Is the European badger a new host for Dirofilaria immitis? The first records in Greece.

Dirofilaria immitis is a ubiquitous nematode parasite with zoonotic potential, transmitted by mosquitoes, that causes heartworm disease in various animal species. Dogs are the parasite's typical final host, and wild carnivores represent the parasite's reservoir in nature. Studies on D. immitis infections in wild animals are essential to assess infection pressure for domestic animals, and until now, there has been only one infection case reported in a European badger (Meles meles). The current report describes the first two European badger cases with cardiovascular dirofilariosis in Greece. Two adult male badgers were rescued in Heraklion and Chania, Crete Island, and admitted to "ANIMA -Wildlife Rehabilitation Centre" in Athens. The detailed clinical examination revealed that the first badger suffered from severe broncho-pneumonitis while the second one displayed clinical signs associated with severe brain trauma. Blood samples were taken for haematology and biochemistry analyses during their short hospitalisation period. In addition, different routine diagnostic tests were carried out, including heartworm antigen testing (ELISA) and the modified Knott's test for microfilariae. Both badgers were positive in both tests. The animals died a few hours after their admission and the detailed necropsies followed, revealed the presence of three parasites in each animal's right heart, morphologically identified as adults of D. immitis. These findings add the European badger in the list of additional potential reservoir hosts for D. immitis and highlight the potential role of wildlife for companion animals and human health.

Fecal and skin microbiota of two rescued Mediterranean monk seal pups during rehabilitation.

IMPORTANCE: This study showed that during the rehabilitation of two rescued Mediterranean monk seal pups (Monachus monachus), the skin and fecal bacterial communities showed similar succession patterns between the two individuals. This finding means that co-housed pups share their microbiomes, and this needs to be considered in cases of infection outbreaks and their treatment. The housing conditions, along with the feeding scheme and care protocols, including the admission of antibiotics as prophylaxis, probiotics, and essential food supplements, resulted in bacterial communities with no apparent pathogenic bacteria. This is the first contribution to the microbiome of the protected seal species of M. monachus and contributes to the animal's conservation practices through its microbiome.

Abortion Related to Toxoplasma gondii Infection in a Bactrian Camel (Camelus bactrianus) in Greece: A Case Report.

A female, 5 yr old Bactrian camel was presented to the Exotic and Wildlife Medicine Unit, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece, with severe dehydration, depression, anorexia, mild dyspnea and diarrhea. Supportive treatment immediately initiated with fluids, electrolytes and broad-spectrum antibiotics. The general condition of the animal was stable for the next 3 days, but at 4th day became worse, since the camel remained in sternal recumbency, denied to drink water and abortion of a mummified fetus was noticed. The aborted fetus and fetal membranes were submitted for laboratory examinations (bacterial cultures, MZN, cytology, PCR) that revealed Toxoplasma gondii infection. Treatment with sulfadimidine improved the situation of the animal that returned to its farm 1 week later. This seems to be the first reported case in the literature of confirmed toxoplasmic abortion in camels.

The Effects of Intravitreal Administration of Antifungal Drugs on the Structure and Mechanical Properties Peripheral Blood Erythrocyte Surface in Rabbits.

BACKGROUND: Fungal infections can pose great threat to sight. Immediate treatment is usually required; antifungal agents are widely accepted and are effective in most cases. The present experimental study aims to investigate the probable effects of intravitreal injection of antifungal agents on the structure and mechanical properties of the surface of peripheral blood erythrocytes. METHODS: Nine albino New Zealand white rabbits, aged five months old, were chosen for the experiment. Solutions of micafungin, voriconazole, or balanced salt solution (BSS) were injected into the midvitreous. Animals were divided into two experimental groups and one control group. Blood sampling from an intravenous (IV) line was performed after 10 days from the last IV injection. An atomic force microscope (AFM) was used to study the structural and mechanical properties of cell surfaces. RESULTS: The analysis results showed that the parameters of the cytoskeleton's spatial organization changed insignificantly with the antifungal drug treatment. CONCLUSIONS: Our findings suggest that locally administered antifungal drugs can cause significant changes to the structure and frictional properties of the erythrocyte surface. These effects occur in the long-term period after administration of the drugs and represent a potential possibility for violation of blood supply to tissues, and the further development of negative side effects.

Intravitreal Administration Effect of Adipose-Derived Mesenchymal Stromal Cells Combined with Anti-VEGF Nanocarriers, in a Pharmaceutically Induced Animal Model of Retinal Vein Occlusion.

Antiangiogenic therapeutic agents (anti-VEGF) have contributed to the treatment of retinal vein occlusion (RVO) while mesenchymal stromal cell- (MSCs-) mediated therapies limit eye degeneration. The aim of the present study is to determine the effect of adipose-derived MSCs (ASCs) combination with nanocarriers of anti-VEGF in a pharmaceutically induced animal model of RVO. Nanoparticles (NPs) of thiolated chitosan (ThioCHI) with encapsulated anti-VEGF antibody were prepared. ASCs were isolated and genetically modified to secrete the green fluorescence GFP. Twenty-four New Zealand rabbits were divided into the I-IV equal following groups: ASCs, ASCs + nanoThioCHI-anti-VEGF, RVO, and control. For the RVO induction, groups I-III received intravitreal (iv) injections of MEK kinase inhibitor, PD0325901. Twelve days later, therapeutic regiments were administered at groups I-II while groups III-IV received BSS. Two weeks later, the retinal damage evaluated via detailed ophthalmic examinations, histological analysis of fixed retinal sections, ELISA for secreted cytokines in peripheral blood or vitreous fluid, and Q-PCR for the expression of related to the occlusion and inflammatory genes. Mild retinal edema and hemorrhages, limited retinal detachment, and vasculature attenuation were observed in groups I and II compared with the pathological symptoms of group III which presented a totally disorganized retinal structure, following of positive immunostaining for neovascularization and related to RVO markers. Important reduction of the high secreted levels of inflammatory cytokines was quantified in groups I and II vitreous fluid, while the expression of the RVO-related and inflammatory genes has been significantly decreased especially in group II. GFP+ ASCs, capable of being differentiated towards neural progenitors, detected in dissociated retina tissues of group II presenting their attachment to damaged area. Conclusively, a stem cell-based therapy for RVO is proposed, accompanied by sustained release of anti-VEGF, in order to combine the paracrine action of ASCs and the progressive reduction of neovascularization.

In Situ Gelling Electrospun Ocular Films Sustain the Intraocular Pressure-Lowering Effect of Timolol Maleate: In Vitro, Ex Vivo, and Pharmacodynamic Assessment.

Most common intraocular pressure (IOP) reduction regimens for the management of glaucoma include the topical use of eye drops, a dosage form that is associated with short residence time at the site of action, increased dosing frequency, and reduced patient compliance. In situ gelling nanofiber films comprising poly(vinyl alcohol) and Poloxamer 407 were fabricated via electrospinning for the ocular delivery of timolol maleate (TM), aiming to sustain the IOP-lowering effect of the ß-blocker, compared to conventional eye drops. The electrospinning process was optimized, and the physicochemical properties of the developed formulations were thoroughly investigated. The fiber diameters of the drug-loaded films ranged between 123 and 145 nm and the drug content between 5.85 and 7.83% w/w. Total in vitro drug release from the ocular films was attained within 15 min following first-order kinetics, showing higher apparent permeability (Papp) values across porcine corneas compared to the drug's solution. The fabricated films did not induce any ocular irritation as evidenced by both the hen's egg test on chorioallantoic membrane and the in vivo Draize test. In vivo administration of the ocular films in rabbits induced a faster onset of action and a sustained IOP-lowering effect up to 24 h compared to TM solution, suggesting that the proposed ocular films are promising systems for the sustained topical delivery of TM.

Zoonotic Thelazia callipaeda eyeworm in brown bears (Ursus arctos): A new host record in Europe.

Thelazia callipaeda (Spirurida, Thelaziidae) eyeworm, a zoonotic parasite transmitted by lachryphagus drosophilids, causes subclinical to clinical ocular disease in a wide range of vertebrates, including humans. In the past 20 years, the infection spread primarily in areas where the vector thrives, and now is endemic in many European countries. Recently, this nematode has been reported also in the United States. Here, we describe the first record of the brown bear (Ursus arctos) as a host of T. callipaeda. Eight (26.7%) out of 30 bears were found to be infected with T. callipaeda adult worms. In addition, the parasite was detected in 13 wolves (Canis lupus) sharing the same environment with the brown bears. At the molecular characterization, all nematodes belonged to the haplotype 1, which is the only one recorded in Europe, as yet. This finding demonstrates that brown bears may act as hosts for this parasite, suggesting its potential role in the maintenance of the cycle not only in the wild but also in captivity. Under the above circumstances, the brown bear may represent a potential zoonotic risk for humans, both in rural and urban areas where other reservoirs may be infected.

First Report of Uncinaria hamiltoni in Orphan Eastern Mediterranean Monk Seal Pups in Greece and Its Clinical Significance.

The Mediterranean monk seal (Monachus monachus) is classified by the IUCN as "endangered," with a global population estimated to number fewer than 800 individuals. Our understanding of the biology and health status of the species is still limited, rendering every medical case a challenge for conservationists and veterinary clinicians. Although studying and managing disease in wild marine hosts is complex and challenging, studying and mitigating the effects of any disease to the Mediterranean monk seal is of utmost importance for conservation. The aim of this study was to document for the first time the presence of the hookworm Uncinaria hamiltoni in rehabilitated Mediterranean monk seal pups in Greece. A detailed examination protocol was followed for all pups that live-stranded over 30 years in 22 different locations, including physical, parasitological, and other examinations. Hookworms (adults and/or eggs) were detected in all the fecal samples, from all animals. Molecular identification using MtDNA (COI) and ribosomal DNA (D2/D3 28S and internal transcribed spacer [ITS] regions) identified the nematode species as Uncinaria hamiltoni. The clinical impacts and the benefits of anthelmintic treatment as a tool for the conservation management of the species are discussed.

Evaluation of Clinical and Histological Outcomes of Adipose-Derived Mesenchymal Stem Cells in a Rabbit Corneal Alkali Burn Model.

To assess effects of adipose-derived mesenchymal stem cells (AMSCs) in corneal alkali injuries in an experimental animal model. Twenty white New Zealand rabbits were included in the study. The animal models were randomly divided into 2 groups. Rabbits in the AMSC group (n = 10) received an intrastromal, a subconjunctival injection, and topical instillation of 0.5 ml totally of phosphate-buffered saline (PBS) containing 2 × 106 AMSCs. In the control group (n = 10), rabbits received only 0.5 ml of PBS using the same methods. A masked investigator measured the corneal sensation, anterior chamber Inflammation (ACI), and conjunctival congestion. Additionally, a blind histological and immunohistochemical evaluation was made. In the AMSC group, the central corneal sensation was increased whereas ACI and conjunctival congestion were reduced compared to the control group in the 28 days of follow-up (p < 0.05). A statistically significant difference (p < 0.05) was noted between the two groups as recorded in the above parameters. Histological analysis showed that pathological vascularization was markedly reduced in the AMSC group which was consistent with the absence of factor VIII in the immunohistochemistry sections. There is a trend towards improved clinical outcomes including corneal sensation as well as acceleration in the restoration of normal corneal architecture in corneal alkali burns treated with AMSCs, results that support the need for further research in the field.

Histological Effects of Intravitreal Injection of Antifungal Agents in New Zealand White Rabbits: An Electron Microscopic and Immunohistochemical Study.

Fungal endophthalmitis is a serious and vision-threatening infection which requires an immediate and effective treatment approach. Our research aims to elucidate the histological effects of the intravitreal injection of the maximum safe dosage of voriconazole and micafungin on retina. Six albino New Zealand White Rabbits were used. In experimental animals, a solution of voriconazole (Group V) or micafungin (Group M) was intravitreally injected in the right eye, while in control animals, balanced salt solution was intravitreally injected in the left eye (Group C). Euthanasia was performed ten days post injection and the retina was removed and prepared for histological examination with a light and electron microscope. Eosin-hematoxylin staining did not reveal any pathological changes in any of the samples examined. The immunohistochemical staining for Tumor Necrosis Factor alpha (TNF-a) marker was detected as negative in all samples, while Interleukin 6 (IL-6) marker was detected as mild only in the group injected with voriconazole. Electron microscopy revealed several ultrastructural alterations in retinal layers in both groups of experimental animals. Histological retinal lesions, revealed with electron microscopy in the present investigation, raises the question of the safe usage of these antifungal agents in the treatment of fungal intraocular infections in the future.

The Antiangiogenic Properties of Adipose-Derived Mesenchymal Stem/Stromal Cells in Corneal Neovascularization in a Rabbit Model.

The purpose was to study the anti-angiogenic effect of adipose-derived mesenchymal stem/stromal cells (ADMSCs) on experimentally induced corneal injuries. Corneal neovascularization (NV) was induced by incising and subsequently suturing the corneal surface in 32 New Zealand rabbits. Following suturing, the rabbits were randomly allocated into 2 groups, and received either phosphate-buffered saline (PBS) (control) or ADMSCs, both administered via three different routes. Digital images of the cornea were obtained two weeks post-incision to measure the area of neovascularized cornea. Tumor necrosis factor (TNF) was immunohistochemically assessed in the both groups. The corneal tissue was evaluated for vascular endothelial growth factor (VEGF). The extent of corneal NV in all eyes was assessed photographically by an independent observer. Fourteen days after the incisions, the degree of corneal NV was substantially decreased in the ADMSC-treated group (1.87 ± 0.9 mm2, 1.4 % ± 0.67 % of corneal surface) compared to the control and PBS-treated group (4.66 ± 1.74 mm2, 3.51 % ± 1.31 %, p < 0.001). ADMSCs significantly decreased injury-induced corneal NV in New Zealand rabbits two weeks post-treatment. This strategy has potential for use in the control of corneal NV in vivo.

Evidence of ingested plastics in stranded loggerhead sea turtles along the Greek coastline, East Mediterranean Sea.

Plastic debris has become a major threat to the marine environment and wildlife. Sea turtles are particularly vulnerable, and are known to ingest plastic debris globally; however, information from Greek waters is still absent. In this study, 36 stranded dead loggerhead turtles (Caretta caretta) were collected from the Greek coastline area, and their gastrointestinal content was analysed for ingested plastic debris. Twenty-six individuals (72%) were found to have ingested plastic, with an average of 7.94 ± 3.85 (SE) plastic items per turtle. In total, 286 plastic items were counted and categorised by size, shape, colour, and polymer type. Fourier Transform Infrared Spectrometry revealed that polypropylene and polyethylene were the dominant polymer plastic types found. Results indicated a variation in plastic ingestion amongst life stages of the loggerhead specimens. This study provides evidence of plastic ingestion by loggerhead turtles in Greek waters.

Self-Assembling Peptide Nanofiber Hydrogels for Controlled Ocular Delivery of Timolol Maleate.

The self-assembling peptides Ac-(RADA)4-CONH2 and Ac-(IEIK)3I-CONH2, which form hydrogels in physiological conditions, were evaluated as carriers for ocular delivery of the ß-blocker timolol maleate. Electron microscopy studies revealed that hydrogels contain nanofibers, whereas rheological studies showed that the Ac-(IEIK)3I-CONH2 self-assembles in a stiffer hydrogel compared with the Ac-(RADA)4-CONH2 peptide. The in vitro release and ex vivo permeation studies demonstrated controlled release and transport of the drug through the cornea, which depended on the self-assembling peptide sequence. In vivo studies in rabbits showed significant increase in the area under the concentration-time curve (AUC) after administration of the drug through the Ac-(RADA)4-CONH2 hydrogel compared to drug solution, whereas a sustained reduction of intraocular pressure for up to 24 h after instillation was achieved for both drug-loaded hydrogels. Histological studies revealed good ocular tolerability upon application of the formulations, suggesting that self-assembling peptide hydrogels are promising systems for sustained ocular drug delivery.

Intraocular localization of Onchocerca lupi adult worm in a dog with anterior uveitis: A case report.

OBJECTIVE: Canine ocular onchocercosis may represent an important ocular disease with zoonotic potential and widespread geographical distribution. An uncommon localization of Onchocerca lupi adult worm in the anterior chamber of the eye is described for the first time. ANIMAL STUDIED: A 4-year-old, intact, male, mixed-breed dog was admitted with profuse lacrimation and severe squinting. On the right eye, there were mucopurulent discharge, chemosis, and all typical signs of anterior uveitis which were attributed to the presence of a moving filarial worm within the anterior chamber. PROCEDURE: Removal of the parasite from the anterior chamber by a limbal incision revealed a mature adult male, 7.65-cm-long O. lupi worm. Periocular skin scrapings for the corresponding microfilariae were negative. Postoperatively, apart from systemic prednisolone and doxycycline hyclate and a topical antibiotic/steroid solution, parenteral melarsomine, followed by ivermectin, was given until the complete disappearance of the ocular lesions. CONCLUSIONS: Intraocular onchocercosis should be included in the differential diagnosis of canine anterior uveitis in endemic areas. This case highlights the atypical localization of O. lupi in the canine eye emphasizing on the possibility for a complete cure after the surgical removal of the parasite and the filaricidal medication subsequently applied.

Mesenchymal stem cells improve healing of the cornea after alkali injury.

PURPOSE: To evaluate the efficacy of mesenchymal stem cells (MSCs) to ameliorate the consequences of corneal alkali injuries. METHODS: Corneal alkali injuries were created in 30 rabbit eyes. The MSC group (n = 15) were treated with intrastromal and subconjunctival injections of phosphate-buffered saline (PBS) containing 2 × 10(6) MSCs and topical application. The control group (n = 15) was treated with PBS by the same applications forms. Drops of standard treatment (ascorbate 10 %, citrate 10 %, tobramycin, dexamethasone, Cyclogyl) were instilled for 2 weeks. Rabbits underwent slit-lamp examination, fluorescein staining, photography, and were evaluated for corneal neovascularization, opacification, and epithelial defects. Tear secretion and IOP were also evaluated. Furthermore, the concentration of Serumglutamic-pyruvic transaminase (SGPT) and vascular endothelial factor (VEGF) were measured. Immunohistochemistry was also performed for a-SMA and Ki-67. RESULTS: Eyes treated with MSCs showed better recovery. The mean neovascularized area was significantly smaller in the MSC group (p < 0.05). A significant difference in the degree of corneal opacification and re-epithelialization was also observed, as well as the IOP at 21 and 28 posttraumatic days (p < 0.05). Histology showed that MSCs resulted in almost normal architecture of eye tissues. After the MSCs infusion, SGPT and VEGF levels in cornea were significantly reduced. Immunohistochemistry demonstrated a reduction of a-SMA in the MSC group with higher mitotic-regenerative activity with the presence of Ki67. CONCLUSIONS: Our study represents a first step in understanding the possibilities of the MSC approach to treatment of alkali injuries of the cornea and shows that such an approach improves clinical outcomes and leads to better prognosis.

Onchocerciasis caused by Onchocerca lupi: an emerging zoonotic infection. Systematic review.

Globalization has increased circulation of people, their food, livestock and pets in the world, and changes in the environment, climate and human behaviour have led to the rapid expansion of emerging infections throughout the world. One of the reasons of a new pathogen affecting humans is the passage from an animal to a human being. Onchocerca (O.) lupi, a filarial worm first described in a wolf in 1967, is an emerging pathogen which has been incriminated as the etiological agent for 205 canine, 2 feline and 18 human infections in Europe, Tunisia, Turkey, Iran and the USA. Most frequent findings in animals and humans are monolateral or asymmetrical variably painful subconjunctival swellings and nodules containing immature or mature worms affecting the eye and/or adjacent tissues accompanied by conjunctival hyperemia. Occasionally, subcutaneous nodules and masses affecting the spinal cord have been observed in humans. Diagnosis of O. lupi is achieved by microscopy of excised adult female worms which exhibit a particular cuticular structure and molecular analysis. Treatment consists in worm removal accompanied by antihelminthic, antibiotic and anti-inflammatory therapy.

Anesthetic management of a 4-month-old red fox (Vulpes vulpes) for orthopedic surgery.

A 4-mo-old red fox (Vulpes vulpes) was found recumbent after a vehicular accident. Radiology revealed several limb fractures and the fox underwent surgery after 24 hr of initial stabilization. Premedication consisted of dexmedetomidine and morphine. Anesthesia was induced with ketamine and midazolam and maintained with isoflurane. Lidocaine, bupivacaine, and morphine were administered epidurally and further analgesia was provided with meloxicam. The heart rate and respiratory rate of the fox remained stable during surgery and, except for a mild hypothermia, the recovery from anesthesia was uneventful. The postoperative pain scores were low and the animal was transported to a rehabilitation facility and eventually released to the wild. The low pain scores postoperatively should be attributed to the successful application of epidural anesthesia and analgesia.

Estimation of normal tear production in free-living Eurasian black vultures (Aegypius monachus) and griffon vultures (Gyps fulvus) in Dadia National Park, Greece.

The aim of this study was to record the Schirmer tear test I (STT I) measurements in free-living vultures in order to estimate normal values. The Eurasian black vulture (Aegypius monachus), which breeds in the Mediterranean region and Asia, is listed as near threatened; it is also classified as vulnerable at the European level and endangered in Greece. The griffon vulture (Gyps fulvus), once widespread across the continent, has undergone a dramatic decline which has led to its extinction in many regions. Sixty-two animals were examined in total including 54 black vultures and 8 griffon vultures. The birds were classified into five age groups while four age groups were then combined into one large group: free-flying. STT I measurements and complete ophthalmic examinations were performed. Mean STT I value for black vultures was 10.9 +/- 3.3 mm/min (right eye, oculus dexter, OD) and 11.9 +/- 3.3 mm/min (left eye, oculus sinister, OS) and for griffon vultures was 6.4 +/- 1.8 mm/min OD and 6.5 +/- 1.8 mm/min OS. In both eyes, STT I values in black vultures were significantly higher than those recorded in griffon vultures. Intraspecific comparisons yielded a significant difference between eyes of black vultures but not between those of griffon vultures, with OS producing higher STT I readings than did OD. When STT I was compared between OD and OS for each age group separately, a statistically significant difference was detected in the immature and free-flying black vultures. In addition, black vulture hatchlings had a significantly higher tear production than did free-flying juveniles, immatures, subadults, and adults. STT I values in black vultures are similar to those reported in other Accipitriformes but are lower in griffon vultures. This difference is probably related to anatomic, evolutionary, and feeding factors and requires further investigation.