Iwan A. Burgener

Hepatocyte-derived microRNAs as sensitive serum biomarkers of hepatocellular injury in Labrador retrievers

Common parenchymal liver diseases in dogs include reactive hepatopathies and primary hepatitis (acute or chronic). In chronic hepatitis, there is usually a long subclinical phase. Specific clinical signs become overt only when liver damage is severe and in this phase, treatment is usually less effective. Limited data are available regarding the sensitivity of liver enzyme activity or biomarkers for early detection of subclinical hepatitis. Hepatocyte-derived microRNAs (HDmiRs) were recently identified as promising biomarkers for hepatocellular injury in multiple species. Here, the potential of the HDmiRs miR-122 and miR-148a as sensitive diagnostic biomarkers for hepatocellular injury in Labrador retrievers was investigated. Samples from 66 Labrador retrievers with histologically normal livers, high hepatic copper, and with various forms of liver injury were evaluated for serum alanine aminotransferase (ALT) activity and microRNA values. Median values of HDmiR-122 were 34.6 times higher in dogs with liver injury and high ALT than in normal dogs (95% confidence intervals [CI], 13-95; P <0.001). HDmiR-122 values were significantly increased in dogs with liver injury and normal ALT (4.2 times; 95% CI, 2-12; P <0.01) and in dogs with high hepatic copper concentrations and unremarkable histopathology (2.9 times; 95% CI, 1.1-8.0; P <0.05). Logistic regression analyses demonstrated that miR-122 and miR-148a were both predictors of hepatocellular injury. The sensitivity of miR-122 was 84% (95% CI, 73-93%), making it superior to ALT (55%; 95% CI, 41-68%) for the detection of hepatocellular injury in Labrador retrievers (P <0.001). This study demonstrated that serum HDmiR, particularly miR-122, is a highly sensitive marker for the detection of hepatocellular injury in Labrador retrievers and is a promising new biomarker that may be used for early detection of subclinical hepatitis in dogs.

Use of Serum MicroRNAs as Biomarker for Hepatobiliary Diseases in Dogs

Background Current biochemical indicators cannot discriminate between parenchymal, biliary, vascular, and neoplastic hepatobiliary diseases. MicroRNAs are promising new biomarkers for hepatobiliary disease in humans and dogs. Objective To measure serum concentrations of an established group of microRNAs in dogs and to investigate their concentrations in various types of hepatobiliary diseases. Animals Forty‐six client‐owned dogs with an established diagnosis of hepatobiliary disease and stored serum samples and eleven client‐owned healthy control Labrador Retrievers. Methods Retrospective study. Medical records of dogs with parenchymal, biliary, vascular, or neoplastic hepatobiliary diseases and control dogs were reviewed. Concentrations of miR‐21, miR‐122, miR‐126, miR‐148a, miR‐200c, and miR‐222 were quantified in serum by real‐time polymerase chain reaction. Results No different microRNA concentrations were found in the adenoma and congenital portosystemic shunt groups. In all other diseases, miR‐122 concentrations were elevated with the highest concentration in the mucocele group (267‐fold, CI: 40–1,768, P < .001). In dogs with biliary diseases, miR‐21 and miR‐222 were only increased in dogs with mucoceles (26‐fold, CI: 5–141, P = .005 and 13‐fold, CI: 2–70, P = .025, respectively). Uniquely increased microRNAs were found in the hepatocellular carcinoma group (miR‐200c, 35‐fold increase, CI: 3–382, P = .035) and the chronic hepatitis group (miR‐126, 22‐fold increase, CI: 5–91, P = .002). Conclusions and Clinical Importance A microRNA panel consisting of miR‐21, miR‐122, miR‐126, miR‐200c, and miR‐222 can distinguish between parenchymal, biliary, and neoplastic hepatobiliary diseases. Serum microRNA profiling is a promising new tool that might be a valuable addition to conventional diagnostics to help diagnose various hepatobiliary diseases in dogs.

Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) among employees and in the environment of a small animal hospital

The aim of the study was to investigate methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) among employees of a small animal hospital and the hospital environment. In total, 96 swabs from employees and 73 swabs from the clinic environment were investigated. Cation-adjusted-Mueller-Hinton broth (CAMHB) + 6.5% NaCl was used for enrichment before plating on Mueller-Hinton (MH) agar with 2% NaCl and 0.25 mg/L oxacillin. The staphylococcal species was determined using MALDI-TOF MS. The isolates were subjected to mecA-PCR, macrorestriction analysis, and antimicrobial susceptibility testing. MRSA were present in five nasal swabs of the 55 employees tested and in six environmental samples, MRSP in two employees (nasal and hand swabs, each) and in three environmental samples. All isolates harboured mecA. Susceptibility testing revealed that all but one of the isolates were multiresistant. All isolates were resistant to β-lactams and fluoroquinolones. All but one of the isolates were resistant to macrolides and lincosamides. A single MRSA was resistant to gentamicin. All MRSP were resistant to trimethoprim/sulfamethoxazole and non-susceptible to gentamicin. One isolate was also resistant to tetracycline. Macrorestriction analysis revealed three main SmaI patterns for MRSA and two main SmaI patterns for MRSP. All environmental isolates were found in areas of high people and animal traffic, such as dog ward areas, waiting and triage rooms. The finding of indistinguishable MRSA or MRSP among employees and in the environment of the small animal hospital suggests the possibility of transfer of these bacteria between humans, animals, and the hospital environment.

Gene expression patterns in the progression of canine copper-associated chronic hepatitis

Copper is an essential trace element, but can become toxic when present in abundance. The severe effects of copper-metabolism imbalance are illustrated by the inherited disorders Wilson disease and Menkes disease. The Labrador retriever dog breed is a novel non-rodent model for copper-storage disorders carrying mutations in genes known to be involved in copper transport. Besides disease initiation and progression of copper accumulation, the molecular mechanisms and pathways involved in progression towards copper-associated chronic hepatitis still remain unclear. Using expression levels of targeted candidate genes as well as transcriptome micro-arrays in liver tissue of Labrador retrievers in different stages of copper-associated hepatitis, pathways involved in progression of the disease were studied. At the initial phase of increased hepatic copper levels, transcriptomic alterations in livers mainly revealed enrichment for cell adhesion, developmental, inflammatory, and cytoskeleton pathways. Upregulation of targeted MT1A and COMMD1 mRNA shows the liver’s first response to rising intrahepatic copper concentrations. In livers with copper-associated hepatitis mainly an activation of inflammatory pathways is detected. Once the hepatitis is in the chronic stage, transcriptional differences are found in cell adhesion adaptations and cytoskeleton remodelling. In view of the high similarities in copper-associated hepatopathies between men and dog extrapolation of these dog data into human biomedicine seems feasible.

Sensitivity and Specificity of Plasma ALT, ALP, and Bile Acids for Hepatitis in Labrador Retrievers

Background Biochemical indicators for diagnosing liver disease are plasma alanine aminotransferase activity (ALT), alkaline phosphatase activity (ALP), and bile acid concentration (BA). Objectives To determine the sensitivity and specificity of ALT, ALP, and BA for detecting primary hepatitis (PH) in clinically healthy Labrador retrievers and investigate whether ALT and ALP can discriminate between dogs with PH and nonspecific reactive hepatitis (RH). Animals 191 clinically healthy and 51 clinically ill Labrador retrievers with hepatic histopathology. Methods Retrospective study. Medical records were reviewed for ALT, ALP, preprandial BA, liver histopathology, and hepatic copper concentrations. Results In 64% (122/191) of the clinically healthy Labrador retrievers, hepatic histology revealed inflammatory infiltrates. This frequency might be biased because part of them was included as first‐line relatives of dogs with copper‐associated hepatitis. Sensitivity of ALT, ALP, and BA in this population for detecting acute hepatitis was 45, 15, and 15%, respectively. For chronic hepatitis, sensitivity was 71, 35, and 13%, respectively. Specificity of ALT, ALP, and BA was >90% for AH, CH, and RH. When increased liver enzymes were present, median ALT was significantly higher in PH cases (312 U/L, range 38–1,369) compared to RH cases (91 U/L, range 39–139) (P < .001). There was no difference in ALP between dogs with a PH and a RH (P = .361). Conclusions and Clinical Importance Histopathologic abnormalities in the liver were present in the majority of apparent clinically healthy Labrador retrievers. The sensitivity of ALT, ALP, and BA for detecting acute and chronic hepatitis in this population was low. More sensitive biomarkers are needed for early detection of liver disease in apparent clinically healthy dogs.

Genome-wide based model predicting recovery from portosystemic shunting after liver shunt attenuation in dogs

Background In dogs with congenital portosystemic shunt (CPSS), recovery after surgical CPSS attenuation is difficult to predict. Objectives Our aim was to build a model with plasma albumin concentration and mRNA expression levels of hepatic gene products as predictors of recovery from portosystemic shunting after surgery. Animals Seventy‐three client‐owned dogs referred for surgical attenuation of CPSS. Methods A prediction model was constructed using 2 case‐control studies of recovered and nonrecovered dogs after surgical CPSS attenuation. In the 1st study, a dog‐specific gene expression microarray analysis was used to compare mRNA expression in intraoperatively collected liver tissue between 23 recovered and 23 nonrecovered dogs. In the 2nd study, preoperative plasma albumin concentration and the expression of microarray‐selected genes were confirmed by RT‐qPCR in intraoperatively collected liver samples of 31 recovered and 31 nonrecovered dogs, including 35 dogs from the 1st study. Results In the 1st study, 43 genes were differently expressed in recovered and nonrecovered dogs. The mean preoperative plasma albumin concentration in recovered dogs was higher compared to nonrecovered dogs (23 and 19 g/L, respectively; P = .004). The best fitting prediction model in the 2nd study included preoperative plasma albumin concentration and intraoperative DHDH, ERLEC1, and LYSMD2 gene expression levels. Conclusion and Clinical Importance A preclinical model was constructed using preoperative plasma albumin concentration and intraoperative hepatic mRNA expression of 3 genes that were unbiasedly selected from the genome to predict recovery from portosystemic shunting after shunt ligation. Further development is essential for clinical application.

Aberrant hepatic lipid storage and metabolism in canine portosystemic shunts

Non-alcoholic fatty liver disease (NAFLD) is a poorly understood multifactorial pandemic disorder. One of the hallmarks of NAFLD, hepatic steatosis, is a common feature in canine congenital portosystemic shunts. The aim of this study was to gain detailed insight into the pathogenesis of steatosis in this large animal model. Hepatic lipid accumulation, gene-expression analysis and HPLC-MS of neutral lipids and phospholipids in extrahepatic (EHPSS) and intrahepatic portosystemic shunts (IHPSS) was compared to healthy control dogs. Liver organoids of diseased dogs and healthy control dogs were incubated with palmitic- and oleic-acid, and lipid accumulation was quantified using LD540. In histological slides of shunt livers, a 12-fold increase of lipid content was detected compared to the control dogs (EHPSS P<0.01; IHPSS P = 0.042). Involvement of lipid-related genes to steatosis in portosystemic shunting was corroborated using gene-expression profiling. Lipid analysis demonstrated different triglyceride composition and a shift towards short chain and omega-3 fatty acids in shunt versus healthy dogs, with no difference in lipid species composition between shunt types. All organoids showed a similar increase in triacylglycerols after free fatty acids enrichment. This study demonstrates that steatosis is probably secondary to canine portosystemic shunts. Unravelling the pathogenesis of this hepatic steatosis might contribute to a better understanding of steatosis in NAFLD.

Intestinal Organoids—Current and Future Applications

Recent technical advances in the stem cell field have enabled the in vitro generation of complex structures resembling whole organs termed organoids. Most of these approaches employ culture systems that allow stem cell-derived or tissue progenitor cells to self-organize into three-dimensional (3D)-structures. Since organoids can be grown from different species (human, mouse, cat, dog), organs (intestine, kidney, brain, liver), and from patient-derived induced pluripotent stem cells, they create significant prospects for modelling development and diseases, for toxicology and drug discovery studies, and in the field of regenerative medicine. Here, we report on intestinal stem cells, organoid culture, organoid disease modeling, transplantation, specifically covering the current and future uses of this exciting new insight model to the field of veterinary medicine.

Erythrocyte copper chaperone for superoxide dismutase and superoxide dismutase as biomarkers for hepatic copper concentrations in Labrador retrievers

Hereditary hepatic copper accumulation in Labrador retrievers leads to hepatitis with fibrosis and eventually cirrhosis. The development of a non-invasive blood-based biomarker for copper status in dogs could be helpful in identifying dogs at risk and to monitor copper concentrations during treatment. In this study, two cellular copper metabolism proteins, Cu/Zn superoxide dismutase (SOD1) and its chaperone (copper chaperone for SOD1, CCS) were measured in erythrocytes and tested for association with hepatic copper concentrations in 15 Labrador retrievers with normal or increased hepatic copper concentrations. Antibodies against CCS and SOD1 were applicable for use in canine specimens. This was demonstrated by the loss of immune-reactive bands for CCS and SOD1 in siRNA treated canine bile duct epithelial cells. Erythrocyte CCS and CCS/SOD1 ratios were decreased 2.37 (P <0.001) and 3.29 (P <0.001) fold in the high copper group compared to the normal copper group. Erythrocyte CCS and CCS/SOD1 ratio are potential new biomarkers for hepatic copper concentrations in Labrador retrievers and could facilitate early diagnosis and treatment monitoring for copper-associated hepatitis in dogs.

Tetrathyridiosis in a domestic shorthair cat

Case summary This report describes the clinical and parasitological findings in a domestic shorthair cat with isolated thoracic tetrathyridiosis. The cat was a stray from Malta that had lived in Germany for several years since as an indoor-only cat. Therefore, the process of infection remains very unusual. In this case it must be considered that the cat had been infected years previously while in Malta, and had lived at least 4 years without any clinical signs. It was possible to diagnose this uncommon disease and initiate an effective treatment with fenbendazole, praziquantel and supportive care. Clinical signs, as well as radiographic findings, were regressive with this treatment. Relevance and novel information Tetrathyridiosis is a rare finding in cats, especially in Germany, but it seems to be a potential differential diagnosis of pleural effusion. Mesocestoides corti, which was the causative parasite in this case, has not previously been isolated in Germany. Because tetrathyridiosis is only diagnosed post mortem in most cases, little is known about effective therapeutic options. Furthermore, clinical signs of this disease can be absent for several years and can potentially be triggered by neoplastic conditions or immunosuppression. Tetrathyridiosis seems to be a treatable disease that can be controlled by adequate antiparasitic therapy.