Sarcocystis cruzi (Hasselmann, 1923) Wenyon, 1926: redescription, molecular characterization and deposition of life cycle stages specimens in the Smithsonian Museum.

Currently, 7 named Sarcocystis species infect cattle: Sarcocystis hirsuta, S. cruzi, S. hominis, S. bovifelis, S. heydorni, S. bovini and S. rommeli; other, unnamed species also infect cattle. Of these parasites of cattle, a complete life cycle description is known only for S. cruzi, the most pathogenic species in cattle. The life cycle of S. cruzi was completed experimentally in 1982, before related parasite species were structurally characterized, and before the advent of molecular diagnostics; to our knowledge, no archived frozen tissues from the cattle employed in the original descriptions remain for DNA characterization. Here, we isolated DNA from a paraffin-embedded kidney of a calf experimentally infected with S. cruzi in 1980; we then sequenced portions of 18S rRNA, 28S rRNA, COX1 and Acetyl CoA genes and verified that each shares 99­100% similarity to other available isolates attributed to S. cruzi from naturally infected cattle. We also reevaluated histological sections of tissues of calves experimentally infected with S. cruzi in the original description, exploiting improvements in photographic technology to render clearer morphological detail. Finally, we reviewed all available studies of the life cycle of S. cruzi, noting that S. cruzi was transmitted between bison (Bison bison) and cattle (Bos taurus) and that the strain of parasite derived from bison appeared more pathogenic than the cattle strain. Based on these newfound molecular, morphological and physiological data, we thereby redescribed S. cruzi and deposited reference material in the Smithsonian Museum for posterity.

Human impact on the diversity and virulence of the ubiquitous zoonotic parasite Toxoplasma gondii.

A majority of emerging infectious diseases in humans are zoonoses. Understanding factors that influence the emergence and transmission of zoonoses is pivotal for their prevention and control. Toxoplasma gondii is one of the most widespread zoonotic pathogens known today. Whereas only a few genotypes of T. gondii dominate in the Northern Hemisphere, many genotypes coexist in South America. Furthermore, T. gondii strains from South America are more likely to be virulent than those from the Northern Hemisphere. However, it is not clear what factor(s) shaped modern-day genetic diversity and virulence of T. gondii Here, our analysis suggests that the rise and expansion of farming in the past 11,000 years established the domestic cat/mouse transmission cycle for T. gondii, which has undoubtedly played a significant role in the selection of certain linages of T. gondii Our mathematical simulations showed that within the domestic transmission cycle, intermediately mouse-virulent T. gondii genotypes have an adaptive advantage and eventually become dominant due to a balance between lower host mortality and the ability to superinfect mice previously infected with a less virulent T. gondii strain. Our analysis of the global type II lineage of T. gondii suggests its Old World origin but recent expansion in North America, which is likely the consequence of global human migration and trading. These results have significant implications concerning transmission and evolution of zoonotic pathogens in the rapidly expanding anthropized environment demanded by rapid growth of the human population and intensive international trading at present and in the future.

Hepatitis E Virus in Pigs from Slaughterhouses, United States, 2017-2019.

Hepatitis E virus (HEV) RNA was detected in 6.3% and HEV IgG in 40% of 5,033 serum samples from market-weight pigs at 25 slaughterhouses in 10 US states. The prevalent HEV genotype was zoonotic genotype 3, group 2. Blood of HEV-viremic pigs from slaughterhouses may contaminate pork supply chains.

Comparative demography elucidates the longevity of parasitic and symbiotic relationships.

Parasitic and symbiotic relationships govern vast nutrient and energy flows, yet controversy surrounds their longevity. Enduring relationships may engender parallel phylogenies among hosts and parasites, but so may ephemeral relationships when parasites colonize related hosts. An understanding of whether symbiont and host populations have grown and contracted in concert would be useful when considering the temporal durability of these relationships. Here, we devised methods to compare demographic histories derived from genomic data. We compared the historical growth of the agent of severe human malaria, Plasmodium falciparum, and its mosquito vector, Anopheles gambiae, to human and primate histories, thereby discerning long-term parallels and anthropogenic population explosions. The growth history of Trichinella spiralis, a zoonotic parasite disseminated by swine, proved regionally specific, paralleling distinctive growth histories for wild boar in Asia and Europe. Parallel histories were inferred for an anemone and its algal symbiont (Exaiptasia pallida and Symbiodinium minutum). Concerted growth in potatoes and the agent of potato blight (Solanum tuberosum and Phytophthora infestans) did not commence until the age of potato domestication. Through these examples, we illustrate the utility of comparative historical demography as a new exploratory tool by which to interrogate the origins and durability of myriad ecological relationships. To facilitate future use of this approach, we introduce a tool called C-PSMC to align and evaluate the similarity of demographic history curves.

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.

Histopathological, morphological, and molecular characterization of Sarcocystis species in elk (Cervus elaphus) from Pennsylvania, USA.

Sarcocystis sarcocysts are common in many species of domestic and wild animals. Here, we report sarcocystosis in muscles from 91 free range elk (Cervus elaphus) from Pennsylvania, USA, tested by histopathology, transmission electron microscopy (TEM), and DNA sequencing. Sarcocysts were detected in hematoxylin and eosin (HE)-stained sections from 83 of 91 (91.2%) elk, including 83/91 (91.2%) tongues and 15/17 (88.2%) hearts. With respect to age, sarcocysts were found in 0/5 calves, 8/9 (88.8%) yearlings, and 75/77 (97.4%) adults. Sarcocysts were identified in 62/69 (89.4%) females and 21/22 (91.2%) males. Associated lesions were mild and consisted of inflammatory foci around degenerate sarcocysts. There were two morphologically distinct sarcocysts based on wall thickness, thin (< 0.5 µm) and thick-walled (> 4.0 µm). Thin-walled sarcocysts had a TEM "type 2" and villar protrusions (vps), identical to Sarcocystis wapiti previously described from elk in western USA. This species was present both in tongue and heart samples and was detected in all infected elk. Thick-walled sarcocysts consisted of three morphologic variants, referred to herein as subkinds A, B, C. Subkind A sarcocysts were rare; only four sarcocysts were found in three elk. Histologically, they had a 5-8-µm thick wall with tufted vp. By TEM, the sarcocyst wall was "type 12" and appeared similar to Sarcocystis sybillensis, previously described from elk in USA. Subkind B, Sarcocystis sp.1 sarcocysts were also rare, found in only 1 elk. These sarcocysts had 6.7-7.3-µm-thick wall with TEM "type 15b" vp. Subkind C Sarcocystis sp.2 sarcocysts were more common (22/91). Morphologically, the sarcocyst wall was 6.1-6.8 µm thick and contained "type 10b" vp. Comparisons of ribosomal DNA loci with published sequences indicated all sarcocysts were similar to what has previously been isolated from cervid hosts across the northern hemisphere. Phylogenetic analysis placed the thin-walled S. wapiti within a strongly supported clade with S. linearis and S. taeniata, while the thick-walled cysts were very closely related to S. truncata, S. elongata, S. silva, and S. tarandi. Further sequencing is needed to produce molecular diagnostics to distinguish among these species. North American elk are hosts to multiple Sarcocystis species with diverse morphology, deriving from two separate evolutionary lineages.

Long-read sequencing improves assembly of Trichinella genomes 10-fold, revealing substantial synteny between lineages diverged over 7 million years.

Genome assemblies can form the basis of comparative analyses fostering insight into the evolutionary genetics of a parasite's pathogenicity, host-pathogen interactions, environmental constraints and invasion biology; however, the length and complexity of many parasite genomes has hampered the development of well-resolved assemblies. In order to improve Trichinella genome assemblies, the genome of the sylvatic encapsulated species Trichinella murrelli was sequenced using third-generation, long-read technology and, using syntenic comparisons, scaffolded to a reference genome assembly of Trichinella spiralis, markedly improving both. A high-quality draft assembly for T. murrelli was achieved that totalled 63·2 Mbp, half of which was condensed into 26 contigs each longer than 571 000 bp. When compared with previous assemblies for parasites in the genus, ours required 10-fold fewer contigs, which were five times longer, on average. Better assembly across repetitive regions also enabled resolution of 8 Mbp of previously indeterminate sequence. Furthermore, syntenic comparisons identified widespread scaffold misassemblies in the T. spiralis reference genome. The two new assemblies, organized for the first time into three chromosomal scaffolds, will be valuable resources for future studies linking phenotypic traits within each species to their underlying genetic bases.

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.

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.

Globally diverse Toxoplasma gondii isolates comprise six major clades originating from a small number of distinct ancestral lineages.

Marked phenotypic variation characterizes isolates of Toxoplasma gondii, a ubiquitous zoonotic parasite that serves as an important experimental model for studying apicomplexan parasites. Progress in identifying the heritable basis for clinically and epidemiologically significant differences requires a robust system for describing and interpreting evolutionary subdivisions in this prevalent pathogen. To develop such a system, we have examined more than 950 isolates collected from around the world and genotyped them using three independent sets of polymorphic DNA markers, sampling 30 loci distributed across all nuclear chromosomes as well as the plastid genome. Our studies reveal a biphasic pattern consisting of regions in the Northern Hemisphere where a few, highly clonal and abundant lineages predominate; elsewhere, and especially in portions of South America are characterized by a diverse assemblage of less common genotypes that show greater evidence of recombination. Clustering methods were used to organize the marked genetic diversity of 138 unique genotypes into 15 haplogroups that collectively define six major clades. Analysis of gene flow indicates that a small number of ancestral lineages gave rise to the existing diversity through a process of limited admixture. Identification of reference strains for these major groups should facilitate future studies on comparative genomics and identification of genes that control important biological phenotypes including pathogenesis and transmission.

Acute muscular sarcocystosis: an international investigation among ill travelers returning from Tioman Island, Malaysia, 2011-2012.

BACKGROUND: Through 2 international traveler-focused surveillance networks (GeoSentinel and TropNet), we identified and investigated a large outbreak of acute muscular sarcocystosis (AMS), a rarely reported zoonosis caused by a protozoan parasite of the genus Sarcocystis, associated with travel to Tioman Island, Malaysia, during 2011-2012. METHODS: Clinicians reporting patients with suspected AMS to GeoSentinel submitted demographic, clinical, itinerary, and exposure data. We defined a probable case as travel to Tioman Island after 1 March 2011, eosinophilia (>5%), clinical or laboratory-supported myositis, and negative trichinellosis serology. Case confirmation required histologic observation of sarcocysts or isolation of Sarcocystis species DNA from muscle biopsy. RESULTS: Sixty-eight patients met the case definition (62 probable and 6 confirmed). All but 2 resided in Europe; all were tourists and traveled mostly during the summer months. The most frequent symptoms reported were myalgia (100%), fatigue (91%), fever (82%), headache (59%), and arthralgia (29%); onset clustered during 2 distinct periods: "early" during the second and "late" during the sixth week after departure from the island. Blood eosinophilia and elevated serum creatinine phosphokinase (CPK) levels were observed beginning during the fifth week after departure. Sarcocystis nesbitti DNA was recovered from 1 muscle biopsy. CONCLUSIONS: Clinicians evaluating travelers returning ill from Malaysia with myalgia, with or without fever, should consider AMS, noting the apparent biphasic aspect of the disease, the later onset of elevated CPK and eosinophilia, and the possibility for relapses. The exact source of infection among travelers to Tioman Island remains unclear but needs to be determined to prevent future illnesses.

NextGen sequencing reveals short double crossovers contribute disproportionately to genetic diversity in Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii is a widespread protozoan parasite of animals that causes zoonotic disease in humans. Three clonal variants predominate in North America and Europe, while South American strains are genetically diverse, and undergo more frequent recombination. All three northern clonal variants share a monomorphic version of chromosome Ia (ChrIa), which is also found in unrelated, but successful southern lineages. Although this pattern could reflect a selective advantage, it might also arise from non-Mendelian segregation during meiosis. To understand the inheritance of ChrIa, we performed a genetic cross between the northern clonal type 2 ME49 strain and a divergent southern type 10 strain called VAND, which harbors a divergent ChrIa. RESULTS: NextGen sequencing of haploid F1 progeny was used to generate a genetic map revealing a low level of conventional recombination, with an unexpectedly high frequency of short, double crossovers. Notably, both the monomorphic and divergent versions of ChrIa were isolated with equal frequency. As well, ChrIa showed no evidence of being a sex chromosome, of harboring an inversion, or distorting patterns of segregation. Although VAND was unable to self fertilize in the cat, it underwent successful out-crossing with ME49 and hybrid survival was strongly associated with inheritance of ChrIII from ME49 and ChrIb from VAND. CONCLUSIONS: Our findings suggest that the successful spread of the monomorphic ChrIa in the wild has not been driven by meiotic drive or related processes, but rather is due to a fitness advantage. As well, the high frequency of short double crossovers is expected to greatly increase genetic diversity among progeny from genetic crosses, thereby providing an unexpected and likely important source of diversity.

Refrigeration provides a simple means to synchronize in vitro cultures of Plasmodium falciparum.

Plasmodium falciparum is usually asynchronous during in vitro culture. Highly synchronized cultures of P. falciparum are routinely used in malaria research. Here, we describe a simple synchronization procedure for P. falciparum asexual erythrocytic culture, which involves storage at 4°C for 8-24 h followed by routine culture. When cultures with 27-60% of ring stage were synchronized using this procedure, 70-93% ring stages were obtained after 48 h of culture and relative growth synchrony remained for at least two erythrocytic cycles. To test the suitability of this procedure for subsequent work, drug sensitivity assays were performed using four laboratory strains and four freshly adapted clinical P. falciparum isolates. Parasites synchronized by sorbitol treatment or refrigeration showed similar dose-response curves and comparable IC50 values to four antimalarial drugs. The refrigeration synchronization method is simple, inexpensive, time-saving, and should be especially useful when large numbers of P. falciparum culture are handled.

Comparative genomics of Giardia duodenalis sub-assemblage AI beaver (Be-2) and human (WB-C6) strains show remarkable homozygosity, sequence similarity, and conservation of VSP genes.

Giardia duodenalis, a major cause of waterborne infection, infects a wide range of mammalian hosts and is subdivided into eight genetically well-defined assemblages named A through H. However, fragmented genomes and a lack of comparative analysis within and between the assemblages render unclear the molecular mechanisms controlling host specificity and differential disease outcomes. To address this, we generated a near-complete de novo genome of AI assemblage using the Oxford Nanopore platform by sequencing the Be-2 genome. We generated 148,144 long-reads with quality scores of > 7. The final genome assembly consists of only nine contigs with an N50 of 3,045,186 bp. This assembly agrees closely with the assembly of another strain in the AI assemblage (WB-C6). However, a critical difference is that a region previously placed in the five-prime region of Chr5 belongs to Chr4 of Be-2. We find a high degree of conservation in the ploidy, homozygosity, and the presence of cysteine-rich variant-specific surface proteins (VSPs) within the AI assemblage. Our assembly provides a nearly complete genome of a member of the AI assemblage of G. duodenalis, aiding population genomic studies capable of elucidating Giardia transmission, host range, and pathogenicity.

Genome sequencing identifies two nearly unchanged strains of persistent Listeria monocytogenes isolated at two different fish processing plants sampled 6 years apart.

Listeria monocytogenes is a food-borne human-pathogenic bacterium that can cause infections with a high mortality rate. It has a remarkable ability to persist in food processing facilities. Here we report the genome sequences for two L. monocytogenes strains (N53-1 and La111) that were isolated 6 years apart from two different Danish fish processers. Both strains are of serotype 1/2a and belong to a highly persistent DNA subtype (random amplified polymorphic DNA [RAPD] type 9). We demonstrate using in silico analyses that both strains belong to the multilocus sequence typing (MLST) type ST121 that has been isolated as a persistent subtype in several European countries. The purpose of this study was to use genome analyses to identify genes or proteins that could contribute to persistence. In a genome comparison, the two persistent strains were extremely similar and collectively differed from the reference lineage II strain, EGD-e. Also, they differed markedly from a lineage I strain (F2365). On the proteome level, the two strains were almost identical, with a predicted protein homology of 99.94%, differing at only 2 proteins. No single-nucleotide polymorphism (SNP) differences were seen between the two strains; in contrast, N53-1 and La111 differed from the EGD-e reference strain by 3,942 and 3,471 SNPs, respectively. We included a persistent L. monocytogenes strain from the United States (F6854) in our comparisons. Compared to nonpersistent strains, all three persistent strains were distinguished by two genome deletions: one, of 2,472 bp, typically contains the gene for inlF, and the other, of 3,017 bp, includes three genes potentially related to bacteriocin production and transport (lmo2774, lmo2775, and the 3'-terminal part of lmo2776). Further studies of highly persistent strains are required to determine if the absence of these genes promotes persistence. While the genome comparison did not point to a clear physiological explanation of the persistent phenotype, the remarkable similarity between the two strains indicates that subtypes with specific traits are selected for in the food processing environment and that particular genetic and physiological factors are responsible for the persistent phenotype.

RNA-Seq of Phenotypically Distinct Eimeria maxima Strains Reveals Coordinated and Contrasting Maturation and Shared Sporogonic Biomarkers with Eimeria acervulina.

Strains of Eimeria maxima, an enteric parasite of poultry, vary in virulence. Here, we performed microscopy and RNA sequencing on oocysts of strains APU-1 (which exhibits more virulence) and APU-2. Although each underwent parallel development, APU-1 initially approached maturation more slowly. Each strain sporulated by hour 36; their gene expression diverged somewhat thereafter. Candidate biomarkers of viability included 58 genes contributing at least 1000 Transcripts Per Million throughout sporulation, such as cation-transporting ATPases and zinc finger domain-containing proteins. Many genes resemble constitutively expressed genes also important to Eimeria acervulina. Throughout sporulation, the expression of only a few genes differed between strains; these included cyclophilin A, EF-1α, and surface antigens (SAGs). Mature and immature oocysts uniquely differentially express certain genes, such as an X-Pro dipeptidyl-peptidase domain-containing protein in immature oocysts and a profilin in mature oocysts. The immature oocysts of each strain expressed more phosphoserine aminotransferase and the mature oocysts expressed more SAGs and microneme proteins. These data illuminate processes influencing sporulation in Eimeria and related genera, such as Cyclospora, and identify biological processes which may differentiate them. Drivers of development and senescence may provide tools to assess the viability of oocysts, which would greatly benefit the poultry industry and food safety applications.

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.

Genetic diversity of Plasmodium vivax populations from the China-Myanmar border identified by genotyping merozoite surface protein markers.

BACKGROUND: Parasite diversity and population structure influence malaria control measures. Malaria transmission at international borders affects indigenous residents and migrants, defying management efforts and resulting in malaria re-introduction. Here we aimed to determine the extent and distribution of genetic variations in Plasmodium vivax populations and the complexity of infections along the China-Myanmar border. METHODS: We collected clinical P. vivax samples from local and migrant malaria patients from Laiza and Myitsone, Kachin State, Myanmar, respectively. We characterized the polymorphisms in two P. vivax merozoite surface protein markers, Pvmsp-3α and Pvmsp-3ß, by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. We sought to determine whether these genetic markers could differentiate these two neighboring parasite populations. RESULTS: PCR revealed three major size variants for Pvmsp-3α and four for Pvmsp-3ß among the 370 and 378 samples, respectively. PCR-RFLP resolved 26 fragment-size alleles by digesting Pvmsp-3α with Alu I and Hha I and 28 alleles by digesting Pvmsp-3ß with Pst I. PCR-RFLP analysis of Pvmsp-3α found that infections in migrant laborers from Myitsone bore more alleles than did infections in residents of Laiza, while such difference was not evident from genotyping Pvmsp-3ß. Infections originating from these two places contained distinct but overlapping subpopulations of P. vivax. Infections from Myitsone had a higher multiplicity of infection as judged by the size of the Pvmsp-3α amplicons and alleles after Alu I/Hha I digestion. CONCLUSIONS: Migrant laborers from Myitsone and indigenous residents from Laiza harbored overlapping but genetically distinct P. vivax parasite populations. The results suggested a more diverse P. vivax population in Myitsone than in the border town of Laiza. PCR-RFLP of Pvmsp-3α offers a convenient method to determine the complexity of P. vivax infections and differentiate parasite populations.

Chromosomal scale assembly reveals localized structural variants in avian caecal coccidian parasite Eimeria tenella.

Eimeria tenella is a major cause of caecal coccidiosis in commercial poultry chickens worldwide. Here, we report chromosomal scale assembly of Eimeria tenella strain APU2, a strain isolated from commercial broiler chickens in the U.S. We obtained 100× sequencing Oxford Nanopore Technology (ONT) and more than 800× Coverage of Illumina Next-Seq. We created the assembly using the hybrid approach implemented in MaSuRCA, achieving a contiguous 51.34 Mb chromosomal-scale scaffolding enabling identification of structural variations. The AUGUSTUS pipeline predicted 8060 genes, and BUSCO deemed the genomes 99% complete; 6278 (78%) genes were annotated with Pfam domains, and 1395 genes were assigned GO-terms. Comparing E. tenella strains (APU2, US isolate and Houghton, UK isolate) derived Houghton strain of E. tenella revealed 62,905 high stringency differences, of which 45,322 are single nucleotide polymorphisms (SNPs) (0.088%). The rate of transitions/transversions among the SNPs are 1.63 ts/tv. The strains possess conserved gene order but have profound sequence heterogeneity in a several chromosomal segments (chr 2, 11 and 15). Genic and intergenic variation in defined gene families was evaluated between the two strains to possibly identify sequences under selection. The average genic nucleotide diversity of 2.8 with average 2 kb gene length (0.145%) at genic level. We examined population structure using available E. tenella sequences in NCBI, revealing that the two E. tenella isolates from the U.S. (E. tenella APU2 and Wisconsin, "ERR296879") share a common maternal inheritance with the E. tenella Houghton. Our chromosomal level assembly promotes insight into Eimeria biology and evolution, hastening drug discovery and vaccine development.

Infections with Sarcocystis wenzeli are prevalent in the chickens of Yunnan Province, China, but not in the flocks of domesticated pigeons or ducks.

The distribution and prevalence of infections with species of Sarcocystis in domestic fowl in Asia are poorly known. Here, ducks, pigeons, and chickens from Yunnan Province, China were examined for evidence of parasitic infection with Sarcocystis spp. One hundred and ninety one chickens, 514 ducks, and nine pigeons were investigated. Whereas the ducks and pigeons lacked tissue cysts in their muscle, brain or peripheral nervous system, cysts of Sarcocystis wenzeli were identified in 17 of 191 chickens (8.9%). Morphologically, the cysts were thread-like, ranging in size from 334-3169 × 41-117 µm (mean 1093 × 65 µm). Cysts were septate with dense, short finger-like protrusions which appeared radially striated. The cyst wall was 1.4-3.5 µm (mean 2.4 µm) thick. The bradyzoites were lancet shaped and measured 12.2-17.7 × 1.8-2.9 µm (mean 14.6 × 2.5 µm). Ultrastucturally, the primary sarcocyst wall had stubby villar protrusions, corresponding to the 'type 9' class previously designated. The protrusions measured 0.87-1.89 × 0.47-0.91 µm (mean 1.27 × 0.59 µm; n = 57). These findings confirm previous work from the vicinity of Kunming concerning the occurrence of S. wenzeli in chickens, and its use of both cats and dogs as definitive hosts, but indicate that corresponding infections may not occur in the regional domestic flocks of other types of fowl.