The gold standard for diagnosis of leishmaniasis is the microscopic detection of amastigotes/Leishman Donovan (LD) bodies, but its moderate sensitivity necessitates the development of molecular approaches. This study aimed to quantify in experimental animal models and human leishmaniasis the expression of amastigote-specific virulence genes, A2 and amastin by droplet digital polymerase chain reaction (ddPCR). Total RNA was isolated from L. donovani-infected hamsters or murine peritoneal macrophages and lesional biopsies from patients with post kala-azar dermal leishmaniasis (PKDL). Following cDNA conversion, EvaGreen-based ddPCR was performed using specific primers for A2 or amastin and parasite load expressed in copies per μL. Assay was optimized and the specificity of amastigote-specific A2 and amastin was confirmed. In hepatic and splenic tissues of L. donovani-infected hamsters and peritoneal macrophages, ddPCR demonstrated a greater abundance of A2 than amastin. Treatment of L. donovani-infected peritoneal macrophages with conventional anti-leishmanials, miltefosine and amphotericin B translated into a dose-dependent reduction in copies per μL of A2 and amastin, and the extrapolated IC50 was comparable with results obtained by counting LD bodies in Giemsa-stained macrophages. Similarly, in dermal biopsies of patients with PKDL, A2 and amastin were detected. Overall, monitoring of A2 by ddPCR can be an objective measure of parasite burden and potentially adaptable into a high throughput approach necessary for drug development and monitoring disease progression when the causative species is L. donovani.

Bats are mammalian hosts to a large diversity of eukaryotic protozoan blood parasites, including different genera of haemosporidians and diverse species of trypanosomes. Phylogenetic studies suggest that bats, particularly in Africa, have played an important role in the evolutionary histories of these parasite groups. However, our understanding of the diversity and distribution of chiropteran haemosporidians and trypanosomes in Africa remains tenuous. We investigated the prevalence and phylogenetic relationships of the blood parasites in different bat species in Northern Nigeria using molecular methods. A low prevalence of Hepatocystis parasites was detected in a potentially rare host species, the African straw-coloured fruit bat (Eidolon helvum) confirming yet another fruit bat species in the diverse range of African bat hosts. Trypanosome infections were identified in 3 different bat species. The trypanosomes of Mops cf. pumilus were recovered as a distinct lineage that is related to Trypanosoma erneyi, a species which is closely related to Trypanosoma dionisii and Trypanosoma cruzi. Nycteris cf. macrotis bats were infected with trypanosomes that are related to the distinct lineage of Trypanosoma cf. livingstonei parasites. Further, 2 different lineages of trypanosomes in E. helvum bats share highest nucleotide identities with Trypanosoma livingstonei and a group of Trypanosoma sp. parasites that are closely related to T. cf. livingstonei and T. livingstonei, respectively. The findings of this study confirm the notion that trypanosomes of African bats are phylogenetically diverse and that African bats might harbour a variety of yet undescribed trypanosome species.

Understanding the determinants of parasite infection in different hosts is one of the main goals of disease ecology. Evaluating the relationship between parasite–host specificity and infection parameters within host communities and populations may contribute to this understanding. Here we propose two measures of specificity that encompasses phylogenetic and ecological relatedness among hosts and investigated how such metrics explain parasite infection prevalence and mean infection intensity (MII). We analysed the parasites associated with an anuran community in an area of Atlantic Forest and used the number of infected hosts and the net relatedness index to calculate the phylogenetic and ecological specificities of the parasites. These specificity measures were related to infection metrics (prevalence and MII) with generalized linear mixed models at community (all hosts) and population (infected host species) scales. Parasite prevalence was correlated with the number of infected hosts and, when considering only multi-host parasites, was positively related to parasite ecological specificity at community and population scales. Thus, parasite species have similar prevalences in ecologically closer hosts. No relationship was found for parasite MII. Incorporating ecological characteristics of hosts in parasite specificity analyses improves the detection of patterns of specificity across scales.

There was little information regarding the occurrence of canine vector-borne disease (CVBDs) in shelter dogs in Thailand. This work is the first report regarding a molecular method used to determine the occurrence and genetic diversity of three canine tick-borne pathogens (TBPs) (Hepatozoon canis, Anaplasma platys and Ehrlichia canis) in blood samples from 275 shelter dogs in the north and central areas of Thailand. The PCR results based on the 18S rRNA and 16S rRNA genes showed that 71 (25.82%) dogs were positive for at least a TBP. The overall occurrence rates of H. canis, A. platys and E. canis infections were 1.81, 16.36 and 7.64%, respectively. For the phylogenetic analysis, A. platys 16S rRNA gene was genetically diverse, while H. canis 18S rRNA and E. canis 16S rRNA genes were conserved. The haplotype diversity exhibited 12 and 2 haplotypes as well as 78 and 178 polymorphic sites of A. platys and E. canis 16S rRNA genes, respectively. Our findings could be used to improve the understanding of phylogeny and genetic diversity of TBP rRNA genes and used to ameliorate the diagnosis and control programmes for the diseases in Thailand.

Aetiologic agents of diseases such as malaria and toxoplasmosis are found in representatives of the phylum Apicomplexa. Therefore, apicomplexan parasites are known to have a significant impact on public health. Epigenetic factors such as histone acetylation/deacetylation are among the main mechanisms of gene regulation in these parasites. Histone deacetylases (HDACs) have aroused a great deal of interest over the past 20 years for being promising targets in the development of drugs for treating several diseases such as cancer. In addition, they have also been shown to be effective for parasitic diseases. However, little is known about the structure of these proteins, as well as their interactions with specific ligands. In this paper, we modelled 14 HDACs from different apicomplexan parasites and performed molecular docking with 12 ligands analogous to the HDAC inhibitors FR235222 and apicidin, which had previously been tested against Toxoplasma gondii and Plasmodium falciparum. In this in silico study, we were able to gather relevant structural data regarding these proteins as well as insights into protein–ligand interactions for testing and developing drugs for these diseases.

In the present study, all published data on the epidemiology and molecular characters of Taenia multiceps were systematically collected from relevant databases (e.g. PubMed, Scopus, National Center for Biotechnology Information), and combined in various statistical and genetic analyses as a contribution to a better understanding of the epidemiology of this ubiquitous taeniid worldwide. While 5.8% of the key hosts (dogs) from various countries had T. multiceps, grey wolves displayed the highest prevalence (21.6%) among the definitive hosts. Small ruminants are the main intermediate hosts and carry the coenuri in various locations, but most commonly in the central nervous system (CNS). Cerebral coenuri were confirmed in 53% of sheep exhibiting neurological symptoms, and infected animals often had only a single coenurus in the brain. Sheep had a higher prevalence (8.8%) of CNS coenuri than goats (5.8%); however, extra-CNS coenuri were detected more frequently in goats than in sheep. In either case, the difference between sheep and goats was statistically insignificant. Analysis of 233 partial cytochrome oxidase subunit I nucleotide sections for T. multiceps revealed high haplotype and low nucleotide diversities. Fifty-one haplotypes were detected circulating in 6 geographic populations. China, Iran and Turkey had 2 major haplotypes, whereas Italy and Egypt shared 3. Haplotypes from Greece circulate worldwide, and displayed similar gene flow values when compared with the other populations. There were no distinct patterns for haplotype distribution in relation to the infected hosts or coenuri locations. The existence of genetic variants in T. multiceps was highlighted, but needs further studies.

Cystic echinococcosis is a zoonotic disease caused by the larval stage of the parasite Echinococcus granulosus sensu lato. The available anti-parasitic treatment is mostly limited to a continuous administration of albendazole. However, due to its numerous side-effects and efficacy of around 50%, there is a need to find new drugs to improve the treatment for this disease. In the current study, the in vitro and in vivo efficacy of a Stevia multiaristata extract against E. granulosus sensu stricto (s.s.) was demonstrated. Stevia multiaristata extract (100 and 50 μg mL−1) caused a quick viability decrease on protoscoleces which was consistent with the observed tegumental alterations. Loss of turgidity was detected in 95 ± 3.4% of cysts incubated with S. multiaristata extract during 2 days (100 μg mL−1) and the collapse of the germinal layer was observed in 60 ± 9.3% of cysts treated with 100 μg mL−1 of the S. multiaristata extract during 4 days. The half maximal effective concentration value was 69.6 μg mL−1 and the selectivity index for E. granulosus s.s. cysts was 1.9. In this clinical efficacy study, the treatment of infected mice with the S. multiaristata extract (50 mg kg−1) caused a significant decrease in the weight of the cysts compared with the control group. These results coincided with the tissue damage observed in the cysts at the ultrastructural level. In conclusion, we observed high protoscolicidal and cysticidal effects, and significant reduction in the weight of the cysts in experimentally infected mice following treatment with the S. multiaristata extract.

The taxonomy of species of Bivesicula Yamaguti, 1934 is analysed for samples from holocentrid, muraenid and serranid fishes from Japan, Ningaloo Reef (Western Australia), the Great Barrier Reef (Queensland), New Caledonia and French Polynesia. Analysis of three genetic markers (cox1 mtDNA, ITS2 and 28S rDNA) identifies three strongly supported clades of species and suggests that Bivesicula as presently recognized is not monophyletic. On the basis of combined morphological, molecular and biological data, 10 species are distinguished of which five are proposed as new. Bivesicula Clade 1 comprises seven species of which three are effectively morphologically cryptic relative to each other; all seven infect serranids and four also infect holocentrids. Bivesicula Clade 2 comprises three species of which two are effectively morphologically cryptic relative to each other; all three infect serranids and one also infects a muraenid. Bivesicula Clade 3 comprises two known species from apogonids and a pomacentrid, and forms a clade with species of Paucivitellosus Coil, Reid & Kuntz, 1965 to the exclusion of other Bivesicula species. Taxonomy in this genus is made challenging by the combination of low resolving power of ribosomal markers, the existence of regional cox1 mtDNA populations, exceptional and unpredictable host-specificity and geographical distribution, and significant host-induced morphological variation.

In response to annual outbreaks of human cercarial dermatitis (HCD) in Lake Wanaka, New Zealand, ducks and snails were collected and screened for avian schistosomes. During the survey from 2009 to 2017, four species of Trichobilharzia were recovered. Specimens were examined both morphologically and genetically. Trichobilharzia querquedulae, a species known from four continents, was found in the visceral veins of the duck Spatula rhynchotis but the snail host remains unknown. Cercaria longicauda [i.e. Trichobilharzia longicauda (Macfarlane, 1944) Davis, 2006], considered the major aetiological agent of HCD in Lake Wanaka, was discovered, and redescribed from adults in the visceral veins of the duck Aythya novaeseelandiae and cercariae from the snail Austropeplea tomentosa. Recovered from the nasal mucosa of Ay. novaeseelandiae is a new species of Trichobilharzia that was also found to cycle naturally through Au. tomentosa. Cercariae of a fourth species of Trichobilharzia were found in Au. tomentosa but the species remains unidentified.

South Africa boasts a rich diversity of small mammals of which several are commensal and harbour parasites of zoonotic importance. However, limited information is available on the parasite diversity and distribution associated with rodents in South Africa. This is particularly relevant for Micaelamys namaquensis (Namaqua rock mouse), a regionally widespread and locally abundant species that is often commensal. To address the paucity of data, the aims of the study were to record the ectoparasite diversity associated with M. namaquensis and develop distribution maps of lice and mites associated with M. namaquensis and other rodents in South Africa. Micaelamys namaquensis individuals (n = 216) were obtained from 12 localities representing multiple biomes during 2017–2018. A total of 5591 ectoparasites representing 5 taxonomic groups – fleas, lice, mesostigmatid mites, chiggers and ticks was recorded. These consisted of at least 57 taxa of which ticks were the most speciose (20 taxa). Novel contributions include new host and locality data for several ectoparasite taxa and undescribed chigger species. Known vector species were recorded which included fleas (Ctenocephalides felis, Dinopsyllus ellobius and Xenopsylla brasiliensis) and ticks (Haemaphysalis elliptica, Rhipicephalus appendiculatus and Rhipicephalus simus). Locality records indicate within-taxon geographic differences between the 2 louse species and the 2 most abundant mite species. It is clear that M. namaquensis hosts a rich diversity of ectoparasite taxa and, as such, is an important rodent species to monitor in habitats where it occurs in close proximity to humans and domestic animals.

The parasite Fasciola hepatica is an important zoonotic parasite. The development of an animal model of F. hepatica's life cycle is critical for studying the biological characteristics of the parasite in snails and mammals. Eggs of F. hepatica of bovine origin were cultured, and metacercariae were obtained after infection of Galba pervia snails. The life cycle system of F. hepatica was initiated in 2 different animals by orally infecting rabbits, SD rats and Kunming mice with the metacercariae. The animals' survival after infection, parasite migration in the animals and pathological damage to the liver were observed. We discovered that rabbits died due to acute suppurative hepatitis 60–69 days after infection, and eggs were found in the feces on day 63 of infection. The liver of SD rats showed punctate lesions on day 3 of infection, and further changes occurred as the infection progressed. However, liver repair was observed at week 9. SD rats survived for more than a year after infection and continued the F. hepatica life cycle. The liver lesions in Kunming mice after infection were similar but more severe than those in SD rats. Death was observed on the 31st post-infection day. We discovered that while rabbits, SD rats and Kunming mice can all be used as animal models of F. hepatica, SD rats are more suitable experimental animals in terms of tolerance and pathological response.

The transmission of adult parasites from prey to predatory hosts has been demonstrated for some acanthocephalan and one cestode species. Derogenes lacustris (Digenea: Hemiuroidea: Derogenidae) is a generalist parasite that infects, as an adult, the stomach of native and introduced freshwater fishes in Andean Patagonia. In the present work, the post-cyclic transmission of D. lacustris from native Galaxias maculatus (Galaxiidae) to introduced Oncorhynchus mykiss (Salmonidae) was proved experimentally. The observed transmission rate for this experimental infection was 19%. The body length of D. lacustris on day 14 post-infection was significantly greater than before transmission. The number of eggs also increased significantly after transmission, showing that D. lacustris can survive, grow and continue with egg production for at least 2 weeks in predatory salmonids. This study provides the first experimental evidence of post-cyclic transmission of trematodes and the results suggest that post-cyclic parasitism enables this species to broaden its range of hosts and distribution ranges in Argentinean Patagonia.

The vectorial role of blood-sucking arthropods, other than mosquitoes, has been explored for Dirofilaria immitis and Dirofilaria repens which are filarioids of increasing interest due to their zoonotic potential. Given the expansion of canine heartworm infection in southern Europe due to D. immitis and the report of the infection in Linosa Island (Sicily, Italy), we investigated and focused on the potential involvement of Culicoides spp. in Dirofilaria spp. transmission as well as the blood-meal source in Dirofilaria spp. molecularly positive biting midges. Out of 1791 specimens collected 1773 were morphologically and molecularly identified as Culicoides paolae and 18 as Culicoides obsoletus complex. Of the 1596 females, 4 (0.26%) parous C. paolae tested positive for D. immitis and D. repens (i.e. 2 Culicoides specimens for each filarial species), of which D. immitis-positive samples fed on both humans and dogs and D. repens-positive samples fed only on humans. The detection of D. immitis and D. repens DNA in Culicoides midges suggests their potential involvement in the epidemiology of the filarial nematodes, which may be of medical and veterinary importance in hyper-endemic areas. Findings of this study shed new light on the epidemiology of Dirofilaria spp. infections indicating that further investigations are needed.

The combined use of molecular and microscopic techniques has become an increasingly common and efficient practice for the taxonomic and evolutionary understanding of single-celled parasites such as haemogregarines. Based on this integrative approach, we characterized Hepatozoon found in Helicops angulatus snakes from the Eastern Amazonia, Brazil. The gamonts observed caused cell hypertrophy and were extremely elongated and, in some cases, piriform (mean dimensions: 25.3 ± 1.9 × 8.6 ± 1.3 μm). These morphological features correspond to Hepatozoon carinicauda, described 50 years ago in the snake Helicops carinicaudus in the southeast region of Brazil. Phylogenetic and genetic divergence analyses, performed with the sequence obtained from the amplification of a 590 bp fragment of the 18S rRNA gene, revealed that Hepatozoon in He. angulatus differed from the other lineages retrieved from GenBank, and was clustered singly in both the phylogenetic tree and the haplotype network. The integration of these data allowed the identification of H. carinicauda in a new aquatic host, and increased the knowledge of its geographical distribution. Therefore, the present study included the first redescription of a Hepatozoon species in a snake from the Brazilian Amazon.

Caryophyllideans are intestinal parasites of freshwater fishes, occupying a basal position among the ‘true’ tapeworms. We performed detailed cytogenetic analyses of the well-known caryophyllidean species Caryophyllaeus laticeps. For comparison, we also examined for the first time the chromosomes of Paracaryophyllaeus gotoi, a specific parasite of loaches in China. Both species showed a diploid chromosome number of 2n = 20, n = 10m. Chromomycin A3 (CMA3)/diamidino-2-phenylindole (DAPI) staining performed for the first time in the class Cestoda revealed CMA3+/DAPI− bands in the pericentromeric regions of the short arms of chromosome pair no. 7 in the karyotype of C. laticeps. Fluorescence in situ hybridization with the 18S rDNA probe confirmed the presence of a single cluster of major rDNA near the centromere on a pair of small chromosomes in both species. These findings support the hypothesis that the ancestral state in the family Caryophyllaeidae is a single interstitial cluster of major rDNA genes and thus one nucleolar organizer region per haploid genome. Our results, which we presented together with literature data plotted on a phylogenetic tree, show stability of caryophyllidean karyotypes at the genus level, but showed differences between genera without a clear phylogenetic signal. The data allowed us to at least formulate a hypothesis about the ancestral haploid chromosome number of n = 10 for the family Caryophyllaeidae and possibly for the sister family Capingentidae. In addition, we compared two populations of C. laticeps from water bodies with different levels of polychlorinated biphenyl contamination, showing a slightly increased incidence of chromosomal abnormalities at the contaminated site.

Migratory birds are implicated in dispersing haemosporidian parasites over great geographic distances. However, their role in sharing these vector-transmitted blood parasites with resident avian host species along their migration flyway is not well understood. We studied avian haemosporidian parasites in 10 localities where Chilean Elaenia, a long-distance Neotropical austral migrant species, spends part of its annual cycle to determine local parasite transmission among resident sympatric host species in the elaenia's distributional range across South America. We sampled 371 Chilean Elaenias and 1,818 birds representing 243 additional sympatric species from Brazilian wintering grounds to Argentinian breeding grounds. The 23 haemosporidian lineages found in Chilean Elaenias exhibited considerable variation in distribution, specialization, and turnover across the 10 avian communities in South America. Parasite lineage dissimilarity increased with geographic distance, and infection probability by Parahaemoproteus decreased in localities harbouring a more diverse haemosporidian fauna. Furthermore, blood smears from migrating Chilean Elaenias and local resident avian host species did not contain infective stages of Leucocytozoon, suggesting that transmission did not take place in the Brazilian stopover site. Our analyses confirm that this Neotropical austral migrant connects avian host communities and transports haemosporidian parasites along its distributional range in South America. However, the lack of transmissive stages at stopover site and the infrequent parasite lineage sharing between migratory host populations and residents at breeding and wintering grounds suggest that Chilean Elaenias do not play a significant role in dispersing haemosporidian parasites, nor do they influence local transmission across South America.

The poultry red mite (PRM), Dermanyssus gallinae, is one of the most detrimental ectoparasite on poultry farms worldwide. The blood fed on birds provides the mites with nutrition and energy for their activities, development and reproduction. In the evaluation of the efficacy of novel drugs or vaccines against PRMs, their effects on blood digestion are generally used as a key parameter. The blood digestion of haematophagous arthropods (including D. gallinae) is usually assessed by weighing; however, this method shows some limitations. The main objective of the present study was to develop a scoring method that can quickly and visually evaluate the blood digestion status of PRMs. A 0–4 point scoring criterion was established to describe the blood digestion status of D. gallinae based on the changes in appearance in the intestinal tract of PRMs during the blood digestion process. There was a good consistency between the results obtained by the blood digestion scoring and the weighing, indicating the reliability of this new method. The results obtained from volunteers were consistent with the results from researchers with low coefficient of variation, indicating that the scoring method has good practicability. The applicability of the scoring method was confirmed in an efficacy study, where it was found that doramectin could significantly inhibit the blood digestion of PRMs, lowering the blood digestion score.

The vast majority of trypanosome species is vector-borne parasites, with some of them being medically and veterinary important (such as Trypanosoma cruzi and Trypanosoma brucei) and capable of causing serious illness in vertebrate hosts. The discovery of trypanosomes in bats emphasizes the importance of bats as an important reservoir. Interestingly, there is a hypothesis that bats are ancestral hosts of T. cruzi. Trypanosome diversity has never been investigated in bats in Thailand, despite being in a biodiversity hot spot. To gain a better understanding of the diversity and evolutionary relationship of trypanosomes, polymerase chain reaction-based surveys were carried out from 2018 to 2020 in 17 sites. A total of 576 bats were captured, representing 23 species. A total of 38 (6.6%) positive samples was detected in ten bat species. Trypanosoma dionisii and Trypanosoma noyesi were identified from Myotis siligorensis and Megaderma spasma, respectively. The remaining 18S rRNA sequences of trypanosomes were related to other trypanosomes previously reported elsewhere. The sequences in the current study showed nucleotide identity as low as 90.74% compared to those of trypanosomes in the GenBank database, indicating the possibility of new species. All bat trypanosomes identified in the current study fall within the T. cruzi clade. The current study adds to evidence linking T. noyesi to a bat trypanosome and further supports the bat host origin of the T. cruzi clade. To the best of authors' knowledge, this is the first study on bat trypanosomes in Thailand and their phylogenetic relationships with global isolates.

Oxyurid nematodes (Syphacia spp.) from bank (Myodes glareolus) and field/common (Microtus spp.) voles, from disparate geographical sites in the British Isles, were examined morphologically and genetically. The genetic signatures of 118 new isolates are provided, based primarily on the rDNA internal transcribed spacers (ITS1-5.8S-ITS2) region and for representative isolates also on the small subunit 18S rDNA region and cytochrome c oxidase subunit 1 (cox-1) gene locus. Genetic data on worms recovered from Microtus spp. from the European mainland and from other rodent genera from the Palaearctic, North America and West Africa are also included. We test historical hypotheses indicating that S. nigeriana is a generalist species, infecting a range of different rodent genera. Our results establish that S. nigeriana is a parasite of both bank and field voles in the British Isles. An identical genotype was also recorded from Hubert's multimammate mouse (Mastomys huberti) from Senegal, but Mastomys spp. from West Africa were additionally parasitized by a related, although genetically distinct Syphacia species. We found no evidence for S. petrusewiczi in voles from the British Isles but isolates from Russia and North America were genetically distinct and formed their own separate deep branch in maximum likelihood molecular phylogenetic trees.

The ichthyofauna of the Atlantic and Pacific coasts off the American continent is very rich. Consequently, a high biodiversity of nematodes parasitizing these vertebrates is also expected. Currently, data on nematode parasites of marine fish off the Americas are fragmented. A review of all adult nematode species reported parasitizing marine fish from off the American continent is herein presented, as well as comments on their patterns of diversity, life cycles and advances in the taxonomic and phylogenetic knowledge. A total of 209 valid species, 19 species inquirendae and 6 dubious records have been recorded, the majority from the fish taxa Eupercaria and Perciformes. The families Sciaenidae, Serranidae and Lutjanidae, as well as the tropical and temperate Atlantic waters, exhibited the highest records of parasitic nematodes. The Cucullanidae, Philometridae and Cystidicolidae were the most speciose families of nematodes, which may be related to technological advances and relatively recent efforts of taxonomists, resulting in description of new taxa and the resolution of taxonomic problems. Numerous taxonomic questions still need resolution and, even though genetic data have been important for this process, the database is very scarce. This is the first review on all currently known nematode species parasitizing marine fish off the Americas and may serve as an important basis of reference for future approaches on these organisms.