I. INTRODUCTION
To successfully develop a challenge model to simulate actual infection as seen in production practices, a seeder challenge model was proposed and previously shown to be an effective method to infect poultry (Cox et al., 1996, 2020). However, applying it in this way to young layers is novel. This pilot study was developed with the objective of determining the success of this challenge method. A second objective was to determine the base line effectiveness of the vaccine against the seeder challenge method. Once established, the methodology could be used to assess any confounding effect of a live Salmonella vaccine protection against wild Salmonella. Identifying the best challenge period, day 0 or week 3, in young birds will set the basis for a successful challenge and control with the current vaccine programs available in Australia.
II. METHOD
The intention was to demonstrate if seeding birds at day old or 3 weeks will achieve colonisation of the control birds and if the vaccination protocol demonstrates protection at this point (a colonisation inhibition effect from the oral vaccine was anticipated following application). Pilot study design (looking at different challenge times under vaccination) was: live Aro-A deletion S. enterica serovar Typhimurium vaccine administered by coarse spray at day 0 and orally (gavage) at day 19 at single label dose rate per bird.
There were two floor pens allocated for each group of 8 birds (day old layer chicks). Pens for groups 1 and 3 were separated from groups 2 and 4 and biosecurity practices enacted between these to prevent cross contamination before the three-week challenge. Seeder birds were housed two per pen in suspended cages above the birds in each challenge pen for 5 days. The seeders were given an oral inoculation with 106 live S. Typhimurium DT 135 at either day 0 or at week 3. After 5 days, the seeder birds were released into the pens. Cloacal swabs were collected at 10, 17, 24, 31 days of age with caeca collected at 35 days of age and cultured for the presence of S. Typhimurium.
All microbiological testing was performed at NATA accredited laboratory (Birling Avian Laboratories, Bringelly, NSW). Rapid finder Multiplex PCR was used to detect and implemented for testing. A selection of positive results was culture confirmed (XLD/Hektoen agar plates).
III. RESULTS
Horizontal transmission from the seeder birds resulting in infection of the challenge birds is shown in Table 1.
Table 1 - Floor pen birds; The number of cloacal or caecal samples positive for Salmonella Typhimurium. Control groups challenged at day 0 or 3 weeks of age (106 cfu). Vaccinated groups challenged at day 0 or 3 weeks of age (106 cfu).
Seeder birds challenged at day 0 clearly showed better uptake than at 3 weeks for unvaccinated and vaccinated birds in this trial, Table 2.
Table 2 - Seeder Birds; The number of cloacal or caecal samples positive for Salmonella Typhimurium. Groups challenged at day 0 or 3 weeks of age (106 cfu).
The day 0 coarse spray vaccination program used in this study was unsuccessful against the seeder challenge at day 0 with no significance difference in the proportion of vaccinated and control birds returning a positive cloacal swab at each sampling point. Oral vaccination at 19 days did not provide any further protection against challenge from day 0.
The week 3 challenge achieved colonisation in only three out of 16 control birds (19%) at 31 days and only two birds from 16 birds at day 35 from the caeca. This was not significantly different from the vaccinated group. The colonisation of the seeder birds at 3 weeks was successful (7/8 birds with positive cloacal swabs at 4 days post infection) but this did not transmit effectively to the treatment birds.
IV. DISCUSSION
Horizontal transmission results from seeder birds have been shown to facilitate gradual intestinal colonization (Muir et al., 1998). Isolation of S. Typhimurium from the cloacal swabs of the control and vaccinated birds showed that there was consistently successful uptake by day 0 challenged birds. Young chicks (under 3 weeks of age) are regarded as much more susceptible to Salmonella infection than older birds and an infection establishing at this early age can allow continuous presence of the organism for long periods (Gast and Porter, 2020). Overall, the study demonstrated that the model of using seeder birds from day 0 could successfully horizontally transmit S. Typhimurium mimicking an early natural infection. Although infection of the seeders in cages at 3 weeks of age was successful, this did not transmit successfully to the birds on litter at 3 weeks of age. The caged seeders remained susceptible to the infection while the birds reared on the floor achieved resistance to infection, perhaps due to differences in establishment of a gut flora due to having access to the floor. The day 0 administration of the live Aro-A deletion S. enterica serovar Typhimurium vaccine was overpowered by this challenge method.
Presented at the 34th Annual Australian Poultry Science Symposium 2023. For information on the next edition, click here. 
