A comparison of UVb compact lamps in enabling cutaneous vitamin D synthesis in growing bearded dragons.

The effect of exposure to different UVb compact lamps on the vitamin D status of growing bearded dragons (Pogona vitticeps) was studied. Forty-two newly hatched bearded dragons (<24 h old) were allocated to six treatment groups (n = 7 per group). Five groups were exposed to different UVb compact lamps for two hours per day, with a control group not exposed to UVb radiation. At 120 days of age, blood samples were obtained and concentrations of 25(OH)D3 , Ca, P and uric acid were determined. In addition, plasma 25(OH)D3 concentration was determined in free-living adult bearded dragons to provide a reference level. Only one treatment resulted in elevated levels of 25(OH)D3 compared to the control group (41.0 ± 12.85 vs. 2.0 ± 0.0 nmol/L). All UVb-exposed groups had low 25(OH)D3 plasma levels compared to earlier studies on captive bearded dragons as well as in comparison with the free-living adult bearded dragons (409 ± 56 nmol/L). Spectral analysis indicated that all treatment lamps emitted UVb wavelengths effective for some cutaneous vitamin D synthesis. None of these lamps, under this regime, appeared to have provided a sufficient UVb dose to enable synthesis of plasma 25(OH)D3 levels similar to those of free-living bearded dragons in their native habitat.

Widespread Usutu virus outbreak in birds in the Netherlands, 2016.

We report a widespread Usutu virus outbreak in birds in the Netherlands. Viral presence had been detected through targeted surveillance as early as April 2016 and increased mortality in common blackbirds and captive great grey owls was noticed from August 2016 onwards. Usutu virus infection was confirmed by post-mortem examination and RT-PCR. Extensive Usutu virus activity in the Netherlands in 2016 underlines the need to monitor mosquito activity and mosquito-borne infections in 2017 and beyond.

Diagnosis and treatment of a congenital portosystemic shunt in a ferret (Mustela putorius furo).

A 3-year-old female neutered ferret presented with progressive weight loss was diagnosed with portosystemic shunting based on increased fasting bile acids, rectal ammonia tolerance testing and advanced imaging. Ammonia reference values were determined in 16 healthy ferrets. A congenital extrahepatic spleno-caval shunt was visualised with ultrasonography and CT angiography of the abdomen. Complete surgical shunt closure by suture ligation was performed, without clinical improvement after surgery. Euthanasia was elected 4 months postoperatively because the clinical condition deteriorated. This is a case report of advanced diagnostics and surgical treatment of a congenital extrahepatic portosystemic shunt in a ferret, demonstrating rectal ammonia tolerance testing and imaging as feasible techniques for the diagnosis.

Detection of novel divergent arenaviruses in boid snakes with inclusion body disease in The Netherlands.

Arenaviruses are bi-segmented negative-stranded RNA viruses, which were until recently only detected in rodents and humans. Now highly divergent arenaviruses have been identified in boid snakes with inclusion body disease (IBD). Here, we describe the identification of a new species and variants of the highly divergent arenaviruses, which were detected in tissues of captive boid snakes with IBD in The Netherlands by next-generation sequencing. Phylogenetic analysis of the complete sequence of the open reading frames of the four predicted proteins of one of the detected viruses revealed that this virus was most closely related to the recently identified Golden Gate virus, while considerable sequence differences were observed between the highly divergent arenaviruses detected in this study. These findings add to the recent identification of the highly divergent arenaviruses in boid snakes with IBD in the United States and indicate that these viruses also circulate among boid snakes in Europe.

Tularaemia in a brown hare (Lepus europaeus) in 2013: first case in the Netherlands in 60 years.

Tularaemia has not been reported in Dutch wildlife since 1953. To enhance detection, as of July 2011, brown hares (Lepus europaeus) submitted for postmortem examination in the context of non-targeted wildlife disease surveillance, were routinely tested for tularaemia by polymerase chain reaction (PCR). Francisella tularensis subspecies holarctica infection was confirmed in a hare submitted in May 2013. The case occurred in Limburg, near the site of the 1953 case. Further surveillance should clarify the significance of this finding.

Effects of root plates, pits and mounds following windthrow events on the biodiversity of plants and lichens in Central European mountain forests.

Windthrows lead to the formation of a characteristic land microtopography and they provide new habitats and substrata for species. The present study is novel, because it concerns different taxonomic groups of organisms and effects of various variables on species composition, richness and abundance within root plate-pit-mound (RPM) complexes. For the first time we studied lichens, liverworts, mosses and vascular plants on RPM complexes across major vegetation types, from deciduous to coniferous forests. We analyzed 164 RPM complexes which included in total 268 components: 115 root plates, 80 pits and 73 mounds. Besides the vegetation types and component types the following variables were also considered: dimensions (area, size), age (decomposition degree), species of the uprooted tree, shade, and elevation. The research revealed that root plates, pits and mounds had their own indicator species, but the key factors for the species composition were the species of uprooted trees and the vegetation type. Furthermore, the type of component and the vegetation type were also the most main variables determining the richness and abundance of the studied taxonomic groups of species. Analyses revealed the greatest species richness and abundance of lichens on root plates, and the highest richness of liverworts and vascular plants in pits. The type of component had no effect on the species richness of mosses. However, they were most abundant on mounds. The response of individual groups to the vegetation types and other variables varied. Findings from the study indicated that the presence of uprooted trees with different features (size and age, species of uprooted tree) is important for the biodiversity of mountain forests. We recommend leaving uprooted trees in forest communities for natural colonization, preferably large objects. Moreover, in managed forests a mosaic of forest communities that mimic the structure of natural unmanaged forests should be supported.

Comparative study of depth-dependent characteristics of equine and human osteochondral tissue from the medial and lateral femoral condyles.

Articular cartilage defects are common after joint injuries. When left untreated, the biomechanical protective function of cartilage is gradually lost, making the joint more susceptible to further damage, causing progressive loss of joint function and eventually osteoarthritis (OA). In the process of translating promising tissue-engineering cartilage repair approaches from bench to bedside, pre-clinical animal models including mice, rabbits, goats, and horses, are widely used. The equine species is becoming an increasingly popular model for the in vivo evaluation of regenerative orthopaedic approaches. As there is also an increasing body of evidence suggesting that successful lasting tissue reconstruction requires an implant that mimics natural tissue organization, it is imperative that depth-dependent characteristics of equine osteochondral tissue are known, to assess to what extent they resemble those in humans. Therefore, osteochondral cores (4-8 mm) were obtained from the medial and lateral femoral condyles of equine and human donors. Cores were processed for histology and for biochemical quantification of DNA, glycosaminoglycan (GAG) and collagen content. Equine and human osteochondral tissues possess similar geometrical (thickness) and organizational (GAG, collagen and DNA distribution with depth) features. These comparable trends further underscore the validity of the equine model for the evaluation of regenerative approaches for articular cartilage.

The sea louse Lepeophtheirus acutus (Caligidae, Siphonostomatoida, Copepoda) as a pathogen of aquarium-held elasmobranchs.

Lepeophtheirus acutus Heegaard, 1943 (Caligidae, Siphonostomatoida, Copepoda), was collected from or observed on four of six elasmobranch species held at Burgers' Zoo (Arnhem, The Netherlands). Circumstantial evidence suggested that a zebra shark, Stegostoma fasciatum (Hermann), from the wild carried the infection into the facility, where copepods reproduced and colonized additional hosts. Copepods typically attached on and about the eyes, in the mouth and occasionally about the cloaca and on the claspers. Severe ocular lesions were associated with infections on zebra sharks, a grey reef shark, Carcharhinus amblyrhynchos (Bleeker), whitetip reef sharks, Triaenodon obesus (Rüppell), and giant shovelnose ray, Rhinobatos typus Bennett, while blacktip reef sharks, Carcharhinus melanopterus (Quoy & Gaimard), and blacktip sharks, Carcharhinus limbatus (Valenciennes), living in infested aquaria showed no sign of infection. Water treatments using trichlorfon were considered primarily responsible for the eradication of copepods from hosts and infested aquaria. This case is the first report of a copepod infection being closely associated with disease and death of an aquarium-held elasmobranch. Given its ability to infect a wide variety of elasmobranchs and promote life-threatening lesions on some hosts, L. acutus should be considered a dangerous pathogen of captive elasmobranchs.

Association of red-mark syndrome with a Rickettsia-like organism and its connection with strawberry disease in the USA.

Red-mark syndrome (RMS), a disease seen mostly in rainbow trout, Oncorhynchus mykiss, is of unknown aetiology. The research presented here indicates the presence of an intracellular bacterium in RMS-affected fish. A positive reaction was observed using immunohistochemistry (IHC) with skin lesions, liver, kidney and spleen of affected fish sampled from several locations within the United Kingdom using two different polyclonal antisera raised against Piscirickettsia salmonis. The same reaction was also seen with a number of different anti-P. salmonis monoclonal antibodies (MAbs). A disease with similar clinical signs to RMS, referred to as strawberry disease (SD), has been reported in the USA. A Rickettsia-like organism (RLO) has recently been associated with SD based on analysis of 16S rDNA sequences. Using the same panel of anti-P. salmonis antibodies used to screen the RMS samples, similar staining was obtained in tissue of SD-affected fish by IHC. A polymerase chain reaction (PCR) using RLO-specific primers was also performed on RMS-affected fish from the United Kingdom, and the samples were positive for the RLO 16S rRNA sequence. These findings suggest that the same aetiological agent may be responsible for RMS in the United Kingdom and SD in the USA.

The nocturnal leopard gecko (Eublepharis macularius) uses UVb radiation for vitamin D3 synthesis.

Vitamin D is an important regulator of calcium and phosphorus homeostasis in animals. It can be acquired from the diet or synthesised de novo when skin is exposed to UVb. Vitamin D deficiency can lead to a complex of diseases collectively called metabolic bone disease (MBD). Diurnal lizards without access to UVb are prone to develop vitamin D deficiency, even when dietary vitamin D3 is provided. A trial was conducted to determine whether juvenile nocturnal lizards require access to UVb to prevent vitamin D deficiency. All leopard geckos (Eublepharis macularius) were supplemented with dietary vitamin D3. One group was exposed to low level UVb radiation (33-51 µW/cm2) from hatching until 6 months of age and a second group remained unexposed. Animals were fed ad libitum and their growth and weight gain compared with non-exposed controls. At the end of the trial, blood samples were analysed for vitamin D3 metabolites. The concentration of the vitamin D3 metabolite, 25(OH)D3, was higher in UVb exposed animals (61 ± 20 vs. 38 ± 8 nmol/L), confirming cutaneous synthesis with UVb exposure. Growth and weight gain were similar in both groups, and this, together with the absence of clinical symptoms, suggests that dietary vitamin D3 alone can meet the vitamin D requirements for growth of this nocturnal gecko, during the first six months of life. It remains to be investigated whether the higher vitamin D metabolite levels holds other health benefits for this species, such as improved bone density or immune response.

Genomic monitoring to understand the emergence and spread of Usutu virus in the Netherlands, 2016-2018.

Usutu virus (USUV) is a mosquito-borne flavivirus circulating in Western Europe that causes die-offs of mainly common blackbirds (Turdus merula). In the Netherlands, USUV was first detected in 2016, when it was identified as the likely cause of an outbreak in birds. In this study, dead blackbirds were collected, screened for the presence of USUV and submitted to Nanopore-based sequencing. Genomic sequences of 112 USUV were obtained and phylogenetic analysis showed that most viruses identified belonged to the USUV Africa 3 lineage, and molecular clock analysis evaluated their most recent common ancestor to 10 to 4 years before first detection of USUV in the Netherlands. USUV Europe 3 lineage, commonly found in Germany, was less frequently detected. This analyses further suggest some extent of circulation of USUV between the Netherlands, Germany and Belgium, as well as likely overwintering of USUV in the Netherlands.

Influence of surface modification of Ti6Al7Nb alloy on adhesion of poly (lactide-co-glycolide) coating (PLGA).

The paper presents the results of studies on the influence of surface topography and wettability of a Ti6Al7Nb alloy substrate on the adhesion of a PLGA coating. The alloy surface was modified using a mechanical pre-treatment including: grinding, vibration treatment, mechanical polishing, sandblasting and anodic oxidation. The polymer coating was applied to the metal substrate by a dip-coating method. The scope of the research included microscopic observations of the substrate and polymer coating using SEM and acoustic microscopy. In addition, studies on the wettability and topography of the polymer coating and the metal substrate, the thickness of the polymer coating as well as qualitative and quantitative testing of the adhesion of the polymer coating to the substrate were carried out. Coating adhesion tests were conducted for samples in the initial state and after 6 weeks exposure to Ringer's solution. Analysis of the results indicates the influence of the method used to modify the metal substrate on its topography and wettability. These parameters affect the thickness of the obtained polymer coating. Regardless the parameters of the metal substrate, a qualitative analysis of the adhesion of the coating applied to the substrate of the Ti6Al7Nb alloy did not show any delamination for both samples exposed and non-exposed to Ringer's solution. On the other hand, quantitative scratch-test studies showed different adhesion of the polymer coating to the substrate depending on the surface topography obtained by various modification methods. The cytotoxicity test conducted by the indirect method using extracts confirmed that the surface modification does not affect cell growth. The complex methods of surface pre-treatment of the alloy together with the kind of polymer selected for the study allowed to develop well adhered PLGA layers on Ti6Al7Nb intended for short term implants. The lack of delamination of the layer during 6 weeks was proved, what allows for maintaining the protection function of the layer during this period and contribute to improving biocompatibility.

Expression of hepatocyte growth factor and the proto-oncogenic receptor c-Met in canine osteosarcoma.

Hepatocyte growth factor (HGF) and the proto-oncogenic receptor c-Met are implicated in growth, invasion, and metastasis in human cancer. Little information is available on the expression and role of both gene products in canine osteosarcoma. We hypothesized that the expression of c-Met is associated with malignant histologic characteristics, a short survival time, and a reduced disease-free interval in canine osteosarcoma. Quantitative real-time polymerase chain reaction was used to analyze the messenger RNA (mRNA) expression of both HGF and c-Met in 59 canine osteosarcoma samples. The relationship between HGF and c-Met expression, patient outcome, and histologic characteristics of the tumor were studied. Western blot analysis was performed to investigate the presence of active HGF protein. The expression pattern of c-Met in 16 slides of canine osteosarcoma was identified by immunohistochemistry. Coexpression of HGF and c-Met mRNA in all canine osteosarcoma samples suggested autocrine or paracrine receptor activation. A significant, moderately positive correlation was found between c-Met and HGF mRNA expression. c-Met mRNA expression was not associated with survival time or disease-free interval. Expression of c-Met was significantly associated with metastasis via the lymphogenic route. Immunolabeling with c-Met revealed a cytoplasmic staining pattern in all osteosarcoma cell types. In this study, c-Met mRNA expression in canine osteosarcoma was found to be of no influence on survival time and disease-free interval. Further studies are necessary to confirm the involvement of the c-Met pathway in the lymphogenic route of metastasis.

Towards high quality real-time whole genome sequencing during outbreaks using Usutu virus as example.

Recently, protocols for amplicon based whole genome sequencing using Nanopore technology have been described for Ebola virus, Zika virus, yellow fever virus and West Nile virus. However, there is some debate regarding reliability of sequencing using this technology, which is important for applications beyond diagnosis such as linking lineages to outbreaks, tracking transmission pathways and pockets of circulation, or mapping specific markers. To our knowledge, no in depth analyses of the required read coverage to compensate for the error profile in Nanopore sequencing have been described. Here, we describe the validation of a protocol for whole genome sequencing of USUV using Nanopore sequencing by direct comparison to Illumina sequencing. To that point we selected brain tissue samples with high viral loads, typical for birds which died from USUV infection. We conclude that the low-cost MinION Nanopore sequencing platform can be used for characterization and tracking of Usutu virus outbreaks.

Effects of body mass on microstructural features of the osteochondral unit: A comparative analysis of 37 mammalian species.

Since Galileo's days the effect of size on the anatomical characteristics of the structural elements of the body has been a subject of interest. However, the effects of scaling at tissue level have received little interest and virtually no data exist on the subject with respect to the osteochondral unit in the joint, despite this being one of the most lesion-prone and clinically relevant parts of the musculoskeletal system. Imaging techniques, including Fourier transform infrared imaging, polarized light microscopy and micro computed tomography, were combined to study the response to increasing body mass of the osteochondral unit. We analyzed the effect of scaling on structural characteristics of articular cartilage, subchondral plate and the supporting trabecular bone, across a wide range of mammals at microscopic level. We demonstrated that, while total cartilage thickness scales to body mass in a negative allometric fashion, thickness of different cartilage layers did not. Cartilage tissue layers were found to adapt to increasing loads principally in the deep zone with the superficial layers becoming relatively thinner. Subchondral plate thickness was found to have no correlation to body mass, nor did bone volume fraction. The underlying trabecular bone was found to have thicker trabeculae (r=0.75, p<0.001), as expected since this structure carries most loads and plays a role in force mitigation. The results of this study suggest that the osteochondral tissue structure has remained remarkably preserved across mammalian species during evolution, and that in particular, the trabecular bone carries the adaptation to the increasing body mass.

Upper respiratory tract infection caused by Cryptosporidium baileyi in three mixed-bred falcons (Falco rusticolus x Falco cherrug).

Three mixed-bred raptors (Falco rusticolus x Falco cherrug) from a German falcon breeder were presented with a history of respiratory distress. In one bird a laryngeal stridor was noted, and oral examination revealed an epiglottal swelling. In the other two birds, nasal discharge and sneezing were the main clinical symptoms. Nasal flushing samples and biopsies were collected for pathologic, bacteriologic, and parasitologic examination. Results confirmed a cryptosporidial infection. Polymerase chain reaction (PCR) and DNA analysis identified the causative agent to be Cryptosporidium baileyi. No cryptosporidia were detected in fecal samples, indicating the infection was confined to the respiratory system. Analysis of prey animals (pigeons, quail) failed to identify the source of infection. Treatment was initiated with paromomycin in all three birds, whereas in two birds an additional therapy with azithromycin was given. However, no clinical improvement was seen after several weeks of treatment, and the birds either died or were euthanatized. To the authors' knowledge, these are the first confirmed cases of disease caused by cryptosporidia in the order of Falconiformes.

Use of a gonadotropin releasing hormone agonist implant as an alternative for surgical castration in male ferrets (Mustela putorius furo).

Surgical castration in ferrets has been implicated as an etiological factor in the development of hyperadrenocorticism in this species due to a castration-related increase in plasma gonadotropins. In search for a suitable alternative, the effect of treatment with the depot GnRH-agonist implant, deslorelin, on plasma testosterone concentrations and concurrent testes size, spermatogenesis, and the typical musky odor of intact male ferrets was investigated. Twenty-one male ferrets, equally divided into three groups, were either surgically castrated, received a slow release deslorelin implant or received a placebo implant. Plasma FSH and testosterone concentrations, testis size and spermatogenesis were all suppressed after the use of the deslorelin implant. The musky odor in the ferrets which had received a deslorelin implant was less compared to the ferrets which were either surgically castrated or had received a placebo implant. These results indicate that the deslorelin implant effectively prevents reproduction and the musky odor of intact male ferrets and is therefore considered a suitable alternative for surgical castration in these animals.

Isolation of Mycobacterium mageritense from the lung of a harbor porpoise (Phocoena phocoena) with severe granulomatous lesions.

Post-mortem investigation of a harbor porpoise (Phocoena phocoena) found dead on the beach of the island of Vlieland, The Netherlands, revealed severe granulomatous changes in the right lung lobe. Ziehl Neelsen staining demonstrated relatively large acid-fast rods. Mycobacterial culture yielded a fast-growing mycobacterium, which was identified by molecular biological methods as Mycobacterium mageritense. Autolysis prevented histopathology. It was tentatively concluded that the granulomatous changes were the cause of porpoise's death and that M. mageritense was the causative agent. This is the first report of the isolation and molecular identification of this mycobacterium in a nonhuman animal species and the first association with the marine environment.

Controlled release of celecoxib inhibits inflammation, bone cysts and osteophyte formation in a preclinical model of osteoarthritis.

Major hallmarks of osteoarthritis (OA) are cartilage degeneration, inflammation and osteophyte formation. COX-2 inhibitors counteract inflammation-related pain, but their prolonged oral use entails the risk for side effects. Local and prolonged administration in biocompatible and degradable drug delivery biomaterials could offer an efficient and safe treatment for the long-term management of OA symptoms. Therefore, we evaluated the disease-modifying effects and the optimal dose of polyesteramide microspheres delivering the COX-2 inhibitor celecoxib in a rat OA model. Four weeks after OA induction by anterior cruciate ligament transection and partial medial meniscectomy, 8-week-old female rats (n = 6/group) were injected intra-articular with celecoxib-loaded microspheres at three dosages (0.03, 0.23 or 0.39 mg). Unloaded microspheres served as control. During the 16-week follow-up, static weight bearing and plasma celecoxib concentrations were monitored. Post-mortem, micro-computed tomography and knee joint histology determined progression of synovitis, osteophyte formation, subchondral bone changes, and cartilage integrity. Systemic celecoxib levels were below the detection limit 6 days upon delivery. Systemic and local adverse effects were absent. Local delivery of celecoxib reduced the formation of osteophytes, subchondral sclerosis, bone cysts and calcified loose bodies, and reduced synovial inflammation, while cartilage histology was unaffected. Even though the effects on pain could not be evualated directly in the current model, our results suggest the application of celecoxib-loaded microspheres holds promise as novel, safe and effective treatment for inflammation and pain in OA.