Interaction Between Pelleting Temperature and Amasil™ NA Inclusion Rate on Recovery of Salmonella from Experimentally Contaminated Feed



Contaminated feed is a known vector for the transmission of various pathogens onto the farm (Li et al., 2012). In recent years there has been a move toward the use of higher pelleting temperatures in the hopes of bolstering biosecurity by decontaminating feed. However, this practice can add cost both in terms of energy required to attain higher pelleting temperatures, but also in terms of nutrient overages necessary to account for the concomitant increase in pelleting related loss of nutrients like vitamins (Coelho, 2000) and enzymes (Abdollahi et al., 2013). Unfortunately, the efficacy of pelleting as a biosecurity measure is limited to a single point in the feed supply chain, and is not expected to protect against recontamination. Davies and Wray (1997) found that 20.2% of samples collected from pellet coolers at 10 different feed mills were contaminated with Salmonella, and the within mill positive rate ranged from 0 to 85.7%. This was the second most frequently contaminated sampling point, with the intake pits being first (24.1% of samples positive).

Formic acid, an FDA approved feed acidifier, has been consistently shown to be one of the most potent organic acids for killing potential feed microbial contaminants such as Salmonella, E. coli, Clostridia, and Campylobacter (Strauss and Hayler, 2001; Navarro et al, 2015). And unlike for pelleting, formic acid acidified feed would be expected to resist re-contamination. Therefore, the present study was conducted in order to understand the relationship between pelleting temperature and inclusion of Amasil NA on recovery of Salmonella from experimentally contaminated broiler feed.


Salmonella enterica subsp. enterica Serovar Enteritidis (ATCC 13076) was grown in trypticase soy broth (TSB) and inoculated into the previously Amasil NA-treated feed at 1 L per 50 kg of feed. Separate inoculum was prepared for each Amasil NA treatment level (0 g/kg, 4 g/ kg, 7 g/kg and 10 g/kg). Following inoculation of the treated diets, the contaminated feed was pelleted at conditioning temperatures of 60°C, 75°C and 90°C. From the pelleted feed, two sets of subsamples were collected to be used for analysis. Hot pellets were collected aseptically directly from the pellet mill and were immediately placed on ice for use in the Challenge Study. Remaining hot pellets were cooled in a room air pellet cooler and subsamples collected for the Initial Study. Samples collected for the Initial Study were evaluated for Salmonella on days 0, 1, 2, 7, and 14 post-processing. Samples collected for the Challenge Study were reinoculated with Salmonella in the lab and evaluated for growth on days 0, 1, 2, 7, and 14 post-re-inoculation. Enumeration of Salmonella was done through serial dilution and spread plating to Xylose Lysine Desoxycholate agar (XLD; Becton, Dickinson and Company, Franklin Lakes, NJ).

Results and Discussion

Pre-Pelleting Contamination Only
Feed samples in the Initial Study were contaminated with approximately 4.0 log10 CFU/g prior to pelleting. The mash sub-samples that were not pelleted showed a dose and time-dependent reduction in Salmonella recovery, as can be seen in Figure 1. The highest dose reduced the recovery Salmonella by 2.7 log10 CFU/g verses the control by d7. This is roughly twice the reduction in S. gallinarum reported by Al-Natour and Alshawabkeh (2005) in turkey feed with the same dose and at the same time point post-manufacture.

Figure 1

Figure 1. Salmonella recovery over time from broiler mash feed containing increasing concentrations of Amasil NA.

Figure 2

Figure 2. Effect of feed form (pellet or mash) on recover of Salmonella from broiler feed after simulated recontamination over time. Pellet columns with a * are significantly different from the mash at the same time point (P < 0.01).

Figure 3

Figure 3. Effect of Amasil NA concentration and time on the level of Salmonella decontamination of broiler feed after simulated recontamination. P-values are for differences relative to the control (0 Log10 CFU/g).

Regardless of temperature, pelleting of feed in the Initial Study was able to reduce the amount of viable Salmonella in the feed to zero. This is in agreement with the work of Burns et al (2016) who showed that the amount of time required to achieve a 10-fold reduction in viable cell numbers for 5 feed-derived Salmonella strains dropped from between 398 and 544 seconds to between 1.1 and 6.8 seconds as the temperature increased from 55°C to 65°C. The relatively low pelleting temperatures of 60 or even 75°C used in the present study are believed to be relatively benign with regard to amino acid availability and enzyme stability.

Post-Pelleting Recontamination

Mash and pelleted feed samples in the Challenge Study were contaminated with approximately 7 log10 CFU/g of Salmonella after the pellets had cooled to simulate a post-pelleting recontamination consistent with a contaminated pellet cooler or similar down-stream feed handling equipment. There was no significant interaction between Amasil NA and feed form. However, there was a Form × Day effect as well as an Amasil NA × Day effect (P < 0.001). Pellets were slightly more resistant to recontamination with Salmonella, although this difference does not become apparent until up to a week post-pelleting (Figure 2). The temperature at which the feed was pelleted did not matter (data not shown), and the pelleted feed was still positive for Salmonella at d 14 unless treated with Amasil NA. This difference is likely the result of mash feed having a higher surface area for the liquid Salmonella-containing culture media to impregnate when the mash was re-inoculated as opposed to the pellets.

The highest dose of Amasil NA on d0 was able to decrease the recovery of Salmonella by approximately 0.7 log10 CFU/g (P = 0.0261; Figure 3). One day later the difference almost doubled to 1.2 log10 CFU/g for the highest level of Amasil NA (10 g/kg; P < 0.001), and the next highest level (7 g/kg) achieved the same 0.7 log10 CFU/g (P = 0.0346). At 1 week post-recontamination, all 3 concentrations of Amasil NA were able to significantly reduce Salmonella compared with the control (0.7, 2.0, and 3.7 log10 CFU/g lower than the control, respectively; P < 0.05). By day 14, the difference between each of the acid containing diets and the control with regard to Salmonella recovery widened, with formic acid virtually eliminating the Salmonella contamination of the feed (0.23 vs 4.16 log10 CFU/g).

While most of the existing data on Salmonella contamination of feed is focused on how frequently it is detected, as opposed to how much is present when it is positive, the data that is available suggests that the levels of Salmonella kill demonstrated in the present study are far in excess of the naturally occurring levels. For example, Patterson (1971) reported average Salmonella levels in feed and feed ingredients below 20 CFU/100g and Franco et al. (2005) surveyed rendered animal protein meals for Salmonella and reported levels ranged between 0.2 and 78 MPN/g.


Salmonella in the present study can be viewed as a surrogate for a range of different microbial feed contaminants. Based on this data, it is clear that the value of pelleting temperatures in excess of 60°C pertaining to biosecurity is low both in terms of decontaminating already Salmonella-positive feed (Initial Study) and prevention of re-contamination (Challenge Study). On the other hand, Amasil NA was shown to quickly reduce Salmonella detection in already contaminated feed, as well as under recontamination. Ultimately these 2 approaches to feed hygiene are not incompatible with each other, and are even complementary in that lower temp pelleting can clean feed at a point and formic acid can protect against recontamination later on.


Li, X., L. A. Bethune, Y. Jia, R. A. Lovell, T. A. Proescholdt, S. A. Benz, T. C. Schell, G. Kaplan, and D. G. McChesney. 2012. Surveillance of salmonella prevalence in animal feeds and characterization of the Salmonella isolates by serotyping and antimicrobial susceptibility. Foodborne pathogens and disease 9:692-698.

Coelho, M. B. (Editor), 2000. Vitamins – one of the most important discoveries of the century. BASF Corporation, Mount Olive, NJ, 141 pp.

Abdollahi, M. R., V. Ravindran, and B. Svihus. 2013. Pelleting of broiler diets: An overview with emphasis on pellet quality and nutritional value. Anim. Feed Sci. Tech. 179:1-23.

Davies, R. H., and C. Wray. 1997. Distribution of salmonella contamination in ten animal feedmills. Veterinary Microbiology 51:159-169.

Burns, A. M., G. Duffy, D. Walsh, B. K. Tiwari, J. Grant, P.G. Lawlor, and G. E. Gardiner. 2016. Survival characteristics of monophasic salmonella typhimurium 4,[5],12:I:- strains derived from pig feed ingredients and compound feed. Food Control 64:105-114.

Al-Natour, M. Q., and K. M. Alshawabkeh. 2005. Using varying levels of formic acid to limit growth of salmonella gallinarum in contaminated broiler feed. Asian-Australas. J. Anim. Sci. 18:390-395.

Navarro, M., R . Stan ley, A. Cusack, andY.Sultanbawa.2015. Combinations of plant-derived compounds against campylobacter in vitro. Poult. Sci. doi:10.3382/japr/pfv035

Franco, D. A. 2005. A survey of Salmonella serovars and most probable numbers (MPN) in rendered animal protein meals:  inferences for animal and human health. J. Environ. Health. 67:18-22.

Patterson, J. T. 1971. Salmonellae in animal feeding stuffs. Rec. Agric. Res. 20:27-33.