In this paper, seasonal samples of wild versus cultured groupers, Epinephelus spp., from Daya Bay, South China Sea were examined to survey the seasonality of two important species, Pseudorhabdosynochus coioidesis and P. serrani (Monogenea: Diplectanidae), and to analyze the interspecific relationships between these two parasites. Between April 2008 and January 2009, P. coioidesis and P. serrani were found to be parasitic only on E. coioides Hamilton during summer and winter in the natural waters of Daya Bay, exhibiting a high degree of host specificity, whereas they co-occurred and persisted on several species of hosts, such as E. coioides, E. bruneus Block and E. awoara Temminck & Schlegel, in an experimental polyculture pond during several seasons. E. coioides is the main host for both of these two monogenean species. The overall prevalences and mean intensities of these two parasites on polycultured Epinephelus spp. showed the same pattern of seasonal fluctuations, with the maximum values during autumn, except for the overall prevalence of P. serrani, which reached its maximum values during summer and winter and the minimum values during spring and autumn. Prevalence and mean intensity were found to be related to host size. In the wild, medium-sized fishes harboured higher infections, whereas under cultured conditions the small-sized and large-sized fishes were more heavily infected. Simultaneous infections of P. coioidesis and P. serrani were common, and there was a significant positive interspecific correlation between these two parasites.
Introduction: Vascular cell adhesion molecule 1 (VCAM-1) is a member of Ig superfamily. The aim of this study was to prepare highly specific polyclonal antibodies against bovine VCAM-1 and to evaluate the expression of VCAM-1 in the mammary lymph nodes of cows with subclinical mastitis.
Material and Methods: The VCAM-1 gene was cloned from bovine Peyer’s patches and inserted into the pGEX-4T-1 and pET-28a vectors. The recombinant plasmids pGEX-4T-1/VCAM-1 and pET-28a/VCAM-1 were transferred into Escherichia coli BL21 and the recombinant strains were induced by isopropyl-D-thiogalactoside to produce fusion proteins tagged with polyhistidine (His) and glutathione S-transferase (GST), respectively. The expressed fusion proteins His-VCAM-1 and GST-VCAM-1 were identified by SDS-PAGE and Western blot. His-VCAM-1 protein was used as an antigen to immunise Wistar rats and polyclonal antibody serum against VCAM-1 was obtained.
Results: The serum titre tested by indirect ELISA was 128,000 using GST-VCAM-1 as the well coating antigen. Western blots indicated that the antibody recognised recombinant VCAM-1 protein as well as endogenous VCAM-1. In addition, using qPCR and Western blot, VCAM-1 mRNA and protein expression levels were measured in dairy cows with subclinical mastitis. It was demonstrated that VCAM-1 levels in the mammary lymph nodes of the cows were significantly higher than those from healthy controls (P < 0.05).
Conclusion: These results are to our knowledge the first report that VCAM-1 expression in the mammary lymph nodes is elevated in dairy cows with subclinical mastitis.
This study aimed to characterise the effects of ketosis on milk yield and composition and digestive capacity in transition dairy cows.
Material and Methods
Seven ketotic and seven healthy cows were housed in individual stalls for six days. Samples of plasma, milk, refused total mixed ration, and faeces were collected, and the blood biochemical parameters, milk yield and composition, dry matter intake, and faecal dry matter (FDM) production were determined.
Compared with healthy cows, the ketotic cows had significantly higher concentrations of milk fat and citrate, but lower levels of milk protein and lactose. The cows exhibited a need for acid detergent fibre in forage and better digestion of neutral detergent fibre, starch, crude protein, and phosphorus than healthy cows, but more fat and gross energy were excreted in their faeces. Ketotic cows had higher energy-corrected milk yields and lower FDM than healthy cows.
Lower feed intake coinciding with the requirement to maintain high milk production is considered to be the cause of ketosis in dairy cows. Ketotic cows exhibited lower dry matter fat digestion.
Yi-Xuan Hou, Chun Xie, Kang Wang, Yu-Ting Zhao, Yang-Yang Xie, Hong-Yan Shi, Jian-Fei Chen, Li Feng, Guang-Zhi Tong, Xiu-Guo Hua, Cong-Li Yuan, Yan-Jun Zhou and Zhi-Biao Yang
Introduction: A real-time RT-PCR method for identification and quantification of porcine epidemic diarrhoea virus (PEDV) strains in China was developed.
Material and Methods: Based on the conserved sequence of the PEDV nucleocapsid (N) gene, a primer pair and probe were designed to establish a TaqMan-MGB real-time RT-PCR assay for quantitative detection of the virus. The sequence was cloned into the pMD18-T vector and a series of diluted recombinant plasmids were used to generate a standard curve with an R2 value of 0.999.
Results: The developed quantitative PCR assay detected viral titres as low as 0.1 TCID50 with high specificity and no cross-reaction with other porcine viruses (PoRV, TGEV, PRRSV, or CSFV). The intra-batch and inter-batch coefficients of variation were both less than 1%, which indicated good reproducibility. Thirty clinical diarrhoea samples obtained from pigs in Shanghai and Fujian were analysed using this quantitative PCR assay. Out of these samples, 93.3% were found to be PEDV positive.
Conclusion: This approach is suitable for clinical sample identification and pathogenesis studies.
Chang-Liang He, Qiong Yi, Yuan-Fang Li, Hang Yang and Lu Wang
Mammary epithelial cells (MECs) from Kunming mice were isolated and stimulated in vitro with 10 μg/mL of Escherichia coli lipopolysaccharide (LPS). The release of tumour necrosis factor α (TNF-α) and interleukin-8 (IL-8) into culture supernatants was measured by ELISA. Furthermore, blocking experiments with Toll-like receptor 2 (TLR2) and TLR4 antibodies were performed to verify whether cytokine secretion depended on LPS-induced activation of TLR2 or TLR4. The results revealed that LPS-stimulated mouse MECs significantly secreted TNF-α and IL-8. Blocking of the TLR4 pathway inhibited the secretion of TNF-α and IL-8, while inhibition of LPS-induced TNF-α and IL-8 production was not observed when TLR2 was blocked. Thus, TLR4 can mediate the LPS-induced expression of cytokines such as TNF-α and IL-8 in mouse MECs.
Haoju Wang, Li Ni, Hongjun Yang, Limin Xu, Ning Ma and Honglei Ding
In order to evaluate the prevalence of the Mycoplasma mycoides cluster in goats in Chongqing, China, an epidemiological survey in this area was carried out. A total of 68 samples were subjected to bacteria isolation on Hartley’s medium. Four isolates (three from lung tissue and one from nasal discharges) were recovered from the samples and identified as the Mycoplasma species by their morphological and biochemical characteristics. They were further confirmed by PCR using 16S rRNA specific primer pairs and by restriction enzyme analysis. In vitro antimicrobial susceptibility of the isolates indicated that some strains had developed resistance to the antibiotics tested. This is the first report on the isolation, identification, and molecular characterisation of Mycoplasma species isolated from goats in Chongqing. This study also revealed a prevalence of Mycoplasma species infection in goats in this area.
Haifeng Yang, Xiaolan Chen, Chunmao Jiang, Kongwang He and Yiyi Hu
Introduction: The aim of the research was to investigate the antiviral and immunoregulatory effects of saikosaponin A, saikosaponin D, Panax notoginseng saponins, notoginsenoside R1, and anemoside B4 saponins commonly found in Chinese herbal medicines.
Material and Methods: control mice were challenged intramuscularly (im) with 0.2 mL of porcine circovirus 2 (PCV2) solution containing 107 TCID50 of the virus/mL. Mice of high-, middle-, and low-dose saponin groups were initially challenged im with 0.2 mL of PCV2 solution and three days later treated intraperitoneally (ip) with one of five saponins at one of three doses (10, 5, or 1 mg/kg b.w.). In the drug control group, mice were dosed ip with 10 mg/kg b.w. of a given saponin, and mice in a blank control group were administered the same volume of normal saline.
Results: The results revealed that the saponins could reduce the incidence and severity of PCV2-induced immunopathological damage, e.g. body temperature elevation, weight loss, anaemia, and internal organ swelling. In addition, it was seen that the saponins could affect the immunoglobulin levels and protein absorption.
Conclusion: The data suggested that the saponins might effectively regulate immune responses.
Jing Shen, Xiao-Ming Lei, Yang Song, Xing Tan, Qin Liu, Li-Wen Dai and Jie Yu
Objective: To observe the effects of electro-acupuncture (EA) on GRP78 and Caspase-12 gene expression in rats with ischemia- reperfusion injury (IRI) by stimulation on Nei Guan (PC6) and Bai Hui (GV20) points, so that to understand whether or not the protective effects of acupuncture is related to endocytoplasmic reticulum (ER) stressapoptosis passage.
Methods: 50 rats were randomly assigned to five groups (10 in each group): normal control(A), pseudo-operation(B), operation(C), Edaravone(D) and EA(E). The ischemia/reperfusion model of middle cerebral artery occlusion (MCAO) was established by suture embolic method. TUNEL staining method was employed to measure the apoptosis index of nerve cells in rats. Real-time polymerase chain reaction (RT-PCR) was employed to measure the mRNA expression of GRP78 and Caspase-12.
Results: Compared with normal group and pseudo-operation group, the apoptosis indexes and mRNA expression of GRP78 and Caspase-12 in operation group, Edaravone group and EA group were increased, with statistical significance（P＜0.05 or P＜0.01）; compared with operation group, the apoptosis indexes and Caspase-12 mRNA expression in Edaravone group and EA group were decreased（P＜0.05 or P＜0.01）, but GRP78 mRNA expression were increased（P＜0.01）; there were no significant difference between Edaravone group and EA group on the above indexes（P＞0.05).
Conclusion: Acupuncture on Nei Guan and Bai Hui points could effectively suppress the nerve cell apoptosis in cerebral ischemia. The underlying mechanism might be related to upregulation of the ERS-protective GRP78 expression and downregulation of apoptosis-promotion Caspase-12 expression.
Qiong Yi, Xin Li, Yuan-Fang Li, Hang Yang, Xiao-Yi Zhang, Zhe Ma and Lu Wang
Introduction: The effects of Jin-Ying-Tang (JYT) on Toll-like Receptor 4 (TLR4) signalling transduction of lipopolysaccharide (LPS)-stimulated mouse mammary epithelial cells (MECs) in vitro were examined. Material and Methods: The cytotoxicity of JYT (0.06-62.50 mg/mL) on mouse MECs was determined by MTT assay. The MECs were co-cultured with LPS in the presence or absence of JYT (39.10 μg/mL, 391 μg/mL, 3910 μg/mL). The concentrations of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) in the culture supernatants were detected by ELISA. The mRNA expression of TLR4 and downstream TLR4 signalling molecules such as myeloid differentiation factor 88 (MyD88), tumour necrosis factor receptor associated factor 6 (TRAF-6), inhibitor κB (IκB), and nuclear factor κB inducing kinase (NIK) were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: The results showed that the IC50 of JYT on MECs was 12.25 mg/mL and JYT could significantly decrease the concentrations of IL-6 and TNF-α in LPS-stimulated MECs (P < 0.05). The mRNA expression of TLR4, MyD88, TRAF-6, IκB, and NIK was also significantly decreased when the LPS-stimulated MECs were cocultured at appropriate concentrations of JYT (P < 0.05, P < 0.01). Conclusion: These observations indicate a potential mechanism through which JYT attenuates the systemic inflammatory response to LPS-stimulated mouse mammary epithelial cells by inhibiting the activation of TLR4/MyD88/ TRAF-6/NIK pathway at the mRNA level.
Yu Cao, Jiang Zhang, Wei Yang, Cheng Xia, Hong-You Zhang, Yan-Hui Wang and Chuang Xu
Introduction: The predictive value of selected parameters in the risk of ketosis and fatty liver in dairy cows was determined.
Material and Methods: In total, 21 control and 17 ketotic Holstein Friesian cows with a β-hydroxybutyrate (BHBA) concentration of 1.20 mmol/L as a cut-off point were selected. The risk prediction thresholds for ketosis were determined by receiver operating characteristic (ROC) curve analysis.
Results: In the ketosis group, paraoxonase-1 (PON-1) activity and concentration of PON-1 and glucose (GLU) were decreased, and aminotransferase (AST) activity as well as BHBA and non-esterified fatty acid (NEFA) contents were increased. The plasma activity and concentration of PON-1 were significantly positively correlated with the level of plasma GLU. The plasma activity and concentration of PON-1 were significantly negatively correlated with the levels of AST and BHBA. According to ROC curve analysis, warning indexes of ketosis were: plasma PON-1 concentration of 46.79 nmol/L, GLU concentration of 3.04 mmol/L, AST concentration of 100 U/L, and NEFA concentration of 0.82 mmol/L.
Conclusion: This study showed that the levels of PON-1, GLU, AST, and NEFA could be used as indicators to predict the risk of ketosis in dairy cows.