Control of Pasteurellosis in pigs and poultry

• Veterinary Research Institute, Sri Lanka
• National Institute of Veterinary Research, Vietnam
• National Veterinary Company, Vietnam
• Monash University, Australia

The bacterium Pasteurella multocida is the causal agent of a range of diseases generally known as pasteurellosis (in chickens, ducks, turkeys it is also termed fowl cholera and in pigs porcine haemorrhagic septicaemia).
Vietnam has over 80 million chickens, about 23 million ducks, and around 15 million pigs. These animals contribute substantially to almost all country households, and pigs are also raised intensively on farms near Ho Chi Minh City. In Sri Lanka too, poultry are major sources of food and income for rural families, but there are also large commercial farms.
Australians eat millions of chickens and eggs every year. Most Australian chickens and pigs are raised in intensive enterprises where virulent forms of pasteurellosis could cause severe losses. Haemorrhagic septicaemia is common in pigs in Vietnam, but rarely seen in Australia. However, in its first serious appearances, around 1994, it killed over 2200 animals.
These diseases are usually fatal, and outbreaks can lead to large losses of stock. Vietnam, Sri Lanka, and Australia would all benefit greatly if there were effective vaccines against virulent strains/serotypes of the bacterium.

This project aimed to develop live vaccines against all forms of virulent pasteurellosis, and to improve on the bacterins (vaccines made from killed bacteria or non-living products of bacteria) that were currently available. The main objective was to gain greater control of these diseases, thereby benefiting many rural communities in Vietnam, Sri Lanka and Australia.

Firstly, researchers examined the problems encountered in each country, seeking to ascertain what triggers outbreaks of disease and what forms of P. multocida are endemic in each place. Tissues from diseased pigs, ducks and chickens were regularly sampled in Vietnam. Researchers in Sri Lanka with special expertise identified the strains of the bacterium from those samples. They also used electrophoresis and molecular-based techniques to distinguish the patterns of infection due to each strain.
Secondly, researchers ran vaccine trials that involved preparation of a new vaccine, injecting it into chickens, administering booster shots, and finally exposing the chickens to the live bacterium to test their immunity. Australian strains of P. multocida, which have extraordinarily low virulence, were tested as one experimental live vaccine. Project scientists also attempted to decrease the virulence of other strains by growing them at relatively high temperatures.
Thirdly, researchers manipulated genes within the bacterium, endeavouring to produce a mutant that would be effective as a vaccine. These three approaches were necessary because the initial bacterins were not derived from appropriate strains of P. multocida, so vaccinated animals could not generate the most effective antibodies to fight an outbreak of disease in each region.

Over 300 isolates of Pasteurella multocida from cases of haemorrhagic septicaemia in pigs and fowl cholera in poultry and ducks were serotyped. The work confirmed that bacteria from particular serogroups are responsible for specific disease syndromes, and that there is an extremely virulent subset of Serotype A1 causing fowl cholera in Vietnam.
In particular, it was shown for the first time that isolates from concurrent outbreaks of haemorrhagic septicaemia in pigs and cattle/buffalo were identical. This had been suspected, but never confirmed. The information obtained allowed the principal vaccine manufacturer in Vietnam to rationalise vaccine production and to choose from (local) strains of bacteria with the best potential to make protective vaccines.
Existing and new bacterins were evaluated. All had shortcomings in the degree of immunity they conferred and in the persistence of even minimal immunity. An aluminium-hydroxide-based adjuvant was shown to be as effective as the traditional aluminium sulfate adjuvant, and it proved to be a more easily administered product that caused fewer tissue reactions post-vaccination. However, because of the cost, the improved adjuvants have not yet been widely adopted.
An Australian isolate of P. multocida with a very low virulence for chickens was selected as a live vaccine candidate. Subjecting it to in vitro metabolic stress produced a genetic drift mutant with an RNA polymerase defect. This form was shown to be less tissue-invasive but still able to invoke very solid immunity when challenged with highly virulent strains isolated from disease outbreaks in Vietnam. The live vaccine candidate was tested in the laboratory and in preliminary field tests where it performed well.
Researchers confirmed that live vaccines elicited an antibody response in chickens that was more protective than that elicited by bacterins, but they could not identify the actual antigens inducing the protection using the techniques available. Molecular studies at Monash University identified several proteins expressed by the bacteria in vivo and in vitro, which had theoretical potential as vaccines. Small quantities of these proteins were expressed in vectors, and showed some protective effect when used as vaccines in mice. (Research subsequent to the project, however, has not confirmed any protective effects in chickens.)
The project has helped Vietnamese institutes to greatly improved their ability to diagnose P. multocida-related diseases, to isolate the causative bacteria, and to classify them more thoroughly than previously. Research continues beyond the project at all the institutes involved. At UQ, several postgraduate students have undertaken research into aspects of the pathogenesis of Pasteurella-induced diseases.
In Vietnam, both NIVR and NAVETCO have undertaken considerable further research with the mutant live strain. A strong relationship persists between all institutes involved in the Pasteurella project. At least three separate projects were initiated following formation of project partnerships, in response to specific animal health problems encountered during field trips and contact with the farming community.
A particularly strong research partnership between members of the veterinary school at UQ and the Molecular Pathogenesis section of Medical Microbiology, Monash, has developed. Collaborative research on the molecular pathogenesis of Pasteurella infection has been particularly productive, and has resulted in many scientific papers, multiple PhD theses, a book chapter, and joint presentations at several international conferences, since the end of the ACIAR project. This activity has established a significant national and international profile for these institutes in this area of research.