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.     

Hemagglutinin of Haemophilus paragallinarum serotype 2 organisms: occurrence and immunologic properties of hemagglutinin

Hemagglutinating properties of Haemophilus paragallinarum serotype 2 and serotype C against freshly collected and glutaraldehyde (GA)- fixed chicken RBC were investigated. Different from serotype 1, the nontreated organisms of serotype 2 and serotype C lacked hemagglutinating activity. However, when the organisms were treated with potassium thiocyanate (KSCN) and/or sonication, activity occurred not only against GA-fixed chicken RBC, but also against GA-fixed RBC of various animal species. The maximum hemagglutination titer (1:64 to 1:256) was obtained against GA-fixed RBC with the KSCN-treated organisms that were also sonicated. The activity was inactivated by heating at 100 C or by treatments with formalin or trypsin, but not by treatments with hyaluronidase or neuraminidase. By using KSCN-treated and sonicated organisms and GA-fixed chicken RBC, a detection of hemagglutination-inhibition (HI) antibody was possible. The HI tests showed that serotype 2 hemagglutinin was immunologically distinct from serotype 1 and that the HI antibody correlated to protective activity against challenge exposure with serotype 2 and serotype C. Chicks having HI antibody greater than 1:5 were protected against challenge exposure with homologous type, but were not protected with serotype 1. Applicability of the HI test was also shown for evaluating protective potency in the serotype 2 vaccine, as well as in the serotype 1 vaccine.     

Efficacy studies on Haemophilus gallinarum bacterin preparations.

Broth cultures inactivated and potentiated by selected methods were tested in chickens for efficacy against homologous and heterologous challenge inoculation, using 2 serotype A strains of Haemophilus gallinarum. Although the 2 strains were within the same serotype, they failed to cross protect. One dose of thimerosal-inactivated bacterin was protective against homologous challenge, but 2 doses of formalin-inactivated bacterin were required. A bivalent bacterin protected chickens well against 1 strain, but not the other, at the 1-dose level.     

Antigenic structure and relationship between serotypes 1 and 2 of Haemophilus paragallinarum.

Antigenic structure and relationship between serotypes 1 and 2 of Haemophilus paragallinarum were analyzed by the rapid plate agglutination and cross-absorption tests. Encapsulated strain forming iridescent colony type of both serotypes 1 and 2 had at least three antigens: heat-labile and trypsin-sensitive (L), heat-labile and trypsin-resistant (HL), and heat-stable and trypsin-resistant (HS). The L was a major antigen located in a surface and divided serologically into three parts: L1, L2, and L3. The L1 was specifici to serotype 1, the L2 was specific to serotype 2, and the L3 was a common antigen shared by serotypes 1 and 2. The HL and HS were common antigens between serotypes. By trypsinization or heating at 121 C, L antigen lost its agglutinability and agglutinin-producing ability. Nonencapsulated organisms forming noniridescent colony type lacked the L antigen. These results suggested that antigenic structure of H paragallinarum serotypes 1 was L1, L3, HL and HS, while serotype 2 was L2, L3, HL, and HS.     

Cross-protection factor(s) of Pasteurella multocida: passive immunization of turkeys against fowl cholera caused by different serotypes

An antiserum cross-protective against different serotypes of Pasteurella multocida was made in turkeys by inoculating them with killed serotype 3 organisms grown in vivo and then exposing them to live serotype 3 organisms. In passive-immunization studies, the antiserum protected young turkeys against the homologous and heterologous serotypes 1, 4, 5, 9, and 12. In addition, the antiserum protected against P. multocida of a heterologous capsule serogroup, serogroup F. A globulin and two IgG fractions purified from the antiserum protected against heterologous challenge with serotype 1. Turkey-grown P. multocida were chemically lysed and separated into soluble and insoluble components to make immunoadsorbents. Antibodies from the cross-protective antiserum isolated by the immunoadsorbents passively protected young turkeys against heterologous serotype I challenge.     

Protection of chickens by ribosomal vaccines from Pasteurella multocida: dependence on homologous lipopolysaccharide

Chickens were protected against fowl cholera by ribosomal vaccines prepared from noncapsulated Pasteurella multocida. Passive hemagglutination (PHA) titers to lipopolysaccharide (LPS) and the degree of protection conferred by ribosomal vaccines were diminished or abolished when ribosomes were chromatographed on an immunoadsorbent column. Addition of subimmunogenic amounts of serotype 1 (homologous) LPS to highly purified ribosomes resulted in vaccines that protected against challenge exposure and produced PHA titers to homologous LPS. Addition of serotype 5 LPS to highly purified ribosomes did not protect chickens against challenge exposure with serotype 1 P multocida, but produced PHA titers to serotype 5 LPS. Combinations of serotype 1 ribosomal RNA and serotype 1 (homologous) LPS did not protect chickens or produce PHA titers to LPS. Purified ribosomes from Brucella abortus, Aspergillus fumigatus, and chicken liver were combined with LPS from P multocida and were evaluated as vaccines. Brucella abortus and A fumigatus ribosomes combined with LPS protected chickens as well as did bacterin made from whole cells of P multocida. Chicken liver ribosomes combined with LPS did not provide protection. To determine whether a protein carrier would substitute for ribosomes, methylated bovine albumin (MBA) was combined with LPS and evaluated as a vaccine. A serologic response to LPS was induced by MBA-LPS vaccine, but the vaccine offered no better protection than when LPS was used alone as vaccine. Ribosome-LPS vaccines produced serologic responses to LPS that were at least 5-fold greater than those produced by MBA-LPS vaccine.     

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.     

New serotype 2 and attenuated serotype 1 Marek's disease vaccine viruses: comparative efficacy

Attempts were made, through selection of optimum viral strains, to develop improved vaccines against Marek's disease (MD). Seven attenuated serotype 1 strains and 22 avirulent serotype 2 strains, both alone and in combination with the FC126 strain of serotype 3, were screened for protective efficacy against challenge with virulent and very virulent MD viral strains. The three viruses selected as most promising were evaluated alone and in various combinations and compared with commercially available vaccines, including FC126, bivalent (FC126 + SB-1), and CV1988/C, in 12 separate assays. Two of these new viruses--301B/1 (serotype 2) and Md11/75C/R2 (serotype 1)--were exceptionally protective compared with prototype vaccine strains. Four new monovalent and polyvalent vaccines based on these two isolates protected chickens better than FC126 alone or CV1988/C alone. Three of these new vaccines provided better protection than the bivalent (FC126 + SB-1) vaccine. Protective synergism was noted commonly between viruses of serotypes 2 and 3 but only sporadically between serotypes 1 and 2 or between serotypes 1 and 3. Strain CVI988/C was protective but was no better than FC126 alone, and it was less effective than bivalent (FC126 + SB-1) vaccine, even when used as a bivalent vaccine with FC126 or SB-1.     

Immunogenicity and antigenicity of infectious bursal disease virus serotypes I and II in chickens

Chickens were inoculated with infectious bursal disease virus serotype I or serotype II to determine if their immune system can distinguish between the two serotypes. Chickens had neutralizing antibodies to only serotype I viruses following exposure to serotype I viruses, and chickens had antibodies to only serotype II viruses following exposure to serotype II viruses. No cross-reactions were observed between antisera prepared to each of these two serotypes using a cross-virus-neutralization assay. Signs of disease were detected only in birds exposed to a virulent serotype I isolate. Chicks exposed to the serotype II viruses were not protected from challenge with a virulent serotype I isolate. In one experiment, antibodies to a serotype II isolate, which were detected before challenge, did not protect chicks from challenge with a virulent serotype I isolate.     

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.     

Embryo vaccination against Marek's disease with serotypes 1, 2 and 3 vaccines administered singly or in combination

Marek's disease virus (MDV) vaccines of serotypes 1 and 2 administered in 18-day-old embryonated eggs induced better protection against post-hatch challenge at 3 days with virulent MDV than vaccines given at hatch. Embryonal vaccination with a polyvalent vaccine containing equal quantities of serotypes 1 and 2 of MDV and serotype 3 virus (turkey herpesvirus, HVT) was also significantly more effective than post-hatch vaccination. These and earlier results indicate that protective efficacy of single or combined Marek's disease vaccine serotypes against post-hatch challenge at 3 days can be substantially improved if the vaccines are injected into 18-day embryos rather than at hatch. Injection of vaccines of serotypes 1 or 2 into embryonated eggs or hatched chicks did not cause detectable gross or microscopic lesions in chickens. Vaccine viruses of serotypes 1 and 2 could be isolated from spleen cells of chickens 1 week post-vaccination, and the titer of recoverable viruses was higher in chickens that received the vaccines at the 18th day of embryonation than in chickens vaccinated at hatch. Although embryo vaccination with HVT usually provided better protection than post-hatch vaccination against early post-hatch challenge with variant pathotypes of MDV, the protection was poor regardless of vaccination protocol. If challenge with variant pathotypes of MDV was delayed until embryonally or post-hatch HVT-vaccinated chickens were 21 days of age, protection of chickens by HVT was not enhanced. Thus, resistance induced by embryonal vaccination with HVT was qualitatively similar to that induced by post-hatch vaccination with this virus.     

Serological differentiation of five bluetongue virus serotypes in indirect ELISA.

The serological reactivity in indirect ELISA of five different bluetongue virus (BTV) serotypes (4, 10, 15, 16 & 20) was compared using polyclonal antisera raised against virus particles and an outer structural protein, VP2. Rabbit and sheep antisera against BTV-10 produced higher ELISA values with their homologous antigens than with heterologous serotypes. A hyperimmune rabbit serum specific for virus particles was able to distinguish heterologous serotypes from each other, but a sheep serum from an infected animal was not. An antiserum directed against VP2, the protein responsible for serotype specificity in neutralization tests, was not serotype-specific in ELISA and cross-reacted with other serotypes. The discriminatory ability of a BTV-4 antiserum was improved by cross-absorption with heterologous antigens. This greatly reduced the ELISA signals with heterologous serotypes and produced an antiserum that was effectively serotype-specific.     

Comparison of ciliary activity and virus recovery from tracheas of chickens and humoral immunity after inoculation with serotypes of avian infectious bronchitis virus

Cross-protection tests with homologous and heterologous serotypes of infectious bronchitis virus (IBV) were used to compare ciliary activity and virus recovery from tracheas of chickens. Validation of this technique included correlating the neutralization indices of antiserum obtained from some infected birds. Chickens were inoculated intratracheally with either the JMK or Connecticut (Conn) serotype of IBV. Three weeks later, infected and uninfected groups were challenged by the same route with homologous and heterologous virus. The JMK strain provided immunity against homologous challenge and the Conn strain, as indicated by good ciliary activity and lack of challenge virus recovery. The Conn strain provided only homologous protection, as ciliostasis occurred and virus was recovered after challenge with the JMK strain. In each case, antiserum to immunizing virus neutralized only the homologous virus. Controls were uniformly susceptible and lacked neutralizing antibody. A similar experiment with the Ark 99 serotype and a recent isolate (397) of IBV revealed complete cross-protection of the tracheas. Antiserum to each virus neutralized the homologous and heterologous virus in each case in reciprocal tests. The results indicate that these two viruses are closely related. The complete agreement between ciliary activity and virus isolation indicates that ciliary activity is a reliable, objective criterion upon which tracheal immunity can be judged in cross-protection tests.     

Immune response to avian reovirus in chickens and protection against experimental infection

Objectives To assess the efficacy of the vaccination procedure and the effect of the transfer of maternal antibodies to progeny chickens on reovirus pathogenicity.
Design To vaccinate chickens and challenge progeny chickens with high doses of homologous and heterologous viruses.
Procedure High doses of reovirus strains RAM-1, 1091 and 724 were used to induce tenosynovitis lesions. High doses were produced by concentration of viruses grown in cell culture. Then similar doses of viruses were used to challenge immunised chickens progeny.
Result Vaccination of breeding hens with the RAM-1 strain of avian reovirus, which resulted in the passive transfer of neutralising antibody to progeny chickens, completely prevented the development of tenosynovitis in 80% of progeny chickens infected with the homologous virus. Even though multiple injection of hens resulted in broadening of the normal type-specificity of the neutralising antibody response against heterologous serotypes of avian reovirus, only marginal protection against strains of two heterologous serotypes of avian reovirus was obtained.
Conclusions A model for assessing the efficacy of vaccination against avian reovirus strains on clinical signs such as tenosynovitis was developed that overcome the normal low virulence of Australian strains of avian reovirus. Breeding hens can be immunised with Australian strain of avian reovirus with passive transfer of antibody via the yolk to the progeny chickens. Although the neutralising antibody response to three injections of inactivated virus decreased the specificity of the neutralising antibody response against antigenically heterologous strains of avian reovirus, the protective immunity appeared to retain type-specificity.     

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).     

Production and characterization of monoclonal antibodies to three infectious bronchitis virus serotypes.

Three panels of monoclonal antibodies (MAbs) were prepared against the spike (S) proteins of infectious bronchitis virus (IBV) strains Arkansas 99, Connecticut 46, and Massachusetts 41. Based on enzyme-linked immunosorbent assay (ELISA), the MAbs were grouped into three categories: 1) group-specific, which reacted with a broad spectrum of homologous and heterologous IBV serotypes; 2) serotype-specific, which reacted only with strains of the homologous serotype; and 3) strain-specific, which reacted "selectively" with only certain strains of homologous and heterologous serotypes. MAbs that displayed serotype specificity were all specific to S1 fractions of the homologous serotype, confirming that epitopes that determine virus serotype are associated with the S1 protein. An excellent correlation was found when the results of IBV serotyping by MAb-based indirect ELISA were compared with those from the conventional virus-neutralization test. This confirms that the MAbs described here will serve as valuable tools in epizootiological studies and serotype-specific diagnosis of IBV infection.     

Experimental immunisation of lambs against pneumonic pasteurellosis

Methods of immunising lambs against pneumonic pasteurellosis, caused by several serotypes of Pasteurella haemolytica, were assessed in specific pathogen free lambs. Lambs were vaccinated intramuscularly with sodium salicylate extract (SSE) of P haemolytica, either alone or in combination with heat-killed organisms (HKO). SSE of P haemolytica type A1 protected vaccinated lambs against pneumonia resulting from challenge with the homologous serotype. SSE of type A2 also provided some protection but this was improved by vaccination with a combination of SSE and HKO.     

Assessment of protective efficacy of live and killed vaccines based on a non-encapsulated mutant of Streptococcus suis serotype 2.

The protective efficacy of a live and killed non-encapsulated isogenic mutant of Streptococcus suis serotype 2 was determined in pigs, and compared with the efficacy of the capsulated wild-type strain. SPF pigs were vaccinated twice intramuscularly at 4 and 7 weeks of age with a dose of 1 x 10(9) formalin-killed CFU of the wild-type (WT-BAC), formalin-killed non-encapsulated mutant (CM-BAC) or live non-encapsulated mutant (CM-LIVE) strain. After 2 weeks, vaccinated pigs and non-vaccinated controls were challenged intravenously with 1 x 10(7) CFU of the homologous, wild-type S. suis serotype 2 strain. Protection was evaluated by clinical, bacteriological, serological and post-mortem examinations. All pigs vaccinated with WT-BAC were completely protected against challenge with the homologous serotype. Pigs vaccinated with CM-BAC were partially protected. Although all pigs vaccinated with CM-BAC survived the challenge, four out of five pigs developed clinical signs of disease for several days. Compared to the WT-BAC and CM-BAC, the CM-LIVE vaccine was less protective. Two out of five pigs vaccinated with CM-LIVE died in the course of the experiment and all of them developed specific clinical signs of disease for several days. The protective efficacy of the vaccines could be associated with serum antibody titers. Antibody titers against cells of wild-type and non-encapsulated mutant strains as well as against muramidase-released proteins (MRP) were high in pigs vaccinated with WT-BAC and CM-BAC. Pigs vaccinated with CM-LIVE showed lower antibody titers. Antibody titers against purified capsular polysaccharides (CPS) of S. suis serotype 2 were only found in pigs vaccinated with WT-BAC. These findings indicate that CPS and other bacterial components of WT-BAC are probably essential for full protection against homologous challenge.     

Eimeria mitis: immunogenicity and cross immunity of two isolates

The ability of two isolates of Eimeria mitis to provide protection against homologous or heterologous challenge was examined. Based on weight gain and feed efficiency of male chickens, the B4 and C2 isolates protected against both homologous and heterologous challenge. The unprotected chickens suffered severe growth depression and impairment of feed utilization. Birds were inoculated with a series of increasing immunizing doses and became immune by the sixth dose. The C2 isolate (strain) appeared to be more immunogenic than the B4 isolate (strain).     

Immunogenicity of Mycoplasma gallisepticum

The serological response and protective immunity elicited in the chicken by the pathogenic Ap3AS strain and the moderately pathogenic 80083 strain of Mycoplasma gallisepticum and variants of strain 80083 attenuated by repeated passage in mycoplasma broth were investigated. Strain 80083 elicited a substantial serum antibody response after administration either in drinking water or by conjunctival sac instillation to 7-week-old SPF chickens. No vaccinated chickens developed air sac lesions when challenged by intra-abdominal (IA) injection with the virulent Ap3AS strain. Chickens vaccinated with strain 80083M (50 broth passages) showed only a weak serological response but were substantially protected when challenged 4 weeks after vaccination. Chickens vaccinated with 80083H (100 broth passages) were serologically negative 4 weeks after vaccination and developed severe air sac lesions after challenge. Thirty-seven-week-old hens vaccinated 6 months previously with strain 80083 had high serum antibody levels and were completely protected against IA challenge with the homologous strain. However, 4/6 showed mild air sac lesions when challenged intra-abdominally with strain Ap3AS. Another group showed high M. gallisepticum serum antibody levels 6 months after vaccination with strain Ap3AS but 4/6 and 2/6 showed mild lesions after IA challenge with strains Ap3AS or 80083, respectively. Strains 80083 or 80083M were administered by conjunctival sac instillation to susceptible 11-week-old commercial pullets at the time of fowl pox vaccination. The concurrent use of both vaccines had no apparent adverse effect on the health of the chickens. Similar protection against IA challenge with strain Ap3AS was produced with the M. gallisepticum vaccines whether used alone or in combination with fowl pox.