Pasteurellaceae oral vaccine vector for economically …...Pasteurellaceae oral vaccine vector for...
Transcript of Pasteurellaceae oral vaccine vector for economically …...Pasteurellaceae oral vaccine vector for...
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Pasteurellaceae oral vaccine vector for economically important diseases of cattle.
Robert E. Briggs and Fred M. TatumNational Animal Disease Center, Ames, IA, USA.
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VirusesMixingTransportHandlingHousingWeather
Shipping Fever:
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Control of bacterial respiratory disease
• Antimicrobials (tx, metaphylaxis, prophylaxis)• Reduce stress• Control Viruses
Reduce mixing of calves, vaccination, cull PI• Vaccination
Timely use difficult, limited efficacy
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Mannheimia haemolytica
• M. haemolytica is a commensal which colonizes tonsils and nasal passages of cattle, sheep, and goats.
• Few isolations from healthy, unstressed calves at farm and order-buyer barn.
• Many isolations / high numbers at feedyard after transport.• Readily spreads among stressed and non-stressed calves.• Colonization elicits local and systemic immune response
and resistance to further colonization.• Once colonized, a calf tends to retain the same strain for
the duration of colonization.
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The Anatomical Record: Advances in Integrative Anatomy and Evolutionary BiologyVolume 294, Issue 11, pages 1939-1950, 1 AUG 2011 DOI: 10.1002/ar.21448http://onlinelibrary.wiley.com/doi/10.1002/ar.21448/full#fig14
• Mannheimia colonizes deep in palatine tonsillar crypts• M-cells, etc. similar to Peyer’s patches are present• Antigen processing and presenting cells are present• Potential induction site for mucosal/systemic vaccination
http://onlinelibrary.wiley.com/doi/10.1002/ar.v294.11/issuetochttp://onlinelibrary.wiley.com/doi/10.1002/ar.21448/full#fig14
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lktC lktA lktB lktD
Leukotoxin operon:
lktC - acylates leukotoxin structural gene to activatelktA - leukotoxin structural genelktB/D - involved in leader-independent leukotoxin exportCommon promotor for entire operon
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NgoMIV NgoMIV
1084 bp 1029 bp
M. haemolytica lktCA3.15 kb
Digest NaeI, ligate.
1035 bp (345 aa) deletion
aa33 aa379T Q A | G S V
ACC CAA GCC | GGC TCG GTT
1084 bp 1029 bp
1035 bp
lktC lktA
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9766
45
312114
kDa
Western blot of native leukotoxin and ∆LktAusing anti-Lkt monoclonal antibody.
∆Lkt Lkt
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Lung exposed to virulent M. haemolytica
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Lung exposed to modified M. haemolytica
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Efficacy of oral and injectable ∆lktA modified-live in calves.
Control 1 Oral2 Injectable2 n=6 n=5 n=5 Lung lesions 32% 4.3%** 7.2%* IHA titer 11 194 28 Lkt neut. titer 72 169 169 Lung bacteria 106.2 102.0 102.2
*P
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Efficacy of injectable ∆lktA modified-live in sheep and goats.
Control1 Vaccinate2 Lung lesions
n=5 40%
n=5 2%*
IHA titer St5:St6 6:3 73:111** Lkt neut. titer 2 21 Lung bacteria 107.8 101.1
*P
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Field trials of mucosalvaccines in transported beef calves
- Tested were Mannheimia-only and Mannheimia/Pasteurella combined- n=84 to 220 calves per study balanced for vaccinated and control- Vaccine delivered single dose on feed or intranasal- Vaccine delivered point-of-first-assembly or at experimental feedlot- Calves monitored for approximately first 5 weeks on feed
- Delivery at point-of-first-assembly enhances weight gain- Increased serum titer in vaccinates- Reduced infectious load of Mannheimia- Reduced morbidity and re-treats
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0102030405060708090
fy0 fy7 fy14 fy35
vacccntlam
Nasal Shedding M. haemolytica
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Weekly M. haemolytica IHA titers of vaccinatedand unvaccinated low-risk calves.
0123456789
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OBB -1 FY 6 FY 12 FY 19 FY 33
NM-NV
NM-V
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Weight gain for days on feed
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0
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7-day 14-day 35-day
Non-VaccVaccAuctionP
ound
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a
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a Greater than non-vaccinated, p
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Summary of oral vaccination field trials
Trial 1 Trial 2 Trial 3 Trial 4
Ark - 2565# /50 head 506# /50 head 570# /42 head 492# /42 head 51.3# / calf 10.1# / calf 13.6# / calf 11.7# / calf
Nmex - 698# / 60 head11.6# / calf
Significantly reduced re-treatment among vaccinates.
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Modified-live bacteria seem to be fast and effective delivered on mucosa…
Other important bovine pathogen’s port-of-entry is often the naso-pharynx or the oro-pharynx…
What if heterologous immunogens were delivered carried and expressed by such bacterial vaccines?
Brucella abortusBVDVMoraxella bovis (pinkeye)Mycobacterium bovisMycoplasma bovisRipicephalus spp. ticks
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MW 1 2 3 4 5 6
1. M. hemolytica expressing carrier protein from chromosome (cells)2. M. haemolytica expressing chimeric carrier and Mycoplasma target protein from chromosome (cells)3. M. haemolytica expressing chimeric carrier and Mycoplasma target protein from chromosome (supernatant)4. M. haemolytica alone (cells)5. M. haemolytica expressing chimeric carrier and Mycoplasma target protein from plasmid (cells)6. M. haemolytica expressing chimeric carrier and Mycoplasma target protein from plasmid (supernatant)
kDa
70
50
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Western blot of M. haemolytica strains expressing carrier protein with or without Mycoplasma target
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MW 1 2 3 4 5
Blot hybridized with M. haemolytica carrier protein monoclonal antibody
1. M. haemolytica cells expressing carrier via plasmid pD80ori
2. M. haemoltica cells expressing chimeric carrier and M bovis protein 1 via plasmid
3. M. haemolytica cells with M. bovis fragment in opposite orientation to carrier protein
4. M. haemolytica cells expressing chimeric carrier and M bovis protein 2 via plasmid (cells)
5. M. haemolytica cells expressing chimeric carrier and M bovis protein 2 via plasmid (supernatant)
Mycobacterium bovis antigen expression via the Μ. haemolytica platform
kDa
10075
50
25
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Chimera of LktAand surface-display carrier
Intracellular LktA
No LktA Control
Confocal imaging of antigen surface-display in E. coli
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1. lktA intracellular2. carrier-lktA
Western Blot of the E coli imaged cells expressing LktA or carrier-LktA probed with the anti-LktA monoclonal used for imaging
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• Modified-live bacteria seem to be fast and effective delivered on mucosa…
• Brucella abortus antigens express in both Mh and Pm, elicit local and systemic humoral response and systemic CMI.
• Moraxella bovis (pinkeye) antigen expresses in both Mhand Pm, elicits local and systemic humoral response.
• BVDV, Mycobacterium bovis, and Mycoplasma bovisexpress in Mh and Pm.
• Surface-display of antigen is possible, potentially influencing the nature of immune response.
• Further study is necessary to determine if the approach has merit to actually mitigate disease or carriage of heterologous agents.
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