CpG oligodeoxynucleotides augment the immune responses of piglets to swine Pasteurella multocida living vaccine in vivo

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) prevent development of T-helper type 2 (Th2) immune response and reverse established allergic responses in mouse models. However, little work on immune responses in piglets has been conducted in vivo. In this report, the ability of a porcine-specific CpG ODN to act as an immunostimulant and enhance immune responses of piglets to swine Pasteurella multocida living vaccine (SPML vaccine) was determined. The titre of IgG and IgG1/IgG2 isotype to SPML vaccine in serum, the proliferation of lymphocytes, SPML-specific interferon-gamma (IFN-gamma) and IL-6, TNF-alpha, IL-4 production of PBMCs in vitro and IFN-gamma, IL-6, TNF-alpha, IL-4, IL-10 in piglets serum were examined to identify the immune responses of the piglets. Immune responses of the piglets vaccinated with SPML and CpG ODN were significantly stronger than responses of piglets vaccinated with SPML alone. All these data summarized that immunostimulatory CpG ODN could modulate the immune response towards a Th1-like response when co-administered to piglets during SPML vaccination, which suggested that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals.     

The molecular biology of pasteurella multocida

Pasteurella multocida is an important veterinary and opportunistic human pathogen. The species is diverse and complex with respect to antigenic variation, host predeliction and pathogenesis. Certain serological types are the aetiologic agents of severe pasteurellosis, such as fowl cholera in domestic and wild birds, bovine haemorrhagic septicaemia and porcine atrophic rhinitis. The recent application of molecular methods such as the polymerase chain reaction, restriction endonuclease analysis, ribotyping, pulsed-field gel electrophoresis, gene cloning, characterisation and recombinant protein expression, mutagenesis, plasmid and bacteriophage analysis and genomic mapping, have greatly increased our understanding of P. multocida and has provided researchers with a number of molecular tools to study pathogenesis and epidemiology at a molecular level.     

种鸭多杀性巴氏杆菌病的诊治

多杀性巴氏杆菌病在鸭、鸡、鹅、火鸡等家禽中易发,尤其鸭易感,对未接种禽霍乱疫苗的鸭子致病率很高,日死亡率可高达10%以上,对已接种禽霍乱疫苗的鸭子仍可感染多杀性巴氏杆菌病。笔者在实际工作中遇到一起病例,现将诊治经过报道如下。一、发病情况雷州市某种鸭养殖场,饲养樱桃谷后备种母鸭4000多只,配套种番鸭公鸭200多只,在90日龄时肌注禽霍乱———大肠杆菌二联活菌弱毒苗,每只2毫升,在160日龄再肌注上述二联苗,每只1.5毫升。当饲养到190日龄时发现种鸭拉黄绿色稀便,个别鸭嘴巴、鼻孔、泄殖腔流出淡红色液体,种公鸭发病180只,死亡11只,致…     

The type 4 fimbrial subunit gene of pasteurella multocida

Colonisation of host tissue by Gram- negative bacteria is facilitated by various adhesins, one of which is type 4 fimbriae (pili). These structures have been associated with pathogenesis in several bacterial species, and have been shown to mediate colonisation of epithelial surfaces. Recently, type 4 fimbriae were identified and characterised from P. multocida strains A, B and D. The type 4 fimbrial subunit protein (PtfA) was identified as an 18-kDa protein which was isolated from whole membrane fractions.We report here the isolation and characterisation of the gene (ptfA) encoding the PtfA protein from P. multocida VP161 (serotype A:1). Part of the gene was cloned on a 2-kb genomic DNA fragment. The complete ptfA gene was obtained using inverse PCR. The gene and its flanking regions were characterised, and the deduced PtfA amino acid sequence was compared to type 4 subunit protein sequences from other bacterial species. The ptfA gene was amplified and sequenced from several P. multocida strains. Comparison of these sequences revealed variation within the type 4 subunit gene of P. multocida.     

犊牛肺炎多杀性巴氏杆菌的分离与鉴定

新疆石河子6个规模化奶牛场相继出现出生1～9周龄的犊牛发生体温升高、呼吸困难以肺部感染为主,部分犊牛发生腹泻的疾病,造成90余头犊牛因肺炎死亡.从其中2个发病牛场死亡犊牛采集痛料,经涂片镜检、细菌分离培养、生化鉴定及动物试验,初步鉴定为多杀性巴氏杆菌,分别命名为Pm142-x-3、Pm142-x-4、Pm149-xby、Pm149-x,参考多杀性巴氏杆菌种特异性基因kmtl和荚膜血清型特异性基因hyaD-hyaC、bcbD、dcbF、ecbJ和fcbD序列,合成引物,进行PCR扩增.选取Pm149-x-3的目标PCR产物进行序列测定并分析比较,结果表明,Pm149-x-3的kmtl基因片段全长为460 bp,与GenBank中各血清型kmtl基因核苷酸同源性为99.4%;荚膜血清型A菌株特异性基因核苷酸同源性为97%;而扩增其他荚膜血清型B、D、E、F的型特异性基因均未获得目的条带.由此确认,分离的4株多杀性巴氏杆菌均为荚膜血清A型.药敏试验表明分离的多杀性巴氏杆菌对氧氟沙星、庆大霉素敏感.     

Protection by toxoid-induced antibody of gnotobiotic piglets challenged with the dermonecrotic toxin of Pasteurella multocida

A crude dermonecrotic toxin (DNT) of Pasteurella multocida (P.m.) type D was prepared by repeated sonication and freezing. It was sterilized by filtration. A toxoid was then made and pigs were hyperimmunized with it to get an antiserum. A control serum was obtained by hyperimmunization of pigs with a preparation derived from nontoxigenic P.m. type D in the same manner as the toxoid. Three gnotobiotic piglets were injected with the antiserum. This resulted in neutralization indices (NI) of 25 in their sera, as tested on mice. Three litter-mated controls were given the control serum. Their NI remained 1. All piglets were challenged intramuscularly 4 times, every third day, with 30 mouse LD50 of the DNT. When euthanized 15 days after the last DNT administration no snout lesions were found in passively immunized piglets, whereas control animals showed severe turbinate atrophy and other changes typical for atrophic rhinitis. The next experiment was identical to the previous one except for the challenge, which was given intranasally (4 times 300 mouse LD50). Also in this case circulating antitoxin protected the piglets from damage of the nasal turbinates caused by the DNT.     

Clinical and pathological effects of the dermonecrotic toxin of Bordetella bronchiseptica and Pasteurella multocida in specific-pathogen-free piglets

The role of dermonecrotic toxin (DNT) of Bordetella bronchiseptica and Pasteurella multocida, purified by repeated chromatography in Sephacryl S-200 gel, in the pathogenesis of atrophic rhinitis (AR) of swine was studied bacteriologically, clinically and pathologically. Two-week-old specific pathogen-free (SPF) piglets were parenterally treated with 30 micrograms of DNT 3 times at 2-day interval and 7-week-old piglets were treated with 15 micrograms of DNT twice a week for 5 weeks. In 2- to 3-week-old piglets, both B. bronchiseptica DNT and P. multocida DNT produced nasal turbinate lesions with similar severity, characterized by damage of the cilia, epithelial metaplasia, intensive proliferation of osteoblasts, regressive changes, and diffuse osteocytic osteolysis. In 7- to 12-week-old piglets, treatment with B. bronchiseptica DNT failed to produce progressive changes in the nasal turbinates. Histopathological examination revealed osteogenic processes and osteoid synthesis besides the proliferation of osteoblasts and mild osteocytic osteolysis. Moreover, severe gross pathological lesions developed in the stomach, liver, kidneys, and lymphoid organs. The piglets' appetite and body weight gain gradually decreased during the DNT treatment and in the last week when the toxic signs appeared. Treatment of 7- to 12-week-old piglets with P. multocida DNT resulted in progressive AR. Histopathologically, diffuse osteocytic osteolysis was observed in the nasal turbinates. Neither clinical signs nor pathological lesions of the visceral organs developed in these piglets. The authors emphasize that the DNT of B. bronchiseptica basically differs from that of P. multocida in biological properties, though there are certain similarities between the DNTs.     

Protective effect of Pasteurella multocida cell-free antigen and toxoid against challenge with toxigenic strains of pasteurella multocida in mice

The cell-free antigen (CFA) obtained from the culture supernatant of Pasteurella multocida (P. multocida) and the toxin (PMT) purified from CFA were inactivated and mixed with oil adjuvant to prepare a trial vaccine. Both of the mice immunized with CFA and PMT toxoid vaccine were noticeably protected against intratracheal challenge with toxigenic strains of P. multocida. Nevertheless, the protective indices of the mice immunized with CFA vaccine indicate that it is more protective and clears away the bacteria more promptly than in the mice immunized with PMT vaccine. The results suggested that CFA would possibly be good as an effective antigen to toxigenic strains of P. multocida infection.     

鹅巴氏杆菌油乳剂灭活苗的研制与应用

禽巴氏杆菌病(Avian pasteurellosis）是由多杀性岂氏杆菌(Pasteurella multoeida)引起的禽类一种接触性传染病。此病对鹅造成的危害较大,一年四季均可发生,主要经呼吸道感染,一般呈散发或地方性流行,发病率和死亡率都较高。病症以急性为主．不食,精神萎顿,拉稀,咽喉部有分泌物,喙和蹼发紫,1～2d内可发生死亡。     

多杀性巴氏杆菌毒素的研究进展

多杀性巴氏杆菌毒素(Pasteurella multocida toxin,PMT)是一种由D型多杀性巴氏杆菌分泌的毒素蛋白,作为猪萎缩性鼻炎的主要致病因子而备受关注。PMT作为一种有丝分裂原,通过细胞内多条信号通路影响细胞的生长和分化。PMT可分为N端和C端两大结构域,N端被认为与受体结合以及跨膜转运有关,C端具有催化活性。近年来,也有人认为PMT与致癌因素有关。随着PMT研究的深入,PMT疫苗也正成为研究的一个热点。本研究就PMT胞内活性、结构与功能等最新研究进展进行概述。     

复方中草药制剂对仔猪免疫机能和抗氧化能力的影响

在基础日粮中添加不同剂量的复方中草药制剂,研究其对仔猪免疫机能的影响。选取9窝(共90头)体重相近的杜×长×大三元杂交仔猪,随机分为3组,每组30头,分别饲喂基础日粮、基础日粮+复方中草药制剂1%、基础日粮+复方中草药制剂2%。试验期45 d。每组随机选取10头仔猪,分别在14、24、32、45日龄进行前腔静脉采血,测定部分免疫指标。结果表明,日粮中添加复方中草药制剂,能显著提高仔猪白细胞吞噬率、T-淋巴细胞转化率、血清免疫球蛋白含量和抗氧化酶活性,提高仔猪免疫力,减轻仔猪断奶应激。     

Atypical Pasteurella strains producing a toxin similar to the dermonecrotic toxin of Pasteurella multocida subspecies multocida

This paper is the first report of the production of a dermonecrotic toxin by pasteurella strains that do not belong to the species Pasteurella multocida subspecies multocida. Four strains, isolated from cattle with atrophic rhinitis, were characterised phenotypically. The strains were related to pasteurellaceae, but their taxonomic position remained unclear. The strains produced a toxin that caused a haemorrhagic dermonecrosis in guinea pigs and was lethal to mice. Both effects were neutralised by an antiserum against the purified dermonecrotic toxin of P multocida subspecies multocida. Western blot analysis of culture filtrates of the bovine strains revealed a protein, with the same molecular weight as dermonecrotic toxin, which reacted with both polyclonal and monoclonal antibodies against the toxin. In an immunodiffusion test, anti-dermonecrotic toxin serum did not discriminate between the toxin of the bovine strains and the toxin of P multocida subspecies multocida. It is concluded that these atypical pasteurella strains produce a toxin that is closely related to the dermonecrotic toxin of P multocida subspecies multocida.     

Effect of CLA on performance and immune response of weanling piglets

An experiment was conducted to evaluate the effect of CLA on the immune response and performance of piglets when subjected to an immune challenge. A total of 32 weanling pigs (17 to 23 d of age) with an initial BW of 8.9 kg were allotted to a 3 × 2 factorial arrangement of treatments. There were 3 levels of dietary CLA (0%, 1%, and 2%) and 2 levels of lipopolysaccharide (LPS) challenge (unchallenged and challenged). Challenged pigs were challenged on d 7 and 21. On d 4 and 18, all pigs were inoculated with BSA for assessment of IgG production. There was no difference in growth performance among piglets receiving different CLA supplementation levels. However, LPS-challenged piglets had poorer BW (P < 0.05), ADFI (P < 0.01), and ADG (P < 0.001) compared with the control group at d 35 postweaning. Lipopolysaccharide-challenged piglets also had increased respiratory rate (P < 0.001) and rectal temperature (P < 0.001), and decreased plasma proteins, hematocrit, and white blood cell counts (P < 0.05). Production of IgG against BSA was increased in the 1% CLA supplementation group (P < 0.001), indicating that CLA has an immunomodulatory effect. Supplementation with CLA did not affect lymphocyte proliferation, percentage of CD4(+) and CD8(+) cells, plasma proteins, red and white blood cell count, respiratory rate, or rectal temperature after LPS challenge. Although CLA supplementation did not influence growth performance or certain immune system measurements, the increased IgG titers with 1% CLA dietary supplementation indicate that it has a beneficial effect on the humoral immune system of weaned piglets.     

CpG序列对猪伪狂犬疫苗免疫效果的影响

将CpG DNA同猪伪狂犬疫苗联合免疫接种初生仔猪,检测仔猪的体液及细胞免疫反应,试验结果表明,CpG DNA与猪伪狂犬疫苗共同免疫的仔猪,其特异性抗体滴度、淋巴细胞白介素-2诱生活性以及淋巴细胞增殖反应均显著高于伪狂犬疫苗免疫组和对照组,证明CpG DNA能显著增强仔猪对常规疫苗的细胞和体液免疫反应.     

The virulence and protective efficacy for chickens of pasteurella multocida administered by different routes

The relative virulence for chickens of five strains of Pasteurella multocida was evaluated. Twenty groups, each of ten chickens, were inoculated with a standard dose of 10(5) of each of five strains by the intramuscular (I.m.), intravenous (I.v.), intratracheal (I.tr.) or conjunctival (Co) routes. The highest mortality occurred in the groups dosed I.m. and I.v., followed by I.tr. inoculation. The relative virulence of each strain did not change when inoculated by the different routes. The most virulent strain, VP161, caused 100% mortality by all except the Co route. The least virulent strain, VP17, caused a single mortality by the I.v. route, but gave a high level of protection to birds inoculated by both the I.m. and I.v. routes, when challenged by intramuscular injection with (VP161). There was no protection against I.m. challenge in the birds inoculated by the I.tr. or Co routes. Serum antibody levels measured by ELISA correlated with the level of protection against virulent challenge for groups inoculated I.m. or I.v., but not I.tr. Western blots of pooled sera from each group did not show any specific antigen recognition that might explain the observed differences in protection. Inoculation with strain VP17, (both I.m. and I.tr.) also gave a high level of protection to birds challenged with strain VP161 by intratracheal instillation.     

Effect of hyperlipidemia on cardiovascular responses to adrenergic stimulation in piglets.

This study was designed to assess the effect of hyperlipidemia on cardiovascular responses to adrenergic stimulation in a porcine model. Four-week-old piglets (n = 10) were divided into two groups; one fed a control diet and the other was fed an atherogenic diet for 8 weeks. Cardiovascular responses were evaluated to both norepinephrine (NE; 0.5 and 2.5 micrograms/kg) and isoproterenol (ISO; 0.1 and 0.5 microgram/kg) from simultaneous recordings of femoral arterial pressure, heart rate, left intraventricular pressure and left intraventricular dP/dt. It was found that no significant difference in the baseline values of cardiovascular function was observed between the control and hyperlipidemic groups. However, in the hyperlipidemic group as compared with the control group: 1) arterial blood pressure responses to NE were significantly increased (P less than 0.05), 2) cardiac contractile responses to NE and ISO were significantly potentiated (P less than 0.05), and 3) reflex bradycardia in response to increasing arterial blood pressure did not occur. These findings indicate that hyperlipidemia can potentiate the cardiovascular responses to adrenergic stimulation, whereas reflex cardiovascular regulation is somewhat altered by hyperlipidemia. Conceivably, these observations may have relevance to the possible role of the mechanism of the interaction of hyperlipidemia and hypertension in atherogenesis.     

复方中草药“猪康散”对仔猪生产性能及细胞免疫功能的影响

为了研究复方中药“猪康散”对断奶仔猪细胞免疫功能及生产性能的影响。选取20日龄杜长大三元杂交仔猪40头,随机分为4组,每组10头。27-31日龄时分别在基础日粮中添加0％、0．5％、110％、2．0％“猪康散”。在35、45、55、65日龄时每组随机选取4头仔猪,前腔静脉采血测定相关免疫指标。1．0％中药组在提高仔猪生产性能、淋巴细胞转化率、细胞亚群方面均显著优于0．5％中药组、2．0％中药组、对照组（P〈0．05或0．01）。结果表明,在断奶仔猪日粮中添加1．0％“猪康散”能增强仔猪细胞免疫功和提高仔猪生产性能。     

应用16S rRNA基因测序法鉴定禽多杀性巴氏杆菌的研究

应用 16SrRNA基因测序法对以疫苗标准强毒株C4 8_1和弱毒疫苗代表株G190E4 0为阳性对照株和分离鉴定的 2株禽源多杀性巴氏杆菌Chicken/guangxi/ 2 0 0 0_1、Chicken/guangdong / 2 0 0 2_1株 ,进行 16SrRNA基因序列分析。结果表明 ,2株分离菌与对照的强弱毒株之间的同源性为 10 0 %。后经Blastn分析Chicken/guangxi/ 2 0 0 0_1、Chicken/guangdong /2 0 0 2_1、C4 8_1、G190E4 0株与已发表 11株多杀性巴氏杆菌同源性高达 10 0 % ,与已发表的 34株多杀性巴氏杆菌同源性均超过 98% ,同时与其它巴氏杆菌种如溶血巴氏杆菌等同源性均低于 96 % ,进一步证实Chicken/guangxi/ 2 0 0 0_1、Chicken/guangdong / 2 0 0 2_1分离株均为多杀性巴氏杆菌。生化实验鉴定表明Chicken/guangxi/ 2 0 0 0_1、Chicken/guangdong / 2 0 0 2_1、C4 8_1、G190E4 0菌株均为subsp .Multocida亚种。