Rickettsia massiliae circulation in sheep and attached Rhipicephalus sanguineus in Central Portugal.

Rickettsiosis is considered an emerging/re-emerging vector-borne disease that causes significant public health threats. Ticks are reservoirs and vectors of Rickettsia having a significant role in the transmission of rickettsiae. In Portugal, little is known about tick-borne Rickettsia species in sheep. Therefore, this study aimed to investigate rickettsiae infection in ticks and their sheep host from 27 farms in four districts of central Portugal, to clarify the role of the sheep host in the circulation of this zoonotic agent. Between March and May 2021, EDTA blood samples (n = 100) of healthy grazing sheep and their ticks (n = 100, one tick per animal) were collected during a herd health program in central Portugal. Obtained ticks were identified as Rhipicephalus sanguineus sensu lato by PCR targeting a partial sequence of 16S rRNA gene followed by sequence analysis. Rhipicephalus sanguineus s.l. and host sheep blood were tested for the presence of Rickettsia spp. by PCR targeting a partial sequence of ompB and ompA genes. From a total of 100 paired R. sanguineus s.l. and host sheep, Rickettsia massiliae was detected in 62 ticks and 35 grazing sheep blood samples, collected in central Portugal, 2021. All 35 positive sheep had attached positive R. sanguineus s.l., with matching nucleotidic sequences. These findings suggest that sheep may develop rickettsiemia and are likely capable of transmitting and amplifying the infection to uninfected ticks maintaining rickettsiae in circulation in the domestic cycle.

Animal models in peripheral nerve transection studies: a systematic review on study design and outcomes assessment.

Aim: Peripheral nerve injury regeneration studies using animal models are crucial to different pre-clinical therapeutic approaches efficacy evaluation whatever the surgical technique explored. Materials & methods: A 944 articles systematic review on 'peripheral nerve injury in animal models' over the last 9 years was carried out. Results: It was found that 91% used rodents, and only 9% employed large animals. Different nerves are studied, with generated gaps (10,78 mm) and methods applied for regeneration evaluation uniformed. Sciatic nerve was the most used (88%), followed by median and facial nerves (2.6%), significantly different. Conclusion: There has not been a significant scale-up of the in vivo testing to large animal models (anatomically/physiologically closer to humans), allowing an improvement in translational medicine for clinical cases.

Hybrid scaffolds for bone tissue engineering: Integration of composites and bioactive hydrogels loaded with hDPSCs.

Bone tissue regeneration remains a significant challenge in clinical settings due to the complexity of replicating the mechanical and biological properties of bone environment. This study addresses this challenge by proposing a hybrid scaffold designed to enhance both bioactivity and physical stability for bone tissue regeneration. This research is the fisrt to develop a rigid 3D structure composed of polycaprolactone (PCL) and hydroxyapatite nanoparticles (nHA) integrated with a bioink containing human dental pulp stem/stromal cells (hDPSCs), alginate, nHA and collagen (Col). The biofabricated constructs were extensively characterized through cytocompatibility tests, osteogenic differentiation assessment, and biocompatibility evaluation in a rat model. In vitro results demontrated that the hybrid scaffolds presented significantly higher cell viability after 168 h compared to the control group. Furthermore, the hybrid scaffolds showed increased osteogenic differentiation relative to other groups. In vivo evaluation indicated good biocompatibility, characterized by minimal inflammatory response and successful tissue integration. These findings highlight the scaffold's potential to support bone tissue regeneration by combining the mechanical strength of PCL and nHA with the biological activity of the alginate-nHA-Col and hDPSCs bioink. The current study provides a promising foundation for the development of biomaterials aimed at improving clinical outcomes in bone repair and regeneration, particulary for the treatment of critical-size bone defects, targeted drug administration, and three-dimensional models for bone tissue engineering.

Advancements and Insights in Exosome-Based Therapies for Wound Healing: A Comprehensive Systematic Review (2018-June 2023).

Exosomes have shown promising potential as a therapeutic approach for wound healing. Nevertheless, the translation from experimental studies to commercially available treatments is still lacking. To assess the current state of research in this field, a systematic review was performed involving studies conducted and published over the past five years. A PubMed search was performed for English-language, full-text available papers published from 2018 to June 2023, focusing on exosomes derived from mammalian sources and their application in wound healing, particularly those involving in vivo assays. Out of 531 results, 148 papers were selected for analysis. The findings revealed that exosome-based treatments improve wound healing by increasing angiogenesis, reepithelization, collagen deposition, and decreasing scar formation. Furthermore, there was significant variability in terms of cell sources and types, biomaterials, and administration routes under investigation, indicating the need for further research in this field. Additionally, a comparative examination encompassing diverse cellular origins, types, administration pathways, or biomaterials is imperative. Furthermore, the predominance of rodent-based animal models raises concerns, as there have been limited advancements towards more complex in vivo models and scale-up assays. These constraints underscore the substantial efforts that remain necessary before attaining commercially viable and extensively applicable therapeutic approaches using exosomes.

Assessment of the circulation of Dirofilaria immitis in dogs from northern Portugal through combined analysis of antigens, DNA and parasite forms in blood.

Dirofilariasis is a vector-borne disease frequent in many countries. Not only infected dogs, but also cats and wild canids (including wolves and foxes), represent important sources of infection for mosquitoes, which are the pathogen vectors. The disease is endemic in Mediterranean countries with increasing incidence in Italy, France, Greece and Spain, but limited epidemiological data is available from Portugal regarding its distribution and impact. Aiming to clarify this, canine whole blood samples (n = 244) from the north of Portugal were tested for Dirofilaria spp. antigens by use of a commercial rapid immunomigration test. Polymerase chain reaction (PCR) and the modified Knott test were also used to assess the presence of microfilariae. Results were also compared to assess the performance of each test used. Of the 244 animals tested, 118 (48.4%) were positive for Dirofilaria immitis (heartworm) in the serological adult worm rapid antigen detection test, and 36 (14.8%) had circulating microfilariae, identified as D. immitis. A combined positivity of 51.6% (126/244) was found. Results indicate that the risk of exposure to D. immitis in dogs is high in this region of Portugal, and that prophylaxis against the parasite is advisable to decrease the occurrence of canine infection and disease. The present study highlights the diagnostic value of serological and molecular tests in determining the prevalence of D. immitis.

Ultrasound Landmarks in the Approach to the Common Peroneal Nerve in a Sheep Model-Application in Peripheral Nerve Regeneration.

Peripheral nerve injury (PNI) remains a medical challenge with no easy resolution. Over the last few decades, significant advances have been achieved in promoting peripheral nerve regeneration, and new assessment tools have been developed, both functional and imaging, to quantify the proportion and quality of nerve recovery. The exploration of new animal models, larger, more complex, and with more similarities to humans, has made it possible to reduce the gap between the results obtained in classic animal models, such as rodents, and the application of new therapies in humans and animals of clinical interest. Ultrasonography is an imaging technique recurrently used in clinical practice to assess the peripheral nerves, allowing for its anatomical and topographic characterization, aiding in the administration of anesthesia, and in the performance of nerve blocks. The use of this technique in animal models is scarce, but it could be a useful tool in monitoring the progression of nerve regeneration after the induction of controlled experimental lesions. In this work, sheep, a promising animal model in the area of peripheral nerve regeneration, were subjected to an ultrasonographic study of three peripheral nerves of the hind limb, the common peroneal, and tibial and sciatic nerves. The main aim was to establish values of dimensions and ultrasound appearance in healthy nerves and landmarks for their identification, as well as to perform an ultrasound evaluation of the cranial tibial muscle, an effector muscle of the common peroneal nerve, and to establish normal values for its ultrasound appearance and dimensions. The results obtained will allow the use of these data as control values in future work exploring new therapeutic options for nerve regeneration after induction of common peroneal nerve injuries in sheep.

Intensive neurorehabilitation and allogeneic stem cells transplantation in canine degenerative myelopathy.

Introduction: Degenerative myelopathy (DM) is a neurodegenerative spinal cord disease with upper motor neurons, with progressive and chronic clinical signs, similar to amyotrophic lateral sclerosis (ALS). DM has a complex etiology mainly associated with SOD1 gene mutation and its toxic role, with no specific treatment. Daily intensive rehabilitation showed survival time near 8 months but most animals are euthanized 6-12 months after clinical signs onset. Methods: This prospective controlled blinded cohort clinical study aims to evaluate the neural regeneration response ability of DM dogs subjected to an intensive neurorehabilitation protocol with mesenchymal stem cells (MSCs) transplantation. In total, 13 non-ambulatory (OFS 6 or 8) dogs with homozygous genotype DM/DM and diagnosed by exclusion were included. All were allocated to the intensive neurorehabilitation with MSCs protocol (INSCP) group (n = 8) or to the ambulatory rehabilitation protocol (ARP) group (n = 5), which differ in regard to training intensity, modalities frequency, and MSCs transplantation. The INSCP group was hospitalized for 1 month (T0 to T1), followed by MSCs transplantation (T1) and a second month (T2), whereas the ARP group was under ambulatory treatment for the same 2 months. Results: Survival mean time of total population was 375 days, with 438 days for the INSCP group and 274 for the ARP group, with a marked difference on the Kaplan-Meier survival analysis. When comparing the literature's results, there was also a clear difference in the one-sample t-test (p = 0.013) with an increase in time of approximately 70%. OFS classifications between groups at each time point were significantly different (p = 0.008) by the one-way ANOVA and the independent sample t-test. Discussion: This INSCP showed to be safe, feasible, and a possibility for a long progression of DM dogs with quality of life and functional improvement. This study should be continued.

Dirofilaria immitis antigenemia and microfilaremia in Iberian wolves and red foxes from Portugal.

BACKGROUND: Dirofilaria immitis is a parasitic nematode endemic in the Mediterranean countries, which causes cardiopulmonary dirofilariosis in wild and domestic animals. Despite being recognized hosts of D. immitis, wild carnivores such as wolves and foxes are frequently disregarded when considering a potential role in the transmission of these zoonotic nematodes. In Portugal, studies available regarding D. immitis circulation are scarce, likely underestimating its relevance. To add knowledge on this, we sought to assess Iberian wolves (Canis lupus signatus) and red foxes (Vulpes vulpes) from northern Portugal for D. immitis antigenemia and microfilaremia. METHODS: Blood samples from 42 Iberian wolves and 19 red foxes were collected, during 2010-2012, in Peneda-Gerês National Park. Antigenemia was searched for by rapid antigen detection test kits (Uranotest Dirofilaria ®). Microfilaremia was assessed by polymerase chain reaction (PCR). Nucleic acids were extracted from blood using QIAamp® DNA Mini Kit (Qiagen), and DNA was screened for the presence of microfilaria using a conventional PCR targeting the 5.8S-internal transcribed spacer 2-28S regions, followed by bidirectional sequencing, Basic Local Alignment Search Tool analysis and phylogenetic analysis. RESULTS: Three red foxes had antigenemia, with an occurrence of 15.8% (95% confidence interval [CI] 3.4-39.6), while showing no evidence for the presence of microfilaremia. No wolf samples presented evidence for D. immitis antigenemia. Nevertheless, two wolves were positive for D. immitis microfilaremia (4.8%; 95% CI 0.6-16.2%) as revealed by PCR and confirmed by bidirectional sequencing. CONCLUSIONS: Although Dirofilaria microfilaremia in wolves does not necessarily correlate to an endangerment of the infected animal's health, positive individuals can act as a reservoir for further infection if the intermediate mosquito hosts are present. To the best of our knowledge, one single study had reported that wolves were suitable Dirofilaria hosts, but microfilaremia have never been reported.

Epidemiology of Porcine Circovirus Type 2 Circulating in Wild Boars of Portugal during the 2018-2020 Hunting Seasons Suggests the Emergence of Genotype 2d.

Porcine circovirus type 2 (PCV-2) is associated with several syndromes affecting swine, also known as porcine-circovirus-associated diseases, of which post-weaning multi-systemic wasting syndrome stands out due to its high economic impact on swine production. Recent data suggest the increasing circulation of the PCV-2d genotype in several countries worldwide. To provide updated data on PCV-2 genotypes currently circulating in swine in Portugal, we screened wild boar stools collected from several districts across Portugal, during the 2018-2020 hunting seasons, for PCV-2 and genetically characterized detected strains. From a total of 76 stool samples of wild boar tested by PCR for the partial PCV-2 ORF2 gene, two sequences were obtained (2/76; 2.6%, 95% confidence interval: 0.032-9.18). Bidirectional sequencing showed that the sequences were 100% identical and both of the PCV-2d genotype, showing for the first time the presence of this genotype in Portugal. Monitoring wild PCV-2 reservoirs is important for both veterinary public health and economic reasons, since PCV-2 infection has a strong economic impact on the swine industry.

First report of human Thelazia callipaeda infection in Portugal.

The zoonotic parasitic nematode Thelazia callipaeda, also known as the oriental eye worm, is endemic in several European countries, including Portugal. Infections may result in ocular disease in domestic and wild animals as well as humans, with more or less severe manifestations. We report the first human case of ocular thelaziosis by T. callipaeda in Portugal, a country where the parasite had already been found to infect dogs, cats, red foxes, wild rabbits and a beech marten. An 80-year-old patient from east-central Portugal, who had been suffering from tearing for a few years, had whitish filiform fragments removed from the left eye. Polymerase chain reaction of partial cytochrome c oxidase subunit 1 and 18S small subunit rRNA genes followed by bidirectional sequencing and BLAST analysis confirmed T. callipaeda haplotype 1, the only haplotype previously reported in Europe. The endemicity of T. callipaeda in domestic and wild animals in east-central Portugal makes it very likely that infection of the human patient had occurred locally. In east-central and other geographical areas of Portugal, veterinarians and physicians, especially ophthalmologists, should regard T. callipaeda as a cause of ocular pathology in animals and humans.