Holstein Cow

On the 28th day of the feeding regimen, a single rumen sample (both solid and liquid fractions) from each Holstein and Jersey cow was collected at 1300 h via esophageal tubing. The esophageal tubing apparatus was prepared by coupling one end of the esophageal tube to a metal strainer as described by Raun and Burroughs (1962) (link) and the other end was connected to a 125-mL Nalgene bottle (Thermo Scientific Inc., Waltham, MA, USA) using a “Tee” connection. The remaining end of the “Tee” was connected to a Masterflex vacuum pump (model 7015-10; Cole Parmer, Vernon Hills, IL) (Figure S1). Rumen samples were collected by passing the tube containing the metal strainer through a Frick speculum into the ventral sac. The first 200-mL of rumen fluid were discarded to minimize saliva contamination. Then 40-mL of rumen fluid were collected and placed into a 50-mL propylene conical tube (Thermo Scientific Inc., Waltham, MA, USA). After removal of the esophageal tube, particles attached to the metal strainer were recovered and added into the conical tube to collect a sample more adequately representative of the rumen content and then samples were snap-frozen in liquid nitrogen. Across samples, particles ranged from 10 to 15% of the total sample. Between sampling, the metal strainer and Frick speculum were thoroughly washed with warm water and water was run through the stomach tube to prevent cross contamination from the previous animal. Additionally, the removal of the first 200-mL also prevented any cross contamination.
Immediately after the collection of the esophageal sampling, another sample was collected from the Holstein cows via the rumen cannula. Ruminal contents were collected from the dorsal, ventral, and caudal areas of the rumen. The digesta were mixed and a representative sample was collected and snap-frozen in liquid nitrogen for bacterial community analysis. All samples collected via esophageal tubing or via the cannula were stored in an -80°C freezer until used for DNA extraction and bacterial community analysis.

The experimental design was evaluated by the Animal Welfare Body of the University of Bologna and found as not falling within the Directive 63/2010 of the European Parliament and of the Council on the protection of animals used for scientific purposes (transposed into Italian law by Legislative Decree 26/2014), thus not requiring any authorization from the national competent Authorities. The study was approved by the Animal Ethics Committee of the University of Bologna and conducted at the dairy cattle farm of the same university. Animals were 8 lactating Holstein–Friesian cows (average weight 752 ± 52 kg) in their late lactation period (262 ± 49 days in milk), with an average milk yield of 19.6 ± 6.6 kg/day. Cows were housed in a naturally ventilated free stall barn and had free access to feed and water. Rations were formulated to mimic total mixed rations used in the Parmigiano Reggiano cheese production area, in Italy, which consisted of all dry and nonfermented components, as detailed in previous studies [50 (link),51 (link)].
Animals were divided into two groups, consisting of 4 cows each, and two different treatments of 10 days’ duration were performed. Group AFB1 received, from day 0 to day 2 only, the basal diet (AFB1 content less than 2 ng/g, see Section 4.3), while group TP was offered the basal diet supplemented with 20 g turmeric powder dissolved in linseed oil/head/day for 10 consecutive days. At day 3, all cows received the basal diet containing naturally contaminated maize with a final AFB1 concentration of 5 ± 1 μg/kg for 8 consecutive days. A crossover experimental design was applied: each cow received both treatments sequentially after of a 4-day washout period, during which all animals were offered the basal diet. The used TP contained 2.5% curcumin, desmethoxycurcumin and bis-desmethoxycurcumin (85/10/5). With an average feed intake of 20 kg/cow/day, the cows approximately received 0.5 g active substances/head/day; this dosage is higher than the recommended inclusion level for flavoring purposes but below the maximum safe concentration of 0.72 g/day calculated by the EFSA [7 (link)].
Animals were milked twice a day, namely at 08:00 h (referred to as M) and 19:30 h (referred to as E) in a double-5 herringbone milking parlour. Individual milk samples (around 100 mL) collected at T0, T2, T4, T6, T8 and T10 were used for the determination of somatic cell count (SCC) (Fossomatic 7, Foss Electric A/S, Hillerød, Denmark) and milk composition (fat, lactose, protein, urea) by means of infrared spectroscopy (MilkoScan FT+, Foss Electric A/S, Hillerød, Denmark). The determination of AFB1 metabolites was performed on further milk samples (around 100 mL) collected at T0 (M), T4, T5 and T6 (M and E), and T7, T8, T9 and T10 (M only), which were stored frozen (−20 °C) pending analysis. The experimental design is outlined in Figure 2.

Animal care and experimental procedures were conducted according to the guidelines of the University of Nebraska-Lincoln (UNL) Animal Care and Use Committee. Five ruminally cannulated Holstein lactating cows averaging 6.2 ± 0.70 (mean ± SE) years of age (range = 3.9 years) and four Jersey lactating cows (not cannulated) averaging 4.5 ± 0.39 years of age (range = 1.9 years) were used in the experiment. All Holstein cows had been part of the UNL dairy herd throughout their lives, whereas Jersey cows were purchased from a commercial dairy farm and had been part of the UNL dairy herd for 230 days under the same management as the Holstein cows before the start of the experiment. Cows were housed in a temperature-controlled room in individual tie stalls, and were fed the same diet once daily at 1000 h for ad libitum consumption at 110% of expected intake for four weeks. On a dry matter (DM) basis, the diet was comprised of 51% forage and 49% concentrate and was formulated to meet or exceed the requirements of a lactating cow (Table S1).

The reference population consisted of 75 Spanish Holstein cows from a single farm located in the Basque Country. In 2022, only five cows from this herd had an ELISA positive result and all the animals had a negative fecal PCR result. Only adult cows (2 years or older, 4.5 years mean age) were included in the study. All the animals included in this study were registered with the Spanish Federation of Holstein cattle (CONAFE; www.conafe.com.) and had negative fecal PCR results when blood samples were collected and in subsequent annual samplings. In addition, the animals used in this study were not diagnosed with any other pathogen. Blood samples were collected in groups of 16 per sampling day. The validation population consisted of 16 cows from the same farm.

MGIA were performed as previously described (22 (link)–24 (link)). Briefly, fifteen milliliters of peripheral blood were drawn from the tail vein of healthy Holstein cows into heparinized Vacutainer tubes (Becton, Dickinson and Company, Sparks, MD, USA) and diluted 1:2 in Hanks balanced salt solution (HBSS). Leucosep tubes were filled with 15 ml of Ficoll-Paque (1.084 g/cm³) (GE Healthcare, Uppsala, Sweden) and centrifuged at 1,000 rpm for 30 seconds at room temperature. Subsequently, the diluted blood was overlaid on the top of the Ficoll-Paque and centrifuged at 800 g for 15 minutes at room temperature. The plasma layer was removed and the cell interphase containing peripheral blood mononuclear cells (PBMCs) was collected and transferred to a clean tube. PBMCs were washed twice in HBSS and centrifuged at 400 g for 10 minutes to remove platelets. Supernatants were aspirated and the purified PBMCs were resuspended in RPMI-1640 supplemented with 20 mM L-glutamine, 10% heat-inactivated bovine serum (Lonza, Spain), 100 U ml-1 penicillin G, and 100 mg ml-1 streptomycin sulfate (Lonza, Spain). PBMCs were cultured at a concentration of 1 x 10⁶ cells/ml into 24-well plates and incubated at 37°C in a humidified 5% CO₂ incubator for 2 h. Non-adherent cells were removed by washing, and adherent cells were incubated in fresh medium for 7 days at 37°C to allow differentiation to MDMs. Differentiated MDMs were inoculated in triplicate with a single-cell suspension of MAP K10 strain at a multiplicity of infection (MOI) of 10:1 (bacteria:cells). After 2 h, the supernatant was removed, and the cells were washed twice with HBSS to remove extracellular bacteria. Infected MDMs were lysed at this time (2 h p. i.) or were cultured at 37°C for 7 days in fresh medium. At each time point, the supernatant was aspirated and infected MDMs were lysed by vigorous pipetting with 0.5 ml of 0.1% Triton X-100 (Sigma-Aldrich) in sterile water for 10 min.

All the experimental procedures were approved by the University of Illinois (Urbana-Champaign) Institutional Animal Care and Use Committee (no. 17166). In total, 16 multiparous Holstein cows past peak lactation (>60 DIM) were used in a randomized complete block design conducted from February to April 2020. All cows were housed in a freestall system equipped with individual feeding gates (American Calan Inc., Northwood, NH) and were fed once daily at approximately 0800 hours. Milking occurred two times per day at 0400 and 1530 hours. Cows were blocked by DIM (97.1 ± 7.6 d) and parity (3.4 ± 0.62), MY, BCS, and SCC (Supplemental Table 1). After 1 wk of adaptation, cows were randomly assigned within block to one of two treatments (eight per treatment): 1) a control group receiving the basal TMR with no supplementation (CON) or 2) basal TMR with 19 g/d of a Saccharomyces cerevisiae fermentation product (NTK; NutriTek®, Diamon V, Cedar Rapids, IA), top-dressed. This is the commercially recommended dose for the type of animal used in this trial. The ration was formulated to meet NRC (2001) requirements and is presented in Table 1. The diet was formulated for cows at 180 DIM, BW of 703 kg, MY of 35.4 kg/d with a target of 3.50% milk fat and 3.12 milk protein and predicted DMI of 24.9 kg/d. The diet included corn silage, alfalfa hay, canola and corn gluten meal, ground shelled corn, rumen-protected lysine and methionine, rumen-inert fat, rumen-bypass protein, vitamin and mineral mix, and the ionophore Rumensin (Elanco, Greenfield, IN). The experimental period lasted 68 d and was divided into two phases: 1) Phase 1 (P1) from the beginning of the experimental period until d 63, and 2) Phase 2, from d 64 to 68, which represents the FR period. Cows were euthanized at the end of FR for a separate experiment. During Phase 2, cows were restricted to 40% of their ad libitum intake of the previous 5 d. This amount of restriction was chosen based off previous work in which 40% restriction was validated as a method to induce intestinal barrier dysfunction in dairy cattle (Kvidera et al., 2017b (link)).