Belgacem Mihi

Galectin-11 induction in the gastrointestinal tract of cattle following nematode and protozoan infections.

Galectin‐11 (LGALS11) has been suggested to play an important role in protective immunity against gastrointestinal nematodes in ruminants. However, in cattle, this molecule has not been characterized in detail. In the current study, it was shown that transcription of LGALS11 was highly inducible in the bovine abomasal mucosa after an Ostertagia ostertagi infection. LGALS11 protein expression was also increased in the abomasal mucosa following O. ostertagi infection and localized to the nucleus and cytoplasm of epithelial cells and the mucus. Using in vitro abomasal epithelial cell cultures, it was shown that LGALS11 induction was associated with the proliferative and dedifferentiated status of cells. However, LGALS11 was not induced following stimulation with O. ostertagi excretory–secretory products. These results suggest that LGALS11 induction in vivo may be an indirect rather than a direct effect of the parasite on the epithelium. In addition, LGALS11 transcript was also detected in the abomasal lymph nodes where it was shown to be transcribed in MHCII+ cells; however, transcription levels in the lymph nodes were not altered after O. ostertagi infection. In addition, LGALS11 was also induced in the small intestine by different types of parasites, including the nematode Cooperia oncophora and the protozoan parasite Giardia duodenalis.

Digital dermatitis in cattle is associated with an excessive innate immune response triggered by the keratinocytes.

BackgroundDigital Dermatitis (DD) is a common disease of dairy cows, the pathogenesis of which is still not clear. This study examined some host responses associated with the typical lesions, in an attempt to further elucidate the pathogenesis of the disease. Twenty four samples representing the 5 different clinical stages of DD (M0-M4) were collected from slaughtered cattle for histopathological and immunological analyses.ResultsSignificant increases in total epidermal thickness were found in M2, M3, and M4 when compared with M0 and M1. M3 samples, when compared with M0 and M1, were characterized by a significant increase in the thickness of the keratin layer. Counts of both eosinophils and neutrophils were at a maximum in the M2 stage and decreased in the M3 and M4 stage. A significant increase in IL8 expression was observed in the M2-M3 stages of the disease and immunohistochemical staining showed the source as keratinocytes, suggesting an important role for keratinocyte-derived IL8 in the pathogenesis of DD.ConclusionResults of the present study point to a strong stimulation of the innate immune response at the level of the keratinocytes throughout most of the clinical stages, and a delayed response of the adaptive immune reaction.

Analysis of the mucosal immune responses induced by single and trickle infections with the bovine abomasal nematode Ostertagia ostertagi

The purpose of this study was to provide more information on the kinetics of the immunological changes occurring in the abomasal mucosa after single and trickle infections with the bovine parasite Ostertagia ostertagi. The time course analysis of gene expression revealed that the major changes coincided with the emergence of adult worms from the gastric glands. These changes consisted of a simultaneous upregulation of Th1‐ and Th2‐type cytokines. In addition, a single O. ostertagi infection elicited an upregulation of the epithelial‐derived cytokine IL33, while TSLP expression levels were not impacted. Apart from the massive increase in inflammatory cytokines IL6, IL17 and IL21, O. ostertagi infection also elicited an upregulation of the immunosuppressors TGFB, IL10 and ARG1, as well as NK and γδ‐T cell markers. Furthermore, the cytotoxic factors granulysin, perforin and granzyme B were upregulated following an O. ostertagi infection. Analysis of cytokine transcript levels in animals receiving trickle infections for 60 days showed a similar trend as observed following a single infection except for IL33, IL6, GATA‐3, TBX21 and NCR1, which were no longer upregulated after trickle infections. Finally, the long trickle infections were associated with mucosal eosinophilia and mastocytosis.

Effect of an Ostertagia ostertagi infection on the transcriptional stability of housekeeping genes in the bovine abomasum.

Quantitative Real-Time PCR (qRT-PCR) is a widely used tool to study host responses against parasites. A crucial step in the gene quantification process is the normalization of the expression data against stable housekeeping genes (HKGs). However, in recent years, several reports have showed that the transcriptional levels of such HKGs can change dramatically, especially when cellular changes appear in the tissues investigated. The aim of the current study was to assess the variability of 11 putative HKGs in bovine abomasal tissue during an infection with the parasitic nematode Ostertagia ostertagi. Gene transcription levels of selected potential HKGs were measured by qRT-PCR and the expression stabilities evaluated using geNorm, NormFinder, and The Mann-Whitney-U test. The analysis showed that all the putative HKGs considered in this study, including the ones selected by geNorm and NormFinder, were found to be significantly upregulated in infected animals compared to the controls, clearly suggesting that none of these genes can actually be used as a HKG. The greatest alterations in gene transcription levels appeared at 24 dpi, which might be due to the dramatic changes in cell populations occurring in the abomasal tissue at this infection time point. To demonstrate the effect of normalizing target gene transcription levels with unstable HKGs, IL4 transcription levels were assessed using different normalization procedures. Our findings clearly showed that gene expression levels determined using HKGs differed significantly from those determined without normalization. The potential for HKG selection to impact candidate transcript levels is therefore an important consideration for studies of parasite infected tissue.

Helicobacter suis affects the health and function of porcine gastric parietal cells

The stomach of pigs at slaughter age is often colonized by Helicobacter (H.) suis, which is also the most prevalent gastric non-H. pylori Helicobacter (NHPH) species in humans. It is associated with chronic gastritis, gastric ulceration and other gastric pathological changes in both hosts. Parietal cells are highly specialized, terminally differentiated epithelial cells responsible for gastric acid secretion and regulation. Dysfunction of these cells is closely associated with gastric pathology and disease. Here we describe a method for isolation and culture of viable and responsive parietal cells from slaughterhouse pigs. In addition, we investigated the interactions between H. suis and gastric parietal cells both in H. suis-infected six-month-old slaughter pigs, as well as in our in vitro parietal cell model. A close interaction of H. suis and parietal cells was observed in the fundic region of stomachs from H. suis positive pigs. The bacterium was shown to be able to directly interfere with cultured porcine parietal cells, causing a significant impairment of cell viability. Transcriptional levels of Atp4a, essential for gastric acid secretion, showed a trend towards an up-regulation in H. suis positive pigs compared to H. suis-negative pigs. In addition, sonic hedgehog, an important factor involved in gastric epithelial differentiation, gastric mucosal repair, and stomach homeostasis, was also significantly up-regulated in H. suis positive pigs. In conclusion, this study describes a successful approach for the isolation and culture of porcine gastric parietal cells. The results indicate that H. suis affects the viability and function of this cell type.

Analysis of cell hyperplasia and parietal cell dysfunction induced by Ostertagia ostertagi infection.

Infections in cattle with the gastric nematode Ostertagia ostertagi are associated with decreased acid secretion and profound physio-morphological changes of the gastric mucosa. The purpose of the current study was to investigate the mechanisms triggering these pathophysiological changes. O. ostertagi infection resulted in a marked cellular hyperplasia, which can be explained by increased transcriptional levels of signaling molecules related to the homeostasis of gastric epithelial cells such as HES1, WNT5A, FGF10, HB-EGF, AREG, ADAM10 and ADAM17. Intriguingly, histological analysis indicated that the rapid rise in the gastric pH, observed following the emergence of adult worms, cannot be explained by a loss of parietal cells, as a decrease in the number of parietal cells was only observed following a long term infection of several weeks, but is likely to be caused by an inhibition of parietal cell activity. To investigate whether this inhibition is caused by a direct effect of the parasites, parietal cells were co-cultured with parasite Excretory/Secretory products (ESP) and subsequently analyzed for acid production. The results indicate that adult ESP inhibited acid secretion, whereas ESP from the L4 larval stages did not alter parietal cell function. In addition, our data show that the inhibition of parietal cell activity could be mediated by a marked upregulation of inflammatory factors, which are partly induced by adult ESP in abomasal epithelial cells. In conclusion, this study shows that the emergence of adult O. ostertagi worms is associated with marked cellular changes that can be partly triggered by the worm’s Excretory/secretory antigens.