The U.S. National Animal Health Monitoring System reported that 32% of calf mortality in 2018 was caused by diarrhea during the first 3 weeks of life (1). Calf diarrhea is predominantly caused by infectious agents, such as viruses, bacteria, and protozoa. The main causative agents of calf diarrhea include bovine rotavirus group A, bovine coronavirus, Salmonella spp., Enterotoxigenic Escherichia coli (ETEC), Clostridium perfringens type C, and Cryptosporidium parvum (2). Regardless of the cause of diarrhea, reduced bacterial diversity and changes in the gut bacterial populations from normal have reported in diarrheic calves (3). In calves, diarrhea is associated with an increase in taxa from the phylum Proteobacteria, especially increased abundance of Enterobacteria (4–6) and decreased abundance of butyrate-producing bacteria (e.g., Bifidobacterium and Faecalobacterium) (3, 7). In humans, a reduction in butyrate producing obligate anaerobes such as Faecalibacterium prausnitzii (8–10) and an increase in facultative anaerobes such as Escherichia coli appears to be a feature of gastrointestinal diseases (8–10). It has been hypothesized that this shift from obligate anaerobes to facultative anaerobes results from an increased release of hemoglobin carrying oxygen and reactive oxygen species into the lumen of the gastrointestinal tract (oxygen hypothesis), which favor the growth of facultative anaerobes (11). Therefore, the first objective of this study was to investigate the fecal bacterial alterations of calves with acute diarrhea (<48 h (h) duration) to determine whether a shift from obligatory to facultative anaerobes occurs in diarrheic calves.

Metabolic acidosis (decreased blood pH and HCO3- concentration) is the most common acid-base disorder occurring in calves with diarrhea (12). Metabolic acidosis is usually accompanied by an increased anion gap (AG), which represents the presence of unmeasured anions (UA) in plasma, commonly D- and L-lactate (6, 7, 12–14). The mechanisms resulting in increased concentrations of UA in diarrheic calves are not fully understood; however, the concentration of D- and L-lactate in fecal samples of diarrheic calves is higher than that of healthy calves (13). These findings suggest that gut bacterial fermentation could contribute to the development of AG acidosis in diarrheic calves. Therefore, the second objective of this study was to investigate if there is a relationship between the fecal microbial alterations of diarrheic calves and the changes in blood pH, bicarbonate, and the load of unmeasured ions calculated using the Anion Gap (AG).

This study investigated the fecal bacterial alterations of calves with diarrhea and examined the association of those bacterial changes with changes in blood pH, HCO3- concentration and AG. In diarrheic calves, richness, evenness and diversity were reduced compared to healthy calves, and both community structure and membership were different between healthy and diarrheic calves. These alterations indicated that dysbiosis occurred in the diarrheic calves included in our study. In our study, a shift from obligate anaerobes to facultative anaerobes was characterized by a decrease in Bacteroidales, Bacteroides, Phocaeicola, Prevotella, Odoribacter Lachnospiraceae, Ruminococcaeceae, Butyricicoccus, Blautia and Faecalibacterium and an enrichment of Streptococcus, Enterococcus, Gallibacterium, and Escherichia/Shigella in diarrheic calves. This shift from obligate anaerobes to facultative anaerobes have been regarded as a hallmark of dysbiosis of gastrointestinal microbiota in different species (3, 11, 24–27). The gastrointestinal tract is characterized by a low oxygen concentration harboring many obligate anaerobes (10⁷ to 10¹¹ g⁻¹ colonies), however during dysbiosis in diarrheic calves it is possible that a reduction or disappearance of oxygen-sensitive bacteria (e.g., Faecalibacterium) occurs (7, 11, 27). The reduction of taxa (e.g., Blautia) that maintain anaerobic conditions in the GIT could have led to a higher oxygen availability in the intestine favoring the proliferation of facultative anaerobes (e.g., Streptococcus, Enterococcus Gallibacterium, and Escherichia/Shigella) (28). Thus, in diarrheic calves, the reduction in Lachnospiraceae, Ruminococcaeceae, Butyricicoccus, Blautia and Faecalibacterium could have contributed to the shift from obligate anaerobes to facultative anaerobes. Proliferation of facultative anaerobes in animal models is associated with a proinflammatory mucosal immune response and plays a causative role in gastrointestinal inflammation (3–5, 7, 11, 26–28). During gastrointestinal inflammation, an increased release of hemoglobin carrying oxygen and reactive oxygen species into the lumen of the gastrointestinal tract appear to occur, which could favor the growth of facultative anaerobes (11). Low oxygen tension conditions facilitate proliferation of Enterobacteriaceae and other potentially pathogenic facultative anaerobes (e.g., Enterococcus, Pseudomonas), which can elicit an inflammatory response (28). Changes in the luminal pH of the GIT could have also contributed to the bacterial alteration observed in the diarrheic calves, because pH causes selective pressure on bacterial growth and metabolism (29). In horses with grain overload and cattle with ruminal acidosis the increase in facultative anaerobes, especially Enterobacteriaceae along with an acidic luminal environment is associated with cecal and ruminal mucosal damage and facilitate translocation of bacteria and their products into systemic circulation (30–34). This is of interest because bacteremia (35–37) and endotoxemia (38) has been reported in diarrheic calves with bacteremia occurring in 10 to 30% of diarrheic calves with E. coli and other facultative anaerobic Enterobacteriaceae bacteria (e.g., Salmonella, Enterobacter, Klebsiella) being the most common bacteria isolated (35–37). These findings highlight the crucial role of gastrointestinal bacterial communities in local and systemic inflammatory responses observed in diarrheic calves.

Diarrheic calves had a lower blood pH and bicarbonate concentrations and higher AG values than healthy calves. Increased concentrations of L- and especially D-lactate in the lumen of the GIT and subsequent absorption into systemic circulation play an important role in the local and systemic abnormalities observed in calves with diarrhea (13, 39–41). In horses with carbohydrate overload (30, 31), cattle with ruminal acidosis (42, 43) and humans with SBD (42, 43), luminal concentrations of D- and L-lactate are increased. These alteration are associated with decrease lactic acid fermentation from both proliferation of lactate-producing bacteria and inhibition of lactate fermenting bacteria because of the availability of carbohydrates (30, 34). In our study, lactate-producing bacteria including Lactobacillus, Streptococcus, Veillonella, Ligilactobacillus and Olsenella were enriched in diarrheic calves. Therefore, the lower blood pH and bicarbonate concentration and the high AG values identified in diarrheic compared to healthy calves was likely due to increased absorption of D- and L-lactate from the lumen of the gastrointestinal tract. The main source of energy and carbohydrates in cow milk is lactose, which is hydrolyzed by lactase in the small intestine to glucose and galactose (44). Lactase is produced and secreted at the brush border of the small intestine (45). Most pathogens associated with calf diarrhea destroy the brush border of the small intestine leading to a decreased activity of lactase (2, 46). In healthy calves under normal lactase activity, only a small percent of ingested lactose will reach the colon (47, 48). When intestinal lactase is low (e.g., during diarrhea) lactose is not metabolized in the small intestine, thus a large amount of lactose enters in the colon (45, 47, 48). Lactate-producing bacteria, such as Lactobacillus, possess β -galactosidase activity and hydrolyze lactose to glucose and galactose (49, 50). Following this hydrolyzation reaction, galactose is then fermented into products such as short chain fatty acids, and D- and L-lactate (50). In diarrheic calves, impaired lactose metabolism has been demonstrated (13, 48). Therefore, the increased luminal concentrations of D- and L-lactate probably resulted from an increased passage of lactose to the distal small intestine and colon due to maldigestion and malabsorption of milk (the only source of food for pre-weaned calves) in the gastrointestinal tract, which may have caused an overgrowth of lactate-producing bacteria (13).

In our study, a single time point fecal sample at the onset of diarrhea was assessed for microbiota analysis, therefore we could not determine whether the differences in fecal microbiota between healthy and diarrheic were already pre-existing and indeed contributed to the susceptibility of the calves to enteropathogens. This is of importance because different farm environmental conditions and management practices such as colostrum administration (51–53), diet (54) or antimicrobial usage at the farm level (55, 56) contribute to the development of the immune system and the colonization and establishment of the bacterial communities in the GIT microbiota (57, 58). Therefore, a longitudinal study involving a large number of farms with multiple management practices is needed to confirm whether the shift from obligated to facultative anaerobes results from the gastrointestinal inflammation or it is a predisposing factor for diarrhea associated with certain management farm practices.

There are some limitations that should be considered when interpreting the results of our study. First, calves of different ages (i.e., 3–7 days) were included in the study. This is of importance because the fecal microbiota of newborn calves changes rapidly during the first days of live (0 to 7 days). However, during sampling, the calf's ages ranged from 10 to 14 days. At that age, the changes of the fecal microbiota are less dramatic than those observed during the first week of life (59–62). Finally, the load of unmeasured anions was estimated using the anion gap which could be affected by changes in blood concentration of total plasma proteins (i.e., albumin and globulin) and phosphates (12) of calves, especially in markedly dehydrated diarrheic calves. Therefore, studies determining the individual concentration of D- and L-lactate in blood and feces of diarrheic calves and their relationship with alterations in the microbiota are warranted. Nonetheless, the results of this study indicate that calf diarrhea is associated with a shift from obligated to facultative anaerobes and expansion of lactate-producing bacteria which are related to acidemia and increased concentrations of unmeasured anions (L- and D-lactate) estimated using the AG.

The original contributions presented in the study are publicly available. This data can be found here: https://www.ncbi.nlm.nih.gov/sra, BioProject ID: PRJNA795094.

The animal study was reviewed and approved by Animal Care Committee, University of Guelph (Animal Use Protocol no. #4238).

DG and DR contributed to the conception and design of the study. DG, LL, HG, LG, and DR contributed to drafting and critically revising the manuscript for important intellectual content. LL and HG contributed to acquisition of data. DG and LL performed the analysis of data. All authors approved the final version to be submitted.

Funding for this study was provided by Veal Farmers of Ontario (Guelph, ON, Canada).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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