Responses in splanchnic and mammary amino acid metabolism to short-term graded removal of methionine in lactating goats.

Four multi-catheterized lactating goats were used in a 4 × 4 Latin square experiment to investigate the responses of amino acid metabolism in portal-drained viscera (PDV), liver, and mammary glands to short-term varying supplies of methionine (Met). During the last 45 h in each experimental period, goats were fasted for 12 h and then abomasally infused with an amino acid (AA) mixture plus glucose for 33 h. Treatments consisted of graded removal of Met from an infused AA mixture to achieve Met content in the infusate of 100% (complete), 60%, 30%, or 0% that in casein. Graded Met removal decreased the production of milk, milk protein, lactose, and fat linearly whilst also decreasing arterial Met concentration linearly (P < 0.05). Meanwhile, net PDV uptake and liver removal of Met decreased linearly (P < 0.05) due to decreased Met affinity of PDV and liver (P < 0.05). Net mammary uptake of Met (P > 0.1) was maintained as Met supply declined. This was achieved through increased mammary affinity (P < 0.05) and increased mammary blood flow (P < 0.05) totally offsetting the negative effect of decreased circulating Met concentration. Graded removal of Met from the infusate linearly decreased mammary uptake-to-milk output ratios of Met (P < 0.05) and tended to decrease essential amino acid (EAA) linearly (0.05 < P < 0.1). Treatments also linearly decreased circulating concentration of prolactin and linearly increased insulin concentration (P < 0.05). In conclusion, results of the present study indicated there were several mechanisms used to mitigate a Met deficiency, including reduced catabolism of Met in PDV, liver, and peripheral tissue (including mammary glands) and a linear increase in mammary blood flow. The observed decreases in milk protein production as Met supply decreased appear to be a result of regulatory events which may have been driven by decreased circulating prolactin, rather than as a result of decreased mammary Met uptake.

Alleviation effect of conjugated linoleic acid on estradiol benzoate induced fatty liver hemorrhage syndrome in Hy-line male chickens.

The purpose of this study was to explore whether conjugated linoleic acid (CLA) could alleviate fatty liver hemorrhagic syndrome (FLHS) induced by estradiol benzoate intramuscular injection in laying hens. One hundred male Hy-Line white chickens were randomly divided into two groups, namely, the control (CON) and estradiol benzoate (E) groups, and both groups were fed the same basal diet. After injections of estradiol benzoate at 2 mg/kg every two days for a total of 7 times, chickens in the E group showed FLHS symptoms, including liver enlargement, hemorrhage, and steatosis. Then half of the chickens in the E group received an additional diet containing 5000 mg/kg CLA for 8 weeks. The results of morphological observations, hematoxylin and eosin staining, and Oil Red O staining showed that CLA alleviated liver enlargement, hemorrhage, and lipid accumulation in FLHS chickens. In addition, we measured liver function and lipid metabolism indicators, including ALT, AST, TG, TCH, HDL-C, and LDL-C, which further suggested that CLA mitigated the disturbance of serum and liver metabolism in FLHS chickens. Mechanistically, CLA inhibited hepatic de novo lipogenesis, cholesterol synthesis, and TG accumulation and increased TG hydrolysis in FLHS chickens by regulating the gene expression of CD36, ACC, FAS, SCD 1, DGAT2, LIPE, ATGL, CPT1A, SREBP-1c, SREBP-2, PPARγ, and PPARα. Furthermore, CLA ameliorated hepatic oxidative stress and inhibited NF-κB signaling pathway-mediated inflammation in FLHS chickens. In conclusion, CLA regulated lipid metabolism, thus further alleviating oxidative stress and inflammation to alleviate FLHS induced by estrogen in chickens.

Fatty liver hemorrhagic syndrome (FLHS) has become one of the most common noninfectious diseases that contribute to laying hen mortality. Conjugated linoleic acid (CLA) is a functional polyunsaturated fatty acid with antioxidant and anti-inflammatory properties The purpose of this study was to investigate the effect of CLA on FLHS induced by estradiol benzoate in laying hens. We successfully replicated the FLHS pathological model by intramuscular injection of estradiol benzoate. The results of morphological and histopathological observations showed that CLA alleviated liver lipid accumulation in FLHS chickens. In addition, we measured liver function and lipid metabolism indicators, which further suggested that CLA mitigated the disturbance of serum and liver metabolism in FLHS chickens. Moreover, CLA inhibited hepatic de novo lipogenesis, cholesterol synthesis, and TG accumulation and increased TG hydrolysis in FLHS chickens by regulating related gene expression. Furthermore, CLA ameliorated hepatic oxidative stress and inhibited inflammation in FLHS chickens. In conclusion, CLA regulated lipid metabolism, thus further alleviating oxidative stress and inflammation to alleviate FLHS induced by estrogen in chickens. Our results provide new evidence and insights for applying CLA as an effective treatment for FLHS.

Porcine Epidemic Diarrhea Virus Infection Subverts Arsenite-Induced Stress Granules Formation.

Stress granules (SGs) are dynamic cytoplasmic protein-RNA structures that form in response to various stress conditions, including viral infection. Porcine epidemic diarrhea virus (PEDV) variant-related diarrhea has caused devastating economic losses to the swine industry worldwide. In this study, we found that the percentage of PEDV-infected cells containing SGs is nearly 20%; meanwhile, PEDV-infected cells were resistant to sodium arsenite (SA)-induced SGs formation, as demonstrated by the recruitment of SGs marker proteins, including G3BP1 and TIA1. Moreover, the formation of SGs induced by SA treatment was suppressed by PEDV papain-like protease confirmed by confocal microscopy. Further study showed that PEDV infection disrupted SGs formation by downregulating G3BP1 expression. Additionally, PEDV replication was significantly enhanced when SGs' assembly was impaired by silencing G3BP1. Taken together, our findings attempt to illuminate the specific interaction mechanism between SGs and PEDV, which will help us to elucidate the pathogenesis of PEDV infection in the near future.

Effects of forage source and particle size on chewing activity, ruminal pH, and saliva secretion in lactating Holstein cows.

The present study investigated the effects of dietary forage source (quality) and particle size on chewing activity, saliva secretion, and ruminal pH. Twelve multiparous lactating Holstein cows, four of which were ruminally cannulated, were used in a replicated 4 × 4 Latin square experimental design with a 2 × 2 factorial arrangement of treatments. Cows fed wild-rye hay diets had longer daily eating times than cows fed oaten hay diets. Treatments had no effect on ruminating time; therefore, resting time varied inversely to eating time. Neither the rate nor the amount of saliva secretion while eating, ruminating, or resting was affected by diet, resulting in similar total daily saliva secretions across treatments (231 L/day). Total volatile fatty acids (VFAs) in the ruminal fluid from animals fed oaten hay diets were higher than those from animals fed wild-rye hay diets; further, VFAs increased with decreasing forage particle size (FPS). Consistent with elevated VFA concentrations, reducing FPS and including oaten hay in the diet decreased mean ruminal pH and increased the daily time of ruminal pH under 5.8. Results of this study suggest that forage source and particle size affect ruminal pH might be via variations in VFA production rather than increased salivary recycling of buffering substrates.

MiR-24-3p regulates cell proliferation and milk protein synthesis of mammary epithelial cells through menin in dairy cows.

MiR-24-3p, a broadly conserved, small, noncoding RNA, is abundantly expressed in mammary tissue. However, its regulatory role in this tissue remains poorly understood. It was predicted that miR-24-3p targets the 3' untranslated region (3'-UTR) of multiple endocrine neoplasia type 1 (MEN1), an important regulatory factor in mammary tissue. The objective of this study was to investigate the function of miR-24-3p in mammary cells. Using a luciferase assay in mammary epithelial cells (MAC-T), miR-24-3p was confirmed to target the 3'-UTR of MEN1. Furthermore, miR-24-3p negatively regulated the expression of the MEN1 gene and its encoded protein, menin. miR-24-3p enhanced proliferation of MAC-T by promoting G1/S phase progression. MiR-24-3p also regulated the expression of key factors involved in phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin and Janus kinase/signal transducer and activators of transcription signaling pathways, therefore controlling milk protein synthesis in epithelial cells. Thus, miR-24-3p appears to act on MAC-T by targeting MEN1. The expression of miR-24-3p was controlled by MEN1/menin, indicating a negative feedback loop between miR-24-3p and MEN1/menin. The negatively inhibited expression pattern of miR-24-3p and MEN1 was active in mammary tissues at different lactation stages. The feedback mechanism is a new concept to further understand the lactation cycle of mammary glands and can possibly to be manipulated to improve milk yield and quality.

Effects of forage source and particle size on feed sorting, milk production and nutrient digestibility in lactating dairy cows.

The objectives of this study were to determine the effects of forage source (quality) and particle size on feed sorting, milk production and nutrient digestibility in lactating dairy cows. Twelve multiparous lactating Holstein cows were used in a replicated 4 × 4 Latin square experiment with a 2 × 2 factorial arrangement of treatments. Treatments were as follows: (a) feeding long oaten hay (OL), (b) feeding short oaten hay (OS), (c) feeding long wild-rye hay (WL) and (d) feeding short wild-rye hay (WS). The sorting activity of cows fed wild-rye hay diets was greater than that of cows fed oaten hay diets. Sorting activity decreased with reduced forage particle size (FPS) for wild-rye hay diets but was not affected for oaten hay diets. Cows fed oaten hay diets had a similar dry matter intake (DMI), but higher total tract nutrient digestibility, and hence higher milk yield than cows fed wild-rye hay diets. The increase in DMI as a result of reduced FPS was significant in cows fed wild-rye hay diets. Feed efficiency (4% fat-corrected milk (FCM)/DMI) decreased from 1.18 to 1.11 when FPS decreased, but was not affected by the forage source. The digestibility of DM, crude protein (CP) and organic matter (OM) in the total tract was decreased by a reduction in FPS for wild-rye hay diets, but was not affected for oaten hay diets. In conclusion, cows fed high-quality forage (oaten hay) had a lower sorting activity and higher production performance than those fed poor-quality forage (wild-rye hay). The optimal dietary FPS in lactating dairy cows should take the effect of forage source into account.

Preliminary Characterization, Antioxidant and Hepatoprotective Activities of Polysaccharides from Taishan Pinus massoniana Pollen.

The objectives of the present study were to characterize the chemical composition, antioxidant activity and hepatoprotective effect of the polysaccharides from Taishan Pinus massoniana pollen (TPPPS). HPLC analysis showed that TPPPS was an acidic heteropolysaccharide with glucose and arabinose as the main component monosaccharides (79.6%, molar percentage). Fourier transform-infrared spectroscopy (FT-IR) analysis indicated that the spectra of TPPPS displayed infrared absorption peaks characteristic of polysaccharides. In in vitro assays TPPPS exhibited different degrees of dose-dependent antioxidant activities , and this was further verified by suppression of CCl4-induced oxidative stress in the liver with three tested doses of TPPPS (100, 200, and 400 mg/kg bw) in rats. Pretreatment with TPPPS significantly decreased the levels of alanine aminotransferase (AST), aspartate aminotransferase (ALT), alkaline phosphatase (ALP), lactic dehydrogenase (LDH) and malondialdehyde (MDA) against CCl4 injuries, and elevated the activities of superoxide dismutase (SOD) as well as glutathione peroxidase (GSH-Px). Histopathological observation further confirmed that TPPPS could protect the liver tissues from CCl4-induced histological alternation. These results suggest that TPPPS has strong antioxidant activities and significant protective effect against acute hepatotoxicity induced by CCl4. The hepatoprotective effect may partly be related to its free radical scavenging effect, increasing antioxidant activity and inhibiting lipid peroxidation.

Effect of hay supplementation timing on rumen microbiota in suckling calves.

An animal feeding trial was conducted on 18 seven-day-old Holstein dairy bull calves weighing 42 ± 3 kg each. Calves were randomly assigned into three groups (n = 6 each). The dietary treatments were as follows: (1) milk and starter for the control group (MS), (2) supplementation of oat hay from week 2 on the basis of milk and starter (MSO2), and (3) supplementation of oat hay from week 6 on the basis of milk and starter (MSO6). All animals were fed starter and oat hay ad libitum. The major phyla in the different groups of rumen fluid included Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, and Euryarchaeota. The major genera were identified, and major genera proportions in the three groups were as follows: Methanobrevibacter (Euryarchaeota), 2.1%, 1.7%, and 2.1%; Olsenella (Actinobacteria), 23.9%, 17.7%, and 12.8%; Prevotella (Bacteroidetes), 10.5%, 16.5%, and 19.2%; Dialister (Firmicutes), 3.3%, 4.1%, and 2.8%; Succiniclasticum (Firmicutes), 3.8%, 4.7%, and 9.2%; and Sharpea (Firmicutes), 0.4%, 2.5%, and 0.2%, respectively. There were no significant differences in the various phyla among the three groups (p > .05). The results showed that calves hay supplementation time did not affect the diversity of the rumen microbiota in the suckling calves. However, the hay supplementation altered the proportion of the various microbial populations, supplementation of oat hay from week 2 on the basis of milk and starter could improve calves rumen pH.

Menin Modulates Mammary Epithelial Cell Numbers in Bovine Mammary Glands Through Cyclin D1.

Menin, the protein encoded by the MEN1 gene, is abundantly expressed in the epithelial cells of mammary glands. Here, we found MEN1/menin expression slowly decreased with advancing lactation but increased by the end of lactation. It happened that the number of bovine mammary epithelial cells decreases since lactation, suggesting a role of menin in the control of mammary epithelial cell growth. Indeed, reduction of menin expression through MEN1-specific siRNA transfection in the bovine mammary epithelial cells caused cell growth arrest in G1/S phase. Decreased mRNA and protein expression of Cyclin D1 was observed upon MEN1 knockdown. Furthermore, menin was confirmed to physically bind to the promoter region of Cyclin D1 through a ChIP assay, indicating that menin plays a regulatory role in mammary epithelial cell cycle progression. Moreover, lower expression of MEN1/menin induced increased epithelial cell apoptosis and caused extracellular matrix remodeling by down-regulating its associated genes, such as DSG2 and KRT5, suggesting that menin's role may also be involved in the control of cell-cell adhesion in normal mammary glands. Taken together, our data revealed an unknown molecular function of menin in epithelial cell proliferation, which may be important in the regulation of lactation behavior of mammary glands.

MEN1/Menin regulates milk protein synthesis through mTOR signaling in mammary epithelial cells.

The MEN1 gene, which encodes the protein Menin, was investigated for its regulatory role in milk protein synthesis in mammary glands. Menin responds to nutrient and hormone levels via the PI3K/Akt/mTOR pathway. Bovine mammary epithelial cells and tissues were used as experimental models in this study. The results revealed that the milk protein synthesis capacity of mammary epithelial cells could be regulated by MEN1/Menin. The overexpression of Menin caused significant suppression of factors involved in the mTOR pathway, as well as milk protein κ-casein (CSNK). In contrast, a significant increase in these factors and CSNK was observed upon MEN1/Menin knockdown. The repression of MEN1/Menin on the mTOR pathway was also observed in mammary gland tissues. Additionally, MEN1/Menin was found to elicit a negative response on prolactin (PRL) and/or insulin (INS), which caused a similar downstream impact on mTOR pathway factors and milk proteins. Collectively, our data indicate that MEN1/Menin could play a regulatory role in milk protein synthesis through mTOR signaling in the mammary gland by mediating the effects of hormones and nutrient status. The discovery of Menin's role in mammary glands suggests Menin could be potential new target for the improvement of milk performance and adjustment of lactation period of dairy cows.

Autophagy blockade and lysosomal membrane permeabilization contribute to lead-induced nephrotoxicity in primary rat proximal tubular cells.

Lead (Pb) is a known nephrotoxicant that causes damage to proximal tubular cells. Autophagy has an important protective role in various renal injuries, but the role of autophagy in Pb-elicited nephrotoxicity remains largely unknown. In this study, Pb promoted the accumulation of autophagosomes in primary rat proximal tubular (rPT) cells, and subsequent findings revealed that this autophagosome accumulation was caused by the inhibition of autophagic flux. Moreover, Pb exposure did not affect the autophagosome-lysosome fusion in rPT cells. Next, we found that Pb caused lysosomal alkalinization, may be through suppression of two V-ATPase subunits. Simultaneously, Pb inhibited lysosomal degradation capacity by affecting the maturation of cathepsin B (CTSB) and cathepsin D (CTSD). Furthermore, translocation of CTSB and CTSD from lysosome to cytoplasm was observed in this study, suggesting that lysosomal membrane permeabilization (LMP) occurred in Pb-exposed rPT cells. Meanwhile, Pb-induced caspase-3 activation and apoptosis were significantly but not completely inhibited by CTSB inhibitor (CA 074) and CTSD inhibitor (pepstatin A), respectively, demonstrating that LMP-induced lysosomal enzyme release was involved in Pb-induced apoptosis in rPT cells. In conclusion, Pb-mediated autophagy blockade in rPT cells is attributed to the impairment of lysosomal function. Both inhibition of autophagic flux and LMP-mediated apoptosis contribute to Pb-induced nephrotoxicity in rPT cells.

Immuno-enhancement of Taishan Pinus massoniana pollen polysaccharides on recombinant Bordetella avium ompA expressed in Pichia pastoris.

Bordetellosis, caused by Bordetella avium, continues to be an economic problem in the poultry industry of China. Vaccines with good protective ability are lacking. Thus, developing a novel vaccine against the B. avium infection is crucial. Here, we constructed a recombinant Pichia pastoris transformant capable of expressing the outer membrane protein A (ompA) of B. avium to prepare the recombinant ompA subunit vaccine and then evaluated its immune effects. To further investigate the immunomodulation effects of Taishan Pinus massoniana pollen polysaccharides (TPPPS) on this subunit vaccine, three concentrations (20, 40, and 60 mg/mL) of TPPPS were used as the adjuvants of the ompA subunit vaccine respectively. The conventional Freund's incomplete adjuvant served as the control of TPPPS. Chickens in different groups were separately vaccinated with these vaccines thrice. During the monitoring period, serum antibody titers, concentrations of serum IL-4, percentages of CD4(+) and CD8(+) T-lymphocytes in the peripheral blood, lymphocyte transformation rate, and protection rate were detected. Results showed that the pure ompA vaccine induced the production of anti-ompA antibody, the secretion of IL-4, the increase of CD4(+) T-lymphocytes counts and lymphocyte transformation rate in the peripheral blood. Moreover, the pure ompA vaccine provided a protection rate of 71.67% after the B. avium challenge. Notably, TPPPS adjuvant vaccines induced higher levels of immune responses than the pure ompA vaccine, and 60 mg/mL TPPPS adjuvant vaccine showed optimal immune effects and had a 91.67% protection rate. Our findings indicated that this recombinant B. avium ompA subunit vaccine combined with TPPPS had high immunostimulatory potential. Results provided a new perspective for B. avium subunit vaccine research.

Immunological and protective effects of Bordetella bronchiseptica subunit vaccines based on the recombinant N-terminal domain of dermonecrotic toxin.

Dermonecrotic toxin (DNT) produced by Bordetella bronchiseptica (B. bronchiseptica) can cause clinical turbinate atrophy in swine and induce dermonecrotic lesions in model mice. We know that the N-terminal of DNT molecule contains the receptor-binding domain, which facilitates binding to the target cells. However, we do not know whether this domain has sufficient immunogenicity to resist B. bronchiseptica damage and thereby to develop a subunit vaccine for the swine industry. In this study, we prokaryotically expressed the recombinant N-terminal of DNT from B. bronchiseptica (named DNT-N) and prepared it for the subunit vaccine to evaluate its immunogenicity. Taishan Pinus massoniana pollen polysaccharide (TPPPS), a known immunomodulator, was used as the adjuvant to examine its immune-conditioning effects. At 49 d after inoculation, 10 mice from each group were challenged with B. bronchiseptica, and another 10 mice were intradermally challenged with native DNT, to examine the protection imparted by the vaccines. The immune parameters (T-lymphocyte counts, cytokine secretions, serum antibody titers, and survival rates) and skin lesions were determined. The results showed that pure DNT-N vaccine significantly induced immune responses and had limited ability to resist the B. bronchiseptica and DNT challenge, whereas the mice administered with TPPPS or Freund's incomplete adjuvant vaccine could induce higher levels of the above immune parameters. Remarkably, the DNT-N vaccine combined with TPPPS adjuvant protected the mice effectively to prevent B. bronchiseptica infection. Our findings indicated that DNT-N has potential for development as an effective subunit vaccine to counteract the damage of B. bronchiseptica infection, especially when used conjointly with TPPPS.

Protective effects of Ginkgo biloba leaf polysaccharide on nonalcoholic fatty liver disease and its mechanisms.

A water-soluble polysaccharide fraction extracted from the leaf of Ginkgo biloba was named GBLP. The protective effect of GBLP on nonalcoholic fatty liver disease (NAFLD) was observed and underlying mechanism was explored. Wistar male rats were randomly divided into five groups, namely, normal control group, model control group and GBLP groups (100, 200 and 400 mg/kg/d). A rat model of NAFLD was established in male Wistar rats by feeding with high-fat diet (HFD) for 8 weeks. On day 57, the intragastric administration of GBLP started once daily for 4 weeks. The results showed that GBLP supplementation significantly and dose-dependently lowered the weight gain of body, liver index and serum lipid parameters in HFD-fed rat. Meanwhile, GBLP attenuated HFD-induced liver injury through reducing hepatic steatosis, TG accumulation, serum ALT, AST and ALP levels. GBLP had a positive effect on obesity-associated insulin resistance (IR) via reducing serum glucose and insulin levels. Furthermore, GBLP enhanced the activities of antioxidant enzymes and reduced MDA levels in serum and liver. These results indicate that GBLP can play a certain protective role against HFD-induced NAFLD, and the protective effects may be associated with attenuating IR, preserving liver function, enhancing antioxidant defense system, and reducing lipid peroxidation.

Immune-enhancing activity of polysaccharides isolated from Atractylodis macrocephalae Koidz.

This study evaluated the immune-enhancing activity of polysaccharides from the rhizoma of Atractylodis macrocephalae Koidz (RAMPS) in vitro. Lymphocyte proliferation, cell cycle distribution, and percentages of CD4(+) and CD8(+) cells were determined. Different concentrations of RAMPS were added to peripheral blood T lymphocytes. Results showed that RAMPStp and RAMPS60c could significantly enhance T lymphocyte proliferation individually or synergistically with phytohemagglutinin at most concentrations. The active sites of RAMPStp and RAMPS60c were then selected. Lymphocyte cell cycle distribution and percentages of CD4(+) and CD8(+) cells were determined by flow cytometry. At most time points, RAMPS60c and RAMPStp could promote lymphocytes enter into S and G2/M phases. RAMPStp and RAMPS60c effectively improved the percentages of CD4(+) and CD8(+) T cells. RAMPStp produced optimal effects. Therefore, RAMPStp could be used as a component of novel immunopotentiators.

Bovine mammary gene expression profiling during the onset of lactation.

BACKGROUND: Lactogenesis includes two stages. Stage I begins a few weeks before parturition. Stage II is initiated around the time of parturition and extends for several days afterwards. METHODOLOGY/PRINCIPAL FINDINGS: To better understand the molecular events underlying these changes, genome-wide gene expression profiling was conducted using digital gene expression (DGE) on bovine mammary tissue at three time points (on approximately day 35 before parturition (-35 d), day 7 before parturition (-7 d) and day 3 after parturition (+3 d)). Approximately 6.2 million (M), 5.8 million (M) and 6.1 million (M) 21-nt cDNA tags were sequenced in the three cDNA libraries (-35 d, -7 d and +3 d), respectively. After aligning to the reference sequences, the three cDNA libraries included 8,662, 8,363 and 8,359 genes, respectively. With a fold change cutoff criteria of ≥ 2 or ≤-2 and a false discovery rate (FDR) of ≤ 0.001, a total of 812 genes were significantly differentially expressed at -7 d compared with -35 d (stage I). Gene ontology analysis showed that those significantly differentially expressed genes were mainly associated with cell cycle, lipid metabolism, immune response and biological adhesion. A total of 1,189 genes were significantly differentially expressed at +3 d compared with -7 d (stage II), and these genes were mainly associated with the immune response and cell cycle. Moreover, there were 1,672 genes significantly differentially expressed at +3 d compared with -35 d. Gene ontology analysis showed that the main differentially expressed genes were those associated with metabolic processes. CONCLUSIONS: The results suggest that the mammary gland begins to lactate not only by a gain of function but also by a broad suppression of function to effectively push most of the cell's resources towards lactation.

Synthesis, cloning, and expression of Mycoplasma suis inorganic pyrophosphatase gene using PCR-based accurate synthesis and overlap-extension PCR, and its immunogenicity analysis.

Mycoplasma suis (M. suis), a hemotrophic pathogen of pigs, causes economic losses in swine production throughout the world. Inorganic pyrophosphatase (ppa) is a very important gene in M. suis. The ppa gene of M. suis was synthesized by PCR-based accurate synthesis (PAS) and overlapextension PCR, inserted into vector pMD18-T, and then subcloned to the prokaryotic expression vector pET28c.The recombinant plasmid pET28c_ppa was transformed to E. coli BL21 for expression under induction of isopropyl thiogalactoside. The expressed product was identified by SDS-PAGE and Western blot, which suggested that the recombinant protein has good antigenicity. Piglets were immunised with purified recombinant protein, and specific antibodies to the recombinant protein were detected in piglet serum. The results show that the ppa gene can be efficiently expressed in E. coli and that the expressed recombinant protein can elicit a specific serum antibody response in piglets. PAS and overlap-extension PCR were first used to synthesize the ppa of M. suis. They provide simple, rapid, reliable and relatively inexpensive methods to synthesize, clone, and express genes. The experiment conducted in this paper will enable future research into the role and function of the ppa gene.

Effects of Taishan Pinus massoniana pollen polysaccharide on immune response of rabbit haemorrhagic disease tissue inactivated vaccine and on production performance of Rex rabbits.

Varied doses of Taishan Pinus massoniana pollen polysaccharide (TPPPS) and Astragalus polysaccharide (APS) extracted by hot water extraction and ethanol precipitation method were added to the vaccine in order to prepare polysaccharide-rabbit haemorrhagic disease (RHD) tissue inactivated vaccine. The purpose was to study effects of TPPPS on immune response of RHD tissue inactivated vaccine and on production performance of Rex rabbits. Results showed that each index in groups I, II, III and IV was higher than that in group V, especially groups I, II and IV, the difference between which and group V was much more significant (P<0.05); each index in group I was extremely higher than that in group V (P<0.01); each index in group I was significantly higher than that in groups II, III (P<0.05), and generally no significant difference was observed between groups II and III. The overall level in group IV was slightly lower than that in group I. Each index in the polysaccharide groups reached its peak value later than that in the non-polysaccharide groups did. Results suggested that any dose of TPPPS can enhance immunologic function and production performance of rabbits, and the amount of 400mg per rabbit has the most obvious efficacy. Furthermore, it can extend the immune peak period of RHD tissue inactivated vaccine and the growing peak period of Rex rabbits. TPPPS has generally higher efficiency than APS.

Treatment of Mycoplasma wenyonii infection in cows with imidocarb dipropionate injection-acupuncture.

Sixteen shorthorn cows from Xiazhuang farm were admitted to the Veterinary Teaching Hospital at the College of Animal Science and Veterinary Medicine, Shandong Agricultural University for evaluation of poor appetite, listlessness, fever, tachycardia, tachypnea, lethargy, positive jugular venous pulse and anemia. Blood smear examination and polymerase chain reaction analysis in these cows revealed an infection with Mycoplasma wenyonii. The subjects were divided into two groups: control group (three cows) treated with intramuscular injection with imidocarb dipropionate (3 mg/kg/day for 2 days) and the experimental group (13 cows), treated with injection-acupuncture (Imidocarb Dipropionate, 1 mg/kg, once every 3 days for 6 days) at BL17, BL18, BL20, BL25, ST36, SP06 and CV04. At day 15, negative results were found using blood smear examination in all control and experimental groups.