Comparative accessory gene fingerprinting of surface water Escherichia coli reveals genetically diverse naturalized population.

AIMS: To utilize comparative accessory gene fingerprinting to discriminate between naturalized and faecal Escherichia coli, with particular emphasis on strains from phylogroup B1. METHODS AND RESULTS: Fourteen accessory genes that were potentially ecotype-specific were selected on the basis of comparative genomic DNA sequence analysis between faecal and environmental strains and also using a literature-based strategy. PCR assays were designed for each gene, and used to screen 107 faecal strains from various hosts and 106 environmental strains from surface water and sediment. While none of the 14 accessory genes were ecotype-specific, six of the genes were ecotype-enriched. Specifically, toxin-antitoxin system genes were more abundant among faecal strains, whereas genes involved in iron acquisition, complement resistance/surface exclusion, and biofilm formation were more abundant among environmental strains. These six genes were used to form composite fingerprints which revealed the presence of several ecotype-specific and -enriched fingerprints. Notably, some of the environmental strain-specific or -enriched fingerprints consisted of strains putatively belonging to clade ET-1, which has been previously recognized as a naturalized subpopulation. CONCLUSIONS: Unlike single genes which did not reliably distinguish between faecal and naturalized phylogroup B1 E. coli strains, composite fingerprints of ecotype-enriched accessory genes may offer a novel method for distinguishing between these two populations. SIGNIFICANCE AND IMPACT OF THE STUDY: Accessory gene fingerprinting may have important practical implications for improving the specificity of methods that are widely used for quantifying and identifying the sources of faecal contamination in surface water.

The role of drinking water in the transmission of antimicrobial-resistant E. coli.

To determine whether drinking water contaminated with antimicrobial-resistant E. coli is associated with the carriage of resistant E. coli, selected households sending water samples to Ontario and Alberta laboratories in 2005-2006 were asked to participate in a cross-sectional study. Household members aged ≥12 years were asked to complete a questionnaire and to submit a rectal swab. In 878 individuals, 41% carried a resistant strain of E. coli and 28% carried a multidrug-resistant strain. The risk of carriage of resistant E. coli was 1·26 times higher for users of water contaminated with resistant E. coli. Other risk factors included international travel [prevalence ratio (PR) 1·33], having a child in nappies (PR 1·33), being male (PR 1·33), and frequent handling of raw red meats (PR 1·10). Protecting private water sources (e.g. by improving systems to test and treat them) may help slow the emergence of antimicrobial resistance in E. coli.

Intergenic sequence comparison of Escherichia coli isolates reveals lifestyle adaptations but not host specificity.

Establishing the risk of human infection is one of the goals of public health. For bacterial pathogens, the virulence and zoonotic potential can often be related to their host source. Escherichia coli bacteria are common contaminants of water associated with human recreation and consumption, and many strains are pathogenic. In this study, we analyzed three promoter-containing intergenic regions from 284 diverse E. coli isolates in an attempt to identify molecular signatures associated with specific host types. Promoter sequences controlling production of curli fimbriae, flagella, and nutrient import yielded a phylogenetic tree with isolates clustered by established phylogenetic grouping (A, B1, B2, and D) but not by host source. Virulence genes were more prevalent in groups B2 and D isolates and in human isolates. Group B1 isolates, primarily from nonhuman sources, were the most genetically similar, indicating that they lacked molecular adaptations to specific host environments and were likely host generalists. Conversely, B2 isolates, primarily from human sources, displayed greater genetic distances and were more likely to be host adapted. In agreement with these hypotheses, prevalence of σ(S) activity and the rdar morphotype, phenotypes associated with environmental survival, were significantly higher in B1 isolates than in B2 isolates. Based on our findings, we speculate that E. coli host specificity is not defined by genome-wide sequence changes but, rather, by the presence or absence of specific genes and associated promoter elements. Furthermore, the requirements for colonization of the human gastrointestinal tract may lead to E. coli lifestyle changes along with selection for increased virulence.

Deactivation of primed respiratory burst response of goldfish macrophages by leukocyte-derived macrophage activating factor(s).

Macrophage activation factors (MAF), induced maximal priming of the respiratory burst response in GMCL after 6 h of stimulus, but by 24 or 48 h no priming effect was observed. Bacterial lipopolysaccharide (LPS) also primed the respiratory burst of goldfish macrophages, but the kinetics of priming were different from that induced by MAF. LPS induced a gradual increase in priming potential over 48 h of cultivation. Co-stimulation of macrophages with MAF and LPS resulted in enhanced priming of respiratory burst activity compared to either factor alone; however, the kinetics of priming were similar to those induced by MAF only. The MAF antagonized the ability of LPS to prime the respiratory burst over extended cultivation. The priming kinetics of the respiratory burst induced by MAF and/or LPS were not unique to GMCL, but were also similar for primary cultures of IVDKM. Respiratory burst deactivated macrophages-mounted potent nitric oxide response, indicating that this deactivation event was selective for respiratory burst activity. Autocrine factors produced by MAF-activated macrophages augmented priming of the respiratory burst, suggesting that deactivation of primed respiratory burst responses was not due to cytokine mediators produced by activated macrophages, but was most likely an intracellular deactivation event. Furthermore, production of reactive intermediates by activated fish macrophages was biphasic; with maximal ROI production occurring 6 h after stimulus, and maximal RNI occurring 72 h after stimulus. Our results indicate that activated fish macrophages mount sequential antimicrobial responses that are selectively deprogrammed once maximal induction has occurred. The ability to selectively deactivate ROI production without affecting subsequent RNI production may play an important role in host defense: regulating the duration of ROI production, and thus minimizing host tissue damage in an otherwise futile attempt to eliminate ROI resistant pathogens.

Products of proteolytic cleavage of transferrin induce nitric oxide response of goldfish macrophages.

Enzymatic cleavage product of transferrin induced the production of nitric oxide (NO) by LPS-stimulated goldfish macrophages. A NO-inducing factor was purified from the supernatants of mitogen-stimulated goldfish kidney leukocytes using fast performance liquid chromatography (FPLC) and the purified proteins analyzed by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry. The proteins were identified as truncated forms of transferrin, having approximate molecular weights (MW) of 33, 35, and 37kDa (kilodaltons). The precursor form (i.e. full-length) of transferrin did not enhance NO production by LPS-stimulated goldfish macrophages, but enzymatic cleavage of this precursor form correlated with enhanced production of NO by goldfish macrophages. Enzymatic cleavage of transferrin was dependent on the presence of stimulated kidney leukocytes and was shown to occur in response to both mixed lymphocyte reactions (MLR) and the mitogenic stimulation of goldfish kidney leukocytes. Time course analysis revealed that 24h after kidney leukocyte MLR or mitogen stimulation, cleaved transferrin products appeared in the supernatants of cultured cells, which was related to the on-set of NO-inducing activity of these preparations. To confirm these findings, bovine transferrin was digested in vitro using protease XXVII. The resulting cleavage products had approximate MW of 33, 35, and 37kDa. When these peptides were subjected to the purification protocols used to purify a NO-inducing factor from goldfish leukocyte supernatants, they were shown to elute to identical fractions. To examine the potential role of fish transferrin in mediating goldfish NO production, carp transferrin was purified from serum and following protease-digestion and purification by FPLC, the truncated proteins were found to elute to similar fractions as bovine transferrin. Furthermore, mitogen-stimulated leukocyte supernatants prepared in the absence of bovine serum (carp serum only) retained NO-inducing activity, indicating that this response was not an artifact of bovine serum components (i.e. bovine transferrin). Anti-bovine and anti-carp transferrin polyclonal antibodies identified the presence of truncated forms of transferrin in the active fractions of FPLC-separated mitogen-stimulated leukocyte supernatants prepared in the presence of bovine or carp serum, respectively. Thus, our results suggest a novel role for fish transferrin as one of the factors that mediates teleost macrophage antimicrobial functions.

Macrophage activating factor(s) secreted by mitogen stimulated goldfish kidney leukocytes synergize with bacterial lipopolysaccharide to induce nitric oxide production in teleost macrophages.

Recent studies in our laboratory demonstrated that fish macrophages produce nitric oxide. To elucidate the mechanisms which regulate nitric oxide production in teleosts, we examined whether macrophage activating factors (MAFs) secreted by mitogen stimulated leukocytes, induced nitric oxide production in a long-term cultured macrophage cell line and in primary cultures of kidney macrophages from the goldfish. The results indicate that both primary and long term cultured goldfish macrophages produce nitric oxide in response to MAF or bacterial lipopolysaccharide (LPS), and co-stimulation with both factors results in a synergistic induction of nitric oxide production. MAF that induced nitric oxide production were present in leukocyte supernatants as early as 24 h after addition of mitogens to cell cultures. The production of MAF was dependent upon the incubation temperature, presence of serum in the culture medium and duration of incubation: maximal MAF activity was detected in 72-96 h supernatants raised in media with serum at 30 degrees C. MAF-induced nitric oxide production by long term cultured macrophages was inhibited by 1000 microM NG-monomethyl-L-arginine or amino-guanidine, indicating an L-arginine-dependent metabolic pathway for the production of the reactive nitrogen intermediates in teleosts. The biochemical events of cytokine induced nitric oxide production by teleost macrophages appear to be similar to those of mammalian macrophages.

Flow cytometric analysis of PKH26-labeled goldfish kidney-derived macrophages.

We recently demonstrated that a goldfish macrophage cell line (GMCL) and primary in vitro-derived kidney macrophage (IVDKM) cultures contain three distinct macrophage subpopulations. Morphological, cytochemical, functional, and flow cytometric characterization of these sub-populations suggested that they may represent cells of the macrophage lineage temporally arrested at distinct differentiation junctures of fish macrophage development (putative early progenitors, monocytes, and macrophages). In this study, we examined the proliferation and differentiation events leading to the generation of mature macrophage-like cells from goldfish kidney hematopoietic tissues. The flow cytometric studies were done after labeling macrophages with PKH26 fluorescent dye and analysis of the data using the MODFIT software. Our results showed that IVDKM cultures proliferated non-synchronously, suggesting the presence of a temporal control mechanism regulating the number of cells entering the paths towards maturation. Such control is most evident during early progenitor proliferation and differentiation events. Our results showed that proliferation may not be a requirement for differentiation of early progenitors to putative monocyte and macrophage subsets. Detailed observation of the mature macrophage-like subpopulation indicated that: 1) they appear to develop from both, the differentiation of monocyte-like cells, and direct differentiation of early progenitors in the absence of a monocyte-like stage; and (2) mature macrophage-like cells appeared to be capable of self-proliferation. Our results suggest the presence of alternate pathways of fish macrophage development other than the classical hematopoietic pathway.

Production of a macrophage growth factor(s) by a goldfish macrophage cell line and macrophages derived from goldfish kidney leukocytes.

We recently established a spontaneously proliferating macrophage cell line from the goldfish (GMCL), and in this report demonstrate the production of a macrophage-specific growth factor(s) (MGFs) by these cells. The supernatants from GMCL cultures induced proliferation and differentiation of macrophage-like cells from kidney hematopoietic tissues of goldfish. Kidney leukocytes cultured at 6.25 x 10(4)cells/ml in the presence of GMCL-derived MGFs proliferated during two weeks of cultivation, whereas those cultured without the MGFs did not. Leukocytes cultured at higher densities (2.5 x 10(5) cells/ml) proliferated in the absence of exogenous growth factor, but not to the same extent as those stimulated with GMCL-derived MGFs, suggesting that kidney leukocytes may produce endogenous MGFs. At higher cell density (1 x 10(6) cells/ml), kidney leukocytes multiplied extensively over a two-week cultivation period in the absence of exogenous GMCL-derived MGFs. The supernatants from these cultures restored the proliferative ability of leukocytes cultured at low densities, providing direct evidence of MGFs production by kidney leukocytes. The predominant cell-type in cultures grown in the presence of GMCL or kidney leukocyte-MGFs was the macrophage based on the following criteria: (1) non-specific esterase staining; (2) morphologic similarity to GMCL; (3) phagocytosis of the bacterium, A. salmonicida; (4) production of reactive oxygen and nitrogen intermediates in response to stimulation with macrophage activating factors and/or bacterial lipopolysaccharide; and (5) flow cytometric analyses. Both in vitro-derived kidney macrophage (IVDKM) and GMCL cultures contained three distinct populations of cells, (determined by flow cytometry), suggesting that these macrophage cultures are comprised of cells arrested at distinct differentiation junctures in macrophage development. Production of MGFs by macrophages and kidney leukocytes may play an important role in regulating macrophage hematopoiesis in fish.

Inhibition of macrophage activity by mitogen-induced goldfish leukocyte deactivating factor.

Macrophage activating and deactivating cytokines have been characterized in mammalian systems but little is known about these immunoregulatory molecules in fish. Using gel permeation and chromatofocusing fast performance liquid chromatography (GP-FPLC and C-FPLC) we partially purified a macrophage deactivating factor (MDF) from mitogen-induced goldfish kidney leukocytes. Inhibition of the macrophage-derived nitric oxide (NO) response induced by this MDF was time-, dose- and temperature-dependent. Macrophages pre-treated for 6 or 24 h with MDF before activation with macrophage activating factors (MAF) and/or bacterial lipopolysaccharide (LPS) exhibited a down-regulation in their NO response, while those treated with MDF 24 h after activation with MAF and LPS did not. MDF treatment also impaired the NO response of goldfish macrophages infected with the mammalian protozoan parasite Leishmania major. These results suggest that MDF exhibits its inhibitory effect downstream of the converging intracellular pathways induced by LPS and/or L. major. The novel teleost MDF has an approximate Mr of 15 kD and a pI < 4, and is the first endogenous molecule of teleosts known to down regulate macrophage antimicrobial responses.

Antimicrobial mechanisms of fish phagocytes and their role in host defense.

Phagocytosis is a primitive defense mechanism in all multicellular animals. Phagocytes such as macrophages and neutrophils play an important role in limiting the dissemination of infectious agents, and are responsible for the eventual destruction of phagocytosed pathogens. These cells have evolved elaborate killing mechanisms for destroying pathogens. In addition to their repertoire of degradative enzymes and antimicrobial peptides, macrophages and neutrophils can be activated to produce a number of highly toxic molecules. Production of reactive oxygen and nitrogen intermediates by these cells are potent cytotoxic mechanisms against bacteria and protozoan pathogens. Studies in fish suggest that the biological basis of these inducible killing mechanisms is similar to those described in mammals. More recent work suggest novel roles for regulating these killing responses in fish. In this review, we describe the biological basis of these killing mechanisms and how they are regulated in fish.

Nucleic acid stains as indicators of Cryptosporidium parvum oocyst viability.

We developed nucleic acid dye staining methodology for untreated, heat-treated and chemically inactivated C. parvum oocysts. The nucleic acid staining was compared to in vitro excystation and animal infectivity using split samples of oocysts. Among the nucleic acid stains tested, SYTO-9, hexidium and SYTO-59 stained the oocysts consistently, and the staining was related to the infectivity of the oocysts to neonatal CD-1 mice but not to in vitro excystation. The nucleic acid viability assay was used to determine log-inactivations of the oocysts after treatment with ozone, chlorine, chlorine dioxide and combinations of different chemical disinfectants, and was found to indicate log-inactivation levels similar to that of animal infectivity. A combined immunofluorescence-nucleic acid staining assay was developed for the oocysts of C. parvum and this assay will be invaluable for the detection and viability of oocysts in the laboratory and in environmental samples.

Intact Cryptosporidium parvum oocysts isolated after in vitro excystation are infectious to neonatal mice.

In vitro excystation is often used as a measure of viability of encysted protozoan parasites. Parasites that do not excyst in vitro are assumed to be non-viable and non-infectious, whereas those that do excyst are assumed viable. To test the validity of these assumptions, Cryptosporidium parvum oocysts were excysted in vitro using two different excystation protocols, and the non-excysted intact oocysts were isolated using flow cytometry. Non-excysted sorted oocysts readily infected neonatal CD-1 mice. Increasing the duration of the excystation assays from 1 h to 3 h resulted in a higher percent of excysted oocysts, but the remaining non-excysted parasites were still capable of infecting neonatal CD-1 mice. Our results suggest that in vitro excystation is not an accurate measure of the viability or infectious potential of C. parvum oocysts.

Studies on the resistance/reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts exposed to medium-pressure ultraviolet radiation.

The ex vivo and in vivo reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts after exposure to different doses of ultraviolet (UV) radiation was determined using animal infectivity. The infectivity of UV-treated parasites stored for 1-4 days (G. muris) or 1-17 days (C. parvum) at room temperature in the dark was similar to that of organisms administered immediately after UV treatment, indicating that the parasites did not reactivate ex vivo. In contrast, we observed in vivo reactivation of G. muris in three of seven independent animal infectivity experiments, when parasites were treated with relatively low doses of medium-pressure UV (<25 mJ/cm(2)). Our observations indicate that G. muris cysts and C. parvum oocysts exposed to medium-pressure UV doses of 60 mJ/cm(2) or higher did not exhibit resistance to and/or reactivation following treatment. This suggests that when appropriate doses of UV are used, significant and permanent inactivation of these parasites may be achieved.

Antigens of adults and third-stage larvae of the meningeal worm, Parelaphostrongylus tenuis (Nematoda, Metastrongyloidea).

We studied the antigens of adults and third-stage larvae of the meningeal worm, Parelaphostrongylus tenuis, in an attempt to identify potential serodiagnostic molecules for this important infection of wild ungulates. Soluble extracts of P. tenuis adult worms and third-stage larvae were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional (2D) electrophoresis and analyzed by immunoblotting using purified rabbit anti-P. tenuis immunoglobulin G (IgG). The IgG antibodies were obtained from animals immunized with P. tenuis adult worm or third-stage larva soluble extract and serum from elk infected with P. tenuis. Out of more than 75 antigens (as shown by 2D electrophoresis and immunoblotting), 7 antigens from adults (four 170-120-kD molecules with isoelectric points between 6.0 and 6.6, two 55-kD molecules with isoelectric points of 5.6 and 5.8, and one 13-kD molecule) and 2 antigens from third-stage larvae (one 25-30-kD molecule with an isoelectric point of 6.3 and one 13-kD molecule) distinguished P. tenuis from two other nematodes, Dictyocaulus viviparus and Trichinella spiralis. Initial results using serum from experimentally infected elk indicate that this serum recognized a similar profile of P. tenuis antigens when compared with the serum from immunized rabbits. This research has set the foundation for the development of a test for P. tenuis infections in wild and recently domesticated elk and and other ungulates.

Meningeal worm evokes a heterogeneous immune response in elk.

Meningeal worm (Parelaphostrongylus tenuis) is a neurotropic nematode of ungulates in eastern North America. Lack of an effective diagnostic test increases the concern of translocating potentially infected ungulates into western North America, where P. tenuis does not occur naturally. In an attempt to identify serodiagnostic molecules, we determined (1) whether elk (Cervus elaphus) experimentally infected with P. tenuis produce antibodies against infective larvae or adult worms, and (2) if sera consistently recognize antigens that distinguish P. tenuis from a common nematode parasite of elk, the lungworm Dictyocaulus viviparus. Each of 10 elk were exposed to 15 or 300 infective P. tenuis larvae. Serum was collected (0, 41, and 83 days post-exposure and at necropsy) and monitored for antibodies using the enzyme-linked immunosorbent assay (ELISA) and immunoblot. When reactivity of sera with larval P. tenuis protein was compared (day 0 versus 83), ELISA values were significantly higher on day 83 for elk exposed to 15 or 300 parasites. Likewise, ELISA values using protein of adult P. tenuis were higher for elk exposed to 300 larvae. Immunoblots showed that sera from elk, with adult worms in the central nervous system, consistently recognized the 25-27, 28-30, and 34-36 kDa antigens of infective larvae after 83 days. However, many D. viviparus molecules were found to cross-react with antibodies formed against meningeal worm antigens. Use of adult worm proteins for serodiagnosis appears limited, because no protein was consistently recognized by sera collected from elk exposed to 15 larvae. We believe that development of a reliable diagnostic test for meningeal worm requires more research addressing cross-reactivity and detection of P. tenuis during the incubation stage.

Associations among pathogenic bacteria, parasites, and environmental and land use factors in multiple mixed-use watersheds.

Over a five year period (2004-08), 1171 surface water samples were collected from up to 24 sampling locations representing a wide range of stream orders, in a river basin in eastern Ontario, Canada. Water was analyzed for Cryptosporidium oocysts and Giardia cyst densities, the presence of Salmonella enterica subspecies enterica, Campylobacter spp., Listeria monocytogenes, and Escherichia coli O157:H7. The study objective was to explore associations among pathogen densities/occurrence and objectively defined land use, weather, hydrologic, and water quality variables using CART (Classification and Regression Tree) and binary logistical regression techniques. E. coli O157:H7 detections were infrequent, but detections were related to upstream livestock pasture density; 20% of the detections were located where cattle have access to the watercourses. The ratio of detections:non-detections for Campylobacter spp. was relatively higher (>1) when mean air temperatures were 6% below mean study period temperature values (relatively cooler periods). Cooler water temperatures, which can promote bacteria survival and represent times when land applications of manure typically occur (spring and fall), may have promoted increased frequency of Campylobacter spp. Fifty-nine percent of all Salmonella spp. detections occurred when river discharge on a branch of the river system of Shreve stream order = 9550 was >83 percentile. Hydrological events that promote off farm/off field/in stream transport must manifest themselves in order for detection of Salmonella spp. to occur in surface water in this region. Fifty seven percent of L. monocytogenes detections occurred in spring, relative to other seasons. It was speculated that a combination of winter livestock housing, silage feeding during winter, and spring application of manure that accrued during winter, contributed to elevated occurrences of this pathogen in spring. Cryptosporidium and Giardia oocyst and cyst densities were, overall, positively associated with surface water discharge, and negatively associated with air/water temperature during spring-summer-fall. Yet, some of the highest Cryptosporidium oocyst densities were associated with low discharge conditions on smaller order streams, suggesting wildlife as a contributing fecal source. Fifty six percent of all detections of ≥ 2 bacteria pathogens (including Campylobacter spp., Salmonella spp., and E. coli O157:H7) in water was associated with lower water temperatures (<∼ 14 °C; primarily spring and fall) and when total rainfall the week prior to sampling was >∼ 27 mm (62 percentile). During higher water temperatures (>∼ 14 °C), a higher amount of weekly rainfall was necessary to promote detection of ≥ 2 pathogens (primarily summer; weekly rainfall ∼>42 mm (>77 percentile); 15% of all ≥ 2 detections). Less rainfall may have been necessary to mobilize pathogens from adjacent land, and/or in stream sediments, during cooler water conditions; as these are times when manures are applied to fields in the area, and soil water contents and water table depths are relatively higher. Season, stream order, turbidity, mean daily temperature, surface water discharge, cropland coverage, and nearest upstream distance to a barn and pasture were variables that were relatively strong and recurrent with regard to discriminating pathogen presence and absence, and parasite densities in surface water in the region.

Agroenvironmental determinants associated with the presence of antimicrobial-resistant Escherichia coli in beach waters in Quebec, Canada.

Exposure to microorganisms resistant to antimicrobials may constitute a health risk to human populations. It is believed that one route of exposure occurs when people engage in recreational activities in water contaminated with these microorganisms. The main objective of this study was to explore population-level and environmental determinants specifically associated with the presence of antimicrobial resistant (AMR) generic Escherichia coli isolated from recreational waters sampled from beaches located in southern Quebec, Canada. Water samples originated from the Quebec provincial beach surveillance program for the summers of 2004 and 2005. This study focused on three classes of determinants, namely: agricultural, population-level and beach characteristics for a total of 19 specific factors. The study was designed as a retrospective observational analysis and factors were assessed using logistic regression methods. From the multivariable analysis, the data suggested that the percentage of land used for spreading liquid manure was a significant factor associated with the presence of AMR E. coli (OR=27.73). Conceptually, broad factors potentially influencing the presence of AMR bacteria in water must be assessed specifically in addition to factors associated with general microbial contamination. Presence of AMR E. coli in recreational waters from beaches in southern Quebec may represent a risk for people engaging in water activities and this study provides preliminary evidence that agricultural practices, specifically spreading liquid manure in agricultural lands nearby beaches, may be linked to the contamination of these waters by AMR E. coli.

Generation and functional analysis of distinct macrophage sub-populations from goldfish (Carassius auratus L.) kidney leukocyte cultures.

Three distinct sub-populations of macrophages derived from goldfish kidney leukocyte cultures were generated and characterised. The sub-populations designated as R1, R2 and R3-type macrophages had distinct morphological, cytochemical and flow cytometric profiles, and also differed in their anti-microbial functions after activation with macrophage activation factors (MAF) and bacterial lipopolysaccharide (LPS). The R1-type macrophages were small cells that contained acid phosphatase, but lacked myeloperoxidase and non-specific esterase. The R2-type macrophages were morphologically similar to mature tissue macrophages of mammals, and were positive for acid phosphatase, myeloperoxidase and non-specific esterase. The R3-type macrophages were round cells with eccentrically placed nuclei and resembled mammalian monocytes. This sub-population stained for acid phosphatase, myeloperoxidase and non-specific esterase. The R2 and R3-type macrophages exhibited distinct functional responses after activation with MAF and/or LPS. R2-type macrophages were potent producers of nitric oxide, while R3-type macrophages produced little or no nitric oxide after activation with MAF and LPS. The R2 and R3-type macrophages also exhibited unique respiratory burst responses (ROI) after treatment with MAF and/or LPS. After treatment with MAF and LPS, activated R2 macrophages were primed for ROI after only 6 h of stimulation with the activating agents, and continued to exhibit a strong ROI response for an extended cultivation period (48 h). In contrast, activated R3-type macrophages showed an early ROI response (6 h after treatment with MAF and LPS), which decreased significantly by 48 h after treatment with the activating agents. Our results suggest that the analysis of the mechanisms of induction of fish anti-microbial responses may be dependent upon the concerted actions of functionally distinct macrophage sub-populations.

Biochemical and functional characterisation of macrophage stimulating factors secreted by mitogen-induced goldfish kidney leucocytes.

Mitogen-stimulated goldfish kidney leucocytes secrete a number of different macrophage activation factors (MAF) that induce profound physiological changes in macrophages. MAF produced by goldfish kidney leucocytes was characterised using fast performance liquid chromatography (FPLC) and bioassays that measured MAF-induced respiratory burst (RB) and nitric oxide (NO) responses of activated macrophages. Mitogen-induced fish kidney leucocyte supernatants were fractionated using gel permeation FPLC (GP-FPLC) and the ability of different fractions to induce NO or RB measured. A MAF of M(r) 50 kD, that induced a potent nitric oxide response in both a long-term goldfish macrophage cell line (GMCL) and in in vitro-derived fish kidney macrophages (IVDKM) was identified. The GP-FPLC partially purified 50 kD MAF activity occasionally induced significantly higher nitric oxide production than that of the crude MAF preparations. This increase in the NO-inducing activity was due to segregation of the 50 kD MAF from a novel macrophage deactivating molecule of M(r) 10-12 kD present in crude MAF preparations. This 10-12 kD molecule was shown to inhibit nitric oxide production in cytokine-activated goldfish macrophages. Mitogen-induced fish kidney leucocyte supernatants contained two distinct MAFs that induced the respiratory burst in GMCL and IVDKM: the 50 kD and 30 kD proteins. The partially purified 30 kD MAF primed goldfish macrophage for increased RB activity after only 6 h of treatment, and continued to augment the RB activity after 24 h of stimulation. In contrast, the GP-FPLC partially purified 50 kD molecule also primed the RB after only 6 h of stimulation, but subsequently deprimed the RB after 24 h of stimulation, an effect similar to that observed for crude MAF preparations. The 50 kD MAF activity was further purified using chromatofocusing FPLC (C-FPLC) using basic pH gradients and was shown to consist of two distinct NO-inducing molecules (> pI 9.3). Mitogen-stimulated fish kidney leucocytes secrete several factors that profoundly affect the anti-microbial responses of teleost macrophages and which undoubtedly are responsible for regulating teleost macrophage function in vivo.

Comparison of animal infectivity and nucleic acid staining for assessment of Cryptosporidium parvum viability in water.

Cryptosporidium parvum oocysts were stained with the fluorogenic dyes SYTO-9 and SYTO-59 and sorted by flow cytometry in order to determine whether the fluorescent staining intensity correlated with the ability of oocysts to infect neonatal CD-1 mice. Oocysts that did not fluoresce or that displayed weak fluorescent intensity when stained with SYTO-9 or SYTO-59 readily established infections in mice, whereas those oocysts that fluoresced brightly did not. Although fluorescent staining profiles varied among different batches of oocysts, a relative cutoff in fluorescent staining intensity that correlated with animal infectivity was observed for all batches.