Yolkin, a Polypeptide Complex from Egg Yolk, Affects Cytokine Levels and Leukocyte Populations in Broiler Chicken Blood and Lymphoid Organs after In Ovo Administration.

Yolkin is a polypeptide complex isolated from hen egg yolk that exhibits immunomodulating properties. The aim of the present study was to determine whether in-ovo-delivered yolkin affects leukocyte populations and cytokine levels in broiler chickens. The experiment was carried out on eggs from Ross 308 broiler breeder birds. Yolkin was administered in ovo on the 18th day of incubation, once, at the following three doses: 1, 10, or 100 µg/egg. The immunological parameters were assessed in 1-, 7-, 14-, 21-, 28-, 35-, and 42-day-old birds kept under farming conditions and routinely vaccinated. The leukocyte populations were determined in the thymus, spleen, and blood. The cytokine (IL-1ß, IL-2, IL-6, and IL-10) levels were determined in the plasma of the broiler chickens. Each experimental group included eight birds. The most pronounced effect of yolkin was an increase in the population of T cells, both CD4+ and CD8+, mainly in the blood. This effect on the lymphocyte subsets may be valuable regarding chicken immune responses, mainly against T-dependent antigens, during infection or after vaccination.

Antioxidant polysaccharide/gelatin blend films loaded with curcumin - A comparative study.

Curcumin (CUR; 0, 0.005, 0.01, 0.02 %) was loaded into binary 75/25 blend films based on polysaccharides (carboxymethyl cellulose (CMC), gum Arabic (GAR), octenyl succinic anhydride modified starch (OSA), water-soluble soy polysaccharides (WSSP)) and gelatin (GEL). The GAR-based system was the least rough and, consequently, the most transparent of the films. An opposite result was found for the WSSP-based film. Despite the phase separation, the CMC75/GEL25 film exhibited excellent mechanical strength and stiffness. CUR improved the UV/VIS light-barrier characteristics of the films, but did not affect most of other physiochemical properties. X-ray diffractograms revealed that CUR provoked the rearrangement of the triple helical structure of GEL. As highly erodible, the CMC75/GEL25 carrier ensured the fastest and the most complete release of CUR. The OSA75/GEL25 system exhibited an opposite behavior. The kinetic profiles of the antiradical activity of the films did not reflect CUR release. A comparison of 2,2-diphenyl-1-picrylhydrazyl (DPPH*) scavenging on the plateau revealed that the CUR-supplemented films had quite comparable antiradical potential. The CMC75/GEL25 system exhibited the highest colorimetric stability, likely as a result of complete encapsulation of CUR in the GEL-rich microspheres. Weak symptoms of physical aging (enthalpy relaxation) were found in the films.

Comparative Nanopore Sequencing-Based Evaluation of the Midgut Microbiota of the Summer Chafer (Amphimallon solstitiale L.) Associated with Possible Resistance to Entomopathogenic Nematodes.

Root-feeding Amphimallon solstitiale larvae and certain other scarab beetles are the main soil-dwelling pests found in Europe, while entomopathogenic nematodes (EPN) have been used as a biocontrol agent against these species. Our study provides the first detailed characterization of the bacterial community of the midgut in wild A. solstitiale larvae, based on the nanopore sequencing of the 16S rRNA gene. In the whole dataset, we detected 2586 different genera and 11,641 species, with only 83 diverse bacterial genera shared by all studied individuals, which may represent members of the core midgut microbiota of A. solstitiale larvae. Subsequently, we compared the midgut microbiota of EPN-resistant and T0 (prior to EPN exposure) individuals, hypothesizing that resistance to this parasitic infection may be linked to the altered gut community. Compared to the control, the resistant insect microbiota demonstrated lower Shannon and Evenness indices and significant differences in the community structure. Our studies confirmed that the gut microbiota alternation is associated with resistant insects; however, there are many processes involved that can affect the bacterial community. Further research on the role of gut microbiota in insect-parasitic nematode interaction may ultimately lead to the improvement of biological control strategies in insect pest management.

Screening and Molecular Identification of Bacteria from the Midgut of Amphimallon solstitiale Larvae Exhibiting Antagonistic Activity against Bacterial Symbionts of Entomopathogenic Nematodes.

Entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) are a group of organisms capable of infecting larvae of insects living in soil, including representatives of the family Scarabaeidae. Their insecticidal activity is related to the presence of symbiotic bacteria Xenorhabdus spp. or Photorhabdus spp. in the alimentary tract, which are released into the insect body, leading to its death caused by bacterial toxins and septicemia. Although the antibacterial activities of symbionts of entomopathogenic nematodes have been well described, there is insufficient knowledge of the interactions between these bacteria and microorganisms that naturally inhabit the alimentary tract of insects infested by nematodes. In this study, 900 bacterial strains isolated from midgut samples of Amphimallon solstitiale larvae were tested for their antagonistic activity against the selected five Xenorhabdus and Photorhabdus species. Cross-streak tests showed significant antibacterial activity of 20 isolates. These bacteria were identified as Bacillus [Brevibacterium] frigoritolerans, Bacillus toyonensis, Bacillus wiedmannii, Chryseobacterium lathyri, Chryseobacterium sp., Citrobacter murliniae, Enterococcus malodoratus, Paenibacillus sp., Serratia marcescens and Serratia sp. Since some representatives of the intestinal microbiota of A. solstitiale are able to inhibit the growth of Xenorhabdus and Photorhrhabdus bacteria in vitro, it can be assumed that this type of bacterial interaction may occur at certain stages of insect infection by Steinernema or Heterorhabditis nematodes.

Steinernema sandneri n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Poland.

A new species of entomopathogenic nematodes, Steinernema sandneri n. sp., was recovered by baiting from Poland. Its morphological traits indicate that the new species is a member of the feltiae-kraussei group. A body length of 843 (708-965) µm, a more anterior position of excretory pore (56 µm), and the lower D% value (40 vs > 46) discriminate this species from most of the other group members. The first-generation males of S. sandneri n. sp. can be distinguished from the other clade members by a 60 µm long spicule, a relatively long gubernaculum (GS% = 79), and the position of the excretory pore (80 µm). Phylogenetic analysis of the ITS rDNA, D2D3 of 28 S rDNA, and cox1 sequences confirmed that S. sandneri n. sp. is a new species of the feltiae-kraussei group, closely related to S. kraussei and S. silvaticum.

Nanopore-Sequencing Characterization of the Gut Microbiota of Melolontha melolontha Larvae: Contribution to Protection against Entomopathogenic Nematodes?

This study focused on the potential relationships between midgut microbiota of the common cockchafer Melolontha melolontha larvae and their resistance to entomopathogenic nematodes (EPN) infection. We investigated the bacterial community associated with control and unsusceptible EPN-exposed insects through nanopore sequencing of the 16S rRNA gene. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the most abundant bacterial phyla within the complex and variable midgut microbiota of the wild M. melolontha larvae. The core microbiota was found to include 82 genera, which accounted for 3.4% of the total number of identified genera. The EPN-resistant larvae differed significantly from the control ones in the abundance of many genera belonging to the Actinomycetales, Rhizobiales, and Clostridiales orders. Additionally, the analysis of the microbiome networks revealed different sets of keystone midgut bacterial genera between these two groups of insects, indicating differences in the mutual interactions between bacteria. Finally, we detected Xenorhabdus and Photorhabdus as gut residents and various bacterial species exhibiting antagonistic activity against these entomopathogens. This study paves the way to further research aimed at unravelling the role of the host gut microbiota on the output of EPN infection, which may contribute to enhancement of the efficiency of nematodes used in eco-friendly pest management.

Effects of Selected Prebiotics or Synbiotics Administered in ovo on Lymphocyte Subsets in Bursa of the Fabricius, Thymus, and Spleen in Non-Immunized and Immunized Chicken Broilers.

The effects of in ovo-delivered prebiotics and synbiotics on the lymphocyte subsets of the lymphoid organs in non-immunized 7-day-old broiler chickens and in non-immunized, sheep red blood cells (SRBC)-immunized, and dextran (DEX)-immunized 21- and 35-day-old birds were studied. The substances were injected on the 12th day of egg incubation: Prebiotic1 group (Pre1) with a solution of inulin, Prebiotic2 group (Pre2) with a solution of Bi2tos (non-digestive transgalacto-oligosaccharides), Synbiotic1 group (Syn1) with inulin and Lactococcus lactis subsp. lactis IBB SL1, and Synbiotic2 group (Syn2) with Bi2tos and Lactococcus lactis subsp. cremoris IBB SC1. In 7-day-old chicks, a decrease in T splenocytes was noticed in all groups. The most pronounced effect in 21- and 35-day-old birds was an increase in TCRγδ+ cells in Syn1 and Syn2 groups. A decrease in bursal B cells was observed in DEX-immunized Pre1 group (21-day-old birds), and in the Syn1 group in non-immunized and SRBC-immunized 35-day-old birds. An increase in double-positive lymphocytes was observed in Pre1 (35-day-old birds) and Pre2 (immunized 21-day-old birds) groups. In Pre1 and Syn1 groups (21- and 35-day-old), an increase in B splenocytes and a decrease in T splenocytes were observed. We concluded that Syn1 was the most effective in the stimulation of the chicken immune system.

Selegiline and clomipramine effects on lymphocyte subsets, regulatory T cells and sheep red blood cell (SRBC)-induced humoral immune response after in vivo administration in mice.

We aimed at investigating the influence of clomipramine and selegiline administered in vivo in mice on lymphocyte subsets in lymphoid organs and SRBC-induced humoral immune response. Balb/c mice were given 7 or 14 oral doses (1 mg/kg) of selegiline or clomipramine. Lymphocyte B and T subsets and splenic regulatory T cell (Treg) subset were determined in non-immunized mice 24 and 72 h after the last dose of the drugs. Some mice treated with 7 doses were immunized with sheep red blood cells (SRBC) 2 h after the last dose, and their number of antibody forming cells, haemagglutinin titers and splenocyte subsets were determined. An increase in T lymphocytes and a decrease in B cells were visible in peripheral lymphoid organs, especially after 14 doses of selegiline or clomipramine in non-immunized mice, as well as in spleens of SRBC-immunized mice. The most pronounced change was a decrease in CD4+/CD8+ ratio resulting mainly from an increase in CD8+ subset after seven doses of the drugs in the non-immunized mice. However, it was of a transient nature, as it disappeared after 14 doses of the drugs. The tested drugs only slightly affected thymocyte maturation and did not alter Treg subset. Selegiline and clomipramine transiently stimulated IgG production in SRBC-immunized mice. Both selegiline and clomipramine administered in vivo modulated lymphocyte subsets. This immunomodulatory effect depended on the drug as well as duration of administration.

Clomipramine, a tricyclic antidepressant, and selegiline, a monoamine oxidase-B inhibitor, modulate the activity of phagocytic cells after oral administration in mice.

OBJECTIVES: Our aim was to find out whether clomipramine, a tricyclic antidepressant, and selegiline, a monoamine oxidase-B inhibitor, influence the activity of phagocytic cells after in-vivo administration in mice. METHODS: Clomipramine and selegiline were administered to Balb/c mice orally at a dose of 1 mg/kg, 7 or 14 times. IL-1ß and nitric oxide (NO) levels were measured in supernatants of the peritoneal macrophage cultures stimulated in vitro with lipopolysaccharide from Escherichia coli. The phagocytic activity of the granulocytes and monocytes was determined using a commercial Phagotest 24 and 72 h after the last dose of the investigated drugs. KEY FINDINGS: Seven doses of clomipramine or selegiline decreased IL-1ß production, while a rise in its synthesis was observed after 14 doses of selegiline. Clomipramine administered 14 times increased NO production. Clomipramine and selegiline administered seven times reduced the percentage of phagocytosing granulocytes. The drugs administered 14 times increased the percentage of phagocytosing granulocytes and decreased the percentage of phagocytosing monocytes. CONCLUSIONS: Both clomipramine and selegiline administered in vivo changed the phagocytic activity of blood cells and IL-1ß and NO production by murine peritoneal macrophages. This effect depended on the drug, the number of doses and the type of phagocytic cells.

Bacteria from the Midgut of Common Cockchafer (Melolontha melolontha L.) Larvae Exhibiting Antagonistic Activity Against Bacterial Symbionts of Entomopathogenic Nematodes: Isolation and Molecular Identification.

The mechanisms of action of the complex including entomopathogenic nematodes of the genera Steinernema and Heterorhabditis and their mutualistic partners, i.e., bacteria Xenorhabdus and Photorhabdus, have been well explained, and the nematodes have been commercialized as biological control agents against many soil insect pests. However, little is known regarding the nature of the relationships between these bacteria and the gut microbiota of infected insects. In the present study, 900 bacterial isolates that were obtained from the midgut samples of Melolontha melolontha larvae were screened for their antagonistic activity against the selected species of the genera Xenorhabdus and Photorhabdus. Twelve strains exhibited significant antibacterial activity in the applied tests. They were identified based on 16S rRNA and rpoB, rpoD, or recA gene sequences as Pseudomonas chlororaphis, Citrobacter murliniae, Acinetobacter calcoaceticus, Chryseobacterium lathyri, Chryseobacterium sp., Serratia liquefaciens, and Serratia sp. The culture filtrate of the isolate P. chlororaphis MMC3 L3 04 exerted the strongest inhibitory effect on the tested bacteria. The results of the preliminary study that are presented here, which focused on interactions between the insect gut microbiota and mutualistic bacteria of entomopathogenic nematodes, show that bacteria inhabiting the gut of insects might play a key role in insect resistance to entomopathogenic nematode pressure.

Effects of yolkin on the immune response of mice and its plausible mechanism of action.

Yolkin is a product of proteolytic degradation of vitellogenin, a protein contained in eggs' yolk, with already described procognitive properties. Here, we investigated effects of yolkin on the humoral and cellular immune response in mice, phenotype of cells from lymphoid organs and function of innate immunity cells. In vitro studies included effects of yolkin on mitogen-induced thymocyte proliferation, percentage of CD19 cells in bone marrow cells culture, expression of signaling molecules in Jurkat cells, interleukin 2 receptor (IL-2R) subunits in WEHI 231 cells and susceptibility of these cells to anti-Ig-induced cell death. The results showed that repeatable i.p. injections of yolkin stimulated the humoral immune response to sheep red blood cells (SRBC) irrespective of the time of the treatment. On the other hand, yolkin inhibited contact sensitivity to oxazolone. Treatment of mice with yolkin diminished the percentage of double positive cells and increasing the content of single positive CD4+ and CD8+ cells in the thymus. At the same time an increase of percentage of CD19 + B cells in the spleen and mesenteric lymph nodes was observed. In addition, the protein, given i.p., diminished ex vivo ability to synthesize nitric oxide by resident, peritoneal macrophages, stimulated with lipopolisaccharide (LPS). In vitro studies showed that yolkin increased CD19+ cell content in bone marrow cell population. The protein also enhanced proliferation of thymocytes to concanavalin A and stimulated expression of MAP kinases in Jurkat cells. In WEHI 231 B cell line yolkin caused a loss of IL-2R gamma chain expression, correlated with an increased resistance of these cells to proapoptotic action of anti-Ig antibodies. In conclusion, this is a first demonstration of immunotropic properties of yolkin in in vitro and in vivo tests. The results provide evidence for induction of maturation and stimulatory signals in immature T and B cells by the protein, suggesting its potential role in the development of an embryo's immune system.

Hawthorn (Crataegus monogyna) Phenolic Extract Modulates Lymphocyte Subsets and Humoral Immune Response in Mice.

This study investigated the effect of hawthorn (Crataegus monogyna) phenolic extract on lymphocyte subsets in the lymphoid organs in nonimmunized mice and on humoral immune response in sheep red blood cell-immunized mice. Hawthorn phenolic extract (50, 100, 200 mg/kg) was administered orally five or ten times. Sheep red blood cells were injected 24 h after administration of the last extract dose. The lymphocyte subsets were assessed 24 and 72 h after the last dose. Humoral immune response was determined 4 and 7 days after immunization. Five doses of the extract decreased the percentage of CD4-CD8- and CD4+ thymocytes but elevated the percentage of CD4+CD8+ and CD8+ thymic cells. The extract increased the total number, percentage, and absolute count of T and B splenocytes. When administered five times, it lowered the percentage of T lymphocytes, but boosted the population of B lymphocytes of mesenteric lymph nodes (after 24 h). However, a rise in the population of T lymphocytes was observed 72 h after five and ten doses. The extract administered ten times elevated the number of plaque-forming cells and total anti-sheep red blood cell hemagglutinin titer but reduced the 2-ME-resistant antibody titer (day 7). At the same time, five doses of the extract increased antibody titers. Considering its impact on lymphocyte subsets and humoral immune response, hawthorn extract may be used as an immunomodulator.

Modulating effect of a new ester, 28-O-phosphatidylbetulin (DAPB), obtained from hen egg yolk lecithin and betulin on lymphocyte subsets and humoral immune response in mice.

Context: Leaf extracts of plants of the genus Betula have traditionally been used as diuretic, anti-rheumatic and diaphoretic preparations. One of the main active ingredients of Betula bark is betulin, lupane-type triterpene alcohol, with multiple biological activities. Objectives: The aim of this study was to investigate in vitro and in vivo immunomodulatory effects of a newly synthesized ester of betulin: 28-O-phosphatidylbetulin [28-O-(1,2-diacyl-sn-glycero-3-phospho)-betulin, DAPB] in comparison with betulin in mice. Materials and methods: Cytotoxic activity of DAPB or betulin was tested against non-cancer (D10.G4.1 and J774E.1) and cancer (GL-1; CL-1 and Jurkat) cell lines. The in vivo part assessed total lymphocyte count, weight ratio and subsets of lymphocytes in the lymphatic organs, and humoral immune response to sheep erythrocytes (SRBC). Results: In vitro assay showed that DAPB, contrary to betulin, had no antiproliferative activity. Exposure to four doses of DAPB increased the absolute count of immature CD4+CD8+ thymic cells as well as the percentage and absolute count of mature CD4+ and CD8+ thymocytes. DAPB enhanced the percentage or absolute count of CD3+ cells in spleen and lymph nodes with corresponding decrease in the percentage and/or absolute count of CD19+ cells. Both DAPB and betulin enhanced the percentage and absolute count of CD8+ lymphocytes in lymph nodes. In SRBC-immunized mice, betulin contrary to DAPB enhanced the number of splenocytes producing anti-SRBC antibodies (PFC). Both DAPB and betulin increased the level of total (IgM + IgG) and IgG titers. Conclusion: Despite the lack of cytotoxic activity, DAPB shows valuable immunomodulatory properties.

Steinernema poinari (Nematoda: Steinernematidae): a new symbiotic host of entomopathogenic bacteria Xenorhabdus bovienii.

Three strains of symbiotic bacteria were isolated from an entomopathogenic nematode Steinernema poinari retrieved from soil in eastern Poland. Using 16S rDNA, recA, gltX, gyrB, and dnaN gene sequences for phylogenetic analysis, these strains were shown to belong to the species Xenorhabdus bovienii. The nucleotide identity between the studied S. poinari microsymbionts and other X. bovienii strains calculated for 16S rDNA and concatenated sequences of four protein-coding genes was 98.7-100% and 97.9-99.5%, respectively. The phenotypic properties of the isolates also supported their close phylogenetic relationship with X. bovienii. All three tested X. bovienii strains of different Steinernema clade origin supported the recovery of infective juveniles and subsequent development of the nematode population. However, the colonization degree of new infective juvenile generations was significantly affected by the bacterial host donor/recipient. The colonization degree of infective juveniles reared on bacterial symbionts deriving from a non-cognate clade of nematodes was extremely low, but proved the possible host-switching between non-related Steinernema species.

Propentofylline, phosphodiesterase and adenosine reuptake inhibitor modulates lymphocyte subsets and lymphocyte activity after in-vivo administration in non-immunized and SRBC-immunized mice.

OBJECTIVES: The aim of the study was to investigate immunomodulatory effect of in-vivo administered propentofylline on the subsets and activity of murine lymphocytes. METHODS: Propentofylline (3 mg/kg) was administered orally to 8-week-old Balb/c mice, once or six times at 12-h intervals. The lymphocyte subsets, regulatory T cells, IL-5 and TNF levels were determined 12 h and 24 h after a single dose or after the sixth dose of the drug in non-immunized mice. Humoral immune response in sheep red blood cells (SRBC)-immunized mice was determined 4, 7 and 14 days after immunization. KEY FINDINGS: Propentofylline inhibited thymocyte maturation (increase in CD4- CD8- thymocyte subset and decrease in the percentage of CD4+ CD8+ thymocytes) and modulated the lymphocyte subsets in spleen and mesenteric lymph nodes. An increase in the absolute count and percentage of splenic regulatory T cells (CD4+ CD25+ Foxp3+ cells) was noticed 24 h after single administration of the drug. Propentofylline lowered serum level of IL-5 and did not affect TNF concentration. Only a weak inhibitory effect on anti-SRBC humoral immune response was observed. CONCLUSIONS: Propentofylline administration induced inhibition of thymocyte maturation and an increase in Treg subset that might be beneficial for an inhibition of immune response.

The effects of bestatin on humoral response to sheep erythrocytes in non-treated and cyclophosphamide-immunocompromised mice.

The effects of bestatin on humoral immune response to sheep erythrocytes (SRBC) and restoration of the response impaired by a single cyclophosphamide dose (350 mg/kg) were tested on mice. Bestatin (at doses of 10, 1, and 0.1 mg/kg) was administered intraperitoneally (i.p.) 5 or 10 times. The pharmacological immunosuppression was induced by a single i.p. injection of cyclophosphamide (350 mg/kg) administered 24 h before the first bestatin dose. The mice were immunized i.p. with SRBC 24 h after the last dose of bestatin. It was found that multiple administration of bestatin at all three doses potentiated the humoral response to SRBC in non-treated mice, resulting in an increased number of plaque-forming cells (PFC) and 2-mercaptoethanol (2-ME)-resistant anti-SRBC antibodies. However, five times administration of bestatin at the doses under investigation caused further decreases in total anti-SRBC hemagglutinins. A single injection of cyclophosphamide (350 mg/kg) suppressed humoral response of mice to the antigen. Administration of bestatin after pharmacological immunosuppression partially prevented the suppressive action of cyclophosphamide in the in vivo model of the humoral immune response to SRBC. The protective action of bestatin was both dose- and schedule-dependent. Ten times' exposure to a bestatin dose of 0.1 mg/kg after a high cyclophosphamide dose partially reduced the suppressive effect of this drug on humoral response of SRBC-immunized mice, increasing PFC on days 4 and 7 after immunization, which coincided with restored ability of the lymphocytes to produce the 2-ME-resistant hemagglutinins on day 7 and the total anti-SRBC hemagglutinins on day 14 after priming.

Effects of bestatin on phagocytic cells in cyclophosphamide-treated mice.

The low-molecular weight dipeptide bestatin is a potent inhibitor of aminopeptidase N and has been demonstrated to have antitumor and immunomodulatory effects. The effects of bestatin on interleukin (IL)-1ß synthesis and release by peritoneal macrophages stimulated in vitro with lipopolysaccharide (LPS) from E. coli, the phagocytic and oxidative burst activity from peripheral blood monocytes and granulocytes and the number of blood leukocytes and blood picture in cyclophosphamide-treated mice were tested. Bestatin at doses of 1 and 0.1 mg/kg was injected into cyclophosphamide-treated mice ip five times on alternating days or ten times at 24 h intervals. The first dose of bestatin was administered 24 h after a single injection of cyclophosphamide at a dose of 350 mg/kg. It was found that bestatin administered at doses of 1 and 0.1 mg/kg five times on alternating days increased the synthesis and release of IL-1ß by resident peritoneal murine macrophages stimulated in vitro with LPS in cyclophosphamide-treated mice. The immunocorrecting action of bestatin on the picture of peripheral blood in cyclophosphamide-treated mice was primarily observed with young forms of neutrophilic granulocytes. The changes were observed irrespective of the dosage and the number of subsequent doses applied. Moreover, the administration of bestatin after pharmacological immunosuppression partially prevented the suppressive effects of cyclophosphamide on the oxidative burst activity of peripheral blood monocytes and stimulated the phagocytic activity of granulocytes.