This study was performed in accordance with the Justus Liebig University Giessen Animal Care Committee Guidelines. Protocols were approved by the Ethics Commission for Experimental Animal Studies of the Federal State of Hesse (Regierungspräsidium Giessen; GI 18/10 Nr. V 2/2022; JLU-No. 0002_V) and are in accordance with European Animal Welfare Legislation: ART13TFEU and currently applicable German Animal Protection Laws.

Healthy adult dairy cows (n = 3-6) were bled by puncturing the jugular vein, and peripheral blood was collected in heparinized sterile plastic tubes (Kabe Labortechnik). Further, 20 mL of heparinized blood was diluted in 20 mL sterile PBS with 0.02% EDTA (ethylenediaminetetraacetic acid, Carl Roth), carefully layered on top of 12 ml Histopaque-1077 separating solution (density = 1.077 g/L; 10771, Sigma-Aldrich) and centrifuged (800 × g, 45 min) without brake. After removal of plasma and peripheral blood mononuclear cells (PBMC), the cell pellet was suspended in 20 mL of lysis buffer (5.5 mM NaH₂PO₄, 10.8 mM KH₂PO₄, and pH 7.2) and gently mixed for 60 s to lyse erythrocytes. Osmolarity was then restored with 10 mL hypertonic buffer (462 mM NaCl, 5.5 mM NaH₂PO4, 10.8 mM KH₂PO₄, and pH 7.2) and 10 mL of Hank’s balanced salt solution (14065-049, Gibco). The lysis step was repeated twice until no erythrocytes were visible. Bovine neutrophils were then suspended in 5 mL of HBSS, counted in a Neubauer chamber, and allowed to rest on ice for 30 min prior to any experimental use (34).

Rumen samples of naturally C. daubneyi-infected cattle (two 14-month old animals) were collected from a local butchery and immediately transported at 4°C to the Institute of Parasitology, Justus Liebig University Giessen. After arrival, C. daubneyi adults were immediately collected, washed twice in sterile PBS 1X and frozen at -20°C until further use.

Rumen fluke eggs were recovered from frozen adults. Briefly, five adult parasites were incubated in PBS overnight in a water bath at 37°C. Then, after agitation, the solution was allowed to sediment for 3 min thrice. The remaining pellet was filtered with a 100 µm filter (PluriSelect), followed by centrifugation (400 × g, 5 min). Eggs were resuspended in HBSS, counted and conserved at 4°C until confrontation with bovine neutrophils.

To identify cattle paramphistomes sequence data of ribosomal DNA region (ITS2) and complete mitochondrial cytochrome oxidase subunit 1 gene (cox1) were generated and compared to GenBank entries. Genomic DNA was isolated from five adult flukes using DNeasy blood and tissue Kit (Qiagen), following the manufacturer´s instructions. The rDNA region was amplified using primer combinations ITS2-F and ITS2-R (35). The cox1 gene was amplified using the primer combination JB3 (nematode COI)/trem.cox1rrnl (trematode mitochondrial rrnl reverse) (36–38). PCRs were performed in 50 μL reactions using HOT FIREPol Blend Master Mix 7.5 mM MgCl₂ (Solis BioDyne), 200 nM of forward and reverse primers each, and 100 ng of flukes DNA under the following conditions: 2 min 94°C initial denaturation, 35 cycles 30 s 94°C, 30 s 53°C, 45 s 72°C and 5 min 72°C final extension. Amplicons were gel-purified, cloned, and sequenced by an external service provider (LGC Genomics GmbH). Complete sequences of rDNA and mitochondrial region were assembled from overlapping amplicons and analyzed by BLAST search against GenBank database. ITS2 sequences are available under accession number PQ821198-PQ821199.

For soluble C. daubneyi antigen (CdAg) preparation, 6 adult flukes were frozen in liquid nitrogen and grounded in a previously UV-sterilized and cooled mortar (-80°C overnight) on ice. Then, 400 μL sterile PBS 1X supplemented with protease inhibitor cocktail (1:200; Sigma-Aldrich) were added into the mortar and collected. The suspension was then sonicated in ice bath with a Sonorex Super RK31^® bath-type sonicator (Bandelin) for 5 cycles of 15 s. The sonicated material was centrifuged at 10,000 × g for 20 min at 4°C. The protein concentration of the supernatant was measured using the Pierce™ BCA Protein Assay Kit (Thermo Scientific™) and final CdAg solutions were stored aliquoted at -80°C until further use.

Bovine neutrophils (n = 3; 1 × 10⁶ neutrophils) were centrifuged at 300 × g, 10 min, RT. Cell pellets were suspended in 2 mL of imaging medium containing 0.1% bovine serum albumin (BSA; Sigma–Aldrich) and 2 µM Hoechst 34580 (Invitrogen™). One mL of cell suspension was seeded in a 35 mm Ibidi^® low profile plastic cell plate and incubated in a top-stage incubation chamber (Ibidi^®) at 37°C and 5% CO₂ atmosphere. After 30 min, 100 µg/mL CdAg were added to the isolated bovine neutrophils. Image acquisition was set for refractive index (RI; 3D tomography) and blue channel detection, applying timelapse settings (image acquisition over 120 min) using a 3D Cell Explorer-fluo (Nanolive). At the end of the experiment, each channel was exported separately using Steve software v.1.6 (Nanolive) and managed with Image J software (Fiji version 1.7, NIH). RI holotomographic reconstruction was obtained using Z project. For the 3D reconstruction images, the software Steve (Nanolive) was used.

NAE-based quantification of C. daubneyi-induced NETosis was performed as described elsewhere (16). Briefly, bovine neutrophils (n = 3; 2 ×10⁵ neutrophils) were co-cultured with CdAg 100 µg/mL or plain medium, as negative control, (120 min, 37°C, 5% CO₂). After incubation, neutrophils were fixed in 2% paraformaldehyde (Merk), washed three times with PBS 1X, and mounted in Fluoromount G™ Mounting Medium (Invitrogen™) for 24 h at RT in the dark. Using an inverted Olympus IX81^® epifluorescence microscope equipped with a XM10^® digital camera (Olympus) and Olympus CellSens Imaging Software, five images were captured randomly for each condition and NAE of single cells was analyzed via DANA I and DANA II software. Single cells presenting decondensed nucleus and exceeding a threshold of 90 µm² were considered as undergoing the process of NETosis. Over 570 neutrophils were analyzed per experimental condition, from three different blood donors.

Spectrofluorometric-based analyses was performed as classical NETosis quantification technique. Bovine neutrophils (n = 3; 2 ×10⁵ neutrophils) were co-cultured with CdAg 100 µg/mL, CdAg 10 µg/mL or plain medium for 120 min in 96-well plastic flat-bottom plates (Greiner), at 37°C and 5% CO₂ atmosphere. After incubation, samples were treated with micrococcal nuclease (0.1 U/µL, New England Biolabs, 15 min, 37°C, 5% CO₂) and later centrifuged (300 × g, 5 min). Supernatants were transferred into new wells and PicoGreen^® (50 µL/sample, Thermo Scientific, diluted 1:200 in 10 nM Tris/1 nM EDTA buffer) was added in the dark. NETosis was estimated via spectrofluorometric analyses at an excitation wavelength of 484 nm and an emission wavelength of 520 nm in an automated multiplate reader (Varioskan^® Flash, Thermo Scientific) (16). Bovine neutrophils in plain medium (negative control) or with zymosan (1 mg/mL, Invitrogen) as positive control were also considered.

Given that neutrophil degranulation was observed in immunofluorescence analyses, it was measured by means of myeloperoxidase (MPO) release, in presence of cytochalasin B (cytoB; Sigma-Aldrich) (39). For total MPO content, CTAB (cetyltrimethylammonium bromide, 0.02%, Sigma-Aldrich) was used. Medium (125 µL) containing CdAg (100 µg/mL), calcium ionophore A23187 (CaI, 5 µg/mL, Sigma-Aldrich) or CTAB (lysed neutrophils) was added to a 96-well plate (Greiner). Background wells containing only medium were added. Bovine neutrophils (n = 3; 1.25 × 10⁶ in 25 µL) were added to each well, including negative control and background wells (neutrophils and HBSS). The plate was incubated at 37°C for 90 min. At the end of the incubation period, 50 µL of pre-warmed 2.5 mM TMB (3,3’,5,5’-tetramethylbenzidine hydrochloride, Sigma-Aldrich) were added to each well, and immediately followed by 50 µL of pre-warmed 5 mM H₂O₂ (Roth). The reaction was allowed to develop for 2 min than 50 µL of 4 M sulphuric acid (Sigma-Aldrich) were added to each well to stop the reaction. The plate was centrifuged (600 × g, 10 min) and 200 µL of supernatant were transferred to a new flat-bottom 96-well plate (Greiner). Optical density (OD) was determined at 405 nm in an automated multiplate reader (Varioskan^® Flash, Thermo Scientific). Reactions were tested in duplicates of three biological replicates. The percentage of MPO release was calculated by the following formula:

Activation of bovine neutrophils was monitored using the Seahorse XF analyzer (Agilent). A total of 1×10⁶ bovine neutrophils from three blood donors were pelleted [500 × g, 10 min, RT]. Cell pellets were re-suspended in 250 µL of XF assay medium (Agilent) supplemented with 2 mM of L-glutamine, 1 mM pyruvate, and 10 mM glucose. A total of 2×10⁵ cells were gently placed in each well of an eight-well XF analyzer plate (Agilent) pre-coated with 0.001% poly-_L-lysine (Sigma-Aldrich) for 30 min. Later, the XF assay medium (Agilent) was adjusted to 180 ml total volume per well and cells were incubated at 37°C without CO₂ supplementation for 45 min before Seahorse XF measurements. CdAg (0.01-100 µg/mL) were suspended in XF assay medium (final volume 20 µL) and supplemented to the cells via instrument-own injection ports after baseline measurements. Plain XF assay medium served as negative control. The total assay duration was 200-240 min. Background subtraction and determination of OCR/ECAR registries, as well as area under the curve (AUC) calculations were performed by using the Seahorse Agilent analytics software Wave^® (Desktop Version, Agilent) and GraphPad Prism^® versions 8.3.1 for Windows 10, GraphPad^® Software, San Diego, California USA.

Intracellular ROS production of bovine neutrophils was assessed by oxidation of 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Sigma-Aldrich) to fluorescent DCF following previous reports (40, 41). Freshly isolated neutrophils (n = 3) were resuspended in sterile 1X HBSS containing Ca²⁺ and incubated with soluble CdAg (10 or 100 μg/mL) (4 ×10⁵ cells/well; 37°C, 30 min, in duplicates), final volume of the reaction 200 μL. Afterwards, DCFH-DA (10 μg/mL) was added to each duplicate. For positive control zymosan was used (final concentration 1 mg/mL (42);). The relative fluorescence units (RFU) were detected every 15 min for a period of 120 min applying 485 nm excitation and 530 nm emission wavelengths (Varioskan^® Flash, Thermo Scientific).

Amplex Red^® reagent (Invitrogen) was used to assay extracellular ROS production. Bovine neutrophils (2 ×10⁵ cells in 100 μL; n = 3, in duplicates) were seeded in a plastic 96-well plate (Greiner). Pre-warmed (66.6 µL, 37°C, 10 min) Amplex Red^® reaction mixture [37.5 μL Amplex Red^® (20 mM) and 75 μL horseradish peroxidase (HRP; 20 U/mL, Merk), in 5.9 mL RPMI 1640 medium without phenol red (Sigma-Aldrich)] were added to each well. Immediately before starting measurements, the stimuli were added in 33.5 μL [10 or 100 µg/mL CdAg; zymosan (1 mg/mL) or plain medium]. Fluorescence intensity was measured in an automated multiplate reader (Varioskan^® Flash, Thermo Scientific) at 530 nm excitation wavelength and 590 nm emission wavelength (43) at determined time points (0, 15, 30, 60, 90, 120 and 150 min of stimulation) and being incubated (37°C, 5% CO₂) between measurements.

Bovine neutrophils (n = 3; 1×10⁶ cells) from three healthy donors were co-cultured with CdAg (10 - 100 µg/mL) in HBSS 1X (14065-049; Gibco) for 15 min (37°C, 5% CO₂). Cells were stimulated with phorbol myristate acetate (PMA)/ionomycin (100 nM/5 M; Sigma-Aldrich) as a positive control. After resting on ice for 5 min, cells were centrifuged at 600 × g for 5 min and extracellular adenosine triphosphate (ATP) concentrations were determined in supernatants using an ATP Determination kit (A22066; Invitrogen) according to manufacturer’s instructions. Total ATP concentration was quantified in whole cell pellets using CellTiter-Glo luminescent. All samples were analyzed by luminometry with an automated reader (Luminoskan Flash).

Statistical analyses and all graphs (mean ± SD) were performed using GraphPad Prism^® versions 8.3.1-9.2.0 for Windows 10, GraphPad^® Software, San Diego, California USA, www.graphpad.com. For comparison of two groups, unpaired two-tailed Mann-Whitney tests were applied, and for comparing three or more groups, no pairing non-parametric Kruskal-Wallis test was performed with Dunn’s multiple comparison test. For ATP analyses ordinary one-way ANOVA and Dunnett’s test for multiple comparisons were applied. Statistical significance was defined at p < 0.05.

Following genetic analysis confirmation of rumen flukes as Calicophoron daubneyi specimens ( Supplementary Table 1 ), C. daubneyi-induced NETosis was analyzed via different methods, i.e., SEM, immunofluorescence, nuclear area expansion (NAE) and spectrofluorometric quantification. SEM analyses revealed that bovine neutrophils co-cultured with 100 µg/mL CdAg extruded short length NET filaments ( Figures 2A–C , arrows), i.e., short spread-like NET structures (sprNETs), while preserving their characteristic round configuration. Other neutrophils remain unchanged ( Figures 2B, C ). Immunofluorescence analyses of co-cultured bovine neutrophils with CdAg 100 µg/mL confirmed the presence of classical components of NETs ( Figures 2D, E ) in short sprNETs, while no extruded NET filaments were visible in non-exposed controls ( Figures 2D2–4 ). Co-localization of extracellular DNA ( Figure 2E1 , blue), neutrophil elastase ( Figure 2E2 , green) and histone (H1-4, Figure 2E3 , red) were identified in CdAg-induced NETosis ( Figure 2E4 ). Also, early NETosis was studied via NAE analyses, as one of the earliest events of NET formation is the decondensation of neutrophil nuclear chromatin. After co-culture of CdAg (100 µg/mL) or Cd-eggs with bovine neutrophils, exposed neutrophils presented a significant increase in NAE when compared to non-exposed controls ( Figures 2F, G , p < 0.0001). In total, more than 570 neutrophils were analyzed individually per condition (n = 3). Additionally, extracellular spectrofluorometric DNA-based quantification of NETosis via PicoGreen^®-derived fluorescence intensities was measured after 120 min co-culture of bovine neutrophils and CdAg 10 and 100 µg/mL ( Figure 2H ). In both cases a rise in PicoGreen^® RFU was observed when compared to neutrophils alone. However, only CdAg 100 µg/mL concentration revealed to be significantly higher ( Figure 2H , p = 0.0341). All these results confirm that C. daubneyi is weak inducer of bovine NETosis.

Immunofluorescence analyses of CdAg-exposed bovine neutrophils (n = 3) unveiled not only the formation of short sprNETs but also granular structures similar to the observed in degranulation process of stimulated neutrophils ( Figures 3A–D ). During neutrophil activation and degranulation, cytoplasmatic granules fuse with the cell membrane thereby discharging their cargo. Here, we observed small round granules in the vicinity of activated neutrophils casting NETs ( Figure 3D ), resembling discharged granules, which led to further investigations. Therefore, as degranulation assay, the myeloperoxidase (MPO) release test was performed showing no alteration on the percentage of MPO release on confronted bovine neutrophils ( Figure 3E ). Noteworthy, during live cell 3D-holotomographic microscopy analyses of bovine neutrophils stimulated with CdAg 100 µg/mL similar degranulation images were observed within activated neutrophils ( Figures 4A, B , arrows). As early as 30 min after stimulation of bovine neutrophils with CdAg 100 µg/mL, it was observed a displacement of neutrophils within the microscopic field ( Figure 4C , yellow arrow). Displacements were also observed via SEM analyses of bovine neutrophils co-cultured with C. daubneyi-eggs ( Figures 4D, E , yellow arrows), confirming that C. daubneyi is capable of bovine neutrophil activation.

Oxygen consumption rate (OCR) measurements reflect neutrophil oxidative responses due to oxidative burst activity by assessing NADPH oxidase (NOX)-related oxygen consumption and reflects mitochondrial respiratory activity, while extracellular acidification rates (ECAR) reflect production and release of lactate as a product of glycolysis. The AUC (area under the curve) analyses revealed CdAg 100 µg/mL induced increase of OCR (p = 0.0152, n = 6) and ECAR (p = 0.0022, n = 6) levels on stimulated bovine neutrophils when compared to controls ( Figures 5A–D ). OCR increase is detected after 100 min of experiment which might be related to glycolysis involvement. High variability in cellular immune responses between individual bovine donors could be observed. Here, only three out of the six individuals responded strongly upon antigen stimulation. The same experiments with CdAg 10 µg/mL were performed but no significant changes were reported, as for lower concentrations ( Supplementary Figure 1 ).

Intracellular ROS production (n = 3) was measured by oxidation of 2′,7′-dichlorofluorescein diacetate to fluorescent DCF ( Figure 6A ). During 120 min, ROS production was only slightly increased by stimulation of bovine neutrophils with CdAg 100 µg/mL ( Figure 6B , p = 0.600), but CdAg 10 µg/mL failed to induce neutrophil-derived ROS production ( Figure 6A ), indicating a dose dependent induction of intracellular ROS production. Zymosan, a well-known inducer of neutrophil respiratory burst activity, was used as positive control. For extracellular ROS production, the highly specific and sensitive Amplex Red quantification assay was performed to detect extracellular hydrogen peroxide (H₂O₂). The H₂O₂ production was not altered by stimulation with CdAg 10 µg/mL over time ( Supplementary Figure 2 ). CdAg 100 µg/mL induced a change in H₂O₂ production, even higher than zymosan effect ( Supplementary Figure 2 ). However, the same magnitude of effect was observed when CdAg 100 µg/mL alone was tested, showing that CdAg 100 µg/mL is also not capable of inducing any H₂O₂ production. These results show that C. daubneyi antigen does not induce ROS production, commonly associated with mammalian NET formation.

To investigate the effects of CdAg on total- and extracellular ATP concentrations of stimulated neutrophils (n = 3), ATP concentrations were quantified by luminometry at 15 min of stimulation ( Figure 7 ). For positive controls, PMA/ionomycin were used. Upon CdAg 10 µg/mL stimulation, both total and extracellular ATP measurements showed no differences between stimulated and non-stimulated neutrophils ( Figure 7 ). With CdAg 100 µg/mL, total ATP concentration was significantly lower than negative control ( Figure 7A , p = 0.0198), while ATP levels measured on supernatants remained unaltered ( Figure 7B , p = 0.4722). Nevertheless, bovine neutrophils stimulated with PMA and ionomycin showed a significant drop in ATP levels ( Figure 7A , p < 0.0001) related to ATP consumption, and an increasing extracellular ATP concentration ( Figure 7B , p = 0.0363).