Browsing by Author "von Dohlen, Alexa Rosypal"

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    Confirmation of Sarcocystis jamaicensis Sarcocysts in IFN-γ Gene Knockout Mice Orally Inoculated With Sporocysts From a Red-Tailed Hawk (Buteo jamaicensis)
    Dubey, Jitender P.; Cerqueira-Cezar, Camila K.; Murata, Fernando H. A.; Mowery, J. D.; Scott, D.; von Dohlen, Alexa Rosypal; Lindsay, David S. (2019-02-22)
    Here, we report confirmation of sarcocysts of Sarcocystis jamaicensis in an experimental intermediate host, IFN-gamma gene knockout (KO) mice orally inoculated sporocysts from its natural definitive host, a red-tailed hawk (Buteo jamaicensis) (RTH). A RTH submitted to the Carolina Raptor Center, Huntersville, North Carolina, was euthanized because it could not be rehabilitated and released. Fully sporulated sporocysts from intestinal scrapings of the RTH were orally fed to 2 laboratory-reared outbred Swiss Webster mice (SW; Mus musculus) and to 2 KO mice. The sporocysts were infective for KO mice but not to SW mice. Both SW mice remained asymptomatic, and neither schizonts nor sarcocysts were found in their tissues when euthanized on day 54 post-inoculation (PI). The KO mice developed neurological signs and were necropsied 38-54 days PI. Schizonts/merozoites were found in both KO mice euthanized and they were confined to the brain. The predominant lesion was meningoencephalitis. Microscopic sarcocysts were found in muscles of both KO mice. When viewed with light microscopy, the sarcocyst wall appeared thin (<1 mu m thick) and smooth. Ultrastructural details of sarcocysts are described.
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    Gamogony of Sarcocystis Strixi in Mammalian Cell Cultures
    Lindsay, David S.; Verma, S. K.; Dubey, Jitender P.; Scott, David; von Dohlen, Alexa Rosypal (2021-07)
    We are interested in the disease ecology of Sarcocystis species that infect birds of prey as definitive and intermediate hosts. The present study was done to test our hypothesis that a laboratory model can be developed for sarcocystis infection in mammals using gamma interferon gene knockout (KO) mice as a source of Sarcocystis strixi bradyzoites and mammalian cell cultures as a source of sporulated S. strixi oocysts. Sporocysts of S. strixi from a naturally infected barred owl (Strix varia) were fed to KO mice to produce sarcocysts, and the enclosed bradyzoites were obtained by acid-pepsin digestion of abdominal and thigh muscles. Bradyzoites, metrocytes, and an unusual spherical stage were seen in digest before the inoculation of host cells. The spherical stages stained dark with Giemsa stain, but no nucleus was observed, and they were seen free and associated with the concave portion of some bradyzoites. Examination of infected cell cultures demonstrated that macrogamonts and microgamonts were present at 24 hr post-inoculation. Since sporulated oocysts were not observed, we had to reject our current hypothesis.
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    Isolation, molecular characterization, and in vitro schizogonic development of Sarcocystis sp ex Accipiter cooperii from a naturally infected Cooper's hawk (Accipiter cooperii)
    Lindsay, David S.; Verma, Shiv K.; Scott, David; Dubey, Jitender P.; von Dohlen, Alexa Rosypal (2017-04)
    Raptors serve as the definitive host for several Sarcocystis species. The complete life cycles of only a few of these Sarcocystis species that use birds of prey as definitive hosts have been described. In the present study, Sarcocystis species sporocysts were obtained from the intestine of a Cooper's hawk (Accipiter cooperii) and were used to infect cell cultures of African green monkey kidney cells to isolate a continuous culture and describe asexual stages of the parasite. Two clones of the parasite were obtained by limiting dilution. Asexual stages were used to obtain DNA for molecular classification and identification. PCR amplification and sequencing were done at three nuclear ribosomal DNA loci; 18S rRNA, 28S rRNA, and ITS-1, and the mitochondrial cytochrome c oxidase subunit 1 (cox1) locus. Examination of clonal isolates of the parasite indicated a single species related to S. columbae (termed Sarcocystis sp. ex Accipiter cooperii) was present in the Cooper's hawk. Our results document for the first time Sarcocystis sp. ex A. cooperii occurs naturally in an unknown intermediate host in North America and that Cooper's hawks (A. cooperii) are a natural definitive host. (C) 2016 Elsevier Ireland Ltd. All rights reserved.
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    Prevalence of Sarcocysts in the Muscles of Raptors From a Rehabilitation Center in North Carolina
    von Dohlen, Alexa Rosypal; Scott, David; Dubey, Jitender P.; Lindsay, David S. (2019-01-22)
    The life cycle of Sarcocystis species is heteroxenous (2-host), with carnivores being the definitive host and herbivores serving as intermediate hosts in predator-prey relationships. Raptors (eagles, hawks, falcons, and owls) are apex predators and are not consumed routinely by other carnivores, making the occurrence of sarcocysts in their muscles unusual. Recent reports of sarcocysts in eagles and owls with Sarcocystis encephalitis suggests that this condition may be becoming more frequent, and Sarcocystis falcatula has been implicated as the agent of encephalitis in golden (Aquila chrysaetos) and bald eagles (Haliaeetus leucocephalus) as well as great horned owls (Bubo virginianus). The present study was done to determine the prevalence of sarcocysts of Sarcocystis species in the muscles of raptors from the southeastern United States. Pectoral and heart muscle from 204 raptor patients from the Carolina Raptor Center, Huntersville, North Carolina were tested for the presence of Sarcocystis species using histology. Only a few sarcocysts were seen in sections of pectoral muscle from 39 of 204 raptors (19.1%) and heart muscle from 9 that also had sarcocysts in their pectoral muscle. Two structural types of sarcocysts, thin-walled (1 mu m; 62%) or thick-walled (>2 mu m, 38%), were seen. Statistical analysis of raptor age and gender was done by Fisher's exact test on samples from raptors with 20 or more samples per group. The prevalence of sarcocysts by age (2 yr or more) was significant for red-shouldered hawks (Buteo lineatus) (P = 0.022) and Cooper's hawks (Accipiter cooperii) (P = 0.028). Sarcocyst prevalence in male raptors from these groups evaluated statistically were always less than in females. Prevalence in female red-tailed hawks (Buteo jamaicensis) (42.1%) was significantly greater than in males (6.7%) using Fisher's exact test (P = 0.047). Examination of case histories from the 39 sarcocyst-positive raptors did not reveal an association with sarcocysts in raptor pectoral or heart muscle and in a diagnosis of encephalitis. Additional studies are needed to determine the epidemiology and relationships of Sarcocystis spp. that use raptors as intermediate hosts and the importance of Sarcocystis spp. in the overall wellbeing of raptors in their natural environments.
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    Sarcocystis Jamaicensis N. Sp., From Red-Tailed Hawks (Buteo Jamaicensis) Definitive Host and Ifn-Gamma Gene Knockout Mice as Experimental Intermediate Host
    Verma, S. K.; von Dohlen, Alexa Rosypal; Mowery, J. D.; Scott, D.; Rosenthal, B. M.; Dubey, Jitender P.; Lindsay, David S. (2017-10)
    Here, we report a new species of Sarcocystis with red-tailed hawk (RTH, Buteo jamaicensis) as the natural definitive host and IFN-gamma 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 x 9.9-mu m 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 mu m 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.
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    Sarcocystis Strixi N. Sp From a Barred Owl (Strix varia) Definitive Host and Interferon Gamma Gene Knockout Mice as Experimental Intermediate Host
    Verma, S. K.; von Dohlen, Alexa Rosypal; Mowery, J. D.; Scott, D.; Cerqueira-Cezar, Camila K.; Rosenthal, B. M.; Dubey, Jitender P.; Lindsay, David S. (2017-12)
    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 12.539.9 lm 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 lm wide. By light microscopy, the sarcocyst wall, 2 lm 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 lm 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 3 2.2 lm. 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.