Pasteurellaceae oral vaccine vector for economically important diseases of cattle.

Robert E. Briggs and Fred M. TatumNational Animal Disease Center, Ames, IA, USA.

VirusesMixingTransportHandlingHousingWeather

Shipping Fever:

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

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.

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

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

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

9766

45

312114

kDa

Western blot of native leukotoxin and ∆LktAusing anti-Lkt monoclonal antibody.

∆Lkt Lkt

Lung exposed to virulent M. haemolytica

Lung exposed to modified M. haemolytica

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

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

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

0102030405060708090

fy0 fy7 fy14 fy35

vacccntlam

Nasal Shedding M. haemolytica

Weekly M. haemolytica IHA titers of vaccinatedand unvaccinated low-risk calves.

0123456789

10

OBB -1 FY 6 FY 12 FY 19 FY 33

NM-NV

NM-V

Weight gain for days on feed

-20

0

20

40

60

80

100

7-day 14-day 35-day

Non-VaccVaccAuctionP

ound

s

a

a

a

a Greater than non-vaccinated, p

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.

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

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

30

Western blot of M. haemolytica strains expressing carrier protein with or without Mycoplasma target

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

Chimera of LktAand surface-display carrier

Intracellular LktA

No LktA Control

Confocal imaging of antigen surface-display in E. coli

1 2

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

• 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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