Griet Vercauteren

Developing a safe antifungal treatment protocol to eliminate Batrachochytrium dendrobatidis from amphibians

Batrachochytrium dendrobatidis is one of the most pathogenic microorganisms affecting amphibians in both captivity and in nature. The establishment of B. dendrobatidis free, stable, amphibian captive breeding colonies is one of the emergency measures that is being taken to save threatened amphibian species from extinction. For this purpose, in vitro antifungal susceptibility testing and the development of efficient and safe treatment protocols are required. In this study, we evaluated the use of amphotericin B and voriconazole to treat chytridiomycosis in amphibians. The concentration at which the growth of five tested B. dendrobatidis strains was inhibited was 0.8 μg/ml for amphotericin B and 0.0125 μg/ml for voriconazole. To completely eliminate a mixture of sporangia and zoospores of strain IA042 required 48 h of exposure to 8 μg/ml of amphotericin B or 10 days to 1.25 μg/ml of voriconazole. Zoospores were killed within 0.5 h by 0.8 μg/ml of amphotericin B, but even after 24 h exposure to 1.25 μg/ml of voriconazole they remained viable. Amphotericin B was acutely toxic for Alytes muletensis tadpoles at 8 μg/ml, whereas toxic side effects were not noticed during a seven-day exposure to voriconazole at concentrations as high as 12.5 μg/ml. The voriconazole concentrations remained stable in water during this exposure period. On the basis of this data, experimentally inoculated postmetamorphic Alytes cisternasii were sprayed once daily for 7 days with a 1.25 μg/ml solution of voriconazole in water which eliminated the B. dendrobatidis infection from all treated animals. Finally, treatment of a naturally infected colony of poison dart frogs (Dendrobatidae) using this protocol, combined with environmental disinfection, cleared the infection from the colony.

Haemorrhagic diathesis in neonatal calves: an emerging syndrome in Europe.

In 2008 and 2009 a large number of cases of haemorrhagic diathesis (HD) in neonatal calves were reported in different European countries. In Flanders, 84 cases of neonatal HD in 30 herds were reported in this period. The disease typically affects calves younger than 1 month old from different breed and gender. Prominent clinical signs are cutaneous bleeding, petechiae on all mucosae, melena and often high fever. Early in the disease, the mental state of the animals is uncompromised. The typical haematological finding is pancytopenia, with severe to complete thrombocytopenia being the cause of the increased susceptibility to bleeding. In seven of the affected herds blood samples of calves of the same age group as the clinical case were collected and on six of those farms at least one subclinical case could be identified. Necropsy findings were generalized petechiae, ecchymoses or haemorrhages and variable lymphadenopathy. Histopathology of haemorrhagic lesions revealed multifocal extravasation of red blood cells (haemorrhage) with preservation of tissue architecture and absence of other abnormalities. Total bone marrow aplasia and depletion of all lymphoid tissue was the most prominent finding on histology. Activated macrophages and haemophagocytosis were seen on bone marrow cytology from two live calves. Polymerase chain reaction for bovine viral diarrhoea virus, bluetongue and epizootic haemorrhagic disease virus was negative. Several attempts to isolate a viral agent were unsuccessful.

Antibacterial therapeutics for the treatment of chytrid infection in amphibians: Columbus’s egg?

BackgroundThe establishment of safe and effective protocols to treat chytridiomycosis in amphibians is urgently required. In this study, the usefulness of antibacterial agents to clear chytridiomycosis from infected amphibians was evaluated.ResultsFlorfenicol, sulfamethoxazole, sulfadiazine and the combination of trimethoprim and sulfonamides were active in vitro against cultures of five Batrachochytrium dendrobatidis strains containing sporangia and zoospores, with minimum inhibitory concentrations (MIC) of 0.5-1.0 μg/ml for florfenicol and 8.0 μg/ml for the sulfonamides. Trimethoprim was not capable of inhibiting growth but, combined with sulfonamides, reduced the time to visible growth inhibition by the sulfonamides. Growth inhibition of B. dendrobatidis was not observed after exposure to clindamycin, doxycycline, enrofloxacin, paromomycin, polymyxin E and tylosin. Cultures of sporangia and zoospores of B. dendrobatidis strains JEL423 and IA042 were killed completely after 14 days of exposure to 100 μg/ml florfenicol or 16 μg/ml trimethoprim combined with 80 μg/ml sulfadiazine. These concentrations were, however, not capable of efficiently killing zoospores within 4 days after exposure as assessed using flow cytometry. Florfenicol concentrations remained stable in a bathing solution during a ten day period. Exposure of Discoglossus scovazzi tadpoles for ten days to 100 μg/ml but not to 10 μg florfenicol /ml water resulted in toxicity. In an in vivo trial, post metamorphic Alytes muletensis, experimentally inoculated with B. dendrobatidis, were treated topically with a solution containing 10 μg/ml of florfenicol during 14 days. Although a significant reduction of the B. dendrobatidis load was obtained, none of the treated animals cleared the infection.ConclusionsWe thus conclude that, despite marked anti B. dendrobatidis activity in vitro, the florfenicol treatment used is not capable of eliminating B. dendrobatidis infections from amphibians.

Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination.

Helicobacter suis (H. suis) is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund’s Complete and Incomplete, Cholera toxin), administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund’s complete (FC)/lysate and intranasal immunization with Cholera toxin (CT)/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually) is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.

Two Atypical Cases of Granulosa Cell Tumours in Belgian Blue Heifers

This paper describes two atypical cases of granulosa cell tumours (GCTs) in Belgian Blue heifers. In the first case, an 18-month-old heifer died after ovariectomy, and a granulosa-theca cell tumour was diagnosed post-mortem. The second heifer (31 months of age) was near term and was successfully treated surgically for a malignant granulosa cell tumour. Unfortunately, no further reproductive data are available, as the heifer was sold for slaughter. This is the first report describing GCTs in Belgian Blue beef cattle.

Expression of P-Glycoprotein in the Intestinal Epithelium of Dogs with Lymphoplasmacytic Enteritis

Inflammatory bowel disease (IBD) is an idiopathic chronic inflammatory disease of the stomach, the small intestine and/or the large intestine. Loss of integrity of the intestinal barrier may be an important factor in the pathogenesis of IBD. In dogs, lymphoplasmacytic enteritis (LPE) is one of the recognized forms of IBD. P-glycoprotein (P-gp) is a membrane-bound efflux pump constituting an important component of the intestinal barrier. Changes in P-gp expression at the level of the intestinal barrier may be important in the pathogenesis of canine LPE, as this may lead to variable protection against xenobiotics and bacterial products in the intestine. The aim of the present study was to evaluate the expression of epithelial P-gp in the intestine in dogs with LPE compared with disease-free animals. Formalin-fixed intestinal biopsy samples from 57 dogs with histopathological evidence of LPE were immunolabelled with anti-P-gp antibodies (C494 and C219). Endoscopic biopsy samples of the duodenum and colon from 16 healthy beagles were used as controls. None of the control dogs had P-gp expression in the apical membrane of duodenal enterocytes, but all had P-gp labelling at the colonic epithelial surface. Twenty out of 57 dogs with LPE had P-gp expression at the apical surface membrane of villus epithelial cells in the duodenum, jejunum and/or ileum. Six out of 16 colonic samples from dogs with LPE had decreased P-gp expression at the epithelial surface compared with controls. It is unclear whether these changes in P-gp expression in dogs with LPE are a cause or a consequence of the inflammation. The observed changes could affect bioavailability of therapeutic drugs used in LPE.

Safety assessment of the butyrate-producing Butyricicoccus pullicaecorum strain 25-3T, a potential probiotic for patients with inflammatory bowel disease, based on oral toxicity tests and whole genome sequencing

Inflammatory bowel disease (IBD) is a chronic inflammation of the digestive tract, characterized by dysbiosis of the intestinal microbiota. Probiotics have been suggested as a strategy to reduce active disease or extend remission. We isolated and characterized the butyrate-producing strain Butyricicoccus pullicaecorum 25-3(T) and identified it as a potential probiotic for patients with IBD. To evaluate the safety of 25-3(T) for use in humans, we conducted a standard acute oral toxicity test and a 28-day repeated oral dose toxicity test. The complete genome of B. pullicaecorum 25-3(T) was sequenced to search for virulence factors and antibiotic resistance determinants. The minimum inhibitory concentration (MIC) of 21 antimicrobials was determined. Results showed no adverse effects in the oral toxicity tests. B. pullicaecorum 25-3(T) is resistant against aminoglycosides and trimethoprim. The genome of 25-3(T) contains no virulence factors, one gene related to harmful metabolites and 52 sequences with high similarity to antimicrobial and toxic compound resistance genes, that did not correspond with a resistant phenotype. This first report of a safety assessment of a butyrate-producing strain from Clostridium cluster IV shows that B. pullicaecorum 25-3(T) is a non-pathogenic strain, but carries antibiotic resistance genes with the risk of transfer, that need further investigation.

Uterine fibrosarcoma in a Warmblood mare.

This paper describes a case of uterine fibrosarcoma in an 18-year-old Warmblood mare. The mare had exhibited bloody fluid accumulation inside the uterus and vaginal haemorrhagic discharge since the previous foaling. The mare was euthanized, and on pathological examination, in addition to the uterine neoplasia, multiple metastases were found in the lungs, liver and spleen. The histological and immunohistochemical examination determined that the tumour was a fibrosarcoma. To our knowledge, this is the first paper to describe a uterine fibrosarcoma in a mare.

Inspiratory stridor secondary to palatolingual myokymia in a Maltese dog

A nine-year-old male Maltese dog was presented with an eight-month history of inspiratory stridor leading to exertional dyspnoea and cyanosis. Myokymic contractions in the palatolingual muscles were noticed and confirmed by electromyography. Brain computer tomography-scan showed ventricular dilatation. Cerebrospinal fluid analysis revealed a slightly elevated protein level. Treatment with slow-release phenytoin was unsuccessful and symptoms gradually worsened over the next nine months. At post-mortem examination a small pituitary adenoma was found. Apart from a single canine report of facial myokymia, this is the only other description of spontaneous focal myokymia in animals. Palatolingual myokymia has only been reported in one human being. Although the co-occurrence with a pituitary adenoma might be incidental, a paraneoplastic pathogenetic mechanism is proposed. Its unique clinical presentation adds a new, albeit uncommon, syndrome to the differential diagnosis of upper airway complaints in dogs.

Laryngeal rhinosporidiosis in a Belgian warmblood horse.

In Belgium and even in northern Europe Rhinosporidium seeberi has not been reported in autochtonous people or animals. In this paper, the authors report the first observation of laryngeal masses, caused by Rhinosporidium seeberi, in a Belgian Warmblood horse. Moreover, laryngeal rhinosporidiosis is extremely rare since this localisation is only described in four human cases.