease activity, and cytotoxicity, which revealed that the SNc region is essential for nuclear translocation and induction of apoptosis in chicken cells. The above results suggest that MGA_0676 is an important virulence factor in cellular pathology and may play a unique role in the life cycle events of M. gallisepticum.

Early detection of Toxoplasma gondii-infected cats by interferon-gamma release assay.

Felines, the only definitive hosts that shed the environmentally-durable oocysts, are the key in the transmission of Toxoplasma gondii to all warm-blooded animals. They seroconvert as late as the third week and begin to shed oocysts as early as 3-8 days after being fed tissue cysts. Early detection of Toxoplasma-infected cats is crucial to evaluate Toxoplasma-contaminated environment and potential risks to public health. Moreover, it is fundamental for Toxoplasma infection control. Interferon-gamma release assay (IGRA) is a blood-based test assessing the presence of IFN-γ released by the T-lymphocytes directed against specific antigens, which is an ideal assay for early detection of Toxoplasma-infected cats. Here, cats were orally infected with the tissue cysts and blood was collected for toxoplasmic antigen stimulation, and the released IFN-γ was measured by ELISA. Results showed that Toxoplasma-infection was detected by IGRA as early as 4 days post-infection (dpi); while serum Toxoplasma IgM and IgG were detected by ELISA at 10 dpi and 14 dpi, respectively. Our findings demonstrated that IGRA-positive and ELISA-negative samples revealed an early Toxoplasma infection in cats, indicating a new strategy for the early diagnosis of Toxoplasma infection by combining IGRA and ELISA. Therefore, IGRA could emerge as a reliable diagnostic tool for the exploration of cat toxoplasmosis prevalence and its potential risks to public health.