[Establishment of human colon cancer transplantation tumor model in normal immune mice].

Objective: Establishment of a new model of human primary colon cancer transplantation tumor in normal immune mice and to provide a reliable experimental animal model for studying the pathogenesis of colon cancer under normal immunity. Methods: Human colon cancer cells come from colon cancer patients who underwent surgery in the Affiliated Hospital of Jining Medical College in 2017. The mice in the cell control group were inoculated with phosphate buffered solution (PBS) containing colon cancer cells, the microcarrier control group was inoculated with PBS containing microcarrier 6, and the cell-microcarrier complex group was inoculated with the PBS containing colon cancer cell-microcarrier complex. The cells of each group were inoculated under the skin of the right axilla of mice by subcutaneous injection, and the time, size, tumor formation rate and pathological changes under microscope were recorded. The transplanted tumor tissue was immunohistochemically stained with the EnVisiion two-step method, and the tumor formation rate of the transplanted tumor was judged according to the proportion of positive cells in the visual field. The polymerase chain reaction (PCR) method was used to detect the expression of human-specific Alu sequence in mice tumor tissue. Results: After inoculation with tumor cells, the mice in the cell control group and the microcarrier control group did not die and did not form tumors; the mice in the cell-microcarrier complex group had palpable subcutaneous tumors in the right axillary subcutaneously on the 5th to 7th days after inoculation, and tumor formation rate is 67% (10/15), and the tumor volume can reach about 500 mm(3) 2 to 3 weeks after vaccination. The immunohistochemistry results showed that CK20, CDX-2 and carcinoembryonic antigen were all positively expressed. The PCR results showed that the expression of human-specific Alu sequence can be detected in the transplanted tumor tissue of tumor-bearing mice. Conclusion: Human primary colon cancer cells used microcarrier 6 as a carrier to form tumors in normal immunized mice, and successfully established a new model of human colon cancer transplantation tumor in normal immune mice.

Xanthophylls increased HDLC level and nuclear factor PPARγ, RXRγ and RARα expression in hens and chicks.

This study was designed to investigate effects of xanthophylls on serum lipid profile (triglyceride, TG; cholesterol, CHO; high-density lipoprotein cholesterol, HDLC; and low-density lipoprotein cholesterol, LDLC) and nuclear factor (peroxisome proliferator-activated receptor gamma, PPARγ; PPAR gamma coactivator 1 alpha, PGC1α; retinoid X receptor gamma, RXRγ; and retinoic acid receptor alpha, RARα) gene expression of breeding hens and chicks. In experiment 1, 432 hens were divided into three groups and fed diets supplemented with 0 (as control group), 20 or 40 mg/kg xanthophylls. Blood was sampled at 7, 14, 21, 28 and 35 days of trial. Liver, duodenum, jejunum and ileum were sampled at 35 days of trial. Results showed that serum HDLC level of hens was increased after dietary 40 mg/kg xanthophyll addition for 21, 28 and 35 days, while serum TG, CHO and LDLC were not affected. Xanthophyll addition also increased PPARγ expression in jejunum, RXRγ expression in duodenum and jejunum, and RARα expression in liver and duodenum. Experiment 2 was a 2 × 2 factorial design. Male chicks hatched from 0 or 40 mg/kg xanthophyll diet of hens were fed diet containing either 0 or 40 mg/kg xanthophylls. Liver, duodenum, jejunum and ileum were sampled at 0, 7, 14 and 21 days after hatching. Blood samples were also collected at 21 days. Results showed that in ovo xanthophylls elevated PPARγ in duodenum and jejunum, and RXRγ and RARα in liver of chicks mainly within 1 week after hatching, while dietary xanthophylls increased serum HDLC level and PPARγ and RXRγ in liver from 2 weeks onwards. In conclusion, our research suggested xanthophylls can regulate serum lipid profile and nuclear factor expression in hens and chicks.

[Protective effect of intraperitoneal transplantation of human liver-derived stem cells at different times against concanavalin A-induced acute liver injury in mice].

Objective: To investigate the protective effect of intraperitoneal transplantation of human liver-derived stem cells at different times against concanavalin A (ConA)-induced acute liver injury in mice. Methods: A total of 88 male C57BL/6 mice were randomly divided into normal control group (group C), ConA model group (group M), and human liver-derived stem cells (HYX1)+ConA group (group E); according to the interval between phosphate buffer/HYX1 injection and ConA injection, Groups M and E were further divided into 3-hour groups (M1 and E1 groups), 6-hour groups (M2 and E2 groups), 12-hour groups (M3 and E3 groups), 24-hour groups (M4 and E4 groups), and 48-hour groups (M5 and E5 groups). The levels of alanine aminotransferase (ALT), aspartate transaminase (AST), and total bilirubin (TBil) in peripheral blood were measured, liver tissue sections were used to observe pathological changes, and the Ishak score for liver inflammation was determined. The independent samples t-test was used for comparison between groups, and P < 0.05 was considered statistically significant. Results: The levels of ALT, AST, and TBil in group C were (36.25±1.16) U/L, (120.20±5.77) U/L, and (2.20±0.23) µmol/L, respectively; the levels of ALT, AST, and TBil and Ishak score were (8 721.23±837.39) U/L, (8 110.31±290.10) U/L, (8.41±0.10) µmol/L, and (13.32±1.30), respectively, in group M1, (8 334.31±666.50) U/L, (7 560.20±760.34) U/L, (10.40±0.80) µmol/L, and (12.67±0.81), respectively, in group M2, (8 960.75±551.93) U/L, (8 535.62±675.14) U/L, (10.95±1.43) µmol/L, and (14.57±0.65), respectively, in group M3, (8 618.57±886.40) U/L, (11 440.54 ± 1 327.86) U/L, (13.30±1.86) µmol/L, and (13.21±1.06), respectively, in group M4, and (10 170.13±1 112.37) U/L, (11 470.56±1 108.40) U/L, (12.75±1.55) µmol/L, and (15.07±1.58), respectively, in group M5. The levels of ALT, AST, and TBil and Ishak score were (1 016.35±163.47) U/L, (952.30±103.91) U/L, (7.77±0.62) µmol/L, and (3.50±0.21), respectively, in group E1, (42.10±6.20) U/L, (126.72±13.33) U/L, (3.41±0.53) µmol/L, and (2.01±0.40), respectively, in group E2, (44.21±4.30) U/L, (216.71±35.88) U/L, (3.47±0.44) µmol/L, and (2.13±0.25), respectively, in group E3, (2 909.69±212.14) U/L, (2 988.43±333.70) U/L, (7.03±0.93) µmol/L, and (4.70±0.50), respectively, in group E4, and (7 874.26±799.60) U/L, (10 940.54±947.35) U/L, (10.53±1.09) µmol/L, and (8.60±0.83), respectively, in group E5. Groups M1-M5 had significantly higher levels of ALT, AST, and TBil than group C (all P < 0.01), and groups M1-M4 had significantly higher levels of AST and ALT than groups E1-E4 (all P < 0.01), while there were no significant differences in the levels of AST and ALT between groups M5 and E5 (both P > 0.05). The pathological sections of liver tissue showed that compared with group M, group E had significant reductions in the degree of necrosis and Ishak score (both P < 0.05). Conclusion: Intraperitoneal transplantation of human liver-derived stem cells has a protective effect against ConA-induced acute liver injury in mice, and the injection at 6 and 12 hours in advance has the best protective effect.

Xanthophyll supplementation reduced inflammatory mediators and apoptosis in hens and chicks.

This study investigated effects of xanthophylls (containing 40% lutein and 60% zeaxanthin) on gene expression of inflammatory mediators ( [] and []) and apoptosis ( [] and ) of breeding hens and chicks. In Exp. 1, 432 hens were divided into 3 groups and fed diets supplemented with 0 (as the control group), 20, or 40 mg/kg xanthophylls. The liver, duodenum, jejunum, and ileum were sampled after 35 d. Results showed that 40 mg/kg of xanthophyll addition decreased in the liver, in the liver and duodenum, and in the liver and jejunum while increasing level in the liver and jejunum. Experiment 2 was a 2 × 2 factorial design. Male chicks hatched from hens fed 0 or 40 mg/kg xanthophyll diets were fed diets containing either 0 or 40 mg/kg xanthophylls. The liver, duodenum, jejunum, and ileum were sampled at 0, 7, 14, and 21 d after hatching. Results showed that in ovo xanthophylls reduced inflammatory mediators and apoptosis in the liver, duodenum, and jejunum of chicks mainly within 1 wk after hatching, whereas dietary xanthophylls only decreased expression in the liver from 2 wk onward. These results underlined important anti-inflammatory and antiapoptotic effects of maternal but not progeny dietary xanthophylls. In conclusion, xanthophylls can suppress inflammatory mediators and apoptosis in different tissues of hens and chicks.

Different combinations of probiotics improve the production performance, egg quality, and immune response of layer hens.

To evaluate the effects of different combinations of probiotics on performance, egg quality, and immune response of layer hens, a trial was carried out with 1,800 white feather layer hens of the Lohmann variety. The experiment was conducted by using a completely randomized design with 9 treatments, 4 replicates, and 50 hens in each replicate. Compared with the control group, group F, which added a composition of heat-inactivated Lactobacillus salivarius(CB) and Bacillus subtilis to the diets of layer hens, caused highly significant (P < 0.05) increases in egg production, daily egg yield, damaged egg ratio, combined with a significant (P < 0.05) decrease in feed conversion and damaged egg ratio. Group G, adding a combination of inactivated Lactobacillus salivarius and sodium butyrate, resulted in a significant increase (P < 0.05) in daily egg yield, feed conversion, damaged egg ratio and Haugh unit. Meanwhile, groups D and H had significantly decreased feed conversion (P < 0.05), and groups B, H, and I had a significantly decreased damaged egg ratio. In serum levels, no significant difference was observed except a significant decrease (P < 0.05) in total cholesterol (groups D, E, and G) and low-density lipoprotein cholesterol (group E and G) and a significant increase (P < 0.05) in total cholesterol (groups D, E, and G) compared with group A. According to the hemagglutination inhibition test, the antibody titer of antibody against the avian influenza virus was significantly (P < 0.05) higher in most treated groups such as groups B, C, E, G, and I after d 15 fed to layers with probiotics and groups B, C, D, E, F, G, and H after d 45 compared with the control group. No significant difference was observed in the antibody titer against the Newcastle disease virus at d 15, but significantly (P < 0.05) higher at d 45 in groups F and G. These results demonstrate that several combinations of probiotics used in this experiment have a positive impact on the performance, egg quality, and immune response of layer hens, and the following work will continue to focus on these groups.

Development of a fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification assay for rapid detection of seasonal Japanese B encephalitis outbreaks in pigs.

The standardization and validation of a one-step, single-tube, accelerated fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the NS3 gene of Japanese B encephalitis virus (JEV) is described for rapid, simple, and high-throughput detection of JEV. The amplification can be completed in 35 min under isothermal conditions at 63°C by employing a set of six primers targeting the NS3 gene of JEV. The RT-LAMP assay described demonstrated high sensitivity for detecting JEV, with a detection limit in swine samples of 8.13 PFU/ml. The specificity of the selected primer sets was established by cross-reactivity studies with pathogens that exhibit similar clinical signs and testing of samples from healthy animals. The clinical applicability of the RT-LAMP assay was validated using either spiked samples or samples from seasonal outbreaks. The comparative evaluation of the RT-LAMP assay revealed 79.59 % concordance with conventional RT-PCR targeting the E gene of JEV. The RT-LAMP assay reported here is a valuable tool for rapid real-time and high-throughput seasonal infection surveillance and quarantine after outbreak through blood sampling by using ordinary real-time PCR thermocyclers without purchasing an expensive Loopamp real-time turbidimeter.

Cloning and phylogenetic analysis of hemagglutinin gene of H9N2 subtype avian influenza virus from different isolates in China during 2002 to 2009.

Forty-seven strains of H9 subtype avian influenza viruses identified by specific reverse transcription-PCR method were isolated from the chicken and duck flocks in different areas of China during the 2002 to 2009 epizootic period. Hemagglutinin (HA) genes of these strains were sequenced and analyzed with the representative strains published in GenBank. The results indicated that the HA genes of these strains and the vaccine strains displayed nucleotide homologies ranging from 91.7 to 96.6% and amino acid homologies ranging from 92.3 to 95.7%, respectively. Analysis of the mature peptide sequences of these HA genes showed that the presence of leucine at position 216 (corresponding to residue 226 in H3 numbering) indicated a preference to the binding of alpha (2-6) sialic acid receptors, which was the same as human isolates. Extra potential glycosylation sites appeared in the HA genes of most tested isolations compared with the vaccine strains. The HA cleavage sites of most of the strains were the 335PSRSSR downward arrow GLF341, but all of the strains met the characteristics of low-pathogenic avian influenza. The results of phylogenetic analysis indicated that all 47 strains and the current vaccine strains belong to the same phylogenetic lineage h9.4.2, but they had some genetic deviation in the last decade. Compared with the vaccine strains, 7 mutations were found in the antigen epitope region of the HA genes of the field strains. These results suggested that the commercial vaccine might not induce satisfactory prevention against infection of H9N2 avian influenza virus.

Immunopathological effects of ochratoxin A and T-2 toxin combination on broilers.

The purpose of this study was to investigate the immunopathological effects of combinations of ochratoxin A (OTA) and T-2 toxin on broilers. Four hundred eighty 1-d-old broilers were randomly assigned to 4 groups, each group consisting of 4 duplicates each with 30 broilers. The 4 groups were fed the following diets for 4 wk: group 1=basal diet (control, mycotoxin-free); group 2=basal diet+2,000 mg/kg of Mycofix Plus; group 3=basal diet+0.25 mg/kg of OTA and 0.5 mg/kg of T-2; and group 4=basal diet+0.25 mg/kg of OTA and 0.5 mg/kg of T-2+2,000 mg/kg of Mycofix Plus. The feeding of OTA-T-2 toxin diets reduced (P<0.05) the level of anti-Newcastle disease virus antibody titers by 10.4%. When broilers were administered lipopolysaccharide, the results of real-time PCR showed that broilers fed OTA-T-2 toxin reduced the cytokine mRNA expression levels of interleukin-2 and interferon-gamma to some extent but not significantly (P>0.05). The concentrations of interleukin-2 and interferon-gamma in serum were significantly decreased (P<0.05) by OTA-T-2 toxin combination. Histopathological studies demonstrated that OTA-T-2 toxin combination caused abnormalities in the thymus, bursa of Fabricius, spleen, and liver. Ochratoxin A-T-2 toxicity could be counteracted by Mycofix Plus partially but not significantly (P>0.05). The concentrations of OTA and T-2 toxin used in this study are under the maximum tolerated levels recommended by Canadian Food Inspection Agency. Our study clearly put the standard and detoxification method for these toxins into question. We suggest that it may be time to reduce the maximum allowable limits of OTA and T-2 mycotoxins in feeds to improve animal health and the safety of the food chain.

Molecular detection of Muscovy duck parvovirus by loop-mediated isothermal amplification assay.

Muscovy duck parvovirus (MDPV) usually causes high morbidity and mortality in 1- to 3-wk-old Muscovy ducklings due to serious infections, which is an imminent threat to the commercial duck industry in China. The objectives of this study were to develop and evaluate a simple, rapid, and inexpensive loop-mediated isothermal amplification (LAMP) method for specific detection of MDPV and to compare it with the PCR method in rapidity, sensitivity, and accuracy. The novel LAMP assay used a set of 4 specific primers to recognize 6 distinct genomic sequences of capsid protein (VP3) from MDPV, which could be completed within 50 min at 63 degrees C in a simple water bath. The diagnostic results demonstrated that the LAMP assay detected all 7 preserved MDPV isolates, had no cross-reactivity with other duck pathogens (i.e., goose parvovirus, duck plague virus, H9N2 avian influenza virus, duck hepatitis type virus I, and Muscovy duck reovirus). The LAMP assay was at least 10-fold more sensitive than the routine PCR assay and obtained more sensitivity in 61 clinical samples. Therefore, the newly developed LAMP assay provides a specific and sensitive means for detecting MDPV and can be simply applied both in field conditions and in laboratory operations in a cost-effective manner with primary care facilities.

Preparation and immune activity analysis of H5N1 subtype avian influenza virus recombinant protein-based vaccine.

Avian influenza is a severe disease among farmed poultry and free-living birds and a constant threat to the commercial chicken industry around the world. Hemagglutinin (HA) is the major immunogen on the envelope of influenza A virus and is the predominant inducer of neutralizing antibody. To obtain the bioactive antigen proteins in large quantities, a new protein expression vector pBCX was constructed, which is based on the pET32a vector. The HA gene of the H5N1 subtype of avian influenza virus (AIV) was inserted into the pBCX vector and expressed efficiently in Escherichia coli BL21 (DE3). Fused expression of the exogenous gene and msyB produced a 97-kDa msyB-HA fusion protein. Sodium dodecyl sulfate-PAGE combined with scanning analysis demonstrated that the msyB-HA fusion protein accounted for 29.5% of the total bacterial protein, 90.5% being soluble. The msyB-HA fusion protein was purified with nondenaturing 50% Ni-NTA column chromatography, and the result showed that 24 mg of purified msyB-HA fusion protein could be obtained from 1 L of induced expression bacterial culture medium. The comparative results in the present study showed that pBCX was superior to pET32a as a protein expression vector. Western blotting showed the recombinant msyB-HA (rHA) to have better antigenic activity, which may be the result from the better posttranslation protein modification and folding in the pBCX expression system. With the rHA fusion protein as antigen, we successfully prepared and screened specific monoclonal antibodys against the H5N1 subtype AIV, which indicated that the rHA had antigen epitopes and biofunctions. The immune test confirmed that the rHA protein vaccine could also induce high neutralizing antibodies, and the AIV challenge test proved that the rHA protein-based vaccine could prevent the corresponding infection. This study demonstrates that the recombinant HA protein produced by the pBCX expression system could be used as a recombinant protein-based vaccine and has potential for further development for diagnosis.

Effects of combinations of ochratoxin A and T-2 toxin on immune function of yellow-feathered broiler chickens.

The study was to investigate the effects of combinations of ochratoxin A (OTA) and T-2 toxin on immune function of yellow-feathered broiler chickens. Three-hundred sixty 21-d-old broiler chickens were randomly assigned to 3 groups, each group consisting of 4 duplicates each with 30 chickens. The 3 groups were fed the following diets for 3 wk: C, basal diet (control, mycotoxin-free); L, basal diet + 0.25 mg/kg of OTA, 0.5 mg/kg of T-2 toxin; and H, basal diet + 0.5 mg/kg of OTA, 1 mg/kg of T-2 toxin. Body weight and feed consumption of chickens in the H group decreased significantly (P < 0.05) during the study, but their efficiency of feed utilization was not affected. The feeding of OTA-T-2 toxin diets decreased not only the relative weight of spleen, thymus, and bursa of Fabricius, but also serum concentrations of total protein, albumin, and globulin. Meanwhile, the feeding of OTA-T-2 toxin diets elevated the activities of serum gamma-glutamyltransferase, asparate aminotransferase, and alanine aminotransferase. The results of methyl thiazolyl tetrazolium reduction assay indicated that the mitogenic responses of peripheral blood lymphocytes were diminished significantly (P < 0.05 for L group; P < 0.01 for H group). Flow cytometry was employed to determine 3 indexes in peripheral blood lymphocyte of broilers, including CD4(+)/CD3(+), CD8(+)/CD3(+), and CD4(+)/CD8(+). Both toxin treatments significantly decreased (P < 0.01) CD4(+)/CD3(+) and CD4(+)/CD8(+) ratios. In summary, the combination of OTA and T-2 toxin impaired chick immune function even at combined concentrations as low as 0.25 mg/kg of OTA and 0.5 mg/kg of T-2 toxin.

Orally delivered foot-and-mouth disease virus capsid protomer vaccine displayed on T4 bacteriophage surface: 100% protection from potency challenge in mice.

An orally delivered foot-and-mouth disease (FMD) vaccine has not previously been reported. By using a T4 bacteriophage nanoparticle surface gene-protein display system (T4-S-GPDS), we created a foot-and-mouth disease virus (FMDV) entire capsid protein vaccine candidate. On the T4 phage surface SOC site, a full length FMDV capsid precursor polyprotein (P1, 755 aa) and proteinase 3C (213 aa) derived from an infected pig of serotype O strain GD-10 (1999), were separately displayed on different T4 phage particle surfaces through inserting their coding region DNAs into the T4 phage genome, yielding phage strains T4-P1 and T4-3C. We also constructed a series of FMDV sub-full length capsid structural protein (subunit) containing T4 phage recombinant vaccines. Both sucking and young BALB/c mice were used as two kinds of FMDV vaccine potency evaluation models. Many groups of both model mice were vaccinated orally or by subcutaneous injection with varying FMDV-T4 phage recombinant vaccines, with and without addition of adjuvant, then challenged with a lethal dose of cattle source virulent FMDV. In the case of immunization with a mixture of phage T4-P1 and phage T4-3C particles without any adjuvant added, all mice were 100% protected following either oral or injection immunization, whereas 100% of the control, non-immunized mice and mice immunized with only T4 phage vector Z1/Zh(-) or wild-type T4(+)D phage died; in contrast, with FMDV subunit vaccine, less than 75% protection followed the same potency challenge in both mice model groups. In addition, two pigs immunized with a phage T4-P1 and phage T4-3C mix were protected upon housing together with infected pigs. This study represents a clear example of how FMD and other pathogenic disease vaccines can be prepared by a simple and efficient bacteriophage route.

Effects of immunisation against leptin on feed intake, weight gain, fat deposition and laying performance in chickens.

1. Three experiments were conducted to study the effects of leptin on weight gain and body composition in laying hens. 2. The effects of immunisation against chicken leptin on feed intake (FI), fat deposition and laying rate were observed in laying Guangdong yellow-feathered hens. Ten hens were inoculated with leptin immunogen on d 3, 31, 63 and 84, together with 10 control hens immunised with bovine serum albumin (BSA). In the 100-d experiment, immunisation against leptin increased blood anti-leptin antibody titres, slightly reduced plasma T3 concentrations, slightly decreased FI and increased live weight; however, laying rate was significantly depressed and abdominal fat mass was increased by the end of the 100-d experiment. 3. Passive immunisation of 50-d-old pullets with yolk extract containing anti-leptin antibody IgY significantly increased FI within 6 h of treatment compared with physiological saline treated controls. 4. In growing 70-d-old pullets, inoculation with 0.5 (group 1) or 1 (group 2) ml leptin immunogen on d 1 and 28 of the experiment slightly increased FI and significantly increased daily gain compared with BSA-immunised control pullets. Abdominal fat mass on d 49 increased from 48+/-4.5 g in controls to 66+/-3.5 and 80+/-3.1 g in groups 1 and 2, respectively. 5. It was suggested that immunisation against leptin mimicked loss of leptin bioactivity and might become a novel technique to stimulate fat growth in certain types of animal production.

Molecular characterization of seven Chinese isolates of infectious bursal disease virus: classical, very virulent, and variant strains.

Seven infectious bursal disease virus (IBDV) strains isolated from China have been characterized in this study, including a classical strain CJ801, an attenuated strain GZ911, a variant strain GZ902, and four very virulent strains G9201, G9302, F9502, and HK46. With the use of reverse transcription-polymerase chain reaction, the full-length VP2 genes were amplified and the hypervariable regions were sequenced. Protein sequences of the hypervariable region (a.a. 143-382) of the field isolates confirmed their identities. CJ801 has the highest identity to the classical strains STC and 52/70. GZ902 has the highest identity to the American variant strains A, E, and GLS, and they share unique amino acid residue at positions 249K and 254S, which are not present in standard serotype 1 strains. Attenuated strain GZ911, like other cell culture-adapted strains, has substitutions at positions 279(D to N) and 284 (A to T) as well as in the serine-rich heptapeptide region. Hence, these substitutions may take an important role in the reduced virulence of these strains. The four very virulent strains have the highest identity to the European very virulent strain UK661 and Japanese strain OKYM. These strains share unique amino acid residues at positions 222A, 256I, and 294I, which are not present in other less virulent strains. The very virulent strains isolated in Guangdong (G9201, G9303) and Fujian (F9502) Provinces have one to five amino acid substitutions at the two hydrophilic domains of VP2 comparing with UK661 and OKYM, indicating that new very virulent strains are evolving. Phylogenetic analysis suggests that Chinese very virulent IBDVs and European very virulent strains are derived from similar origin.