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The micropore contains two distinct morphological types, with type I being present in most apicomplexans. The micropore is a functional nutrient uptake organelle and is involved in artemisinin resistance in Plasmodium. Although possessing distinct morphologies, type I and II micropores share a core set of components across apicomplexan species. A new model for micropore assembly is proposed which involves sequential recruitment of EPS15 (epidermal growth factor receptor substrate-15), Kelch13, and UBP1 (ubiquitin binding protein-1). Future studies should consider the transformation between type I and type II micropores. The micropore, a mysterious structure found in apicomplexan species, was recently shown to be essential for nutrient acquisition in Plasmodium falciparum and Toxoplasma gondii. However, the differences between the micropores of these two parasites questions the nature of a general apicomplexan micropore structure and whether the formation process model from Plasmodium can be applied to other apicomplexans. We analyzed the literature on different apicomplexan micropores and found that T. gondii probably harbors a more representative micropore type than the more widely studied ones in Plasmodium. Using recent knowledge of the Kelch 13 (K13) protein interactome and gene depletion phenotypes in the T. gondii micropore, we propose a model of micropore formation, thus enriching our wider understanding of micropore protein function. [ABSTRACT FROM AUTHOR]

Genetic exchange in Trypanosoma brucei is now well characterized. It is a key tool that has enabled an understanding of important parasite genetic traits and underpinned the Trypanosoma brucei genome project. However, a key aspect that has eluded us is the point in the trypanosome life cycle where genetic exchange occurs. Research using green and red fluorescent trypanosomes for visualizing genetic crosses has now identified this stage. [Copyright &y& Elsevier]

The apicoplast is a four-membrane plastid found in the apicomplexans, which harbors biosynthesis and organelle housekeeping activities in the matrix. However, the mechanism driving the flux of metabolites, in and out, remains unknown. Here, we used TurboID and genome engineering to identify apicoplast transporters in Toxoplasma gondii. Among the many novel transporters, we show that one pair of apicomplexan monocarboxylate transporters (AMTs) appears to have evolved from a putative host cell that engulfed a red alga. Protein depletion showed that AMT1 and AMT2 are critical for parasite growth. Metabolite analyses supported the notion that AMT1 and AMT2 are associated with biosynthesis of isoprenoids and fatty acids. However, stronger phenotypic defects were observed for AMT2, including in the inability to establish T. gondii parasite virulence in mice. This study clarifies, significantly, the mystery of apicoplast transporter composition and reveals the importance of the pair of AMTs in maintaining the apicoplast activity in apicomplexans. [ABSTRACT FROM AUTHOR]

Background: Vacuolar H+-ATPase (V-ATPase) is a highly conserved protein complex which hydrolyzes ATP and pumps protons to acidify vacuolar vesicles. Beyond its role in pH maintenance, the involvement of V-ATPase in endocytosis is well documented in mammals and plants but is less clear in Trypanosoma brucei. Methods: In this study, the subcellular localization of V-ATPase subunit B (TbVAB) of T. brucei was assessed via in situ N-terminal YFP-tagging and immunofluorescence assays. Transgenic bloodstream forms (BSF) of T. brucei were generated which comprised either a V-ATPase subunit B (TbVAB) conditional knockout or a V-ATPase subunit A (TbVAA) knockdown. Acridine orange and BCECF-AM were employed to assess the roles of V-ATPase in the pH regulation of BSF T. brucei. The endocytic activities of three markers were also characterized by flow cytometry analyses. Furthermore, trypanosomes were counted from trypanolysis treatment groups (either containing 1% or 5% NHS) and endocytosed trypanosome lytic factor (TLF) was also analyzed by an immunoblotting assay. Results: TbVAB was found to localize to acidocalcisomes, lysosomes and probably also to endosomes of BSF of T. brucei and was demonstrated to be essential for cell growth. TbVAB depletion neutralized acidic organelles at 24 hours post-tetracycline depletion (hpd), meanwhile the steady state intracellular pH increased from 7.016 ± 0.013 to 7.422 ± 0.058. Trypanosomes with TbVAB depletion at 24 hpd were found to take up more transferrin (2.068 ± 0.277 fold) but less tomato lectin (49.31 ± 22.57%) by endocytosis, while no significant change was detected in dextran uptake. Similar endocytic dysregulated phenotypes were also observed in TbVAA knockdown cells. In addition, TbVAB depleted trypanosomes showed a low uptake of TLF and exhibited less sensitive to lysis in both 1% and 5% NHS treatments. Conclusions: TbVAB is a key component of V-ATPase and was found to play a key function in endocytosis as well as exhibiting different effects in a receptor/cargo dependent manner in BSF of T. brucei. Besides vacuolar alkalinization, the dysregulation of endocytosis in TbVAB depleted T. brucei is considered to contribute to the reduced sensitivity to lysis by normal human serum. [ABSTRACT FROM AUTHOR]

A response by Joanna Cunnington and colleagues to a letter to the editor about their own article "A randomized, double-blind, controlled study of ultrasoundguided corticosteroid injection into the joint of patients with inflammatory arthritis" in the July 2010 issue is presented.

Outbreaks of fish trypanosomiasis are regularly reported in cultured groupers (Epinephelus spp.), largemouth bass (Micropterus salmoides), and many other species of Cyprinidae, but only rarely reported in ornamental fish. This study reports a trypanosomiasis outbreak in farmed blood parrot cichlids (Vieja melanura ♀ × Amphilophus citrinellus ♂) in the Nansha district, Guangzhou, Guangdong province. From May 2021 to January 2022, up to 100 fish died daily with an estimated overall mortality of 20%. Blood samples were collected from 21 sick fish, characterized by reduced feeding, slow movement and swimming close to the surface of the water, and 5 asymptomatic fish for parasite examination. Wet blood mounts and Giemsa-stained smears showed that trypanosomes were found in 20 of 21 sick fish (95.2%) and 4 of 5 (80%) asymptomatic fish. Parasitemias were recorded at levels of up to approximately 108 trypanosomes /ml of blood. Morphological and genetic analysis showed that the trypanosome infecting blood parrot cichlids was Trypanosoma carassii danilewskyi. In addition, histopathological analysis showed a severe steatosis in liver and an aggregation of pigments in the spleen and kidneys, which are likely to be melanomacrophages. To our knowledge, this is the first report of an outbreak of trypanosomiasis found in farmed blood parrot cichlids. • The first outbreak of trypanosomiasis caused high mortality in farmed ornamental fish. • Trypanosoma carassii danilewskyi is highly prevalent in both sick fish and asymptomatic fish. • Trypanosomiasis is linked with severe steatosis and melanomacrophage aggregation. [ABSTRACT FROM AUTHOR]

Humans and a wide range of mammals are generally susceptible to Schistosoma infection, while some rodents such as Rattus rats and Microtus spp are not. We previously demonstrated that inherent high expression levels of nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), plays an important role in blocking the growth and development of Schistosoma japonicum in wild-type rats. However, the potential regulatory effects of NO on the immune system and immune response to S. japonicum infection in rats are still unknown. In this study, we used iNOS-knockout (KO) rats to determine the role of iNOS-derived NO in the immune system and immunopathological responses to S. japonicum infection in rats. Our data showed that iNOS deficiency led to weakened immune activity against S. japonicum infection. This was characterized by the impaired T cell responses and a significant decrease in S. japonicum-elicited Th2/Th1 responses and cytokine and chemokine-producing capability in the infected iNOS-KO rats. Unlike iNOS-KO mice, Th1-associated cytokines were also decreased in the absence of iNOS in rats. In addition, a profile of pro-inflammatory and pro-fibrogenic cytokines was detected in serum associated with iNOS deficiency. The alterations in immune responses and cytokine patterns were correlated with a slower clearance of parasites, exacerbated granuloma formation, and fibrosis following S. japonicum infection in iNOS-KO rats. Furthermore, we have provided direct evidence that high levels of NO in rats can promote the development of pulmonary fibrosis induced by egg antigens of S. japonicum, but not inflammation, which was negatively correlated with the expression of TGF-β3. These studies are the first description of the immunological and pathological profiles in iNOS-KO rats infected with S. japonicum and demonstrate key differences between the responses found in mice. Our results significantly enhance our understanding of the immunoregulatory effects of NO on defensive and immunopathological responses in rats and the broader nature of resistance to pathogens such as S. japonicum. Author summary: Schistosomiasis is a zoonosis that affects more than 200 million people worldwide. A wide range of mammals, including mice, are permissive hosts of Schistosoma and develop chronic disease characterized by egg-granuloma formation and fibrosis after infection. Rats, on the other hand, are non-permissive hosts and develop efficient immune responses to eliminate the worms. Interestingly, schistosome eggs elicit a dominant Th2 immune response within mouse hosts, whereas rats with schistosomiasis develop a significant Th2 response in the absence of available egg production. The Th2 response in rats seems to play an essential role in the protection of the host against Schistosoma. So far, the factors that lead to the different immune responses to Schistosoma infection in both hosts have not been demonstrated. In this study, our results show that an iNOS-dependent mechanism maintains the function of the immune system in rats by modulating CD4+ T cell-mediated Th1/Th2-associated cytokine responses and chemokine production. Additionally, the absence of iNOS led to slow clearance of parasites, increases in the development of worms, and an exacerbation of granuloma formation and fibrosis in rats. Furthermore, high levels of NO in rats can promote the development of fibrosis induced by inflammation (rapid inflammatory repair). Therefore, this study demonstrates that the difference in iNOS levels between mice and rats is responsible for the different immune responses and outcomes induced by schistosome infection in both hosts. [ABSTRACT FROM AUTHOR]

Septic subacromial bursitis is an uncommon disorder with only a few reported cases in the literature. The most common causative organism is Staphylococcus aureus. We report the case of a 61-year-old female with a septic subacromial bursitis where the causative organism was found to be Mycobacterium avium-intracellulare (MAI). The diagnosis was only made following a biopsy, and we use this case to highlight the importance of recognising the need to consider a biopsy and aspiration in atypical situations. [ABSTRACT FROM AUTHOR]

[Display omitted] • The trypanosomes isolated from a freshwater leech, Hirudinaria manillensis , belong to a new species. • This trypanosome is a new TthIII phylogenetic lineage in the Trypanosoma (Megatrypanum) clade. • Water buffalo is most likely the mammalian host of this trypanosome. • Results highlight the potential role of freshwater leeches in the transmission of mammalian trypanosomes. Leeches have long been considered potential vectors for the aquatic lineage of trypanosomes, while bloodsucking insects are generally considered as the vectors for the terrestrial lineage of trypanosomes. The freshwater leech, Hirudinaria manillensis , is a widely distributed species in southern China and could potentially act as the vector for trypanosomes. Prior to this study, no trypanosomes had been reported from this leech. However, in this study, leeches were collected from three different places in Guangdong province, China, and a large number of flagellates were isolated and successfully cultured in vitro. Based on morphology, these flagellates looked like a typical trypanosome species. Analysis was carried out on the molecular sequences of the 18S rRNA gene and the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene. To our surprise, these flagellates were identified as likely to be a mammalian trypanosome belonging to the clade containing Trypanosoma (Megatrypanum) theileri but they are significantly different from the typical TthI and TthII stocks. Analyses of blood composition indicated that the source of the blood meal in these leeches was from the water buffalo (Bubalus bubalis). To further test if this flagellate from the freshwater leech was indeed a mammalian trypanosome, we transferred the trypanosomes cultured at 27–37 °C and they were able to successfully adapt to this mammalian body temperature, providing further supporting evidence. Due to the significant genetic differences from other related trypanosomes in the subgenus Megatrypanum , we propose that this flagellate, isolated from H. manillensis , is a new species and have named it Trypanosoma bubalisi. Our results indicate that freshwater leeches may be a potential vector of this new mammalian trypanosome. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) has been declared as one of the six major tropical diseases by the World Health Organization. This disease has been successfully controlled in China, except for some areas in the western region, such as the Xinjiang Autonomous Region, where both anthroponotic VL (AVL) and desert type zoonotic VL (DT-ZVL) remain endemic with sporadic epidemics. Methodology/Principal findings: Here, an eleven-year survey (2004–2014) of Leishmania species, encompassing both VL types isolated from patients, sand-fly vectors and Tarim hares (Lepus yarkandensis) from the Xinjiang Autonomous Region was conducted, with a special emphasis on the hares as a potential reservoir animal for DT-ZVL. Key diagnostic genes, ITS1, hsp70 and nagt (encoding N-acetylglucosamine-1-phosphate transferase) were used for phylogenetic analyses, placing all Xinjiang isolates into one clade of the L. donovani complex. Unexpectedly, AVL isolates were found to be closely related to L. infantum, while DT-ZVL isolates were closer to L. donovani. Unrooted parsimony networks of haplotypes for these isolates also revealed their relationship. Conclusions/Significance: The above analyses of the DT-ZVL isolates suggested their geographic isolation and independent evolution. The sequence identity of isolates from patients, vectors and the Tarim hares in a single DT-ZVL site provides strong evidence in support of this species as an animal reservoir. Author summary: Black faver, also known as visceral leishmaniasis (VL), is caused by pathogens of Leishmania species, spread by the bites of infected sand flies. This disease has been successfully controlled in China, except for some areas in the western region, such as Xinjiang. However, the knowledge on Leishmania in these areas remains a few important gaps. Particularly, what is the animal reservoir for desert type zoonotic VL (DT-ZVL), as sand flies get infected in areas free of patient or infected dogs? To address this question, an eleven-year survey (2004–2014) in Xinjiang for Leishmania species was carried out. We found that VLs in Xinjiang are contributed to Leishmania donovani complex, and Tarim hares is likely the reservoir animal for DT-ZVL. [ABSTRACT FROM AUTHOR]

Background: Diagnosis of blood borne infectious diseases relies primarily on the detection of the causative agent in the blood sample. Molecular techniques offer sensitive and specific tools for this although considerable difficulties exist when using these approaches in the field environment. In large scale epidemiological studies, FTA®cards are becoming increasingly popular for the rapid collection and archiving of a large number of samples. However, there are some difficulties in the downstream processing of these cards which is essential for the accurate diagnosis of infection. Here we describe recommendations for the best practice approach for sample processing from FTA®cards for the molecular diagnosis of trypanosomiasis using PCR. Results: A comparison of five techniques was made. Detection from directly applied whole blood was less sensitive (35.6%) than whole blood which was subsequently eluted from the cards using Chelex®100 (56.4%). Better apparent sensitivity was achieved when blood was lysed prior to application on the FTA cards (73.3%) although this was not significant. This did not improve with subsequent elution using Chelex®100 (73.3%) and was not significantly different from direct DNA extraction from blood in the field (68.3%). Conclusions: Based on these results, the degree of effort required for each of these techniques and the difficulty of DNA extraction under field conditions, we recommend that blood is transferred onto FTA cards whole followed by elution in Chelex®100 as the best approach. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Most corticosteroid injections into the joint are guided by the clinical examination (CE), but up to 70% are inaccurately placed, which may contribute to an inadequate response. The aim of this study was to investigate whether ultrasound (US) guidance improves the accuracy and clinical outcome of joint injections as compared with CE guidance in patients with inflammatory arthritis. METHODS: A total of 184 patients with inflammatory arthritis and an inflamed joint (shoulder, elbow, wrist, knee, or ankle) were randomized to receive either US-guided or CE-guided corticosteroid injections. Visual analog scales (VAS) for assessment of function, pain, and stiffness of the target joint, a modified Health Assessment Questionnaire, and the EuroQol 5-domain questionnaire were obtained at baseline and at 2 weeks and 6 weeks postinjection. The erythrocyte sedimentation rate and C-reactive protein level were measured at baseline and 2 weeks. Contrast injected with the steroid was used to assess the accuracy of the joint injection. RESULTS: One-third of CE-guided injections were inaccurate. US-guided injections performed by a trainee rheumatologist were more accurate than the CE-guided injections performed by more senior rheumatologists (83% versus 66%; P = 0.010). There was no significant difference in clinical outcome between the group receiving US-guided injections and the group receiving CE-guided injections. Accurate injections led to greater improvement in joint function, as determined by VAS scores, at 6 weeks, as compared with inaccurate injections (30.6 mm versus 21.2 mm; P = 0.030). Clinicians who used US guidance reliably assessed the accuracy of joint injection (P < 0.001), whereas those who used CE guidance did not (P = 0.29). CONCLUSION: US guidance significantly improves the accuracy of joint injection, allowing a trainee to rapidly achieve higher accuracy than more experienced rheumatologists. US guidance did not improve the short-term outcome of joint injection. [ABSTRACT FROM AUTHOR]

The article presents a study which compared the use of ultrasound (US) and clinical examination (CE) guidance in improving the clinical outcome of injection of corticosteroids into the joint of patients with inflammatory arthritis. According to the authors, US-guided injections performed by a novice rheumatologist were more accurate than CE-guided injections performed by a senior rheumatologist. They add that US-guided injections provided higher accuracy but did not improve the short-term outcome of joint injection.

Purpose: Robot-assisted total knee arthroplasty (rTKA) remains in its infancy, is expensive but offers the promise of improved kinematic performance through precise bone cuts, with minimal soft tissue disruption, based on pre-resection soft tissue behaviour. This cadaveric study examined load transfer, soft tissue performance and radiographic indices for conventional (sTKA) versus rTKA. The null hypothesis was there would be no difference between the two modes of implantation.Methods: Whole (ten) cadaveric limbs were randomised to receive either robotic (rTKA, N = 5) or conventional measured resection (sTKA, N = 5) knee arthroplasty. Laxity patterns were established using validated fixed sensors (Verasense) with manual maximum displacement for six degrees of freedom. Tibiofemoral load and contact points were determined dynamically using remote sensor technology for medial and lateral compartments through a functional arc of motion (0-110 degrees of motion). Final component position was assessed using pre- and post-implantation CT.Results: No significant intergroup differences for laxity were found (n.s.). The rTKA group exhibited consistently balanced mediolateral load throughout the full arc with significantly reduced overall total load across the joint (for distinct points of measurement, p < 0.05). Despite using flexion-extension and mediolateral gap balancing with measured resection, the sTKA group failed to achieve balance in at least three points of the flexion arc. Post-operative CT confirmed satisfactory component alignment with no significant differences for positioning between the two groups.Conclusion: This work found improved load sharing for rTKA when compared to conventional surgery for same donor knees. Laxity and CT determined final component positioning was not significantly different. The work supports the contention that robot-assisted TKA delivers improved tibiofemoral load sharing in time zero studies under defined conditions but such offers the promise of improved clinical performance and reduced implant wear. [ABSTRACT FROM AUTHOR]

Introduction and Aim: Toxoplasma gondii is an intracellular protozoan parasite infecting approximately 30% of the global human population. It has often been suggested that chronic infection with T. gondii is related to personality changes and various mental disorders including depression. It is not known whether this includes post-partum blues or depression. In this study, we test the hypothesis that there is a relationship between T. gondii infection and post-partum blues by measuring the association between infection and postpartum blues.Methods: A total of 475 Chinese women who have just given birth were detected serology for Toxoplasma IgG and IgM antibodies, and evaluated the degree of depression by Hamilton Depression Scale (HAMD) score. Data were analyzed by Chi-square or Fisher's Exact tests using SPSS software.Results: We found an overall Toxoplasma seroprevalence of 5.68% (27/475; 95% CI: 3.59-7.77) which was broken down into a prevalence of 6.60% (7/106; 95% CI: 1.80-11.41) in mothers with post-partum blues and 5.42% (20/369; 95% CI: 3.10-7.74) in non-affected mothers. There was no significant association between infection and post-partum blues (p = 0.64).Conclusion: The results suggest that there is no relationship between T. gondii infection and postpartum blues, at least in this sample of patients from China. [ABSTRACT FROM AUTHOR]

Trypanosoma musculi, a common blood flagellate found in mice, is similar in morphology and life cycle to the rat trypanosome T. lewisi . Both species belong to the subgenus Herpetosoma , and as T. lewisi has recently been shown to be a zoonotic pathogen, there is concern that T. musculi could also be potentially infective to humans. To test this hypothesis, a well-established method, the normal human serum (NHS) incubation test, was carried out which distinguishes human and non-human infective trypanosomes. We found that T. musculi could grow in 0.31% NHS in vitro , and even kept their infectivity to mice after incubation with 10% NHS for 24 h. In in vivo experiments, T. musculi were only slightly affected by NHS injection, confirming that it was less sensitive to the NHS than T. b. brucei , but more sensitive than T. lewisi . This resistance probably does not rely on a restricted uptake of ApoL-1. Due to this partial resistance, we cannot definitively confirm that T. musculi has the potential for infection to humans. As resistance is less than that of T. lewisi , our data suggest that it is unlikely to be a zoonotic pathogen although we would advise caution in the case of immunocompromised people such as AIDS and cancer patients. [ABSTRACT FROM AUTHOR]

Background: Human trichomoniasis caused by Trichomonas vaginalis is one of the most common sexually transmitted diseases with more than 200 million cases worldwide. It has caused a series of health problems to patients. For prevention and control of infectious diseases, vaccines are usually considered as one of the most cost-efficient tools. However, until now, work on the development of T. vaginalis vaccines is still mainly focused on the screening of potential immunogens. Alpha-actinin characterized by high immunogenicity in T. vaginalis was suggested as a promising candidate. Therefore, the purpose of this study was to evaluate the protective potency of recombinant α-actinin against T. vaginalis infection in a mouse intraperitoneal model. Methods: Two selected coding regions of α-actinin (ACT-F, 14-469 aa and ACT-T, 462-844 aa) amplified from cDNA were cloned into pET-32a (+) expression vector and transfected into BL21 cells. After induction with IPTG and purification with electroelution, the two recombinant fusion proteins were emulsified in Freunds adjuvant (FA) and used to immunize BALB/C mice. Following intraperitoneal inoculation with T. vaginalis, the survival rate of mice was monitored for the assessment of protective potency. After immunization, the antibody level in mouse serum was assessed by ELISA, splenocyte proliferation response was detected with CCK8 and cytokines in the supernatant of splenocytes were quantified with a cytometric bead-based assay. Results: We successfully obtained purified ACT-F (70.33 kDa) and ACT-T (61.7kDa). Both recombinant proteins could provide significant protection against T. vaginalis challenge, especially ACT-T (with 100% protection within one month). Meanwhile, high levels of specific total IgG and subtypes (IgG1 > IgG2a) were detected in sera from the immunized mice. Our results also revealed a statistically significant increase in splenocyte proliferation and related cytokine (IFN-γ, IL-6, IL-17A and IL-10) production after repeated stimulation with the corresponding antigens in vitro. Conclusions: Immunization with both ACT-F and ACT-T could confer partial to complete protection and trigger strong Th1/Th2 mixed humoral and cellular immune responses in the mouse host. This suggested that recombinant α-actinin subunit antigens may be promising vaccine candidates against trichomoniasis. [ABSTRACT FROM AUTHOR]

Trehalose, a non-permeating cryoprotective agent (CPA), has been documented as less toxic and highly efficient at cryopreserving different kinds of cells or organisms. In the present study, trehalose was evaluated for its application in cryopreservation of both Trypanosoma brucei procyclic and bloodstream form cells. The cryopreservation efficiency was determined by the motility of trypanosomes after thawing, as well as a subsequent recovery and infectivity assessment. The viability of trypanosomes from cultivation that were frozen in a serial concentrations of trehalose showed similar results to classical CPAs of glycerol and DMSO. Nevertheless, trypanosomes cryopreserved in 0.2 M trehalose showed the best growth characteristic during subsequent cultivation. In addition, CPA cocktails with trehalose and permeating CPA glycerol or DMSO were developed and evaluated. Interestingly, trypanosomes in host (mouse) blood cryopreserved in 0.4 M trehalose plus 5% glycerol showed higher infectivity than those preserved in trehalose/DMSO cocktails as well as individually. Further investigations showed that, in comparison with slow freezing at −80 °C, flash freezing in liquid nitrogen provided better cryopreservation for bloodstream form cells than slow freezing. In conclusion, trehalose is an easy, safe and efficient CPA for cryopreservation of T. brucei and potentially for other protozoan species and cells. [ABSTRACT FROM AUTHOR]

Trypanosomes are haemoflagellates found in vertebrate species and many of them can cause death in infected hosts including fish and humans. With the development of high-density farming in marine and freshwater fish aquaculture systems, severe disease or death, caused by trypanosomiasis, has been frequently reported. However, due to the lack of a model system, particularly for marine fish trypanosomes, and a paucity in the understanding of the biology and pathogenesis of these parasites, effective treatment for fish trypanosomiasis is significantly hampered. The goldfish is the common model system for freshwater fish trypanosomes, mainly of the species Trypanosoma carassii , while a similar model for marine fish trypanosomes has not yet been established. To address this issue, we found that Nile tilapia (Oreochromis niloticus) could be easily infected with a marine fish trypanosome, Trypanosoma epinepheli isolated from Lates calcarifer. Obvious clinical symptoms, associated with a high parasitemia (>108/ml), were found in the infected tilapias and more than 70% mortality was recorded in individuals within 20 days of infection. Interestingly, we also found that the Nile tilapia could also be infected with a freshwater fish trypanosome isolated from the largemouth bass (Micropterus salmoides) and caused significant death (more than 13%) in infected fish. This system not only provides an economical and effective laboratory model to study the biology and pathogenesis of marine and freshwater fish trypanosomes, but also provides a useful platform to develop vaccines and screen compounds for the protection and treatment of fish trypanosomiasis. [Display omitted] • This is the first test of Nile tilapia infected by both marine fish trypanosome and freshwater fish trypanosome separately. • Parasitemia and mortality of Nile tilapia infected by these two species of trypanosomes separately were recorded in detail. • An animal model of fish trypanosome with strong application is provided. [ABSTRACT FROM AUTHOR]

The subgenus Trypanozoon includes three species Trypanosoma brucei , Trypanosoma evansi and Trypanosoma equiperdum , which are morphologically identical and indistinguishable even using some molecular methods. In this study, PCR-based single strand conformation polymorphism (PCR-SSCP) was used to analyze the ribosomal DNA of the Trypanozoon species. Data indicate different patterns of ITS2 fragments between T. brucei , T. evansi and T. equiperdum by SSCP. Furthermore, analysis of total ITS sequences within these three members of the subgenus Trypanozoon showed a high degree of homology using phylogenetic analysis but were polyphyletic in haplotype networks. These data provide novel nuclear evidence to further support the notion that T. evansi and T. equiperdum should be subspecies or even strains of T. brucei. [ABSTRACT FROM AUTHOR]

Background: The haemoflagellate Trypanosoma lewisi is a kinetoplastid parasite which, as it has been recently reported to cause human disease, deserves increased attention. Characteristic features of all kinetoplastid flagellates are a uniquely structured mitochondrial DNA or kinetoplast, comprised of a network of catenated DNA circles, and RNA editing of mitochondrial transcripts. The aim of this study was to describe the kinetoplast DNA of T. lewisi. Methods/Results: In this study, purified kinetoplast DNA from T. lewisi was sequenced using high-throughput sequencing in combination with sequencing of PCR amplicons. This allowed the assembly of the T. lewisi kinetoplast maxicircle DNA, which is a homologue of the mitochondrial genome in other eukaryotes. The assembly of 23,745 bp comprises the non-coding and coding regions. Comparative analysis of the maxicircle sequence of T. lewisi with Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma brucei and Leishmania tarentolae revealed that it shares 78 %, 77 %, 74 % and 66 % sequence identity with these parasites, respectively. The high GC content in at least 9 maxicircle genes of T. lewisi (ATPase6; NADH dehydrogenase subunits ND3, ND7, ND8 and ND9; G-rich regions GR3 and GR4; cytochrome oxidase subunit COIII and ribosomal protein RPS12) implies that their products may be extensively edited. A detailed analysis of the non-coding region revealed that it contains numerous repeat motifs and palindromes. Conclusions: We have sequenced and comprehensively annotated the kinetoplast maxicircle of T. lewisi. Our analysis reveals that T. lewisi is closely related to T. cruzi and T. brucei, and may share similar RNA editing patterns with them rather than with L. tarentolae. These findings provide novel insight into the biological features of this emerging human pathogen. [ABSTRACT FROM AUTHOR]

Background: Wildlife can be important sources and reservoirs for pathogens. Trypanosome infections are common in many mammalian species, and are pathogenic in some. Molecular detection tools were used to measure trypanosome prevalence in a well-studied population of wild European badgers (Meles meles). Findings: A nested ITS-PCR system, that targeted the ribosomal RNA gene locus, has been widely used to detect pathogenic human and animal trypanosomes in domestic animals in Africa and some wildlife hosts. Samples from a long-term DEFRA funded capture-mark-recapture study of wild badgers at Woodchester Park (Gloucestershire, SW England) were investigated for trypanosome prevalence. A total of 82 badger blood samples were examined by nested ITS-PCR. Twenty-nine of the samples were found to be positive for trypanosomes giving a prevalence of 35.4 % (25.9 % - 46.2 %; 95 % CI). Infection was not found to be linked to badger condition, sex or age. Analysis of DNA sequence data showed the badgers to be infected with Trypanosoma (Megatrypanum) pestanai and phylogenetic analysis showed the Woodchester badger trypanosomes and T. pestanai to cluster in the Megatrypanum clade. Conclusions: The results show that the ITS Nested PCR is an effective tool for diagnosing trypanosome infection in badgers and suggests that it could be widely used in wildlife species with unknown trypanosomes or mixed infections. The relatively high prevalence observed in these badgers raises the possibility that a significant proportion of UK badgers are naturally infected with trypanosomes. [ABSTRACT FROM AUTHOR]

Cancer is a general name for more than 100 malignant diseases. It is postulated that all cancers start from a single abnormal cell that grows out of control. Untreated cancers can cause serious conse- quences and deaths. Great progress has been made in cancer research that has significantly improved our knowledge and understanding of the nature and mechanisms of the disease, but the origins of cancer are far from being well understood due to the limitations of suitable model systems and to the complexities of the disease. In view of the fact that cancers are found in various species of vertebrates and other metazoa, here, we suggest that cancer also occurs in parasitic protoz- oans such as Trypanosoma brucei, a blood parasite, and Toxoplasma gondii, an obligate intracellular pathogen. Without treatment, these protozoan cancers may cause severe disease and death in mammals, including humans. The simpler genomes of these single-cell organ- isms, in combination with their complex life cycles and fascinating life cycle differentiation processes, may help us to better understand the origins of cancers and, in particular, leukemias. [ABSTRACT FROM AUTHOR]

Mouse models differ considerably from humans with regard to clinical symptoms of toxoplasmosis caused by Toxoplasma gondii and, by comparison, the rat model is more representative of this disease in humans. In the present study, we found that different strains of adult and newborn rats (Lewis, Wistar, Sprague Dawley, Brown Norway and Fischer 344) exhibited remarkable variation in the number of brain cysts following inoculation with the T.   gondii Prugniaud strain. In adult rats, large numbers of cysts (1231 ± 165.6) were observed in Fischer 344, but none in the other four. This situation was different in newborn rats aged from 5 to 20 days old. All Fischer 344 and Brown Norway newborns were cyst-positive while cyst-positive infection in Sprague Dawley neonates ranged from 54.5% to 60% depending on their age at infection. In Wistar and Lewis rat neonates, however, cyst-positivity rates of 0–42.9% and 0–25% were found respectively. To investigate whether rat strain differences in infectivity could be related to inherent strain and genetic differences in the host immune response, we correlated our data with previously reported strain differences in iNOS/Arginase ratio in adult rats and found them to be linked. These results show that interactions between host genetic background and age of rat influence T.   gondii infection. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is a highly successful parasite with a worldwide prevalence. Small rodents are the main intermediate hosts, and there is growing evidence that T. gondii modifies their behaviour. Chronically infected rodents show impaired learning capacity, enhanced activity, and, most importantly, a reduction of the innate fear towards cat odour. This modification of host behaviour ensures a successful transmission of T. gondii from rodents to felids, the definitive hosts of the parasite. Given the negative fitness consequences of this behavioural manipulation, as well as an increased mortality during the acute phase of infection, we expect rodents to evolve potent resistance mechanisms that prevent or control infection. Indeed, studies in laboratory mice have identified candidate genes for T. gondii resistance. Of particular importance appear to be the innate immune receptors Toll-like receptor 11 (TLR11) and Toll-like receptor 12 (TLR12), which recognise T. gondii profilin and initiate immune responses against the parasite. Here we analyse the genetic diversity of TLR11 and TLR12 in a natural population of wood mice ( Apodemus sylvaticus ), and test for associations between TLR11 and TLR12 polymorphisms and T. gondii infection, as well as for epistatic interactions between TLR11 and TLR12 on infection status. We found that both TLR11 and TLR12 were polymorphic in wood mice, with four and nine amino acid haplotypes, respectively. However, we found no evidence that TLR11 or TLR12 genotypes or haplotypes were significantly associated with Toxoplasma infection. Despite the importance of TLR11 and TLR12 in T. gondii recognition and immune defence initiation, naturally occurring polymorphisms at TLR11 and TLR12 thus appear to play a minor role in mediating qualitative resistance to T. gondii in natural host populations of A. sylvaticus . This highlights the importance of assessing the role of candidate genes for parasite resistance identified in a laboratory setting in an ecologically meaningful context to quantify their role in mediating host–parasite interactions in the wild. [ABSTRACT FROM AUTHOR]

Highlights: [•] Two UK bat species, Pipistrellus spp, were infected with Toxoplasma gondii (10.39%). [•] P. pipistrellus prevalence: 9.9% (±7.0%; n =71); P. pygmaeus: 16.67% (±29.8%; n =6). [•] Microsatellite genotyping revealed one major interbreeding group in P. pipistrellus. [•] No significant association was observed between T. gondii infection and genotype. [ABSTRACT FROM AUTHOR]

It is well known that toxoplasmosis can be life threatening to immunocompromised individuals such as AIDS and organ transplantation patients. Glucocorticoids (GCs) are widely used in the clinic for the treatment of autoimmune diseases and organ transplantation resulting in acute toxoplasmosis in these patients. However, the interaction and mechanism between the development of acute toxoplasmosis and GC therapy are still unknown. The aims of this study were to investigate the infection of Toxoplasma gondii in the peritoneal macrophages of rats treated with glucocorticoids. Our results showed that the growth rate of T. gondii RH strain was significantly increased in the peritoneal macrophages of rats treated with glucocorticoids in vivo. For instance, 242 (±16) tachyzoites were found in 100 macrophages from the rats treated with methylprednisolone (MP), while only 16 (±4) tachyzoites were counted in the macrophages from the non-treated control rats 24 h after infection ( P < 0.01). We also demonstrated that a significant inhibition of nitric oxide (NO) production was detected in the macrophages collected from the rats post-treated with GCs with 12.90 μM (±0.99 μM) of nitrite production from the rats treated with MP, while 30.85 μM (±1.62 μM) was found in the non-treated control rats 36 h after incubation ( P < 0.01). Furthermore, glucocorticoids could significantly inhibit the expression of inducible nitric oxide synthase mRNA and its protein in the rat peritoneal macrophages. Our results strongly indicate that the decrease of NO in the rat peritoneal macrophages is closely linked to the cause of acute toxoplasmosis in the host. Additionally, there was a significant increase in the number of cysts produced by the naturally cyst forming, T. gondii Prugniaud strain with an average of 2,795 (±422) cysts of the parasite being detected in the brains of the rats treated with dexamethasone, while only 1,356 (±490) cysts were found in the non-treated control animals ( P < 0.01). As rats and humans are both naturally resistant to T. gondii infection, these novel data could lead to a better understanding of the development of acute toxoplasmosis during glucocorticoid therapy in humans. [ABSTRACT FROM AUTHOR]

Rats are naturally resistant to Toxoplasma gondii infection, particularly the RH strain, while mice are not. Previous studies have demonstrated that inducible nitric oxide synthase (iNOS) and arginase-1 of rodent peritoneal macrophages are linked to the mechanism of resistance. As an increasing number of studies on human and animal infections are showing that pulmonary toxoplasmosis is one of the most severe clinical signs from T. gondii infection, we are interested to know whether T. gondii infection in alveolar macrophages of rats is also linked to the levels of iNOS and arginase-1 activity. Our results demonstrate that T. gondii could grow and proliferate in rat alveolar macrophages, both in vitro and in vivo, at levels higher than resistant rat peritoneal macrophages and at comparable levels to sensitive mouse peritoneal macrophages. Lower activity and expression levels of iNOS and higher activity and expression levels of arginase-1 in rat alveolar macrophages were found to be linked to the susceptibility of T. gondii infection in these cells. These novel findings could aid a better understanding of the pathogenesis of clinical pulmonary toxoplasmosis in humans and domestic animals. [ABSTRACT FROM AUTHOR]

Abstract: Trypanosoma lewisi has widely been considered as a non-pathogenic rat trypanosome. However, more and more cases of humans infected with T. lewisi have been reported around the world, indicating that it can infect humans in some undetermined circumstances. Quick and sensitive diagnosis of infection by T. lewisi is important for both treatment of patients and epidemiological studies of this parasite. In this paper, three methods i.e. wet blood smear (diagnosis by microscopy), PCR and LAMP were used to detect T. lewisi from 238 wild rats (Rattus norvegicus) collected from the field in Huadu, Guangdong province, China. Infection rates of these samples detected by the 3 methods was 6.7% (16/238), 12.6% (30/238), and 18.9% (45/238), respectively. LAMP could detect all samples shown positive by microscopical observation of wet smear and by single PCR indicating good potential for application in the detection of T. lewisi. So far as we know, this is the first report of the LAMP method being used to detect T. lewisi in wild rats. The specific T. lewisi LAMP primers were able to amplify the target fragment from the genomic DNA of 19 T. lewisi strains isolated from Huadu, Guangdong province (n =16), Changchun, Jilin province of China (n =1) and from Thailand (n =2). Based on the analyses of ITS1 (internal transcribed spacer 1) and ITS2 sequences, these 19 strains show a very close genetic relationship with over 96–97% similarity to the other corresponding sequences of T. lewisi published in Genbank. Phylogenetic trees of the species in the subgenus Herpetosoma were constructed, based on the ITS1 and ITS2 sequences, and these results also indicate that they are closely related and in the same clade. [Copyright &y& Elsevier]

Background: In East Africa, animal trypanosomiasis is caused by many tsetse transmitted protozoan parasites including Trypanosoma vivax, T. congolense and subspecies of T. brucei s.l. (T. b. brucei and zoonotic human infective T. b. rhodesiense) that may co-circulate in domestic and wild animals. Accurate species-specific prevalence measurements of these parasites in animal populations are complicated by mixed infections of trypanosomes within individual hosts, low parasite densities and difficulties in conducting field studies. Many Polymerase Chain Reaction (PCR) based diagnostic tools are available to characterise and quantify infection in animals. These are important for assessing the contribution of infections in animal reservoirs and the risk posed to humans from zoonotic trypanosome species. New matrices for DNA capture have simplified large scale field PCR analyses but few studies have examined the impact of these techniques on prevalence estimations. Results: The Whatman FTA matrix has been evaluated using a random sample of 35 village zebu cattle from a population naturally exposed to trypanosome infection. Using a generic trypanosome-specific PCR, prevalence was systematically evaluated. Multiple PCR samples taken from single FTA cards demonstrated that a single punch from an FTA card is not sufficient to confirm the infectivity status of an individual animal as parasite DNA is unevenly distributed across the card. At low parasite densities in the host, this stochastic sampling effect results in underestimation of prevalence based on single punch PCR testing. Repeated testing increased the estimated prevalence of all Trypanosoma spp. from 9.7% to 86%. Using repeat testing, a very high prevalence of pathogenic trypanosomes was detected in these local village cattle: T. brucei (34.3%), T. congolense (42.9%) and T. vivax (22.9%). Conclusions: These results show that, despite the convenience of Whatman FTA cards and specific PCR based detection tools, the chronically low parasitaemias in indigenous African zebu cattle make it difficult to establish true prevalence. Although this study specifically applies to FTA cards, a similar effect would be experienced with other approaches using blood samples containing low parasite densities. For example, using blood film microscopy or PCR detection from liquid samples where the probability of detecting a parasite or DNA molecule, in the required number of fields of view or PCR reaction, is less than one. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is a protozoan parasite capable of infecting almost all warm-blooded animals. The cat is the definitive host and becomes infected by consuming contaminated meat or infected prey. Humans can act as intermediate hosts and in healthy individuals the infection is mild and self-limiting. In pregnant women it can cause spontaneous abortions and foetal abnormalities and is capable of inducing serious illness in immuno-compromised patients. In infested dwellings, mice could act as intermediate hosts and play a role in the persistence/propagation of the disease. A total of 200 mice were trapped alive in 27 infested properties in Manchester, UK, and screened for Toxoplasma infection; 59% tested positive. Evidence of vertical transmission from infected dams to foetus was found, possibly maintaining the infection in urban areas. These findings have important implications when considering approaches to rodent control. [ABSTRACT FROM AUTHOR]

Abstract: Direct PCR analysis of trypanosome infected blood samples in the quantities required for large scale epidemiological study has always been problematic. Current methods for identifying and differentiating trypanosomes typically require several species-specific reactions, many of which rely on mouse passaged samples to obtain quality concentrated genomic DNA. As a consequence important epidemiological information may be lost during the sample preparation stage. Here, we report a PCR methodology that reduces processing and improves on the sensitivity of present screening methods. The PCR technique targets the gene encoding the small ribosomal subunit in order to identify and differentiate all clinically important African trypanosome species and some subspecies. The method is more economical, simple, and sensitive than current screening methods, and yields more detailed information, thereby making it a viable tool for large-scale epidemiological studies. [Copyright &y& Elsevier]

Animal trypanosomiasis, caused by the members of subgenus Trypanozoon (Trypanosoma brucei brucei, T. evansi and T. equiperdum), has reduced animal productivity leading to significant negative economic impacts in endemic regions. Due to limited drug discovery and the emergence of drug-resistance over many recent decades, novel and effective compounds against animal trypanosomiasis are urgently required. This study was conducted to evaluate the antitrypanosomal potential of a batch of carbazole aminoalcohol derivatives. Among them, we found that the most effective compound was H1402, which exhibited potent trypanocidal efficacy against the bloodstream-form of T. b. brucei (EC 50 = 0.73 ± 0.05 µM) and presented low cytotoxicity against two mammalian cell lines with CC 50 > 30 µM. Using a murine model of acute infection, oral administration with H1402 demonstrated a complete clearance of T. b. brucei and all the infected mice were cured when they were treated twice daily for 5 days at a dose of 100 mg/kg. Furthermore, parasites were not detected in mice infected with T. evansi and T. equiperdum (the causative agents of surra and dourine, respectively, in animals) within 30 days following the same regimen with H1402. In addition, H1402 caused severe morphological and ultrastructural destruction to trypanosomes, as well as causing phosphatidylserine externalization, which are suggested to be the most likely cause of cell death. Overall, the present data demonstrated that H1402 could be promising as a rapid, safe and orally active lead compound for the development of new chemotherapeutics for animal trypanosomiasis. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii has long been considered a ubiquitous parasite possessing the capacity of infecting virtually all warm-blooded animals globally. Occasionally, this parasite can also infect cold-blooded animals such as fish if their body temperature reaches 37 °C. However, we are currently lacking an understanding of key details such as the minimum temperature required for T. gondii invasion and proliferation in these cold-blooded animals and their cells. Here, we performed in vitro T. gondii infection experiments with rat embryo fibroblasts (REF cells), grouper (Epinephelus coioides) splenocytes (GS cells) and zebra fish (Danio rerio) hepatocytes (ZFL cells), at 27 °C, 30 °C, 32 °C, 35 °C and 37 °C, respectively. We found that T. gondii tachyzoites could penetrate REF, GS nd ZFL cells at 27 °C but clear inhibition of multiplication was observed. Intriguingly, the intracellular tachyzoites retained the ability to infect mice after 12 days of incubation in GS cells cultured at 27 °C as demonstrated by bioassay. At 30 °C, 32 °C and 35 °C, we observed that the mammalian cells (REF cells) and fish cells (GS and ZFL cells) could support T. gondii invasion and replication, which showed a temperature-dependent relationship in infection and proliferation rates. Our data demonstrated that the minimum temperature for T. gondii invasion and replication was 27 °C and 30 °C respectively, which indicated that temperature should be a key factor for T. gondii invasion and proliferation in host cells. This suggests that temperature-dependent infection determines the differences in the capability of T. gondii to infect cold- and warm-blooded vertebrates. Image 1 • Toxoplasma gondii tachyzoites are capable to invade fish cells and maintain infectivity for 12 days post infection at 27°C. • Among 27°C, 30°C, 32°C and 35°C, the minimum temperature for T. gondii invasion is 27°C and for replication is 30°C. • Temperature determines the capability of T. gondii to infect cold- or warm- blooded vertebrates, but not cell types. [ABSTRACT FROM AUTHOR]

A correction to the article "Analysis of the mitochondrial maxicircle of Trypanosoma lewisi, a neglected human pathogen" that was published online on January 13, 2016.