Exploitation of intestinal colonization-inhibition between salmonella organisms for live vaccines in poultry: potential and limitations

Immunization represents one of the most important methods to increase the resistance of chickens against Salmonella infection. In addition to the development of an adaptive immune response, oral administration of live Salmonella strains to day-old chicks provides protection against infection within hours by intestinal colonization-inhibition. For the exploitation of this phenomenon, practical information on colonization-inhibition between Salmonella organisms is needed. Colonization-inhibition capacity between Salmonella strains from serogroups B, C1, C2, D and G was assessed in chickens. The most profound level of intestinal colonization-inhibition occurred between isogenic strains. Inhibition between strains of the same serovar was greater than that between strains of different serovars. The degree of inhibition between different serovars was not sufficiently high to identify a single strain which might inhibit a wide range of other Salmonella organisms. However, as Salmonella Enteritidis is the dominant serovar in poultry in many countries and because of the profound colonization-inhibition within this serovar there is a considerable potential to exploit this phenomenon in the development of novel live S. Enteritidis vaccines. Treatment of young chicks with mixtures of different Salmonella serovars resulted not only in a very strong growth inhibition of the isogenic strains but also in a substantial inhibition of heterologous serovars. The potential of mixtures of heterologous Salmonella strains as a 'Salmonella Inhibition Culture' and as a 'live Salmonella vaccine' should be further explored.     

Differential responses of macrophages to Salmonella enterica serovars Enteritidis and Typhimurium

Macrophages are major effectors against Salmonella infection, and also transport bacteria between host tissues and provide a protected site for intracellular bacterial replication. We hypothesized that differences in chicken macrophage responses to Salmonella enterica serovar Enteritidis (SE) and serovar Typhimurium (ST) played a role in preferential infection of eggs by SE compared with ST. To test this hypothesis, we determined bacterial phagocytosis and intracellular viability and macrophage nitric oxide (NO) production following in vitro infection with SE or ST in the presence or absence of interferon-gamma (IFN-gamma). The effects of bacterial components, lipopolysaccharide (LPS), outer membrane proteins (OMP) and flagella, on NO production were also assessed. Our results showed: (1) in the presence or absence of IFN-gamma, the percentage macrophages phagocytizing SE and ST was similar; (2) the number of intracellular viable SE was significantly reduced compared with ST in the presence or absence of IFN-gamma; (3) increased macrophage necrosis was seen in the presence of IFN-gamma and ST; (4) Salmonella infection acted synergistically with IFN-gamma in induction of nitric oxide production; and (5) in the absence of IFN-gamma, macrophages produced significantly greater NO following treatment with SE outer membrane protein or flagella compared with ST OMP or flagella, while in the presence of IFN-gamma significantly less NO was produced following treatment with SE-LPS compared with ST-LPS. These results suggest that differential responses of chicken macrophages to SE versus ST may result in increased macrophage death with ST, which could result in an increased inflammatory response as compared to SE.     

Evaluation of a French ELISA for the detection of Salmonella Enteritidis and Salmonella Typhimurium in flocks of laying and breeding hens

In France, the regular and compulsory detection of Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) in flocks of breeding and laying hens is based on bacteriological examination of environmental swabs and faeces samples. The aim of this study was to compare this bacteriological examination with a serological method (ELISA) developed in our laboratory. This ELISA was first evaluated by use of artificially infected hens. During these experimental infection studies, several groups of hens were inoculated with SE, ST, different vaccines and different Salmonella serovars to calculate the experimental parameters of our ELISA. Then, in a field study, 43 flocks were followed monthly using two bacteriological samples (environmental swab and pool of faeces) and 20 serological samples (sera or yolks). Twenty-seven flocks without SE or ST gave a negative serological response throughout their surveillance. Among the 10 various serovars different from SE and ST isolated in this study, S. Heidelberg, S. Agona and S. Hadar gave seropositive results in seven flocks. Consequently, this ELISA was not specific of SE and ST as it detected serovars sharing or not common antigens with SE and ST. Seropositive results were also obtained each month for two flocks where no Salmonella could be isolated. Finally, in seven flocks found infected with SE or ST, the positive ELISA results appeared later than the bacteriological detection. Therefore, for the detection of chicken flocks recently infected with SE or ST, bacteriological examination currently used in France seems to be more appropriate than this ELISA.     

Detection and identification of salmonellas from poultry-related samples by PCR

A polymerase chain reaction (PCR) assay was developed for the generic detection of Salmonella sp. and the identification of S. Enteritidis (SE), S. Gallinarum (SG), S. Pullorum (SP) and S. Typhimurium (ST) in material collected in the field from poultry. The specificity and sensitivity of the assay combined with Rappaport-Vassiliadis selective enrichment broth (PCR-RV) were determined, and field samples were analyzed to verify the validity of the method application. Specificity of the assay was tested using 29 SE, 11 SG, 10 ST and 10 SP strains, along with 75 strains of 28 other Salmonella serovars and 21 strains of other bacterial genera. The assay was 100% specific for Salmonella detection and ST identification. The primer pair for SE, SG and SP also detected S. Berta. PCR detection limits for Salmonella at the genus level were 2 ST, 8 SE, 1.1x10(3) SG and 1.8x10(5) SP cells. At the serovar level, detection limits were 7 ST, 1.2x10(3) SE, 4.4x10(7) SG and 1.8x10(6) SP cells. At the genus level, PCR-RV detected approximately 128% more positive field samples than the standard microbiological techniques and results were ready in 48h instead of 7 days. PCR-RV method is diagnostic of Salmonella at the genus level and ST at the serovar level, although other tests are needed to identify SE, SG and SP to serovar level.     

Cell-mediated immune responses to a killed Salmonella enteritidis vaccine: lymphocyte proliferation, T-cell changes and interleukin-6 (IL-6), IL-1, IL-2, and IFN-gamma production

Two experimental approaches were used to investigate the immunological responses of chickens to a commercial killed Salmonella enteritidis (SE) vaccine. In the first, the effects of host age on antigen-specific proliferative responses and cytokine production were examined. Compared with non-vaccinated controls, 4-wk-old vaccinated chickens showed higher proliferation to SE LPS and flagella. The lymphoproliferation responses to these antigens of 8-mo-old vaccinated chickens were not different compared to the non-vaccinated controls. Increased production of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) by antigen-stimulated splenocytes following vaccination were, in general, more often observed in 4-wk-old compared with 8-mo-old chickens, whereas serum levels of these cytokines were consistently higher in the vaccinated birds compared with controls regardless of age. The second set of experiments were designed to determine the effects of SE vaccination on mitogen- or antigen-induced splenocyte proliferation and serum nitric oxide (NO) and cytokine levels. Splenocytes from vaccinated chickens stimulated with SE flagella showed significantly increased numbers of TCRgammadelta+ cells at 7 days post-vaccination compared with non-vaccinated birds. In contrast, no differences were noted with CD4+, CD8+, or TCRalphabeta+ cells at any time points examined. Higher levels of NO production were observed following stimulation with SE flagella at 4, 7, 11, and 14 days after SE vaccination while serum levels of IFN-gamma, IL-1, IL-6, and IL-8 were elevated only at day 7 post-vaccination. In conclusion, younger chickens mounted a more robust antigen-specific immune response to the SE vaccine compared with older birds and vaccination induced not only T-cell-mediated responses but also host innate and pro-inflammatory responses.     

A cross-protection experiment in pigs vaccinated with Haemophilus parasuis serovars 2 and 5 bacterins, and evaluation of a bivalent vaccine under laboratory and field conditions.

Cross-protection between Haemophilus parasuis serovars 2 and 5 was examined in pigs using a bacterin based vaccine, and subsequently the safety and efficacy of a bivalent vaccine were evaluated. Upon intratracheal challenge of a serovar 2 or 5 strain, pigs immunized with a monovalent vaccine were protected against challenge with a homologous serovar strain, but not with a heterologous serovar strain. Immunization with a bivalent vaccine containing both serovars 2 and 5 bacterins conferred protection in pigs against lethal challenge with each of the serovar strains. A total of 86 pigs from two SPF herds were injected with the bivalent vaccine intramuscularly twice at a four-week interval. No adverse reactions following the vaccination were observed. On day 7 after the second vaccination, vaccinated and non-vaccinated control pigs from herd A were transferred to herd B, where Glasser's disease had broken out. Pigs in the control group developed clinical signs of the disease, and 6 of 8 (75%) pigs died until slaughter, in contrast with only 4 of 46 (9%) pigs in the vaccinated group. In herd C, where there was no outbreak of Glasser's disease, complement fixation antibody titer was raised only in the vaccinated group. A challenge experiment on days 20 and 79 after the second vaccination showed that only the vaccinated pigs were protected. From these findings, the safety and efficacy of the bivalent vaccine were confirmed under laboratory and field conditions.     

Adenovirus vaccination of pregnant ewes and studies on the colostral immunity of their lambs

Pregnant ewes were vaccinated 1 month before parturition with mono or bivalent adenovirus vaccines. Vaccination resulted in increased levels of homologous and heterologous antibody in ewes, with corresponding increases in passive immunity of lambs. Challenge of lambs with homologous or heterologous virus at 21 days of age was associated with significant resistance to development of lesions in lambs challenged with homologous virus, and partial resistance in those challenged with heterologous virus. Bivalent vaccines gave comparable protection to challenge with both virus types.     

Evaluation of Erysipelothrix rhusiopathiae vaccines in pigs by intradermal challenge and immune responses

In a vaccine trial, pigs were challenged intradermally with eight E. rhusiopathiae strains of serovars 1a, 1b or 2 given concurrently. The strains were derived from six herds affected with vaccine breakdowns in 1997-1999, one herd without vaccine breakdown and a serovar 2 reference strain. Responses to two commercial bacterins (one implicated in the vaccine breakdowns), and two experimental bacterins (based on field isolates from affected herds) showed distinct differences in protection, particularly in clinical responses measured at 72 h. Less protection was afforded against serovar 1 challenge by the vaccine implicated in the vaccine breakdowns. Antibody and cell-mediated immune (CMI) responses were significantly different between treatments, and highlighted a similar post-vaccinal antibody response was produced against serovar 2 lysate by all vaccines, but only those providing significant protection against serovar 1 [corrected] produced significantly elevated antiserovar I lysate [corrected] antibodies. Vaccination in general significantly reduced CMI responses to the mitogens concanavalin A and phytohaemagglutinin. This experimental pig challenge system was readily able to confirm suboptimal performance of a commercial bacterin that had passed potency tests in mice but was associated with vaccine failure in commercial herds. This vaccine was also the most immunosuppressive to CMI responses associated with E. rhusiopathiae-specific and non-specific stimulation. The best vaccine response was associated with the highest mean serovar 1 antibody response and the highest CMI response (by lymphoproliferation assay) to serovar 2.     

Differences among six Salmonella serovars in abilities to colonize reproductive organs and to contaminate eggs in laying hens

The abilities of Salmonella serovars to colonize the reproductive organs of chickens and to contaminate eggs were compared. Mature laying hens were inoculated intravenously with 10(5) colony-forming units of Salmonella enteritidis, Salmonella typhimurium, Salmonella infantis, Salmonella hadar, Salmonella heidelberg, or Salmonella montevideo to cause the systemic infection. Salmonella enteritidis was recovered from three yolks of the laid eggs (7.0%), suggesting egg contamination from the transovarian transmission of S. enteritidis. The liver, spleen, and cecum were colonized by each serovar similarly at 4 or 7 days postinoculation (PI), whereas the ovary and preovulatory follicles were colonized by S. enteritidis with significantly (P < 0.05) higher levels than by the other serovars at 4 and 7 days PI. Salmonella enteritidis was recovered from the cloaca and vagina at 2, 4, and 7 days PI and from the other portions of the oviduct at 4 and 7 days PI. In addition, S. enteritidis had been persistent in the peripheral blood for 7 days PI. These results suggest that S. enteritidis is the predominant serovar to colonize the reproductive organs of mature laying hens among six serovars used in this study, reflecting the field situatibn in which the predominant outbreaks of human salmonellosis were caused by S. enteritidis-contaminated eggs recently. The ability of S. enteritidis to colonize the reproductive organs may be one of the reasons that egg contamination with S. enteritidis has increased.     

Salmonella enteritidis clearance and immune responses in chickens following Salmonella vaccination and challenge

Our previous work showed that the cell-mediated immunity (CMI) was enhanced by live Salmonella vaccine (LV). The objective of this study was to evaluate the impact of live and killed Salmonella vaccines on Salmonella enteritidis (SE) clearance and to determine if the clearance was mediated by cell-mediated and/or humoral immunity. Chickens were first immunized at 2 weeks of age followed by a booster dose at 4 weeks, challenged with live SE 2 weeks later (6-week-old) and tested for CMI, antibody response and SE clearance 1-week post SE-challenge (7-week-old). Spleen cell proliferation induced by SE-flagella and Concanavalin A (Con A) were significantly higher and SE shedding was significantly lower in the LV group. The splenic CD3 population was significantly lower and B cells were higher in the control group compared to all the SE-challenged groups (with and without vaccination). Serum antibody to SE-flagella and envelope were significantly higher in the KV group compared to all the other groups. These results suggest that LV protects against SE infection, probably by enhancing the CMI.     

Frequency of Salmonella enteritidis and other salmonellae in the ceca of spent hens at time of slaughter.

A study was conducted to determine the frequency of Salmonella enteritidis (SE) and other Salmonella serovars in the cecal contents of spent laying hens at a hen-processing plant in the southeastern United States over a 4 1/2-month period, from October 1990 through February 1991. A total of 1920 pooled cecal samples (three ceca per sample) from 38 flocks representing 23 producers were obtained and tested for the presence of SE and other Salmonella serovars. A total of 359 samples (18.7%) from 37 of the 38 flocks (97.4%) showed characteristic reactions for salmonellae on triple sugar iron agar (TSIA) slants. Twenty-nine of the 359 Salmonella-positive samples (8.1%) were Group D-positive, all of which were found to be SE on further serotyping. The SE-positive samples were from seven of the 38 flocks (18.4%); four flocks originated from the USDA/APHIS-designated Northern Region of the United States, and three were from the Southeastern Region. Serotyping of the 330 TSIA-positive Group-D negative Salmonella revealed 37 different serovars. S. heidelberg, the predominant serovar, was identified in 49.1% of these isolates.     

Efficacy of an outer membrane protein of Pasteurella haemolytica A2, A7 or A9-enriched vaccine against intratracheal challenge exposure in sheep

The outer membrane proteins (OMP) were extracted from the P. haemolytica A2, A7 and A9 to determine their potential as immunogens and their capability for cross-protection. Sixty lambs of approximately 9 months old were divided into four main groups. Animals in Group 1 were vaccinated with 2ml vaccine containing 100microg/ml of the outer membrane proteins of P. haemolytica A2. Animals in Group 2 were similarly vaccinated with the OMPs of P. haemolytica A7 while Group 3 with OMPs of P. haemolytica A9. Animals in Group 4 were unvaccinated control. During the course of the study, serum was collected to evaluate the antibody levels toward each OMP. There appeared to be good immune responses. However, high antibody levels did not necessarily result in good protection of the animals, particularly against cross-infection with P. haemolytica A9 in animals vaccinated with the OMPs of P. haemolytica A2. It seemed that the antibody responses were more specific toward the homologous challenge but generally did not cross-protect against heterologous serotype challenge. However, the OMPs of P. haemolytica A7 produced good in vivo cross-protection and excellent correlations when good antibody responses against all serotypes led to successful reductions of the extent of lung lesions following homologous and heterologous challenge exposures. Thus, the OMPs of P. haemolytica A7 was effective in protecting animals against homologous and heterologous infection by live P. haemolytica A2, A7 and A9.     

Experimentally induced infectious bovine keratoconjunctivitis: resistance of vaccinated cattle to homologous and heterologous strains of Moraxella bovis.

In studies to determine whether vaccination with one strain of Moraxella bovis would protect against challenge with virulent homologous or heterologous strains, calves were intramuscularly inoculated 3 times with formalin-killed M bovis, with 14 days between inoculations. Fourteen days after the 3rd vaccinal dose was given, all calves were exposed to homologous or heterologous virulent cultures of M bovis. The results indicated that vaccination with one strain of M bovis may induce protective immunity against homologous and heterologous challenge exposure; however, because vaccinated cattle resisted infection and disease produced by a homologous strain to a greater extent than they resisted those produced by heterologous strains, polyvalent vaccines or highly immunogenic common antigens may be needed to protect cattle against the numerous strains they might encounter under natural field conditions. There was minimal correlation between the presence of precipitating antibodies against the heterologous strains and the establishment of infection and disease.     

Equine influenza vaccine efficacy: the significance of antigenic variation

To investigate the level of cross-protection induced by equine influenza H3N8 vaccines derived from different lineages, two studies have been carried out with ponies vaccinated with 'American-like' and 'European-like' vaccines and experimentally challenged with a European-like strain. The results demonstrated that equine influenza vaccines clearly protect against challenge with homologous virus if serum antibody titres are sufficiently high. On the other hand, protection is incomplete even when animals vaccinated with heterologous strains have comparative antibody levels. Nevertheless, the protection afforded by heterologous viruses can be improved by stimulating high levels of antibody. It would be advisable to update equine influenza vaccine strains regularly so that they contain similar strains to variants that are circulating in the field.     

Diagnostic specificity, sensitivity and cross-reactivity of an enzyme-linked immunosorbent assay for the detection of antibody against Leptospira interrogans serovars pomona, sejroe and hardjo in cattle.

Outer sheath antigen was prepared from Leptospira interrogans serovars pomona, sejroe and hardjo by treating the organisms with 1.0M NaC1 followed by 0.04% sodium dodecyl sulfate (SDS). Sodium dodecyl sulfate was removed from the SDS-protein complexes by the extraction of dodecyl sulfate anions as ion pairs with triethylammonium cations into an organic solvent. The outer sheath antigen was recovered from the organic solvent as a precipitate and used as the source of leptospiral enzyme-linked immunosorbent assay (ELISA) antigen. Utilizing this antigen, ELISA was adapted to detect bovine serum antibody to L. interrogans serovars pomona, sejroe and hardjo. The specificity of this assay in 344 bovine sera, which were negative in the microscopic agglutination test (MAT) for seven serovars, was 99.4%. In sera from 37 and 87 cattle which revealed MAT titers greater than or equal to 1:50 for L. interrogans serovars pomona and sejroe, the relative sensitivity of the test was 100%. The ELISA also showed a considerable degree of low level cross-reactivity with other serovars. Sixty-six (75.9%) out of 87 bovine sera which were MAT-positive (MAT titer of greater than or equal to 1:50) with serovars sejroe and hardjo only were ELISA positive with heterologous pomona antigen; 16 (43.2%) and six 16.2%) out of 37 bovine sera which were MAT positive MAT titer of greater than or equal to 1:50) with serovar pomona only were ELISA positive with heterologous sejroe and hardjo ELISA antigen respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Failure of protection and enhanced pneumonia with a US H1N2 swine influenza virus in pigs vaccinated with an inactivated classical swine H1N1 vaccine

Two US swine influenza virus (SIV) isolates, A/Swine/Iowa/15/1930 H1N1 (IA30) and A/Swine/Minnesota/00194/2003 H1N2 (MN03), were evaluated in an in vivo vaccination and challenge model. Inactivated vaccines were prepared from each isolate and used to immunize conventional pigs, followed by challenge with homologous or heterologous virus. Both inactivated vaccines provided complete protection against homologous challenge. However, the IA30 vaccine failed to protect against the heterologous MN03 challenge. Three of the nine pigs in this group had substantially greater percentages of lung lesions, suggesting the vaccine potentiated the pneumonia. In contrast, priming with live IA30 virus provided protection from nasal shedding and virus replication in the lung in MN03 challenged pigs. These data indicate that divergent viruses that did not cross-react serologically did not provide complete cross-protection when used in inactivated vaccines against heterologous challenge and may have enhanced disease. In addition, live virus infection conferred protection against heterologous challenge.     

An evaluation of inactivated infectious coryza vaccines containing a double-emulsion adjuvant system.

The efficacy of experimental inactivated infectious coryza vaccines produced by a commercial vaccine manufacturer was evaluated. The vaccines, containing as the adjuvant phase either a double-emulsion mineral oil system or aluminum-hydroxide gel, were administered to 6-week-old chickens as a single dose. Some vaccines were a monovalent product containing a Page serovar C Haemophilus paragallinarum strain, and others were a bivalent product containing both Page serovar A and serovar C strains. After 3 weeks, all chickens were challenged by infraorbital sinus inoculation of virulent H. paragallinarum, either Page serovar C (strain HP31) or Page serovar A (strain HP14). The monovalent serovar C double-emulsion-based vaccines gave significant protection against a serovar C challenge, with the level of protection varying from 60% to 100%. The monovalent serovar C aluminum-hydroxide-gel vaccine also gave significant protection (94%) against a serovar C challenge. The bivalent double-emulsion vaccine gave significant protection against challenge from both serovars (100% for serovar C and 83% for serovar A). Although no major adverse reactions were detected, some chickens receiving both the double-emulsion vaccines and the aluminum-hydroxide vaccine developed relatively minor granulomatous reactions at the site of injection.     

A serosurvey for antibodies to Leptospira in dogs in the lower North Island of New Zealand

AIMS: To investigate the prevalence of antibodies to endemic and exotic Leptospira serovars in samples from a serum bank, collected from dogs in the lower North Island of New Zealand. METHODS: Sera (n=466), which had been collected from apparently healthy dogs, were screened using the microscopic agglutination test (MAT) for antibodies to serovars L. borgpeterseni serovar hardjo, L. interrogans serovars pomona, copenhageni and canicola, and L. kirschneri serovar grippotyphosa. RESULTS: Antibody to Leptospiral antigen was found in 14.2% of dogs tested. The highest level of reactivity was with serovar copenhageni, to which 9.5% (41/433) of sera were positive. Antibodies to serovars grippotyphosa and canicola were not detected in this population of dogs. CONCLUSIONS: Leptospira infection is relatively common in dogs in the lower North Island . CLINICAL RELEVANCE: Vaccination of dogs against leptospirosis should be considered using vaccine containing antigen to serovars hardjo, pomona and copenhageni. The presence of moderate levels of copenhageni antibody in dogs in the lower North Island raises the possibility that this serovar has become established in rodent populations in this region.     

Antigen-specific lymphocyte proliferation and interleukin production in chickens immunized with killed Salmonella enteritidis vaccine or experimental subunit vaccines

Lymphocyte proliferation and interleukin (IL)-2 and IL-6 levels in serum were measured as indicators of cell-mediated immunity after immunization of chickens with a commercial killed Salmonella enteritidis (SE) vaccine or experimental subunit vaccines of crude protein (CP) extract or the outer membrane protein (OMP). Significantly increased proliferative responses to SE flagella, but not lipopolysaccharide, porin, CP, or OMP, were observed at 1 wk postimmunizarion in the three vaccination groups. The responses to flagella were specific because flagella-induced proliferation was not seen in chickens immunized with adjuvant alone. Of the three immunization protocols, use of the killed SE vaccine appeared most effective because it induced higher flagella-stimulated lymphocyte proliferation at 1 and 2 wk postvaccination compared with the CP- and OMP-vaccinated groups. Significantly increased IL-2 and IL-6 levels in serum were seen at 1 wk postimmunization in the three vaccination groups compared with adjuvant alone, but there were no differences between the killed vaccine and the subunit vaccines at this time, and the levels of both lymphokines returned to baseline at 2 wk postimmunization. We conclude that cell-mediated immunity to SE after vaccination with the killed bacterial vaccine or subunit vaccines is transient and mainly limited to flagella.