Vaccination of gnotobiotic primary specific pathogen-free pigs against Haemophilus parasuis.

Three trials were conducted to establish if young primary specific pathogen free (SPF) pigs could be protected from Glasser's disease by vaccination. Three age groups of cesarean-derived isolator-reared gnotobiotic pigs were vaccinated twice at 4 and 6, 3 and 5, and 2 and 4 wk of age respectively with a formalin killed aluminum hydroxide adsorbed bacterin prepared from three strains of Haemophilus parasuis isolated from Ontario pigs affected with Glasser's disease. When challenged two weeks later with the homologous strains of virulent bacteria, all the vaccinated pigs remained healthy, while 17/18 nonvaccinated pigs became severely sick or died between three and seven days postchallenge. The one surviving nonimmunized pig was retarded in growth. All of the nonimmunized pigs had visible lesions of polyserositis, the most common being polyarthritis (14/18). Other lesions were fibrinous meningitis, pericarditis, pleurisy and/or peritonitis. Two of the pigs died with a septicemia. Haemophilus parasuis was isolated from 15/18 nonimmunized pigs, usually from several of the affected sites. The organisms were not isolated from the immunized pigs, nor from the surviving nonimmunized pig. Attempts to detect the presence of specific antibodies against the H. parasuis strains in the sera of the immunized or exposed pigs by the passive hemagglutination test or by enzyme linked immunoassay were unsuccessful. The results of this work indicate that primary SPF pigs can be protected from Glasser's disease by vaccination as early as 2 and 4 wk of age. The nature of this protective mechanism was not established in this study.     

Blood cellular immune response in pigs immunized and challenged with Haemophilus parasuis

The cellular immune response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässer’s disease in pigs, was characterized studying changes in peripheral blood mononuclear cells (PBMC) in colostrum-deprived pigs. Five groups were studied, four of those were previously immunized with different formulations and the fifth was maintained as non-immunized control. All groups were challenged with 5 × 10⁹ CFU of H. parasuis serotype 5. The non-commercial bacterin conferred a complete protection, while the OMP-vaccine and the exposure to a subletal dose of 10⁵ CFU of H. parasuis protected only partially, and the recombinant Tbp B-vaccine induced no protection. PBMC were analyzed using monoclonal antibodies against porcine CD45⁺, CD3⁺, CD4⁺, CD8α⁺, CD25⁺, CD4⁺ naïve, αIgM⁺ and SWC3⁺ cells in single-colour fluorescence, and CD4⁺/CD8α⁺ and CD8α⁺/CD8β⁺ combinations in two-colour fluorescence. The different groups showed no significant changes in PBMC subsets following vaccination, and only minor changes were encountered after challenge, consisting mainly of significant increases (P < 0.05) in the relative proportions of monocytes and granulocytes (SWC3⁺) and B cells (αIgM⁺), as well as a significant reduction in CD3⁺ cells (P < 0.05). These changes were similar for the five groups compared, except for the significant increase of CD25⁺ cells, which was only observed for the bacterin-vaccinated group. These results suggest an increase of trafficking of inflammatory cells and the onset of the adaptive antibody response against H. parasuis infection; in addition, the blood cellular response developed by the different groups was not relevant to protection.     

Differences in phagocytosis susceptibility in Haemophilus parasuis strains

Haemophilus parasuis is a colonizer of the upper respiratory tract of healthy pigs, but virulent strains can cause a systemic infection characterized by fibrinous polyserositis, commonly known as Glässer’s disease. The variability in virulence that is observed among H. parasuis strains is not completely understood, since the virulence mechanisms of H. parasuis are largely unknown. In the course of infection, H. parasuis has to survive the host pulmonary defences, which include alveolar macrophages, to produce disease. Using strains from different clinical backgrounds, we were able to detect clear differences in susceptibility to phagocytosis. Strains isolated from the nose of healthy animals were efficiently phagocytosed by porcine alveolar macrophages (PAM), while strains isolated from systemic lesions were resistant to this interaction. Phagocytosis of susceptible strains proceeded through mechanisms independent of a specific receptor, which involved actin filaments and microtubules. In all the systemic strains tested in this study, we observed a distinct capsule after interaction with PAM, indicating a role of this surface structure in phagocytosis resistance. However, additional mechanisms of resistance to phagocytosis should be explored, since we detected different effects of microtubule inhibition among systemic strains.     

Differences in susceptibility to Haemophilus parasuis infection in pigs

In animal breeding programs, deoxyribonucleic acid (DNA) markers can be used to identify sires that are less susceptible to disease. These DNA markers are typically discovered in populations that display differences in susceptibility. To find those differences, it was hypothesized that sires influence their offspring responses to infection with H. parasuis. To identify differences in susceptibility, colostrum-deprived pigs derived from 6 sires were inoculated with a virulent strain of H. parasuis serovar 5. Pigs were infected at 21-d of age and euthanized 1, 2, or 3 days post-infection. Rectal temperatures, bacterial detection, clinical signs, and lesions were measured by comparing disease susceptibility in the offspring from each sire. The effect of the sire on the severity of disease in the offspring was statistically analyzed using to a 2-way ANOVA with sire and test day as fixed effects. Significant differences among sires were found for lesions, rectal temperatures from days 0-1 and 0-2 (P < 0.05) and marginal effects for clinical signs (P = 0.08). On average, the offspring of sire H94 was the most susceptible to challenge. Responses to infection were categorized to determine the clinical responses and analyzed by Chi square. Overall, 10% of all pigs infected were fully resistant to H. parasuis infection. Boar H94 didn't produce any fully resistant offspring. Differences in susceptibility to H. parasuis were observed, and the results support the hypothesis that sires influence their offspring's response to infection. Tissues from this population could be used to identify DNA markers for genetic selection of sires that produce offspring more resistant to H. parasuis infection.     

豚鼠副猪嗜血杆菌感染动物模型的建立

为建立副猪嗜血杆菌(Hps)的感染动物模型,本试验用Hps血清5型标准菌株(Nagasaki),以2.0×10~9CFU剂量腹腔感染豚鼠,观察豚鼠发病及死亡情况.取死亡豚鼠的主要器官组织,观察其病理和组织病理变化,与猪Classer's病痛变进行比较.并同时对死亡豚鼠进行细茵分离,分离菌经PCR鉴定.实验结果显示:在接种14 h后试验组豚鼠(5/8)出现死亡,死亡豚鼠剖检时出现了与猪Classer's病相似的病变;主要组织器官组织学变化以炎性细胞浸润、纤维蛋白和红细胞渗出等变化;并通过细茵分离培养,在豚鼠大脑、心血、肺、肝、脾和肾主要器官中分离到Hps血清5型茵.实验结果表明豚鼠可以作为Hps的感染动物模型.这一结果为研究其致病机制、诊断和免疫研究奠定基础.     

Prevalence of Haemophilus parasuis serovars among isolates from swine.

Two hundred sixty Haemophilus spp isolates that had been obtained from the respiratory tract and other sites of swine were acquired from diagnostic laboratories, primarily in the United States and Canada. The majority of isolates (243/260) were biochemically characterized as H parasuis; however, a few isolates of taxa distinct from H parasuis (taxa "minor group," D, E, and F) were identified. Fourteen H parasuis serovars were identified, and of those previously described, the most prevalent were 5 (24.3% of isolates), 4 (16.1%), 2 (8.2%), and 7 (3.7%). Three new serovars that were also prevalent included ND4 (11.1%), ND3 (8.6%), and ND5 (6.6%). Serovars 1, 3, 6, C, D, and new serovars ND1 and ND2 were infrequently identified, and 15.2% of isolates were nontypeable. It was not uncommon to isolate multiple serovars from swine of the same herd or related herds. Distribution of serovars among isolates from the United States and Canada was generally similar; however, a higher prevalence of serovar 5 and a lower prevalence of serovars 2, ND3, and ND5 were evident in isolates from Canada. Comparison of isolates obtained from the respiratory tract of swine without polyserositis with those obtained from swine with polyserositis revealed an increased frequency of serovars 4 and 5, and a decreased frequency of serovar 2, among isolates from swine with polyserositis. However, all prevalent serovars were isolated from swine with polyserositis, and data were not indicative of an association between serovar, site of isolation, or pathogenic potential.     

Serological characterisation of Haemophilus parasuis isolates from Australian pigs

A total of 31 isolates of Haemophilus parasuis obtained from Australian pigs were serotyped by the Kielstein-Rapp-Gabrielson scheme. The isolates were assigned to serovar 1 (1 isolate), serovar 2 (1 isolate), serovar 4 (4 isolates), serovar 5 (7 isolates), serovar 9 (2 isolates), serovar 10/7 (4 isolates), serovar 12 (1 isolate) and serovar 13 (6 isolates). The remaining 5 isolates could not be assigned to a serovar. Two different serovars (5 and 13) were detected in one herd. The only 2 isolates obtained from clinically normal pigs (from the same herd) were serovar 9. The common serovars were isolated from pigs with pneumonia as well as from pigs with conditions of the Glässer's disease type. The serological heterogeneity amongst Australian isolates of H parasuis has important implications for the use of vaccines to control Glässer's disease.     

Comparative virulence of Haemophilus parasuis serovars 1 to 7 in guinea pigs.

Reference strains for Haemophilus parasuis serovars 1 to 7 were examined for virulence by inoculation of guinea pigs. Guinea pig response to intraperitoneal inoculation was similar for the 7 reference strains. However, apparent differences in virulence were detected after intratracheal inoculation. Cells of the references strains for serovars 1 and 5 were most invasive, causing moribundity or death at higher doses and a persistent septicemia at lower doses. Haemophilus parasuis could be isolated from respiratory and systemic sites; purulent bronchopneumonia, pericarditis, and pleuritis were apparent in infected guinea pigs. Inoculation of cells of the reference strains for serovars 2 and 6 also resulted in bronchopneumonia and moribundity or death in some guinea pigs; however, reisolation of H parasuis and microscopic lesions at necropsy were less pronounced than those observed with serovars 1 and 5. Inoculation of cells of serovars 3, 4 and 7 induced only transient clinical signs and minimal evidence of H parasuis infection at necropsy. The data from intratracheal inoculation of guinea pigs are similar to data from other investigations in swine, indicating differences in the pathogenic potential of H parasuis strains. Thus, guinea pigs may be useful as a laboratory animal model for examining cellular factors associated with virulence and immunogenicity of H parasuis.     

Systemic antibody response in colostrum-deprived pigs experimentally infected with Haemophilus parasuis

The serum antibody response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässer’s disease in pigs, was characterized by ELISA measuring IgM and IgGt levels against whole-cells and outer-membrane-proteins (OMPs) as antigens. Five groups of pigs were studied, four of those were previously immunized with different formulations, and the fifth was maintained as non-immunized control. All groups were challenged with 5 × 10⁹ CFU of H. parasuis. The non-commercial bacterin induced a full protection against disease, the OMP-vaccine and the exposure to a sublethal dose of 10⁵ CFU protected only partially, and the recombinant TbpB-vaccine conferred no protection. The humoral response in the pigs that died after infection (all controls, all those vaccinated with the recombinant TbpB, and two of both those inoculated with OMPs and those exposed to the sublethal dose) could be only measured before it, but it was irrelevant in all cases. However, a specific IgM and IgGt production was observed before challenge in all the surviving pigs, irrespective of the type of immunization received. This antibody response was even greater after H. parasuis infection, especially in those survivors receiving the sublethal dose. These results suggest a role of the antibodies developed after the different immunization protocols in preventing infection and death; therefore, the humoral immunity is protective against experimental Glässer’s disease.     

副猪嗜血杆菌对豚鼠和小鼠的致病力研究

为了验证副猪嗜血杆菌分离株FS0307株、XX0306株和标准株HS1079株的致病力,本文以豚鼠和Balb/c小鼠作为模型动物,分别腹腔接种副猪嗜血杆菌FS0307株、XX0306株和HS1079株,观察临床症状和死亡情况,并进行Hps的再分离和血清抗体检测。结果显示,2株副猪嗜血杆菌分离株均能引起豚鼠和Balb/c小鼠100%发病,以及对豚鼠和小鼠的致死率均分别达到100%和20%;标准株HS1079株能引起豚鼠和Balb/c小鼠100%发病,并对豚鼠有80%的致死率,而不致死小鼠。可见,副猪嗜血杆菌FS0307株、XX0306株和HS1079株的致病力均较强,这为副猪嗜血杆菌病疫苗的研究提供了可靠的技术参数。     

Molecular analysis of lungs from pigs immunized with a mutant transferrin binding protein B-based vaccine and challenged with Haemophilus parasuis

The molecular analysis of pigs vaccinated with a mutant transferrin-binding protein B (Y167A) from Haemophilus parasuis was compared with that performed for unvaccinated challenged (UNCH) and unvaccinated unchallenged (UNUN) pigs. Microarray analysis revealed that UNCH group showed the most distinct expression profile for immune response genes, mainly for those genes involved in inflammation or immune cell trafficking. This fact was confirmed by real-time PCR, in which the greatest level of differential expression from this group were CD14, CD163, IL-8 and IL-12. In Y167A group, overexpressed genes included MAP3K8, CD14, IL-12 and CD163. Proteomics revealed that collagen α-1 and peroxiredoxins 2 and 6 were overexpressed in Y167A pigs. Our study reveals new data on genes and proteins involved in H. parasuis infection and several candidates of resistance to infection that are induced by Y167A vaccine. The expression of proinflammatory molecules from Y176A pigs is similar to their expression in UNUN pigs.     

6株副猪嗜血杆菌基因组DNA的PCR指纹图谱研究

根据肠道菌基因闻重复一致序列，设计了一对特异性引物，采用ERIC-PCR和RAPD技术，研究了副猪嗜血杆菌6个分离菌株的指纹图谱和DNA多态性。结果表明，6个分离株的PCR指纹图谱与15个标准血清型指纹图谱相比较可分辨出4种血清型；6个分离株的RAPD研究结果均表现出多态性。有意义的是，6个菌株的多态性DNA片段也能明显将其分为4种类型的副猪嗜血杆菌，与特异性引物PCR结果相一致。该研究可作为流行病学调查和该菌的分子分型快速诊断方法的基础。     

副猪嗜血杆菌病研究进展

副猪嗜血杆菌为革兰氏阴性的条件性致病菌,常引起断奶前后仔猪以多发性浆膜炎、关节炎、呼吸困难、高热等临床特征的传染性疾病,给养猪业带来重大经济损失。文章综述了近年来副猪嗜血杆菌病的病原学,流行病学,致病机理,临床症状与病理变化,临床诊断和防治等方面的研究进展。     

副猪嗜血杆菌流行优势菌株调查和耐药性分析

为调查研究河南规模化猪场副猪嗜血杆菌(HPS)流行优势菌株及其耐药性情况,2017-2018年从河南规模化猪场分离到30株HPS,根据菌株分离部位统计,肺脏、气管是HPS分离的首要组织部位,肺脏分离14株,占46.67%;气管分离11株,占36.67%。通过PCR分子血清型鉴定,河南流行的优势菌株为血清5,7,4型,其中血清型5型有9株,占30%;血清型7型有5株,占16.67%;血清型4型有4株,占13.33%。通过K-B纸片琼脂法药敏试验,30株分离菌株除对头孢噻呋100%敏感外,对其他17种常用抗生素都有不同程度的耐药现象,多重耐药现象突出,预防控制HPS病选药、用药方面更需慎重和规范。     

副猪嗜血杆菌的分离鉴定及药敏试验

从病猪中分离到4株细菌,经染色镜检、生化试验以及PCR等方法鉴定为副猪嗜血杆菌,。通过药敏试验确定其对药物的敏感性,以期对临床用药有一定的指导作用。     

Biofilm formation by field isolates and reference strains of Haemophilus parasuis

The ability to form biofilms for a total of 80 field isolates and 15 reference strains of Haemophilus parasuis, the etiological agent of Glasser's disease, was tested by glass tube and polystyrene microtiter plate assays. A total 43% of field isolates, including strains representing 13 serovars (except serovars 3 and 8) and non-typable strains, exhibited the ability to form biofilms at different levels via polystyrene microtiter plate assays. Among the reference strains representing 15 serovars, only serovars 2, 9, 12, 13 and 15 could not form biofilms on the polystyrene surface. A total of 85% of the strains forming biofilms at air-liquid interfaces in glass tubes also formed biofilms on polystyrene surfaces. Generally, non-virulent serovars showed a higher degree of biofilm formation than virulent serovars. The biofilm formation phenotype of most strains was maintained when cultures were passaged on agar and in broth. H. parasuis from the nasal cavities of pigs experimentally infected with biofilm-positive bacteria maintained the biofilm formation phenotype, whereas bacteria recovered from the lung and brain lost the ability to form biofilms. The biofilm-negative strains did not recover the ability to form biofilms via experimental infection. Our data indicate that most serovars of H. parasuis could form biofilms in vitro, and the biofilm formation phenotype is associated with the recovery site of the strains and is maintained when bacteria are passaged in vitro and in the upper respiratory tract.