Hemophilia A in a litter of Border Collies caused by a one base pair deletion in the F8 gene.

In dogs, hemophilia A is known to affect different breeds. This is a case report describing hemophilia A in a litter of Border Collies. A privately owned bitch and her puppies (n = 7) were presented to the referring veterinarian after acute hematoma formation in the male offspring (n = 3) following microchip implantation. Global coagulation testing, as well as determination of factor VIII and IX activity, were carried out. Based on the results, factor VIII deficiency was suspected. Two of the affected male puppies were euthanized within a few days. Genetic testing of the mother and the surviving male puppy resulted in the description of a deletion in exon 14 of the F8 gene. This c.3206delA variant leads to a frameshift in amino acid sequence and a premature stop codon (p.Asn1069IlefsTer7). The detection of the mutation and consequent testing of related dogs revealed that the deletion most likely had occurred spontaneously in the mother and had been transmitted to several of her offspring in different litters. Identified carriers were taken out of the breeding scheme. It is concluded that genetic testing in the context of suspected genetic disease can lead to preventive measures, including timely exclusion of carriers from breeding.

Salmonella in reptiles: a review of occurrence, interactions, shedding and risk factors for human infections.

Salmonella are considered a part of the normal reptile gut microbiota, but have also been associated with disease in reptiles. Reptile-associated salmonellosis (RAS) can pose a serious health threat to humans, especially children, and an estimated 6% of human sporadic salmonellosis cases have been attributed to direct or indirect contact with reptiles, although the exact number is not known. Two literature searches were conducted for this review. The first evaluated reports of the prevalence of Salmonella in the intestinal tracts of healthy reptiles. Salmonella were most commonly detected in snakes (56.0% overall), followed by lizards (36.9%) and tortoises (34.2%), with lower detection rates reported for turtles (18.6%) and crocodilians (9%). Reptiles in captivity were significantly more likely to shed Salmonella than those sampled in the wild. The majority of Salmonella strains described in reptiles belonged to subspecies I (70.3%), followed by subspecies IIIb (29.7%) and subspecies II (19.6%). The second literature search focused on reports of RAS, revealing that the highest number of cases was associated with contact with turtles (35.3%), followed by lizards (27.1%) and snakes (20.0%). Reptiles associated with RAS therefore did not directly reflect prevalence of Salmonella reported in healthy representatives of a given reptile group. Clinical symptoms associated with RAS predominantly involved the gastrointestinal tract, but also included fever, central nervous symptoms, problems with circulation, respiratory symptoms and others. Disease caused by Salmonella in reptiles appears to be dependent on additional factors, including stress, inadequate husbandry and hygiene, and other infectious agents. While it has been suggested that reptile serovars may cause more severe disease than human-derived strains, and some data is available on invasiveness of individual strains in cell culture, limited information is available on potential mechanisms influencing invasiveness and immune evasion in reptiles and in RAS. Strategies to mitigate the spread of Salmonella through reptiles and to reduce RAS focus mostly on education and hygiene, and have often been met with some success, but additional efforts are needed. Many aspects regarding Salmonella in reptiles remain poorly understood, including the mechanisms by which Salmonella persist in reptile hosts without causing disease.

Establishment of a Real-Time PCR Assay for the Detection of Devriesea agamarum in Lizards.

(1) Background: Devriesea (D.) agamarum is a potential cause of dermatitis and cheilitis in lizards. The aim of this study was to establish a real-time PCR assay for the detection of D. agamarum. (2) Methods: Primers and probe were selected targeting the 16S rRNA gene, using sequences of 16S rRNA genes of D. agamarum as well as of other bacterial species derived from GenBank. The PCR assay was tested with 14 positive controls of different D. agamarum cultures as well as with 34 negative controls of various non-D. agamarum bacterial cultures. Additionally, samples of 38 lizards, mostly Uromastyx spp. and Pogona spp., submitted to a commercial veterinary laboratory were tested for the presence of D. agamarum using the established protocol. (3) Results: Concentrations of as low as 2 × 104 colonies per mL were detectable using dilutions of bacterial cell culture (corresponding to approximately 200 CFU per PCR). The assay resulted in an intraassay percent of coefficient of variation (CV) of 1.31% and an interassay CV of 1.80%. (4) Conclusions: The presented assay is able to detect D. agamarum in clinical samples, decreasing laboratory turn-around time in comparison to conventional culture-based detection methods.

miRNA Profiles of Canine Intestinal Carcinomas, Lymphomas and Enteritis Analysed by Digital Droplet PCR from FFPE Material.

Most canine intestinal tumours are B-cell or T-cell lymphomas or carcinomas. They have to be distinguished from cases of enteritis. Non-invasive biomarkers such as miRNAs would be a step towards faster diagnosis. The aim of this study was to investigate shifts in miRNA expression in tissue samples collected from cases of enteritis, carcinoma and lymphoma of the small and large intestine to better understand the potential of miRNA as biomarkers for tumour diagnosis and classification. We selected two oncogenic miRNAs (miR-18b and 20b), two tumour suppressive miRNAs (miR-192 and 194) and two potential biomarkers for neoplasms (miR-126 and 214). They were isolated from FFPE material, quantified by ddPCR, normalised with RNU6B and compared with normal tissue values. Our results confirmed that ddPCR is a suitable method for quantifying miRNA from FFPE material. Expression of miR-18b and miR-192 was higher in carcinomas of the small intestine than in those of the large intestine. Specific miRNA patterns were observed in cases of enteritis, B-cell and T-cell lymphoma and carcinoma. However, oncogenic miR-18b and 20b were not elevated in any group and miR-126 and 214 were down-regulated in T-cell and B-cell lymphoma, as well as in carcinomas and lymphoplasmacytic enteritis of the small intestine.

KITLG Copy Number Germline Variations in Schnauzer Breeds and Their Relevance in Digital Squamous Cell Carcinoma in Black Giant Schnauzers.

Copy number variations (CNVs) of the KITLG gene seem to be involved in the oncogenesis of digital squamous cell carcinoma (dSCC). The aims of this study were (1) to investigate KITLG CNV in giant (GS), standard (SS), and miniature (MS) schnauzers and (2) to compare KITLG CNV between black GS with and without dSCC. Blood samples from black GS (22 with and 17 without dSCC), black SS (18 with and 4 without dSSC; 5 unknown), and 50 MS (unknown dSSC status and coat colour) were analysed by digital droplet PCR. The results are that (1) most dogs had a copy number (CN) value > 4 (range 2.5-7.6) with no significant differences between GS, SS, and MS, and (2) the CN value in black GS with dSCC was significantly higher than in those without dSCC (p = 0.02). CN values > 5.8 indicate a significantly increased risk for dSCC, while CN values < 4.7 suggest a reduced risk for dSCC (grey area: 4.7-5.8). Diagnostic testing for KITLG CNV may sensitise owners to the individual risk of their black GS for dSCC. Further studies should investigate the relevance of KITLG CNV in SS and the protective effects in MS, who rarely suffer from dSCC.

Challenges in microbiological identification of aerobic bacteria isolated from the skin of reptiles.

INTRODUCTION: Bacterial pathogens are often involved in dermatitis in reptiles. Exact identification of reptile-specific but otherwise uncommon bacterial species may be challenging. However, identification is crucial to evaluate the importance of the detected bacterial species. OBJECTIVE: The aim of this study was to assess the number of aerobic bacterial isolates cultured from skin-derived samples of reptiles which were not reliably identified by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), and to determine their identity. MATERIAL AND METHODS: Routine bacterial diagnostics were performed on 235 skin samples, and 417 bacterial isolates were analysed by MALDI-TOF MS. The isolates were grouped into categories based on their first score: category I (≥ 2.00), category II (≥ 1.70 and < 2.00), and category III (< 1.70). Isolates from category III were further investigated by 16S rRNA gene sequencing and the following criteria were applied: query cover 100%, e-value rounded to 0.0 and sequence identity (%) > 98.00% for genus identification, and > 99.00% for species identification. RESULTS: The majority of bacterial isolates were in category I (85.1%) or category II (8.4%). In category III (6.5%) results achieved at first by MALDI-TOF MS corresponded to the results of the molecular analysis in 8.0% of isolates at the species level and in 24.0% at the genus level. Bacterial isolates classified as category III were heterogenic in genus (e.g. Chryseobacterium, Devriesea, Pseudomonas, Staphylococcus, Uruburuella), and some have only been described in reptiles so far. CONCLUSIONS: Most of the aerobic bacterial isolates cultured from reptile skin achieved high scores by MALDI-TOF MS. However, in the majority of category III isolates MALDI-TOF MS results were different from those of the molecular analysis. This strengthens the need to carefully examine low-scored results for plausibility and to be familiar with the occurrence and morphology of relevant reptile-specific bacterial species (e.g. Devriesea agamarum) as well as with the limits of the database used.

[Aerobic bacteria from skin lesions in reptiles and their antimicrobial susceptibility]. / Aerobes Keimspektrum und Resistenzlage bei Hautläsionen von Reptilien.

OBJECTIVE: Bacterial skin infections are common in reptiles. Although many such infections are influenced by multifactorial problems, specific treatment of bacterial infections is an important consideration. The objective of this study was to evaluate the range of aerobic bacteria in skin lesions of reptiles and to determine their antimicrobial susceptibility. MATERIAL AND METHODS: Swabs of skin lesions from 219 reptiles were cultured for aerobic bacteria between January 2017 and June 2018. Isolates were identified based on growth on selective agar plates, biochemical parameters, as well as MALDI-TOF MS. Antibiotic susceptibility testing was carried out using the microdilution method. RESULTS: A total of 306 isolates were identified, mostly gram-negative, including Pseudomonas spp. (n = 48), Citrobacter spp. (n = 31, only in chelonians), aerobic spore-forming bacteria (n = 30), Aeromonas spp. (n = 20), Acinetobacter spp. (n = 20), Proteus spp. (n = 15), Staphylococcus spp. (n = 15), Klebsiella spp. (n = 13), Enterococcus spp. (n = 13), Morganella spp. (n = 11) as well as 78 other gram-negative and 12 other gram-positive bacteria. Colonization with 2 (n = 80) or more (n = 16) bacterial isolates was seen in 96 animals. Antibiotic susceptibility testing was carried out with 208 of the 306 isolated bacteria. Many isolates were sensitive (minimal inhibitory concentration [MIC] in µg/ml ≤ breakpoint) to enro- (E) and marbofloxacin (M): 86.4 % MIC ≤ 0.5 (E) and 95.5 % MIC ≤ 1 (M) for Pseudomonas spp., 86.4 % MIC ≤ 0.5 (E) and 90.9 % MIC ≤ 1 (M) for Citrobacter spp., 75.0 % MIC ≤ 0.5 (E) and 100 % MIC ≤ 1 (M) for Aeromonas spp. Trimethoprim/sulfamethoxazol proved to be effective against most of the Citrobacter spp. (90.9 % MIC ≤ 2/38) and Aeromonas spp. (75.0 % MIC ≤ 2/38). Amikacin was effective against nearly all Pseudomonas spp. (97.7 % MIC ≤ 16), Citrobacter spp. (95.5 % MIC ≤ 16) and Aeromonas spp. (93.8 % MIC ≤ 16). CONCLUSION AND CLINICAL RELEVANCE: The majority of isolates were gram-negative; the clinical relevance of individual isolates must, however, be evaluated on a case by case basis. Many of the isolated bacteria were sensitive to fluoroquinolones as well as aminoglycosides. Susceptibility testing is recommended since use of these antibiotics should be limited and for every tested group of antibiotics resistant isolates were found.