The importance of mucus as a host defensive mechanism against intestinal nematode infections has been reported in several animal species, including sheep, cattle and rodents [13, 15, 25, 26], but the role of mucus in the response to abomasal parasites remains largely unclear. Apart from the recently reported changes in TFF3, GCNT3 and gastrokine 2 transcription levels during an O. ostertagi infection , little is known about the effect of this parasite on abomasal mucus composition and synthesis.
In the current study, increased gene expression of mucins, which are normally present in the abomasum, was observed for MUC1, MUC6 and MUC20. Although the transcriptional upregulation started early during infection, the highest changes were found when adult worms were present on the surface of the abomasal mucosa, after emergence from the infected glands. Transcription level of MUC5AC, another mucin normally present in abomasal tissues, did not change during infection. Previous studies in humans have shown that the gastric mucus layer is able to react to invading pathogens, such as Helicobacter pylori, through the modification of the expression profile of MUC1, MUC5AC and MUC6, but the role of these changes still remains unknown [28–30].
Qualitative changes of mucins, related to sugar composition, were also observed. Histochemistry of neutral and acidic (sialylated and sulfated) mucins in uninfected animals was found to be similar to what has been described in previous studies in abomasum of sheep . Consistent with previous observations [19, 32], the abomasal mucosa was found to be thickened at 24 dpi and in animals exposed for 60 days to a natural infection, compared to uninfected control animals. The length of the fundic glands was increased, due to the hyperplasia of the mucus secreting epithelium, as shown by PAS staining. The hyperplasia of these mucus-secreting cells may be the reason for the detected increase in mucin gene transcription levels during the infection. At 14 dpi, AB-PAS and HID-AB stainings showed that the abomasal mucosa was characterized by depletion of both neutral and acidic mucins, confined to the area invaded by the parasite larvae, indicating a strictly localized response to the parasite. The limited modification observed at this stage of the infection may be an attempt of the parasite to create an optimal environment for itself through the production of enzymes that break down mucin protein and carbohydrate structures, as previously suggested . In the rest of the mucosa, during the infection, the alteration in mucin composition, in particular in the hyperplastic glands, was characterized by a decrease of neutral and an increase in acidic mucins produced by the MNCs in the neck region of the gland. These alterations in mucin composition are very similar to what has been described in sheep during a primary infection with H. contortus . Since the modification of the glycosylation status of mucins has been reported to relate with alteration in the viscoelastic properties of mucus [34, 35] and the possible attachment of the parasite [16, 31], these alterations may be an attempt of the host to eliminate the parasite.
Genes involved in mucin core structure synthesis and branching were observed to be altered in infected animals compared to controls. Biosynthesis of mucin O-linked glycan is a complicated process, which starts with the formation of N-acetylgalactosamine (GalNAC), followed by the synthesis of four main core structures (core 1, 2, 3 and 4) that can be branched to form a high variety of glycans. GCNT enzymes are involved in these processes and among the genes analyzed, GCNT3 and GCNT4 were found to be upregulated. Interestingly, in a previous study on cattle infected with C. oncophora, strong upregulation of GCNT3 in intestinal goblet cells and in columnar epithelial cell was noticed throughout the infection. As observed in the current study, the increased transcription level started early during the infection (6 dpi). GCNT3 catalyzes a key rate-limiting step in mucins biosynthesis. An early upregulation during nematode infections suggests an enhancement in mucin secretion and an early capacity of the host to respond to the presence of the parasite, before the major alterations and damages in the mucosa appear, independently from the species of the invading parasite. During infection with O. ostertagi some of the sugar residues of the mucins in the abomasal mucosa were also found to be altered. Staining with the UEA-I lectin, which binds to α-L-Fucosyl residues, was increased during infection, in particular in the secreted mucus and in SMCs, compared to control animals. This observation is in contrast with the results of Hoang et al.  where a reduced UEA binding was observed in the abomasal fundus during infection with T. circumcincta. Almost all fucose in mucus is found on mucins where it has an important effect on the viscosity of the mucus [34, 37]. The increased levels of fucose in the tissue during infection are consistent with the observed increase in transcription levels of FUT2 and FUT4, coding for enzymes transfering Fuc α-1,2 and Fuc α-1,3 respectively, on mucins. Similarly in the small intestine of rats infected with N. brasiliensis, an induced fucosylation, due to an upregulation of Fut2 gene expression, has been reported . Disulphide isomerases are a family of enzymes that play an important role in the process of disulphide bond formation of gel forming mucins [39, 40]. They are expressed in several tissues, including the stomach and the intestine where they are produced by mucus secreting cells [40, 41]. Increased transcription levels of 3 disulphide isomerases (AGR2, PDIA3 and PDIA4) were observed after an O. ostertagi infection. Since the polymerization of mucin monomers is crucial in the formation of viscoelastic mucus, an increase of disulphide isomerases level may increase the gastric mucus viscosity. Although further studies analyzing the rheological properties of mucus during infection need to be done, it is possible that all the modification observed in mucus may be related to an attempt of the host to eliminate the invading pathogens.
Trefoil factor peptides are normally synthesized and secreted in human gastric and intestinal mucosa . In humans, TFF1 is predominantly located in the foveolar cells of the gastric mucosa, TFF2 in the MNCs and deep in the pyloric glands, while TFF3, also called intestinal trefoil factor, is expressed mainly by goblet cells of the large and small intestine [43, 44]. TFF3 has recently also been localized in the human gastric cardia . In the current study, a strong upregulation of TFF3 was observed from 9 dpi onward. Immunofluorescence confirmed the increased expression of TFF3 in the mucosa of infected animals and showed that SMCs produce this peptide in the bovine fundus of uninfected calves, similar to what has been described in man . In infected animals the hyperplastic mucus secreting cells appear to be the ones producing TFF3. The TFF3 upregulation is consistent with a recent study of Li et al.  that have showed an increase of TFF3 mRNA levels in primary and repeated infections with O. ostertagi. TFF3 upregulation has also been observed during H. contortus and T. colubriformis infections in sheep . TFF1 was also observed to be upregulated at 24 dpi. To our knowledge this is the first time that TFF1 upregulation is observed during a GI nematode infection. In man, gastric pit cells (SMCs) in the cardia are able to synthesize TFF1, TFF3 and MUC5AC . It is possible that the hyperplastic TFF3-expressing cells in the abomasum of infected calves also produce TFF1. The potential role of TFF3 during nematode infections has been related to mucosal defense and tissue restitution [15, 27] but it still remains unknown if TFF1 may also contribute to tissue repair.
In conclusion, this study has shown that the abomasal mucin, TFFs and glycogenes transcription levels, as well as mucin glycosylation patterns are significantly altered during an Ostertagia infection in cattle. These changes are likely caused by the alterations in mucosal cell populations, characterized by hyperplasia of mucus secreting cells. The effect of these changes on the protective mucus barrier is still unclear. Studies on host immune response against O. ostertagi have highlighted that expulsion of adult worms during primary infection is uncommon [46, 47], therefore alteration in mucin sugar composition and mucus viscosity does not seem to be an efficient method to eliminate this parasite from the abomasum in this stage of the infection. Further studies comparing the mucosal changes in primary infected animals versus animals with an acquired immunity against O. ostertagi, will be helpful in clarifying if mucus has a protective role against this parasite.