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Jin L.,Dalian University of Technology | Jin L.,Ministry of Education Center for Food Safety of Animal Origin | Li X.,Dalian University of Technology | Li X.,Ministry of Education Center for Food Safety of Animal Origin | And 5 more authors.
Aquaculture Research | Year: 2013

Egg yolk immunoglobulins (IgY) were obtained from laying hens immunized with inactivated Aeromonas hydrophila. The purified IgY was shown to inhibit the growth of A. hydrophila in vitro and the optimum concentration for inhibition of A. hydrophila-specific IgY was 75 mg mL-1. In a subsequent challenge trial, 100 carp (200~250 g) were assigned to one of ten tanks with ten carp per tank. The fish in one tank were unchallenged whereas the remaining 90 fish were injected intraperitoneally with 100 μL of A. hydrophila at a concentration of 108 cfu mL-1. For the next 21 days, all fish were moved in their respective groups to a clean tank for 20 min day-1. The fish in four tanks (one unchallenged tank and three challenged tanks) received no treatment whereas the fish in the remaining six tanks were immersed in either 0.5 g L-1 aqueous nonspecific IgY (N = 3) or 0.5 g L-1 aqueous specific IgY (N = 3). Haemoglobin concentrations, white and red blood cell numbers as well as the mortality of specific IgY-treated fish were significantly different from those of the control. © 2012 Blackwell Publishing Ltd.

Li X.,Dalian University of Technology | Li X.,Ministry of Education Center for Food Safety of Animal Origin | Wang L.,Dalian University of Technology | Wang L.,Ministry of Education Center for Food Safety of Animal Origin | And 4 more authors.
Journal of Animal Science and Biotechnology | Year: 2015

In recent years, the use of in-feed antibiotics for growth and disease prevention in livestock production has been under severe scrutiny. The use and misuse of in-feed antibiotics has led to problems with drug residues in animal products and increased bacterial resistance. Chicken egg yolk antibodies (IgY) have attracted considerable attention as an alternative to antibiotics to maintain swine health and performance. Oral administration of IgY possesses many advantages over mammalian IgG such as cost-effectiveness, convenience and high yield. This review presents an overview of the potential to use IgY immunotherapy for the prevention and treatment of swine diarrhea diseases and speculates on the future of IgY technology. Included are a review of the potential applications of IgY in the control of enteric infections of either bacterial or viral origin such as enterotoxigenic Escherichia coli, Salmonella spp., rotavirus, porcine transmissible gastroenteritis virus, and porcine epidemic diarrhea virus. Some potential obstacles to the adoption of IgY technology are also discussed. © 2015 Li et al.

Cao Z.,Dalian University of Technology | Zhang J.,Dalian University of Technology | Niu Y.D.,Agriculture Center | Cui N.,Shanxi Datong University | And 7 more authors.
PLoS ONE | Year: 2015

January The objective of this study was to investigate the potential of using phages as a therapy against hemorrhagic pneumonia in mink both in vitro and in vivo. Five Pseudomonas aeruginosa (P. aeruginosa) strains were isolated from lungs of mink with suspected hemorrhagic pneumonia and their identity was confirmed by morphological observation and 16S rDNA sequence analysis. Compared to P. aeruginosa strains isolated from mink with hemorrhagic pneumonia in 2002, these isolates were more resistant to antibiotics selected. A lytic phage vB-PaeP-PPA-ABTNL (PPA-ABTNL) of the Podoviridae family was isolated from hospital sewage using a P. aeruginosa isolate as host, showing broad host range against P. aeruginosa. A one-step growth curve analysis of PPA-ABTNL revealed eclipse and latent periods of 20 and 35 min, respectively, with a burst size of about 110 PFU per infected cell. Phage PPA-ABTNL significantly reduced the growth of P. aeruginosa isolates in vitro. The genome of PPA-ABTNL was 43,227 bp (62.4% G+C) containing 54 open reading frames and lacked regions encoding known virulence factors, integration-related proteins and antibiotic resistance determinants. Genome architecture analysis showed that PPAABTNL belonged to the "phiKMV-like Viruses" group. A repeated dose inhalational toxicity study using PPA-ABTNL crude preparation was conducted in mice and no significantly abnormal histological changes, morbidity or mortality were observed. There was no indication of any potential risk associated with using PPA-ABTNL as a therapeutic agent. The results of a curative treatment experiment demonstrated that atomization by ultrasonic treatment could efficiently deliver phage to the lungs of mink and a dose of 10 multiplicity of infection was optimal for treating mink hemorrhagic pneumonia. Our work demonstrated the potential for phage to fight P. aeruginosa involved in mink lung infections when administered by means of ultrasonic nebulization. © 2015 Cao et al.

Li J.,Dalian University of Technology | Xu Y.,Dalian University of Technology | Xu Y.,Ministry of Education Center for Food Safety of Animal Origin | Wang X.,Dalian University of Technology | And 5 more authors.
International Immunopharmacology | Year: 2016

The purpose of this study was to construct a single-chain variable fragment (scFv) antibody from chicken egg yolk immunoglobulin (IgY) by means of genetic engineering and subsequent panning for a specific antibody against Staphylococcus aureus. Methods and results We amplified the scFv using blood and spleen obtained from 100-day-old Roman chickens immunized with inactivated S. aureus and subsequently constructed a T7 phage display antibody library using phage display technology. Four non-repeated blood scFv and 6 spleen scFv were obtained following 3 rounds of panning of the T7 phage display antibody library, enzyme-linked immunosorbent assay and sequencing. These 10 scFv were cloned into the prokaryotic expression vector pCold I with expression induced at a low temperature. Four soluble proteins were obtained. Among them, soluble protein SFV6 derived from the spleen showed good reactivity against S. aureus using indirect ELISA and produced a particularly strong antibacterial effect in vitro. Conclusion We were successful in isolating a highly specific scFv antibody against S. aureus from the spleen phage display library. This study provides a simple and rapid method for the quick preparation of a large number of antibodies against S. aureus and provides the foundation for the positioning of antibodies in the organism and the study of the antibacterial mechanism through which the antibody functions. © 2016 Elsevier B.V. All rights reserved.

Wang X.,Dalian University of Technology | Wang L.,Dalian University of Technology | Li X.,Dalian University of Technology | Li X.,Ministry of Education Center for Food Safety of Animal Origin | And 2 more authors.
Electronic Journal of Biotechnology | Year: 2016

Background: The alga Laminaria japonica is the most economically important brown seaweed cultured in China, which is used as food and aquatic animal feedstuff. However, the use of L. japonica as a feedstuff in Apostichopus japonicas farming is not ideal because A. japonicas does not produce enough enzyme activity for degrading the large amount of algin present in L. japonica. In this study, semi solid fermentation of the L. japonica feedstuff employing a Bacillus strain as the microbe was used to as a mean to degrade the algin content in L. japonica feedstuff. Results: The Bacillus strain, Bacillus amyloliquefaciens WB1, was isolated by virtue of its ability to utilize sodium alginate as the sole carbon source. Eight factors affecting growth and algin-degrading capacity of WB1 were investigated. The results of Plackett–Burman design indicated that fermentation time, beef extract, and solvent to solid ratio were the significant parameters. Furthermore, the mutual interaction between the solvent to solid ratio and beef extract concentration was more significant than the other pairs of parameters on algin degradation. Optimal values obtained from Central-Composite Design were 113.94 h for fermentation time, 0.3% (w/v) beef extract and 44.87 (v/w) ratio of solvent to feedstuff. Under optimal conditions, 56.88% of the algin was degraded when a 50-fold scale-up fermentation was carried out, using a 5-L fermenter. Conclusions: This study provides an alternative and economical way to reduce the algin content in L. japonica through degradation by WB1, making it a promising potential source of feed for cultured L. japonica. © 2016 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved.

Li Z.,Dalian University of Technology | Li X.,Dalian University of Technology | Zhang J.,Dalian University of Technology | Wang X.,Dalian University of Technology | And 4 more authors.
Fish and Shellfish Immunology | Year: 2016

In the present study, we isolated 3 bacteriophages with the ability to control Vibrio splendidus, a bacterium known to cause disease in the juvenile sea cucumber. These bacteriophages were designated as vB_VspS_VS-ABTNL-1 (PVS-1), vB_VspS_VS-ABTNL-2 (PVS-2) and vB_VspS_VS-ABTNL-3 (PVS-3). The ability of the 3 phages to inhibit the growth of V. splendidus VS-ABTNL was tested in vitro using each of the 3 phages individually or in the form of a cocktail of all 3 phages in the proportion of 1:1:1. All treated cultures produced a significant (P < 0.05) inhibition of growth of V. splendidus VS-ABTNL compared with untreated V. splendidus VS-ABTNL with the cocktail being superior to any of the 3 phages used individually. The lytic capability of the 3 phages was subsequently determined with a Spot Assay Technique performed with 4 isolates of V. splendidus, 3 other Vibrio species and 2 environmental isolates. Both PVS-1 and PVS-2 were lytic to all 4 isolates of V. splendidus while PVS-3 only inhibited the growth of 3 of them. V. splendidus VS-ABTNL was more susceptible to phage PVS-2 than the other 2 phages. In an in vivo performance trial, 360 sea cucumbers (23 ± 2 g) were randomly assigned to 1 of 6 treatments. Each treatment was housed in 3 PVC tanks (38 cm × 54 cm × 80 cm) with 20 sea cucumbers per tank. Six diets were prepared including an unsupplemented control diet, antibiotic treatment diet, 3 diets containing 1 of the 3 phages individually and a diet containing a cocktail of all 3 phages. After 60 days of feeding, all sea cucumber were challenged with V. splendidus VS-ABTNL by immersion in sea water containing a bacterial concentration of 6 × 106 CFU/mL for 2 days. The survival rate of sea cucumbers during the next 10 days was 18% for the unsupplemented diet, 82% for the antibiotic treatment, 82% for the phage cocktail, 65% for phage PVS-1, 58% for phage PVS-2 and 50% for phage PVS-3. There were no significant differences in weight gain, ingestion rate or feed conversion among sea cucumber fed the 4 phage treatments compared with those fed the unsupplemented diet (P > 0.05). The levels of nitric oxide synthase and acid phosphatase of sea cucumbers fed phage-containing diets were significantly (P < 0.05) increased compared with those fed the control diet. However, no significant differences (P > 0.05) were detected among the 4 phage-fed treatments. An additional study was conducted in which 60 healthy sea cucumbers (23 ± 2 g) were randomly assigned to a control, an untreated group and a test group to investigate the effects of injecting phages by coelomic injection on the survival rate and enzyme activities in the coelomic fluid of the sea cucumbers. The control was injected with 1 ml of sterilized seawater while the untreated group and the test group were injected with the same volume of V. splendidus-ABTNL culture (3 × 105 CFU/mL). Then, the test group was injected with 1 ml of the 3 phage cocktail (MOI = 10). After 48 h, the activities of lysozyme, acid phosphatase and superoxide dismutase were elevated in the untreated group while the levels of these enzymes in the test group were similar to the blank control. After 10-day observation, the survival rate of the sea cucumber was 100% for the blank control, 80% for the test group and 20% for the negative control. The overall results of this experiment indicate that phage therapy increased the survival of sea cucumber infected with V. splendidus VS-ABTNL. The above results demonstrate that using phages, especially a combination of different phages, may be a feasible way to control Vibrio infection in the sea cucumber industry. © 2016 Elsevier Ltd.

Wang T.,Dalian University of Technology | Wang T.,Dalian University | Xu Y.,Dalian University of Technology | Xu Y.,Ministry of Education Center for Food Safety of Animal Origin | And 8 more authors.
Protein Expression and Purification | Year: 2011

Apostichopus japonicus (sea cucumber) is one of the economically important farmed echinoderm species in Northern China. As a crucial enzyme in innate immunity, lysozyme plays a key role in the overall defense against pathogens in A. japonicus. In the present study, a lysozyme gene from A. japonicus was cloned by PCR and expressed in Pichia pastoris using the expression vector pPIC9K. The expressed lysozyme had a molecular mass of ∼14 kD, as shown by SDS-PAGE and Western-blotting. The expression condition was optimized, and the highest expression level was achieved by induction with 1% methanol at pH 5.0 for 120 h. The recombinant lysozyme was purified by affinity chromatography using a Ni-NTA column. The specific activity of the purified lysozyme was 34,000 U/mg using Micrococcus lysodeikticus as substrates. It exhibited antimicrobial activity toward M. lysodeikticus, as detected by growth inhibition on agar plate and turbidity assay, suggesting a potential application of A. japonicus lysozyme as an antimicrobial agent in A. japonicus aquaculture. © 2011 Elsevier Inc. All rights reserved.

Sha L.,Dalian University of Technology | Xu Y.-P.,Ministry of Education Center for Food Safety of Animal Origin
Indian Journal of Animal Research | Year: 2013

Fatty liver syndrome is a metabolic disorder that occurs in mink (Mustela vison). The aim of this study was to compare the effects of dietary fat sources (chicken oil, fish oil and soybean oil) on the recovery fatty liver syndrome in female mink in response to short term fasting for 5 days and subsequent re-feeding for 7-28 days. Food deprivation resulted in the rapid mobilization of body fat, causing an elevated hepatosomatic index and increased liver lipid content. Re-feeding resulted in a more pronounced recovery of liver health in the mink fed the fish oil diet than chicken oil or soybean oil diet. The fish oil diet was found to be useful to relieve the severity of hepatic lipidosis in mink.

Li Z.,Dalian University of Technology | Zhang J.,Dalian University of Technology | Li X.,Dalian University of Technology | Wang X.,Dalian University of Technology | And 5 more authors.
Aquaculture | Year: 2016

Vibro cyclitrophicus is thought to be responsible for the cause of severe infection in juvenile sea cucumber (Apostichopus japonicus). Increases in the prevalence of antibiotic resistant pathogens have promoted us to develop effective agents to antibiotics for combating microbial infection among animals as well as human. In the present study, we isolated a bacteriophage with the ability to cleave V. cyclitrophicus present in the sewage of sea cucumber farms. This bacteriophage was designated as phage vB_VcyS_Vc1 (Vibrio phage Vc1). A one-step growth curve analysis of the phage revealed eclipse and latent periods of 25 and 45 min, respectively, with a burst size of 215 PFU/infected cell. Morphological analysis revealed the phage belongs to family of Siphoviridae. Furthermore, genomic sequencing results revealed a double-stranded DNA containing 44,541 bp with a G + C content of 44.16%. Forty four coding sequences were annotated in the genome, and nineteen of these were associated with a known function. Genes related to virulence and toxins were not detected in the genome. In addition, a prevention experiment conducted in a marine environment demonstrated that the phage increased the survival rate of juvenile sea cucumbers (18 ± 2 g) from 18% to 81% when the sea cucumber were fed with feedstuff containing the freeze-dried phage powder, 58% when the sea cucumber was injected with purified phages (MOI = 10), and 63% when the sea cucumbers were immersed in a suspension containing purified phages. Notably, when the phage provided nearly the same protection to the sea cucumbers as antibiotic when it was fed to the sea cucumbers in the form of freeze-dried powder mixed with feedstuff. Taken together, the results demonstrated that the use of phage to control the infection of V. cyclitrophicus in sea cucumber may be a feasible alternative to antibiotics. Statement of relevance: The A. japonicus (sea cucumber) is the most profitable aquaculture animal with the highest output as single variety in China, which is of high nutritional value and economic value. In the past decade, as the growth of the market demand, A. japonicus's artificial breeding scale expands rapidly and the sea cucumber industry has become a vigorous sector in China aquaculture. However, with the high density and intensive development model of A. japonicus breeding, the various diseases have resulted in serious economic losses. Skin ulceration syndrome (SUS) is one of the most epidemic and serious diseases that affect sea cucumbers. At present, sanitizer and antibiotics are still commonly used in farms or hatcheries to control the bacterial disease in juvenile sea cucumbers, which resulted in the environment pollution and antibiotic residues. Nowadays, increased appearance of antibiotic resistant phenomenon promoted us to develop effective agents. Hence, alternative strategies to antibiotics should be further developed. This study was the first to monitor the effectiveness of using feedstuff mixed with freeze-dried phage powder as a form of protection for sea cucumber against vibrio infection, and demonstrated the effectiveness of phage in the control of pathogen in aquaculture. © 2015 Elsevier B.V.

Li X.,Dalian University of Technology | Li X.,Ministry of Education Center for Food Safety of Animal Origin | Jing K.,Dalian University of Technology | Wang X.,Dalian University of Technology | And 8 more authors.
Fish and Shellfish Immunology | Year: 2016

Vibrio splendidus is one of the most harmful pathogens associated with skin ulceration syndrome in the sea cucumber (Apostichopus japonicus) due to its high virulence and frequency of appearance. The objective of this study was to determine the effectiveness of chicken egg yolk antibody (IgY) against V. splendidus infection in the sea cucumber. Whole V. splendidus cells were used as an immunogen to immunize 20 White Leghorn hens (25 weeks old). IgY was produced from egg yolks obtained from these immunized hens using water dilution, two-step salt precipitation and ultrafiltration. The purity of the IgY produced was approximately 83%. Enzyme-linked Immunosorbent Assay indicated a high specificity for IgY with a maximum antibody titer of 320,000. The growth of V. splendidus in liquid medium was significantly inhibited by IgY in a dose-dependent manner at concentrations ranging from 1 to 10 mg/mL. The protective effects of IgY were evaluated in sea cucumber by intraperitoneally injecting anti- V. splendidus IgY antibodies (10 mg/mL) or immersing the sea cucumber in aqueous IgY (1 g/L) after an intraperitoneal injection of V. splendidus. Intraperitoneal injection resulted in an 80% survival while immersion resulted in a 75% survival during the 11-day experimental period. The survival rates were significantly higher than the positive control and the non-specific IgY group (P < 0.05). As well, the bacterial burden in the respiratory tree, intestine and coelomic liquid was significantly (P < 0.05) lower in sea cucumber treated with specific IgY than those treated with non-specific IgY. The phagocytosis of coelomocytes for V. splendidus in the presence of specific IgY was significantly (P < 0.05) higher than that obtained with non-specific IgY or without IgY, suggesting that specific IgY enhanced phagocytic activity. The current work suggests that specific IgY has potential for protecting sea cucumbers against V. splendidus infection. © 2015 Elsevier Ltd.

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