Tropical Density Estimation of Phylogenetic Trees.

Much evidence from biological theory and empirical data indicates that, gene trees, phylogenetic trees reconstructed from different genes (loci), do not have to have exactly the same tree topologies. Such incongruence between gene trees might be caused by some "unusual" evolutionary events, such as meiotic sexual recombination in eukaryotes or horizontal transfers of genetic material in prokaryotes. However, most of the gene trees are constrained by the tree topology of the underlying species tree, that is, the phylogenetic tree depicting the evolutionary history of the set of species under consideration. In order to discover "outlying" gene trees which do not follow the "main distribution(s)" of trees, we propose to apply the "tropical metric" with the max-plus algebra from tropical geometry to a non-parametric estimation of gene trees over the space of phylogenetic trees. In this research we apply the "tropical metric," a well-defined metric over the space of phylogenetic trees under the max-plus algebra, to non-parametric estimation of gene trees distribution over the tree space. Kernel density estimator (KDE) is one of the most popular non-parametric estimation of a distribution from a given sample, and we propose an analogue of the classical KDE in the setting of tropical geometry with the tropical metric which measures the length of an intrinsic geodesic between trees over the tree space. We estimate the probability of an observed tree by empirical frequencies of nearby trees, with the level of influence determined by the tropical metric. Then, with simulated data generated from the multispecies coalescent model, we show that the non-parametric estimation of the gene tree distribution using the tropical metric performs better than one using the Billera-Holmes-Vogtmann (BHV) metric developed by Weyenberg et al. in terms of computational times and accuracy. We then apply it to Apicomplexa data.

Temporal gene expression during asexual development of the apicomplexan Sarcocystis neurona.

Asexual replication in the apicomplexan Sarcocystis neurona involves two main developmental stages: the motile extracellular merozoite and the sessile intracellular schizont. Merozoites invade host cells and transform into schizonts that undergo replication via endopolygeny to form multiple (64) daughter merozoites that are invasive to new host cells. Given that the capabilities of the merozoite vary significantly from the schizont, the patterns of transcript levels throughout the asexual lifecycle were determined and compared in this study. RNA-Seq data were generated from extracellular merozoites and four intracellular schizont development time points. Of the 6,938 genes annotated in the S. neurona genome, 6,784 were identified in the transcriptome. Of these, 4,111 genes exhibited significant differential expression between the merozoite and at least one schizont development time point. Transcript levels were significantly higher for 2,338 genes in the merozoite and 1,773 genes in the schizont stages. Included in this list were genes encoding the secretory pathogenesis determinants (SPDs), which encompass the surface antigen and SAG-related sequence (SAG/SRS) and the secretory organelle proteins of the invasive zoite stage (micronemes, rhoptries, and dense granules). As anticipated, many of the S. neurona SPD gene transcripts were abundant in merozoites. However, several SPD transcripts were elevated in intracellular schizonts, suggesting roles unrelated to host cell invasion and the initial establishment of the intracellular niche. The hypothetical genes that are potentially unique to the genus Sarcocystis are of particular interest. Their conserved expression patterns are instructive for future investigations into the possible functions of these putative Sarcocystis-unique genes. IMPORTANCE: The genus Sarcocystis is an expansive clade within the Apicomplexa, with the species S. neurona being an important cause of neurological disease in horses. Research to decipher the biology of S. neurona and its host-pathogen interactions can be enhanced by gene expression data. This study has identified conserved apicomplexan orthologs in S. neurona, putative Sarcocystis-unique genes, and gene transcripts abundant in the merozoite and schizont stages. Importantly, we have identified distinct clusters of genes with transcript levels peaking during different intracellular schizont development time points, reflecting active gene expression changes across endopolygeny. Each cluster also has subsets of transcripts with unknown functions, and investigation of these seemingly Sarcocystis-unique transcripts will provide insights into the interesting biology of this parasite genus.

Systemic Caryospora-like coccidiosis in a clutch of hatchling red-eared slider turtles (Trachemys scripta elegans).

Caryospora-like organisms (CLOs) form a clade of at least 11 genotypes of related coccidia that can cause epizootic mortality in marine turtles. The biology, transmission, host species range, and host cell tropism of these organisms are still largely unknown. The goal of this study was to characterize the host cell tropism, pathologic and ultrastructural features, and phylogeny associated with the first report of a mortality event due to CLO in the freshwater red-eared slider turtle (Trachemys scripta elegans). Sudden mortalities within a clutch of captive-raised red-eared slider hatchlings (n = 8) were recorded, and deceased animals had severe segmental to diffuse, transmural, fibrinonecrotic enterocolitis and multifocal to coalescing hepatic necrosis, among other lesions associated with numerous intracytoplasmic developing stages of intralesional coccidia. Among the different developmental stages, merozoites were ultrastructurally characterized by an apical complex. A pan-apicomplexan polymerase chain reaction (PCR) yielded a 347 bp-amplicon matching the Schellackia/Caryospora-like clade with 99.1% identity to the US3 strain from green sea turtles (Chelonia mydas) and 99.1% identity to Schellackia sp. Isolate OC116. Surviving hatchlings were treated with toltrazuril sulfone (ponazuril) but were subsequently euthanized due to the risk of spreading the parasite to other chelonids in the collection. The ponazuril-treated hatchlings (n = 4) had mild proliferative anterior enteritis, with few intraepithelial coccidia in one hatchling confirmed as CLO by PCR. This is the first report of Caryospora-like coccidiosis in non-cheloniid turtles, highlighting the relevance of this disease as an emerging highly pathogenic intestinal and extra-intestinal form of coccidiosis of turtles with potential cross-species infectivity.

Equine Protozoal Myeloencephalitis.

Advances in the understanding of equine protozoal myeloencephalitis (EPM) are reviewed. It is now apparent that EPM can be caused by either of 2 related protozoan parasites, Sarcocystis neurona and Neospora hughesi, although S neurona is the most common etiologic pathogen. Horses are commonly infected, but clinical disease occurs only infrequently; the factors influencing disease occurrence are not well understood. Epidemiologic studies have identified risk factors for the development of EPM, including the presence of opossums and prior stressful health-related events. Attempts to reproduce EPM experimentally have reliably induced antibody responses in challenged horses, but have not consistently produced neurologic disease. Diagnosis of EPM has improved by detecting intrathecal antibody production against the parasite. Sulfadiazine/pyrimethamine (ReBalance) and the triazine compounds diclazuril (Protazil) and ponazuril (Marquis) are effective anticoccidial drugs that are now available as FDA-approved treatments for EPM.

Transcriptional dynamics in the protozoan parasite Sarcocystis neurona and mammalian host cells after treatment with a specific inhibitor of apicomplexan mRNA polyadenylation.

In recent years, a class of chemical compounds (benzoxaboroles) that are active against a range of parasites has been shown to target mRNA polyadenylation by inhibiting the activity of CPSF73, the endonucleolytic core of the eukaryotic polyadenylation complex. One particular compound, termed AN3661, is active against several apicomplexan parasites that cause disease in humans. In this study, we report that AN3661 is active against an apicomplexan that causes disease in horses and marine mammals (Sarcocystis neurona), with an approximate IC50 value of 14.99 nM. Consistent with the reported mode of action of AN3661 against other apicomplexans, S. neurona mutants resistant to AN3661 had an alteration in CPSF73 that was identical to a mutation previously documented in AN3661-resistant Toxoplasma gondii and Plasmodium falciparum. AN3661 had a wide-ranging effect on poly(A) site choice in S. neurona, with more than half of all expressed genes showing some alteration in mRNA 3' ends. This was accompanied by changes in the relative expression of more than 25% of S. neurona genes and an overall 5-fold reduction of S. neurona transcripts in infected cells. In contrast, AN3661 had no discernible effect on poly(A) site choice or gene expression in the host cells. These transcriptomic studies indicate that AN3661 is exceedingly specific for the parasite CPSF73 protein, and has the potential to augment other therapies for the control of apicomplexan parasites in domestic animals.

Pathologic and immunohistochemical findings in an outbreak of systemic toxoplasmosis in a mob of red kangaroos.

Toxoplasma gondii is a zoonotic protozoan pathogen that infects many endothermic vertebrates, including humans; the domestic cat and other felids serve as the definitive host. Macropodids are considered highly susceptible to toxoplasmosis. Here, we describe the clinical, pathologic, and immunohistochemical findings of an outbreak of systemic toxoplasmosis in a mob of 11 red kangaroos (Macropus rufus), with high morbidity (73%) and mortality (100%) rates. Affected animals had either severe and rapidly deteriorating clinical conditions or sudden death, which was correlated with widespread necrotizing lesions in multiple organs and intralesional T. gondii organisms identified via MIC3-specific immunohistochemistry and confirmed by REP529-specific rtPCR. Quantification of parasites demonstrated the highest parasite density in pulmonary parenchyma compared with other tissues. Our study highlights the continued importance of this severe condition in Australian marsupials.

Fussing About Fission: Defining Variety Among Mainstream and Exotic Apicomplexan Cell Division Modes.

Cellular reproduction defines life, yet our textbook-level understanding of cell division is limited to a small number of model organisms centered around humans. The horizon on cell division variants is expanded here by advancing insights on the fascinating cell division modes found in the Apicomplexa, a key group of protozoan parasites. The Apicomplexa display remarkable variation in offspring number, whether karyokinesis follows each S/M-phase or not, and whether daughter cells bud in the cytoplasm or bud from the cortex. We find that the terminology used to describe the various manifestations of asexual apicomplexan cell division emphasizes either the number of offspring or site of budding, which are not directly comparable features and has led to confusion in the literature. Division modes have been primarily studied in two human pathogenic Apicomplexa, malaria-causing Plasmodium spp. and Toxoplasma gondii, a major cause of opportunistic infections. Plasmodium spp. divide asexually by schizogony, producing multiple daughters per division round through a cortical budding process, though at several life-cycle nuclear amplifications stages, are not followed by karyokinesis. T. gondii divides by endodyogeny producing two internally budding daughters per division round. Here we add to this diversity in replication mechanisms by considering the cattle parasite Babesia bigemina and the pig parasite Cystoisospora suis. B. bigemina produces two daughters per division round by a "binary fission" mechanism whereas C. suis produces daughters through both endodyogeny and multiple internal budding known as endopolygeny. In addition, we provide new data from the causative agent of equine protozoal myeloencephalitis (EPM), Sarcocystis neurona, which also undergoes endopolygeny but differs from C. suis by maintaining a single multiploid nucleus. Overall, we operationally define two principally different division modes: internal budding found in cyst-forming Coccidia (comprising endodyogeny and two forms of endopolygeny) and external budding found in the other parasites studied (comprising the two forms of schizogony, binary fission and multiple fission). Progressive insights into the principles defining the molecular and cellular requirements for internal vs. external budding, as well as variations encountered in sexual stages are discussed. The evolutionary pressures and mechanisms underlying apicomplexan cell division diversification carries relevance across Eukaryota.

Histologically, immunohistochemically, ultrastructurally, and molecularly confirmed neosporosis abortion in an aborted equine fetus.

Neosporosis is a common cause of abortion in cattle worldwide but is rare in horses. Here, the first case of histologically, ultrastructurally, immunohistochemically, and molecularly confirmed equine abortion caused by neosporosis is reported. Samples of lung, heart, liver, skeletal muscle, tongue, brain, and the placenta from a female fetus aborted at 280 days of gestation were fixed in formalin and submitted for diagnosis. Histologically, there was disseminated neosporosis with severe lesions in lungs, liver and the heart. Protozoal tachyzoites in all tissues reacted with polyclonal anti-Neospora caninum rabbit antibodies. Transmission electron microscopic observation on lung tissue revealed tachyzoites consistent with Neospora, including many rhoptries. Polymerase-chain reaction (PCR) using primers designed to amplify the rRNA gene internal transcribed spacer 1 (ITS1) of the Sarcocystidae was performed on DNA extracted from fetal tissues. Comparison of the ITS1 amplified from the foal tissue to sequences available in GenBank revealed 100% sequence identity to the ITS1 from three isolates of Neospora hughesi.

Characterization of mRNA polyadenylation in the apicomplexa.

Messenger RNA polyadenylation is a universal aspect of gene expression in eukaryotes. In well-established model organisms, this process is mediated by a conserved complex of 15-20 subunits. To better understand this process in apicomplexans, a group of unicellular parasites that causes serious disease in humans and livestock, a computational and high throughput sequencing study of the polyadenylation complex and poly(A) sites in several species was conducted. BLAST-based searches for orthologs of the human polyadenylation complex yielded clear matches to only two-poly(A) polymerase and CPSF73-of the 19 proteins used as queries in this analysis. As the human subunits that recognize the AAUAAA polyadenylation signal (PAS) were not immediately obvious, a computational analysis of sequences adjacent to experimentally-determined apicomplexan poly(A) sites was conducted. The results of this study showed that there exists in apicomplexans an A-rich region that corresponds in position to the AAUAAA PAS. The set of experimentally-determined sites in one species, Sarcocystis neurona, was further analyzed to evaluate the extent and significance of alternative poly(A) site choice in this organism. The results showed that almost 80% of S. neurona genes possess more than one poly(A) site, and that more than 780 sites showed differential usage in the two developmental stages-extracellular merozoites and intracellular schizonts-studied. These sites affected more than 450 genes, and included a disproportionate number of genes that encode membrane transporters and ribosomal proteins. Taken together, these results reveal that apicomplexan species seem to possess a poly(A) signal analogous to AAUAAA even though genes that may encode obvious counterparts of the AAUAAA-recognizing proteins are absent in these organisms. They also indicate that, as is the case in other eukaryotes, alternative polyadenylation is a widespread phenomenon in S. neurona that has the potential to impact growth and development.

Protozoal coinfection in horses with equine protozoal myeloencephalitis in the eastern United States.

BACKGROUND: Infection by 2 or more protozoa is linked with increased severity of disease in marine mammals with protozoan encephalitis. HYPOTHESIS/OBJECTIVES: To assess whether horses with equine protozoal myeloencephalitis (EPM) caused by Sarcocystis neurona also have evidence of infection with Neospora hughesi or Toxoplasma gondii. We hypothesized that horses with EPM would be more likely than horses with cervical vertebral stenotic myelopathy (CVSM) to be positive for antibodies to multiple protozoan parasites. ANIMALS: One hundred one horses with neurologic disease: 49 with EPM and 52 with CVSM. METHODS: Case review. Archived serum and cerebrospinal fluid (CSF) from 101 horses were examined. Inclusion criteria included neurologic disease, antemortem or postmortem diagnosis of EPM or CVSM, and availability of serological results or archived samples for testing. Additional testing for antibodies was performed on serum for T. gondii, as well as serum and CSF for N. hughesi. RESULTS: Horses with EPM were more likely than horses with CVSM to have positive immunologic results for S. neurona on serum (95.9% versus 76.9%, P = .0058), CSF (98.0% versus 44.2%, P < .00001), and serum : CSF titer ratio (91.8% versus 0%, P < .00001). Positive results for Neospora and Toxoplasma were uncommon, with total seroprevalence rates of 12.9% and 14.9%, respectively. The proportions of EPM cases testing positive for Neospora and Toxoplasma (16% and 12%) were not different from the proportions of CVSM cases testing positive (10% and 17%, P = .31 and .47, respectively). CONCLUSION: Results do not indicate an important role for protozoal coinfection in EPM in the eastern United States.

Molecular Genetic Manipulation of Sarcocystis neurona.

Sarcocystis neurona is a member of the important phylum Apicomplexa and the primary cause of equine protozoal myeloencephalitis (EPM). Moreover, S. neurona is the best-studied species in the genus Sarcocystis, one of the most successful parasite taxa, as virtually all vertebrate animals may be infected by at least one species. Consequently, scientific investigation of S. neurona will aid in the control of EPM and neurologic disease in sea mammals, while also improving our understanding of a prominent branch on the apicomplexan phylogenetic tree. These protocols describe methods that expand the capabilities to study this prominent member of the Apicomplexa. © 2018 by John Wiley & Sons, Inc.

Testing the Sarcocystis neurona vaccine using an equine protozoal myeloencephalitis challenge model.

Equine protozoal myeloencephalitis (EPM) is an important equine neurologic disorder, and treatments for the disease are often unrewarding. Prevention of the disease is the most important aspect for EPM, and a killed vaccine was previously developed for just that purpose. Evaluation of the vaccine had been hampered by lack of post vaccination challenge. The purpose of this study was to determine if the vaccine could prevent development of clinical signs after challenge with Sarcocystis neurona sporocysts in an equine challenge model. Seventy horses that were negative for antibodies to S. neurona and were neurologically normal were randomly assigned to vaccine or placebo groups and divided into short-term duration of immunity (study #1) and long-term duration of immunity (study #2) studies. S. neurona sporocysts used for the challenge were generated in the opossum/raccoon cycle isolate SN 37-R. Study #1 horses received an initial vaccination and a booster, and were challenged 34days post second vaccination. Study #2 horses received a vaccination and two boosters and were challenged 139days post third vaccination. All horses in study #1 developed neurologic signs (n=30) and there was no difference between the vaccinates and controls (P=0.7683). All but four horses in study #2 developed detectable neurologic deficits. The neurologic signs, although not statistically significant, were worse in the vaccinated horses (P=0.1559). In these two studies, vaccination with the S. neurona vaccine failed to prevent development of clinical neurologic deficits.

Differential Roles for Inner Membrane Complex Proteins across Toxoplasma gondii and Sarcocystis neurona Development.

The inner membrane complex (IMC) of apicomplexan parasites contains a network of intermediate filament-like proteins. The 14 alveolin domain-containing IMC proteins in Toxoplasma gondii fall into different groups defined by their distinct spatiotemporal dynamics during the internal budding process of tachyzoites. Here, we analyzed representatives of different IMC protein groups across all stages of the Toxoplasma life cycle and during Sarcocystis neurona asexual development. We found that across asexually dividing Toxoplasma stages, IMC7 is present exclusively in the mother's cytoskeleton, whereas IMC1 and IMC3 are both present in mother and daughter cytoskeletons (IMC3 is strongly enriched in daughter buds). In developing macro- and microgametocytes, IMC1 and -3 are absent, whereas IMC7 is lost in early microgametocytes but retained in macrogametocytes until late in their development. We found no roles for IMC proteins during meiosis and sporoblast formation. However, we observed that IMC1 and IMC3, but not IMC7, are present in sporozoites. Although the spatiotemporal pattern of IMC15 and IMC3 suggests orthologous functions in Sarcocystis, IMC7 may have functionally diverged in Sarcocystis merozoites. To functionally characterize IMC proteins, we knocked out IMC7, -12, -14, and -15 in Toxoplasma. IMC14 and -15 appear to be involved in switching between endodyogeny and endopolygeny. In addition, IMC7, -12, and -14, which are all recruited to the cytoskeleton outside cytokinesis, are critical for the structural integrity of extracellular tachyzoites. Altogether, stage- and development-specific roles for IMC proteins can be discerned, suggesting different niches for each IMC protein across the entire life cycle. IMPORTANCE The inner membrane complex (IMC) is a defining feature of apicomplexan parasites key to both their motility and unique cell division. To provide further insights into the IMC, we analyzed the dynamics and functions of representative alveolin domain-containing IMC proteins across developmental stages. Our work shows universal but distinct roles for IMC1, -3, and -7 during Toxoplasma asexual division but more specialized functions for these proteins during gametogenesis. In addition, we find that IMC15 is involved in daughter formation in both Toxoplasma and Sarcocystis. IMC14 and IMC15 function in limiting the number of Toxoplasma offspring per division. Furthermore, IMC7, -12, and -14, which are recruited in the G1 cell cycle stage, are required for stress resistance of extracellular tachyzoites. Thus, although the roles of the different IMC proteins appear to overlap, stage- and development-specific behaviors indicate that their functions are uniquely tailored to each life stage requirement.

Small sarcocysts can be a feature of experimental infections with Sarcocystis neurona merozoites.

Several reports indicate the presence of small tissue cysts associated with Sarcocystis neurona infections. Several failed attempts to develop tissue cysts in potential intermediate host using in vitro derived parasites originally isolated from horses with equine protozoal myeloencephalitis suggest that the experimental methods to achieve bradyzoites with those isolates was not possible. Those prior studies reported the lack of detectable sarcocysts based on histology and in vivo feeding trials. A recent report of successful production and detection of small sarcocysts triggered us to review archived tissues from earlier experimental infection studies. The retrospective review sought to determine if small sized sarcocysts were not detected due to their relatively smaller size and infrequency as compared to larger sized sarcocysts produced with other isolates in these experimental inoculation trials. Tissues from two prior in vivo inoculation studies, involving in vitro-produced parasites inoculated into laboratory-reared cats and raccoons, were re-examined by immunohistochemistry staining to more easily detect the tissue cysts. In the experimental cat study no small tissue cysts were seen, consistent with the original publication results. However, in the experimental raccoon study, one raccoon inoculated with an EPM-derived isolate, SN-UCD1, had small sarcocysts not reported in the original publication. This retrospective study suggests that much closer scrutiny of tissues, including the use of immunohistochemistry on tissue sections is required to detect the smaller S. neurona sarcocysts associated with the experimental inoculations of the isolates originally derived from horses with EPM.

Seroepidemiology of Sarcocystis neurona and Neospora hughesi infections in domestic donkeys (Equus asinus) in Durango, Mexico.

There is currently no information regarding Sarcocystis neurona and Neospora hughesi infections in donkeys in Mexico. Here, we determined the presence of antibodies against S. neurona and N. hughesi in donkeys in the northern Mexican state of Durango. Serum samples of 239 domestic donkeys (Equus asinus) were assayed for S. neurona and N. hughesi antibodies using home-made enzyme-linked immunoassays; six (2.5%) of the 239 donkeys tested seropositive for S. neurona. The seroprevalence of S. neurona infection was comparable among donkeys regardless of their origin, health status, or sex. Multivariate analysis showed that seropositivity to S. neurona was associated with increased age (OR = 2.95; 95% CI: 1.11-7.82; p = 0.02). Antibodies to N. hughesi were found in two (0.8%) of the 239 donkeys. Both exposed donkeys were healthy, 3- and 6-year-old females. This is the first evidence of S. neurona and N. hughesi infections in donkeys in Mexico.

A serosurvey of selected cystogenic coccidia in Spanish equids: first detection of anti-Besnoitia spp. specific antibodies in Europe.

BACKGROUND: Equine besnoitiosis, caused by Besnoitia bennetti, and equine protozoal myeloencephalitis (EPM), caused by Sarcocystis neurona and Neospora hughesi are relevant equine diseases in the Americas that have been scarcely studied in Europe. Thus, a serosurvey of these cystogenic coccidia was carried out in Southern Spain. A cross-sectional study was performed and serum samples from horses (n = 553), donkeys (n = 85) and mules (n = 83) were included. An in-house enzyme-linked immunosorbent assay (ELISA) was employed to identify a Besnoitia spp. infection and positive results were confirmed by an a posteriori western blot. For Neospora spp. and Sarcocystis spp., infections were detected using in-house ELISAs based on the parasite surface antigens N. hughesi rNhSAG1 and S. neurona rSnSAG2/3/4. Risk factors associated with these protozoan infections were also investigated. RESULTS: Antibodies against Besnoitia spp., Neospora spp. and Sarcocystis spp. infections were detected in 51 (7.1%), 46 (6.4%) and 20 (2.8%) of 721 equids, respectively. The principal risk factors associated with a higher seroprevalence of Besnoitia spp. were the host species (mule or donkey), the absence of shelter and the absence of a rodent control programme. The presence of rodents was the only risk factor for Neospora spp. infection. CONCLUSIONS: This study was the first extensive serosurvey of Besnoitia spp. infection in European equids accomplished by two complementary tests and gives evidence of the presence of specific antibodies in these populations. However, the origin of the infection is still unclear. Further parasite detection and molecular genotyping are needed to identify the causative Besnoitia and Neospora species. Finally, cross-reactions with antibodies directed against other species of Sarcocystis might explain the positive reactions against the S. neurona antigens.

Sarcocystis neurona manipulation using culture-derived merozoites for bradyzoite and sporocyst production.

Equine protozoal myeloencephalitis (EPM) remains a significant central nervous system disease of horses in the American continents. Sarcocystis neurona is considered the primary causative agent and its intermediate life stages are carried by a wide host-range including raccoons (Procyon lotor) in North America. S. neurona sarcocysts mature in raccoon skeletal muscle and can produce central nervous system disease in raccoons, mirroring the clinical presentation in horses. The study aimed to develop laboratory tools whereby the life cycle and various life stages of S. neurona could be better studied and manipulated using in vitro and in vivo systems and compare the biology of two independent isolates. This study utilized culture-derived parasites from S. neurona strains derived from a raccoon or from a horse to initiate raccoon infections. Raccoon tissues, including fresh and cryopreserved tissues, were used to establish opossum (Didelphis virginiana) infections, which then shed sporocyts with retained biological activity to cause encephalitis in mice. These results demonstrate that sarcocysts can be generated using in vitro-derived S. neurona merozoites, including an isolate originally derived from a naturally infected horse with clinical EPM. This study indicates the life cycle can be significantly manipulated in the laboratory without affecting subsequent stage development, allowing further purification of strains and artificial maintenance of the life cycle.

Normalizing Kernels in the Billera-Holmes-Vogtmann Treespace.

As costs of genome sequencing have dropped precipitously, development of efficient bioinformatic methods to analyze genome structure and evolution have become ever more urgent. For example, most published phylogenomic studies involve either massive concatenation of sequences, or informal comparisons of phylogenies inferred on a small subset of orthologous genes, neither of which provides a comprehensive overview of evolution or systematic identification of genes with unusual and interesting evolution (e.g., horizontal gene transfers, gene duplication, and subsequent neofunctionalization). We are interested in identifying such "outlying" gene trees from the set of gene trees and estimating the distribution of trees over the "tree space". This paper describes an improvement to the kdetrees algorithm, an adaptation of classical kernel density estimation to the metric space of phylogenetic trees (Billera-Holmes-Vogtman treespace), whereby the kernel normalizing constants, are estimated through the use of the novel holonomic gradient methods. As in the original kdetrees paper, we have applied kdetrees to a set of Apicomplexa genes. The analysis identified several unreliable sequence alignments that had escaped previous detection, as well as a gene independently reported as a possible case of horizontal gene transfer. The updated version of the kdetrees software package is available both from CRAN (the official R package system), as well as from the official development repository on Github. ( github.com/grady/kdetrees).

Extended-spectrum antiprotozoal bumped kinase inhibitors: A review.

Many life-cycle processes in parasites are regulated by protein phosphorylation. Hence, disruption of essential protein kinase function has been explored for therapy of parasitic diseases. However, the difficulty of inhibiting parasite protein kinases to the exclusion of host orthologues poses a practical challenge. A possible path around this difficulty is the use of bumped kinase inhibitors for targeting calcium-dependent protein kinases that contain atypically small gatekeeper residues and are crucial for pathogenic apicomplexan parasites' survival and proliferation. In this article, we review efficacy against the kinase target, parasite growth in vitro, and in animal infection models, as well as the relevant pharmacokinetic and safety parameters of bumped kinase inhibitors.

Gotta catch'em all! Pokémon GO and physical activity among young adults: difference in differences study.

OBJECTIVE:  To estimate the effect of playing Pokémon GO on the number of steps taken daily up to six weeks after installation of the game. DESIGN:  Cohort study using online survey data. PARTICIPANTS:  Survey participants of Amazon Mechanical Turk (n=1182) residing in the United States, aged 18 to 35 years and using iPhone 6 series smartphones. MAIN OUTCOME MEASURES:  Number of daily steps taken each of the four weeks before and six weeks after installation of Pokémon GO, automatically recorded in the "Health" application of the iPhone 6 series smartphones and reported by the participants. A difference in difference regression model was used to estimate the change in daily steps in players of Pokémon GO compared with non-players. RESULTS:  560 (47.4%) of the survey participants reported playing Pokémon GO and walked on average 4256 steps (SD 2697) each day in the four weeks before installation of the game. The difference in difference analysis showed that the daily average steps for Pokémon GO players during the first week of installation increased by 955 additional steps (95% confidence interval 697 to 1213), and then this increase gradually attenuated over the subsequent five weeks. By the sixth week after installation, the number of daily steps had gone back to pre-installation levels. No significant effect modification of Pokémon GO was found by sex, age, race group, bodyweight status, urbanity, or walkability of the area of residence. CONCLUSIONS:  Pokémon GO was associated with an increase in the daily number of steps after installation of the game. The association was, however, moderate and no longer observed after six weeks.