黄芪多糖和低浓度嗜水气单胞菌对中华绒螯蟹免疫相关因子基因表达的影响

采用逆转录-聚合酶链反应(RT-PCR)方法研究黄芪多糖和低浓度嗜水气单胞菌对中华绒螯蟹免疫相关因子基因表达的影响。结果表明:注射低浓度嗜水气单胞菌后,河蟹血细胞抗脂多糖因子(ALF)、热休克蛋白(HSP)、抗菌肽(Crus1)、脂肪酸结合蛋白酶(EsFABP3)、JAK通路细胞受体因子(EsDOME)、JAK通路信号转导与转录激活因子(EsSTAT)、河蟹肝胰腺HSP、EsFABP3和EsSTAT及河蟹鳃组织HSP的基因表达水平显著上调(P<0.05),黄芪多糖能够显著提高河蟹血细胞中ALF、酚氧化酶(PO)、HSP、Crus1、Toll样受体基因(TLR)、EsFABP3、EsJAK、EsDOME、EsSTAT、河蟹肝胰腺中ALF、PO、谷胱甘肽过氧化物酶(GPX)、HSP、TLR、EsFABP3、EsJAK、EsSTAT及河蟹鳃组织中ALF、GPX、HSP、Crus1、masquerade样蛋白(MasL)、TLR、EsJAK、EsDOME和EsSTAT的基因表达水平(P<0.05)。除了HSP和MasL外,注射黄芪多糖的河蟹免疫相关因子的基因表达水平普遍高于嗜水气单胞菌组河蟹。     

嗜水气单胞菌弹性蛋白酶基因的克隆表达

为了研究嗜水气单胞菌重组弹性蛋白酶的酶学性质,试验根据GenBank中的嗜水气单胞菌弹性蛋白酶基因ahyB设计1对含酶切位点的特异引物,以嗜水气单胞菌J-1(AhJ-1)株为模板,经PCR扩增得到不含信号肽的成熟弹性蛋白酶基因片段(787 bp),并与pMD18-T载体连接、测序,再用DNAStar软件分析。结果表明:该基因片段与豚鼠气单胞菌胞外蛋白酶同源性高达95%,与嗜水气单胞菌AG2株弹性蛋白酶ahyB基因同源性为92%,与铜绿假单胞菌LasB基因同源性为82%;将PCR产物连入表达载体pET-32a,转化至大肠杆菌BL21菌株中进行诱导表达,出现50 ku的融合表达蛋白,该表达产物纯化复性后表现出酶的活性。     

Experimental Staphylococcus aureus infection of the mammary gland induces region-specific changes in innate immune gene expression

Staphylococcus aureus is a prolific mastitis-causing bacterium that resides naturally in the environment of the dairy cow. The aim of this study was to profile immune gene expression in tissue from the alveolar, ductal, gland cistern and teat canal regions of the bovine mammary gland following intramammary infection with S. aureus. Quantitative real-time PCR (qPCR) was used to profile expression of innate immune genes including pattern recognition receptors (PRRs), cytokines, antimicrobial peptides (AMPs) and acute phase proteins (APPs). Consistent expression of Toll-like receptors (TLRs) 1-10 and NOD-like receptors (NODs) 1-2 was detected in all four tissue regions. Pro-inflammatory cytokines (IL6, IL17A and IL8) and anti-inflammatory cytokine (IL10) were induced in all 4 tissues. APP (SAA3 and HP) and AMP (DEFB4 and DEFB5) genes showed the greatest induction throughout the mammary gland in response to S. aureus, with particularly high expression in alveolar tissue (SAA3 and HP >133- and >80-fold respectively, P<0.05; DEFB4 and DEFB5 >9- and >27-fold respectively, P<0.05). Collectively, our data show both sentinel and effector immune functions throughout the mammary gland in response to S. aureus challenge.     

Cloning, nucleotide sequence and phylogenetic analyses, and tissue-specific expression of the transferrin gene in Cirrhinus mrigala infected with Aeromonas hydrophila

Transferrin partial complementary DNAs were cloned from the livers of five species in four genera of Indian carps (Indian major carp species: Labeo rohita, Catla catla and Cirrhinus mrigala; medium carp: Puntius sarana; minor carp: Labeo bata) subsequent to polymerase chain reaction amplification with published heterologous primers or self-designed primers derived from conserved regions of transferrin cDNA sequences. The partial transferrin cDNAs of the five species of carps had sizes from 624 to 633 bp (487 bp for L. rohita) and encoded an open reading frame consisting of 206–211 (162 for L. rohita) amino acids. The alignments of carp cDNA sequences showed 85–97% homology and 71–93% homology in deduced amino acid sequences. A phylogenetic tree of amino acid sequences of transferrin cDNAs from carps showed that the relationship among the four genera of Indian carps is well correlated with that derived from classic morphologic analyses. The hypothesized cleavage site and interdomain bridge of transferrin molecule were predicted for the above carp species and interestingly the cleavage site amino acid sequence was found to be conserved among all the carps. To study the tissue-specific expression of the transferrin gene, various tissues (liver, kidney, spleen, brain, muscle, testis, heart, intestine, gill and fin) from apparently healthy (control), moribund and survived C. mrigala experimentally infected with Aeromonas hydrophila infection were analyzed. Transferrin mRNA was detected only in liver RNA and to lesser extent in brain tissue out of the 10 tissues analyzed irrespective of bacterial infection.     

Biochemical changes in blood and uterine fluid of fowl following experimental EDS'76 virus infection

The pH, pCO2, and pO2 values and the concentrations of sodium, potassium, calcium, magnesium, chloride, phosphorus, bicarbonate, base excess, protein, glucose, as well as the activity of alkaline phosphatase and malate dehydrogenase were determined in the venous blood and uterine fluid of control and EDS'76 virus-infected fowl. Moreover, the pH of the mucosa of different parts of the oviduct was measured. Hens were examined in the period from 10 to 24 days following infection; blood and uterine fluid samples were collected approximately 14 hours after oviposition, provided a plumped egg was present in the uterus. Examination of blood and pH measurement of oviduct mucosa did not yield significant differences between infected and noninfected hens. In comparison with noninfected control birds, the mean sodium concentration of the uterine fluid of infected hens producing soft shelled or shell-less eggs had evidently increased, while the mean concentration of potassium, calcium, magnesium and glucose had decreased. Similar differences were also observed between infected hens producing normally shelled eggs and infected hens producing abnormally shelled eggs. No significant differences between infected and not infected hens were observed concerning the other values determined in the uterine fluid. It is concluded that functional disturbances which account for shell aberrations following EDS'76 virus infection are located in the surface epithelial cells of the uterine mucosa. These disturbances are very probably initiated by a depressed function of the sodium pump. All changes observed in the uterine fluid of infected hens could be explained by this depressed function.     

Clinical, haematological and blood biochemical changes in goats after experimental infection with tick-borne fever

Tick-borne fever in goats caused by Ehrlichia (Cytoecetes) phagocytophila was characterised by high fever, dullness, anorexia, tachycardia and a slight to moderate inhibition of rumen motility. The animals developed a gradual decline in the total number of circulating white blood cells. There was a decrease in lymphocytes over a short period, followed by an increase. The number of neutrophils was higher on the 3rd day, causing considerable change in the lymphocyte:neutrophil ratios. The number of eosinophils increased slightly. Serum alkaline phosphatase (ALP) decreased during the febrile episodes, and a marked decline was observed in both plasma zinc and iron concentrations. Furthermore, there was a small but progressive decrease of haemoglobin and haematocrit values. Circulating endogenous pyrogen/leucocyte endogenous mediator could not be detected in plasma from febrile goats. Tick-borne fever was passively transmitted to kids with plasma obtained from these febrile animals.     

Role of microfilaments and microtubules in the invasion of EPC cells by Aeromonas hydrophila

The influence of the cytoskeleton on the invasion of Aeromonas hydrophila strain AhJ-1, isolated from diseased fish, in the monolayer cell of epithelioma papillosum cells of carp (EPC) was evaluated by the recovery of gentamicin-resistant bacteria from Triton X-100 cell lysates. The depolymerization of microfilaments (MF) by cytochalasin B and D inhibited the uptake of A. hydrophila in a dose-dependent manner and that of microtubules (MT) by colchicines and nocodazole did not affect the invasion of A. hydrophila in EPC cells significantly. The invasion frequency decreased approximately 62% with the addition of 0.1 microg/ml cytochalasin D and nearly 86% by the addition of 5.0 microg/ml. Invasion decreased approximately 49% and 83% by addition of cytochalasin B in a concentration of 2.5 microg/ml and 10.0 microg/ml. Colchicine and nocodazole, inhibitors of MT formation appears to have little effect on the invasion of EPC cells by strain Ah J-1. Thus MF formation, but not MT formation seems to play an important role in the internalization of A. hydrophila J-1.     

嗜水气单胞菌不同分离株生化特性及胞外蛋白酶的检测

嗜水气单胞菌可引起多种宿主患病,该菌致病性与其产生的多种毒力因子特别是胞外蛋白酶有关。本研究检测了26株嗜水气单胞菌不同分离株的生化指标,采用脱脂乳平板法检测了胞外蛋白酶的产生情况,并利用PCR技术检测了2种胞外蛋白酶基因ahp-A(编码丝氨酸胞外蛋白酶)和eprCAI(编码温敏胞外蛋白酶)。结果表明,大部分菌株生化指标表现为蔗糖(+)、阿拉伯糖(+)、葡萄糖(+)、甘露醇(+)、鸟氨酸脱羧酶(-)、肌醇(-)、七叶苷(+);脱脂乳平板法检测发现,91.7%(22/24)的致病菌株均能产生胞外蛋白酶,而2株无毒株则不能产生,对2个胞外蛋白酶基因的检测也得到相似的结果。本研究将为嗜水气单胞菌的流行病学调查和致病机制的深入研究奠定基础。     

Influence of elevated protein and tannin-rich peanut skin supplementation on growth performance,blood metabolites,carcass traits and immune-related gene expression of grazing meat goats

The aim of the present study was to define whether elevated rumen-undegradable protein (RUP) and tannin-rich peanut skin (PS) supplementation would affect animal growth performance, average daily gain (ADG), blood metabolites, carcass traits associated with lipogenic and immune-related gene expressions in meat goats grazing winter wheat (WW). Thirty-six Kiko-crossbreed male goats at approximately 6 months of age were blocked by body weight (BW; 25.6 ± 1.1 kg) and randomly assigned to one of the four treatments with two replicates based on a 2 × 2 factorial design. Diets contained PS replacing alfalfa meal (ALM), without or with RUP supplementation. Both PS and ALM were incorporated into grain mix portion of the diet and pelletized, with remaining diets fed ad libitum of WW forage for a period of 51 days. Lipogenic genes examined included SCD, ACLY, YWHAZ, PPIA and FABP4, while immune-related genes examined included ACTB (as a control gene), H3F3A, PPIA, IRF3, STAT2, HERC3 and IFIT3 antibody genes. The meat goats on PS-pellet-supplemented group with or without RUP supplementation grew 38.5% faster ADG (p < .001) when compared to control-supplemented group. When goats received PS diet, empty body weight, hot carcass, cold carcass, shoulder, hind shank, rack, loin and fat thickness were greater (p < .05) than control diet. Animals on PS-pellet had higher ACLY, YWHAZ, PPIA and FABP4 gene expression (p < .05) when compared to ALM-pellet control, with RUP by PS-pellet interactions (p < .01). Goats receiving additional RUP supplementation had increased (p < .05) STAT2 gene expression, whereas goats receiving PS-pellet supplementation showed increased STAT2 (p < .05) and a tendency to increase IRF3 (p = .07) gene expressions. In conclusion, the addition of PS-pellet or RUP supplementation has the potential to improve ADG and altered selected lipogenic and immune-related gene expressions.     

In vitro and in vivo studies of the use of some medicinal herbals against the pathogen Aeromonas hydrophila in goldfish

Aeromonas hydrophila is a ubiquitous and opportunistic bacterial pathogen that produces ulcerative dermatitis under stress conditions and inflicts severe losses on global fisheries and fish culture. This study evaluates the antimicrobial potency of aqueous and ethanolic decoction (individual extract) and concoction (mixed extract) of three common medicinal herbs, turmeric Curcuma longa, Tulsi plant Ocimum sanctum, and neem Azadirachta indica, against the in vitro growth of A. hydrophila. Among the decoctions, A. indica exhibited the most potent antibacterial property (P < 0.05) against A. hydrophila. Among the concoctions, both the aqueous and ethanolic triherbal extracts mixed in the ratio of 1:1:1 had higher antibacterial activity (P < 0.05) than the other concoctions and decoctions. Goldfish Carassius auratus (10 +/- 2 g) were challenged with A. hydrophila intramuscularly in the caudal region with two separate doses (days 1 and 3) of 50 microL/fish (1.8 x 10(3) colony-forming units per milliliter). On days 9 (early) and 15 (late) of infection, fish were held in a net and dip treated for 5 min daily in a 1-L solution of 1% aqueous triherbal concoction. Red blood cell (RBC) count, hemoglobin, and hematocrit levels of the infected group were significantly higher (P < 0.05) than those of the control group. In the early treated group, all of the affected profile values returned to near normal, while the late-treated group registered a partial recovery, such as improved RBC count. The derived hematological values, such as mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, of the early and late-treated groups also significantly declined (P < 0.05) but were restored to near normal (P > 0.05) only in the early treated group. The results suggest that dip treatment of A. hydrophila-infected goldfish in an aqueous triherbal concoction had a synergistic restorative effect on the hematological variables.     

Histopathology of the early phase during experimental Corynebacterium pseudotuberculosis infection in lambs.

Histological responses during experimental Corynebacterium pseudotuberculosis infection in lambs were investigated in parotid lymph nodes for ten days following inoculation. Lambs were infected by the subcutaneous route into the right eyelid with a virulent strain of C. pseudotuberculosis. Multiple microscopic acute abscesses, predominantly infiltrated with polymorphonuclear (PMN) leucocytes, were seen in the right parotid lymph node on the 1st day post-inoculation (PI). This massive PMN infiltration coincided with a peripheral blood granulocytosis. On day 3 PI, an influx of histiocytes was observed, while the microabscesses became confluent. From day 3 to day 10 PI, these lesions became enlarged and transformed into typical pyogranulomas with a central necrosis and a peripheral mantle of mononuclear cells composed of macrophages, epithelioid cells and lymphocytes; these histological changes were associated with a bacterial dissemination limited to the superficial lymph nodes. A lymphoid hyperplasia with prominent germinal centers was observed in the draining lymph nodes from day 3 PI. These results illustrate the dual role of granulomatous lesions in chronic bacterial infections: although they limit bacterial dissemination, the granulomas do not impair the persistence of infectious organisms in the host, leading to focal tissue damage.     

Host responses during experimental infection with Fasciola gigantica or Fasciola hepatica in Merino sheep I. Comparative immunological and plasma biochemical changes during early infection

This study reports the early biochemical changes in plasma, comparative host-immune responses and parasite recovery data in Merino sheep during the first 10 weeks of infection with Fasciola gigantica and Fasciola hepatica. One group of sheep were uninfected, four groups of sheep received incremental challenge doses of F. gigantica metacercariae (50, 125, 225 and 400, respectively) and the sixth group was challenged with 250 F. hepatica metacercariae. At 10 weeks post infection (wpi), sheep challenged with F. hepatica showed the greatest fluke recovery (mean 119, range 84-166); a significantly higher biomass of parasites recovered (2.5-fold greater than the highest dose of F. gigantica); and a greater mean % parasite recovery (39.3%, range 27-55%) than any group challenged with F. gigantica. Within the groups dosed with F. gigantica a strong dose-dependent response was observed in both fluke recovery and fluke biomass with increasing dose of metacercariae. The mean % parasite recovery of F. gigantica infected groups 1-5 were 26, 23, 26 and 25%, respectively, suggesting a uniform viability of parasite establishment independent of infection dose. At 6 wpi, elevated levels of plasma GLDH were observed in the F. gigantica infected groups compared to the uninfected sheep (p<0.005) whereas the F. hepatica challenged group had four-fold higher levels of GLDH compared to the F. gigantica infected group (p<0.001). Elevated levels of GGT as an indicator of epithelial damage in the bile duct was only seen in the group challenged with F. hepatica at 10 wpi when it rose from below 100 IU/l to approximately 250 IU/l (p<0.0001) whereas no detectable increase in GGT was observed in any of the groups challenged with F. gigantica. The white blood cell response to F. hepatica infection was biphasic with the initial peak at 4 wpi and a second peak at 9 wpi, corresponding to the period of migration of juvenile fluke in the liver and the time when adult flukes are migrating into the bile duct, respectively. This biphasic response was also evident in the changes in the eosinophil counts and serum haemoglobin levels. There was a trend toward higher parasite-specific IgG2 titres in sheep infected with lower worm burdens, suggesting that higher F. gigantica or F. hepatica burdens suppress IgG2 responses. The findings of this study suggest that, in early infection in a permissive host, F. hepatica appears to be more pathogenic than F. gigantica because of its rapid increase in size and the speed of its progression through the migratory phases of its life cycle.     

维氏气单胞菌TH0426株ompW基因的克隆、生物信息学分析及其原核表达

为研究维氏气单胞菌(Aeromonas veronii)ompW基因的生物信息学特性及相关功能,试验克隆了维氏气单胞菌ompW基因片段,通过生物信息学软件分析其编码蛋白的理化性质、疏水性、信号肽、跨膜区、二级及三级结构,并对其进行原核表达及反应原性检测。结果显示,ompW基因全长594 bp,编码197个氨基酸,TH0426株ompW基因与A.veronii B5B6株属于同一分支;该蛋白属于外膜蛋白,在19～29位氨基酸之间有一个典型的疏水区域,不存在跨膜区、信号肽,二级结构以β折叠与无规则卷曲为主。Western blot结果显示OmpW重组蛋白能与鼠抗维氏气单胞菌(A.veronii)TH0426株血清发生特异性结合。综上,OmpW重组蛋白具有一定的免疫原性,为进一步研究A.veronii疫苗奠定基础。     

Is there a relationship between haemoglobin genotype and the innate resistance to experimental Haemonchus contortus infection in Merino lambs?

Responses to a single or repeated infection with 7000 infective larvae of Haemonchus contortus were studied in an experiment using a total of 106 3-month-old lambs with AA, AB or BB haemoglobin (Hb) genotypes. Results were assessed by faecal egg counts, adult worm counts, haematocrit values, haemoglobin concentrations, total serum protein and serum antibody IgG1 and IgA ELISA titres. None of these parameters showed a strong relationship to the Hb type. The prevalence of low responder (greater than 500 worms) and of high responder (less than 50 worms) animals in groups AA, AB and BB Hb types was 3.8 and 34.6, 20.6 and 35.2, 28.1 and 43.7%, respectively, suggesting that the responsiveness to nematode infection is under the control of gene(s) not closely linked with those determining the Hb genotype. Worm counts of a primary infection are more subject to variation than those of a secondary infection. There is a strong relationship between adult worm counts and faecal egg counts taken close to the time of slaughter. In living animals low and high responder discrimination can be based on individual faecal egg counts around 50 days after a secondary infection. Haematocrit values proved to be of little value in the low and high responder selection. In this regard neither Hb concentration nor total serum protein values are of practical significance. In 3-month-old lambs primary infection induced partial immunity which could prevent the establishment of a part of the secondary infection, irrespective of the presence or absence of the primary worm population. The development of immunity was not associated with an increase of serum IgG1 and IgA antibody levels. Specific antibody production was not influenced by Hb types. Mean antibody levels of low responder lambs showed no difference from those of high responders. Thus, serum IgG1 and IgA levels are of no predictive value in identifying lambs which are genetically resistant to Haemonchus infection.