Maja Ruetten

Environmental marine pathogen isolation using mesocosm culture of sharpsnout seabream: striking genomic and morphological features of novel Endozoicomonas sp.

Aquaculture is a burgeoning industry, requiring diversification into new farmed species, which are often at risk from infectious disease. We used a mesocosm technique to investigate the susceptibility of sharpsnout seabream (Diplodus puntazzo) larvae to potential environmental pathogens in seawater compared to control borehole water. Fish exposed to seawater succumbed to epitheliocystis from 21 days post hatching, causing mortality in a quarter of the hosts. The pathogen responsible was not chlamydial, as is often found in epitheliocystis, but a novel species of the γ-proteobacterial genus Endozoicomonas. Detailed characterisation of this pathogen within the infectious lesions using high resolution fluorescent and electron microscopy showed densely packed rod shaped bacteria. A draft genome sequence of this uncultured bacterium was obtained from preserved material. Comparison with the genome of the Endozoicomonas elysicola type strain shows that the genome of Ca. Endozoicomonas cretensis is undergoing decay through loss of functional genes and insertion sequence expansion, often indicative of adaptation to a new niche or restriction to an alternative lifestyle. These results demonstrate the advantage of mesocosm studies for investigating the effect of environmental bacteria on susceptible hosts and provide an important insight into the genome dynamics of a novel fish pathogen.

Emerging pathogens of gilthead seabream: characterisation and genomic analysis of novel intracellular β-proteobacteria

New and emerging environmental pathogens pose some of the greatest threats to modern aquaculture, a critical source of food protein globally. As with other intensive farming practices, increasing our understanding of the biology of infections is important to improve animal welfare and husbandry. The gill infection epitheliocystis is increasingly problematic in gilthead seabream (Sparus aurata), a major Mediterranean aquaculture species. Epitheliocystis is generally associated with chlamydial bacteria, yet we were not able to localise chlamydial targets within the major gilthead seabream lesions. Two previously unidentified species within a novel β-proteobacterial genus were instead identified. These co-infecting intracellular bacteria have been characterised using high-resolution imaging and genomics, presenting the most comprehensive study on epitheliocystis agents to date. Draft genomes of the two uncultured species, Ca. Ichthyocystis hellenicum and Ca. Ichthyocystis sparus, have been de novo sequenced and annotated from preserved material. Analysis of the genomes shows a compact core indicating a metabolic dependency on the host, and an accessory genome with an unprecedented number of tandemly arrayed gene families. This study represents a critical insight into novel, emerging fish pathogens and will be used to underpin future investigations into the bacterial origins, and to develop diagnostic and treatment strategies.

The effect of orally administered ranitidine and once-daily or twice-daily orally administered omeprazole on intragastric pH in cats.

Background Gastric acid suppressants frequently are used in cats with acid‐related gastric disorders. However, it is not known if these drugs effectively increase intragastric pH in cats. Objectives To examine the effects of PO administered ranitidine and omeprazole on intragastric pH in cats and to compare the efficacy of once‐daily versus twice‐daily dosage regimens for omeprazole. Animals Eight domestic shorthair cats. Methods Using a randomized 4‐way cross‐over design, cats were given enteric‐coated omeprazole granules (1.1–1.3 mg/kg q24h and q12h), ranitidine (1.5–2.3 mg/kg q12h), and placebo. Intragastric pH was monitored continuously for 96 hours using the Bravo™ system1, starting on day 4 of treatment, followed by a median washout period of 12 days. Mean percentage of time pH was ≥3 and ≥4 was compared among groups using repeated measures ANOVA. Results Mean ± SD percentage of time intragastric pH was ≥3 and ≥4 was 67.0 ± 24.0% and 54.6 ± 26.4% for twice‐daily omeprazole, 24.4 ± 22.8% and 16.8 ± 19.3% for once‐daily omeprazole, 16.5 ± 9.0% and 9.6 ± 5.9% for ranitidine, and 9.4 ± 8.0% and 7.0 ± 6.6% for placebo administration. Twice‐daily omeprazole treatment significantly increased intragastric pH, whereas pH after once‐daily omeprazole and ranitidine treatments did not differ from that of placebo‐treated cats. Conclusion and Clinical Importance Only twice‐daily PO administered omeprazole significantly suppressed gastric acidity in healthy cats, whereas once‐daily omeprazole and standard dosages of ranitidine were not effective acid suppressants in cats.

A frameshift mutation in the cubilin gene (CUBN) in Beagles with Imerslund–Gräsbeck syndrome (selective cobalamin malabsorption)

Mammals are unable to synthesize cobalamin or vitamin B12 and rely on the uptake of dietary cobalamin. The cubam receptor expressed on the intestinal endothelium is required for the uptake of cobalamin from the gut. Cubam is composed of two protein subunits, amnionless and cubilin, which are encoded by the AMN and CUBN genes respectively. Loss-of-function mutations in either the AMN or the CUBN gene lead to hereditary selective cobalamin malabsorption or Imerslund-Gräsbeck syndrome (IGS). We investigated Beagles with IGS and resequenced the whole genome of one affected Beagle at 15× coverage. The analysis of the AMN and CUBN candidate genes revealed a homozygous deletion of a single cytosine in exon 8 of the CUBN gene (c.786delC). This deletion leads to a frameshift and early premature stop codon (p.Asp262Glufs*47) and is, thus, predicted to represent a complete loss-of-function allele. We tested three IGS-affected and 89 control Beagles and found perfect association between the IGS phenotype and the CUBN:c.786delC variant. Given the known role of cubilin in cobalamin transport, which has been firmly established in humans and dogs, our data strongly suggest that the CUBN:c.786delC variant is causing IGS in the investigated Beagles.

Feasibility and safety of endoscopic ultrasound-guided fine needle aspiration of the pancreas in dogs

BACKGROUND Endoscopic ultrasound (EUS)-guided fine needle aspiration (EUS-FNA) has proven a useful and safe diagnostic tool for assessing pancreatic disease in human medicine. No information about pancreatic EUS-FNA is available in dogs. OBJECTIVES To assess the feasibility and safety of pancreatic EUS-FNA in healthy dogs. ANIMALS Thirteen beagles with a median body weight of 13.4 kg. METHODS Experimental study. An ultrasound endoscope (insertion tube outer diameter 11.8 mm) was used, and FNA was carried out with 19 G needles. The optimal puncture site was chosen with the aid of Doppler imaging. Complete clinicopathologic assessments including pain scoring and pancreas-specific lipase measurements were obtained before EUS as well as on day 1 and day 2 after EUS-FNA. RESULTS The pancreatic body was identified in all dogs, the left lobe was clearly identified in 9/13 and appeared indistinctly marginated in 4/13 dogs, and the distal third of the right lobe could not be identified in 7/13 dogs. EUS-FNA was carried out in 12/13 dogs. Cellularity of smears was adequate for evaluation in 8/12 cases, in which samples were obtained transgastrically (n = 4) or transduodenally (n = 4). All dogs recovered uneventfully and no clinical and laboratory abnormalities occurred during the 48 hour monitoring period after the procedure. CONCLUSION AND CLINICAL IMPORTANCE Although the healthy canine pancreas is difficult to visualize in its entirety with EUS, pancreatic EUS-FNA with a 19 G needle is feasible in medium-sized dogs and can be considered a safe procedure. Its diagnostic usefulness should be evaluated in dogs with pancreatic disease.

Blood plasma lipidome profile of dairy cows during the transition period

BackgroundThe transition period of dairy cows, around parturition and the onset of lactation, involves endocrine and metabolic changes to compensate for an increased energy requirement aggravated by reduced feed intake. Transition cows adjust to the resulting negative energy balance with the mobilization of lipids from the adipose tissues yielding increased blood levels of non-esterified fatty acids and ketone bodies like β-hydroxybutyrate.ResultsTo study the biochemical adaptations underlying this physiologic adjustment and possible pathologic derangements, we analyzed the blood plasma lipidome of transition cows by ultra-pressure liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry. The resulting data were processed by principal component analysis, revealing over 60 lipid masses that change in abundance over the test period ranging from two weeks before calving to four weeks postpartum. Further characterization of analytes by tandem mass spectrometry demonstrated that the concentration of triacylglycerides in plasma drops at the day of parturition whereas the plasma level of many phosphatidylcholines and two sphingomyelins increases steadily during early lactation.ConclusionThis newly identified shift in phospholipid composition delivers a potential biomarker to detect aberrant metabolic pathways in transition cows and also provides insights into how to prevent and treat associated disorders like fatty liver disease.

Rectal duplication cyst in a cat

Enteric duplication is a rare developmental malformation in people, dogs and cats. The purpose of the present report is to describe the first case of a rectal duplication cyst in a 7-year-old domestic shorthair cat presenting for acute constipation and tenesmus. On rectal palpation a spherical mass compressing the lumen of the rectum could be felt in the dorsal wall of the rectum. A computed tomography (CT) scan confirmed the presence of a well demarcated cystic lesion in the pelvic canal, dorsal to the rectum. The cyst was surgically removed via a perineal approach. No communication with the rectal lumen could be demonstrated. Histopathological examination was consistent with a rectal duplication cyst. Clinical signs resolved completely after excision of this conjoined non-communicating cystic rectal duplicate.

Evaluation of zebrafish as a model to study the pathogenesis of the opportunistic pathogen Cronobacter turicensis

Bacteria belonging to the genus Cronobacter spp. have been recognized as causative agents of life-threatening systemic infections, primarily in premature, low-birth weight and/or immune-compromised neonates. Knowledge remains scarce regarding the underlying molecular mechanisms of disease development. In this study, we evaluated the use of a zebrafish model to study the pathogenesis of Cronobacter turicensis LMG 23827T, a clinical isolate responsible for two fatal sepsis cases in neonates. Here, the microinjection of approximately 50 colony forming units (CFUs) into the yolk sac resulted in the rapid multiplication of bacteria and dissemination into the blood stream at 24 h post infection (hpi), followed by the development of a severe bacteremia and larval death within 3 days. In contrast, the innate immune response of the embryos was sufficiently developed to control infection after the intravenous injection of up to 104 CFUs of bacteria. Infection studies using an isogenic mutant devoid of surviving and replicating in human macrophages (ΔfkpA) showed that this strain was highly attenuated in its ability to kill the larvae. In addition, the suitability of the zebrafish model system to study the effectiveness of antibiotics to treat Cronobacter infections in zebrafish embryos was examined. Our data indicate that the zebrafish model represents an excellent vertebrate model to study virulence-related aspects of this opportunistic pathogen in vivo.

Molecular characterization and virus neutralization patterns of severe, non-epizootic forms of feline calicivirus infections resembling virulent systemic disease in cats in Switzerland and in Liechtenstein

Feline calicivirus (FCV) infections are associated with oral ulceration, chronic stomatitis and a limping syndrome. Epizootic outbreaks of virulent systemic disease (VSD) have been reported in the USA and Europe. Here, the molecular characterization and neutralization patterns of FCV isolates from cases of severe, non-epizootic infection associated with skin ulceration and edema are presented. Samples from eleven symptomatic cats, four in-contact cats and 27 cats with no contact with symptomatic cats were collected and tested for FCV, feline herpesvirus-1 (FHV-1), feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV). Phylogenetic analyses based on the capsid (VP1) gene of FCV and virus neutralization with antisera raised against four FCV vaccine strains were performed. Nine kittens and two adult cats in two shelters and two veterinary clinics in four geographically distinct locations in Switzerland and Liechtenstein were affected. The cats showed fever, tongue and skin ulceration, head and paw edema, and occasionally jaundice, generalized edema and dyspnea. All symptomatic cats tested FCV-positive but were negative for FHV-1, FeLV and FIV, with the exception of one FIV-positive kitten. All kittens of one litter and both adult cats died. The disease did not spread to cats in the environment. Cats in the environment displayed phylogenetically distinct, but related, FCV strains. Virus neutralization patterns suggested that some cases might have been potentially prevented by vaccination with the optimal vaccine strain. In conclusion, clinicians should be aware of severe, non-epizootic forms of FCV infections with initial clinical presentations similar to VSD.

Degenerative Liver Disease in Young Beagles with Hereditary Cobalamin Malabsorption Because of a Mutation in the Cubilin Gene

Cobalamin is an essential cofactor for enzyme systems, and adequate amounts are required for nucleic acid synthesis and hematopoiesis. The 2 most important reactions involving cobalamin are the conversion of methylmalonyl-coenzyme A to succinylcoenzyme A and the remethylation of homocysteine. Cobalamin deficiency leads to decreased activity of these enzyme systems, resulting in increased concentrations of urinary methylmalonic acid and total plasma homocysteine. Dogs are unable to synthesize cobalamin and rely on uptake of dietary cobalamin, which is bound in the small intestine to the secreted protein, intrinsic factor (IF). The cobalamin-IF complex binds to the membrane-bound cubam receptor, which mediates endocytosis. Cubam consists of 2 separate protein subunits, amnionless (AMN) and cubilin (CUBN). In infants, mutations in either the AMN or CUBN genes lead to Imerslund-Gr€asbeck syndrome (IGS). This is a rare autosomal recessive disorder which, if left untreated, results in failure to thrive, megaloblastic anemia, proteinuria, and neurological damage. In dogs, primary cobalamin malabsorption, which is analogous to IGS in humans, has been reported in young Australian Shepherds, a Beagle, Border Collies, and Giant Schnauzers. The genetic defects in affected Border Collies and Beagles recently have been identified as 2 independent mutations in the CUBN gene. Similar to human patients, dogs typically present at a young age with inappetence, weakness, and failure to thrive. Although liver disease is recognized in cobalamin-deficient farm animals, especially lambs, it has not been reported in dogs suffering from hereditary cobalamin malabsorption. The aim of the present case series was to describe the clinical and histopathologic findings of liver disease in 2 client-owned beagles suffering from genetically confirmed cobalamin malabsorption.