George A. Ingram

Noncellular nonspecific defence mechanisms of fish

Abstract Fish tissues and body fluids contain naturally occurring proteins or glycoproteins of non-immunoglobulin (Ig) nature that react with a diverse array of environmental antigens and may confer an undefined degree of natural immunity to fish. They consist of microbial growth inhibitory compounds that include “acute phase” proteins such as transferrins, caeruloplasmin, and metallothionein. Their action is simply to chelate metal ions and deprive bacteria and other parasites of essential inorganic ion sources. Both serum and cellular interferons are found in fish, and this anti-virus protein has been demonstrated mainly in salmonids during viral disease studies. Enzyme-inhibitors (α2-macroglobulin and other α-globulins) thus far detected in fish appear to be antibacterial proteinases, and are involved in the inhibition of extracellular proteases secreted by fish pathogens. Fish also possess a variety of relatively specific lytic molecules that cause cell lysis, and some of these materials are hydrolase enzymes (lysozyme, chitinase, chitobiase) whose main actions are against bacteria and fungi. In addition, mucus contains trypsin-like proteinases which destroy gram-negative bacteria. Nonspecific lysins and agglutinins against erythrocytes and other cellular antigens are found in serum, eggs, and skin mucus. The lysins, including toxins, some of which are bacteriolytic in activity, are, in their mode of action, natural or spontaneous, antibody-independent and noncomplement-mediated. In contrast, specific hemolytic antibodies (Ig), which complex with antigens, bind complement, and cause complement-mediated immune lysis, are reported to exist. The agglutinins are generally reactive toward certain sugar residues on erythrocyte or bacterial cell walls, and in most cases act as lectins or lectin-like molecules. Natural lysins and agglutinins behave in a similar way as antigen-induced antibodies or Igs, but exhibit a high degree of cross-reactions, due to the occurrence of similar carbohydrate determinants on many types of microbial cell surface. As with mammals, both C-type (calcium-dependent) and S-type (thiol-dependent) lectins are present in fish. They more resemble invertebrate lectins than those of higher animals. Fish lectins appear to play antibacterial or antifungal roles and in some instances seem to be involved in egg-sperm fusion, polyspermy prevention, and embryo development. Natural, non-Ig precipitins (e.g. α-precipitin and C-reactive proteins) are found largely, but not exclusively, in fish serum and precipitate with simple monosaccharides or long chain polysaccharides of certain stereochemistry and glycosidic linkages. Their functions remain unknown, but C-reactive protein is induced following stress-induction and exposure to inflammatory agents. Many of the above mentioned “defence” substances are present in skin mucus and possess the capacity to react with potentially infective microorganisms including parasites. Mucus thus acts as an immediate defence barrier to invasion and/or colonisation of pathogens.

Lectins (haemagglutinins) in the haemolymph of Glossina fuscipes fuscipes: Isolation, partial characterization, selected physico-chemical properties and carbohydrate-binding specificities

Abstract Glossina fuscipes fuscipes haemolymph contained agglutinins (lectins), titre range 2 −11 –2 −18 , against red blood cells (RBC) of human ABO(H) blood group with highest values detected against “AB” RBC. The use of protease- and neuraminidase-treated RBC in many cases increased titres whilst treatment with galactosidases or glucosidases caused decreased levels. Haemolymph adsorption with “O” RBC reduced titres against “O” and “AB” but to a lesser extent anti-A or -B activity indicating lectin heterogeneity. The carbohydrate-binding specificities for human RBC were directed towards N -acetylated and deoxy derivatives of glucose and/or galactose. In addition the haemagglutinins were reactive against some oligosaccharides, ribose, deoxymannose, deoxygalactose, xylose and xylan with certain of the RBC types. The agglutinins were glycoprotein in nature, thermo-labile, affected by storage, freezing and thawing treatments and exposure to a high dosage of γ-radiation, possessed limited disulphide and hydrogen bonds, and depended upon slightly acid to neutral conditions for optimum agglutination. The haemag-glutinins did not require the presence of divalent cations (Ca 2+ , Mn 2+ or Cu 2+ ions) for activity although an elevated concentration of Mg 2+ ions resulted in increased endpoint titres. However heavy metal ions (Pb 2+ and Fe 2+ ) in the buffer lowered agglutinin levels. The intact lectin molecule had an isoelectric point of 6.2, a relative molecular weight of 710 kDa and comprised approx. 70 kDa subunits.

Sugar specificities of anti-human ABO(H) blood group erythrocyte agglutinins (lectins) and haemolytic activity in the haemolymph and gut extracts of three Glossina species

Relatively heat-labile, human ABO(H) blood group non-specific lectins or lectin-like agglutinins, titre range 2−9–2−16, were detected in Glossina morsitans morsitans, Glossina palpalis gambiensis and Glossina tachinoides haemolymph. The haemagglutinins exhibited wide specificities for carbohydrate residues on the surface of human erythrocytes, indicative of heterogeneity, which varied according to the tsetse species examined and the type of erythrocyte used. Haemolymph agglutinin reactivities were directed mainly towards sorbose, trehalose, glucose, 2-deoxygalactose and to a lesser extent the deoxy, [1–4]- and/or [1–6]-linked derivatives of glucose. Occasionally fructose, mannose, sucrose, turanose, stachyose and melezitose minimally inhibited agglutination. Midgut haemagglutinins, titres 2−6 or 2−7, were only found in G. m. morsitans exclusively against “AB” erthrocytes whilst hindgut extracts in all threee Glossina species caused agglutination (titres 2−1–2−7) of most erythrocyte types. Heat-labile, possibly protease but not trypsin, haemolytic molecules were present in most gut preparations. Conversely, a non-proteolytic, partially thermostable haemolysin(s) was detected in G. m. morsitans midgut samples. Gut haemagglutinin specificities were less diverse than those of haemolymph and effective agglutination inhibitors were glucose, galactose or mannose and their deoxy, aminated and N-acetylated derivatives. Additionally sorbose, sucrose, turanose, gluconic acid and methyl glucoside inhibited in G. m. morsitans. Both agglutinin and lytic activities were either negated or reduced following freezing and thawing treatments of gut extracts and haemolymph.

Naturally occurring agglutinins against trypanosomatid flagellates in the haemolymph of insects

In vitro studies of the behaviour of the trypanosomatid flagellates Trypanosoma brucei and Leishmania hertigi in the presence of cell-free haemolymph of locusts, Schistocerca gregaria and cockroaches, Periplaneta americana revealed the presence of parasite agglutinins. The range of normal values of agglutination titres was 2(-4) to 2(-13). Physico-chemical treatment of haemolymph indicated that these agglutinins are protein or glycoprotein in nature and are only partially affected by heat treatment below 65 degrees C, at which temperature incubation of haemolymph for 30 min abrogated all agglutination. Agglutination was not dependent on the presence of Ca2+ or Mg2+. Prior injection of locusts and cockroaches with T. brucei and L. hertigi significantly increased agglutinin titres between Days 4 and 6 in cockroaches (P less than 0.05) and from Days 2 to 4 when L. hertigi was inoculated into locusts. The induced differences in titres observed in locusts infected with T. brucei were not significant. Lysozyme levels were significantly increased after inoculation of T. brucei into cockroaches compared with placebo-inoculated and uninoculated controls. L. hertigi inoculation produced significant increases in lysozyme levels compared with controls between Days 1 and 7 in locusts and 3 to 6 in cockroaches. These studies indicate that, at least in easily manipulated model systems, induced responses to intrahaemocoelic inoculation to trypanosomes and Leishmania can occur. As far as we are aware this is the first report of an induced response of an insect to such important parasites. The possibility that induced responses in natural vector to this parasites occurs requires investigation.

The humoral immune response of the spiny-tailed agamid lizard (Agama caudospinosum) to injection with Leishmania agamae promastigotes

Spiny-tailed agamid lizards (Agama caudospinosum) were given a single intraperitoneal injection of Leishmania agamae promastigotes. Direct agglutinins (DA), indirect haemagglutinins (IHA) and complement-fixing antibodies (CFA) produced against the parasites were non-precipitating, relatively thermostable and dithiothreitol sensitive. Antibodies were also detected by the immobilisation test (IMM) and by enzyme-linked immunosorbent assay (ELISA). The most sensitive method was the ELISA one. Antibodies were detected 7 days post-injection and maximum IMM (2(-6)), DA (2(-8)), CFA (2(-10)), IHA (2(-12)) and ELISA (2(-16)) titres were obtained from 35 to 49 days. In all cases, the levels of antibody following antigenic stimulation were significantly different from the controls (P less than 0.001). Serum lysozyme levels increased three-fold (P less than 0.001) with the highest value of 46 micrograms/ml occurring after 42 days.

Carbohydrate-binding specificities of anti-erythrocyte lectins (haemagglutinins) in Anopheles gambiae gut extracts and haemolymph.

Abstract. Lectins that agglutinate red blood cells (RBC) were demonstrated in Anopheles gambiae mosquito haemolymph and gut extracts. No apparent differences in haemagglutinin titres were detected between male and female mosquitoes and overall agglutinin levels were not increased following a bloodmeal.

Lectins and lectin-like molecules in lower plants — I. Marine algae

Marine algae possess agglutinins, generally of unknown carbohydrate specificity, against a diverse array of erythrocytes but in a small number of species these haemagglutinins react with certain human blood group types. In seaweeds, lectins or lectin-like molecules are involved in gamete recognition and consequent reproductive cell fusion. Although some substances produced by algae are antiviral, antifungal, antibacterial, haemolytic and toxic, no similarity to mammalian immunoglobulins either structurally or physico-chemically has been observed for algal agglutinins. Whether such compounds have a defence or immune function and perform an antibody-like role to enable algal species to survive and counteract infections within their environment remains tentative.

Comparative study of haemagglutination activity in the haemolymph of three tsetse fly Glossina species

1. 1. Glossina morsitans morsitans (Gmm), G. palpalis gambiensis (Gpg) and G. tachinoides (Gt) haemolymph possessed multiple, glycoproteinaceous haemagglutinins (HGN). 2. 2. Tsetse HGN bind to human erythrocyte surface glycoprotein/glycopeptide residues or, with Gmm and Gpg anti-0 activity, glycolipid moieties. 3. 3. Variations in HGN physico-chemical properties occurred between the morsitans (Gmm) and palpalis (Gpg and Gt), and amongst the palpalis, groups of flies with respect to relative heat-lability, susceptibility to dithiothreitol reduction, resistance to γ-radiation exposure and sensitivity to urea treatment. 4. 4. Gt and Gmm required acid and acid to neutral conditions respectively, and Ca2+ ion presence, for optimum agglutination activity whilst Gpg required neutral to alkaline pH and Mg2+ ions. 5. 5. The findings reported here provide further information regarding HGN (lectin) properties in different species of the genus Glossina, member of the Diptera, a little studied order with respect to insect vector immunity.

Use of fluorescein-labelled lectin binding of salivary glands to distinguish between Anopheles stephensi and An. albimanus species and strains

Abstract Fluorescein isothiocyanate (FITC)-conjugated lectins were used as markers to distinguish different carbohydrate moieties on the surface of the salivary glands of Anopheles stephensi and An. albimanus species and strains. Six FITC-conjugated lectins showed interspecific differences between the two Anopheles species, and intraspecific variations between An. stephensi strains. Both fluorescence and electron microscopy demonstrated clear binding of these lectins. The salivary glands of Plasmodium-infected and uninfected An. stephensi were also examined in order to determine whether salivary gland surface carbohydrates change following infection. However, no variations were observed and it appears that the surface sugars are involved in vector tissue recognition by Plasmodium sporozoites.

Responses of european green lizards Lacerta viridis following administration of Leishmania agamae promastigotes

European green lizards (Lacerta viridis) were injected intraperitoneally, subcutaneously or orally with viable Leishmania agamae promastigotes. Neither promastigotes nor amastigotes were later found in blood and tissue impression smears, or in blood and selected organ cultures. However, by the use of an immunoperoxidase technique, parasite antigens were detected in the liver, stomach, small intestine, kidney, gonad, heart, lung and skin but not in the bone marrow, brain or spleen. Non-precipitating antibodies with beta 2-electrophoretic mobility were induced against L. agamae. They were detected in the sera by enzyme-linked immunosorbent assay 3-7 days post-infection. The titres increased significantly above background levels (P less than 0.001) and reached maxima after 6-7 weeks, with 27 out of 29 lizards producing antibodies. The mean serum protein concentration significantly increased after infection (P less than 0.005) with no significant differences in mean values between male and female animals. Lizard sera separated into 7 components on cellulose acetate membranes with migration rates comparable to albumin, alpha- and beta-globulins of human serum; gamma-globulins were absent. Significant decreases occurred (P less than 0.05) in the albumin fraction, with significant increases in the beta-globulin region of anti-L. agamae sera. C-reactive protein was not detected in either normal or immune lizard sera.