Chagas Disease: A Silent Threat for Dogs and Humans.

Chagas disease (CD) is a vector-borne Neglected Zoonotic Disease (NZD) caused by a flagellate protozoan, Trypanosoma cruzi, that affects various mammalian species across America, including humans and domestic animals. However, due to an increase in population movements and new routes of transmission, T. cruzi infection is presently considered a worldwide health concern, no longer restricted to endemic countries. Dogs play a major role in the domestic cycle by acting very efficiently as reservoirs and allowing the perpetuation of parasite transmission in endemic areas. Despite the significant progress made in recent years, still there is no vaccine against human and animal disease, there are few drugs available for the treatment of human CD, and there is no standard protocol for the treatment of canine CD. In this review, we highlight human and canine Chagas Disease in its different dimensions and interconnections. Dogs, which are considered to be the most important peridomestic reservoir and sentinel for the transmission of T. cruzi infection in a community, develop CD that is clinically similar to human CD. Therefore, an integrative approach, based on the One Health concept, bringing together the advances in genomics, immunology, and epidemiology can lead to the effective development of vaccines, new treatments, and innovative control strategies to tackle CD.

Exploiting Leishmania-Primed Dendritic Cells as Potential Immunomodulators of Canine Immune Response.

Dendritic cells (DCs) capture pathogens and process antigens, playing a crucial role in activating naïve T cells, bridging the gap between innate and acquired immunity. However, little is known about DC activation when facing Leishmania parasites. Thus, this study investigates in vitro activity of canine peripheral blood-derived DCs (moDCs) exposed to L. infantum and L. amazonensis parasites and their extracellular vesicles (EVs). L. infantum increased toll-like receptor 4 gene expression in synergy with nuclear factor κB activation and the generation of pro-inflammatory cytokines. This parasite also induced the expression of class II molecules of major histocompatibility complex (MHC) and upregulated co-stimulatory molecule CD86, which, together with the release of chemokine CXCL16, can attract and help in T lymphocyte activation. In contrast, L. amazonensis induced moDCs to generate a mix of pro- and anti-inflammatory cytokines, indicating that this parasite can establish a different immune relationship with DCs. EVs promoted moDCs to express class I MHC associated with the upregulation of co-stimulatory molecules and the release of CXCL16, suggesting that EVs can modulate moDCs to attract cytotoxic CD8+ T cells. Thus, these parasites and their EVs can shape DC activation. A detailed understanding of DC activation may open new avenues for the development of advanced leishmaniasis control strategies.

Detection and molecular characterization of Cryptosporidium spp. in dairy calves in Lisbon and Tagus Valley, Portugal.

Cryptosporidium is a protozoan parasite with worldwide distribution, infecting a wide range of hosts with some zoonotic species. Calves have been identified as one of the most common reservoirs of this parasite. However, little is known about the genetics of Cryptosporidium in calves in Portugal. This study aimed to molecularly characterize infections of Cryptosporidium in pre-weaned calves from the Lisbon and Tagus Valley (LTV) in Portugal. Fifty-two samples were collected from calves from eight dairy and two beef farms in LTV, Portugal. Cryptosporidium oocysts were detected by Modified Ziehl-Neelsen staining (MZN) and direct immunofluorescent assay (DFA). MZN and DFA revealed the presence of Cryptosporidium oocysts in 40.4% (21/52) and 67.3% (35/52) samples, respectively. Positive samples were analyzed by PCR-RFLP of the 18 s rRNA gene for species identification. DNA amplification of the 18S rRNA gene was successful for 88.6% (31/35) of samples. Cryptosporidium parvum was identified in 96.8% (30/31) of the samples, and from one sample Cryptosporidium bovis was identified. Cryptosporidium parvum positive samples were subtyped by sequencing the PCR product of a partial fragment of the 60 kDa glycoprotein (gp60) gene. Subtype analysis of the C. parvum isolates revealed that all isolates belonged to subtype family IIa. Four subtypes were recognized within this subtype family, including the hyper-transmissible IIaA15G2R1 subtype that is the most frequently reported worldwide (27/30), IIaA14G2R1 (1/30), IIaA16G2R1 (1/30) and IIaA19G2R1 (1/30). To our knowledge, this is the first report of C. bovis, and C. parvum subtypes IIaA14G2R1 and IIaA19G2R1 in cattle in LTV, Portugal. The presence of the zoonotic C. parvum subtype in this study suggests that pre-weaned calves are likely to be a significant reservoir of zoonotic C. parvum, highlighting the importance of animal-to-human infection transmission risk. Further molecular studies are required to better understand the epidemiology of cryptosporidiosis in Portugal.

Competitive adsorption of acetaminophen and caffeine onto activated Tingui biochar: characterization, modeling, and mechanisms.

Tingui biochar (TB) activated with potassium hydroxide (TB-KOH) was synthesized in the present study. The adsorption capacity of TB-KOH was evaluated for the removal of acetaminophen and caffeine in monocomponent and bicomponent solutions. As a result, the study of the TB-KOH characterization as well as the adsorption kinetics, isotherm, thermodynamics, and a suggestion of the global adsorption mechanism are presented. TB-KOH was characterized through physical-chemical analysis to understand its surface morphology and how it contributes to the adsorption of these drugs. Furthermore, modelling using advanced statistical physical models was performed to describe how acetaminophen and caffeine molecules are adsorbed in the active sites of TB-KOH. Through the characterizations, it was observed that the activation with KOH contributed to the development of porosity and functional groups (-OH, C-O, and C = O) on the surface of TB. The monocomponent adsorption equilibrium was reached in 90 min with a maximum adsorption capacity of 424.7 and 350.8 mg g-1 for acetaminophen and caffeine, respectively. For the bicomponent solution adsorption, the maximum adsorption capacity was 199.4 and 297.5 mg g-1 for acetaminophen and caffeine, respectively. The isotherm data was best fitted to the Sips model, and the thermodynamic study indicated that acetaminophen removal was endothermic, while caffeine removal was exothermic. The mechanism of adsorption of acetaminophen and caffeine by TB-KOH was described by the involvement of hydrogen bonds and π-π interactions between the surface of TB-KOH and the molecules of the contaminants.

Predicting the distribution of Ixodes ricinus and Dermacentor reticulatus in Europe: a comparison of climate niche modelling approaches.

BACKGROUND: The ticks Ixodes ricinus and Dermacentor reticulatus are two of the most important vectors in Europe. Climate niche modelling has been used in many studies to attempt to explain their distribution and to predict changes under a range of climate change scenarios. The aim of this study was to assess the ability of different climate niche modelling approaches to explain the known distribution of I. ricinus and D. reticulatus in Europe. METHODS: A series of climate niche models, using different combinations of input data, were constructed and assessed. Species occurrence records obtained from systematic literature searches and Global Biodiversity Information Facility data were thinned to different degrees to remove sampling spatial bias. Four sources of climate data were used: bioclimatic variables, WorldClim, TerraClimate and MODIS satellite-derived data. Eight different model training extents were examined and three modelling frameworks were used: maximum entropy, generalised additive models and random forest models. The results were validated through internal cross-validation, comparison with an external independent dataset and expert opinion. RESULTS: The performance metrics and predictive ability of the different modelling approaches varied significantly within and between each species. Different combinations were better able to define the distribution of each of the two species. However, no single approach was considered fully able to capture the known distribution of the species. When considering the mean of the performance metrics of internal and external validation, 24 models for I. ricinus and 11 models for D. reticulatus of the 96 constructed were considered adequate according to the following criteria: area under the receiver-operating characteristic curve > 0.7; true skill statistic > 0.4; Miller's calibration slope 0.25 above or below 1; Boyce index > 0.9; omission rate < 0.15. CONCLUSIONS: This comprehensive analysis suggests that there is no single 'best practice' climate modelling approach to account for the distribution of these tick species. This has important implications for attempts to predict climate-mediated impacts on future tick distribution. It is suggested here that climate variables alone are not sufficient; habitat type, host availability and anthropogenic impacts, not included in current modelling approaches, could contribute to determining tick presence or absence at the local or regional scale.

The potential role of scavenging flies as mechanical vectors of Lagovirus europaeus/GI.2.

The European rabbit (Oryctolagus cuniculus) populations of the Iberian Peninsula have been severely affected by the emergence of the rabbit haemorrhagic disease virus (RHDV) Lagovirus europaeus/GI.2 (RHDV2/b). Bushflies and blowflies (Muscidae and Calliphoridae families, respectively) are important RHDV vectors in Oceania, but their epidemiological role is unknown in the native range of the European rabbit. In this study, scavenging flies were collected between June 2018 and February 2019 in baited traps at one site in southern Portugal, alongside a longitudinal capture-mark-recapture study of a wild European rabbit population, aiming to provide evidence of mechanical transmission of GI.2 by flies. Fly abundance, particularly from Calliphoridae and Muscidae families, peaked in October 2018 and in February 2019. By employing molecular tools, we were able to detect the presence of GI.2 in flies belonging to the families Calliphoridae, Muscidae, Fanniidae and Drosophilidae. The positive samples were detected during an RHD outbreak and absent in samples collected when no evidence of viral circulation in the local rabbit population was found. We were able to sequence a short viral genomic fragment, confirming its identity as RHDV GI.2. The results suggest that scavenging flies may act as mechanical vectors of GI.2 in the native range of the southwestern Iberian subspecies O. cuniculus algirus. Future studies should better assess their potential in the epidemiology of RHD and as a tool for monitoring viral circulation in the field.

Insights on Host-Parasite Immunomodulation Mediated by Extracellular Vesicles of Cutaneous Leishmania shawi and Leishmania guyanensis.

Leishmaniasis is a parasitic disease caused by different species of Leishmania and transmitted through the bite of sand flies vector. Macrophages (MΦ), the target cells of Leishmania parasites, are phagocytes that play a crucial role in the innate immune microbial defense and are antigen-presenting cells driving the activation of the acquired immune response. Exploring parasite-host communication may be key in restraining parasite dissemination in the host. Extracellular vesicles (EVs) constitute a group of heterogenous cell-derived membranous structures, naturally produced by all cells and with immunomodulatory potential over target cells. This study examined the immunogenic potential of EVs shed by L. shawi and L. guyanensis in MΦ activation by analyzing the dynamics of major histocompatibility complex (MHC), innate immune receptors, and cytokine generation. L. shawi and L. guyanensis EVs were incorporated by MΦ and modulated innate immune receptors, indicating that EVs cargo can be recognized by MΦ sensors. Moreover, EVs induced MΦ to generate a mix of pro- and anti-inflammatory cytokines and favored the expression of MHCI molecules, suggesting that EVs antigens can be present to T cells, activating the acquired immune response of the host. Since nano-sized vesicles can be used as vehicles of immune mediators or immunomodulatory drugs, parasitic EVs can be exploited by bioengineering approaches for the development of efficient prophylactic or therapeutic tools for leishmaniasis.

Morphological and Molecular Identification of Physaloptera alata (Nematoda: Spirurida) in a Booted Eagle (Aquila pennata) from Portugal.

Physaloptera spp. are parasitic nematodes that infect the gastrointestinal tracts of many carnivores and omnivores. Although they are distributed worldwide, Physaloptera spp. have not been studied in raptors in Portugal. In this study, we report Physaloptera alata in a booted eagle (Aquila pennata) in Portugal. Adult nematodes were discovered in the gizzard of a young booted eagle, and morphological features were consistent with those of the genus Physaloptera. DNA was extracted and a PCR assay performed to amplify a region of the 18S small subunit of the ribosomal RNA gene and the cytochrome c oxidase subunit I gene. The resulting PCR products were Sanger-sequenced, and comparison with the available sequences in the GenBank database confirmed the initial morphological classification as Physaloptera sp. Phylogenetic analysis clustered the sequence within the Physaloptera group. The presence of this parasite in raptors from Portugal is of particular importance to wildlife rehabilitation centers, disease ecologists, and wildlife professionals. Furthermore, we produced a new genetic sequence and have added it to the GenBank database of parasites in birds of prey.

Size does matter: intraspecific geometric morphometric analysis of wings of the blowfly Chrysomya albiceps (Diptera: Calliphoridae).

Blowflies have forensic, sanitary and veterinary importance, as well as being pollinators, parasitoids and ecological bioindicators. There is still little work with real data and from experiments assessing the relationship between blowflies' morphologic features and environmental and demographic factors. The present work tests whether the variation, in the shape and size, of Chrysomya albiceps (Wiedemann, 1819) wings is influenced by the following factors: 1) time; 2) temperature; 3) sex and; 4) different types of carcasses (pig, dog/cat and whale). Male and female wings from four different sites collected in six different years were used to obtain wing size and shape of C. albiceps. Analyses between wing shape and the variables tested had low explanatory power, even though they had statistical support. However, it was possible to identify differences in wing shape between males and females, with good returns in sex identification. The comparison between wing size and the variables tested showed that wing size has a negative relationship with temperature, significant differences between sexes, slight variation over time and no influence by carcass types. Furthermore, wing size influenced wing shape. Understanding population-specific characteristics of C. albiceps provide important insights about how the species reacts under specific conditions.

Dogs with leishmaniosis: how are we managing proteinuria in daily practice? A Portuguese questionnaire-based study.

BACKGROUND: Proteinuria is a common finding in dogs with leishmaniosis. Although antileishmanial therapeutic protocols are widely implemented, little information is available on which treatments are most adequate for identifying proteinuria in patients with canine leishmaniosis (CanL), especially regarding the use of immunosuppressants. The aim of this study was to explore the current paradigm regarding the antiproteinuric approach adopted by veterinary practitioners in Portugal to treat dogs with CanL. METHODS: A questionnaire-based study was developed using Google Forms®. The questionnaire presented a number of different hypothetical scenarios of CanL, and the topics surveyed included the general features of the respondents and the protocols preferred by these respondents to manage proteinuria in the presented scenarios, including choice of therapeutic drugs, appropriate diet and use of immunosuppressants, in dogs with immune-mediated glomerulonephritis. The questionnaire was internally prevalidated and diffused online over a 2-month period through Portuguese veterinary social networking groups, and data were collected for descriptive analysis. RESULTS: A total of 86 veterinary practitioners responded to the survey. When exposed to theoretical scenarios of proteinuria in dogs with CanL at stages IIb, III and IV (LeishVet guidelines), 16.3%, 62.8% and 93.8% of the respondents, respectively, answered that they would treat it. The dog was started on a renal diet as therapy by 28.6%, 83.3% and 97.4% of respondents, respectively. Angiotensin-converting enzyme inhibitors (ACEI) were prescribed by 100%, 85.2% and 78.9% of respondents as first-choice drugs for CanL at stages IIb, III and IV, respectively, with ACEI used in monotherapy by 64.3%, 40.7% and 46.1%. In comparison, protocols using ACEI in combination with other compounds were chosen by 7.1%, 33.3% and 39.5% of respondents, and combination therapy which did not include ACEI was the choice of 0.0%, 12.9% and 14.5%. Regarding immunosuppressants, 44.2% of the respondents answered they would prescribe them, with 97.4% electing for prednisolone and 5.3% choosing mycophenolate mofetil. CONCLUSIONS: Among the veterinary practitioners who responded, proteinuria treatment was considered since stage IIb CanL, although implementation of a therapeutic approach was more evident in advanced CanL stages. ACEI were the first-choice drugs, particularly for the treatment of stage IIb CanL; in advanced stages, a combination of antiproteinuric drugs was more often used. Immunosuppressant use was controversial, although when applied, prednisolone was the preferred choice. These findings reinforce the small body of evidence that supports the use of such drugs and the need to further explore their role in CanL.

Parasite island syndromes in the context of nidicolous ectoparasites: Fleas (Insecta: Siphonaptera) in wild passerine birds from Azores Archipelago.

Island syndrome, previously established for isolation process of insular vertebrates' populations, have been adapted to insular parasites communities, termed parasite island syndromes. In this work, were studied for the first time the insular syndromes for nidicolous ectoparasites of the bird species, Turdus merula, Sylvia atricapilla, Fringilla coelebs and Erithacus rubecula from Azores and the mainland Portugal. Flea species were only recorded on Azorean birds, namely Dasypsyllus gallinulae and Ctenocephalides felis felis, known as not host-specific parasites. In the absence of shared flea species between mainland and islands birds, a comparison among our fleas prevalence to Azores Islands and mainland fleas prevalence, recorded to others European studies, showed that Azorean host populations undergo higher prevalence than the mainland one. This result was consistent with parasite island syndromes predictions recorded to ectoparasites, hippoboscid flies and chewing lice, that fleas have higher prevalence on the Azores Islands compared to mainland Portugal. However, our results provide a new perspective to parasite island syndromes assumptions, namely in the context of nidicolous ectoparasites that spend only brief periods on the hosts' body.

Zoonotic Visceral Leishmaniasis: New Insights on Innate Immune Response by Blood Macrophages and Liver Kupffer Cells to Leishmania infantum Parasites.

L. infantum is the aetiological agent of zoonotic visceral leishmaniasis (ZVL), a disease that affects humans and dogs. Leishmania parasites are well adapted to aggressive conditions inside the phagolysosome and can control the immune activation of macrophages (MØs). Although MØs are highly active phagocytic cells with the capacity to destroy pathogens, they additionally comprise the host cells for Leishmania infection, replication, and stable establishment in the mammal host. The present study compares, for the first time, the innate immune response to L. infantum infection of two different macrophage lineages: the blood macrophages and the liver macrophages (Kupffer cells, KC). Our findings showed that L. infantum takes advantage of the natural predisposition of blood-MØs to phagocyte pathogens. However, parasites rapidly subvert the mechanisms of MØs immune activation. On the other hand, KCs, which are primed for immune tolerance, are not extensively activated and can overcome the dormancy induced by the parasite, exhibiting a selection of immune mechanisms, such as extracellular trap formation. Altogether, KCs reveal a different pattern of response in contrast with blood-MØs when confronting L. infantum parasites. In addition, KCs response appears to be more efficient in managing parasite infection, thus contributing to the ability of the liver to naturally restrain Leishmania dissemination.

Diagnosis and clinical management of canine leishmaniosis by general veterinary practitioners: a questionnaire-based survey in Portugal.

BACKGROUND: Canine leishmaniosis (CanL) can be appropriately managed following international recommendations. However, few studies have assessed the preferred protocols in real-life veterinary practice and whether these are in line with the guidelines. This survey aimed to investigate the current trends in the clinical management of CanL among veterinary practitioners in Portugal, taking into consideration different scenarios of infection/disease and the awareness of and application by veterinary practitioners of the current guidelines. METHODS: A questionnaire-based survey was conducted online using an electronic platform. The following topics were surveyed: (i) general characteristics of the responding veterinarian; (ii) the preferred protocols used for the diagnosis, treatment and prevention of CanL, considering different theoretical scenarios of infection/disease; and (iii) the responding veterinarian's current knowledge and application of the existing guidelines on CanL. After internal validation, the survey was distributed online, for 2 months, via Portuguese social network veterinary groups. Data were collected for descriptive analysis. RESULTS: Eighty-six replies were obtained. Analysis of the results showed that the preferred diagnostic techniques varied widely according to the theoretical scenario of infection/disease. In general daily practice, serology testing (enzyme-linked immunosorbent assay [ELISA]) was the most used tool (67.4%). The preferred matrices used for PCR test were lymph nodes (62.3%) and/or bone marrow (59.0%). Regarding treatment, for subclinical infection/stage I CanL, 51.2% of the respondents did not prescribe any medical treatment, but 98.8% proceeded with both monitoring and preventive measures. Among those who prescribed a treatment (n = 42), most chose domperidone (47.6%). For the treament of stages IIa, IIb and III CanL, allopurinol/meglumine antimoniate (MA) was chosen by 69.8, 73.3 and 51.2% of respondents, respectively, followed by allopurinol/miltefosine (20.9, 19.8 and 38.4%, respectively). In contrast, dogs with stage IV CanL were mostly treated with allopurinol/miltefosine (48.8%) rather than with allopurinol/MA (23.3%). The use of repellents was the preferred preventive strategy (98.8%). About 93.0% of responders were aware of the existence of guidelines, and most of these veterinarians consulted the guidelines of the LeishVet group and the Canine Leishmaniosis Working Group; however, 31.3% reported that they did not follow any specific recommendations. CONCLUSIONS: Of the veterinarians responding to the survey, most reported following international guidelines for the clinical management of CanL. While allopurinol/MA was the preferred therapeutic protocol for the treatment of stages II/III CanL, allopurinol/miltefosine was the first choice for the treatment of stage IV CanL, possibly due to the unpredictable effect of MA on renal function. This study contributes to a better understanding of the trends in practical approaches to the treatment of CanL in Portugal.

Molecular survey of vector-borne diseases in two groups of domestic dogs from Lisbon, Portugal.

BACKGROUND: Canine vector-borne diseases (CVBDs) are caused by a wide range of pathogens transmitted by arthropods. They have been an issue of growing importance in recent years; however, there is limited information about the vector-borne pathogens circulating in Portugal. The aim of the present study was to detect canine vector-borne bacteria and protozoa of veterinary and zoonotic importance using molecular methods. METHODS: One hundred and forty-two dogs from Lisbon, southern Portugal, were tested: 48 dogs from a veterinary hospital clinically suspected of vector-borne diseases and 94 apparently healthy dogs from shelters. Anaplasma spp./Ehrlichia spp., Babesia/Theileria spp., Hepatozoon spp., and Mycoplasma spp. infections were detected by PCR from blood samples and examined under light microscopy. Other information including clinical status and diagnostic test results were collected for each animal. RESULTS: Infections were detected by PCR in 48 (33.80%) dogs. Single infections were found in 35 dogs (24.64%), and co-infections were found in 13 (9.15%) dogs. Twenty-nine (20.42%) dogs were positive for Hepatozoon spp., 15 (10.56%) for Mycoplasma spp., 11 (7.75%) for Anaplasma spp./Ehrlichia spp., and six (4.21%) for Babesia spp. DNA sequencing was used to identify Babesia vogeli (2.81%), Babesia canis (1.40%), Hepatozoon canis (20.42%), Mycoplasma haematoparvum (2.11%), Mycoplasma haemocanis (8.45%), Anaplasma platys (7.04%), and Ehrlichia canis (0.70%). CONCLUSIONS: This is the first molecular identification of B. canis and M. haematoparvum in dogs from southern Portugal. This study highlights the importance of molecular methods to identify CVBD pathogens in endemic areas and helps to guide the clinical approach of veterinarians in practice.

Presence of one ecto- and two endoparasite species of the black stork (Ciconia nigra) in Portugal.

BACKGROUND: The black stork (Ciconia nigra Linnaeus, 1758) is a recognized endangered species in Europe and most of the specimens from the Western Palearctic region breed in the Iberian Peninsula. Available works regarding parasites in black storks are scarce. This work reports the presence one ecto- and two endoparasite species from a black stork in Portugal. CASE PRESENTATION: A black stork was found in southern Portugal after colliding against electric cables. The specimen did not survive its sustained injuries and a post-mortem exam was performed. During the procedure, several ecto- and endoparasite specimens were found. The collected parasites were lice (Neophilopterus tricolor), nematodes (Desportesius sagittatus) and trematodes (Cathaemasia hians). CONCLUSIONS: Three different species of parasites are reported from a black stork in Portugal. Ecto- and endoparasites of C. nigra have not frequently been described in the literature, and this case report is a contribution to the field. Additional studies will be important to better understand the impact that parasites can have on C. nigra health and survival.

Louse flies in Azorean and mainland populations of four Passeriformes species: A new perspective to parasite Island syndromes.

Hippoboscid flies, also known as louse flies, are obligate blood-feeders ectoparasites of birds and mammals. By studying louse fly parasites of four Passeriformes species, Eurasian blackbird (Turdus merula), Eurasian blackcap (Sylvia atricapilla), common chaffinch (Fringilla coelebs) and European robin (Erithacus rubecula), with dissimilar time of colonization of Azores islands, we tested whether: (i) island host populations have lower parasite richness than the mainland one; (ii) island host populations undergo higher parasite prevalence, mean intensities and mean abundance than the mainland one; (iii) island parasite diversity are composed exclusively by specific parasites and (iv) parasite richness is positively correlated with the island area and proximity to the continent. For these purposes, 775 birds were sampled for presence of louse flies, by modified fumigation chamber method, from Azores Islands (São Miguel, Terceira and Flores) and Portugal mainland. Insular and mainland parasite assemblages were statistically compared. We record for the first time to Azores, Ornithomya fringillina and Icosta minor from Portugal mainland. Louse flies had highest prevalence and abundance from Azores Islands compared to those observed in mainland birds, especially blackbirds. The insular parasite diversity of Azores blackbirds, blackcaps and chaffinches was richer than the one observed in mainland population. None of the hippoboscid flies observed on the islands and mainland were host-specific. Thus, our findings provide an upgrade of parasite island syndromes knowledge, in the context of the ectoparasites, namely to the hippoboscid flies case.

First report of Ericotrombidium ibericense in domestic dogs.

PURPOSE: Larval stages of trombiculid mites infest a wide variety of wild and domestic animals. The most common clinical signs related with the presence of these parasites are alopecia, crusts, erythema, excoriation, erosion, papules, pustules and vesicles. Most of trombiculid infestations may not be perceived by the clinician due to their small size. Although Ericotrombidium ibericense has been recorded on cats, it has not been found on dogs. METHODS: In August 2019, three domestic dogs presenting pruritic dermatological lesions in the ventral area of the body and interdigital spaces were presented at a veterinary clinic in Santarém, central Portugal. Trombiculid mites were extracted from the skin and preserved in 70% ethanol. Specimens were prepared in slides with Hoyer's medium and observed with optical microscopy. RESULTS: After morphological examination of the specimens, mites were identified as E. ibericense (Acariformes: Trombiculidae). CONCLUSIONS: Most of the trombiculids recorded in European clinical practice are generally identified as Neotrombicula autumnalis by default, since, in most cases, mites are not examined morphologically. This is the first record of E. ibericense in domestic dogs. More studies are needed to evaluate the distribution of these mites in Portugal. Veterinary clinicians must be aware of this parasitosis, as trombiculids can cause exuberant clinical signs, but are often misdiagnosed.

3D-Hepatocyte Culture Applied to Parasitology: Immune Activation of Canine Hepatic Spheroids Exposed to Leishmania infantum.

The application of innovative three-dimensional (3D) spheroids cell culture strategy to Parasitology offers the opportunity to closely explore host-parasite interactions. Here we present a first report on the application of 3D hepatic spheroids to unravel the immune response of canine hepatocytes exposed to Leishmania infantum. The liver, usually considered a major metabolic organ, also performs several important immunological functions and constitutes a target organ for L. infantum infection, the etiological agent of canine leishmaniasis (CanL), and a parasitic disease of major veterinary and public health concern. 3D hepatic spheroids were able to sense and immunologically react to L. infantum parasites, generating an innate immune response by increasing nitric oxide (NO) production and enhancing toll-like receptor (TLR) 2 and interleukin-10 gene expression. The immune response orchestrated by canine hepatocytes also lead to the impairment of several cytochrome P450 (CYP450) with possible implications for liver natural xenobiotic metabolization capacity. The application of meglumine antimoniate (MgA) increased the inflammatory response of 3D hepatic spheroids by inducing the expression of Nucleotide oligomerization domain (NOD) -like receptors 1 and NOD2 and TLR2, TLR4, and TLR9 and enhancing gene expression of tumour necrosis factor α. It is therefore suggested that hepatocytes are key effector cells and can activate and orchestrate the immune response to L. infantum parasites.

Correction to: The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus complex (Diptera: Ceratopogonidae) at the European level.

An amendment to this paper has been published and can be accessed via the original article.

Immunophenotyping of Peripheral Blood, Lymph Node, and Bone Marrow T Lymphocytes During Canine Leishmaniosis and the Impact of Antileishmanial Chemotherapy.

Dogs are a major reservoir of Leishmania infantum, etiological agent of canine leishmaniosis (CanL) a zoonotic visceral disease of worldwide concern. Therapeutic protocols based on antileishmanial drugs are commonly used to treat sick dogs and improve their clinical condition. To better understand the impact of Leishmania infection and antileishmanial drugs on the dog's immune response, this study investigates the profile of CD4+ and CD8+ T cell subsets in peripheral blood, lymph node, and bone marrow of sick dogs and after two different CanL treatments. Two CanL groups of six dogs each were treated with either miltefosine or meglumine antimoniate combined with allopurinol. Another group of 10 clinically healthy dogs was used as control. Upon diagnosis and during the following 3 months of treatment, peripheral blood, popliteal lymph node, and bone marrow mononuclear cells were collected, labeled for surface markers CD45, CD3, CD4, CD8, CD25, and intracellular nuclear factor FoxP3, and T lymphocyte subpopulations were immunophenotyped by flow cytometry. CanL dogs presented an overall increased frequency of CD8+ and CD4+CD8+ double-positive T cells in all tissues and a decreased frequency of CD4+ T cells in the blood. Furthermore, there was a higher frequency of CD8+ T cells expressing CD25+FoxP3+ in the blood and bone marrow. During treatment, these subsets recovered to levels similar to those of healthy dogs. Nevertheless, antileishmanial therapy caused an increase of CD4+CD25+FoxP3+ T cells in all tissues, associated with the decrease of CD8+CD25-FoxP3- T cell percentages. These findings may support previous studies that indicate that L. infantum manipulates the dog's immune system to avoid the development of a protective response, ensuring the parasite's survival and the conditions that allow the completion of Leishmania life cycle. Both treatments used appear to have an effect on the dog's immune response, proving to be effective in promoting the normalization of T cell subsets.