Optimization of serum EVELISA for milk testing of Johne's disease.

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.

Motility of macrogamonts of Eimeria magna (Coccidia) in cell culture.

Amoeboid movement of spheroid or ovoid immature or mature macrogamonts within cultured Madin-Darby bovine cells usually began with the formation of a pseudopodium-like protrusion at the margin of the gamont. The protrusion enlarged as the gamont cytoplasm and nucleus moved into the protruded area. During movement, macrogamonts had an elongate shape. The body of the gamont was constricted as it moved through the cell cytoplasm or from one cell to an adjacent one. After movement had ceased, the gamont resumed its ovoid or spheroidal shape.

Absence of the tight junctional protein AF-6 disrupts epithelial cell-cell junctions and cell polarity during mouse development.

BACKGROUND: The establishment, maintenance and rearrangement of junctions between epithelial cells are extremely important in many developmental, physiological and pathological processes. AF-6 is a putative Ras effector; it is also a component of tight and adherens junctions, and has been shown to bind both Ras and the tight-junction protein ZO-1. In the mouse, AF-6 is encoded by the Af6 gene. As cell-cell junctions are important in morphogenesis, we generated a null mutation in the murine Af6 locus to test the hypothesis that lack of AF-6 function would cause epithelial abnormalities. RESULTS: Although cell-cell junctions are thought to be important in early embryogenesis, homozygous mutant embryos were morphologically indistinguishable from wild-type embryos through 6.5 days post coitum (dpc) and were able to establish all three germ layers. The earliest morphological abnormalities were observed in the embryonic ectoderm of mutant embryos at 7.5 dpc. The length of the most apical cell-cell junctions was reduced, and basolateral surfaces of those cells were separated by multiple gaps. Cells of the embryonic ectoderm were less polarized as assessed by histological criteria and lateral localization of an apical marker. Mutant embryos died by 10 dpc, probably as a result of placental failure. CONCLUSIONS: AF-6 is a critical regulator of cell-cell junctions during mouse development. The loss of neuroepithelial polarity in mutants is consistent with a loss of efficacy of the cell-cell junctions that have a critical role in establishing apical/basolateral asymmetry.

Ultrastructural differentiation of Toxoplasma gondii schizonts (types B to E) and gamonts in the intestines of cats fed bradyzoites.

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.

Comparative ultrastructure and expression of L-selectin on bovine alphabeta and gammadelta T cells.

Compared to most alphabeta T cells, bovine gammadelta T cells express two to five times the level of L-selectin and have a much higher capacity to roll on monolayers of endothelial cells, platelets, and leukocytes in assays done under physiological flow. To gain additional insight into the basis for these differences, scanning (SEM) and transmission (TEM) electron microscopy were used to compare the cellular ultrastructure of bovine gammadelta and alphabeta T cells and to study the expression of L-selectin on these cells. It is interesting that gammadelta T cells had more than twice as many microvilli and other surface projections on a per cell basis as alphabeta T cells. This was not due to the gammadelta T cell being larger; after adhesion and fixation procedures used for the EM studies, gammadelta T cells averaged 3.9 +/- 0.01 microm in diameter, whereas alphabeta T cells were slightly larger (5.7 +/- 0.01 microm in diameter). As previously shown for human neutrophils and lymphocytes, L-selectin was preferentially localized to the tips of the microvilli. In contrast, WC1, a lineage-specific antigen on gammadelta T cells, was localized on the plasmalemma between the microvilli. Our findings suggest that more effective rolling of gammadelta T cells in various in vitro flow assays may be due to the greater number of microvilli on gammadelta T cells leading to a higher number of contact sites during adhesion events. In addition, this physical parameter may explain the increased level of L-selectin expression on gammadelta versus alphabeta T cells because L-selectin is clustered at the tips of microvilli.

Developmental gene expression in Eimeria bovis.

By differential screening of stage-specific cDNA libraries of Eimeria bovis, we have identified and isolated a large set of genes that are regulated during development of the sporozoites and merozoites. Duplicate lifts of cDNA libraries constructed from partially sporulated oocysts and merozoites were probed with radioactively labeled first-strand cDNA prepared from partially sporulated oocyst and merozoite mRNA. Out of 60,000 plaques screened in each case, over 250 plaques from the partially sporulated oocyst library preferentially hybridized with the oocyst cDNA probe and 67 plaques from the merozoite library preferentially hybridized with the merozoite cDNA probe. Three of the oocyst phage and 7 of the merozoite phage were selected for further characterization. Northern analysis revealed a common pattern of mRNA expression for the oocyst cDNA clones. Consistent with the results of the differential screen, no hybridization to merozoite RNA was detected with any of these 3 oocyst cDNA clones. The expression of the merozoite cDNA clones was more complex, with 3 different classes of merozoite genes being identified based on their pattern of developmental regulation. Although each of the merozoite clones was expressed to some extent during sporulation, in all cases, expression was higher in merozoites than in partially sporulated oocysts, consistent with the restriction of expression defined by the differential screen. Sequence analysis revealed that 2 of the merozoite cDNA clones encode elongation factor 1 alpha and the ubiquitin/ribosomal protein fusion, and 1 of the sporozoite cDNAs displays a significant identity to insulin-degrading enzyme. The developmental expression of E. bovis genes involved in protein synthesis and degradation provides additional evidence for the importance of regulation of protein metabolism during parasite development.

Sporozoites of Toxoplasma gondii lack dense-granule protein GRA3 and form a unique parasitophorous vacuole.

The invasion of host cells by sporozoites of Toxoplasma gondii leads to the formation of parasitophorous vacuoles that are distinctly different from those surrounding tachyzoites. In sporozoite-infected cells, the fluid-filled space surrounding the sporozoite is many times larger in volume than the sporozoite, essentially lacks granular or tubular structures, and has no detectable continuous parasitophorous vacuolar membrane when prepared by conventional electron microscopic methods. Consistent with the ultrastructural differences, dense-granule protein GRA3, which associates with the parasitophorous vacuolar membrane of tachyzoites, was not detected by indirect immunofluorescence in sporozoite-infected cells 2-12 h post-inoculation or by Western blot analysis of sporozoite extracts. Western blots incubated with the alpha ROP/DG antiserum, which recognizes tachyzoite rhoptry and dense-granule proteins, revealed numerous other antigenic differences between sporozoites and tachyzoites. Cell cultures inoculated with sporozoites were monitored at various intervals for the expression of GRA3 and the developmentally-regulated tachyzoite surface protein SAG1. Expression of SAG1 and GRA3 was first observed in 30% of the sporozoite-infected cells at 12 and 15 h post-inoculation, respectively, and in all intracellular parasites at 24 h. Parasite replication was only observed in sporozoite-infected cells that were positive for GRA3 and SAG1. Thus, these data indicate that sporozoites and their interaction with host cells differ substantially from tachyzoites and the expression of tachyzoite-specific proteins is likely required for parasite replication.

Effect of gamma irradiation on unsporulated and sporulated Toxoplasma gondii oocysts.

The effect of 137Cs irradiation on unsporulated and sporulated Toxoplasma gondii oocysts was investigated as a model system for sterilisation of fruit contaminated with other coccidia such as Cyclospora or Cryptosporidium. Unsporulated oocysts irradiated at > or = 0.4 to 0.8 kGy sporulated but were not infective to mice. Sporulated oocysts irradiated at > or = 0.4 kGy were able to excyst, and sporozoites were infective but not capable of inducing a viable infection in mice. Toxoplasma gondii was detected in histologic sections of mice up to 5 days but not at 7 days after feeding oocysts irradiated at 0.5 kGy. Transmission electron microscopy revealed that sporozoites from irradiated oocysts penetrated enterocytes and all cells in the lamina propria except for red blood cells. Sporozoites appeared normal ultrastructurally and formed a typical parasitophorous vacuole containing a well-developed tubulovesicular membrane network. Raspberries inoculated with sporulated T. gondii oocysts were rendered innocuous after irradiation at 0.4 kGy. Results indicate that irradiation at 0.5 kGy is effective in "killing" coccidian oocysts on fruits and vegetables.

Comparative ultrastructure of tachyzoites, bradyzoites, and tissue cysts of Neospora caninum and Toxoplasma gondii.

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.

Canine neosporosis: clinical signs, diagnosis, treatment and isolation of Neospora caninum in mice and cell culture.

Clinical signs, diagnosis, treatment and isolation of Neospora caninum from two littermate dogs are described. Three of six pups from a Labrador bitch developed paralysis. Neosporosis was diagnosed ante mortem by serological examination in two of the affected pups. At necropsy, tissue cysts were seen in unstained smears and in histologic sections of their brains. Tissue cysts were often thin-walled (approximately 1 micron) but antigenically and ultrastructurally identified as N. caninum. Furthermore, N. caninum (isolates NC-4, NC-5) was isolated in mice and in cell cultures inoculated with neural tissues of these two dogs. Serological diagnosis of neosporosis using a variety of tests is discussed.

Redescription of Neospora caninum and its differentiation from related coccidia.

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.

Early diagnosis of Johne's disease in the American bison by monoclonal antibodies directed against antigen 85.

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.

Surface interactions between macrophages and Trypanosoma cruzi.

Macrophages from the peritoneal cavities of normal mice or mice previously immunized with epimastigotes of Trypanosoma cruzi were incubated with sera from normal or immunized mice and then infected with T. cruzi epimastigotes or trypomastigotes. After 2 hours of incubation the specimens were prepared for scanning electron microscopy. Macrophages from immunized mice were observed to be qualitatively and quantitatively more effective in binding either parasite form than were macrophages from normal mice. Preincubation with specific antibody appeared to enhance parasite binding in all cases, and extracellular destruction of both epimastigote and trypomastigote forms was noted in these preparations. While extensive destruction of epimastigotes was seen with either normal or immune macrophages pre-incubated with antibody, lysis of trypomastigotes was typically seen only in the presence of both immune macrophages and specific antibody.

Prevalence of chloroquine-resistant falciparum malaria in the Brazilian Amazon.

The prevalence of chloroquine-resistant falciparum malaria was determined for humans living at 28 different sites in the Brazilian Amazon. Blood samples obtained from each patient were defibrinated, placed in vials containing 0.5% glucose and or chloroquine and incubated for 24 hours at 39-40 degrees C without agitation. In vitro sensitivity of the parasite to four different concentrations of chloroquine was determined for each sample. After 24 hours of incubation, trophozoites of Plasmodium falciparum developed to schizonts in all control cultures (no chloroquine) as well as in 80.6, 48.4, 11.8 and 7.5% of the cultures containing 0.5, 1.0, 2.0, and 3.0 nmol chloroquine/ml blood, respectively. Chloroquine-resistant P. falciparum was found in blood samples from all 28 locations, indicating that such resistance is widely spread in the Brazilian Amazon.

Ultrastructure of schizonts and merozoites of Sarcocystis neurona.

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.

Ultrastructure of sarcocysts from water buffalo in India.

The ultrastructure of sarcocysts of macro- and microscopic species of Sarcocystis was compared from naturally infected water buffalo from India. Grossly visible sarcocysts had walls consisting of cauliflower-like villar protrusions, typical of S. fusiformis. The sarcocyst wall of the microscopic species of Sarcocystis was 6.4 microns thick and consisted of tightly packed conical villar protrusions that were 9.6 microns long and 3.7 microns wide at the base. At approximately 3 microns above the base, the distal two-thirds of the villar protrusion became conical shaped and was bent laterally at an angle of 45 degrees to the sarcocyst surface. The granular layer beneath the villar protrusions was 0.9 microns thick. In S. levinei the granular layer was 1.9 microns thick, the villar protrusions were narrow and it had a highly undulating primary cyst wall. Whether the microscopic S. levinei-like sarcocysts of Indian and Malaysian water buffalo are distinct species of Sarcocystis will require further investigation.

Ultrastructural differentiation between sarcocysts of Sarcocystis hirsuta and Sarcocystis hominis.

The ultrastructure of macroscopic (2-7 mm) Sarcocystis hirsuta sarcocysts from naturally infected cattle from New Zealand was compared with the ultrastructure of 222-day-old S. hominis in experimentally infected cattle in the United States. The villar protrusions of S. hirsuta were approximately 8 microns long, constricted at the base, expanded laterally in the mid-region and tapered distally. Some of the villar tips were folded to form two to four conical projections. The distal portion of the villar protrusions was bent at an angle of 45-90 degrees to the sarcocyst surface. The villar core contained numerous microfilaments and rows of electron-dense granules. The villar protrusions of S. hominis were cylindrical, oriented nearly perpendicularly to the sarcocyst surface, not constricted at their base and contained relatively few electron-dense granules. Although the sarcocysts of S. hirsuta were indistinguishable from those of S. hominis by light microscopy, they were distinguishable ultrastructurally.

Ovine sporozoan encephalomyelitis linked to Sarcocystis infection.

The unidentified sporozoan causing encephalomyelitis in sheep described by Hartley and Blakemore (1974) was restudied, and the parasite was identified as a Sarcocystis species based on its location and structure. The parasite was located free in the host-cell cytoplasm, divided by endopolygeny and mature merozoites lacked rhoptries.

A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM).

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.

Characteristics of a recent isolate of Sarcocystis neurona (SN7) from a horse and loss of pathogenicity of isolates SN6 and SN7 by passages in cell culture.

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.