Effect of the aggregated protein dye YAT2150 on Leishmania parasite viability.

The problems associated with the drugs currently used to treat leishmaniasis, including resistance, toxicity, and the high cost of some formulations, call for the urgent identification of new therapeutic agents with novel modes of action. The aggregated protein dye YAT2150 has been found to be a potent antileishmanial compound, with a half-maximal inhibitory concentration (IC50) of approximately 0.5 µM against promastigote and amastigote stages of Leishmania infantum. The encapsulation in liposomes of YAT2150 significantly improved its in vitro IC50 to 0.37 and 0.19 µM in promastigotes and amastigotes, respectively, and increased the half-maximal cytotoxic concentration in human umbilical vein endothelial cells to >50 µM. YAT2150 became strongly fluorescent when binding intracellular protein deposits in Leishmania cells. This fluorescence pattern aligns with the proposed mode of action of this drug in the malaria parasite Plasmodium falciparum, the inhibition of protein aggregation. In Leishmania major, YAT2150 rapidly reduced ATP levels, suggesting an alternative antileishmanial mechanism. To the best of our knowledge, this first-in-class compound is the only one described so far having significant activity against both Plasmodium and Leishmania, thus being a potential drug for the treatment of co-infections of both parasites.

Nodular pyogranulomatous panniculitis due to Leishmania infantum infection in a domestic ferret (Mustela putorius furo).

Leishmaniosis is a vector-borne disease caused by different Leishmania species and transmitted by phlebotomine sand flies under natural conditions in Europe. Scientific information related to Leishmania infantum in dogs is extensive, where less information is available in cats and other companion animals. Recently, first clinical cases of L.infantum infection in domestic ferrrets (Mustela putorius furo) have been described. However, clinical information on leishmaniosis in this species is limited A 15-month-old male neutered domestic ferret was presented with chronic weight loss and the presence of coalescent, erythematous and firm subcutaneous nodules in the ventral abdominal subcutis. A fine-needle aspiration of these nodules was performed and the cytological examination revealed a granulomatous inflammation with the presence of macrophages contained a number of oval organisms with an eccentric nucleus and pale cytoplasm, compatible with Leishmania spp. amastigotes compatible with Leishmania spp. amastigotes. The nodules were surgically excised and histological examination showed a severe multifocal pyogranulomatous panniculitis. Specific immunohistochemistry and qPCR for L. infantum from excised nodules were positive. Additionally, L. infantum was cultured and isolated from the nodules by a fine-needle aspiration. An in-house Western Blot test for L. infantum was performed in serum sample and a positive result was obtained. This is the first reported case of nodular pyogranulomatous panniculitis due to L. infantum infection in a domestic ferret. Further studies are necessary to determine the relevance of domestic ferrets in the transmission of leishmaniosis. The description of new clinical forms of the disease is important as it can assist veterinarians in identifying these new clinical presentations.

Anisakid Presence in the European Conger, Conger conger, from Spanish Mediterranean Waters.

The European conger, Conger conger, is a benthic marine fish species with a geographical distribution extending through the northeastern Atlantic and the Mediterranean. Despite being extensively distributed and widely appreciated by Spanish consumers, studies regarding parasite presence in this fish are scarce. In the present work, a hundred and eight specimens from the Mediterranean coast of northeastern Spain (Catalan waters) were surveyed for the presence of nematode parasites. Several species were morphologically identified: third-stage larvae of Anisakis type I (sensu Berland, 1961) (n = 131), third-stage larvae of Anisakis type II (sensu Berland, 1961) (n = 18), third- and fourth-stage larvae and adults of Hysterothylacium spp. (n = 48), adults of Cucullanus sp. (n = 391), and adults of Cristitectus congeri (n = 69). Moreover, some fish and decapode species were also observed as part of the host's diet, with the most detected preys being Micromesistius poutassou, Sardina pilchardus, Macropipus sp., and Goneplax rhomboides. This represents the first survey of nematode parasites infecting C. conger from the northeastern Spanish Mediterranean waters. Among the parasite species detected, the presence of Anisakis species should be highlighted as the ingestion of C. conger parasitized with these larvae could potentially lead to anisakiasis in consumers.

Effect of Local Administration of Meglumine Antimoniate and Polyhexamethylene Biguanide Alone or in Combination with a Toll-like Receptor 4 Agonist for the Treatment of Papular Dermatitis due to Leishmania infantum in Dogs.

Papular dermatitis is a cutaneous manifestation of canine Leishmania infantum infection associated with mild disease. Although it is a typical presentation, nowadays, there is still no established treatment. This study evaluated the safety and clinical efficacy of local meglumine antimoniate, locally administered polyhexamethylene biguanide (PHMB) alone or PHMB in combination with a Toll-like receptor 4 agonist (TLR4a) for the treatment of papular dermatitis due to L. infantum and assessed parasitological and immunological markers in this disease. Twenty-eight dogs with papular dermatitis were divided randomly into four different groups; three of them were considered treatment groups: PHMB (n = 5), PHMB + TLR4a (n = 4), and meglumine antimoniate (n = 10)), and the remaining were considered the placebo group (n = 9), which was further subdivided into two sub-groups: diluent (n = 5) and TLR4a (n = 4). Dogs were treated locally every 12 h for four weeks. Compared to placebo, local administration of PHMB (alone or with TLR4a) showed a higher tendency towards resolution of papular dermatitis due to L. infantum infection at day 15 (χ2 = 5.78; df = 2, p = 0.06) and day 30 (χ2 = 4.; df = 2, p = 0.12), while local meglumine antimoniate administration demonstrated the fastest clinical resolution after 15 (χ2 = 12.58; df = 2, p = 0.002) and 30 days post-treatment (χ2 = 9.47; df = 2, p = 0.009). Meglumine antimoniate showed a higher tendency towards resolution at day 30 when compared with PHMB (alone or with TLR4a) (χ2 = 4.74; df = 2, p = 0.09). In conclusion, the local administration of meglumine antimoniate appears to be safe and clinically efficient for the treatment of canine papular dermatitis due to L. infantum infection.

In Vitro Evaluation of Aerosol Therapy with Pentamidine-Loaded Liposomes Coated with Chondroitin Sulfate or Heparin for the Treatment of Leishmaniasis.

The second-line antileishmanial compound pentamidine is administered intramuscularly or, preferably, by intravenous infusion, with its use limited by severe adverse effects, including diabetes, severe hypoglycemia, myocarditis and renal toxicity. We sought to test the potential of phospholipid vesicles to improve the patient compliance and efficacy of this drug for the treatment of leishmaniasis by means of aerosol therapy. The targeting to macrophages of pentamidine-loaded liposomes coated with chondroitin sulfate or heparin increased about twofold (up to ca. 90%) relative to noncoated liposomes. The encapsulation of pentamidine in liposomes ameliorated its activity on the amastigote and promastigote forms of Leishmania infantum and Leishmania pifanoi, and it significantly reduced cytotoxicity on human umbilical endothelial cells, for which the concentration inhibiting 50% of cell viability was 144.2 ± 12.7 µM for pentamidine-containing heparin-coated liposomes vs. 59.3 ± 4.9 µM for free pentamidine. The deposition of liposome dispersions after nebulization was evaluated with the Next Generation Impactor, which mimics human airways. Approximately 53% of total initial pentamidine in solution reached the deeper stages of the impactor, with a median aerodynamic diameter of ~2.8 µm, supporting a partial deposition on the lung alveoli. Upon loading pentamidine in phospholipid vesicles, its deposition in the deeper stages significantly increased up to ~68%, and the median aerodynamic diameter decreased to a range between 1.4 and 1.8 µm, suggesting a better aptitude to reach the deeper lung airways in higher amounts. In all, nebulization of liposome-encapsulated pentamidine improved the bioavailability of this neglected drug by a patient-friendly delivery route amenable to self-administration, paving the way for the treatment of leishmaniasis and other infections where pentamidine is active.

Genetic diversity of Contracaecum rudolphii sp. A (Nematoda: Anisakidae) parasitizing the European Shag Phalacrocorax aristotelis desmarestii from the Spanish Mediterranean coast.

Sibling species of the Contracaecum rudolphii (s.l.) complex are habitual endoparasites of cormorants of the Phalacrocoracidae family, worldwide. In Europe, the two species, C. rudolphii sp. A and C. rudolphii sp. B, have been identified. However, information regarding the occurrence and distribution of these anisakids in cormorants from Spain is scarce. In the present study, 20 specimens of the European Shag, Ph. aristotelis desmarestii, from the western Mediterranean Spanish marine coast were parasitologically analyzed for the presence of nematodes. All hosts were found parasitized with Contracaecum specimens (n = 1,517). A representative subsample was genetically identified as C. rudolphii sp. A by sequence analysis of the mtDNA cox2 gene and the ITS1 and ITS2 regions of the rDNA. This represents the first report of C. rudolphii sp. A from the Spanish Mediterranean waters. Population genetic analysis was performed including other C. rudolphii sp. A specimens from the west Sardinian and the Tyrrhenian Sea. At the intraspecific level, a significant genetic differentiation (Fst ≈ 0.08, p < 0.00001) between the metapopulation from the Spanish Mediterranean coast and that from the Sardinian waters was observed; whereas, no differentiation was found between metapopulations of the parasite from the Spanish and the Tyrrhenian Italian coast. The findings highly support the hypothesis of the adaptation of the life cycle of C. rudolphii sp. A in brackish and marine ecosystems. Furthermore, the results on the population genetics of C. rudolphii sp. A suggest the possible role of the migration routes of wintering populations of cormorants in the Mediterranean Sea in influencing the parasite genetic structure.

Underestimation of Human Cutaneous Leishmaniasis Caused by Leishmania infantum in an Endemic Area of the Mediterranean Basin (Balearic Islands).

Leishmaniasis is an infectious zoonotic disease caused by protozoan parasites of the genus Leishmania. In the Mediterranean basin, leishmaniasis is caused by Leishmania infantum and transmitted by bites of sandflies of the genus Phlebotomus, with the dog as the main reservoir host. The most common form is cutaneous leishmaniasis (CL), although visceral cases also occur. The aim of this study was to assess the underestimation of CL in an endemic Mediterranean region. Thus, a retrospective study was performed on all CL cases diagnosed and treated in the Dermatology Service of Manacor Hospital (Majorca, Balearic Islands), and the data obtained were compared with those of local government epidemiological bulletins for the same period. The different clinical presentations were compiled, and data related to sex, age, and lesion type and number were analyzed. The results reveal a clear sub-notification, which indicates that the real incidence of human CL in this area is unknown.

Identification of Leishmania infantum Epidemiology, Drug Resistance and Pathogenicity Biomarkers with Nanopore Sequencing.

The emergence of drug-resistant strains of the parasite Leishmania infantum infecting dogs and humans represents an increasing threat. L. infantum genomes are complex and unstable with extensive structural variations, ranging from aneuploidies to multiple copy number variations (CNVs). These CNVs have recently been validated as biomarkers of Leishmania concerning virulence, tissue tropism, and drug resistance. As a proof-of-concept to develop a novel diagnosis platform (LeishGenApp), four L. infantum samples from humans and dogs were nanopore sequenced. Samples were epidemiologically typed within the Mediterranean L. infantum group, identifying members of the JCP5 and non-JCP5 subgroups, using the conserved region (CR) of the maxicircle kinetoplast. Aneuploidies were frequent and heterogenous between samples, yet only chromosome 31 tetrasomy was common between all the samples. A high frequency of aneuploidies was observed for samples with long passage history (MHOM/TN/80/IPT-1), whereas fewer were detected for samples maintained in vivo (MCRI/ES/2006/CATB033). Twenty-two genes were studied to generate a genetic pharmacoresistance profile against miltefosine, allopurinol, trivalent antimonials, amphotericin, and paromomycin. MHOM/TN/80/IPT-1 and MCRI/ES/2006/CATB033 displayed a genetic profile with potential resistance against miltefosine and allopurinol. Meanwhile, MHOM/ES/2016/CATB101 and LCAN/ES/2020/CATB102 were identified as potentially resistant against paromomycin. All four samples displayed a genetic profile for resistance against trivalent antimonials. Overall, this proof-of-concept revealed the potential of nanopore sequencing and LeishGenApp for the determination of epidemiological, drug resistance, and pathogenicity biomarkers in L. infantum.

First epidemiological survey of Leishmania infantum in the domestic ferret (Mustela putorius furo) in a canine leishmaniosis endemic area using serology and PCR.

BACKGROUND: Leishmaniosis, a vector-borne disease caused by Leishmania infantum, is one of the most important parasitic zoonoses in Europe. The transmission cycle of leishmaniosis is maintained by both domestic and wild animals. However, few data are available on the role of wild mammals in transmitting the parasite in the European Mediterranean basin. As feline leishmaniosis, diagnosis of the infection in ferrets can be a challenge, the use of different serological and molecular methods combined is a recommended approach. Our aim was to investigate the prevalence of infection of L. infantum in apparently healthy domestic ferrets (Mustela putorius furo) in an endemic region of Spain (Community of Valencia), using serological and molecular methods and to evaluate the results comparing the different techniques. METHODS: The prevalence of Leishmania infection was studied in domestic ferrets. Blood was collected from each animal for serology and molecular analysis. Two serological methods, enzyme-linked immunosorbent assay (ELISA) and western blot (WB), were used for the detection of L. infantum antibodies, and real-time polymerase chain reaction (qPCR) was used for the detection of L. infantum DNA. RESULTS: Blood samples from 102 apparently healthy ferrets were analyzed. In the serological study, 25.5% of the animals tested positive by western blot, and 9.0% by enzyme-linked immunosorbent assays. The seroprevalence of L. infantum infection, based on a positive result in any serological test, was 28.4% (95% confidence interval [CI] 20.6-S37.9%). No kinetoplast DNA (kDNA) was detected by qPCR in peripheral blood samples from the ferrets tested. CONCLUSIONS: The immunological response revealed by these tests indicates that the ferrets are exposed to repeated inoculations with the endemic parasite L. infantum. Although the low population of domestic ferrets means their reservoir potential is limited in the absence of a primary host, it would be of interest to carry out further studies using xenodiagnosis to determine whether they are accidental or reservoir host species capable of spreading infection.

Serum protein electrophoretogram profile detected in apparently healthy cats infected with Leishmania infantum - Short communication.

The information about the clinical features of Leishmania infantum infection in cats is scarce. In this study, we evaluated the serum protein electrophoresis of samples from 19 infected but apparently healthy cats. To detect L. infantum infection, two serological tests, i.e. western blot (WB) and enzyme-linked immunosorbent assay (ELISA) as well as quantitative polymerase chain reaction (qPCR) on the blood samples were performed. Eventual infection by several selected bacterial and viral pathogens was also tested. All but one of the cats were found positive with WB. The WB-negative cat was positive by ELISA only. From the 18 WB-positive cats, only three were positive also by ELISA and eight with qPCR, including the only animal which was positive in all the three tests. No concomitant infections were detected in any of the cats. The main alteration of the proteinogram was characterised by an increase of the α-2 fraction. In the five cats with hypergammaglobulinaemia, the pattern detected was polyclonal. None of the cats were seropositive to any other pathogens tested. The presence of polyclonal gammopathy and elevation of the α-2 fraction could suggest the presence of active infection. In contrast, the only detection of an increase of the α-2 fraction alone with the presence of positive serological result could be associated by immune response activation against L. infantum.

Leishmaniosis caused by Leishmania infantum in ferrets: Update review.

Leishmaniosis in domestic ferrets (Mustela putorius furo) is a disease caused by Leishmania infantum, a parasite transmitted through the bite of an infected female phlebotomine sand fly. Among vertebrates, the dog is the primary domestic reservoir of the parasite; however, other domestic animals can be implicated such as cats. The first description of a clinical case of leishmaniosis in domestic ferrets was reported recently. As a result, new knowledge has been published including empirically based treatment protocols, confirmatory techniques to detect the presence of the parasite infection and seasonal variation in the antibodies against Leishmania in apparently healthy domestic ferrets. The most common clinical signs observed are enlargement of peripheral lymph nodes and skin lesions such as papular and/or ulcerative dermatitis. Additionally, the most frequent laboratory alterations seen are hyperproteinaemia with hyperglobulinaemia and biochemical analytes alterations depending on the affected tissue. Two different therapeutic protocols have been described to treat domestic ferrets with leishmaniosis: meglumine antimoniate plus allopurinol protocol or miltefosine plus allopurinol protocol. These treatment protocols seemed to be able to control the Leishmania infection, although the presence of xanthinuria could be detected. The susceptibility of domestic ferrets to Leishmania infantum, the clinical picture, treatment of infected animals and prevention are poorly understood, due to the scarcity of recent description in the literature. Different proposed diagnostic algorithms have been included for domestic ferrets with suspected leishmaniosis, clinically healthy domestic ferrets and animals as blood donors. In this sense, the present review provides updated data on scientific knowledge of leishmaniosis in ferrets.

Clinical leishmaniosis in a domestic ferret (Mustela putorius furo) treated with miltefosine plus allopurinol: Serological and clinical follow-up.

The published information on the treatment of mustelid leishmaniosis is extremely scarce because there are only two case reports available. In one case, a domestic ferret (Mustela putorius furo) was treated with a combination of meglumine antimoniate plus allopurinol and, in the other case, a therapeutic regimen with allopurinol was administrated to a Eurasian otter (Lutra lutra). This article describes for the first time a combined therapeutic protocol with miltefosine (2 mg/kg once a day during 28 days per os), and allopurinol (10 mg/kg twice a day PO sine die) in a domestic ferret with splenomegaly, lymphadenomegaly and a facial pyogranulomatous dermatitis, with a moderate level of antibodies to Leishmania infantum.

Hybrid Genotype of Anisakis simplex (s.s.) and A. pegreffii Identified in Third- and Fourth-Stage Larvae from Sympatric and Allopatric Spanish Marine Waters.

The sibling species Anisakis simplex (s.s.) and Anisakis pegreffii are parasites of marine mammals and fish worldwide and the main causative agents of human anisakiasis. In sympatric areas, a hybrid genotype between the two species has been identified, mainly in third-stage larvae, but rarely in fourth-stage and adult forms. The aim of this study was to confirm the presence of hybrid genotypes in larvae parasitizing fish caught in sympatric and allopatric Spanish marine waters, the North-East Atlantic and West Mediterranean, respectively, and to study possible differences in the growth behaviour between genotypes. Of the 254 molecularly analysed larvae, 18 were identified as hybrids by PCR-RFLP analysis of the rDNA ITS region, 11 of which were subsequently confirmed by EF1 α-1 nDNA gene sequencing. These results therefore indicate an overestimation of hybrid genotypes when identification is based only on the ITS region. We also report the detection of a hybrid specimen in a host from the West Mediterranean, considered an allopatric zone. Additionally, fourth-stage larvae with a hybrid genotype were obtained in vitro for the first time, and no differences were observed in their growth behaviour compared to larvae with A. simplex (s.s.) and A. pegreffii genotypes.

Leishmaniosis in Rodents Caused by Leishmania infantum: A Review of Studies in the Mediterranean Area.

Leishmaniosis infection begins when a phlebotomine sand fly vector inoculates pathogenic protozoan parasites of the genus Leishmania into a mammalian host. In the case of Leishmania infantum, the domestic dog is considered to be the main parasite reservoir, and canine leishmaniosis (CanL) has a high mortality rate in untreated dogs. Hundreds of cases of human leishmaniosis (HL) are reported in the world each year, the incidence in Europe being relatively low. Leishmaniosis control is primarily focused on the dog, combining methods that prevent sand fly bites and boost host resistance to infection. However, these measures are only partially effective and new solutions need to be found. One of the main factors limiting CanL and HL control is the existence of a sylvatic Leishmania transmission cycle that interacts with the domestic cycle maintained by dogs. It is suspected that the main reservoir of infection in wildlife are rodents, whose expansion and rapid population growth worldwide is increasing the risk of human and zoonotic pathogen transfer. The aim of this review is therefore to analyze reports in the literature that may shed light on the potential role of rodents in the leishmaniosis transmission cycle in the Mediterranean area. Following the general methodology recommended for reviews, six databases (Google Scholar, Ovid, PubMed, Science Direct, Scopus and Web of Science) were explored for the period January 1995 to December 2020. The results extracted from 39 publications that met the established inclusion criteria were analyzed. It was found that 23 species of rodents have been studied in nine countries of the Mediterranean basin. Of the 3,643 specimens studied, 302 tested positive for L. infantum infection by serology, microscopy and/or molecular techniques.

Antibodies to Leishmania in naturally exposed domestic ferrets (Mustela putorius furo) in Spain.

Zoonotic leishmaniosis due to Leishmania infantum is a vector-borne disease endemic in southern Europe and dogs are the main reservoir for this infection. Seasonal variations in antibody titers in this species in areas where phlebotomine vectors have seasonal patterns of activity are important for epidemiological, preventive and clinical studies related with canine leishmaniosis. It has been suggested that cats, rabbits and ferrets may act as peridomestic reservoirs and not only as accidental hosts. The aim of this study was to determine if seropositive ferrets (Mustela putorius furo) to Leishmania could be affected by seasonal variations of anti-Leishmania antibodies. A group of seropositive clinically healthy ferrets (n = 21) were included in this study. A significant reduction in anti-Leishmania infantum antibodies was detected during non-transmission period (December 2020-February 2021) in comparison to transmission period (April-October 2020). This study describes for the first time a seasonal variation in the anti-Leishmania antibodies detected in domestic ferrets following natural exposure during sand fly transmission period and the following non-sand fly transmission period in a Mediterranean area considered as an area where L. infantum is endemic.

A cross-sectional study of Leishmania infantum infection in stray cats in the city of Zaragoza (Spain) using serology and PCR.

BACKGROUND: Feline leishmaniosis is a vector-borne parasitic disease caused by Leishmania spp. Leishmania infection in dogs is prevalent in the Mediterranean basin, but in other animals, such as cats, it could also play a role in the epidemiology of the disease. Information on the geographical distribution and epidemiological features of L. infantum infection in cats is scarce, particularly in urban stray cats living in regions where canine leishmaniosis is endemic. As diagnosis can be challenging, combining different serological and molecular methods is a useful approach. Our aim was to investigate the prevalence of infection of L. infantum in apparently healthy stray cats in an endemic region of Spain (Zaragoza city) using serological and molecular methods, and to compare the results of the different techniques. METHODS: The prevalence of Leishmania infection was studied in stray cats captured in urban and peri-urban areas of Zaragoza. Blood was collected from each animal for serology and molecular analysis. Three serological methods, namely the immunofluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and western blot (WB), were used to detect L. infantum antibodies and a real-time PCR (qPCR) assay was used to detect L. infantum DNA. The results were analyzed by Fisher's exact test and Cohen's kappa statistic (κ) to assess the level of agreement between the diagnostic techniques. RESULTS: Serological analysis of blood samples from 180 stray cats revealed 2.2% (4/179) Leishmania infection positivity by IFAT, 2.8% (5/179) by ELISA and 14.5% (26/179) by WB. Leishmania DNA was detected by qPCR in 5.6% (10/179) of the cats. Sixteen cats (8.9%) tested positive by only one serological technique and four tested positive by all three serological methods used. The overall rate of infected cats (calculated as the number of cats seropositive and/or qPCR positive) was 15.6%, and only two cats tested positive by all the diagnostic methods. A significant association was found between male cats and a positive qPCR result. Comparison of the techniques revealed a fair agreement in seropositivity between blood qPCR and IFAT (κ = 0.26), blood qPCR and ELISA (κ = 0.24), WB and ELISA (κ = 0.37) and WB and IFAT (κ = 0.40). The highest agreement between seropositive results was between IFAT and ELISA (κ = 0.89), and the lowest was between blood qPCR and WB (κ = 0.19). The prevalence of the feline leukemia virus antigen was 4.49% (8/178 cats) and that of the feline immunodeficiency virus (FIV) antibody was 6.74% (12/178), while co-infection with both retroviruses was observed in one female cat (1/178). Leishmania ELISA and IFAT seropositivity were statistically associated with FIV status by the chi-square test. CONCLUSIONS: The results obtained in this study, using serological tests and qPCR, indicate the existence of L. infantum asymptomatic infection in apparently healthy stray cats in the city of Zaragoza, an endemic area in Spain.

Diagnostic usefulness of immunohistochemical evaluation of CD1a antigen and polyclonal anti-leishmania antibodies in cutaneous leishmaniasis.

BACKGROUND: Different immunohistochemical markers to detect amastigotes in cutaneous leishmaniasis have been proposed with variable diagnostic usefulness. OBJECTIVES: To evaluate the diagnostic usefulness of immunohistochemical amastigotes identification by specific polyclonal anti-Leishmania antibodies and CD1a expression (clone EP3622) in a series of PCR confirmed cutaneous leishmaniasis. MATERIALS AND METHODS: Thirty-three skin samples corresponding to PCR confirmed cutaneous leishmaniasis were included in the study. All samples were stained with Hematoxylin-eosin and Giemsa. Moreover, immunohistochemical studies with anti-CD1a and anti-Leishmania antibodies were performed. The patients clinical features and the observed histopathological features were also recorded. RESULTS: From the selected 33 biopsies, Leishmania spp. amastigotes were detected in 48.4% of cases with conventional Hematoxylin-eosin stain and in 57.5% of cases by Giemsa staining. In 31/33 cases, anti-CD1a allowed us to identify parasitic structures, and in 33/33 cases amastigotes were detected with anti-Leishmania antibodies. Concordance between both techniques, anti-CD1a and anti-Leishmania, was 94% [CI 95%: (79,8%-99,3%)] ; p value <0.05. The sensitivity of anti-CD1a in comparison with the PCR was 94%, with a positive predictive value of 100%. Two cases of low parasitic index were negative for CD1a immunostaining. In cases with high parasitic index, anti-CD1a stained amastigotes in superficial and deep dermis. Only a few cases were originally diagnosed with the available histological techniques, needing PCR for Leishmania spp. CONCLUSIONS: Anti-CD1a antibody seems to be a useful technique to identify amastigotes when PCR and anti-Leishmania antibodies are not available. The sensitivity to detect amastigotes is increased when the CD1a immunostaining is added to the classical Haematoxylin - eosin and Giemsa staining.

Estimation of the number of Anisakis larvae in commercial fish using a descriptive model based on real-time PCR.

BACKGROUND: Seafood parasitation by Anisakis (Anisakidae) larvae has been reported in most of the oceans and seas worldwide. The presence of these nematodes in commonly consumed fish represents a potential hazard for consumers as they can provoke gastrointestinal symptoms and allergic reactions. In the present work, the capacity of a SYBR Green qPCR protocol to quantify Anisakis larvae in commercial fish was evaluated using experimentally spiked samples with different numbers (0-50) of A. simplex third-stage larvae (L3). To verify the agreement of the obtained results, 25 naturally infected fish specimens of Atlantic blue whiting underwent a parallel visual inspection. RESULTS: The logarithmic behavior of the Cq data obtained from the experimentally spiked samples allowed the development of a descriptive mathematical model that correlates the Cq value with the number of Anisakis larvae (R2 = 0.9908, CV = 2.37%). In the commercial blue whiting specimens there was a high correlation between the results of the molecular technique and the visual inspection (R2 = 0.9912); the Bland-Altman analysis showed that 94% of the differences were within the limits of agreement (-4.98 and 6.68), indicating the reliability of the descriptive mathematical model based on the SYBR Green qPCR technique. CONCLUSION: The descriptive function presented based on the SYBR Green qPCR assay is promising as a sensitive and accurate tool for measuring the Anisakis larval load in commercial fish, with a potential application not only in the food industry but also in prevention programs for public health. © 2020 Society of Chemical Industry.

Serological and molecular survey of Leishmania infection in dogs from Venezuela.

Venezuela is a country where human and canine leishmaniosis due to Leishmania infantum, Leishmania braziliensis and other Leishmania spp. is endemic. However, only limited data is available on canine Leishmania infection in Venezuela. The aim of this cross-sectional study was to evaluate the prevalence of Leishmania infection in dogs (n = 152) from the states of Lara (n = 91) and Yaracuy (n = 61) in Venezuela by means of serological and molecular methods. Physical examination was performed and blood samples were collected from all dogs. Serology for antibodies reactive with L. infantum and L. braziliensis antigens was assessed by the enzyme-linked immunosorbent assay (ELISA) and detection of Leishmania DNA from blood samples was evaluated by kinetoplast Leishmania real-time polymerase chain reaction (RT-PCR). In addition, Leishmania internal transcribed spacer (ITS-1) RT-PCR was performed on the samples positive by kinetoplast RT-PCR. The prevalence of Leishmania infection based on serological and/or molecular techniques was 11.8%. The seroprevalence for L. infantum and L. braziliensis antigens were 2.1% (3/144) and 8.3% (12/144), respectively. All dogs from the state of Yaracuy were serologically negative to L. infantum while 4.6% (4/86) of the dogs were reactive to L. braziliensis antigen. Fourteen percent (8/58) of the dogs from the state of Lara were positive to L. infantum and 5.2% (3/58) to L. braziliensis antigen. Three dogs were positive to both Leishmania spp. antigens. By RT-PCR, 6.5% (4/61) and 4.4% (4/91) of the dogs were positive for infection in the states of Lara and Yaracuy, respectively. The RT-PCR product of one dog from the state of Yaracuy was sequenced revealing a 100% identity with L. infantum. However, all RT-PCR positive dogs were seronegative to both Leishmania spp. antigens. In conclusion, the positivity for Leishmania spp. infections observed indicates that dogs are frequently infected by L. infantum, L. braziliensis or related Leishmania spp. in Venezuela.

Treatment and follow-up of a domestic ferret (Mustela putorius furo) with clinical leishmaniosis caused by Leishmania infantum.

Leishmania infantum infection including treatment and follow up in domestic animals other than dogs and cats has not been described at this moment. This article describes the anti-Leishmania treatment and follow-up of a ferret (Mustela putorius furo) with leishmaniosis. A combined therapeutic protocol established for the patient, not yet approved for ferrets, was a combination of meglumine antimoniate plus allopurinol. A follow-up was established monthly during the first year in order to monitor the health condition of the patient. Six months after commencing allopurinol therapy, xanthine crystalluria was observed in urine sediment with no other urine alterations detected by urine analysis. The ferret worsened progressively with diarrhoea and weight loss after cohabiting with another ferret diagnosed with cryptosporidiosis. Cryptosporidium parvum was isolated in faecal samples from the patient detected by three different methods including Ziehl-Neelsen staining, a qualitative test to detection of C. parvum antigens and finally a specific molecular analysis to characterize the species. To the best of the authors´ knowledge, this is the first report providing information about anti-Leishmania protocol therapy used and follow-up in a domestic ferret with clinical leishmaniosis. Veterinarians practicing in endemic areas should be aware of this infection in ferrets at risk and their susceptibility especially when immunosuppressive conditions are present.