The biological and genetic diversity of Neospora caninum is very limited because of availability of only a few viable isolates worldwide. This study describes the isolation and biological and molecular characterization of a new viable isolate of N. caninum (NC-SP1), from a cattle in Brazil. Approximately 400 g of brain from a naturally infected adult male cattle from an abattoir was fed to a 2-month-old dog. Neospora-like oocysts were observed on day 7 post-inoculation (PI) and the duration of oocyst shedding was 14 days. The DNA obtained from oocysts was characterized molecularly and the final sequence was 99% identical to homologous sequences of N. caninum available in GenBank®. For bioassay, gerbils (Meriones unguiculatus) were orally inoculated with 10 100 and 1000 oocysts; all gerbils remained clinically normal but developed N. caninum antibodies 14 days PI. Cell culture isolation was successful using the brain homogenate from one of the gerbils and tachyzoites were observed 24 days PI. Microsatellite genotyping revealed a unique genetic profile for this new reference isolate.

Trichinella spiralis is a parasitic helminth that can infect almost all mammals, including humans. Trichinella spiralis infection elicits a typical type 2 immune responses, while suppresses type 1 immune responses, which is in favour of their parasitism. DNA vaccines have been shown to be capable of eliciting balanced CD4+ and CD8+ T cell responses as well as humoral immune responses in small-animal models, which will be advantage to induce protective immune response against helminth infection. In this study, serine protease (Ts-NBLsp) was encoded by a cDNA fragment of new-born T. spiralis larvae, and was inserted after CMV promoter to construct a DNA vaccine [pcDNA3·1(+)-Ts-NBLsp]. Ts-NBLsp expression was demonstrated by immunofluorescence. Sera samples were obtained from vaccinated mice, and they showed strong anti-Ts-NBLsp-specific IgG response. Mice immunized with the pcDNA3·1(+)-Ts-NBLsp DNA vaccine showed a 77·93% reduction in muscle larvae (ML) following challenge with T. spiralis ML. Our results demonstrate that the vaccination with pcDNA3·1(+)-Ts-NBLsp plasmid promoted the balance of type 1 and 2 immune responses and produced a significant protection against T. spiralis infection in mice.

Numerous studies have been conducted on the cellular morphology of Cryptocaryon irritans. However, details regarding the tomont stage of its life cycle remain lacking. In this study, we investigated the morphology of the tomont stage throughout encystment and cell division using light and electron microscopy. Results showed that there was no secretion of encystation-specific secretory vesicles or extrusomes during formation of the cyst wall. Instead, the synthesis and construction of the C. irritans cyst wall materials may involve molecular events at the pellicle. The somatic cilia and the cytostome were present during encystment and covered by the newly formed cyst wall. New somatic cilia were continuously created between old cilia and showed various lengths during cell division, a process that was similar to morphogenesis in many free-living ciliates. During cell division inside the tomont, dividing daughter cells formed temporary cell chains with no oral primordia before separating from each other into dissociative tomite precursors. The process of cell division may not be accompanied by stomatogenesis, and new oral primordia in offspring cells likely formed before the dividing cell chains split into dissociative spherical tomites. Mitochondrial autophagy was observed in encysting C. irritans cells. Numerous endosymbionts and Golgi structures were observed in the tomont cytoplasm. Cellular metabolic activity in the C. irritans tomont was quite high, with large amounts of materials or cellular organelles potentially being synthesized and prepared for the following infective theront stage.

The distribution of Hepatozoon canis mainly encompasses areas where its main tick vector, Rhipicephalus sanguineus sensu lato, is present. However, the detection of this pathogen in dogs, foxes and golden jackals well outside the areas inhabited by this tick species reinforced the hypothesis that additional ixodids are involved in the life cycle and transmission of this protozoon. The present study provides, for the first time, data supporting the sporogonic development of H. canis in specimens of Rhipicephalus turanicus collected from a naturally infected fox from southern Italy. The epidemiological role of R. turanicus as a vector of H. canis is discussed, along with information on the potential use of cell cultures for the experimental infection with H. canis sporozoites. The in vitro infection of canine leucocytes by sporozoites from ticks is proposed as a potential tool for future in-depth studies on the biology of H. canis.

This study reports an outbreak of oriental theileriosis in dairy cattle imported to Vietnam from Australia. Following clinical and pathological diagnoses, a total of 112 cattle blood samples were divided into three groups and tested using multiplexed tandem PCR. Group 1 were from aborted heifers in Vietnam; group 2 were from cattle before shipment from group 1 cattle and group 3 were from the same batch of cattle but transported to Taiwan. Theileria orientalis DNA was detected in 72·3% cattle. The prevalences of T. orientalis in groups 1, 2 and 3 were 77·6, 86·9 and 57·5%, respectively, and the difference in prevalence was significant between groups 1 and 3 (P < 0·0001). The infection intensities of genotypes chitose and ikeda of T. orientalis were higher in groups 1 (57 721 and 33 709, respectively) and 3 (5897 and 61 766, respectively) than those in group 2 (2071 and 6331, respectively). Phylogenetic analyses of the major piroplasm surface protein sequences revealed that genotypes chitose and ikeda determined herein were closely related to those previously reported from Australia. This first report of an outbreak of oriental theileriosis in imported cattle emphasizes improved measures for the export and import of cattle infected with T. orientalis.

Xanthohumol (Xan) is a natural constituent of human nutrition. Little is known about its actions on leishmanial parasites and their mitochondria as putative target. Therefore, we determined the antileishmanial activity of Xan and resveratrol (Res, as alternative compound with antileishmanial activity) with respect to mitochondria in Leishmania amazonensis promastigotes/amastigotes (LaP/LaA) in comparison with their activity in peritoneal macrophages from mouse (PMM) and macrophage cell line J774A.1 (J774). Mechanistic studies were conducted in Leishmania tarentolae promastigotes (LtP) and mitochondrial fractions isolated from LtP. Xan and Res demonstrated antileishmanial activity in LaA [half inhibitory concentration (IC50): Xan 7 µm, Res 14 µm]; while they had less influence on the viability of PMM (IC50: Xan 70 µm, Res >438 µm). In contrast to Res, Xan strongly inhibited oxygen consumption in Leishmania (LtP) but not in J774 cells. This was based on the inhibition of the mitochondrial electron transfer complex II/III by Xan, which was less pronounced with Res. Neither Xan nor Res increased mitochondrial superoxide release in LtP, while both decreased the mitochondrial membrane potential in LtP. Bioenergetic studies showed that LtP mitochondria have no spare respiratory capacity in contrast to mitochondria in J774 cells and can therefore much less adapt to stress by mitochondrial inhibitors, such as Xan. These data show that Xan may have antileishmanial activity, which is mediated by mitochondrial inhibition.

Chagas disease is one of the most important vector-borne zoonotic diseases in Latin America. Control strategies could be improved if transmissibility patterns of its aetiologic agent, Trypanosoma cruzi, were better understood. To understand transmissibility patterns of Chagas disease in Mexico, we inferred potential vectors and hosts of T. cruzi from geographic distributions of nine species of Triatominae and 396 wild mammal species, respectively. The most probable vectors and hosts of T. cruzi were represented in a Complex Inference Network, from which we formulated a predictive model and several associated hypotheses about the ecological epidemiology of Chagas disease. We compiled a list of confirmed mammal hosts to test our hypotheses. Our tests allowed us to predict the most important potential hosts of T. cruzi and to validate the model showing that the confirmed hosts were those predicted to be the most important hosts. We were also able to predict differences in the transmissibility of T. cruzi among triatomine species from spatial data. We hope our findings help drive efforts for future experimental studies.

The present study determined the prevalence, hematological findings and genetic diversity of Bartonella spp. in domestic cats from Valdivia, Southern Chile. A complete blood count and nuoG gene real-time quantitative PCR (qPCR) for Bartonella spp. were performed in 370 blood samples from cats in Valdivia, Southern Chile. nuoG qPCR-positive samples were submitted to conventional PCR for the gltA gene and sequencing for species differentiation and phylogenetic analysis. Alignment of gltA gene was used to calculate the nucleotide diversity, polymorphic level, number of variable sites and average number of nucleotide differences. Bartonella DNA prevalence in cats was 18·1% (67/370). Twenty-nine samples were sequenced with 62·0% (18/29) identified as Bartonella henselae, 34·4% (10/29) as Bartonella clarridgeiae, and 3·4% (1/29) as Bartonella koehlerae. Bartonella-positive cats had low DNA bacterial loads and their hematological parameters varied minimally. Each Bartonella species from Chile clustered together and with other Bartonella spp. described in cats worldwide. Bartonella henselae and B. clarridgeiae showed a low number of variable sites, haplotypes and nucleotide diversity. Bartonella clarridgeiae and B. koehlerae are reported for the first time in cats from Chile and South America, respectively.

Current theories on diversity–disease relationships describe host species diversity and species identity as important factors influencing disease risk, either diluting or amplifying disease prevalence in a community. Whereas the simple term ‘diversity’ embodies a set of animal community characteristics, it is not clear how different measures of species diversity are correlated with disease risk. We therefore tested the effects of species richness, Pielou's evenness and Shannon's diversity on bovine tuberculosis (bTB) risk in cattle in the Afar Region and Awash National Park between November 2013 and April 2015. We also analysed the identity effect of a particular species and the effect of host habitat use overlap on bTB risk. We used the comparative intradermal tuberculin test to assess the number of bTB-infected cattle. Our results suggested a dilution effect through species evenness. We found that the identity effect of greater kudu – a maintenance host – confounded the dilution effect of species diversity on bTB risk. bTB infection was positively correlated with habitat use overlap between greater kudu and cattle. Different diversity indices have to be considered together for assessing diversity–disease relationships, for understanding the underlying causal mechanisms. We posit that unpacking diversity metrics is also relevant for formulating disease control strategies to manage cattle in ecosystems characterized by seasonally limited resources and intense wildlife–livestock interactions.

Trichinella spiralis (T.spiralis) muscle-larva (ML) excretory–secretory proteins (ESPs) contain antitumour-active substances. ESPs have been shown to inhibit tumour growth. To explore the effects of these proteins on small cell lung cancer cells and the possible mechanisms of their antineoplastic action, H446 SCLC cells were co-cultured with different concentrations of T. spiralis ML ESPs for 12, 24 and 48 h. Our results showed that T. spiralis ML ESPs significantly inhibited H446 cell proliferation, which was dose-and time-dependent. The results of flow cytometry testing indicate a clear apoptosis trend in H446 cells co-cultured with ESPs for 24 h. Reverse transcription polymerase chain reaction and Western blotting results showed increased expression of pro-apoptosis genes Bax, Cyt-C, Apaf-1, caspase-9 and caspase-3, compared with the negative control group, and decreased the expression of anti-apoptosis genes Bcl-2 and Livin. Our results suggest that T. spiralis ML ESPs can induce apoptosis in H446 cells through a mitochondrial pathway, which may be a mechanism of antineoplastic action in T. spiralis ML ESPs.

Parasites can influence host population dynamics, community composition and evolution. Prediction of these effects, however, requires an understanding of the influence of ecological context on parasite distributions and the consequences of infection for host fitness. We address these issues with an amphibian – trematode (Digenea: Echinostomatidae) host–parasite system. We initially performed a field survey of trematode infection in first (snail) and second (larval green frog, Rana clamitans) intermediate hosts over 5 years across a landscape of 23 ponds in southeastern Michigan. We then combined this study with a tadpole enclosure experiment in eight ponds. We found echinostomes in all ponds during the survey, although infection levels in both snails and amphibians differed across ponds and years. Echinostome prevalence (proportion of hosts infected) in snails also changed seasonally depending on host species, and abundance (parasites per host) in tadpoles depended on host size and prevalence in snails. The enclosure experiment demonstrated that infection varied at sites within ponds, and tadpole survival was lower in enclosures with higher echinostome abundance. The observed effects enhance our ability to predict when and where host–parasite interactions will occur and the potential fitness consequences of infection, with implications for population and community dynamics, evolution and conservation.

Strongyloides stercoralis is a parasite that can cause death in immunocompromised people. A proper diagnosis is hence essential. The real-time polymerase-chain reaction (RT–PCR) is a novel, promising diagnostic method, that detects the DNA of the parasite in stool samples. In this retrospective study, we compared the sensitivity of agar plate coproculture (APC), an in-house immunofluorescence test (IFAT) and an in-house RT–PCR for the diagnosis of S. stercoralis infection. The study sample was composed by 223 samples. Samples resulting positive to APC, IFAT and RT–PCR were 20, 140 and 25, respectively. When sensitivity was calculated against a composite reference standard, serology confirmed the best performance (sensitivity 95%), followed by RT–PCR (57%) and APC (45%). In conclusion, in a non-endemic setting, serology is the best screening method, while the combination of APC and RT–PCR does not seem a reasonable approach to increase sensitivity. Both methods can have a role as confirmatory tests for selected cases.

Across host–parasite systems, there is evidence that pesticide exposure increases parasite loads and mortality following infection. However, whether these effects are driven by reductions in host resistance to infection or slower rates of parasite clearance is often unclear. Using controlled laboratory experiments, we examined the ability of larval northern leopard frogs (Lithobates pipiens) and American toads (Anaxyrus americanus) to resist and clear trematode (Echinoparyphium sp.) infections following exposure to the insecticide carbaryl. Northern leopard frogs exposed to 1 mg L−1 of carbaryl had 61% higher parasite loads compared with unexposed individuals, while there was no immediate effect of carbaryl on parasite encystment in American toads. However, when tadpoles were exposed to carbaryl and moved to freshwater for 14 days before the parasite challenge, we recovered 37 and 63% more parasites from carbaryl-exposed northern leopard frogs and American toads, respectively, compared with the control. No effects on clearance were found for either species. Collectively, our results suggest that pesticide exposure can reduce the ability of amphibians to resist parasite infections and that these effects can persist weeks following exposure. It is critical for researchers to incorporate species interactions into toxicity studies to improve our understanding of how contaminants affect ecological communities.

The dilution effect (DE) has been reported in many diseases, but its generality is still highly disputed. Most current criticisms of DE are related to animal diseases. Particularly, some critical studies argued that DE is less likely to occur in complex environments. Here our meta-analyses demonstrated that the magnitude of DE did not differ between animal vs plant diseases. Moreover, DE generally occurs in all three subgroups of animal diseases, namely direct-transmitted diseases, vector-borne diseases and diseases caused by parasites with free-living stages. Our findings serve as an important contribution to understanding the generality of DE.

Neospora caninum is an apicomplexan parasite strongly related to reproductive problems in cattle. The neosporosis control is not well established and several fronts are under development, predominantly based on immune protection, immunomodulation and chemotherapy. The use of anti-malarial drugs as therapeutic sources has, in theory, considerable potential for any apicomplexan. Drugs such as methylene blue (MB) and pyrimethamine (Pyr) represent therapeutic options for malaria; thus, their use for neosporosis should be assessed. In this work, we tested the effects of MB and Pyr on N. caninum proliferation and clearance, using LacZ-tagged tachyzoites. The drugs inhibited at nanomolar dosages and its combination demonstrated an antagonistic interaction in proliferation assays, according to the Chou and Talalay method for drug combination index. However, the drug combination significantly improved the parasite in vitro clearance. The repositioning of well-established drugs opens a short-term strategy to obtain low-cost therapeutics approaches against neosporosis.

In this investigation, the host–parasite relationship of ninespine stickleback fish Pungitius pungitius and the cestode parasite Schistocephalus pungitii was studied using samples from Dog Bone Lake, Kenai Peninsula, Alaska, to test the hypothesis that S. pungitii is a castrator of ninespine stickleback. Infected, adult females of all sizes (ages) were capable of producing clutches of eggs. S. pungitii had a negative effect on the ability of host females to produce a clutch, which was related to increasing parasite:host mass ratio (parasite index, PI). Among infected females with egg clutches, both clutch size and egg size were reduced; and the reduction increased with greater PI. The results of this study are consistent with the hypothesis that S. pungitii causes host sterility as a result of simple nutrient theft and is not a true castrator as hypothesized in earlier reports. The degree of parasite-induced sterility appears to vary among populations of the ninespine stickleback, perhaps reflecting differences in resource availability. Populations of ninespine stickleback appear to show a greater reduction in host reproductive capacity with PI than populations of the threespine stickleback infected by Schistocephalus solidus, possibly owing, in part, to the length-adjusted somatic mass of the threespine stickleback being greater.

Trypanosoma cruzi epimastigote reservosomes store nutrients taken up during the intense endocytic activity exhibited by this developmental form. Reservosomes were classified as pre-lysosomal compartments. In contrast, trypomastigote forms are not able to take up nutrients from the medium. Interestingly, trypomastigotes also have acidic organelles with the same proteases contained in epimastigote reservosomes. Nevertheless, the origin and function of these organelles have not been disclosed so far. Given the similarities between the compartments of epimastigotes and trypomastigotes, the present study aimed to investigate the origin of metacyclic trypomastigote protease-containing organelles by tracking fluorospheres or colloidal gold particles previously stored in epimastigotes’ reservosomes throughout metacyclogenesis. Using three-dimensional reconstruction of serial electron microscopy images, it was possible to find trypomastigote compartments containing the tracer. Our observations demonstrate that the protease-containing compartments from metacyclic trypomastigotes may originate directly from the reservosomes of epimastigotes.