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.

Pyogranulomatous pleuropneumonia and mediastinitis in ferrets (Mustela putorius furo) associated with Pseudomonas luteola Infection.

Between 2008 and 2009, three pet ferrets from different sources presented with acute episode of dyspnoea. Cytological examination of pleural exudates revealed severe purulent inflammation with abundant clusters of rod-shaped microorganisms with a clear surrounding halo. Treatment was ineffective and the ferrets died 2-5 days later. Two ferrets were subjected to necropsy examination, which revealed pyothorax, mediastinal lymphadenopathy and multiple white nodules (1-2mm) in the lungs. Microscopical examination showed multifocal necrotizing-pyogranulomatous pleuropneumonia and lymphadenitis with aggregates of encapsulated microorganisms, some of which were positively stained by periodic acid-Schiff and alcian blue. In-situ hybridization for Pneumocystis spp., Ziehl-Neelsen staining and immunohistochemistry for distemper, coronavirus and influenza antigen were negative in all cases. Electron microscopically, the bacteria were 2-3 µm long with a thick electron-lucent capsule. Microbiology from one ferret yielded a pure culture of gram-negative bacteria identified phenotypically as Pseudomonas luteola. This speciation was later confirmed by 16S RNA gene amplification.

Phagosomal maturation and intracellular survival of Mycobacterium avium subspecies paratuberculosis in J774 cells.

The mechanisms by which Mycobacterium avium subspecies paratuberculosis (M. a. ptb) survives within macrophages are not well characterized. One strategy for intracellular survival developed by Mycobacterium tuberculosis is inhibition of phagosomal maturation. In this study it was hypothesized that M. a. ptb is capable of survival within macrophages by residing within a phagosomal compartment that does not mature into a functional phagolysosome. To test this hypothesis the following objectives were determined. Phagosomal maturation was evaluated by comparison of stage specific markers on the membranes of phagosomes containing live M. a. ptb with those containing killed M. a. ptb, Mycobacterium smegmatis, and zymosan A using immunofluorescent labeling and confocal microscopy. Intracellular survival of live M. a. ptb within J774 macrophages was compared to that of M. smegmatis by direct determination of bacterial viability by differential live/dead staining. The results of this study show that the phagosomes containing live M. a. ptb had increased levels of an early marker (transferrin receptor [TFR]) and decreased levels of a late maturation marker (lysosome associated membrane protein one [Lamp-1]), relative to those containing killed M. a. ptb, M. smegmatis, and zymosan A. Additionally, compared to M. smegmatis, M. a. ptb has enhanced ability to survive within cultured macrophages. These data indicate that M. a. ptb resists intracellular killing by residing within a phagosomal compartment that retains the characteristics of early phagosomes and resists maturation into functional phagolysosome.

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.

Intracellular trafficking of Mycobacterium avium ss. paratuberculosis in macrophages.

The granulomatous enteric lesions of cattle with Johne's disease are composed of infected macrophages, and grow by accumulation, re-infection, and expansion of macrophage populations in the intestinal wall. We have examined the growth of bacteria in macrophages to define characteristics of intracellular trafficking for exocytosis, replication, and antigen presentation. Using immunocytochemical markers for light, confocal and electron microscopy, we have examined potential pathway tropisms using data for bacterial attachment, phagosomal acidification, phagolysosomal degradation and apoptosis. Our hypotheses are that pathogenic/wild-type strains block phagosomal acidification so that the phagosome fails to obtain markers of the late phagosome and phagolysosome, and this leads to the replication pathway within bacteriophorous vacuoles. Non-pathogenic strains appear to be processed to exocytosis, and avirulent mutant strains may be degraded and have preference of antigen processing pathways that involve transport vesicles bearing MHC II antigens. Pathogenicity in a nude mouse model of intestinal infection reveals lesion development and confirms pathway preferences of virulent strains for bacteriophorous vacuole formation.

Experimental Brucella abortus induced abortion in a llama: pathologic effects.

Brucella abortus infection has not been documented in llamas. This report describes the abortion of the only pregnant animal in a group of 12. The llama was infected by inoculating 1 x 10(8) viable B. abortus organisms into the conjunctival sac. Forty-three days postinfection, the llama aborted a fetus of approximately 8 months gestational age. Brucella organisms were isolated from the placenta and all fetal specimens examined. These organisms were also isolated from the dam's mammary gland and numerous lymph nodes when the llama was necropsied 42 days later. Microscopically, there was a moderate, multifocal, lymphocytic and histiocytic, subacute placentitis with marked loss of trophoblastic epithelial cells. The superficial chorioallantoic stroma contained abundant necrotic and mineralized debris as well as numerous swollen capillaries protruding multifocally from the denuded surface. Immunohistochemistry revealed that these capillaries, as well as sloughed and intact trophoblasts, were expanded by large numbers of Brucella organisms. Brucellar antigen was also detected in occasional macrophages in the fetal kidney and lung. Ultrastructurally, bacteria labeled by an antibody-based colloidal gold procedure were located within degenerate capillaries, within necrotic leukocytes, and extracellularly in the placental stroma.

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.

Survival of Brucella abortus strain RB51 lyophilized and as liquid vaccine under different storage conditions.

Brucella abortus strain RB51 (SRB51) is a new cattle vaccine that is approved for use in the U.S. for prevention of brucellosis. At the present time, other countries are implementing or considering the use of SRB51 vaccine in their brucellosis control programs. In the current study, the effect of three stabilizing media, two fill volumes (1 and 3 ml), and three storage temperatures (-25, 4 and 25 degrees C) on the viability of lyophilized SRB51 over a 52 week period was determined. The effects of three concentrations of bacteria (5 x 10(8), 1 x 10(9), or 5 x 10(9) cfu/ml) and two storage temperatures (4 or 25 degrees C) on viability of liquid SRB51 vaccine were also determined. For lyophilized strain RB51 vaccine, fill volume did not influence viability (P> 0.05) during lyophilization. Although fill volume did not influence viability during storage in World Health Organization (WHO) media or media containing both WHO and Lactose Salt (LS) media, 1 ml fill volumes of SRB51 in LS media had greater (P< 0.05) viability when compared to 3 ml fill volumes. Lyophilized SRB51 vaccine stored at 25 degrees C had a more rapid decline in viability (P< 0.05) when compared to vaccine stored at -25 or 4 degrees C. With the exception of the 3-ml fill volumes of LS media, all three stabilizing media were similar in maintaining viability of SRB51 at -25 degrees C storage temperatures. However, when compared to WHO or WHO/LS media, stabilization in LS media was associated with a more rapid decline in viability during storage at 4 or 25 degrees C (P< 0.05). Initial SRB51 concentration in liquid vaccine did not influence (P> 0.05) viability during storage at 4 or 25 degrees C. When compared to liquid SRB51 vaccine stored at 25 degrees C, storage at 4 degrees C was associated with a slower decline in viability (P< 0.05) during 12 weeks of storage. Biochemical and morphological characteristics of SRB51 were stable under the storage conditions utilized in the present study. This study suggests that viability of SRB51 can be readily maintained during storage as a lyophilized or liquid brucellosis vaccine.

Brucella-induced abortions and infection in bottlenose dolphins (Tursiops truncatus).

Two bottlenose dolphins (Tursiops truncatus) aborted fetuses that died as a result of Brucella infection. Brucella placentitis occurred in both cases. Infected placenta and vaginal/uterine fluids may transmit Brucella species to other cetaceans. In a third case, an identical organism was cultured from lung necropsy tissue of an adult female T. truncatus. Microbiology, specific polymerase chain reaction, and pulsed-field gel electrophoresis results supported the designation of an additional genomic group(s), Brucella delphini, for isolates adapted to T. truncatus. Current serologic diagnostic tests reliable for known Brucella species are unreliable in detecting dolphin brucellosis. Our findings, together with previous reports, suggest that dolphin brucellosis is a naturally occurring disease that can adversely impact reproduction in cetaceans. The zoonotic significance of cetacean brucellosis is unknown, although the disease has not been reported in people who have frequent contact with dolphins. Further studies on the zoonotic aspects, distribution, prevalence, virulence, and impact of this disease in cetaceans and other marine mammal species are needed.

Responses of cattle to two dosages of Brucella abortus strain RB51: serology, clearance and efficacy.

A new brucellosis vaccine, Brucella abortus strain RB51 (SRB51), is currently recommended for use as a calfhood vaccine in the US at dosages between 1 x 10(10)and 3.4 x 10(10)colony-forming units (CFU). The purpose of the study reported here was to compare responses to minimal and maximal recommended SRB51 dosages. Eighteen heifer calves were vaccinated subcutaneously with 1.6 x 10(10)CFU of SRB51, 3.2 x 10(10)CFU of SRB51, or saline (n = 6 per treatment). The vaccine strain was recovered from the superficial cervical lymph node 14 weeks after vaccination in two of six animals that received 1.6 x 10(10)CFU SRB51, but not from any cattle vaccinated with 3.2 x 10(10)CFU SRB51. The higher SRB51 dosage stimulated greater antibody titres. Protection against abortion or infection following B. abortus strain 2308 (S2308) challenge was similar for both SRB51 dosages and greater than resistance of non-vaccinates. The vaccine strain was recovered from one heifer and her fetus at necropsy 1 week prior to estimated parturition. Data from this study suggests that SRB51 induces similar protective immunity across the recommended dosage range. The SRB51 vaccine may persist in some cattle into adulthood but the incidence and significance of this persistence remains unknown.

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.

Expression and distribution of leptospiral outer membrane components during renal infection of hamsters.

The outer membrane of pathogenic Leptospira species grown in culture media contains lipopolysaccharide (LPS), a porin (OmpL1), and several lipoproteins, including LipL36 and LipL41. The purpose of this study was to characterize the expression and distribution of these outer membrane antigens during renal infection. Hamsters were challenged with host-derived Leptospira kirschneri to generate sera which contained antibodies to antigens expressed in vivo. Immunoblotting performed with sera from animals challenged with these host-derived organisms demonstrated reactivity with OmpL1, LipL41, and several other proteins but not with LipL36. Although LipL36 is a prominent outer membrane antigen of cultivated L. kirschneri, its expression also could not be detected in infected hamster kidney tissue by immunohistochemistry, indicating that expression of this protein is down-regulated in vivo. In contrast, LPS, OmpL1, and LipL41 were demonstrated on organisms colonizing the lumen of proximal convoluted renal tubules at both 10 and 28 days postinfection. Tubular epithelial cells around the luminal colonies had fine granular cytoplasmic LPS. When the cellular inflammatory response was present in the renal interstitium at 28 days postinfection, LPS and OmpL1 were also detectable within interstitial phagocytes. These data establish that outer membrane components expressed during infection have roles in the induction and persistence of leptospiral interstitial nephritis.

Production and characterization of canine osteosarcoma cell lines that induce transplantable tumors in nude mice.

OBJECTIVE: To produce and characterize cell lines from canine primary appendicular osteosarcomas that induce transplantable tumors in athymic nude mice. ANIMALS: 57 six- to 8-week-old female athymic nude mice. PROCEDURE: Canine primary appendicular osteosarcoma tumors were harvested and cell lines were produced. Canine osteosarcoma (COSCA)-Toby (COSCA-T; 10 mice), COSCA-Princess (COSCA-Pr; 16) or canine osteosarcoma D-17 (ATCC CCL-183; 31) cells were injected into the proximal portion of the left tibia of nude mice to evaluate tumor production from each cell line; the right tibia served as the control. Tibial measurements were taken on alternating days to evaluate tumor growth during a 6-month period. Student's t-tests were used to determine whether size of the proximal portion of the left and right tibias differed significantly during the observation period. RESULTS: 88% of mice receiving COSCA-Pr and 50% of mice receiving COSCA-T cells developed a tumor at the injection site by 9 days after implantation. The D-17 cells induced tumors in 50% of injected tibias; however, tumors were not detected for 79 days. Tumors generated from COSCA-Pr and COSCA-T cells in nude mice were histologically similar to the canine tumor from which they were developed. CONCLUSION: New osteosarcoma cell lines that can reliably and rapidly induce transplantable tumors in nude mice were developed. CLINICAL RELEVANCE: Use of cell lines will allow evaluation of new treatments of canine primary appendicular osteosarcoma in a nude mouse model.

Tumor necrosis factor-alpha in pregnant cattle after intravenous or subcutaneous vaccination with Brucella abortus strain RB51.

OBJECTIVE: To determine the influence of brucellosis vaccination on tumor necrosis factor-alpha (TNF-alpha) concentrations in pregnant cattle and the possible role of the bovine placenta in TNF-alpha production. ANIMALS: Polled Hereford heifers obtained from a nonvaccinated, brucellosis-free herd and bred at 16 to 27 months at age. All cattle were seronegative for Brucella abortus by results of the standard tube agglutination test. PROCEDURE: At 6 months' gestation, cattle were vaccinated i.v. with B abortus strain RB51 (n = 10), s.c. with B abortus strain RB51 (n = 5), or s.c. with B abortus strain 19 (n = 5); controls received pyrogen-free saline solution s.c. (n = 2). Blood samples were collected periodically for TNF-alpha assays. At necropsy, 8 to 12 weeks after vaccination, placental fluids and fetal blood were collected for TNF-alpha analysis and placental tissues were collected for immunohistochemical detection of TNF-alpha. RESULTS: Radioimmunoassays indicated no increase in TNF-alpha concentration in blood from i.v. or s.c. vaccinated cattle, compared with controls. Similarly, TNF-alpha concentrations in amniotic and allantoic fluids from s.c. vaccinated cattle were not different from values for controls. Although only i.v. vaccinated cattle developed placentitis, immunohistochemical analysis for TNF-alpha revealed increased immunoreactivity within placental trophoblastic epithelial cells of s.c. and i.v. vaccinated cattle. CONCLUSIONS: s.c. vaccination for prevention of brucellosis, using recommended adult dosages, does not result in increase of TNF-alpha concentration in plasma, serum, or placental fluids; however, vaccination of pregnant cattle stimulates trophoblastic epithelial cells to express TNF-alpha, although the physiologic and quantitative importance of this expression remains unknown.

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.

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.

Effects of oral or intravenous inoculation with Brucella abortus strain RB51 vaccine in beagles.

OBJECTIVES: To determine whether the vaccine Brucella abortus strain RB51 (SRB51) would infect dogs, be shed in urine or feces, or cause placentitis and abortion. ANIMALS: 18 Beagles. PROCEDURE: Males (n = 3), nonpregnant females (n = 3), and pregnant females (n = 4) were inoculated orally with SRB51; control dogs (n = 2) were fed sterile saline solution. A separate group of pregnant females (n = 5) received SRB51 i.v., and their controls (n = 1) received sterile saline solution i.v.. Dogs were observed twice daily for evidence of abortion. Urine and feces were collected periodically for bacteriologic culture, and blood was collected for bacteriologic culture and serologic analysis. At full gestation (oral and i.v. inoculated pregnant females) or on postinoculation day 49 (nonpregnant females and males), dogs were euthanatized and samples were collected for bacteriologic culture and microscopic examination. RESULTS: Abortion was not apparent during the study, and SRB51 was not found in samples of urine of feces from any dog. Strain RB51 was isolated from retropharyngeal lymph nodes from all orally inoculated dogs (9/9). One orally inoculated and 1 i.v. inoculated pregnant dog had SRB51 in placental tissues. Strain RB51 was also isolated from 1 fetus from the orally inoculated female dog with placentitis, but lesions were not detected in the fetus. CONCLUSIONS AND CLINICAL RELEVANCE: Oral inoculation of nonpregnant female or male dogs with SRB51 did not result in shedding in urine or feces, although oropharyngeal lymph nodes became infected; in pregnant females, it caused infection of the placenta, with resulting placentitis and fetal infection, but abortion was not apparent. Intravenous inoculation resulted in infection of maternal spleen, liver, and placenta; however, fetal infection and abortion were not observed. Infected canine placental membranes or fluids may be a source of infection for other animals and human beings.