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1.
Foodborne Pathog Dis ; 9(8): 749-54, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22845627

ABSTRACT

Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium ssp. paratuberculosis (MAP), is one of the most economically important diseases of dairy cattle. Control of JD could be achieved by good herd management practices, and diagnosis; however, this approach has been hampered by the low sensitivity of currently available enzyme-linked immunosorbent assay (ELISA) tests. In our previous study, we developed a sensitive serum ELISA test, ethanol-vortex enzyme-linked immunosorbent assay (EVELISA), using ethanol extract of MAP. The objective of this study is to demonstrate that the EVELISA can be used for detection of anti-MAP antibodies in milk samples. In this study, we tested and optimized concentrations of antigen, milk, and secondary antibody for better differentiation of milk samples of cattle with MAP infections from those of cattle in JD-free herds. We evaluated five environmental mycobacteria as absorbents of cross-reactive antibodies in milk and found that the mycobacteria had no significant effect on EVELISA results. Using the optimized conditions, a total of 57 milk samples from Holstein dairy cattle (37 animals found positive on the fecal polymerase chain reaction test and 20 animals from JD-free herds) were tested for anti-MAP antibody in milk by using the EVELISA method. The average of ELISA values in the JD-positive milk samples (mean±SD=0.355±0.455) was significantly higher than that in the JD-negative milk samples (mean±SD=0.071±0.011). These results warrant further studies for evaluation and validation of the EVELISA for milk testing of cattle for JD.


Subject(s)
Antibodies, Bacterial/isolation & purification , Enzyme-Linked Immunosorbent Assay/veterinary , Milk/microbiology , Mycobacterium avium subsp. paratuberculosis/isolation & purification , Paratuberculosis/diagnosis , Animals , Cattle , Enzyme-Linked Immunosorbent Assay/methods , Feces/chemistry , Feces/microbiology , Mycobacterium avium subsp. paratuberculosis/immunology , Mycobacterium avium subsp. paratuberculosis/pathogenicity , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/veterinary
2.
Clin Vaccine Immunol ; 13(8): 837-44, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16893982

ABSTRACT

Johne's disease (JD), or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Control of JD could be accomplished by diagnosis and good animal husbandry, but this is currently not feasible because commercially available diagnostic tests have low sensitivity levels and are incapable of diagnosing prepatent infections. In this study, a highly sensitive and subspecies-specific enzyme-linked immunosorbent assay was developed for the diagnosis of JD by using antigens extracted from the surface of M. avium subsp. paratuberculosis. Nine different chemicals and various intervals of agitation by vortex were evaluated for their ability to extract the surface antigens. Various quantities of surface antigens per well in a 96-well microtiter plate were also tested. The greatest differences in distinguishing between JD-positive and JD-negative serum samples by ethanol vortex enzyme-linked immunosorbent assay (EVELISA) were obtained with surface antigens dislodged from 50 microg/well of bacilli treated with 80% ethanol followed by a 30-second interval of agitation by vortex. The diagnostic specificity and sensitivity of the EVELISA were 97.4% and 100%, respectively. EVELISA plates that had been vacuum-sealed and then tested 7 weeks later (the longest interval tested) had diagnostic specificity and sensitivity rates of 96.9 and 100%, respectively. In a comparative study involving serum samples from 64 fecal culture-positive cattle, the EVELISA identified 96.6% of the low-level fecal shedders and 100% of the midlevel and high-level shedders, whereas the Biocor ELISA detected 13.7% of the low-level shedders, 25% of the mid-level shedders, and 96.2% of the high-level shedders. Thus, the EVELISA was substantially superior to the Biocor ELISA, especially in detecting low-level and midlevel shedders. The EVELISA may form the basis for a highly sensitive and subspecies-specific test for the diagnosis of JD.


Subject(s)
Antigens, Surface/chemistry , Cattle Diseases/diagnosis , Enzyme-Linked Immunosorbent Assay , Mycobacterium avium subsp. paratuberculosis/immunology , Mycobacterium avium/immunology , Paratuberculosis/diagnosis , Animals , Antigens, Surface/biosynthesis , Cattle , Cattle Diseases/immunology , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/veterinary , Ethanol/chemistry , Female , Immunochemistry , Immunoglobulin G/analysis , Immunoglobulin G/metabolism , Male , Paratuberculosis/immunology , Sensitivity and Specificity , Sonication , Time Factors
3.
Clin Vaccine Immunol ; 13(5): 535-40, 2006 May.
Article in English | MEDLINE | ID: mdl-16682472

ABSTRACT

Enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis, were developed using whole bacilli treated with formaldehyde (called WELISA) or surface antigens obtained by treatment of M. avium subsp. paratuberculosis bacilli with formaldehyde and then brief sonication (called SELISA). ELISA plates were coated with either whole bacilli or sonicated antigens and tested for reactivity against serum obtained from JD-positive and JD-negative cattle or from calves experimentally inoculated with M. avium subsp. paratuberculosis, Mycobacterium avium subsp. avium, or Mycobacterium bovis. Because the initial results obtained from the WELISA and SELISA were similar, most of the subsequent experiments reported herein were performed using the SELISA method. To optimize the SELISA test, various concentrations (3.7 to 37%) of formaldehyde and intervals of sonication (2 to 300 s) were tested. With an increase in formaldehyde concentration and a decreased interval of sonication, there was a concomitant decrease in nonspecific binding by the SELISA. SELISAs prepared by treating M. avium subsp. paratuberculosis with 37% formaldehyde and then a 2-s burst of sonication produced the greatest difference (7x) between M. avium subsp. paratuberculosis-negative and M. avium subsp. paratuberculosis-positive serum samples. The diagnostic sensitivity and specificity for JD by the SELISA were greater than 95%. The SELISA showed subspecies-specific detection of M. avium subsp. paratuberculosis infections in calves experimentally inoculated with M. avium subsp. paratuberculosis or other mycobacteria. Based on diagnostic sensitivity and specificity, the SELISA appears superior to the commercial ELISAs routinely used for the diagnosis of JD.


Subject(s)
Antibodies, Bacterial/blood , Cattle Diseases/diagnosis , Mycobacterium avium subsp. paratuberculosis/immunology , Mycobacterium avium/immunology , Paratuberculosis/diagnosis , Animals , Antibody Specificity , Antigens, Bacterial/chemistry , Antigens, Bacterial/immunology , Cattle , Cattle Diseases/blood , Cattle Diseases/microbiology , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/veterinary , Formaldehyde/chemistry , Mycobacterium bovis/immunology , Paratuberculosis/blood , Paratuberculosis/microbiology , Sensitivity and Specificity , Sonication
4.
Foodborne Pathog Dis ; 2(3): 250-62, 2005.
Article in English | MEDLINE | ID: mdl-16156706

ABSTRACT

Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Attempts to control JD have proven inordinately difficult due to low levels of sensitivity by currently available diagnostic tests, which are also incapable of detecting prepatent MAP infections. In the present work, we describe the use of a flow cytometry method (FCM) for serological diagnosis of subclinical and clinical JD in cattle. The FCM was capable of distinguishing MAP-infected from MAP-non-infected cattle as well as MAP from M. scrofulaceum and M. avium subsp. avium. Results of the FCM were compared to that of a commercially available ELISA using 82 serum samples from JD-positive and JD-negative dairy and beef cattle farms that were separated into the following groups: (1) sera from a JD-free farm; (2) sera from JD-positive farms that had tested negative by ELISA; and (3) sera from JD-positive farms that tested JD-positive by ELISA. The FCM found that groups 1-3 were 6.6%, 73.3%, and 97.3% positive for MAP infections, respectively. By using 30 fecal culture-negative samples from a JD-free farm and 21 fecal culture-positive samples from JD-positive farms, diagnostic sensitivity and specificity of the FCM were calculated to be 95.2% and 96.7%, respectively. A retrospective study of 10 JD-positive cows showed that the FCM detected MAP infections 6-44 months earlier than the fecal culture test. Further, the FCM specifically detected MAP infections in serum samples as early as 170 days after experimental inoculation of calves with MAP and did not react with calves inoculated with other mycobacteria. Production of IgG against MAP was detected by FCM in all the calves inoculated with MAP 240 days after inoculation, whereas positive anti-MAP IgG production was not detected in control calves or calves experimentally infected with M. avium subsp. avium or M. bovis. The FCM assay is rapid and is completed in less than 4 h. Moreover, the FCM is objective, technically easy and can be automated for handling large numbers of samples. This novel assay might form the basis of a highly sensitive and subspecies-specific test for the diagnosis of JD.


Subject(s)
Cattle Diseases/diagnosis , Flow Cytometry/veterinary , Mycobacterium avium subsp. paratuberculosis/isolation & purification , Paratuberculosis/diagnosis , Animals , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Cattle , Consumer Product Safety , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/veterinary , Feces/microbiology , Flow Cytometry/methods , Food Microbiology , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Mycobacterium avium subsp. paratuberculosis/immunology , Paratuberculosis/blood , Sensitivity and Specificity , Species Specificity
5.
Int J Parasitol ; 35(2): 193-206, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15710440

ABSTRACT

The ultrastructural characterisitics of four types of Toxoplasma gondii schizonts (types B, C, D and E) and their merozoites, microgamonts and macrogamonts were compared in cats killed at days 1, 2, 4 and 6 after feeding tissues cysts from the brains of mice. Schizonts, merozoites and gamonts contained most of the ultrastructural features characteristic of the phylum Apicomplexa. All four types of schizonts developed within enterocytes or intraepithelial lymphocytes. Occasionally, type B and C schizonts developed within enterocytes that were displaced beneath the epithelium into the lamina propria. Type D and E schizonts and gamonts developed exclusively in the epithelium. Tachyzoites occurred exclusively within the lamina propria. Type B schizonts formed merozoites by endodyogeny, whereas types C to E developed by endopolygeny. The parasitophorous vacuoles surrounding type B and C schizonts consisted of a single membrane, whereas those surrounding types D and E schizonts were comprised of two to four electron-dense membranes. The parasitophorous vacuole of type B schizonts had an extensive tubulovesicular membrane network (TMN); the TMN was reduced or absent in type C schizonts and completely absent in types D and E schizonts and gamonts. Type B merozoites were ultrastructurally similar to tachyzoites, except that they were slightly larger. Type C merozoites exhibited a positive periodic acid-Schiff reaction by light microscopy and ultrastructurally contained amylopectin granules. Rhoptries were labyrinthine in type B merozoites but were electron-dense in types C-E. The development of microgamonts, macrogamont and oocysts is also described.


Subject(s)
Cat Diseases/parasitology , Intestine, Small/parasitology , Toxoplasma/ultrastructure , Toxoplasmosis, Animal/parasitology , Amylopectin/analysis , Animals , Cats , Enterocytes/parasitology , Enterocytes/ultrastructure , Intestinal Mucosa/parasitology , Intestinal Mucosa/ultrastructure , Intestine, Small/cytology , Intestine, Small/ultrastructure , Life Cycle Stages , Lymphocytes/parasitology , Lymphocytes/ultrastructure , Membranes/ultrastructure , Mice , Microscopy, Electron/methods , Oocysts/ultrastructure , Toxoplasma/growth & development , Vacuoles/ultrastructure
6.
J Eukaryot Microbiol ; 50(6): 476-82, 2003.
Article in English | MEDLINE | ID: mdl-14733440

ABSTRACT

The intermediate hosts for Sarcocystis rileyi (Stiles 1893) Minchin 1913 are ducks (Anas spp.), and the striped skunk (Mephitis mephitis) is its definitive host. The structure of sarcocysts from an experimentally infected shoveler duck (Anas cylpeata) fed sporocysts from an experimentally-infected M. mephitis was studied and compared with type specimens from a naturally infected duck. The experimentally infected duck was killed 154 d after feeding sporocysts. By light microscopy the sarcocyst wall was 3-5 microm thick with indistinct villar protrusions. Ultrastructurally, the sarcocyst wall was a type-23 cyst wall with anastomosing villar protrusions that were up to 7.5 microm long. The villar projections contained filamentous structures. The bradyzoites were 12-14 microm long. Structurally, the sarcocyst from the naturally infected and experimentally infected ducks appeared similar.


Subject(s)
Sarcocystis/classification , Sarcocystis/ultrastructure , Animals , Disease Models, Animal , Ducks/parasitology , Microscopy, Electron , Sarcocystosis/parasitology , Sarcocystosis/pathology
7.
Ann N Y Acad Sci ; 969: 66-72, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12381565

ABSTRACT

Several monoclonal antibodies derived from hybridomas from mice that had been immunized with recombinant Mycobacterium bovis antigen 85 (Ag85) were tested for reactivity against antigen 85 (Ag85) from M. bovis and against sera from 100 bison inoculated with M. paratuberculosis and from 100 control bison from a disease-free herd. Monoclonal antibodies mAb85.1, mAb85.44.1, mAb85.44.9, and mAb85.96 reacted against three or four 30-33-kDa bands of the Ag85 complex of M. bovis. Importantly, these mAbs also reacted with bands of similar molecular weight in the sera of bison inoculated with M. paratuberculosis. Additionally, when sera from 198 bison in four herds were reacted against mAb85.1 and mAb85.96, 26 bison reacted positively for the presence of Ag85 by either mAb or by both. These preliminary results indicate that monoclonal antibodies may eventually lead to a reliable diagnostic test for the early detection of M. paratuberculosis infections in ruminants as well as to a means for identifying contaminated dairy products.


Subject(s)
Antibodies, Monoclonal , Antigens, Bacterial/immunology , Bison , Mycobacterium bovis/immunology , Paratuberculosis/diagnosis , Animals , Antibodies, Monoclonal/analysis , Antigen-Antibody Reactions , Antigens, Bacterial/analysis , Blotting, Western/veterinary , Female , Hybridomas , Immunization/veterinary , Mice , Mice, Inbred BALB C , Paratuberculosis/immunology
8.
Cell Immunol ; 215(2): 219-31, 2002 Feb.
Article in English | MEDLINE | ID: mdl-12202158

ABSTRACT

L-selectin is constitutively expressed on most leukocytes and is responsible for the initial events in cell trafficking termed tethering and rolling. Recently, L-selectin has been shown to associate with the actin-based cytoskeleton under a variety of conditions. In an effort to better understand L-selectin cytoskeletal association and the ultrastructural nature of the cytoskeleton itself, we provide a comparison of the cytoskeletal association of various human and bovine surface proteins in relation to L-selectin. Electron microscopic examination of the cytoskeleton provided further data on the ultrastructure of freshly isolated peripheral lymphocytes as well as demonstrated L-selectin localization to the periphery of the cytoskeleton following low dose detergent treatment of the cell. Clusters of colloidal-gold-stained L-selectin were found on the surface of the detergent-treated lymphocytes, even though these particles completely lacked microvilli. By flow cytometry, we have defined three distinct patterns of cytoskeletal association; constitutive, inductive, and mAb crosslink-induced, and assigned human and bovine CD2, CD3, CD4, CD5, CD8, CD18, CD19, CD44, CD45RA, CD45RO, alphabeta TCR, gammadelta TCR, E-selectin ligands, and L-selectin surface antigens to one of these respective patterns. SDS-PAGE analyses confirmed most of the flow cytometry results. Depending upon its conformation, L-selectin fell into the inductive or mAb crosslink-induced pattern of association, similar to E-selectin ligand(s). Our data provide additional insight into the functional role of L-selectin and the cytoskeleton in immunological events.


Subject(s)
Antigens, Surface/metabolism , Cytoskeleton/metabolism , L-Selectin/metabolism , Lymphocytes/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antigens, Surface/immunology , Cattle , Cytoskeleton/ultrastructure , Detergents/chemistry , Flow Cytometry , Humans , Lymphocytes/metabolism , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/ultrastructure
9.
Int J Parasitol ; 32(8): 929-46, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12076623

ABSTRACT

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.


Subject(s)
Coccidia/classification , Neospora/classification , Neospora/cytology , Animals , Biological Specimen Banks , Coccidia/cytology , Coccidia/physiology , Coccidiosis/parasitology , Coccidiosis/pathology , Dogs/parasitology , Foxes/parasitology , Microscopy , Museums , Neospora/genetics , Neospora/physiology , Phylogeny , Species Specificity
10.
J Eukaryot Microbiol ; 48(5): 595-603, 2001.
Article in English | MEDLINE | ID: mdl-11596925

ABSTRACT

A new species, Sarcocystis lindsayi n. sp., is proposed for a parasite resembling Sarcocystis falcatula. It was obtained from the lungs and muscles of budgerigars (Melopsittacus undulatus) fed sporocysts from a naturally-infected South American opossum, Didelphis albiventris, from Jaboticabal, Brazil. Sarcocysts of S. lindsayi n. sp. in budgerigars are microscopic, up to 600 microm long and up to 50 microm wide. The cyst wall is up to 2 microm thick. Ultrastructurally, the sarcocyst wall consists of numerous slender villar protrusions (up to 2.0 microm long and up to 0.3 microm wide), each with a stylet at its tip. Schizonts in cell culture divide by endopolygeny leaving a residual body. Sporocysts are approximately 12 x 7 microm. The parasite is genetically distinct from other organisms that also cycle between opossums and avian species and resemble S. falcatula. Diagnostic genetic variation has been observed in the nuclear large subunit ribosomal RNA gene, the internal transcribed spacer (ITS-1), and each of two other genetic loci. Although the structure of the sarcocyst wall may not provide sufficient grounds for differential diagnosis, several other attributes including schizont morphology and genetic variation at each of these genetic loci permit identification of S. lindsayi n. sp.. Natural intermediate hosts for S. lindsayi n. sp. are not known, and fuller characterization of these and other Sarcocystis species would benefit from experimental avian hosts that are more permissive to the maturation of sarcocysts.


Subject(s)
Opossums/parasitology , Sarcocystis/classification , Sarcocystis/isolation & purification , Sarcocystosis/veterinary , Animals , Bird Diseases/parasitology , Brazil , DNA, Protozoan/analysis , DNA, Ribosomal Spacer/genetics , Molecular Sequence Data , Parrots/parasitology , Phylogeny , RNA, Ribosomal/genetics , Sarcocystis/pathogenicity , Sarcocystis/ultrastructure , Sarcocystosis/parasitology , Sequence Analysis, DNA
11.
Vet Parasitol ; 95(2-4): 89-131, 2001 Feb 26.
Article in English | MEDLINE | ID: mdl-11223193

ABSTRACT

Equine protozoal myeloencephalitis (EPM) is a serious neurological disease of horses in the Americas. The protozoan most commonly associated with EPM is Sarcocystis neurona. The complete life cycle of S. neurona is unknown, including its natural intermediate host that harbors its sarcocyst. Opossums (Didelphis virginiana, Didelphis albiventris) are its definitive hosts. Horses are considered its aberrant hosts because only schizonts and merozoites (no sarcocysts) are found in horses. EPM-like disease occurs in a variety of mammals including cats, mink, raccoons, skunks, Pacific harbor seals, ponies, and Southern sea otters. Cats can act as an experimental intermediate host harboring the sarcocyst stage after ingesting sporocysts. This paper reviews information on the history, structure, life cycle, biology, pathogenesis, induction of disease in animals, clinical signs, diagnosis, pathology, epidemiology, and treatment of EPM caused by S. neurona.


Subject(s)
Encephalomyelitis/veterinary , Horse Diseases/parasitology , Sarcocystis , Sarcocystosis/veterinary , Animals , Antiprotozoal Agents/therapeutic use , Cats , Encephalomyelitis/drug therapy , Encephalomyelitis/parasitology , Horse Diseases/drug therapy , Horses/parasitology , Life Cycle Stages , Mephitidae/parasitology , Mink/parasitology , Opossums/parasitology , Otters/parasitology , Raccoons/parasitology , Sarcocystosis/drug therapy , Sarcocystosis/parasitology , Seals, Earless/parasitology
12.
Vet Parasitol ; 95(2-4): 155-66, 2001 Feb 26.
Article in English | MEDLINE | ID: mdl-11223196

ABSTRACT

An isolate of Sarcocystis neurona (SN7) was obtained from the spinal cord of a horse with neurologic signs. The parasite was isolated in cultures of bovine monocytes and equine spleen cells. The organism divided by endopolygeny and completed at least one asexual cycle in cell cultures in 3 days. The parasite was maintained by subpassages in bovine monocytes for 10 months when it was found to be non-pathogenic to gamma interferon knockout (KO) mice. Revival of a low passage (10th passage) of the initial isolate stored in liquid nitrogen for 18 months retained its pathogenicity for KO mice. Merozoites (10(6)) of the late passage (22nd passage) were infective to only one of four KO mice inoculated. Similar results were obtained with SN6 isolate of S. neurona. No differences were found in Western blot patterns using antigens from the low and high passage merozoites of the SN7 and SN6 isolates. These results suggest that prolonged passage in cell culture may affect the pathogenicity of some isolates of S. neurona.


Subject(s)
Horse Diseases/parasitology , Sarcocystis/isolation & purification , Sarcocystis/pathogenicity , Sarcocystosis/veterinary , Animals , Antibodies, Protozoan/biosynthesis , Blotting, Western/veterinary , Cells, Cultured , Electrophoresis, Polyacrylamide Gel/veterinary , Horses , Mice , Mice, Inbred BALB C , Mice, Knockout , Sarcocystosis/immunology , Spinal Cord/parasitology
13.
Vet Parasitol ; 95(2-4): 263-71, 2001 Feb 26.
Article in English | MEDLINE | ID: mdl-11223206

ABSTRACT

The ultrastructure of Sarcocystis neurona schizonts and merozoites was studied in specimens derived from cell culture and from the brains of infected mice. Schizonts and merozoites were located in the host cell cytoplasm without a parasitophorous vacuole at any stage of development. Merozoites divided by endopolygeny. Fully formed merozoites had a pellicle, numerous polysomes and ribosomes, smooth and rough endoplasmic reticulum, 22 subpellicular microtubules, 9-16 dense granules, 25-75 micronemes, a plastid, a Golgi complex, 1-3 mitochondria, a conoid, 2 apical rings, 2 polar rings, 0-6 lipid bodies, a nucleus and nucleolus, but no rhoptries. Most micronemes were located anterior to the nucleus including 1-6 micronemes in the conoid. Merozoites were either slender (7.3 microm x 1.7 microm) or stumpy (7.7 microm x 3.1 microm). Dense granules appeared to arise from the maturation face of the Golgi complex. The ultrastructure of in vitro derived schizonts and merozoites were similar to in vivo derived organisms.


Subject(s)
Sarcocystis/ultrastructure , Animals , Brain/parasitology , Cattle , Coturnix , Horses , Interferon-gamma/physiology , Mice , Mice, Knockout , Microscopy, Electron , Rodent Diseases/parasitology , Sarcocystosis/parasitology , Sarcocystosis/veterinary
14.
J Parasitol ; 87(6): 1323-7, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11780816

ABSTRACT

The ultrastructure of Sarcocystis neurona sarcocysts was studied from muscle of an experimentally infected cat. The cat was killed 144 days after being fed sporocysts from a naturally infected opossum. Sarcocysts were microscopic, up to 700 microm long, and up to 50 microm wide. By light microscopy, the sarcocyst wall was 1-2 microm thick. Ultrastructurally, the sarcocyst wall consisted of numerous villar protrusions. The villar protrusions were up to 2.8 microm long and 0.4 microm wide, with a tapered end. Microtubules extended from the tip of the villus to the base and occasionally extended deep into the granular layer. The granular layer was approximately 0.5 microm thick. Longitudinally cut bradyzoites were 5.2 by 1.2 (4.8-6.5 by 1.0-1.3) microm in size. Micronemes in bradyzoites were numerous and located in the anterior 1/3 of the conoidal end.


Subject(s)
Cat Diseases/parasitology , Muscle, Skeletal/pathology , Sarcocystosis/veterinary , Animals , Cat Diseases/pathology , Cats , Muscle, Skeletal/parasitology , Muscle, Skeletal/ultrastructure , Opossums , Sarcocystis/ultrastructure , Sarcocystosis/pathology
15.
J Parasitol ; 86(4): 671-8, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10958438

ABSTRACT

Schizonts of Sarcocystis speeri Dubey and Lindsay, 1999 were cultured in vitro in bovine monocyte and equine kidney cell cultures inoculated with infected tissues of nude and gamma-interferon knockout mice fed sporocysts from opossums, Didelphis albiventris. At least 1 asexual cycle was completed in 3 days. In vitro-grown merozoites were structurally and antigenically distinct from those of Sarcocystis neurona and Sarcocystis falcatula. Culture-derived merozoites of S. speeri were not infective to budgerigars (Melopsittacus undulatus).


Subject(s)
Sarcocystis/growth & development , Sarcocystosis/parasitology , Animals , Brain/parasitology , Cattle , Cell Line , Chlorocebus aethiops , Histocytochemistry , Horses , Immunohistochemistry , Kidney/cytology , Kidney/parasitology , Liver/parasitology , Mice , Mice, Knockout , Mice, Nude , Microscopy, Phase-Contrast , Monocytes/cytology , Monocytes/parasitology , Opossums/parasitology , Parrots , Sarcocystis/pathogenicity
16.
J Parasitol ; 86(3): 624-7, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10864267

ABSTRACT

Sarcocystis speeri Dubey and Lindsay, 1999 from the South American opossum Didelphis albiventris was successfully transmitted to the North American opossum Didelphis virginiana. Sporocysts from a naturally infected D. albiventris from Argentina were fed to 2 gamma-interferon knockout (KO) mice. The mice were killed 64 and 71 days after sporocyst feeding (DAF). Muscles containing sarcocysts from the KO mouse killed 71 DAF were fed to a captive D. virginiana; this opossum shed sporocysts 11 days after ingesting sarcocysts. Sporocysts from D. virginiana were fed to 9 KO mice and 4 budgerigars (Melopsittacus undulatus). Schizonts, sarcocysts, or both of S. speeri were found in tissues of all 7 KO mice killed 29-85 DAF; 2 mice died 39 and 48 DAF were not necropsied. Sarcocystis stages were not found in tissues of the 4 budgerigars fed S. speeri sporocysts and killed 35 DAE These results indicate that S. speeri is distinct from Sarcocystis falcatula and Sarcocystis neurona, and that S. speeri is present in both D. albiventris and D. virginiana.


Subject(s)
Opossums/parasitology , Sarcocystis/pathogenicity , Sarcocystosis/veterinary , Animals , Argentina , Brain/parasitology , Feces/parasitology , Interferon-gamma/genetics , Intestine, Small/parasitology , Liver/parasitology , Mice , Mice, Knockout , Microscopy, Electron , Muscle, Skeletal/parasitology , North America , Parrots , Sarcocystis/ultrastructure , Sarcocystosis/transmission
17.
J Parasitol ; 86(1): 25-32, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10701559

ABSTRACT

The development and merozoite production of Sarcocystis falcatula and 2 isolates (SN6 and SN2) of Sarcocystis neurona were studied in various cultured cell lines inoculated with culture-derived merozoites. All 3 parasites underwent multiple cycles of schizogony in VERO cells, bovine monocytes (M617 cells), and bovine pulmonary artery endothelial cells (CPA). Sarcocystis neurona strains SN6 and SN2 formed schizonts in rat myoblasts (L6) but not in quail myoblasts (QM7); S. falcatula formed schizonts in QM7 cells but not in L6 cells. Merozoites did not develop to sarcocysts in the myoblast cells lines. During a 47-day culture period in VERO cells, SN6 produced substantially more merozoites than did SN2 or S. falcatula. M617 cells produced substantially more merozoites of SN6 than did VERO or CPA cells. During a 17-day culture period of SN6, M617 cells produced mean totals of 4.7 x 10(8) merozoites, VERO cells produced 1.9 x 10(8) merozoites, and CPA cells produced 5.9 x 10(7) merozoites. At 4-12 days after inoculation of cultured cells with SN6, M617 cells cultured in the presence of 10% fetal bovine serum (FBS) produced a mean merozoite total of 5.1 x 10(8) compared to 3.6 x 10(8) for culture medium containing 1% FBS.


Subject(s)
Sarcocystis/growth & development , Animals , Cattle , Cell Line , Chlorocebus aethiops , Endothelium, Vascular/cytology , Endothelium, Vascular/parasitology , Horse Diseases/parasitology , Horses , Monocytes/parasitology , Pulmonary Artery , Quail , Rats , Sarcocystis/classification , Sarcocystosis/parasitology , Sarcocystosis/veterinary , Spinal Cord/parasitology , Vero Cells
18.
J Parasitol ; 86(1): 160-3, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10701583

ABSTRACT

Sarcocystis sporocysts from the intestines of 2 opossums (Didelphis albiventris) from Argentina were fed to gamma-interferon knockout (KO) and nude mice. Protozoal schizonts were seen in brain, liver, spleen, and adrenal glands of mice examined 33-64 days after feeding sporocysts. Sarcocysts were seen in skeletal muscles of KO mice 34-71 days after feeding sporocysts. Schizonts and sarcocysts were structurally similar to Sarcocystis speeri Dubey and Lindsay, 1999 seen in mice fed sporocysts from the North American opossum Didelphis virginiana from the United States.


Subject(s)
Intestinal Diseases, Parasitic/veterinary , Opossums/parasitology , Sarcocystis/isolation & purification , Sarcocystosis/veterinary , Animals , Argentina , Female , Horses , Host-Parasite Interactions , Intestinal Diseases, Parasitic/parasitology , Intestinal Diseases, Parasitic/transmission , Intestine, Small/parasitology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Muscle, Skeletal/parasitology , Sarcocystis/physiology , Sarcocystis/ultrastructure , Sarcocystosis/parasitology , Sarcocystosis/transmission
19.
Int J Parasitol ; 29(10): 1509-19, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10608436

ABSTRACT

The ultrastructure of tachyzoites, bradyzoites and tissue cysts of the NC-1, NC-5 and NC-Liverpool strains of Neospora caninum are reviewed and compared with those of the VEG and ME-49 strains of Toxoplasma gondii. While each stage of N. caninum and T. gondii shared many ultrastructural characteristics, each parasite stage also had certain features or organelles that could be used to distinguish the two parasites. Some of the most prominent ultrastructural differences occurred in the number, appearance and location of rhoptries, looped-back rhoptries, micronemes, dense granules, small dense granules and micropores. The tissue cysts of both parasites were also basically similar, being surrounded by a cyst wall and not compartmentalised by septa. The cyst wall of N. caninum was irregular and substantially thicker, 0.5-4 microm, than those of T. gondii which were smooth and 0.5 microm thick.


Subject(s)
Coccidiosis/veterinary , Neospora/ultrastructure , Toxoplasma/ultrastructure , Toxoplasmosis, Animal/parasitology , Animals , Cattle , Coccidiosis/parasitology , Dog Diseases/parasitology , Dogs , Mice , Microscopy, Electron , Neospora/growth & development , Toxoplasma/growth & development
20.
J Eukaryot Microbiol ; 46(5): 500-6, 1999.
Article in English | MEDLINE | ID: mdl-10519218

ABSTRACT

An isolate of Sarcocystis neurona (SN6) was obtained from the spinal cord of a horse from Oregon with neurologic signs. The parasite was isolated in cultures of bovine monocytes and equine spleen cells. The parasite divided by endopolygeny and completed at least one asexual cycle in cell cultures in three days. Two gamma interferon knockout mice inoculated with cell culture-derived merozoites became ill 35 d later and S. neurona schizonts and merozoites were found in encephalitic lesions. The parasite in tissue sections of mice reacted with S. neurona-specific antibodies and S. neurona was reisolated from the brain of knockout mice.


Subject(s)
Encephalomyelitis/veterinary , Horse Diseases/parasitology , Sarcocystis/isolation & purification , Sarcocystosis/veterinary , Spinal Cord/parasitology , Animals , Cattle , Cells, Cultured , Encephalomyelitis/parasitology , Horses/parasitology , Immunoblotting , Immunocompromised Host , Male , Mice , Oregon , Rabbits , Sarcocystis/growth & development , Sarcocystis/pathogenicity , Sarcocystosis/parasitology
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