A Long-Term Efficacy Trial of a Live, Attenuated Salmonella Typhimurium Vaccine in Layer Hens

Abstract

Salmonella remains one of the most common causes of bacterial foodborne gastrointestinal disease in humans. Raw eggs or food items containing undercooked eggs are frequently identified as the source of Salmonella. Salmonella Typhimurium contamination of table eggs most commonly occurs when they are laid in a contaminated environment. Several control strategies, including vaccination, are widely used to mitigate the total Salmonella load. It is unclear, however, whether live attenuated Salmonella vaccines are efficacious over the life span of a layer hen. Live attenuated Salmonella vaccines have been favored due to their ability to illicit a strong humoral immune response. The lifespan of a layer hen ranges between 60 and 80 weeks and the long term efficacy of attenuated vaccine strains has not been investigated. In this study, commercial brown layer chicks were vaccinated at day old, 6 weeks of age, and again at 10 weeks of age with the Bioproperties Vaxsafe^TM STM1 aroA mutant vaccine. Birds were challenged at 18 weeks of age with Salmonella Typhimurium DT9 (MLVA 03 15 08 11 550). Feces and eggs were monitored for S. Typhimurium for 40 weeks post-infection. Birds produced a strong immune response following the final dose which was administered intramuscularly. The serum antibody response to S. Typhimurium DT9 infection did not differ between challenged groups. Fecal shedding and egg contamination was highly variable and did not differ significantly between vaccinated and unvaccinated birds that had been challenged with S. Typhimurium DT9. Total bacterial load in feces was quantified using qPCR. No significant difference was detected between unvaccinated and vaccinated birds after challenge.

Keywords: Salmonella Typhimurium; Salmonella vaccine; egg contamination; layer hen; persistent infection.

FIGURE 1

Shedding pattern of S. Typhimurium DT9 in feces. Fecal samples were collected and tested for the presence of S. Typhimurium DT9 using standard culture methods over a 41 week period. Data are presented as mean proportion of S. Typhimurium positive fecal samples ± the standard error. Samples collected from challenged only (black line) as well as vaccinated and challenged (red line) birds were positive for S. Typhimurium. A two-way ANOVA test revealed significant effects of both treatment and time (P < 0.0001). A significant interaction was also observed between both factors (P = 0.018). Multiple comparison of the means revealed the proportion of positive feces in vaccinated and challenged birds was significantly greater proportion than challenged only birds at weeks 4 and 17 p.i.

FIGURE 2

Detection and quantification of S. Typhimurium DT9 by PCR. The prevalence of S. Typhimurium shedding was characterized using standard PCR (A). Consistent with culture results, S. Typhimurium positive samples were highly variable over the experimental time course. No significant difference in the proportion of positive fecal samples was observed. The abundance of S. Typhimurium present in fecal samples collected from both challenged only and vaccinated and challenged birds was determined by qPCR (B). Peak S. Typhimurium DT9 load in feces was observed at days 3 and 6 p.i. No significant difference in bacterial load was observed between the two treatment groups.

FIGURE 3

Proportion of egg shells positive for S. Typhimurium DT9. Egg shells were washed with buffered peptone water which was then cultured for S. Typhimurium DT9. Data are presented as the proportion of egg shells positive for S. Typhimurium DT9. The number of positive egg shells was variable for both treatment groups. A significant effect of time was detected (P < 0.001). No significant difference was observed between treatment groups.

FIGURE 4

Salmonella group B antibody response following vaccination Serum samples were collected and tested for group B LPS antibodies. Samples were taken from birds 1 week after vaccination with STM1. The first STM1 dose was administered by ocular inoculation at day old (A). The second dose was administration in drinking water at 6 weeks of age (B). The final dose was administered by IM injection into the pectoralis muscle at 10 weeks of age (C). Data are presented as mean ± standard error of the mean. The mean antibody titre following the first (A) and second (B) doses of STM1 vaccine was not significantly different from control unvaccinated birds and was below the positive threshold. Following IM inoculation, the mean antibody titre was significantly higher than control (P < 0.001). The post-challenge IgG response from day 3 p.i. to week 39 p.i is shown in (D). Antibody titres from control (uninfected and non-vaccinated) (gray line) and vaccinated only (blue line) birds were not significantly different from each other. Peak antibody titres were observed in the challenged only (black line) and vaccinated and challenged (red line) treatment groups at day 11 p.i. Both challenged only and vaccinated and challenged birds exhibited significantly higher antibody responses than either control or vaccinated only birds (P < 0.001). The black hashed line indicates the threshold for positive samples.

FIGURE 5

Avidity of group B antibodies. Antibody avidity was tested by washing ELISA plates with varying concentrations of sodium thiocyanate following incubation with test plasma. Avidity was tested at 3 weeks p.i. (A) and at week 39 (B). Avidity of final vaccination (at 10 weeks of age prior to challenge) is shown in both A and B by the hashed line. Vaccinated birds (gray), vaccinated and challenged (red), and challenged only (black).

FIGURE 6

Comparative titres of mucosal IgA. Ileal scrapings were collected at the end of the experiment. Secreted IgA was quantified using a chicken specific ELISA. Mean sIgA titres ranged between 122.3 ± 24.9 μg IgA/mg tissue and 422.8 ± 72.6 μg IgA/mg tissue. The mean sIgA titre observed in the challenged only group (S. Typhimurium DT9; indicated as STM9) was significantly higher than all other treatment groups (P < 0.05).