Dr Jodi 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 milk1. When feeding equipment is not properly cleaned there is more opportunity for disease causing bacteria and pathogens to grow 2. By feeding calves with “dirty” equipment we are inadvertently feeding and exposing calves, and their fragile immune systems, to large amounts pathogens 3,4. Even with best practices cleaning protocols implemented on farm after each use, procedural drift can result in higher than recommended bacterial numbers. Cleanliness is key for healthy calves and the equipment used for feeding preweaning calves must be kept as clean as possible 5.
Contamination of feeding equipment for preweaning calves can be described using adenosine triphosphate (ATP) as expressed as relative light units, (RLU) 6,7,8. ATP allows for on-site assessment of cleanliness by quantifying the amount of ATP, present in every life form, into RLU by a chemical luminescent reaction with an enzyme, luciferase 6. Previous research has demonstrated a wide variability of different cleaning protocols and RLU measurements as many farms did not consistently and properly clean feeding equipment 8.
Some dairy farms have invested in pasteurizers to reduce bacterial numbers for colostrum and milk. However, the use of on-farm pasteurizers to reduce bacterial numbers on hard-to-clean milk 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 milk feeding and 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 multipurpose heavy-duty cleaner and degreaser to remove biofilm from already “cleaned” colostrum and milk feeding equipment.

Materials and methods:

Colostrum and milk feeding equipment from three farms was used. Herds were selected based on their willingness to participate in the study and additionally were clients of the Ormstown Veterinary Hospital. The equipment tested included both nipple, bottles and esophageal feeders (Figure 1). The equipment had been used for either colostrum or milk feeding on-farm was deemed “clean” by each of the farm.

Figure 1. Milk feeding equipment used in study. Two bottles and their respective nipples or esophageal feeders were used from each of the three farms.

The feeding equipment was then randomly divided in two experimental groups: cleaning via a pasteurizer or cleaning with a multipurpose heavy-duty cleaner and degreaser (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). 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 6. To mimic an on-farm investigation, all ATP luminometry measurements were performed once. All data collection was performed by the same trained operator (author JAW). 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. 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.

Figure 2: Image of milking feeding equipment; nipple, esophageal tube feeder (ET) and bottle in the pasteurizer.

In the multi-purpose degreaser cleaning group, feeding equipment washed in a sink with a dosage of 15 ml of BiosolvePlus ™ per liter of water (medium soil level dosage). As per label instructions, the equipment was soaked for 10 min in the BiosolvePlus ™ 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.
Data was recorded as RLU and is presented in tables as before and after with the percent RLU reduction calculated for each piece of feeding equipment. Data analysis was performed in Excel using the OpenAI. A Welch’s t-test was conducted to account for independent samples. A Cohen’s test evaluated the size of the effect and a non-inferiority analysis was also performed. A one-sided T test to demonstrate non-inferiority of the pasteurizer as compared BiosolvePlus ™. All data were stored in an Excel file (Excel 2016, Microsoft Corp., Redmond, WA). Statistical analyses, including Welch’s t-tests, effect size calculations (Cohen’s d), and non-inferiority testing, were performed using OpenAI’s ChatGPT (version 4.0, 'Excel AI' configuration).

Results:

In this study, the efficacy of a novel pasteurization method compared to the standard multi-purpose degreaser cleaning procedure in reducing ATP across multiple sample types was evaluated. After removing an outlier (Tube 1) with an increased RLU count post-treatment, the Pasteurizer group demonstrated a higher mean RLU reduction percentage (96.52%) than the BiosolvePlus ™ (78.77%). A Welch’s t-test was conducted to account for unequal variances, yielding a t-statistic of 1.79 and a one-sided p-value of 0.062. Although this did not meet the traditional threshold for statistical significance (p < 0.05), the result indicates a strong trend favoring the Pasteurizer.


Table 1: ATP luminometry results as expressed as relative Light Units (RLU) and percent reduction after pasteurization at 60C for 60 minutes.
Pasteurizer RLU count Before RLU count After % reduction
Tube 1 23 240 -943.50%
Bottle 2 20,000 73 99.60%
Nipple 2 20,000 155 99.20%
Bottle 3 46 8 82%
Tube 4 20,000 102 99%
Bottle 5 20,000 27 99.80%
Nipple 5 5575 27 99.50%

Table 2: ATP luminometry results as expressed as relative Light Units (RLU) and percent reduction after cleaning colostrum and milk feeding equipment with 15 ml of BiosolvePlus™ per liter of water.
BiosolvePlus™ RLU count Before RLU count After % reduction
Bottle 1 3384 12 99.60%
Tube 2 36 10 72%
Tube 3 15585 9805 37%
Bottle 4 60 12 82%
Bottle 6 362 0 100%
Nipple 6 20,000 2116 82%

Additionally, the effect size (Cohen’s d = 1.03) suggests a large practical difference between the two methods. A non-inferiority analysis was also performed using a margin (Δ) of -5%, representing the maximum acceptable difference for clinical equivalence. The 95% confidence interval for the difference in mean RLU reduction ranged from [60.19%, 97.35%], indicating that Pasteurizer is not inferior to BiosolvePlus ™ and may offer enhanced decontamination performance.
Traditionally total bacteria counts (TBC) were used to assess if the feeding equipment was contaminated (> 100,000 cfu/ml TBC) or not contaminated (< 100,000 cfu/ml TBC). The relative cut-off values for contaminated versus non-contaminated feeding equipment using RLU was described by Van Driessche et al. (2023). The cut-off values for RLU of non-contaminated nipples is under 380 RLU. This data suggests that the “cleaned” nipples to start the current study were extremely contaminated.
In the current study, pasteurization was effective to reduction RLU in bottles, however practically, 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 calf equipment can effectively reduce surface bacterial counts on already “clean” milk feeding equipment. This trial demonstrates that the novel pasteurization method is at least as effective, and potentially superior, to the standard multi-purpose cleaner and degreaser in reducing RLU levels across multiple equipment types. The pasteurizer consistently outperformed the multi-purpose cleaner and degreaser in most samples tested. Although the difference did not reach conventional statistical significance, non-inferiority analysis confirmed that Pasteurizer meets performance thresholds deemed operationally acceptable. These findings support pasteurization of feeding equipment as a promising addition to current sanitation programs.