Background Acute diarrhea is a common clinical condition where clinical parameters are used to assess disease severity, course, and prognosis. Objectives The aim of this study was to investigate procalcitonin (PCT) and beta‐defensin2 (Bdef2) as biomarkers for disease severity, course, and prognosis of dogs with acute diarrhea. Methods Dogs with acute diarrhea (enteritis group [EG], n = 35) were compared with 30 healthy controls. The dogs in the EG were scored using the Canine Acute Diarrhea Severity (CADS) index and grouped by bacterial fecal culture results. Procalcitonin and Bdef2 were analyzed in serum and feces. Results Dogs with acute diarrhea showed higher serum PCT concentrations (P < 0.0001) and lower fecal Bdef2 concentrations (P = 0.0001) than unaffected dogs. Serum PCT was moderately and positively related to the extent of disease classified by the CADS score. Dogs with Clostridium perfringens or hemolyzing Escherichia coli as predominant pathogen had increased serum Bdef2 concentrations (P < 0.01). Differentiation between uncomplicated (≤3 days) and complicated (>3 days) disease courses, determined by receiver operating characteristic (ROC) curves, resulted in a sensitivity of 0.74 and a specificity of 0.69 for serum PCT at a cutoff of 3.9 ng/mL. The serum PCT and fecal Bdef2 quotient resulted in a sensitivity of 0.80 and a specificity of 0.92, with a cutoff of 80.5. Conclusions The results of the present study indicate that PCT and Bdef2 are potential biomarkers that can provide information on the severity, course, and prognosis of acute diarrhea in dogs.

Hemophilia B is a classical monogenic, X-chromosomal, recessively transmitted bleeding disorder caused by genetic variants within the coagulation factor IX gene (F9). Although hemophilia B has been described in dogs, it has not yet been reported in the Hovawart breed. Here we describe the identification of a Hovawart family transmitting typical signs of an X-linked bleeding disorder. Five males were reported to suffer from recurrent hemorrhagic episodes. A blood sample from one of these males with only 2% of the normal concentration of plasma factor IX together with samples from seven relatives were provided. Next-generation sequencing of the mother and grandmother revealed a single nucleotide deletion in the F9 promoter. Genotyping of the deletion in 1,298 dog specimens including 720 Hovawarts revealed that the mutant allele was only present in the aforementioned Hovawart family. The deletion is located 73 bp upstream of the F9 start codon in the conserved overlapping DNA binding sites of hepatocyte nuclear factor 4α (HNF-4α) and androgen receptor (AR). The deletion only abolished binding of HNF-4α, while AR binding was unaffected as demonstrated by electrophoretic mobility shift assay using human HNF-4α and AR with double-stranded DNA probes encompassing the mutant promoter region. Luciferase reporter assays using wildtype and mutated promoter fragment constructs transfected into Hep G2 cells showed a significant reduction in expression from the mutant promoter. The data provide evidence that the deletion in the Hovawart family caused a rare type of hemophilia B resembling human hemophilia B Leyden.

Hemophilia B is a classical monogenic X-chromosomal recessively transmitted bleeding disorder caused by genetic variants within the coagulation factor IX gene (F9). Although hemophilia B has been described in 32 dog breeds hitherto, it has not yet been reported in the Hovawart. Here we describe the identification of a Hovawart family transmitting typical signs of an X-linked bleeding disorder. Five males had been reported to suffer from recurrent hemorrhagic episodes, four of them had to be euthanized finally and one died due to severe blood loss. A blood sample of one of these males with only 2% of the normal concentration of plasma factor IX (FIX) together with samples of seven relatives including the mother and grandmother were provided for further analysis. Next generation sequencing of DNA of the mother and grandmother revealed a single nucleotide deletion in the F9 promoter (NC_006621.3:g.109,501,492delC; CanFam3.1). Genotyping of the deletion in 1,298 dog specimens (83 different breeds) including 720 Hovawarts revealed that the mutation was only present in the aforementioned Hovawart family. The deletion is located 73 bp upstream of the F9 start codon in the highly conserved overlapping DNA binding sites of hepatocyte nuclear factor 4α (HNF4α) and androgen receptor (AR). The deletion only abolishes binding of HNF4α as demonstrated by electrophoretic mobility shift assay (EMSA) using purified recombinant human HNF4α and a transient overexpression lysate of human AR with double-stranded DNA probes encompassing the mutated promoter region. Luciferase reporter assays using wild type and mutated promoter fragment constructs transfected into Hep G2 cells showed a 65.3% reduction in expression of the mutated promoter. The data presented here provide evidence that the deletion identified in the Hovawart family caused a rare type of hemophilia B resembling human hemophilia B Leyden.