Antileishmanial Effect of 1,5- and 1,8-Substituted Fused Naphthyridines.

In the absence of a vaccine, there is a need to find new drugs for the treatment of neglected tropical diseases, such as leishmaniasis, that can overcome the many drawbacks of those currently used. These disadvantages include cost, the need to maintain a cold chain, the route of administration, the associated adverse effects and the generation of resistance. In this work we have evaluated the antileishmanial effect of 1,5- and 1,8-substituted fused naphthyridines through in vitro and ex vivo assays, using genetically modified axenic and intramacrophagic Leishmania infantum amastigotes. The toxicity of these compounds has been tested in the mammalian host cell using murine splenic macrophages, as well as in murine intestinal organoids (miniguts) in order to assess their potential for oral administration. The 1,8- derivatives showed greater leishmanicidal activity and the presence of a nitrogen atom in the fused ring to the naphthyridine was important to increase the activity of both types of molecules. The aromatization of the pyridine ring also had marked differences in the activity of the compounds.

Differences within Churra breed sheep in the early immune response to the infection by Teladorsagia circumcincta.

This study describes early immunological mechanisms that underlie resistance to Teladorsagia circumcincta infection in adult Churra sheep. After a first experimental infection, 6 animals were classified as resistant (RG) and 6 as susceptible (SG) to T. circumcincta infection based on their cumulative faecal egg count (cFEC) at the end of the infection. RG showed higher IgA levels against somatic antigen of T. circumcincta fourth-larvae stage (L4) in serum at day 3 post-infection (pi) (p < 0.05) and close to significance at day 21 pi (p = 0.06). Moreover, a strong negative correlation between cFEC and specific IgA was only significant in RG at day 3 pi (r = - 0.870; p < 0.05), but absent in SG. At the end of this infection, sheep were treated with moxidectin and infected again 3 weeks later to be slaughtered at day 7 pi. At necropsy, the specific IgA levels in gastric mucosa were similar between groups; the absence differences at day 7 pi could be due to a previous increase in the IgA response, probably around day 3 pi, as described during the first infection. L4 burden, 68% lower in RG than in SG, was influenced by the specific IgA in gastric mucus and the number of γδ T cells. RG group showed a positive correlation between γδ T cells and eosinophils (r = 0.900; p = 0.037); however, this correlation was not found in SG. These results show that these two phenotypes show different early immune response pattern to T. circumcincta infection in Churra sheep.

Drug discovery technologies: Caenorhabditis elegans as a model for anthelmintic therapeutics.

Helminthiasis is one of the gravest problems worldwide. There is a growing concern on less available anthelmintics and the emergence of resistance creating a major threat to human and livestock health resources. Novel and broad-spectrum anthelmintics are urgently needed. The free-living nematode Caenorhabditis elegans could address this issue through automated high-throughput technologies for the screening of large chemical libraries. This review discusses the strong advantages and limitations for using C elegans as a screening method for anthelmintic drug discovery. C elegans is the best model available for the validation of novel effective drugs in treating most, if not all, helminth infections, and for the elucidation the mode of action of anthelmintic candidates. This review also focuses on available technologies in the discovery of anthelmintics published over the last 15 years with particular attention to high-throughput technologies over conventional screens. On the other hand, this review highlights how combinatorial and nanomedicine strategies could prolong the use of anthelmintics and control resistance problems.

Screening Marine Natural Products for New Drug Leads against Trypanosomatids and Malaria.

Neglected Tropical Diseases (NTD) represent a serious threat to humans, especially for those living in poor or developing countries. Almost one-sixth of the world population is at risk of suffering from these diseases and many thousands die because of NTDs, to which we should add the sanitary, labor and social issues that hinder the economic development of these countries. Protozoan-borne diseases are responsible for more than one million deaths every year. Visceral leishmaniasis, Chagas disease or sleeping sickness are among the most lethal NTDs. Despite not being considered an NTD by the World Health Organization (WHO), malaria must be added to this sinister group. Malaria, caused by the apicomplexan parasite Plasmodium falciparum, is responsible for thousands of deaths each year. The treatment of this disease has been losing effectiveness year after year. Many of the medicines currently in use are obsolete due to their gradual loss of efficacy, their intrinsic toxicity and the emergence of drug resistance or a lack of adherence to treatment. Therefore, there is an urgent and global need for new drugs. Despite this, the scant interest shown by most of the stakeholders involved in the pharmaceutical industry makes our present therapeutic arsenal scarce, and until recently, the search for new drugs has not been seriously addressed. The sources of new drugs for these and other pathologies include natural products, synthetic molecules or repurposing drugs. The most frequent sources of natural products are microorganisms, e.g., bacteria, fungi, yeasts, algae and plants, which are able to synthesize many drugs that are currently in use (e.g. antimicrobials, antitumor, immunosuppressants, etc.). The marine environment is another well-established source of bioactive natural products, with recent applications against parasites, bacteria and other pathogens which affect humans and animals. Drug discovery techniques have rapidly advanced since the beginning of the millennium. The combination of novel techniques that include the genetic modification of pathogens, bioimaging and robotics has given rise to the standardization of High-Performance Screening platforms in the discovery of drugs. These advancements have accelerated the discovery of new chemical entities with antiparasitic effects. This review presents critical updates regarding the use of High-Throughput Screening (HTS) in the discovery of drugs for NTDs transmitted by protozoa, including malaria, and its application in the discovery of new drugs of marine origin.

Fasciola hepatica and Fasciola gigantica coexistence in domestic ruminants in Nigeria: application of a PCR-based tool.

The distinction between Fasciola hepatica and Fasciola gigantica has been traditionally based on morphological criteria, although accurate recognition of the two flukes is usually difficult because of substantial variations in morphological features. The main aim of this study was to develop a PCR-based assay for discrimination between both species collected in sheep and cattle from Nigeria. A total of 47 animals, 33 cattle and 14 sheep, were sampled, and a single adult fluke was collected from each animal. DNA was extracted from flukes, and primers were designed based on mitochondrial DNA sequences to amplify a 304 bp fragment for the identification of F. hepatica and 752 bp for F. gigantica. PCR products from 12 flukes were sequenced for phylogenetic analysis. A total of 29 out of 47 flukes were identified as F. hepatica and 18 as F. gigantica. Within each host, the percentage of each fluke species was as follows: In cattle, 18/33 (54.5%) and 15/33 (45.5%) were F. hepatica and F. gigantica, respectively. In sheep, 11/14 (78.6%) were F. hepatica and 3/14 F. gigantica (21.4%). The phylogenetic analysis confirmed these results. Although the number of flukes collected in sheep was limited, it seems that F. hepatica is more prevalent in sheep than F. gigantica, whereas the percentage of each species was similar in cattle. This study confirms the presence of F. hepatica in Nigeria.

Exploring the mechanisms of resistance to Teladorsagia circumcincta infection in sheep through transcriptome analysis of abomasal mucosa and abomasal lymph nodes.

The present study exploited the RNA-seq technology to analyze the transcriptome of target tissues affected by the Teladorsagia circumcincta infection in two groups of adult ewes showing different statuses against gastrointestinal nematode (GIN) infection with the aim of identifying genes linked to GIN infection resistance in sheep. For this, based on the accumulated faecal egg count of 18 adult Churra ewes subjected to a first experimental infection with T. circumcincta, six ewes were classified as resistant and six others as susceptible to the infection. These 12 animals were dewormed and infected again. After humanitarian sacrifice of these 12 animals at day 7 post-infection, RNA samples were obtained from abomasal mucosa and lymph node tissues and RNA-Seq datasets were generated using an Illumina HiSeq 2000 sequencer. The distribution of the genes based on their expression level were very similar among the two different tissues and conditions. The differential expression analysis performed with two software (DESeq and EdgeR) only identified common differentially expressed genes (DEGs), a total of 106, in the lymph node samples which were considered as GIN-activated. The enrichment analysis performed for these GIN-activated genes identified some pathways related to cytokine-mediated immune response and the PPARG signaling pathway as well as disease terms related to inflammation and gastro-intestinal diseases as enriched. A systematic comparison with the results of previous studies confirmed the involvement of genes such as ITLN2, CLAC1 and galectins, in the immune mechanism activated against T. circumcincta in resistant sheep.

Quantification of resistant alleles in the ß-tubulin gene of field strains of gastrointestinal nematodes and their relation with the faecal egg count reduction test.

BACKGROUND: Benzimidazole (BZ) resistance in gastrointestinal nematodes is associated with a single nucleotide polymorphism (SNP) at codons 167, 198 and 200 in the isotype 1 of beta-tubulin gene although in some species these SNPs have also been associated with resistance to macrocyclic lactones. In the present study we compared the levels of resistance in Teladorsagia circumcincta and Trichostrongylus colubriformis by means of the faecal egg reduction test (FECRT) and the percentage of resistant alleles obtained after pyrosequencing. The study was conducted in 10 naturally infected sheep flocks. Each flock was divided into three groups: i) group treated with albendazole (ABZ); ii) group treated with ivermectin (IVM); iii) untreated group. The number of eggs excreted per gram of faeces was estimated at day 0 and 14 post-treatment. RESULTS: Resistance to ABZ was observed in 12.5% (1/8) of the flocks and to IVM in 44.4% (4/9) of them. One flock was resistant to both drugs according to FECRT. Coprocultures were performed at the same dates to collect L3 for DNA extraction from pooled larvae and to determine the resistant allele frequencies by pyrosequencing analysis. In T. circumcincta, SNPs were not found at any of the three codons before treatment; after the administration of ABZ, SNPs were present only in two different flocks, one of them with a frequency of 23.8% at SNP 167, and the other 13.2% % at SNP 198. In relation to T. colubriformis, we found the SNP200 before treatment in 33.3% (3/9) of the flocks with values between 48.5 and 87.8%. After treatment with ABZ and IVM, the prevalence of this SNP increased to 75 and 100% of the flocks, with a mean frequency of 95.1% and 82.6%, respectively. CONCLUSION: The frequencies observed for SNP200 in T. colubriformis indicate that the presence of resistance is more common than revealed by the FECRT.

Effect of dietary supplementation with carnosic acid or vitamin E on animal performance, haematological and immunological characteristics of artificially reared suckling lambs before and after road transport.

To elucidate the influence of dietary carnosic acid (CA) and vitamin E on animal performance, immune response indicators and haematological parameters before and after transport stress, 24 lambs were individually fed ad libitum with milk replacer (MR) using an auto-feeder. Once daily the lambs received MR alone (Group CON, n = 8), MR + 0.096 g CA/kg live weight (LW) (Group CARN, n = 8) or MR + 0.024 g of α-tocopheryl acetate per kg LW (Group VitE, n = 8). After reaching the target slaughter weight (12 ± 0.5 kg), blood samples were collected to measure haematological and immunological parameters. Then, lambs were subjected to 4-h road transport and blood samples were collected again for haematological assessment. The animals were subsequently slaughtered. Before road transport, dietary CA supplementation promoted a descent of circulating white blood cells (WBC), red blood cells (RBC), haematocrit and haemoglobin concentration when compared with Groups CON and VitE (p < 0.05), but it did not affect production of cytokines by blood mononuclear cells. Road transport did not affect either RBC or haematocrit significantly. Nevertheless, transport affected leucocyte profile similarly in all the treatments, increasing granulocytes and monocytes proportions and decreasing lymphocytes. In contrast, after transport, WBC was increased in Group CARN, reaching similar values than Groups CON and VitE. However, under conditions of the present study, those modifications did not influence animal performance or immunity parameters of artificially reared suckling lambs.

Detection and replication of QTL underlying resistance to gastrointestinal nematodes in adult sheep using the ovine 50K SNP array.

BACKGROUND: Persistence of gastrointestinal nematode (GIN) infection and the related control methods have major impacts on the sheep industry worldwide. Based on the information generated with the Illumina OvineSNP50 BeadChip (50 K chip), this study aims at confirming quantitative trait loci (QTL) that were previously identified by microsatellite-based genome scans and identifying new QTL and allelic variants that are associated with indicator traits of parasite resistance in adult sheep. We used a commercial half-sib population of 518 Spanish Churra ewes with available data for fecal egg counts (FEC) and serum levels of immunoglobulin A (IgA) to perform different genome scan QTL mapping analyses based on classical linkage analysis (LA), a combined linkage disequilibrium and linkage analysis (LDLA) and a genome-wide association study (GWAS). RESULTS: For the FEC and IgA traits, we detected a total of three 5 % chromosome-wise significant QTL by LA and 63 significant regions by LDLA, of which 13 reached the 5 % genome-wise significance level. The GWAS also revealed 10 significant SNPs associated with IgAt, although no significant associations were found for LFEC. Some of the significant QTL for LFEC that were detected by LA and LDLA on OAR6 overlapped with a highly significant QTL that was previously detected in a different half-sib population of Churra sheep. In addition, several new QTL and SNP associations were identified, some of which show correspondence with effects that were reported for different populations of young sheep. Other significant associations that did not coincide with previously reported associations could be related to the specific immune response of adult animals. DISCUSSION: Our results replicate a FEC-related QTL located on OAR6 that was previously reported in Churra sheep and provide support for future research on the identification of the allelic variant that underlies this QTL. The small proportion of genetic variance explained by the detected QTL and the large number of functional candidate genes identified here are consistent with the hypothesis that GIN resistance/susceptibility is a complex trait that is not determined by individual genes acting alone but rather by complex multi-gene interactions. Future studies that combine genomic variation analysis and functional genomic information may help elucidate the biology of GIN disease resistance in sheep.

Screening anthelmintic resistance to triclabendazole in Fasciola hepatica isolated from sheep by means of an egg hatch assay.

BACKGROUND: In the present study, the detection of anthelmintic resistance to triclabendazole (TCBZ) in sheep infected by Fasciola hepatica was studied using an egg hatch assay (EHA). F. hepatica eggs were recovered from bile and faeces of infected animals by isolates with different grade of anthelmintic resistance to TCBZ: i) a resistant isolate (RT); ii) a susceptible isolate (ST); iii) naturally infected sheep by a susceptible field strain (FST). In the EHA the percentage of hatched eggs were calculated according to the following concentrations of TCBZ diluted in dimethyl-sulfoxide (DMSO): 0.05, 0.2, 1, 5, and 25 µg/ml. RESULTS: In relation to the EHAs carried out with the eggs from bile of sheep infected by ST, differences were found in the percentage of hatched eggs between the control well, only with DMSO, and the two highest concentrations of TCBZ (5 and 25 µg/m) (p < 0.05). However, when we tested the drug with the eggs from the bile of sheep infected by RT, the percentage of hatched eggs was similar among all concentrations. Since the range of hatching varied between isolates, we calculated the ratio of the results of each concentration to its control value confirming the higher hatching in RT than in ST. We developed an EHA with eggs recovered from faeces in order to avoid the slaughter of sheep. The results of the EHAs with the isolate ST showed differences in the percentage of hatching between the highest concentration (25 µg/ml) and the control well (p < 0.05); however, these differences were not confirmed under field conditions with the strain FST. CONCLUSIONS: The ovicidal effect of TCBZ in F. hepatica eggs from bile was shown using a commercial formulation diluted in DMSO with a minimum concentration of 5 µg/ml. However, in eggs recovered from faeces the results are not conclusive. The cleaning of eggs recovered from faeces is an important issue that should be reviewed and standardized before comparing results between susceptible and resistant isolates in this kind of EHA.

Intraspecific mitochondrial DNA variation of Fasciola hepatica eggs from sheep with different level of anthelmintic resistance.

In the current study, Fasciola hepatica strains of sheep with different degrees of resistance to anthelmintics were analyzed by sequencing the cytochrome C oxidase (COX1) and the NADH dehydrogenase (NAD1) subunits. The strains were as follows: LS, susceptible to all drugs tested; CS, resistant to albendazole and triclabendazole; and SV, resistant to albendazole and clorsulon. The molecular characterization was done in eggs recovered from sheep infected by LS and CS. In relation to SV, eggs were recovered before (SV0) and after a treatment with albendazole (SVA) and clorsulon (SVC). Nested PCRs were carried out to amplify a fragment of 798 bp of the COX1 subunit and 870 bp of the NAD1 subunit. The pairwise sequence identity between eggs was analyzed for each strain. Population diversity indices, neutrality indices, and the degree of gene flow among the strains were evaluated. As a result, we have shown that there was homogeneity in the demographic expansion of the studied strains, and, according to the pairwise fixation index, these were not genetically differentiated. Although we found that the resistant strains had lower pairwise percentage similarities, higher haplotype diversity, and higher frequencies of specific SNPs, especially in the COX1 subunit, these differences were not very significant. Therefore, we conclude that the presence of adult flukes resistant to anthelmintics does not result in significant higher genetic diversity in the mtDNA of their eggs.

Polyamine Metabolism for Drug Intervention in Trypanosomatids.

Neglected tropical diseases transmitted by trypanosomatids include three major human scourges that globally affect the world's poorest people: African trypanosomiasis or sleeping sickness, American trypanosomiasis or Chagas disease and different types of leishmaniasis. Different metabolic pathways have been targeted to find antitrypanosomatid drugs, including polyamine metabolism. Since their discovery, the naturally occurring polyamines, putrescine, spermidine and spermine, have been considered important metabolites involved in cell growth. With a complex metabolism involving biosynthesis, catabolism and interconversion, the synthesis of putrescine and spermidine was targeted by thousands of compounds in an effort to produce cell growth blockade in tumor and infectious processes with limited success. However, the discovery of eflornithine (DFMO) as a curative drug against sleeping sickness encouraged researchers to develop new molecules against these diseases. Polyamine synthesis inhibitors have also provided insight into the peculiarities of this pathway between the host and the parasite, and also among different trypanosomatid species, thus allowing the search for new specific chemical entities aimed to treat these diseases and leading to the investigation of target-based scaffolds. The main molecular targets include the enzymes involved in polyamine biosynthesis (ornithine decarboxylase, S-adenosylmethionine decarboxylase and spermidine synthase), enzymes participating in their uptake from the environment, and the enzymes involved in the redox balance of the parasite. In this review, we summarize the research behind polyamine-based treatments, the current trends, and the main challenges in this field.

Treatment and Healing of Leishmaniasis in a Wolf in Semi-Captivity Regime from an Educational Center of Zamora Province (Spain).

Leishmaniasis in wild canids is a vector-borne disease caused in Europe by the protozoan parasite Leishmania infantum. To date, there is limited information on clinical signs and laboratory abnormalities in wolves due to leishmaniasis. The current clinical case report described a female Iberian wolf (Canis lupus signatus) housed in semi-captivity conditions at the Centro del Lobo Ibérico "Félix Rodríguez de la Fuente", in Robledo de Sanabria, Zamora (Spain), with an interdigital ulcerous wound at the right forepaw, hyper-gammaglobulinemia, and abnormal liver blood parameters. Definitive serodiagnosis of leishmaniasis was established using antileishmanial serum antibodies and PCR analysis of different biological samples. A gold-standard anti-L. infantum treatment protocol consisting in subcutaneous meglumine antimoniate and oral allopurinol combination was installed. However, the presence of pain at the site of injection due to meglumine antimoniate administration forced its substitution by oral miltefosine. A progressive reduction of the levels of anti-L. infantum serum antibodies and the concentrations of gamma-globulin fraction was detected after antileishmanial treatment as well as a decline of liver GPT. To our knowledge, this is the first case of leishmaniasis diagnosed in a wolf housed in semi-captivity conditions, with the condition subsequently treated and successfully cured.

Towards precision parasite management for livestock gastrointestinal nematodes in 2030.

The management of parasitic nematodes calls for a shift from conventional, indiscriminate, anthelmintic use to a more precise approach, directed by diagnostics. We should accept those parasite infection intensities that have minimal impact on production and welfare rather than attempt to eliminate them. The diagnostic toolbox for gastrointestinal nematodes (GINs) faces challenges due to anthelmintic resistance (AR), which is species-specific, drug-class-specific, and varies by region. We discuss which traditional tools may become obsolete and which tools need development to gain widespread use. Social science research highlights the need for dialogue between farmers and veterinarians that emphasises effective parasite management and upskilling the veterinary workforce for more sustainable practices centred on diagnostics to be adopted in practice by 2030.

In Vitro and Ex Vivo Synergistic Effect of Pyrvinium Pamoate Combined with Miltefosine and Paromomycin against Leishmania.

One of the major drawbacks of current treatments for neglected tropical diseases is the low safety of the drugs used and the emergence of resistance. Leishmaniasis is a group of neglected diseases caused by protozoa of the trypanosomatidae family that lacks preventive vaccines and whose pharmacological treatments are scarce and unsafe. Combination therapy is a strategy that could solve the above-mentioned problems, due to the participation of several mechanisms of action and the reduction in the amount of drug necessary to obtain the therapeutic effect. In addition, this approach also increases the odds of finding an effective drug following the repurposing strategy. From the previous screening of two collections of repositioning drugs, we found that pyrvinium pamoate had a potent leishmanicidal effect. For this reason, we decided to combine it separately with two clinically used leishmanicidal drugs, miltefosine and paromomycin. These combinations were tested in axenic amastigotes of Leishmania infantum obtained from bone marrow cells and in intramacrophagic amastigotes obtained from primary cultures of splenic cells, both cell types coming from experimentally infected mice. Some of the combinations showed synergistic behavior, especially in the case of the combination of pyrvinium pamoate with paromomycin, and exhibited low cytotoxicity and good tolerability on intestinal murine organoids, which reveal the potential of these combinations for the treatment of leishmaniasis.

Evaluating alternative compounds for strongyloidiasis therapy: Novel insights from larval migration inhibition test.

Strongyloidiasis is a neglected tropical disease estimated to affect more than 600 million people worldwide. Recently, the World Health Organization road map on neglected tropical diseases 2021-2030 has put the focus on strongyloidiasis, including this disease within its mass drug administration campaigns. With the use of ivermectin in extensive treatment of all populations at-risk, identifying effective therapeutic alternatives is crucial in case ivermectin resistance arises. The objective of the present study was the development of a larval migration inhibition assay to evaluate the anthelmintic efficacy of commercial drugs and diamine and aminoalcohol derivatives against infective Strongyloides ratti third stage larvae. Through this technique, we successfully screened and estimated the in vitro anthelmintic efficacy of six commercial drugs, seven diamine derivatives and eight aminoalcohol derivatives. Unexpectedly, the half-maximal effective concentration of ivermectin and moxidectin (2.21 and 2.34 µM, respectively) were observed as the highest value obtained among all commercial drugs tested by this in vitro technique. Moreover, some diamine and aminoalcohol derivatives showed superior efficacy inhibiting S. ratti motility compared to ivermectin, with five compounds (AA23, AA34, AO2 AO7 and AO14b) also displaying selectivity indexes on HepG2 and Caco2 higher than 1. These findings underscore the potential of these derivatives as promising alternatives for strongyloidiasis treatment, warranting further investigation and in vivo efficacy assessment.

New benzimidazole derivative compounds with in vitro fasciolicidal properties.

BACKGROUND: Control of the zoonotic food-borne parasite Fasciola hepatica remains a major challenge in humans and livestock. It is estimated that annual economic losses due to fasciolosis can reach US$3.2 billion in agriculture and livestock. Moreover, the wide distribution of drug-resistant parasite populations and the absence of a vaccine threaten sustainable control, reinforcing the need for novel flukicides. METHODS: The present work analyses the flukicidal activity of a total of 70 benzimidazole derivatives on different stages of F. hepatica. With the aim to select the most potent ones, and screenings were first performed on eggs at decreasing concentrations ranging from 50 to 5 µM and then on adult worms at 10 µM. Only the most effective compounds were also evaluated using a resistant isolate of the parasite. RESULTS: After the first screenings at 50 and 10 µM, four hit compounds (BZD31, BZD46, BZD56, and BZD59) were selected and progressed to the next assays. At 5 µM, all hit compounds showed ovicidal activities higher than 71% on the susceptible isolate, but only BZD31 remained considerably active (53%) when they were tested on an albendazol-resistant isolate, even with values superior to the reference drug, albendazole sulfoxide. On the other hand, BZD59 displayed a high motility inhibition when tested on adult worms from an albendazole-resistant isolate after 72 h of incubation. CONCLUSIONS: BZD31 and BZD59 compounds could be promising candidates for the development of fasciolicidal compounds or as starting point for the new synthesis of structure-related compounds.

Secretory IgA as Biomarker for Gastrointestinal Nematodes Natural Infection in Different Breed Sheep.

Specific IgA antibody has been shown to play an important role in resistance to gastrointestinal nematode (GIN) infections in sheep, particularly in Teladorsagia circumcincta parasitosis. In some breeds, negative associations have been shown between IgA levels and worm burden in experimentally infected sheep. In the present study, we have studied the relationship between IgA levels in naturally infected sheep (582 ewes in total; 193 younger than one year old and 389 older than one year old) and fecal egg count (FEC) in the Assaf, Castellana, and Churra breeds. ELISA assays were performed to measure IgA levels against the somatic antigen of T. circumcincta third larval stage (L3) and a 203-amino-acid fragment of the protein disulfide isomerase from the same GIN species. A multilevel random intercept model was developed to predict the infection risk according to age or breed. Spearman's correlation rank was used for statistical analysis. The prediction model showed that breed was not an influential factor in this study, although the Assaf breed could be considered slightly more susceptible than the others. In addition, age affected the infection risk, with the young ewes more susceptible to infection than the adult groups, except for the Castellana breed, whose risk of infection was similar at all ages. The most significant positive association was found between FEC and IgA measured in the nasal secretions of young ewes using both antigens (Rho = 0.5; p = 0.00); the correlation of FEC with IgA in serum was moderately significant (Rho = 0.306; p = 0.00). Comparing both antigens, the protein disulfide isomerase antigen was less reactive than the somatic antigen from L3. In conclusion, under natural conditions, specific IgA against GIN was positively associated with FEC in sheep, with nasal secretions from young animals being the sample where this association is stronger, which, therefore, could be used as a marker of infection in further studies.

Occurrence of Leishmaniasis in Iberian Wolves in Northwestern Spain.

Canine leishmaniasis is an important vector-borne protozoan disease in dogs that is responsible for serious deterioration in their health. In the Iberian Peninsula, as in most countries surrounding the Mediterranean Sea, canine leishmaniasis is caused by Leishmania infantum (zymodeme MON-1), a digenetic trypanosomatid that harbors in the parasitophorous vacuoles of host macrophages, causing severe lesions that can lead to death if the animals do not receive adequate treatment. Canine leishmaniasis is highly prevalent in Spain, especially in the Mediterranean coastal regions (Levante, Andalusia and the Balearic Islands), where the population of domestic dogs is very high. However, the presence of this disease has been spreading to other rural and sparsely populated latitudes, and cases of leishmaniasis have been reported for years in wildlife in northwestern Spain. This work describes for the first time the presence of wolves that tested positive for leishmaniasis in the vicinity of the Sierra de la Culebra (Zamora province, northwestern Spain), a protected sanctuary of this canid species, using PCR amplification of L. infantum DNA from different non-invasive samples such as buccal mucosa and those from both ears and hair. In addition to live animals (21), samples from carcasses of mainly roadkill animals (18) were also included and analyzed using the same technique, obtaining a positivity rate of 18 of the 39 wolves sampled (46.1%) regardless of their origin.

Further Investigations of Nitroheterocyclic Compounds as Potential Antikinetoplastid Drug Candidates.

Due to the lack of specific vaccines, management of the trypanosomatid-caused neglected tropical diseases (sleeping sickness, Chagas disease and leishmaniasis) relies exclusively on pharmacological treatments. Current drugs against them are scarce, old and exhibit disadvantages, such as adverse effects, parenteral administration, chemical instability and high costs which are often unaffordable for endemic low-income countries. Discoveries of new pharmacological entities for the treatment of these diseases are scarce, since most of the big pharmaceutical companies find this market unattractive. In order to fill the pipeline of compounds and replace existing ones, highly translatable drug screening platforms have been developed in the last two decades. Thousands of molecules have been tested, including nitroheterocyclic compounds, such as benznidazole and nifurtimox, which had already provided potent and effective effects against Chagas disease. More recently, fexinidazole has been added as a new drug against African trypanosomiasis. Despite the success of nitroheterocycles, they had been discarded from drug discovery campaigns due to their mutagenic potential, but now they represent a promising source of inspiration for oral drugs that can replace those currently on the market. The examples provided by the trypanocidal activity of fexinidazole and the promising efficacy of the derivative DNDi-0690 against leishmaniasis seem to open a new window of opportunity for these compounds that were discovered in the 1960s. In this review, we show the current uses of nitroheterocycles and the novel derived molecules that are being synthesized against these neglected diseases.