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AUSTRALIAN POULTRY CRC

FINAL REPORT

Program: Application and Commercialisation; Sub-Program 2B; Strategy 2.5.4

Project No: 03-12

FINAL REPORT

PROJECT LEADER:

Dr Peter Scott

July 2010

PROJECT NO: 03-12

Towards rapid Registration of a live Pasteurella multocida vaccine

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© 2010 Australian Poultry CRC Pty Ltd. All rights reserved.

Towards rapid Registration of a live Pasteurella multocida vaccine

Publication No:

Project No. 03-12

The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable industries. The information should not be relied upon for the purpose of a particular matter. Specialist and/or appropriate legal advice should be obtained before any action or decision is taken on the basis of any material in this document. The Australian Poultry CRC, the authors or contributors do not assume liability of any kind whatsoever resulting from any person's use or reliance upon the content of this document.

This publication is copyright. However, Australian Poultry CRC encourages wide dissemination of its research, providing the Centre is clearly acknowledged. For any other enquiries concerning reproduction, contact the Communications Officer on phone 02 6773 3767.

Researcher Contact Details

Dr. Peter C. Scott Senior Research Fellow Veterinary School of Microbiology University of Melbourne 250 Princes Highway Werribee VIC 3030 In submitting this report, the researcher has agreed to the Australian Poultry CRC publishing this material in its edited form.

Australian Poultry CRC Contact Details

PO Box U242

University of New England

ARMIDALE NSW 2351

Phone: 02 6773 3767

Fax: 0267733050 Email: info@poultrycrc.com.au Website: http://www.poultrycrc.com.au

Published in: July 2010

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Executive Summary

Pasteurella multocida (PM) is the causative agent of fowl cholera (FC), a disease of chickens as well as

other avian species including turkeys and ducks. The disease results in high mortalities and severe production

losses. Current vaccines against FC are based on inactivated vaccine preparations which offer limited

(homologous) protection against P. multocida. Bioproperties Pty Ltd has developed a novel live attenuated

vaccine (Vaxsafe PM) against PM. The majority of the animal studies were conducted at the University of

Melbourne (APCAH) facility in collaboration with Dr. Peter Scott. The attenuation of Vaxsafe PM is based

on an aroA gene deletion which renders the virulent parent strain (serotype 1) auxotrophic. Hence, the

vaccine strain becomes dependent on aromatic amino acids and that are not freely available in vivo. The

deletion therefore renders the vaccine strain non-pathogenic.

This report outlines the series of registration based studies necessary to demonstrate both efficacy and safety

of the vaccine candidate. The studies are guided by both US and EU guidelines appropriate to the registration

of a PM vaccine.

The main difficulty in these sets of studies was in the establishment of a reproducible challenge model. An

intratracheal administration of a heterologous strain of PM was used in the majority of the efficacy studies.

This challenge model was important as it allowed determination of cross protection of the vaccine strain

(serotype 1) with a heterologous challenge (serotype 4). However, variability was observed in the %

mortality rates from some of the studies which required them to be repeated. Further to the heterologous

challenge, a homologous challenge model based on intramuscular injection was also established. This model

has been used in the US and appeared to be more consistent. It was therefore used to experimentally

determine duration of immunity of the vaccine strain.

During the course of these studies which took place over several years, improvements were also being made

to the manufacturing process. As such, a number of bridging studies were also performed to establish

efficacy of the vaccine produced from the improved process. This is important to determine that fermentation

or bioprocessing conditions did not adversely impact the final vaccine product.

In vivo studies using Vaxsafe PM have demonstrated that this vaccine is both safe and effective in the face of

a strong heterologous challenge with a virulent strain (serotype 4). Vaxsafe PM is an injectable vaccine for

the protection of long lived chickens (breeders and layers). Its use in turkeys and ducks has also been

explored in this study.

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Table of Contents

Executive Summary ......................................................................................................................................... 3

Table of Contents ............................................................................................................................................. 4

Introduction ...................................................................................................................................................... 8

Objectives ...................................................................................................................................................... 10

Part A: Chickens ...........................................................................................................................................10 Part B: Turkeys .............................................................................................................................................10 Part C: Ducks ................................................................................................................................................10

Regulatory Guidelines ................................................................................................................................... 11

Methodology .................................................................................................................................................. 11

Nutrient Broth plus Yeast Extract (NBYE) ..................................................................................................11 Nutrient Agar plus Yeast Extract (NAYE) ...................................................................................................11 Total viable counts (TVC) ............................................................................................................................11 Heterologous Challenge preparation (PM206) .............................................................................................11 Homologous Challenge preparation (X73) ...................................................................................................12 Chickens: ......................................................................................................................................................12 Turkeys: ........................................................................................................................................................13 Ducks: ...........................................................................................................................................................13 Intratracheal administration (PM206) ..........................................................................................................13 Intramuscular administration (X73) .............................................................................................................13 Determination of Protective Index (PI): .......................................................................................................13 Dissemination study (PMP R0715) – Microbial sampling of various organs ..............................................14 Processing of swab samples .........................................................................................................................14 Processing of organ/tissue samples: .............................................................................................................14 Reversion to virulence trial (PMP R0807) sample collection and processing .............................................14 PCR analysis .................................................................................................................................................15 Optimisation of vaccine manufacture ...........................................................................................................15 Summary of trial work undertaken ...............................................................................................................15

PART A: Vaxsafe® PM for use in chickens................................................................................................... 16

(i) Efficacy studies ......................................................................................................................................... 16

PMP R0501: Development of a laboratory challenge model to evaluate the efficacy of Vaxsafe® PM

Vaccine (living) ............................................................................................................................................ 16

PMP R0502: A Dose Response Study to Determine the Minimum Protective Dose of Vaxsafe® PM

Vaccine (living) ............................................................................................................................................ 17

PMP R0601: A study to determine the onset of protective immunity after vaccination with

Vaxsafe® PM Vaccine (living) ..................................................................................................................... 18

PMP R0603: Efficacy of Vaxsafe® PM in commercial chickens .................................................................18

PMP R0605: Vaxsafe® PM Vaccine (living) – Duration of immunity in chickens...................................... 19

PMP R0801: A study to determine the clinical effects of a Pasteurella multocida (strain PM206)

challenge when administered to chickens at various doses .......................................................................... 21

PMP R0802: A study to determine the efficacy of three batches of Vaxsafe® PM in chickens ................... 21

PMP R0809: Development of a master seed of Pasteurella multocida challenge strain X-73 ..................... 22

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PMP R0803a: A study to determine the duration of immunity following vaccination of chickens with

Vaxsafe® PM ................................................................................................................................................ 23

PMP R0803b: Efficacy of Vaxsafe® PM – equivalence of two formulations .............................................. 23

PMP R0803c: Determination of the compatibility of Vaxsafe® PM with concurrently administered

vaccines in chickens ..................................................................................................................................... 24

(ii) Safety Studies ........................................................................................................................................... 25

PMP R0502: A Dose-Response Study to Determine the Minimum Protective Dose of Vaxsafe® PM

Vaccine (living) ............................................................................................................................................ 25

PMP R0503: A study to determine the safety of Vaxsafe® PM Vaccine (living) in chickens ..................... 25

PMP R0601: A study to determine the onset of protective immunity after vaccination with

Vaxsafe® PM Vaccine (living) ..................................................................................................................... 28

PMP R0602: Safety of Vaxsafe® PM Vaccine – a dose response study to evaluate endotoxic effects

following administration to 6-week-old chickens ........................................................................................ 28

PMP R0604: A study to determine the spread and dissemination of the Vaxsafe® PM Vaccine (living)

strain of Pasteurella multocida in chickens ................................................................................................. 29

PMP R0607: Evaluation of the genetic stability of Pasteurella multocida strain PMP-1, an aroA

deletion mutant derived from P. multocida X-73 ......................................................................................... 29

PMP R0611: Measurement of endotoxin content relative to viable cell count of five batches of

Vaxsafe® PM Vaccine (living) ..................................................................................................................... 29

PMP R0715: A study to determine the safety and dissemination of Vaxsafe® PM in chickens .................. 30

PMP R0803c: Determination of the compatibility of Vaxsafe® PM with concurrently administered

vaccines in chickens ..................................................................................................................................... 32

PMP R0807: A study to assess the reversion to virulence of Vaxsafe® PM after 5 passages in SPF

chickens ........................................................................................................................................................ 33

PART B: Vaxsafe® PM for use in Turkeys .................................................................................................. 35

(i) Efficacy studies ......................................................................................................................................... 35

PMP R0804: A dose response study using Pasteurella multocida (X-73) challenge strain in turkeys ......... 35

PMP R0808: A dose confirmation study to determine efficacy of Vaxsafe® PM Vaccine (living) in

commercial turkeys vaccinated with the chicken end-of-shelf-life-titre (EOSLT) dose .............................. 35

PMP R0902: Efficacy of Vaxsafe® PM Vaccine (living) in turkeys, using alternative routes of

administration ............................................................................................................................................... 36

(ii) Safety studies ........................................................................................................................................... 37

PMP R0806: A study to determine the safety of Vaxsafe® PM Vaccine (living) in turkeys........................ 37

PMP R0901: A study to determine the safety of Vaxsafe® PM Vaccine (living) in turkeys........................ 38

PART C: Vaxsafe® PM for use in Ducks ...................................................................................................... 40

(i) Efficacy ..................................................................................................................................................... 40

PMP R0903: A dose response study using Pasteurella multocida (X-73) challenge strain in ducks .......... 40

(ii) Safety ....................................................................................................................................................... 41

PMP R0904: A study to determine the safety of Vaxsafe® PM Vaccine (living) in ducks .......................... 41

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PMP R1001: A study to determine the safety of a 10X overdose of Vaxsafe® PM Vaccine (living) in

ducks ............................................................................................................................................................. 42

OVERALL SUMMARY ............................................................................................................................... 42

Plain English Compendium Summary .........................................................................................................44

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TABLES

Table 3: PMP R0501 ........................................................................................................................................16

Table 4: PMP R0502 ........................................................................................................................................17

Table 5: PMP R0601 ........................................................................................................................................18

Table 6: PMP R0603 ........................................................................................................................................19

Table 7: PMP R0605 ........................................................................................................................................20

Table 8: PMP R0801 ........................................................................................................................................21

Table 9: PMP R0802 ........................................................................................................................................22

Table 10: PMP R0803b ....................................................................................................................................23

Table 11: Treatment groups for the safety study PMP R0503 .........................................................................25

Table 12: Trial PMP R0715 .............................................................................................................................30

Table 13. Total Viable Counts (TVC) at each passage level. ...........................................................................34

Table 14: PMP R0808 ......................................................................................................................................36

Table 15: PMP R0902 ......................................................................................................................................37

Table 17: PMP R0806 ......................................................................................................................................38

Table 18: PMP R0901 (Original study design) ................................................................................................39

Table 19: PMP R0901 (Modified study design) ...............................................................................................39

Table 16: PMP R0903 ......................................................................................................................................40

Table 21: PMP R0904 ......................................................................................................................................41

FIGURES

Figure 1: Shikimate pathway outlining the gene deleted in Vaxsafe®PM (aroA). ...........................................9

Figure 2: P. multocida induced mortality in chickens following IT administration of strain PM206. ..........17

Figure 3. Lesion at injection site at 2 weeks post vaccination. ......................................................................26

Figure 4. Remnants of a lesion in chicken pectoral muscle (site of injection) at 9 weeks post

vaccination at an overdose of Vaxsafe® PM. ..................................................................................27

Figure 5. Effect of single and 10X overdose on body weights in spf chickens at 6, 8 and 9 weeks post

vaccination. .....................................................................................................................................27

Figure 6. Dissemination of Vaxsafe® PM in the injection site and spleen of chickens at various times

post vaccination. .............................................................................................................................31

Figure 7. Dissemination of Vaxsafe® PM in lungs and liver of chickens at various times post

vaccination. .....................................................................................................................................31

Figure 8. Gel electrophoresis analysis of PCR products obtained from amplification of the (aroA

gene delete) region of Pasteurella multocida X-73 (wild type), the initial inoculum of

Vaxsafe® PM (P0), and products of P1 to P5 tissue homogenate material. ...................................35

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Introduction Avian pasteurellosis or fowl cholera is an endemic disease in commercial and wild birds.Fowl cholera is

caused by infection with Pasteurella multocida (PM). PM is a gram negative rod and transmission of this

organism is via inhalation or ingestion. In its acute form, birds may experience depression, ruffled feathers,

fever, anorexia, mucous discharge from the mouth, diarrhea and increased respiratory rate. These clinical

signs may arise only a few hours prior to death (Rhoades and Rimler, 1989). In its chronic form, exudative

conjunctivitis and pharyngitis may be seen, as well as localized infections such as swelling of the wattle,

sinuses, periorbital subcutaneous tissues, leg or wing joints, sternal bursea and foot pads (Rhoades and

Rimler, 1989).

Early classification of PM is based on the existence of a capsule. There are five capsular serogroups; A, B,

D, E and F. An analysis of the LPS antigen has identified 16 unique serotypes. However, there are still many

others that are classified as non-typeable.

FC may be controlled by chemical intervention. However, there is a strong movement away from this type of

program for control of FC disease. There are presently a number of killed vaccines [e.g. MBL®FC3 and

MBL®FC4 Gold (L.A.H.I), Multimume

® K and K5 (Biomune), Pabac

® and Poulvac

® Pabac

® IV (Fort

Dodge) as well as a number of live FC vaccines registered (e.g. Avichol®, M-Ninevax, M-Ninevax

®-C,

PM-Onevax and PM-Onevax®-C (Schering-Plough), Choleramune

® CU, Choleramune

® M, Multimune

®

CU and Multimune® M (Biomune)]. The killed (formalin inactivated whole bacterial cell preparations) can

be effective so long as an autogenous strain is prepared for each outbreak. The existence of strain variation

diminishes the efficacy of killed vaccines as a universal approach to control. There have been a number of

live vaccines used. The earliest one was the Clemson University (CU) strain. This strain was found to be

insufficiently attenuated and had the potential to cause disease following vaccination (Hopkins and Olson,

1977). M-9, PM#1 and PM#3 strains are less virulent than the CU strain. The CU and the M-9 strains have

been used in many of the registered vaccines outlined above, however, they still cause mortality and can

cause outbreaks through reversion to virulence (Hofacre et al, 1989b; Snipes et al, 1990).

The development pathway for Vaxsafe® PM now dates back to over a decade (Scott et al., 1999). The parent

strain of Vaxsafe® PM is Pasteurella multocida strain X-73 which is a highly virulent strain that had been

isolated from a peracute case of fowl cholera in the USA. A deletion of 302 bp in size was created within the

aroA gene of X-73 to create the PMP-1 strain, i.e. Vaxsafe® PM. This work was conducted in the laboratory

of Professor Ben Adler (Monash University, Australia). The deletion within the aroA gene renders the

organism dependent upon aromatic amino acids and vitamins. These substances are not freely available in

the avian host. As the mutant strain is incapable of sequestering these essential components from the chicken

tissue, it becomes unable to replicate and will be eliminated in vivo by macrophages. However, it is assumed

that the mutant strain will undergo a round of two of replication utilizing intracellular nutrients absorbed

during culturing in growth media enhanced with these essential components. Figure 1 below outlines the

shikimate biochemical pathway for the metabolism of the essential aromatic compounds and their reliance of

the gene product of aroA (3–enolpyruvylshikimate-5-phospate synthetase). Elimination of the aroA gene

product will prevent the synthesis of the aromatic amino acids, phenylalanine, tyrosine and tryptophan as

well as the aromatic vitamins.

The work described in the report will cover the registration based studies that will show Vaxsafe® PM to be a

safe and efficacious live vaccine for chickens. Work conducted on the use of Vaxsafe® PM in turkeys and

ducks will also be described.

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Figure 1: Shikimate pathway outlining the gene deleted in Vaxsafe®PM (aroA).

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Objectives The overall objective of this program is:

To develop and register a live attenuated vaccine for Fowl Cholera that is safe and provides effective cross-

protection between serotypes. This objective extends to chickens, turkeys and ducks.

Part A: Chickens

The objectives that must be met in order to achieve the overall objective for chickens are:

1. Development of a suitable heterologous challenge model for Pasteurella multocida

2. Demonstrate safety of the vaccine candidate by following the APVMA and EU guidelines

3. Demonstrate efficacy of the vaccine candidate by following the APVMA and EU guidelines

Part B: Turkeys

The objectives that must be met in order to achieve the overall objective for turkeys are:

1. Development of a suitable homologous challenge model for Pasteurella multocida

2. Demonstrate safety of the vaccine candidate by following the guidelines for minor species

3. Demonstrate efficacy of the vaccine candidate by following the guidelines for minor species

Part C: Ducks

The objectives that must be met in order to achieve the overall objective for ducks are:

1. Development of a suitable homologous challenge model for Pasteurella multocida

2. Demonstrate safety of the vaccine candidate by following the guidelines for minor species

3. Demonstrate efficacy of the vaccine candidate by following the guidelines for minor species

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Regulatory Guidelines The series of registration based studies described in this report were conducted under OECD

a and VICH

b

guidelines: GLPc (safety studies) and GCP

d (efficacy studies)

All studies were planned in accordance with:

APVMA guidelines (Australian Pesticides & Veterinary Medicines Authority) – MORAG Guideline 47

European Union pharmacopeia – Appendix XV K (Vet), monographs 04/2005/1945, 01/2008:1945.

USDA Monograph (9CFR 113.70) – live avian PM vaccines

Studies are aimed for both Australian and International registration.

All animal studies had received approval prior to their commencement by the University of Melbourne

animal ethics committee and were conducted at the APCAH (Werribee).

Specific pathogen free (SPF) chickens were used for all registration based studies (unless otherwise

specified).

Methodology

Nutrient Broth plus Yeast Extract (NBYE)

Twenty-five gram (25g) of Nutrient broth number 2 (Oxoid, CM67) and three (3) gram of Oxoid Yeast

Extract (Oxoid, L21) is added to 1000 mL of distilled H2O and mixed to dissolved. The broth is then

autoclaved at 121C for 15 mins.

Nutrient Agar plus Yeast Extract (NAYE)

Twenty-five gram (25g) of Nutrient broth number 2 (Oxoid, CM67), three (3) gram of Oxoid Yeast Extract

(Oxoid, L21) and 10 g of Bacteriological grade agar (Oxoid, L11) is added to 1000 mL of distilled H2O and

mixed to dissolved. The broth is then autoclaved at 121C for 15 mins. Cool to 50 – 55C. Working within a

laminar flow cabinet, pour ~20mL into sterile petri dishes and allow plates to set for 15 – 30 mins. Once agar

has set, stack plates on top of each other and place into zip lock plastic bags. Store at 4C.

Total viable counts (TVC)

TVC were conducted following 10-1

serial dilutions (100 μl culture into 900 μL diluent or broth) and plating

100 μL of the appropriate dilution levels onto horse blood agar (HBA) plates. The plates were incubated

overnight at 37C prior to being counted.

Heterologous Challenge preparation (PM206)

The heterologous challenge strain PM206 was prepared as follows: A master seed vial of PM206 was plated

out onto a NA+YE plate. Following 16 hours growth at 37C, the growth was washed off the surface of the

plate with NB+YE media and placed into

a OECD: Organisation for Economic Co-Operation and Development

b VICH: International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary

Medicinal Products (EU, Japan, USA) c GLP: Good Laboratory Practice

d GCP: Good Clinical Practice

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One day 1, a vial of PM206 working seed was thawed at room temperature. One loop-full (~10 µL) of

culture was streaked onto nutrient agar plus yeast extract plate using standard bacteriological procedure. The

plate was incubated at 37C for 16±1hr.

One day 2, two (2) mL of nutrient broth supplemented with yeast extract was poured onto the agar plate

containing the PM206 colonies. The colonies were resuspended in NB+YE using a disposable sterile plastic

spreader, lifting the colonies gently. A 10-2

dilution was prepared by adding 1 mL of cell suspension into 99

mL of NB+YE in a sterile 200 mL Schott bottle. The mixture was swirled to ensure homogeneity and the

optical density (OD600) was measured in a spectrophotometer. The OD600 of the culture should fall within the

range of 0.04 to 0.1 absorbance units (A). The culture was then incubated in a pre-heated water bath set at

30°C. After 2 hr of incubation, the OD was measured. The OD should fall within the vicinity of 0.2A. The

culture concentration was then adjusted to 0.017A. For PM206, this OD is consistent with ~7.0 x 107 cfu/mL.

The dilution was prepared in a 50 mL plastic falcon tube. An example of the dilution is shown below.

A culture with an initial OD of 0.2 would be diluted in the following manner to give a final volume of 15

mL: If ODinitial = 0.2, then the DF = 0.21/0.017 = 12.35

Volsample = 15/12.35 = 1.21 Voldiluent = 13.79 mL

Therefore, 1.21 mL of the preparation is added to 13.79 mL of NB+YE to give an

OD600 of 0.017A. Once prepared, vaccine syringes were prepared by drawing 1 mL of the diluted culture into

1.0 mL syringes. This provided a single dose of ~5x107 (log 10

7.7) cfu. The vaccine was then delivered

through a catheter via intratracheal (IT) administration (see below).

Homologous Challenge preparation (X73)

Chickens:

The homologous challenge strain X73 was prepared as follows: One day 1, a vial of X73 working seed was

thawed at room temperature. One loop-full (~10 uL) of culture was streaked onto nutrient agar plus yeast

extract plate using standard bacteriological procedure. The plate was incubated at 37C for 16±1hr.

One day 2, two (2) mL of nutrient broth supplemented with yeast extract was poured onto the agar plate

containing the X73 colonies. The colonies were resuspended in NBYE using a disposable sterile plastic

spreader, lifting the colonies gently. A 10-2 dilution was prepared by adding 1 mL of cell suspension into 99

mL of NB+YE in a sterile 200 mL Schott bottle. The mixture was swirled to ensure homogeneity and the

optical density (OD600) was measured in a spectrophotometer. The OD600 of the culture should fall within the

range of 0.04 to 0.1 absorbance units (A). The culture was then incubated in a pre-heated water bath set at

30C. After 2 hr of incubation, the OD was measured. The OD should fall within the vicinity of 0.2A. The

culture concentration was then adjusted to 0.017A. For X73, this OD is consistent with ~7.0 x 107 cfu/mL.

The dilution was prepared in a 50 mL plastic falcon tube. An example of the dilution is shown below.

A culture with an initial OD of 0.2 would be diluted in the following manner to give a final volume of 15

mL: If ODinitial = 0.2, then the DF = 0.21/0.017 = 12.35

Volsample = 15/12.35 = 1.21 Voldiluent = 13.79 mL

Therefore, 1.21 mL of the preparation is added to 13.79 mL of NB+YE to give an

OD600 of 0.017A. Once prepared, vaccine syringes were prepared by drawing 0.5 mL of the diluted culture

into 1.0 mL syringes. This provided a single dose of X73 at ~7x107 cfu/mL.

In order to prepare dilutions other than the 7x 107 cful/mL, the following dilutions were prepared as shown in

the table below:

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Turkeys:

The X73 is grown as described for chickens and prepared at an OD of 0.017A. Additional dilutions were

prepared from the original OD 0.017A culture to give doses of 102.6

, 103.3

and 104.0

cfu/0.5mL/dose.

Once dilutions were prepared, the following volumes were drawn into vaccine syringes:

a. Draw 0.5 mL of 10-3.5

dilution into each of 20 syringes. Each dose corresponds to 104.0

cfu/0.5mL/dose.

b. Draw 0.5 mL of 10-4.2

dilution into each of 20 syringes. Each dose corresponds to 103.3

cfu/0.5mL/dose.

c. Draw 0.5 mL of 10-4.9

dilution into each of 20 syringes. Each dose corresponds to 102.6

cfu/0.5mL/dose.

The syringes were stored at room temperature and administered into chickens within 1 hr.

Ducks:

The X73 is grown as described for chickens and prepared at an OD of 0.017A. This culture was used as neat

to give a dose of 107.5

ccu/0.5mL/dose.Additional dilutions were prepared from the original OD 0.017A

culture to give doses of 104.0

and 105.5

ccu/0.5mL/dose.

Once dilutions were prepared, the following volumes were drawn into vaccine syringes:

a. Draw 0.5 mL of 10-3.5

dilution into each of 20 syringes (15 doses + 5 spare). Each dose

corresponds to 104.0

cfu/0.5mL/dose.

b. Draw 0.5 mL of 10-2

dilution into each of 20 syringes (15 doses + 5 spare). Each dose

corresponds to 105.5

cfu/0.5mL/dose.

c. Draw 0.5 mL of NEAT into each of 20 syringes (15 doses + 5 spare). Each dose corresponds

to 107.5

cfu/0.5mL/dose.

The syringes were stored at room temperature and administered into chickens within 1 hr.

Intratracheal administration (PM206)

Two operators were present for intratracheal (IT) vaccination. One operator held the bird upright with one

hand placed firmly around the legs and the other hand placed about the body to support its weight while

holding the wings in place. The second operator held the bird’s head and pulled the head out slightly to

straighten and lengthen the neck. This operator then opened the bird’s mouth to expose the trachea (which is

evident by moving as the bird breathes). The plastic catheter was then placed down into the trachea. The

required volume of inoculum (1 mL) was then delivered and the catheter withdrawn.

Intramuscular administration (X73)

Two operators must be present for intramuscular (IM) vaccination. One operator will hold the bird on its

back while the second operator removes cap on the needle and injects the bird. The feathers on the breast of

the bird (right or left side as appropriate) are separated and the needle carefully inserted into the breast

muscle near the sternum. The angle of the needle should be 45° and a depth of 1 – 2 cm depending on the

size of the bird. Once the needle is inserted, the plunger should be drawn back slightly to ensure that the

needle has not entered a vein. If blood is visualized, then the needle is drawn out slightly and the procedure

repeated. When no blood is observed then the full inoculum is delivered and the needle withdrawn. The

needle cap should be very carefully replaced and the entire syringe and needle unit disposed of in a biohazard

bin.

Determination of Protective Index (PI):

The critical formula used for the determination of efficacy (protective index):

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PI = 100 x % mortality in unvaccinated group - % mortality in vaccinated group

% mortality in unvaccinated group

Dissemination study (PMP R0715) – Microbial sampling of various organs

At various time intervals post vaccination with the P. multocida live vaccine “Vaxsafe® PM”, chickens were

subjected to post-mortem examination, during which swab samples were collected from the pharyngeal

mucosa, tracheal mucosa, cloacal mucosa, and site of injection. Swabs were taken by using sterile swabs

previously moistened with nutrient broth containing yeast extract. Also during post-mortem examination,

small pieces of tissues were excised from the site of injection, liver, lung and spleen; and bone marrow

scooped out directly from the leg bone.

Once the pharyngea of the chicken were exposed during necropsy, a moistened swab was rubbed against the

pharyngeal surface. The swab was quickly inserted back into its container and the lid replaced to prevent

contamination. An identical procedure was employed to collect swab samples from the tracheal (inner or

mucosal surface), the site of injection between the upper and deep pectoral muscles (following incision into

the breast muscle at the site of injection) and the cloaca.

Tissue samples were also collected for microbial examination. Care was taken to ensure the sterility of the

scalpel (using 70% ethanol and a Bunsen burner to flame sterilize the blade and tissue surfaces when

appropriate). A piece of muscle tissue was excised (~0.5 – 1.0 cm3) and placed into a 10 mL labelled tube.

Once the abdominal cavity was opened, the spleen was removed as aseptically as possible and placed into a

sterile labelled tube. In a similar manner, the lung tissue and the liver tissue (~0.5 – 1.0 cm3) were also

aseptically removed and placed into appropriately labelled tubes.

For access to the bone marrow the skin was removed from the thigh of the chicken. The lower leg bone was

snapped and the bone pushed through the muscle to expose the bone marrow in the lumen of the broken

bone. Using a sterile thin spatula, the marrow was scooped out.

Processing of swab samples

Working in a biosafety cabinet, remove the swab from its container and swab one corner of the HBA plate.

Starting from the primary inoculum the plate was streaked out for single colonies using a sterile loop. The

plate was then incubated at 37°C overnight. This procedure was used for processing of all swab samples.

Processing of organ/tissue samples:

In a biosafety cabinet, 2 mL of NB+YE culture were added to each tube containing tissue samples. The

tissue samples were macerated using a 1 mL pipette tip and jabbing the tissues while drawing broth up and

down several times. The tissue samples should be come macerated, though not necessarily completely

homogenised. Samples (100 μl) were taken directly from the macerated tissue and spread onto plates.

Plates were checked after 24 hr and colonies typical of PM were counted. For dissemination studies, the

plates were scored as +, ++, +++ or – depending on the number of PM organisms on the plate. To confirm

that the colonies were PM, a select number of colonies were analysed directly by PCR.

Reversion to virulence trial (PMP R0807) sample collection and processing

At various time points post vaccination, muscle tissue at the site of injection as well as the spleen was

collected as described above. The muscle and the spleen were collected based on the previous dissemination

study which indicated that the vaccine strain accumulated predominately in these two areas. The tissues were

macerated and briefly spun to remove large tissue pieces. The supernatant was then spun down at 10,000 rpm

in a centrifuge and the pellet washed in diluent. This procedure was repeated twice. The pellet was then

resuspended in 0.4 mL diluent per sample. An aliquot of the sample was retained for TVC determination. A

15

0.2mL aliquot of the sample was then administered by IM administration into a fresh group of chickens (2

birds per passage) for passages 2 to 5. This procedure was conducted for each of the 5 passages.

At each sample preparation time point, dilutions were prepared as described above. Dilutions at 10-4

to 10-7

were plated out onto HBA plates and the plates incubated overnight at 37°C. At 24 hr post incubation, the

plates were counted for colonies characteristic of PM. Total viable counts were then tabulated for each

passage level.

Furthermore the prepared aliquots at each passage levels were also examined by PCR analysis to determine

that it was indeed the PM vaccine strain PMP-1.

PCR analysis

PCR was conducted by using PCR primers (PMP1-F1 and PMP1-R1) flanking the deletion site within the

aroA gene. The PCR cycle conditions included denaturing at 94°C, annealing temperature at 52°C and

extension at 72°C. The PCR was run for 35 cycles.

For the reversion to virulence study (PMP R0807) the material obtained from P0 (original 1X MaxRT

Vaxsafe® PM inoculum) as well as P1 to P5 (Passages 1 to 5) tissue homogenate material were individually

subjected to PCR analysis. This PCR assay detects the aroA gene of P. multocida as well as differentiates

between wild type and vaccine strain. PCR amplification of the vaccine strain DNA will produce a 1035 bp

size fragment. PCR amplification of a wild type P. multocida strain will produce a 1337 bp size fragment.

Hence, any genetic changes within the aroA deletion region can be detected by a shift of the resultant DNA

band away from the expected 1035 bp PCR fragment size.

Optimisation of vaccine manufacture

During the development of Vaxsafe® PM, the production process for the vaccine changed in order to

facilitate commercial-scale handling. Initially, this involved a change from a chemically-defined medium

(CDM) to a semi-defined medium (SDM), which then became the basis for subsequent batches of vaccine.

Later, the buffer exchange and concentration method was changed from centrifugation to tangential-flow

filtration, and a new stabiliser formulation was added during the buffer exchange step.

These changes required a number of bridging studies to be performed, in order to demonstrate the

equivalence of vaccine manufactured after these changes to previous batches of vaccine. In the study

summaries that follow, the final production method is generally referred to as the “commercial method”, and

previous production methods as “pre-commercial”.

Summary of trial work undertaken

The following summary will consist of individual summaries of the studies undertaken, focusing initially on

studies to determine the efficacy of Vaxsafe® PM, followed by studies examining the safety of the vaccine.

Each study was assigned a study code, PMP RXXYY, with XX denoting the year and YY the sequential

study number for that year. The study number was assigned at the time the study was first proposed, and

therefore some studies took place some years later than the study number would indicate. The following

summaries are ordered by study number under the “Efficacy” and “Safety” sub-headings.

The vaccine is often referred to in the following summaries as Vaxsafe® PM, the proposed commercial name

for the vaccine by Bioproperties Pty Ltd. It is also sometimes referred to simply by the name of its active

constituent, Pasteurella multocida strain PMP-1.

16

PART A: Vaxsafe® PM for use in chickens

(i) Efficacy studies

PMP R0501: Development of a laboratory challenge model to evaluate the efficacy of Vaxsafe® PM Vaccine (living)

In order to determine the efficacy of the Vaxsafe® PM vaccine, it was necessary to establish a challenge

strain of P. multocida that would consistently produce Fowl Cholera (FC) in unvaccinated chickens. As a

guide to the level of disease required, reference was made to two regulatory monographs. European

Pharmacopoeia (EP) monograph 04/2005:1945 (later succeeded by 01/2008:1945) describes the

requirements for an inactivated Fowl Cholera vaccine; the EP has no monograph for a live FC vaccine. From

this monograph, administration of the challenge strain must cause 70% of the unvaccinated birds to either die

or show signs of infection. The USDA provides a monograph (9CFR 113.70) for a live P. multocida vaccine,

which requires that administration of the challenge strain causes death in at least 80% of unvaccinated birds.

It was further proposed that ideally the challenge strain should be heterologous to vaccine strain PMP-1, to

permit demonstration of the vaccine’s protection across serotypes. The parent strain for PMP-1 is Heddleston

serotype 1.

A total of 70 12 week old commercial layer pullets were enrolled in the trial, and assigned into trial groups as

shown in Table 1 below.

Table 1: PMP R0501

Group Challenge

(serotype) Route

Actual dose

(cfu/bird)

Number of

birds

Mortality

rate

1 PM221 (1) Intra-tracheal

(IT) 10

7.59 20 5%

2 PM206 (4) IT 107.69

20 70%

3a PM995 (3) IT 107.89

10 30%

3b PM1073 (3) IT 107.77

10 10%

4 Medium only IT 1 mL 10 0

Doses of the challenge strains were administered intra-tracheally at a target dose of 107.70

colony-forming

units (cfu) per bird in each case, with the retained titres for each group listed in Table 1. The birds were

monitored post-challenge for 7 days.

The only strain to approach usable levels of morbidity and mortality was serotype 4 strain PM206, with

mortality of 70%. As this strain also fulfilled the heterologous criteria, this was the strain selected for

subsequent challenge in efficacy trials for Vaxsafe® PM.

An example of a typical mortality profile for PM206 delivered by IT in chickens is shown in Figure 2.

17

Figure 2: P. multocida induced mortality in chickens following IT administration of strain PM206.

PMP R0502: A Dose Response Study to Determine the Minimum Protective Dose of Vaxsafe® PM Vaccine (living)

The minimum protective dose (MPD) is an important dose to establish. Once this dose is determined, other

important parameters such as end of shelf life titre (EOSLT), minimum release titre (MinRT) and maximum

release titre (MaxRT) can be set.

In order to determine the MPD of Vaxsafe® PM, 110 commercial layer pullets, 11 wk of age, were assigned a

treatment group as shown in Table 2 below.

Table 2: PMP R0502

Grou

p

Vaccination

Group PMP-1

(CFU/0.4 mL)

IT Challenge

Dose PM206

(CFU/mL)*

Number of

birds

Protective

Index (PI)

1 106.03

107.40

19# 69.4%

2 107.03

107.40

20 76.5%

3 108.03

107.40

20 100.0%

4 109.03

107.40

20 94.1%

5 Diluent only 107.40

20 N/A

6 Diluent only Nil 10 N/A

* challenge dose given in 1 mL by intra-tracheal inoculation 28 days

after vaccination.

# One bird died unrelated to treatment or challenge

Groups 1 to 4 were vaccinated with doses of Vaxsafe® PM ranging in one log10 intervals from 10

6.0 colony

forming units (cfu) to 109.0

cfu/ 0.4mL bird dose, with the actual doses listed in Table 2 above. The birds

were then challenged with strain PM206 at 28 days after vaccination.

The protective index shown in the table was derived by comparison of the mortality in the unvaccinated,

challenged group (85%) and the mortality in the respective vaccinated groups, as described in the

Typical Mortalities in Unvaccinated Chickens Post-Challenge with PM206

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7

Days Post-Challenge

Mo

rtaliti

es (

%)

Mortalities

(deaths & culls)

18

Methodology section above. From the protective indices, the method of Reed and Muench (1938) was used

to derive a protective dose 75% (PD75), calculated to be 106.7

cfu per dose. From this, a vaccine End of Shelf

Life Titre (EOSLT) was proposed. In line with the 9CFR 113.70 monograph, the EOSLT was set at twice the

PD75, or 107.0

cfu/dose.

PMP R0601: A study to determine the onset of protective immunity after vaccination with Vaxsafe® PM Vaccine (living)

The onset of protective immunity allows us to determine the length of time at which the animal is expected

to be protected post vaccination.

A total of 130 commercial layer pullets, 14 weeks of age, were enrolled into this trial, and assigned into

treatment groups as shown in Table 3 below.

Table 3: PMP R0601

Group

Vaxsafe® PM

dose (cfu,

actual)

Challenge

(weeks after

vaccination)

Number of

birds

Protective

index (PI)

1 107.64

1 10 67%

2 10 56%

3 10 25%

4 10 33%

5 10 44%

2 108.05

4 10 75%

5 10 67%

3 Nil 10 birds each

challenge 50 N/A

4 Nil Nil 10 N/A

The vaccine doses were targeted at the proposed EOSLT of 107.0

cfu for Group 1, and 108.0

cfu for Group 2;

however, the actual doses received by Group 1 were somewhat higher. Although the lower dose of

Vaxsafe® PM did not produce a protective index above the 75% PI threshold set by this trial, protective

immunity was clearly present from even 7 days after vaccination.

It is unclear why Vaxsafe® PM failed to produce the required level of protective immunity in this trial.

PMP R0603: Efficacy of Vaxsafe® PM in commercial chickens

During the course of this project, improvements in the manufacture of Vaxsafe PM were taking place. When

significant changes are made to the manufacturing process, it is important to conduct a bridging study to

ensure that the changes did not impact efficacy of the vaccine.

A total of 110 commercial layer pullets were enrolled into this trial, and allocated into four treatment groups

as shown in Table 4 below.

19

Table 4: PMP R0603

Group Vaccine growth

medium

Vaccine

dose (cfu,

actual)

Number

of birds

Protective

index

1 SDM

107.1

20 81%

2 107.8

20 75%

3 CDM

106.7

20 81%

4 107.5

20 94%

5 (Diluent only) N/A

20 N/A

6 10

CDM: Chemically defined medium

SDM: Semi-defined medium

Groups 1 to 4 were vaccinated at 14 wk of age, and challenge with strain PM 206, at a target dose of 107.9

cfu, occurred four weeks after vaccination. As part of the manufacturing development process, vaccine

fermented in a semi-defined medium was compared with vaccine manufactured using a chemically-defined

medium. Each vaccine batch (one produced in SDM and one in CDM) was administered to two groups, one

receiving a target EOSLT dose (107.0

cfu) and one a more likely commercial release dose targeted at 107.7

cfu. The actual doses received are documented in Table 4.

Based on the mortality rates after challenge of the unvaccinated Group 5 (80%, Group 6 was an unchallenged

control group in which no mortalities were seen) and the four vaccinated groups, Protective Indices (PI) were

again derived.

All four vaccinated groups demonstrated a PI sufficient to pass the thresholds for efficacy of both the EP

(threshold for efficacy PI ≥ 57%) and 9CFR (threshold for efficacy PI ≥ 75%) monographs, confirming the

acceptability of both CDM and SDM formulations of the vaccine.

PMP R0605: Vaxsafe® PM Vaccine (living) – Duration of immunity in chickens

To demonstrate the duration of immunity of Vaxsafe® PM in chickens, 615 commercial layers, 9 weeks of

age, were housed in a pen facility and vaccinated once with the anticipated end of shelf life titre for Vaxsafe®

PM, 107.0

cfu/dose. In order to demonstrate the equivalence in terms of efficacy of a batch of Vaxsafe® PM

manufactured using the commercial production method with a batch manufactured using a pre-commercial

method (see “Optimisation of vaccine manufacture” above), each batch was used to vaccinate a separate

group. Sub-groups of 20 chickens from each group were moved to isolators and challenged with virulent

heterologous Pasteurella multocida strain PM206 at intervals from 4 weeks after vaccination.

20

Table 5: PMP R0605

Group No. of

Pens Chickens Group Purpose

Experimental

Treatments Dose

Chicken

Age(s) at

Treatments

Chicken Ages for

Challenge Period

A 2 190 Negative

Controls

Sterile Diluent

(20 birds only) 0.2mL IM 9 weeks 13-22 weeks

B 1 70 Bioequivalence Vaxsafe

® PM

PMP 071021A

107.0

cfu per

0.4mL 9 weeks 13-22 weeks

C 2 185

Single dose

regimen

(+/- booster)

Vaxsafe® PM

PMP 071022B

107.0

cfu per

0.2mL on 1

occasion

9 weeks 13-22 weeks

D 2 170

Two dose

regimen

(+/- booster)

Vaxsafe® PM

PMP 071022B

107.0

cfu per

0.2mL on 2 (or

3) occasions

9 and 16

weeks 13-22 weeks

The initial challenge 4 weeks after vaccination failed to produce many mortalities amongst the unvaccinated

control birds, and led to changes in the challenge procedure. A challenge 7 weeks after vaccination produced

95% mortality amongst the unvaccinated birds, and produced protective indices (PI) of 42% and 37% for

pre-commercial and commercial production method batches respectively. These PI dropped to 11% and 22%

respectively two weeks later.

One further group of chickens received a second dose of Vaxsafe® PM, 10

7.0 cfu/dose, 6 weeks after the

initial vaccination. This group was challenged with strain PM206 at 5 weeks after the second dose and

showed a PI of 50%, with 100% of the unvaccinated controls dying within 2 days. At a challenge for this

group two weeks later, the PI had dropped to 32%.

In the face of an extremely strong heterologous challenge, Vaxsafe® PM did provide protective immunity at

7 weeks after a single dose, and at least 5 weeks after two doses. This trial provides some evidence that a two

dose regime of Vaxsafe® PM evokes better protection than a single dose. The duration of immunity may well

be prolonged beyond the times measured in this trial in the field situation, where normal field challenges do

not result in greater than 75% mortality within the first 48 hours, as is required in the challenge model.

Nevertheless, the PI recorded for this trial did not met the thresholds required for demonstration of efficacy

by the European or US monographs most applicable to this product. The lack of mortality amongst the non-

vaccinates at four weeks after initial vaccination made demonstration of peak efficacy unattainable.

The PI produced by the previous and current production method batches of Vaxsafe® PM were similar at

each timepoint. Given neither reached the monograph threshold levels of efficacy, it is difficult to suggest

that this trial shows equivalence between the two production methods, but supporting evidence was

provided.

21

PMP R0801: A study to determine the clinical effects of a Pasteurella multocida (strain PM206) challenge when administered to chickens at various doses

Due to the unpredictable results from administration of strain PM206 in previous trials, it was proposed to

undertake a further dose-response trial with this challenge strain to select an optimum dose that would

produce mortality rates suitable for regulatory trials, without causing overwhelming disease that could

potentially mask the efficacy of the candidate vaccine.

A total of 90 commercial layer hens, 25 to 27 weeks of age, were enrolled and assigned one of five

experimental groups as shown in Table 6 below.

Table 6: PMP R0801

Group No.

Chickens

PM206

Dose (cfua)

Mortality rate

A 20 106

60%

B 20 105 60%

C 20 104

70%

D 20 103

75%

E 10

Nil 0

a. cfu = colony-forming units

PM206 doses administered were close to the target titres in each case. The expected dose-response

relationship between PM206 dose and mortalities was not seen. Doses of 103, 10

4, 10

5 and 10

6 cfu produced

mortality rates of 75%, 70%, 60% and 60% respectively. When clinical signs and post-mortem re-isolation of

PM206 was included, at least 80%, 80%, 100% and 65% of birds were affected respectively. There was no

statistically significant difference between mortality rates in the four groups, and the totals including clinical

signs and bacterial re-isolation were not suitable for statistical analysis. No birds in the control group

experienced clinical signs of disease or death.

Doses of 103, 10

4 and 10

5 cfu of PM206 met the European challenge guidelines for an inactivated Fowl

Cholera Vaccine. None of the doses met the 80% mortality requirement for challenge in the US 9CFR

monograph.

PMP R0802: A study to determine the efficacy of three batches of Vaxsafe® PM in chickens

Again due to the unpredictable results seen in previous challenge trials, it was proposed to increase the

EOSLT for Vaxsafe® PM from 10

7.0 cfu/dose to 10

7.2 cfu/dose. Therefore, this trial was undertaken to

demonstrate the efficacy of this new EOSLT. Further, it was to investigate the equivalence of vaccine

manufactured using the final commercial method with vaccine manufactured using a pre-commercial

method, as this was not well demonstrated by trial PMP R0605. Efficacy was to be demonstrated against the

heterologous challenge strain PM206, using the dose determined optimal by trial PMP R0801, being 104.0

cfu.

A total of 140 commercial layer birds, 8 to 10 weeks of age were assigned into one of the trial groups shown

in Table 7 below. Birds were administered 107.2

cfu/dose Vaxsafe® PM from one of three batches and

challenged 28 days later using strain PM206. An additional challenge group using PM206 at a dose of 107.7

22

cfu was included to correlate to the dose used in earlier work. An additional group was challenged with an

intratracheal dose of the medium (nutrient broth plus yeast extract, NB+YE) used for preparation of PM206,

to investigate the level of pathology caused by the intratracheal challenge route aside from that caused by the

challenge strain.

Table 7: PMP R0802

Group No. of

Chickens

Vaxsafe® PM

Batch

Vaxsafe® PM

Dose

PM206 Challenge

Dose

A 20 Nil Nil 104 cfu

B 20 PMP 061481A 107.2

cfu 104 cfu

C 20 PMP 071021B 107.2

cfu 104 cfu

D 20 PMP 071022C 107.2

cfu 104 cfu

E 10 Nil Nil 1 mL NB+YE

only

F 10 Nil Nil Nil

G 20 PMP 061481A 107.2

cfu 107.7

cfu

H 20 Nil Nil 107.7

cfu

Only one bird in the unvaccinated control group challenged at 104 cfu (Group A) died, with only transient

clinical signs of disease in other birds. Re-isolation of the challenge strain was not attempted.

Therefore the challenge at this dose was not valid as per regulatory guidelines, and efficacy of the vaccine

could not be evaluated. Challenge at 107.7

cfu produced 80% mortality.

PMP R0809: Development of a master seed of Pasteurella multocida challenge strain X-73

As detailed above, P. multocida strain PM206, heterologous to vaccine strain PMP-1, was primarily used for

challenge work in the development of Vaxsafe® PM. However, the unpredictability of results after

administration of strain PM206, and the attraction of measuring the duration of immunity of the vaccine

against both heterologous and homologous challenge, led to the need to develop a master seed bank of

homologous P. multocida strain X-73. This strain was the parent strain for the manipulations that produced

the vaccine strain PMP-1.

This report detailed the history of strain X-73, from its import from the USA until a sufficient quantity was

banked down for use in the duration of immunity trial PMP R0803a, summarised below, as well as

subsequent turkey and duck trials. This included an in vivo passage in birds. During trial PMP R0803a, the

resulting X-73 seed demonstrated a consistent level of clinical signs and mortality in the commercial layer

chickens to which it was administered, indicating the master seed banking process was successful.

23

PMP R0803a: A study to determine the duration of immunity following vaccination of chickens with Vaxsafe® PM

A single study protocol for PMP R0803 was written, to investigate the duration of immunity of

Vaxsafe® PM, compare the efficacy of vaccine manufactured using a pre-commercial method with that

produced using the final commercial method, and examine the compatibility of Vaxsafe® PM with other

vaccines commonly administered to commercial chickens. These investigations were run concurrently using

chickens housed on a single commercial layer farm. For clarity, this trial was reported in three separate Final

Study Reports: PMP R0803a reports the duration of immunity investigation, PMP R0803b reports the

comparison of efficacy, and PMP R0803c the examination of compatibility. Please see below for summaries

of reports PMP R0803b and PMP R0803c.

Trial PMP R0803a was designed to demonstrate the duration of immunity following vaccination of chickens

with Vaxsafe® PM at the end of shelf life titre (EOSLT) dose.

A group of approximately 1,000 commercial layer chickens, housed in a commercial layer shed, were

vaccinated with Vaxsafe® PM at 9 weeks of age. A second dose was administered 4 weeks later. This is the

recommended vaccination protocol for this product. The vaccine was administered at the end of shelf life

titre (EOSLT), 107.2

cfu/dose. An untreated control group was housed in a separate production shed under

identical management conditions. At intervals after vaccination, subgroups of 20 birds from each of the

groups were removed from the production facility and transferred to isolators at the Asia-Pacific Centre for

Animal Health (APCAH). Here they were challenged with either a heterologous strain (PM206) or

homologous strain (X-73, the parent strain for Vaxsafe® PM) of P. multocida to demonstrate the duration of

protective immunity induced by Vaxsafe® PM.

Achieving reproducibility with the disease model using the heterologous challenge strain PM206 has proven

to be difficult. However, the challenge 4 weeks after the second dose of Vaxsafe® PM was valid,

demonstrating a Protective Index (PI) of 81%. At 8 weeks after the second dose of Vaxsafe® PM, the PI for

heterologous challenge had declined to 18%. Subsequent challenges using the homologous strain X-73

showed significant levels of efficacy up to 36 weeks following the second dose of Vaxsafe® PM. The PI at

26, 32 and 36 weeks after the second vaccine dose were 70%, 60% and 45% respectively.

Thus, Vaxsafe® PM provided protection against challenge with a heterologous (serotype 4) strain of P.

multocida for at least four weeks after two doses of vaccine, exceeding the EP monograph threshold for

efficacy with a PI of 81%. However, protection against heterologous challenge had declined by eight weeks

after the second dose of vaccine. Protection against a homologous (serotype 1) strain exceeded the EP

threshold for efficacy at 30 weeks after vaccination. On the basis of the PI, a reasonable degree of protective

immunity was still evident at 36 weeks after the second vaccination (PI of 45%).

PMP R0803b: Efficacy of Vaxsafe® PM – equivalence of two formulations

As noted in the summary of PMP R0803a above, trial PMP R0803b was undertaken to demonstrate the

equivalence of efficacy between vaccine manufactured using a pre-commercial method and that produced

using the final commercial method. A demonstration of this equivalence was sought in trials PMP R0605 and

PMP R0802, summarised above, but due to issues with the challenge strain or vaccine efficacy at a lower

dose, it had not been possible to demonstrate this equivalence. See “Optimisation of vaccine manufacture”

above for a summary of the differences between these production methods. This trial was run concurrently

with trials PMP R0803a and PMP R0803c. The batches of Vaxsafe® PM used were as for trial PMP R0605.

Table 8: PMP R0803b

Group Vaxsafe® PM batch

Protective index

1st challenge 2

nd challenge

24

A PMP 061481B

(pre-commercial) 94% 33%

B PMP 071422D

(commercial) 81% 18%

C None N/A N/A

1st challenge 4 wk after second dose of vaccine

2nd

challenge 8 wk after second dose of vaccine

Two groups each of approximately 1000 commercial layer chickens, housed in a commercial layer shed,

were vaccinated with one of two batches of Vaxsafe® PM at 9 weeks of age, with a second dose administered

approximately 4 weeks later. This is the proposed vaccination protocol for this product. Both doses were at

the planned end of shelf life titre (EOSLT, 107.2

cfu/dose). One of these batches of Vaxsafe® PM was

manufactured using the commercial method, the other using the pre-commercial method. A control group

was housed in a separate production shed under identical management conditions. At approximately four

week intervals, 20 birds from each of the groups were removed from the production facility and placed in

isolators at the Asia-Pacific Centre for Animal Health (APCAH). Birds were challenged with a virulent

heterologous strain of P. multocida, strain PM206.

Challenges with strain PM206 were unpredictable in their validity; however, at the challenge that took place

4 weeks after the second dose of Vaxsafe® PM, both batches of Vaxsafe

® PM exceeded the European

monograph threshold for efficacy, with Protective Indices (PI) of 81% and 94% for the commercial and pre-

commercial batches respectively. At 8 weeks after the second dose of Vaxsafe® PM, protective immunity

produced by the two batches of Vaxsafe® PM was waning.

These two challenge events demonstrated the equivalent efficacy of the two formulations of the vaccine.

PMP R0803c: Determination of the compatibility of Vaxsafe® PM with concurrently administered vaccines in chickens

Trial PMP R0803 had three concurrent parts, as discussed above. Trial PMP R0803c focused on the

compatibility of Vaxsafe® PM with other vaccinations commonly given at the same age as Vaxsafe

® PM,

specifically live Fowl Pox and Avian Encephalomyelitis Virus (AEV) vaccines and a combined inactivated

Newcastle Disease plus Egg Drop Syndrome vaccine (ND+EDS). This trial was run concurrently with trials

PMP R0803a and PMP R0803b, summarised above.

A total of 24,000 commercial layer chickens, housed in a commercial layer shed, received two doses of

Vaxsafe® PM at an interval of 4 weeks. The birds were 9 weeks old at the time of the first dose. Two groups

of 1,000 birds each received an end of shelf life titre (EOSLT) of Vaxsafe® PM, with the remainder receiving

a commercial release dose. A control group was housed in a separate production shed under identical

management conditions. Concurrent with the first Vaxsafe® PM vaccination, birds received ND+EDS

vaccine intramuscularly, Fowl Pox vaccination by wing stab, and approximately 15% of birds received AEV

vaccination by eye drop. Blood sampling of 20 birds was undertaken at 13 weeks of age to demonstrate

serological evidence of the response to these vaccinations, and repeated at 20 weeks of age. A further

sampling of 30 birds was undertaken at 60 weeks of age for ND serology only.

No significant differences in serological titres between vaccinated and unvaccinated chickens were found for

AEV at 13 and 20 weeks, EDS at 20 weeks (not tested at 13 weeks), or NDV at 13 weeks. NDV titres at 20

weeks were significantly lower in the vaccinated group than the unvaccinated group; however, no significant

difference was present at 60 weeks. Titres in both groups were in excess of those required by the National

25

Newcastle Disease Management Plan (Animal Health Australia, 2008) to produce protective immunity

against Newcastle Disease.

Comparison of the efficacy data produced by the duration of immunity arm of this trial (PMP R0803a) with

previous data indicates that the use of other vaccines concurrent with Vaxsafe® PM has not decreased the

efficacy of Vaxsafe® PM.

Vaxsafe® PM is compatible with the vaccines commonly administered close to its recommended age of

administration.

(ii) Safety Studies Some studies summarised in the Efficacy Studies section above also provided data on the safety of

Vaxsafe® PM. Those studies are thus re-summarised below, with an emphasis on the safety data revealed.

PMP R0502: A Dose-Response Study to Determine the Minimum Protective Dose of Vaxsafe® PM Vaccine (living)

As summarised above, this trial involved 110 commercial layer chickens, 11 wk of age, administered

Vaxsafe® PM at doses ranging from 10

6.03 cfu to 10

9.03 cfu in 1 log10 increments. The birds were observed for

28 days after vaccination, and then challenged to examine vaccine efficacy.

No clinical signs of disease or ill health were noted in the birds after vaccination with Vaxsafe® PM, even at

the highest dose of 109.03

, which is well in excess of the final proposed maximum release titre of 108.1

cfu/dose.

PMP R0503: A study to determine the safety of Vaxsafe® PM Vaccine (living) in chickens

For regulatory purposes, the safety of a candidate vaccine must be demonstrated using the maximum release

titre (MaxRT) – the highest titre per dose permissible for release of commercial batches of vaccine. Live

vaccines such as Vaxsafe® PM must demonstrate safety a single MaxRT dose (1xMaxRT), at ten times the

MaxRT (10xMaxRT), and after repeated 1xMaxRT doses. At this point in the vaccine’s development, it was

proposed that the MaxRT be 108.0

cfu/dose. These doses must be administered to the most susceptible group

of the target species – in this case considered to be the youngest birds likely to be vaccinated.

A total of 80 specific pathogen free chickens (SPF, hybrid white Leghorn strain) were took part in the trial to

demonstrate the safety of Vaxsafe® PM. They were assigned into one of four trial groups as documented in

Table 9 below.

Table 9: Treatment groups for the safety study PMP R0503

Gro

up

No

.

No

. o

f

Ch

ick

ens

Group

Purpose

Vaxsafe® PM

Treatment

Vaccine

Dose

(cfua)

Observations

Ob

s. P

erio

d

(day

s)b

1 20 Single Dose MaxRT 108.0

Body Weights 63

26

2 20 10x Overdose 10xMaxRT 109.0

Clinical Signs

Post-Mortem Lesions

Body Temperatures*

63

3 20 3 Repeat

Doses

MaxRT

(repeat x3)

108.0

(x 3)d

70

4 20 Negative

Controls

Nil (Sterile

Diluent)

Nil

(x3)d

72

a. cfu = colony-forming units

b. Days post-vaccination (or after initial treatment for Groups 3 & 4)

c. The letter designations of isolator numbers given in the study protocol

correspond to the following isolator numbers appearing in the study records:

A = L4, B = L3, C = L2 & D = L1

d. Three treatments repeated at 4-week intervals

* Non-pivotal data

Of the chickens administered a 1xMaxRT dose, a small number demonstrated mild clinical signs which were

acute and transient in nature. A significant proportion of the chickens administered the 10xMaxRT overdose

demonstrated similar clinical signs and in this group they persisted slightly longer (i.e. 2-3 days post-

vaccination). Three of eighteen chickens in the 10xMaxRT overdose group were culled due to progressive

disease at least partially attributable to the high vaccine doses administered.

Typical injection site lesion 2 weeks after administration of a MaxRT is shown in Fiigure 3. The left panel

shown discolouration visible through superficial pectoral muscle and the right panel shows an incision made

through the same lesion. Yellow caseous material is evident between the superficial and deep pectoral

muscles.

Figure 3. Lesion at injection site at 2 weeks post vaccination.

Below is a typical example of the most severe injection site lesions detectable 9 weeks after administration

of a 1xMaxRT or a 10xMaxRT (Figure 4).

27

Figure 4. Remnants of a lesion in chicken pectoral muscle (site of injection) at 9 weeks post

administration of an overdose of Vaxsafe® PM.

When corrected for sex and initial weight, rate of weight gain in the 10x MaxRT and repeat dose groups was

decreased compared to the unvaccinated controls. There was evidence of compensatory growth in the repeat

dose group, with their mean body weight not significantly different from the control group at the second last

timepoint. The mean body weight in the group receiving a single 1xMaxRT dose was not significantly

different from the unvaccinated controls at any timepoint.

Figure 5. Effect of 1xMaxRT and 10xMaxRT on body weights in SPF chickens at 6, 8 and 9 weeks post

vaccination.

28

The decreases in weight gain seen were not considered to endanger the utility of the vaccine, as the changes

were seen only in the birds given either three doses instead of the recommended two, or the very high

10xMaxRT dose. Further, the decrease in weight gain was seen in comparison to birds unchallenged by P.

multocida. It is expected in the commercial situation, Vaxsafe® PM would primarily be administered to

poultry flocks expected to experience field challenge with P. multocida.

PMP R0601: A study to determine the onset of protective immunity after vaccination with Vaxsafe® PM Vaccine (living)

As summarised above, this study involved 130 commercial layer pullets administered Vaxsafe® PM at two

doses: 107.64

cfu and 108.05

cfu, the latter being very close to the proposed MaxRT for Vaxsafe® PM. The

birds administered the higher dose can therefore provide information on the safety of the vaccine.

The birds administered the higher dose of Vaxsafe® PM were monitored for at least 4 weeks after vaccine

administration. No clinical signs of disease or ill health were seen in these birds during the observation

period.

PMP R0602: Safety of Vaxsafe® PM Vaccine – a dose response study to evaluate endotoxic effects following administration to 6-week-old chickens

As a Gram-negative organism, the cell wall of P. multocida contains significant amounts of endotoxin. The

acute nature of clinical signs seen after administration of Vaxsafe® PM to chickens in trial PMP R0503

indicated that release of endotoxin could be an important factor in the response to Vaxsafe® PM. Therefore,

in association with quantification of the endotoxin levels of several batches of Vaxsafe® PM (trial PMP

R0611 summarised below), this trial was undertaken to correlate endotoxin levels with degree of clinical

signs seen.

Four groups of 20 commercial layer chickens, 6 weeks of age, were administered doses of Vaxsafe® PM

containing 106.2

, 105.7

, 105.2

or 104.7

endotoxin units (EU) by intramuscular (IM) injection. A further group of

20 chickens were administered the vaccine diluent only, by the same route. All chickens were observed for

clinical signs consistent with endotoxicosis over a period of 4 days post-vaccination. Changes to body

temperature and the identification of gross pathology at post mortem examination were also recorded.

The chickens administered vaccine diluent only exhibited neither clinical signs nor gross post mortem

pathology. They demonstrated a mild, transient increase in body temperature at 2 hours post-vaccination,

possibly due to the handling associated with treatment administration (mean cloacal temperature of 42.2 °C).

In contrast, all vaccinated chickens exhibited clinical signs consistent with endotoxicosis. The clinical signs

were generally more severe with larger doses of endotoxin. Chickens administered the two lowest doses of

endotoxin ceased to exhibit any clinical abnormalities by 24-48 hours post-vaccination.

During post mortem examination, all vaccinated chickens demonstrated gross pathology at the site of

injection only. Transient elevations in body temperature were identified in the two highest dose groups

(mean cloacal temperatures of 43.1 °C and 43.4 °C, respectively). Qualitative assessment of the body

temperature data for the two lowest dose groups did not identify an obvious departure from the expected

body temperature range.

Overall, it was concluded that clinical signs consistent with endotoxicosis were demonstrated by chickens

following intramuscular administration of Vaxsafe® PM doses containing 10

4.7 to 10

6.2 EU. Recovery from

the acute clinical signs of endotoxicosis due to doses as high as 105.7

EU can be expected within four days

post-vaccination. Chickens receiving the lowest dose of 104.7

EU, equivalent to ten-times the currently

proposed maximum release titre of 108.0

colony-forming units (cfu) per dose, exhibited the least persistent

clinical and pathological effects.

29

PMP R0604: A study to determine the spread and dissemination of the Vaxsafe® PM Vaccine (living) strain of Pasteurella multocida in chickens

Six-week old, mixed-sex Specific Pathogen Free (SPF) chickens received a dose equivalent to the initially

proposed maximum release titre (MaxRT) of Vaxsafe® PM (10

8.0 colony-forming units [cfu] per dose). Eight

chickens were periodically removed from one group and euthanised. These underwent gross morphological

and microbiological examination to determine the post-administration dissemination of the vaccine

organisms within the chickens. Another group of vaccinated chickens were placed in cohabitation with

unvaccinated chickens (non-vaccinates). The non-vaccinates were removed periodically for euthanasia and

gross morphological and microbiological examinations. This was intended to determine the spread of the

vaccine organisms to unvaccinated chickens.

Gross morphological examination of the vaccinated chickens in the dissemination study yielded observations

of inflammatory lesions adjacent to the site of injection. However, the sensitivity of the microbiological

analysis was not adequate to yield valid results. Consequently the study objectives were not achieved and the

extent of dissemination and spread of Vaxsafe® PM was not determined.

PMP R0607: Evaluation of the genetic stability of Pasteurella multocida strain PMP-1, an aroA deletion mutant derived from P. multocida X-73

Strain PMP-1 is the vaccine strain of P. multocida found in Vaxsafe® PM. It was derived by genetic

manipulation from parent strain X-73, specifically by deletion of a segment of the aroA gene, rendering this

gene non-functional.

In order to examine the stability of this gene deletion during in vitro passaging, the PMP-1 pre-master seed

was grown to a maximum of 16 passages, and then subjected to restriction endonuclease (RE) digestion and

sequencing. Comparison was made with the parent organism, P. multocida strain X-73.

RE digestion patterns revealed no evidence of genetic alterations within the aroA gene in either the pre-

master seed 2/95 (i.e. least propagated sample) or after 16 passages through culture (equivalent to vaccine

passage level) which represents the most propagated sample. Similarly, sequencing analysis, which offers the

highest level of stringency with respect to identifying genetic changes, also showed no evidence of genetic

alterations between the least and the most propagated samples of P. multocida strain PMP-1. This study

demonstrates that the 302 base pair deletion of the aroA gene of P. multocida strain PMP-1 is indeed

genetically stable.

PMP R0611: Measurement of endotoxin content relative to viable cell count of five batches of Vaxsafe® PM Vaccine (living)

Like other Gram-negative bacteria, P. multocida expresses endotoxins on its outer membrane. Such

endotoxins, also known as lipopolysaccharides (LPS), are complex molecules that contain both lipid and

carbohydrates and are major constituents of the bacterial cell wall (e.g. a single Escherichia coli cell contains

about 2 million LPS molecules). Similar to other Gram-negative bacteria, PM can release small amounts of

endotoxins into their surrounding environment during active cell growth, but large amounts are released

when cells are disrupted or destroyed as a result of cell lysis, or when they are engulfed by the host immune

cells (e.g. phagocytes). Systemic exposure of chickens to high levels of endotoxins can cause septicaemia

and as a result sudden death. Considering that Vaxsafe® PM may contain considerable amounts of

endotoxins, its safety has to be thoroughly demonstrated before release. Also, different batches may contain

varying amounts of endotoxins. Therefore the safety of each batch has to be demonstrated independently

from other batches. Very little data are available to define the safe endotoxin limits for poultry vaccines. In

particular, a quantitative relationship between endotoxin content and possible systemic or local reactions in

chickens has yet to be established.

The Limulus Amebocyte Lysate (LAL) test is widely used for in vitro detection and quantification of

bacterial endotoxins, where LPS amounts are expressed in endotoxin units (EU). Typically, 1 nanogram (ng)

30

of LPS corresponds to 10 EU. LAL is commonly used for end-product testing of human and animal

injectable drugs for validation, lot release, or measurement of the amount of endotoxin in a sample.

Five batches of Vaxsafe® PM were submitted to an external laboratory for quantification of endotoxin using

the LAL test. The same batches were also subject to viable cell count in order to express the mean endotoxin

dose per dose of vaccine. The batches were shown to have a mean endotoxin concentration of 2.90 x 106

EU/mL, which equated to 3.9 x 104 EU per 1xMaxRT dose.

This information was then used as a basis for trial PMP R0602, summarised above.

PMP R0715: A study to determine the safety and dissemination of Vaxsafe® PM in chickens

Three groups of six-week old commercial layer chickens (females) received the following treatments with

Vaxsafe® PM (injected intramuscularly into the pectoral muscles):

Table 10: Trial PMP R0715

Group Treatment

A A negative control group not administered any vaccine or

sham treatment (i.e. nil treatment)

B

One dose at the maximum release titre (1xMaxRT). These

birds were also examined by post mortem at regular intervals

for dissemination of the vaccine

C One dose at five-times the proposed maximum release titre

(5xMaxRT)

D One dose at 10xMaxRT

E One dose at an intermediate titre of approximately 107.4

cfu

F

A positive control group administered single doses of an

inactivated Pasteurella vaccine (CFCV) by intramuscular

injection (IM) into the pectoral muscles or subcutaneous

injection (SC) into the back of the neck

This safety study was performed using vaccine manufactured by the commercial method and was performed

in 6 week old chickens. Note that the original proposed maximum release titre (MaxRT) was 108.0

cfu/dose.

However, the dose delivered to the 1xMaxRT group in this trial was 108.1

cfu, and therefore this is the final

proposed MaxRT.

1xMaxRT Group

The chickens were assessed for the development of clinical disease (including faecal abnormalities) using a

scoring system, and subject to post-mortem examination for gross pathological lesions attributable to the

vaccine treatments. Activity scores in this group dropped to 4 (“Quiet but responsive”) for 1 day following

vaccination, and thereafter returned to 5 (“Normal activity”) for the remainder of the 14 day observation

period. Birds in the negative control group showed activity scores of 5 throughout the study. 1/30 birds (ie 1

bird of 30 in group) showed ruffled feathers and hunched posture 1 day after vaccination. Thereafter, no

abnormalities of posture or appearance were observed. No faecal abnormalities were noted compared to

controls.

In the 1xMaxRT group, birds were culled at regular intervals for the dissemination element of the study so

injection site and other lesions were monitored in this group throughout the 14 day period. No deaths or culls

occurred in this trial due to the vaccine.

31

In the five birds culled at 2 hours post-vaccination, small haemorrhage about the injection site was the only

finding in 4/5 birds. Similar results were found in the 5 birds culled at 6 hours post-vaccination. In the 5

birds culled 24 hours post-vaccination, pallor and cellulitis of the deep muscle tissue was seen with

thickening of the interfascial region in all birds. The 5 birds culled 48 hours post-vaccination similarly

showed cellulitis around the pectoral muscles with evidence of local hyperaemia. At seven days post-

vaccination, internal injection site lesions were noted to be resolving with some fascial thickening and fibrin

present, and in most cases no external lesion was visible. Internal injection site lesions in those birds culled

at 14 days were recorded as minor, with some organised fibrin and congestion of pectoral musculature. Three

of the five birds culled at 14 days post-vaccination were noted to have severely congested lungs. No other

lesions were noted at this or other time points in this group.

To assess dissemination of the vaccine strain eight different sites were analysed - Site of injection,

pharyngeal mucosa, tracheal mucosa, cloacal mucosa, pulmonary tissue, splenic tissue, hepatic tissue, and

femoral/tibial bone marrow.

Figure 6. Dissemination of Vaxsafe® PM in the injection site and spleen of chickens at various times

post vaccination.

Figure 7. Dissemination of Vaxsafe® PM in lungs and liver of chickens at various times post

vaccination

In vivo dissemination of Vaxsafe PM (living)

0

1

2

3

4

5

pre-vaccination 2 hr 6 hr 1d 2d 7d 14d

Time post vaccination

Po

sit

ive

PM

(5

bir

ds

pe

r g

rou

p) Injection Site

Spleen

In vivo dissemination of Vaxsafe PM (living)

0

1

2

3

4

5

pre-vaccination 2 hr 6 hr 1d 2d 7d 14d

Time post vaccination

Po

sit

ive

PM

(5

bir

ds

pe

r g

rou

p) Lungs

Liver

32

5xMaxRT Group

Some interesting findings were noted in this 5xMaxRT treatment group compared to the single overdose

group, as they were studied for a longer period after vaccination.

The chickens were assessed for the development of clinical disease including faecal abnormalities using a

scoring system, and subject to post mortem examination for gross pathological lesions attributable to the

vaccine treatments.

Activity scores in this group dropped to 3 (“Mostly sitting, will stand if stimulated”) for 1 day following

vaccination, and were then between 4 and 5 until the 5th day following vaccination. After this time, activity

scores were 5 for the remainder of the 14 day observation period. A maximum of 8/20 birds showed a

hunched posture at 1 day following vaccination, decreasing in number over the following observations until

no abnormalities were noted on the 5th day following vaccination.

The post-mortem examinations undertaken on Day 28 showed resolving internal injection site lesions, with

healing more advanced than in Group B (1xMaxRT) at 14 days post-vaccination, with no external site

lesions. Day 42 examinations showed further improvement with 1/5 birds showing no lesions. Day 56

examinations showed no visible lesions, internal or external, on 8/10 birds, with 2/10 showing very minor

internal lesions.

One bird culled at Day 42 showed a shortening of the upper beak, which was not a vaccine-related lesion. No

other lesions were found in this group.

10xMaxRT Group

The target dose for this group was 109.0

cfu, but the actual titre dose was 109.1

cfu, supporting a MaxRT of

108.1

cfu/dose.

Activity scores in the group receiving 10xMax RT dropped to 3 (“Mostly sitting, will stand if stimulated”)

for 1 day following vaccination, were then between 4 (“Quiet but responsive”) and 5 (“Normal”) until the 5th

day following vaccination, and thereafter returned to 5 for the remainder of the 14 day observation period.

Ten of 19 birds showed a hunched posture 1 day post vaccination (pv) which lasted up to 5-9 days pv. No

faecal abnormalities or breathing abnormalities were noted in any birds.

No birds given this dose were culled or died as a result of the vaccine. One bird in this group was culled due

to morbidity and was diagnosed with a needle-stick injury to the liver on post-mortem examination.

The post-mortem examinations undertaken on this group 14 days post-vaccination showed a 1 x 2 cm area of

necrosis and fibrosis in the pectoral musculature of the injection site and thickening of the interfascial region

in 6/20 birds. Nine of 20 birds showed organising fibrin, pallor and thickening in the area of the injection

site. Three of 20 birds showed no injection site lesions. One bird showed dilatation of the oesophageal

proventricular gizzard on post-mortem examination, otherwise no other lesions were recorded.

No significant clinical abnormalities were noted in the group receiving a 10xMaxRT overdose. Transient

clinical signs were noted only for a few days after vaccination. There were no significant post mortem

lesions other than an area of fibrosis and thickening at the injection site in the majority of birds.

PMP R0803c: Determination of the compatibility of Vaxsafe® PM with concurrently administered vaccines in chickens

As described in the Efficacy Studies section above, this study took place on a commercial caged layer farm,

with a total of 24 000 birds vaccinated at 9 and 13 weeks of age with a commercial dose of Vaxsafe® PM.

Concurrent with the first Vaxsafe® PM vaccination, birds received a combined inactivated Newcastle

Disease and Egg Drop Syndrome (ND+EDS) vaccine intramuscularly, live Fowl Pox vaccination by wing

stab, and approximately 15% of birds received live AEV vaccination by eye drop. Blood sampling of 20

birds was undertaken at 13 weeks of age to demonstrate serological evidence of the response to these

33

vaccinations, and repeated at 20 weeks of age. A further sampling of 30 birds was undertaken at 60 weeks of

age for ND serology only. This would thus demonstrate, by comparison to a matching shed of birds not

vaccinated with Vaxsafe® PM, whether the administration of Vaxsafe

® PM had a negative effect on the

efficacy of the concurrently administered vaccines.

No significant differences in serological titres between vaccinated and unvaccinated chickens were found for

AEV at 13 and 20 weeks, EDS at 20 weeks (not tested at 13 weeks), or NDV at 13 weeks. NDV titres at 20

weeks were significantly lower in the vaccinated group than the unvaccinated group, however, no significant

difference was present at 60 weeks. Titres in both groups were in excess of those required by the National

Newcastle Disease Management Plan (Animal Health Australia, 2008) to produce protective immunity

against Newcastle Disease.

The ND+EDS, Fowl Pox and AEV vaccines are therefore considered compatible for concurrent use with

Vaxsafe® PM.

PMP R0807: A study to assess the reversion to virulence of Vaxsafe® PM after 5 passages in SPF chickens

This trial was undertaken in accordance with VICH Guideline 41, which describes the procedure for

examining the potential for reversion to virulence of candidate live vaccines for the purposes of registration

in Europe.

Two six-week old SPF chickens were vaccinated with a 1xMaxRT dose of Vaxsafe® PM. Twenty-four hours

later, the chickens were euthanized and a section of the pectoral muscle injected with Vaxsafe® PM and the

spleen were harvested at necropsy. These tissues were selected for sampling as being those most likely to

permit re-isolation of the vaccine strain, on the basis of the dissemination results from trial PMP R0715. The

samples were homogenised and the bacterial strain recovered. This was then inoculated into the pectoral

muscle of a further two six-week-old SPF chickens. This process was repeated for a total of five passages. In

the final passage, the inoculate was injected into 8 six-week-old SPF chickens which were then observed for

21 days. The bacterial strain recovered after each of the first four passages was also compared to Vaxsafe®

PM and wild-type P. multocida strain X-73 by PCR to detect any variation in the DNA segment containing

the aroA gene.

Viable bacteria were recovered after each of the 24-hour passages. Viability of the auxotrophic bacterial

strain was potentially enhanced by the presence of homogenised muscle and spleen tissue, which may well

have supplied the organism with exogenous aromatic acids. One bird died immediately following injection of

second passage homogenate, with a necropsy examination unable to determine an obvious cause of death.

Another bird was inoculated to allow the trial to continue. One of the eight chickens injected with final

passage homogenate also showed an adverse reaction, was subsequently unwell for 6 days and then normal

for the remainder of the 21 day observation period. Given the peracute nature of the reactions seen in two

birds, it is unlikely the reactions were indicative of a lack of vaccine safety. Potential anaphylactoid reactions

to the tissue homogenate inoculation have been suggested as a possible cause.

The remaining chickens at each passage level showed no clinical signs, including those of the final passage

monitored for 21 days. Vaxsafe® PM therefore showed no reversion to virulence when passaged in vivo

according to VICH Guideline 41.

The P. multocida vaccine strain was re-isolated from passages 1 to 5 (see Table 13).

34

Table 11. Total Viable Counts (TVC) at each passage level.

Passage Number

TVC (cfu/mLa)

Target Retained Returned

P0 108.7 109.0 108.9

P1 N/A 107.4 N/A

P2 N/A 107.3 N/A

P3 N/A 107.0 N/A

P4 N/A 108.7 N/A

P5 N/A 108.3 N/A

a cfu/mL – colony forming units per millilitre

At each passage level, the re-isolated PM strain showed an identical result following PCR analysis of the

aroA gene region as Vaxsafe® PM. This demonstrates that the genetic mutation (i.e. deletion) remained

stable through the in vivo passaging.

Figure 8 below shows an electrophoresis gel of DNA products obtained from PCR analysis of X-73 (wild

type), the initial inoculum of Vaxsafe® PM (P0), and products of P1 to P5 tissue homogenate material. The

X73 wild type control shows the expected 1337 bp sized PCR fragment. The Vaxsafe® PM inoculum (P0)

and products of P1 to P5 on the other hand shows a noticeably smaller band size of 1035 bp indicating that

they are derived from the Vaxsafe® PM (aroA gene delete) vaccine strain. This gel also demonstrates that no

macro genetic changes had taken place around the aroA deletion site over the course of the five in vivo

passages.

35

Figure 8. Gel electrophoresis analysis of PCR products obtained from amplification of the (aroA gene

delete) region of Pasteurella multocida X-73 (wild type), the initial inoculum of Vaxsafe® PM (P0), and

products of P1 to P5 tissue homogenate material.

PART B: Vaxsafe® PM for use in Turkeys

(i) Efficacy studies

PMP R0804: A dose response study using Pasteurella multocida (X-73) challenge strain in turkeys

It was proposed to extend the use of Vaxsafe® PM from chickens to turkeys. In order to demonstrate the

efficacy of Vaxsafe® PM in turkeys, it is necessary to have a challenge strain of P. multocida that produces a

level of mortality in unvaccinated birds that is compatible with regulatory requirements. US and European

monographs require mortality or severe signs of disease in at least 70 to 80% of unvaccinated birds for a

challenge to be considered valid. P. multocida strain X-73 is homologous to Vaxsafe® PM, and has produced

consistent challenge results in chickens, and was therefore the challenge strain selected for evaluation in

turkeys.

Four groups of 15 turkeys, 6 weeks of age, were placed in separate isolators. One group received no

treatment and remained as a control group. The other three groups received P. multocida challenge strain X-

73 at one of three doses: 102.6

, 103.3

or 104.0

cfu. Each dose was given as a 0.5 mL intramuscular injection,

and the birds were observed for deaths or signs of disease.

All three doses produced mortality rates of 100% within three days of challenge administration. The highest

dose produced the quickest deaths, with all birds in that group dying by the end of the first day after

challenge administration. The control turkeys remained healthy.

Any of the three doses would meet the regulatory requirements for a challenge model to demonstrate the

efficacy of Vaxsafe® PM in turkeys.

PMP R0808: A dose confirmation study to determine efficacy of Vaxsafe® PM Vaccine (living) in commercial turkeys vaccinated with the chicken end-of-shelf-life-titre (EOSLT) dose

It was proposed to extend the use of Vaxsafe® PM to turkeys, and thus this trial was undertaken to examine

the efficacy of the vaccine in this species. For simplicity, a vaccine dose and administration regime identical

to that already defined for chickens was chosen. This required the use of two intramuscular (IM) doses

delivered four weeks apart, with the first dose administered to turkeys six weeks of age. The target dose was

the current end of shelf life titre (EOSLT), 107.2

cfu in 0.2 mL. Furthermore, the use of subcutaneous

administration, at the back of the neck, was investigated, using the same dose and volume, in order to allow

producers to avoid potential damage to the valuable breast meat. Approximately four weeks after the first

and second doses of vaccine, sub-groups of turkeys were challenged with virulent P. multocida strain X-73,

to allow determination of vaccine efficacy.

36

Table 12: PMP R0808

Group Bird # Purpose

Dose 1 Dose 2

Vacc.

(Day 0)

Challenge X-73

(Day 28)

(Day 25)

Vacc

(Day 28)

Challenge X-73

(Day 53)

Inject

Left side

Inject

Right side

Inject

Right side

Inject

Left side

A 15 Negative controls Diluent IM N/A Diluent IM N/A

B 36 Efficacy by IM

injection

EOSLT

IM 15 birds EOSLT 15 birds

C 30 Efficacy by SC

injection

EOSLT

SC 15 birds EOSLT 15 birds

D 30 Positive controls Diluent IM 15 birds Diluent IM 15 birds

Administration of the vaccine produced a higher incidence and severity of adverse effects than had been

expected. Both groups administered IM and SC groups demonstrated signs of depression for at least a week

after vaccination, with one death in the IM group and three in the SC group. Although unexpected from

previous experience with chickens, these signs were consistent with the observations of the concurrently-run

safety trial PMP R0806 (summarised below). The second dose of vaccine produced a shorter duration of

adverse effects.

Challenge after a single dose of vaccine produced a Protective Index (PI) of 53% for birds administered the

vaccine IM, and 33% for those administered an SC dose. The PI rose after two doses of vaccine to 100 % for

IM administration, and 73% for SC administration. Examination of the injection sites in five birds necropsied

two weeks after the second dose of vaccine showed no lesions in most birds.

Although the efficacy of the vaccine was satisfactory after two doses of the vaccine, the adverse effects seen

after vaccine administration, in parallel with the results of trial PMP R0806, indicate that these

administration regimes are not satisfactory for the turkey. This led to the investigation of alternative routes of

administration for the vaccine in this species, in trials PMP R0901 and PMP R0902 (summarised below).

PMP R0902: Efficacy of Vaxsafe® PM Vaccine (living) in turkeys, using alternative routes of administration

This trial investigated the efficacy of Vaxsafe® PM when administered to 9 week old turkeys by inoculation

into the wing web (WW). This route of administration was chosen after previous trials (PMP R0806 and

PMP R0808) demonstrated that intramuscular or subcutaneous administration alone led to an unacceptably

high level of adverse effects.

Initially, it was planned that the first dose of Vaxsafe® PM would be administered to these birds by the oral

route, in order to provide a “priming” effect. However, this route was shown by accompanying safety study

PMP R0901 to also cause an unacceptably high level of adverse effects. Therefore, the design of this trial

was revised to the following: a total of 56 turkeys were enrolled into 4 groups, as shown in Table 13 below.

A negative control group, receiving neither vaccine nor challenge, was shared with the revised design of

PMP R0901, run concurrently. A second unvaccinated group provided the control birds for two separate

challenges. Two groups received Vaxsafe® PM: one group received two WW doses before challenge, whilst

37

the other group received two WW doses followed by a subcutaneous dose at the nape of the neck. Each dose

was at the end of shelf life titre (EOSLT 107.2

cfu), and the interval between each dose, and between last dose

and challenge, was 28 days in each case. Challenge was undertaken using virulent strain X-73, at a target

dose of 104.0

cfu administered intramuscularly.

Table 13: PMP R0902

Group Number of

turkeys

Treatment Challenge Termination

Day 0 Day 28 Day 56 Day 84

A 11 Nil Nil Nil Nil

Day 98 if

PMP R0901

also complete

B 12 WW WW Challenge

X-73 N/A

Day 98

C 13 WW WW SC Challenge

X-73

D 20 Nil Nil

Challenge

X-73

(half)

Nil (half)

Challenge

X-73

(remaining)

Birds showed no systemic signs of disease after administration of vaccine. However, local lesions at the

wing web site of inoculation were significant, with open wounds seen 1 to 2 weeks after inoculation, and

although resolving, lesions were still detectable at the conclusion of the study in some cases.

The challenge was effective, causing 100% mortality in the unvaccinated challenged birds at both challenges,

albeit at doses somewhat higher than targeted. The vaccine demonstrated a Protective Index (PI) of 64% for

the group administered two WW doses, and a PI of 54% for the group administered two WW doses and a

subsequent SC dose.

Whilst wing web inoculation did produce significant lesions at the site of inoculation, this route of

administration provided for far fewer systemic adverse effects whilst maintaining reasonable efficacy.

(ii) Safety studies

PMP R0806: A study to determine the safety of Vaxsafe® PM Vaccine (living) in turkeys

As part of the proposal to extend the use of Vaxsafe® PM to turkeys, a safety trial in this species was

undertaken, in association with an efficacy trial PMP R0808 (as summarised above in Efficacy Studies).

A total of 54 turkeys were the subject of the study, and assigned into trial groups as shown in Table 14

below.

38

Table 14: PMP R0806

Group Pen

# # of

Turkeys Group Purpose

Treatment Vaccine Dose

(cfua/0.2

mL)

Termination (days post-

vaccination)b Day 0 Day 28

Ac 1 15 Negative Controls

Sterile Diluent

Sterile Diluent

Nil 59

B 2 15 Safety of

10xMaxRT

Vaxsafe® PM

(10x MaxRT) Nil 10

9.1 59

C 3 24 Repeat

d dose of

1xMaxRT

Vaxsafe® PM

(1x MaxRT)

Vaxsafe® PM

(1x MaxRT) 10

8.1 59

a. cfu = colony-forming units. b. Days post initial vaccination. c. Group A turkeys will also act as negative control birds for the PMP R0808 study which will run concurrently with R0806

(but commence 3 days later). d. Repeat dose given four (4) weeks post dose 1.

Fifteen of these were a control group which received no vaccine treatment. A further fifteen received an

intramuscular dose of Vaxsafe® PM equivalent to 10 times the maximum release titre proposed for chickens

(i.e. approximately 109.1

cfu). A third group of 24 turkeys were to receive two intramuscular doses of

Vaxsafe® PM, four weeks apart, with each dose equivalent to the maximum release titre proposed for

chickens (i.e. approximately 108.1

cfu/mL). The birds were six weeks of age at the first dose of vaccine.

The turkeys administered a dose of Vaxsafe® PM exhibited a level of adverse effects that was unexpectedly

high, in comparison to chickens which received the same doses. The pattern of adverse effects was also

different from that seen in chickens. After initially showing signs of depression, the turkeys appeared healthy

until four to eight days later, when effectively all turkeys became moribund and required euthanasia.

Necropsy examination revealed evidence of multiplication of the vaccine strain of Pasteurella multocida

around the site of injection and in the hock joints of several birds.

The vaccine administration regime as used in chickens is clearly not useful in turkeys. These results led to

trials PMP R0901 and PMP R0902 to investigate alternative routes of administration as a means of

ameliorating the adverse effects

.

PMP R0901: A study to determine the safety of Vaxsafe® PM Vaccine (living) in turkeys

Given the unacceptable safety profile seen in trial PMP R0806 when Vaxsafe® PM was administered to

turkeys by intramuscular injection, it was proposed to examine the safety profile of the vaccine when

administered using an oral “priming” dose, followed by wing-web (WW) administration and/or subcutaneous

(SC) injection.

A total of 65 commercial turkeys, approximately 6 weeks of age, were enrolled into the trial, and assigned

one of the trial groups shown in Table 15 below (original trial design). All groups with the exception of the

control groups N and A received an oral dose of Vaxsafe® PM on trial day 0. It was considered that the oral

dose would likely “prime” the birds, giving them a low level of protective immunity against the vaccine

strain which would in turn then protect them against the adverse effects seen when the vaccine was

administered either by the wing-web or subcutaneous routes.

39

Table 15: PMP R0901 (Original study design)

Group # of

Turkeys

Treatment Termination

Day 0 Day 28 Day 56

N

10 Nil Nil Nil

Day 70 or when PMP R0902

complete A 10

Oral

Water + SMP only

WW

Diluent only (RIGHT)

SC

Diluent only

B 15 Oral

10xMaxRT

WW

10xMaxRT

(RIGHT)

WW

1xMaxRT

(LEFT)

Day 70 C 15

Oral

10xMaxRT

Oral

1xMaxRT

WW

10xMaxRT

(LEFT)

D 15 Oral

10xMaxRT

Oral

1xMaxRT

SC

10xMaxRT

Unfortunately, birds in groups B, C and D developed clinical signs of disease following the initial oral dose.

The signs were very similar to those seen in previous trial PMP R0806, consistent with systemic

pasteurellosis. Significant morbidity and occasional mortality was seen in these groups, with deaths

occurring from six days after administration. As this safety profile was clearly unacceptable, Groups B, C

and D were culled and the remaining birds (Groups N and A) re-assigned into a modified study design, as

shown in Table 16 below.

Table 16: PMP R0901 (Modified study design)

Group # of

Turkeys

Treatment Termination

Day 0 Day 28 Day 56

N* (A)

11 Nil Nil Nil

Day 70 or when PMP R0902

complete A* (1) 10

WW

10xMaxRT

(LEFT)

WW

1xMaxRT

(RIGHT)

SC

10xMaxRT

*: Group N was renamed Group A, and Group A renamed Group 1

40

No systemic signs of disease were noted after administration of any of the three doses of Vaxsafe® PM to

Group 1 (two WW doses, one SC). However, significant local lesions in the WW became present within a

few days of administration, generally noted as open, necrotic wounds. These slowly resolved, although

chronic granulomatous inflammation was still present at the conclusion of the trial in several birds, and those

with no obvious lesions generally showed notable contracture of the wing, consistent with a resolved

inflammatory process. Culture of these wounds 7 days after administration showed vaccine strain PMP-1 to

be still present and viable. Lesions at the SC site of injection were not as pronounced, but inflammatory

masses were often present at this site at the termination of the trial.

Whilst the WW route of administration, followed by a SC dose, showed an improved safety profile in terms

of systemic disease, compared to IM administration, the local lesions were still a significant concern, and

would jeopardise commercial acceptance of the product.

PART C: Vaxsafe® PM for use in Ducks

(i) Efficacy

PMP R0903: A dose response study using Pasteurella multocida (X-73) challenge strain in ducks

In order to extend the use of Vaxsafe® PM to ducks, it was necessary to establish a challenge system that

would provide an appropriate level of morbidity and mortality in unvaccinated ducks to conform with the

European and US regulatory monographs. It was proposed to use the homologous challenge strain X-73 for

this purpose, as this strain produced a consistent level of clinical signs in challenges administered to

chickens.

A total of 51 commercial Pekin ducks, six weeks of age, were enrolled in the trial and assigned a trial group

as shown in Error! Reference source not found. below. As ducks are known to be relatively resistant to the

effects of P. multocida, the doses used were equal to and higher than the effective dose used in chickens. The

highest dose directly available from the banked seed of X-73 was 107.5

cfu per 0.5 mL dose.

Table 17: PMP R0903

Group Number of ducks X-73 Dose

(cfu/0.5mL) Mortality

A 10 Nil 0/10

B 11 104.0

7% (1/15)

C 15 105.5

13% (2/15)

D 15 107.5

20% (3/15)

41

As can be seen from Error! Reference source not found., the maximum level of mortality seen with these

doses of X-73 was 20%, well below the 70 % to 80% minimum required by the regulatory monographs.

This trial demonstrates that X-73 will not be a suitable challenge strain for demonstration of Vaxsafe® PM

efficacy in ducks. Efficacy trials in this species can not commence until a suitable challenge strain is sourced

and characterised.

(ii) Safety

PMP R0904: A study to determine the safety of Vaxsafe® PM Vaccine (living) in ducks

In order to determine the safety of Vaxsafe® PM in ducks, a total of 50 commercial Pekin ducks, five to

seven weeks of age, were enrolled into the trial and assigned one of the trial groups shown in Table 18

below.

Table 18: PMP R0904

Group Pen No.

No. of Ducks

Group Purpose

Treatment Vaccine Dose

(cfu/0.2 mL)

Termination

Day 0 Day 28 Day 56 (days post-vaccination)

A 1 10 Negative Controls

NIL NIL NIL Nil 70

B 2 10 ORAL ORAL

5x MaxRT

NIL NIL 108.8

14

C 3 10 IM IM 5X

MaxRT NIL NIL 10

8.8 14

D 4 10 WW WW 5X

MaxRT NIL NIL 10

8.8 14

Ec 5 10 Repeat dose

TBD 1X

MaxRT

TBD 1X

MaxRT

IM 1X

MaxRT 10

8.1 70

Given the significant adverse effects seen in turkeys when administered the 10xMaxRT dose, it was

considered suitable to administer initially a 5xMaxRT dose to the ducks and observe their response.

No adverse effects were observed in any of the treatment groups. Ducks appear to be highly resistant to any

adverse effects (e.g. endotoxicity) induced by the vaccine.

42

PMP R1001: A study to determine the safety of a 10X overdose of Vaxsafe® PM Vaccine (living) in ducks

As trial PMP R0904, summarised above, demonstrated that a 5xMaxRT dose of Vaxsafe® PM was safe in

ducks, it was proposed to satisfy regulatory requirements by demonstrating the safety of a full 10xMaxRT

dose of the vaccine in a small number of ducks.

A total of 12 ducks were enrolled into this trial. Ten ducks were administered Vaxsafe® PM at a dose of

approximately 109.1

cfu (10xMaxRT), with a further two ducks held as untreated controls. All 12 ducks were

held in the same pen of the APCAH Animal House and observed for 16 days. Holding both groups in the

same pen was considered acceptable as previous trial PMP R0715 had demonstrated in the chicken that

lateral transmission of the vaccine was very unlikely. Given the similar safety results to date in the duck, it

was considered that the likelihood of lateral transmission in the duck was also very low.

All ten vaccinated ducks showed no signs of adverse effects after administration. The safety profile of

Vaxsafe® PM appears to be very satisfactory in ducks.

OVERALL SUMMARY All registration based studies for chickens, and commercial bioprocessing work has now been completed on

the candidate PMP-1 vaccine strain. Vaxsafe® PM has been developed as a freeze dried product to protect

against fowl cholera disease in chickens. Vaxsafe® PM has been demonstrated to be a safe product in

chickens. There are no persistent clinical signs, no signs of systemic pathology and no adverse effect on body

weights even when a large overdose is administered to chickens. It has been shown not to persist in vivo

beyond a few days in chickens and is shown to be genetically stable. Safety studies have demonstrated no

reversion to virulence as has been the case for other commercial live attenuated P. multocida vaccines.

Hence, Vaxsafe® PM will not cause the vaccine-induced Fowl Cholera in chickens that has been associated

with other live Fowl Cholera vaccines.

Numerous efficacy studies performed have shown that Vaxsafe® PM affords protection against FC in

chickens following homologous and heterologous challenges.

Vaxsafe® PM will be available in 500, 1000 and 2000 dose sizes. The product is to be resuspended in sterile

diluent prior to application by IM delivery into the pectoral muscle of chickens 8 weeks and older. The

vaccination regimen will be a 2 dose vaccination with first vaccine delivered at around 8 weeks and the

booster at 4 weeks post dose 1.

Vaxsafe® PM is currently under review by the APVMA with results of the review expected by September

2010.

The safety profile for Vaxsafe® PM in turkeys did not prove to be as good as for chickens. Adverse effects

induced by the vaccine (i.e. lameness and death) show that turkeys are clearly far more sensitive to the

effects of the vaccine. Hence registration for turkeys will not be pursued.

The safety profile for Vaxsafe® PM in ducks was excellent. In fact, no adverse effects were noted even after

a 10X overdose. This resistance to the effects of P. multocida was demonstrated by the failure to induce

disease or death in ducks despite even a high titre inoculation with wild type X73. Bioproperties is currently

pursuing the use of other more virulent strains of PM in order to establish a suitable challenge model for

ducks.

43

44

Plain English Compendium Summary

Project Title:

Towards rapid Registration of a live Pasteurella multocida vaccine

Project No.: CRC 03-12

Researcher: Principal Investigator Dr Peter C. Scott

Organisation: University of Melbourne

Phone: (03) 9731 2265

Fax: (03) 9732 7576

Email: pcscott@unimelb.edu.au

Objectives To demonstrate that a live Pasteurella multocida candidate vaccine, Vaxsafe PM, is safe and efficacious in the control of Fowl Cholera and to successfully commercialise the vaccine by the establishment of experimental protocols and outcomes that meet both domestic and international regulatory requirements to achieve registration of Vaxsafe PM

Background Fowl Cholera is disease of commercial poultry caused by the bacteria Pasteurella multocida. This disease causes significant production losses in commercial birds and particularly those that are housed in alternate systems such as barn and free range. Control has been by the use of therapeutic antibiotics and killed autogenous vaccines. The increasing restrictions on the use of antibiotics and the limited efficaciousness of killed vaccines has limited the ability to control this disease in the increasing numbers of birds that are being maintained under barn or free range conditions. Fowl Cholera is also a disease of other poultry such as turkeys and ducks.

Research The candidate vaccine, Vaxsafe PM, was tested to establish that it was safe to used in chickens and safe for release into the environment. Experimental protocols were established to develop fowl cholera challenge models in chickens. Studies were then undertaken to see if Vaxsafe PM protected chickens against a number of strains of Pasteurella multocida and for what period of time after vaccination this protection lasted. Laboratory studies were concurrently undertaken to optmise the methods involved in the commercial manufacture of the vaccine.

45

Outcomes Extensive studies have demonstrated that Vaxsafe PM is both safe and effective in protecting chickens against fowl cholera. In turkeys the vaccine when administered by a number of different routes was found to cause illness and thus was not considered safe. Preliminary work in ducks indicated that the vaccine was safe but further studies are required to demonstrate if it will protect against fowl cholera.

Implications The project has achieved its primary aim of developing a registrable vaccine that is safe and protects commercial chickens against fowl cholera.

Publications Scott, P.C., Markham, J.F. and Whithear, K.G. (1998). Safety and Efficacy of Two Live Pasteurella multocida aro-A mutant Vaccines in Chickens. Avian Dis. 43:83-88