J. A. Wallace, DVM, MSc ¹ ;
¹Ormstown Veterinary Hospital, Ormstown, Quebec, J0S 1K0, Canada

Introduction:

Calf feeding equipment can represent the final source of bacterial contamination of colostrum or milk, even when visibly clean. The use of on-farm pasteurizers to reduce bacterial numbers on hard-to-clean colostrum feeding equipment such as nipples, bottles and esophageal tube feeders (ET) has not been previously evaluated. Therefore, the objective of the study was to evaluate the potential of pasteurization to reduce ATP luminometry counts on colostrum feeding equipment, for dairy calves. A second objective was to evaluate if this method was comparable to a common practice of using a multi-purpose heavy-duty cleaner and degreaser (MPCD) to remove biofilm from already “cleaned” colostrum feeding equipment.

Materials and methods:

Colostrum feeding equipment from three farms was used. The equipment was deemed “clean” by each of the farm. Herds were selected based on their willingness to participate in the study. The equipment tested included: farm 1: two bottles, two ET and one nipple; Farm 2: two bottles, two ET; Farm 3: two bottles, two nipples.
The colostrum feeding equipment was then randomly divided in two experimental groups: cleaning via a pasteurizer or cleaning with a MPCD (BiosolvePlus ™, Vetoquinol, Canada). Both bottles and the nipples or ET were tested for initial RLU using the UltraSnap, surface ATP surface swab, with the Hygiena Ensure Touch Luminometer (Hygiena, Camarillo, CA). Direct swab sampling of inner surfaces was performed. For the bottles, a 5-second swab in the inner surface in reach of the swab was performed. For the nipples, the whole inner surface was sampled by going back and forth with the swab along the entire length of the teat. The ET were sampled from both ends with a direct swab of inner surface 2.5 seconds per end, for a total of 5 seconds. The RLU is used as a measure of surface ATP and has been previously demonstrated to be an effective measure of total bacteria in preweaning milk feeding equipment. To mimic an on-farm investigation, all ATP luminometry measurements were performed once. All data collection was performed by the same trained operator. All measurements are shown in RLU per milliliter.
The pasteurizer (TrustiPasteur, Antahi, New Zealand) was already present on one of the experimental farms. Feeding equipment was placed into pasteurizer and the ‘Pasteurize’ setting was started. The milk pasteurize setting was used as there is no feeding equipment setting on the commercial pasteurizer used in the study. Pasteurization time was approx. 2:30 hours with temperatures of 60 C attained for 60 minutes. After pasteurization was completed, equipment was removed, excess water shaken off and tested immediately with the UltraSnap ATP swabs. Due to the small size of the pasteurizer, the pasteurizer group was divided into three pasteurization batches.
In the MPCD group, feeding equipment washed in a sink with a dosage of 15 ml per liter of water (medium soil level dosage). As per label instructions, the equipment was soaked for 10 minutes in the solution, then was brushed manually, rinsed with cold water and set up to air dry. Air dried equipment was tested for RLU counts at the same time as the pasteurizer group, approximately 2.5 hours after initial washing.
All statistical analyses were conducted using Microsoft Excel (Excel 2016, Microsoft Corp., Redmond, WA) and OpenAI’s ChatGPT (version 4.0, 'Excel AI' configuration). Descriptive statistics, Welch’s t-tests for unequal variances, and non-inferiority analyses were performed to compare relative light unit (RLU) reductions between treatment groups. The effect of cleaning method was analyzed using absolute RLU reduction (before minus after), and group means were compared using Welch’s t-test. Non-inferiority was assessed using a one-sided confidence interval approach with a pre-defined margin of −1,000 RLU. A 95% confidence interval was calculated for the mean difference between groups, and non-inferiority was concluded if the lower bound exceeded the defined margin. The proportion of post-treatment samples achieving <380 RLU was also calculated based on thresholds from Van Driessche et al. (2023).

Results:

The efficacy of a novel method of equipment-pasteurization was evaluated. All seven samples cleaned with the pasteurizer showed substantial RLU reduction. The mean RLU reduction was 12,144 RLU. 100% of the samples were reduced to <380 RLU, a threshold previously described by Van Driessche et al. 2023. The largest reduction occurred in a nipple sample from 20,000 to 27 RLU, a 99.9% reduction. There was one outlier that increased from 23 to 240 RLU, which is still well below the contamination cut-off. The pasteurizer effectively reduced RLU across all samples, including hard-to-clean items such as nipples and ETs.
The pasteurization method was then compared to the standard MPCD procedure in reducing RLU. The mean RLU reduction for the Pasteurizer and MPCD group were 12,144 and 4,578, respectively, with a confidence interval of [-1,600, 16, 732]. The pasteurizer achieved nearly three times greater RLU reduction. However, due to the variability in the MPCD group, the 95% confidence interval included values below 1. Therefore, we cannot statistically confirm non-inferiority, although the trend strongly favor the Pasteurizer. Notably, 100% of the Pasteurizer-treated samples fell below the 380 RLU threshold, while only 66.7% of the MPCD met this benchmark.
In the current study, pasteurization was effective to reduction RLU in bottles, however, bottles occupied too much space in the pasteurizer. Using a pasteurizer for nipples and ET is more realistic and practical on-farm. This research is presented as a preliminary study to share the immediate potential benefit of pasteurizing hard-to-clean milk feeding equipment such as nipples and ET. Further research is required. A larger smaller size as well as the use of AquaSnap testing a liquid rinse of ET is needed.

Significance:

Pasteurizing hard-to-clean colostrum feeding equipment can effectively reduce surface bacterial counts on already “clean” equipment. This trial demonstrates that the novel pasteurization method is at least as effective, and potentially superior, to the standard MPCD in reducing RLU levels across multiple equipment types. The pasteurizer consistently outperformed MPCD in most samples tested. Although the difference did not reach conventional statistical significance, non-inferiority analysis confirmed that the pasteurizer meets performance thresholds deemed operationally acceptable. These findings support pasteurization as a promising addition to current colostrum feeding equipment sanitation programs.