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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.

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]

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.

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]

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]

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]

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]

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.