A year-long pilot longitudinal field study was carried out to assess MAP transmission to dairy calves through naturally contaminated milk. The study was carried out in a farm located in the Los Ríos region, southern Chile, between November 2017 and October 2018. The study was done on a dairy farm instead of at experimental animal research facilities for animal welfare concerns as well as economics. Seventeen newborn male calves were randomly assigned upon arrival to one of four experimental groups and each animal received a dairy diet of 4L milk/once day for 12 weeks. Group A calves (n=5) received milk naturally contaminated with MAP (collected from 5 infectious nurse cows), group B calves (n=5) were fed the same MAP-infected milk after first being treated with a novel decontamination tool based on copper ions. As controls for thermic-treated milk, group C calves (n=4) received commercial milk replacer (Sprayfo™), and group D calves (n=3) were fed UHT milk marketed for human consumption (COLUN).
We selected a small sample size considering mainly animal welfare 3Rs, reducing the number of animals used to a minimum. In addition, one of the treatments was evaluated in vivo for the first time. Although, the latter was not the main goal of the study, we wanted to gather basic evidence regarding the use of this novel treatment in field conditions.
Only the two main investigators (PS and MS) were aware of the groups allocation, but the samples were collected, processed and analyzed in a blind manner, since the technical staff and the rest of the co-authors did not know the individual identification of each calf and to which group belongs each one until the data were analyzed.
Calves. The seventeen newborn calves were acquired, within a month, from 3 small dairy herds (herd size between 50 to 80 lactating cows), all located in different districts within the Los Ríos region, southern Chile, with history of negative paratuberculosis diagnostic results for at least 5 years based on culture and ELISA, and absence of clinical cases. The only exclusion criterion was if the calf had nursed. Calves were immediately separated from their mothers (before they were able to nurse on their own), fed colostrum from their own dam (first day of life) in a hygienic environment, kept in individual pens, and then transferred to the experimental farm (a completely separate farm) once calves had a dry hair coat and navel cord, according to international animal regulations regarding animal welfare . Afterwards, as the animals arrived the experimental farm, they were randomly separated, into one of four experimental groups as described above, ensuring as a first priority that there were five animals for groups A and B. Also, during the first 12 weeks, the calves were also offered hay, concentrate and water ad libitum. Finally, from 12 weeks onwards, the animals also received grass silage. The weight of the calves was monitored monthly. Each group of calves was kept in separated pens (without direct contact between groups) until the end of the one-year study period. From the fourth month to the end of the study period, the calves had access to an open yard, while maintaining group separation, which guaranteed the minimum area for each calf for animal welfare, i.e. minimum than 2.6 m2 per animal . Technical staff entered the pens wearing clean coveralls, boots, and gloves used exclusively for each pen. Likewise, each pen had dedicated cleaning equipment to eliminate the risk of cross contamination.
Nurse cows. Five MAP-infected and infectious lactating cows (confirmed by serum ELISA and fecal culture) were acquired from a herd with a history of high within-herd paratuberculosis prevalence and several clinical cases per year. These cows were selected as nurse cows, transferred to the experimental farm, and the milk from them was pooled fresh and used to feed experimental calf groups A and B.
The naturally MAP-contaminated milk was decontaminated using a copper treatment adapted from Steuer et al. , modified for field conditions. Briefly, the treatment device consisted of an aluminum receptacle containing 50 L of milk naturally contaminated with MAP in which two high purity copper cylinders were immersed. The copper cylinders were stimulated with a low voltage (24V) electric current (3A) to quickly release copper ions for 8 minutes. The receptacle was carefully shaken during treatment to allow constant mixing.
The milk replacer (Sprayfo™) used for feeding calves from group C, was pasteurized and then high-pressure homogenized during its industrial processing, according to the manufacturer's instructions (Trouw Nutrition, the Netherlands).
The milk for human consumption (COLUN) used for feeding group D calves, as part of its manufacturing process, was subjected to cooling, pasteurization and standardization before the UHT (Ultra High Temperature) treatment, at 138° C for 4 seconds.
Bacteriological analyses of milk and environmental samples
Milk was sampled once a week for a total of 12 weeks (duration of milk feeding), in order to detect MAP and estimate the viable numbers in milk. To achieve this, milk samples were cultured in the BACTEC-MGIT 960 liquid culture system (Becton Dickinson, Sparks, MD). For all milk samples except those treated with copper ions, a decontamination step before inoculation of liquid culture media, was carried out, according to Dundee et al. . Briefly, 50 mL milk samples were centrifuged at 2500 x g for 15 min and the pellets were resuspended in 10 mL 0.75% (w/v) hexadecylpyridinium chloride (HPC, Sigma) and incubated for 5 h at room temperature (20-21°C). Then, the samples were centrifuged again at 2500 x g for 15 min and the pellets resuspended in 1 mL phosphate buffered saline (PBS). Finally, 100 µL of the resuspended pellets inoculated into MGIT tubes (Becton Dickinson, Sparks, MD), supplemented with 0.8 mL of ParaTB supplement (Becton Dickinson, Sparks, MD), 0.5 mL of egg yolk (Becton Dickinson, Sparks, MD) and 0.1 mL of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA) antibiotic mixture (Becton Dickinson, Sparks, MD). Copper-treated milk samples were processed similarly but without the HPC decontamination step and without addition of PANTA in the culture medium to avoid antibacterial effects other than that caused by copper ions.
MGIT tubes flagged as positive (indicating a change in oxygen concentration in the MGIT tube) were subjected to DNA extraction and purification according to a published protocol , and then tested by IS900-qPCR to confirm MAP presence. In addition, direct MAP detection in milk was performed by qPCR and the total MAP load in milk (live and dead cells) was estimated, as explained below.
Along with the bacteriological analyses of milk, hay, concentrate, silage and water were also cultured in the BACTEC-MGIT 960 system (Becton Dickinson, Sparks, MD) and confirmed by IS900 qPCR. Milk was collected prior to feeding in 50 mL sterile Falcon tubes and cultured once a week during the first 12 weeks. Environmental samples (hay, water, concentrate and silage) were cultured monthly until the end of the study period. Domestic tap quality water was offered to the animals in hygienic drinking containers. Two to three grams of food and water samples were weighed, suspended in sterile distilled water, vortexed and then allowed to stand for 30 minutes. After this time, the supernatant was collected and processed as a sample in the above-mentioned culture system protocol.
Estimation of MAP load in milk samples
Bacterial load (total live and dead cells) from naturally contaminated milk samples before and after copper ions treatment, and from milk replacer and UHT milk were estimated by qPCR (Roche 2.0 real-time PCR) using a standard curve as previously described . Briefly, this standard curve was based on the concentration of MAP DNA measured in a Nanoquant spectrophotometer (TECAN group, Männedorf, Schweiz), adjusted to a 108 dilution, the number of copies of the IS900 target gene, and the reference of the molecular weight of the genome of MAP ATCC strain 19698. The copy numbers of the target region were expressed as MAP-specific bacterial cell equivalents (bce), according to Dzieciol et al. .
Evaluation of the infection progression in the study animals
Infectious status determination. MAP infection in calves were evaluated before the first milk intake and then monthly both by fecal cultures, using the BACTEC-MGIT system, and serology using an ELISA kit (IDEXX, Westbrook ME). For culture, 5 to 10 g of fecal material was obtained directly from the rectum using individual palpation sleeves. Blood samples (5 to 10 mL) were obtained by jugular venipuncture up to the first month, and from then on from the coccygeal vein, using Vacutainer™ tubes (BD Diagnostics, Franklin, NJ). All samples were transported to the Laboratorio de Enfermedades Infecciosas, Instituto de Medicina Preventiva Veterinaria, Facultad de Ciencias Veterinarias, Universidad Austral de Chile. Fecal and blood samples were kept at room temperature until processed the following day. The serum was stored at -20°C.
Post-mortem analyses. At the end of the study period (12 months), a necropsy was performed on all calves. The pathologist was blind as to treatment group for all animals. Calves were euthanized by a veterinarian pathologist using a retained projectile pistol, in accordance with the criteria of the Bioethics Committee of the Research and Development Department (DID) of the Universidad Austral de Chile (Validation report Nº263-2016). Ileum and lymph nodes samples were cultured in the BACTEC-MGIT 960 system for MAP detection, as previously described.
Ancillary data: monitoring potential copper toxicosis in calves
Copper concentrations in both untreated and copper-treated milk samples were measured by atomic absorption spectrophotometry at each milk sampling during the 12 weeks of feeding. Once the milk diet was completed at 12 weeks of age, and with the aim to confirm or rule out whether a copper accumulation or intoxication effect was produced by the copper-treatment of milk, monthly blood samples were used to evaluate copper plasma concentration and the plasma activity of liver enzymes gamma-glutamyl transferase (GGT), glutamate dehydrogenase (GD) and aspartate aminotransferase (AST). In addition, liver samples were collected at necropsy to determine hepatic copper concentrations.
Normality of the data were checked using the Shapiro-Wilk normality test. A comparison of MAP load (using the log10 of the bce estimates) between treated and untreated milk was conducted using the Wilcoxon–Mann–Whitney test. Survival analysis using the Kaplan-Meier estimator  was used to assess differences in the time of the first fecal culture positive between groups. Finally, a Bayesian fitted zero-inflated Poisson mixed model (ZIPMM) was fitted to compare MAP fecal load between groups, where the log10 of the MAP bce estimates was rounded to the closest integer (Yijk), representing the MAP load (response variable) for the ith calf in, in the jth group (Xj), at the kth sampling time. Control groups (C and D) were defined as the reference for comparison to the groups receiving MAP-contaminated milk (A and B). In general, ZIPMM is used when data are over-dispersed due to an excess of zero counts in the response variable . All statistical analyses were run using the statistical software R version 3.6.3 (R core team 2020). In the particular cases of the survival analysis and ZIPMM, those analyses were run using the packages “survival”  and “GLMMadaptive” .