F4 receptor-independent priming of the systemic immune system of pigs by low oral doses of F4 fimbriae

Oral administration of F4 fimbriae of Escherichia coli induces intestinal mucosal immune responses in F4 receptor-positive (F4R(+)) pigs, but not in F4R(-) pigs. We examined whether F4 fimbriae in F4R(-) animals behave like a food antigen and can induce oral tolerance. Therefore, F4R(+) and F4R(-) pigs were fed 2mg of F4 and challenged i.m. to evaluate the effect of oral F4 on the systemic immune system. As control antigen, two different oral doses (2 and 600 mg) of OVA were used. Thirty days after the i.m. OVA challenge, the OVA-specific serum IgG titre in 600 mg-fed pigs was lower than that in non-fed animals, indicating that tolerance was induced. Conversely, in the 2mg-fed pigs a rapid increase of OVA-specific IgG with higher titres than those in non-fed pigs was seen following challenge, indicating a priming of the systemic immune system. A similar priming was seen in both F4-fed F4R(-) and F4R(+) pigs. Upon challenge, non-fed pigs displayed a primary immune response with a slow increase of F4-specific serum IgG, whereas F4-fed F4R(-) and F4R(+) pigs showed secondary responses with a rapid increase of serum IgG. This was expected in F4R(+) pigs, as in these animals oral F4 induces F4-specific antibody-secreting cells in the spleen, suggesting a priming of the systemic immune system. However, also the F4-fed F4R(-) pigs displayed a secondary response, despite the failure to detect a response upon oral F4 administration. These findings suggest that the F4 antigen, at a dose of 2 mg, behaves like a common food antigen in F4R(-) pigs, namely it induces a systemic priming.     

Maternal immunity enhances systemic recall immune responses upon oral immunization of piglets with F4 fimbriae

F4 enterotoxigenic Escherichia coli (ETEC) cause diarrhoea and mortality in piglets leading to severe economic losses. Oral immunization of piglets with F4 fimbriae induces a protective intestinal immune response evidenced by an F4-specific serum and intestinal IgA response. However, successful oral immunization of pigs with F4 fimbriae in the presence of maternal immunity has not been demonstrated yet. In the present study we aimed to evaluate the effect of maternal immunity on the induction of a systemic immune response upon oral immunization of piglets. Whereas F4-specific IgG and IgA could be induced by oral immunization of pigs without maternal antibodies and by intramuscular immunization of pigs with maternal antibodies, no such response was seen in the orally immunized animals with maternal antibodies. Since maternal antibodies can mask an antibody response, we also looked by ELIspot assays for circulating F4-specific antibody secreting cells (ASCs). Enumerating the F4-specific ASCs within the circulating peripheral blood mononuclear cells, and the number of F4-specific IgA ASCs within the circulating IgA⁺ B-cells revealed an F4-specific immune response in the orally immunized animals with maternal antibodies. Interestingly, results suggest a more robust IgA booster response by oral immunization of pigs with than without maternal antibodies. These results demonstrate that oral immunization of piglets with F4-specific maternal antibodies is feasible and that these maternal antibodies seem to enhance the secondary systemic immune response. Furthermore, our ELIspot assay on enriched IgA⁺ B-cells could be used as a screening procedure to optimize mucosal immunization protocols in pigs with maternal immunity.     

Active oral immunization of suckling piglets to prevent colonization after weaning by enterotoxigenic Escherichia coli with fimbriae F18

Immunoprophylaxis of porcine oedema disease and post-weaning diarrhoea caused by strains of Escherichia coli expressing fimbriae F18 is an unsolved problem. The study was designed to examine whether vaccination with a live F18ac vaccine of unweaned pigs born to sows with F18ac antibody in the colostrum requires preformed fimbriae in the vaccine, and whether protection against the heterologous fimbrial variant F18ab is induced as well. Genetically susceptible pigs were vaccinated orally on three consecutive days, beginning 10 days before weaning with 10(11) CFU of an F18ac culture. Challenge with a dose of 10(7) CFU of E. coli F18 on three consecutive days was initiated 9 or 11 days after weaning. Eighteen pigs given the fimbriated F18ac vaccine and challenged with a strain of the homologous fimbrial variant were protected against colonization; mean faecal viable counts of the challenge strain were >3 log10 lower than those from the 17 non-vaccinated control pigs. The vaccinated pigs developed a significant rise of F18ac IgA serum antibodies. The 23 pigs which had received the non-fimbriated vaccine showed no significant protection and exhibited much lower serum F18ac IgA ELISA reactivities. Eighteen pigs vaccinated with the fimbriated F18ac and challenged with an F18ab strain had faecal viable counts nearly as high as those from 16 non-vaccinated control pigs. It is concluded that only oral vaccines having preformed fimbriae induce protection limited to the homologous fimbrial variant.     

Cholera toxin improves the F4(K88)-specific immune response following oral immunization of pigs with recombinant FaeG

Oral immunization of both humans and animals with non-replicating soluble antigens often results in the induction of oral tolerance. However, receptor-dependent uptake of orally administered soluble antigens can lead to the induction of an antigen-specific immune response. Indeed, oral immunization of pigs with recombinant FaeG (rFaeG), the adhesin of the F4(K88) fimbriae of enterotoxigenic Escherichia coli (ETEC), induces an F4-specific humoral and cellular immune response. This response is accompanied with a reduction in the excretion of F4(+)E. coli following challenge. To improve the immune response against F4, rFaeG was orally co-administered with the mucosal adjuvant cholera toxin (CT). Oral immunization of pigs with rFaeG and CT significantly improved the induction of an F4-specific humoral and cellular immune response and also significantly reduced the faecal F4(+)E. coli excretion following F4(+) ETEC challenge as compared to rFaeG-immunized pigs. Therefore, the present study demonstrates that CT can act in pigs as a mucosal adjuvant for antigens that bind to the intestinal epithelium by a CT-receptor-independent mechanism.     

Influence of porcine intestinal pH and gastric digestion on antigenicity of F4 fimbriae for oral immunisation

Newly weaned piglets can be orally immunised against F4+ enterotoxigenic Escherichia coli (ETEC) infection with F4 fimbriae. However, to efficiently develop a vaccine against ETEC induced postweaning diarrhoea, knowledge of the stability of the F4 fimbriae to different pH and gastric digestion is needed. The gastrointestinal pH in suckling and recently weaned piglets was measured and the stability of F4 fimbriae to different pH and to pepsin was assessed in vitro. In the stomach the lowest pH was found in the fundus gland region. Gastric pH values below 2.5 were not found in suckling piglets or at the day of weaning, in contrast to piglets 1 and 2 weeks postweaning. Along the first half of the small intestine and in the caecum, a negative correlation was found between pH and age. The F4 fimbriae were stable to pH 1.5 and 2 for 2 h, whereas longer incubation periods resulted in conversion of the multimeric forms into monomers. The F4 fimbriae were partially degraded by incubation for 15-30 min in simulated gastric fluid at pH 1.5 and 2, and completely digested from 3 h onwards. At pH 3, the fimbriae maintained their antigenicity for at least 4h. The results demonstrate that gastric digestion will only have a limited impact on oral immunisation since liquid passes through the stomach relatively quickly (50% within 2 h). However, we previously demonstrated that the transit times are prolonged shortly after weaning. Shortly after weaning it could be necessary to protect the F4 fimbriae against gastric digestion to obtain efficient oral immunisation of the piglets.     

Methodology and application of Escherichia coli F4 and F18 encoding infection models in post-weaning pigs

The enterotoxigenic Escherichia coli(ETEC) expressing F4 and F18 fimbriae are the two main pathogens associated with post-weaning diarrhea(PWD) in piglets. The growing global concern regarding antimicrobial resistance(AMR)has encouraged research into the development of nutritional and feeding strategies as well as vaccination protocols in order to counteract the PWD due to ETEC. A valid approach to researching effective strategies is to implement piglet in vivo challenge models with ETEC infection. Thus, the proper application and standardization of ETEC F4 and F18 challenge models represent an urgent priority. The current review provides an overview regarding the current piglet ETEC F4 and F18 challenge models; it highlights the key points for setting the challenge protocols and the most important indicators which should be included in research studies to verify the effectiveness of the ETEC challenge.Based on the current review, it is recommended that the setting of the model correctly assesses the choice and preconditioning of pigs, and the timing and dosage of the ETEC inoculation. Furthermore, the evaluation of the ETEC challenge response should include both clinical parameters(such as the occurrence of diarrhea,rectal temperature and bacterial fecal shedding) and biomarkers for the specific expression of ETEC F4/F18(such as antibody production, specific F4/F18 immunoglobulins(Igs), ETEC F4/F18 fecal enumeration and analysis of the F4/F18 receptors expression in the intestinal brush borders). On the basis of the review, the piglets' response upon F4 or F18 inoculation differed in terms of the timing and intensity of the diarrhea development, on ETEC fecal shedding and in the piglets' immunological antibody response. This information was considered to be relevant to correctly define the experimental protocol, the data recording and the sample collections. Appropriate challenge settings and evaluation of the response parameters will allow future research studies to comply with the replacement, reduction and refinement(3 R) approach, and to be able to evaluate the efficiency of a given feeding, nutritional or vaccination intervention in order to combat ETEC infection.     

口服硫酸安普霉素在猪体内的药代动力学和生物利用度研究

对 5头健康猪口服和静脉注射国产硫酸安普霉素 ,研究其在猪体内的药代动力学和生物利用度。用微生物法测定血清药物浓度 ,结果平均回收率为 99.0 3%,血清最低检测浓度为 0 .0 5 μg/ ml,日内日间变异系数为 2 .2 %～ 5 .0 %,且血清浓度在0 .0 5～ 3μg/ m l范围呈良好线性关系 (r=0 .996 5 )。以 2 0 mg/ kg口服和以 2 0 mg/ kg静脉注射硫酸安普霉素后 ,经 Mcpkp药代动力学计算机程序处理 ,体内药物运转分别符合开放型一室和二室模型 ,生物半衰期 t1 / 2 分别为 (7.36± 1 .5 2 ) h和 (3.1 7± 0 .75 )h;CLB分别为 4 .82 L / kg· h和 0 .1 6 L / kg· h;AUC值分别为 4 .1 4和 1 30 .6 2。口服 :Cmax为 (0 .2 4± 0 .0 3)μg/ ml;Tp为 (5 .1 2±0 .6 1 ) h;T1 / 2 K为 (7.36± 1 .5 2 ) ;生物利用度 (AUCp.0 / AUCi.v)为 (3.1 92 8± 0 .70 4 4 ) %。上述药代动力学数据为动物临床用药提供有价值的理论依据     

Immune responses elicited in mice with recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis> expressing F4 fimbrial adhesin FaeG by oral immunization

Enterotoxigenic Escherichia coli (ETEC) is a major pathogenic agent causing piglet diarrhea. The major subunit and adhesin FaeG of F4⁺ ETEC is an important virulence factor with strong immunogenicity. To determine whether Lactococcus lactis (L. lactis) could effectively deliver FaeG to the mucosal immune system, recombinant L. lactis expressing FaeG was constructed, and immune responses in mice following oral route delivery of recombinant L. lactis were explored. The production of FaeG expressed in L. lactis was up to approximately 10% of soluble whole-cell proteins, and recombinant FaeG (rFaeG) possessed good immunoreactivity by Western blot analysis. Oral immunization with recombinant L. lactis expressing FaeG induced F4-specific mucosal and systemic immune responses in the mice. In addition, high dose recombinant L. lactis or co-administration of high dose recombinant L. lactis with CTB enhanced the immune responses. These results suggested that L. lactis expressing FaeG was a promising candidate vaccine against ETEC.     

Identification of infant and adult swine susceptible to enterotoxigenic Escherichia coli by detection of receptors for F4(K88)ac fimbriae in brush borders or feces.

We attempted to determine F4(K88)-adhesive and non-adhesive phenotypes of infant (neonatal <3 day old and weaned <4 week old pigs) and adult (>6 month old) swine by ELISA using immobilized F4(K88)ac fimbrial antigen or whole F4(K88) + E. coli cells (strains M1823 and 1476) and isolated small intestinal brush borders or easily-obtainable fecal samples from the same animals. Nineteen of 22 neonates (86%), 17 of 20 weaners (85%), and 26 of 39 adults (67%) were classified identically as F4(K88) receptor-positive or negative by the ELISA. The ELISA with feces from adult swine was found to be almost equally specific (87%) as that with feces from neonatal (90%) and weaned (91%) pigs. However, the sensitivity of the assay was low (38%), indicating that fecal samples from adults contained less receptor-material than necessary for comparable phenotyping. The receptor-positive brush borders from neonates and weaners reacted significantly better (P < 0.02, < 0.001 respectively) with purified F4(K88) antigen than did those from adults. There was good agreement between the average ELISA values for feces from infant and adult swine regardless the source of coating antigen applied. With this assay we can determine F4(K88) phenotypes of infant swine using easily-collected fecal samples rather than isolated brush borders. It was also concluded that tested feces is not an acceptable alternate source of the receptor-material to brush borders from F4(K88)-susceptible adult swine.     

Comparison of production traits between pigs with and without the Escherichia coli F4 receptors in a White Duroc x Erhualian intercross F2 population

To evaluate the influence of enterotoxigenic Escherichia coli (ETEC) F4 receptors on production traits in pigs, ETEC F4ab, F4ac, and F4ad adhesion phenotypes and 27 traits related to growth, carcass, meat quality, and length of the small intestine in a White Duroc x Erhualian intercross population were measured. Performance data revealed that pigs with the F4ab or F4ac receptor (adhesive phenotypes) had greater (P < 0.01) ADG during the fattening period (from 46 to 240 d) and carcass weight and length at 240 d than pigs lacking the receptors (nonadhesive phenotype). Conversely, animals having the F4ad receptor had less (P < 0.01) ADG during the fattening period and carcass weight than those lacking the receptor. In total, 8 adhesion patterns (A to H) for the 3 F4 strains were observed in this experimental population. Pigs with both F4ab and F4ac receptors (phenotype B) had greater (P < 0.01) ADG, carcass weight, and length at 240 d compared with pigs without the F4 receptors. No difference was found (P > 0.05) in traits related to meat quality, fatness, and length of the small intestine between pigs with or without the receptors. On the basis of the antagonistic relationship between susceptibility to F4ab/ac and production traits, we speculate that the prevalence of the ETEC F4ab/ac adhesive phenotype in pig populations is attributable to balanced natural and artificial selection.     

Characterization of polymorphisms of transferrin receptor and their association with susceptibility to ETEC F4ab/ac in pigs

Diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 (F4ab, F4ac and F4ad) fimbriae is a significant cause of mortality and morbidity in newborn and weaned pigs. The locus controlling susceptibility towards ETEC F4ab/ac has been mapped to SSC13q41, in which TFRC (transferrin receptor) was localized and considered as a positional candidate gene for ETEC F4ab/ac receptor. In this study, we determined susceptibility/resistance to ETEC F4ab/ac in a total of 755 F2 animals from a White Duroc x Erhualian intercross using a microscopic enterocyte adhesion assay. We identified two TFRC polymorphisms (SNPs 591 A>G and 632 A>G) in a single exon after comparative sequencing analysis of 2371-bp amplicons containing the complete coding region of TFRC using RNA of eight full-sib F2 animals with susceptible and resistant phenotypes. The intron sequences flanking the two exon polymorphisms were obtained, revealing an intron polymorphism (SNP 291 C>T). We genotyped the 19 founder animals of the White Duroc x Erhualian intercross for the identified polymorphisms, showing that only the 291 C>T polymorphism is a highly informative marker. We further genotyped all 59 F1 and 755 F2 animals for the 291 C>T polymorphism, and the association of this polymorphism with susceptibility/resistance to ETEC F4ab/ac in these F2 animals was evaluated by the transmission disequilibrium test. The result showed that the 291 C>T polymorphism is not a causal mutation, however, has a significant linkage disequilibrium with the ETEC F4ab/ac, especially F4ac receptor locus.     

Pharmacokinetics of amoxicillin administered in drinking water to recently weaned 3- to 4-week-old pigs with diarrhea experimentally induced by Escherichia coli O149:F4

OBJECTIVE: To measure effects of Escherichia coli O149:F4-induced diarrhea on water consumption and pharmacokinetics of amoxicillin after administration in drinking water. ANIMALS: 24 recently weaned 24- to 28-day-old crossbred pigs. PROCEDURE: 10 pigs were inoculated with E. coli O149:F4; all 10 pigs subsequently developed diarrhea. Pigs were medicated by administration of amoxicillin in the drinking water (0.75 mg/mL) for a 4-hour period on 2 consecutive days. Fourteen age-matched noninfected healthy pigs (control group) were medicated in a similar manner. Blood samples were obtained from both groups daily, and plasma concentrations of amoxicillin were analyzed by use of high-performance liquid chromatography. RESULTS: Diarrhea reduced the area under the plasma concentration-versus-time curve (AUC) and maximum plasma concentration (C(max)) of amoxicillin on the first day of medication by 56% and 63%, respectively. The AUC of amoxicillin on the second day of medication for diarrheic pigs did not differ significantly from that of control pigs on the first day of medication. CONCLUSIONS AND CLINICAL RELEVANCE: E. coli-induced diarrhea reduced the AUC of amoxicillin and time that plasma concentration of amoxicillin was > 0.025 microg/mL and, hence, less likely to have a therapeutic effect on the first day of administration in drinking water. On the assumption that plasma concentrations may indirectly reflect concentrations at the site of infection, analysis of our results suggests that higher doses of amoxicillin may be appropriate for administration in drinking water during a 4-hour period on the first day that pigs have diarrhea attributable to E. coli O149:F4.     

Protection against neonatal Escherichia coli diarrhoea in pigs by vaccination of sows with a new vaccine that contains purified enterotoxic E. coli virulence factors F4ac, F4ab, F5 and F6 fimbrial antigens and heat-labile E. coli enterotoxin (LT) toxoid

The efficacy of a new vaccine against neonatal Escherichia coli diarrhoea in piglets containing purified F4ab, F4ac, F5 and F6 fimbriae and detoxified heat-labile toxin (LT) was tested in challenge experiments by the method described by the European Pharmacopoeia (3rd edn, EDQM, Council of Europe, Strasbourg, France). A group of 11 young sows from a herd without E. coli problems was vaccinated 6-8 and 2-4 weeks prior to expected farrowing and another group of nine young sows were non-vaccinated controls. Escherichia coli antibody titres were determined in serum samples taken from the sows before first vaccination and before farrowing and in colostrum samples. The newborn piglets were allowed to suckle colostrum from their mother immediately after birth. The piglets were marked with individually numbered ear tags. Approximately 12 h after birth, 118 piglets from vaccinated sows and 79 piglets from non-vaccinated control sows were challenged by oral instillation of 5 ml of a freshly prepared culture of one of the challenge strains [O8:K87:F4ab (LT+) or O149:K91:F4ac (LT+) or O9:K30:F5 or O9:K103:F6 respectively]. The challenge cultures contained as a mean 6.8x10(9) CFU/ml. After challenge the piglets were observed for 7 days and mortality and morbidity were recorded. Vaccinated sows developed significant levels of antibody titres in colostrum and serum. Control sows stayed at a low/seronegative level. The protective efficacy was excellent because 66.7-87.5% of the piglets from vaccinated sows remained without clinical signs after challenge. Only 0.0-28.0% of the piglets from non-vaccinated sows remained healthy and more than 47.1% of the piglets in this group died after challenge. It is concluded that the new vaccine is very effective in protection of piglets against neonatal E. coli diarrhoea.     

Administration of probiotics influences F4 (K88)-positive enterotoxigenic Escherichia coli attachment and intestinal cytokine expression in weaned pigs

ABSTRACT: This study evaluated the effect of the probiotics Pediococcus acidilactici and Saccharomyces cerevisiae boulardii on the intestinal colonization of O149 enterotoxigenic Escherichia coli harbouring the F4 (K88) fimbriae (ETEC F4) and on the expression of ileal cytokines in weaned pigs. At birth, different litters of pigs were randomly assigned to one of the following treatments: 1) control without antibiotics or probiotics (CTRL); 2) reference group in which chlortetracycline and tiamulin were added to weanling feed (ATB); 3) P. acidilactici; 4) S. cerevisiae boulardii; or 5) P. acidilactici + S. cerevisiae boulardii. Probiotics were administered daily (1 × 109 CFU per pig) during the lactation period and after weaning (day 21). At 28 days of age, all pigs were orally challenged with an ETEC F4 strain, and a necropsy was performed 24 h later. Intestinal segments were collected to evaluate bacterial colonization in the small intestine and ileal cytokine expressions. Attachment of ETEC F4 to the intestinal mucosa was significantly reduced in pigs treated with P. acidilactici or S. cerevisiae boulardii in comparison with the ATB group (P = 0.01 and P = 0.03, respectively). In addition, proinflammatory cytokines, such as IL-6, were upregulated in ETEC F4 challenged pigs treated with P. acidilactici alone or in combination with S. cerevisiae boulardii compared with the CTRL group. In conclusion, the administration of P. acidilactici or S. cerevisiae boulardii was effective in reducing ETEC F4 attachment to the ileal mucosa, whereas the presence of P. acidilactici was required to modulate the expression of intestinal inflammatory cytokines in pigs challenged with ETEC F4.     

Genotypic prevalence of the fimbrial adhesins (F4, F5, F6, F41 and F18) and toxins (LT, STa, STb and STx2e) in Escherichia coli isolated from postweaning pigs with diarrhoea or oedema disease in Korea

A PCR was used to determine the genotypic prevalence of five fimbrial adhesins (F4, F5, F6, F41 and F18), two heat-stable enterotoxins (STa and STb), the heat-labile enterotoxin (LT), and the shiga toxin 2e (Stx2e) in 230 isolates of Escherichia coli from postweaning pigs with diarrhoea or oedema disease. Ninety-four (40.9 per cent) of the isolates carried genes for at least one of the fimbrial adhesins or toxins. Genes for the F18 fimbrial adhesin were detected in 18.3 per cent, and genes for F4, F6, F5 and F41 were detected in 10.0 per cent, 4.3 per cent, 1.7 per cent and 0.8 per cent of the isolates, respectively. Genes for STa, STb and LT were detected in 25.7 per cent, 15.2 per cent and 8.7 per cent of the isolates, respectively. Genes for Stx2e were detected in 36 (15.6 per cent) of the isolates, and among them 24 also contained the gene for F18ab and four also contained the gene for F18ac.     

Effect of susceptibility to enterotoxigenic Escherichia coli F4 and of dietary tryptophan on gut microbiota diversity observed in healthy young pigs

Healthy weaned pigs susceptible to enterotoxigenic Escherichia coli F4 (ETEC) require more tryptophan (Trp) to maximize their performance. This may be related to an effect on intestinal microbiota. We studied the intestinal bacterial diversity of healthy pigs with different susceptibility to ETEC and fed different Trp levels. Thirty-six littermate weaned pigs were selected to obtain a set potentially formed of 50% ETEC-susceptible and 50% non-susceptible pigs, based on a Mucin 4 gene polymorphism. Pigs were fed a diet with 0.17 (TrpL) or 0.22 (TrpH) standardized ileal digestible Trp:Lys ratio for 21 days. Slaughtered pigs were classified into non-susceptible, mildly susceptible, and susceptible, by testing ETEC adhesion to intestinal villi. Bacterial diversity in jejunum content was assessed by the 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting analysis and expressed by the Shannon index. Susceptible pigs had a reduced bacterial diversity, particularly with TrpL diet (p = 0.003). The ETEC adhesion class affected the quantification of enterobacteria DNA (p = 0.027). One DGGE band, which referred to Clostridium bartlettii, was not evidenced in all the susceptible pigs; less DNA from this microbe was quantified by RT-PCR in the jejunum from TrpH susceptible pigs (p = 0.025) compared to TrpL. The gene expression for β-galactoside α-2,3-sialyltransferase 1 was higher in jejunal tissue of ETEC-susceptible pigs (p = 0.019). In studies on pig gut microbiota, the presence of intestinal receptors for ETEC should be considered because of their contribution to a reduced bacterial diversity. This effect could be partially reversed by dietary Trp addition.     

奶牛乳腺中4F2hc基因的表达模式及亮氨酸对其表达的调控研究

为了研究4F2hc在奶牛乳腺中的表达模式及调控方式,进一步明确氨基酸在奶牛乳腺上皮细胞中的跨膜转运过程,本研究采用Western blotting和实时荧光定量PCR技术检测了4F2hc在泌乳期和干奶期奶牛乳腺组织中的表达变化;在体外培养的泌乳期奶牛乳腺上皮细胞中添加亮氨酸,采用Western blotting和实时荧光定量PCR技术检测其对奶牛乳腺上皮细胞中4F2hc表达的影响;采用雷帕霉素抑制剂抑制mTOR信号通路,使用Western blotting方法检测mTOR信号抑制后奶牛乳腺上皮细胞中4F2hc表达以及乳蛋白合成的变化。结果显示,在泌乳期的奶牛乳腺组织中4F2hc的mRNA和蛋白表达水平均显著或极显著高于干奶期（P<0.05,P<0.01）;在体外培养的奶牛乳腺上皮细胞中添加亮氨酸可以极显著提高乳腺上皮细胞中4F2hc的mRNA和蛋白质表达水平（P<0.01）;亮氨酸刺激可以激活细胞内的mTOR信号通路（P<0.05）,而雷帕霉素处理则可以显著抑制mTOR信号分子的磷酸化并极显著抑制亮氨酸诱导的4F2hc的表达（P<0.05,P<0.01）,进而极显著抑制β-Casein的合成（P<0.01）。以上研究结果表明,4F2hc基因的表达与奶牛乳腺的泌乳活性之间呈正相关,亮氨酸可以通过激活mTOR信号通路来调节4F2hc基因的表达,进而影响乳蛋白的合成。     

Protection Against Neonatal Escherichia coli Diarrhoea in Pigs by Vaccination of Sows with a New Vaccine that Contains Purified Enterotoxic E. coli Virulence Factors F4ac,F4ab,F5 and F6 Fimbrial Antigens and Heat‐Labile E. coli Enterotoxin (LT) Toxoid

The efficacy of a new vaccine against neonatal Escherichia coli diarrhoea in piglets containing purified F4ab, F4ac, F5 and F6 fimbriae and detoxified heat‐labile toxin (LT) was tested in challenge experiments by the method described by the European Pharmacopoeia (3rd edn, EDQM, Council of Europe, Strasbourg, France). A group of 11 young sows from a herd without E. coli problems was vaccinated 6–8 and 2–4 weeks prior to expected farrowing and another group of nine young sows were non‐vaccinated controls. Escherichia coli antibody titres were determined in serum samples taken from the sows before first vaccination and before farrowing and in colostrum samples. The newborn piglets were allowed to suckle colostrum from their mother immediately after birth. The piglets were marked with individually numbered ear tags. Approximately 12 h after birth, 118 piglets from vaccinated sows and 79 piglets from non‐vaccinated control sows were challenged by oral instillation of 5 ml of a freshly prepared culture of one of the challenge strains [O8:K87:F4ab (LT+) or O149:K91:F4ac (LT+) or O9:K30:F5 or O9:K103:F6 respectively]. The challenge cultures contained as a mean 6.8 × 10⁹ CFU/ml. After challenge the piglets were observed for 7 days and mortality and morbidity were recorded. Vaccinated sows developed significant levels of antibody titres in colostrum and serum. Control sows stayed at a low/seronegative level. The protective efficacy was excellent because 66.7–87.5% of the piglets from vaccinated sows remained without clinical signs after challenge. Only 0.0–28.0% of the piglets from non‐vaccinated sows remained healthy and more than 47.1% of the piglets in this group died after challenge. It is concluded that the new vaccine is very effective in protection of piglets against neonatal E. coli diarrhoea.     

Evaluation of immune responses in sheep induced by DNA immunization with genes encoding GRA1, GRA4, GRA6 and GRA7 antigens of Toxoplasma gondii

The dense granule proteins of Toxoplasma gondii are investigated as possible vaccine candidates against the parasite. The aim of this research was to evaluate the immune responses of sheep injected twice, intramuscularly, with DNA plasmids encoding T. gondii dense granule antigens GRA1, GRA4, GRA6 and GRA7 formulated into liposomes. Control sheep were injected with an empty vector or received no injections. The injection of sheep with DNA plasmids encoding for GRA1, GRA4, GRA6 or GRA7 elicited an immune response after the first and the second injections as indicated by the moderate to high antibody responses. The injection of pGRA7 induced a significant level of anti-GRA7 IgG2 antibody and IFN-γ responses indicating a Th1-like immune response whereas injection with pGRA1, pGRA4 and pGRA6 stimulated a IgG1 type antibody response with a limited, if any, IFN-γ response. The results demonstrate that the intramuscular injection of sheep with a DNA liposome formulated plasmid coding for GRA proteins is an effective system that induces a significant immune response against T. gondii.