First report of a Mikrocytos-like parasite in European oysters Ostrea edulis from Canada after transport and quarantine in France.

As part of a disease resistance experiment, 112 apparently healthy European flat oysters Ostrea edulis L. were exported from Canada (Nova Scotia) into France to test their susceptibility to Bonamia ostreae infection. Twelve oysters died in transit and 17 others died within 2 wk of laboratory quarantine acclimation. All oysters were examined histologically, and the 17 that died during quarantine were assayed for microcells (Bonamia sp. and Mikrocytos mackini) using molecular techniques. A microcell parasite was detected in the connective tissue of 5 of the 112 oysters. Morphological appearance, tissue affinity and molecular characterization through PCR, in situ hybridization (ISH), fluorescence in situ hybridization (FISH) and sequencing revealed a protist related to M. mackini. This is the first report of a parasite of the genus Mikrocytos in a species belonging to the genus Ostrea from the Atlantic Ocean.

Regulatory light-chain Cys-55 sites on the two heads of myosin can come within 2A of each other.

The di-thiol reagent, 5,5'-dithiobis (2-nitrobenzoic acid) is shown to induce disulfide bond formation between Mercenaria regulatory light-chain Cys-55 sites on either head of scallop hybrid myosin. This indicates that these two sites on opposite heads of myosin can come within 2A of each other and this confirms a prediction based on earlier data [Chantler, Tao and Stafford (1991) Biophys. J. 59, 1242-1250]. Results demonstrate that myosin heads in solution show a considerable mutual freedom of movement which can be monitored by probes in the vicinity of regulatory light-chain residue 55. Implications for light-chain movement on the myosin head are discussed.

Acquired and innate resistance to the haemoflagellate cryptobia salmositica in sockeye salmon (Oncorhynchus nerka).

Juvenile sockeye salmon (Oncorhynchus nerka, Fulton River stock) were protected from otherwise lethal challenges with the haemoflagellate Cryptobia salmositica by acclimation to elevated water temperatures (20 degrees C). Fish treated in this manner displayed increased immunity to C. salmositica and yielded plasma showing enhanced lytic activity against the parasite. The acquired lytic activity was antibody- and complement-mediated. In contrast, a stock of naive O. nerka from Weaver Creek, previously identified as having a high innate resistance to the lethal effects of C. salmositica, also had plasma factors that destroyed the parasite in vitro. This anti-Cryptobia activity also involved complement because 1) it resulted in lysis of the parasite, 2) it was heat-labile (40 degrees C for 20 min), and 3) it was largely removed from the plasma by substances capable of activating (binding) complement by the classical pathway (an antigen:antibody complex of Renibacterium salmoninarum and its specific antibody) and the alternate pathway (Escherichia coli lipopolyssacharide). The complement-mediated lysis associated with innate resistance was apparently the result of activation by the alternate pathway because it occurred in fish lacking antibodies against the parasite. The reaction was unusual in that a long incubation period (about 2 days) was required for maximum lysis of the parasite. At least one component of the innate lytic system depended on disulphide bonds because lytic activity was destroyed by 2-mercaptoethanol.

Ultrastructure of Mikrocytos mackini, the cause of Denman Island disease in oysters Crassostrea spp. and Ostrea spp. in British Columbia, Canada.

An ultrastructural study was carried out on Mikrocytos mackini, the cause of Denman Island disease in Pacific oysters Crassostrea gigas in western Canada. Three forms were identified, quiescent cells (QC), vesicular cells (VC) and endosomal cells (EC). QC occurred in the vesicular connective tissue (VCT), haemocytes (hyalinocytes), adductor and heart myocytes, and extracellularly. They had a central round to ovoid nucleus, < 7 cisternae of inactive nuclear membrane-bound Golgi, few vesicles and lysosome-like bodies. VC were rarely extracellular and usually occurred in adductor and heart myocytes, in close association with host cell mitochondria. The contents of the host cell mitochondria appeared to pass through a tubular extension into the cytoplasm of the parasite. Cytoplasmic vesicles resembled the tubular structure in appearance and size. EC occurred in the VCT, in haemocytes and extracellularly. They had a dilated nuclear membrane, sometimes containing a looped membranous structure that appeared to derive from the nucleus, and pass into the cytoplasm. A well-developed anastomosing endoplasmic reticulum connected the nuclear and plasma membranes, and endosomes were present in the cytoplasm. QC and EC cells were frequently observed tightly against, or between, the nuclear membranes of the host cell. Few organelles occurred in all forms of M. mackini, especially QC. The lack of organelles found in most eukaryotic cells, including mitochondria or their equivalents, may be due to obligate parasitism and the utilization of host cell organelles reducing the need for parasite organelles. Alternatively, perhaps M. mackini is a primitive eukaryote. Although phylogenetic affinities could not be determined, it is not a haplosporidian. A developmental cycle is proposed from these findings.

Detection of infection and susceptibility of different Pacific salmon stocks (Oncorhynchus spp.) to the haemoflagellate Cryptobia salmositica.

The haematocrit centrifugation technique, modified by keeping the haematocrit tubes cold (between 1 and 10 C), was sensitive for detecting light infections of Cryptobia salmositica (as few as 75 flagellates per ml of blood). In wet mount preparations, infections lighter than 7.5 X 10(3) flagellates per ml of blood could not be detected consistently. Different Pacific salmon stocks from British Columbia demonstrated differences in susceptibility to C. salmositica in experimental studies using laboratory reared juvenile fish. Oncorhynchus keta and Oncorhynchus tshawytscha from the Big Qualicum River stocks (Vancouver Island), and Oncorhynchus nerka from the Fulton River stock (Skeena River system), were all equally susceptible and suffered high mortalities at low exposures (100 flagellates in 0.1 ml physiological saline inoculated intraperitoneally per fish). Oncorhynchus nerka from the Weaver Creek stock (Fraser River system) was the most resistant with no mortalities even at exposures of 10(6) flagellates (in 0.1 ml physiological saline) per fish. Oncorhynchus kisutch seemed to be slightly less resistant than the Weaver Creek O. nerka, but fewer than 16% of the inoculated fish died. Oncorhynchus kisutch from the Big Qualicum River seemed to be slightly more resistant than O. kisutch from the Capilano River stock (a coastal river near Vancouver), with fewer mortalities and lighter infections when the experiments were terminated. Differences in susceptibility are believed to be associated with innate, genetically transmitted resistance.

The distribution, prevalence, and morphological features of the cystic stage of an apicomplexan parasite of native littleneck clams (Protothaca staminea) in British Columbia.

Sixty-nine of 98 native littleneck clams (Protothaca staminea) collected from Cooper's Cove, Sooke Basin, British Columbia during November 1995 contained apicomplexan cysts. The cysts, which measured 20-150 microns in diameter occurred in several tissues, particularly in the kidney and in connective tissue surrounding the intestine and contained closely packed, banana-shaped zoites that measured about 25 x 4 microns. A pronounced fibrillar layer underlain by labyrinthine structures separated the host tissues from the cyst wall. The apical region of the zoites was reinforced and appeared as an electron-dense, caplike structure. The cysts probably represent a stage of a heteroxenous coccidian life cycle, with a predator of clams serving as the definitive host in which gamogony and sporogony occur.

A simple technique to concentrate the protozoan Mikrocytos mackini, causative agent of Denman Island Disease in oysters.

This report describes a simple filtration technique to isolate the parasite Mikrocytos mackini from oyster tissue. The technique is based on successive filtration through filter papers and polycarbonate membrane filters of decreasing mesh using a low vacuum (<8 cm Hg). This technique allows for the recovery of about 1 x 10(8) parasites (microcells) from about 2 g of heavily infected oyster tissue. About 99% of the particulate material in the final preparation consisted of intact M. mackini.

The importance of choice of visualization technique in the use of indirect immunodetection methods: specific reference to the detection of light chain movement on a regulatory myosin.

This paper addresses a practical problem associated with the use of visual detection systems used in immunoblotting. Western blot analyses from the same experiment, differing only at the level of the secondary antibody used and the means of visualization employed, have produced apparently different results which, in isolation, could lead to different conclusions at both the qualitative and quantitative level. Indirect immunofluorescence, using a fluorescein isothiocyanate labeled secondary antibody and visualized by fluorescence excitation, was excellent for detecting the major species present but could not detect minor components. Indirect immunoperoxidase staining, on the other hand, appeared to detect all immunoreactive species present, both major and minor, presumably reflecting a more realistic picture of the experimental situation. All results were obtained during the observation of photocross-link formation between regulatory light chains and regulatory and essential light chains after hybridization of scallop myosin with benzophenone-4-maleimide labeled regulatory light chains. Results were obtained under conditions designed to simulate the physiological states of rest and rigor; the implication of these results with respect to myosin-linked regulation is discussed.

Margolisiella kabatai gen. et sp. n. (Apicomplexa: Eimeriidae), a parasite of native littleneck clams, Protothaca staminea, from British Columbia, Canada, with a taxonomic revision of the coccidian parasites of bivalves (Mollusca: Bivalvia).

Two of 98 native littleneck clams, Protothaca staminea Conrad, from Cooper's Cove, Sooke Basin were infected with an eimeriorin coccidian parasite. Merogonic gamontogonic and sporogonic development were observed in renal tubular epithelial cells. Sporulation of the oocysts occurred within the host. Mature oocysts were spherical mean 41 microns (range 30-44), and contained about 32 subspherical sporocysts (9 x 10 microns), each of which contained 4 sporozoites. Spherical 19 microns (18-20), cyst-like structures and smaller multinucleate bodies, some of which resembled sporocysts, were also seen. A review of the coccidian parasites of bivalves led to the erection of the new genus, Margolisiella (family Eimeriidae Minchin, 1903) to accommodate M. kabatai sp. n., the parasite in Protothaca staminea described herein. Four previously described monoxenous species (Pseudoklossia patellae Debaisieux, P. chitonis Debaisieux, P. tellinovum Buchanan and P. haliotis Friedman, Gardner, Hedrick, Stephenson, Cawthorn et Upton) were also transferred to the new genus. The 2 remaining possibly heteroxenous species (P. pelseneeri Léger and P. glomerata Léger et Duboscq) were retained in the genus Pseudoklossia Léger et Duboscq (family Aggregatidae Labbé, 1899).

The prevalence of Trypanosoma catostomi in white sucker (Catostomus commersoni) from southern Ontario.

During the summer of 1975, 285 white suckers (Catostomus commersoni) from 10 localities in southern Ontario were examined for trypanosomes. Trypanosoma catostomi Daly and DeGiusti, 1971 was found in the blood of 11.6% of the fish examined using the haematocrit centrifugation technique. Infected juvenile fish (33%) and infected adult fish (4%) were found in 6 of 10 locations.

A method for the temperature calibration of an infrared camera using water as a radiative source.

Presented here is an effective low-cost method for the temperature calibration of infrared cameras, for applications in the 0-100 degrees C range. The calibration of image gray level intensity to temperature is achieved by imaging an upwelling flow of water, the temperature of which is measured with a thermistor probe. The upwelling flow is created by a diffuser located below the water surface of a constant temperature water bath. The thermistor probe is kept immediately below the surface, and the distance from the diffuser outlet to the surface is adjusted so that the deformation of the water surface on account of the flow is small, yet the difference between the surface temperature seen by the camera and the bulk temperature measured by the thermistor is also small. The benefit of this method compared to typical calibration procedures is that, without sacrificing the quality of the calibration, relatively expensive commercial blackbodies are replaced by water as the radiative source (epsilon approximately 0.98 for the wavelengths considered here). A heat transfer analysis is provided, which improves the accuracy of the calibration method and also provides the user with guidance to further increases in accuracy of the method.

The burden of inpatient neurologic disease in two Ethiopian hospitals.

OBJECTIVES: To define the burden of inpatient neurologic disease seen in Ethiopian teaching hospitals. METHODS: We reviewed records of all medical inpatients admitted over a 6-month period to two teaching hospitals, one with and one without neurologists. RESULTS: Neurologic cases made up 18.0% and 24.7% of all medical admissions. The mortality rates were 21.8% and 34.7%. Noninfectious diseases were 36.7% and 31.7% of neurologic cases, but unknown etiologies made up 42.2% and 29.0% of all cases. Of total cases, only 42.9% and 24.1% had at least a high level of diagnostic certainty. CONCLUSIONS: Patients with neurologic disease make up a substantial minority of medical inpatients in Ethiopia. Noninfectious neurologic disease is at least as common as infectious neurologic disease. Reaching a well-defined final diagnosis occurs in only a minority of cases. Areas for improving the mortality rate include improving the barriers to diagnostic certainty and increasing treatment options for Ethiopian patients.

Cross-linking between translationally equivalent sites on the two heads of myosin. Relationship to energy transfer results between the same pair of sites.

Mercenaria myosin and scallop pure hybrid myosin possessing Mercenaria regulatory light chains were reacted with various concentrations of 4-4'-dimaleimidylstilbene-2-2'-disulfonic acid (DMSDS). Regulatory light chain homodimers are formed with great efficiency (20-50%). Dimers incorporating essential light chains were not formed upon reaction of DMSDS with Mercenaria myosin but some (less than 5%) essential light chain homodimers were obtained in the case of scallop hybrid myosin, probably occurring through relatively specific intermolecular associations within small myosin aggregates. Results were invariant, irrespective of the presence or absence of calcium and/or ATP. No radioactivity is incorporated into regulatory light chain homodimers upon post-labeling DMSDS-reacted myosin with 14C-labeled N-ethylmaleimide, irrespective of the original labeling ratio of DMSDS to myosin heads. This indicates the absence of free sulfhydryl groups in the regulatory light chain homodimer and suggests, therefore, that DMSDS links the two light chains together between translationally equivalent residues (Cys-50 of the Mercenaria regulatory light chain). These results imply that translationally equivalent sites on the two heads of myosin can come within 18 A of each other, the span of reacted DMSDS. Because energy transfer results between identical pairs of translationally equivalent sites on hybrid myosins indicated a low efficiency of energy transfer between these sites (Chantler, P.D., and Tao, T. (1986) J. Mol. Biol. 192, 87-99), it would appear that even though the two cysteines can come within 18 A of each other, their mean separation is much greater than this distance (greater than 50 A), a result consistent with a considerable flexibility of the two myosin heads with respect to each other.

Immunological comparison of four Trypanosoma spp. (sub-genus Schizotrypanum) from bats.

The antigenic differences and similarities between culture forms of Trypanosoma (Schizotrypanum) hedricki, Trypanosoma (Schizotrypanum) myoti, Trypanosoma (Schizotrypanum) vespertilionis and Trypanosoma (Schizotrypanum) dionisii, all isolated from bats, were compared using the double-diffusion technique. Antiserum to each trypanosome species was produced in rabbits by inoculating them with sonicated culture forms. Two isolates of T. hedricki were antigenically identical to each other, as were 2 isolates of T. myoti. There were several common antigens between the species. However, at least 1 antigen of each species was distinct from those of the other species.

Detection, isolation, and experimental transmission of Mikrocytos mackini, a microcell parasite of Pacific oysters Crassostrea gigas (Thunberg).

Denman Island disease, characterized by clinical signs of focal green lesions on the body surface or within the mantle, palps, and adductor muscle of Pacific oysters (Crassostrea gigas), is caused by a protozoan parasite of unknown taxonomic affiliations, Mikrocytos mackini. Detection of M. mackini was more sensitive and rapid by use of tissue imprints than histological sections. Of several isolation procedures investigated, centrifugation of homogenized infected tissues through a 15% sucrose solution enabled the isolation of the highest number of M. mackini with the lowest amount of oyster debris. Experimental transmission showed that oysters exposed to M. mackini by inoculation with isolated parasites had shorter prepatent periods and higher prevalences and intensities of infection than those incubated in homogenates from infected oysters (bath exposure) or those naturally exposed in the field. Experimental transmission was also used to propagate M. mackini in vivo in the laboratory year round. For the development of the disease, exposed oysters required prolonged incubation at low temperatures (about 10 degrees C).