Plasma proteome signature of canine acute haemorrhagic diarrhoea syndrome (AHDS)

Acute haemorrhagic diarrhoea is a common complaint in dogs. In addition to causes like intestinal parasites, dietary indiscretion, intestinal foreign bodies, canine parvovirus infection, or hypoadrenocorticism, acute haemorrhagic diarrhoea syndrome (AHDS) is an important and sometimes life-threatening differential diagnosis. There is some evidence supporting the link between Clostridium perfringens toxins and AHDS. These toxins may be partially responsible for the epithelial cell injury, but the pathogenesis of AHDS is still not fully understood. Recent studies have suggested that severe damage to the intestinal mucosa and associated barrier dysfunction can trigger chronic gastrointestinal illnesses. Besides bloodwork and classical markers for AHDS such as protein loss and intestinal bacterial dysbiosis, we focused mainly on the plasma-proteome to identify systemic pathological alterations during this disease and searched for potential biomarkers to improve the diagnosis. To accomplish the goals, we used liquid chromatography-mass spectrometry. We compared the proteomic profiles of 20 dogs with AHDS to 20 age-, breed-, and sex-matched control dogs. All dogs were examined, and several blood work parameters were determined and compared, including plasma biochemistry and cell counts. We identified and quantified (relative quantification) 207 plasmatic proteins, from which dozens showed significantly altered levels in AHDS. Serpina3, Lipopolysaccharide-binding protein, several Ig-like domain-containing proteins, Glyceraldehyde-3-phosphate dehydrogenase and Serum amyloid A were more abundant in plasma from AHDS affected dogs. In contrast, other proteins such as Paraoxonase, Selenoprotein, Amine oxidases, and Apolipoprotein C-IV were significantly less abundant. Many of the identified and quantified proteins are known to be associated with inflammation. Other proteins like Serpina3 and RPLP1 have a relevant role in oncogenesis. Some proteins and their roles have not yet been described in dogs with diarrhoea. Our study opens new avenues that could contribute to the understanding of the aetiology and pathophysiology of AHDS

Plasma proteome signature of canine acute haemorrhagic diarrhoea syndrome (AHDS).

Acute haemorrhagic diarrhoea is a common complaint in dogs. In addition to causes like intestinal parasites, dietary indiscretion, intestinal foreign bodies, canine parvovirus infection, or hypoadrenocorticism, acute haemorrhagic diarrhoea syndrome (AHDS) is an important and sometimes life-threatening differential diagnosis. There is some evidence supporting the link between Clostridium perfringens toxins and AHDS. These toxins may be partially responsible for the epithelial cell injury, but the pathogenesis of AHDS is still not fully understood. Recent studies have suggested that severe damage to the intestinal mucosa and associated barrier dysfunction can trigger chronic gastrointestinal illnesses. Besides bloodwork and classical markers for AHDS such as protein loss and intestinal bacterial dysbiosis, we focused mainly on the plasma-proteome to identify systemic pathological alterations during this disease and searched for potential biomarkers to improve the diagnosis. To accomplish the goals, we used liquid chromatography-mass spectrometry. We compared the proteomic profiles of 20 dogs with AHDS to 20 age-, breed-, and sex-matched control dogs. All dogs were examined, and several blood work parameters were determined and compared, including plasma biochemistry and cell counts. We identified and quantified (relative quantification) 207 plasmatic proteins, from which dozens showed significantly altered levels in AHDS. Serpina3, Lipopolysaccharide-binding protein, several Ig-like domain-containing proteins, Glyceraldehyde-3-phosphate dehydrogenase and Serum amyloid A were more abundant in plasma from AHDS affected dogs. In contrast, other proteins such as Paraoxonase, Selenoprotein, Amine oxidases, and Apolipoprotein C-IV were significantly less abundant. Many of the identified and quantified proteins are known to be associated with inflammation. Other proteins like Serpina3 and RPLP1 have a relevant role in oncogenesis. Some proteins and their roles have not yet been described in dogs with diarrhoea. Our study opens new avenues that could contribute to the understanding of the aetiology and pathophysiology of AHDS

The figure illustrates the Gene Ontology (GO) analysis results conducted on the plasma proteome of dogs affected by AHDS.

The analysis categorized the proteins according to their pathways using Panther software [19]. Each slice represents the set of genes (hits) for each specific functional category or pathway. Only genes that had a functional category described are represented.</p

Boxplot analysis of significant blood cell markers in the AHDS and control groups.

This figure presents boxplots showcasing the significant variations in blood cell markers observed between the AHDS and control groups. Statistical differences were determined using either the student’s t-test for samples with equal variance or the Welch test for samples exhibiting differing variances. Significance levels were established as p p p **, very significant.</p

The figure illustrates a boxplot analysis of non-significant biochemistry parameters in both the AHDS and control groups.

The boxplots highlight noteworthy variations in specific plasma biochemistry markers between the AHDS and control groups. Statistical differences were evaluated using either the student’s t-test for samples with equal variance or the Welch test for samples exhibiting varying variances. Statistical significance was attributed to values with p (TIF)</p

S1 Table presents the results of the proteomic experiment analysis utilizing Perseus export, detailing the detection and quantification of plasma proteins in dogs affected by Acute Hemorrhagic Diarrhea Syndrome (AHDS) in comparison with a control group.

The dataset encompasses five replicates from six distinct independent pools of plasma samples, comprising both AHDS-affected dogs and healthy controls. Notice that the LFQ intensity values were log2 transformed. Statistical analysis was conducted employing the student t-test, and p-values were subjected to correction using the false discovery rate (FDR) methodology. The analysis was performed utilizing MaxQuant and Perseus software, both computational platforms for the analysis of label-free quantification of proteins through Liquid Chromatography-Mass Spectrometry (LC-MS). (XLSX)</p

Fig 5 -

Network visualization of proteins overrepresented (green) and depleted (red) in plasma of dogs with AHDS compared to the plasma of healthy dogs using STRING database [21]. The network diagram depicts the interactions between various canine plasma proteins using as inclusion a significant FDR-adjusted p-value regardless of the level of expression. Each node represents a unique protein, and the edges connecting nodes indicate known or predicted interactions. Node colour indicates the type of interaction evidence, ranging from experimental data to curated databases and text mining.</p

Boxplot analysis of significant biochemistry parameters in the AHDS and control groups.

This figure provides a boxplot analysis depicting significant variations in select plasma biochemistry markers between the AHDS and control groups. Statistical differences were assessed using either the student’s t-test for samples with equal variance or the Welch test for samples displaying varying variances. Significance levels were established as p p p ***, extremely significant.</p

The figure displays a boxplot analysis of non-significant blood cell markers in both the AHDS and control groups.

The boxplots illustrate notable variations in blood cell markers between these two groups. Statistical distinctions were assessed using the student’s t-test for samples with equal variance or the Welch test for samples with differing variances. Statistical significance was attributed to values with p (TIF)</p