Effects and immune responses of probiotic treatment in ruminants

Abstract

Gut microbial colonization and establishment are vital to ruminant health and production. This review article focuses on current knowledge and methods used to understand and manipulate the gut microbial community in ruminant animals, with a special focus on probiotics treatment. This review highlights the most promising of studies in this area, including gut microbial colonization and establishment, effect of gastrointestinal tract microbial community on host mucosal innate immune function, impact of feeding strategies on gut microbial community, current probiotic treatments in ruminants, methods to manipulate the gut microbiota and associated antimicrobial compounds, and models and cell lines used in understanding the host immune response to probiotic treatments. As a lot of work in this area is done in humans and mice, this review article also includes up-to-date knowledge from relevant studies in human and mouse models. This review is a useful resource for scientists working in the areas of ruminant nutrition and health, and to researchers investigating the microbial ecology and its relation to animal health.

Gut microbial colonization and establishment

During fetal development, intestinal tissues proliferate while the rumen is underdeveloped at birth. The rumen proliferates from 30% to 70% gastrointestinal tract (GIT) capacity (Warner, 1956), while the intestinal tissues develop earlier to enable the digestion and absorption of nutrients from colostrum. The initial colonization of the ruminant GIT is suggested to derive from the environment and maternal feces, saliva, skin, and vaginal canal (Meale et al., 2016), while on-farm practices (e.g., housing, contact with dam/ewe), diet, and development contribute to GIT microbial establishment. As the ruminant ages, diet and GIT development are key contributors to the changes in the GIT bacterial communities (Malmuthuge et al., 2014). Bacteria, archaea, and fungi are the first microbiota to colonize the GIT, whereas rumen ciliates are not present within the first week after birth. Rumen ciliates may colonize by mouth to mouth contact with a faunated adult. However, specific ruminal conditions are required for their establishment (Eadie and Hobson, 1962). Archaea and fibrolytic bacteria colonize the GIT of the young ruminant within 20 minutes after parturition (Guzman et al., 2015).

The bacteria belonging to the genera Bifidobacterium, Faecalibacterium, and Lactobacillus are suggested to be of great importance to the young ruminant as they aid in GIT development and have demonstrated their effectiveness as probiotics. Specifically, bacteria belonging to the genus Faecalibacterium produce butyrate, a volatile fatty acid (VFA) that promotes ruminal and intestinal development. In vitro experiments suggest that butyrate enhances the gut barrier function through increased expression of tight junction protein-related genes (Wang et al., 2012). The lactic acid-producing bacterial genera Bifidobacterium and Lactobacillus are typically more prevalent in the pre-weaned ruminant because of an abundance of oligosaccharides in milk. Rey et al. (Rey et al., 2014) reported the relative abundances of the bacterial genus Bifidobacterium in the rumen to be stable from birth to 83d in age. The bacterial genus Lactobacillus was undetectable in ruminal fluid from 2-day old and 15–83d old calves, while low relative abundances (1.2%) were present at 3–15d in age (Rey et al., 2014). Fecal relative abundances of the genera Bifidobacterium, Faecalibacterium, and Lactobacillus were shown to be greater in pre-weaned (1.21, 4.07, and 2.87%, respectively) than post-weaned (0.34, 0.94, and 0.20%, respectively) calves (Meale et al., 2016). Song et al. characterized the hindgut digesta and mucosa-attached microbiota from calves at birth, 7, 21, and 42 days of age. Newborn and 7d-old calves had greater relative abundances of mucosa-associated Lactobacillus and potential pathogenic bacteria Escherichia-Shigella and Salmonella than 21 and 42-d old calves. There was a positive correlation between the relative abundance of Faecalibacterium and molar proportions of butyrate, the both of which peaked at 21d of age (Song et al., 2018). McFarland et al. showed that fecal bacterial operational taxonomic units (OTU) related to the genera Faecalibacterium and Lactobacillus decreased or disappeared as calves aged (Dill-McFarland et al., 2017). As the ruminant reaches adulthood, the rumen contains greater relative abundances of the genus Prevotella and members of the bacterial family Succinivibrionaceae along with increased microbial diversity (Dill-McFarland et al., 2017).

Effects of GI tract microbial community on host mucosal innate immune function

GIT commensal microbiota are involved in the development of the intestinal architecture and immunomodulatory processes in a symbiotic and beneficial way. Immediately after birth, ruminants go through a process of colonization of foreign microorganisms. The archetypal molecules produced by the commensal microorganisms mediate the development of the host immune system. For example, Bacteroides fragilis, a gram-negative anaerobe, colonizes the mammalian lower GIT, with beneficial properties in ameliorating inflammatory symptoms. It was reported that the Zwitterionic polysaccharides produced by B. fragilis played several immunomodulatory roles in directing the maturation of the developing immune system, including correcting systemic T cell deficiencies, adjusting T helper cell 1 (Th1)/T helper cell 2(Th2) imbalances and directing Lymphoid tissue biogenesis (Mazmanian et al., 2005). On the other hand, the host immune response helps the beneficial microbes to establish a robust colonization and spatial segregation between the host and the microbes. In a recent study by Donaldson and co-authors (Donaldson et al., 2018), the immunoglobulin A (IgA) antibody produced by the host in response to the B. fragilis capsule helps anchor the bacteria to the epithelial surface, providing a colonization advantage. In comparison, in mice lacking IgA, the bacterium was less successful at colonizing the intestinal surface and subsequently less efficient in maintaining long-term stability. RegIIIγ is an antibacterial lectin expressed in intestinal epithelial cells. It promotes the spatial separation of commensal microbiota and host in the intestine, and maintains a lectin-mediated, symbiotic host-microbial relationship (Cash et al., 2006; Vaishnava et al., 2011). Consistent with these findings, studies in germ-free animals revealed that significant immune system deficiency is associated with a lack of gut microbiota composition and metabolic activity of the intestinal microbiome (Okada et al., 1994; Bouskra et al., 2008).

Specific microbes having demonstrated roles in promoting inflammatory (Ivanov et al., 2008; Ivanov et al., 2009), or anti-inflammatory (Mazmanian et al., 2008; Round and Mazmanian, 2010) responses in the gut. As a prominent human commensal species, B. fragilis, mediates the conversion of CD4⁺ T cell into Foxp3(+) regulatory T cells, which suppress IL-17 production, and therefore, protect the host against inflammatory insults (Round and Mazmanian, 2010; Round et al., 2011). Furthermore, polysaccharide A produced by B. fragilis, is capable of protecting animals from intestinal inflammation through the suppression of IL-17 production (Mazmanian et al., 2008). Thus, when selecting beneficial probiotics with the aim to manipulate the GIT microbiome, the immunoregulatory properties are important factors to consider (Kechaou et al., 2013).

Effects of feeding regime on gut microbiota

The composition of the intestinal microbiota is dependent on diet, antibiotic use, host genetics and other environmental factors (De Filippo et al., 2010; Maslowski and Mackay, 2011). Alterations in the commensal bacterial populations have significant effects on fiber digestion and fermentation, vitamin synthesis, and regulation of inflammatory responses (Maslowski and Mackay, 2011). The microbiota enable the host to absorb nutrients from complex dietary carbohydrates that can’t be digested by mammalian enzymes (Zebeli and Metzler-Zebeli, 2012). Specifically, microbial fermentation produces VFAs, which have effects on immunoregulation, metabolism and ruminal epithelium maturation (Baldwin and McLeod, 2000; Maslowski and Mackay, 2011; Laarman et al., 2012). Maintaining the ecological balance among the gut microbiota via nutrition is crucial for the health of the host (Zebeli and Metzler-Zebeli, 2012).

The quantity of carbohydrate in the feed has significant effects on the ruminal microbiome. The main digestible component in grain-based cattle diets is starch, which rapidly ferments in the rumen and generates a large amount of VFA (Klevenhusen et al., 2017). Feeding cattle concentrates rich in starches increases the risk of subacute ruminal acidosis, which is due to the accumulation of VFA in excess of the rumen buffering capacity (Aschenbach et al., 2011). Recent studies have demonstrated that feeding highgrain diets modifies the rumen microbial population in favor of amylolytic and lactic acid-producing populations at the expense of fibrolytic microbiota (Fernando et al., 2010; Petri et al., 2013; McCann et al., 2016). These changes to the ruminal microbiome can lead to a reduction of fiber degradation that can result in decreased feed efficiency (Klevenhusen et al., 2017).

In adult cattle, it has been shown that changes in dietary carbohydrates (inclusion of high quality hay containing higher sugar and metabolizable energy instead of concentrates with lower quality hay) shift the rumen epimural microbial population from predominantly Firmicutes to Proteobacteria with a higher abundance of Campylobacter (Petri et al., 2018). The effect of high-quality hay or high neutral detergent fiber feed versus starch-rich feed on the rumen microbial community diversity has varied among studies (Belanche et al., 2012; Petri et al., 2013; Klevenhusen et al., 2017; Petri et al., 2018). In a recent study, replacing fiber-rich hay with sugar-rich hay caused significant changes in the rumen bacterial communities at every taxonomic level; these alterations led to improvements in feed degradability and nutrient absorption (Klevenhusen et al., 2017). Changes to the ruminal microbiome, induced by feeding high quality hay, stimulated rumination, stabilized the ruminal pH, and improved total feed digestibility (Kleefisch et al., 2017). In beef cattle, the concentrate-based diet has been shown to cause an increase in the relative abundance of Proteobacteria; a high ratio of Proteobacteria to (Firmicutes + Bacteroidetes) was shown to be a good indicator of ruminal dysbiosis (Auffret et al., 2017). The changes to the rumen microbiota are therefore influenced by both diet and management practices (Shanks et al., 2011).

Early feeding regimes in calves have also been shown to have significant effects on the gut microbiota. In dairy calves, the gut microbiota is affected by colostrum administration in the first 2 to 6 hours of life. It has been shown that feeding heat-treated colostrum, compared to fresh colostrum or no colostrum, increases the relative abundance of Bifidobacterium and decreases the colonization of Escherichia coli in the small intestine (Malmuthuge, 2016). These results suggest that the impact of heat-treated colostrum is to enhance the establishment of beneficial microbiota and prevent pathogen colonization of the GIT (Malmuthuge, 2016). Following colostrum administration, calves on commercial dairies are fed whole milk or milk replacer with varying access to concentrates and forages. Pre-weaned dairy calves that were consuming calf starter, in addition to milk replacer, were shown to have increased ruminal fermentation capacity when compared to calves consuming milk replacer alone (Laarman et al., 2012). The increase in ruminal fermentation did not appear to cause a detrimental decrease in rumen pH in calves, which may indicate adaptation of the rumen epithelium to calf starter fermentation (Laarman and Oba, 2011). Additionally, the diet containing starter promoted greater diversity of bacterial taxa known to readily utilize fermentable carbohydrates (Dias et al., 2017), and the changes in the ruminal archaeal community were exclusively dependent on diet (Dias et al., 2017). Overall, diet and age were found to have simultaneous effects on the microbial commensal population (Dias et al., 2017). Access to concentrates in addition to milk and grass hay (compared to grass hay and milk alone) during the pre-weaning period, was found to alter the ruminal microbial population in lambs; some of these differences persisted 4 months after weaning while both groups were receiving the same diet (Yanez-Ruiz et al., 2010). These studies indicate the potential feasibility of manipulating the microbial populations early in life using diets or dietary additives to promote health and productivity benefits in adults (Yanez-Ruiz et al., 2015).

Antibiotic administration may also have an effect on the GIT microbiota. Antibiotics are commonly used to treat bacterial infections in all animal species, but the effects of antimicrobial drugs on the microbial communities of the GIT are not well described (Oultram et al., 2015). In previous human studies, antibiotic treatment significantly decreased taxonomic richness, diversity and evenness of the fecal microbiota and increased the number of antibiotic resistance genes found (Jernberg et al., 2007; Dethlefsen et al., 2008). A recent study in humans, however, demonstrated that while fluoroquinolone and β-lactam antibiotics caused significantly decreased microbial diversity in fecal samples, there was an increase in the average microbial load (due to an increase in Gram-negative bacteria) in people treated with β-lactam antibiotics (Panda et al., 2014). In a recent study evaluating the effect of antibiotics on the fecal microbiota of dairy calves, calves treated with parenteral oxytetracycline had a reduced amount of Lactobacillus species in their fecal microbiota one week after treatment and calves treated with florfenicol had reduced species richness that persisted two weeks post-treatment (Oultram et al., 2015). A recent metagenomic study in feedlot steers demonstrated that administration of antibiotic feed additives (e.g. monensin, tylosin) reduced the abundance of Gram-positive bacteria; however, there was no correlation between the administration of antimicrobial feed additives and the presence of antibiotic resistance genes (Thomas et al., 2017). Overall, it is evident that parenteral and oral antibiotic administration modifies the GIT microbiota. Further studies will be required to investigate the impact of these changes on animal and human health as well as the development of antimicrobial resistance.

Current bacterial probiotics research in ruminants

There has been variable terminology used to describe microbial additives used as dietary interventions to affect rumen function and improve animal health and performance (Yoon and Stern, 1995). The Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) definition of a probiotic was recently clarified in an expert consensus document: “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al., 2014). The most frequently studied probiotics include species of Saccharomyces yeast (Newbold et al., 1996), the filamentous fungus Aspergillus oryzae (Mathieu et al., 1996; Jouany et al., 1998), Bifidobacterium and lactic acid bacteria (Chiquette et al., 2008; Weimer, 2015). Additional probiotics studied in ruminants have included Bacillus, Enterococcus and Faecalibacterium species (Uyeno et al, 2015). Probiotics have been associated with enhanced intestinal health through the stimulation of beneficial gut microbiota, improved mucosal immunity, and the prevention of enteric pathogens that typically colonize in the intestines of young ruminants (Uyeno et al., 2015). In addition to targeting the lower GIT, probiotics have been used to enhance ruminal fermentation. It was reported that the administration of the lactate-utilizing bacterium, Megasphaera elsdenii, increased ruminal butyrate production and feed intake in neonatal calves (Muya et al., 2015). However, treatment of dairy calves with Lactobacillus plantarum and Bacillus subtilis were shown not to alter ruminal fermentation characteristics (Zhang et al., 2017). Current research continues to demonstrate the positive responses to early probiotic administration on animal performance and health, despite inconsistent effects on ruminal fermentation capacity (Table 1). Probiotics have the potential to enhance intestinal health by stimulating the development of a balanced microbiota with a predominance of beneficial bacteria, preventing the colonization of enteropathogens, increasing digestive capacity and improving mucosal immunity (Uyeno et al., 2015). Furthermore, demonstrated benefits of probiotic administration include increased body weight gain and reduced incidence of diarrhea (Timmerman et al., 2005; Goto et al., 2016).

Table 1.

Probiotic Treatment in ruminants

Probiotic	Host	Component affected by probiotic administration			Main Conclusions	Reference
		Fecal consistency	Feed efficiency	Body weight (BW) gain
Lactobacillus acidophilus	buffalo calves	+	+	+	Improved feed efficiency, final BW gain, dry matter intake, and average daily gain	(Sharma et al., 2018)
Mixture of Lactobacillus species	dairy calves	+	n/a	+	The overall health index, including fecal consistency, was greater in treated calves. Calf morbidity and mortality tended to be lower in treated animals.	(Maldonado et al., 2017)
Lactobacillus plantarum	dairy calves	ND	n/a	-	High doses of the probioticdecreased BW gain and abundance of other fecal lactobacilli.	(Rodriguez-Palacios et al., 2017)
Pediococcus acidilactici and Pediococcus pentosaceus	lambs	n/a	ND	ND	No differences in pre- or postweaning average daily gains, milk intakes, or feed conversion ratios between treatments.	(Saleem et al., 2017)
Mixture of Bacillus subtilis, Enterococcus faecium, Lactobacillus acidophilus, and Saccharomyc es cerevisiae	dairy calves	ND	ND	ND	No difference in BW gain, dry matter intake or feed efficiency of calves during pre- or post-weaning periods. Fecal scores tended to be improved in treated animals.	(M. I. Marcondes, 2016)
Kefir (Acetobacter, Lactobacillus spp., Lactococcus spp., and yeasts)	dairy calves	+	ND	ND	Dry matter intake, BW gain, and gain to feed ratios did not differ between treatments. Fecal scores were improved and days with diarrhea during the first 2 weeks of life were reduced in kefir-fed calves.	(Fouladgar et al., 2016)
Bacillus, Paenibacillus, and Staphylococc us isolates from moose	lambs	n/a	+	ND	BW gain and wool quality did not differ between treatments. Feed intake was decreased in animals provided the probiotic mixture.	(Ishaq et al., 2015)
Megasphaera elsdenii	dairy calves	n/a	n/a	ND	Greater dry matter intake, ruminal papillae width, density and butyrate, and no difference in average daily gain	(Muya et al., 2015)
Faecalibacte rium prausnitzi	dairy calves	ND	n/a	+	Decreased mortality rate associated with severe diarrhea	(Foditsch et al., 2015)

^*ND: not determined

Probiotic treatment based antimicrobial compounds

Several probiotic strains have been shown to produce a variety of antimicrobial compounds (Table 2) (e.g., VFA, hydrogen peroxide, nitric oxide, and bacteriocins) (Atassi and Servin, 2010; Chenoll et al., 2011). The production of potent antimicrobial compounds enables the probiotic bacteria to compete with native or pathogenic species (Eschenbach et al., 1989; Roos and Holm, 2002). The most commonly produced antimicrobial compounds are bacteriocins, which are produced by major lineages of Bacteria and, more recently, have been reported as universally produced by some members of the Archaea (Riley and Wertz, 2002b, a; Shand RF, 2008). Bacteriocins, antimicrobial peptides synthesized in the ribosome, are different from antibiotics in that they inhibit the growth of other closely related bacterial species or bacteria of the same strain, while antibiotics have a much broader killing spectrum that doesn’t often affect closely related strains (Riley and Wertz, 2002b; Kralik et al., 2018) The bacteriocin family includes a diverse group of peptides, differing in size, microbial targets, immunity mechanisms, and modes of action and release.

Table 2.

Bacteriocins and their functions.

Producing strain	Bacteriocins	Function	Reference
Bacillus thuringiensis DPC 6431	Thuricin CD	Active against Clostridium difficile	(Rea et al., 2011)
Enterococcus faecium	lactococcin G, plantaricin A, enterocin X	Active against other lactic acid bacteria	(Hummel et al., 2007)
Enterococcus mundtii ST4SA	Enterocin	Inhibit Enterococcus faecalis, S.aureus&P.aeruginosa, K.pneumonia	(Granger et al., 2008)
Pediococci	pediocins	Active against Listeria monocytogenes	(Shin et al., 2008)
Escherichia	colicins, microcins	Prevent Salmonella contamination in poultry industry	(Gillor et al., 2004)
Lactobacillus salivarius UCC118	Abp118	Active against Listeria monocytogenes	(Corr et al., 2007)
Lactobacillus johnsonii strain LA1	UN	Inhibit against Helicobacter pylori in	(Michetti et al., 1999; Gotteland et al., 2008)
L.acidophilus strain LB	UN	Inhibit H. pylori, H. felis	(Coconnier et al., 1998)
L. salivarius strain CRL 1328	BLIS	Inhibit Enterococcus spp, Neisseria gonorrhoeae	(Ocana et al., 1999)
Streptococcus mutans	Mutacin B-Ny266	Active Staphylococci, Streptococci, Neisseria	(Mota-Meira et al., 1997; Mota-Meira et al., 2000)
Streptococcus salivarius K12	Salivaricin A &B	Inhibit S. pyogenes, S. sobrinus, S. mutans	(Balakrishnan et al., 2000)

Bacteriocin production has been considered an important trait in probiotic selection; however, there are few studies documenting the ability of bacteriocin production to influence the strain’s ability to positively impact the health of the host (Corr et al., 2007). Bacteriocins may utilize a variety of mechanisms to reduce or prevent microbial infections within the GIT (Corr et al., 2009; Dobson et al., 2012). A potential function includes acting as colonizing peptides, allowing the probiotic strain to integrate with an already occupied niche (Hillman et al., 1987; Dawid et al., 2007; Gillor et al., 2009). Alternatively, bacteriocins may function by directly eliminating pathogens within the GIT via bacteriocidal activity (Corr et al., 2007; Le Blay et al., 2007). Recently, it was shown that lectin-like bacteriocins eliminate microbial rivals via contact-dependent disruption of outer membrane protein assembly machinery (Ghequire et al., 2018). Finally, bacteriocins may act as signaling peptides, inducing the host immune system or other bacteria via quorum sensing (Kleerebezem et al., 1997; Meijerink et al., 2010; van Hemert et al., 2010). The mechanisms of bacteriocin production and ecological function within the mammalian GIT are complex and will require further investigation.

Isolated bacteriocins have potential applications in ruminants. Possible uses include as an alternative to antibiotic treatment, as a tool for reducing environmental contamination of zoonotic pathogens, and as a means to regulate ammonia production within the rumen. In the cow’s rumen, Streptococcus bovis is a dominant bacterial species when the diet contains large amounts of soluble starch (Wells et al., 1997; Zhang et al., 2015). Whitford et al. screened S. bovis cultures and found 20% of these strains inhibited other Streptococci (Whitford et al., 2001). Consistent with this finding, Mantovani et al. reported that 50% of S. bovis isolates from cattle fed hay- or grain-based diets inhibited the growth of S. bovis JB1 (Mantovani et al., 2001). The colicin-producer, E. coli strain Nissle 1917 (EcN), was found to produce microcin H47 and microcin M, both of which were shown to have antagonistic activity against enterobacteria (Patzer et al., 2003). Administering the EcN strain as a probiotic to dairy calves resulted in a significant reduction of neonatal diarrhea (von Buenau et al., 2005), which may have been due to antagonism of pathogenic enterobacteria. Compared to the control group, calves orally inoculated with a mixture of eight colicin E7-producing E. coli strains were reported to have significantly reduced shedding of E. coli O157:H7, a foodborne pathogen carried by at least 20% of the cattle. This observation suggested that the colicins may have the capability to inhibit or exclude E. coli O157:H7 in the GIT of cattle (Schamberger et al., 2004). Butyrivibrio fibrisolvens are ruminal microbes that are important for fiber digestion (Hungate, 1966), and many strains of B. fibrisolvens have been shown to produce bacteriocins that inhibit other Butyrivibrio species (Kalmokoff and Teather, 1997). Specifically, B. fibrisolvens JL5 was found to reduce the growth of obligate amino-acid fermenting bacteria, which may reduce ammonia production within the rumen (Rychlik and Russell, 2002).

Current understanding of host’s immune response to probiotic treatment

The host mucosal immune system contributes to the maintenance of intestinal homeostasis and protective responses against pathogen infection (Yan and Polk, 2011). Probiotics play an intricate role in maintaining the delicate balance between necessary and excessive defense mechanisms. They can be used to stimulate or regulate epithelial and immune cells of the intestinal mucosa, which generates beneficial immunomodulatory effects. Specifically, the effects of probiotics on host intestinal mucosal defense system include: blocking pathogenic bacterial effects by producing bactericidal substances (Ocana and Elena Nader-Macias, 2004; EF et al., 2012), competing with pathogens and toxins for adherence to the intestinal epithelium (Perea Velez et al., 2007), and promoting intestinal epithelial cell survival(Yan et al., 2007; Yan et al., 2011).

The mechanisms of probiotic-induced immunomodulation mainly involve regulation of gene expression and signaling pathways in host cells. In human clinical studies, several probiotic genes/families involved in the regulation of host immune responses have been identified. Using isolated strains of a model probiotic organism, Lactobacillus plantarum, Meijerink et al (Meijerink et al., 2010) employed comparative genome hybridization and a random forest algorithm to successfully identify eight candidate genes in the L. plantarum genome that may modulate the cytokine response (IL-10 and IL-12) of dendritic cells to L. plantarum. Six of these genes (lp_0422, lp_0423, lp_0424a, lp_0424, lp_0425and lp_0429) were involved in bacteriocin secretion and production. The immunomodulatory effects of these genes were confirmed by comparing the cytokine production between targeted gene deletion mutants and wildtype strains. Additionally, probiotic strains have shown varied responses in different GIT locations in the host. In an in vitro GIT model, Weiss et al (Weiss and Jespersen, 2010) studied differentially expressed genes in Lactobacillus acidophilus during passage through the GIT. They found that stress-response related genes, GroEL, DnaK and ClpP, were significantly up-regulated during gastric digestion, followed by a significant decrease in subsequent duodenal digestion. Genes encoding mucin-binding and fibronectin-binding showed significant up-regulation during incubation of duodenal contents and bile, indicating that probiotic strains led to different gene expression profiles in different locations of the GIT. Thus, it may be an ideal strategy to find a specific niche for each probiotic strain when designing probiotic-based feed treatment.

Probiotic-strain secreted compounds or proteins have been identified with beneficial effects to the host. p40, a Lactobacillus rhamnosus GG-derived soluble protein, was shown to prevent dextran sulfate sodium-induced intestinal injury and acute colitis (Yan et al., 2011). Intestinal epithelial apoptosis and the disruption of barrier function in a mouse model of colitis were decreased after treatment with p40. p40 treatment was also associated with reduced production of tumor necrosis factor (TNF-∝), IL-6, keratinocyte chemoattractant, and interferon-γ production, suggesting p40’s role in regulating innate immunity and the Th1 immune response (Yan et al., 2011). In the study of Lactobacillus reuteri RC-14, a human vaginal isolate (Reid et al., 1987), was found to be capable of inhibiting the staphylococcal quorum-sensing system agr. And Li et al (Li et al., 2011) found two compounds secreted by L. reuteri RC-14 that were able to inhibit the expression of toxic shock syndrome toxin-1. The beneficial effects of probiotic bacterial strain secreted compounds were manifested by targeted genome modification. The phosphoglycerol transferase gene mediates lipoteichoic acid (LTA) biosynthesis in Lactobacillus acidophilus NCFM (NCK56). Oral administration of LTA-deficient L. acidophilus NCFM significantly protected mice against experimentally induced colitis (Khan et al., 2012). Along with other studies on LTA (Grangette et al., 2005; Claes et al., 2010), it was proposed that LTA synthesized in L. acidophilus induces the inflammatory response, and its absence significantly attenuates intestinal inflammation.

Probiotics affect the host innate and adaptive immune responses through modulating the functions of dendritic cells, macrophages, and T and B lymphocytes (Kwon et al., 2010; Evrard et al., 2011). A mixture of probiotics, consisting of B. bifidium, L. acidophilus, L. casei, L. reuteri and Streptococcus thermophilus, stimulated regulatory dendritic cells that expressed high levels of IL-10, TGF-β and COX-2, and also induced both T-cell and B-cell hypo-responsiveness and downregulated Th1, Th2, and Th17 cytokines without inducing apoptosis (Kwon et al., 2010). In addition, the probiotic L. rhamnosus Lcr35 was shown to induce a dose-dependent increase in dendritic cell secretion of pro-Th1/Th17 cytokines (e.g., TNF, IL-1β, IL-12p70, IL-12p40, and IL-23) (Evrard et al., 2011).

Models and cell lines used to study host response to probiotic treatment

The establishment of in vitro models is critical for the understanding of molecular and cellular interactions between probiotic and pathogenic microorganisms within the host. Bovine intestinal epithelial cells (IEC) have important roles in nutrient absorption and regulation of immune barriers. Primary cell cultures of bovine IEC are ideal models for investigating the beneficial effects of probiotic strains on the bovine host (Zhan et al., 2017). Using a novel clone cell method, Zhan et al. (Zhan et al., 2017) successfully cultured primary bovine IEC collected from duodenal, jejunal, and ileal tissue of a healthy lactating Holstein cow. Cultured bovine IEC can be passed for about five generations, with cellular aggregates and clusters loosely adherent at day two of culture. More importantly, the cultured bovine IEC displayed positive reactions against IEC markers, cytokeratin 18 and E-cadherin. Another set of epithelial cell specific markers include cell to cell adhesion molecules (Hollande et al., 2003), ZO-1 and β-catenin, both of which were also expressed in bovine IEC (Miyazawa et al., 2010).

The immunology of bovine IEC has been evaluated by several groups. In the study to validate bovine IEC as a model system to study antiviral responses, Chiba et al (Chiba et al., 2012) confirmed toll-like receptor (TLR) 3 expression. Additionally, bovine IECs showed a significant response to stimulation with the TLR3 agonist poly(I:C). This involved a pronounced up-regulation of pro-inflammatory cytokines and type I interferons. These responses were consistent with various intestinal viral infections of cattle and other hosts (Rollo et al., 1999; Xu et al., 2009). By evaluating the expression of TLRs in bovine IEC in response to a challenge with heat-stable Enterotoxigenic E. coli (ETEC), Takanashi et al. (Takanashi et al., 2013) observed recognition of pathogen associated molecular patterns (PAMP) by IEC, demonstrated by production of cytokines and activation of MAPK and NF-κB pathways. The authors also found that Lactobacilli casei OLL2768 had the capacity to attenuate the heat-stable ETEC PAMP induced pro-inflammatory response in bovine IEC (Takanashi et al., 2013). Findings from this study concluded that bovine IEC are suitable models for studying the molecular mechanisms underlying the protective activity of probiotics against pathogen-induced inflammation and for selecting lactic acid bacteria with immunoregulatory functions (Takanashi et al., 2013).

Future directions

Probiotics have great potential in effective management of ruminant health. Multiple studies have shown that the microbial community in ruminants’ GIT can be altered by various factors, including diet, probiotic treatments, age, and stress. Artificially introduced community changes may be achieved by well-defined probiotic treatments. And ideally, such GI microbial community changes may lead to increased production efficiency or strengthened host immunity. In depth, in vivo and in vitro studies of the dynamics and functional effects initiated by probiotic treatments can greatly enrich our understanding of when and how these treatments can benefit ruminants. The major areas of future studies include describing the structure and interactions of the gut microbiota, and functional relationships between the microbial community in the gut mucosa and the host cells. This would include comprehensive investigations that employ a “meta-omics” approach (i.e. metagenomic, metatranscriptomic, and metaproteomic) to investigate the dynamics between the GIT microbial community and host metabolism. Results from these studies can further the identification of a core set of well-defined microbial species for targeted health improvement and early development in ruminants.

The host mucosal innate immune function is largely affected by the GIT microbial community. The ruminal epithelium plays a major role in host mucosal innate immune function. Studies that specifically focus on the interaction between the mucosal microbial community and the host ruminal epithelium will most likely facilitate the identification of key genes critical for host immune homeostasis. Ideally, with the application of targeted gene editing technology, one could engineer the genetic makeup of the microbiome to potentially enable optimal host health and productivity.