A.Jerald Ainsworth

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.

Fish granulocytes: Morphology, distribution, and function

Abstract In the last 20 years interest in fish granulocyte identification, distribution, and function has burgeoned, as evidenced by the increasing number of reports in scientific literature. Neutrophils, eosinophils, and basophils are present in peripheral blood and certain organs of fish. Some species of fish have all three cell types, while other species may only possess one cell type. Granulopoiesis in fish occurs in the spleen, kidneys, epigonal organ, organ of Leydig, and other specialized tissue, with the specific locations depending on the species. In some fish, neutrophils are actively phagocytic. In vitro assays indicate phagocytosis, mobilization in response to chemotaxins, detectable chemiluminescence, and an active respiratory burst when appropriately stimulated. In fish species without neutrophils, the eosinophil may be responsible for phagocytosis. Eosinophils also function in antiparasite immunity in certain species. Basophil function has not been investigated. Responses of fish neutrophils and eosinophils can be altered by season of the year, environmental pollutants, disease, and others stressors. Differences among fish species in type, cellular distribution, and function of granulocytes are the focus of this review.

Differences in the phagocytosis of four bacteria by channel catfish neutrophils

The purpose of this study was to compare the phagocytosis of Aeromonas hydrophila, Edwardsiella ictaluri, Edwardsiella tarda and Micrococcus luteus by channel catfish neutrophils. Various aspects of opsonization effect and bactericidal ability of channel catfish neutrophils were investigated. Percent phagocytosis ranged from a low of 1% to a high of 91%. The highest percent phagocytosis and phagocytic indices routinely occurred with normal serum and were highest for M. luteus (91.78%, 55.25) and A. hydrophila (87.52%, 43.60). In all cases, the percent phagocytosis and phagocytic index was lowest in assays without serum. Channel catfish neutrophils displayed a bactericidal/static ability for each bacterium tested except E. tarda. Neutrophils exhibited a greater inhibitory capacity for A. hydrophila and M. luteus than for the other bacteria when in the presence of normal or inactivated catfish serum.

Listeria monocytogenes Subgroups IIIA, IIIB, and IIIC Delineate Genetically Distinct Populations with Varied Pathogenic Potential

ABSTRACT Listeria monocytogenes lineage III strains belonging to subgroups IIIA (n = 8), IIIB (n = 5), and IIIC (n = 6) were examined along with other known serotype strains (n = 11) by PCR and Southern hybridization using several recently described species-, virulence-, and serotype-specific primers and probes. The virulence of seven representative lineage III strains was then evaluated in mice via the intraperitoneal route. The results suggest that subgroup IIIA consists of typical rhamnose-positive avirulent serotype 4a and virulent serotype 4c strains, subgroup IIIC consists of atypical rhamnose-negative virulent serotype 4c strains, and subgroup IIIB consists of atypical rhamnose-negative virulent non-serotype 4a and non-serotype 4c strains, some of which may be related to serotype 7. It is possible that subgroup IIIB (including serotype 7) may represent a novel subspecies within L. monocytogenes.

Effect of temperature on the immune system of channel catfish (Ictalurus punctatus). I : Leucocyte distribution and phagocyte function in the anterior kidney at 10°C

1. Temperatures of 18 degrees C for acclimation or assay had minimal or no effect on channel catfish phagocyte function. Significant suppression was observed at 10 degrees C acclimation and assay temperature. 2. According to the results of a multiple acclimation/assay temperature combination study, the primary impact of temperature on phagocyte function was due to the assay temperature. 3. The only functional change caused by acclimation temperature was the possible adaptation of the respiratory burst. However, 10 degrees C acclimation did cause a decline in the number of lymphocytes in the anterior kidney but not the number of neutrophils. In a temperature-kinetic study, the suppressive effect of 10 degrees C assay temperature was confirmed. 4. Results of our study indicated that phagocytosis in channel catfish is temperature-mediated. However, phagocytes appeared to be more resistant to low temperature than lymphocytes, which implies the importance of phagocytosis in the defense mechanisms of channel catfish at low temperatures.

A β-glucan inhibitable zymosan receptor on channel catfish neutrophils

In mice and humans zymosan binds to the complement receptor three/Mac-1 receptor; however, identification of this receptor in channel catfish (Ictalurus punctatus) has not been accomplished. Soluble fluorescein isothiocyanate (FITC) conjugated beta-glucan, a component of zymosan, was found to bind to channel catfish anterior kidney (AK) neutrophils but not to B-lymphocytes. Serum activated zymosan (SAZ)-mediated chemiluminescence responses of channel catfish AK neutrophils could be inhibited by beta-glucan but not by mannan, and inhibition of chemiluminescence responses by beta-glucan was dose dependent. Similarly, phagocytosis of FITC-SAZ could be inhibited by beta-glucan in a dose-dependent manner. Treatment of channel catfish AK neutrophils with various concentrations of trypsin resulted in inhibition of phagocytosis of FITC-SAZ but not of Aeromonas hydrophila indicating that A. hydrophila phagocytosis was mediated by a trypsin-resistant receptor. Deleting serum or using heat-inactivated serum in the mixtures for the chemiluminescence and FITC-SAZ phagocytosis assays resulted in baseline readings. These data indicate that the beta-glucan component of zymosan is responsible for zymosan phagocytosis. Furthermore, the recognition of zymosan by a specific receptor is evident based on trypsin sensitivity assays. Based on these results it is proposed that a complement receptor 3, Mac-1-like receptor, is present on channel catfish AK neutrophils.

Listeria monocytogenes Serotype 4b Strains Belonging to Lineages I and III Possess Distinct Molecular Features

ABSTRACT A collection of Listeria monocytogenes serotype 4b strains belonging to lineages I and III were examined by PCR and Southern blot analysis using species-, virulence-, and serotype-specific primers and probes. Whereas four serotype 4b lineage I strains reacted in PCR with the serotype 4b-, 4d-, and 4e-specific ORF2110 and virulence-specific lmo1134 and lmo2821 primers, all nine serotype 4b lineage III strains were negative by ORF2110 and lmo1134 primers. In addition, the nine serotype 4b lineage III strains formed two separate groups through their reactions in PCR with virulence-specific lmo2821 primers. Southern blot analysis using species-specific lmo0733 and virulence-specific lmo2821 gene probes largely confirmed the PCR results. These findings indicate that L. monocytogenes serotype 4b strains belonging to lineages I and III possess distinct molecular features.

Identification of Listeria innocua by PCR targeting a putative transcriptional regulator gene

Listeria innocua is a common, non-pathogenic bacterial species that shares morphological, biochemical and molecular characteristics with the pathogenic species L. monocytogenes. The presence of L. innocua may cause difficulty or confusion in the laboratory identification of L. monocytogenes or other Listeria spp. In this report, through examining the recently published genome sequence of L. innocua strain CLIP 11262 (serovar 6a), we identified a L. innocua-specific gene (lin0464) encoding a putative transcriptional regulator and evaluated its efficacy for species-specific detection by polymerase chain reaction (PCR). The specificity of the oligonucleotide primers (lin0464F and lin0464R) derived from this gene was confirmed with the formation of a 749-bp fragment in PCR from genomic DNA of L. innocua strains only. We expect that this assay will be useful in confirming identification of L. innocua or in studies where rapid detection of L. innocua is necessary.

Differential cytochemical staining characteristics of channel catfish leukocytes identify cell populations in lymphoid organs

This is one of the first characterizations of channel catfish (Ictalurus punctatus) leukocytes by enzyme cytochemistry. Leukocytes demonstrated cytoplasmic staining patterns very similar to mammalian leukocytes when stained with acid phosphatase, alpha-naphthyl butyrate esterase, beta-glucuronidase, alpha-naphthyl acetate esterase, Sudan Black B and anti-immunoglobulin specific immunohistochemistry. Lymphocytes, monocytes, macrophages, neutrophils, and surface immunoglobulin positive (surface Ig+) cells were present in channel catfish renal hematopoietic tissue and spleen and demonstrated distinctive cytoplasmic foci staining patterns, cytoplasmic blushing or cell membrane staining. Monocytes, macrophages, lymphocytes and surface Ig+ cells were present in the thymus. Thymic and splenic cellular organization appeared very similar to these same mammalian tissues. In the thymus, acid phosphatase positive cells were distributed throughout the parenchyma, while alpha-naphthyl butyrate esterase and beta-glucuronidase positive cells were concentrated in the cortex and the medulla, respectively. Surface immunoglobulin positive cells occurred in the cortex. In the spleen, acid phosphatase positive cells were scattered throughout the parenchyma, while alpha-naphthyl butyrate esterase positive cells were scattered throughout the parenchyma and adjacent to splenic arterioles. Beta-glucuronidase and surface immunoglobulin positive cells were restricted to immediately adjacent to splenic arterioles. Sudan Black B positive cells were scattered throughout the parenchyma, while alpha-naphthyl acetate esterase positive cells occurred adjacent to peri-arteriole lymphoid sheaths and appear very similar to mammalian metallophils.

Serum Cortisol Levels in Channel Catfish, from Production Ponds

Abstract Serum cortisol levels were measured in pond cultured channel catfish to determine if time of year or stocking density influenced this serum parameter. A significant effect of sampling month was found on cortisol concentration with values obtained in September and December being the highest. Stocking density also significantly influenced coritsol concentration with 9,800 fish/ha having the lowest mean cortisol concentration when compared to densities of 14,800/ha and 19,800/ha. Care must be exercised when fish are sampled due to the rapidity with which cortisol concentrations will change in response to stress.