Passive immunization against Histomonas meleagridis does not protect turkeys from an experimental infection

Histomonosis or blackhead is a disease of gallinaceous birds, caused by the protozoan Histomonas meleagridis. As recent regulatory action has removed almost all drugs against this disease from the European market, the development of new prophylactics has become crucial. Identification of the protective immune mechanism would facilitate the choice and development of a vaccination strategy to prevent histomonosis. In this study, turkeys were either actively or passively immunized and were then challenged to assess the role of antibody-mediated immunity in the protection form this disease. Active immunization was performed either by experimental infection and treatment or by intramuscular injection with lysed H. meleagridis. Passive immunization was attempted by intraperitoneal administration of pooled, concentrated, neutralizing antisera from immunized donor animals to naive turkeys. A significantly higher IgG response was observed after infection and treatment than after intramuscular injection, which in turn was higher than the responses of placebo and control birds. While active immunization of turkeys by intramuscular injection of dead H. meleagridis antigens appeared not to be protective against histomonosis, immunization by infection and treatment did induce protection. However, no significant level of protection could be observed in the passively immunized birds. These results suggest that serum antibodies to H. meleagridis may not be a key component in the protection against this parasite. It is, however, possible that the concentration of antibodies at the mucosal site is insufficient. Therefore, further investigation on mucosal immune responses is necessary

Role of lysozyme inhibitors in the virulence of avian pathogenic Escherichia coli

Lysozymes are key effectors of the animal innate immunity system that kill bacteria by hydrolyzing peptidoglycan, their major cell wall constituent. Recently, specific inhibitors of the three major lysozyme families occuring in the animal kingdom (c-, g- and i-type) have been discovered in Gram-negative bacteria, and it has been proposed that these may help bacteria to evade lysozyme mediated lysis during interaction with an animal host. Escherichia coli produces two inhibitors that are specific for c-type lysozyme (Ivy, Inhibitor of vertebrate lysozyme; MliC, membrane bound lysozyme inhibitor of c-type lysozyme), and one specific for g-type lysozyme (PliG, periplasmic lysozyme inhibitor of g-type lysozyme). Here, we investigated the role of these lysozyme inhibitors in virulence of Avian Pathogenic E. coli (APEC) using a serum resistance test and a subcutaneous chicken infection model. Knock-out of mliC caused a strong reduction in serum resistance and in in vivo virulence that could be fully restored by genetic complementation, whereas ivy and pliG could be knocked out without effect on serum resistance and virulence. This is the first in vivo evidence for the involvement of lysozyme inhibitors in bacterial virulence. Remarkably, the virulence of a ivy mliC double knock-out strain was restored to almost wild-type level, and this strain also had a substantial residual periplasmic lysozyme inhibitory activity that was higher than that of the single knock-out strains. This suggests the existence of an additional periplasmic lysozyme inhibitor in this strain, and indicates a regulatory interaction in the expression of the different inhibitors

Evaluation of the persistence and gene expression of an anti-Chlamydophila psittaci DNA vaccine in turkey muscle

BACKGROUND: DNA vaccination has been shown to elicit specific cellular and humoral immune responses to many different agents in a broad variety of species. However, looking at a commercial use, the duration of the immune response against the vaccine is critical. Therefore the persistence of the DNA vaccine, as well as its expression, should be investigated. We conducted these investigations on a DNA vaccine against Chlamydophila psittaci, a Gram-negative intracellular bacterium which causes respiratory disease in turkeys and humans. Previous studies showed that the DNA vaccine confers partial protection against C. psittaci infection in turkeys. Turkeys were injected intramuscularly with the DNA vaccine : a eukaryotic expression vector (pcDNA1::MOMP) expressing the major outer membrane protein (MOMP) of an avian C. psittaci serovar D strain. Over a period of 11 weeks, cellular uptake of the DNA vaccine was examined by PCR, transcription of the insert by reverse transcript-PCR (RT-PCR) and mRNA translation by immunofluorescence staining of muscle biopsies. RESULTS: The results indicate that the DNA vaccine persists in turkey muscle for at least 10 weeks. Moreover, during this period of time MOMP was continuously expressed, as evidenced by the immunofluorescence staining and RT-PCR. CONCLUSION: Since C. psittaci infections occur at the age of 3 to 6 and 8 to 12 weeks, a vaccine persistence of 10 weeks seems adequate. Therefore, further research should concentrate on improving the elicited immune response, more specifically the cell-mediated immune response, rather than prolonging the lifespan of the plasmid

Evaluation of 'Bacterial ghosts' with iron receptors for protection against avian pathogenic Escherichia coli (APEC)

Aviaire pathogene Escherichia coli (APEC) zijn Gram-negatieve bacteriën die wereldwijd verscheidene ziektes bij pluimvee veroorzaken. Een van deze aandoeningen, colibacillosis, wordt beschouwd als één van de belangrijkste oorzaken van economische verliezen in de pluimvees ector. De bacteriën tasten eerst de ademhalingswegen aan waarna ze de bl oedbaan binnendringen (septicemie) en interne organen infecteren hetgeen leidt tot perihepatitis, pericarditis, peritonitis, splenitis en salpin gitis. Dit resulteert in koorts, verminderde groei, verhoogde mortalitei t en een lagere eiproductie. Een ander belangrijk ziektesyndroom is cell ulitis of necrotische dermatitis waarbij APEC de diepere weefsellagen ro nd de dijen koloniseren. Cellulitis is gekarakteriseerd door een diffuse , oedemateuze ontsteking en wordt meestal vastgesteld in het slachthuis met karkas afkeuringen tot gevolg. Deze gegevens illustreren de behoefte aan preventieve maatregelen. Het doel van deze doctoraatsthesis was dan ook de ontwikkeling van een breedspectrum vaccin tegen APEC infecties. Een van de virulentiefactoren geassocieerd met APEC is het ijzeropnamesy steem dat gebruik maakt van ferri-siderofoor receptoren en receptoren vo or heem. Met behulp van 5 nieuw ontwikkelde multiplex polymerase ketting reacties werden 239 APEC stammen onderzocht op het voorkomen van 12 ijze rreceptorgenen. De volgende prevalenties werden gevonden: 100% voor fhuE en fepA, 96.2% voor fiu, 92.9% vo or cir, 92.5% voor iroN, 87.4% voor iutA</ &gt;, 63.2% voor fecA, 53.1% voor fyuA, 46.9% voor&amp;nb sp;fhuA, 45.6% voor ireA, 41.8% voor chuA en 4.6% voor iha. In elke onderzochte stam werden minstens 4 ij zerreceptorgenen gedetecteerd hetgeen het belang van het ijzeropname sys teem voor APEC virulentie benadrukt. Gebaseerd op deze prevalenties en/o f op eerdere publicaties die melding maakten van succesvolle vaccinatiep roeven werden de ferri-siderofoorreceptoren FhuE, FepA, IroN en IutA wee rhouden als beloftevolle kandidaat antigenen. Voor de ontwikkeling van een breedspectrum vaccin is het belangrijk dat een kandidaat antigen geconserveerd is tussen de verschillende stammen. Aangezien werd aangetoond dat APEC sterk verwant is met uropathogene&amp;nbs p;E. coli (UPEC) en neonatale meningitis geassocieerde E. coli (NMEC) werd de graad van conservering voor FhuE, FepA, IroN en I utA tussen deze pathotypes nagegaan met behulp van meervoudige proteïnea lignering. De proteïnesequenties geassocieerd met deze receptoren werden afgeleid voor de APEC stam CH2 (en/of APEC1) en vergeleken met de seque nties voor APEC, UPEC of NMEC terug te vinden op NCBI. Alle ferri-sidero foorreceptoren waren voor minstens 99% geconserveerd tussen de geselecte erde stammen. Twee tentatieve vaccins, levende E. coli en E. coli</ &gt; bacterial ghosts die FepA, FhuE, IroN en IutA uitdrukken als recombi nante proteïnen in hun buitenmembraan, werden geëvalueerd in een vaccina tie-experiment. Twee expressievectoren die elk 2 ferri-siderofoor-recept orgenen bevatten, pACYC::fepA,iutA en pRSF::fhuE,iroN, werde n gebruikt om E. coli BL21 Star DE3 te transformeren. Express ie werd geïnduceerd met behulp van isopropyl-ß-D-thiogalactopyranos ide (IPTG) en resulteerde in het tentatieve levende E. coli v accin. Hiernaast werden E. coli BL21 Star DE3 getransformeerd met het lysisplasmide pBAD::lysE gevolgd door transformatie met p ACYC::iutA,fepA of pRSF::iroN,fhuE. Expressie van de ijzerre ceptoren door inductie met IPTG gevolgd door initiatie van lysis met beh ulp van L-arabinose resulteerde in 2 recombinante bacterial ghost cons tructen die samengevoegd werden tot het tentatieve geïnactiveerde vaccin . Immunisatie van kippen, intramusculair met de bacterial ghosts of in tranasaal met levende E. coli, induceerde een E. coli -specifieke IgG immuunrespons, die vlak voor challenge significant ho ger was dan de respons in de controle groepen, die PBS of LB toegediend kregen. Hoewel deze tentatieve vaccins noch letsels noch mortaliteit kon den verhinderen na APEC challenge, lag de mortaliteit na immunisatie met recombinante (23%) of niet-recombinante (30%) bacterial ghosts lager dan in de controle groep (60%). Niettemin resulteerde enkel immunisatie met de recombinante bacterial ghosts in een trend naar significantie.Dankwoord I Samenvatting V Summary VII List of abbreviations IX Table of contents XIII Chapter 1 General introduction on avian pathogenic Escherichia coli, its iron acquisition system and bacterial ghosts 1 Part 1 Avian pathogenic Escherichia coli (APEC) 3 Part 2 Bacterial iron acquisition, a necessity for bacterial survival 15 Part 3 Bacterial ghosts, a new versatile approach in vaccine development 37 Aims of study 47 Chapter 2 High prevalence iron receptor genes of avian pathogenic Escherichia coli. 49 Chapter 3 The ferri-siderophore receptors FepA, FhuE, IroN and IutA are highly conserved between avian pathogenic Escherichia coli (APEC), uropathogenic E. coli (UPEC) and neonatal meningitis-associated E. coli (NMEC) 59 Chapter 4 Immunization of chickens with Escherichia coli ghosts or live E. coli expressing the ferri-siderophore receptors FepA, FhuE, IroN and IutA does not protect chickens against colibacillosis 71 Chapter 5 General discussion and future perspectives 95 References 101 List of publications 121status: publishe

Passive immunization against Histomonas meleagridis does not protect turkeys from an experimental infection

Histomonosis or blackhead is a disease of gallinaceous birds, caused by the protozoan Histomonas meleagridis. As recent regulatory action has removed almost all drugs against this disease from the European market, the development of new prophylactics has become crucial. Identification of the protective immune mechanism would facilitate the choice and development of a vaccination strategy to prevent histomonosis. In this study, turkeys were either actively or passively immunized and were then challenged to assess the role of antibody-mediated immunity in the protection form this disease. Active immunization was performed either by experimental infection and treatment or by intramuscular injection with lysed H. meleagridis. Passive immunization was attempted by intraperitoneal administration of pooled, concentrated, neutralizing antisera from immunized donor animals to naive turkeys. A significantly higher IgG response was observed after infection and treatment than after intramuscular injection, which in turn was higher than the responses of placebo and control birds. While active immunization of turkeys by intramuscular injection of dead H. meleagridis antigens appeared not to be protective against histomonosis, immunization by infection and treatment did induce protection. However, no significant level of protection could be observed in the passively immunized birds. These results suggest that serum antibodies to H. meleagridis may not be a key component in the protection against this parasite. It is, however, possible that the concentration of antibodies at the mucosal site is insufficient. Therefore, further investigation on mucosal immune responses is necessary.status: publishe

Evaluation of the persistence and gene expression of an anti-<it>Chlamydophila psittaci </it>DNA vaccine in turkey muscle

Abstract Background DNA vaccination has been shown to elicit specific cellular and humoral immune responses to many different agents in a broad variety of species. However, looking at a commercial use, the duration of the immune response against the vaccine is critical. Therefore the persistence of the DNA vaccine, as well as its expression, should be investigated. We conducted these investigations on a DNA vaccine against Chlamydophila psittaci, a Gram-negative intracellular bacterium which causes respiratory disease in turkeys and humans. Previous studies showed that the DNA vaccine confers partial protection against C. psittaci infection in turkeys. Turkeys were injected intramuscularly with the DNA vaccine : a eukaryotic expression vector (pcDNA1::MOMP) expressing the major outer membrane protein (MOMP) of an avian C. psittaci serovar D strain. Over a period of 11 weeks, cellular uptake of the DNA vaccine was examined by PCR, transcription of the insert by reverse transcript-PCR (RT-PCR) and mRNA translation by immunofluorescence staining of muscle biopsies. Results The results indicate that the DNA vaccine persists in turkey muscle for at least 10 weeks. Moreover, during this period of time MOMP was continuously expressed, as evidenced by the immunofluorescence staining and RT-PCR. Conclusion Since C. psittaci infections occur at the age of 3 to 6 and 8 to 12 weeks, a vaccine persistence of 10 weeks seems adequate. Therefore, further research should concentrate on improving the elicited immune response, more specifically the cell-mediated immune response, rather than prolonging the lifespan of the plasmid.</p

Evaluation of the persistence and gene expression of an anti-Chlamydophila psittaci DNA vaccine in turkey muscle

BACKGROUND: DNA vaccination has been shown to elicit specific cellular and humoral immune responses to many different agents in a broad variety of species. However, looking at a commercial use, the duration of the immune response against the vaccine is critical. Therefore the persistence of the DNA vaccine, as well as its expression, should be investigated. We conducted these investigations on a DNA vaccine against Chlamydophila psittaci, a Gram-negative intracellular bacterium which causes respiratory disease in turkeys and humans. Previous studies showed that the DNA vaccine confers partial protection against C. psittaci infection in turkeys. Turkeys were injected intramuscularly with the DNA vaccine : a eukaryotic expression vector (pcDNA1::MOMP) expressing the major outer membrane protein (MOMP) of an avian C. psittaci serovar D strain. Over a period of 11 weeks, cellular uptake of the DNA vaccine was examined by PCR, transcription of the insert by reverse transcript-PCR (RT-PCR) and mRNA translation by immunofluorescence staining of muscle biopsies. RESULTS: The results indicate that the DNA vaccine persists in turkey muscle for at least 10 weeks. Moreover, during this period of time MOMP was continuously expressed, as evidenced by the immunofluorescence staining and RT-PCR. CONCLUSION: Since C. psittaci infections occur at the age of 3 to 6 and 8 to 12 weeks, a vaccine persistence of 10 weeks seems adequate. Therefore, further research should concentrate on improving the elicited immune response, more specifically the cell-mediated immune response, rather than prolonging the lifespan of the plasmid.status: publishe

High prevalence iron receptor genes of avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli are known to cause significant losses in the poultry industry worldwide. Although prophylactic measures based on vaccination are advisable, until now no full heterologous protection against colibacillosis has been achieved. Since iron is an essential nutrient to these bacteria, the aim of this study was to investigate the prevalence of 12 outer-membrane iron receptor genes in 239 pathogenic strains isolated from clinical cases of colibacillosis in chickens. Five multiplex polymerase chain reactions were developed as a tool for efficient screening. Among the 239 avian E. coli isolates, 100% were positive for fhuE and fepA, 96.2% for fiu, 92.9% for cir, 92.5% for iroN, 87.4% for iutA, 63.2% for fecA, 53.1% for fyuA, 46.9% for fhuA, 45.6% for ireA, 41.8% for chuA and 4.6% for iha.status: publishe

High hydrostatic pressure sensitivity of virulent avian pathogenic Escherichia coli

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Non-curative, but prophylactic effects of paromomycin in Histomonas meleagridis-infected turkeys and its effect on performance in non-infected turkeys

Histomonosis (blackhead or infectious enterohepatitis) is a disease of gallinaceous birds, especially of turkeys, and is caused by the protozoan Histomonas meleagridis. Since the ban of all chemoprophylactic and chemotherapeutic products against this disease in the European Union, this parasite causes a considerable amount of economical problems in the poultry industry. Research which could ultimately lead to the discovery of new drugs against this disease is thus highly necessary. Hence, in this study, the efficacy of paromomycin against histomonosis in turkeys was investigated. First, the prophylactic and therapeutic efficacy of this drug against H. meleagridis and its effect on the weight gain of turkeys was determined. Adding paromomycin to the feed (400 ppm as well as 200 ppm paromomycin) or to the drinking water (420 mg paromomycin per liter water, added prior to or on the day of challenge) significantly lowered the mortality rate and the caecal and liver lesion scores after an intracloacal infection compared to infected untreated birds. However, when paromomycin was administered to turkeys in the drinking water after the challenge, no significant differences in mortality or in lesion scores could be observed compared to the infected untreated control group. This demonstrates that paromomycin exerts a purely preventive action against histomonosis in turkeys. Additionally, the weight gain of the treated birds was positively influenced by the use of the drug, as the average weight gain of all treated groups (except for the group treated at the day of first mortality) was significantly higher than that of the untreated control group. Finally, the target site of paromomycin was detected in the SS rRNA gene of H. meleagridis. Consequently, the susceptibility to paromomycin can be correlated to the presence of the binding site of the drug at the 3' end of the small subunit rRNA gene of the parasite. In conclusion, paromomycin can be used as a new prophylactic measure in the control of histomonosis in turkeys.status: publishe