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Aquatic macroaggregates (flocs ≥ 0.5 mm) provide an important mechanism for vertical flux of nutrients and organic matter in aquatic ecosystems, yet their role in the transport and fate of zoonotic pathogens is largely unknown. Terrestrial pathogens that enter coastal waters through contaminated freshwater runoff may be especially prone to flocculation due to fluid dynamics and electrochemical changes that occur where fresh and marine waters mix. In this study, laboratory experiments were conducted to evaluate whether zoonotic pathogens (Cryptosporidium, Giardia, Salmonella) and a virus surrogate (PP7) are associated with aquatic macroaggregates and whether pathogen aggregation is enhanced in saline waters. Targeted microorganisms showed increased association with macroaggregates in estuarine and marine waters, as compared with an ultrapure water control and natural freshwater. Enrichment factor estimations demonstrated that pathogens are 2-4 orders of magnitude more concentrated in aggregates than in the estuarine and marine water surrounding the aggregates. Pathogen incorporation into aquatic macroaggregates may influence their transmission to susceptible hosts through settling and subsequent accumulation in zones where aggregation is greatest, as well as via enhanced uptake by invertebrates that serve as prey for marine animals or as seafood for humans.

The coastal ecosystems of California are highly utilized by humans and animals, but the ecology of fecal bacteria at the land-sea interface is not well understood. This study evaluated the distribution of potentially pathogenic bacteria in invertebrates from linked marine, estuarine, and freshwater ecosystems in central California. A variety of filter-feeding clams, mussels, worms, and crab tissues were selectively cultured for Salmonella spp., Campylobacter spp., Escherichia coli-O157, Clostridium perfringens, Plesiomonas shigelloides, and Vibrio spp. A longitudinal study assessed environmental risk factors for detecting these bacterial species in sentinel mussel batches. Putative risk factors included mussel collection near higher risk areas for livestock or human sewage exposure, adjacent human population density, season, recent precipitation, water temperature, water type, bivalve type, and freshwater outflow exposure. Bacteria detected in invertebrates included Salmonella spp., C. perfringens, P. shigelloides, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio alginolyticus. Overall, 80% of mussel batches were culture positive for at least one of the bacterial species, although the pathogens Campylobacter, E. coli-O157, and Salmonella were not detected. Many of the same bacterial species were also cultured from upstream estuarine and riverine invertebrates. Exposure to human sewage sources, recent precipitation, and water temperature were significant risk factors for bacterial detection in sentinel mussel batches. These findings are consistent with the hypothesis that filter-feeding invertebrates along the coast concentrate fecal bacteria flowing from land to sea and show that the relationships between anthropogenic effects on coastal ecosystems and the environmental niches of fecal bacteria are complex and dynamic.

Humoral and cell-mediated immune (CMI) responses [i.e. proliferative responses and gamma interferon (IFN-gamma) production], were elicited in five cows infected between 159 and 169 days of gestation by a combined intravenous-intramuscular inoculation of Neospora caninum tachyzoites. Analysis of antigen-specific immunoglobulin (IgG) subclasses revealed a predominant IgG2 response in two cows, a mixed IgG1-IgG2 response in two other cows and a predominant IgG1 response in one cow. No correlation was found between IgG2 titers and IFN-gamma levels. CD4-T cells were responsible for the CMI responses in peripheral blood mononuclear cells from three infected cows. All five fetuses removed from infected dams at week 9 post-infection (219-231 days of gestation) mounted strong Neospora-specific humoral responses and had a predominant IgG1 response, regardless of their ability to produce IFN-gamma. However, CMI responses were highly variable between fetuses. These data indicate the complexity of the immune mechanisms associated with Neospora infection in both the dams and their fetuses.

The development of a rodent model for the study of Sarcocystis neurona encephalitis is described. Animal models have been developed for a number of protozoal parasites; however, no such model exists for S. neurona. The approach used in this study is similar to that employed for other closely related protozoal parasites such as Neospora caninum and Toxoplasma gondii. A time course of infection was examined, and histopathology, immunohistochemistry, and parasite isolation were used to examine the pathogenesis and follow the infection from 1 to 6 weeks postinoculation. S. neurona was associated with the development of encephalitis in these mice, and the immune status determined the susceptibility of these mice to S. neurona-associated encephalitis.

Techniques have been established for the continuous culture of a newly recognized salmonid microsporidian parasiteEnterocytozoon salmonis in salmonid mononuclear leukocytes. Utilization of human recombinant interleukin-2 (HrIL-2) and polyclonal mitogens in the culture medium supported viability of salmonid mononuclear leukocytes which allowed proliferation of the intracellular microsporidian parasite. The microsporidian maintained in primary culture for 45 days was infective both in cultures of mononuclear leukocytes and in juvenile salmonids. Additional passages of the agents from primary cultures to new cultures of uninfected mononuclear leukocytes have allowed continuous in vitro propagation ofE. salmonis. The parasites from these cultures retained developmental stages from early meronts to mature spores that were identical to those observed in naturally infected fish. Also, after cryopreservation in liquid nitrogen the parasites maintained their infectivity for juvenile chinook salmon and cell cultures of mononuclear leukocytes. The development of methods for the in vitro culture ofE. salmonis has made possible investigation of the effect of this microsporidian on immune functions of its key target cell, the mononuclear leukocyte.

To determine the identity of the tick vector of enzooticBabesia gibsoni in California, two common ixodid ticks were allowed to engorge uponB. gibsoni infected dogs. Sporozoites were observed in the salivary glands of prefed nymphalRhipicephalus sanguineus ticks that fed as larvae onB. gibsoni-infected dogs. A higher proportion (31%) of nymphal ticks that prefed on an uninfected dog for 48 hours contained sporozoites in their salivary glands than did ticks which had fed for 24 hours (13%). Sporozoites were not observed in the salivary glands of prefedR. sanguineus nymphs which were derived from the eggs of adult females that fed on an infected dog, in adults that were fed as nymph on an infected dog, or in the nymphal and adult uninfected controls.Dermacentor variabilis ticks appeared not to become infected. Although attempts to transmitB. gibsoni to susceptible, splenectomized dogs were unsuccessful,R. sanguineus would appear to be the most likely tick vector to maintain this piroplasm in California.

The ability of Theileria parva-specific DNA probes to detect T. parva sporoblasts and sporozoites in samples prepared from the salivary glands of infected Rhipicephalus appendiculatus ticks was evaluated. The two DNA probes used, pgTpM-23 and IgTpM-58, were selected from a genomic library of T. parva (Muguga) piroplasm DNA. In all, 25-200 adult ticks infected with each of 6 different T. parva stocks were tested. One salivary gland from each tick was processed for DNA hybridization, whereas the other was stained and examined by light microscopy to determine the number of infected acini. The correlation for the detection of infected acini between the two methods was 90%-100% for both probes, except when the pgTpM-23 probe was hybridised to salivary glands from ticks infected with the Mariakani stock of T. parva (84% correlation). The discrepancy lay within the range expected, based on the observation that in 12.5% of the ticks, only one salivary gland was infected. The probes did not hybridize to salivary glands from uninfected ticks.

The southern sea otter (Enhydra lutris nereis) is listed as “threatened” under the Endangered Species Act (ESA) and is a “keystone species,” strongly influencing the abundance and diversity of the other species within its kelp forest ecosystem. This is accomplished primarily by preying upon urchins that eat the kelp stipe and holdfast, which can reduce a kelp forest to an urchin barren. Sea otters are very susceptible to marine pollutants such as petroleum, which may be directly toxic and/or alter their fur’s insulating properties. Sea otters are an excellent sentinel species. They eat approximately 25% of their body weight per day in shellfish and other invertebrates, and can concentrate and integrate chemical contaminants. In addition, they appear to be susceptible to a number of diseases and parasites that may have anthropogenic origins, and shellfish may serve as an intermediary for some of these infections. Many of the shellfish the otters eat are also harvested for human food. In their role as sentinels, sea otter health has implications for human health, economic sustainability of shellfisheries, as well as overall marine ecosystem health. The recent southern sea otter decline has been viewed with some alarm by conservationists and, indeed, recovery seems a long way off. High mortality rather than depressed recruitment appears to underlie the decline. A good deal of debate has centered on the role of infectious diseases and parasites, exposure to contaminants, nutrition and prey availability, net and pot fishery interactions, and other sources of mortality. Current research is being done related to major classes of mortality, various types of pollutants and some specific organisms causing southern sea otter mortality, and their implications for marine ecosystem health and sustainability.

Callus-derived suspension cultures of oats dramatically increase the viscosity of the culture media after one month in culture. Colorimetric assays for sugars and protein, as well as measurements of viscosity, suggest that the released material is a long-chain polysaccharide, probably a pectinaceous substance. These cells grow slowly in liquid culture, yet despite their low cell density, they are able to increase the viscosity of the media several fold within seven days after media transfer. Ultrastructural observations show that oat cells have features common to actively-secreting cells; especially evident are numerous dictyosomes with hypertrophied cisternae. Using a combination of filtering and centrifugation techniques we were able to recover large numbers of intact secretory vesicles. The interior of the vesicles stain with periodic acid-silver hexamine, and colormetric analysis of the vesicle pellet for total sugars confirms the presence of polysaccharides in this vesicle fraction. Because of the uniformity of these cells, the high rate of secretion, and the accessability of a large vesicle population, this culture system is'a useful model for studying the secretory process in plant cells.

Cytokinin stimulates caulonemata ofFunaria to undergo an asymmetric division leading to the gametophore. The earliest detectable event is a small protuberance at the distal portion of the cell accompanied by the reorganization of the underlying organelles into a polarized distribution reminiscent of a tip growing cell. Dictyosomes and associated vesicles accumulate in the protuberance directly beneath the plasma membrane with mitochondria subjacent to the vesicular layer. Endoplasmic reticulum lies beneath the mitochondrial zone directly above the large central vacuole, while chloroplasts are outside the bud. As development continues the bud elongates causing the outer cell wall to exfoliate. During the above events the nucleus migrates toward the bud site concomitant with an increase in the number of microtubules between the nucleus and the base of the outgrowth. Nucleoli, extruded from the nucleus during a previous division, persist as diffuse fragments within the protuberance. Upon reaching the bud site, division occurs with the developing phragmoplast being initiated distal to the caulonema tip cell. The former polarized distribution of the cytoplasm is altered as mitochondria, chloroplasts and small vacuoles become evenly dispersed throughout the cytoplasm; dicytosomes and endoplasmic reticulum occupy a cortical position. These events indicate a change from 2-D tip growth to 3-D diffuse growth. To quantify the ultrastructural changes associated with bud formation we performed a morphometric analysis of cells in various stages of budding. The relative volumes of dictyosomes and vesicles adjacent to the bud apex decrease during bud development coincident with an increase in these organelles in lower portions of the cytoplasm. Mitochondria and chloroplasts follow this same pattern although their highest relative volumes initially are 4 μm from the bud apex and outside the bud site, respectively. These data, as well as density profile topographic maps for vesicle fractions, support the contention that cytokinin induces a change in morphological symmetry and polarity in the fine structure ofFunaria.

Biosynthetically radiolabelled Theileria parva schizonts were purified from bovine lymphoblastoid cells and their proteins were analyzed by two-dimensional gel electrophoresis and autoradiography. The protein spot patterns of schizont proteins from three stocks of T. parva parva indicated that the phenotypic diversity among the stocks was minimal, with the Mariakani and Uganda stocks being identical and the Muguga stock showing only a few differences in minor spots. Comparison of the spot patterns of schizonts of three T. parva subspecies showed that T.p. parva and T.p. bovis differed in only one protein and thus could not be reliably distinguished on the basis of their protein differences. However, T.p. lawrencei showed several protein differences and could be distinguished easily from the other subspecies. Differences in schizont-protein spot patterns were also seen when two different cell lines were infected with the same Theileria stabilate, when one cell line was infected with two different stabilates of the same stock and when uncloned and cloned infected cell lines were used. These results suggest the possibility that selection of phenotypically different parasites could occur in vivo or in vitro.

In the mouse, potent primary in vitro proliferation of T cells can be induced by allelic variants of cell-surface glycoproteins, Ia antigens, the genes for which are located in the I region of the major histocompatibility complex (MHC) on chromosome 17. The only other potent primary proliferative response known is induced by mixing MHC-identical lymphocytes from strains that differ at the locus termed Mls (ref. 1) (for mixed lymphocyte stimulating), which has been mapped to chromosome 1. While it is relatively easy to raise antibodies against Ia antigens, and thus determine both their chemical nature and their role in T-cell stimulation, the nature of the product of the Mls locus has remained obscure. It has been proposed that the Mls locus product is a minor antigen recognized in association with self-Ia antigens2,3, a translocated Ia-like element4, or a mitogenic molecule found on the surface of antigen presenting cells (APC)5. Here, we demonstrate that APCs from mice carrying stimulatory Mls locus alleles present antigen more efficiently to cloned, antigen-specific, Ia-restricted T cells than APCs from mice carrying nonstimulating Mls locus alleles. We propose that the Mls locus does not encode a unique cell-surface antigen at all; we suggest instead that the T-cell proliferative response induced by Mls-locus disparate cells reflects recognition of self-Ia molecules on APCs. If our interpretation is correct, it provides further evidence both for the quantitative nature of self tolerance and for the existence of a distinct recognition site for self-Ia molecules on antigen-specific T lymphocytes.