Blending of a conventional Mycoplasma hyopneumoniae vaccine with a positive marker: tracking of immunised pigs by peptide-specific antibodies raised to the marker component

Highly immunodominant marker antigens simply blended to existing veterinary vaccines may represent a smart approach for addressing the still open issue of vaccination compliance. This approach was evaluated by blending a widely deployed Mycoplasma hyopneumoniae vaccine with a peptide-KLH (Keyhole Limpet Hemocyanin) conjugate as marker. Piglets were vaccinated twice with: (i) a combination of the M. hyopneumoniae-specific vaccine and the marker, (ii) M. hyopneumoniae-specific vaccine, (iii) marker alone or (iv) placebo dose only. All piglets which received the M. hyopneumoniae-specific vaccine/marker formulation or, as control, the marker blended with Montanide IMS1313 adjuvant responded to the respective immunisation from day 21 to 77 post vaccination as seropositive for the appropriate peptide and KLH. However, the responder rate to M. hyopneumoniae of piglets administered with M. hyopneumoniae-specific vaccine/marker was slightly reduced at day 35 and 49 post immunisation in comparison with piglets vaccinated with M. hyopneumoniae-specific vaccine alone. Accordingly, we conclude that this marker technology could be successfully applied to label a whole set of vaccines prevented that the blending process will be optimised.     

Protective effects of an oral microencapsulated Mycoplasma hyopneumoniae vaccine against experimental infection in pigs.

Oral microencapsulated Mycoplasma hyopneumoniae vaccines were tested for their ability to prevent mycoplasmal pneumonia in pigs. Eighteen four-week-old specific pathogen free pigs were divided into six groups of three. Each pig of groups 1 to 4 was inoculated intramuscularly with formalin-inactivated M hyopneumoniae in adjuvant and boosted orally 14 days later with four different microencapsulated vaccine microspheres. Group 5 was used as a positive control. All 15 pigs of groups 1 to 5 were challenged at 28 days after the first vaccination by an intratracheal inoculation of pneumonic lung suspension. The three pigs of group 6 were used as a negative control. All four vaccinated groups showed some protection when challenged, but the protection was more solid in pigs boosted with vaccine D (group 4) which contained less porcine serum in the microsphere. The study indicates that oral vaccination with M hyopneumoniae could play a role in prevention and eradication of mycoplasmal pneumonia in the pig.     

皂树皂苷QS21增强猪支原体肺炎活疫苗滴鼻免疫小鼠效果评价

为提高猪支原体肺炎活疫苗的滴鼻免疫效果,以猪支原体肺炎(168株)活疫苗为抗原,以QS21为免疫佐剂混合后滴鼻免疫小鼠,ELISA 法检测免疫效果,包括血清和支气管肺泡灌洗液中抗原特异性抗体水平,并测定了Th1型细胞因子IFN-γ、Th2型细胞因子IL-4和Th17型细胞因子IL-17的分泌情况。结果表明,QS21能激活黏膜和全身性的体液免疫,显著升高血清和支气管肺泡灌洗液中IgG,IgG1、IgG2a抗体含量,并能提高支气管肺泡灌洗液中黏膜免疫抗体sIgA的含量。QS21还能显著增加支气管肺泡灌洗液中IL-4、IFN-γ和IL-17的分泌,激活Th1、Th2和Th17型细胞免疫应答。以上结果表明QS21能全面激活免疫系统,具有开发为猪支原体肺炎活疫苗黏膜免疫佐剂的应用前景。     

Levamisole mucosal adjuvant activity for a live attenuated Escherichia coli oral vaccine in weaned pigs

The present study tested the hypothesis that levamisole exerts its immunopotentiating activity in weaned pigs vaccinated against colibacillosis by priming the lymphocytes and macrophages in the mesenteric lymph node (MLN). Ten weaned piglets were used and allocated into two equal groups. The experimental group was intramuscularly primed with levamisole at an immunostimulatory dose of 2.5 mg/kg given daily, in three consecutive days, and controls received saline according to the same schedule. Both groups were orally vaccinated with the vaccinal Escherichia coli strain on day 0 and challenged with the virulent E. coli strain 7 days later. All pigs were killed on postchallenge day 6. Upon virulent challenge the health status of the two groups was evaluated by clinical observations, and expression of CD25, SWC7 and SWC9 activation antigens by MLN and spleen T and B cells and macrophages, respectively, was tested using flow cytometry. Priming by levamisole significantly contributed to the effectiveness of a live attenuated oral vaccine against porcine postweaning colibacillosis, as evidenced by a good health status of primed vaccinated vs. un-primed vaccinated pigs. The CD3+, CD25+ and SWC9+ MLN but not spleen T cells and macrophages increased in experimental vs. control pigs, implying that levamisole exerts its potentiating activity in the MLN by augmenting both recruitment and activation of cells that participate in cell-mediated immunity.     

Studies of the field efficacy and safety of a single-dose Mycoplasma hyopneumoniae vaccine for pigs

The field efficacy and safety of a single-dose Mycoplasma hyopneumoniae vaccine were evaluated in three-to five-week-old pigs. Two field efficacy studies were conducted, one in England with 673 pigs, and one in Germany with 719 pigs. The pigs were injected intramuscularly with either the vaccine or saline (control) at a ratio of 2:1 and reared under commercial conditions to slaughter weight. The efficacy of the vaccine was evaluated by comparing the lung lesions associated with infection with M. hyopneumoniae in the control and vaccinated animals postmortem. In both countries the vaccinated pigs had a significantly lower percentage of lung lesion scores, in England 5.7 v 10.2 per cent (P = 0.0022) and in Germany 3.9 v 7.7 per cent (P = 0.0056). In Germany the average daily weight gain (ADG) of the vaccinated pigs was significantly higher (639 g v 616 g) (P = 0.0205). In both countries and in both the treated and control animals there was a significant negative correlation between the ADG and the lung lesion score (P = 0.0001). Two safety trials were conducted, one in England and one in Germany, each with 75 pigs, and in each case 50 pigs were given the maximum batch release antigen titre of the vaccine and 25 were given saline. The safety of the vaccine was evaluated by observation for local and systemic reactions and any increases in rectal temperature. No abnormal reactions were observed in the vaccinated pigs and there was no significant difference between the mean peak rectal temperatures of the vaccinated and control pigs in either trial.     

Systemic and local immune response in pigs intradermally and intramuscularly injected with inactivated Mycoplasma hyopneumoniae vaccines

The systemic and respiratory local immune response induced by the intradermal administration of a commercial inactivated Mycoplasma hyopneumoniae whole-cell vaccine (Porcilis^® MHYO ID ONCE – MSD AH) in comparison with two commercial vaccines administered via the intramuscular route and a negative control (adjuvant only) was investigated. Forty conventional M. hyopneumoniae-free pigs were randomly assigned to four groups (ten animals each): Group A = intradermal administration of the test vaccine by using the needle-less IDAL^® vaccinator at a dose of 0.2 ml; Group B = intramuscular administration of a commercially available vaccine (vaccine B); Group C = intramuscular administration of the adjuvant only (2 ml of X-solve adjuvant); Group D = intramuscular administration of a commercially available vaccine (vaccine D). Pigs were vaccinated at 28 days of age. Blood and bronchoalveolar lavage (BAL) fluid samples were collected at vaccination (blood only), 4 and 8 weeks post-vaccination. Serum and BAL fluid were tested for the presence of antibodies by ELISA test. Peripheral blood monomorphonuclear cells (PBMC) were isolated to quantify the number of IFN-γ secreting cells by ELISpot. Moreover, cytokine gene expression from the BAL fluid was performed. Total antibodies against M. hyopneumoniae and specific IgG were detected in serum of intradermally and intramuscularly (vaccine B only) vaccinated pigs at 4 and 8 weeks post-vaccination. M. hyopneumoniae specific IgA were detected in BAL fluid from vaccinated animals (Groups A and B) but not from controls and animals vaccinated with the bacterin D (p < 0.05). Significantly higher gene expression of IL-10 was observed in the BAL fluid at week 8 post-vaccination in the intradermally vaccinated pigs (p < 0.05). The results support that the intradermal administration of an adjuvanted bacterin induces both systemic and mucosal immune responses. Moreover, the intramuscularly administered commercial vaccines each had a different ability to stimulate the immune response both systemically and locally.     

Quantification of the spread of Mycoplasma hyopneumoniae in nursery pigs using transmission experiments

Mycoplasma hyopneumoniae (M. hyopneumoniae) is present in almost all swine herds worldwide, but transmission of the pathogen through herds is not yet fully clarified. The aim of this study, performed in 2003, was to investigate and to quantify the transmission of M. hyopneumoniae under experimental conditions by means of an adjusted reproduction ratio (R_n). This R_n-value, calculated according to the final size method, expresses the mean number of secondary infections due to one typical infectious piglet during the nursery period. The period lasted from 4 to 10 weeks of age, corresponding with the nursery period used in most European production systems. Additionally, a comparison was made between transmissions of highly virulent and low virulent isolates.

Forty-eight weaned piglets, free of M. hyopneumoniae, were housed in six separate pens. During 6 weeks, two animals experimentally infected with M. hyopneumoniae were housed together with six susceptible piglets. At the end of the study, the number of contact-infected animals was determined by the use of nPCR on bronchoalveolar lavage fluid. The R_n-values of the highly virulent and the low virulent isolates were estimated to be 1.47 (0.68–5.38) and 0.85 (0.33–3.39), respectively. No significant difference between the groups was found (P = 0.53). The overall R_n was estimated to be 1.16 (0.94–4.08). Under the present experimental conditions, the transmission of M. hyopneumoniae, assessed for the first time by a reproduction ratio, shows that one piglet infected before weaning will infect on average one penmate during the nursery phase.     

Serum and mucosal antibody responses and protection in pigs vaccinated against Mycoplasma hyopneumoniae with vaccines containing a denatured membrane antigen pool and adjuvant

Objective To investigate the protective efficacy of a pool of denatured membrane protein antigens of Mycoplasma hyopneumoniae (J strain) in the molecular size range 70 to 85 kDa (F3 antigen) in combination with adjuvants for pigs challenged with M hyopneumoniae .
Design A vaccine efficacy experiment with assessment of serum and respiratory tract antibody responses.
Procedure F3 antigens were emulsified with five different adjuvants. To groups of three pigs per vaccine, four vaccines were given by intramuscular injection, and two vaccines, including one of those given intramuscularly, were given by intraperitoneal injection.
Results Compared to six unvaccinated pigs, animals vaccinated with F3 antigen displayed significantly reduced pneumonia (54% reduction in mean lung score) following experimental challenge. Analysis of post-vaccination, pre-challenge IgG and IgA ELISA antibody absorbances in serum and respiratory tract washings revealed no correlation with lung score. Six weeks after challenge, pigs previously vaccinated intramuscularly mostly demonstrated greater IgG and IgA responses in respiratory tract washings, and greater IgG serum antibody responses, than those vaccinated by intraperitoneal injection.
Conclusion Pigs vaccinated with M hyopneumoniae antigens in the molecular size range of 70 to 85 kDa showed a significant reduction in lung lesions compared with unvaccinated control animals after experimental challenge. IgG and IgA antibody concentrations in serum and respiratory tract washings after vaccination do not provide a useful prognostic indicator of protection from enzootic pneumonia.     

Evaluation of Mycoplasma hyopneumoniae inactivated vaccine in pigs under field conditions

An inactivated vaccine prepared from broth culture supernatant of Mycoplasma hyopneumoniae with an aluminum adjuvant was evaluated in three herds (herd A: specific pathogen-free herd, herd B: high health status herd with no clinical signs of respiratory infection, herd C: low health status herd with serious epidemiological and economical problems). A total of 212 pigs from the three herds were divided into two groups. One group was injected twice with the vaccine at 4-week intervals and the other was a control group. No adverse reactions were noted following the vaccinations either systematically or locally in any of the vaccinated pigs from any of the herds. In herd A, the vaccination provided antibody response within 4 weeks after the second vaccination and antibody responses continued for more than 12 weeks. In herds B and C, the number of pigs with lung lesions, mean percentage of lung lesions, and the numbers of M. hyopneumoniae recovered from pigs at slaughter in the vaccinated group were significantly (P < 0.05) reduced compared to the control group. Furthermore, vaccination resulted in improved average daily weight gain (ADG), improved feed conversion ratio (FCR), and improved days to market weight in herd C, whereas no difference in growth performance was shown in herd B. It is suggested that the inactivated vaccine prepared from broth culture supernatant of M. hyopneumoniae is effective in reducing clinical signs and lung lesions. Also, vaccination resulted in improved growth performance in herds where clinical signs and economic losses were significant.     

副猪嗜血杆菌灭活疫苗佐剂的筛选

为了评价铝胶、蜂胶及白油佐剂对副猪嗜血杆菌的免疫增强效果,我们将铝胶、蜂胶和白油3种佐剂分别与副猪嗜血杆菌血清5型菌株联合制备灭活疫苗,然后接种雄性成年家兔,采用ELSIA方法分别检测3种灭活苗在免疫期间副猪嗜血杆菌P5蛋白的抗体效价。结果显示:3次免疫后,白油佐剂免疫组的平均效价为1∶4000,铝胶佐剂免疫组的平均效价为1∶700,蜂胶佐剂免疫组的平均效价为1∶100,无佐剂对照组的平均效价为1∶100。说明这3种佐剂中免疫增强效果最好的为白油,铝胶次之。     

A modified live PRRSV vaccine and the pathogenic parent strain induce regulatory T cells in pigs naturally infected with Mycoplasma hyopneumoniae

Rhodococcus equi is an important respiratory pathogen of young foals for which a vaccine has long been sought. Two major impediments to effective vaccination are the functionally immature type I immune responses of neonatal foals and early exposure to the bacterium via the environment. Despite these obstacles, it appears that under specific circumstances foals can develop a protective immune response. In this study we investigated the protective mechanisms behind oral inoculation of foals with virulent R. equi bacteria. Two foals receiving an oral inoculum demonstrated accelerated development of R. equi specific cytotoxic T lymphocytes (CTL) as evidenced by significant lysis of R. equi infected, ELA-A mismatched cells at 3 weeks of age. As in a previous study, CTL were not detected until 5-6 weeks of age in two control foals. At each time point the ability of foal peripheral blood mononuclear cells (PBMC) to produce IFN-γ following stimulation with live R. equi or extracted cell wall lipids was similar to that of an adult horse control and between foals, regardless of treatment. These results provide a potential mechanism of protection which has previously been shown to occur following oral inoculation, and suggest that the early detection of CTL may be a useful marker for induction of protective immunity.     

Efficacy of an inactivated aqueous vaccine for the control of enzootic pneumonia in pigs infected with Mycoplasma hyopneumoniae

The efficacy of an inactivated aqueous vaccine against Mycoplasma hyopneumoniae was evaluated at two M hyopneumoniae-infected farrow-to-finish commercial farms (A and B) in Greece. In a prospective, randomised double-blind study, two groups on each farm received intramuscular doses of either the vaccine or the adjuvant when they were one and four weeks of age. The pigs were observed daily for clinical signs of disease; morbidity and mortality were recorded; and bodyweight was recorded at intervals. At slaughter, the lungs of the animals were examined and the chest cavities were examined for signs of pleuritis. No adverse reactions to the treatments were observed in any of the pigs. On farm A the vaccinated pigs were on average 6 kg heavier at slaughter, and on farm B they were on average 4 kg heavier; on both farms the average daily gain of the pigs was greater than that of the unvaccinated pigs. The prevalence and severity of enzootic pneumonia in the affected lungs were significantly lower in the vaccinated than in the unvaccinated pigs.     

Control of Mycoplasma hyopneumoniae infections in pigs

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, occurs worldwide and causes major economic losses to the pig industry. The organism adheres to and damages the ciliated epithelium of the respiratory tract. Affected pigs show chronic coughing, are more susceptible to other respiratory infections and have a reduced performance. Control of the disease can be accomplished in a number of ways. First, management practices and housing conditions in the herd should be optimized. These include all-in/all-out production, limiting factors that may destabilize herd immunity, maintaining optimal stocking densities, prevention of other respiratory diseases, and optimal housing and climatic conditions. Strategic medication with antimicrobials active against M. hyopneumoniae and, preferably, also against major secondary bacteria may be useful during periods when the pigs are at risk for respiratory disease. Finally, commercial bacterins are widely used to control M. hyopneumoniae infections. The main effects of vaccination include less clinical symptoms, lung lesions and medication use, and improved performance. However, bacterins provide only partial protection and do not prevent colonization of the organism. Different vaccination strategies (timing of vaccination, vaccination of sows, vaccination combined with antimicrobial medication) can be used, depending on the type of herd, the production system and management practices, the infection pattern and the preferences of the pig producer. Research on new vaccines is actively occurring, including aerosol and feed-based vaccines as well as subunit and DNA vaccines. Eradication of the infection at herd level based on age-segregation and medication is possible, but there is a permanent risk for re-infections.     

In vitro and in vivo efficacy of a live attenuated Salmonella Typhimurium vaccine at preventing intestinal colonization in chicks

Vaccination of chicks with Salmonella (S .) Typhimurium aroA deletion mutants has previously been shown to inhibit intestinal colonization of wild‐type S.  Typhimurium strains. In Australia, Bioproperties VaxSafe? STM1 strain is the only licensed and commercially available S . Typhimurium vaccine. This vaccine is a live attenuated aroA deletion mutant. Currently, it is recommended that the first dose of the STM1 vaccine is administered through coarse spray. It is unclear whether this mode of administration effectively permits intestinal colonization. Furthermore, it is not known whether the STM1 strain prevents or inhibits Salmonella colonization of chicks following this first dose. This study investigated both in vitro and in vivo colonization parameters. Invasiveness was assessed using an in vitro invasion assay into sections of ileum and caecum collected from day‐old chicks. The S.  Typhimurium definitive types (DT) 9 and 44 exhibited the greatest invasion into both intestinal segments. STM1 was invasive but was significantly less so than both isolates of S.  Typhimurium. In dual and triple infections, no competitive microbial interactions between STM1 and wild‐type Salmonella were observed. In vivo colonization inhibition was also tested. Vaccinated and nonvaccinated day‐old chicks were challenged with S.  Typhimurium DT9. Both STM1 and S.  Typhimurium DT9 were found in spleen, liver, ileum, caecum and caecal contents from day 2 postinfection. No significant exclusion effect was observed in vaccinated and challenged chicks.     

环丙沙星在支原体肺炎猪体内的药动学

18头健康杜洛克×长白×大白杂交猪,分3组,每组6头,通过气管内接种含有猪肺炎支原体的病肺悬液复制疾病模型后,以5.0mg/kg静注、肌注及内服给药进行环丙沙星药物动力学研究.高效液相色谱法测定血浆中药物浓度.MCPKP药物动力学程序处理药时数据.结果显示感染猪静注给药的药时数据适合二室开放模型,t1/2α为0.59 h,t1/2β为3.52 h,Vd(area)为3.21 L/kg,ClB为0.645 L/(ks·h).感染猪肌注和内服给药后的药时数据则适合一级吸收一室模型,t1/2ka分别为0.09、0.31 h;tmax分别为0.46、1.41 h;Cmax分别为1.67、0.35 mg/L;F分别为97.30%、34.66%.上述结果表明,支原体性肺炎对环丙沙星静注给药在猪体内的分布有一定影响,但对其消除过程的影响不大;与健康猪比较,感染猪肌注给药的峰浓度、药时曲线下面积及生物利用度均显著提高,而内服给药的峰浓度、药时曲线下面积及生物利用度均显著降低.     

“猪地方性肺炎及其防治控制”专栏四：猪肺炎支原体的防治措施

猪肺支原体是猪地方性肺炎的主要病原体,其普遍存在于世界各地,会给养猪业造成巨大的经济损失。此病原体感染猪后通常黏附于猪呼吸道纤毛上皮,并会造成纤毛上皮的损伤。猪感染后主要表现为慢性咳嗽症状、容易发生其他呼吸道感染和生产性能下降。该病的控制可通过多种措施实现,首先应该完善管理措施,改善畜舍饲养环境,这包括实行全出/全进的饲养方式、减少会破坏群体免疫水平的因素、维持最佳的饲养密度、防止其他呼吸道疾病的感染,以及提供最佳的畜舍及环境条件。其次,当猪群处于呼吸道疾病感染的威胁之下时,战略性使用能够有效地预防猪肺炎支原体和最好还能预防大多数继发感染细菌的药物对预防本病非常有效。最后,商用疫苗已被广泛地用来控制猪肺炎支原体感染,接种疫苗的优点在于其能够减少临床症状、减轻肺脏损伤、减少药物的使用和提高猪群的生产性能。但是,疫苗仅能提供部分保护作用,并且不能防止病原体在猪体内的定殖。因此,应根据猪群的种类、猪场的生产系统和管理体制、感染的类型及猪农的喜好选择不同的免疫策略（免疫时机、母猪免疫、免疫接种再结合抗菌素治疗）。新疫苗正在紧锣密鼓的研究之中,如气雾苗、通过饲料接种的疫苗以及亚单位苗和DNA疫苗。按年龄进行隔离饲养和药物治疗在猪群水平上根除猪肺炎支原体感染是可能的,但该病复发的威胁将永久存在。     

猪肺炎支原体的防治措施

猪肺支原体是猪地方性肺炎的主要病原体,其普遍存在于世界各地,会给养猪业造成巨大的经济损失.此病原体感染猪后通常黏附于猪呼吸道纤毛上皮,并会造成纤毛上皮的损伤.猪感染后主要表现为慢性咳嗽症状、容易发生其他呼吸道感染和生产性能下降.该病的控制可通过多种措施实现.首先应该完善管理措施.改善畜舍饲养环境,这包括实行全出/全进的饲养方式、减少会破坏群体免疫水平的因素、维持最佳的饲养密度、防止其他呼吸道疾病的感染,以及提供最佳的畜舍及环境条件.其次,当猪群处于呼吸道疾病感染的威胁之下时,战略性使用能够有效地预防猪肺炎支原体和最好还能预防大多数继发感染细菌的药物对预防本病非常有效.最后,商用疫苗已被广泛地用来控制猪肺炎支原体感染,接种疲苗的优点在于其能够减少临床症状、减轻肺脏损伤、减少药物的使用和提高猪群的生产性能.但是,疫苗仅能提供部分保护作用,并且不能防止病原体在猪体内的定殖.因此,应根据猪群的种类、猪场的生产系统和管理体制、感染的类型及猪农的喜好选择不同的免疫策略(免疫时机、母猪免疫、免疫接种再结合抗菌素治疗).新疫苗正在紧锣密鼓的研究之中.如气雾苗、通过饲料接种的疫苗以及亚单位苗和DNA疫苗.按年龄进行隔离饲养和药物治疗在猪群水平上根除猪肺炎支原体感染是可能的,但该病复发的威胁将永久存在.     

Protection against enzootic pneumonia of pigs: intraperitoneal inoculation with live LKR strain of Mycoplasma hyopneumoniae

Pigs obtained from a mycoplasma-free piggery were randomised into 4 groups of 9. Groups 1 and 2 were injected by the intraperitoneal route with liquid culture of the LKR strain of Mycoplasma hyopneumoniae. Group 1 was injected once and group 2 twice. Group 3 was made up of pigs inoculated by the intranasal route with the virulent Beaufort strain of M. hyopneumoniae; they served as the source of infection for the challenge. Group 4 were uninfected, uninjected controls. Six weeks after the last injection, groups from 1 to 4 were placed in contact. Seven of the pigs in the 1-dose group and 6 in the 2-dose group were free of lesions at necropsy 6 weeks after challenge. Of the two pigs with lesions in the 1-dose group one had only a small lesion but the other had extensive lesions; it had not shown an antibody response after injection of culture. The lesions in the 3 pigs in the 2-dose group were all small. All 9 control pigs had lesions which varied from medium to large in size. The difference in the incidence of pneumonia between the injected and control groups was significant (P less than 0.05) and the proportion of severely affected pigs in the vaccinated groups was significantly lower (P greater than 0.01). There was no difference between those given one dose of vaccine and those receiving 2 doses.     

Safety of multiple,submucosal inoculations of a live attenuated strangles vaccine in pregnant mares

A live attenuated vaccine against Streptococcus equi was administered submucosally in the upper lip to 224 pregnant and healthy mares to evaluate its safety. After a primary immunisation the mares were inoculated every 3 months until foaling. As control group, 206 mares of the same breeding farm were administered the solvent of the vaccine submucosally. None of the 430 mares presented any clinical evidence of strangles and neither local nor systemic reactions to vaccination were noticed. There was no association between abortion and vaccination. Furthermore no case of S. equi infection was revealed at post mortem examinations of aborted foals.