The primary active transporters are ATP-dependent, including the family of ABC transporters and ATPases (ion pumps) that transport ions, lipids, carbohydrates, and xenobiotics against a concentration gradient (Schumann et al., 2020). ABC transporters play a crucial role in the pathophysiology of infectious diarrhea for two reasons: they facilitate secretion of various ions such as chloride and modulate xenobiotic absorption, distribution, metabolism, and secretion, but they can also interact with pathogens, potentially contributing to host protection (Mercado-Lubo and McCormick, 2010).

A number of ABC transporter genes were expressed in our data set ( Supplementary Table 2 ), but only ABCC2, ABCC6, and ABCD1 expressions were reduced by infection. Nies and Keppler (2007) previously reviewed the substrate specificity of human and rat ABCC2. In the gut epithelium, ABCC2 localizes exclusively in the apical brush border membrane of villi, serving to efflux anions such as chloride and excrete endogenous and xenobiotic substances, especially in conjunction with glutathione, glucuronate, or sulfate into the intestinal lumen (Nies and Keppler, 2007). During intestinal infection (by Salmonella typhimurium), ABCC2 was shown to be upregulated (Pazos et al., 2008), probably to facilitate the apical efflux of the neutrophil chemoattractant hepoxilin A3 (HXA3) (Mrsny et al., 2004). Even though C. parvum induces intestinal inflammation, we have previously shown that neutrophil chemotaxis and differentiation associated pathways were downregulated (Veshkini et al., 2024), suggesting a minor role of neutrophils in neonatal calves right after the peak of infection. ABC transporter expression is under the control of transcription factors such as the retinoid X receptor (RXR), pleomorphic adenoma gene (PLAG), and zinc finger transcription factor specificity protein 1 (SP1) which imprints a tissue-specific expression (Abruzzese et al., 2022). In contrast to ABCC2, the ABCC6 transporter is located at the basolateral membrane and serves multiple functions, however the precise nature of its substrate(s) is unknown (Vanakker et al., 2013). It can be postulated that nutrient drainage by C. parvum interferes with the ABCC2 and ABCC6 function and expression, thereby disrupting the secretion and homeostasis of one or more (yet unknown) substrates. Furthermore, ABCD1, which is located at the peroxisome membrane, facilitates the import of coenzyme A-activated saturated very long-chain fatty acids (VLCFA; >C22:0) into peroxisomes for break down by β-oxidation, a crucial step in activating innate immunity, especially pro-inflammatory signaling in monocytes and macrophages (Zierfuss et al., 2022). Since fatty acids are required for both glycolysis and lipogenesis to cover the rapid energy demands and produce pro-inflammatory mediators, a downregulation of ABCD1 appears to interfere with the immune response and lead to the accumulation of VLCFA in plasma and tissues.

In addition, C. parvum infection affected members of ATPase transporters, resulting in higher expression of ATPase Ca²⁺ transporting 2 (ATP2A2) and ATPase Copper Transporting Alpha (ATP7A). ATP2A2 is an intracellular pump primarily located in the sarcoplasmic reticula and endoplasmic reticula of muscle tissues (Dhitavat et al., 2003). It functions as a regulator of the contraction/relaxation cycle by hydrolyzing ATP and translocating calcium from the cytosol into the sarcoplasmic reticulum lumen, thus probably playing a role in the intestinal motility to compensate diminished absorption. This claim is, however, unsupported by any evidence, and further research is required to approve it.

Solute carrier proteins (SLC) are a large and diverse group of membrane proteins which mediate passive and secondary active transport of ions, nucleotides, and sugars across biological membranes. In this study 240 solute carrier proteins (SLC) were identified in our data set ( Supplementary Table 2 ), of which 18 genes had higher and 14 genes had lower expression levels ( Figure 2 ). Bioinformatics analysis suggested that the transport and homeostasis of cation, anion, and metal ion, carboxylic acid, sulfur, pyrimidine containing compounds, nucleobase containing compounds, purine containing compounds, and carbohydrate derivate were disturbed due to the lower expression levels of SLC48A1, SLC22A17, SLC40A1, SLC11A2, SLC30A10, SLC37A2, SLC29A2, SLC24A3, SLC4A8, SLC5A4, SLC19A3 and the higher expression levels of SLC3A2, SLC20A1, SLC6A19, SLC31A1, SLC4A4, SLC5A8, SLC10A2, SLC6A8 ( Figure 3 ; Supplementary Table S5 ).

In the pathophysiology of diarrhea, dysregulation of intestinal ion transport is a critical factor (Das et al., 2018). Some of the differentially expressed SLC transporters are involved in the ion transport system across (in or out) cell membranes. In particular, influx divalent metal transporter 1 (DMT1), also known as SLC11A2, is the solute transporter responsible for transport of ferrous iron across the brush border membrane of intestinal epithelial cells (Kayaaltı et al., 2015). Afterwards, the basolateral efflux channel ferroportin (FPN, SLC40A1) mediates the intracellular translocation and export of iron into the circulation in conjunction with multicopper ferroxidase, including hephaestin (Heph) and/or ceruloplasmin (Cp), and hepcidin (Dlouhy et al., 2019). These two SLC transporters are exclusively iron transporters, and their downregulation in our study indicates diminished iron turnover. Iron availability is crucial not only to support erythropoiesis, immune cell differentiation and function, metabolic processes, and cellular respiration but also essential for pathogens’ proliferation and pathogenicity (Grander et al., 2022). There is evidence indicating a close relationship between DMT1 and FPN transporters and inflammation. Previous work reported that intestinal inflammation interferes with iron metabolism and DMT1 expression, affecting erythropoietic (Urrutia et al., 2013) and macrophage function (Grander et al., 2022). Activated Toll-like receptors (TLRs) and inflammatory factors such as IL-6 are known to induce the production of hepcidin, which consequently reduces iron availability by suppressing the expression of FPN1 through the hepcidin-FPN1 axis (Cai et al., 2021). TNF exposure reduced DMT1 expression in human intestinal cells (in vitro) (Johnson et al., 2004) and reduced duodenal iron transport in mice (in vivo) (Laftah et al., 2006). In accordance, we reported earlier that TNF-α is induced in response to C. parvum infection but neither erythrocytes and immune cell count nor hemoglobin and hematocrit concentrations were significantly affected (Veshkini et al., 2024).

The other group of differentially expressed SLC transporters in our study are involved into transport of more complex compounds such as sugars, amino acids, vitamins, nucleotides, carbohydrates, oligopeptides, and drugs. In the small intestine, thiamine (Vitamin B1) is transported across cell membranes by SLC19A3, where it is phosphorylated to its active form, thiamine pyrophosphate (TPP), and acts as a cofactor of transketolase. TPP is later transported by SLC25A19 to mitochondria, where it is a cofactor of pyruvate dehydrogenase complexes, branched chain ketothiolase dehydrogenases, and alpha-ketoglutarate dehydrogenases (Plecko and Steinfeld, 2017). SLC19A3 downregulation while SLC25A19 remains unchanged may affect the pyruvate oxidation in TCA cycle as well as energy and amino acids metabolism. SLC25A13, encoding a citrin protein, is an important mitochondrial solute transporter involved in the urea metabolism by swapping mitochondrial aspartate for cytosolic glutamate, where it can be used later for nucleotide synthesis pathways (Lin et al., 2011). Thus, it can be postulated that downregulation of SLC25A13 may affect the malate-aspartate shuttle, gluconeogenesis, amino acid homeostasis, TCA cycle, and protein turnover.

The higher expression levels of SLC transporters appears to be due to feedback mechanisms initiated by inflammation as compensation for vital compounds and also to regulate the activity of immune cells, but less information is available in cattle. ASBT is an active sodium-dependent bile acid transporter (encoded by SLC10A2) responsible for ileal bile acid (BA) reabsorption and has also been linked to intestinal disorders like diarrhea (Jung et al., 2004). Moreover, it plays an important role in regulating lipid and cholesterol homeostasis as well as determining the size of the BAs pool (Han et al., 2015). ASBT gene expression is primarily controlled by transcription factors such as caudal-type homeobox-1 (CDX1) and -2 (CDX2) and hepatocyte nuclear factor 1-α (HNF1-α), nuclear receptors such as retinoid X receptor (RXR) and farnesoid x receptor (FXR), and intestinal flora, which plays a critical role in both pathological and physiological processes as reviewed by (Yang et al., 2020). The reasons for the SLC10A2 higher expression is not clear but it seems to be a compensatory mechanism to reabsorb BA and decrease the excretion of fecal BAs and keep the liver cholesterol homeostasis in check.

Solute carrier family 1, member 1 (SLC1A1; also known as EAAT3 or EAAC1) is a major epithelial transporter of L-glutamate and D/L-aspartate especially in the intestines (Bailey et al., 2011; Sheng et al., 2022). The sodium bicarbonate cotransporter (NBCe1) encoded by SLC4A4 plays a role in pH regulation and homeostasis by transporting the bicarbonate from the blood to the lumen (Cappellesso et al., 2022). Positive correlations were found between SLC1A1 expression and levels of infiltrating CD8+T cells and dendritic cells, and between SLC4A4 expression and CD8+T cell infiltration levels (Zhou et al., 2020). A number of organic anion transporting polypeptides (OATPs) substrates are transported by SLCO4A1, including steroid hormone conjugates, pro-inflammatory prostaglandin PGE2, thyroid hormones, and xenobiotics. SLCO4A1 expression is under control of inflammation-associated pathways such as NF-κB, and TNF-receptor 2 signaling cascades (Koller et al., 2022). As immune cells depend on SLCs to induce rapid and robust metabolic reprogramming, thereby controlling their expression through major immune associated nuclear transcription factors, this seems to be a consequence of an activated immune response.

In our study, a subset of genes was identified to function as ion channels, notably chloride channels, potassium channels (KCN family), magnesium transporters, copper transporters, and heme transporters ( Supplementary Table 2 ). Among those, the infected calves had lower expression of KCNJ15, KCNK16, and KCNK17, as well as chloride voltage-gated channel 6 (CLCA6), whereas calcium-sensitive chloride transporting channel 4 (CLCA4) had higher expression.

During infectious diarrhea, disruption of ion channel function may lead to alterations in electrolyte, nutrient, and fluid transport, potentially causing acid-base imbalances. In this regard, a good example would be the complex relationship between ion channels and lactose absorption in the intestinal epithelium under diarrheal conditions (Misselwitz et al., 2019). C. parvum-induced diarrhea leads to reduced lactase activity at the brush border, lower absorption of lactose metabolites and sodium via sodium/glucose cotransporter 1 (SGLT1), and increased passage of lactose to the distal small intestine and colon (Gomez et al., 2022). Due to the coupled transport of sodium and potassium with glucose and galactose, reduced lactose absorption means less glucose is available for SGLT1, potentially decreasing sodium absorption.

The function of potassium channels is closely connected with the (re)absorption of Na⁺, Cl⁻, and water as well as secretion of K⁺, HCO₃ ⁻ in the basolateral and luminal membranes of the epithelial cells (Heitzmann and Warth, 2008). Chloride channels regulate intestinal Cl− secretion and have emerged as potential targets in the diarrhea treatment (Ko et al., 2014; Thiagarajah et al., 2015). At the crypt base, cells predominantly secrete fluid containing Cl⁻ and HCO₃ ^– but the surface epithelial cells’ function is to reabsorb Na⁺ and to secrete HCO₃ ⁻ and K⁺ to preserve the ion gradient at the crypt-villus axis (Heitzmann and Warth, 2008). Therefore, ion channel deregulation, leading to acid-base imbalances, as a part of physiologic and metabolic pathways leading to bacterial D-lactate production, could partly explain diarrheic calf metabolic (D-lactic) acidosis.

The absorption of chloride is essential for fluid absorption, and when dysregulated by infection, it affects the absorption of water and other solutes. In this regard, another cluster of DEG was associated with the transportation of solutes such as water and cholesterol ( Supplementary Table 4 ). In response to infection, aquaporin (AQP11) expression was reduced, whereas ATPase transporters (ATP2A2, ATP7A) expression were significantly elevated. AQPs are water channel proteins that facilitate the absorption of fluid (primarily water) in many tissues including the small intestine and colon and are responsive to osmotic gradients (Zhu et al., 2016; Tomita et al., 2017; Zhang et al., 2019). The osmoregulation and mucosal fluid fluxes associated with ion and aquaporin channels make them a potential target for diarrhea treatment.