White-tailed deer (Odocoileus virginianus) are a reservoir of a diversity of Toxoplasma gondii strains in the USA and pose a risk to consumers of undercooked venison.

To assess the role of white-tailed deer (Odocoileus virginianus, WTD) in the epidemiology of toxoplasmosis, we conducted a national survey of WTD across the USA for Toxoplasma gondii infection. To do this, we combined serology with parasite isolation to evaluate the prevalence and genetic diversity of T. gondii in this game species. From October 2012 to March 2019, serum and tissues were collected from 914 WTD across the USA. Serum samples were screened for antibodies to T. gondii, and then the tissues of seropositive WTD were bioassayed in mice. Antibodies were detected in 329 (36%) of 914 WTD tested by the modified agglutination test (positive reaction at 1:25 or higher). Viable T. gondii was isolated from the heart of 36 WTD from 11 states. Three of the 36 isolates were pathogenic but not highly virulent to outbred Swiss Webster mice and all 36 isolates could be propagated further in cell culture and were genotyped. For genotyping, DNA extracted from cell culture-derived tachyzoites was characterized by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using the genetic markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. Genotyping revealed seven ToxoDB PCR-RFLP genotypes, including 24 isolates for genotype #5 (haplogroup 12), four isolates for #2 (type III, haplogroup 3), three isolates for genotypes #1 (type II, haplogroup 2), two isolates for genotypes #3 (type II, haplogroup 2) and one isolate each for #39, #221 and #224. Genotype #5 was the most frequently isolated, accounting for 66.6% (24 of 36) of the isolates. Combining the 36 isolates from this study with previously reported 69 isolates from WTD, 15 genotypes have been identified. Among these, 50.4% (53/105) isolates belong to genotype #5. Our results indicate moderate genetic diversity of T. gondii in WTD. The results also indicate that undercooked venison should not be consumed by humans or fed to cats.

Genotyping of viable Toxoplasma gondii from the first national survey of feral swine revealed evidence for sylvatic transmission cycle, and presence of highly virulent parasite genotypes.

Feral swine are known reservoirs of various pathogens, including Toxoplasma gondii. Here, we report the first national survey of viable T. gondii in feral swine in the USA. We paired serological surveys with parasite isolation and bioassay to evaluate the prevalence and genetic diversity of these parasites. From 2012-2017, sera and tissues from 1517 feral swine across the USA were collected for the isolation of viable T. gondii. Serum samples were initially screened for antibodies to T. gondii, and then the tissues of seropositive feral swine were bioassayed in mice. Antibodies were detected in 27.7% of feral swine tested by the modified agglutination test (1:25 or higher). Antibody positive rates increased significantly with age, with 10.1% of juveniles, 16.0% of sub-adults and 38.4% of adults testing seropositive. Myocardium (50 g) from 232 seropositive feral swine was digested in pepsin and bioassayed in mice. Viable T. gondii was isolated from 78 feral swine from 21 states. Twelve of the 78 isolates were pathogenic to outbred Swiss Webster mice and 76 of the 78 isolates could be propagated further in cell culture and were genotyped. For genotyping, deoxyribonucleic acid extracted from cell culture-derived tachyzoites was characterized by polymerase chain reaction restriction fragment length polymorphism using the genetic markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. Genotyping revealed 15 ToxoDB genotypes, including 43 isolates for genotype #5 (haplogroup 12), 11 isolates for #24, four isolates for #2 (haplogroup 3), two isolates for each of genotypes #3 (haplogroup 2), #4 (haplogroup 12), #216, #221, #289 and #297 and one isolate for each of genotypes #1 (haplogroup 2), #39, #66, #260, #261 and #299. Genotype #5 was the most frequently isolated, accounted for 57% (43/76) of the isolates, followed by #24, accounted for 14% (11/76). Genotypes #260, #289, #297 and #299 are new types. Genotype #289 was highly virulent to mice and originated from feral swine collected in Louisiana on the same day at the same location. Genotype #216 was previously demonstrated to be highly virulent to mice. Our results indicate moderate genetic diversity of T. gondii in feral swine in the USA, with the genotype #5 (haplogroup 12) dominant in the continental USA, whereas genotype #24 (10/14) was dominant in Hawaii, suggesting different population structures of the parasites among the two distinct geographical locations.

Molecular characterization and development of Sarcocystis speeri sarcocysts in gamma interferon gene knockout mice.

The North American opossum (Didelphis virginiana) is the definitive host for at least three named species of Sarcocystis: Sarcocystis falcatula, Sarcocystis neurona and Sarcocystis speeri. The South American opossums (Didelphis albiventris, Didelphis marsupialis and Didelphis aurita) are definitive hosts for S. falcatula and S. lindsayi. The sporocysts of these Sarcocystis species are similar morphologically. They are also not easily distinguished genetically because of the difficulties of DNA extraction from sporocysts and availability of distinguishing genetic markers. Some of these species can be distinguished by bioassay; S. neurona and S. speeri are infective to gamma interferon gene knockout (KO) mice, but not to budgerigars (Melopsittacus undulatus); whereas S. falcatula and S. lindsayi are infective to budgerigars but not to KO mice. The natural intermediate host of S. speeri is unknown. In the present study, development of sarcocysts of S. speeri in the KO mice is described. Sarcocysts were first seen at 12 days post-inoculation (p.i.), and they became macroscopic (up to 4 mm long) by 25 days p.i. The structure of the sarcocyst wall did not change from the time bradyzoites had formed at 50-220 days p.i. Sarcocysts contained unique villar protrusions, 'type 38'. The polymerase chain reaction amplifications and sequences analysis of three nuclear loci (18S rRNA, 28S rRNA and ITS1) and two mitochondrial loci (cox1 and cytb) of S. speeri isolate from an Argentinean opossum (D. albiventris) confirmed its membership among species of Sarcocystis and indicated an especially close relationship to another parasite in this genus that employs opossums as its definitive host, S. neurona. These results should be useful in finding natural intermediate host of S. speeri.

In the United States, negligible rates of zoonotic sarcocystosis occur in feral swine that, by contrast, frequently harbour infections with Sarcocystis miescheriana, a related parasite contracted from canids.

Transmission of pathogens between domestic and wild life animals plays an important role in epidemiology. Feral pig populations are increasing and expanding in the USA, and may constitute a risk to non-biosecure domestic pig facilities by serving as reservoirs for pathogens. We surveyed, for Sarcocystis infection, the myocardium of 1006 feral pigs (Sus scrofa) trapped or hunted in 29 states during the Comprehensive Feral Swine Disease Surveillance Program of the USDA's Animal and Plant Health Inspection Service, Wildlife Services unit during 2012-2014. Sarcocysts were detected in histological sections of 25% (251/1006) of myocardium with an average parasitic load/intensity of infection of 3.03 sarcocysts/section (1.5×0.7 cm), and higher prevalence of myocarditis in severe infections. Microscopic examination of pepsin digests of 147 hearts revealed a higher prevalence of Sarcocystis bradyzoites (49%, 72/147) than when diagnosed by histology. A fragment of Sarcocystis 18S rRNA was amplified and digested with a restriction endonuclease, revealing a pattern consistent with Sarcocystis miescheriana in all 44 selected samples. Sequencing 31 of these 44 isolates confirmed their correspondence to S. miescheriana. Thus, S. miescheriana infection, but not the zoonotic parasite Sarcocystis suihominis, appears to be prevalent and widespread in feral pigs in the USA.

TRICHINELLA AND AT LEAST THREE SPECIES OF SARCOCYSTIS PARASITIZE THE MUSCLES OF BOBCATS (LYNX RUFUS) FROM MISSISSIPPI.

Muscles of 25 bobcats (Lynx rufus) from remote areas of Mississippi in 2017 were tested for parasites. Testing for Sarcocystis infections included microscopic examination of fresh unstained muscle squashes, pepsin digestion of hearts and tongues, and histological sections of paraffin-embedded tissues. Sarcocystis spp. infections were detected in the muscles of 21 (84%) by a combination of methods. Sarcocysts were detected in the unstained tongue squashes of 2 bobcats. Sarcocystis sp. bradyzoites were detected in the pepsin digests of 3 of 19 hearts, and 12 of 19 tongues. In paraffin-embedded histological sections, sarcocysts were detected in 7 of 25 hearts, 17 of 25 tongues, and 5 of 23 limb muscles. Based on the character of the cyst wall, at least 3 morphologic types of sarcocysts were detected: those with small spikes on the cyst wall, corresponding to Sarcocystis felis, those with long villar protrusions, corresponding to Sarcocystis neurona, and those lacking visible cyst wall protrusions, representing an unidentified type of sarcocyst. Myositis associated with sarcocysts was seen in the tongues of 3, and in the limb muscles of 1 bobcat. Multilocus genotyping of the DNA extracted from paraffin-embedded sections from 2 bobcats, employing 18S, 28S, COI, ITS-1, and 5.8S and rpoB genes, diagnosed Sarcocystis caninum, S. felis, Sarcocystis lutrae, and S. neurona. An encapsulated species of Trichinella was identified in the tongue of 1; it represents the first documented occurrences in bobcats from Mississippi. Taken together, these observations suggest intensive exposure of these wild carnivores to Trichinella tissue cysts, implies predation or scavenging on these tissues promotes parasite transmission, and raises caution concerning zoonotic risk when such meat is rendered for human consumption.

MOLECULAR AND MORPHOLOGICAL CHARACTERIZATION OF SARCOCYSTIS INFECTIONS IN THE MUSCLES OF GRAY WOLVES (CANIS LUPUS) FROM MINNESOTA SUGGEST THEY MAY SERVE AS RESERVOIRS FOR INFECTION IN DOMESTICATED DOGS.

ABSTRACT: Sarcocystis infections were found for the first time in the muscles of 3 of 3 gray wolves (Canis lupus) from Minnesota. Two kinds (thin-walled and thick-walled) of sarcocysts were detected, based on the appearance of the sarcocyst wall. In wolf 1, sarcocysts were thin-walled (<0.5 µm), and without any visible protrusions. Ultrastructurally, the sarcocyst wall was type 1a and identical to Sarcocystis svanai of the domestic dog (Canis familiaris). The second kind of sarcocyst, with a relatively thicker (>1 µm) sarcocyst wall, was detected in wolves 2 and 3. Ultrastructurally, the sarcocyst wall had undulating, pleomorphic villar protrusion of type 9c; these sarcocysts were identical to Sarcocystis caninum from the domestic dog. Molecularly, the 2 Sarcocystis species were characterized using 18S, 28S, COI, ITS-1, and rpoB genetic markers. All these markers showed 100% identity to either of the 2 species previously described from the domestic dog. The thick-walled sarococyst corresponded to Sarcocystis caninum, whereas the thin-walled sarcocyst corresponded to Sarcocystis svanai.

Filters comprised of sand and Zero Valent Iron hold promise as tools to mitigate risk posed by Cyclospora cayetanensis oocysts.

Irrigation water contaminated by human fecal material may elevate the risk of produce contamination with the enteric parasite Cyclospora cayetanensis. Oocysts of C. cayetanensis are resistant to commonly used disinfectants and a method of removing C. cayetanensis from irrigation water would mitigate this risk. We evaluated zero valent iron (ZVI) sand filtration as one such method. We sought to determine if sand filters containing ZVI outperformed those without ZVI. We first evaluated the abundant poultry parasites Eimeria maxima, E. tenella and E. acervulina as surrogates for C. cayetanensis. We determined if a miniaturized gravity fed ZVI-sand filter, scaled to evaluate scarce supplies of C. cayetanensis oocysts, provided useful information about the performance of larger filtration systems. Filters were inoculated with oocysts, rinsed, and the resulting filtrate examined microscopically for oocysts. We performed experiments to measure the effect of varying ZVI concentrations, repeated filter use, simulated agricultural water, and oocyst size and condition. We then compared the performance of mini filters to that of larger, gravity-fed pool filters and found that ZVI-sand filtration was far more effective at removing Eimeria spp. from water when compared to sand filtration, at both scales. Sand mini filters retained 13-54 % of E. acervulina oocysts, and pool filters retained 82 %, but when combined with 50 % (mini filter) or 35 % (pool filter) v/v ZVI, mini filters retained 89-99 % of oocysts and pool filters retained >99 %. The effectiveness of the mini filters increased with increasing ZVI concentration, and the addition of ZVI far outweighed the influence of any other measured variable. We then performed experiments including C. cayetanensis, which provided similar results to those utilizing Eimeria; 59 % of inoculated C. cayetanensis oocysts were retained in sand mini filters, and 97 % in mini filters composed of 35 % v/v ZVI. In sum, ZVI is highly effective in removing oocysts from water and Eimeria is a useful surrogate for C. cayetanensis to assess filtration. ZVI-sand filtration shows promise as a tool to mitigate the risk of C. cayetanensis contamination of irrigation water. Further studies should evaluate the performance of ZVI-sand in pressurized fast filtration systems under a range of field conditions.

Bovine sarcocystosis: Sarcocystis species, diagnosis, prevalence, economic and public health considerations, and association of Sarcocystis species with eosinophilic myositis in cattle.

Infections by Sarcocystis in cattle are ubiquitous worldwide. There is considerable debate concerning the identity of Sarcocystis spp. in cattle. Proper diagnosis of Sarcocystis spp. is important to assess their economic and public health importance. Currently there are seven named species: Sarcocystis hirsuta, Sarcocystis cruzi, Sarcocystis hominis, Sarcocystis bovifelis, arcocystis heydorni, Sarcocystis bovini and Sarcocystis rommeli. Additionally, there are unnamed Sarcocystis spp. Two species, S. hominis and S. heydorni, are zoonotic. One out of seven species (S. hirsuta, contracted from cats) forms macroscopic cysts which can be visible during carcass inspection. Current molecular characterization is based on DNA extracted from sarcocysts from naturally infected cattle because DNA was not characterized from tissues of experimentally infected cattle or feces of experimentally infected definitive hosts. Sarcocystis cruzi (transmitted via canids) is recognized as the most pathogenic species and it causes abortion, low milk yield, poor body growth, and outbreaks of clinical sarcocystosis and death. Additionally, Sarcocystis infections have been linked to an inflammatory condition of striated muscles termed bovine eosinophilic myositis (BEM). Cattle affected by BEM appear clinically normal. Diagnosis of BEM at slaughter occurs when inspecting the carcass surface, or once the carcass has been divided into prime cuts or quarters. Sex and breed have no apparent influence on prevalence of BEM. The condition evidently occurs with equal frequency in steers, cows, and heifers. Virtually all striated muscles can be affected including skeletal muscles, the muscles of the eye, larynx, and the heart. In the USA, regulations require condemnation of BEM-affected parts, or (in severe cases) the entire carcass. These aesthetic considerations result in economic losses. Cattle experimentally infected with Sarcocystis did not have BEM at slaughter. Here, we review the status of Sarcocystis spp. and BEM in cattle including prevalence, lesions, epidemiology, and association of BEM with different species of Sarcocystis.

Modest genetic differentiation among North American populations of Sarcocystis neurona may reflect expansion in its geographic range.

Sarcocystis neurona is an important cause of neurological disease in horses (equine protozoal myeloencephalitis, EPM) and sea otters in the United States. In addition, EPM-like disease has been diagnosed in several other land and marine mammals. Opossums are its only definitive hosts. Little genetic diversity among isolates of S. neurona from different hosts has been reported. Here, we used 11 microsatellites to characterize S. neurona DNA isolated from natural infections in 22 sea otters (Enhydra lutris) from California and Washington and in 11 raccoons (Procyon lotor) and 1 striped skunk (Mephitis mephitis) from Wisconsin. By jointly analyzing these 34 isolates with 26 isolates previously reported, we determined that geographic barriers may limit S. neurona dispersal and that only a limited subset of possible parasite genotypes may have been introduced to recently established opossum populations. Moreover, our study confirms that diverse intermediate hosts share a common infection source, the opossum (Didelphis virginiana).

Rare but evolutionarily consequential outcrossing in a highly inbred zoonotic parasite.

Recurrent self-mating can result in nearly clonal propagation of biological lineages, but even occasional outcrossing can serve to redistribute variation in future generations, providing cohesion among regional populations. The zoonotic parasite Trichinella spiralis has been suspected to undergo frequent inbreeding, resulting in genetically uniform larval cohorts which differ markedly from one another. Here, we explored the extent of inbreeding for this parasite by determining how genetic variation (at variable microsatellite markers) is distributed among 1379 larvae derived from 41 wild boars in Extremadura, Spain. In particular, we sought to determine how much of the genetic variation in this region's parasites occurs among the larvae of any given wild boar, and whether each derives from one, or more, parental lineages. We found strong evidence for inbreeding, resulting in genetically distinct parasite subpopulations among the parasites derived from many pairs of wild boar. Fully two-thirds of these parasite cohorts appear to derive from inbred parents; in 10% of the wild boars, parasites were so inbred as to become absolutely fixed in all of the assayed genetic loci. In spite of this, more than one pair of parents appear to have given rise to the infections in one-third of the sampled wild boars, resulting in mixed infections. These mixed infections should slow losses of heterozygosity and multi-locus polymorphism in any given parasite lineage. Such outcrossing should limit distinctions that would otherwise accumulate among transmission chains, thereby enforcing cohesion through the region's population in spite of its marked departure from panmixia. Conditions of transmission may differ in other regions, where such epidemiological features may engender different evolutionary outcomes.

Hsp60 is targeted to a cryptic mitochondrion-derived organelle ("crypton") in the microaerophilic protozoan parasite Entamoeba histolytica.

Entamoeba histolytica is a microaerophilic protozoan parasite in which neither mitochondria nor mitochondrion-derived organelles have been previously observed. Recently, a segment of an E. histolytica gene was identified that encoded a protein similar to the mitochondrial 60-kDa heat shock protein (Hsp60 or chaperonin 60), which refolds nuclear-encoded proteins after passage through organellar membranes. The possible function and localization of the amebic Hsp60 were explored here. Like Hsp60 of mitochondria, amebic Hsp60 RNA and protein were both strongly induced by incubating parasites at 42 degreesC. 5' and 3' rapid amplifications of cDNA ends were used to obtain the entire E. histolytica hsp60 coding region, which predicted a 536-amino-acid Hsp60. The E. histolytica hsp60 gene protected from heat shock Escherichia coli groEL mutants, demonstrating the chaperonin function of the amebic Hsp60. The E. histolytica Hsp60, which lacked characteristic carboxy-terminal Gly-Met repeats, had a 21-amino-acid amino-terminal, organelle-targeting presequence that was cleaved in vivo. This presequence was necessary to target Hsp60 to one (and occasionally two or three) short, cylindrical organelle(s). In contrast, amebic alcohol dehydrogenase 1 and ferredoxin, which are bacteria-like enzymes, were diffusely distributed throughout the cytosol. We suggest that the Hsp60-associated, mitochondrion-derived organelle identified here be named "crypton," as its structure was previously hidden and its function is still cryptic.

Sarcocystis pantherophisi n. sp., from Eastern Rat Snakes (Pantherophis alleghaniensis) as Definitive Hosts and Interferon Gamma Gene Knockout Mice as Experimental Intermediate Hosts.

Here, we report a new species, Sarcocystis pantherophisi n. sp., with the Eastern rat snake (Pantherophis alleghaniensis) as natural definitive host and the interferon gamma gene knockout (KO) mouse as the experimental intermediate host. Sporocysts (n = 15) from intestinal contents of the snake were 10.8 × 8.9 µm. Sporocysts were orally infective to KO mice but not to laboratory-raised albino outbred house mice (Mus musculus). The interferon gamma KO mice developed schizont-associated neurological signs, and schizonts were cultivated in vitro from the brain. Mature sarcocysts were found in skeletal muscles of KO mice examined 41 days postinoculation (PI). Sarcocysts were slender, up to 70 µm wide and up to 3.5 mm long. By light microscopy, sarcocysts appeared thin-walled (<1 µm) without projections. By transmission electron microscopy, the sarcocyst wall was a variant of "type 1" (type 1i, new designation). The parasitophorous vacuolar membrane (pvm) had approximately 100-nm-wide × 100-nm-long bleb-like evaginations interspersed with 100-nm-wide × 650-nm-long elongated protrusions at irregular distances, and invaginations into the ground substance layer (gs) for a very short distance (6 nm). The gs was smooth, up to 500 nm thick, without tubules, and contained a few vesicles. Longitudinally cut bradyzoites at 54 days PI were banana-shaped, 7.8 × 2.2 µm (n = 5). Molecular characterization using 18S rRNA, 28S rRNA, ITS-1, and cox1 genes indicated a close relationship with other Sarcocystis parasites that have snake-rodent life cycles. The parasite in the present study was molecularly and biologically similar to a previously reported isolate (designated earlier as Sarcocystis sp. ex Pantherophis alleghaniensis) from P. alleghaniensis, and it was structurally different from other Sarcocystis species so far described.

Sarcocystis jamaicensis n. sp., from Red-Tailed Hawks (Buteo jamaicensis) Definitive Host and IFN-γ Gene Knockout Mice as Experimental Intermediate Host.

Here, we report a new species of Sarcocystis with red-tailed hawk (RTH, Buteo jamaicensis) as the natural definitive host and IFN-γ gene knockout (KO) mice as an experimental intermediate host in which sarcocysts form in muscle. Two RTHs submitted to the Carolina Raptor Center, Huntersville, North Carolina, were euthanized because they could not be rehabilitated and released. Fully sporulated 12.5 × 9.9-µm sized sporocysts were found in intestinal scrapings of both hawks. Sporocysts were orally fed to laboratory-reared outbred Swiss Webster mice (SW, Mus musculus) and also to KO mice. The sporocysts were infective for KO mice but not for SW mice. All SW mice remained asymptomatic, and neither schizonts nor sarcocysts were found in any SW mice euthanized on days 54, 77, 103 (n = 2) or 137 post-inoculation (PI). The KO mice developed neurological signs and were necropsied between 52 to 68 days PI. Schizonts/merozoites were found in all KO mice euthanized on days 52, 55 (n = 3), 59, 61 (n = 2), 66, and 68 PI and they were confined to the brain. The predominant lesion was meningoencephalitis characterized by perivascular cuffs, granulomas, and necrosis of the neural tissue. The schizonts/merozoites were located in neural tissue and were apparently extravascular. Brain homogenates from infected KO mice were infective to KO mice by subcutaneous inoculation and when seeded on to CV-1 cells. Microscopic sarcocysts were found in skeletal muscles of 5 of 8 KO mice euthanized between 55-61 days PI. Only a few sarcocysts were detected. Sarcocysts were microscopic, up to 3.5 mm long. When viewed with light microscopy, the sarcocyst wall appeared thin (<1 µm thick) and smooth. By transmission electron microscopy, the sarcocyst wall classified as "type 1j" (new designation). Molecular characterization using 18S rRNA, 28S rRNA, ITS-1, and cox1 genes revealed a close relationship with Sarcocystis microti and Sarcocystis glareoli; both species infect birds as definitive hosts. The parasite in the present study was biologically and molecularly different from species so far described in RTHs and we therefore propose a new species name, Sarcocystis jamaicensis n. sp.

Sarcocystis strixi n. sp. from a Barred Owl ( Strix varia) Definitive Host and Interferon Gamma Gene Knockout Mice as Experimental Intermediate Host.

Here we report a new species of Sarcocystis with a barred owl ( Strix varia) as the natural definitive host and interferon gamma gene knockout (KO) mice as an experimental intermediate host. A barred owl submitted to the Carolina Raptor Center, Huntersville, North Carolina, was euthanized because of paralysis. Fully sporulated 12.5 × 9.9 µm sporocysts were found in intestinal scrapings from the owl. Sporocysts from the barred owl were orally fed to 4 laboratory-reared outbred Swiss Webster (SW) ( Mus musculus) and 8 KO mice. All mice remained asymptomatic. Microscopic sarcocysts were found in all 5 KO mice euthanized on day 32, 59, 120, 154, and 206 post-inoculation (PI), not in KO mice euthanized on day 4, 8, and 14 PI. Sarcocysts were not found in any SW mice euthanized on day 72, 120, 206, and 210 PI. Sarcocysts were microscopic, up to 70 µm wide. By light microscopy, the sarcocyst wall < 2 µm thick had undulating, flat to conical, protrusions of varying dimensions. Numerous sarcocysts were seen in the histological sections of tongue and skeletal muscles from the abdomen, limbs, and eye but not in the heart. By transmission electron microscopy, the sarcocyst wall was "type 1j." The ground substance layer (gs) was homogenous, up to 2 µm thick, with very fine granules, and a few vesicles concentrated toward the villar projections. No microtubules were seen in the gs. Longitudinally cut bradyzoites at 206 days PI were 7.8 × 2.2 µm. Based on molecular characterization using 18S rRNA, 28S rRNA, and cox1 genes and morphology of sarcocysts, the parasite in the present study was biologically and structurally different from species so far described, and we therefore propose a new species name, Sarcocystis strixi n. sp.

Acute fatal sarcocystosis hepatitis in an Indo-Pacific bottlenose dolphin (Tursiops aduncus) in Hong Kong.

Unlike most species in the genus Sarcocystis, Sarcocystis canis has a broad intermediate host range. Its life cycle is incompletely known and most reports are from the USA. Here we report fatal hepatitis in a 4year old male Indo-Pacific bottlenose dolphin (Tursiops aduncus) from Hong Kong associated with a S. canis-like infection. Diagnosis was made based on clinical presentation, histopathology, transmission electron microscopy (TEM), and molecular characterization. Microscopically, S. canis-like like infection was confined to the liver. Immature and mature schizonts were found in hepatocytes and the parasite was associated with generalized hepatic necrosis. By TEM, schizonts divided by endopolygeny, and merozoites lacked rhoptries. Molecular characterization of parasites present in liver and brain tissues at the cox1 gene showed a high degree of identity (97-98%) and clustered together with Sarcocystis canis, S. lutrae, S. arctica, S. speeri, S. turdusi, and S. rileyi in a phylogenetic study. This is the first report of S. canis-like infection from Asia.

Generally applicable methods to purify intracellular coccidia from cell cultures and to quantify purification efficacy using quantitative PCR.

The objective of this study was to evaluate the utility of a simple, efficient, and rapid method for the isolation of Sarcocystis neurona merozoites and Besnoitia darlingi tachyzoites from cultured cells. The efficacy of this purification method was assessed by microscopy, SDS-PAGE, Western blotting, immuno-fluorescence, and three novel quantitative PCR assays. Culture medium containing host cell debris and parasites was eluted through PD-10 desalting columns. This purification method was compared to alternatives employing filtration through a cellulose filter pad or filter paper. The estimated recovery of S. neurona merozoites purified by the column method was 82% (+/-3.7) of the original merozoites with 97.5% purity. In contrast, estimated recovery of S. neurona merozoites purified by filter pad and filter paper was 40% and 30% with 76% and 83% purity, respectively. The same procedures were applied to purify B. darlingi tachyzoites from cultured cells. Of the original cultured B. darlingi tachyzoites, 94% (+/-2.5) were recovered from the PD-10 column with 96.5%, purity whereas percentage recovery of B. darlingi tachyzoites purified by filter pad and filter paper were 51% and 35% with 84% and 88% purity, respectively. All described methods maintained sterility so that purified parasites could be subsequently cultured in vitro. However, purification using a PD-10 column minimized parasite loss and the loss of viability as determined by the trypan blue dye exclusion assay, the rate of parasite production, and plaque forming efficiency in cell culture. Moreover, column-purified parasites improved the sensitivity of an immuno-fluorescent (IFA) analysis and real-time quantitative PCR assays targeted to parasite 18S ribosomal DNA and hsp70 genes. This technique appears generally applicable for purifying coccidia grown in cell cultures.

Developmental profile of select immune cells in mice infected with Trichinella spiralis during the intestinal phase.

Trichinella spiralis can cause immunosuppression during the intestinal phase of early infection. However, changes in the peripheral blood during T. spiralis early infection remain unclear. Here, select immune cells in mice infected with 500 muscle larvae (ML) of T. spiralis during the intestinal phase of infection were studied. First, the recovery rates of the intestinal worms and female fecundity were determined, and the results showed that the intestinal worms were completely eliminated at 17 days post-infection (dpi) and that large numbers of new-born larvae (NBL) were generated from 5 to 9dpi. Using flow cytometry, it was shown that the number of CD4+ T cells and CD8+ T cells increased over the entire intestinal phase, except on 7dpi when CD4+ T cells decreased significantly compared to the control groups. Although both CD4+ and CD8+ T cells increased, CD8+ T cells increased more than CD4+ T cells, leading to a lower CD4+/CD8+ ratio compared to the control group. Subsequently, a depression of the proliferative response of T cells to concanavalin A (Con A) was noticed at 7 and 11dpi. Although the proliferative response of B cells to LPS was enhanced, the number of B cells from mouse peripheral blood stimulated by T. spiralis antigens showed no differences with the control group prior to 11dpi. The expression of CD14 on monocyte-macrophages decreased during the same period, which meant that the antigen-presenting response was reduced in the immune system of the infected mice. Moreover, the alternatively activated macrophages were induced in T. spiralis early infection. These data provide a better understanding of the development of the intestinal immune response in mice infected with T. spiralis.

The roles of supernatant of macrophage treated by excretory-secretory products from muscle larvae of Trichinella spiralis on the differentiation of C2C12 myoblasts.

The excretory-secretory products (ESPs) released by the muscle-larvae (ML) stage of Trichinella spiralis have been suggested to be involved in nurse cell formation. However, the molecular mechanisms by which ML-ESPs modulate nurse cell formation remain unclear. Macrophages exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for skeletal muscle repair, notably, via their actions on myogenic precursor cells. However, these interactions during T. spiralis infection have not been characterized. In the present study, the ability of conditioned medium (CM) from J774A.1 macrophages treated with ML-ESPs to influence the differentiation of murine myoblasts, and the mechanisms of this influence, were investigated in vitro. The results showed that the expression of Myogenic Regulatory Factors (MRFs) MyoD and myogenin, myosin heavy chain (MyHC), and the p21 cyclin-dependent kinase inhibitor were reduced in CM treated cells compared to their expression in the control group. These findings indicated that CM inhibited myoblast differentiation. Conversely, CM promoted myoblast proliferation and increased cyclin D1 levels. Taken together, results of our study suggested that CM can indirectly influence myoblast differentiation and proliferation, which provides a new method for the elucidation of the complex mechanisms involved in cell-parasite and cell-cell interactions during T. spiralis infection.

Redescription of Besnoitia bennetti (Protozoa: Apicomplexa) from the donkey (Equus asinus).

Besnoitia bennetti tissue cysts were found in four naturally-infected donkeys (Equus asinus) from the USA. Infectivity of its bradyzoites and tachyzoites to animals and cell culture was studied. The bradyzoites were not infectious to out-bred Swiss Webster mice, rabbits or gerbils. When fed tissue cysts, cats did not excrete oocysts. However, the parasite was infectious to interferon-gamma gene knock out mice. The parasite from tissues of two donkeys was grown successfully in bovine monocyte monolayers for the first time. Non-dividing, uninucleate tachyzoites were approximately 6 x 1.5 microm in size. Longitudinally-cut bradyzoites in tissue sections measured 8.7 x 1.9 microm. Ultrastructurally, tachyzoites and bradyzoites were similar to those in other Besnoitia species, and in particular to parasites described from cattle (Besnoitia besnoiti) and reindeer (Besnoitia tarandi), in that their bradyzoites lacked enigmatic bodies. Based on comparative analysis of three portions of nuclear ribosomal DNA (the small and large subunits and the first internal transcribed spacer) B. bennetti was found to be more closely related to the other congeners described from ungulates. The parasite was formally redescribed and specimens deposited in the US National Parasite Collections.