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During ontogeny, macrophages develop from CD115+precursors, including erythro-myeloid progenitors (EMP). EMP arise in the embryonic yolk sac, the primary site of early haematopoiesis. In adults, CD115+bone marrow-derived monocytes represent essential macrophage precursors. Herein, we identify a CD115-macrophage precursor within the adult bone marrow that is unrelated to the classical monocyte lineage but rather shares transcriptomic and functional characteristics of embryonic EMP. These EMPROR (forErythroMyeloidPrecursor) cells are capable of efficiently generating macrophages in disease settings. During early development, EMPROR cells were largely absent from the yolk sac but were instead found at the embryonic-maternal interface in the uterine wall. Unexpectedly, the latter site contains robust haematopoietic activity and harbours defined embryonic haematopoietic progenitor cells, including classical CD115+EMP. Our data suggest the existence of an alternative pathway of macrophage generation in the adult. Further, we uncover a hitherto unknown site of earliest blood cell development.

Objectives: The immunologic events that build up to the fatal neurological stage of experimental cerebral malaria (ECM) are incompletely understood. Here, we dissect immune cell behaviour occurring in the central nervous system (CNS) when Plasmodium berghei ANKA (PbA)-infected mice show only minor clinical signs. Methods: A 2-photon intravital microscopy (2P-IVM) brain imaging model was used to study the spatiotemporal context of early immunological events in situ during ECM. Results: Early in the disease course, antigen-specific CD8+ T cells came in contact and arrested on the endothelium of post-capillary venules. CD8+ T cells typically adhered adjacent to, or were in the near vicinity of, perivascular macrophages (PVMs) that line post-capillary venules. Closer examination revealed that CD8+ T cells crawled along the inner vessel wall towards PVMs that lay on the abluminal side of large post-capillary venules. 'Activity hotspots' in large post-capillary venules were characterised by T-cell localisation, activated morphology and clustering of PVM, increased abutting of post-capillary venules by PVM and augmented monocyte accumulation. In the later stages of infection, when mice exhibited neurological signs, intravascular CD8+ T cells increased in number and changed their behaviour, actively crawling along the endothelium and displaying frequent, short-term interactions with the inner vessel wall at hotspots. Conclusion: Our study suggests an active interaction between PVM and CD8+ T cells occurs across the blood-brain barrier (BBB) in early ECM, which may be the initiating event in the inflammatory cascade leading to BBB alteration and neuropathology.

When nanoparticles interact with human blood, a multitude of plasma components adsorb onto the surface of the nanoparticles, forming a biomolecular corona. Corona composition is known to be influenced by the chemical composition of nanoparticles. In contrast, the possible effects of variations in the human blood proteome between healthy individuals on the formation of the corona and its subsequent interactions with immune cells in blood are unknown. Herein, we prepared and examined a matrix of 11 particles (including organic and inorganic particles of three sizes and five surface chemistries) and plasma samples from 23 healthy donors to form donor-specific biomolecular coronas (personalized coronas) and investigated the impact of the personalized coronas on particle interactions with immune cells in human blood. Among the particles examined, poly(ethylene glycol) (PEG)-coated mesoporous silica (MS) particles, irrespective of particle size (800, 450, or 100 nm in diameter), displayed the widest range (up to 60-fold difference) of donor-dependent variance in immune cell association. In contrast, PEG particles (after MS core removal) of 860, 518, or 133 nm in diameter displayed consistent stealth behavior (negligible cell association), irrespective of plasma donor. For comparison, clinically relevant PEGylated doxorubicin-encapsulated liposomes (Doxil) (74 nm in diameter) showed significant variance in association with monocytes and B cells across all plasma donors studied. An in-depth proteomic analysis of each biomolecular corona studied was performed, and the results were compared against the nanoparticle-blood cell association results, with individual variance in the proteome driving differential association with specific immune cell types. We identified key immunoglobulin and complement proteins that explicitly enriched or depleted within the corona and which strongly correlated with the cell association pattern observed across the 23 donors. This study demonstrates how plasma variance in healthy individuals significantly influences the blood immune cell interactions of nanoparticles.

Serpinb9 (Sb9, also called Spi6) is an intracellular inhibitor of granzyme B (grB) that protects cytotoxic lymphocytes from grB-mediated death. In addition, Sb9 is also expressed in accessory immune cells, including dendritic cells (DCs), although its role is debated. Recently, we have demonstrated that Sb9 plays a grB-independent role in cross-presentation of antigens by CD8+ DCs. Here, using a mouse line expressing green fluorescent protein knocked in under the control of the Sb9 promoter, we demonstrate that Sb9 expression is highest in those tissue-resident and migratory DC subsets capable of cross-presentation. Further, we show that CD8+ DCs can be divided into two subsets based on Sb9 expression, and that only the subset expressing higher levels of Sb9 is capable of cross-presentation. These findings add support for role for Sb9 cross-presentation, and indicate that high Sb9 expression is a novel marker of cross-presentation capable DCs.

The kynurenine pathway of tryptophan metabolism has been implicated in brain function, immunoregulation, anti-microbial mechanisms and pregnancy. Some of these actions are due to depletion of tryptophan and others to the formation of biologically active metabolites. This review focuses on the roles of the kynurenine pathway in host responses during two parasitic diseases of major health and economic importance, malaria and toxoplasmosis, with an emphasis on their impacts on CNS function. This article is part of the Special Issue entitled ‘The Kynurenine Pathway in Health and Disease’.

During pneumococcal meningitis, clearance of bacteria by recruited neutrophils is crucial for host protection. However, these innate immune mechanisms are often insufficient and treatment with antibiotics is necessary to prevent death. Despite this antibiotic treatment, approximately half of all survivors suffer lifelong neurological problems. There is growing evidence indicating the harmful effects of neutrophils on CNS integrity. Therefore, the present study investigated the roles of neutrophils in the acute inflammatory response and the resulting long-term neuropsychological effects in murine pneumococcal meningitis. Long-term behavioural and cognitive functions in mice were measured using an automated IntelliCage system. Neutrophil depletion with antibody 1A8 as adjunctive therapy was shown to remarkably impair survival in meningitic C57BL/6J mice despite antibiotic (ceftriaxone) treatment. This was accompanied by increased bacterial load in the cerebrospinal fluid (CSF) and an increase in IL-1β, but decrease in TNF, within the CSF at 20h after bacterial inoculation. In the longer term, the surviving neutrophil-depleted post-meningitic (PM) mice displayed reduced diurnal hypolocomotion compared to PM mice treated with an isotype antibody. However, they showed nocturnal hyperactivity, and greater learning impairment in a patrolling task that is believed to depend upon an intact hippocampus. The data thus demonstrate two important mechanisms: 1. Neutrophil extravasation into the CNS during pneumococcal meningitis influences the pro-inflammatory response and is central to control of the bacterial load, an increase in which may lead to death. 2. Neutrophil-mediated changes in the acute inflammatory response modulate the neuropsychological sequelae in mice that survive pneumococcal meningitis.

Gamma interferon (IFN-γ) drives antiparasite responses and immunopathology during infection with Plasmodium species. Immunity-related GTPases (IRGs) are a class of IFN-γ-dependent proteins that are essential for cell autonomous immunity to numerous intracellular pathogens. However, it is currently unknown whether IRGs modulate responses during malaria. We have used the Plasmodium berghei ANKA (PbA) model in which mice develop experimental cerebral malaria (ECM) to study the roles of IRGM1 and IRGM3 in immunopathology. Induction of mRNA for Irgm1 and Irgm3 was found in the brains and spleens of infected mice at times of peak IFN-γ production. Irgm3 −/− but not Irgm1 −/− mice were completely protected from the development of ECM, and this protection was associated with the decreased induction of inflammatory cytokines, as well as decreased recruitment and activation of CD8 + T cells within the brain. Although antigen-specific proliferation of transferred CD8 + T cells was not diminished compared to that of wild-type recipients following PbA infection, T cells transferred into Irgm3 −/− recipients showed a striking impairment of effector differentiation. Decreased induction of several inflammatory cytokines and chemokines (interleukin-6, CCL2, CCL3, and CCL4), as well as enhanced mRNA expression of type-I IFNs, was found in the spleens of Irgm3 −/− mice at day 4 postinfection. Together, these data suggest that protection from ECM pathology in Irgm3 −/− mice occurs due to impaired generation of CD8 + effector function. This defect is nonintrinsic to CD8 + T cells. Instead, diminished T cell responses most likely result from defective initiation of inflammatory responses in myeloid cells.

A study was conducted to compare the effects of two chemical therapeutants on channel catfish (CCF) Ictalurus punctatus concurrently infected with Flavobacterium columnare and Ichthyobodo necator. Copper sulfate (CuSO4) and potassium permanganate (KMnO4) were investigated for their ability to reduce the bacterial load, parasite load, and subsequent mortality. Treatment rates of CuSO4 or KMnO4 were 2.1 mg/L and 3.0 mg/L, respectively, and were applied at 24 h intervals on three consecutive days and control fish were untreated. Fin and gill samples were taken on day 4 (24 h after the final treatment) and day 10 (one week after the final treatment) for quantification of parasite and bacterial load. The survival rate of CuSO4-treated fish (73.0 %) was significantly different from the untreated control fish (41.5%). KMnO4-treated fish was (53.6%) and not significantly different from untreated control or CuSO4-treated fish. I. necator loads were significantly reduced by both CuSO4 and KMnO4, but only CuSO4 sign...

Cerebral malaria (CM) is a severe complication of and a leading cause of death due toPlasmodium falciparuminfection. CM is likely the result of interrelated events, including mechanical obstruction due to parasite sequestration in the microvasculature, and upregulation of Th1 immune responses. In parallel, blood-brain-barrier (BBB) breakdown and damage or death of microglia, astrocytes, and neurons occurs. We found that a novel formulation of a liposome-encapsulated glucocorticosteroid,β-methasone hemisuccinate (nSSL-BMS), prevents experimental cerebral malaria (ECM) in a murine model and creates a survival time-window, enabling administration of an antiplasmodial drug before severe anemia develops. nSSL-BMS treatment leads to lower levels of cerebral inflammation, expressed by altered levels of corresponding cytokines and chemokines. The results indicate the role of integrated immune responses in ECM induction and show that the new steroidal nanodrug nSSL-BMS reverses the balance between the Th1 and Th2 responses in malaria-infected mice so that the proinflammatory processes leading to ECM are prevented. Overall, because of the immunopathological nature of CM, combined immunomodulator/antiplasmodial treatment should be considered for prevention/treatment of human CM and long-term cognitive damage.

As a synthetic androgen, 17α-methyltestosterone (17MT) is frequently used to redirect the course of sex differentiation by exposing the undifferentiated gonad to a sufficient dosage. This hormone has been widely accepted as a safe and effective treatment for sex reversal in many fish species, and it is administered to larval tilapia (3–12 d old) for ∼28 consecutive days to produce populations of >90% males. This study assessed the safety of 17MT-treated feed when administered to larval Nile Tilapia Oreochromis niloticus at one, three, and five times (i.e., 1×, 3×, and 5×) the proposed dosage of 9 mg 17MT/kg fish daily for 28 consecutive days. Despite elevated total ammonia nitrogen levels measured during the last 5 d of the study, environmental conditions were acceptable for rearing tilapia. Fish fed aggressively regardless of the concentration of 17MT in the feed, behavior was considered normal with no dose-related differences detected, and no mortality was observed in the 3× treatment group. Fi...

Concurrent ectoparasite infections on fishes are commonly encountered in the wild and in aquaculture (Rohde 1984; Hoffman 1999), for example, infections of both Ichthyophthirius multifiliis Fouquet, 1876 (Hymenostomatida: Ichthyopththiriidae; Colorni 2008) and gill‐infecting monogenoids (Platyhelminthes: Monogenoidea; Whittington and Chisholm 2008). The decision to aggressively treat infections of either of these parasites can be prudent in aquaculture systems. Both taxa have direct life cycles and short generation times, which may result in high infection intensities that can debilitate or kill the fish host (Noga 2010). Various chemical therapies have been evaluated to prevent, reduce, or eliminate such infections. Typical treatments for killing monogenoids include: freshwater or saltwater baths, formalin, copper sulfate (CuSO4), hydrogen peroxide, mebendazole, trichlorphon, and praziquantel (Whittington and Chisholm 2008). Treatments for infections of I. multifiliis (Ich) include: temporary exposure to high temperature, formalin, malachite green, CuSO4, potassium permanganate, and sodium chloride (Colorni 2008). Despite the common occurrence of these parasites on fish, little species‐specific information exists for the vast majority of fish–parasite combinations across the diversity of culture systems and captive settings. Formalin is approved by the US Food and Drug Administration (US FDA) for use as a treatment for external monogenetic trematodes in fish at up to 250 mg/L for up to 1 h (US FWS/AFS 2008). The ambiguity of this recommendation suggests a wide therapeutic range with no interpretation of repeated applications. Likewise, little information exists on the safety or efficacy of repeated formalin treatments on fish, and CuSO4 has an indeterminate approval status but is widely accepted as having high efficacy. In October 2009, white bass, Morone chrysops (Rafinesque, 1820; Perciformes: Moronidae), in a flow‐through culture system were diagnosed with concurrent infections of the gill‐infecting monogenoid Onchocleidus mimus (Mueller, 1936; Monogenoidea: Ancyrocephalidae) and Ich. O. mimus has a direct life cycle with no intermediate host required and as the intensity of infection increases can become problematic to a population. This study reports an opportunistic evaluation of the efficacy of repeated applications of formalin and CuSO4 to reduce, control, or eradicate these infections in cultured white bass.

The complete extent to which the human genome is translated into polypeptides is of fundamental importance. We report a peptidomic strategy to detect short open reading frame (sORF)-encoded polypeptides (SEPs) in human cells. We identify 90 SEPs, 86 of which are previously uncharacterized, which is the largest number of human SEPs ever reported. SEP abundances range from 10-1,000 molecules per cell, identical to abundances of known proteins. SEPs arise from sORFs in noncoding RNAs as well as multicistronic mRNAs, and many SEPs initiate with non-AUG start codons, indicating that noncanonical translation may be more widespread in mammals than previously thought. In addition, coding sORFs are present in a small fraction (8 out of 1,866) of long intergenic noncoding RNAs. Together, these results provide strong evidence that the human proteome is more complex than previously appreciated.

The pathology associated with Streptococcus pneumoniae meningitis results largely from activation of immune-associated pathways. We systematically investigated the production of IFN subtypes, as well as their influence on pathology, in a mouse model of S. pneumoniae meningitis. Despite the occurrence of a mixed IFN type I/II gene signature, no evidence for production or involvement of type I IFNs in disease progression was found. In contrast, type II IFN (IFN-γ) was strongly induced, and IFN-γ−/− mice were significantly protected from severe disease. Using intracellular cytokine staining and targeted cell-depletion approaches, NK cells were found to be the dominant source of IFN-γ. Furthermore, production of IFN-γ was found to be dependent upon ASC and IL-18, indicating that an ASC-dependent inflammasome pathway was responsible for mediating IFN-γ induction. The influence of IFN-γ gene deletion on a range of processes known to be involved in bacterial meningitis pathogenesis was examined. Although neutrophil numbers in the brain were similar in infected wild-type and IFN-γ−/− mice, both monocyte recruitment and CCL2 production were less in infected IFN-γ−/− mice compared with infected wild-type controls. Additionally, gene expression of NO synthase was strongly diminished in infected IFN-γ−/− mice compared with infected controls. Finally, bacterial clearance was enhanced in IFN-γ−/− mice, although the underlying mechanism remains unclear. Together, these data suggest that inflammasome-dependent IFN-γ contributes via multiple pathways to pathology during S. pneumoniae meningitis.

Peracetic acid (PAA) is a relatively new compound suggested for use to treat pathogens in aquaculture. It is approved for use in Europe, but not in the United States. This study determined the effectiveness of PAA for fungus control on channel catfish, Ictalurus punctatus (Rafinesque), eggs. The study consisted of five PAA concentrations (2.5, 5, 10, 15 and 20 mg L)1) and an untreated control in a flowthrough system. A single spawn was used for each replication (N = 4). Eggs were treated twice daily until the embryos developed eyes. When hatching was complete for all viable eggs, fry were counted to determine the percent survival in each treatment. Fungal growth was severe in the untreated controls resulting in 11% survival. Treatments of 2.5, 5 and 10 mg L)1 PAA were significantly different from the controls (P < 0.05). The highest percent survival of hatched fry was with 5 mg L)1 PAA administered twice daily; the 2.5 mg L)1 PAA treatment had slightly less survival, but gives a higher margin of safety in case of treatment error. Very little fungus was present in treatments receiving 2.5 mg L)1 PAA or higher, and concentrations of 15 and 20 mg L)1 PAA were toxic to the eggs. The mean survivals in the 0, 2.5, 5, 10, 15 and 20 mg L)1 PAA treatments were 11%, 60%, 63%, 62%, 32% and 0%, respectively. Therefore, PAA may be a compound that merits further investigations regarding its use in U.S. aquaculture.

Ichthyobodo necator is a single-celled biflagellate parasite, which in high density can cause significant mortality in young fish. Copper sulphate (CuSO4), potassium permanganate (KMnO4) and peracetic acid (PAA) were evaluated for effectiveness against ichthyobodosis. Treatments were: untreated control, 2.1 mg L−1CuSO4, 3.0 mg L−1 KMnO4, 1.5 mg L−1 PAA and 3.0 mg L−1 PAA, and were applied to flow-through tanks on three consecutive days. The study was designed to simulate the flow-through systems utilized in the commercial rearing of juvenile channel catfish (Ictalurus punctatus). Mortality was monitored daily to compare survival rate among treatments. Parasite intensity was assessed pre chemical exposure and 20–24 h after the third application to determine effectiveness of the treatment. An assessment was also done 7 days post application to investigate possible reoccurrence. Copper sulphate, KMnO4 and PAA (3.0 mg L−1) significantly reduced the infestation rate of I. necator. Copper sulphate significantly improved the survival of I. necator infested channel catfish after three flow-through applications compared with the untreated control. The 3.0 mg L−1 PAA resulted in significantly lower survival than the untreated control, the 1.5 mg L−1 PAA and the KMnO4 were not statistically different from the untreated control.

Copper sulfate (CuSO4) is used in the catfish industry to control saprolegniasis (caused by the watermolds Achlya spp. or Saprolegnia spp.) on eggs. This study was designed to establish the margin of safety of CuSO4 at 10, 30, and 50 mg/L when applied for three times the normal treatment duration to hatching troughs containing channel catfish Ictalurus punctatus eggs in 26°C, flow-through well water. The safety of CuSO4 was indicated by the percentage of fry that hatched from the eggs. Eggs were treated daily until the embryos reached the eyed stage. The least-squares means ± SE of hatch rates were 40.8 ± 9.1, 80.9 ± 6.4, 64.2 ± 8.6, and 80.3 ± 6.6% for the 0-, 10-, 30-, and 50-mg/L CuSO4 treatments, respectively. The hatch rate for the 0-mg/L CuSO4 treatment was significantly different from that of all other CuSO4 treatments. Treating channel catfish eggs with five times the recommended dose of 10-mg/L CuSO4 for three times the recommended duration did not adversely affect the hatch rates in thi...

Aquaflor (florfenicol, 50% type A medicated article) is a relatively new antibiotic used in U.S. aquaculture and has been widely accepted as a safe and effective therapeutant. Some peer-reviewed studies have suggested that 15 mg florfenicol (FFC)·kg−1 body weight (BW)·d−1 for 10 d controls mortality to a greater extent than 10 mg FFC·kg−1 BW·d−1 for 10 d. This study evaluated the safety of Aquaflor to sunshine bass (female white bass Morone chrysops × male striped bass M. saxatilis) when administered in feed at 15 (1× the maximum proposed therapeutic dose), 45 (3×), and 75 (5×) mg FFC·kg−1 BW·d−1 for 20 d (2× the currently approved 10-d treatment duration). The medicated feed was top-coated with Aquaflor and fed at 2% BW·d−1 divided equally between the morning and afternoon feedings. Juvenile sunshine bass (13.6 ± 1.6 g [mean ± SD]) were stocked into 100-L flow-through tanks at 20 fish per tank. Diets were randomly assigned to three replicate tanks per treatment; fish in three additional nonstudy...

Ammonia concentrations in water can affect the severity of Flavobacterium columnare infections in fish. Two trials lasting 7 d each were conducted to determine the effect of a single immersion flush treatment of total ammonia nitrogen (TAN; 15 mg/L) on the survival of channel catfish Ictalurus punctatus infected with F. columnare; the chemical was added while the water flowed continuously through the tanks. Both trials consisted of four treatments: (1) no ammonia exposure and no bacterial challenge (control), (2) ammonia exposure only, (3) bacterial challenge only, and (4) both ammonia exposure and bacterial challenge. Two hours after exposure to ammonia, the highest un-ionized ammonia level was 0.43 mg/L. The percent un-ionized ammonia is based on TAN, temperature, and pH. Caudal fins from three fish in each treatment were sampled at 24 h posttreatment to be analyzed by quantitative real-time polymerase chain reaction (qPCR). No significant difference in survival (mean ± SE) was noted between th...

This study at a commercial hatchery was required by the U.S. Food and Drug Administration to provide independent substantiation of the results of previous laboratory dose-confirmation studies on the use of copper sulfate (CuSO4) to control fungus (Saprolegnia spp.) on the eggs of channel catfish, Ictalurus punctatus. The study compared an untreated control group of eggs to eggs treated with 10 mg/L CuSO4 in a flow-through system; mean water temperature was 23.5°C. Eggs were treated once daily until the embryos reached the eyed stage (5 treatments). Hatching was complete by day 11, and fry were counted to determine the percentage of survival in each treatment. Fungus was identified by polymerase chain reaction (PCR) as Saprolegnia spp. The mean survival in the control treatments was 4% and 40% in the CuSO4 treatments; the latter survival was significantly higher, but still lower than normal. This study confirms that 10 mg/L CuSO4 is an effective treatment to control fungus on catfish eggs when used daily u...

MicroRNAs (miRNAs) are posttranscriptional regulatory molecules that have been implicated in the regulation of immune responses, but their role in the immune response to Plasmodium infection is unknown. We studied the expression of selected miRNAs following infection of CBA mice with Plasmodium berghei ANKA (PbA), which causes cerebral malaria (CM), or Plasmodium berghei K173 (PbK), which causes severe malaria but without cerebral complications, termed non-CM. The differential expression profiles of selected miRNAs (let-7i, miR-27a, miR-150, miR-126, miR-210, and miR-155) were analyzed in mouse brain and heart tissue by quantitative reverse transcription-PCR (qRT-PCR). We identified three miRNAs that were differentially expressed in the brain of PbA-infected CBA mice: let7i, miR-27a, and miR-150. In contrast, no miRNA changes were detected in the heart, an organ with no known pathology during acute malaria. To investigate the involvement of let-7i, miR-27a, and miR-150 in CM-resistant mice, we assessed the expression levels in gamma interferon knockout (IFN-γ −/− ) mice on a C57BL/6 genetic background. The expression of let-7i, miR-27a, and miR-150 was unchanged in both wild-type (WT) and IFN-γ −/− mice following infection. Overexpression of these three miRNAs during PbA, but not PbK, infection in WT mice may be critical for the triggering of the neurological syndrome via regulation of their potential downstream targets. Our data suggest that in the CBA mouse at least, miRNA may have a regulatory role in the pathogenesis of severe malaria.

Catfish hatcheries use copper sulphate pentahydrate (CuSO4·5H2O) as an economical control for saprolegniasis on eggs. This study determines hatch rate of channel catfish, Ictalurus punctatus (Rafinesque 1818), eggs in hatching troughs containing 23.8 °C flow-through well water when treated with 100 mg L−1 CuSO4·5H2O (10 times the proposed therapeutic dose). Eggs were treated daily until the embryos reached the eyed stage. Fry survival in the control and 100 mg L−1 CuSO4·5H2O treatments was significantly different (15% and 71% respectively). This study demonstrates that there is a considerable margin of safety in using CuSO4·5H2O as a catfish egg treatment to control saprolegniasis.

Streptococcus pneumoniae is a major meningitis-causing pathogen globally, bringing about significant morbidity and mortality, as well as long-term neurological sequelae in almost half of the survivors. Subsequent to nasopharyngeal colonisation and systemic invasion, translocation across the blood‒brain barrier (BBB) by S. pneumoniae is a crucial early step in the pathogenesis of meningitis. The BBB, which normally protects the central nervous system (CNS) from deleterious molecules within the circulation, becomes dysfunctional in S. pneumoniae invasion due to the effects of pneumococcal toxins and a heightened host inflammatory environment of cytokines, chemokines and reactive oxygen species intracranially. The bacteria‒host interplay within the CNS likely determines not only the degree of BBB pathological changes, but also host survival and the extent of neurological damage. This review explores the relationship between S. pneumoniae bacteria and the host inflammatory response, with an emphasis on the BBB and its roles in CNS protection, as well as both the acute and long-term pathogenesis of meningitis.

Graphical abstract, Lung complications during malaria infection can range from coughs and impairments in gas transfer to the development of acute respiratory distress syndrome (ARDS). Infecting C57BL/6 mice with Plasmodium berghei K173 strain (PbK) resulted in pulmonary oedema, capillaries congested with leukocytes and infected red blood cells (iRBCs), and leukocyte infiltration into the lungs. This new model of malaria-associated lung pathology, without any accompanying cerebral complications, allows the investigation of mechanisms leading to the lung disease. The activity of the amiloride-sensitive epithelial sodium channel (ENaC) in alveolar epithelial cells is decreased by several respiratory tract pathogens and this is suggested to contribute to pulmonary oedema. We show that PbK, a pathogen that remains in the circulation, also decreased the activity and expression of ENaC, suggesting that infectious agents can have indirect effects on ENaC activity in lung epithelial cells. The reduced ENaC activity may contribute to the pulmonary oedema induced by PbK malaria.

Reduced survival of fish eggs is often a result of infestation with fungi Saprolegnia spp. However, timely chemical treatments often limit these infestations and increase survival. The effect of copper sulfate pentahydrate (CSP; 10 mg of CSP/L of water), diquat bromide (25 mg of diquat cation/L), formalin (433 mg/L), and hydrogen peroxide (H2O2; 250 mg/L) on percent hatch and fungal infestation in eggs of channel catfish Ictalurus punctatus was compared in two identical trials (five replicates for each chemical treatment per trial). The percent hatch in all chemical treatments was significantly better than percent hatch of the controls. The amount of fungal coverage on egg masses treated with CSP, formalin, and H2O2 was significantly less than that observed on the controls. Although not statistically different from the other treatments, H2O2 at 250 mg/L exhibited the highest average percent hatch (64%; control hatch = 34%), the lowest fungal coverage (1.5 cm; control coverage = 7.2 cm), and the l...

Two dose confirmation studies are required by the U.S. Food and Drug Administration to verify the effectiveness of a candidate before approval as a new animal drug is awarded; the two studies provide independent substantiation of the results. This laboratory study was designed to compare an untreated control and a 10-mg/L copper sulfate (CuSO4) treatment to control fungus (Saprolegnia spp.) on eggs of channel catfish Ictalurus punctatus at 23.5°C in a flow-through system. The eggs were treated once daily until the embryos reached the eyed stage (five treatments). When hatching was complete for all viable eggs (day 10), the fry were counted to determine the percent survival in each treatment. Infestation by a fungus identified morphologically and by polymerase chain reaction as Saprolegnia spp. was severe in the control fish, resulting in 8% survival. The mean percent survival of fry hatched from the CuSO4-treated eggs was significantly higher (mean, 55%; range, 27–71%).

The free-living infective theront of Ichthyophthirius multifiliis historically has been thought to be the only stage susceptible to treatment. Here we introduce a technique to determine the toxicity of compounds to the newly released tomont, the encysted tomont and the developing tomites within the tomont that emerge as theronts. The toxicity of Wofasteril E400 (40% peracetic acid, PAA) to free-living forms of I. multifiliis was determined shortly after tomonts were physically removed from the surface of the fish and at 2.5 and 24 h after removal. Results indicate that 0.6 to 0.9 mg l(-1) PAA killed 39 to 82% of the newly released tomonts within 48 h when treated immediately. In a second experiment, tomonts were allowed to settle for 2.5 h after sampling from the skin and then treated for 12 h; concentrations > or =0.5 mg l(-1) PAA produced significantly fewer theronts than the controls. In a third experiment, encysted tomonts that were exposed to PAA 24 h after sampling from the skin and treated for 2 or 4 h produced a variable amount of theronts, but the concentrations tested (0.5 to 3.0 mg l(-1)) did not halt theront production. This research demonstrates that encysted I. multifiliis are less susceptible to chemical treatments.

Macrophages are central players in both lipid metabolism and innate immunity. Their determinant role in the pathogenesis of atherosclerosis is under the control of the ATP-binding cassette transporter (ABCA1), which by minimizing cellular lipid content, limits development of pro-inflammatory foam cells. Considering the differential contribution of monocyte subsets to the generation of vascular lesions we analyzed the immunophenotype of ABCA1-expressing cells in the myeloid lineage, by the combined use of flow cytometry and real-time quantitative RT-PCR. ABCA1 expression is limited to "non-inflammatory" Ly6C(lo) circulating monocytes and tissue-resident macrophages expressing markers of alternative activation. In ABCA1(-/-) peritoneal macrophages the transcriptional programs induced by LPS/IFN-gamma or IL-4 cytokines are altered and deviated phosphorylation patterns of STAT transcriptional regulators in response to stimuli are observed.

Fungal infestations of channel catfish Ictalurus punctatus eggs can lower hatch rate, which requires the producer to spawn more channel catfish or risk fingerling shortages. Hydrogen peroxide (H2O2) treatments (0.0, 15.6, 31.3, 62.5, 125, 250, 500, 1,000, 2,000, and 4,000 mg/L) were evaluated to determine their effect on channel catfish hatch rate (number of fry/number of eggs × 100) and control of naturally occurring infestations of fungi Saprolegnia spp. in an experimental compartmentalized trough hatching system. Experiments were run at water temperatures of 23.2–24.0°C, dissolved oxygen concentrations of 4.5–6.8 mg/L (53–79% saturation), total alkalinity of 209–217 mg/L, and total hardness of 91–110 mg/L. Treatments were applied to hatching compartments while well water was flowing at a rate of one water exchange every 30 min and egg density was 2.5 g eggs/L of water. Observations for fungal development were made daily, and the maximum measure of fungal growth was recorded. An initial range-f...

Ice-water and salt treatments were evaluated for disinfection of small immersible fisheries equipment contaminated with a nonindigenous tropical snail, the red-rim melania Melanoides tuberculatus. This introduced species can displace native snails and transmit trematodes directly to fish and indirectly to other animals, including humans. The red-rim melania has a well-developed operculum that protects it from desiccation and allows it to remain viable for days on dry fisheries equipment. Treatments were produced by adding 10 kg of salt and 33.3 kg of ice to 66.6 L of water (salt–ice-water [SIW]) or by adding 40 kg of ice to 32 L of water (ice-water only [IW]) and were tested for various periods (0.17 min to 24 h) to find exposures that would kill 100% of the treated snails. Temperatures produced in the test containers ranged from −6.3°C to −2.4°C for SIW and from 0°C to 4.9°C for IW. The survival of snails in saltwater-only (SW) treatments (10 kg of NaCl in 100 L of water) was also tested. Three ...

The biflagellated, single-celled parasite Ichthyobodo necator can cause significant losses among fish populations, particularly those cultured in tanks. Treatments of KMnO4 and CuSO4 were evaluated against a naturally occurring I. necator infestation on sunshine bass (female white bass Morone chrysops x male striped bass M. saxatilis) raised in tanks. Four-hour static treatments with 3 mg of KMnO4/L of water (2.5 mg/L above the determined KMnO4 demand) or 2 mg of CuSO4/L of water (total alkalinity = 207 mg/L; total hardness = 95 mg/L) were randomly applied to 4 tanks/treatment (23 fish/tank); the same treatments were reapplied 2 d later. Four tanks were used as positive controls. By 2 d posttreatment (after the second treatment), only 17.4% of the untreated control fish survived, and a sample of the remaining fish was heavily infested with I. necator. All remaining control fish were dead by 5 d posttreatment. The KMnO4 treatment significantly curtailed the initial mortality (survival = 92.4%) and slightly reduced the high parasite loads at 2 d posttreatment. However, fish mortalities increased dramatically over the next 3 d (survival at 5 d posttreatment = 37.5%), and parasite loads from sampled fish remained high. The CuSO4 treatment was effective in significantly lowering the parasite load (almost eliminating I. necator) and maintaining a high fish survival (87.5%) by 5 d posttreatment. The findings in this study clearly demonstrate that CuSO4 is a viable treatment for ichthyobodosis in tanks.

Previous studies have shown that IFN-gamma is essential for the pathogenesis of cerebral malaria (CM) induced by Plasmodium berghei ANKA (PbA) in mice. However, the exact role of IFN-gamma in the pathway (s) leading to CM has not yet been described. Here, we used 129P2Sv/ev mice which develop CM between 7 and 14 days post-infection with PbA. In this strain, both CD4(+) and CD8(+) T cells were involved in the effector phase of CM. When 129P2Sv/ev mice deficient in the IFN-gamma receptor alpha chain (IFN-gammaR1) were infected with PbA, CM did not occur. Migration of leucocytes to the brain at the time of CM was observed in wild type (WT) but not in deficient mice. However, in the latter, there was an accumulation of T cells in the lungs. Analysis of chemokines and their receptors in WT and in deficient mice revealed a complex, organ-specific pattern of expression. Up-regulation of RANTES/CCL5, IP-10/CCL3 and CCR2 was associated with leucocyte migration to the brain and increased expression of MCP-1/CCL2, IP-10/CCL3 and CCR5 with leucocyte migration to the lung. This shows that IFN-gamma controls trafficking of pathogenic T cells in the brain, thus providing an explanation for the organ-specific pathology induced by PbA infection.

The faucet snail Bithynia tentaculata, a nonindigenous aquatic snail from Eurasia, was introduced into Lake Michigan in 1871 and has spread to the mid-Atlantic states, the Great Lakes region, Montana, and most recently, the Mississippi River. The faucet snail serves as intermediate host for several trematodes that have caused large-scale mortality among water birds, primarily in the Great Lakes region and Montana. It is important to limit the spread of the faucet snail; small fisheries equipment can serve as a method of snail distribution. Treatments with chemical disinfection, pH extremes, and heated water baths were tested to determine their effectiveness as a disinfectant for small fisheries equipment. Two treatments eliminated all test snails: (1) a 24-h exposure to Hydrothol 191 at a concentration of at least 20 mg/L and (2) a treatment with 50°C heated water for 1 min or longer. Faucet snails were highly resistant to ethanol, NaCl, formalin, Lysol, potassium permanganate, copper sulfate, Ba...

An experiment was performed to evaluate the in vitro and in vivo efficacy of potassium permanganate (KMnO4) against Flavobacterium columnare. In vitro, F. columnare treated with KMnO4 at 2 mg/L for 8 h exhibited a 70% reduction in colony-forming units (CFU). A minimum KMnO4 concentration of 10 mg/L was needed to inhibit bacterial growth. An acute and systemic experimental infection was produced in channel catfish Ictalurus punctatus by waterborne exposure to the bacteria after mechanical cutaneous abrasion to remove mucus and epithelium. At 22 h postchallenge, an 8-h treatment with KMnO4 at 2.3 mg/L (2.0 mg/L above the average KMnO4 demand of 0.3 mg/L) was initiated. This did not reduce mortality in experimentally infected fish. The infection model utilized in the experiment was evaluated by examining the clinical signs and histopathology of infected fish. Fish in the model showed columnaris signs similar to those of a natural infection, including skin depigmentation and ulceration and gill necro...

The red-rim melania Melanoides tuberculata, a subtropical and tropical snail, is a nonindigenous species that has become established and is spreading in the United States. Of concern is the potential of the red-rim melania to displace native snail populations and to transmit trematodes that cause serious problems. One of these, a fish gill trematode, Centrocestus formosanus, has negatively affected U.S. commercial and wild fish stocks, including some endangered species. The snail has an operculum that can protect it from desiccation and can remain viable for days on dry fisheries equipment. Thus, contaminated fisheries equipment is suspected as one of the ways the snail is being moved from place to place. A study was conducted to find chemical treatments that would kill 100% of the red-rim melania. Thirteen different chemicals and chemical combinations were evaluated at different concentrations and for various exposure periods. Roccal-D-Plus, Hydrothol 191, niclosamide, and Virkon showed promise ...

Trematode parasites can cause massive infections in commercially raised fish. The most promising approach for the control of these infections is the reduction or elimination of snails that serve as vectors for the trematodes. A recent approach, the application of high concentrations of slurried hydrated lime (SHL) or copper sulfate pentahydrate (CSP) along pond margins (shoreline treatment), has shown promise in reducing populations of the marsh rams-horn snail Planorbella trivolvis (the snail vector of Bolbophorus damnificus and Clinostomum marginatum) in experimental ponds. While CSP has been shown to effectively reduce snail numbers, treatment efficacy and application methods using SHL have not been well defined. Four SHL treatments were evaluated for their ability to kill marsh rams-horn snails in experimental units designed to mimic commercial aquaculture ponds. The treatments were applied at water temperatures of 75–79°F (24–26°C), total alkalinity of 137–257 mg CaCO3/L of water, and total ...

The acute toxicity and highest nonlethal concentration of praziquantel (LC0) were determined in the laboratory for grass carp Ctenopharyngodon idella and golden shiner Notemigonus crysoleucas, two cyprinids known to harbor the Asian tapeworm Bothriocephalus acheilognathi. Praziquantel is an anthelmintic used to treat fish with tapeworms. The 24-h and 96-h LC50 values were 55.1 and 49.7 mg/L for golden shiners (1.3 g) and 63.4 and 60.6 mg/L for grass carp (9.1 g). The 24-h and 96-h LC0 values were 50.0 and 45.0 mg/L for golden shiners and 60.0 and 60.0 mg/L for grass carp.

The proinflammatory cytokine interferon-gamma (IFNγ) recently was shown to play a crucial role in experimental pneumococcal meningitis (PM) pathogenesis, and we aimed in this study to investigate IFNγ-driven nitric oxide synthase-2 (NOS2)-mediated pathogenesis of murine PM. We demonstrate that costimulation of toll-like receptors and IFNγ receptors was synergistic for NOS2 expression in cultured murine microglia. Using an experimental PM model, wild-type mice treated with anti-IFNγ antibody, as well as IFNγ and NOS2 gene knockout (GKO) mice, were inoculated intracerebroventricularly with 10(3) colony-forming units of Streptococcus pneumoniae (WU2 strain). Mice were monitored daily during a 200-h disease course to assess survival rate and blood-brain barrier (BBB) permeability measured at 48 h. IFNγ deficiency was protective in PM, with an approximate 3-fold increase in survival rates in both antibody-treated and IFNγ GKO mice compared to controls (P

A digenetic trematode identified as Bolbophorus sp. has been implicated as a cause of severe mortality and reduced production in commercially raised channel catfish Ictalurus punctatus in the southeastern United States. Currently, chemical control measures to limit the impact of this disease focus on pond shoreline treatments with hydrated lime or copper sulfate to kill the marsh ram's-horn snail Planorbella trivolvis, an intermediate host that inhabits aquatic vegetation around the pond perimeter. Although effective, these treatments have little impact on snails that are not within close proximity to the pond bank. Tests were conducted to determine the feasibility of using elevated levels of copper sulfate pentahydrate (CSP; CuSO4·5H2O), uniformly applied to the surface of the pond water, to eradicate snails from the pond environment. Results demonstrated that uniform application to deliver between 2.5- and 5.0-ppm CSP (0.64–1.27-ppm Cu) was effective in killing snails around the margins of the ...

The most severe complication of Plasmodium falciparum infection is cerebral malaria (CM). Cerebral malaria implies the presence of neurological features, especially impaired consciousness. The treatment of CM is limited to: (i) a few conventional anti-malarial drugs (quinine or artemisinins), (ii) adjunctive treatments (initial stabilisation, blood exchange transfusion, osmotic diuretics and correction of hypoglycaemia, acidosis and hypovolaemia) and (iii) immunomodulation. There are clear procedures concerning treatment of CM, which include the use of the anti-plasmodial drugs. Adjunctive treatments are permissible but there is no single official guideline and immune intervention is a possibility currently being examined in rodent models only. The suggested immunomodulation approach is based on the strong likelihood that CM is the result of an immunopathological process. P. falciparum initiates the multifactorial chain of events leading to lethal CM and, after a certain stage, it is impossible to stop the progression even by using anti-malarial drugs. We present evidence that CM is a result of a dysregulated immune response. Therefore, it might be prevented by early modulation of discrete factors that participate in this process. In experimental systems, some immunomodulators delay or prevent CM without affecting the parasitaemia. Therefore, in the future the ultimate treatment of CM may be a combination of an anti-malarial and an immunomodulator. However, the overall effect of an immunomodulator would need to be carefully examined in view of concomitant infections, especially in malaria endemic areas.

On 16 November 1963, the U. S. Fish and Wildlife Service Fish Farming Experimental Station at Stuttgart, Arkansas, became the first institution to import grass carp (Ctenopharyngodon idella) into the United States. This introduction was the result of at least seven years of effort to find an effective biological control for problematic aquatic weeds. The introduction was in keeping with a strong environmental and political mandate of that day to replace the broad use of chemicals with biological controls. For about 10 years, federal and state agencies and university systems strongly promoted introductions, spawning, and nationwide stocking of the grass carp. In 1966, the USFWS laboratory at Stuttgart, Arkansas, was apparently responsible for the first accidental release of grass carp to the environment. By 1972, grass carp were stocked in open water systems, documented in 16 states, and established in the Mississippi River system. All this occurred before the first private-sector commercial produ...

Centrocestus formosanus, the gill trematode, has caused serious losses among fish raised by tropical fish producers since the early 1980s and is believed to be harmful to wild fish populations, including the federally listed endangered fountain darter (Etheostoma fonticola), in the Comal River near San Marcos, Texas. The parasite appears to infect in many fishes from Hawaii, Florida, Texas, and Utah. The gill trematode has a complex life cycle involving definitive hosts (aquatic birds and occasionally some mammals) and intermediate hosts (aquatic snails and several fish species). In the United States, the green heron (Butorides virescens) and the great egret (Ardea alba) serve as definitive hosts. The first intermediate host, the red-rim melania (Melanoides tuberculatus), an exotic snail, has been found in 15 southern and western states. This snail exhibits resistance to desiccation, molluscicides, and disinfectants and has been documented to out-compete established mollusks. Both the gill tremat...

The red-rim melania Melanoides tuberculatus (family Thiaridae), a tropical, nonindigenous aquatic snail, has become established and is spreading in the United States. Concerns associated with the spread of this snail include its potential to displace native snail populations and to transmit trematodes. Of particular concern is the gill trematode Centrocestus formosanus now found in U.S. commercial and wild fish stocks. The snail survives year-round in subtropical ponds and constant-temperature spring runs in Florida and Texas and in geothermal waters of several western and midwestern states. Knowledge of the temperature tolerances of the snail will increase the ability to predict its potential range in North American waters and to control its spread. Under constant temperature conditions in the laboratory, all red-rim melania (15–25 mm shell height (SH)) were killed by exposure to 5°C for 1 d, 9°C for 2 d, 11°C for 8 d, and 13°C for 12 d. At 17°C and 32.5°C, about 10% of the snails died within 27...

Products of the kynurenine pathway of tryptophan metabolism have been implicated in the pathogenesis of murine and human cerebral malaria. Indoleamine 2,3-dioxygenase is the first and rate-limiting enzyme in this pathway and we have developed an immunohistochemical method for its detection in tissues from normal and malaria-infected mice. Mice were infected with Plasmodium berghei ANKA, a murine model of cerebral malaria, or P. berghei K173, a non-cerebral malaria model. Vascular endothelial cells were the primary sites of indoleamine 2,3-dioxygenase expression in both types of malaria infection and this response was systemic, with positive staining of vascular endothelium in all tissues examined. No indoleamine 2,3-dioxygenase expression was detected in uninfected or interferon-gamma-/- mice. Corroborative data were obtained using quantitative reverse transcription PCR for indoleamine 2,3-dioxygenase mRNA. These results suggest that interferon-gamma-dependent indoleamine 2,3-dioxygenase expression is part of a normal systemic host response to the parasite, perhaps performing some tissue protective functions that may become deranged under some circumstances and contribute to the pathogenesis of cerebral malaria. On the other hand, constitutive indoleamine 2,3-dioxygenase expression in the epididymis and the placenta was detected in both C57Bl/6 wild-type and interferon-gamma-/- mice, suggesting a distinct regulatory mechanism for its induction in these normal physiological situations. Although increased indoleamine 2,3-dioxygenase production during murine malaria infection may not by itself cause cerebral pathology, metabolites of the kynurenine pathway may combine with other features of cerebral malaria, such as breakdown of the blood-brain barrier, to influence CNS function and contribute to the symptoms and pathology observed.

Many states ban the importation of fish infected with the Asian tapeworm Bothriocephalus acheilognathi. Treatment with an effective tapeworm parasiticide prior to shipment would allow fish to be imported that might otherwise be rejected. In this study, extended praziquantel bath treatments of infected grass carp Ctenopharyngodon idella were tested to determine their efficacy against Asian tapeworms. Preliminary praziquantel treatments of 0.7 mg/L or more for an exposure period of 24 h at grass carp densities of 69 g/L were effective in eliminating all tapeworms from 22-g fish. When the treatment duration was lowered to 12 h, a praziquantel concentration of 2.8 mg/L was required for effective treatment. In replicated studies, significantly reduced tapeworm numbers (0–5 tapeworms per treated fish compared with 30+ tapeworms per control fish) were observed following 24-h exposures to praziquantel at 0.5, 0.7, 1.0, and 1.5 mg/ L. Tapeworm elimination occurred only at the maximum concentration of 1.5 ...

Tests were run to determine whether any refinements were warranted in an established copper sulfate–citric acid pond shoreline treatment (Environmental Protection Agency registration number 1278–8) for aquatic marsh rams-horn snails Planorbella trivolvis. The treatments applied in these studies consisted of two levels (full and half) of copper sulfate (CuSO4; 589 and 294.5 g) with and without citric acid (CA; 58.9 and 0 g) that were delivered in a 2-m swath 10-m length of shoreline. In research ponds, shoreline treatments without CA were significantly more effective against the snails than the treatments with CA. The full CuSO4 treatment without CA was significantly more effective than the half treatment without CA at all temperatures tested below 35°C. There was a significant reduction in the effectiveness of the full treatment without CA at 17 ± 0.5°C (40% snail survival), while snail survival (average, 3.2%) in the trials run at 21.5, 23, 26, and 35°C was not significantly different from each ...

Largemouth bass Micropterus salmoides are piscivorous fish raised on farms then sold live in Asian fish markets on the east and west coasts of the United States. In the winter of 1998, a major producer of feed-trained bass suffered a significant increase in fish mortality both during shipping and while the fish were still in ponds. No bacterial, viral, or significant parasitic pathogens were found at necropsy. Livers of affected fish were pale and translucent with 3–10 mm pink nodules on their surface and deeper in the parenchyma. Histological examination of these livers showed that the translucent regions of the liver contained few hepatocytes and were composed of tissue consistent with a chronic inflammatory response. Also present were eosinophils, islands of pancreatic and biliary cells, and granulomas that did not stain positively for mycobacteria. The pink nodules were areas of multifocal regeneration of normal hepatocytes. Sequential studies of bass in ponds revealed that the bass were progressively accumulating glycogen in their hepatocytes to an extent sufficient to explain the massive necrosis of that organ. In order to determine the effect of diets varying in available carbohydrate on fish growth, survival, and liver glycogen content, a 12-wk feeding trial was conducted in aquaria with juvenile largemouth bass. Nitrogen-free extract values indicated that an extruded trout diet, a steelhead trout diet, and a diet designed to contain 45% protein and 25% fat, contained 35, 27, and 21% carbohydrate, respectively. Weight gain was lowest in fish fed the extruded trout diet, while liver glycogen was significantly higher in fish fed diets with >27% carbohydrate than in fish fed the diet with 21% carbohydrate. The farmer switched to a diet similar to the 45–25 diet used in our trial. Subsequently, 16-mo-old fish examined in October 2000 had no hepatic nodules or necrosis, were significantly larger and less variable in size than previous crops, and the farmer experienced no significant losses in ponds or during shipping.

The rams-horn snail Planorbella trivolvis carries at least two important digenetic trematodes that infect propagated fish species in the southeastern United States. These snails are found in fish production ponds, and there are no proven chemical methods for eliminating them that would not also kill the fish. Application of an aqueous solution of 589 g of copper sulfate and 58.9 g of citric acid per 10 linear meters in a 2-m-wide swath along the pond shoreline produced an effective treatment. This would give an instantaneous treatment rate of about 59 ppm if the water in the 2-m swath averaged 0.5 m deep. In two separate trials, there was a significant difference in snail survival in the treated and sham-treated ponds. Average survival was 2.2% and 0% in the treatment ponds and 63.3% and 77.8% in the sham treatment ponds.