Serology of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5 strains: establishment of subtypes a and b

The serological characteristicis of 14 strains assigned to serotype 5 were examined by the slide agglutination test, the indirect haemagglutination (IHA) test and by gel diffusion.All the strains possessed identical capsular antigenic determinants of polysaccharide (PS) nature and lipopolysaccharide (LPS) nature. However, based upon a capsular antigenic determinant of PS nature the strains could be divided in two subtypes. It is therefore proposed to refer to the H. pleuropneumoniae strains of serotype 5 to two subtypes: subtype a with strain K17 as the subtype strain and subtype b with strain L20 as the subtype strain.     

Serological characterization of Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae) strains and proposal of a new serotype: serotype 9

Ten strains of H. pleuropneumoniae isolated from 10 herd outbreaks of pleuropneumonia were studied by means of the slide agglutination test, the indirect haemaggluitiniation (IHA) test and by gel diffusion. The strains were antigenically homogeneous and serologically distinct from serotypes 1 through 8. It is therefore proposed to refer these strains to a new serotype: serotype 9, with strain CVJ 13261 as the type strain.In addition to the serotype-specific capsular antigens, capsular antigen of serotype 1 (strain 4074) could be demonstrated in the 10 strains by means of gel diffusion analyses.In cross protection studies it was shown that the antigenic determinants shared by serotypes 9 and 1 were unable to yield a sufficient protection against disease. Thus, parenteral immunization with a killed 6-h culture of serotype 9 did not afford an acceptable protection against challenge with serotype 1 since only 3 of the 5 vaccinates were protected. The reverse experiment showed that parenteral immunization with serotype 1 only protected 1 out of 4 vaccinates.     

Haemophilus pleuropneumoniae serotypes--cross protection experiments

Pigs vaccinated with a killed 6-hour culture of Haemophilus pleuropneumoniae serotype 2 with Freund's incomplete adjuvant were not protected against challenge with serotypes 1, 5, 6 or 8. Equivalent results were obtained when pigs were vaccinated with serotypes 4 or 5 and challenged with serotype 2. In earlier studies of immunity induced by intranasal immunization with live H. pleuropneumoniae organisms, it was clearly shown that intranasal inoculation with one serotype of H. pleuropneumoniae would induce a strong immunity to both homologous and heterologous serotypes (Nielsen 1979). The present study has shown that cross immunity is not obtained with parenteral immunization. The results strongly suggest that the immune response of the pig to parenteral vaccination is different from the response seen after natural infection, and indicate that an important part of the defence mechanism against H. pleuropneumoniae infection is a local immune-barrier which is effective in preventing the bacterium from penetrating the mucosa. In earlier vaccination experiments 90 per cent of vaccinates were protected against homologous challenge (Nielsen 1976). In the present work a vaccine containing serotypes 1 through 6 was fully protective against serotypes 2 and 3 and also against serotype 8, which shares antigenic determinants with serotypes 3 and 6. These results indicate that the protection obtained by parenteral immunization is serotype-specific. Vaccines must therefore contain the serotypes existing in the swine population.     

Characterization of monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5.

Two monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5, designated as 5MAb-1 and 5MAb-6, were characterized. Enzyme-linked immunosorbent assay-inhibition tests with whole-cell antigens obtained from strains of serotype 1 through 12 of A pleuropneumoniae revealed that 5 MAb-1 bound to only serotype-5 strains. The epitope recognized by 5MAb-1 was a carbohydrate that was sensitive to periodate oxidation and resided on the structure of beta-1,6-linked D-galactose in an O-antigen polysaccharide of serotype-5 lipopolysaccharide. Analysis of these results revealed that the O-antigen polysaccharide of lipopolysaccharide was 1 of the antigenic determinants responsible for the serotype specificity of A pleuropneumoniae. On the other hand, 5MAb-6 reacted with strains of serotype 1 through 10 in varying degrees and its epitope was located on polypeptides sensitive to proteinase K. In an immunoblotting analysis, 5MAb-6 reacted with 2 polypeptide bands, with molecular weights of approximately 41,500 and 28,000, in the outer membrane protein-rich fraction obtained from strains of serotype 1 through 10. These results indicated that outer membrane proteins from several serotype strains of A pleuropneumoniae possessed common antigenic determinants.     

Serological characterization of Actinobacillus pleuropneumoniae serotype 2 strains by using polyclonal and monoclonal antibodies

The antigenic differences between strains of serotype 2 of both biotypes I and II of Actinobacillus pleuropneumoniae were studied by using several serological techniques. Monoclonal antibodies (MAbs) against A. pleuropneumoniae biotype I serotype 2 were produced by fusion of spleen cells of BALb/c mice immunized with whole-cell (WC) suspension with SP2/O-Ag14 murine myeloma cells. Desirable MAbs were selected by enzyme-linked immunosorbent assay (ELISA) using WC as antigen. MAbs MK-7 and MK-10 identified multiple bands of lipopolysaccharide in Western-blot. Treatment of WC with proteinase K and sodium periodate indicated that both MAb binding sites were carbohydrates in nature. In both ELISA and Western-blot, MAbs MK-7 and MK-10 recognized only biotype I serotype 2 isolates. Neither MAb MK-7 nor MK-10 reacted with reference strains of remaining serotypes of A. pleuropneumoniae and other Gram-negative bacteria tested. The results obtained with various serological tests showed that strains of serotype 2 biotype I shared antigenic determinants with strain N-282 of serotype 2 biotype II, but not with strain N-273 of serotype 1 biotype II. It is suggested that data obtained from this study may be helpful in the development of specific serotyping and serodiagnostic reagents of A. pleuropneumoniae strains.     

Safety and efficacy of two live Pasteurella multocida aro-A mutant vaccines in chickens

Two auxotrophic aro-A mutants of Pasteurella multocida designated PMP1 (serotype 1) and PMP3 (serotype 3) were tested as vaccine candidates to protect chickens against fowl cholera. A reliable intratracheal challenge method was established that resulted in > or = 75% mortality in both specific-pathogen-free chickens and commercial broiler breeders 24 hr after challenge. Dose protection studies indicated that at least 10(6) colony-forming units (CFU) of PMP1 and 10(8) CFU of PMP3 were required to provide complete protection against challenge in all birds. Although high doses of 10(9) CFU of the vaccine strains produced some endotoxinlike reactions, lower but protective dose levels produced no clinical sign or lesion in any chicken. Both vaccine strains provided cross-protection with a heterologous challenge strain PM206 (serotype 4). Future studies will examine the duration of protective immunity induced by the two vaccine candidates, PMP1 and PMP3, and cross-protection against other serovars.     

Identification of the cross-reacting antigen among Actinobacillus pleuropneumoniae strains of serotype 1, 9 and 11 by use of monoclonal antibodies.

Two monoclonal antibodies (MAbs), lMAb-1 and lMAb-5, against Actinobacillus pleuropneumoniae serotype 1 were obtained. In enzyme-linked immunosorbent assay-inhibition tests with whole cell antigens obtained from serotype 1 to 12 strains of A. pleuropneumoniae, lMAb-1 reacted to only a serotype 1, strain 4074. The epitope recognized by lMAb-1 was a carbohydrate sensitive to periodate oxidation and resided on capsular polysaccharide (CP) of A. pleuropneumoniae serotype 1. On the other hand, lMAb-5 reacted with serotype 1, 9 and 11 strains at the same degree and its epitope was found to be located on O-polysaccharide of serotype 1, 9 or 11 lipopolysaccharide (LPS). These results showed that CP was one of the serotype-specific antigens of A. pleuropneumoniae, and that O-polysaccharide of LPS obtained from serotype 1, 9 or 11 strain was the cross-reacting antigen among these strains.     

Production and characterization of monoclonal antibodies against Actinobacillus (Haemophilus) pleuropneumoniae serotype 2

Four hybridoma cell lines producing monoclonal antibodies (MAbs) against Actinobacillus (Haemophilus) pleuropneumoniae were established by fusion of mouse myeloma and spleen cells obtained from mice immunized with a serotype 2, strain SH-15. Enzyme-linked immunosorbent assay-inhibition tests with antigens obtained from 12 serotype strains of A. pleuropneumoniae and 9 other gram-negative bacteria showed that all the MAbs bound to only serotype 2 strains of A. pleuropneumoniae. The epitopes recognized by the MAbs were located on a carbohydrate moiety of lipopolysaccharide (LPS) of the organism, which was sensitive to periodate oxidation. In immunoblotting analyses of LPS obtained from A. pleuropneumoniae serotype 2, all the four MAbs reacted specifically with the characteristic ladder bands of LPS detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that O-antigen side chains of the LPS are one of the antigenic determinants responsible for the serotype-specificity of A. pleuropneumoniae.     

胸膜肺料放线杆菌溶血毒素特性鉴定及免疫原性研究

本文报道了胸膜肺炎防线杆菌（APP）血清型7型菌株深－8株分泌的溶血素。研究证明该株至少可分泌两种溶血素，一种为热稳定的溶血素，另一种为热不稳定的溶血素。本研究没有对热稳定的溶血素进行提纯分析，而对热不稳定的溶血素经过盐析及凝胶过滤层析提纯后，进行SDS－PAGE分析，表明该溶血素为分子量65kDa的蛋白。本研究利用纯化的65kDa的溶血素蛋白与APP所有型的阳性血清做交叉中和实验和琼脂扩散实验及动物实验，证明该溶血素蛋白具有一定的免疫原性及保护力，但其诱导的免疫反应对同源菌株的攻击只能提供部分保护。     

Characterization of an attenuated strain of Actinobacillus pleuropneumoniae, serotype 1

Pleuropneumonia is an important disease of swine caused by Actinobacillus pleuropneumoniae. Putative virulence determinants include capsule, lipopolysaccharide, and cytotoxin. We studied the virulence and virulence determinants of 2 strains: CM5 and CM5A of serotype 1. Strain CM5 was isolated from a pig with pleuropneumonia and passaged once in vitro; strain CM5A was a substrain of CM5 passaged 70 times in vitro. Pigs challenge exposed to an aerosol of 1.3 x 10(7) colony-forming units of CM5/ml died within 30 hours; pigs challenge exposed to an aerosol of 1.6 x 10(8) colony-forming units of CM5A/ml survived. The average thickness of the capsular layer was 137 nm in strain CM5 and 53 nm in strain CM5A in bacteria treated with homologous antibody and examined by transmission electron microscopy. Similarly, capsular material binding polycationic ferritin was found in colonies of strain CM5, but not in strain CM5A. The ratio of hexosamine to protein in extracted capsule of CM5 was more than twice that of CM5A. The polyacrylamide gel electrophoretic profile of the lipopolysaccharide, outer membrane proteins, and whole cell proteins did not differ between the 2 strains. Also, the amount of cytotoxin or endotoxin produced by the 2 strains during the logarithmic growth phase was not different. The electrophoretic profile of restriction endonuclease digested DNA was similar, with the exception of bands in the 750- and 620-basepair regions. It was concluded that attenuation of strain CM5A during in vitro passage was a result of reduced capsule production and that encapsulation is an important virulence determinant of A pleuropneumoniae, serotype 1.     

Type IV fimbrial subunit protein ApfA contributes to protection against porcine pleuropneumonia

ABSTRACT: Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae accounts for serious economic losses in the pig farming industry worldwide. We examined here the immunogenicity and protective efficacy of the recombinant type IV fimbrial subunit protein ApfA as a single antigen vaccine against pleuropneumonia, or as a component of a multi-antigen preparation comprising five other recombinant antigens derived from key virulence factors of A. pleuropneumoniae (ApxIA, ApxIIA, ApxIIIA, ApxIVA and TbpB). Immunization of pigs with recombinant ApfA alone induced high levels of specific serum antibodies and provided partial protection against challenge with the heterologous A. pleuropneumoniae serotype 9 strain. This protection was higher than that engendered by vaccination with rApxIVA or rTbpB alone and similar to that observed after immunization with the tri-antigen combination of rApxIA, rApxIIA and rApxIIIA. In addition, rApfA improved the vaccination potential of the penta-antigen mixture of rApxIA, rApxIIA, rApxIIIA, rApxIVA and rTbpB proteins, where the hexa-antigen vaccine containing rApfA conferred a high level of protection on pigs against the disease. Moreover, when rApfA was used for vaccination alone or in combination with other antigens, such immunization reduced the number of pigs colonized with the challenge strain. These results indicate that ApfA could be a valuable component of an efficient subunit vaccine for the prevention of porcine pleuropneumonia.     

Immunogenicity of infectious bursal disease viruses in chickens.

Cross-protective properties of infectious bursal disease viruses (IBDVs) were studied. Viruses represented different subtypes of serotype 1, including recently isolated viruses (variants), and a serotype 2 virus. Chickens were vaccinated at 3 weeks of age with inactivated vaccines containing 10(5), 10(6), 10(7), or 10(8) mean tissue-culture infectious dose of a given virus and challenged 2 weeks later using either 10(2) or 10(3.5) mean embryo infectious dose (EID50) of either a standard virus or a variant serotype 1 virus. Protection was evaluated at 5 and 10 days post-challenge, based on gross and microscopic lesions, body weight, and bursa/body-weight ratios. The serotype 2 virus did not confer protection on birds challenged with the serotype 1 viruses. Vaccines made of variant viruses at the low doses protected chickens challenged with the high or low doses of either the standard or the variant viruses. Vaccines made of the standard or variant strains at low doses protected against high or low challenge doses of the standard strain. Vaccines made of the high dose of any of the standard strains protected chickens against the variant virus when the low challenge dose (10(2) EID50) was used, but not when the high challenge dose (10(3.5) EID50) was used. The lowest dose of the standard viruses vaccines required to confer protection against the variant virus varied depending on the strain. Results indicated that protection depended on the strain and dose of both the vaccine and challenge viruses and that the variant strains and standard strains share a common protective antigen(s).     

鸡新城疫、传染性支气管炎、禽流感(H9亚型)三联灭活疫苗对禽流感H9亚型流行株攻毒的保护作用

为了监测鸡新城疫、传染性支气管炎、禽流感(H9亚型)三联灭活疫苗(LaSota株+M41株+SS/94株)对H9亚型禽流感病毒流行毒株的免疫保护效果,采用H9亚型禽流感病毒SS/94株及2009—2010年现地分离的3株H9亚型禽流感病毒对已免疫上述三联灭活苗的SPF鸡进行攻毒试验。结果显示,试验鸡以0.3 mL/只的剂量免疫三联灭活苗后21 d,其H9亚型禽流感病毒的HI抗体效价可达8～11log2,此抗体水平可抵抗2×106EID50的H9亚型禽流感病毒SS/94株、BLCN09株、WDZ09株、YT10株的攻击,攻毒保护率均达90%(9/10)以上。可见,以SS/94株作为禽流感疫苗抗原制备的三联灭活苗具有良好的免疫原性,能使免疫鸡抵抗2009—2010年期间现地分离的多株H9亚型禽流感病毒的攻击。     

Relationship between protective activity and antigen structure of Haemophilus paragallinarum serotypes 1 and 2

The object in this investigation was to determine the relationship between protective activity and antigenic structure of Haemophilus paragallinarum, serotypes 1 and 2. A close relationship exists in both serotypes between protective activity and colonial phenotypic form (iridescent and noniridescent). Protective activities of both serotypes were related to a heat-labile, trypsin-sensitive (L) antigen of iridescent form that produced serotype-specific agglutinin to chickens. The chickens having the agglutinins were protected against challenge exposure with homologous strain, but not with heterologous strain. The chickens injected with unencapsulated organisms of noniridescent form that were derived from encapsulated organisms of iridescent form failed to produce both serotype-specific agglutinins and protection against challenge exposure with homologous strain. Most of the chickens injected with serotype 1 strain produced both hemagglutination-inhibition antibody and serotype 1-specific agglutinin, whereas those injected with serotype 2 produced serotype 2-specific agglutinin and protected against homologous challenge exposure. The protective activity was found in saline extract derived from encapsulated organisms of serotype 1, but was absent in those of serotype 2.     

Evaluation of heat-sensitive, neutrophil-toxic, and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae

Cytotoxic and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae serotype 1 strain CM5 was investigated because of the potential role as a virulence determinant. Viable bacteria were toxic for porcine and bovine neutrophils, whereas bacteria killed by heat treatment at 60 C for 1 hour were not. Similarly, bacteria-free culture supernatant was cytotoxic and hemolytic in assays that used porcine neutrophils and erythrocytes, whereas supernatant treated at 60 C for 1 hour had no activity. Erythrocytes from various species were susceptible to the hemolytic activity of bacteria-free culture supernatant, with ovine and bovine erythrocytes being most sensitive. The neutrophil-toxic and hemolytic activity of bacteria-free culture supernatant was inhibited by cholesterol and oxygen and abolished after trypsin digestion. The neutrophil-toxic and hemolytic activity was preserved during storage at or less than 4 C, but was lost rapidly at 56 C or 80 C. Neutralizing antibodies were demonstrated in serum of pigs and rabbits immunized with 10-fold concentrated culture supernatant of strain CM5 and in field pigs that had recovered from natural infection with H pleuropneumoniae serotype 1. Bacteria-free culture supernatants of 18 strains, including H pleuropneumoniae serotypes 1 through 10, Actinobacillus suis, and Haemophilus taxon minor group, were tested for heat-sensitive, neutrophil-toxic, and hemolytic activity. Fifteen strains were neutrophil toxic, but only 10 of these were hemolytic. Haemophilus pleuropneumoniae, serotype 1, strain VLS557; serotype 5, strain K17; and Haemophilus taxon minor group strain 33PN were neither cytotoxic nor hemolytic.     

Immunity to toxoplasmosis in hamsters

Protective immunity to Toxoplasma was studied in hamsters. Immunity developed in 2 to 3 weeks after vaccinations were performed. Vaccination with live RH, T-45, and ts-4 strains afforded the best protection against challenge exposure with the most pathogenic RH strain used. Even a killed-toxoplasma vaccine protected all hamsters against the slightly less pathogenic T-1 strain through 24 weeks, but it did not protect hamsters against challenge exposure with the RH strain. Both ts-4, a nonpersistent strain, and killed-toxoplasma vaccine provided protective immunity in hamsters that was not dependent upon premunition. Toxoplasma antibody titers in hamsters given the 2 vaccines were similar. However, there was a difference in the quality of immunity: fever and body weight loss were seen in hamsters vaccinated with the killed-toxoplasma vaccine after they were challenge exposed with T-1 strain, whereas these changes were rarely seen in hamsters given the live-toxoplasma vaccine and then challenge exposed with RH strain. Delayed-type hypersensitivity to Toxoplasma antigen always appeared before protective immunity and was detected in all hamsters by 4 days after vaccination with live-toxoplasma strains. Although the development of delayed-type hypersensitivity preceded protective immunity, it was not indicative that protective immunity was present or would develop.     

Evaluation of immunogenicity and protective efficacy of Actinobacillus pleuropneumoniae HB04C(-) mutant lacking a drug resistance marker in the pigs

Previously, we reported the construction and characterization of a genetically defined Actinobacillus pleuropneumoniae (A. pleuropneumoniae) apxIIC gene mutant, HB04C(-), which conferred protection to mice against infection with A. pleuropneumoniae. In this study, we further evaluated HB04C(-) for safety and its ability to elicit protective immunity in pigs. It was demonstrated that a dose of 2 x 10(8) CFU HB04C(-) was safe to the pigs via intranasal or intramuscular injection. Immunization with a dose of 2 x 10(8) HB04C(-) by both intranasal and intramuscular routine could yield equal protective efficacy and elicited significant protection against experiment challenge with homologous or heterologous serotypes of a virulent A. pleuropneumonia. Taken together, HB04C(-) might serve as a promising vaccine candidate against infection with A. pleuropneumoniae.     

Streptococcus suis serotype 9 bacterin immunogenicity and protective efficacy

Streptococcus suis diseases in pigs, most importantly meningitis, are worldwide responsible for major economic losses in the pig industry. About one fourth of invasive S. suis diseases are caused by S. suis serotype 9 strains in Europe. However, little is known about serotype 9 since most studies were performed with serotype 2. The objective of this study was to determine the immunogenicity and protective efficacy of a serotype 9 bacterin in piglets. Challenge was conducted with a reference serotype 9 strain, belonging to the same clonal complex but to a different sequence type as the bacterin strain. The bacterin induced protection against mortality but not morbidity. Eleven days post infection, 3 of 7 vaccinated survivors were not fully convalescent and had not eliminated the challenge strain from inner organs completely. In accordance with the clinical findings, the majority of piglets showed fibrinous-suppurative lesions in at least one inner organ or tissue. In contrast to the placebo group such lesions were not detected in one third of bacterin-vaccinated piglets. Determination of specific serum IgG titers revealed that the bacterin elicited seroconversion against muramidase-released protein and basic membrane lipoprotein. Furthermore, vaccination was associated with induction of opsonizing antibodies against the serotype 9 challenge strain. However, titers of opsonizing antibodies were rather low in comparison to those found in our previous serotype 2 vaccination trial. Piglets developed substantially higher titers of opsonizing antibodies after challenge. Opsonizing antibodies were absorbable with the serotype 9 challenge strain but not with an unencapsulated isogenic mutant of a serotype 2 strain indicating their specificity. The results indicate that a serotype 9 bacterin is less protective than a serotype 2 bacterin, most likely due to inducing only low titers of opsonizing antibodies. This might contribute to emergence of serotype 9 strains, in particular strains of this clonal complex, in Europe.     

Serological cross-reactivity between a porcine Actinobacillus strain and Haemophilus pleuropneumoniae.

During serological screening of a closed SPF-herd free of pleuropneumonia, more than half of the pigs were positive for complement-fixing antibodies to Haemophilus pleuropneumoniae. Actinobacillus bacteria closely related to A. suis were isolated from tonsillar tissue of 14 out of 20 slaughtered pigs submitted for pathological and bacteriological evaluation. None of the pigs had evidence of respiratory disease. Two pigs inoculated endobronchially with a selected Actinobacillus strain developed mild focal pneumonia and complement-fixing antibodies cross-reacting with H. pleuropneumoniae. Five pigs exposed and vaccinated with the Actinobacillus strain and five pigs spontaneously infected with the strain also developed complement-fixing antibodies against H. pleuropneumoniae and appeared to be less susceptible to experimental Haemophilus pleuropneumonia than pigs not exposed to the Actinobacillus infection. The agglutination test applied on serum treated with 2-mercaptoethanol detected antibodies against H. pleuropneumoniae serotype 5 but not against serotype 1 in pigs exposed to the Actinobacillus strain. Antibodies reactive with the Actinobacillus strain were also found in pigs hyperimmunized against H. pleuropneumoniae serotypes 1-5 in 2-mercaptoethanol tube agglutination test and rabbits hyperimmunized against serotypes 1,2 and 7, and strain 73567 in the immunodiffusion test. Conversely rabbits immunized against the Actinobacillus strain had antibodies against H. pleuropneumoniae serotypes 1, 3, 4, 5 and 6. It is concluded that pigs infected with Actinobacillus organisms may become false positive reactors against H. pleuropneumoniae.     

Effect of endobronchial challenge with Actinobacillus pleuropneumoniae serotype 9 of pigs vaccinated with a vaccine containing Apx toxins and transferrin-binding proteins

The efficacy of a subunit vaccine containing the Apx toxins of Actinobacillus pleuropneumoniae and transferrin-binding proteins was determined. Ten pigs were vaccinated twice with the vaccine. Eight control animals were injected twice with a saline solution. Three weeks after the second vaccination, all pigs were endobronchially inoculated with 10(6.5) colony-forming units (CFU) of an A. pleuropneumoniae serotype 9 strain. In the vaccine group, none of the pigs died after inoculation. Only one pig of the control group survived challenge. Surviving pigs were killed at 7 days after challenge. The mean percentage of affected lung tissue was 64% in the control group and 17% in the vaccine group. Actinobacillus pleuropneumoniae was isolated from the lungs of all animals. The mean bacterial titres of the caudal lung lobes were 5.0 x 10(8) CFU/g in the control group and 3.0 x 10(6) CFU/g in the vaccine group. It was concluded that the vaccine induced partial protection against severe challenge.