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 Hodgson Dr. J. Chris Hodgson Leader Leader Pasteurella multocidaPasteurella multocida research group research group

Moredun Research Institute Moredun Research Institute Scotland UKScotland UK

Merial forum on BRD29th Sept to 1st Oct 2010 Lisbon, Portugal

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

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

Pneumonic pasteurellosis in young calves

Pneumonic pasteurellosis in young calves

Causes:Causes:

MannheimiaMannheimia ((PasteurellaPasteurella) ) haemolyticahaemolytica

PasteurellaPasteurella multocidamultocida

FocusFocus

Pasteurella multocida

Carriage by clinically normal calves

What happens during infection

The potential role of biofilms

Antibiotic effectiveness

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

Ablaze and obvious dangerAblaze and obvious danger

AftermathAftermath

Apparent calm, unseen dangerApparent calm, unseen danger

A closer lookA closer look

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

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

AdhesionsAdhesions

Lung damage, fibrin depositsLung damage, fibrin deposits

Lung abscessesLung abscesses

Clinical signsClinical signs

Dullness Pyrexia Laboured breathing Nasal discharge Anorexia

Part of an annual cost of ~ £60M in UK

Incidence of bovine respiratory disease in UK due to P. multocida or M. haemolyticaIncidence of bovine respiratory disease in UK due to P. multocida or M. haemolytica

Pneumonic outbreaks in calves, UK

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P. multocidaM. haemolytica

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

Scottish farm surveyScottish farm survey

Random sample of beef and dairy calvesRandom sample of beef and dairy calves Throughout ScotlandThroughout Scotland Feb Feb –– Jun 2008Jun 2008

Prevalence of P. multocida in “healthy” Scottish calvesPrevalence of P. multocida in “healthy” Scottish calves

Farm type Infected/total Infected/total(n=68) farms % calves %

BEEF 12/33 36 28 / 321 9DAIRY 20/35 57 77 / 295 26

TOTALS 32/68 47 105 / 616 17

Some unanswered questions relating to bovine respiratory disease

Some unanswered questions relating to bovine respiratory disease

1.1. Can Can commensalcommensal carriage lead to clinical carriage lead to clinical disease?disease?

2.2. What are the dynamics of spread of What are the dynamics of spread of infection to nainfection to naïïve animals? ve animals?

3.3. Can Can commensalcommensal carriage be controlled?carriage be controlled?4.4. Carriage Carriage –– where and in what form?where and in what form?

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

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. multocidacarriage, 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. multocidacarriage, age and origin

Drank metered milk replacer and weighed weekly

Nine carrier animals at the start of the experiment

Calves and management

Result?Result?

Infection in bought-in calvesInfection in bought-in calves

Infection in bought-in calves

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Number infected PmaNumber infected MhmNumber killed PmaNumber killed Mhm

Why does this happen?Why does this happen?

Predisposing factorsPredisposing factors

Viral infectionViral infection MycoplasmaMycoplasma infectioninfection Management stressManagement stress TransportTransport

Viral resultsViral results

18 PM samples +18 PM samples +veve for RSVfor RSV

1 PM sample +1 PM sample +veve for PI3for PI3

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

Reduced antibacterial activity of blood and lung neutrophils

Reduced antibacterial activity of blood and lung neutrophils

Bacterial killing by lung immune cellsBacterial killing by lung immune cells

Defence cell recruitment, death and engulfment by other lung cellsDefence cell recruitment, death and engulfment by other lung cells

PathologyPathology

Causes acute bronchopneumoniaCauses acute bronchopneumonia Small abscesses developSmall abscesses develop Lung tissue dies and becomes Lung tissue dies and becomes

consolidatedconsolidated

Progress of disease and decrease in Progress of disease and decrease in lung function is rapid and severelung function is rapid and severe

PathologyPathology

Lung pathology appeared worse in many Lung pathology appeared worse in many cases than that observed after cases than that observed after experimental challenge with experimental challenge with P. P. multocidamultocida alone. Disease alone. Disease multifactorialmultifactorial–– monitoring for monitoring for P. P. multocidamultocida, , MannheimiaMannheimia haemolyticahaemolytica, , mycoplasmamycoplasmaand virusesand viruses

Overall summaryOverall summary4 calves developed systemic 4 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 calvesDays 12Days 12--14d (mean=13d) 14d (mean=13d) –– 10 calves10 calvesDays 15Days 15--17d (mean=16d) 17d (mean=16d) –– 9 calves9 calves

7 calves remained healthy and were killed on 7 calves remained healthy and were killed on schedule, average age 21.5dschedule, average age 21.5d

Overall bacteriological resultsOverall bacteriological results

10 calves tested positive only for 10 calves tested positive only for P. P. multocidamultocida,,on at least one occasionon at least one occasion

19 calves tested 19 calves tested postivepostive for both for both P. P. multocidamultocidaand and M. M. haemolyticahaemolytica, , on at least one occasionon at least one occasion

4 calves tested 4 calves tested postivepostive only for only for M. M. haemolyticahaemolytica, , on at least one occasionon at least one occasion

7 calves tested positive for neither 7 calves tested positive for neither P. P. multocidamultocida nor nor M. M. haemolyticahaemolytica

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

Transmission between calvesTransmission between calves

Bacterial accumulation in the nasal cavities

Bacterial accumulation in the nasal cavities

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 on 10 colonies per isolated picked and grown on SBA.SBA.

DNA extracted using DNA extracted using DNeasyDNeasy ((QiagenQiagen).).

RAPD performed using ReadyRAPD performed using Ready--toto--go RAPD go RAPD analysis beads (GE Healthcare) primer 2.analysis beads (GE Healthcare) primer 2.

Phylogenetic analysisPhylogenetic analysis

RAPD run on 1.5% TAE gel, photographed RAPD run on 1.5% TAE gel, photographed under UV under UV transilluminationtransillumination..

Profiles Profiles analysedanalysed using using BionumericsBionumericsSoftware.Software.

PhylogeneticPhylogenetic relationships compared with relationships compared with time of positive swab and animal groupingstime of positive swab and animal groupings

Molecular characterisation using RAPDMolecular characterisation using RAPD

Farm A isolate Farm B, C, D isolate

L L

L= 100bp ladder

Farm A

Farm B, C, D

2 positiveanimals

7 positiveanimals

10% of infected animals

90% of infected animals

Spread of P. multocidafrom initial carriers

Spread of P. multocidafrom initial carriers

PreventionPrevention

ManagementManagement–– HousingHousing–– TransportTransport

VaccinesVaccines

AntibioticsAntibiotics

TreatmentTreatment

ColostrumColostrum

AntibioticAntibiotic

Prevention better than curePrevention better than cure

Adequate and early intake of colostrumAdequate and early intake of colostrum

Prophylaxis and treatment using antimicrobials

Prophylaxis and treatment using antimicrobials

Relevant antibiotic classesRelevant antibiotic classes

• Penicillins (ampicillin)• Macrolides (tilmicosin)• Tetracyclines (oxytetracycline)• Aminoglycosides (streptomycin)• Cephalosporins (ceftiofur)• Sulphonamides (trimethoprim)• Quinolones (danofloxacin)

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?

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

Does antibiotic clear nasal carriage?Does antibiotic clear nasal carriage?

Nasal swabs from 2 calves continued positive Nasal swabs from 2 calves continued positive for for P. P. multocidamultocida

Three others became positive 3, 6 and 7d laterThree others became positive 3, 6 and 7d laterThree calves remained negative 6 Three calves remained negative 6 –– 7d after 7d after

treatmenttreatmentTwo treated calves killed for welfare reasonsTwo treated calves killed for welfare reasons

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

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 structureb) Biofilm-associated genes are present in

bovine isolates of P. multocidac) Poor response to systemic antibiotic

suggests biofilm protection

What are Biofilms?What are Biofilms?

• A community of microorganisms with altered rates of gene transcription and growth

• Encapsulated in a self-synthesisedmatrix of extracellular polysaccharide (EPS) and attached irreversibly to a (biological) surface

• 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

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 of Can form with multiple species of

bacteria bacteria –– for example, for example, P. P. multocidamultocida and and HistophilusHistophilus somnisomni

•• Bacteria can exchange genetic Bacteria can exchange genetic information in information in biofilmbiofilm structuresstructures

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

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

Congo red agar assayCongo red agar assay

Biofilm break-up?Biofilm break-up?

• Basic response by host to bacterial infection is to limit iron availability

• Decrease in transcription of adhesingenes

• Dispersal of biofilm• Bacteria migrate to lungs• Cause influx of neutrophils

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 bacterial Basic 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 by may also be affected by stress and iron restrictionstress and iron restriction

Effect of iron restriction and noradrenalin

or transferrin on P. multocida growth

Effect of iron restriction and noradrenalin

or transferrin on P. multocida growth

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Iron depleted +holoTf

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Stress and biofilmsStress and biofilmsNoradrenalin causes an increase in iron

regulated genes and other virulence factors

Noradrenalin in the presence of transferrinincreases biofilm formation

Noradrenalin-stimulated growth of biofilmsmay lead to dissemination of infection

Commensal carriage, a time bomb?Commensal carriage, a time bomb?

Current targetsCurrent targets

Develop new vaccines and Develop 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

Current situationCurrent situation Established prevalence of Established prevalence of P. P. multocidamultocida on on

Scottish dairy and beef farmsScottish dairy and beef farms Produced molecular fingerprints to help Produced molecular fingerprints to help

identify dangerous identify dangerous P. P. multocidamultocida isolatesisolates Identified new vaccine candidatesIdentified new vaccine candidates Commercial links to test potential control Commercial links to test potential control

strategies for strategies for P. P. multocidamultocida International collaborations to assess International collaborations to assess

geographical differences in disease geographical differences in disease

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)