A veternarian takes a nasal swab for testing.
The vaccination programme is being rolled out across New South Wales, Queensland and Victoria.
It is understood the second shipment will be 130,000 doses, completing the total order of 150,000 vials by federal authorities.
Veterinarian were injecting horses in NSW and Queensland over the weekend, with Victoria's programme - which centres only on horses involved in the upcoming spring carnival - expected to begin today. Victoria is hoping for enough vaccine to eventually inoculate all racehorses in the state.
NSW began its programme by establishing the southern buffer zone near Mittagong, south of Sydney, vaccinating almost 300 horses.
"This is part of our plan to create a wall of immunity to prevent the disease creeping out into unaffected regions," said Department of Primary Industries deputy chief veterinary officer Steve Dunn.
"Ponies, donkeys, miniature horses and companion horses are part of the first wave of animals vaccinated," he said.
Mr Dunn said the state's horse owners needed to understand that vaccine would not be made available widely. "There will not be a free-for-all when it comes to this vaccine," he said.
"We are using it strategically and with precision to prevent the spread of the disease from known infection sites."
"Coupled with zoning arrangements now in place across NSW, the equine flu control and eradication campaign still hinges on vigilance from horse owners and the general community."
The state's vaccination programme continued throughout the weekend.
Queensland also began inoculations, with 1990 horses receiving the vaccine by the end of the weekend.
The state has 7940 does in its possession with a further 1300 still in Sydney on standby.
All horses receiving the vaccine will require a second injection to create complete immunity.
The number of infected properties in NSW stood at 2653 at the weekend, with an extra 323 considered dangerous, and 356 suspect.
"The disease containment situation remains encouraging and there is still reason to be optimistic about the prospects for eradication," a spokesman said, "although a sustained response effort will be required over the next six or more months to achieve this goal."
The president of the Equestrian Federation of Australia (EFA), Geoff Sinclair, has assured members that non-racing horse interests are being heard over the outbreak.
"Racing may get most of the publicity because the public identifies with this but I can assure you the personal representation to Government and the inclusion of federation representatives on industry committees working to solve the problem has been very strong and well received.
"Franz Venhaus, our EFA CEO is a representative on the National Management Group that decides on what action will be taken. Barry Smyth represents the whole horse industry on this group on behalf of the Australian Horse Industry Council and he and Franz have been fantastic in ensuring that our sport is understood and heard."
Mr Sinclair said there is much debate as to whether Australia should just get on with permanently vaccinating the whole horse population.
"We can assure you there is no guarantee that vaccination stops the disease. The cost to do this compared to the possibility of becoming EI-free is around $A100 million a year - about $200 per horse - which each of us will have to bear."
Meanwhile, it is possible the 250 horses confined to Morgan Park, Warwick, may be allowed home soon.
The horses and about 100 caregivers have been under lockdown since late August. It is understood the official quarantine period ends this week, but there is no formal word on when the horses and their owners can return home.
The makeshift settlement has been named Morgantown, and even has its own temporary school.
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