Influence of microbiome in intraprostatic inflammation and prostate cancer.

BACKGROUND: Chronic infection and inflammation have been linked to the development of prostate cancer. Dysbiosis of the oral and gut microbiomes and subsequent microbial translocation can lead to pathogenic prostate infections. Microbial-produced metabolites have also been associated with signaling pathways that promote prostate cancer development. A comprehensive discussion on the mechanisms of microbiome infection and the prostate microenvironment is essential to understand prostate carcinogenesis. METHODS: Published studies were used from the National Center for Biotechnology Information (NCBI) database to conduct a narrative review. No restrictions were applied in the selection of articles. RESULTS: Microbiome-derived short-chain fatty acids (SCFAs) have been found to upregulate multiple signaling pathways, including MAPK and PI3K, through IGF-1 signaling and M2 macrophage polarization. SCFAs can also upregulate Toll-like receptors, leading to chronic inflammation and the creation of a pro-prostate cancer environment. Dysbiosis of oral microbiota has been correlated with prostate infection and inflammation. Additionally, pathogenic microbiomes associated with urinary tract infections have shown a link to prostate cancer, with vesicoureteral reflux potentially contributing to prostate infection. CONCLUSIONS: This review offers a comprehensive understanding of the impact of microbial infections linked to intraprostatic inflammation as a causative factor for prostate cancer. Further studies involving the manipulation of the microbiome and its produced metabolites may provide a more complete understanding of the microenvironmental mechanisms that promote prostate carcinogenesis.

Humidity-Induced Protein-Based Artificial Synaptic Devices for Neuroprosthetic Applications.

Neuroprosthetics and brain-machine interfaces are immensely beneficial for people with neurological disabilities, and the future generation of neural repair systems will utilize neuromorphic devices for the advantages of energy efficiency and real-time performance abilities. Conventional synaptic devices are not compatible to work in such conditions. The cerebrospinal fluid (CSF) in the central part of the nervous system is composed of 99% water. Therefore, artificial synaptic devices, which are the fundamental component of neuromorphic devices, should resemble biological nerves while being biocompatible, and functional in high-humidity environments with higher functional stability for real-time applications in the human body. In this work, artificial synaptic devices are fabricated based on gelatin-PEDOT: PSS composite as an active material to work more effectively in a highly humid environment (≈90% relative humidity). These devices successfully mimic various synaptic properties by the continuous variation of conductance, like, excitatory/inhibitory post-synaptic current(EPSC/IPSC), paired-pulse facilitation/depression(PPF/PPD), spike-voltage dependent plasticity (SVDP), spike-duration dependent plasticity (SDDP), and spike-rate dependent plasticity (SRDP) in environments at a relative humidity levels of ≈90%.

Ligand-gated ion channels as potential biomarkers for ADT-mediated cognitive decline in prostate cancer patients.

Men with prostate cancer are at increased risk of developing cognitive decline by the use of second-generation androgen signaling inhibitors. To date, reliable and sensitive biomarkers that could distinguish men at high risk of cognitive dysfunction under androgen deprivation therapy (ADT) have not been characterized. We used high-throughput transcriptional profiling utilizing human prostate cancer cell culture models mimicking ADT, biomarker selection using minimal common oncology data elements-cytoscape, and bioinformatic analyses employing Advaita® iPathwayGuide and DisGeNET for identification of disease-related gene associations. Validation analysis of genes was performed on brain neuronal and glial cells by quantitative real-time polymerase chain reaction assay. Our systematic analysis of androgen deprivation-associated genes involved multiple biological processes, including neuroactive ligand-receptor interaction, axon guidance, cytokine-cytokine receptor interaction, and metabolic and cancer signaling pathways. Genes associated with neuroreceptor ligand interaction, including gamma-aminobutyric acid (GABA) A and B receptors and nuclear core proteins, were identified as top upstream regulators. Functional enrichment and protein-protein interaction network analysis highlighted the role of ligand-gated ion channels (LGICs) and their receptors in cognitive dysfunction. Gene-disease association assigned forgetfulness, intellectual disability, visuospatial deficit, bipolar disorder, and other neurocognitive impairment with upregulation of type-1 angiotensin II receptor, brain-derived neurotrophic factor, GABA type B receptor subunit 2 (GABBR2), GABRA3, GABRA5, GABRB1, glycine receptor beta, glutamate ionotropic receptor N-methyl-D-aspartate receptor (NMDA) type subunit 1, glutamate ionotropic receptor NMDA type subunit 2D, 5-hydroxytryptamine receptor 1D, interferon beta 1, and nuclear receptor subfamily 3 group C member 1 as top differentially expressed genes. Validation studies of brain glial cells, neurons, and patients on ADT demonstrated the association of these genes with cognitive decline. Our findings highlight LGICs as potential biomarkers for ADT-mediated cognitive decline. Further validation of these biomarkers may lead to future practical clinical use.

Inhibition of Wnt/ß-catenin pathway overcomes therapeutic resistance to abiraterone in castration-resistant prostate cancer.

Abiraterone acetate has been clinically approved for the treatment of patients with advanced-stage prostate cancer. It reduces testosterone production by blocking the enzyme cytochrome P450 17 alpha-hydroxylase. Despite improved survival outcomes with abiraterone, almost all patients develop therapeutic resistance and disease recurrence, progressing to a more aggressive and lethal phenotype. Bioinformatics analyses predicted activation of canonical Wnt/ß-catenin and involvement of stem cell plasticity in abiraterone-resistant prostate cancer. Increased expression of androgen receptor (AR) and ß-catenin and their crosstalk causes activation of AR target genes and regulatory networks for which overcoming acquired resistance remains a major challenge. Here we show that co-treatment with abiraterone and ICG001, a ß-catenin inhibitor, overcomes therapeutic resistance and significantly inhibited markers of stem cell and cellular proliferation in abiraterone-resistant prostate cancer cells. Importantly, this combined treatment abrogated the association between AR and ß-catenin; diminished SOX9 expression from the complex more prominently in abiraterone-resistant cells. In addition, combined treatment inhibited tumor growth in an in vivo abiraterone-resistant xenograft model, blocked stemness, migration, invasion, and colony formation ability of cancer cells. This study opens new therapeutic opportunity for advanced-stage castration-resistant prostate cancer patients.

Increased cytokine gene expression and cognition risk associated with androgen deprivation therapy.

BACKGROUND: Androgen deprivation therapy (ADT) is a standard treatment modality for locally advanced, high-risk, and metastatic hormone-sensitive prostate cancer. Long-term ADT treatment likely develops side-effects that include changes in cognition or onset of dementia. However, the molecular understanding of this effect remains elusive. We attempt to establish a link between ADT and changes in cognitive function using patient databases and bioinformatics analyses. METHODS: Gene expression profiling was performed using RNA sequencing data from Alzheimer patient cohort and compared with the data from advanced-stage prostate cancer patients receiving neoadjuvant antiandrogen therapy. Differentially expressed genes (DEGs) were analyzed using the Ingenuity knowledge database. RESULTS: A total of 1952 DEGs in the Alzheimer patient cohort and 101 DEGs were identified in ADT treated prostate cancer patients. Comparing both data sets provided a subset of 33 commonly expressed genes involving cytokine-cytokine signaling with an over representation of cytokine-cytokine receptor interaction, inflammatory cytokines, signaling by interleukins together with alterations in the circulating lymphocyte repertoire, adaptive immune responses, regulation of cytokine production, and changes in T-cell subsets. Additionally, lipopolysaccharide, tumor necrosis factor, and toll-like receptors were identified as upstream transcriptional regulators of these pathways. The most commonly expressed genes viz. IL-17A, CCL2, IL-10, IL-6, IL-1RN, LIF/LIFR were further validated by quantitative RT-PCR exhibited higher expression in antiandrogen treated neuronal, glial, and androgen-responsive prostate cancer cells, compared to no-androgen antagonist treatment. CONCLUSIONS: Our findings suggest that changes in cytokine signaling under the influence of ADT in prostate cancer patients may be linked with cognitive impairment presenting new avenues for diagnostic and therapeutic development in combating brain deficits.

Role of solute carrier transporters SLC25A17 and SLC27A6 in acquired resistance to enzalutamide in castration-resistant prostate cancer.

Enzalutamide (XTANDI®), an antiandrogen, is used for the treatment of advanced-stage prostate cancer. Approximately, 60% of patients receiving enzalutamide show initial remission followed by disease relapse with the emergence of highly aggressive castration-resistant prostate cancer. Solute carrier (SLC) proteins play a critical role in the development of drug resistance by altering cellular metabolism. Transcriptome analysis revealed the predominance of SLC25A17 and SLC27A6 in enzalutamide-resistant prostate cancer cells; however, their role in antiandrogen resistance has not been elucidated. sgRNA-mediated knockdown of SLC25A17 and SLC27A6 suppressed cell proliferation and migration in enzalutamide-resistant cells. An induction of G1/S cell cycle arrest and abundance of hypo-diploid cells along with the reduction in the protein expression CyclinD1 and CDK6, the checkpoint factors, was observed including increased cell death as evident by BAX upregulation in knockdown cells. Inhibition of SLC25A17 and SLC27A6 resulted in downregulation of fatty acid synthase and acetyl-CoA carboxylase with parallel decrease in the levels of lactic acid in enzalutamide resistant cells. However, downregulation of triglyceride and citric acid was only observed in SLC25A17 silenced cells. The protein-protein interaction of SLC25A17 and SLC27A6 revealed alteration in some common drug-resistant and metabolism-related genes. Analysis of The Cancer Genome Atlas database exhibiting high SLC25A17 and SLC27A6 gene expression in prostate cancer patients were associated with poor survival than those with low expression of these proteins. In conclusion, SLC25A17 and SLC27A6 and its interactive network play an essential role in the development of enzalutamide resistance through metabolic reprogramming and may be identified as therapeutic target(s) to circumvent drug resistance.

Role of ZBTB7A zinc finger in tumorigenesis and metastasis.

The zinc finger and BTB (broad-complex, tramtrack and bric a brac) domain containing protein 7A (ZBTB7A) is a pleiotropic transcription factor that plays an important role in various stages of cell proliferation, differentiation, and other developmental processes. ZBTB7A is a member of the POK family that directly and specifically binds to short DNA recognition sites located near their target genes thereby acting as transcriptional activator or repressor. ZBTB7A overexpression has been associated with tumorigenesis and metastasis in various human cancer types, including breast, prostate, lung, ovarian, and colon cancer. However in some instances downregulation of ZBTB7A results in tumor progression, suggesting its role as a tumor suppressor. ZBTB7A is involved with complicated regulatory networks which include protein-protein and protein-nucleic acid interactions. ZBTB7A involvement in cancer progression and metastasis is perhaps enabled through the regulation of various signaling pathways depending on the type and genetic context of cancer. The association of ZBTB7A with other proteins affects cancer aggressiveness, therapeutic resistance and clinical outcome. This review focuses on the involvement of ZBTB7A in various signaling pathways and its role in cancer progression. We will also review the literature on ZBTB7A and cancer which could be potentially explored for its therapeutic implications.

Role of class I histone deacetylases in the regulation of maspin expression in prostate cancer.

Maspin repression is frequently observed in prostate cancer; however, the molecular mechanism(s) causing the loss is not completely understood. Here, we demonstrate that inhibition of class I histone deacetylases (HDACs) mediates re-expression of maspin which plays an essential role in suppressing proliferation and migration capability in prostate cancer cells. Human prostate cancer LNCaP and DU145 cells treated with HDAC inhibitors, sodium butyrate, and trichostatin A, resulted in maspin re-expression. Interestingly, an exploration into the molecular mechanisms demonstrates that maspin repression in prostate tumor and human prostate cancer cell lines occurs via epigenetic silencing through an increase in HDAC activity/expression, independent of promoter DNA hypermethylation. Furthermore, transcriptional activation of maspin was accompanied with the suppression of HDAC1 and HDAC8 with significant p53 enrichment at the maspin promoter associated with an increase in histone H3/H4 acetylation. Our results provide evidence of maspin induction as a critical epigenetic event altered by class I HDACs in the restoration of balance to delay proliferation and migration ability of prostate cancer cells.

Identification & characterization of leucine-rich repeat kinase 2 & parkin RBR E3 ubiquitin protein ligase variants in patients with Parkinson's disease.

BACKGROUND & OBJECTIVES: Parkinson's disease (PD) is a motor disorder that affects movement. More than 24 loci and 28 associated genes have been identified to be associated with this disease. The present study accounts for the contribution of two candidates, leucine-rich repeat kinase 2 ( LRRK2) and parkin RBR E3 ubiquitin protein ligase ( PRKN) in the PD patients, and their characterization in silico and in vitro. METHODS: A total of 145 sporadic PD cases and 120 ethnically matched healthy controls were enrolled with their informed consent. Mutation screening was performed by direct DNA sequencing of the targeted exons of LRRK2 and all exons flanking introns of PRKN. The effect of the pathogenic PRKN variants on a drug (MG-132) induced loss of mitochondrial membrane potential (△ΨM) was measured by a fluorescent dye tetramethylrhodamine methyl ester (TMRM). RESULTS: Twelve and 20 genetic variants were identified in LRRK2 and PRKN, respectively. Interestingly, five out of seven exonic LRRK2 variants were synonymous. Further assessment in controls confirmed the rarity of two such p.Y1527 and p.V1615. Among the pathogenic missense variations (as predicted in silico) in PRKN, two were selected (p.R42H and p.A82E) for their functional study in vitro, which revealed the reduced fluorescence intensity of TMRM as compared to wild type, in case of p.R42H but not the other. INTERPRETATION & CONCLUSIONS: About 6.2 per cent of the cases (9/145) in the studied patient cohort were found to carry pathogenic (as predicted in silico) missense variations in PRKN in heterozygous condition but not in case of LRRK2 which was rare. The presence of two rare synonymous variants of LRRK2 (p.Y1527 and p.V1615) may support the phenomenon of codon bias. Functional characterization of selected PRKN variations revealed p.R42H to cause disruption of mitochondrial membrane potential (△ΨM) rendering cells more susceptible to cellular stress.

Low prevalence of viable Toxoplasma gondii in fresh, unfrozen American pasture-raised pork and lamb from retail meat stores in the United States.

In a national survey of fresh, unfrozen, American pasture-raised lamb and pork, the prevalence of viable Toxoplasma gondii was determined in 1500 samples selected by random multistage sampling (750 pork, 750 lamb) obtained from 250 retail meat stores from 10 major geographic areas in the USA. Each sample consisted of a minimum of 500g of meat purchased from the retail meat case. To detect viable T. gondii, 50g meat samples of each of 1500 samples were bioassayed in mice. Viable T. gondii was isolated from 2 of 750 lamb samples (unweighted: 0.19%, 0.00-0.46%; weighted: 0.04%, 0.00-0.11%) and 1 of 750 pork samples (unweighted: 0.12%, 0.00-0.37%; weighted: 0.18%, 0.00-0.53%) samples. Overall, the prevalence of viable T. gondii in these retail meats was very low. Nevertheless, consumers, especially pregnant women, should be aware that they can acquire T. gondii infection from ingestion of undercooked meat. Cooking meat to an internal temperature of 66°C kills T. gondii.

Androgen Deprivation Induces Transcriptional Reprogramming in Prostate Cancer Cells to Develop Stem Cell-Like Characteristics.

Enzalutamide, an antiandrogen, is approved for therapy of castration resistant prostate cancer. Clinical applications have shown that approximately 30% of patients acquire resistance after a short period of treatment. However, the molecular mechanisms underlying this resistance is not completely understood. To identify transcriptomic signatures associated with acquisition of drug resistance we profiled gene expression of paired enzalutamide sensitive and resistant human prostate cancer LNCaP (lymph node carcinoma of the prostate) and C4-2B cells. Overlapping genes differentially regulated in the enzalutamide resistant cells were ranked by Ingenuity Pathway Analysis and their functional validation was performed using ingenuity knowledge database followed by confirmation to correlate transcript with protein expression. Analysis revealed that genes associated with cancer stem cells, such as POU5F1 (OCT4), SOX2, NANOG, BMI1, BMP2, CD44, SOX9, and ALDH1 were markedly upregulated in enzalutamide resistant cells. Amongst the pathways enriched in the enzalutamide-resistant cells were those associated with RUNX2, hedgehog, integrin signaling, and molecules associated with elastic fibers. Further examination of a patient cohort undergoing ADT and its comparison with no-ADT group demonstrated high expression of POU5F1 (OCT4), ALDH1, and SOX2 in ADT specimens, suggesting that they may be clinically relevant therapeutic targets. Altogether, our approach exhibits the potential of integrative transcriptomic analyses to identify critical genes and pathways of antiandrogen resistance as a promising approach for designing novel therapeutic strategies to circumvent drug resistance.

The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat.

The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2 encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rodents less fearful of their definitive Felidae hosts. However, L-DOPA is also a structural precursor of melanins, secondary quinones, and dityrosine protein crosslinks, which are produced by many species. For example, dityrosine crosslinks are abundant in the oocyst walls of Eimeria and T. gondii, although their structural role has not been demonstrated, Here, we investigated the biology of AAH knockout parasites in the sexual reproductive cycle within cats. We found that ablation of the AAH genes resulted in reduced infection in the cat, lower oocyst yields, and decreased rates of sporulation. Our findings suggest that the AAH genes play a predominant role during infection in the gut of the definitive feline host.

G-quadruplex structure at intron 2 of TFE3 and its role in Xp11.2 translocation and splicing.

Transcription Factor E3 (TFE3) translocation is found in a group of different type of cancers and most of the translocations are located in the 5' region of TFE3 which may be considered as Breakpoint Region (BR). In our In silico study by QGRS mapper and non BdB web servers we found a Potential G-quadruplex forming Sequence (PQS) in the intron 2 of TFE3 gene. In vitro G-quadruplex formation was shown by native PAGE in presence of Pyridostatin(PDS), which with inter molecular secondary structure caused reduced mobility to migrate slower. G-quadruplex formation was mapped at single base resolution by Sanger sequencing and Circular Dichroism showed the formation of parallel G-quadruplex. FRET analysis revealed increased and decreased formation of G-quadruplex in presence of PDS and antisense oligonucleotide respectively. PCR stop assay, transcriptional and translational inhibition by PQS showed stable G-quadruplex formation affecting the biological processes. TFE3 minigene splicing study showed the involvement of this G-quadruplex in TFE3 splicing too. Therefore, G-quadruplex is evident to be the reason behind TFE3 induced oncogenesis executed by translocation and also involved in the mRNA splicing.

Sarcocystis cymruensis: discovery in Western Hemisphere in the Brown rat (Rattus norvegicus) from Grenada, West Indies: redescription, molecular characterization, and transmission to IFN-γ gene knockout mice via sporocysts from experimentally infected domestic cat (Felis catus).

Rodents are intermediate hosts for many species of Sarcocystis. Little is known of Sarcocystis cymruensis that uses the Brown rat (Rattus norvegicus) as intermediate hosts and the domestic cat (Felis catus) as experimental definitive host. Here, we identified and described Sarcocystis cymruensis in naturally infected R. norvegicus from Grenada, West Indies. Rats (n = 167) were trapped in various locations in two parishes (St. George and St. David). Microscopic, thin (< 1 µm) walled, slender sarcocysts were found in 11 of 156 (7.0%) rats skeletal muscles by squash examination. A laboratory-raised cat fed naturally infected rat tissues excreted sporocysts that were infectious for interferon gamma gene knockout (KO) mice, but not to Swiss Webster outbred albino mice. All inoculated mice remained asymptomatic, and microscopic S. cymruensis-like sarcocysts were found in the muscles of KO mice euthanized on day 70, 116, and 189 post inoculation (p.i.). Sarcocysts from infected KO mice were infective for cats at day 116 but not at 70 days p.i. By transmission electron microscopy, the sarcocyst wall was "type 1a." Detailed morphological description of the cyst wall, metrocytes, and bradyzoites is given for the first time. Additionally, molecular data on S. cymruensis are presented also for the first time. Molecular characterization of sarcocysts 18S rDNA and 28S rDNA, ITS-1, and cox1 loci showed the highest similarity with S. rodentifelis and S. muris. In conclusion, the present study described the natural infection of S. cymruensis in Brown rat for the first time in a Caribbean country and provided its molecular characteristics.

Prevalence and genetic characterization of Toxoplasma gondii in free-range chickens from grocery stores and farms in Maryland, Ohio and Massachusetts, USA.

Chickens are considered important in the epidemiology of Toxoplasma gondii. Chicken hearts (n = 1185) obtained from grocery stores were tested for T. gondii infection. Antibodies to T. gondii were assayed in fluid removed from the heart cavity using the modified agglutination test (MAT) at 1:5, 1:25, and 1:100 dilutions. MAT antibodies were detected in 222 hearts at 1:5 dilution and 8 hearts at 1:25 dilution, but none were positive at 1:100 dilution. Seropositive (n = 230, 19.4%) chicken hearts were bioassayed in mice and seronegative (n = 157) chickens were bioassayed in cats. Viable T. gondii was not isolated from any hearts by bioassays in mice. The 2 cats fed 60 and 97 hearts did not excrete T. gondii oocysts. The results indicate a low prevalence of viable T. gondii in chickens from grocery stores. Molecular typing of 23 archived T. gondii strains isolated from free-range chickens from Ohio and Massachusetts using the 10 PCR-RFLP markers including SAG1, SAG2 (5'-3'SAG2 and altSAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed that seven were ToxoDB PCR-RFLP genotype #1, 11 were genotype #2, one was genotype #3, three were genotype #170, and one was mixed genotype. These results indicate that the clonal genotypes #1 (type II), #2 (type III), and #3 (type II variant) are common in free-range chickens.

Morphological and molecular characterization of Sarcocystis arctica-like sarcocysts from the Arctic fox (Vulpes lagopus) from Alaska, USA.

The muscles of herbivores commonly harbor sarcocysts of parasites belonging to species in the genus Sarcocystis, but such muscle parasites are rare in carnivores. Here, we report Sarcocystis arctica-like sarcocysts in muscles of Arctic foxes (Vulpes lagopus) from Alaska, USA, for the first time. The tongues of 56 foxes were examined for Sarcocystis infection using several methods. Sarcocystis bradyzoites were detected in pepsin digests of 13 (23.2%), and sarcocysts were found in histological sections stained with hematoxylin and eosin (HE) of 9 (16.0%). By light microscopy, sarcocysts were up to 4 mm long and up to 245 µm wide. In HE-stained sections, the sarcocyst wall appeared smooth and up to 1.5 µm thick without visible protrusions. By transmission electron microscopy, the sarcocyst wall had a wavy parasitophorous vacuolar membrane (pvm) folded as pleomorphic villar protrusions (vp), sometimes with anastomoses of villar tips. The vp and the ground substance (gs) layer were smooth and without microtubules. The gs was up to 2.0 µm thick. The total width of the wall including vp and the gs was up to 4.0 µm. The vp were up to 3.0 µm long and most closely resembled "type 9c." All sarcocysts were mature and contained numerous 8.1 × 2.1 µm sized bradyzoites. Molecular characterization (at 18S rDNA, 28S rDNA, ITS-1, and cox1) showed the highest affinity for S. arctica of the Arctic fox (V. lagopus) from Norway. In the present investigation, we provide evidence that sarcocysts are common in tongues of Alaskan Arctic foxes suggesting that these carnivores are serving as intermediate hosts, and we also provide ultrastructure of S. arctica from the Arctic fox for the first time.

Sarcocystis rommeli, n. sp. (Apicomplexa: Sarcocystidae) from Cattle (Bos taurus) and its Differentiation from Sarcocystis hominis.

Cattle (Bos taurus) are intermediate hosts for three named species of Sarcocystis, S. cruzi, S. hirsuta, and S. hominis. Recently, a fourth species was identified and named S. sinensis. However, S. sinensis originally named a species of Sarcocystis in water buffalo (Bubalus bubalis) in China. Based on unverifiable evidence, it was suggested that the same parasite infects cattle. In addition, S. sinensis was recently declared as nomen nudum because its naming violated the rules of International Code of Zoological Nomenclature. Thus, the fourth species using cattle as an intermediate host does not have a valid name. Here, we propose a new name, Sarcocystis rommeli for the S. sinensis-like parasite from cattle in Argentina, and differentiate it ultrastructurally from S. hominis sarcocysts from experimentally infected cattle. Sarcocystis rommeli sarcocysts were microscopic with a 5-µm-thick wall with slender villar protrusions (Vp); the Vp were up to 5 µm long, up to 0.5 µm wide, and of uneven thickness, often bent at an angle. The ground substance layer (Gs) was up to 0.8 µm thick and smooth. Vesicular structures were seen at the base of the Vp. The bradyzoites were 10-12 µm long. Sarcocystis hominis sarcocysts had Vp that were often upright, up to 7.5 µm long, and up to 1.8 µm wide; the Gs was up to 2 µm thick and without vesicles. Its sarcocyst wall was up to 5.6 µm thick, the vp were bent at an angle, up to 5.8 µm long, the Gs was up to 2 µm thick, but without vesicles seen in S. rommeli. Beef containing sarcocysts of S. rommeli was not orally infectious for two human volunteers and a red fox (Vulpes vulpes). The Sarcocystis described here is molecularly different from S. cruzi, S. hirsuta, and S. hominis based on 18S rRNA and cox1 gene sequences.

Antibody Detection and Molecular Characterization of Toxoplasma gondii from Bobcats (Lynx rufus), Domestic Cats (Felis catus), and Wildlife from Minnesota, USA.

Little is known of the epidemiology of toxoplasmosis in Minnesota. Here, we evaluated Toxoplasma gondii infection in 50 wild bobcats (Lynx rufus) and 75 other animals on/near 10 cattle farms. Antibodies to T. gondii were assayed in serum samples or tissue fluids by the modified agglutination test (MAT, cut-off 1:25). Twenty nine of 50 bobcats and 15 of 41 wildlife trapped on the vicinity of 10 farms and nine of 16 adult domestic cats (Felis catus) and six of 14 domestic dogs resident on farms were seropositive. Toxoplasma gondii oocysts were not found in feces of any felid. Tissues of all seropositive wild animals trapped on the farm were bioassayed in mice and viable T. gondii was isolated from two badgers (Taxidea taxus), two raccoons (Procyon lotor), one coyote (Canis latrans), and one opossum (Didelphis virginiana). All six T. gondii isolates were further propagated in cell culture. Multi-locus PCR-RFLP genotyping using 10 markers (SAG1, SAG2 (5'-3'SAG2, and alt.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico), and DNA from cell culture derived tachyzoites revealed three genotypes; #5 ToxoDataBase (1 coyote, 1 raccoon), #1 (1 badger, 1 raccoon, 1 opossum), and #2 (1 badger). This is the first report of T. gondii prevalence in domestic cats and in bobcats from Minnesota, and the first isolation of viable T. gondii from badger.

Sarcocystis masoni, n. sp. (Apicomplexa: Sarcocystidae), and redescription of Sarcocystis aucheniae from llama (Lama glama), guanaco (Lama guanicoe) and alpaca (Vicugna pacos).

There is considerable confusion concerning the species of Sarcocystis in South American camelids (SAC). Several species names have been used; however, proper descriptions are lacking. In the present paper, we redescribe the macroscopic sarcocyst forming Sarcocystis aucheniae and describe and propose a new name, Sarcocystis masoni for the microscopic sarcocyst forming species. Muscles samples were obtained from llamas (Lama glama) and guanacos (Lama guanicoe) from Argentina and from alpacas (Vicugna pacos) and llamas from Peru. Individual sarcocysts were processed by optical and electron microscopy, and molecular studies. Microscopic sarcocysts of S. masoni were up to 800 µm long and 35-95 µm wide, the sarcocyst wall was 2·5-3·5 µm thick, and had conical to cylindrical villar protrusions (vp) with several microtubules. Each vp had 11 or more rows of knob-like projections. Seven 18S rRNA gene sequences obtained from sarcocysts revealed 95-96% identity with other Sarcocystis spp. sequences reported in the GenBank. Sarcocysts of S. aucheniae were macroscopic, up to 1·2 cm long and surrounded by a dense and laminar 50 µm thick secondary cyst wall. The sarcocyst wall was up to 10 µm thick, and had branched vp, appearing like cauliflower. Comparison of the 11 sequences obtained from individual macroscopic cysts evidenced a 98-99% of sequence homology with other S. aucheniae sequences. In conclusion, 2 morphologically and molecularly different Sarcocystis species, S. masoni (microscopic cysts) and S. aucheniae (macroscopic cysts), were identified affecting different SAC from Argentina and Peru.

Ancient, globally distributed lineage of Sarcocystis from sporocysts of the Eastern rat snake (Pantherophis alleghaniensis) and its relation to neurological sequalae in intermediate hosts.

There is an emerging concern that snakes are definitive hosts of certain species of Sarcocystis that cause muscular sarcocystosis in human and non-human primates. Other species of Sarcocystis are known to cycle among snakes and rodents, but have been poorly characterized in the USA and elsewhere. Although neurological sequalae are known for certain species of Sarcocystis, no such neurological symptoms are known to typify parasites that naturally cycle in rodents. Here, sporocysts of a species of Sarcocystis were found in the intestinal contents of a rat snake (Pantherophis alleghaniensis) from Maryland, USA. The sporocysts were orally infective for interferon gamma gene knockout (KO) mice, but not to Swiss Webster outbred mice. The KO mice developed neurological signs, and were necropsied between 33 and 52 days post-inoculation. Only schizonts/merozoites were found, and they were confined to the brain. The predominant lesion was meningoencephalitis characterized by perivascular cuffs, granulomas, and necrosis of the neuropil. The schizonts and merozoites were located in neuropil, and apparently extravascular. Brain homogenates from infected KO mice were infective to KO mice and CV-1 cell line. DNA extracted from the infected mouse brain, and infected cell cultures revealed the highest identity with Sarcocystis species that employ snakes as definitive hosts. This is the first report of Sarcocystis infection in the endangered rat snake (P. alleghaniensis) and the first report of neurological sarcocystosis in mice induced by feeding sporocysts from a snake. These data underscore the likelihood that parasites in this genus that employ snakes as their definitive hosts constitute an ancient, globally distributed monophyletic group. These data also raise the possibility that neurological sequalae may be more common in intermediate hosts of Sarcocystis spp. than has previously been appreciated.