Deying Ma

Effect of Ligustrum lucidum and Schisandra chinensis on the egg production, antioxidant status and immunity of laying hens during heat stress

Abstract The experiment was conducted to evaluate the effect of two plants belonging to Chinese herbal medicines, Ligustrum lucidum (LL) and Schisandra chinensis (SC), on the laying performance, antioxidant status and immunity of hens during heat stress. The results showed that diets supplement with 1% of either LL or SC had beneficial effects on egg production and FCR of hens during heat stress (p < 0.05), compared with the control group. Either LL or SC significantly reduced malondialdehyde (MDA) concentration of heart, liver, sera and egg yolk. In addition, glutathione reductase (GR) activity of tissues and sera of the birds was significantly elevated by supplementation LL or SC. Furthermore, LL or SC supplementation significantly elevated lymphoblastogenese of the birds and the antibody values against Newcastle disease virus (NDV). The results suggest that diets supplement with 1% of either LL or SC may enhance egg production, immune function, and antioxidant status of hens during heat stress.

Expression and characterization of recombinant gallinacin-9 and gallinacin-8 in Escherichia coli.

Gallinacins (Gal) are antimicrobial peptides that play significant roles in innate immunity in chickens. Two Gal genes--Gal-8 and Gal-9--were cloned and sequenced from chicken liver and tongue, respectively, by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, the mRNA expression of these genes has been demonstrated across a panel of chicken tissues. It was demonstrated that Gal-9 mRNA was highly expressed in the tongue and small intestine and moderately expressed in the chicken proventriculus, lung, liver, heart, spleen, and thymus. However, Gal-8 mRNA was highly expressed in the chick small intestine and liver, and moderately expressed in the chick tongue, and lung. The recombinant fusion proteins containing Gal-9 or Gal-9 and Gal-8, namely rGal-9 and rGal-9-Gal-8, were produced and purified, respectively. Both rGal-9 and rGal-9-Gal-8 were expressed as insoluble bodies and exhibited the expected antimicrobial activity against Escherichia coli and pathogenic Streptococci suis CAB strain, as determined by the measurement of the inhibition zone and a liquid growth inhibition assay.

Three novel Anas platyrhynchos avian β-defensins, upregulated by duck hepatitis virus, with antibacterial and antiviral activities.

Three novel Anas platyrhynchos avian β-defensins (Apl_AvBDs), Apl_AvBD4, 7 and 12, were identified successfully and characterized in tissues from Peking ducks in the present study. The cDNA fragment of Apl_AvBD4 contained 171 bp, and encoded 56 amino acids. The complete nucleotide sequences of Apl_AvBD7 and 12 contained 204 bp and 198 bp open reading frames, which encoded 67 and 65 amino acids, respectively. Both recombinant and synthetic forms of the three Apl_AvBDs showed antibacterial activity against most of the bacteria investigated, including Gram-negative and Gram-positive bacteria, except for Salmonella choleraesuis. In addition, the antibacterial activity of all the three Apl_AvBDs decreased significantly in 150 mM NaCl. Significant antiviral activity of the three Apl_AvBDs was shown against duck hepatitis virus (DHV). However, none of the Apl_AvBDs showed significant hemolytic activity. Additionally, the expressions of the three Apl_AvBDs in response to DHV infection was highly variable, and significant upregulation of Apl_AvBD7 in liver was found in response to infection at different time points. Expression of Apl_AvBD4 in thymus, and of Apl_AvBD7 in bone marrow was induced in a time-dependent fashion by DHV infection. In contrast, expression of Apl_AvBD12 was found to be significantly decreased, and was hard to detect in cecal tonsil, spleen, bursa of Fabricius, and thymus of ducks at some time points after DHV infection. The present results demonstrate that Apl_AvBDs play vital roles in the immune response of ducks against bacterial and viral pathogens.

Identification and characterization of a novel antibacterial peptide, avian β-defensin 2 from ducks

In this study, a novel avian β-defensin (AvBD) was isolated from duck pancreas. The complete nucleotide sequence of the gene contained an 195 bp open reading frame encoding 64 amino acids. Homology, characterization and comparison of the gene with AvBD from other avian species confirmed that it was duck AvBD2. The mRNA expression of the gene was analyzed in 17 tissues from 21-day-old ducks. AvBD2 was highly expressed in the trachea, crop, heart, bone marrow, and pancreas; moderately expressed in the muscular stomach, small intestine, kidney, spleen, thymus, and bursa of Fabricius; and weakly expressed in skin. We produced and purified recombinant AvBD2 by expressing the gene in Escherichia coli. As expected, the recombinant peptide exhibited strong bactericidal properties against Bacillus cereus, Staphylococcus aureus, and Pasteurella multocida, and weak bactericidal properties against E. coli and Salmonella choleraesuis. In addition, the recombinant protein retained antimicrobial activity against S. aureus under different temperatures (range, −20°C to 100°C) and pH values (range, 3 to 12)

Identification and characterization of an avian β-defensin orthologue, avian β-defensin 9, from quails

In this study, a newly identified avian β-defensin (AvBD) orthologue was isolated from Chinese painted quail (Coturnix chinensis) lung and bone marrow tissues. The complete nucleotide sequence of the gene contained a 204-bp open-reading frame encoding 67 amino acids. Homology, characterization, and comparison of the gene with AvBD from other avian species confirmed that it was quail AvBD9. To analyze and compare the expression pattern of AvBD9 in tissues from young and adult quails, layer hens, and broilers, reverse transcription polymerase chain reaction was performed using mRNA isolated from 21 different tissues. The AvBD9 expression pattern distribution was differed among quails of different ages, layer hens, and broilers. It was widely expressed in all the tissues except the trachea, liver, and kidney and was highly expressed in the lung and heart of young quails. Similarly, it was widely expressed in all the tissues of adult quails except for the liver, kidney, spleen, thymus, and Harderian gland. In layer hens, AvBD9 was widely expressed in all the tissues except the trachea, glandular stomach, and cecum. Similarly, it was widely expressed in all the tissues of broilers except for the trachea, glandular stomach, rectum, cecum, bone marrow, and cecal tonsil. Recombinant AvBD9 (rAvBD9) was produced and purified by expressing the gene in Escherichia coli. Additionally, peptide according to quail AvBD9 sequence was synthesized, named sAvBD9. As expected, both rAvBD9 and sAvBD9 exhibited strong bactericidal properties against 11 strains of bacteria, including Gram-positive and Gram-negative forms.

Functional analysis and induction of four novel goose (Anser cygnoides) avian β-defensins in response to salmonella enteritidis infection

In the current study, four novel avian β-defensins (AvBDs) (AvBD2, 5, 9, and 10) were identified in tissues from the Chinese goose (Anser cygnoides). The antibacterial activity of the AvBDs showed that all of these AvBDs exhibited antibacterial activity against most of the bacteria investigated (P<0.01). In addition, antibacterial activity of all of the AvBDs against Staphylococcus aureus and Pasteurella multocida decreased significantly or was completely abolished at 150mM NaCl (P<0.01). None of the AvBDs showed hemolytic activity. AvBD2 and AvBD10 were expressed widely, whereas AvBD5 and AvBD9 mRNAs were expressed in a limited number of geese tissues. AvBD9 was significantly induced in some immune tissues from geese after Salmonella enteritidis infection. The others were significantly upregulated in small intestine and some immune tissues of the geese (P<0.01). The present results suggest that the AvBDs are part of the host defense mechanism of the goose.

Identification, Expression and Activity Analyses of Five Novel Duck Beta-Defensins

In the current study, five novel avian β-defensins (AvBDs) were identified and characterized in tissues from Peking ducks (Anas platyrhynchos). The nucleotide sequences of these cDNAs comprised 198 bp, 182 bp, 201 bp, 204 bp, and 168 bp, and encoded 65, 60, 66, 67, and 55 amino acids, respectively. Homology, characterization and comparison of these genes with AvBD from other avian species confirmed that they were Apl_AvBD1, 3, 5, 6, and 16. Recombinant AvBDs were produced and purified by expressing these genes in Escherichia coli. In addition, peptides were synthesized according to the respective AvBD sequences. Investigation of the antibacterial activity of the Apl_AvBDs showed that all of them exhibited antibacterial activity against all 12 bacteria investigated (P<0.05 or P<0.01). In addition, the antibacterial activity of all of the AvBDs against M. tetragenus and P. multocida decreased significantly in the presence of 150 mM NaCl (P<0.01). None of the AvBDs showed hemolytic activity. Consistent with their broad-spectrum antibacterial activity, the five novel Apl_AvBDs inhibited replication of duck hepatitis virus (DHV) in vitro significantly (P<0.05). The mRNA expression of all five Apl_AvBD in most tissues, including immune organs and the liver, was upregulated in response to DHV infection at different time points. These findings provide evidence that these defensins activate the immune response to combat microbial infection.

Discovery and characterization of Coturnix chinensis avian β-defensin 10, with broad antibacterial activity.

A novel avian β‐defensin (AvBD), AvBD10, was discovered in the liver and bone marrow tissues from Chinese painted quail (Coturnix chinensis) in the present study. The complete nucleotide sequence of quail AvBD10 contains a 207‐bp open reading frame that encodes 68 amino acids. The quail AvBD10 was expressed widely in all the tissues from quails except the tongue, crop, breast muscle, and thymus and was highly expressed in the bone marrow. In contrast to the expression pattern of AvBD10 in tissues from quail, the chicken AvBD10 was expressed in all 21 tissues from the layer hens investigated, with a high level of expression in the kidney, lung, liver, bone marrow, and Harderian glands. Recombinant glutathione S‐transferase (GST)‐tagged AvBD10s of both quail and chicken were produced and purified by expression of the two cDNAs in Escherichia coli, respectively. In addition, peptide according to the respective AvBD10s sequence was synthesized, named synthetic AvBD10s. As expected, both recombinant GST‐tagged AvBD10s and synthetic AvBD10s of quail and chicken exhibited similar bactericidal properties against most bacteria, including Gram‐positive and Gram‐negative forms. However, no significant bactericidal activity was found for quail recombinant GST‐tagged AvBD10 against Salmonella choleraesuis or for chicken recombinant GST‐tagged AvBD10 against Proteus mirabilis. Copyright

Identification of three novel avian beta-defensins from goose and their significance in the pathogenesis of Salmonella.

Here, we report the characterization of three avian β-defensins (AvBDs) from the goose, named anser_AvBD1, AvBD3, and AvBD6, respectively. All of anser_AvBDs exhibited broad antibacterial activity. In addition, the antibacterial activity of all of the AvBDs against Staphylococcus aureus and Proteus mirabilis decreased significantly in the presence of 100mM NaCl (P<0.01). None of the AvBDs showed hemolytic activity. In order to assess the significance of these anser_AvBDs in the infection of Salmonella enteritidis, mRNA expression of Toll-like receptor (TLR) 4 and anser_AvBDs in tissues of both control and infected geese was evaluated. We observed a significant up-regulation of TLR4, anser_AvBD1, 3, and 6 in some immune tissues evaluated, in response to S. enteritidis infection. These data demonstrated that TLR4 may serve a possible role in eliciting host immune responses to pathogens, and β-defensins may play a pivotal role in the host defense mechanisms of the goose.

Influence of mannan oligosaccharide, Ligustrum lucidum and Schisandra chinensis on parameters of antioxidative and immunological status of broilers.

Abstract The study was conducted to evaluate effects of dietary supplementation with Ligustrum lucidum (LL, 10 g/kg), Schisandra chinensis (SC, 10 g/kg), LL (10 g/kg) + mannan oligosaccharides (MOS, 50 mg/kg), or SC (10 g/kg) + MOS (50 mg/kg) on growth performance and parameters of antioxidative and immunological status of broilers. The results showed that feeding LL, SC, LL + MOS, or SC + MOS had no significant effect on growth performance of broilers relative to the control. However, compared to the control, LL, SC, LL + MOS, or SC + MOS significantly decreased malondialdehyde concentration in serum, thigh, and heart of broilers. In addition, glutathione reductase activity of heart and sera of the birds were significantly elevated by supplementation LL, SC, LL + MOS, or SC + MOS. Furthermore, LL, SC, LL + MOS, or SC + MOS significantly improved antibody titres against Newcastle disease virus and lymphocyte proliferation of broilers (p < 0.05). Whereas, no cooperating effect between LL (or SC) and MOS on antioxidant status and immunity of broilers were found.