Techniques in electron microscopy of animal tissue.

Technical improvements in electron microscopy, both instrumental and preparative, permit increasingly accurate analyses. Digital images for transmission electron microscopy (TEM) can be processed by software programs that automate tasks and create custom tools that allow for image enhancement for brightness, contrast and coloration; for creation of rectangular, ellipsoidal or irregular area selections; and for measurement of mean area and standard deviation. Sample preparation remains a source of error since organelles and spatial arrangements of macromolecules rapidly change after anoxia. Guidelines for maintaining consistency in preparation, examination and interpretation are presented for different electron microscopy (EM) modalities.

Monocytic origin and postnatal mitosis of intravascular macrophages in the porcine lung.

Ultrastructural studies of near-term to 2-month-old pigs were done to document characteristics and developmental changes of intravascular macrophages in pulmonary capillaries. Evidence is presented that blood monocytes colonize the porcine lung perinatally, replicate within capillaries postnatally, and attach to endothelium by intercellular junctions during differentiation. Major ultrastructural features of differentiated intravascular macrophages are adhesion to capillary endothelium, pseudopods, phagosomes, and tubular structures of micropinocytosis vermiformis. Ultrastructure indicates that intravascular macrophages are cells of the mononuclear phagocyte system involved in several functions (eg, blood cell sequestration) that are usually attributed to hepatolineal macrophages. In newborn and 3-day-old pigs, the majority of cells closely apposed to endothelium consisted of few differentiated monocytes, but in 7-day-old and older animals, most cells that were joined to endothelium had characteristics of differentiated intravascular macrophages.

Development, testing and commercialization of a new brucellosis vaccine for cattle.

Vaccines used against brucellosis do not generally protect completely against infection or abortion. Genetic analysis has revealed differences in arrangements of DNA sequences between these vaccine strains and the virulent parent strain and permits the specific identification of field isolates of B. abortus as wild-type or vaccine strain. B. abortus strain 19 is a low-virulence, live vaccine developed for use in cattle. Although it is effective, strain 19 vaccine had a tropism for the placenta and caused abortion when given to pregnant cows, was infectious for humans, and caused serologic responses in calves that could not be differentiated from those in cattle infected with natural field strains. In the mid-1980s the need for a new vaccine emerged when the USDA increased its efforts in brucellosis eradication. In the 1990s, research on biosafety, vaccine efficacy and field application rapidly established the fact that strain RB51 is protective in cattle at doses comparable to those of strain 19. Thus, Brucella abortus strain RB51 is the vaccine of choice against brucellosis of cattle in the United States. Studies have established the relative efficacy of strain RB51 vaccine on bison, and the vaccine has also been accepted for use in commercial bison herds in the U.S.

Pathogenicity of equine herpesvirus 1 subtype 2 for foals and adult pony mares.

Three pony mares and 4 pony foals were inoculated with a subtype 2 strain of equine herpesvirus 1. Foals had periods of fever 12 h and 2.5 days after inoculation and leukopenia, involving both neutrophils and lymphocytes, followed by leukocytosis. Mares had transient fever and leukopenia 24 hours after inoculation that were less severe than in foals. An increase in circulating virus-neutralizing antibody was seen in 2 of 3 inoculated mares, but not in foals. Attempts to isolate virus from blood were unsuccessful. These studies show that equine herpesvirus 1 subtype 2 is a mild pathogen for ponies and infection may result in inapparent clinical disease.

Immunity induced in rats vaccinated with toxoid prepared from heat-labile toxin produced by Pasteurella multocida serogroup D.

Rats were vaccinated with a toxoid (D-toxoid) prepared from purified heat-labile toxin (D-toxin) produced by Pasteurella multocida serogroup D. Vaccination of rats with D-toxoid prevented death and other effects of D-toxin (hepatic necrosis, development of elevated leukocyte counts, lymphopenia, neutrophilia, and elevated complement titers) that occurred in phosphate buffered saline (PBS)-vaccinated control rats.

Immune responses to superoxide dismutase and synthetic peptides of superoxide dismutase in cattle vaccinated with Brucella abortus strain 19 or RB51.

Antibody and lymph node cell-mediated immune responses to recombinant Brucella abortus strain 19 Cu-Zn superoxide dismutase (rSOD) and to three synthetic strain 19 Cu-Zn SOD peptides were measured during 2 to 12 weeks following vaccination of cattle with B. abortus strain 19 or RB51. Cattle vaccinated with strain 19 or RB51 did not produce antibody to rSOD and to the SOD peptides. Lymph node cells from cattle vaccinated with strain 19, but not with strain RB51, proliferated when incubated with either rSOD or one of the three tested SOD peptides (GGDNYSDKPEPLGG). These results suggest that neither the strain 19 nor the strain RB51 vaccine induces antibody production to SOD and only the strain 19 vaccine induces lymph node cell-mediated immune responses to SOD.

Comparative effects of bovine cytokines on cattle and bison peripheral blood mononuclear cell proliferation.

Proliferation of peripheral blood mononuclear cells (PBMC) from cattle and bison was measured following stimulation of PBMC with bovine cytokines. Bovine interleukin 1 beta (BoIL-1 beta), interleukin 2 (BoIL-2) or granulocyte-macrophage colony-stimulating factor (BoGM-CSF) at 0.1-100 U/ml were incubated for 48 h with PBMC alone or with PBMC and various mitogens. These included concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) or Escherichia coli 055:B5 lipopolysaccharide (LPS) at 10-0.1 micrograms/ml. BoIL-2 alone, but not BoIL-1 beta and BoGM-CSF alone, induced proliferation of cattle and bison PBMC in the absence of mitogens. In addition, BoIL-1 beta and BoIL-2, but not BoGM-CSF, enhanced proliferation of cattle and bison PBMC induced by mitogens. These results indicate that BoIL-1 beta and BoIL-2 stimulate cattle and bison PBMC proliferation in a similar manner, whereas BoGM-CSF does not appear capable of stimulating either cattle or bison PBMC proliferation.

Serum complement activity and serum enzymes in rats after a subcutaneous injection of toxin prepared from Pasteurella multocida type D.

Toxin produced by Pasteurella multocida type D was investigated for its effect on serum complement and serum biochemistry in rats. Rats were given a sublethal single subcutaneous injection of D toxin equivalent to 0.2 microgram/kg of body weight. Serum obtained 1, 3, 5 and 7 days post-treatment was tested for complement activity, total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). Serum complement titers were significantly elevated (P less than 0.05) at all times after injection of toxin compared to rats injected with diluent and tested at the same intervals. Bilirubin was decreased but both control and D toxin-treated rats had low concentrations of bilirubin in their sera. The other biochemical constituents measured had no consistent pattern that would indicate liver damage in the rats.

Comparative analysis of immune responses in cattle vaccinated with Brucella abortus strain 19 or strain RB51.

Immune responses were measured for 12 weeks following vaccination of cattle with either Brucella abortus strain (S) 19 or SRB51. Cattle vaccinated with S19, but not with SRB51, produced antibodies that agglutinated B. abortus S1119 in the standard tube agglutination test. Cattle vaccinated with S19 or SRB51 produced antibodies to the surface antigens of SRB51 when measured by a dot enzyme-linked immunosorbent assay. Superficial cervical lymph node (LN) cells obtained by biopsy at 10 and 12 weeks from cattle given the S19 or SRB51 vaccine exhibited similar proliferative responses when incubated in vitro with gamma-irradiated B. abortus S2308. At 10 and 12 weeks after vaccination, LN cells obtained from cattle given S19 or SRB51 proliferated to 22 protein fractions (106-18 kDa proteins) of B. abortus S2308 that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Twelve of the same 22 fractions, which contained 49-27 kDa proteins, produced a stimulation index of greater than 10 when incubated with LN cells taken from S19-vaccinated or SRB51-vaccinated cattle. Two factions, which contained 27 kDa proteins of S2308, induced the highest proliferative response (stimulation index 25 or greater) by LN cells in cattle given either S19 or SRB51. These results suggest that cattle vaccinated with S19 or SRB51 have similar LN immune responses to S2308, but unlike S19, SRB51 does not induce positive results in the standard tube agglutination test used to diagnose brucellosis in cattle.

Morphometric analysis of CD4+, CD8+, and gamma/delta+ T-lymphocytes in lymph nodes of cattle vaccinated with Brucella abortus strains RB51 and 19.

T-lymphocyte subpopulations were examined in vivo by computer-assisted morphometry of superficial cervical lymph nodes of cattle vaccinated with Brucella abortus. Twenty-four 8-month-old Hereford heifers were injected subcutaneously in the axillary area with 1 x 10(10) live B. abortus strain RB51 (SRB51, n = 12) or strain 19 (S19, n = 6) suspended in 2 ml of saline. Six control heifers were injected with sterile saline. Lymph nodes were collected at 1, 2, 4, 6, 10 and 12 weeks postvaccination. Both SRB51 and S19 were cultured from lymph nodes, but SRB51 persisted for a longer period after vaccination (10 weeks) than S19 (6 weeks). Cryostat sections were incubated with monoclonal antibody to CD4 (IL-A11), CD8 (IL-A51), or gamma/delta (IL-A29) bovine T-cell surface antigen and processed for immunoperoxidase staining. Numbers of stained lymphocytes in randomly selected fields were calculated using image-analysis software. There were no significant differences in the number (P = 0.07) or relative proportions (P = 0.22) of CD4+, CD8+, and gamma/delta+ lymphocytes in SRB51, S19, and control lymph nodes. There was a statistically significant difference in the distribution of the three T-cell subsets (P = 0.001). The CD4+ cells were most closely grouped and the gamma/delta+ cells had the most widely scattered distribution, regardless of vaccination status. The results support other studies indicating lymphocyte depletion is not a sequela of infection with B. abortus vaccine strains given to conventionally reared cattle.

Use of rats to compare atrophic rhinitis vaccines for protection against effects of heat-labile protein toxin produced by Pasteurella multocida serogroup D.

Four bacterin-toxoid and three bacterin commercial vaccines against atrophic rhinitis were tested in rats for their capacity to immunize against the lethal and systemic effects of purified heat-labile protein toxin (D-toxin) produced by Pasteurella multocida serogroup D. Only one bacterin-toxoid vaccine stimulated sufficient immunity to prevent the death of all rats challenged with D-toxin. None of the vaccines prevented weight loss, leukocytosis or increases in serum complement titers in rats challenged with D-toxin. Rats provide an inexpensive animal model for testing the capacity of vaccines to generate antitoxic immunity against the lethal and systemic effects of D-toxin.

Effect of Rous associated virus number 7 on lymphoid cells and tissues of the chicken.

Infection of chicks or chick embryos with Rous associated virus number 7 (RAV-7) led to a decreased blastogenic response to Concanavalin A (Con A) by lymphocytes isolated from the spleen and thymus. Chicks infected with RAV-7 8 days after hatch manifested decreased Con A blastogenesis 5 weeks postinfection, while chicks infected in ovo at 10 days of incubation showed an unusual pattern of cell density dependent decreased blastogenesis two weeks post-hatch (three weeks post-infection). Histopathological examination of tissues from RAV-7 infected chicks revealed evidence of lymphoid organ involution and widespread lymphoproliferative lesions by 3 weeks of age. The combination of decreased in vitro lymphoid blastogenesis and in vivo lymphoproliferation suggests that RAV-7 interacts with lymphocytes in a fashion that has not previously been described in the chicken.

Generalized Equine Cutaneous Mastocytosis.

A newborn foal developed generalized cutaneous mastocytosis characterized by multiple elevated nodules of mast cells in skin and basophil hyperplasia in bone marrow. Skin lesions began as small aggregates of mast cells that progressively enlarged, ulcerated, and regressed spontaneously. Eosinophil infiltration, collagen necrosis, and fibroplasia were characteristic of advanced lesions. Many new lesions developed during the first month of life but numbers progressively diminished. Large numbers of mast cells were present in biopsies of lymph node, spleen and bone marrow. Discrete aggregates of mast cells were present in the bone marrow postmortem but no other significant change was seen. Mast cells contained large amounts of histamine but little serotonin. Ultrastructurally, their cytoplasmic granules were chiefly granular with few dense forms. In cell culture, mast cells from early lesions maintained mitotic activity through 14 passages. Cells obtained from older lesions were rapidly overgrown with fibroblasts. An equine herpesvirus isolated from cultures of cutaneous mast cell lesions and of spleen was not thought to be related to the disease.

Experimental use of a dot-blot assay to measure serologic responses of cattle vaccinated with Brucella abortus strain RB51.

Brucella abortus strain RB51 was recently approved as an official brucellosis calfhood vaccine for cattle by the Animal and Plant Health Inspection Service branch of the United States Department of Agriculture. Currently available serologic surveillance tests for B. abortus do not detect seroconversion following SRB51 vaccination. The purpose of this study was to evaluate a dot-blot assay using gamma-irradiated strain RB51 bacteria for its specificity and sensitivity to detect antibody responses of cattle vaccinated with strain RB51. Dot-blot titers of sera at a recommended dosage (10(10) colony-forming units) were similar to those of sera from cattle vaccinated with similar numbers of B. abortus strain 19 and greater (P < 0.05) than titers of nonvaccinated cattle. In the first 12 weeks after vaccination with 10(10) colony-forming units of strain RB51, the RB51 dot-blot assay had 100% specificity for titers of 80 or less and a 53% sensitivity for titers of 160 or greater. Sensitivity of the RB51 dot-blot assay peaked at 4 weeks after vaccination with 10(10) colony-forming units of strain RB51. Dot-blot responses of sera from cattle vaccinated with a reduced dosage of strain RB51 (10(9) colony-forming units) did not differ (P > 0.05) from titers of sera from nonvaccinated cattle. Following intraconjunctival challenge with B. abortus strain 2308, titers on the RB51 dot-blot assay did not differ (P > 0.05) between nonvaccinated cattle and cattle vaccinated at calfhood with strain 19 or strain RB51.

Serologic responses of Brucella abortus strain 19 calfhood-vaccinated cattle following adult vaccination with strain RB51.

This study was designed to determine if Brucella abortus strain RB51, which expresses small amounts of the lipopolysaccharide O side chain, would cause positive responses on brucellosis serologic surveillance tests when given to adult cattle that were vaccinated as calves with B. abortus strain 19. Cattle vaccinated as adults with strain RB51 that had been vaccinated as calves with strain 19 (n = 40) had significantly greater antibody titers (P < 0.05) against strain RB51 at 4 and 8 weeks postvaccination in the dot blot assay than did animals (n = 10) not vaccinated with strain RB51. When evaluated using the card or buffered acid plate agglutination presumptive tests, 7 strain RB51 vaccinates tested positive at either 4 or 8 weeks following vaccination as compared with 4 cattle in the control group that were not vaccinated with strain RB51. One strain RB51 vaccinate was scored as suspect on the standard tube agglutination (STA) test at 8 weeks following vaccination. Remaining samples from strain RB51 vaccinates tested negative on the STA, complement fixation (CF), rivanol, and particle concentration fluorescence immunoassay (PCFIA) confirmatory tests. Samples from 2 control cattle were PCFIA positive at time 0; 1 of these animals was CF positive throughout the study. This study suggests that use of strain RB51 in cattle vaccinated with strain 19 as calves will not cause positive responses on confirmatory tests and will not impair brucellosis serologic surveillance efforts.

Genomic fingerprinting and development of a dendrogram for Brucella spp. isolated from seals, porpoises, and dolphins.

Genomic DNA from reference strains and biovars of the genus Brucella was analyzed using pulsed-field gel electrophoresis (PFGE). Fingerprints were compared to estimate genetic relatedness among the strains and to obtain information on evolutionary relationships. Electrophoresis of DNA digested with the restriction endonuclease XbaI produced fragment profiles for the reference type strains that distinguished these strains to the level of species. Included in this study were strains isolated from marine mammals. The PFGE profiles from these strains were compared with those obtained from the reference strains and biovars. Isolates from dolphins had similar profiles that were distinct from profiles of Brucella isolates from seals and porpoises. Distance matrix analyses were used to produce a dendrogram. Biovars of B. abortus were clustered together in the dendrogram; similar clusters were shown for biovars of B. melitensis and for biovars of B. suis. Brucella ovis, B. canis, and B. neotomae differed from each other and from B. abortus, B. melitensis, and B. suis. The relationship between B. abortus strain RB51 and other Brucella biovars was compared because this strain has replaced B. abortus strain 19 for use as a live vaccine in cattle and possibly in bison and elk. These results support the current taxonomy of Brucella species and the designation of an additional genomic group(s) of Brucella. The PFGE analysis in conjunction with distance matrix analysis was a useful tool for calculating genetic relatedness among the Brucella species.

Seminal vesiculitis and orchitis caused by Brucella abortus biovar 1 in young bison bulls from South Dakota.

Specimens of blood, lymph nodes, spleens, and genitalia were collected at slaughter from seven 3- and 4-year-old male bison that had recently become seropositive for brucellosis. The animals were from a captive herd of approximately 3,500 bison located in central South Dakota. Brucella abortus biovar 1 was isolated from 2 or more specimens from each of 6 bison. Severe necrotizing and pyogranulomatous orchitis was present in 1 testicle from 1 bull, and 4 animals had mild to marked seminal vesiculitis. Immunohistochemical staining labeled organisms in seminal vesicles and the testicle with orchitis. Ultrastructurally, intact bacilli were present in cytoplasmic vacuoles of some macrophages; other macrophages contained intracytoplasmic aggregates of calcified coccobacilli.

Isolation of surface tubules of fowlpox virus.

Surface tubules of fowlpox virus were isolated using chemical and physical methods. Suspensions of lipid cytoplasmic inclusion bodies were obtained by treating infected chorioallantoic membranes with 1% trypsin. Inclusions were treated with ultrasonic sound, detergents, and enzymes and were examined by electron microscopy. Although lipase treatment altered the morphology of lipid inclusions, no viral surface tubules were recovered. Treatment with the detergent Nonidet-P40 followed by 2-mercaptoethanol disrupted virions without allowing surface tubules to be recovered. Disruption of lipid inclusions by ultrasonic sound or manual grinding of chorioallantoic membranes produced free virions but only small numbers of tubules. These results indicate that surface tubules can be recovered, but that the lipid nature of cytoplasmic inclusions interferes with procedures commonly used in tubule purification.

T cell-dependent inducible nitric oxide synthase production and ultrastructural morphology in BALB/c mice infected with Mycobacterium avium subspecies paratuberculosis.

Euthymic BALB/c and athymic nude BALB/c mice aged 3-8 days were infected intraperitoneally with Mycobacterium avium subspecies paratuberculosis (ATCC strain 19698). After euthanasia at 5 months post-inoculation, hepatic granulomas were evaluated by morphometric analysis of digital images captured from light microscopy sections, by electron microscopy and by immunohistochemical methods. Euthymic mice differed from athymic mice in that (1) their hepatic granulomas were smaller, contained fewer bacteria, and produced more inducible nitric oxide synthase, and (2) their hepatic macrophages contained fewer bacteria, a higher percentage of degraded bacteria, and increased numbers of primary lysosomes. The study showed that macrophage activation was markedly less in the T cell-deficient athymic mice than in the euthymic mice.