NorthernLights in slide-based cytometry and microscopy.

In recent years, slide-based cytometry has become a key technology for polychromatic cytometric investigations, and many efforts have been made to increase the number of measurable fluorochromes for multiparametric analysis. Sequential photobleaching of fluorochromes next to very photostable dyes is one approach for this technology. As the ALEXA dyes are known to be photostable as compared to the conventional fluorochromes FITC, PE (Riggs et al., Am J Pathol 1958;34:1081-1097), and APC, a differentiation within a fluorochrome pair is possible. Here, we have analyzed the newly available NorthernLights secondary antibodies for use in slide-based cytometry and microscopy. Currently, these fluorochrome-conjugates are now available with three distinct excitation- and emission maxima (NL493, NL557, NL637). Their spectral properties are similar to the frequently used fluorochromes FITC, PE, and APC and can, therefore, be used with most common excitation sources of cytometers or microscopes. As the NorthernLights are bright, resistant to photobleaching, stable in alcohols and xylene and of affordable price, these dyes are promising candidates for use with most laser- and HBO/XBA-based fluorescence microscopy-like techniques.

Imaging of cell trafficking and metastases of paediatric rhabdomyosarcoma.

OBJECTIVE: The aim of this study was to establish a preclinical mouse model to study metastases of paediatric rhabdomyosarcoma at the macroscopic and cellular levels, with different imaging methods. EXPERIMENTAL DESIGN: The alveolar rhabdomyosarcoma cell line Rh30 was stably transfected with the red fluorescent protein (DsRed2) then was xenotransplanted (intravenous injection [n = 8], and footpad injection [n = 8]) into nude mice (NMRI nu/nu). Macroscopic imaging of metastases was performed using DsRed2-fluorescence and flat-panel volumetric computed tomography scan. In a further series of animals (n = 8), in vivo cell trafficking of rhabdomyosarcoma cells using cellular imaging with an Olympus OV100 variable-magnification small-animal imaging system was used. RESULTS: Metastases in the pelvis, thoracic wall and skin were visualized by fluorescence imaging. Pelvic metastases were found after tail vein injection and at other metastatic sites after footpad injection. Flat-panel volumetric computed tomography scan data allowed highly specific analysis of contrast between tumour and surrounding tissue. Correlation between fluorescence and flat-panel volumetric computed tomography scan imaging data was observed. Single-cell imaging visualized tumour cells in the vessels and demonstrated the arrest of tumour cells at vessel junctions followed by extravasation of the tumour cells. CONCLUSION: We established a model for visualization of experimental metastatic invasion and describe relevant tools for imaging childhood rhabdomyosarcoma metastases at the macroscopic and cellular levels. Imaging of cell trafficking visualized the behaviour of tumour cells and development of metastases by accumulation and extravasation of rhabdomyosarcoma cells.

In vivo imaging in experimental preclinical tumor research--a review.

The multiparametric molecular cell and tissue analysis in vitro and in vivo is characterized by rapid progress in the field of image generation technologies, sensor biotechnology, and computational modeling. Fascinating new potentials in unraveling the detailed functions of single cells, organs, and whole organisms are presently emerging and permit the close monitoring i.e. tumor development or basic cell development processes with an unprecedented multiplicity of promising investigative possibilities. To answer basic questions of in vivo tumor development and progression fluorescence based imaging techniques provide new insights into molecular pathways and targets. Genetic reporter systems (eGFP, DsRED) are available and high sensitive detection systems are on hand. These techniques could be used for in vitro assays and quantified e.g. by microscopy and CCD based readouts. The introduction of novel fluorescent dyes emitting in the near infrared range (NIR) combined with the development of sensitive detector systems and monochromatic powerful NIR-lasers for the first time permits the quantification and imaging of fluorescence and/or bioluminescence in deeper tissues. Laser based techniques particularly in the NIR-range (like two-photon microscopy) offer superb signal to noise ratios, and thus the potential to detect molecular targets in vivo. In combination with flat panel volumetric computed tomography (fpVCT), questions dealing e.g. with tumor size, tumor growth, and angiogenesis/vascularization could be answered noninvasively using the same animal. The resolution of down to 150 microm/each direction can be achieved using fpVCT. It is demonstrated by many groups that submillimeter resolutions can be achieved in small animal imaging at high sensitivity and molecular specificity. Since the resolution in preclinical small animal imaging is down to approximately 10 microm by the use of microCT and to subcellular resolutions using ( approximately 1 microm) microscope based systems, the advances of different techniques can now be combined to "multimodal" preclinical imaging and the possibilities for in vivo intravital cytometry now become within one's reach.

Blood parasites of blue grouse (Dendragapus Obscurus) in western North America.

Three hundred thirty-three blue grouse (Dendragapus obscurus) were examined for blood parasites from 11 sites: southern Yukon Territory, southeast coastal Alaska, northern and central interior British Columbia, south coastal British Columbia, northcentral Washington, southcentral Oregon, northwestern California, eastcentral Nevada, northwestern Colorado, and westcentral Montana. Three species of protozoan parasites (Leucocytozoon lovati, Haemoproteus mansoni, Trypanosoma avium) and a splendidofilariid nematode (Microfilaria sp. B) were found in nearly all locations. Prevalence levels were consistently high for L. lovati (92%). The other hematozoa were found less frequently (H. mansoni 29%; T. avium 46%; and microfilaria 29%). The range of these parasites in blue grouse was extended to a more northern (Yukon Territory) and more southern distribution (Nevada than previously reported. Ranges were also extended to blue grouse populations in Alaska, Washington, Oregon and California.

Effects of immune colostrum and orally administered antisporozoite monoclonal antibodies on the outcome of Cryptosporidium parvum infections in neonatal mice.

A neonatal BALB/c mouse model of cryptosporidiosis was used to examine the potential passive transfer of immunity via immune colostrum and oral treatment with anticryptosporidial monoclonal antibodies. Neonates suckled by dams that recovered from Cryptosporidium parvum infections were equally susceptible to infection as their control counterparts suckled by naive dams. Parasite loads among the control and immune colostrum-fed mice were indistinguishable. Neonates receiving orally administered antisporozoite monoclonal antibodies were equally susceptible to infections compared with the control and immune colostrum-fed mice. Parasite loads among the mice receiving daily oral treatment with monoclonal antibody mixtures exhibited significantly lower parasite loads compared with the control mice (P less than 0.05).

Kinetics of murine delayed-type hypersensitivity response to Eimeria falciformis (Apicomplexa: Eimeriidae).

Mice recovering from a primary infection with an intestinal protozoan parasite, Eimeria falciformis (Apicomplexa: Eimeriidae), showed a classic delayed-type hypersensitivity (DTH) reaction to oocyst antigen challenge. This reaction was characterized by a biphasic pattern of footpad swelling. The first swelling peaked at 2 h after antigen challenge, whereas the second swelling peaked at 24 to 48 h after challenge. The DTH reaction was transferable with a T-cell-enriched spleen cell population from mice that had recovered from E. falciformis infection. Cytotoxic depletion of immune T cells with anti-L3T4 antibody and complement abrogated DTH transfer, indicating that L3T4-positive T cells were required. A T-cell-enriched spleen cell population from acutely infected mice suppressed the transfer of DTH with immune cells from recovered animals, implicating the existence of infection-induced immunoregulatory cells controlling the parasite-specific immune response during infection. Immune spleen cells also transferred resistance to infection as measured by oocyst production and death rate of recipients. Together, these results indicate that the DTH reaction, induced by infection with E. falciformis, is mediated by L3T4-positive T cells and is associated with resistance to infection.

Murine major histocompatibility complex and immune response to Eimeria falciformis.

The genetics of the immune response to Eimeria falciformis were investigated in three inbred and six congenic strains of mice. There were significant differences among strains in oocyst production and age-related mortality from parasitic infection. Genes within the H-2 complex and also non-H-2 genes share in the immune response to eimerian infection.

Populations of Leucocytozoon gametocytes in blue grouse (Dendragapus obscurus) from Hardwicke Island, British Columbia.

Population dynamics of round and elongate gametocytes of Leucocytozoon in wild and captive blue grouse (Dendragapus obscurus (Say)) from Hardwicke Island, British Columbia, were studied from 1980 to 1982. Blue grouse chicks were sampled weekly throughout each transmission season. Three patterns in the type of gametocyte produced during primary infection were observed in naturally infected captive and wild blue grouse chicks. Such variation in the expression of the gametocyte stage within a single host population suggests a different interpretation than has been previously reported for species of Leucocytozoon. The data from the primary patterns and profiles coupled with reexposure data and the asynchronous appearance of round and elongate gametocytes can be best interpreted as infection with two concurrent species of Leucocytozoon in blue grouse. More detailed research on the life cycle is necessary to confirm if two species of Leucocytozoon exist in blue grouse.

Development of Sarcocystis alceslatrans Dubey, 1980, in the small intestine of dogs.

Laboratory-reared dogs were fed moose musculature infected with Sarcocystis alceslatrans. These dogs shed sporocysts [15.6 X 11.4 microns (14.4 to 15.8 X 10.8 to 11.5)] 11 to 15 days after inoculation. The prepatent period was 10 to 14 days. Two cats and 1 coyote that also ate infected moose musculature did not pass sporocysts. Histologic examination of intestinal tissue from experimentally infected dogs revealed microgamonts, macrogametes, and oocysts. All stages were present in the lamina propria of the small intestine, usually in the luminal third of the villi. Infections were concentrated in the proximal half of the small intestine. Oocysts were first noticed in dogs killed 7 days after inoculation and a sequence of sporogonic development occurred in dogs killed on subsequent days. Ultrastructural observations were made on the oocyst and sporocyst walls during sporogony.

Ultrastructure of the cyst wall and merozoites of Sarcocystis from moose (Alces alces) in Alberta, Canada.

Two distinct types of cysts of Sarcocystis from the musculature of moose (Alces alces) were compared by electron microscopy. The fusiform Type A cysts differed from the spherical Type B cysts in the appearance and thickness of the primary cyst wall, organization of cyst interior, and the presence of a secondary cyst wall around Type B. The respective merozoites also differed in size as well as in the number of rhoptries and diameter and arrangement of micronemes. Comparison of the ultrastructure of the moose sarcocysts with those described from other ungulates revealed substantial differences. It appears that two hitherto undescribed species of Sarcocystis are present in moose although cross-transmission and additional life cycle studies are necessary for a complete description.

Sarcocystis in wild ungulates in Alberta.

Muscle samples from 557 wild ungulates in Alberta, comprising seven species, were examined grossly and/or histologically for cysts of Sarcocystis. Sarcocystis was found in 100, 96, 94, 75, 75, 73, and 49% of the wapiti (Cervus canadensis), moose (Alces alces), bison (Bison bison), mule deer (Odocoileus hemionus), bighorn sheep (Ovis canadensis), mountain goat (Oreamnos americanus), and white-tailed deer (O. virginianus), respectively.

Isospora neorivolta SP. N. from the domestic dog.

The endogenous development of canine Isospora rivolta (Mahrt, J Protozool 14: 754--759, 1967) was compared with the development of I. ohioensis (Dubey, Parasitology, 1978, In press) in intestines of dogs. A new name, Isospora neorivolta, is proposed for the canine I. rivolta because of developmental differences from I. ohioensis. The major difference between these 2 coccidia is their site of development. Isospora neorivolta develops predominantly in the lamina propria of the posterior half of the small intestine, whereas I. ohioensis develops only in the epithelium and infection occurs throughout the small intestine. Additional information on the development of I. neorivolta in dogs is given.

Blood parasites of mallard and pintail ducks from central Alberta and the Mackenzie Delta, Northwest Territories.

Blood films from 60 mallard (Anas platyrhynchos) and 67 pintail (A. acuta) ducks, collected in Alberta and the Mackenzie Delta, Northwest Territories, during 1973 and 1974, were examined for blood parasites. Twenty-two (37%) of the mallards and fourteen (21%) of the pintails were infected with one or more species of hematozoa. Infections of Leucocytozon simondi occurred more frequently (86%) than Haemoproteus nettionis (22%) in the infected birds. Trypanosoma avium occurred in one individual of each species of duck; one pintail harbored an unidentified microfilaria. Differences of prevalence between species are predicted on the basis of host attractancy to vectors and/or host habitat selection, and are discussed.

[Study of the oocyst of the coccidia Eimeria nieschulzi by freeze-fracturing]. / Etude de l'oocyste de la coccidie Eimeria nieschulzi par cryodécapage

Ultrastructure of Eimeria nieschulzi unsporulated oocyst is revealed by freeze etching. The two layers of the oocyst wall are observed; they are separated from the multilayered cytoplasmic pellicule by an intraoocystic fluid. Nuclear envelope with pores, endoplasmic reticulum and mitochondrias surrounded by amylopectin granules are easy to identify. This technique makes possible the study of sporulation in Eimeria, which cannot be done with the classical Electron Microscope methods.