The synthesis and antiprotozoal activity of some simple dialkyl pyrazole-3,5-dicarboxylates (compounds 2–6) and their sodium salts (pyrazolates) (compounds 7–9) against Trypanosoma cruzi, Leishmania infantum and Leishmania braziliensis are reported. In most cases the studied compounds showed, especially against the clinically significant amastigote forms, in vitro activities higher than those of the reference drugs (benznidazole for T. cruzi and glucantime for Leishmania spp.); furthermore, the low non-specific cytotoxicities against Vero cells and macrophages shown by these compounds led to good selectivity indexes, which are 8–72 times higher for T. cruzi amastigotes and 15–113 times higher for Leishmania spp. amastigotes than those of the respective reference drugs. The high efficiency of diethyl ester 3 and its sodium salt 8 against the mentioned protozoa was confirmed by further in vitro assays on infection rates and by an additional in vivo study in a murine model of acute and chronic Chagas disease. The inhibitory capacity of compounds 3 and 8 on the essential iron superoxide dismutase of the aforementioned parasites may be related to the observed anti-trypanosomatid activity. The low acute toxicity of compounds 3 and 8 in mice is also reported in this article.

Angiostrongylus vasorum is a cardiovascular nematode increasingly found in dogs and foxes in endemic foci throughout Europe. The present study evaluates ELISAs for detection of circulating antigens and specific antibodies against A. vasorum in foxes. Blood and worm burdens (WBs) from carcasses of 215 Swiss wild red foxes (Vulpes vulpes) and from 75 farmed foxes of different age groups experimentally inoculated once or repeatedly with infective doses of 50, 100 or 200 third-stage larvae were obtained. Antigen detection in the naturally infected Swiss foxes had 91·2% sensitivity and 89·4% specificity, whereas the corresponding figures for antibody detection were 42·2 and 92·0%. The experimentally infected foxes became positive for circulating antigens 5–10 weeks post-inoculation (wpi) and remained highly positive up to 22 wpi, irrespectively of further challenge inoculation. The antibody responses in the same foxes were highly variable: high optical density (OD) values were reached 5–7 wpi in all animals, followed by a decrease in over half of the animals despite accumulating and consequently high WBs resulting in persistent infections. After each challenge, a slight increase of OD values was observed 7 weeks later. We hypothesize that infected foxes develop a variable and non-protective immunity. Such parasite tolerance allows long-term survival of A. vasorum in the animals, and may explain why the parasite appears to spread rapidly within a fox population, an epidemiological dynamic that is evident in many parts of Europe where A. vasorum has been found over the last decades.

Little is known about the genetic diversity of the protozoan parasite, Giardia duodenalis, infecting humans in Queensland, Australia. The present study typed 88 microscopically Giardia-positive isolates using assemblage-specific primers at the triose phosphate isomerase (tpi) gene and sequenced a subset of isolates at the glutamate dehydrogenase (gdh) gene (n = 30) and tpi locus (n = 27). Using the tpi-assemblage specific primers, G. duodenalis assemblage A and assemblage B were detected in 50% (44/88) and 38·6% (34/88) of samples, respectively. Mixed infections with assemblages A and B were identified in 4·5% (4/88) and assemblage E was identified in 6·8% (6/88) of samples. Sequence analysis at the gdh and tpi loci also confirmed the presence of assemblage E in these isolates. Cyst numbers per gram of feces (g−1) were determined using quantitative polymerase chain reaction and of the isolates that were typed as assemblage E, cyst numbers ranged 13·8–68·3 × 106 cysts g−1. This is the first report of assemblage E in humans in Australia, indicating that in certain settings, this assemblage may be zoonotic.

Tabanids are haematophagous flies feeding on livestock and wildlife. In the absence of information on the relationship of tabanid flies and protozoan parasites in South Africa and Zambia, the current study was aimed at characterizing tabanid flies collected in these two countries as well as detecting protozoan parasites they are harbouring. A total of 527 tabanid flies were collected whereby 70·2% were from South Africa and 29·8% were from Zambia. Morphological analysis revealed a total of five different genera collected from the sampled areas namely: Ancala, Atylotus, Haematopota, Philoliche and Tabanus. DNA extracted from South African Tabanus par and Tabanus taeniola tested positive for the presence of Trypanosoma congolense (Savannah) and Trypanosoma theileri whilst one member from T. par was positive for Trypanosoma brucei species. DNA extracted from Zambian tabanid flies tested positive for the presence of Besnoitia species at 1·27% (2/157), Babesia bigemina 5·73% (9/157), Theileria parva 30·11% (30/157) and 9·82% (14/157) for Trypanosoma evansi. This study is the first to report on relationship of Babesia and Theileria parasites with tabanid flies. Further investigations are required to determine the role of tabanids in transmission of the detected protozoan parasites in livestock and wildlife in South Africa and Zambia.

Leishmania (Viannia) braziliensis is a protozoan that causes mucocutaneous leishmaniasis, which is an infectious disease that affects more than 12 million people worldwide. The available treatment is limited, has side-effects or is inefficient. In a search for alternative compounds of natural origin, we tested the microbicidal activity of Piper aduncum essential oil (PaEO) on this parasite. Our data showed that PaEO had an inhibitory effect on the growth of L. braziliensis promastigotes with an IC50/24 h=77·9 µg mL−1. The main constituent (nerolidol: 25·22%) presented a similar inhibitory effect (IC50/24 h = 74·3 µg mL−1). Ultrastructural observation of nerolidol-treated parasites by scanning and transmission electron microscopies revealed cell shrinkage and morphological alterations in the mitochondrion, nuclear chromatin and flagellar pocket. Flow cytometry analysis showed a reduction in the cell size, loss of mitochondrial membrane potential, phosphatidylserine exposure and DNA degradation, which when associated with the morphological changes indicated that nerolidol induced incidental cell death in the L. braziliensis promastigotes. The results presented here indicate that nerolidol derivatives are promising compounds for further evaluation against Leishmania parasites.

In South America, visceral leishmaniasis is frequently caused by Leishmania infantum and, at an unknown frequency, by Leishmania amazonensis. Therefore, mixed infections with these organisms are possible. Mixed infections might affect the clinical course, immune response, diagnosis, treatment and epidemiology of the disease. Here we describe the clinical course of mixed infections with L. amazonensis and L. infantum in a hamster model. We show that mixed infections are associated with more severe clinical disease than infection with L. amazonensis or L. infantum alone. In spleens with mixed infections, L. infantum outcompeted L. amazonensis in the tissue, but not in culture from tissue. We found increased levels of IgG in animals infected with L. infantum. Although more than 30 bands were revealed in a Western blot, the highest immunogenicity was observed with proteins having molecular masses of 95 and 90 kDa, whereas proteins with molecular masses of lower than 50 kDa were reactive frequently with serum from hamsters infected with L. amazonensis, and proteins with molecular masses of 80 and 70 kDa were reactive only with serum from hamsters infected with L. infantum. This finding has important implications regarding the biology of Leishmania and humoral immune responses to infections with these organisms.

Babesia are intraerythrocytic parasites of importance worldwide within the fields of human and veterinary medicine, as some Babesia sp., including Babesia microti are potentially zoonotic and can cause fatal disease in both humans and animals. The aims of this study were to use a nested PCR (amplifying the 18S rRNA gene) to determine the presence and species of Babesia parasite DNA found in blood (n = 47) and spleen (n = 47) samples collected from Eurasian badgers (Meles meles) in Scotland. The results showed 28/47 (59·6%) blood and 14/47 (29·8%) spleen samples tested positive for the presence of Babesia DNA. Initial sequence analysis of the Babesia DNA identified three distinct sequence types (submitted to GenBank KX528553, KX528554 and KX528555), which demonstrated ⩾99% identity to Babesia sp. parasites previously identified in badgers in Spain (KT223484 and KT223485). Phylogenetic analysis showed that the three isolates are closely related to Babesia annae, B. microti and other Piroplasmida species found in wildlife. Further sequence analysis of the samples demonstrated that the badgers were routinely infected with more than one parasite isolate and there was also evidence of genetic recombination between the Babesia parasite isolates (submitted to GenBank KY250472 – KY250477)

To reveal factors responsible for spatial variation in parasite community composition we studied patterns of similarity in helminth species composition in two closely-related rodents (Rhabdomys pumilio and Rhabdomys dilectus) that differ in their social and spatial behaviour and live under different environmental conditions across 20 localities in South Africa. We asked whether the two hosts harbour similar assemblages, whether these are more dissimilar between than within hosts and if host social structure, behaviour or environment affects similarity patterns in helminth infracommunities within and among localities. We also investigated whether similarity in species composition of helminth component communities decreases with an increase of geographic distance between host populations. We found that the pattern of space use by the hosts rather than their social behaviour promotes differences in helminth species composition between host species as well as among host populations from different localities. The rate of distance decay of similarity in species composition of helminth component communities differed between the two hosts due to difference in the degree of environmental variation across their geographic ranges. We conclude that patterns of spatial variation in helminth species composition are driven mainly by host spatial behaviour and, to a lesser extent, by environment-associated factors.

Field surveys indicate that host population size, rather than density, is the most important determinant of monogenean infection dynamics. To verify this prediction, epidemic parameters were monitored for 70 days at five host population sizes held at constant density using a goldfish – Gyrodactylus kobayashii laboratory model. During the first 20 days, the rate of increase of prevalence and mean abundance was faster in small host populations. Total mean prevalence and total mean abundance throughout the experiment were not significantly affected by host population sizes. Higher transmission rates were detected in larger host populations. However, there were no significant differences in effective contact rates among the five host populations on each sampling day during the first 20 days, implying that contact rates may be saturated at a sufficiently high host density. These results demonstrate that the epidemic occurs more quickly in smaller host populations at the beginning of the experiment. However, the epidemic is independent of the host population size due to the similar effective contact rates in the five population sizes. Significant negative influence of the initial body condition (Kn) of uninfected goldfish on total mean abundance of parasites suggests that susceptibility of hosts is also a determinant of parasite transmission.

Few studies have focussed on the health and immunity of triploid Atlantic salmon and therefore much is still unknown about their response to commercially significant pathogens. This is important if triploid stocks are to be considered for full-scale commercial production. This study aimed to investigate and compare the response of triploid and diploid Atlantic salmon to an experimental challenge with Neoparamoeba perurans, causative agent of amoebic gill disease (AGD). This disease is economically significant for the aquaculture industry. The results indicated that ploidy had no significant effect on gross gill score or gill filaments affected, while infection and time had significant effects. Ploidy, infection and time did not affect complement or anti-protease activities. Ploidy had a significant effect on lysozyme activity at 21 days post-infection (while infection and time did not), although activity was within the ranges previously recorded for salmonids. Stock did not significantly affect any of the parameters measured. Based on the study results, it can be suggested that ploidy does not affect the manifestation or severity of AGD pathology or the serum innate immune response. Additionally, the serum immune response of diploid and triploid Atlantic salmon may not be significantly affected by amoebic gill disease.

Eggs of chicken ascarids (Ascaridia galli and Heterakis spp.) are believed to be hardy and survive for long periods. However, this has not been evaluated quantitatively and our study therefore aimed to determine development and recovery of chicken ascarid eggs after burying in pasture soil. Unembryonated eggs were mixed with soil, placed in sealed nylon bags and buried at 7 cm depth in pasture plots April (spring, n = 72) and December 2014 (winter, n = 72). Eight randomly selected bags per season were used to estimate pre-burial egg recovery [0 week post-burial (wpb)]. Eight random bags were removed at 5, 12, 23, 38, 52, 71 wpb per season and additionally at 104 wpb for spring burial. The content of each bag was analysed for numbers and development stages of eggs. Eggs buried in spring were fully embryonated within 12 wpb. In contrast, eggs buried in winter were developing between 23 and 38 wpb, so that all viable eggs seemed to be fully developed by 38 wpb. About 90% eggs disappeared within 23 wpb (spring) and 38 wpb (winter). Small proportions (2–3%) of seemingly viable and infective eggs were still recovered up to 2 years after deposition. In conclusion, most eggs buried in temperate pasture soil seem to experience a heavy mortality within a few months after the deposition, especially during warm periods. However, a small proportion of eggs may survive and remain infective for at least 2 years.

The majority of research on Histomonas meleagridis was performed in the first half of the last century, especially those on morphological aspects. In the present study identical monoxenic settings for cultures of the same H. meleagridis clonal strain in its virulent low passage and attenuated high passage form enabled a comparative analysis of parasite characteristics. For the first time, it could be shown that long-term in vitro cultivation led to a severe shift in cell morphology, with the occurrence of a very distinct phenotype expressing a flagellated and highly amoebic cell morphology. Furthermore, the attenuated parasites showed better growth rates and a higher tenacity when confronted with adverse conditions. During these experiments up to 100% of the parasites, both virulent and attenuated, assumed a completely rounded morphology elucidated by electron microscopy. The findings indicate that such previously reported cyst-like stages are a defence strategy of H. meleagridis, independent of the passage level in vitro and pathogenicity in vivo. In conclusion, long-term in vitro passaging of H. meleagridis led not only to an attenuation of the parasite, as previously demonstrated, but also to a shift in the parasite's phenotype regarding morphology, growth behaviour and a higher level of tenacity.

Phosphoenolpyruvate carboxykinase (PEPCK) is involved in glycolysis in the cestode parasite, Raillietina echinobothrida; whereas, it executes a gluconeogenic role in its host, Gallus domesticus. Because of its differing primary function in the cestode parasite and its host, this enzyme is regarded as a plausible anthelmintic target. Hence, the biological significance of PEPCK in the parasite was analysed using siRNA against PEPCK from R. echinobothrida (RePEPCK). In order to find out the functional differences between RePEPCK and GdPEPCK (PEPCK from its host, G. domesticus), PEPCK genes from both sources were cloned, over-expressed, characterized, and some properties of the purified enzymes were compared. RePEPCK and GdPEPCK showed a standard Michaelis-Menten kinetics with Kmapp of 46.9 and 22.9 µm, respectively, for phosphoenolpyruvate and Kmapp of 15.4 µm for oxaloacetate in GdPEPCK decarboxylation reaction. Here, we report antagonist behaviours of recombinant PEPCKs derived from the parasite and its host. In search of possible modulators for PEPCK, few phytoestrogens were examined on the purified enzymes and their inhibitory constants were determined and discussed. This study stresses the potential of these findings to validate PEPCK as the anthelmintic drug target for parasitism management.