Autochthonous Leishmaniasis Caused by Leishmania tropica, Identified by Using Whole-Genome Sequencing, Sri Lanka.

Cutaneous leishmaniasis is atypical in Sri Lanka because Leishmania donovani, which typically causes visceral disease, is the causative agent. The origins of recently described hybrids between L. donovani and other Leishmania spp. usually responsible for cutaneous leishmaniasis remain unknown. Other endemic dermotropic Leishmania spp. have not been reported in Sri Lanka. Genome analysis of 27 clinical isolates from Sri Lanka and 32 Old World Leishmania spp. strains found 8 patient isolates clustered with L. tropica and 19 with L. donovani. The L. tropica isolates from Sri Lanka shared markers with strain LtK26 reported decades ago in India, indicating they were not products of recent interspecies hybridization. Because L. tropica was isolated from patients with leishmaniasis in Sri Lanka, our findings indicate L. donovani is not the only cause of cutaneous leishmaniasis in Sri Lanka and potentially explains a haplotype that led to interspecies dermotropic L. donovani hybrids.

TRIM5α: A Protean Architect of Viral Recognition and Innate Immunity.

The evolutionary pressures exerted by viral infections have led to the development of various cellular proteins with potent antiviral activities, some of which are known as antiviral restriction factors. TRIpartite Motif-containing protein 5 alpha (TRIM5α) is a well-studied restriction factor of retroviruses that exhibits virus- and host-species-specific functions in protecting against cross-primate transmission of specific lentiviruses. This specificity is achieved at the level of the host gene through positive selection predominantly within its C-terminal B30.2/PRYSPRY domain, which is responsible for the highly specific recognition of retroviral capsids. However, more recent work has challenged this paradigm, demonstrating TRIM5α as a restriction factor for retroelements as well as phylogenetically distinct viral families, acting similarly through the recognition of viral gene products via B30.2/PRYSPRY. This spectrum of antiviral activity raises questions regarding the genetic and structural plasticity of this protein as a mediator of the recognition of a potentially diverse array of viral molecular patterns. This review highlights the dynamic evolutionary footprint of the B30.2/PRYSPRY domain in response to retroviruses while exploring the guided 'specificity' conferred by the totality of TRIM5α's additional domains that may account for its recently identified promiscuity.

Human ESCRT-I and ALIX function as scaffolding helical filaments in vivo.

The Endosomal Sorting Complex Required for Transport (ESCRT) is an evolutionarily conserved machinery that performs reverse-topology membrane scission in cells universally required from cytokinesis to budding of enveloped viruses. Upstream acting ESCRT-I and ALIX control these events and link recruitment of viral and cellular partners to late-acting ESCRT-III CHMP4 through incompletely understood mechanisms. Using structure-function analyses combined with super-resolution imaging, we show that ESCRT-I and ALIX function as distinct helical filaments in vivo . Together, they are essential for optimal structural scaffolding of HIV-1 nascent virions, the retention of viral and human genomes through defined functional interfaces, and recruitment of CHMP4 that itself assembles into corkscrew-like filaments intertwined with ESCRT-I or ALIX helices. Disruption of filament assembly or their conformationally clustered RNA binding interfaces in human cells impaired membrane abscission, resulted in major structural instability and leaked nucleic acid from nascent virions and nuclear envelopes. Thus, ESCRT-I and ALIX function as helical filaments in vivo and serve as both nucleic acid-dependent structural scaffolds as well as ESCRT-III assembly templates. Significance statement: When cellular membranes are dissolved or breached, ESCRT is rapidly deployed to repair membranes to restore the integrity of intracellular compartments. Membrane sealing is ensured by ESCRT-III filaments assembled on the inner face of membrane; a mechanism termed inverse topology membrane scission. This mechanism, initiated by ESCRT-I and ALIX, is universally necessary for cytokinesis, wound repair, budding of enveloped viruses, and more. We show ESCRT-I and ALIX individually oligomerize into helical filaments that cluster newly discovered nucleic acid-binding interfaces and scaffold-in genomes within nascent virions and nuclear envelopes. These oligomers additionally appear to serve as ideal templates for ESCRT-III polymerization, as helical filaments of CHMP4B were found intertwined ESCRT-I or ALIX filaments in vivo . Similarly, corkscrew-like filaments of ALIX are also interwoven with ESCRT-I, supporting a model of inverse topology membrane scission that is synergistically reinforced by inward double filament scaffolding.

ToxoNet: A high confidence map of protein-protein interactions in Toxoplasma gondii.

The apicomplexan intracellular parasite Toxoplasma gondii is a major food borne pathogen that is highly prevalent in the global population. The majority of the T. gondii proteome remains uncharacterized and the organization of proteins into complexes is unclear. To overcome this knowledge gap, we used a biochemical fractionation strategy to predict interactions by correlation profiling. To overcome the deficit of high-quality training data in non-model organisms, we complemented a supervised machine learning strategy, with an unsupervised approach, based on similarity network fusion. The resulting combined high confidence network, ToxoNet, comprises 2,063 interactions connecting 652 proteins. Clustering identifies 93 protein complexes. We identified clusters enriched in mitochondrial machinery that include previously uncharacterized proteins that likely represent novel adaptations to oxidative phosphorylation. Furthermore, complexes enriched in proteins localized to secretory organelles and the inner membrane complex, predict additional novel components representing novel targets for detailed functional characterization. We present ToxoNet as a publicly available resource with the expectation that it will help drive future hypotheses within the research community.

Giardia intestinalis reshapes mucosal immunity toward a Type 2 response that attenuates inflammatory bowel-like diseases.

Diarrheal diseases are the second leading cause of death in children worldwide. Epidemiological studies show that co-infection with Giardia intestinalis decreases the severity of diarrhea. Here, we show that Giardia is highly prevalent in the stools of asymptomatic school-aged children. It orchestrates a Th2 mucosal immune response, characterized by increased antigen-specific Th2 cells, IL-25, Type 2-associated cytokines, and goblet cell hyperplasia. Giardia infection expanded IL-10-producing Th2 and GATA3+ Treg cells that promoted chronic carriage, parasite transmission, and conferred protection against Toxoplasma gondii-induced lethal ileitis and DSS-driven colitis by downregulating proinflammatory cytokines, decreasing Th1/Th17 cell frequency, and preventing collateral tissue damage. Protection was dependent on STAT6 signaling, as Giardia-infected STAT6-/- mice no longer regulated intestinal bystander inflammation. Our findings demonstrate that Giardia infection reshapes mucosal immunity toward a Type 2 response, which confers a mutualistic protection against inflammatory disease processes and identifies a critical role for protists in regulating mucosal defenses.

Rapid detection, quantification and speciation of Leishmania using real-time PCR and DNA sequencing at the rRNA Internal Transcribed Spacer 2.

The ability to discriminate infection between closely related Leishmania species within the Viannia species complex, specifically L. braziliensis, L. guyanensis and L. panamensis is critical to inform the clinical diagnosis and determine the most efficacious treatment modality. We designed a nested primer set targeting the rRNA Internal Transcribed Spacer 2 (ITS2), located on Chromosome 27, to distinguish among all human infective Leishmania species. Separate nested and single primer pairs were developed for conventional and quantitative PCR approaches respectively. Species-specific single nucleotide polymorphisms and indels located within the PCR products were identified by Sanger sequencing. This single locus approach provides a sensitive and specific platform to identify the species of Leishmania causing infection.

Far-East Asian Toxoplasma isolates share ancestry with North and South/Central American recombinant lineages.

Toxoplasma gondii is a global protozoan pathogen. Clonal lineages predominate in Europe, North America, Africa, and China, whereas highly recombinant parasites are endemic in South/Central America. Far East Asian T. gondii isolates are not included in current global population genetic structure analyses at WGS resolution. Here we report a genome-wide population study that compared eight Japanese and two Chinese isolates against representative worldwide T. gondii genomes using POPSICLE, a novel population structure analyzing software. Also included were 7 genomes resurrected from non-viable isolates by target enrichment sequencing. Visualization of the genome structure by POPSICLE shows a mixture of Chinese haplogroup (HG) 13 haploblocks introgressed within the genomes of Japanese HG2 and North American HG12. Furthermore, two ancestral lineages were identified in the Japanese strains; one lineage shares a common ancestor with HG11 found in both Japanese strains and North American HG12. The other ancestral lineage, found in T. gondii isolates from a small island in Japan, is admixed with genetically diversified South/Central American strains. Taken together, this study suggests multiple ancestral links between Far East Asian and American T. gondii strains and provides insight into the transmission history of this cosmopolitan organism.

Extant interspecific hybridization among trematodes within the Schistosoma haematobium species complex in Nigeria.

BACKGROUND: Natural interspecific hybridization between the human parasite (Schistosoma haematobium [Sh]) and bovine parasites (Schistosoma bovis [Sb], Schistosoma curassoni [Sc]) is increasingly reported in Africa. We developed a multi-locus PCR DNA-Seq strategy that amplifies two unlinked nuclear (transITS, BF) and two linked organellar genome markers (CO1, ND5) to genotype S. haematobium eggs collected from infected people in Ile Oluji/Oke Igbo, Ondo State (an agrarian community) and Kachi, Jigawa State (a pastoral community) in Southwestern and Northern Nigeria, respectively. PRINCIPAL FINDINGS: Out of a total of 219 urine samples collected, 57 were positive for schistosomes. All patients from Jigawa state possessed an Sh mitochondrial genome and were infected with a genetic profile consistent with an Sh x Sb hybrid based on sequences obtained at CO1, ND5, transITS and BF nuclear markers. Whereas samples collected from Ondo state were more varied. Mitonuclear discordance was observed in all 17 patients, worms possessed an Sb mitochondrial genome but one of four different genetic profiles at the nuclear markers, either admixed (heterozygous between Sh x Sc or Sh x Sb) at both markers (n = 10), Sh at BF and admixed at transITS (Sh x Sc) (n = 5), admixed (Sh x Sc) at BF and homozygous Sc at transITS (n = 1) or homozygous Sh at BF and homozygous Sc at transITS (n = 1). SIGNIFICANCE: Previous work suggested that zoonotic transmission of S. bovis in pastoral communities, where humans and animals share a common water source, is a driving factor facilitating interspecific hybridization. However, our data showed that all samples were hybrids, with greater diversity identified in Southwestern Nigeria, a non-pastoral site. Further, one patient possessed an S. bovis mitochondrial genome but was homozygous for S. haematobium at BF and homozygous for S. curassoni at transITS supporting at least two separate backcrosses in its origin, suggesting that interspecific hybridization may be an ongoing process.

Commensal protist Tritrichomonas musculus exhibits a dynamic life cycle that induces extensive remodeling of the gut microbiota.

Commensal protists and gut bacterial communities exhibit complex relationships, mediated at least in part through host immunity. To improve our understanding of this tripartite interplay, we investigated community and functional dynamics between the murine protist Tritrichomonas musculus and intestinal bacteria in healthy and B-cell-deficient mice. We identified dramatic, protist-driven remodeling of resident microbiome growth and activities, in parallel with Tritrichomonas musculus functional changes, which were accelerated in the absence of B cells. Metatranscriptomic data revealed nutrient-based competition between bacteria and the protist. Single-cell transcriptomics identified distinct Tritrichomonas musculus life stages, providing new evidence for trichomonad sexual replication and the formation of pseudocysts. Unique cell states were validated in situ through microscopy and flow cytometry. Our results reveal complex microbial dynamics during the establishment of a commensal protist in the gut, and provide valuable data sets to drive future mechanistic studies.

Fatal hepatic sarcocystosis in three captive and one free-ranging pinniped.

Fatal hepatic sarcocystosis was diagnosed as the cause of death in four pinnipeds: two captive Hawaiian monk seals (Monachus schauinslandi), a captive, and a free-ranging California sea lion (Zalophus californianus). Based on necropsy, histopathology, electron microscopy and DNA sequencing, intralesional protozoal schizonts were determined to have caused the necrotizing hepatitis observed. Transmission Electron Microscopy (TEM) revealed schizonts similar to Sarcocystis canis in hepatocytes. PCR-DNA sequencing and phylogenetic analysis at the conserved 18S rRNA and variable ITS1 gene markers within the nuclear rRNA gene array from schizont-laden tissue established that the parasites were indistinguishable from Sarcocystis canis at the 18S rRNA locus. However, six distinct single nucleotide polymorphisms (SNPs) were resolved at ITS1 suggesting that the parasites infecting pinnipeds were distinct from S. canis, which commonly infects bears and dogs. We hypothesize that the parasite represents a novel Sarcocystis variant that we refer to as S. canis-like that infects pinnipeds. The definitive host of S. canis is enigmatic and its life cycle incomplete. These findings document a critical need to identify the life cycle(s), definitive host(s), and all susceptible marine and terrestrial intermediate hosts of S. canis and the S. canis-like variant infecting pinnipeds.

The same genotype of Sarcocystis neurona responsible for mass mortality in marine mammals induced a clinical outbreak in raccoons (Procyon lotor) 10 years later.

Here, we report the first known outbreak of clinical protozoal myeloencephalitis in naturally infected raccoons by the parasite Sarcocystis neurona. The North American opossum (Didelphis virginiana) and the South American opossum (Didelphis albiventris) are its known definitive hosts. Several other animal species are its intermediate or aberrant hosts. The raccoon (Procyon lotor) is considered the most important intermediate host for S. neurona in the USA. More than 50% of raccoons in the USA have sarcocysts in their muscles, however clinical sarcocystosis in raccoons is rare. In 2014, 38 free-living raccoons were found dead or moribund on the grounds of the Saint Louis Zoo, Missouri, USA. Moribund individuals were weak, lethargic, and mildly ataxic; several with oculo-nasal discharge. Seven raccoons were found dead and 31 were humanely euthanized. Postmortem examinations were conducted on nine raccoons. Neural lesions compatible with acute sarcocystosis were detected in eight raccoons. The predominant lesions were meningoencephalitis and perivascular mononuclear cells. Histologic evidence for the Canine Distemper Virus was found in one raccoon. Schizonts and merozoites were present in the encephalitic lesions of four raccoons. Mature sarcocysts were present within myocytes of five raccoons. In six raccoons, S. neurona schizonts and merozoites were confirmed by immunohistochemical staining with S. neurona-specific polyclonal antibodies. Viable S. neurona was isolated from the brains of two raccoons by bioassay in interferon gamma gene knockout mice and in cell cultures seeded directly with raccoon brain homogenate. Molecular characterization was based on raccoon no. 68. Molecular characterization based on multi-locus typing at five surface antigens (SnSAG1-5-6, SnSAG3 and SnSAG4) and the ITS-1 marker within the ssrRNA locus, using DNA isolated from bradyzoites released from sarcocysts in a naturally infected raccoon (no. 68), confirmed the presence of S. neurona antigen type I, the same genotype that caused a mass mortality event in which 40 southern sea otters stranded dead or dying within a 3 week period in April 2004 with S. neurona-associated disease. An expanded set of genotyping markers was next applied. This study reports the following new genotyping markers at 18S rRNA, 28S rRNA, COX1, ITS-1, RON1, RON2, GAPDH1, ROP20, SAG2, SnSRS21 and TUBA1 markers. The identity of Sarcocystis spp. infecting raccoons is discussed.

Fine structure and molecular characterization of two new parabasalid species that naturally colonize laboratory mice, Tritrichomonas musculus and Tritrichomonas casperi.

Tritrichomonas muris is a common flagellated protist isolated from the cecum of wild rodents. This commensal protist has been shown previously to alter immune phenotypes in laboratory mice. Other trichomonads, referred to as Tritrichomonas musculis and Tritrichomonas rainier, also naturally colonize laboratory mice and cause immune alterations. This report formally describes two new trichomonads, Tritrichomonas musculus n. sp., and Tritrichomonas casperi n. sp., at the ultrastructural and molecular level. These two protists were isolated from laboratory mice and were differentiated by their size and the structure of their undulating membrane and posterior flagellum. Analysis at the 18S rRNA and trans-ITS genetic loci supported their designation as distinct species, related to T. muris. To assess the true extent of parabasalid diversity infecting laboratory mice, 135 mice bred at the National Institutes of Health (NIH) were screened using pan-parabasalid primers that amplify the trans-ITS region. Forty-four percent of mice were positive for parabasalids, encompassing a total of eight distinct sequence types. Tritrichomonas casperi and Trichomitus-like protists were dominant. T. musculus and T. rainier were also detected, but T. muris was not. Our work establishes a previously underappreciated diversity of commensal trichomonad flagellates that naturally colonize the enteric cavity of laboratory mice.

The commensal protist Tritrichomonas musculus exhibits a dynamic life cycle that induces extensive remodeling of the gut microbiota.

Commensal protists and gut bacterial communities exhibit complex relationships, mediated at least in part through host immunity. To improve our understanding of this tripartite interplay, we investigated community and functional dynamics between the murine protist Tritrichomonas musculus ( T. mu ) and intestinal bacteria in healthy and B cell-deficient mice. We identified dramatic, protist-driven remodeling of resident microbiome growth and activities, in parallel with T. mu functional changes, accelerated in the absence of B cells. Metatranscriptomic data revealed nutrient-based competition between bacteria and the protist. Single cell transcriptomics identified distinct T. mu life stages, providing new evidence for trichomonad sexual replication and the formation of pseudocysts. Unique cell states were validated in situ through microscopy and flow cytometry. Our results reveal complex microbial dynamics during the establishment of a commensal protist in the gut, and provide valuable datasets to drive future mechanistic studies.

Ultrastructural identification and molecular characterization of two new parabasalid species that naturally colonize laboratory mice, Tritrichomonas musculus and Tritrichomonas casperi.

Tritrichomonas muris is a flagellated protist isolated from the cecum of wild mice in the Czech Republic. This commensal protist has been shown previously to alter immune phenotypes in laboratory mice. Other trichomonads, previously referred to as Tritrichomonas musculis and Tritrichomonas rainier , also naturally colonize laboratory mice and cause immune alterations. This report formally describes two new trichomonads, Tritrichomonas musculus n. sp., and Tritrichomonas casperi n. sp., at the ultrastructural and molecular level. These two protists were isolated from laboratory mice, and were differentiated by their size and the structure of their undulating membrane and posterior flagellum. Analysis at the 18S rRNA and trans- ITS genetic loci supported their designation as distinct species, related to T. muris . To further assess the true extent of parabasalid diversity infecting laboratory mice, 135 mice were screened at the NIH using pan-parabasalid primers that amplify the trans- ITS region. Forty-four percent of mice were positive for parabasalids, encompassing a total of 8 distinct sequence types. Tritrichomonas casperi and Trichomitus- like protists were dominant. T. musculus and T. rainier were also detected, but T. muris was not. Our work establishes a previously underappreciated diversity of commensal trichomonad protists that naturally colonize the enteric cavity of laboratory mice.

Congenital ocular toxoplasmosis in consecutive siblings / Toxoplasmose ocular congênita em irmãos consecutivos

ABSTRACT Toxoplasma gondii infection can cause ocular manifestations after acquired and congenital disease. We report two cases of symptomatic congenital toxoplasmosis with ocular involvement in non-twin siblings, with a 2-year interval between pregnancies. Vertical transmission of toxoplasmosis in successive pregnancies, which was once considered impossible, is now found to be plausible even in immunocompetent subjects.

RESUMO A infecção pelo Toxoplasma gondii pode causar manifestações oculares tanto após a sua forma congênita quanto a sua forma adquirida. Reportamos aqui dois casos de toxoplasmose congênita sintomática com envolvimento ocular em irmãos não gêmeos, com intervalo de 2 anos entre gestações. A transmissão vertical da toxoplasmose em gestações sucessivas, outrora considerada impossível, é um evento plausível mesmo em indivíduos imunocompetentes.

A Flow Cytometry-Based Assay to Measure C3b Deposition on Protozoan Parasites Such as Toxoplasma gondii.

Flow cytometry is a powerful tool that can be used to study protozoan parasite interactions with the complement system. We developed a flow cytometric assay to measure the deposition of complement activation product C3b and to assess resistance to complement-mediated lysis. This assay involves exposing cultured parasites to human serum (the source of human complement) and staining parasites with antibodies against complement proteins to detect and quantify complement components on the parasite surface by flow cytometry. The assay can be used to compare complement activation across a variety of different species of protozoan parasites. As a proof of concept, we describe protocols to study C3 deposition on the single-cell protist Toxoplasma gondii. This parasite actively regulates C3 deposition and proteolytic inactivation to eliminate the formation of lytic pores targeted to the parasite surface coat, which is the end-product of the complement cascade. The antibodies used in this assay recognize both active and inactive forms of C3 deposited on parasite surfaces. Hence, the assay facilitates the identification and characterization of parasite resistance factors that regulate complement deposition and catabolic inactivation. © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Culturing human foreskin fibroblasts and Toxoplasma gondii strains Basic Protocol 2: In vitro complement activation assay Support Protocol: Screening of normal human serum Basic Protocol 3: Flow cytometric analysis of C3b deposition.

Intestinal immune responses to commensal and pathogenic protozoa.

The physical barrier of the intestine and associated mucosal immunity maintains a delicate homeostatic balance between the host and the external environment by regulating immune responses to commensals, as well as functioning as the first line of defense against pathogenic microorganisms. Understanding the orchestration and characteristics of the intestinal mucosal immune response during commensal or pathological conditions may provide novel insights into the mechanisms underlying microbe-induced immunological tolerance, protection, and/or pathogenesis. Over the last decade, our knowledge about the interface between the host intestinal mucosa and the gut microbiome has been dominated by studies focused on bacterial communities, helminth parasites, and intestinal viruses. In contrast, specifically how commensal and pathogenic protozoa regulate intestinal immunity is less well studied. In this review, we provide an overview of mucosal immune responses induced by intestinal protozoa, with a major focus on the role of different cell types and immune mediators triggered by commensal (Blastocystis spp. and Tritrichomonas spp.) and pathogenic (Toxoplasma gondii, Giardia intestinalis, Cryptosporidium parvum) protozoa. We will discuss how these various protozoa modulate innate and adaptive immune responses induced in experimental models of infection that benefit or harm the host.

Using the Sleeping Beauty (SB) Transposon to Generate Stable Cells Producing Enveloped Virus-Like Particles (eVLPs) Pseudotyped with SARS-CoV-2 Proteins for Vaccination.

The Sleeping Beauty (SB) transposon system is an efficient non-viral tool for gene transfer into a variety of cells, including human cells. Through a cut-and-paste mechanism, your favorite gene (YFG) is integrated into AT-rich regions within the genome, providing stable long-term expression of the transfected gene. The SB system is evolving and has become a powerful tool for gene therapy. There are no safety concerns using this system, the handling is easy, and the time required to obtain a stable cell line is significantly reduced compared to other systems currently available. Here, we present a novel application of this system to generate, within 8 days, a stable producer HEK293T cell line capable of constitutively delivering enveloped virus-like particles (eVLPs) for vaccination. We provide step-by-step protocols for generation of the SB transposon constructs, transfection procedures, and validation of the produced eVLPs. We next describe a method to pseudotype the constitutively produced eVLPs using the Spike protein derived from the SARS-CoV-2 virus (by coating the eVLP capsid with the heterologous antigen). We also describe optimization methods to scale up the production of pseudotyped eVLPs in a laboratory setting (from 100 µg to 5 mg). © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Generation of the SB plasmids Basic Protocol 2: Generation of a stable HEK293T cell line constitutively secreting MLV-based eVLPs Basic Protocol 3: Evaluation of the SB constructs by immunofluorescence assay Basic Protocol 4: Validation of eVLPs by denaturing PAGE and western blot Alternate Protocol 1: Analysis of SARS-CoV-2 Spike protein oligomerization using blue native gel electrophoresis and western blot Alternate Protocol 2: Evaluation of eVLP quality by electron microscopy (negative staining) Basic Protocol 5: Small-scale production of eVLPs Alternate Protocol 3: Large-scale production of eVLPs (up to about 1 to 3 mg VLPs) Alternate Protocol 4: Large-scale production of eVLPs (up to about 3 to 5 mg VLPs) Support Protocol: Quantification of total protein concentration by Bradford assay.

Congenital ocular toxoplasmosis in consecutive siblings.

Toxoplasma gondii infection can cause ocular manifestations after acquired and congenital disease. We report two cases of symptomatic congenital toxoplasmosis with ocular involvement in non-twin siblings, with a 2-year interval between pregnancies. Vertical transmission of toxoplasmosis in successive pregnancies, which was once considered impossible, is now found to be plausible even in immunocompetent subjects.

INVESTIGATION OF SARCOCYSTIS SPP. INFECTION IN FREE-RANGING AMERICAN BLACK BEARS (URSUS AMERICANUS) AND GRIZZLY BEARS (URSUS ARCTOS HORRIBILIS) IN BRITISH COLUMBIA, CANADA.

Sarcocystis spp. are protozoan parasites that cause a spectrum of lesions in various hosts. Hepatic sarcocystosis and encephalitis have been described in captive American black bears (Ursus americanus) and polar bears (Ursus maritimus), and in a free-ranging grizzly bear (Ursus arctos horribilis), but have not previously been reported in free-ranging American black bears. This study aimed to characterize the presence and lesions associated with Sarcocystis spp. in free-ranging bears in British Columbia, Canada from samples submitted to the provincial diagnostic laboratory. From 2007 to 2019, 102 free-ranging American black bear and grizzly bear tissues were examined postmortem for sarcocystosis using histopathology and follow-up molecular diagnostics. Sarcocystosis was confirmed in 41 (40%) free-ranging bears including 39 American black bears and two grizzly bears. Microscopic lesions included multifocal necrotizing hepatitis, nonsuppurative encephalitis, and/or intramuscular sarcocysts with or without associated inflammation. Sarcocystosis was considered the cause of death in eight (20%) of these bears, exclusively in cubs of the year (<1 yr old). Sarcocystis canis was identified in 22/32 (69%) cases where molecular characterization was performed and was the etiologic agent associated with bears that died of sarcocystosis. Confirmed cases were distributed widely across British Columbia. While there was an alternate proximate cause of death in the other confirmed bears, sarcocystosis may have contributed. Age was a significant risk factor, with yearlings presenting more often with fulminant lesions; however, there was a sampling bias toward juvenile bear submissions due to size and ease of transport. Further research is needed to understand the disease epidemiology and significance to population health.