In an attempt to dissect mechanisms underlying protective immune responses to Ostertagia ostertagi
infections in cattle, which develops very slowly and requires a prolonged exposure before becoming effective, we developed partially immune animals using multiple drug-attenuated infections. While host mechanisms underlying the development of long-term protective immunity have recently been discussed 
, the gut microbiota of ruminants has not been systematically characterized until recently 
. Three-way interactions between the host, its microbiota and parasites are little understood. In this study, we characterized the bovine abomasal microbiota using metagenomic tools. Our results provided the first piece of evidence that a minimal disruption in the bovine abomasal microbiota by the parasitic nematode may contribute equally to the restoration of gastric function in immune animals.
The abomasum is an important yet unique organ. Its low luminal pH environment, normally ranging from pH 1 to 3, is essential to activation of digestive enzymes and absorption of nutrients. The abomasal acidity is also a critical determinant in the pathogenesis of many diseases, including abomasal ulceration, abomasitis, abomasal bloat, and gastric tumors. While this extreme acidic environment serves as a potent barrier against bacterial infection and functions as an abomasal sterilizer, many enteric microorganisms, such as Escherichia coli
and Salmonella typhimurium
, have evolved elegant mechanisms to cope with the potentially lethal effects of acid stress that allows them to tolerate drastic pH fluctuations in their ever-changing surroundings and during pathogenesis 
. Cataloguing biodiversity in this environment will facilitate our understanding of survival strategies of microorganisms under stress, which could have important implications in animal and human health. Ostertagia ostertagi
infections did not seem to impact the abomasal microbial diversity in immune cattle. The number of genera identified from control and infected groups was not statistically different. Only 32 genera were identified in all samples tested, possibly representing the core microbiome of the bovine abomasal microbial community ().
is among the most abundant genera from the rumen and hindgut of ruminants, functioning mainly in the breakdown of protein and carbohydrate substrates. It is conceivable that peptidases secreted by Prevotella
species may play an important role in protein digestion in the bovine gut. Compared to phylogenetic profiles of the human stomach microbiota obtained using 16s rDNA sequencing 
, the bovine abomasal microbiota compositions displayed a unique pattern. The human stomach microbiota is dominated by 5 major phyla: Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Fusobacteria, which is significantly different from the microbial communities in other human organs, suggesting the human stomach harbors a distinct microbial ecosystem. Recent studies suggest that the number of microorganisms in the human stomach is related to luminal pH changes induced by diseases and therapy 
The gastrointestinal tract is the organ with the richest proteases (peptidases) of both endogenous and exogenous origin. Protein degradation in ruminants is controlled by microorganisms and is strongly influenced by gut luminal pH 
. In the rumen, intact dietary proteins are first converted to shorter oligopeptides by proteases. The resultant oligopeptides are degraded via a biphasic mechanism: oligopeptides are cleaved first to dipeptides and/or tripeptides by DPP, which are further degraded by di- or tri-peptidases to amino acids 
. Peptide hydrolysis in the human intestine seems to follow the same biphasic process 
. Protein requirements for ruminants include both dietary protein that escapes ruminal degradation (i.e., ruminally undegraded protein or RUP) and ruminally-synthesized microbial protein reaching the duodenum. Dairy cows require a proper balance of RUP and ruminally degraded protein (RDP) in their diets to best meet nutritional requirements for metabolizable protein and achieve desired nitrogen efficiency. Proteases of microbial origin in the rumen and hindgut play an important role in ruminant nutrition as well. In addition, proteases are able to serve as signaling molecules. Exogenous proteases can activate protease-activated receptors (PAR) in host enterocytes, thereby providing a novel mechanism of the interactions between gut microbiota and host cells 
. Understanding gut protease balance and its regulation represents an important aspect in dissecting the pathophysiology of gastrointestinal diseases. A total of 175 Pfam families with peptidase activities were identified in our metagenome datasets, including prolyl oligopeptidase family, peptidase families C10, S46 and M20 as well as DPP type IV (PF00930). None of these families seemed induced by infection and their abundance remained stable upon reinfection. However, a KEGG pathway on nitrogen metabolism (Acc# 00910) was impacted by infection in immune animals. Nevertheless, it is conceivable that pH-induced changes in the abomasal microbial community composition could have an important impact on host nutrition and physiology. Infection and ensuing elevation of the abomasal pH in immune animals did not appear to affect the abomasal proteolytic capability, suggesting that the development of protective immunity in immune animals may include mechanisms in regulating their gut microbiota and its secretory capabilities.
Two-component regulatory systems (2CRS) enable microorganisms to sense, respond, and adapt to a wide range of adverse environmental conditions. A membrane-bound histidine kinase functions as a sensor and transmits detected stimuli/signals to a corresponding response regulator to mediate the cellular response. This generally leads to differential expression of target genes, resulting in adaption or stress tolerance or even cross-protection responses 
. Many of the two-component systems are involved in signaling systems for sensing the changes of external environment such as temperature, osmolarity, chemo attractants, and pH. The latter regulates genes in many biological processes, including amino acid and sugar catabolism, electron transport, oxidative stress, and periplasmic and envelope proteins in E. coli 
. While many enterobacteria prefer to grow in neutral pH environments, they nevertheless experience dramatic pH fluctuations in their native habitats 
, especially after feeding cycles. On the other hand, uncontrolled metabolic activities of enterobacteria also induce drastic pH changes in their own habitat. For example, rapid fermentation of starch by Streptococcus bovis
in the rumen often leads to an increase in ruminal lactate production and results in a decrease in ruminal pH, which in turn promotes its own proliferation, creating a feedback loop and contributing to the progress of rumen acidosis and many other pathological conditions. As a result, it is often attempted to control S. bovis
growth in the gut microbial ecosystem. Recently a two-component signal transduction system involving the control of S. bovis
growth has been identified 
species were indeed present in the abomasal microbial communities of both control and infected animals; and the two-component system was the second most abundant pathways in these communities. Our data showed that infection induced a significant increase of two 2CRS-related protein families, two component regulator propeller (PF07494) and response regulator receiver domain (PF00072). Our results suggest that the abomasal pH elevation may elicit activation of 2CRS, which in turn promoted survival or even proliferation of acid-tolerant species in the gut of infected animals. Ecophysiological implications of pH-induced changes in the microbiota composition remain largely unknown. However, a slightly elevated pH would allow proliferation of certain acid-tolerant species, such as Streptococcus bovis
, which has been shown to produce an inhibitor of gastrin secretion by the host cell 
, modulating systemic host responses.
Host-parasite interactions in the cattle-Ostertagia
system have been well understood. Parasites display their ability to impact host normal physiology by hijacking their proteolytic pathways via structural mimicry 
. Most notably, parasites are able to modulate acid secretion and pepsinogen production of the host, resulting in a systemic response. During a primary Ostertagia ostertagi
infection in cattle, the acidity of the abomasal content decreases markedly to pH 5 to 6, leading to hypergastrinamia. Similarly, Ostertagia leptospicularis
infection in sheep is able to mediate inhibition of abomasal acid secretion 
, suggesting the parasites have abilities to induce changes in their environment that favors their survival and increase their reproduction. Indeed, the gastric acid secretion pathway was significantly impacted by infection (). On the other hand, the host cell is able to trigger a cascade of events, including activation of the complement system and recruitment of leukocytes to the site of infection. The amplification loop resulting from activation of the alternative pathway could allow for a sustained elevation of inflammatory cytokines. This, along with reactive oxygen species and nitride oxide as well as proteases released by infiltrates, leads to amplification of local inflammation, thus creating a hostile environment for developing worms and providing an efficient containment for parasites 
. In addition, the host cell has abilities to enhance tissue repair and promote its mucin-secreting function. These factors are instrumental in the development of protective immunity.
The host and its microbiota have developed a mutualistic relationship during evolution. Normal gut microflora is essential for the host function. The abomasal microbiota harbors bacteria that produce a potent inhibitor of gastrin secretion in the host 
, resulting in the reduction of gastrin levels by approximately 90%, therefore directly impacting host physiology. The ruminal fluids from the same animals display a similar inhibition of gastrin secretion, suggesting the gastrin inhibitors might be produced by rumen microorganisms that survive in the abomasum, which may account for the bulk of observed increases in anaerobic populations in the abomasal ecosystem when the luminal pH is elevated to 3.5 or above. Gastrin inhibitory activities is pH-dependent, suggesting normal pH environment in the abomasum may not favor production of the inhibitor. Our results indicated that the abomasal microbiota can produce an abundant level of serpins, a large class of protease inhibitors involved in regulation of a wide spectrum of physiological processes. Many gut bacteria, especially those commensal bacteria, are facing constant attack in their native habitat by proteases secreted by host cells, such as neutrophils 
. Serpins produced by the abomasal microbiota prevent attachment of host proteases, playing an important role in the interaction between the abomasal microbiota and the host. In addition, it has been known that intestinal parasite infection tends to increase dietary lysine requirements by up to 50% 
. One possible mechanism is that parasite infection alters gut microbiota composition and thus reduces the production of essential amino acids by the microbiota. Our results suggest that unlike in naive animals, Ostertagia
infection in immune cattle induced a minimal disruption in the abomasal microbiota, which may contribute equally to the development of long-term protective immunity. While immune animals may develop abilities to maintain proper stability of their abomasal microbiota, metagenome plasticity in the bovine abomasum was evident. Approximately 1.3% of Pfam protein families identified in the microbial communities displayed a significant difference in abundance upon reinfection. The microbiota was responsive to external changes in the environment, which allows certain species to fluctuate in the population, to explore newly available niche space, and yet maintain overall function of the entire community by expressing a stable level of various proteins. It is foreseeable that a holistic understanding of the complex three-way interactions between the host, its microflora, and parasites will lead to the improvement of animal and human health.