1. Current concepts of carriage,
spread and control of
Pasteurella multocida in calves
Current concepts of carriage,
spread and control of
Pasteurella multocida in calves
Dr. J. Chris HodgsonDr. J. Chris Hodgson
LeaderLeader Pasteurella multocidaPasteurella multocida research groupresearch group
Moredun Research InstituteMoredun Research Institute
Scotland UKScotland UK
Merial forum on BRD
29th Sept to 1st Oct 2010 Lisbon, Portugal

2. Moredun Research Institute
Division of Control of Bacterial Diseases
Moredun Research Institute
Division of Control of Bacterial Diseases
Our work assigned under following headings:Our work assigned under following headings:
• Pathogen biology
• Host susceptibility and immune response
• Detection and intervention

3. Scope of workScope of work
 Study mechanisms of pathogenesis,
epidemiology and host specificity
 Encompass molecular – whole animal
 Develop and evaluate novel control methods,
diagnostic tests

4. Pneumonic pasteurellosis in young
calves
Pneumonic pasteurellosis in young
calves
Causes:Causes:
 MannheimiaMannheimia ((PasteurellaPasteurella)) haemolyticahaemolytica
 PasteurellaPasteurella multocidamultocida

5. FocusFocus
 Pasteurella multocida
 Carriage by clinically normal calves
 What happens during infection
 The potential role of biofilms
 Antibiotic effectiveness

6. Pasteurella multocida backgroundPasteurella multocida background
 A re-emerging bovine pathogen
 % disease in UK attributed to P. multocida risen
from 35% to 50% over last 14 years
 Present in a proportion of calves that appear
healthy
 No test to tell difference between dangerous and
less harmful forms of the bacterium
 No European vaccine, those in USA of variable
efficacy and need new and effective control
measures

7. Ablaze and obvious dangerAblaze and obvious danger

8. AftermathAftermath

9. Apparent calm, unseen dangerApparent calm, unseen danger

10. A closer lookA closer look

11. Aftermath, 1 day post-challengeAftermath, 1 day post-challenge

12. Aftermath, 10 day post-challengeAftermath, 10 day post-challenge

13. AdhesionsAdhesions

14. Lung damage,
fibrin deposits
Lung damage,
fibrin deposits

15. Lung abscessesLung abscesses

16. Clinical signsClinical signs
 Dullness
 Pyrexia
 Laboured breathing
 Nasal discharge
 Anorexia
Part of an annual cost of ~ £60M in UK

17. Incidence of bovine respiratory disease in
UK due to P. multocida or M. haemolytica
Incidence of bovine respiratory disease in
UK due to P. multocida or M. haemolytica
Pneumonic outbreaks in calves, UK
0
50
100
150
200
250
300
1992
1994
1996
1998
2000
2002
2004
Year
Numberofcases
P. multocida
M. haemolytica

18. Prevalence of P. multocida carriage?Prevalence of P. multocida carriage?

19. Scottish farm surveyScottish farm survey
 Random sample of beef and dairy calvesRandom sample of beef and dairy calves
 Throughout ScotlandThroughout Scotland
 FebFeb –– Jun 2008Jun 2008

20. Prevalence of P. multocida in
“healthy” Scottish calves
Prevalence of P. multocida in
“healthy” Scottish calves
Farm type Infected/total Infected/total
(n=68) farms % calves %
BEEF 12/33 36 28 / 321 9
DAIRY 20/35 57 77 / 295 26
TOTALS 32/68 47 105 / 616 17

21. Some unanswered questions relating
to bovine respiratory disease
Some unanswered questions relating
to bovine respiratory disease
1.1. CanCan commensalcommensal carriage lead to clinicalcarriage lead to clinical
disease?disease?
2.2. What are the dynamics of spread ofWhat are the dynamics of spread of
infection to nainfection to naïïve animals?ve animals?
3.3. CanCan commensalcommensal carriage be controlled?carriage be controlled?
4.4. CarriageCarriage –– where and in what form?where and in what form?

22. Trial in model and target animal
systems
Trial in model and target animal
systems
• Test virulence of different bacterial
isolates
• Test safety and efficacy of new vaccines
and drugs

23. Forty newborn (≤ 4d old) dairy bull calves from 18 farms
All given colostrum and were clinically healthy
Quarantined and nasal swabs cultured on selective SBA
plates for P. multocida
Allocated to 2 groups of 20 according to P. multocida
carriage, age and origin
Drank metered milk replacer and weighed weekly
Nine carrier animals at the start of the experiment
Forty newborn (≤ 4d old) dairy bull calves from 18 farms
All given colostrum and were clinically healthy
Quarantined and nasal swabs cultured on selective SBA
plates for P. multocida
Allocated to 2 groups of 20 according to P. multocida
carriage, age and origin
Drank metered milk replacer and weighed weekly
Nine carrier animals at the start of the experiment
Calves and management

24. Result?Result?

25. Infection in bought-in calvesInfection in bought-in calves
Infection in bought-in calves
0
5
10
15
20
25
30
35
0 3 6 9 13 16 20
Days housed
No.affectedcalves
Number infected Pma
Number infected Mhm
Number killed Pma
Number killed Mhm

26. Why does this happen?Why does this happen?

27. Predisposing factorsPredisposing factors
 Viral infectionViral infection
 MycoplasmaMycoplasma infectioninfection
 Management stressManagement stress
 TransportTransport

28. Viral resultsViral results
18 PM samples +18 PM samples +veve for RSVfor RSV
1 PM sample +1 PM sample +veve for PI3for PI3

29. Effect on lung defencesEffect on lung defences
Cells within the lung that normally fight
infection and keep the lung sterile
 Begin to lose their anti-bacterial
effectiveness
 Recruited cells contribute to the damage
caused

30. Reduced antibacterial activity of
blood and lung neutrophils
Reduced antibacterial activity of
blood and lung neutrophils

31. Bacterial killing by lung immune cellsBacterial killing by lung immune cells

32. Defence cell recruitment, death
and engulfment by other lung cells
Defence cell recruitment, death
and engulfment by other lung cells

33. PathologyPathology
 Causes acute bronchopneumoniaCauses acute bronchopneumonia
 Small abscesses developSmall abscesses develop
 Lung tissue dies and becomesLung tissue dies and becomes
consolidatedconsolidated
Progress of disease and decrease inProgress of disease and decrease in
lung function is rapid and severelung function is rapid and severe

34. PathologyPathology
Lung pathology appeared worse in manyLung pathology appeared worse in many
cases than that observed aftercases than that observed after
experimental challenge withexperimental challenge with P.P.
multocidamultocida alone. Diseasealone. Disease multifactorialmultifactorial
–– monitoring formonitoring for P.P. multocidamultocida,,
MannheimiaMannheimia haemolyticahaemolytica,, mycoplasmamycoplasma
and virusesand viruses

35. Overall summaryOverall summary
4 calves developed systemic4 calves developed systemic colibacillosiscolibacillosis, killed, killed
6.5d6.5d
29 calves developed respiratory disease, killed at:29 calves developed respiratory disease, killed at:
Days 9Days 9--11d (mean=10.5d)11d (mean=10.5d) –– 10 calves10 calves
Days 12Days 12--14d (mean=13d)14d (mean=13d) –– 10 calves10 calves
Days 15Days 15--17d (mean=16d)17d (mean=16d) –– 9 calves9 calves
7 calves remained healthy and were killed on7 calves remained healthy and were killed on
schedule, average age 21.5dschedule, average age 21.5d

36. Overall bacteriological resultsOverall bacteriological results
 10 calves tested positive only for10 calves tested positive only for P.P. multocidamultocida,,
on at least one occasionon at least one occasion
 19 calves tested19 calves tested postivepostive for bothfor both P.P. multocidamultocida
andand M.M. haemolyticahaemolytica,, on at least one occasionon at least one occasion
 4 calves tested4 calves tested postivepostive only foronly for M.M.
haemolyticahaemolytica,, on at least one occasionon at least one occasion
 7 calves tested positive for neither7 calves tested positive for neither P.P.
multocidamultocida nornor M.M. haemolyticahaemolytica

37. How does P. multocida spread?How does P. multocida spread?

38. Transmission between calvesTransmission between calves

39. Bacterial accumulation
in the nasal cavities
Bacterial accumulation
in the nasal cavities

40. RAPD analysis of P. multocida isolatesRAPD analysis of P. multocida isolates
 Nasal swabs plated on selective SBA.Nasal swabs plated on selective SBA.
 10 colonies per isolated picked and grown on10 colonies per isolated picked and grown on
SBA.SBA.
 DNA extracted usingDNA extracted using DNeasyDNeasy ((QiagenQiagen).).
 RAPD performed using ReadyRAPD performed using Ready--toto--go RAPDgo RAPD
analysis beads (GE Healthcare) primer 2.analysis beads (GE Healthcare) primer 2.

41. Phylogenetic analysisPhylogenetic analysis
 RAPD run on 1.5% TAE gel, photographedRAPD run on 1.5% TAE gel, photographed
under UVunder UV transilluminationtransillumination..
 ProfilesProfiles analysedanalysed usingusing BionumericsBionumerics
Software.Software.
 PhylogeneticPhylogenetic relationships compared withrelationships compared with
time of positive swab and animal groupingstime of positive swab and animal groupings

42. Molecular characterisation using RAPDMolecular characterisation using RAPD
Farm A isolate Farm B, C, D isolate
L L
L= 100bp ladder

43. Farm A
Farm B, C, D
2 positive
animals
7 positive
animals
10% of infected
animals
90% of infected
animals
Spread of P. multocida
from initial carriers
Spread of P. multocida
from initial carriers

44. PreventionPrevention
ManagementManagement
–– HousingHousing
–– TransportTransport
VaccinesVaccines
AntibioticsAntibiotics

45. TreatmentTreatment
ColostrumColostrum
AntibioticAntibiotic
Prevention better than curePrevention better than cure

46. Adequate and early intake of colostrumAdequate and early intake of colostrum

47. Prophylaxis and treatment
using antimicrobials
Prophylaxis and treatment
using antimicrobials

48. Relevant antibiotic classesRelevant antibiotic classes
• Penicillins (ampicillin)
• Macrolides (tilmicosin)
• Tetracyclines (oxytetracycline)
• Aminoglycosides (streptomycin)
• Cephalosporins (ceftiofur)
• Sulphonamides (trimethoprim)
• Quinolones (danofloxacin)

49. Antibiotic effectivenessAntibiotic effectiveness
• Planktonic and sessile forms of P.
multocida susceptible to most antibiotics
• Concentration antibiotic required to kill
sessile bacteria may be > 1000x that
required to kill planktonic bacteria
• Persistence due to poor accessibility?

50. Does antibiotic clear nasal carriage?Does antibiotic clear nasal carriage?
Elimination of biofilms by antibiotics in vivo
has not been evaluated
Our experience at Moredun shows
• P. multocida susceptible to antibiotic in the
laboratory
• Difficult to get rid of from the calf’s nose

51. Does antibiotic clear nasal carriage?Does antibiotic clear nasal carriage?
Nasal swabs from 2 calves continued positiveNasal swabs from 2 calves continued positive
forfor P.P. multocidamultocida
Three others became positive 3, 6 and 7d laterThree others became positive 3, 6 and 7d later
Three calves remained negative 6Three calves remained negative 6 –– 7d after7d after
treatmenttreatment
Two treated calves killed for welfare reasonsTwo treated calves killed for welfare reasons

52. Reason?Reason?
Our experience at Moredun shows
 Pasteurella multocida susceptible to
antibiotic in the laboratory
 Difficult to get rid of from the calf’s nose
 Suggests biofilm formation protects
against drugs

53. Carriage where and in what form?Carriage where and in what form?
• Upper respiratory tract
• Planktonic? (free living)
• Sessile? (biofilm)
a) P. multocida can exist as a biofilm structure
b) Biofilm-associated genes are present in
bovine isolates of P. multocida
c) Poor response to systemic antibiotic
suggests biofilm protection

54. What are Biofilms?What are Biofilms?
• A community of microorganisms with
altered rates of gene transcription and
growth
• Encapsulated in a self-synthesised
matrix of extracellular polysaccharide
(EPS) and attached irreversibly to a
(biological) surface

55. • Antibiotics work best against rapidly-
dividing bacterial cells but biofilms are
slow-growing
• The extraordinary resistance to
antimicrobials may be due to ‘persister cells’
that neither grow nor die
in the presence of bactericidal agents
• Persister cells protect against immune cells
or drugs and repopulate the biofilm after
treatment
Protective structure of biofilmsProtective structure of biofilms

56. Characteristics of biofilmsCharacteristics of biofilms
•• May form at early stage (within 1 week)May form at early stage (within 1 week)
of respiratory infectionof respiratory infection
•• Chronic, difficult to treatChronic, difficult to treat
•• Can form with multiple species ofCan form with multiple species of
bacteriabacteria –– for example,for example, P.P. multocidamultocida andand
HistophilusHistophilus somnisomni
•• Bacteria can exchange geneticBacteria can exchange genetic
information ininformation in biofilmbiofilm structuresstructures

57. Biofilm formation by P. multocida
(methyl violet stain of EPS)
Biofilm formation by P. multocida
(methyl violet stain of EPS)

58. Congo red agar assayCongo red agar assay

59. Biofilm break-up?Biofilm break-up?
• Basic response by host to bacterial
infection is to limit iron availability
• Decrease in transcription of adhesin
genes
• Dispersal of biofilm
• Bacteria migrate to lungs
• Cause influx of neutrophils

60. Effect of iron availability and
simulated stress on bacterial
growth and biofilms
Effect of iron availability and
simulated stress on bacterial
growth and biofilms
 Basic host response to bacterialBasic host response to bacterial
infection is to limit iron availabilityinfection is to limit iron availability
 Affects bacterial growthAffects bacterial growth
 BiofilmsBiofilms may also be affected bymay also be affected by
stress and iron restrictionstress and iron restriction

61. Effect of iron restriction and
noradrenalin
or transferrin on P. multocida growth
Effect of iron restriction and
noradrenalin
or transferrin on P. multocida growth
0.07
0.09
0.11
0.13
0.15
0.17
0.19
Time
Absorbance(600nm)
Iron depleted +
Noradrenalin
Iron depleted +
holoTf
Iron depleted +
holoTf +
Noradrenalin
Iron replete

62. Stress and biofilmsStress and biofilms
Noradrenalin causes an increase in iron
regulated genes and other virulence factors
Noradrenalin in the presence of transferrin
increases biofilm formation
Noradrenalin-stimulated growth of biofilms
may lead to dissemination of infection

63. Commensal carriage, a time bomb?Commensal carriage, a time bomb?

64. Current targetsCurrent targets
 Develop new vaccines andDevelop new vaccines and diagnostic
tests against dangerous forms of P.P.
multocidamultocida
 Investigate formation of biofilms during
carriage in upper respiratory tract and
devise methods to eradicate

65. Current situationCurrent situation
 Established prevalence ofEstablished prevalence of P.P. multocidamultocida onon
Scottish dairy and beef farmsScottish dairy and beef farms
 Produced molecular fingerprints to helpProduced molecular fingerprints to help
identify dangerousidentify dangerous P.P. multocidamultocida isolatesisolates
 Identified new vaccine candidatesIdentified new vaccine candidates
 Commercial links to test potential controlCommercial links to test potential control
strategies forstrategies for P.P. multocidamultocida
 International collaborations to assessInternational collaborations to assess
geographical differences in diseasegeographical differences in disease

66. Others involvedOthers involved
 Colin Bayne (Molecular biology & proteomics)
 Bioservices (Care and maintenance of animals)
 Moredun Scientific (commercial arm of Moredun)
(Clinicals)
 Emily Hotchkiss (Calf survey)
 David Kennedy (veterinary advice)
 David McBean (Colostrum analysis)
 Kim Willoughby (Viral analyses)
 Mark Dagleish, Jeanie Finlayson (Pathology)
 Biomathematics & Statistics Scotland (Statistical
advice)
 Scottish Government (Funding)